Tuberculosis

TUBERCULOSIS

Springer Berlin Heidelberg New York Hong Kong London Milan Paris Tokyo

M. Monir Madkour (Editor) A. Al Saif . M. Al Shahed . K. R. Al Moutaery A. Al Kudwah (Associate Editors)

Tuberculosis Foreword by

David A. Warrell

With 444 Figures in 846 Separate Illustrations, 139 in Color and 89 Tables

Springer

Editor: M. MONIR MADKOUR

Department of Medicine Armed Forces Hospital Riyadh 11159 Saudi Arabia

Associate Editors: ABDULAZIZ AL SAIF

MONA AL SHAHED

Department of Medicine Armed Forces Hospital Riyadh 11159 Saudi Arabia

Department of Radiology Armed Forces Hospital Riyadh 11159 Saudi Arabia

KALAF AL MOUTAERY

AIDA AL KUDWAH

Department of Neurosurgery Armed Forces Hospital Riyadh 11159 Saudi Arabia

Department of Dermatology Armed Forces Hospital Riyadh 11159 Saudi Arabia

ISBN 3-540-01441-1 Springer-Verlag Berlin Heidelberg Library of Congress Cataloging-in-Publication Data Tuberculosis I M. Monir Madkour, ed p.cm. Includes bibliographical references and index. ISBN 3-540-01441-1 (alk. paper) 1. Tuberculosis. 1. Madkour, M. Monir. RC311.T4172003 616.9'95--dc22

2003059067

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitations, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer-Verlag is part of Springer Science+Business Media httpllwww.springeronline.com © Springer-Verlag Berlin Heidelberg 2004 Printed in Germany The use of general descriptive names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every case the user must check such information by consulting the relevant literature. Cover-Design: Erich Kirchner, Heidelberg Typesetting: Verlagsservice Teichmann, Mauer 2113150xq - 5432 1 0 - Printed on acid-free paper

Foreword

It was through his superb monograph on brucellosis, first published in 1989, that I came to know Dr. M. Monir Madkour and later to meet him in Riyadh. From this introduction, I was quick to enlist his help as an author of the Oxford Textbook ofMedicine. He has now turned his considerable talents as physician, author and editor to a disease at least as interesting but even more important, tuberculosis. Considered against the background of a large literature on this subject, Dr. Madkour's monumental monograph is distinctive in several respects. As he emphasises in his preface, his book, unlike most others published on tuberculosis in English, has not been written by western experts whose experience of the disease is based in developed countries. The Kingdom of Saudi Arabia has afforded the unusual combination of an abundance of clinical material with state-ofthe-art facilities for laboratory diagnosis, investigation, imaging and medical and surgical management. Dr. Madkour and his four associate editors, all from the prestigious Riyadh Armed Forces Hospital, have assembled a distinguished international cast of 64 authors; 29 from the Kingdom of Saudi Arabia, 16 from Europe, 12 from North America, five from Asia and two from Africa. The result is an exciting, comprehensive and original work incorporating a wide range of clinical and scientific skills and experience and carrying enormous authority. Tuberculosis is the most prevalent of all bacterial infections, existing actively or in latent form in a third of the world's population. In the developing world, almost everyone is infected and the tubercle bacillus might almost be regarded as part of the normal human condition. It is not surprising that the disease has played such an important role in the political, social, industrial, artistic and scientific history of mankind. This commanding historical perspective is clearly identified in the two opening chapters. I was fascinated by Dr. Saleh A. Bedeir's review of tuberculosis in Ancient Egypt, although the examples of Pott's disease in remains from the predynastic period may be antedated by neolithic finds near Heidelberg, Germany (5000 B.C.) and Liguria, Italy (4000 B.C.) (Formicola et al. 1987). So was it Europe that gave the world tuberculosis? In Chap. 2 ("Historical aspects of tuberculosis"), Dr. Madkour and colleagues include an important section on the achievements of Arabian medicine during the period 500-1500 A.D., usually regarded as the "Dark Ages" in Europe. The roles of the Baghdad and Cairo Schools during this era, and notably the contributions of Ibn an-Nafis, Rhazes, Avicenna and Albucasis, have certainly been neglected. Understandably, this account does not dwell on the enormous impact of tuberculosis on the arts, literature and science in Europe, especially during the nineteenth century, when this disease determined the creative life of many outstanding authors, artists and composers and supplied many fictional heroes, heroines and story lines. In Chap. 3 ("Global epidemiology of tuberculosis"), Marcos Espinal and Mario Raviglione of WHO Geneva, provide compelling evidence of the current worldwide crisis in tuberculosis control. An annual incidence of some 8 million new cases, with 2 million deaths and a case notification rate of around 50 per 100,000, shows no evidence of decline

despite the availability of effective anti-tuberculosis chemotherapy. The most chilling evidence for a failure of implementation of chemotherapy, the impact of the HIV-AIDS epidemic and emerging multi-drug resistant disease, is given in their Fig. 3.5, which predicts an increase to 10 million new cases in 2010. The epidemiological situation in the Kingdom of Saudi Arabia (Chap. 4) is complicated by the large influx of pilgrims from all over the world during the Hajj. Molecular fingerprinting of Mycobacterium tuberculosis (Chap. 5) has provided fascinating insights into the epidemiology, history and geographical origins of the pathogen, for example into the Cape coloured population of Cape Town (van HeIden 2002). Tuberculosis has become a major occupational health concern for medical personnel and others who work in high-risk communities. This important area is covered by Jaime Esteban in Chap. 7 ("Tuberculosis in Special Groups and Occupational Hazards"). There is a strong section on the microbiology, immunology, pathogenesis and pathology of tuberculosis, including a substantial review of immunology and pathogenesis by Graham Rook (Chap. 9). Pathological changes in the central nervous system are often dominated by secondary effects of obstructive or communicating hydrocephalus or the involvement of major blood vessels or cranial nerves at the base of the brain (Chap. 10). Environmental and other non-tuberculous mycobacteria, many of which have been regarded as virtually non-pathogenic in immunocompetent people, have caused lifethreatening infections in those with subtle immune deficits, for example in the IL-12dependent interferon-gamma pathway (Lammas et al. 2000; Fieschi et al. 2003) (Chap. 11). These rare deficiency syndromes provide insights into normal immune defence mechanisms, as does reactivation of latent tuberculosis by corticsteroids, anti-TNF antibodies and other agents. The section on diagnosis (Chaps. 12-15) covers conventional and newer methods, including PCR. The last decade has witnessed increasing investment in basic science research into the cell biology, genetics and immunology of tuberculosis. A landmark achievement has been the sequencing of the M. tuberculosis genome. However, these advances in the understanding of the biology of the bacillus and the host response have not yet resulted in any tangible improvements in diagnosis and management of tuberculosis patients and their contacts. One area in which recent scientific advances are beginning to make an impact on clinical practice is the diagnosis of latent tuberculosis infection. Until very recently, this relied upon the century-old tuberculin skin test, introduced by Robert Koch (Chap. 15). Blood tests measuring interferon-gamma secretion in response to purified protein derivative (PPD) may offer a more rapid and convenient approach to detecting latent infection. Cellular immune responses to PPD may, however, cross-react with BCG vaccination, just as with the delayed type hypersensitivity response to PPD detected by skin testing, resulting in poor specificity. Comparative mycobacterial genomics has identified a stretch of DNA (region of difference I, RD1) that is present in M. tuberculosis but absent from all strains of BCG vaccine. The antigens encoded by RD 1 genes thus offer the opportunity to develop more specific tests for M. tuberculosis infection. Two of these antigens, ESAT-6 and CFP10, have now been incorporated into a rapid, sensitive blood test that enumerates individual interferon-gamma-secreting T cells, the enzyme-linked immunospot (ELISPOT) assay. In active tuberculosis, EllSPOT appears to have a higher sensitivity and specificity than the tuberculin skin test (Lalvani et al. 2001). In latent infection, the absence of a gold standard reference test makes evaluation of any new test difficult, but recent studies indicate that ELISPOT successfully distinguishes latent infection from BCG vaccination and thus has a higher specificity than skin testing, while the observation that ELISPOT correlates more

closely with M. tuberculosis exposure than skin testing suggests that it may also have a higher sensitivity (Lalvani et al. 2001; Ewer et al. 2003). From the clinical point of view, tuberculosis is the most protean of all diseases, demanding inclusion in the differential diagnosis of almost every symptom, sign and abnormal result of investigation. The organ-related chapters of the book provide a thorough coverage of disease manifestations in the different age groups (Chaps. 16-18), during pregnancy (Chap. 19), in the face of immunosuppression (Chap. 29) and in the various sytems, organs and tissues (Chaps. 20-43). A great strength of the book is its inclusion of a rich variety of high-resolution pictures of clinical cases (see, for example, Chap. 36 on skin manifestations, and Chap. 40 on the eye). The clinical suspicion that allows the crucial early diagnosis of tuberculous meningitis (Chaps. 32 and 33) is particularly subtle, but the diagnosis may be difficult to confirm in the developing world. I have seen too many patients undergoing inappropriate therapeutic trials of antituberculosis chemotherapy for this condition. Tuberculous meningitis is a prevalent and commonly misdiagnosed condition that deserves a far more detailed and lengthy discussion. Compliance to the necessarily prolonged treatment regimens has been improved by the "DOTS" (directly observed treatment) strategy (Chap. 45). Once the correct treatment has been implemented, its efficacy must be monitored (Chap. 47), as the greatest threat to successful cure is drug resistance. Peter Davies provides an authoritative review of this, the most challenging obstacle to cure and control (Chap. 46). Of the many suggested ancillary treatments, corticosteroids are becoming less controversial for pericardial and pleural effusions and for tuberculous meningitis. It is to be hoped that the current large randomised controlled trial of corticosteroids in tuberculous meningitis, being conducted by The Centre for Tropical Diseases in Ho Chi Minh City, Vietnam, will finally provide decisive evidence in this long-running debate. I congratulate Dr. Madkour and his expert team of contributors for producing a timely, thorough, scholarly and truly fascinating account of one of the world's most important and complex diseases. Their book deserves to be consulted by all medical practitioners and to be read in its entirety by all infectious diseases specialists. It will surely become a classic in the literature of this disease. Oxford, UK

DAVID

A. WARRELL

References Ewer K, Deeks J, Alvarez L et al (2003) Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 361: 1168-1173 Fieschi C, Dupuis S, Catherinot E et al (2003) Low penetrance, broad resistance, and favorable outcome of interleukin-12 receptor beta-l deficiency: medical and immunological implications. J Exp Med 197:527-535 Formicola V, Milanesi Q, Scarsini C (1987) Evidence of spinal tuberculosis at the beginning of the fourth millennium B.C. from Arene Candide Cave (Liguria, Italy). American J Physical Anthropology 72:1-6 Van HeIden PD (2002) Molecular epidemiology of TB: challenging dogmas and asking new questions. IUBMB Life 53:219-223 Lalvani A, Pathan AA, McShane H et al (2001) Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am J Respir Crit Care Medicine 163:824-828 Lammas DA, Casanova J-L, Kumararatne DS (2000) Immunodeficiency review. Clinical consequences of defects in the IL-12-dependent interferon-gamma (IFN-gamma) pathway. Clin Exp Immunol 121:417-425

Preface

In 1993, the World Health Organization declared tuberculosis a global emergency. Yet, the incidence of tuberculosis continues to rise. It is not clear, however, whether the rising incidence can be attributed to failure of control strategies, the recent impact of the HIV pandemic or the increasing problem of multidrug resistance. At present, it is estimated that one-third of the world population is infected with Mycobacterium tuberculosis, with over 8 million new cases of active disease detected every year and almost 2 million annual deaths. The impact of the present situation and the current inadequate efforts necessitates the development of a novel and alternative approach to the control and therapy measures. It is interesting to note that while the incidence of tuberculosis is generally low in developed countries, most of the authoritative textbooks on tuberculosis in the English language have been written by western experts. As an expatriate, working in Saudi Arabia, I found a rare opportunity to work in an endemic area yet enjoy the diagnostic and therapeutic facilities of developed countries. Such a combination of circumstances is rarely present in developing or developed countries. The rich clinical material is clearly reflected in this book in the form of "evidence-based" clinical practice enriched with vast numbers of images of various body organs and systems as well as by a thorough survey of all the relevant literature. The numerous figures that are present in a lot of chapters, many in colour, give a vivid picture of what is seen in clinical practice and comprise a special feature of this textbook. Separate imaging chapters were compiled whenever the inclusion with the text would have rendered the corresponding chapter unwieldy. Some of the world's most distinguished medical scientists from North America, South-East Asia, the Far East, South Africa and many European countries have contributed chapters on global epidemiology, molecular epidemiology, the molecular epidemiology of Mycobacterium bovis, polymerase chain reaction (PCR), serological methods, latent tuberculosis, prevention and control, directly observed therapy (DOT), multidrug-resistant tuberculosis, HIV and tuberculosis, thoracic surgery, new vaccines and many other clinical topics. Animal tuberculosis, which may contribute to human disease, is included in a separate chapter. The main goal of this book is to present a clear account of all aspects of tuberculosis as seen and practiced both in developing and developed countries. All authors took this into consideration while writing their chapters. Recent advances in diagnosis, prevention and control measures, and treatment are also addressed. I am humbled by the honour that was given to me by Professor David A. Warrell, Founding Director of the Centre for Tropical Medicine (Emeritus), University of Oxford and editor of the Oxford Textbook of Medicine, in agreeing to write the foreword in this book, and I am most grateful to him. Riyadh

M. MONIR MADKouR

This work is dedicated to: The renaissance of the ancient glorious Library of Alexandria, Egypt, and to those who contributed to its resurrection over the original site. Alexandria was the chief cultural and commercial center of its time, and when the Library was built in approximately 332 Be it attracted manuscripts and scholars from the entire Hellenistic world. It contained over 700,000 rolls of the work of Socrates, Plato, Aristotle, Hippocrates and all the writing of the known world, but was destroyed by a great fire. Virtual images of the ancient Library are available at UNESCO. The inauguration ceremony of the "new--ancient" Library of Alexandria in September 2002 was attended by heads of state, scientists, artists and dignitaries from all over the world. The World Health Organization, for its enormous, determined and unyielding efforts to control tuberculosis, particularly in endemic, poorly resourced, and developing countries.

Acknowledgements: I am indebted to Gen. Dr. S.M. Al Deeb and the Hospital Management Board for providing the facilities I required for this project. I am grateful to our medical library staff: R. Ahtashemuddi, S.Z. Khan, Y.S. AI-Zharani, M.A. Khushi, S.M. AI-Rakaf, S. Al-Salafi, R. AI-Sous, and A. AI-Sultan; our medical record staff: J. Al Anazi, A. Al Humaid and T. Al Shuriti; and our medical illustration staff: S. Nally, T. Michalak, D. Hargreaves, K. Jefferson, N. Morgan, R. Ponmbath, M. Horaib. Support from our colleagues from the department of microbiology, Dr. S. Bakheshwain and Dr. N. EI-Khizzi, is highly appreciated. I would like to thank Dr. M. Sofi and Dr. S.A. AI-Mohaimeed for their support. My colleagues from the department of medicine helped and supported this project, including Drs. T. AI-Tassan, B. AI-Dosary, S. AI-Faraj, A. AI-Zaid, N. AI-Qahtani and many others, and I am grateful. I would like to thank our secretaries for typing the manuscripts of my chapters and for their secretarial help: I. Andries, J. Misquith, E. Sabbagh. I would like to thank my son Amr Madkour, pre-medical student at Austin, Texas for helping me with his experience in the Internet and other computer facilities for this project during his summer vacations. My other children, Rasha, Monir and Reem, helped with typing and sending correspondence bye-mail and with MedLine searches. Without their help, my handicap in computer technology would have been insurmountable. I am most grateful to Dr. Julia Heidelmann, Dr. Ute Heilmann, Ms. Wilma McHugh and the staff at Springer-Verlag for their meticulous care and efforts in the production of this book. Despite the help I have received from others, I alone stand responsible for any shortcomings this book may contain. M. MONIR MADKouR

Contents

Historical Background

.

Tuberculosis in Ancient Egypt SALEH A. BEDEIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Historical Aspects of Tuberculosis M. MONIR MADKOUR, KITAB E. AL-OTAIBI, R. AL SWAILEM

Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 15

31

3 Global Epidemiology of Tuberculosis MARCOS A. ESPINAL and MARIO C. RAVIGLIONE

33

4 Epidemiology of Tuberculosis in Saudi Arabia ABDULRAHMAN A.ALRAJHI and ALI M.AL-BARRAK

45

5 The Molecular Epidemiology of Tuberculosis KATHRYN DERIEMER and CHARLES L DALEY

57

6 Molecular Epidemiology of Mycobacterium bovis ROBIN A SKUCE and SYDNEY D NEILL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

7 Tuberculosis in Special Groups and Occupational Hazards JAIME ESTEBAN

93

Microbiology, Immunology, Pathogenesis and Pathology

113

8 Microbiology of Tuberculosis A OLU OSOBA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 115 9 The Immunology and Pathogenesis of Tuberculosis GRAHAM A. W. ROOK

133

10 Pathology of Tuberculosis MOHAMMED AKHTAR and HADEEL AL MANA

153

11 Nontuberculous Mycobacteria JAE-JOON YIM and STEVEN M. HOLLAND

163

Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 185 12 Serologic Testing for Tuberculosis CHAKHRADHAR KOTARU and EDWARD D. CHAN. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 187

XIV

Contents

13 PCR and Diagnosis of Tuberculosis DIANA LWILLIAMS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 199 14 Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients GORGON AKPEK

213

15 Immunodiagnostics for Latent Tuberculosis Infection ROHIT K. KATIAL

231

Organ-related Chapters

241

General 16 Childhood Tuberculosis PETER R. DONALD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 243 17 Primary Tuberculosis in Adults M. MONIR MADKOUR

265

18 Miliary/Disseminated Tuberculosis M. MONIR MADKOUR

273

19 Tuberculosis and Pregnancy M. MONIR MADKOUR

301

Thorax 20 Post-primary Pulmonary Tuberculosis M. MONIR MADKOUR, Y. ABUSABAAH, ALI BEN MOUSA, ALI AL MASOUD . . . . . . . .. 313 21 Endobronchial Tuberculosis HEE SOON CHUNG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 329 22 Pleural Tuberculosis M. MONIR MADKOUR, MAJDY IDREEs, M. AL SHAHED

349

23 Radiology of Pulmonary Tuberculosis MONA AL SHAHED, MOHAMMED ABD EL BAGI, M. MONIR MADKOUR . . . . . . . . . . .. 359 24 Pulmonary Function Test and Tuberculosis MAJDY M. IDREES, SIRAJ O. WALl, ABDULLA AL-AMOUDI

385

25 Thoracic Surgery for Tuberculosis YUJI SHIRAISHI

395

26 Radionuclides in Pulmonary and Extra-Pulmonary Tuberculosis DAVID HAMILTON and JAWDA AL-NABULSI

411

27 Tuberculosis of the Heart and Pericardium ERNESTO E SALCEDO and AHMAD S OMRAN

431

Contents

xv

Immune System 28 Mycobacterial Lymphadenitis

M. MONIR MADKOUR and RASHID AL KUHAYMI 29 Tuberculosis and Co-infection with the Human Immunodeficiency Virus ALIMUDDIN ZUMLA and JOHN M GRANGE

445 455

eNS

30 Spinal Tuberculosis M. MONIR MADKOUR, M. WASEF AL SEBAI, KHALAF R. AL MOUTAERY .. . . . . . . . .. 481 31 Surgical Management of Spinal Tuberculosis M. WASEF AL SEBAI, M. MONIR MADKOUR, KAHLAF R. AL MOUTAERY . . . . . . . . . .. 493 32 Tuberculosis of the Central Nervous System M. ZUHEIR AL-KAWI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 535 33 Imaging of Brain and Spinal Cord Tuberculosis FRANCIS MCGUINNESS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 547

Musculo-Skeletal and Soft Tissue 34 Extraspinal Musculoskeletal Tuberculosis M. MONIR MADKOUR, AIDA J. AL-KUDWAH, MOHAMMED ABD EL BAGI

587

35 Imaging of Musculoskeletal Tuberculosis MOHAMMED ABD EL BAGI, MONA AL SHAHED, M. MONIR MADKOUR . . . . . . . . . . .. 605 36 Tuberculosis of the Skin AIDA J. AL KUDWAH

627

Gastrointestinal 37 Abdominal Tuberculosis MOHAMMAD SULTAN KHUROO and NAIRA SULTAN KHUROO

659

38 Imaging of Gastrointestinal Tuberculosis MONA AL SHAHED and MOHAMMED ABD EL BAGI

679

Genitourinary 39 Genitourinary Tuberculosis M. MONIR MADKOUR

699

Head 40 Ocular Manifestations of Tuberculosis SHWU-JIUAN SHEU

731

41 Otorhinolaryngeal Aspects of Tuberculosis SUJATA MURANJAN

741

XVI

Contents

Endocrine 42 Endocrine and Metabolic Manifestations of Tuberculosis SOHAIL INAM and MONA AL-SHAHED

751

Vascular 43 Tuberculous Vasculitis and Mycotic Aneurysms M. MONIR MADKOUR

771

Treatment

779

44 Treatment of Tuberculosis DEAN E SCHRAUFNAGEL

781

45 Directly Observed Treatment for Tuberculosis Control ZHANG LI-XING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 801 46 Multi-Drug-Resistant Tuberculosis PETER D. O. DAVIES

809

47 Monitoring Treatment Efficiacy P. H. LAGRANGE, N. SIMONNEY, A. O. SOUSA, A. WARGNIER, J. L. HERRMANN

839

Control and Prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 853 48 Tuberculosis Control in Developing and Developed Countries KEVIN SCHWARTZMAN .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 855 49 BCG and New Tuberculosis Vaccines ZHOU XING

881

50 Tuberculosis in Animals P. L. NICOLETTI

893

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 903 About the Editor

930

List of Contributors

ABD EL BAGI MOHAMMED, MB, BCh, DMRD, FSRRCSI Department of Radiology Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL-KuDWAH AIDA J, MD, DD, FRCP (Ed) Consultant Dermatologist, Department of Dermatology Riyadh Armed Forces Hospital P.O. Box 7897, C-117 Riyadh 11159 Saudi Arabia

ABUSABAAH Y, ABIM Fellow, Respiratory Medicine Department of Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL KUHAYMI RASHID, FRCS Director of Surgery Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AKHTAR MOHAMMED, MD, FCAP, FRCPA, FRCPath Chairman, Department of Pathology and Laboratory Medicine King Faisal Specialist Hospital and Research Centre P.O. Box 3354, MBCI0 Riyadh 11211 Saudi Arabia

AL MANA HADEEL, MD Fellow, Department of Pathology ad Laboratory Medicine King Faisal Specialist Hospital and Research Centre P.O. Box 3354 Riyadh 11211 Saudi Arabia

AKPEK GORGON, MD Assistant Professor of Medicine Greenebaum Cancer Center at University of Maryland School of Medicine 22 South Greene Street Baltimore MD 21201 USA

AL MAsouD ALI, MD Department of Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL-AMOUDI ABDULLA, MD, FRCP (C) Deputy Head, Department of Medicine King Faisal Specialist Hospital & Research Center Jeddah Saudi Arabia

AL MOUTAERY KHALAF R, MD, FRCS (Ed), FACS Head of Neurosurgery Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL-BARRAK ALI M, AmBIM, DTM & H, FRCP (C) Consultant Infectious Diseases Unit Department of Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL-NABULsI JAWDA, DMRD, MSc NM Nuclear Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

AL-KAwI M. ZUHEIR, MD, FACP Senior Consultant Neurologist & Deputy Chairman Department of Neurosciences King Faisal Specialist Hospital and Research Center P.O. Box 3354 Riyadh 11211 Saudi Arabia

AL-OTAIBI KITAB E, Facharzt (Urology), MD, FRCS (Edin) Director General of Medical Services Department Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia

XVIII ALRAJHI ABDULRAHMAN A, MD, MPH, FIDSA Consultant and Head, Section of Infectious Diseases, Chairman, Department of Medicine (MBC #46), King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia AL SAIF A, MD, PhD, FCCP, FRCP (Lon) Consultant Respiratory Medicine Director of Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 1159 Saudi Arabia AL SEBAI M WASEF, FRCS. FFCS Consultant Spinal Surgeon Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia AL SHAHED MONA, MBBS, FRCR Consultant Radiologist Department of Radiology Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia AL SWAILEM R, MRCP (UK), ABIM Consultant Rheumatologist Department of Medicine Director of Postgraduate and Academic Affairs Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia BEDEIR SALEH A, MD Dean, Faculty of Medicine Cairo University Kasr El-Aini Hospitals Cairo Egypt BEN MOUSA ALI, MD Department of Medicine, Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia CHAN EDWARD D, MD Associate Professor of Medicine K613e Goodman Bldg. National Jewish Medical and Research Center, Denver, CO 1400 Jackson St Denver, CO 80206 USA

List of Contributors CHUNG HEE SOON, MD, FCCP Associate Professor of Medicine Seoul National University College of Medicine and Seoul Municipal Boramae Hospital affiliated to Seoul National University Hospital # 395 Shindaebang - 2 Dong Dongjak-Gu, Seoul, 156-707 Korea DALEY CHARLES L, MD Associate Professor of Medicine Division of Pulmonary and Critical Care Medicine San Francisco General Hospital Room 5K-l 1001 Potrero Ave San Francisco, CA 94110 USA DAVIES PETER D 0, DM, FRCP Director, Tuberculosis Research Unit Cardiothoracic Centre Liverpool Ll4 3PE UK DERIEMER KATHRYN, PhD Senior Research Scientist Division of Infectious Diseases and Geographic Medicine Stanford Medical Center, Stanford California, USA DONALD PETER R, MD Department of Paediatrics and Child Health Faculty of Health Sciences University of Stellenbosch P.O. Box 19063 7505, Tygerberg, South Africa ESPINAL MARCOS A, MD World Health Organization Stop TB Department Tuberculosis Strategy & Operations Ave Appia # 22 1211 Geneva Switzerland ESTEBAN JAIME, MD Department of Medical Microbiology Fundaci6n Jimenez Diaz Av. Reyes Cat6licos 2 28040 Madrid Spain GRANGE JOHN M, MSc, MD (Lon) Visiting Prof, Centre for Infectious Diseases & International Health Royal Free and University College Medical School Windeyer Institute of Medical Sciences 46 Cleveland St London WIP 6DB UK

XIX

List of Contributors HAMILTON DAVID, PhD, FIPEM Department of Nuclear Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia HERRMANN J L, MD Service de Microbiologie H6pital Saint Louis Assistance Publique-H6pitaux de Paris Universite Denis Diderot 1, avenue Claude Vellefaux 75475 Paris France HOLLAND STEVEN M, MD Immunopathogenesis Unit Building 10, Room llN103 10 Center Dr. MSC 1886 Bethesda, MD 20892 1886 USA IDREES MAJDY M, MD, FRCP (C), FCCP Head, Pulmonary Function Laboratory Division of Pulmonary Medicine Department of Medicine Riyadh Armed Forces Hospital ClIO, P.O. Box 7897 Riyadh 11159 Saudi Arabia INAM SOHAIL, MBBS, FRCP (Edin), FRCP Head of Endocrinology Division Department of Medicine Riyadh Armed Forces Hospital P.O. Box 7897 Riyadh 11159 Saudi Arabia KATIAL ROHIT K, MD Associate Professor of Medicine National Jewish Medical and Research Center Denver 1400 Jackson St. Denver, CO 80206 USA KHUROO MOHAMMAD SULTAN, MD, DM, FRCP (Edin), MACP Professor, Consultant Gastroenterologist Department of Medicine - MBC 46 King Faisal Specialist Hospital and Research Centre P.O. Box 3354 Riyadh 11211 Saudi Arabia KHUROO NAIRA SULTAN, MBBS Department of Radiology King Faisal Specialist Hospital and Research Centre P.O. Box 3354 Riyadh 11211 Saudi Arabia

KOTARU CHAKRADHAR, MD Department of Medicine and Program in Cell Biology National Jewish Medical and Research Center Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and Denver Veteran Administration Medical Center K613e, Goodman Building 1400 Jackson Street Denver, CO 80206 USA LAGRANGE P H, MD Service de Microbiologie H6pital Saint Louis Assistance Publique-H6pitaux de Paris Universite Denis Diderot 1, avenue Claude Vellefaux 75475 Paris France LI-XING ZHANG, MD Vice President & Secretary General Chinese Anti-Tuberculosis Association 5, Dong Gang Hu-Tong Beijing 100035 China MADKOUR M MONIR, MD, DM, FRCP (London) Consultant Department of Medicine Riyadh Armed Forces Hospital P.O. Box 7897, C-119 Riyadh 11159 Saudi Arabia MCGUINESS FRANCIS, MD Apt. 169, Al Haurin EI Grande 2912 Malaga Spain MURANJAN SUJATA, MS (ENT), DNB, DORL Consultant ENT Suman Apptartment, 3rd Floor 16B Naushir Bharucha Road Tardeo Mumbai 400007 India NEILL SYDNEY D., BSc, PhD Veterinary Sciences Division Department of Agriculture and Rural Development Stoney Road Stormont, Bellfast BT4 3SD UK NICOLETTI P L, DVM, MS College of Veterinary Medicine Department of Veterinary Pathology P.O. Box 110880 Gainseville Florida 32611 0880 USA

xx Ow OSOBA A, MD, FRCPath (UK), FFPath (Ireland), FWACP Consultant & Head of Microbiology

King Khalid National Guard Hospital P.O. Box 9515 Jeddah 2143 Saudi Arabia OMRAN AHMAD S, MD Consultant Cardiologist King Abdulaziz Cardiac Center, King Abdulaziz Medical City, National Guard, Riyadh, Saudi Arabia P.O. Box 22490 Riyadh 11426 Saudi Arabia RAVIGLIONE MARIO C, MD World Health Organization Stop TB Department Ave Appia # 22 1211 Geneva Switzerland ROOK GRAHAM A.W, BA, MB Bchir, MD Professor, Department Medical Microbiology Royal Free and University College Medical School Windeyer Institute of Medical Sciences 46 Cleveland Street London WIT 4JF UK SALCEDO ERNESTO E, MD Department of Cardiology King Abdulaziz Cardiac Center National Guard Hospital P.O. Box 22490 Riyadh 11426 Saudi Arabia SCHRAUFNAGEL DEAN E, MD Department of Medicine M/C 787 University of Illinois at Chicago 840 S. Wood St Chicago, IL 60612 7323 USA SCHWARTZMAN KEVIN, MD, MPH, FRCPC Assistant Professor Department of Medicine and Epidemiology And Biostatistics Respiratory & Epidemiology Unit McGill University 1110 Pine Avenue West Montreal, Quebec Canada H3A lA3 SHEU SHWU-JIUAN, MD Department of Ophthalmology Kaohsiung Veterans General Hospital 386 Ta-Chung 1st Road, Kaohsiung Taiwan 813

List of Contributors SHIRAISHI YUJI, MD Head, Section of Chest Surgery Fukujuji Hospital 3-1-24 Matsuyama Kiyose Tokyo 204 8522 Japan SIMONNEY N, MD Service de Microbiologie H6pital Saint Louis Assistance Publique-H6pitaux de Paris Universite Denis Diderot 1, avenue Claude Vellefaux, 75475 Paris France SKUCE ROBIN A, BSc, PhD Veterinary Sciences Division Stoney Road Stormont Belfast, BT4 3SD Northern Ireland

SOUSA A O,MD Service de Microbiologie H6pital Saint Louis Assistance Publique-H6pitaux de Paris Universite Denis Diderot 1, avenue Claude Vellefaux 75475 Paris France

WALl SIRAJ 0, MD, FRCP (C), FCCP Head, Division of Pulmonary Medicine Department of Medicine King Khalid Hospital National Guard Jeddah Saudi Arabia

WARGNIER A, MD Service de Microbiologie H6pital Saint Louis Assistance Publique-H6pitaux de Paris Universite Denis Diderot 1, avenue Claude Vellefaux 75475 Paris France

WILLIAMS DIANA L, PhD Molecular Biology Research Department Laboratory Research Branch National Hansen's Disease Programs At LSU-SVM, Rm 3517W Skip Bertman Dr., Baton Rouge, LA 70803 USA

List of Contributors XING ZHOU, MD Associate Prof. Pathology & Molecular Medidine Head, Division of Infectious Diseases Centre of Gene Therapeutics McMaster University Health Sciences 1200 Main Street W Hamilton, Ontario L8N 3Z5 Canada

MD Respiratory and Critical Care Medicine Seoul Natinal University College of Medicine Seoul South Korea 110744

YIM JAE-JOON,

XXI ZUMLA ALIMUDDIN, BSc, MBChB, MSc, PhD, FRCP (Lon), FRCP (Edin) Professor of Infectious Diseases and International Health Director, Centre for Infectious Diseases and International Health Royal Free & University College Medical School Windeyer Institute of Medical Sciences Room G41 46 Cleveland St London WIP 6DB UK

Historical Background

1

Tuberculosis in Ancient Egypt SALEH

A. BEDEIR

CONTENTS 1.1 1.2 1.2.1 1.2.1.1 1.2.1.2 1.2.1.3 1.2.2 1.2.3 1.2.3.1 1.3

1.4 1.5 1.6 1.7 1.8 1.8.1 1.8.1.1 1.8.1.2 1.8.1.3 1.8.1.4 1.8.1.5 1.8.1.6 1.8.1.7 1.9 1.10

Introduction 3 Predynastic Period 3 The Foundation of Egypt 4 Predynastic Period 4 Archaic Period 4 Old Kingdom 4 Development of Egyptian Language 4 A Chronology of Literature 5 From Alexander to Napoleon 5 Ancient Egyptian Art 5 Paleopathology 6 Differential Diagnosis 7 The Author's Case 7 Pulmonary and Osseous Tuberculosis in Ancient Egyptian Mummies 8 PCR and the Diagnosis of Tuberculosis in Ancient Egyptian Population 8 Environment 9 The Nile 9 Climate 9 Housing 9 Diet 9 Religion 10 Sports and Recreation 10 Physique 10 The Medical Papyri 10 Sanatorial Treatment 11 References 12

1.1 Introduction In all probability, tuberculosis is older than the human race, and different species in the genus Mycobacterium may have caused the disease in mammals, birds, reptiles and fish (Breed et al. 1957). Tuberculosis in man and other animals, both warm-blooded and cold-blooded, may have arisen from a common Mycobacterium ancestor many millions of years ago (Cockburn and Cockburn 1980). Other authors believe that tuberculosis in man may be of much more recent origin. The earliest report on presumptive tuberculosis in ancient Old World human remains is an article by Bartels (1907) on a Neolithic skeleton found near Heidelberg, Germany. The fourth and fifth thoracic vertebrae have collapsed and fused with the sixth vertebra, creating an angulation often seen in spinal tuberculosis (Ortner and Putschar 1981).

1.2 Predynastic Period Tuberculosis has existed for thousands of years in Egypt and Nubia, as shown by art forms and skeletal material. The ancient Egyptian obtained his first tuberculosis bacillus from the close contact with livestock that occurred after the Neolithic revolution, as early as 3300 B.C., during the initial herding period of Fayoum (Filer 1995). One of the earliest examples of spinal tuberculosis has been found at excavations at the predynastic site of Adaima, 8 km south of Esna. The chronological order of Ancient Egyptian Dynasties are given here according to:

S. A. BEDEIR, MD Dean, Faculty of Medicine, Cairo University, Kasr El-Aini Hospital, Cairo, Egypt

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

a. b. c. d.

Foundation of Egypt Development of Egyptian Language A Chronology of Literature From Alexander to Napoleon

S.A. Bedeir

4

1.2.1 The Foundation of Egypt 1.2.1.1

Predynastic Period Badarian Period

Naqada I Period

Naqada II Period

Naqada III Period

5000-4000 B.C. First evidence for

4000-3500 B.C. Development of pottery in Egypt

3500-3150 B.C. First major towns culture

3150-3000 B.C. First hieroglyphs

1.2.1.2

Archaic Period Dynasty I

Dynasty II

3050-2815 B.C. Unification of Egypt; royal tombs at Abydos

2815-2600 B.C. Royal tombs at Saqqara

1.2.1.3 Old Kingdom Dynasty III

Dynasty IV

2660-2600 B.C. First pyramids

2600-2470 B.C. Great pyramids at Giza

1.2.2 Development of Egyptian Language Archaic Period

Old Kingdom

First Intermediate Period

Dynasties I-II 3050-2660 B.C. Earliest script

Dynasties III-IV 2660-2200 B.C. Old Egyptian

Dynasties VII-XIa 2200-2070 B.C. Transition to Middle Egyptian, which lasts through the Middle Kingdom, dynasties Xlb-XIII

Second Intermediate Period

New Kingdom

Third Intermediate Period

Dynasties XIV-XVII 1650-1550 B.C. Middle Egyptian

Dynasties XVIII-XX 1550-1070 B.C. Middle Egyptian transition to Late Egyptian

Dynasties XXI-XXV 1070-664 B.C. Late Egyptian Demotic

Late Period

Graeco-Roman Period

Coptic Period

Dynasties XXVII-XXXI 664-332 B.C. Late Egyptian

Dynasties XVIII-XX Ptolemies 332-30 B.C. Demotic

Romans Byzantines 30 B.C.-A.D. 395 A.D. 395-640 Demotic Coptic Demotic

5

Tuberculosis in Ancient Egypt

1.2.3 A Chronology of Literature Archaic Period

Old Kingdom

First Intermediate Period

Middle Kingdom

Dynasties I-II 3050-2660 B.C. Labels; short inscriptions only

Dynasties III-IV 2660-2200 B.C. Pyramid texts, first biographies

Dynasties VII-XIa 2200-2070 B.C. Decrees, biographies

Dynasties XIb-XIII 2070-1650 B.C. Coffin texts, stories, wisdom texts

New Kingdom

Third Intermediate Period

Graeco-Roman Period

Dynasties XVIII-XX 1550-1070 B.C. Book of the Dead; "Historical" texts

Dynasties XXI-XXV 1070-664 B.C. Major mythological texts: continue through Later Period, Dynasties XXVI-XXXI

Ptolemies Romans 332-30 B.C. 30 B.C.-A.D. 395 Increasing Greek influence

1.2.3.1 From Alexander to Napoleon Coptic Period

Ptolemaic Period

Roman Period

332-30 B.C. Egypt ruled by heirs of Alexander the Great

30 B.C.-A.D. 395 A.D. 395-640 Egypt made a Egypt part of Roman province the Eastern Empite, based on Constantinople

Arab Period

Ottoman Period

A.D. 640-1517 GeneralAmr conquers Egypt for the Caliphs

A.D. 1517-1805 The Turkish Sultan Selim I invades and incorporates the country into his empire

Spinal tuberculosis, or Pott's disease, is more likely to be found in an archaeological context. Evidence for pulmonary tuberculosis is less tenable, as the bacilli disappear soon after the death of the victim. The discovery of lung collapse or pleuritic adhesions in some mummies has been mentioned as evidence of its existence, but other conditions may cause these complications.

1.3

Ancient Egyptian Art Pott's disease is well presented in both ancient Egyptian art and human remains. Several clay statuettes of men, dating to the Predynastic period, have been considered to be cases of Pott's disease. The men are depicted with humped spines and an emaciated look that suggests tuberculosis. Yet some of the figures are in large clay bowls, which may reflect a style of burial (Filer 1995). A wooden figurine in the Brussels Museum, probably Predynastic period, shows an angular deformity of the spine and chest, strongly suggesting Pott's disease. A gardening scene from the tomb of Ipwy of the 19th dynasty shows a

gardener with a humped back denoting Pott's disease (Fig. 1.1). In the Cairo Museum, there is an Old Kingdom statue with a humped spine and a concomitant deformity of the chest (Fig. 1.2). A false door from a mastaba tomb of Ankh-my-was of the Old Kingdom (4th to 5th dynasty), now preserved in Glyptotek Ny Carlsberg, Copenhagen, represents the deceased with a deformed upper part of the torso, indicating Pott's disease (Cave 1939). The tomb of Seshen Nufer I from the 4th dynasty near the Giza pyramids compromises the scene of a humped-back serving maid clearly suffering from Pott's disease (Mahmoud 1998). Ruffer (1921) described two drawings denoting tuberculosis of the spine. The first, in Beny Hassan, shows affection of the lower cervical and upper dorsal spine. The other, in Tel EI-Amarna, dates back to the 18th dynasty and shows the kyphosis is in the lower dorsal and upper lumbar region. There are many representations of humped-back servants in the tomb chapels, but it is difficult to distinguish between Pott's disease, Porter's hump, ankylosing spondylitis and simply poor posture. In Pott's disease, the deformity is localized and the angulation is usually in the form of a gibbus. The chest wall may show a corresponding deformity.

6

S.A. Bedeir

Fig. 1.1. A scene from the tomb of Ipwy, 19th dynasty, Deir el-Medinah. A gardener irrigating a garden with a "shadouf". His spine appears to have Pott's disease

Fig. 1.2. An Old Kingdom statue with a humped back and chest deformity denoting Pott's disease

1.4

A 4- to 6-year-old girl from the Ptolemaic period, whose mummy is in the Bolton Museum, appears to have Pott's disease. Lines of growth arrest (Harris line) are shown at the ends of the long bones due to the long period of disease and stress (Filer 1995). When the tomb of Iurudef at Saqqara was excavated in 1991, an intrusive burial of a non-royal female was found to have

Paleopathology There are several reports of lesions attributed to tuberculosis in Egyptian skeletal materials and mummies. The classic, and perhaps the most convincing case of skeletal tuberculosis, comes from the 21st dynasty, or about 1000 B.C. It is the mummy of Nespaheran, a 25 to 30-year-old Egyptian priest of Amun, near Thebes. It was found among a cache of 44 priests of Amun. The spine is acutely angulated due to destruction of the lower thoracic and upper lumbar vertebrae. A huge abscess cavity is present in the sheath of the right psoas muscle (Ruffer 1910, 1921; Cave 1939; Reyad 1960; Kamal 1964; Filer 1995; Nunn 1997). The drawing, by Mrs. Cecil M. Firth, was first introduced by Ruffer in 1910 and is now a classic (Fig. 1.3). Most of the known possible cases from the Predynastic to the 21st dynasty were reviewed by Morse et al. (1964). Buikstra et al. (1993) added new cases. Flinders Petrie and Quibell collected 13 specimens with tuberculosis affection from 2000 graves in Naqada (Nunn 1997). Derry (1938) reported nine cases. The Nubian collection of the Royal College of Surgeons of England contained many cases of skeletal tuberculosis, but many were lost together with their records during an air raid in 1941. Morse et al. (1964) described a single case of a mummy with a collapsed lung and pleural adhesions, which are common complications of tuberculosis.

Fig. 1.3. Nespaheran, a priest of Amun from the 21st dynasty. The side view shows marked kyphosis of the spine and, in the front view, a large cold abscess cavity is seen in the right psoas major muscle. The anterior abdominal wall is removed

Tuberculosis in Ancient Egypt

7

severe degeneration of the spine with marked angulation and a possible paravertebral abscess. Iurudef was a high Memphite official in the household of Princess Tia, sister of Rameses II of the 19th dynasty. It is believed that the woman, who died at approximately the age of 20 years, had lived her life as an invalid and only survived through family care (Walker 1991). It is interesting to note that one of the possible causes of the early death of Tutankhamen is tuberculosis (Reyad 1960).

1.5 Differential Diagnosis Although there is very little doubt that tuberculosis was the cause of the pathological findings in most of the reported cases, it is sometimes difficult to exclude compression fractures of the spine, osteomyelitis, spinal metastasis degenerative spondylosis, fungal infection and bone cysts.

Fig. 1.4. Tuberculosis of the right iliac bone and right sacroiliac joint, of a male aged 35-39 years old at time of death. Giza necropolis. There is ankylosis of the sacroiliac joint, erosion of the iliac bone and the sacrum and a round defect in the iliac bone

1.6 The Author's Case I was privileged to have the opportunity to examine the Giza collection of disarticulated skeletons, which should be considered one of the most important materials in the history of archaeology and archaeopathology. The material belongs to a very important period, the Old Kingdom, which was when the great pyramids were built. The United Nations Educational, Scientific and Cultural Organization (UNESCO) had added the entire archaeological area of Giza to its list of world heritage, as it is the oldest and most famous necropolis in the world. The material includes the skeletal remains of two different classes that lived in the same country during the same period of the third millennium B.C. The high officials and the dignitaries were buried in the western cemetery and the workmen and artisans were buried in the southeast cemetery. The excavations were headed by Reisner in 1942 and Hawass in 1994. Skeleton number 1932 (male) is one of the high officials buried in the western cemetery. His age at the time of death was between 35 years and 39 years. The right iliac bone and the adjoining right side of the sacrum are markedly affected by a chronic infective condition (Fig. 1.4). There is complete bony ankylosis of the right sacroiliac bone (Fig. 1.5). The outer part

Fig. 1.5. Radiograph of the specimen in Fig. 1.4

of the bone is markedly eroded, with bone buildup at the iliac crest and parts of the iliac bone and sacrum. The greater trochanter of right femur shows exuberant bone formation (Fig. 1.6). It seems that the tuberculous cold abscess trickled down along the gluteal muscle to involve the trochanter in the tuberculous process. The sacroiliac joint is the most common site of the pelvis that is affected by tuberculosis (Ortner and Putschar 1981) and chronic tuberculous affection of the greater trochanter is a characteristic lesion that affects young adults more than children. The spine and the hip joints do not show obvious pathological or radiological changes. The differential diagnosis of this case is pyogenic osteomyelitis, which is very rare in ancient Egyptian

8

S.A. Bedeir

1.7 Pulmonary and Osseous Tuberculosis in Ancient Egyptian Mummies

Fig. 1.6. Chronic tuberculosis of the greater trochanter of the right femur. Same case as in Fig. 1.4. There is erosion of the bone and exuberant bone buildup denoting a good healing reaction. There is no involvement of the femoral head

skeletal remains (Wood-Jones 1910). Patients with hematogenous osteomyelitis were expected to die from toxemia and pyemia before the inflammation produced manifested bone changes. The skeletal response to the tubercle bacilli is dependent on the virulence of the organism and the host response. If the host had little ability to defend himself, then tuberculosis could cause a progressive type of acute disease and early death. If the host's resistance was considerable, it is presumed that the disease then would be chronic, with the possibility of extensive healing and exuberant bone reaction. In our case, we believe that the patient's resistance was good, as manifested by the complete bony fusion of the right sacroiliac joint, the marked bone healing reaction of the iliac bone and the excessive bony build-up of the greater trochanter. The patient was one of the high dignitaries of the king. He most likely enjoyed eating a good, nutritive diet and lived in an appropriate house. It is interesting to comment on the presence of three healed fractures of both ulnae and left fibula. Fracture of the ulna is usually considered a "parry" injury, in which the patient tried to fend off a blow using the forearm. These injuries, which were in different stages of healing, indicate that our patient was active and aggressive during his life.

Pulmonary tuberculosis has been suspected by many authors to have occurred in ancient Egyptian populations (Cockburn et al.1975). The first to prove its occurrence, with microscopic confirmation of the presence of Mycobacterium tuberculosis bacilli, was Zimmerman (1979) from Michigan, USA. This author reported his anatomical and microscopic finding on the mummy of an ancient Egyptian child of five years of age belonging to Dra-Abu-el-Naga, found on the road to the Valley of the Kings, the tombs of the officials of 19th and 20th dynasties (1314-1085 B.C.). The author described the anatomical features that suggested pulmonary and osseous tuberculosis with recurrent and finally fatal pulmonary hemorrhage. Ziehl-Neelsen stain of a bone specimen showed scattered acid-fast bacilli. In a more recent report, pulmonary tuberculosis in an ancient Egyptian mummy was diagnosed and confirmed by polymerase chain reaction (PCR) (Nerlich et al. 1997). These German authors described their excavation findings at the tombs of the Nobles at Thebes-West in Upper Egypt. They reported a mummy of 35-year-old male dated from the New Kingdom (1550-1080 B.C.). These authors noted residue of the affected right lung with extensive pleural adhesions while the pleura of the left lung were normal. They also found two lumbar vertebral bodies severely affected anteriorly with lytic lesions and new bone formation. Specimens from both lungs were examined using PCR-amplification ofmycobacterial DNA. A specific positive band was identified from right lung specimens but not from the left unaffected lung. This was probably the first confirmed case of pulmonary tuberculosis using PCR in an ancient Egyptian mummy (Nerlich et al. 1997).

1.8 peR and the Diagnosis of Tuberculosis in Ancient Egyptian Population Many authors have tried to use the relatively recently acquired knowledge and development of DNA molecular technology for various purposes (Leek 1979; Harrison and Connolly 1969, David 1997). Svante Piiiibo (1985) used this technology, aiming at studying the descent and relationship of the ancient pharaonic families, individual relationships between members of these families and population in the Nile Valley. The author sampled 23 mummies from various

Tuberculosis in Ancient Egypt

dynasties between the 6th dynasty (2370-2160 B.C.) up to the Roman times. The ages ofthese mummies ranged between one-year-old boys to older ages, as estimated by the radiocarbon age technique of accelerator mass spectroscopy. Blunt-ended extract material was mixed with E. coli DNA polymerase with labeled radioactive nucleotides. The author concluded that recombinant DNA techniques could allow the study of the descent of the ancient Egyptian pharaonic families and their individual relationships. However, this author failed to recognize what would become the much wider use of this technique-detecting diseases in ancient Egyptian pharaonic population and families. The molecular approaches to DNA probe analysis using a nucleic acid-base methodology was originally described in early 1980s. It has been used extensively and has begun to have a major influence on current clinical management of various diseases. Several methods of PCR assays have been developed and IS6110 PCR can be done with a turnaround time of 24-36 h. It is a rapid and reliable molecular approach that can detect Mycobacterium tuberculosis DNA directly from clinical specimens. Such advances in investigative techniques have been exploited by many scientists and researchers who aim to trace diseases to an ancient time. In a large series of ancient Egyptian cadavers, Zink and colleagues (2001) from Munich, Germany, collected 41 skeletal specimens. Four were from Abydos (3000 B.C.) and thirty-seven were from Thebes-West, Upper Egypt (2120-500 B.C.). Three had typical macromorphological evidence of skeletal tuberculosis, 17 showed probable disease and 21 were without morphological skeletal changes. DNA was extracted from bone specimens and amplified by PCR with a primer pair that recognized the Mycobacterium tuberculosis complex insertion sequence IS611O. Nine of these cases were positive for Mycobacterium tuberculosis DNA. Two of the three cadavers with typical macromorphology of osseous tuberculosis had positive results for Mycobacterium tuberculosis DNA. Five of those with probable and two of those without morphological changes had positive results for Mycobacterium tuberculosis DNA. The authors concluded that their findings confirmed the relatively frequent occurrence of tuberculosis in ancient Egypt, dating back to 3000 B.C.

1.8.1 Environment

The lives of ancient Egyptians were shaped by their environment. The geography of Egypt was, and is still, like that of no other country. The Nile valley

9

was enclosed by deserts that protected Egypt against invasion, which resulted in long periods of stability. 1.8.1.1 The Nile

The Nile played a central role in daily life. Between July and October of each year, the Nile flooded its banks, depositing a rich and fertile layer of alluvial slit on the land. Inundation saved an enormous labor in the transportation of water and renewed the fertility of the land. 1.8.1.2

Climate Herodotus stated that the good health of the ancient Egyptians was due to the lack of variability in the weather. The Egyptian sky was blue almost all year round. The sun's rays nourished the Egyptians'bodies with vitamin D, and decreased the incidence of rickets. The dry, hot climate of Egypt is better suited to the preservation of human remains than that of any other country in the world. However, heat encourages flies, which were responsible for the spread of infections, especially eye infections and diarrhea. The Sahara (Khamasin) winds blew up dust storms and sand particles, resulting in chest infection, pneumoconiosis, laryngitis and sinusitis. 1.8.1.3 Housing The hot and dry climate influenced the design of ancient Egyptian housing. Houses varied from the large, villa-like houses of the top members of society to the small, crowded houses, with limited amount of light and space, of the poor people. In Amarna, smoke-blackened roof timbers indicated a heavy passage of soot. Tuberculosis thrived in such conditions of ill-housing and bad ventilation. 1.8.1.4

Diet Egyptians were already enjoying reasonably healthy and varied diets. Food production was not a major problem in Egypt, provided that the inundation of the Nile occurred every year. The main cereal crops were barley and emmer, with wheat being introduced only in the late period. Barley was used to make loaves and beer. Vegetables and fruits were grown on a large scale. Many of the familiar varieties were known: grapes, figs,

S.A. Bedeir

10

dates,pomegranates, melons, onions, leeks, cucumbers, lettuce, garlic, lentils and chickpeas. These provided many of the vitamins essential for good health. The ancient Egyptians consumed a wide variety of animal products, including cattle, sheep, goats and pigs. They kept sheep for producing wool, milk, cheese and meat. They ate ducks, geese, pigeons and quails. Hens were known only during the Ptolemaic period. The ancient Egyptians consumed fish that was fresh or dried and salted. Pure salt has been found from the sixth dynasty, but the method of preparation is unknown. Dates or honey was used for sweetening. Bee keeping was actively practiced, and honey was used to prepare medicines. The funerary offerings for King Unas of the fifth dynasty included: • Milk, three kinds of beer, five kinds of wine, ten loaves, four of bread, ten cakes, fruit cakes, four meats, different cuts, joints, roast, spleen, limb, breast, tail, goose, pigeon, figs, ten other fruits, three kinds of corn, barley, spelt, five kinds of oil and fresh plants (Nunn 1997). 1.8.1.5 Religion

Religion had a beneficial effect on the general health of the ancient Egyptians. Sacred holidays allowed them to rest and go to the temples to see the King. The poor people took their shares from the offerings. Vacations and ceremonial activities had the effect of raising the morale of the people. They had to take baths using running water and natron to clean their bodies. Men would shave their hair and women removed their unwanted hair. The ancient Egyptians believed that diseases were caused by excessive food, thus they used drugs and suppositories to evacuate their stomachs and bowels three times every month. 1.8.1.6 Sports and Recreation

Athletic sports were an ancient Egyptian invention. The ancient Egyptians were skilled in wrestling, stick combat, gymnastics, ball games, hunting, fishing and bull fighting. Families played cup games and board games (senet). They also enjoyed music, singing, and dancing. 1.8.1.7 Physique

The ancient Egyptians were small; the mean height of the males was 1.67 cm and the females 1.53 cm.

The arithmetic mean age at death was 36 years in the dynastic period. There are very few burials of people over the age of 60 years (Sarry EI-Din 1995). Pepi II and Rameses II survived to well in their eighties.

1.9 The Medical Papyri Although there can be no doubt of the existence of tuberculosis in ancient Egypt, many authors believe that nothing in the medical papyri can be directly related to the disease. The medical papyri do not contain any clear descriptions of tuberculosis (Ortner and Putschar 1981; Steinbock 1976). However, Ebbell (1937) and Kamal (1964) believe that the Ebers Papyrus (Ebers 1875) contains the description of two cases with tuberculosis of the cervical lymph nodes. The first case is described in section 860, column 195; Ebbell entitled the section "Soft Tuberculosis Gland". The section is translated as follows: \'Jl "Information about enlarged cystic node in his neck" - Examination: if you examine a man having an enlarged cystic node in his neck, and you find it like the thymus(?) gland because of its softness at palpation and its white discharge ... (empty space). - Diagnosis: you shall then say concerning him he has enlargement of the cystic node in his neck. An ailment I will treat by surgery to protect the vessels. - Treatment: you shall prepare medicines to treat him by a bandage that makes the cyst open through the skin: acacia seyal, pea, fruit, animal blood, insect's blood, honey, common salt (and other things). Grind. Mix together and bandage. «I The second case, section 861, reads as follows: - Title: information about enlarged infected node in the neck of a man. - Examination: if you examine an enlarged infected node in the neck of a man. After it is enlarged and removed the skin that covered it, and caused suppurating granulation for many years and months. It discharges a secretion like the seminal fluid of synodontis (a type of fish). - Diagnosis: you shall then say concerning him he has an enlarged infected node. An ailment I will treat. - Treatment: you shall prepare the medicines for him: wax or fat, As(?), ink, salt, goose fat, fruit(?) Galena. Heat and bandage the neck (Fig. 1.8),

Tuberculosis in Ancient Egypt

11

Fig.I.7. Healed fractures of the right ulna and left fibula of the same case as in Fig. 1.4

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Fig.1.8. Ebers papyrus, part of paragraph 861, describing a chronic tuberculous node in the neck. Arabic translation by Kamal (1964)

The Ebers Papyrus was purchased in Luxor by Edwin Smith in 1862. George Ebers referred to the Papyrus in 1873, and it is now in the University Library of Leipzig. It was translated into English by Dr. Ebbell in 1836. It comprises 110 pages and is by far the longest of the medical papyri. It is in very good condition and the handwriting is clear. It was written in 1534 B.C. It is claimed that the Ebers' Papyrus (or at least parts of it) date back to an ancient origin.

1.10 Sanatorial Treatment In 1938, Derry excavated four possible cases of spinal tuberculosis from two neighboring graves. In one grave, Derry found the remains of a man and a

woman and, in the other grave, he found the skeletons of two adult males and a nine-year-old boy. This cluster of cases was regarded as evidence of a cemetery for a tuberculosis sanatorium. The other possibility is that the individuals were related and acquired the disease from a common source of infection within the family. The ancient Egyptians knew sanatorial treatment. The sanatorium was a building in which patients could be totally or partially immersed in healing holy water. In certain temples, incubation or "temple sleep" was practiced; the patients were allowed to dream in the hope of receiving a cure from a deity (Quirke 1992). Sanatorial treatment was practiced in the great temples of Hathor and Dendera. Certain basins were supplied with water from the sacred lake, which could be made more curative by allowing it to ft.ow over healing statues and certain texts. The effect of the sacred water was obtained by drinking it (Dunn 1997).

S.A. Bedeir

12

Parts of Deir el-Bahri temple (Fig. 1.9) are considered by certain authorities to be the first sanatorium in history. The temple was established by Queen Hatshepsut in the 18th dynasty, 1500 B.C. Patients and invalids from different countries visited the sanatorium and stayed beside its columns, hoping for recovery. During the Ptolemaic period, Imhotep (Fig.UO) and Amenhotep, the son of Hapu, were worshipped on the upper terrace of the temple at Deir el-Bahri, where the faithful and sick gathered to request grace from the two gods of medicine. In 1923, Sir William Osler said that Imhotep was "the first figure of a physician to stand out clearly

from the mists of antiquity" (Nunn 1997). Milne and Joseph (1914) concluded that the temple was used as a sanatorium, and currently visitors can see the writings of patients who wrote while sitting; the levels of these writings corresponded with the heights of the patients. Frequently, the writings include the name and occupation of the patient as well as the times of his arrival and departure. One of those paragraphs reads as follows: I am Andropachus from Macedonia. I came to visit Amenotis. I was very ill, but now I am cured thanks to the mighty God. I ask you merciful God to give us all good health. Goodbye (Reyad 1960).

References

Fig. 1.9. Deir el-Bahri. Is it the first sanatorium in the world?

Fig.1.10. Statue of Imhotep. He was the patron of science, medicine and writing-one of the "wise men" of the past. Graeco-Roman period, Cairo Museum

Bartels P (1907) Tuberkulose in der Jiingeren Steinzeit. Arch AnthropoI6:243-250 Breed RS, Murray EGD, Smith NB (eds) (1957) Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore Buikstra AE, Baker BJ, Cook DC (1993) What diseases plagued the ancient Egyptians? A century of controversy considered. In: Davies WV, Walker R (eds) Biological anthropology and the study of Ancient Egypt. British Museum Press, London, pp 231-250 Cave AJE (1939) The evidence forthe incidence of tuberculosis in ancient Egypt. Br J TubercoI33:142-152 Cockburn A, Cockburn E (eds) (1980) Mummies, diseases and Ancient cultures. Cambridge University Press, Cambridge Cockburn A, Barraco RA et al (1975) Autopsy of an Egyptian mummy. Science 187:1155-1160 David AR (1997) Disease in Egyptian mummies: the contribution of new technologies. Lancet 349:1760-1763 Derry DE (1938) Pott's disease in ancient Egypt. Med Press Circ 197:196-199 Ebbell B (1973) The Papyrus Ebers. Oxford University Press, London Ebers GM (1875) Papyros Ebers (two vols). Englemann, Leipzig Filer J (1995) Disease. British Museum Press, London Harrison RG, Connolly RC (1969) Microdetermination of blood group substances in ancient human tissue. Nature 224:326 Hawass Z (1996) The workmen's community at Giza plateau. Sonderdruck aus Haus und Palast in alien Agypten. Internationales Symposium, 8 bis 11 April 1992 in Kairo. Kamal H (1964) Ancient Egyptian medicine (two vols). Egyptian Organization for editing, translation and distribution (in Arabic) Leek FF (1979) Teeth and bread in ancient Egypt. J Egyptian ArchaeoI58:126-132 Mahmood MMR (1998) Occupational rehabilitation of the disabled in Ancient Egypt. Thesis, Master's degree, Helwan University Milne M, Joseph R (1914) The sanatorium of Der el-Bahri. J Egypt Archaei 1:96-99 Morse D, Brothwell DR, Ucho PJ (1964) Tuberculosis in Ancient Egypt. Am Rev Respir Dis 90:524-541 Nerlich AG, Haas CJ et al (1997) Molecular evidence for tuberculosis in an ancient Egyptian mummy. Lancet 350:1404

Tuberculosis in Ancient Egypt Nunn JF (1977) Ancient Egyptian medicine. British Museum Press, London Ortner DJ, Putschar WGJ (1981) Identification of pathological conditions in human skeletal remains. Smithsonian Institution Press, Washington DC Quirke SGT (1992) Ancient Egyptian religion. British Museum Press, London Reisner GA (1942) History of Giza necropolis, vol I. Cambridge Harvard University Press, London Reyad N (1960) Ancient Egyptian Medicine. Karnak Press, Cairo (in Arabic) Ruffer MA (1910) Pott'sche Krankheit an einer agyptischer Mumie aus der zeit der 21 Dynastie. Zur historischen Biologie der Krankheitserreger, 3. Heft, Giessen Ruffer MA (1921) Studies in palaeopathology of Egypt. University of Chicago Press, Chicago Sarry El-Din AM (1995) Comparative study of skeletal material from Giza old Kingdom with Sudanese Nubia. Thesis, PhD degree, Cairo University

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Steinbock RT (1976) Paleopathological diagnosis and interpretation. Bone diseases in ancient human populations. Thomas, Springfield, Illinois Svante Piiiibo (1985) Molecular cloning of ancient Egyptian mummy DNA. Nature 314:644-645 Walker R (1991) Skeletal remains. In: Raven MJ (ed) The tomb of Iurudef, a Memphite official of Rameses II. Egyptiam Exploration Society, London, pp 55-76 Wood-Jones F (1910) General pathology (including diseases of the teeth). In: Elliot-Smith G, Wood-Jones F (eds) The Archaeological Survey of Nubia Report for 1907-1908, vol II. Report on human remains. National Printing Department, Cairo, pp 263-292 Zimmerman MR (1979) Pulmonary and osseous tuberculosis in an Egyptian mummy. Bull NY Acad Med 55: 604-608 Zink A et al (2001) Molecular analysis of skeletal tuberculosis in an ancient Egyptian population. J Med Microbiol 50: 355-366

2

Historical Aspects of Tuberculosis M. MONIR MADKouR, KITAB

E. AL-OTAIBI, R. AL SWAILEM

2.1 2.2 2.3

2.25 2.26 2.27 2.28 2.29

M. M. MADKouR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia K. E. AL-OTAlBI, MD, FRCS Director General of Medical Services Department, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia R. AL SWAILEM, MRCP (UK) Consultant Rheumatologist, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia

Tuberculosis was first described by ancient Egyptian doctors who described clearly the clinical features and medical and surgical treatment of scrofula (Fig.8, Chap. 1). The Ancient Egyptian artists contributed to the description through their exquisite paintings, which showed spinal tuberculosis and its gibbous deformity (Fig. 1, Chap. 1). The first known sanatorium in the history of mankind was established by the Ancient Egyptians and is now open to visitors (Fig. 9, Chap. 1). Archaeological and anthropological studies indicated that tuberculosis infected the Chinese, Indo-European and pre-Columbian American tribes (Buikstra 1983; Powell 1992; Verano and Ubelaker 1992; Paulsen 1987). Hippocrates (460-361 B.C.) described tuberculosis as "phthisis" and considered it to be an inherited and non-contagious disease (Chadwick and Mann 1978). Galen of Pergamon (130-200 A.D.) believed that tuberculosis was contagious. From Galen's time until the 8th century, nothing of significant importance in the history of tuberculosis occurred. The Middle Ages in European history, also known as the "Dark Ages" (approximately 500-1500 A.D.) roughly corresponded to the "Golden Age of Islam." Scholars in Islamic medicine, such as Rhazes, Avicenna and Albucasis, described symptomatology, pathology and predisposing factors for the development of tuberculosis. Avicenna, suggested the communicable nature of tuberculosis. Avicenna's "Canon of Medicine" was translated to Latin and served as foundation for university courses in medicine between 1250 and 1600 in Europe (Cruse 1999). Bimaristan (house of the sick) was first established in Baghdad by the famous Harun AI-Rashid in 803. Ibn an-Nafis (AI-Quraishi, d. 1288) discovered pulmonary circulation (Magner 1992).

CONTENTS Islamic Medicine During Europe's Dark Ages 16 The Pulmonary Circulation and Ibn an-Nafis 16 Islamic Medicine and Its Scientific Contribution to Tuberculosis 17 2.4 The Germ Theory 18 2.4.1 Miasma or Contagion 18 2.5 Percussion by an Amateur Musician 19 2.6 Laennec 19 2.6.1 Laennec's Work on Tuberculosis 19 2.6.2 Laennec's Invention of the Stethoscope 19 2.6.3 Laennec's Tubercles 19 2.7 Gaspard Laurent Bayle (1774-1816) 20 2.8 "Tubercle" and "Tuberculosis" 20 2.9 The White Plague 20 2.10 Robert Koch (December 11, 1843-May 27,1910) 21 2.10.1 The Discovery of the Tubercle Bacillus 21 2.10.2 Koch's Secret Cure (Tuberculin) and the Storm That Followed 22 2.10.3 The Ziehl-Neelsen Stain, or Koch-EhrlichRindfleisch-Ziehl-Neelsen Method? 23 2.11 Franz Ziehl (1857-1926) 23 2.12 Friedrich Karl Adolph Neelsen (1854-1894) 24 2.13 Paul Ehrlich 24 2.14 The Evolution of Tuberculosis Treatment 25 2.15 Climatological and Sanatorium Therapy 25 2.16 Sanatorium-Bimaristan or Deir el-Bahri 26 2.17 Artificial Lung Collapse Therapy 26 2.18 Thoracoplasty 26 2.19 Plombage 27 2.20 Pulmonary Resection 27 2.21 The History of BCG 27 2.22 The History of Chemotherapy for Tuberculosis 27 2.23 Streptomycin and Modern Antituberculosis Therapy 27 2.24 Animal and Human Trials on the Efficacy of Streptomycin 28

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Para Aminosalicyclic Acid (PAS) Isoniazid 28 Rifampicin (Rifampin) 28 Drug Therapy for Cure 28 Conclusion 29 References 29

28

M. M. Madkour et aI.

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During the early development of the industrial revolution in Europe (approximately 1780), the incidence of tuberculosis increased and reached epidemic proportions. It was the most common cause of death in the 18th century. In Europe, from the later part of the 18th century throughout the 19th century, progress and advances have been achieved in the diagnosis of tuberculosis. France was the champion, with many French scholars such as Corvisart, Bayle, Laennec and Villemin, greatly advancing our understanding of tuberculosis. The breakthrough in the field of the diagnosis of tuberculosis was made by the Germans. Robert Koch discovered the causative bacillus in 1882 and Ziehl-Neelsen reported the staining method to identify the bacillus in 1882-1888. In 1895, Roentgen discovered Roentgen's X-ray, an advanced investigative method. Despite the identification of the bacillus, the discovery of antibiotics and their use for tuberculosis treatment lagged behind for over 60 years. However, surgical interventions for the treatment of tuberculosis started much earlier than chemotherapy. The human immunodeficiency virus (HIV) epidemic started in 1981 and its association with the resurgence of tuberculosis in areas where the disease had been controlled is a major setback in the fight against tuberculosis. As there is no cure for HIV at present, its management is reminiscent of tuberculosis prior to the antibiotics era.

2.1 Islamic Medicine During Europe's Dark Ages The Middle Ages of European history, from approximately 500 to 1500 A.D., an era also popularly known as the Dark Ages, roughly corresponds to the Golden Age of Islam. The great sages of Islamic medicine were established and studied in their own right and not just in terms of their role in preserving classical Greek medicine. Latin translation of the works of many brilliant Islamic scholars, including Rhazes, Avicenna and Albucasis, were most influential in Europe. Other Arabic scholars held prominent status in medical knowledge and practice in the Muslim world that had no parallel in the west (Magner 1992). During the European Dark Ages, western scholars were merely interested in recovering Greek medical knowledge and had less interest in the practical aspects of Islamic medicine, such as the development of hospitals and clinical training. In 750, Baghdad was

established as the capital of the Islamic Empire and by the end of the 10th century, Baghdad and Cairo had developed into independent centers of scholarship (Magner 1992). The library in Cairo had over 100,000 volumes of manuscripts. Baghdad had 36 public libraries by 1258, the year when the Moguls conquered the city and its great libraries were destroyed.

2.2 The Pulmonary Circulation and Ibn an-Nafis Western scholars long maintained that Arabic medical manuscripts were a Greek translation and lacked originality. However, the strange story of Ibn an-Nafis (AI-Qurashi, d. 1288) and his discovery of the pulmonary circulation demonstrates that such previous assumptions were unsound. The discovery and the writings of Ibn an-Nafis on pulmonary circulation were essentially ignored until 1924, when an Egyptian physician (Dr Muhyi ad-Din at-Tatawi) presented his doctoral thesis to the Medical Faculty of Freiburg, Germany and brought it to the attention of Prof. Max Meyerhof (Magner 1992). Dr. Lois N. Magner (1992), from the Department of History, Indiana, USA, in his book, A History of Medicine; wrote: "In the midst of a fairly conventional discussion of the structure and function of the heart, Ibn an-Nafis departed from the accepted explanation of the movement of the blood. His description of the two ventricles of the heart accepts the Galenic doctrine that the right ventricle is filled with blood and the left ventricle with vital spirit. His next statement, however, boldly contradicted Galen's teachings on the pores in the septum. An-Nafis insisted that there were no visible or invisible passages between the two ventricles and argued that the septum between the ventricles was thicker than other parts of the heart in order to prevent the harmful and inappropriate passage of blood or spirit between them. Thus, to explain the path taken by the blood, he reasoned that after the blood had been refined in the right ventricle, it was transmitted to the lung where it was rarefied and mixed with air. The finest part of this blood was then clarified and transmitted from the lung to left ventricle. That is, the blood can only get into the left ventricle by way of the lungs. Perhaps some still obscure Arabic, Persian, or Hebrew manuscript contains a commentary on the curious doctrines of Ibn an-Nafis, but there is as yet no evidence that later Arab authors or European anatomists were interested

Historical Aspects of Tuberculosis

in these anti-Galenic speculations. Thus, although Ibn an-Nafis did not influence later writers, the fact that his concept was so boldly stated in the thirteenth century should lead us to question our assumptions about progress and originality in the history of science. Since only a small percentage of the pertinent manuscripts have been studied, edited, translated, and printed, the question may go unanswered for quite some time:'

2.3 Islamic Medicine and Its Scientific Contribution to Tuberculosis Nothing of significant importance in the history of tuberculosis occurred until the 9th and 10th centuries, when the Islamic physicians Rhazes, Avicenna and Albucasis wrote excellent descriptions of the symptomatology, pathology and predisposing factors for the development of the disease. The communicable nature of tuberculosis was also suggested by Avicenna, who believed in the curability of the disease (Lyons and Petrucelli 1978). Rhazes (Abu Bakr Muhammad Ibn Zakariya alRazi) (850-925) was a brilliant physician who practiced medicine in Baghdad and at various locations in Iran. He has been known as the greatest physician of the Islamic world. He is considered one of the most scientifically minded physicians of the Middle Ages and one of the greatest physicians of all time (Magner 1992). He was the author of over 200 medical and philosophical treatises, including his massive masterpiece the Continens, or Comprehensive Book of Medicine, which was translated into Latin by the Jewish physician Faraj Ibn Salim (known in Latin as Farragut) for King Charles of Anjou (Magner 1992). Rhazes' manual of the healing arts Kitab al-Mansori (Almansour) was very influential in the West into the 16th century. He was chosen as director of one of the first great hospitals in Baghdad and selected the most healthful location for it. Rhazes provided clear insight into the relationship between patients and physicians, the signs and symptoms that the physician thought significant, the type of treatment given, the family background and occupation of the patient. Rhazes' book on smallpox and measles highlighted an extraordinary ability of his clinical acuteness, diagnosis, therapy and concepts of diseases. Mesue (Yuhanna Ibn-Masawayh) was the court's physician to four Abbasid Caliphs during the late 8th and first half of the 9th centuries. He was a brilliant

17

physician and renowned clinician and teacher. He wrote influential texts on nosology and therapeutics. One of the 9th century physicians was Joannitius (Hunay Ibn-Ishaq AI-Ibadi), who was proficient in Greek, Syriac and Arabic and translated many Greek medical texts. He wrote about many diseases, including a monograph on ophthalmology. Haly Abbas (Ali Ibn AI-Abbas AI-Majusi), who died in 994 and who wrote the famous Kitab AI-Malaki (also known as Kamil AI-Sinaa Filtib), one of the most concise and well-recognized expositions of GrecoIslamic medicine, was a physician at the Buwayhid court (Johnston 1993; Gallagher 1993; Risse 1993, quoted by Bishop and Neuma 1970). Avicenna (Abu-Ali AI-Husayn Ibn-Sina) (9801037) was also known as a "Prince of Physicians" (Cruse 1999). He was the first scholar to create a complete philosophical system in the Arabic language and his influence in medicine cannot be underestimated. Some critics indicated that his influence could not be challenged by other physicians, which therefore inhibited further development in medicine. At the age of ten, Avicenna memorized the Qur'an and turned to the natural sciences and to learning medicine. He soon realized that some things could only be learned by experience rather than solely from books. Early in the 11 th century, he wrote his leading medical encyclopedia, Canon of Medicine, a five-volume study dealing with physiology, nosology, etiology, symptomatology and therapy, pathology and the preparation of compound remedies. His enormous knowledge influenced the Islamic world and was translated into Latin. The Latin translation served as foundation for university courses in medicine between 1250 and 1600 in Europe (Cruse 1999). Significant advances made by Arab physicians served to teach European physicians during the 8th through the 11 th centuries. A well-known, brilliant Jewish physician in Arabic medicine was Maimonides (l135-1204), who translated "Canon of Medicine" into Hebrew and served as a liaison for medical knowledge between East and West (Cruse 1999). Rhazes, Avicenna and Albucasis wrote about the virtues of dry air and consuming fresh milk as climatological therapy. They linked the ulcers of the lungs to those of the extremities. They advised on the values of using camphor in sugar roses and believed in the curability of the disease. Avicenna suggested the communicable nature of tuberculosis. Avicenna gave an excellent description of the clinical features and pathology of tuberculosis in Arabic script. Avicenna, Rhazes and Haly Abbas described the people who were at great risk of developing tuberculosis as

18

having narrow chests, thin bodies and ages between 18 years and 30 years. They described the symptoms of tuberculosis (known in Arabic as; As'sul or Ad'daran) as fever (more so at night), sweating, malaise, anorexia, weight loss, cough, expectoration of sputum, hemoptysis and swelling of the legs. Al Majousi also described clubbing of the fingers in patients with tuberculosis (Al-Amoud 1993).

M. M. Madkour et al.

2.4 The Germ Theory 2.4.1 Miasma or Contagion

The idea that tuberculosis can be transmitted by contact is an ancient folk belief, but it was generally ignored in Hippocratic doctrine (Magner 1992). PhysiAlbucasis or Abu'l-Qasim (936-1013): Abu'l-Qasim cians and scientists have argued since antiquity about KalafIbn-'Abbas al-Zahrawi (Albucasis), an extremely the terms contagion theory and miasma theory as the ascetic man who devoted much of his time to work- cause of diseases. The miasma theory of disease was ing among the poor, wrote a more specialized guide known as disease-inducing noxious and contaminated to Arab Surgery. His book on Surgery and Instruments air and the theory was applied until the 19th century. is one of the first comprehensive illustrated treatises The contagion theory of disease was suspected by on this subject. He practiced in Cordoba, Spain and Fracastoro who published On Contagion in 1546. wrote an encyclopedic study that contained an influ- Girolamo Fracastoro (1478-1553) was an Italian phyential section on surgery based on Greek sources and sician, scientist, mathematician, astronomer, geologist his own findings (Magner 1992). and poet. He created the story of the shepherd named Pharmacology, alchemy and optics were other Syphilis, who brought about the first outbreak of areas of great interest to Arab scientists. Arabic trea- syphilis because he cursed the sun and became the first tises on medical pharmacology, including drug prod- victim of the new pestilence. Fracastoro's contagion ucts and their nomenclature and aromatic medicinal theory of disease interested and inspired the microbiplants, were written in the 8th to the 11th centuries. ologists of the 19th century, and he was regarded as the first to give an exposition of germ theory. Although the AI-Kindi (800-870): Yaaqub Ibn-Ishaq-Al-Kindi wrote microscope was invented at the end of the 16th century, a medical formulary and served as a model for Arabic its use to establish the existence of tiny "animalcules" treatises on pharmacology, botany, zoology and min- was not applied until the 17th century by Antoni Van eralogy. Persian and Indian drugs that were unknown Leeuwenhoek (1632-1723) who described "little anito Hippocrates and Galen appeared in such formular- mals" (Magner 1992). Professor Jacob Henle of Zurich (1809-1895) ies, as did new kinds of drug preparations. The eleventh century chemist and author Abu AI- wrote, "physicians blamed disease on miasma, which Biruni composed a treaties on pharmacy in which they defined as something that mixed with and poihe listed 720 drugs, including metallic compounds, soned the air, but no one had ever demonstrated the for the treatment of diseases. Such expansions led to existence of miasma with scientific instruments". the establishment of a medical pharmacy (Johnston Henle's hypothesis was that "Contagia Animata" 1993; Gallagher 1993; Risse 1993). (living organisms) caused contagious diseases Islam devoted secular institutions to care for (Magner 1992). the sick, named bimaristan, a Persian term meanGiovanni Cosimo Bonomo (d.1697) was the first to ing "house for the sick." The first bimaristan was provide convincing evidence that scabies was caused established by Ibn Barmak in Baghdad before the by a tortoise-like mite. He also provided the contiguyear 803, during the rule of the famous "Harun ous nature of scabies, "the itch", from one person to AI-Rashid". Inmates came from all sectors of the another by means of bedding or clothing. Yet, the population, including psychiatric patients (Galla- itch mite observation was not used as an example gher 1993). Bimaristans were operated by a direc- of other infectious diseases (Magner 1992). Agostino tor, medical staff, pharmacist, servants (as nurses) Bassi (1773-1857) also showed that silkworm disease and had their own libraries, which contained many could be transmitted to healthy silkworms and sugmedical texts. They also offered educational experi- gested its contiguous nature. In 1839, Lucas Schonences for medical students serving apprenticeships. lein (1793-1864) a German professor of medicine Lectures and discussions by famous physicians were reported his finding of the fungus that caused ringoffered for students who were issued licenses for worm. In the second half of the 19th century, Louis practicing medicine if they had successfully com- Pasteur and Robert Koch developed medical microbiology as a new science in the late 19th century. pleted theoretical and clinical courses.

Historical Aspects of Tuberculosis

2.5 Percussion by an Amateur Musician Leopold Joseph Avenbrugger (1722-1809) was an Austrian physician who worked in Vienna, he was also a gifted amateur musician and composer. Using his sensitive ear and musical talent, he invented chest percussion as a diagnostic procedure. His published work, entitled Invention Novum, became a landmark in the history of medicine. He gave an account of a new diagnostic method called "chest percussion", also known as "chest thumping", which could give the physician insight into the internal status of the chest cavity by carefully evaluating the sounds produced by chest percussion (Magner 1992). Only few physicians paid attention to Avenbrugger's invention until Jean Nicolas Covisart (1755-1821) published a translation and commentary in 1808.

2.6 Laennec Rene Theophile Hyacinthe Laennec (1781-1826) was a leading French physician, known for his work on tuberculosis, a disease that he certainly died from at the early age of 45. He postulated that all tubercular phenomena (phthisis, scrofula and miliary) constituted a single disease, which therefore raised the theories of etiology. His invention of the initial primitive stethoscope in 1816 made some of his critics scoff at the perceived value of his instrument. However, in 1825 his instrument was referred to as "Laennec's immortal work". Laennec was born in 1781 at Quimper in Brittany, northwest France. At the age of 14 1/2 years, he studied medicine at Nantes under his uncle, Guillaume Francois Laennec, rector of Nantes University. He later went on to study in Paris in 1801 and qualified in 1804. He was awarded many prizes and was asked to withdraw from some competitive examinations so as not to discourage his fellow-pupils.

2.6.1 Laennec's Work on Tuberculosis The extensive pathological anatomy made by Laennec was recorded in his 800-page manuscript, still in existence in Nantes and Paris, but was never published. He identified the various stages of pulmonary consumption and defined the tubercle that was

19

found in many other body organs. During his work, Laennec was subjected to many cuts to his fingers while cutting through the cadavers, which possibly contributed to his contracting and dying from the disease (Keers 1981; Sakula 1981).

2.6.2 Laennec's Invention of the Stethoscope While examining a stout female patient in 1816 at Necker hospital, he used tightly rolled sheets of paper attached to a wooden model in order to better hear her heart sounds. This primitive stethoscope of his went through many variations of form and material throughout the 19th century. He published his first book on auscultation in 1819, titled Del' Auscultation Mediate etc. This book introduced a diagnostic tool into medicine for the first time. He also wrote the most clear, accurate and almost complete account of chest diseases. While working in Cornwall, UK in 1821, Dr. John Forbes (later Sir John), translated Laennec's book into English, abridged and condensed. The first edition of Forbes' translation seriously underestimated the role and influence of Laennec's stethoscope, but in the second edition he admitted that he had been wrong in this estimation.

2.6.3 Laennec's Tubercles Laennec wrote on "phthisis Pulmonalis" and "of the essential, or anatomical character of tubercles in the lung" and stated: "The existence, in the lungs, of those peculiar productions to which the name of Tubercles has been restricted by modern anatomists, is the cause, and constitutes the true anatomical character, of Consumption". Laennec then went on to describe the appearances of tubercles, tracing them from their first, observable state, in which he wrote that they vary from the size of a millet seed to that of a hemp seed (and may be called miliary tubercles), to the ultimate fibro-cavernous destruction of lung substance (Bishop 1981). Later, he wrote: "There is perhaps no organ free from the attack of tubercles, and wherein we do not, occasionally discover them in our examination of phthisical subjects. The following are the parts in which I have met with these degenerations, and I enumerate them in the order of their frequency: the bronchial, mediastinal, the cervical,

M. M. Madkour et al.

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and the mesenteric glands; the other glands throughout the body; the liver - in which they attain large size, but come rarely to maturation; the prostate - in which they are often found completely softened, and leave, after their evacuation by the urethra, cavities of different sizes; the surface of the peritoneum and pleura, in which situations they are found small and very numerous,... the epididymis, the vasa deferentia, the testicle, spleen, heart, uterus, the brain and cerebellum, the bodies of the cranial bones, the substance of the vertebra or the point of union between these and the ligaments, the ribs, and lastly, tumours of the kind usually denominated scirrus or cancer, in which the tuberculous matter is either intimately combined with, or separated in distinct patches from, the other kinds of morbid degeneration existing in them." (p. 11; quoted from Bishop 1981). Laennec restored the hope and confidence for a cure or suspension of symptoms for patients suffering from tuberculosis. He discussed remedies that has been used since antiquity, such as blood-letting, mercurial salivation, emetics, charcoal, mushrooms, red cabbage, the conserve of roses, crabs, oysters, frogs, chocolate, alcohol, opium, cinchona, preparation of lead, hydrocyanic acid and the use of a swing for the cure of phthisis (Bishop 1981). Laennec never embraced these remedies, and it was said that his approach to medicine was Hippocratic.

2.7 Gaspard Laurent Bayle (1774-1816) A French physician, Laennec's friend and one-time co-worker, published the result of his research on tuberculosis. He carried out extensive pathological studies of 900 post-mortem specimens. He recognized and reported six types of pulmonary consumption including: tubercular phthisis, glandular phthisis, phthisis with melanosis, ulcerous phthisis, calculous phthisis and cancerous phthisis. He noted "Four of his ex-named varieties of phthisis represented one disease, tuberculosis in varying phases of developmene' He contributed ideas to the knowledge of the pathology and the unity of tuberculosis although he failed to grasp the etiological identity of one phase with another (Bishop 1981). Thomas Young (1773-1829), a well-known London physician who initially studied medicine at St. Bartholomew's Hospital in London (Bishop 1973) wrote A Practical and Historical Treatise on Consumptive Diseases published in London in 1815. He devoted nearly 12 pages to Bayle and wrote:

"Mr. Bayle's elaborate work has very lately been introduced to the English public, with no small parade, by his translator Dr. Barrow; and it must not be denied that it contains much valuable matter, although the practical importance of anatomical observations in general may have been somewhat overrated by the author. The translator talks of the propriety of some legislative measures for enforcing the keeping of registers of cases in all public hospitals, observing that, if such a regulation were adopted, France would not 'long exult as at present in her claim to preeminence, either in pathological or practical knowledge'" (p. 447; quoted by Bishop 1973).

2.8 "Tubercle" and "Tuberculosis" The term "tubercle" was first used by Franciscus (Francois) de la Boe, also known as Sylvius of Leyden (1416-1672), an outstanding Dutch anatomist and iatrochemist. He attempted to classify catarrhal diseases into groups distinguished by the chemical qualities of the humor produced-either alkaline or acidic in nature. He established the first university chemical laboratory at Leyden. He was the first to use the term "tubercle" and stated that tubercles were often seen in the lungs of consumptives (Johnston 1993). The term "tuberculosis" was first used by Laennec and his friend and colleague, Bayle. Laennec postulated that all tubercular phenomena (phthisis, scrofula and miliary) constitute a single disease and raised the theory of etiology.

2.9

The White Plague "I decided to entitle this presentation ...by avoiding the unfashionable word 'tuberculosis' and substituting a more romantic appellation:' noted Professor John F. Murray, University of California, San Francisco, when he gave "The 1989 J. Burns Amberson Lecture", entitled: "The White Plague". He hoped to stimulate an interest in and recruit an audience for his lecture, and certainly he did. Prof. Murray went on to say, "I borrowed my title from that of a book by Rene and Jean Dubos, who borrowed it, in turn, from Oliver Wendell Holmes, Sr., who first used 'the white plague' in 1861 to describe the enormity of the problems caused by tuberculosis at the time:' The phrase

21

Historical Aspects of Tuberculosis

"the white plague" has intrigued many historians, such as Dr. Allen B. Weisse (1995) from the University of New Jersey Medical School who wrote: "I wondered if the 'white' in 'white plague' related to race, pathology, the appearance of the patient or perhaps some other aspect of the disease". Although the population of Western Europe and the USA is predominantly white, non-whites are more susceptible to the disease than whites. Pathological appearance of the lesion in early post-mortem descriptions included terms such as "tumor albus, or white swelling" as in Richard M. Burke's book, An Historical Chronology of Tuberculosis (Myers 1977). Others described hepatic lesions as "the white tubercle of the liver", while Matthew Baillie (in 1703) described pulmonary tubercle as gray, or, as noted by the great Laennec, grayish or, following caseation, yellowish (quoted by Weisse 1995). If "white" reflected the patients' appearance, Dr. Weisse explained, clinicians have often commented on flushed skin rather than pallor reflecting the febrile state. The term "plague", from the Latin "to blow or strike", is an epidemic disease causing a high rate of mortality: pestilence. Early prehistoric ancient Greeks knew tuberculosis when over 130,000 persons died over a 5-year period (430-426 B.C.). Hippocrates (460-370 B.C.) was alive in Greece during that plague but he used the term "phthisis" to describe the rapidly progressive pulmonary form of the disease, which later became known as "galloping consumption". In the USA and Western Europe during the 18th and 19th centuries, tuberculosis was one of the most common causes of death (the other was cholera, the black death), and the usual chronic course of the illness was well recognized. Therefore the term "plague" was obviously a misnomer when referring to tuberculosis (Weisse 1995).

2.10 Robert Koch (December 11, 1843-May 27,1910) While working on anthrax in 1877, Robert Koch, a great German physician and bacteriologist, was the first to demonstrate that a specific pathogen caused a specific disease. He discovered tubercle bacillus on 24 March 1882 as the cause of tuberculosis. On 17 September 1883, while working in Alexandria, Egypt, he discovered Vibrio cholerae, but was unsuccessful in transmitting the infection to an experimental animal. This made his critics doubt his "postulates", which were not fulfilled; a criterion that he determined in

order to relate an organism as a causative agent to a disease. He also discovered the method of transmission of bubonic plague and sleeping sickness as well as other tropical diseases. One day after his birthday, on 12 December 1905, Koch received the Nobel Prize. Heinrich Herrmann Robert Koch was born on 11 December 1843 in Clausthal, a small mining city located between Hannover and Gottingen. He was the 3rd in a family with 11 (surviving) children (9 boys and 2 girls) and was closely attached to his mother. He was interested in photography, mathematics and natural sciences as a child. He studied English and French and was fluent. He joined Gottingen University at age 19 years to become a teacher but decided to change to studying medicine. He graduated at the age of 23 years and wanted to become a ship's doctor but gave up his idea in favor of becoming engaged to his sweetheart, Emmy Fraatz. He worked as medical assistant in Hamburg General Hospital as a source of income, but the income was not enough to provide for a family. He married Emmy on 16 July 1867. However, he had financial difficulties as he moved to other jobs. He volunteered in the Franco-German war in 1870 after having been rejected for army service because he was nearsighted. In 1872, he took an exam for District Medical Officer. His work on anthrax began in 1875. At the end of that year, he discovered the causative organism after inoculating a rabbit through the ear and showing the bacteria on microscopic examination (Brock 1999). The discovery of the etiology of anthrax made Koch famous among the intellectuals.

2.10.1 The Discovery of the Tubercle Bacillus Tuberculosis was strongly suspected to be a contagious disease before the discovery of the bacillus. In 1865, it was shown by a French physician, Jean Antoine Villemin (1827-1892), that tuberculosis was transmissible to experimental animals; however, the suspected causal organism had never been seen (Brock 1999). In August, 1881, Koch started his experimental studies, aiming at identifying the causative organism. On 24 March 1882, Koch gave his historic lecture in Berlin on the discovery of the bacillus. He was aware that the presence of the organism did not indicate that it was the cause of the disease. He proposed a criteria in order to make such an association, which became known as "Koch's Postulates". The postulates are a series of steps that should be followed to prove that a specific organism is the cause of a specific disease. The organism

22

must be isolated and grown in a pure culture and the pure culture must induce the disease when injected in experimental animal. Koch himself failed to fulfill his postulate when he discovered Vibrio cholerae as the cause of cholera while working in Alexandria, Egypt on 17 September 1883. He was not successful in establishing an animal model at that time and his postulates were thrown back at him by his critics in a proceeding of the first cholera conference. An editorial that appeared in the «British Medical Journal" in 1884 (quoted by Brock 1999) noted, «Of course, the whole point turns on whether Dr. Koch has made out that the comma-bacillus is really the cause of the disease. In order to demonstrate that a given bacterium is the cause of a disease, it must be proved: 1) that a special bacterium with definite characteristics marking it out from other forms of bacteria, is constantly present in the parts affected 2) that his bacterium is present in sufficient numbers to account for the disease 3) that it is not similarly associated with other diseases 4) that this bacterium can be cultivated apart from the body, and that its introduction into lower animals is followed by the same effects as the introduction of the infective material itself" In response to his critics, Koch wrote,«The comma bacillus is a specific bacterium, found exclusively in association with Asiatic Cholera. As long as this statement is not contradicted, all of my conclusions regarding the diagnostic utility of this bacterium and its relation to the pathology of cholera remain valid. However, my opponents state that a causal relationship between the comma bacillus and cholera has not been established because it has not been possible to induce cholera artificially in experimental animals. This objection is not valid because Professors Rietsch and Nicati succeeded, during the last cholera epidemic in Marseille, to bring about cholera-like symptoms in dogs and guinea pigs, provided the bile duct had been tied off and a pure culture of the comma bacillus injected directly into the duodenum... These studies have been recently repeated here in my laboratory, using a pure culture diluted so much that less than one-hundredth of a drop was injected... With few exceptions, the treated animals died in 1.5-3 days... These animal studies have been extended in other directions and have shown without a doubt that the comma bacillus is pathogenic. Under these circumstances, it would be very advisable to avoid completely any inoculation studies in humans (as has been recently discussed), and restrict all inoc-

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ulation studies to guinea pigs and other experimental animals" (quoted by Brock 1999).

2.10.2 Koch's Secret Cure (Tuberculin) and the Storm That Followed In August, 1890, Koch dramatically reported secret research in a speech given at the 10th International Congress of Medicine in Berlin. He implied that he found a cure for tuberculosis and said: «[after long study of many chemicals 1I have at last found substances which both in the test-tube and in the living body prevent the growth of the tubercle bacilli. All such investigations... are very exhausting and slow; and my experiments with these substances, though lasting more than a year, are not yet concluded, so that all I can say at present is that if guinea pigs are treated they cannot be inoculated with tuberculosis, and guinea pigs which already are in the late stages of the disease are completely cured, although the body suffers no ill effects from the treatment. From these experiments I will draw no other conclusion at present than that it is possible to render pathogenic bacteria within the body harmless without ill effect on the body itself" (quoted from Brock 1999). News spread rapidly and thousands of patients traveled from all over Europe to Berlin, seeking the injections of «Koch's lymph" hoping to get cured. His new «remedy" was tried on a human at the Charite Hospital, but the validity of the cure were doubted. Tuberculous patients commonly showed a strong reaction to injection with tuberculin as a hypersensitivity reaction and became known as «Koch Phenomenon;' Later «tuberculin" was used as an important diagnostic test. Koch injected himself with the tuberculin and reported fever, rigor, joint pains, nausea and vomiting which improved after 12 h to few days. He refused to describe the method of tuberculin preparation and was severely criticized for not revealing the nature of his remedy. In 189011891, it became clear that Koch's tuberculin had no therapeutic value but was of great diagnostic use. The compound was known as «remedy" or «Koch's lymph:'but in 1891 Koch and Libbertz labeled it «tuberculin". The stress of the tuberculin storm created by his cynical critics was intense and Koch arranged for a long vacation to Egypt in 1891, leaving his son-in-law in charge of tuberculin and the Institute for Infectious Diseases (Magner 1992). The «tuberculin" crisis had severely affected Koch's scientific life. He became acquainted with Hedwig Frieberg, a pretty, 17-year-old art student.

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His relationship with his wife Emmy had been deteriorating and they eventually divorced in 1891. He married Hedwig on 13 September 1893, when he was 50 years and she was 20 years of age. They lived together until he died in 1910. Hedwig Koch died on 16 June 1945. "In his lifetime, Robert Koch did more to single handedly advance the world's understanding of microbes as causes of disease than any other man, with the exception only of his great French rival Louis Pasteur" (James Strick, who wrote the foreword to Thomas D. Brock's book, Robert Koch: A Life in

appear brown, while the tubercle bacilli on the other hand, stain a beautiful blue. Moreover, all other bacteria which I have investigated to date, with the exception of the lepra bacilli, take on a brown colour with this staining method. The color contrast between the brown stained tissue and the blue tubercle bacilli is so striking that the latter, which are present often only in very small number, nevertheless, are to be found and identified with the greatest certainty" (pp. 227-228 in the original: Koch 1882; the translation is that of William de Rouville, 1938, pp. 854-55).

Medicine and Bacteriology, 1999). 2.10.3 The Ziehl-Neelsen Stain, or Koch-EhrlichRindfleisch-Ziehl-Neelsen Method? The history of staining the tubercle bacilli started when Koch's made his discovery of the tubercle bacillus on 24 March 1882. The translation of Koch's first publication on the subject on 10 April 1882 Die Aetiologie der Tuberculose was translated by William de Rouville in 1938 and quoted in the Bishop and Neumann (1970) paper on the history of the Ziehl-Neelsen Stain. Koch's method of staining the tubercle bacillus included the three most important factors: the use of a chemical substance that made staining possible (mordant), heat, and decolorizing. He wrote: "The material to be examined is prepared in the usual manner for examining for pathogenic bacteria, and either spread on the cover slip, dried and heated or cut into sections after fixation in alcohol. The cover slips or sections are placed in a staining solution of the following constitution: 200 cc of distilled water are mixed with 1 cc of a concentrated alcoholic solution of methylene blue, shaken up, and then 0.2 cc of a 10% solution of potassium hydroxide is added with repeated shaking. This mixture must show no precipitate after standing for several days. The materials to be stained remain in this solution for 20 to 24 hours. By heating the solution to 400C in a water bath this time can be shortened to a half to one hour. Following this the cover slips are covered with a concentrated aqueous solution of vesuvin (Bismarck brown) which is filtered each time before using, and one to two minutes rinsed with distilled water. When the cover slips come from the methylene blue, the attached layer appears dark blue and markedly (p. 222) overstained. During the treatment with vesuvin this blue colour is lost and it appears stained a faint brown. Under the microscope all the constituents of animal tissue, that is, the cell nuclei and their products of disintegration

2.11

Franz Ziehl (1857-1926)

A neurologist at Lubeck, Franz Ziehl was born in Germany at Wismar in Mecklenburg-Schwerin. He qualified and worked in Heidelberg as assistant in a medical clinic from 1881 until 1886. He moved to Lubeck in 1887 to work as neurologist until his death on 7 April 1926. Ziehl published his first paper on staining the tubercle bacillus on 12 August 1882 and described how he had modified Ehrlich's method by changing the mordant from aniline to carbolic acid. He wrote: "Up to the present we have not been able to obtain the pure constituents of aniline oil. As it was necessary to use a substance belonging to the aromatic series we tested such a one, the reaction of which was known to us from that second aniline oil, namely carbolic acid" (Ziehl 1882 p. 451, quoted by Bishop and Neuman 1970). Description of the acid-fastness of the bacilli by Ziehl was published in another paper on 25 April 1883, entitled "On the staining of tubercle bacilli" (Ziehl 1883b, quoted by Bishop and Neumann 1970). "The importance of finding tubercle bacilli especially with regard to diagnosis and prognosis" was the title of another paper made by Ziehl that appeared on 31 January 1883 and reported the finding of the bacilli in 73 cases of pulmonary tuberculosis and concluded: "1.In most cases of pulmonary tuberculosis tubercle bacilli may be found; exceptions do occur. 2. The finding of tubercle bacilli may confirm the diagnosis of tuberculosis. In some cases the differential diagnosis is possible by this finding. 3. If tubercle bacilli cannot be found, it is not always possible to exclude tuberculosis. 4. The number and kind of tubercle bacilli do not allow any prognostic conclusions." (Ziehl 1883a, quoted by Bishop and Neuman 1970).

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2.12 Friedrich Karl Adolph Neelsen (1854-1894) A German pathologist born at Utersen, Holstein, Neelsen studied, qualified and worked as assistant at the Pathology Institute at Leipzig in 1876 until 1878. Between 1878 and 1885 he had a similar position at Rostock and became a professor in 1884. He moved to Dresden in 1885 and worked as Prosector at the Staedtisches Krankenhaus. He died at the age of 40 years on 11 April 1894. His death was almost certainly due to tuberculosis, as he suffered from emaciation, cough, progressive hoarseness and general deterioration. These symptoms started in the autumn of 1893. In 1883, Neelsen combined ZieW's mordant with Ehrlich's red stain fuchsin in staining the tubercle bacillus. Neelsen published the first article on the subject on 14 July 1883 in a German journal "Centralblattfur die medicinischen Wissenschaften". The title of the article was "A casuistic note on the theory of tuberculosis" and noted: "In this case I used first as a staining fluid a threequarter per cent solution of fuchsin in 5% carbolic acid with admixture of some alcohol and decolorized with 25% sulphuric acid. This method, which I have used almost exclusively for a long time, gives in my hands better results than the other methods (perhaps only because I am most accustomed to it)" (Neelsen 1883 p. 500, quoted by Bishop and Neuman 1970).

2.13

Paul Ehrlich

A German bacteriologist and pathologist born in 1863, Ehrlich made an improvement of Koch's method of staining the tubercle bacillus. His first communication on the subject was made to the Berlin Society ofInternal Medicine on 1 May 1882. He used decolorization with a mineral acid, used a red stain, fuchsin, and changed the mordant to aniline oil instead of alkaline methylene blue. He wrote: "After these remarks I wish to briefly report the method. I have worked almost exclusively with dry preparations, Le. of sputa, but I have done controlled trials which have proved that the method is also applicable to section preparations. I proceed by taking out a small particle with a dissecting needle from the sputum and squeezing it flat between two cover glasses, and I have found that those 0.10 to 0.12 mm are best. Under these conditions it is easy to obtain uniformly thin layers from the small plug of sputum. Then both glasses are torn from each other and one then gets two

M. M. Madkour et aI.

thin layers, which can easily be air-dried. These preparations are not yet suitable. Usually I have done this by keeping the preparations for one hour at 100 to 110oe; however there exists a more convenient method which I have seen used in the Kaiserliches Gesundheitsamt and which consists in passing the air-dried preparations three times through the flame of a Bunsen burner. For staining I use water, saturated by aniline oil, which may be prepared in a few minutes by shaking water with surplus aniline oil and filtering it through a moistened filter. To the water-clear fluid obtained in this way one adds drop by drop a saturated alcoholic solution of fuchsin or methylene violet until a distinct opalescence indicates saturation with the dyestuff. Upon this fluid the preparations are made to float and staining is adequate within a quarter to half and hour. The colour of the tubercle bacilli does not disappear under the influence of vesuvin (Bismarck brown) or only very slowly. It is necessary to use an acid and I soon turned to the use of strong, I may even say heroic ones. A mixture consisting of 1volume of official nitric acid and 2 volumes ofwater can be recommended. Under its influence one sees the preparation grow pale within a few seconds, yellow clouds rise and the preparation becomes white:' "If one examines the preparation at this stage, it is seen to be decolorized and only the bacillus has maintained its intensive staining. It might be possible to examine such a preparation, but the technical difficulties of focusing the bacillus are extremely great. It is recommended to stain the background yellow if the bacilli are violet, and blue if they are red." "I have succeeded in staining the bacillus with all basic aniline dyes, even with Bismarck brown, and hence it follows that the substance of the bacillus itself does not differ from that of other bacilli in its staining properties. But the discrimination of the tubercle bacillus by staining depends on the presence of a surface layer with characteristic and specific properties. The first of these to which Koch's technique is pointing is that the covering layer is permeable only to dyes under the influence of alkalis'." (Ehrlich 1882 pp. 269-270) In 1882, Edvard Georg Rindfleisch, a Wiirzburg pathologist, made an improvement on Koch's method of staining tubercle bacillus by heating the slide instead of putting it in hot water. Historians of tuberculosis made good accounts in the literature of the history of the staining methods of the tubercle bacillus since its first discovery by Robert Koch in 1882. The time between 1882 and 1888 was noted by Predohl, where all the methods of staining Koch's bacillus have been described. He was the first to mention the words, the Ziehl-Neelsen

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method, and noted,"I must describe the modification of Ziehl's method given by Neelsen". The phrase Ziehl-Neelsen method of staining tubercle bacilli was not mentioned in the first edition of Edgar March Crookshank's book of bacteriology in 1886. Although his list included both Ziehl's and Neelsen's methods, the phrase"Ziehl-Neelsen method" was not used. In the fourth edition in 1896, Crookshank noted: "The Ziehl-Neelsen method is preferred by the author for sections and cover-glass preparations;' The phrase and label of the Ziehl-Neelsen method may have become known sometime between 1890 and 1893 (Bishop and Neumann 1970). These two German doctors, Ziehl and Neelsen, described their methods nearly 100 years ago. These methods remain in use at present and will always be in the literature of tuberculosis.

2.14 The Evolution of Tuberculosis Treatment From the time of the Ancient Egyptians, Hippocrates, Galen, and the Middle Ages, up until the early 19th century, little has been added to the therapy of tuberculosis. The ancient Egyptians treated scrofula with surgery and dressings (see Chap. 1). Hippocrates' treatment was simple and related to confinement into the temples of health, resting, praying, drinking milk, dieting, exercising and avoidance of extreme weather. Drug treatment was not emphasized. Five centuries later, Galen added little to the classic Hippocratic treatment for pulmonary tuberculosis. Galen recommended bed rest, gargles, application of plasters to the chest and head and the diet and dry air ofhealth resorts. Such concepts of treatment for tuberculosis remained for more than 1000 years. In medieval Europe, scrofula was more common than the pulmonary form of the disease. It was customary to believe in the "King's touch", in which the kings of France and England were believed to have the power to cure scrofula simply by touching its victims. This custom originated in the 12th century and persisted until the 18th century (Risse 1993; Gallagher 1993; Johnston 1993). The Oxfordshire village of Stanton St. John was burdened with scrofula, "The King's Evil". Its residents desired the cure by the touch. The touching was performed by English sovereigns from Edward the Confessor to Queen Anne. Those who did not respond chose a range of equally efficacious modes of therapy accompanied by prayer. Records at Stanton St. John indicated that certificates attested to the eligibility of parishioners to practice "The King's Touch" (Crawfurd 1911).

In North America, early in the 17th century, the Reverend Cotton Mather, famous for his persecution of witchcraft in Salem, wrote a medical work entitled The Angel of Bethesda, which quoted Galen on therapy and directed the patient to repent his sins. He recommended a concoction obtained by strangling two roosters, beating them to a pulp, boiling them in a mixture of wine and water, straining the mixture through a cloth and adding raisins, hartshorn, maidenhair and saffron (Rosenblatt 1973).

2.15 Climatological and Sanatorium Therapy During the early industrial revolution, around 1780, the recorded mortality rate due to tuberculosis in England was 1,120 per 100,000 population (Rogers 1969). Early in the 19th century the mortality rate started to decline and the 19th century was called the century of tuberculosis. Treatment of tuberculosis at the beginning of the 19th century was summarized in a thesis of Edward Delafield published in 1816 (quoted by Rogers 1969). Delafield recommended blood letting, emetics, mercury, opiates, digitalis and Peruvian bark. He recommended sea voyages and mild climate, particularly in the south of France. He speculated that the nausea of seasickness was the principle that explained the benefits of sailing. In 1834, Samuel Morton, who studied in Paris and was lectured by Laennec, Bayle and Louis, published the first American textbook on tuberculosis (Rogers 1969). Morton deplored blood letting for the treatment of tuberculosis and was in favor of the effect of climate, particularly cold, dry atmosphere. Many other authors in the 19th century were interested in climatotherapy and recommended other areas, such as the Rocky Mountains and Pike's Peak. Rogers (1969) quoted from Disturnell (1867) who quoted reports of surgeon G.K. Wood, of the US Army, stationed at Fort Laramie, Wyoming: "The climate of these broad and elevated table-lands which skirt the base of the Rocky Mountains on the east, is especially beneficial to persons suffering from pulmonary diseases, or with a scrofulous diathesis... It is of great importance that the climate of this region should be generally known..." Daniel Dark (1850), quoted by Rogers (1969), described the Missouri river and its environs around Fort Leavenworth in a work entitled, "Journeys of health on the great plains;' He wrote: 1. The patient escapes from malaria, or that condition of the atmo-

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sphere, which, in the larger portions of the Great Valley, gives origin to intermittent and remittent fevers, too often followed by infirmities for which a change of locality is the only effectual remedy. 2. He is constantly immersed in a dryer air.... 4. He lives on a reduced, solid, and simple diet. ..6. He takes regular saddle exercise...8. He is divested of his old cares..... 9. He is redeemed from the dominion of empiricism and polypharmacy." In the second half of the 19th century, climatography was well established. The American Climatological Association was formed in 1884 and held the first meeting in Washington, D.C. in 1908. The Sixth International Congress on Tuberculosis was held, and W. Jarvis Barlow of Los Angeles said, "The high altitude treatment received great impetus from this congress:' The transactions of the congress of 1908 showed something quite different: "The apogee of high altitude therapy have been reached and its advocacy would slowly subside thereafter" (Rogers 1969). The name of the "American Climatological Association" was changed in 1914 and again in 1933 as the problem of the efficacy of altitude therapy was a classic case of the problem of multiple variables. In 1960, Spriggs (quoted by Rogers 1969) published an article and said, "What statistical evidence there was for the value of rest treatment does not nowadays convince.....". By the time of World War II, climatotherapy in the United States was a dead issue (Rogers 1969; Wilson 1979).

2.16 Sanatorium-Bimaristan or Deir el-Bahri "I am Andropachus from Macedonia. I came to visit Amenotis. I was very ill, but now I am cured thanks to the mighty God. I ask you merciful God to give us all good health. Goodbye:' These were the writings in hieroglyphic by an ancient sick Greek patient on the wall of Deir el-Bahri temple, which was believed by Milne and Joseph (1914) to be the first Sanatorium in history (see Chap. I). Nowadays visitors can see the writing of patients who sought cure in this sanatorium "Temple:' Bimaristan (houses of the sick in Persian) were first established in 803 by the famous "Harun AIRashid" in Baghdad. Sanatorium, is a word that comes from the Latin Sanatorius meaning "an establishment providing therapy" (Wright 1988). In 1836, George Bodington, a practitioner in Birmingham, UK, was the first to propose housing tuberculosis patients in one build-

ing (Rosenblatt 1973). His aim was to provide these patients with diet, physical activities and fresh air, but the proposal failed for lack of support. The first sanatorium for the treatment of patients with pulmonary tuberculosis was opened in 1859 in Gorbersdorf, Germany by Dr. Hermann Breemer (Rosenblatt 1973). He believed that mountain air and exercise were beneficial. Many other sanatoria were opened, and perhaps one of the most famous was Trudeau Sanatorium in Saranac Lake, N.Y. It was established by Edward L. Trudeau in 1885, a young New Yorker who decided on a sudden impulse to become a doctor as he passed by the College of Physicians and Surgeons while walking along Fourth Avenue in 1868. Shortly after graduation, he was struck by tuberculosis. Fifty years later, the number of sanatoria in the USA had increased to 600 with a total bed capacity of 95,000.

2.17 Artificial Lung Collapse Therapy Bourru (1774) in Paris was the first to think of inducing artificial lung collapse,aiming at the arrest ofthe disease, but the concept was not given a clinical trial (Rosenblatt 1973). An attempted trial to induce pneumothorax in two tuberculous patients by Carson had failed, but despite these failures its concept was intriguing. In 1835, McRuer from the USA and Houghton and Strokes from the UK were successful and their patients improved. Others had similar encouraging experiences, including Cayley (1885) who used the therapy in a patient who had had a pulmonary hemorrhage (Rosenblatt 1973). The most significant work on artificial pneumothorax therapy was reported by Carlo Forlanini (b. 1847) in 1882. He used an apparatus to force nitrogen gas through a tube and a needle inserted into the patient's pleural cavity and was successful (Rosenblatt 1973). Pneumothorax remained popular despite its therapeutic limitations and complications, such as development ofpleural effusion, emphysema and increased mortality, and remained in use until the 1940s.

2.18 Thoracoplasty Rib resection to collapse and close tuberculosis cavities (thoracoplasty) was a popular operation in the late 19th and early 20th centuries. The first tho-

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racoplasty was performed by de Cerenville in 1885, and in 1909, Braver introduced a two-stage technique to reduce the surgical shock that was noted in the one-stage thoracoplasty (Rosenblatt 1973). Phrenic nerve crush was used to paralyze the diaphragm and compress the affected lung. Pneumoperitoneum with phrenectomy were popular in the 1930s and early 1940s (Godfrey and Norman 1969).

2.19 Plombage Plombe is a word introduced by Professor Custodis of Dusseldorf, Germany to describe the implants he used to produce infolding of the sclera in cases of retinal detachment. Plombage (a French word meaning stopping) is the procedure of packing the pleural cavity with inert material. Extrapleural pneumothorax by instillation of oil to maintain lung collapse was first suggested by Mayer 1913 (quoted in Magner 1992), but was not used until two decades later. Insertion of plombe from fatty tissue, paraffin or Lucite balls into the extrapleural space was called plombage. The procedure was used to provide permanent compression of the underlying diseased lung, particularly with large peripheral cavities.

2.20 Pulmonary Resection In 1883, Block (quoted in Magner 1992) was the first to perform pulmonary resection for tuberculosis in man. The patient died and at autopsy there was no evidence of tuberculosis. Block shot himself. In 1884 Kronlein (quoted in Magner 1992) resected apical tuberculosis in two patients and both died. Later, despite advances in surgery in the pre-antibiotics era, postoperative mortality was high.

2.21 The History of BCG Leon Charles Albert Calmette (b.l863) and Camille Guerin (b.l872) were French bacteriologists working in the Pasteur Institute in Lille. Their work on the vaccine began in 1908 on a strain of Mycobacterium tuberculosis isolated by Nocard from a cow that had tuberculous

mastitis. An attenuated bovine bacillus was first isolated in 1921 by Calmette and Guerin after years of laboratory cultivations. The attenuated strain of tubercle bacillus was incapable of producing tuberculosis in any laboratory animals (Calmette 1923). In 1930, a tragic incident occurred whereby administration of a virulent tubercle bacilli stored in the same refrigerator as the BCG led to the death of 67 German babies in Lubeck. BCG was initially given to many infants in France as an oral vaccine but the Scandinavians pioneered its administration intradermally in 1950.

2.22 The History of Chemotherapy for Tuberculosis The antibiotics era began in 1928 when Alexander Fleming (1881-1955), a Scottish bacteriologist and immunologist in London, discovered the effect of the mold Penicillium notatum on bacteria. His discovery remained a laboratory curiosity until World War II. "Fleming, recalled that neither bacteriologists nor physicians paid any attention to penicillin until the introduction of sulphonamide" (Quoted from Magner 1992 p. 353). A sulfur containing red dye called Prontosil, which protected mice from staphylococcal and streptococcal infections was discovered by Domagk in 1932. Gerhard Domagk (1895-1964) was a German chemist and pathologist. In 1935, Domagk reported "A contribution to the chemotherapy of bacterial infections;' and with that report he initiated the age of chemotherapy. Prontosil and other sulfonamides, including promizole and diasone, were hailed as "miracle drugs" in the 1930s. In 1939 Domagk was awarded the Nobel Prize for Physiology and Medicine "For the discovery of the antimicrobial effect of Prontosil;' but Nazi officials would not allow him to accept it. He finally received the Nobel Medal in 1947. Sulfonamides were tried on patients with tuberculosis but were found to be of little value (Hinshaw et al. 1944,1946; Hinshaw 1969).

2.23 Streptomycin and Modern Antituberculosis Therapy In 1943, Selman A. Waksman (1888-1973) and coworkers isolated streptomycin from two strains of

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an actinomycete species. Much of the initial work was done by his graduate student, Albert Schatz. Waksman was a Russian immigrant to the USA who, in 1915, graduated from the Agricultural College of Rutgers University in New Jersey (Ryan 1992). He published a book titled, Principles of Soil Microbiol-

ogy.

2.24 Animal and Human Trials on the Efficacy of Streptomycin Experimental trials of streptomycin on animals infected with tuberculosis and on tuberculous patients demonstrated its efficacy (Feldman et al. 1947; Hinshaw et al. 1946; Pfuetze et al. 1955) and by the late 1940s streptomycin's effectiveness was clearly established. Schatz shared royalties with Waksman but did not share the Nobel Prize, which was awarded to Waksman alone. The use of streptomycin as a single agent for the treatment of tuberculosis was difficult (Cooke et al. 1946).

2.25 Para-Aminosalicyclic Acid (PAS) Dr. Jorgen Lehmann was born in Denmark and graduated from a Swedish medical school. He went to New York as a research fellow then returned back to work as Chief of Chemical Pathology in Goteborg, Sweden. He developed PAS in 1943, after synthesizing analogues and various derivatives of salicylic acid in Swedish laboratories. PAS was first used as a topical agent for the treatment of tuberculous fistulous tract caused by cervical scrofula or by intrapleural installation in patients with tuberculous empyema. The first oral tablets of PAS were used in October 1944 and in 1946, Lehmann reported a preliminary laboratory and clinical report on the drug (Lehmann 1946, 1964). Feldman and colleagues (1947) from Mayo clinic tried PAS on animals infected with tuberculosis and found it to be effective (Feldman et al. 1947). The British Medical Research Council (MRC) study on using PAS alone, streptomycin (SM) alone or PAS plus SM found SM alone was superior to PAS alone and that their combination showed clinical and radiological improvement with reduced emergence of resistant organisms (British Medical Research Council 1950).

2.26 Isoniazid Isoniazid (isonicotinic acid hydrazide, INH) was first synthesized by Meyer and Malley in Prague (Meyer and Malley 1912). However, it was not until 1942 that its antituberculous effect was noted by Chorine (1942). In 1951-1952, Hoffman-La Roche, Squibb in the USA, and Bayer in Germany independently and simultaneously developed INH. The first clinical trial of INH alone for the treatment of tuberculous patients was in Seaview Sanatorium, New York in 1951 by Robitzek and Selikoff (Robitzek and Selikoff 1952; McDermott 1969).

2.27 Rifampicin (Rifampin) The initial microbiologically active components of the rifamycin group were obtained from organisms of the species Amycolatopsis mediterranei found in the soil of a French pine forest in 1957 by Lepetit Research Laboratory in Milan. The microbiologically active components were nicknamed "Rififi" after a popular French movie. Rifamycin B had no intrinsic microbiological activity, but the spontaneous breakdown product, rifamycin SV, was active but was not absorbed by the gastrointestinal tract. Collaboration between Lepetit (Milan) and Ciba-Geigy (Basel), laboratories synthesized a product that was better absorbed orally, and more active for a prolonged period (Maggi et al. 1965; Hobby and Lenert 1968; Sensi 1983).

2.28 Drug Therapy for Cure The principle of multi-drug therapy of tuberculosis was found in the 1960s to be the cornerstone of any effective treatment regimen, as it also prevented the development of drug resistance (Canetti 1962; Mitchison 1965). Canetti (1962) proposed the principle of twophase chemotherapy: in the first phase, with a high cavitary load of bacilli, intensive combined chemotherapy is required, and in the second phase, with a small load of bacilli, continued and prolonged therapy with isoniazid alone is sufficient. Mitchison (1965) recommended initial therapy with a threedrug combination.

Historical Aspects of Tuberculosis

The British Medical Research Council trial recommended three drugs for the first 6 weeks and two drugs for a total of 1 year (British Medical Research Council 1962). Long-term therapy, though effective, was expensive and compliance of patients was difficult. Short-course therapy on large-scale trials, first introduced in East Africa by the British Medical Research Council using several 6-month regimens (East African/British Research Council 1973, 1974), clearly established their effectiveness.

2.29 Conclusion Tuberculosis has been known and recorded since antiquity by the Ancient Egyptians and by Greek, Arab, European and other scholars. Despite the development of knowledge on the pathology and clinical features, the cause remained unknown till the discovery of the bacillus late in the 19th century. The discovery of antibiotics lagged behind the identification of the causative organisms. In the 1930s and 1940s, the use of antibiotics in the treatment oftuberculosis marked a new and distinct era in the history of tuberculosis. Despite the application of antibiotics and subsequent developments of other new anti-tuberculous drugs over the following years, tuberculosis remained a common disease in developing countries. The discovery of HIV in 1981 and its effect on the resurgence of tuberculosis both in developing and developed countries together with the development of drug resistance, remains a challenge.

References Al-Amoud M (1993) The history of tuberculosis. Saudi Med J 14:515-520 Bishop PJ (1973) Thomas Young and his 'A practical and historical treatise on consumptive diseases', 1815. Tubercle 54:159-164 Bishop PJ (1981) Laennec: a great student of tuberculosis. Tubercle 62: 129-134, 147-148 Bishop PJ, Neumann G (1970) The history of the Ziehl-Neelsen stain. Tubercle 51:196-206 British Medical Research Council (1950) Treatment of pulmonary tuberculosis with streptomycin and para-aminosalicyclic acid: a Medical Research Council investigation. BMJ 2:1073-1085 British Medical Research Council (1962) Long-term chemotherapy in the treatment of chronic pulmonary tuberculosis with cavitation: a report to the Medical Research Council by their Tuberculosis Chemotherapy Trials Committee. Tubercle 43:201-219

29 Brock TD (1999) Robert Koch - a life in medicine and bacteriology. ASM Press, Washington DC Buikstra JE (1983) Prehistoric tuberculosis in the Americas (Evanston, Ill.: Northwestern University Archeological Program, 1981) (reviewed by William D Sharpe). Trans Stud Coll Phys Phil 5(5):278-281 Calmette A (1923) Tubercle Bacillus Infection and tuberculosis in Man and Animals (trans WB Soper and GH Smith). Williams and Wilkins, Baltimore MD (quoted by Evans CC, 1998, Clinical tuberculosis, 2nd edn. Davies, Chapman and Hall Medical) Canetti G (1962) The eradication of tuberculosis: theoretical problems and practical solutions. Tubercle 43:301-321 Chadwick J, Mann WN (1978) Hippocratic writings. Penguin, NewYork,pp 117-118 Chorine V (1942) Action de l'amide nicotinique sur les bacilles du genre Mycobacterium. CR Acad Sci (Paris) 220: 150-151 Cooke RE, Dunphy DL, Blake FG (1946) Streptomycin in tuberculous meningitis. Yale J Bio Med 18:221-226 Crawfurd R (1911) The king's evil. Oxford Cruse JM (1999) History of medicine: the metamorphosis of scientific medicine in the ever-present past. Am J Med Sci 318:171-180 East African/British Medical Research Council (1973) Controlled clinical trial of four short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis. Second report. Lancet 1:1331-1338 East-African/British Medical Research Council (1974) Controlled clinical trial of four short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis. Third report. Lancet 2:237-240 Feldman WH, Karlson AG, Hinshaw HC (1947) Para-aminosalicyclic acid in experimental tuberculosis in guinea pigs. Proc Sraff Meet Mayo Clin 22:473-479 Gallagher NE (1993) Islamic and Indian Medicine. In: Kiple KF (ed) The Cambridge world history of human disease. Cambridge University Press, Cambridge, pp 27-34 Godfrey LG, Norman CD (1969) Surgical history of pulmonary tuberculosis: the rise and fall of various technical procedures. Can J Surg 12:381-388 Hinshaw HC (1969) Tuberculosis chemotherapy: reminiscences of early clinical trials. Scand J Respir Dis so: 197- 203 Hinshaw HC, Pfuetzek and Feldman WH (1944) Chemotherapy of clinical tuberculosis with promin (Promizole). Am Rev Tuber 50:52-57 Hinshaw HC, Feldman WH, Pfuetze KH (1946) Treatment of tuberculosis with streptomycin. A summary of observations on one hundred cases. JAMA 132:778-782 Hobby GL, Lenert TF (1968) The antimycobacterial activity of Rifampin. Am Rev Respir Dis 97:713 Johnston WD (1993) Tuberculosis. In: Kiple KF (ed) The Cambridge world history of human disease. Cambridge University Press, Cambridge, pp 1059-1068 Keers RY (1981) Laennec: his medical history. Thorax 36:91-94 Lehmann J (1946) Para-aminosalicyclic acid in the treatment of tuberculosis. Lancet 1: 15-16 Lehmann J (1964) Twenty years afterward: historical notes on the discovery of the antituberculous effect of para-aminosalicyclic acid (PAS) and the first clinical trials. Am Rev Respir Dis 90:953-956 Lyons A, Petrucelli R (1978) Medicine, an illustrated history. Abrams, New York

30 Maggi N, Pallanza R, Sensi P (1965) New derivatived of Rifamycin Sv. Antimic. Agents Chemother 5:765 Magner LN (ed) (1992) A history of medicine. Dekker, New York Magner LN (1992a) Islamic medicine. In: Magner LN (ed) A history of medicine. Dekker, New York, pp 133-152 Magner LN (1992b) The scientific revolution and the circulation of the blood. In: Magner LN (ed) A history of medicine. Dekker, New York, pp 189-216 McDermott W (1969) The story of INH. J Infect Dis 119: 678-683 Meyer H, Malley J (1912) Hydrazine derivatives of pyridinecarboxyclic acids. Monatsh 33:393-414 Mitchison DA (1965) Chemotherapy of tuberculosis: a bacteriologist's viewpoint. BMJ 1:1333-1340 Murray JF (1989) The white plague: down and out, or up and coming? Am Rev Respir Dis 140:1788-1795 Myers JA (1977) Captain of all these men of death. Tuberculosis historical highlights. Green, St Louis Paulsen HJ (1987) Tuberculosis in the native American: indigenous or introduced? Rev Infect Dis 9:1180-1186 Pfuetze KH, Pyle MM, Hinshaw HC et al (1955) The first clinical trial of streptomycin in human tuberculosis. Am Rev TubercoI71:752-754 Powell ML (1992) Health and disease in the late prehistoric southeast. In: Verano JW, Ubelaker DH (eds) Disease and demography in the Americas. Smithsonian Institution Press, Washington DC, pp 41-53

M. M. Madkour et al. Risse GB (1993) History of western medicine from Hippocrates to Germ theory. In: Kiple KF (ed) The Cambridge world history of human disease. Cambridge University Press, Cambridge, pp 11-27 Robitzek EH, Selikoff IJ (1952) Hydrazine derivatives of isonicotinic acid (Rimifon, Marsilid) in the treatment of active progressive caseous-penumonic tuberculosis. A preliminary report. Am Rev Tubercol 65:402-428 Rogers FB (1969) The rise and decline of the altitude therapy of tuberculosis. Bull Hist Med 43:1-16 Rosenblatt MB (1973) Pulmonary tuberculosis: evolution of modern therapy. Bull NY Acad Med 49: 163-196 Ryan F (1992) The Forgotten Plague: how the battle against tuberculosis was won and lost. Little Brown, Boston Sakula A (1981) RTH Laennec 1781-1826 his life and work: a bicentenary appreciation. Thorax 36:81-90 Sensi PG, Greco AM, Ballotta R (1959/1960) Rifomycin I. isolation and properties of Rifomycin Band Rifomycin complex. Antibiot Annu 262 Verano JW, Ubelaker DH (eds) (1992) Disease and demography in the Americas. Smithsonian Institution Press, Washington DC Weisse AB (1995) Tuberculosis: why "The white plague"? Perspect Bioi Med 39:132-138 Wilson JL (1979) History of the American Thoracic Society. Am Rev Respir Dis 119:177-184 Wright KW (1988) A history of the Onondaga Sanatorium for the treatment of tuberculosis. NY State J Med 137-145

Epidemiology

3

Global Epidemiology of Tuberculosis MARCOS

A.

ESPINAL

and

MARIO

C. RAVIGLIONE

CONTENTS 3.1 3.2 3.2.1 3.2.2 3.2.3 3.3 3.3.1 3.3.2 3.3.3 3.4 3.5 3.6 3.7

Introduction 33 Exposure and Infection with M. tuberculosis 33 Exposure 33 From Exposure to Infection 34 Magnitude of Infection Worldwide 34 Active Tuberculosis 34 Risk of Disease 34 Morbidity 35 Mortality 36 The Impact of HIV/AIDS on the Epidemiology of Tuberculosis 37 The Impact of Multidrug-Resistant Tuberculosis 38 Future Trends of Tuberculosis 38 Concluding Remarks 40 References 41

3.1 Introduction Tuberculosis has been present in the lives of human beings for many centuries. Evidence of bone lesions suggestive of tuberculosis in mummies of North America, Peru, and Egypt confirms the ancient impact of this disease on early civilizations (Rothschild et al. 2001; Nerlich et al. 2000; Salo et al. 1994). More than 50 years after the introduction of anti-tuberculosis chemotherapy, one-third of the world population is estimated to be infected with Mycobacterium tuberculosis and active disease remains a worldwide pandemic, with more than eight million new cases

M. A. ESPINAL

World Health Organization, Stop TB Department, Tuberculosis Strategy & Operations, Ave Appia 22, 1211 Geneva, Switzerland M. C. RAVIGLIONE World Health Organization, Stop TB Department, Ave Appia 22, 1211 Geneva, Switzerland

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

every year and almost two million deaths annually (Corbett et al. 2003). Despite the existence of a proven comprehensive cost-effective strategy-DOTS-aimed to reduce mortality, morbidity, and transmission of the disease, only 32% of the estimated new smearpositive tuberculosis cases worldwide were managed under this strategy in 2001. The emergence of the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and of multidrug-resistant tuberculosis (MDR-TB-resistance to at least isoniazid and rifampicin) have posed additional challenges to tuberculosis control. In order to accelerate control efforts and overcome these threats, partnership efforts by the international community and a revised framework for tuberculosis control have been launched at the beginning of this new century. This chapter will review the epidemiology of infection and disease with M. tuberculosis globally, present morbidity and mortality data, discuss the impact of HIV and MDR-TB, and examine future trends.

3.2 Exposure and Infection with M. tuberculosis 3.2.1 Exposure

Tuberculosis is caused by bacilli belonging to the M. tuberculosis complex, especially the human variety. Tuberculosis caused by milk contamination with M. bovis is not an issue any longer. Likewise, tuberculosis caused by M. africanum is rarely reported. The natural history of tuberculosis can be described in four different stages: exposure, infection, disease, and death. Exposure to M. tuberculosis is influenced by the number of infectious cases and the duration of their infectiousness. The intensity of exposure will depend on various factors, including population density, family size, climatic changes, and age of sources of infection (Rieder 1999).

34

3.2.2 From Exposure to Infection M. tuberculosis infection is usually airborne. The probability of infection depends on the number of infectious droplet nuclei that remain suspended in the air and the duration of exposure of a susceptible individual. Droplets ofless than 5 f.lIll diameter transmit M. tuberculosis (Sonkin 1951). Coughing is the main source of air droplets, but they can also be generated by sneezing and talking (Wells 1955; Riley and O'Grady 1961). The number of bacilli present in the source case is estimated to be between 102 and 104 in nodular lesions and between 8 107 and 10 in cavitary lesions (Canetti 1965). Smearpositive tuberculosis cases constitute by far the most important source of infection (Styblo 1991). The presence of infection is usually measured with the tuberculin intradermal skin test through the assessment of the size of induration generated by the test. Incidence of infection is usually estimated through the risk of infection (also called annual risk of tuberculosis infection; or ART!) from tuberculin prevalence surveys. The risk of infection also suggests the extent of transmission of M. tuberculosis in the past. The ART! has been defined as the probability that any individual will be infected with M. tuberculosis in 1 year. It has been the basis of several exercises to estimate the global magnitude of tuberculosis (Sudre et al. 1992; Dolin et al. 1994; Murray and Lopez 1997). Styblo estimated that in the absence of tuberculosis control, an annual risk of infection of 1% corresponded roughly to an incidence of new smear-positive tuberculosis cases of 50 per 100,000 population (Styblo 1985). The disadvantages of tuberculin prevalence surveys include high costs and technical problems inherent to the application and reading of the tuberculin test. Furthermore, HIV infection has made the interpretation of tuberculin prevalence surveys very difficult and the previously established fixed relationship between ART! and incidence of smear-positive cases is no longer valid. Thus, in many settings it is not possible to obtain accurate and reliable data of the levels of infection and estimates need to be constructed.

3.2.3 Magnitude of Infection Worldwide

M. A. Espinal and M. C. Raviglione

models suggest various prevalence patterns in different regions of the world. In high-income countries the population most infected is that above 65 years of age. In Europe with the decline of the risk of infection by an estimated 10-12% annually, the young generations are basically free of tuberculosis infection (Styblo et al. 1969; Sutherland et al. 1971; Styblo 1991). In the Netherlands, data from military recruits suggest that between 1910 and 1939, the annual risk of infection was already declining by 5.4% annually, largely due to isolation of cases and better socio-economic conditions. Such a declining trend increased further up to 12.9% annually between 1940 and 1969, concomitant to the introduction of milk pasteurization and antituberculosis drugs (Styblo et al. 1969). Likewise, in the United States the risk of infection has been declining for several decades. Data from Navy recruits show that the proportion of positive reactions to the tuberculin test fell from 6.6% in 1949-1951 to 3.1% in 1967-1968 and to 1.5% in 1980-1986 (Comstock 2000). The scenario is completely different in resourcelimited countries, where young generations and economically productive populations are still currently subject to a high risk of infection with M. tuberculosis. This is due to a slower decline of the annual risk of infection that was estimated at 1-6% per year in the 1980s (Cauthen et aI.1988). Estimates released by the World Health Organization (WHO) in 1991 suggested that one-third of the world population, or 1.7 billion people, were infected with M. tuberculosis (Kochi 1991). Revised estimates suggested that in 19971.86 billion people [32% of the world population] were infected with the tuberculous bacillus (Dye et al.1999). These numbers are not much different from newly revised estimates, which suggest that in 2000 1.8 billion people were infected with M. tuberculosis, again one third of the world population (Corbett et al. 2003). South-East Asia and Africa, with an estimated 46% and 31% of their populations infected with M. tuberculosis, were the most affected regions of the world.

3.3 Active Tuberculosis 3.3.1 Risk of Disease

Prevalence of infection with M. tuberculosis varies according to demographic (age, sex, place) and socioeconomic (crowding, poverty, confinement) determinants and the degree of contact and intensity of exposure. Empirical data available and mathematical

Tuberculosis develops as a result of progression of primary infection, exogenous reinfection, or endogenous reactivation. The highest risk of disease is posed immediately (first 24 months) after infection is acquired. With

35

Global Epidemiology of Tuberculosis

time, the risk declines. In the United States, 82,269 children between 1to 18 years of age who reacted positively to the tuberculin skin test were followed for 20 years (Comstock et al. 1974). Two incidence peaks of active disease where identified: at ages 1-4 years, and during late adolescence and early adulthood. Another cohort of children followed for 20 years in Great Britain showed that almost half of the tuberculosis cases developed in the first 2 years of age and 68% in the first 5 years after infection (D'Arcy Hart and Sutherland 1977). The accepted principle is that the risk of disease is greatest within the first 2 years following infection and after about 7 years starts to level off, remaining low and unchanged over the following decade and, usually, during lifetime. The lifetime risk of progression from infection to disease is estimated to be about 10% if the infection has been acquired during childhood (Comstock et al. 1974). Half of the risk is during the 12-24 months post-infection. Several factors have been identified that are responsible for progression to active disease. These include HIV infection, spontaneously healed fibrotic lesions, malnutrition, age, sex, use of immunosuppression agents, medical conditions including silicosis, a variety of neoplastic disorders, and diabetes. The contribution of many of these factors to the pandemic of tuberculosis is probably not very extensive, because their prevalence is low or they are weakly associated with an increase risk of disease. HIV infection is the most potent risk factor for tuberculosis yet identified. The risk of tuberculosis in people co-infected with M. tuberculosis and HIV has been shown in several studies to range from 5% to 15% per year (Hopewell and Chaisson 2000). Differences in the quantification of the risk of tuberculosis among HIV-infected individuals are especially related to the degree of immunosuppression as measured by the CD4+ lymphocyte counts. Studies conducted in the last 10 years using DNA fingerprinting techniques have suggested that half of tuberculosis cases are attributable to recent or ongoing transmission in some settings (Wilkinson et al. 1997; Godfrey-Faussett et al. 2000). Even in low-prevalence settings, where previously most disease was though to be due to reactivation, one third of cases have been attributed to ongoing transmission (Small et al.1994; Alland et al. 1994; Borgdorff et al. 1997).

3.3.2 Morbidity

Tuberculosis started to decline in Europe by the 19th century, a trend that mirrored the increase in

industrialization, better sanitation, and improvement of living conditions (Evans 1998). This trend was accelerated after the introduction of chemotherapy by mid 20th century. As of today, tuberculosis in Western Europe, Australia, Canada, Japan, and the United States is only a problem of selected groups as the general population of these countries is minimally affected. Only 5% of the world burden of tuberculosis is estimated to be in the industrialized world (Corbett et al. 2003). The highest toll of tuberculosis in industrialized countries is predominantly among immigrants from high-prevalence settings, the homeless, intravenous drug addicts, and HIVinfected people. Since 1980 yearly case notifications to WHO have allowed monitoring and modeling of the future impact of the tuberculosis pandemic (Raviglione et al. 1997a; World Health Organization 2003). Overall, tuberculosis notifications to WHO have remained at around 60 per 100000 people since 1980, with little fluctuations (Fig. 3.1). In industrialized countries data show that tuberculosis continues its declining trend, after a few years of stagnation in the late 1980s and early 1990s. Tuberculosis notifications in Asia and Latin America have remained fairly stable throughout the years, without significant changes. On the other hand, tuberculosis notifications in Africa and Eastern Europe have shown very different epidemiological trends. In sub-Saharan Africa, notifications have been increasing on average 10% per year since 1980, largely due to the HIV/AIDS epidemic. In the former Union of Socialists Soviet Republics (USSR) tuberculosis was declining at 5% per year up to 1990; thereafter, it started to increase at 8% per year due to deteriorating socioeconomic conditions leading to poverty, congestion, malnutrition, and to the collapse of the public health system and of Case notiticafion rate (pe, 100,000)

80

60

40

20

o 1980

1985

1990

1995

2000

Years

Fig. 3.1. Global trend in the tuberculosis case notification rate. Source: Global Tuberculosis Control. World Health Organization Report 2003. WHO/CDS/TB2003316

M. A. Espinal and M. C. Raviglione

36

tuberculosis control efforts. In the recent years rates of increase have started to slow down. The most recent data show that, in the year 2001, 3,813,109 (62 per 100,000 people) cases were notified worldwide, of which 59% were notified in Asia and 21 % in Africa (Fig. 3.2). Of the two million cases notified in Asia, India, China, Bangladesh, the Philippines, and Indonesia accounted for 87%. In Africa, of the 811,172 cases notified, Nigeria, Ethiopia, South Africa, the Democratic Republic of Congo, and Kenya accounted for 53%. Of the 22 high-burden countries of the world responsible for 80% of the global caseload, 18 are located in Asia and Africa. The global notification of smear-positive cases in 2001 was 1,602,153 (26 per 100,000 people), of which 59% were notified in Asia and 23% in Africa. Notification data have been pivotal to track the tuberculosis pandemic; however, they do not reflect the true picture of the global situation. Lack of access to health services, poor capacity to suspect and diagnose cases, and under-reporting are some of the issues that limit the significance of notification data. Therefore, estimates are needed in order to have a clearer picture of the burden of disease. Several figures have been published in the past 25 years, ranging from 4 million new tuberculosis cases in 1977 to 8.3 million cases in 2000 (Bulla 1977; Sudre et al.1992; Murray et al. 1993; Dolin et al. 1994; Raviglione et al. 1995; Murray and Lopez 1997; Dye et al. 1999; Corbett et al. 2003). These estimates have been useful in portraying the global situation of tuberculosis; however, the earlier studies only provided data for regions of the world and not for individual countries. Using several sources of data, expert

opinion and mathematical methods, WHO estimated the burden of disease for 212 countries individually for 1997 (Dye et al.1999). There were 16 million prevalent tuberculosis cases worldwide and 8 million (range: 6.311.1 million) new cases for a case rate of 136 per 100,000 people. Of these, 3.5 million were smear-positive for a case rate of 60 per 100,000 people. Eighty percent of all new cases were in 22 countries. Newly revised estimates for 2000 report 8.3 million (range: 7.3-9.2 million) new tuberculosis cases (Fig. 3.2) for a case rate of 137 per 100,000 people, of which 3.7 million were smear-positive for a case rate of 61 per 100,000 people (Corbett et al. 2003). Incidence in adults 15-49 years was estimated to be 5.4 million for a case rate of 172 per 100,000 people. The greatest incidence rate was in sub-Saharan Africa (290 per 100,000 people). However, the greatest number of cases (more than five million) is estimated to be in Asia including 3.2 million cases in India and China. Between 1997 and 2000, the increase of new tuberculosis cases and incidence rates were at 1.8% and 0.4% per year respectively. The greatest rise was observed in Eastern Europe and sub-Saharan Africa due to reasons explained earlier.

3.3.3 Mortality Today, HIV/AIDS is the most important condition influencing tuberculosis mortality. Other factors include lack of access to chemotherapy, missed diagnosis, late diagnosis, treatment default, and multidrug-resistant tuberculosis.

Cc:nual and Eastc:tn Europe

410000 c.... cslimalCd (87 per 100 000 pop)

326 739 cases notified (70 per 100 000 pop)

'

.....

LatIn America

360 000 ...... CIIiD*Od (70 per 100000 pop) 215842 _ _lIIed (42 per 100 000 pop)

I 900 000 cues Cllilllllled (290 per 100000 pop)

811 In caseSDOlified (115 per 100 000 pop)

Fig. 3.2. Estimated and notified tuberculosis cases in the world,

2000

Global Epidemiology of Tuberculosis

Tuberculosis mortality has decreased to a minimum in the industrialized world. Mortality in European countries was highest between the late 18th century and early 19th century, reaching 50-60% of all tuberculosis cases, after 5 years of diagnosis (Drolet 1938; Thompson 1943). Thereafter, mortality declined with the introduction of chemotherapy and currently is not a major issue. A different pattern, however, is observed in resource-limited countries, regardless of the availability of effective chemotherapy for more than 50 years. In the 1980s and 1990s, various studies estimated that almost three million deaths were due to tuberculosis globally (Styblo 1983; Dolin et al. 1994). The general impression today was that these numbers were overestimating the magnitude of mortality from tuberculosis because of the very high case-fatality ratio, which in the past was at around 30% (Enarson and Rouillon 1998). In the absence of treatment the case-fatality ratio is estimated to be around 50%. However, such a high estimate may not be completely accurate, as already many countries were providing chemotherapy. In 1997 Murray & Lopez new estimates reported two million deaths from tuberculosis worldwide (Murray and Lopez 1997). However, these numbers excluded deaths due to HIV. Recent estimates suggest that mortality from tuberculosis may be lower than previously estimated (Corbett et al. 2003) and that 1.82 million tuberculosis deaths (30 per 100,000 people) occurred in 2000. Ninetyeight percent of such deaths were in resource-limited countries and 226,000 were attributable to HIV.

3.4 The Impact of HIV/AIDS on the Epidemiology of Tuberculosis In a very short period of time, spanning over 20 years, the HIV/AIDS pandemic has rampaged all regions of the world and today more than 60 million people may have been infected with HIY. The impact of HIV/AIDS on the tuberculosis pandemic has been devastating. The Joint United Nations Program on HIV/AIDS (UNAIDS) estimates that 42 million individuals are currently living infected with HIV (UNAIDS 2002). An estimated five million became infected in 2002 and 800,000 of them were children. Three-quarters of the world population infected with HIV live in sub-Saharan Africa and a further 17% in Asia. HIV/AIDS has claimed more than 22 million deaths in the past two decades.

37

Worldwide tuberculosis is the most common HIV-associated disease of public health importance occurring in AIDS patients. As mentioned already, HIV is the strongest risk factor for reactivation of tuberculosis infection. However, the impact of HIV infection on the tuberculosis pandemic is not only limited to reactivation of tuberculosis infection. Consistent evidence shows that people infected with HIY, who later become infected or reinfected with M. tuberculosis, progress faster to full-blown tuberculosis than people who are infected with M. tuberculosis but not with HIV (Di Perri et al. 1989; Daley et al. 1992; Girardi et al. 2000a; Sonnenberg et al. 2001). Fortunately, HIV-infected tuberculosis patients do not appear to be potent transmitters of M. tuberculosis. In fact, household contact studies have consistently shown that HIV-infected people with tuberculosis are less likely to transmit the tubercle bacilli to their close counterparts than HIV-uninfected people with tuberculosis (Klausner et al.1993; Elliot et al.1993; Cauthen et al. 1996; Espinal et al. 2000a). Therefore, the ART! may not be affected in spite of the increasing tuberculosis incidence in high HIV-prevalence settings. Eleven million adults aged 15-49 years (0.36% prevalence) were estimated to be co-infected with HIV and M. tuberculosis in 2000 (Fig. 3.3), of which 9.5 million were in sub-Saharan Africa and 2.3 million in South-East Asia. The overall global prevalence of HIV among new adult tuberculosis cases has been estimated to be 11% (Corbett et al. 2003). However, in sub-Saharan Africa, this figure is 38%, the highest worldwide, compared with only 5.9% in the Americas and 3.2% in South-East Asia. Furthermore, in some African countries such as Cote d'Ivoire, Malawi, South Africa, Uganda, and Zambia prevalence in new tuberculosis cases can be as high as 40-78% (Raviglione et al. 1997b). Also, several sub-Saharan African countries have reported continued increases in tuberculosis notifications (Fig. 3.4), regardless of

Fig. 3.3. Estimated distribution of adults infected with HIV and tuberculosis, 2000. Source: World Health Organization

38

M. A. Espinal and M. C. Raviglione

No~flca~on Rates

(xl00.000)

500

450

ZImbabwe

400 350 300 250

••

•• •,

200 150

! /

Kenye Melewl UR T nzanle

0-------1980

1985

1990

1995

2000

Years

Fig. 3.4. Tuberculosis trends in selected African countries 1980-2001. Source: World Health Organization

the existence of successful national tuberculosis control programs (Narain et al. 1992). The proportion of new tuberculosis cases attributable to HIV/AIDS in sub-Saharan Africa in 2000 was 31 % or 421,000 cases. This proportion is very well above the global estimate of 9% (511,000 cases). This clearly shows the negative impact that HIV/AIDS is posing over tuberculosis in this region of the world.

3.5 The Impact of Multidrug-Resistant Tuberculosis Drug resistance to at least isoniazid and rifampicin, a combination known as MDR-TB, is, after HIV/AIDS, the most important threat to tuberculosis control. Surveillance efforts launched in the 1990s by WHO and the International Union Against Tuberculosis and Lung Disease (IUATLD) have shown that the magnitude of the tuberculosis problem is grossly of drug-susceptible cases (World Health Organization 1997,2000; PablosMendez et al. 1998; Espinal et al. 2001). Data from 67 countries/sites show a global prevalence of MDR-TB of 1% in new cases of tuberculosis and 9.1% in previously treated cases. However, the same data also show that MDR-TB is a severe problem in several countries of Eastern Europe including Estonia, Latvia, and Russia. Areas of India and China also show concerning MDRTB prevalence. The problem appears to be less severe in Africa and Latin America. Several of these countries did not follow internationally recommended guidelines for tuberculosis control-DOTS-until just recently. For instance, in several former Soviet Union countries, interventions that are customarily used for tuberculo-

sis control include long hospitalization of tuberculosis cases, individualized treatment regimen, and unsupervised administration of therapy (Perelman 2000). Hospitalization of tuberculosis cases in the absence of infection control measures can be a major vehicle for the rapid dissemination of MDR-TB. Lack of standardization of treatment and use of inadequate regimens without proper supervision are clearly conducive to creation and spread of drug-resistant bacilli (World Health Organization 1997) On the other hand, countries implementing sound tuberculosis control measures for many years such as Benin, Botswana, Chile, Cuba, Kenya, and Uruguay have reported a very low prevalence of MDR-TB (World Health Organization 2000). Since the available data are limited and more than half of the world countries have not yet been surveyed, a mathematical model has estimated the magnitude of MDR-TB globally (Dye et al. 2001). This model suggests that in 2000 3% (273,000, 95% confidence intervals: 185,000 and 414,000) of all new estimated tuberculosis cases were MDR-TB. Eight of the twenty-two high-burden countries of the world, from which data are not available yet, were estimated to have more than 4000 cases of MDR-TB each. These countries need to be surveyed urgently in order to have a clear picture of the magnitude of the problem. If current estimates prove accurate, the impact of MDR-TB on tuberculosis control in these countries may be overwhelming, as the management of MDRTB is more costly, complex, and lengthy than that of drug-susceptible tuberculosis. MDR-TB is a man-made problem and most countries with a high prevalence have a history of poor tuberculosis control. The spread and evolution of MDR-TB will depend on the efforts these countries are willing to undertake to accelerate tuberculosis control according to recognized and tested international guidelines. The only effective way of preventing MDR-TB from becoming a problem of the magnitude of drug-susceptible tuberculosis is by ensuring the cure of all tuberculosis cases susceptible to first-line drugs under proper management conditions (Dye et al. 2002a). In addition, careful management of MDRTB with second-line drugs will be also needed in settings where the problem is at epidemic levels.

3.6

Future Trends of Tuberculosis Tuberculosis is expected to continue increasing in the first decade of the XXI century. About ten million

Global Epidemiology of Tuberculosis

39

new tuberculosis cases are expected in 2010, if current tional130,OOO extra new smear-positive tuberculosis trends continue and control efforts are not accelerated cases on average every year have been recruited (Fig.3.5) (Dye 2000). Several factors are expected to under DOTS since 1996. In order to achieve the influence such trends. These include lack of political global targets by 2005, an annual average of 360,000 will to effectively expand tuberculosis control, the extra new smear-positive tuberculosis cases needs to HIV/AIDS pandemic, drug resistance, lack of involve- be detected and treated under DOTS. Detection and ment of the private sector in tuberculosis control, provision of short-course chemotherapy to these increasing poverty, demographic changes, the current cases may halve tuberculosis incidence in low HIVburden of M. tuberculosis infection worldwide, and prevalence areas in about 10 years at a rate of decline lack of better tools to speed-up prevention, diagnosis, of 5-10% annually (Dye et al.1998).At the moment, this is not happening; thus, tuberculosis will continue and cure of tuberculosis cases. Strong focus on the expansion of effective tuber- to spread uncontrolled in many settings. culosis control is the key to reverse the current trends HIV/AIDS is another factor that will continue of tuberculosis worldwide. In 1991 WHO adopted impacting the tuberculosis pandemic for several the DOTS strategy for tuberculosis control (World years to come, unless effective measures beyond Health Organization 1991), which was later labeled DOTS and specific to HIV-associated tuberculoas one of the most cost-effective health interventions sis are implemented to reverse the current trends. available (World Bank 1993). The effectiveness of WHO has estimated that there will be 3.5 million DOTS has been successfully tested in the field with new tuberculosis cases in sub-Saharan Africa in impressive results (Suarez et al. 2001; Dye et al. 2000). 2005 (World Health Organization 2001). It is now As of 2001, 155 countries have implemented DOTS widely accepted that in spite of the existence of good , which is increased from only 10 countries in 1991 tuberculosis control programs in this continent, (World Health Organization 2003). However, in many DOTS alone cannot reverse the increasing toll of of these countries population coverage by DOTS is HIV-associated tuberculosis (de Cock and Chaisson low. Today, only half of the world's population lives 1999; Elzinga and Nunn 2002). Therefore, intensive in countries implementing DOTS. Furthermore, only collaboration between tuberculosis and AIDS con32% of the estimated new smear-positive tuberculo- trol programs, exploring novel options designed to sis cases in the world were detected and managed enhance control of the two diseases should be impleunder DOTS conditions in 2001, which means that mented. Useful strategies to test in field conditions 68% of the world infectious cases were managed with include active tuberculosis case-finding and making unclear or inappropriate strategies. voluntary counseling and HIV testing widely availMathematical modeling suggests that if tubercu- able not only to tuberculosis patients but also to the losis control is not expanded effectively and quickly, general population in order to facilitate entry into the global targets of 70% case-detection and 85% preventive and treatment strategies. In particular, cure rate among new tuberculosis cases will not be administration of preventive therapy with isoniazid achieved until 2013 (Dye et al. 2002b). Only an addi- and highly active antiretroviral therapy (HAART) to people with HIV/AIDS needs strong and urgent consideration. The current evidence shows that therapy New TB cases ('OOOs)lyear with isoniazid prevents reactivation of tuberculosis among HIV-infected people who are tuberculin skin 12000 test positive (WHO/UNAIDS 1999). There is also 10000 increasing evidence that HAART can reduce the 8000 _ _ risk of tuberculosis by more than 80% by restoring immunocompetence (Girardi et al. 2000b; Jones et al. 6000 2000; Badri et al. 2002; Santoro-Lopes et al. 2002). The threat of MDR-TB on tuberculosis control 4000 efforts cannot be undermined. Current estimates suggest that at least 80% cure rate will be needed to prevent or control an epidemic of MDR-TB, assum70% of new MDR-TB cases are detected annually ing 1995 2000 2005 2010 Years and that the reproductive fitness of MDR strains Fig. 3.5. Global tuberculosis pandemic. New cases annually to is similar to that of drug-susceptible strains (Dye 2010. Source: World Health Organization and Williams 2000). Such a cure rate may be only

r

2~L

--

40

M. A. Espinal and M. C. Raviglione

achieved by the careful introduction of second-line to control tuberculosis worldwide suggest that US drugs, as short-course chemotherapy cannot achieve $1 billion per year is needed to achieve the global an 80% cure rate among MDR-TB cases (Espinal et targets in the 22 high-burden countries (Floyd et al. al. 2000b). However, most resource-limited countries 2002). A further US $200 million per year are needed lack experience in dealing with longer, more expen- for low- and middle-income countries outside the 22 sive and more toxic treatment regimens employing high-burden countries. Of the estimated total costs second-line drugs (Gupta et al. 200l). Thus, feasible needed for the 22 high-burden countries, 69% is and cost-effective strategies are needed to address currently contributed by the government of these MDR-TB in countries with high MDR-TB prevalence countries and only a minor 4% is contributed by within the current framework of tuberculosis control the international community. The remaining 27% or (Espinal et al.1999). Operational research in this field US $300 million per year is the financial gap that the is ongoing and preliminary evidence suggests that 22 high-burden countries will need to fill to control management of MDR-TB with second-line drugs in tuberculosis. However, new financial resources will resource-limited settings is feasible and cost-effec- not be all that is needed. Innovative approaches to tive when a strong tuberculosis control program is in increase case finding and cure rates are necessary, as place (Suarez et al. 2002). Fortunately, it appears that is a sound strategy to human resource development, MDR-TB is more of a focal problem, limited to some if new funds are to be spent effectively. settings or countries, rather than a global problem of Demographic changes will also play their role in the magnitude of drug-susceptible tuberculosis (Dye keeping tuberculosis incidence at high rates in the current decade. Changes in the age structure of the and Espina1200l). Involving the private sector in tuberculosis control population of the developing world will certainly needs to be strongly pursued. A large proportion of contribute to increasing rates of tuberculosis. Most tuberculosis patients in high prevalence countries of the population of resource-limited countries is including India, Pakistan, Philippines, Vietnam, and young, contrary to the industrialized world where Uganda first approach a private practitioner (Uplekar the population is increasingly aging. Children of et al. 200l). A study in India suggested that 88% of poor countries infected with M. tuberculosis that are rural and 85% of urban patients with tuberculosis entering the economically productive age will defifirst went to a private practitioner (Uplekar et al. nitely be at risk of developing active disease. Since 1998). Strategies to involve the private sector are one third of the world population is infected with currently under testing. The challenge will be their M. tuberculosis infection, the vast majority of which implementation on a wider scale. Nevertheless, with- are in resource-limited countries, the joint effects of out involving private practitioners the international demographic changes and the high burden of M. community will not achieve tuberculosis control in tuberculosis infection will impact negatively on the morbidity and mortality of tuberculosis. many countries. Finally, the lack of new tools to speed-up control Growing poverty is another factor that will prevent tuberculosis incidence from declining in the and elimination of tuberculosis is another major next several years. Poverty has been always associ- obstacle to overcome. New vaccines, drugs and diagated with tuberculosis (Moore-Gillon 1998). The nostics are not expected to be available for at least the decreasing incidence of tuberculosis in Europe in next 10 years (Freire and Roscigno 2002; Perkins and the 19th century was parallel to the reduction of Kritski 2002). For instance, the neglect in the field poverty. Recently, the most clear impact of poverty of new drugs has been so immense that, in the last on the incidence of tuberculosis has been observed in 25 years, only 16 new chemical entities out of 1393 Russia. The economic downfall in Russia, due to the were marketed for tropical diseases, including tubertransition from the socialist economy model to the culosis (Trouiller et al. 2002). market economy one, has been linked to the growing incidence of tuberculosis observed in the last decade (Dye 2000). Tuberculosis is one of the three major diseases of 3.7 poverty along with HIV/AIDS and Malaria. The 22 Concluding Remarks tuberculosis high-burden countries, where increasing poverty is making control of infectious diseases The tuberculosis pandemic will be affecting human an enormous task, are all very limited in resources. lives for many more decades. While much progress Recent estimates regarding the resources needed has been made in the last few years, control of tuber-

Global Epidemiology of Tuberculosis

culosis is only slowly becoming a priority in many countries. Clearly strong financial, political and managerial actions are needed in order to accelerate DOTS coverage, reach the WHO global targets, and incorporate new tools in disease control. To move forward, a Stop TB partnership was created in 1998 to ensure that endemic countries are adequately supported technically and financially to control tuberculosis (Raviglione and Pio 2002). The original framework for tuberculosis control (World Health Organization 1994) has been also recently revised to widen the scope of DOTS, and make it more comprehensive and more patient-centered in order to address some of the current threats to tuberculosis control (WHO 2002). Nevertheless, current efforts are not sufficient. To make tuberculosis control more efficient and efficacious, novel interventions need to be added urgently in high HIV/AIDS and MDR prevalence settings or where private practitioners are not involved in national tuberculosis control efforts, or where health system changes are not addressing tuberculosis control as a top priority. The recent creation of the Global Fund to Fight AIDS, Tuberculosis, and Malaria offers a tremendous opportunity to change the future of the tuberculosis pandemic if resources are properly used. The world should not miss it; otherwise, tuberculosis will continue to be a major scourge to individuals and communities.

Acknowledgements. Thanks to C. Dye and C. Watt for providing some of the maps and figures.

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41 Comstock GW, Livesay VT, Woolpert SF (1974) The prognosis of a positive tuberculin reaction in childhood and adolescence. Am J Epidemiol 99: 131-138 Comstock GW (2000) Epidemiology of tuberculosis. In: Reichman LB, Hershfield ES (eds) Tuberculosis: a comprehensive international approach. Dekker, New York, pp 129-156 Corbett et al (2003) The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 163:1009-1021 Daley C et al (1992) An outbreak of tuberculosis with accelerated progression among persons with the human immunodeficiency virus. An analysis using restriction-fragmentlength polymorphism. N Engl J Med 326:231-235 D'Arcy Hart P, Sutherland I (1977) BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life. Final report to the Medical Research Council. Br Med J 2:293-295 De Cock KM, Chaisson RE (1999) Will DOTS do it? A reappraisal of tuberculosis control in countries with high rates of HIV infection. Int J Tuberculosis Lung Dis 3:457-465 Di Perri et al (1989) Nosocomial epidemic of active tuberculosis among HIV-infected patients. Lancet 21:502-504 Dolin PJ, Raviglione MC, Kochi A (1994) Tuberculosis: a global overview of the situation today. Bull WHO 72:213-220 Drolet GJ (1938) Present trend in case fatality rates in tuberculosis. Am Rev Tuberculosis 37:125-151 Dye C (2000) Tuberculosis 2000-2010: control, but not elimination. Int J Tuberculosis Lung Dis 4:S146-S152 Dye C, Espinal M (2001) Will tuberculosis become resistant to all antibiotics? Proc R Soc Lond Bioi Sci 268:45-52 Dye C, Williams BG (2000) Criteria for the control of drug-resistant tuberculosis. Proc Nat! Acad Sci USA 97: 8180-8185 Dye C et al (1998) Prospects for worldwide tuberculosis control under the WHO DOTS strategy. Lancet 352:1886-1891 Dye C et al (1999) Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. J Am Med Assoc 282:677-686 Dye C et al (2000) Evaluating the impact of tuberculosis control: number of deaths prevented by short-course chemotherapy in China. Int J Epidemiol 29:558-564 Dye C et al (2001) Worldwide incidence of multidrug-resistant tuberculosis. J Infect Dis 185:1197-1202 Dye C et al (2002a) Erasing the world's slow stain: strategies to beat multidrug-resistant tuberculosis. Science 295: 2042-2046 Dye C et al (2002b) Low access to a highly effective therapy: a challenge for international tuberculosis control. Bull WHO 80:437-444 Elliot A et al (1993) The impact of HIV-l on infectiousness of pulmonary tuberculosis: a community study in Zambia. AIDS 7:981-987 Elzinga G, Nunn P (2002) TB and HIV: joint problems, joint solutions? Bull WHO 80:469-470 Enarson DA, Rouillon A (1998) The epidemiological basis of tuberculosis control. In: Davies PDO (ed) Clinical tuberculosis, 2nd edn. Cambridge University Press, Cambridge, pp 35-52 Espinal MA et al (1999) Rational DOTS-Plus for the control of MDR-TB. Int J Tuberculosis Lung Dis 3:561-563 Espinal MA et al (2000a) Infectiousness of Mycobacterium tuberculosis in HIV-1 infected patients with tuberculosis: a prospective study. Lancet 355:275-280

42 Espinal MA et al (2000b) Standard short-course chemotherapy for drug-resistant tuberculosis: treatment outcome in 6 countries. J Am Med Assoc 283:2537-2545 Espinal MA et al (2001) Global trends in resistance to antituberculosis drugs. N Engl J Med 344:1294-1303 Evans CC (1998) Historical background. In: Davies PDO (ed) Clinical tuberculosis, 2nd edn. University Press, Cambridge, pp 1-33 Floyd K et al (2002) Resources required for global tuberculosis control. Science 295:2040-2041 Freire M, Roscigno G (2002) Joining forces to develop new weapons against TB: together we must. Bull WHO 80:429 Girardi E et al (2000a) Impact of the HIV epidemic on the spread of other diseases: the case of tuberculosis. AIDS 14:S47-S56 Girardi E et al (2000b) Impact of combination antiretroviral therapy on the risk of tuberculosis among persons with HIV infection. AIDS 14:1985-1991 Godfrey-Faussett P et al (2000) Tuberculosis control and molecular epidemiology in a South African gold-mining community. Lancet 356:1066-1071 Gupta R et al (2001) Responding to market failures in tuberculosis control. Science 293:1049-1051 Hopewell PC, Chaisson RE (2000) Tuberculosis and human immunodeficieny virus infection. In: Reichman LB, Hershfield ES (eds) Tuberculosis: a comprehensive international approach, 2nd edn. Dekker, New York, pp 525-527 Jones JL, Hanson MS, de Cock KM (2000) HIV-associated tuberculosis in the era of highly active antiretroviral therapy. Int J Tuberculosis Lung Dis 4:1026-1031 Klausner JD et al (1993) Mycobacterium tuberculosis in household contacts of human immunodeficiency virus type-lseropositive patients with active pulmonary tuberculosis in Kinshasa, Zaire. J Infect Dis 168: 106-111 Kochi A (1991) The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 72:1-6 Moore-Gillon JC (1998) Tuberculosis and poverty in the developed world. In: Davies PDO (ed) Clinical tuberculosis, 2nd edn. Cambridge University Press, Cambridge, pp 383-393 Murray CJ, Lopez AD (1997) Mortality by cause of eight regions of the world: global burden of disease study. Lancet 349:1269-1276 Murray q, Styblo K, Rouillon A (1993) Tuberculosis. In: Jamison JT, Mosley WH, Measham AR, Bobadilla JL (eds) Disease control priorities in developing countries. Oxford University Press, New York, pp 233-259 Narain JP, Raviglione MC, Kochi A (1992) HIV-associated tuberculosis in developing countries: epidemiology and strategies for prevention. Tubercle Lung Dis 73:311-321 Nerlich AG et al (2000) Ancient Egyptian prosthesis of the big toe. Lancet 356:2176-2179 Pablos-Mendez A et al (1998) Global surveillance for antituberculosis-drug resistance, 1994-1997. N Engl J Med 338: 1641-1649 Perelman MI (2000) Tuberculosis in Russia. Int J Tuberculosis Lung Dis 4:1097-1103 Perkins M, Kritski AL(2002) Diagnostic testing in the control of tuberculosis. Bull WHO 80:512-513 Raviglione MC et al (1995) Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic. J Am Med Assoc 273:220-226 Raviglione MC et al (1997a) Assessment of worldwide tuberculosis control. Lancet 350:624-629

M. A. Espinal and M. C. Raviglione Raviglione MC et al (1997b) Tuberculosis and HIV: current status in Africa. AIDS II:SI15-S123 Raviglione MC, Pio A (2002) Evolution of WHO policies for tuberculosis control, 1948-2001. Lancet 359:775-780 Rieder HL (1999) Epidemiologic basis of tuberculosis control, 1st edn. International Union Against Tuberculosis and Lung Disease, Paris Riley RL, O'Grady F (1961) Airborne infection: transmission and control. Macmillan, New York Rothschild BM et al (2001) Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present. Clin Infect Dis 33:305-311 Salo WL et al (1994) Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy. Proc Nat! Acad Sci USA 91:2091-2094 Santoro-Lopes G et al (2002) Reduced risk of tuberculosis among Brazilian patients with advanced human immunodeficiency virus infection treated with highly active antiretroviral therapy. Clin Infect Dis 34:543-546 Small PM et al (1994) The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N Engl J Med 330: 1703-1709 Sonkin LS (1951) The role of particle size in experimental airborne infection. Am J Hyg 70:185-196 Sonnenberg P et al (2001) HIV-l and recurrence, relapse, and reinfection of tuberculosis after cure: a cohort study in South African mineworkers. Lancet 358:1687-1693 Styblo K (1983) The epidemiological situation of tuberculosis and the impact of control measures. Bull Int Union Against Tuberculosis 58: 179-186 Styblo K (1985)The relationship between the risk of tuberculous infection and the risk of developing tuberculosis. Bull Int Union Against Tuberculosis 60:117-119 Styblo K (1991) Epidemiology of tuberculosis, 2nd edn. Royal Netherlands Tuberculosis Association, The Hague Styblo K, Meijer J, Sutherland I (1969) The transmission of tubercle bacilli - its trend in a human population. Tuberculosis Surveillance Research Unit report no 1. Bull Int Union Against Tuberculosis 42:1-104 Suarez PG et al (2001) Epidemiological impact of tuberculosis control in Peru. J Infect Dis 184:473-478 Suarez PG et al (2002) Feasibility and cost-effectiveness of standardised second-line drug treatment for chronic tuberculosis patients: a national cohort study in Peru. Lancet 359: 1980-1989 Sudre P, ten Dam G, Kochi A (1992) Tuberculosis: a global overview of the situation today. Bull WHO 70:149-159 Sutherland I et al (1971) Annual risks of tuberculous infection in 14 countries, derived from the results of tuberculin surveys in 1948-1952. Bull Int Union Against Tuberculosis Lung Dis 45:75-114 Thompson BC (1943) Survival rates in pulmonary tuberculosis. Br Med J 2:721 Trouiller P et al (2002) Drug development for neglected diseases: a deficient market and a public-health policy failure. Lancet 359:2188-2194 UNAIDS (2002) Joint United Nations Program on HIV/AIDS I World Health Organization. AIDS epidemic update. 02.46E. UNAIDS, Geneva Uplekar M et al (1998) Tuberculosis patients and practitioners in private clinics in India. Int J Tuberculosis Lung Dis 2: 324-329

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World Health Organization (1994) Global tuberculosis programme. Framework for effective tuberculosis control. WHO/TB/94.179. WHO, Geneva World Health Organization (1997) Anti-tuberculosis drug resistance in the world. WHO/TB/97.229. WHO, Geneva World Health Organization (2000) Anti-tuberculosis drug resistance in the world. Report no 2: prevalence and trends. WHO/CDS/TB/2000.278. WHO, Geneva World Health Organization (2001) Global tuberculosis control. WHO Report 2001. WHO/CDS/TBI2001.287. WHO, Geneva World Health Organization (2002) An expanded DOTS framework for effective tuberculosis control. Int J Tuberculosis Lung Dis 6:378-388 World Health Organization (2003) Global tuberculosis control: surveillance, planning, financing. WHO report 2002. WHOI CDS/TBI2003.316. WHO, Geneva

4

Epidemiology of Tuberculosis in Saudi Arabia ABDULRAHMAN A. ALRAJHI and ALI M. AL-BARRAK

CONTENTS 4.1 4.1.1 4.1.2 4.1.3

4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.4 4.5

General Epidemiology 45 Incidence of Pulmonary Tuberculosis 46 Incidence of Extrapulmonary Tuberculosis 48 Prevalence of Infection 49 Epidemiology of Drug-Resistant M. tuberculosis 51 Tuberculosis in Certain Populations 51 The Elderly 52 Diabetics 52 Persons Infected with Human Immunodeficiency Virus 52 Hemodialysis Patients 53 Cancer and Bone Marrow Transplant Patients 54 Solid Organ Transplant Recipients 54 Mycobacterial Other than M. tuberculosis 54 Conclusion 55 References 55

tion of transmission, number of infected persons without active disease, distribution of drug-resistant tuberculosis, and tuberculosis in certain populations. There are no data on the impact of tuberculosis on community, economy or development. Mortality data are not available either.

4.1 General Epidemiology

Measuring the size of tuberculosis in a community is not as easy as it may appear initially. The disease course and the community it afflicts are constantly changing. Definitions used for various tuberculosis cases are not standardized and surveillance appliTuberculosis is older than recorded history. Typical cation in the field has enormous variations. The lesions of spinal tuberculosis were noted in Neolithic community is also changing its composition, living human remains and paintings. Pre-Islam Arab poetry standards, and exposure between members. There are several measures to indicate the size of had described the disease and its association with a non-cure. The writings of Moslem physicians indi- tuberculosis as a community problem. Before the cate that the disease was well known along with its era of tuberculosis chemotherapy discovery and its risk factors, symptoms and complications. Some of use in treatment, an indirect measure, which could the factors in this part of the world were encourag- also assess the impact of the disease, was mortality. ing the introduction of the organism on a continu- Tuberculosis chemotherapy and control measures ous basis through commerce; as the peninsula was make mortality an imprecise measure of disease involved in commerce roads, and pilgrims were extent in the community. Another measure identified early in the 20th century is tuberculin skin test. It is coming from all corners. The purpose of this chapter is to present the a useful measure to identify the prevalence of infecavailable epidemiology about tuberculosis in Saudi tion in a community at a certain point of time and to Arabia. It includes data on numbers of new cases follow trends. Unfortunately, the value of tuberculin annually, type of tuberculosis involvement, demo- testing is affected by BeG vaccination, exposure to graphics of affected patients, geographic distribu- nontuberculous mycobacteria, and waning immunity (Thompson et al. 1979). A third measure was used in some areas, which is field surveillance by chest X-ray A. A. ALRA1HI, MD, MPH, FIDSA and sputum cultures from community samples. This Consultant and Head, Section of Infectious Diseases, Chairman, Department of Medicine (MBC #46), King Faisal Specialist Hos- method has to be repeated so incidence rates can be pital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi measured. Indirectly, incidence can be estimated to Arabia be half of the prevalence, considering the disease A. M. AL-BARRAK, AmBIM, DTM & H, FRCP (C) Consultant Infectious Diseases Unit, Department of Medicine, lasts for 2 years without treatment. Another epidemiRiyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, ologic concept was developed; annual risk of tuberculosis infection reflects the probability of a person Saudi Arabia M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

A. A. Alrajhi and A. M. AI-Barrak

46

becoming infected within a given year. This measure requires a prevalence assay on multiple occasions in order to be precise. BCG vaccination will influence prevalence rates that are measured using Mantoux tuberculin test. Lastly, the World Bank has attempted to estimate the economic impact of tuberculosis in its 1993 World Development Report though disability-adjusted life years (DALYs), the years that are consumed by various diseases, including tuberculosis (World Bank 1993). This chapter will attempt to present available data on these various measures pertaining to Saudi Arabia. Tuberculosis is a reportable disease in Saudi Arabia. The Ministry of Health (MOH), represented by Preventive Medicine has been collecting data on newly diagnosed cases of tuberculosis annually. These incidence rates are published every year. Some of the information included was gender, age, nationality, and type of tuberculosis involvement. These rates are the only available data at population level. Although they may not include all cases because of reporting deficiency from care providers, they provide valuable means to monitor trends. These data have been considered along with the social, developmental, and economic changes in the country.

Case identification and treatment is considered one of the most important components of tuberculosis control and prevention. The annual incidence rates of pulmonary tuberculosis were higher for foreigners compared with Saudis. In 1996, the rate was 23.9 cases per 100,000 population for non-Saudis compared with 7.4 cases per 100,000 population for Saudis (Ministry of Health 1996). The female and male rates for Saudis were almost similar,6.5 and 8.3 cases per 100,000 population, respectively. For non-Saudis, these rates were 22.7 for males and 26.9 cases per 100,000 population for non-Saudi females. Certain regions of the country consistently scored higher rates. Aseer, ]azan, and Holy Makkah have higher rates than Hail and Gaseem. Table 4.1 summarizes incidence rates for pulmonary tuberculosis in various groups and regions over the period of 1996-1997. Apart from reported pulmonary tuberculosis, investigators have attempted to estimate incidence

Cases per 100.000 population 1600 1400 1200 1000 800

-

I---

-

400

I---

-

200

-

I---

-

600

4.1.1 Incidence of Pulmonary Tuberculosis The first available incidence rate of pulmonary tuberculosis was in 1970. During that year, there were 76,748 new cases of pulmonary tuberculosis, making a rate of 1298.5 cases per 100,000 population per year (Ministry of Health 1979). Over the subsequent years, incidence rate of pulmonary tuberculosis has decreased steadily to 166.1 cases per 100,000 population in 1979. Figure 4.1 depicts the trend of incidence rates over the 10 years of the 1970s. Incidence of reported pulmonary tuberculosis continued to decrease steadily during the 1980s and the 1990s. For the year 1980, the incidence rate was 135 cases per 100,000 population. In 1997, the rate has dropped to 12 cases per 100,000 population (Ministry of Health 1997). When considering other changes in the health care system availability, advances in diagnosis capabilities, improved reporting associated with communications improvement, this steady reduction becomes more impressive, Fig. 4.2. This reduction was attributed to BCG vaccination program that have been started many years ago, and more importantly, the active screening program for foreign workers coming to the country (Ministry of Health 1996).

o

70

71

72

73

74

-

75 Year

.• • •

76

77

78

79

Fig. 4.1. Annual incidence rates of pulmonary tuberculosis in Saudi Arabia, 1970-1979 (Ministery of Health: Annual Health Report)

Reported cases per 100,000 population 140 120 100 80 60 40 20

o

••

III1IIIIII

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Year

Fig. 4.2. Annual incidence rates of pulmonary tuberculosis in Saudi Arabia, 1980-1997 (Ministery of Health: Annual Health Report)

Epidemiology of Tuberculosis in Saudi Arabia Table 4.1. Annual incidence rates of pulmonary tuberculosis in various groups and regions during 1996-1997 (Ministry of Health 1997,1996) Group

Rates per 100,000 population

12.0 Overall Nationality Saudis 7.4 Non-Saudis 23.9 Gender 8.3 Saudi, male Saudi, female 6.5 Non-Saudi, male 22.7 Non-Saudi, female 26.9 Regions 20.5 Jazan Makkah 17.0 Riyadh 13.9 Eastern 10.9 Hail 2.6

rates at community level (Zaman 1991a; Milaat et al. 1994; el-Kassimi 1994). However, these efforts have not provided more reliable data than Ministry of Health annual reports. Milaat et al reviewed all tuberculosis cases in a regional chest hospital in Jeddah for 1 year, 1989-1990. Population data were obtained from MOH records to calculate incidence rates. The overall incidence rate was estimated at 63.4 cases per 100,000 populations. Extrapulmonary tuberculosis was found in 7.6% of all cases. There were 2.6 cases of tuberculosis in non-Saudis for each one case of tuberculosis in Saudis from the same population number (99.6 non-Saudis and 38.8 Saudi tuberculosis cases per 100,000 population). Reporting tuberculosis cases from the private health care facilities has been grossly deficient. This is noted in Jeddah, where private medicine provides care for a large proportion of population in that region. Milaat and colleagues think that the higher estimated rate noted in Jeddah relative to the country in general is related to the crowding and high immigrant population in Jeddah. In addition, Jeddah serves as the major port of entry for visitors to the two holy cities. This is suggested by peaking of cases during the 11th and 12th Hijra months. In a nationwide survey, positive Mantoux reaction was noted to be three times higher in the Western region than the nation as a whole (AI-Kassimi et al. 1993). AI-Kassimi has calculated incidence rates from the annual risk of infection (AI-Kassimi 1994). The result was double the rates from reported cases to MOH. He correctly points to the problem of under-notification by health care providers to MOH. The high rates of tuberculosis as infection and incidence of active disease are reflected in the pro-

47

portion of patients admitted with pneumonia that end up diagnosed as having pulmonary tuberculosis. In a review of 112 patients admitted with pneumonia over the year 1983 at Riyadh Military hospital, tuberculosis was confirmed in 7.1% of them. In fact, it was only second to Streptococcus pneumoniae (Mohamed and Evans 1987). Some of the radiological changes noted in tuberculosis patients could also be seen in patients with community acquired bacterial pneumonia (AI-Dabbagh et aI.1991)."Unusual" chest radiography findings were noted in 13% of 714 patients with pulmonary tuberculosis in two hospitals in Jeddah. Cavities were seen in 78% of all patients, suggesting a prolonged and unrecognized disease. The most common radiological finding, however, was upper or multilobar lesions, seen in 86% of patients. However, more severe pneumonia requiring intensive care had a slightly different etiology (Dahmash et al. 1994; Dahmash and Chowdhury 1994). In a university hospital in Riyadh, 113 patients were admitted to the intensive care unit with pneumonia between 1991 and 1992. Patients were followed prospectively, 50 patients (44%) had hospital-acquired pneumonia. Out of the community acquired pneumonias, 9 (14.3%) were due to M. tuberculosis, which was second only to S. pneumoniae, isolated from 10 patients but far more fatal than S. pneumoniae-5 of 9 (55.6%) compared with 2 of 10 (20%) (Dahmash and Chowdhury 1994). The population at risk for pulmonary tuberculosis remains large in Saudi Arabia. Cases of pulmonary tuberculosis most frequently involve young adults between 15 years and 35 years of age (AI-Dabbagh et al. 1991; AI-Hajjaj et al. 1991). This seems to be the case for last thousand years (AI-Amoud 1993). Some argue that this is related to the number at risk being higher in the age group because of the immigrant work force. However, this is not the reason, as the calculated rates are still highest for the young age group (Ministry of Health 1996). In the large review of 1566 cases of pulmonary tuberculosis at Riyadh Chest Hospital between 1983 and 1987, 41 % of patients were aged 21-30 years (AI-Hajjaj et al. 1991). These patients were admitted for therapy initiation at a rate of 32.6 cases monthly. Around 52% of patients were non-Saudis, mostly from Yemen, 20% of all patients. A concerning phenomenon in this cohort is the high rate of "old" cases, which represent 29% of the cases with most of the patients being Saudis. They had more hemoptysis and were more difficult to cure. Sputum smear positivity was relatively high at 75%. The conversion rate was low at two months (80%). These patients from Riyadh had larger proportion of lower

48

lung field tuberculosis compared with Jeddah-lO.3% compared with 5.3% (AI-Dabbagh et al.199l; Pandya et al. 1990). These percentages represent the post primary infection. The patients from Riyadh had higher rates of sputum positive smears-84% compared with 75% for all pulmonary tuberculosis cases. Pulmonary tuberculosis from the South Region of Saudi Arabia has been characterized in a prospective study managed at the Chest Hospital in Abha between 1989 and 1991 (al-Wabel et al. 1995) One hundred ninety patients were admitted; 69.5% of them had no previous diagnosis of tuberculosis. As in other areas of the country, the group aged 20-40 years constituted more than 34% of all cases. In this region, patients older than 60 years were 25% of all patients with pulmonary tuberculosis. Unlike in the central or western regions, less than 25% of patients were nonSaudis. Another peculiar finding in these patients was the high proportion of diabetics (21 %), reflecting the higher rates of diabetes in elderly nationals. Sputum smears were positive in 42% of patients, posing a considerable risk for disease transmission from these cases. Radiologically, cavitary lung lesions were noted in 38% of patients, lower lung field in 3.2%, and miliary pattern in 1.3%. Saudi Arabia hosts millions of foreigners as part of the workforce, visitors for business, or Hajj and Omrah. Many of the workers come from countries where tuberculosis is highly endemic. Screening for these workers is performed before they obtain their visas and after they arrive. In spite of this, they represent more than 50% of tuberculosis cases in a large city like Riyadh (AI-Hajjaj et al. 1991). The impact of these cases is not limited to economy, but to the extent of increasing the hazard of tuberculosis infection. Drug-resistant M. tuberculosis is also of major concern as these patients come from areas where drug-resistant rates are very high. Screening for tuberculosis is not applied to visitors, including pilgrims for Hajj and Omrah. Alzeer et al. (l998) reviewed the microbiology of pneumonia in patients admitted during Hajj of 1994 (l414 H). Out of 64 admitted patients, M. tuberculosis was the causative agent in 13 (20%) patients. It was the most common agent for pneumonia identified, followed by S. pneumoniae and coliforms in 9% and 11%, respectively. Radiographic features of tuberculous and non-tuberculous pneumonias were the same. This high rate of tuberculosis among pilgrims was thought to be the reason for the above national annual risk of infection noted in pilgrim-receiving cities (AI-Kassimi et al. 1993). An earlier report of the 1991-1992 Hajj season did not attempt to obtain respiratory speci-

A. A. Alrajhi and A. M. AI-Barrak

mens other than expectorated sputum (EI-Sheikh et al.1998). The focus was mainly on bacterial and viral pathogens. Nevertheless, two patients of eighty-six (2.3%) with lower respiratory tract infection had positive smears for acid-fast bacilli.

4.1.2 Incidence of Extrapulmonary Tuberculosis Extrapulmonary tuberculosis requires tissue sampling to confirm diagnosis. This may not be available to many health care facilities. Subsequently, diagnosis is delayed if attempted. The number of reported extrapulmonary tuberculosis cases are even smaller than the number of actual cases. The annual Health Reports of the MOH started presenting data on extrapulmonary tuberculosis in 1989. The annual incidence rate of reported extrapulmonary tuberculosis was 3.7 cases per 100,000 population nation-wide in 1989 (Ministry of Health 1989). This figure decreases to 1.7 per 100,000 population in 1993 (Ministry of Health 1993). This rate has been steadily increasing to 4.7 cases per 100,000 population in 1997, Fig. 4.3 (Ministry of Health 1997). During the same time frame, reported pulmonary tuberculosis rates were decreasing (Ministry of Health 1997). The increasing rates of reported extrapulmonary tuberculosis may have resulted from better diagnostic facilities, which identified more cases, and better reporting by health providers. Earlier reports describe their series in their local hospitals. In 1982, Froude and Kingston reviewed 162 cases diagnosed with extrapulmonary tuberculosis between 1979 and 1981 from King Faisal Specialist Hospital and Research Centre (Froude and Kingston 1982). Although this finding may be due to referral pattern, CNS disease (including spine involvement) was the

New cases per 100 population

5 4.5 4 3.5 3 -

I-I--

f---

2.5 2

f---

1.5

I---

1 0.5

o

I---

-

I---

I---

I---

-

-

I---

I---

I---

-

-

I---

f---

'--r-

~

1989

1991

1992

1993

1994

Year

f---

I--

-

I---

I--

-

f---

I--

I---

I--

I---

I--

I---

I--

f---

I--

-

1995

1996

'-

1997

Fig. 4.3. Annual incidence rates for extrapulmonary tuberculosis in Saudi Arabia, 1989-1997 (Ministery of Health: Annual Health Report)

49

Epidemiology of Tuberculosis in Saudi Arabia

most common, comprising 36% of cases, followed by lymphadenopathy 22%, gastrointestinal disease 16%, and surprisingly, disseminated disease was diagnosed in 14% of the extrapulmonary tuberculosis cases. The morbidity associated with extrapulmonary tuberculosis can be very high. Seventeen patients (11 %) had spinal cord damage related to vertebral involvement. During the same study period of 27 months, six new pulmonary tuberculosis cases were diagnosed every month. Around the same time, a similar report was published about patients from Jeddah. Mokhtar and Salman published details of 125 patients with extrapulmonary tuberculosis in 1983 (Mokhtar and Salman 1983). Lymph node involvement was identified in 54% of the patients, followed by abdominal disease and urogenital disease in 16% each, and bone and joints in 10%. Surprisingly, only one CNS disease was diagnosed as meningitis. During the same period, 20 new cases of pulmonary tuberculosis were diagnosed every month. In their Hospital, Mokhtar and Salman found extrapulmonary tuberculosis in 15% of all tuberculosis cases identified. In 1980, incidence of tuberculosis was 120 cases per 100,000 population in Jeddah (Mokhtar and Salman 1983).

Fig. 4.4. Distribution of involvement in extrapulmonary tuberculosis (Froude and Kingston 1982)

10 CNS 0 Lymph node 0 Urogenital • Abdominal 0 Bone & Joint • Othenl

Fig. 4.5. Distribution of involvement in extrapulmonary tuberculosis (Mokhtar and Salman 1983)

4.1.3 Prevalence of Infection Human exposure to M. tuberculosis will result in infection. No disease may be apparent initially. After a variable period of time and factors related to host immunity, M. tuberculosis will be reactivated, resulting in disease. The prevalence of infection can be measured by the tuberculin test. In 1908, Mantoux described his intradermal test, which was developed to a purified protein derivative (PPD) in 1934 by Seibert (Schein and Huebner 1995). When this test was applied to a large population, it was realized that most people get infected without having active disease. This test, known as Mantoux, tuberculin, and PPD served as good tools to measure the proportion of the population that had been exposed to M. tuberculosis. In a rural area in the western region of Saudi Arabia in 1979, prevalence of pulmonary tuberculosis was as high as 1% of the population (Hammam et al.1979). In Saudi Arabia, the first reference of testing of asymptomatic population for tuberculosis exposure was in 1979. Out of81,006 subjects tested, 48,483 (59.9%) subjects had a positive test. During the same year, 66% of tested males were positive, compared with 40% of tested females. These percentages were higher with the increasing age of the subjects (Ministry of Health 1979). Although no details are available on these subjects as to whether or not they are patient contacts, these percentages give an indication of how common tuberculosis infection is in the area. Problems of the test, its administration, or interpretation may influence the prevalence estimation towards the underestimation side. Subsequent to 1980, many field studies published in peer-reviewed journals have reported the tuberculin skin testing results. Saudi Arabia has been among the middle prevalence countries (2-14%) according to the classification proposed by the International Union Against Tuberculosis (AI-Kassimi et al. 1993). The World Health Organization (WHO) expert committee on tuberculosis recommends continuing early life vaccination until the percentage of tuberculin reactor entering school drops to 2%, which is a low prevalence level (WHO 1964). The protective effect of BCG vaccination does not last beyond 10 years. Saudi Arabia has adopted a BCG vaccine given at birth. In a national immunization coverage survey in 1991, BCG immunization coverage was high (99%) both in urban and rural area settings, but the western and southern area had slightly lower coverage (Farag et al.1995). In early 1990s, community based studies for the prevalence of Mantoux reaction were done at

A. A, Alrajhi and A, M. AI-Barra!<

50

different regions of Saudi Arabia. In a nationwide community survey, about 24% of the children aged 5-14 years were not vaccinated (AI-Kassimi et al. 1993). The prevalence of positive Mantoux reaction (10 mm induration) in children not vaccinated aged 5-14 years was 6%, and was 4% in Saudi nationality. In this age group, there was marked difference in prevalence between urban and rural dwellers (10% and 2%, respectively). This difference was more in western (20% and 1%) and southern (12% and 1.5%) provinces. The authors have attributed this to the presence of settlers after pilgrimage in the western province and Yemeni laborers' exemption from medical screening in the south, adjacent to their country. The prevalence of positive Mantoux reaction increased with age steadily to peak at age 45-64 years at 56% for non-vaccinated and 70% for BCG vaccinated subjects, Fig. 4.6.

a

100%

101W. North Region

Central Region

80%

/

60%

.. ,

......

40%

?'

.•...

....

-

"'~'-"

~

20%

\

'5-14

80%

+------------f-----l

60";'

+-------------.I--.....--l

40";' + - - - - - - - - - : ;......'--------,_-----l

\

20";'

'

0%

Bener and Abdullah confirmed the above finding in another Saudi Arabia population sample (Bener and Abdullah 1993). Positive Mantoux reaction was significantly higher among BCG recipient subjects in all age groups, male gender, nationalities, and occupations. Studies from different regions of Saudi Arabia confirmed the finding of this prevalence (elKassimi et al. 1991; Bener 1990; Abdullah et al. 1991; AI-Kassimi et al. 1991). In the central region, 7% of unvaccinated children aged 4 to 14 years had positive Mantoux reaction, while 15% of those vaccinated were positive. There was increased prevalence of Mantoux positivity with age, more in the BCG-vaccinated population. There was a steep upward increase in prevalence of positivity between age 5 years and 14 years and 25 years and 34 years, probably due to an increased number of a previously infected expatriate population who are workers in the age group

+-----::.-=---:+---'--'-----------1

.......

0% +-~...,....-"""T""-__r-__.--..__----l 15-24

35-44

25·34

45-64

65+

5-14

15-24

100% 80%

.

40% 20%

0";'

•

'

60";'

+-------+----\;--'---1

40"/. t - - - - - : : f - -.....-'---+----I

25-34

35-44

45-64

+------.f---:.-'------4----I +-:....,r--__r-"""T""-...,....-T""'i~

65+

'5·14

100% 80%

35-44

45-64

65+

I··... No-BeG - - - BCG·Recipients 1

Nation-wide

60%

+-----/---.....=-----=----.-=-.•-..-..-..-...

40%

+----/---:;,L,,-..-,..-•-..

20%

+--.,,4---::.,....:....-----------1 ~

0%

15·24 25-34 Age groups

Age groups

b

65+

+ - - - - - - - - -...----1

0% 15-24

45-64

80%

20%

'5-14

35-44

100"1. South Region

Eastern Region

60%

25-34

Age groups

Age groups

•....

'

....

.'

- -1

--:..:----------1

7" ••

+------.--..__--.-----.--...,....---1 5-14

15-24

25-34

35-44

Age groups

45-64

65+

1.. ···No-BCG

--BCG-Recipients

I

Fig.4.6. a: Rates of positive Mantoux test in various regions in BCG-recipient and non-recipient population (el-Kassimi et al. 1991; Bener 1990; Abdullah et aI. 1991; AI-Kassimi et aI. 1991) b: Rates of positive Mantoux tests nationwide in BCG-recipient and non-recipient population (Al-Kassimi et aI. 1993)

Epidemiology of Tuberculosis in Saudi Arabia

51

25-44 years, and an increased opportunity to acquire infection by adolescents as they leave the sheltered life of young age. Saudi nationality again had lower prevalence, but higher prevalence in male gender than in female. Urban dwellers had, in general, higher prevalence. In the northern area, only 4% of unvaccinated children aged 5-14 years had a positive Mantoux reaction. A positive Mantoux reaction was seen in significantly more BCG-vaccinated subjects (manifested only in adults) and in older age groups. The overall prevalence of positive Mantoux reaction in the unvaccinated group was 19% and reached its peak (58%) in the 65+ age group, while in those vaccinated was 15%. The overall rate of positivity in males was 37% which is significantly higher than in women (12%). There was no difference in subjects living status, nationality, level of education, and history tuberculosis in the family or chest symptoms. In the Eastern area, 5% of unvaccinated children aged 5-14 years had a positive Mantoux reaction. Again, a positive Mantoux reaction was seen in significantly more BCG-vaccinated subjects (manifested only in adults) and in older age groups. In the Southern area, 4% of unvaccinated children aged 5-14 years had a positive Mantoux reaction. Again, a positive Mantoux reaction was seen in significantly more BCG-vaccinated subjects and in older age groups. The overall prevalence of a positive Mantoux reaction in the unvaccinated group was 26% and reached its peak (67%) in the 65+ age group, while in those vaccinated was 17%. The overall rate of positivity in male was 35% as opposed to 42% in female with difference between the sexes is more marked in the 25-64 year age group. There was significant difference between urban and rural residents, and in nationality status. In 1992, MOH has established the National Tuberculosis Control Committee. The field application of the National Tuberculosis Control Program was implemented in Riyadh in 1996 according to WHO guidelines (AI-Hajjaj 2000).

pulmonary tuberculosis. Certain "Chest Hospitals" had the facilities to isolate and test susceptibility of M. tuberculosis. Testing in these facilities was not done for all isolates. Health reports did not include the limited data on drug susceptibility. Thus, patterns and distribution of drug-resistance are not known at the community level. Other health care facilities were performing susceptibility on their isolates. Many reports of sensitivity patterns were published (Schiott et al. 1985; AIOrainey 1986; AI-Orainey et al. 1989; Zaman 1991b; Jarallah et al.I992; Ellis et al.1996; AI-Jama et al.1999; Khan et al. 2001; Alrajhi et al. 2002). These reports used different methodologies, definitions, and various ways of presenting the data. So any epidemiological analysis of these reports lacks the necessary details to identify patterns, geographic and population distribution. Rates varied as high as 43.7% resistant to at least one of the first-line agents and as low as 8.7% of isolates being resistant. Table 4.2 summarizes the published data on drug resistance. Two reports on drug resistance rates appeared from the same institutions over two periods of time. Khan et al have shown clearly that resistance rates were increasing between the period of 1993-1995 and the period of 1996-1998 This increase was statistically significant for one or more antituberculous agents and for all first-line agents singularly except for ethambutol and pyrazinamide (Khan et al. 2001). On the other hand, from King Faisal Specialist Hospital and Research Centre, where most of tuberculosis cases are Saudis, the resistance rates were not statistically significant between the period 1989-1994 and the period 1995-1999 (Alrajhi et al. 2002). As most of the reported resistance rates came from referral centers, they may not represent the actual rates in the community as a whole. In a recent report by the WHO, no data from the Eastern Mediterranean Region, where the Kingdom of Saudi Arabia is grouped, were included (WHO 1997).

4.2 Epidemiology of Drug-Resistant M. tuberculosis

4.3 Tuberculosis in Certain Populations

The reporting procedures of all tuberculosis cases in Saudi Arabia did not include the susceptibility results of isolated organisms. Microbiological diagnosis at point of care areas did not attempt to grow the organisms. Diagnosis was limited to smears of respiratory specimens and histopathological findings for extra-

As M. tuberculosis activity is closely related to host immunity, rates of reactivation of a latent infection will depend mostly on host-related factors. Thus reactivation rates will vary widely among various patient and non-patient populations. Over the next few sections, available data of disease prevalence in various populations will be reviewed.

52

A. A. Alrajhi and A. M. AI-Barrak

Table 4.2. Drug-resistance rates against isoniazid and rifampin from various reports Author (Reference year) Year

Region

Total

Schiott et aI. (1985) AI-Orainey (1986) AI-Oraineyet aI. (1989) Zaman (1991b) Jarallah et aI. (1992) Chowdhury and Kambal (1992) Ellis et aI. (1996) AI-Jama et aI. (1999)* Khan et aI. (2001) AIrajhi et aI. (2002) Total**

Not indicated 1979-1982 1986-1988 Not indicated 1986-1988 1987-1991

Jazan Riyadh Riyadh Jeddah Taif Riyadh

103 1968 432 483 678 214

42 82 84 86 44 20

(40.8%) (4.2%) (19.4%) (17.8%) (6.5%) (9.3%)

42 113 104 8

(9.7%) (23.4%) (15.3%) (3.7%)

1989-1994 1989-1998 1996-1998 1995-2000

Mixed Eastern Western Mixed

289 1239 101 320 5827

22 211 29 29 649

(7.6%) (17%) (28.7%) (9.1%) (11.1 %)

9 (3.1 %) 78 (6.3%) 21 (20.7%) 9 (2.8%) 405 (10.5%)

Isoniazid number (%)

Rifampin number (%)

Isoniazid and rifampin, no (%)

21 (20.4%)

20 (19.4%)

**

**

At least resistant to one agent

38 (8.8%) 70 (14.5%) 26 (3.8%) 7 (3.3%)

45 259 92 145 153 24

(43.7%) (13.2%) (21.3%) (30%) (22.6%) (11.2%)

8 (2.8%) 34 (2.7%) 21 (20.7%) 9 (2.8%) 233 (6.0%)

25 302 30 36 1111

(8.7%) (24.4%) (29.7%) (11.3%) (19.1%)

* Report included mycobacteria other than tuberculosis ** Rifampin and MDR rates are calculated without Ref. no. (AI-Orainey 1986)

4.3.1 The Elderly The high prevalence of tuberculosis in Saudi Arabia increases with age. As health care improves and life expectancy is prolonged, more population is at risk of tuberculosis reactivation with advanced age. A report from a teaching hospital in Riyadh has outlined the diagnostic challenges of tuberculosis in the elderly (Dahmash et al. 1995). Dahmash et al. reported on 80 patients older than 60 years admitted with tuberculosis between 1988-1993. Pulmonary tuberculosis accounted for 80% and miliary tuberculosis in 20% of these patients. Diagnosis was delayed in 23% of patients, and 21 % died during the same admission. Of the cohort that had follow-up in the same hospital, 21 % died (9 of 43), seven had miliary tuberculosis and two had pulmonary tuberculosis. Fatality occurred within the first two months of therapy. Another problem in elderly patients with tuberculosis is intolerance to antituberculous chemotherapy. In their series, Dahmash et al. reported adverse effects in 18% of the patients.

4.3.2 Diabetics

Diabetes mellitus is higWy prevalent in Saudi Arabia. Prevalence in the general population ranges between 7% in males of rural areas to 15% in females from urban areas. It increases with age, and one of every two females above 50 years had diabetes (Al-Nuaim 1997). The association of tuberculosis and diabetes mellitus is well documented and is directly proportional to

insulin requirements (Boucot et al. 1952). Pandya et al. (1991) noted that out of 1566 patients with pulmonary tuberculosis at Riyadh Chest Hospital between 1983 and 1987,136 (8.7%) had diabetes mellitus. The proportion was higher for Saudi patients (13%) compared with non-Saudi patients (5%) with tuberculosis. However, prevalence of diabetes mellitus in patients with pulmonary tuberculosis does not seem to be higher than in the general population (AI-Nuaim 1997). Over a 4-year period starting in 1995, diabetes mellitus was found in 6.2-15.5% of active tuberculosis seen in Riyadh Chest Hospital (AI-Hajjaj 2000).

4.3.3 Persons Infected with Human Immunodeficiency Virus

The Human Immunodeficiency Virus (HIV) and its Acquired Immunodeficiency Syndrome (AIDS) global epidemic have had a profound impact on global tuberculosis (Narain et al. 1992). Saudi Arabia is no exception when it comes to interaction between HIV and M. tuberculosis. In fact, the high prevalence of tuberculosis infection at an early age makes the impact of HIV/AIDS on tuberculosis reactivation more pronounced. Among the HIV/AIDS patients at King Faisal Specialist Hospital and Research Centre, 11 out of 233 patients had culture-proven tuberculosis, giving a rate of 4.7% for reactivation. Some patients presented with tuberculosis before they were known to have HIV infection. As of 1999,7 of 171 HIV positive patients (4.1%) had tuberculosis (Aljurf et al. 1999). Screening of all tuberculosis patients for HIV infection has been recommended.

53

Epidemiology of Tuberculosis in Saudi Arabia

4.3.4 Hemodialysis Patients

a center in Abha, southwest of the country, tuberculosis was diagnosed in 13 patients (4.8%) out of 270 dialysis patients after a mean of 19 months on dialysis. Pulmonary tuberculosis was found in 8 patients (62%) in contrast to other reports finding higher percentages of extrapulmonary tuberculosis (al-Homrany 1997). From Jeddah, two dialysis centers reported 17 tuberculosis cases among 210 dialysis patients with a prevalence rate of 8.1% (Shohaib et al. 1999). Again, as in Abha, ten patients had pulmonary involvement and nine had lymph node involvement. Table 4.3 summarizes the numbers of patients on dialysis and tuberculosis in various published reports. Hemodialysis patients who developed tuberculosis and completed therapy did not relapse after kidney transplantation (Hussein et al. 1996). Reactivation oflatent infection seems to be the role rather than a primary infection. However, this depends on the prevalence of disease in the community. The other co-morbid illnesses in hemodialysis patients, such as viral hepatitis, diabetes, and hypertension make chemotherapy in this population complex and in need of close monitoring. PPD test in patients on hemodialysis with tuberculosis is unreliable. Reports of positive test rates ranged from zero to 62% of patients. High level of clinical suspicion for tuberculosis is essential for prompt diagnosis and therapy institution. A unique report has prospectively evaluated the risk of tuberculosis in patients with renal impairment not requiring dialysis (AI Shohaib 2000). Al Shohaib has prospectively followed 80 patients with a creatinine clearance of 40 ml/min or less for 3 years in Jeddah. At enrollment, all patients had a normal chest X-ray examination and 50% of patients had a positive PPD test of more than 10 mm induration. During follow-up, eight patients (10%) developed tuberculosis, four pulmonary, two renal, one meningitis, and one adenitis. Four of them had a positive PPD test at enrollment. AIl had an excellent response to a nine-month course except the meningitis case, who was treated for one year. This study has provided valuable data on patients with renal

As of 1999, there were 5706 patients receiving hemodialysis in Saudi Arabia; 2084 patients started hemodialysis in 1999 (Jondeby et al. 2001). These patients started recently on hemodialysis are older on average, and 16% of them have diabetes mellitus. In fact, in 1999-initiated patients alone, 40% of them have diabetes mellitus. Uremia associated impaired cell mediated immunity is well documented (Wilson et al. 1965). In 1974, the first report of tuberculosis in dialysis patients was published (Pradhan et al. 1974). This increased incidence continues until the late 1990s (Cengiz 1996; Murthy and Pereira 1997). In 1990, the first report from Saudi Arabia on tuberculosis in hemodialysis patients was published by AI-Khader et al. (1990) Forty-two out of fifty dialysis centers in the Kingdom responded to a questionnaire. Tuberculosis was diagnosed in 4.4% of 1450 patients on chronic hemodialysis. Around fifty percent had lymphadenitis and less than one third had pulmonary involvement. On average, tuberculosis patients were older, mean age 42 years compared with general dialysis patients, mean age of 33 years. The report did not indicate the period on hemodialysis that preceded tuberculosis diagnosis. In 1991, Mitwalli reported on tuberculosis in dialysis patients at one hospital in Riyadh (Mitwalli 1991). Patients were diagnosed with tuberculosis after either one or two months on dialysis. Of 26 dialysis patients, 7 (27%) were diagnosed with tuberculosis, predominantly, lymphadenitis (57%), and only one with pulmonary tuberculosis as pleurisy. From another dialysis center in Tau, western province that provides hemodialysis to 325 patients, 30 patients (9.2%) had tuberculosis. Again extrapulmonary tuberculosis was predominant (70%) mostly lymphadenopathy (57%). Pulmonary tuberculosis accounted for 30% of tuberculosis cases (Hussein et al.1990, 1994) Frequently, pulmonary tuberculosis in hemodialysis patients is associated with pleurisy. From

Table 4.3. Number and percentage of dialysis patients with tuberculosis in Saudi Arabia Author, Reference year Al-Khader*, (Al-Khader et aI. 1990) Mitwalli (1991) Hussein et al. (1994) Al-Homrany (1997) Shohaib et al. (1999) Total

Years covered

Region

Total on dialysis

Mixed

1450

64

4.41

26 325 270 210 2281

7 30 13 17 131

26.92 9.23 4.81 8.1 5.74

1989-1990 Riyadh 1980-1993 Al-Hada 1986-1993 Abha 1992-1997 Jeddah

Patients with Percent tuberculosis

* From a survey of 42 dialysis centers, some cases may be reported in other reports

54

impairment without dialysis and risk for tuberculosis. However, time of tuberculosis reactivation in relation to renal impairment course was not indicated, and no attempt was made to identify risk factors for reactivation. An indirect estimate for the tuberculosis reactivation rate in this population can be made at one case for every 30 persons per year. This is equal to 3300 cases annually per 100,000 population, a rate more than 51 times the general population in Jeddah (Milaat et al. 1994). This study also provides evidence that PPD test is unreliable in this population to identify patients at risk for tuberculosis-reactivation.

4.3.5 Cancer and Bone Marrow Transplant Patients

Neoplasia constitute a major immune compromise with and without cytotoxic therapy. In hematologic malignancies, tuberculosis reactivation has been reported from developed countries. However, data from developing countries are limited. The King Faisal Specialist Hospital and Research Centre in Riyadh has been performing bone marrow transplantation and tuberculosis was diagnosed in 4 (0.6%) of 641 adult recipients of bone marrow transplantation between 1986 and 1997 (Aljurf et al. 1999). Diagnosis in these severely immunocompromised hosts was delayed. Two patients died before tuberculosis was diagnosed. Rates of tuberculosis in bone marrow transplantation patients in developed countries ranged between 0.1% and 2.0%. For some reason, tuberculosis reactivation rates in bone marrow transplantation patients in Saudi Arabia are lower than expected given the prevalence rate in the country and degree of immune suppression in these patients. Another group of cancer patients in which tuberculosis rates have been poorly evaluated is lymphoma patients. They have a considerable degree of immunosuppression, even prior to chemotherapy. Lymphoma is one of the most common cancers in Saudi Arabia (Cancer registry report: KFSH-RC). There are no reports on lymphoma cohorts who developed tuberculosis to assess the reactivation risk. Among a group of 3921 lymphoma patients at King Faisal Specialist Hospital and Research Centre, culture-proven tuberculosis was documented in 26 patients (0.7%). Rates were similar between Hodgkin's and non-Hodgkin's disease. Pulmonary tuberculosis was noted in ten patients, extrapulmonary in thirteen patients and three had disseminated disease. Diagnosis was delayed and mortality was high at 46%. Three of the twelve mortalities

A. A. Alrajhi and A. M. AI-Barral<

were diagnosed to have tuberculosis post-mortem (Neamatallah et al. 2000).

4.3.6 Solid Organ Transplant Recipients

The immunosuppressive therapy used in solid organ transplant patients enhances the chances for reactivation of tuberculosis (Barber and Sugar 1994). This has been documented from a kidney transplant series at King Faisal Specialist Hospital and Research Centre in Riyadh. Qunibi et al. (1990) reviewed 403 kidney-transplanted patients over 9 years. Tuberculosis was documented in 14 cases, a rate of 3.5%. This rate was thought to be 50 times higher than the annual incidence rate. Pulmonary tuberculosis was diagnosed in 29% of the cases, disseminated in 64%. One case acquired infection from the transplanted kidney. At the same institution, King Faisal Specialist Hospital and Research Centre, two liver transplant patients developed tuberculosis out of a cohort of 72 patients. The rate is 2.7%, close to the rate in kidney transplant recipients (A. Alrajhi, unpublished data).

4.4

Mycobacteria Other than M. tuberculosis

Mycobacterial species other than M. tuberculosis (MOTT) has not been studied well in Saudi Arabia. This could be due to non-culture methods of identifying tuberculosis in some centers, difficulty in identifying non-tuberculosis mycobacterium, or considered as contamination. Zaman found 9% (47 isolates) of mycobacterium isolates are nontuberculosis at National Guard Hospital in Jeddah over 2 years (Zaman 1991a). M. fortuitum and M. chelonei were the most prevalent. Respiratory and urogenital tracts were most frequently affected. At King Faisal Specialist Hospital and Research Centre in Riyadh, MOTT was found in 396 (64%) of the 620 mycobacteria isolates over a 3-year period between 1987 and 1989 (Brodie and Qadri 1990). M. aviumintracellulare complex was cultured most frequently. Respiratory specimens were the most frequent source of positive source. This high incidence is probably due to bias in patient population selection in this tertiary care center, which treats transplanted and malignancy patients. There is a need for reliable data collection to be able to establish the true incidence and prevalence of

Epidemiology of Tuberculosis in Saudi Arabia

MOTT in Saudi Arabia. Progress in health care has led to an increase of numbers in immunocompromised patients who are predisposed to these organisms. Specimens processed for mycobacteria need to be cultured and processed even if they are MOTT.

4.5 Conclusion Saudi Arabia has been in the middle percentage countries affected by tuberculosis. The incidence of reported pulmonary tuberculosis continued to decrease steadily during the 1980s and the 1990s. However, extra-pulmonary tuberculosis has resurged late in the 1990s. The incidences around the Kingdom differ according to area of high traffic of a population during a short time, e.g., Jeddah area and Hajj or a concentration of a certain population. There is alarming data of increasing multi-drug resistance tuberculosis. There is an increasing demand for nationwide studies on incidence and prevalence of tuberculosis in different populations in the new centuries in order to asses the effect of the national tuberculosis program that was established last decade.

References Abdullah A et al (1991) Tuberculosis epidemiology survey: Mantoux test results from Central region of Saudi Arabia. Saudi Med J 12:107-110 Al-Amoud M (1993) The history of tuberculosis. Saudi Med J 14:515-520 AI-Dabbagh A, El-Deeb H, AI-Baghdadi T (1991) The radiographic features of pulmonary tuberculosis observed for the Western region of Saudi Arabia. Ann Saudi Med 11: 194-200 Al-Hajjaj M (2000) The outcome of tuberculosis treatment after implementation of the national tuberculosis control program in Saudi Arabia. Ann Saudi Med 20:125-128 AI-Hajjaj M et al (1991) Pulmonary tuberculosis in Saudi Arabia: a retrospective study of 1566 patients. Ann Saudi Med 11: 443-447 Al-Homrany M (1997) Successful therapy of tuberculosis in hemodialysis patients. Am J NephroI17:32-35 Al-Jama A, Borgio F, Al-Qatari K (1999) Patterns of resistance to antituberculous drugs in Eastern Province, Saudi Arabia. Saudi Med J 20:927-930 Aljurf M et al (1999) Mycobacterium tuberculosis infection in allogeneic bone marrow transplantation patients. Bone Marrow Transplant 24:551-554 Al-Kassimi F (1994) Review of tuberculosis in Saudi Arabia. Saudi Med J 15:192-195 Al-Kassimi F et al (1991) Mantoux Reaction Survey conducted

55 in the Northern region of Saudi Arabia. Ann Saudi Med 11:315-321 Al-Kassimi F et al (1993) Nationwide community survey of tuberculosis epidemiology in Saudi Arabia. Tubercle Lung Dis 74:254-260 AI-Khader A et al (1990) Prevalence of tuberculosis in the dialysis population in Saudi Arabia. Saudi Kidney Dis Transplant Bull 1:155-157 Al-Orainey I (1986) Resistance to standard antituberculous drugs in Saudi Arabia. Saudi Med J 7:363-368 Al-Orainey let al (1989) Resistance to antituberculosis drugs in Riyadh, Saudi Arabia. Tubercle 70:207-210 Alrajhi AA, et al (2002) Risk factors for drug-resistant Mycobacterium tuberculosis in Saudi Arabia. Saudi Med J 2002; 23:305-310 Al Shohaib S (2000) Tuberculosis in chronic renal failure in Jeddah. J Infect 40:150-153 AI-Wabel A et al (1995) Spectrum of pulmonary tuberculosis in the Asir Region of Saudi Arabia. Saudi Med J 16:105-107 Al-Wabel A et al (1997) Symptomatology and chest roentgenographic changes of pulmonary tuberculosis among diabetics. East Afr Med J 74:62-64 Alzeer A et al (1998) Tuberculosis is the commonest cause of pneumonia requiring hospitalization during Hajj (pilgrimage to Makkah). J Infect 36:303-306 Barber T, Sugar MA (1994) Mycobacteriossis and nocardiosis in the immunocompromised host. In: Robin RH, Young LS (eds) Clinical approach to infection in the compromised host. Plenum, New York, pp 251-253 Bener A (1990) Prevalence of tuberculosis infection in ABHA and BAHA. Eur J EpidemioI6:376-381 Bener A, Abdullah A (1993) Reaction to tuberculin testing in Saudi Arabia. Indian J Publ Health 37:105-110 Boucot K, Cooper D, Dillon E et al (1952) Tuberculosis among diabetics: the Philadelphia survey. Rev Tuberculosis 65:127 Braun M et al (1991) A retrospective cohort study of the risk of tuberculosis among women of childbearing age with HIV infection in Zaire. Am Rev Respir Dis 143:501-504 Brodie L, Qadri SM (1990) Isolation of mycobacteria other than that of tuberculosis at a major tertiary care hospital in Saudi Arabia. Med Sci Res 18:257-258 Cengiz K (1996) Increased incidence of tuberculosis in patients undergoing hemodialysis. Nephron 73:421-424 Chowdhury M, Kambal A (1992) Mycobacterial resistance in a general hospital in Riyadh Saudi Arabia. Med Sci Res 20: 21-22 Dahmash N, Chowdhury M (1994) Re-evaluation of pneumonia requiring admission to an intensive care unit: a prospective study. Thorax 49:71-76 Dahmash N et al (1994) Infections in critically ill patients: experience in MICU at a major teaching hospital. Infection 22:264-270 Dahmash N et al (1995) Diagnostic challenge of tuberculosis of the elderly in hospital: experience at a university hospital in Saudi Arabia. J Infect 31:93-97 El-Kassimi F (1994) The annual risk of tuberculosis infection used as predictor of the future incidence of smear-positive cases. Respir Med 88:589-592 El-Kassimi F et al (1991) Tuberculin survey in the Eastern Province of Saudi Arabia. Respir Med 85:111-116 Ellis M et al (1996) High proportion of multi-drug resistant Mycobacterium tuberculosis in Saudi Arabia. Scand J Infect Dis 28:591-595

56 EI-Sheikh S et al (1998) Bacteria and viruses that cause respiratory tract infections during the pilgrimage Haj season in Makkah, Saudi Arabia. Trop Med Int Health 3:205-209 Farag M et al (1995) National immunization coverage survey Saudi Arabia, 1991. J Trop Pediatr 41:S59-S67 Froude J, Kingston M (1982) Extrapulmonary tuberculosis in Saudi Arabia, a review of 162 cases. King Faisal Special Hosp Med J 2:85-95 Hammam H, Kamel L, Hidayat N (1979) Third Saudi Medical Conference, Dammam Hussein M, Bakir N, Roujouleh H (1990) Tuberculosis in patients undergoing maintainence dialysis. Nephrol Dial Transplant 5:584-587 Hussein M et al (1994) Observations in a Saudi-Arabian dialysis population over a 13-year period. Nephrol Dial Transplant 9: 1072-1076 Hussein M, Mooij J, Roujouleh H (1996) Tuberculosis in hemodialysis patients. Saudi J Kidney Dis Transplant 7:6-9 Jarallah J et al (1992) High rate of rifampicin resistance of Mycobacterium tuberculosis in the Taif region of Saudi Arabia. Tubercle Lung Dis 73:113-115 Jondeby M et al (2001) Caring for hemodialysis patients in Saudi Arabia. Saudi Med J 22:199-204 Khan M et al (2001) Increasing resistance of M. tuberculosis to anti-TB drugs in Saudi Arabia. Int J Antimicrob Agents 17:415-418 Mackay A, Cole R (1984) The problem of tuberculosis in the elderly. Quart J Med 212:497-510 Milaat W et al (1994) Epidemiology of tuberculosis in Jeddah region, Saudi Arabia. Saudi Med J 15:133-137 Ministry of Health (1979) Tuberculosis. First Annual Health Report, 1979. Ministry of Health, Riyadh, pp 199-208 Ministry of Health (1991) Tuberculosis. Annual Health Report, 1991. Ministry of Health, Riyadh, pp 252-258 Ministry of Health (1996) Tuberculosis. Annual Health Report, 1996. Ministry of Health, Riyadh, pp 35-37 Ministry of Health (1997) Tuberculosis. Annual Health Report, 1997. Ministry of Health, Riyadh, pp 46-49 Mitwalli A (1991) Tuberculosis in patients on maintenance dialysis. Am J Kidney Dis 18:579-582 Mohamed A, Evans D (1987) The spectrum of pneumonia in 1983 at the Riyadh Armed Forces Hospital. J Infect 14:31-37 Mokhtar A, Salman K (1983) Extrapulmonary tuberculosis. Saudi Med J 4:317-322 Murthy B, Pereira B (1997) A 1990 s perspective of hepatitis C, human immunodeficiency virus, and tuberculosis infections in dialysis patients. Semin NephroI17:346-363 Narain 1, Raviglione M, Kochi A (1992) HIV-associated tuber-

A. A. Alrajhi and A. M. AI-Barrak culosis in developing countries: epidemiology and startegies for prevention. Tubercle Lung Dis 73:311-321 Neamatallah A, Alrajhi N, Alrajhi A (2000) Mycobacterium tuberculosis infection during lymphoma chemotherapy. 10th European congress of clinical microbiology and infectious diseases, Stockholm, Sweden Pandya L et al (1990) Lower-lung-field tuberculosis in Saudi Arabia. Ann Saudi Med 10:374-377 Pandya L et al (1991) Pulmonary tuberculosis in diabetic patients. Ann Saudi Med 11:293-296 Pradhan R et al (1974) Tuberculosis in dialyzed patients. JAMA 229:798-800 Qunibi W et al (1990) Mycobacterial infection after renal transplantation - report of 14 cases and review of the literature. Quart J Med 77:1039-1060 Schein M, Huebner R (1995) Tuberculin skin testing. McGrrawHill, New York, pp 73-75 Schiott C et al (1985) Incidence of drug resistance amongst isolates of Mycobacterium tuberculosis recovered in the Gizan area, Saudi Arabia. Saudi Med J 6:375-378 Selwyn P et al (1989) A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 320:545-550 Shanks N, Khalifa I, Al Kalai D (1983) Tuberculosis in Saudi Arabia. Saudi Med J 4:151-156 Shohaib S, Scrimgeour E, Shaerya F (1999) Tuberculosis in active dialysis patients in Jeddah. Am J NephroI19:34-37 Styblo K (1980) Recent advances in epidemiological research in tuberculosis. Adv Tuberculosis Res 20:1-63 Thompson N et al (1979) The booster phenomenon in serial tuberculin testing. Am Rev Respir Dis 119:587-597 WHO (1964) WHO technical report series no 290. World Health Organization, Geneva WHO (1997) Anti-tuberculosis drug resistance in the world. The WHO/IUATLD global project on anti-tuberculosis drug resistance surveillance 1994-1997. World Health Organization, Geneva Wilson W, Kirkpatrick C, Talmage D (1965) Suppression of immunologic responsiveness in uremia. Ann Intern Med 62:1-8 World Bank W (1993) World development report 1993: investing in health. Oxford University Press, Oxford Zaman R (199Ia) Tuberculosis in Saudi Arabia: epidemiology and incidence of Mycobacterium tuberculosis and other mycobacterial species. Tubercle 72:43-49 Zaman R (1991b) Tuberculosis in Saudi Arabia: initial and secondary drug resistance among indigenous and nonindigenous populations. Tubercle 72:51-55

5

The Molecular Epidemiology of Tuberculosis KATHRYN DERIEMER and CHARLES 1. DALEY

CONTENTS 5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.3.6 5.4 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.5

Introduction 57 Genotyping Methods 57 Commonly Used Secondary Typing Methods 59 PGRS Typing 59 Spoligotyping 59 Other PCR-Based Methods 59 Comparisons of Different Typing Methods 60 Methodological Issues in Interpreting IS6110 RFLP Typing 60 Whole Genome and Microarray Technologies 62 Lessons Learned Using Molecular Epidemiology 62 Contact and Outbreak Investigations 62 Community Epidemiology and Tuberculosis Control 65 Exogenous Reinfection with M. tuberculosis 66 Geographical Distribution and Dissemination of M. tuberculosis 67 Evaluation of Laboratory Cross-contamination 68 The Future of Molecular Epidemiology 69 References 70

5.1 Introduction The ability to track specific strains of Mycobacterium tuberculosis as they spread through a population is critical for our understanding of the transmission and pathogenesis of tuberculosis. However, until recently, the only phenotypic markers that distinguished different strains of M. tuberculosis were drug resistance patterns and mycobacterial phage typing (Gruft et al. 1984). Molecular genotyping techniques that allow us to differentiate isolates of K. DERIEMER, PhD Senior Research Scientist, Division of Infectious Diseases and Geographic Medicine, Stanford Medical Center, Stanford, California, USA C. 1. DALEY, MD Associate Professor of Medicine, Division of Pulmonary and Critical Care Medicine, San Francisco General Hospital, Room 5K-1, 1001 Potrero Ave, San Francisco, CA 94110, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

M. tuberculosis for the purpose of tracking strains in the community and designing prevention and control strategies to block further transmission of M. tuberculosis are now available. Molecular epidemiology combines the straintyping capabilities of genotyping with conventional epidemiologic approaches in order to study the distribution and determinants of disease occurrence. Molecular techniques can help address epidemiological problems that cannot be approached or would be more difficult to address by conventional methods. Epidemiologic investigations that incorporate genotyping of M. tuberculosis have been used to provide novel information about the spread of tubercle bacilli in mini-epidemics and outbreaks, to determine the risk factors for transmission in a community, to distinguish exogenous re-infection from relapses, to track the geographic distribution and spread of strains ofM. tuberculosis of public health importance, and to identify instances of laboratory contamination (Foxman and Riley 2001). This chapter updates current knowledge about the accomplishments and future directions of this relatively new field.

5.2

Genotyping Methods Until recently, drug susceptibility testing and phage typing were the only bio-markers available for epidemiological studies of M. tuberculosis, both of which have serious limitations. Drug susceptibility test results can change in a strain as the strain acquires resistance to one or more antimicrobials during treatment. Bacteriophage typing of M. tuberculosis is oflimited value for epidemiologic studies as only a few phage types can be recognized and thus, it cannot adequately distinguish between different strains (Rado et al. 1915; Grange et al. 1978; Engel 1978; Crawford and Bates 1984). During the past decade, several nucleic acid-based genotyping methods based on the variations in the

58

K. DeRiemer and C. L. Daley

sequence in bacterial genomes were developed. These new methods allow us to accurately distinguish between different strains of M. tuberculosis. The most widely used method is referred to as restriction fragment length polymorphisms (RFLP) analysis (Fig. 5.1). Specific restriction endonucleases cleave the mycobacterial DNA at the sites of specific repetitive sequences, producing DNA restriction fragments of different lengths. The restriction fragments can be separated by size using conventional agarose gel electrophoresis and the banding patterns of these restriction fragments can be detected by staining the gel with ethidium bromide (Collins and de Lisle 1985, 1987; Swaminathan and Matar 1993). However, the resulting genotype pattern contains thousands of fragments and is very difficult to read and interpret. In order to decrease the number of fragments to be compared, the chromosomal DNA restriction fragments are probed with specific, repetitive, labeled DNA fragments (Eisenach et al. 1988; Reddi et al. 1988). Only the genomic DNA restriction fragments that are complementary to and hybridize with the probe are visible, resulting in fewer bands and a more readable band pattern. An internationally accepted, standardized protocol for RFLP typing of the M. tuberculosis complex Chromosomal DNA

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was published in 1993 and is still used today (van Embden et al. 1993). This method takes advantage of a specific, well-characterized, repetitive element insertion sequence 6110 OS611O) (Thierry et al. 1990). It was originally thought that IS6110 insertions occur randomly (Zhang et al.1992) but this was disproved when the H37Rv strain of M. tuberculosis was sequenced (Philipp et al. 1996; Cole et al. 1998). Between 0 and 25 copies of IS611 0 are found in almost all strains of the M. tuberculosis complex (van Soolingen et al.1993; Agasino et al.1998). The position and number of copies of the IS6110 elements varies from strain to strain; this variability in the chromosome of M. tuberculosis is exploited to distinguish between different strains (Cave et al.1991; van Soolingen et al. 1991). The RFLP band patterns of strains may be compared visually or scanned optically by a computerized reading system and matched to a reference library of strain profiles (Woelffer et al.1996; Heersma et al.1998). When used in conjunction with standardized international databases and computer-assisted analysis, this approach allows comparisons of strains between different laboratories in widely separated geographical regions. However, the standardized method of IS6110 RFLP typing has several disadvantages. It is slow, labor intenDigested DNA

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Fig. 5.1. Method ofI56110-based restriction fragment length polymorphisms (RFLP) analysis. Depicted are two strains of Mycobacterium tuberculosis, labeled 1 and 2. The location and number of the insertion sequence, 156110, is noted by the small black rectangles. A. The first step in the process is the extraction of the chromosomal DNA. B. The extracted DNA is then cleaved with a restriction endonuclease (PvuII). Note that in reality, thousands of fragments are created. C. After digestion, the DNA fragments are separated according to size by gel electrophoresis. In reality, this process results in thousands of bands that are nearly confluent on the gel. D. Hybridization with probe for 156110 results in a gel with bands only containing the 156110 element. Thus, there are fewer bands and the pattern is easier to read. The two strains can be seen to differ in the number of bands (i.e., the number of 156110 copies in the genome) and the location of the bands (i.e., the size of the restriction fragments)

59

The Molecular Epidemiology of Tuberculosis

sive, and technically demanding. It requires a relatively large amount (e.g. 2 j.1g) of high-quality DNA from each strain of M. tuberculosis, an amount that can only be extracted from a large number of bacteria grown from clinical material. For isolates with less than six copies of IS6110 (less than six bands in the RFLP pattern), it has relatively poor discriminatory power and should be supplemented by analyses with other probes. Finally, sophisticated computer software and technical support are required to compare, analyze, and interpret large numbers of IS6110 RFLP band patterns. Despite these limitations, IS6110 RFLP typing remains the most commonly used method in molecular epidemiologic studies of M. tuberculosis and is considered the gold standard technique.

S.3 Commonly Used Secondary Typing Methods Several basic assumptions underlie our interpretation of genotyping results. For example, we assume that two strains with an identical IS6110 RFLP pattern have a common origin, albeit, perhaps, remote. In addition, we assume that strains with identical IS6110 RFLP band patterns with multiple bands are more likely to have a common origin and be clonal than are strains with relatively few bands. Strains with few bands or low copy numbers in the RFLP pattern could appear identical by chance alone. A secondary probe, such as the polymorphic guanine/cytosinerich repetitive sequences (PGRS) or spoligotyping can be used to further differentiate between strains with low copy numbers (Burman et al. 1997b). Early comparative studies showed that using multiple molecular techniques in a single study provided better discrimination between strains and insight for phylogenetic groupings (Bifani et al. 1999). Today, most molecular epidemiologic studies rely on IS611 0 RFLP typing and a secondary typing method, such as PGRS or spoligotyping, for those isolates with less than six bands in the IS6110 RFLP band pattern.

system uses the polymorphic GC-rich repeat sequence contained in the recombinant plasmid pTBN12 as a probe (Cousins et al. 1992; Ross et al. 1992). The main disadvantage of the PGRS typing system is that some of the PGRS regions include many non-perfect repeats and the patterns that PGRS typing generates are complex and difficult to interpret. In addition, PGRS typing is less discriminatory than IS6110-based RFLP genotyping. Despite these limitations, PGRS genotyping is a commonly used secondary typing method.

5.3.2 Spoligotyping Spoligotyping is a polymerase-chain-reaction (PCR)based method that interrogates a small direct repeat (DR) sequence with 36-base pair (bp) repeats interspersed with short, unique, non-repetitive sequences (Hermans et al. 1991; Groenen et al. 1993; Kamerbeek et al. 1997). All of the unique, non-repetitive sequences, or "spacers:' between the direct repeats can be amplified simultaneously using one set of primers. Strains are differentiated by the number and position of the spacers that are missing from the complete spacer set. The complete spacer set is defined by sequencing this region from a large number of isolates of M. tuberculosis. This spacer oligotyping or "spoligotyping" is more rapid and easier to perform than IS6110 RFLP typing, making it an excellent tool in resource-poor settings. However, it has a lower level of discrimination than IS6110 RFLP typing (Diaz et al.1998; Goyal et al.1999). Nevertheless, it can be used to validate the IS6110 RFLP typing results, to further differentiate strains with low IS6110 copy numbers (less than six bands in the RFLP pattern) (Wilson et al.1998; Bauer et al.1999; Goyal et al. 1999; Yang et al. 2000; Soini et al. 2001), and to show more distant phylogenetic relationships among isolates. As recommended in several studies (van Soolingen et al. 1995; Stauffer et al. 2000), spoligotyping could be used as a preliminary screening step, to be followed by another typing method with greater discriminatory power.

5.3.1 PGRSTyping

5.3.3 Other peR-Based Methods

The most common repetitive DNA sequences in the genome of M. tuberculosis are the PGRS, a triplet repeat of GTG, and the major polymorphic tandem repeat (Hermans et al. 1992; Ross et al. 1992; Poulet and Cole 1995; Chaves et al. 1996). The PGRS typing

Relative to IS6110 RFLP genotyping, PCR-based methods are easier to perform, require small amounts of genomic DNA, and can even be performed on nonviable organisms or directly from clinical specimens,

60

K. DeRiemer and C. L. Daley

thereby eliminating the lengthy time required to culture M. tuberculosis (Driscoll et al. 1999; Qian et al. 1999; Taylor et al. 1999). Several other PCR-based typing assays have been developed using IS6110 as the target. Mixer-linked PCR (Haas et al. 1993) ligationmediated PCR (Bonora et al.1999), heminested inverse PCR, IS6110 inverse PCR, IS6110 ampliprinting, and double-repetitive (DR) element PCR (Friedman et al. 1995a) are among the techniques developed to date. None of these techniques have been studied or applied as extensively as IS611O-based RFLP genotyping. Analysis by variable number tandem repeat (VNTR) is a PCR-based method that differentiates between strains by identifying the number and length of exact tandem repeats present in an isolate, independently of the IS6110 polymorphisms (Skuce et al. 2002). A high-resolution typing method based on the VNTRs of mycobacterial interspersed repetitive units (MIRUs) based on 12 specific intergenic regions of the M. tuberculosis genome has been proposed (Mazars et al. 2001; Supply et al. 2002). In a study of 72 clinical isolates, the correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Relative to IS611 0 RFLP typing, MIRU-VNTR profiling is fast, appropriate for strains regardless of their IS611 0 RFLP copy number, and permits rapid comparison of results from independent laboratories using a binary data classification system.

5.3.4 Comparisons of Different Typing Methods Despite the development of these different methods, IS6110 RFLP remains the most widely used genotyping method for the molecular epidemiology of

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5.3.5 Methodological Issues in Interpreting 156110 RFLPTyping While the methods for generating IS6110 RFLP patterns are well-standardized, the interpretation of these patterns depends on the epidemiological question being asked and the stability of the IS6110 element in the mycobacterial genome. Genetic markers that change rapidly obscure epidemiological links, whereas those that are too stable suggest direct links and relatedness where true links do not exist. In well-defined outbreak settings, some tuberculosis patients may have isolates whose RFLP band patterns differ by one or two insertion elements (Fig. 5.3) (Daleyet al. 1992). This latter observation suggests that as a strain spreads through the community, the strain is evolving and produces progeny with subtle variations in their RFLP patterns and the rate at which these changes occur is

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tuberculosis and is the gold standard to which other methods are compared. In an inter-laboratory study, Kremer and colleagues compared most of the currently available molecular typing methods for M. tuberculosis (Fig. 5.2) (Kremer et al. 1999). Five RFLP typing methods including IS611O, IS1081, PGRS, the DR and the (GTGh repeat as probes were highly reproducible. Of the PCR-based methods that were compared, VNTR typing, mixed-linker PCR, and spoligotyping were highly reproducible between different laboratories. However, the double repetitive element PCR, IS611 0 inverse PCR, IS611 0 ampliprinting and arbitrarily primed PCR were not very reproducible. The most discriminatory methods were IS6110 RFLP typing and the mixed-linker PCR.

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Fig. 5.2. Comparison of different genotyping methods. Depicted above are patterns of ten Mycobacterium tuberculosis complex duplicate samples obtained by various typing methods. The methods are ordered from most to least reproducible (left to right). The 156110 RFLP patterns are shown as representatives of the Pvull-based RFLP method. Numbers in vertical direction indicate duplicate samples. (Adapted from Kremer et al. 1999)

61

The Molecular Epidemiology of Tuberculosis

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-0" Fig. 5.3. Restriction fragment length polymorphisms (RFLP) patterns of M. tuberculosis isolates obtained from an outbreak of tuberculosis in an HIV congregate living site (A) and controls living in San Francisco at the same time (B). The first two lanes are the RFLP patterns of M. bovis, BCG, and M. intracellulare. Cases 1 and 2 (A) were receiving antituberculosis therapy when they entered the facility. Tuberculosis developed in the remaining patients while they lived in the facility. Note the shift of the band in the upper part of lane 11. (Adapted from Daley et al. 1992)

important (Bifani et al. 1996). At least two studies have shown that the 156110 RFLP band patterns of different strains of M. tuberculosis are relatively stable, with a half-life of approximately 3-4 years (Yeh et al. 1998; de Boer et al.1999). Therefore, the 156110 element should be useful for studying transmission of M. tuberculosis over relatively short periods of time. To interpret the results of 15611 0 RFLP strain typing and other typing methods, we assume that epidemiologically related strains will have the same genotype pattern and epidemiologically unrelated strains will have different patterns. Ideally, strain typing will provide a clear, objective basis for identifying an outbreak strain and distinguishing it from epidemiologically unrelated isolates. Many studies of tuberculosis have extended these assumptions to define clusters of patients in the community whose isolates had identical or nearly identical genotype patterns. Clustering has often been equated with recent or ongoing transmission, and the factors associated with clustering have been sought as a means to identify and target subpopulations with substantial ongoing transmission

(Glynn et al.1999a). Since the first large-scale molecular epidemiologic studies of tuberculosis transmission using 156110 RFLP typing, it has generally been accepted that tuberculosis cases with identical 156110 RFLP band patterns that are isolated from two or more persons in the same community represent recent or ongoing exogenous infection and rapid progression to disease. By contrast, patients whose isolates of M. tuberculosis have genotype patterns that do not match any other isolates in the community are considered to be unique. Unique isolates likely represent disease caused by reactivation of latent tuberculosis infection (LTBI) (Alland et al. 1994). Molecular techniques enable us to distinguish tuberculosis due to recent or ongoing infection versus reactivation of LTBI and to estimate the fraction of active tuberculosis transmission in a community. However, some strains may be identical for reasons other than recent transmission and there is not always an epidemiological link between patients whose isolates have identical genotype patterns. For example, a large proportion of the isolates from newly diagnosed tuberculosis cases in a relatively stable, rural population in the United States showed high degrees of clustering based on 156110 RFLP typing, but most of the patients had no discernible contact or other epidemiological links among themselves (Braden et al. 1997). Similar findings were reported from Malawi and Kenya; some strains from a large rural area had identical genotype patterns, but there were no discernible epidemiological links between the patients (Godfrey-Faussett and Stoker 1992). Combined, such studies suggest that the epidemiological interpretation of molecular epidemiologic studies may be confusing in areas where it likely that ancestral, endemic, well-conserved strains with stable genotype patterns occur. The amount of transmission represented by clustering using molecular typing techniques will depend on the sampling strategy and duration of the study (Murray 2002; Murray and Narde1l2002b). Undersampling can bias the estimates of the proportion of tuberculosis cases that were likely caused by recent tuberculosis transmission and it can bias the estimates of the risk factors associated with clustering. When an early hospital-based study of tuberculosis transmission in New York City, USA, was re-examined and adjusted for potential sampling bias, the revised estimate of the odds ratio of the association of human immunodeficiency virus (HIV) and clustering increased from 2.7 to 23.6 (Murray and Alland 2002a). No study has 100% case ascertainment, and incomplete sampling will underestimate the proportion of tuberculosis cases that are clustered, even if the number of tuber-

K. DeRiemer and C. 1. Daley

62

culosis cases included in the study is large (Glynn et al.1999a,b). Biases may also be introduced if a molecular epidemiologic study does not cover an adequate time period. 1\vo population-based studies that have been ongoing for more than 10 years (San Francisco [Small et al. 1994; Jasmer et al. 1999] and the Netherlands [van Soolingen et al. 1999]), showed that the percentage of clustered strains was high during the first 2 years and declined thereafter. Clustering based on less than 2 years of sampling is unlikely to identify the source and secondary cases in a chain of transmission, and will likely underestimate the amount of recent transmission.

genomic deletions can be detected relative to the reference strain, depending on the array. Because deletions in the M. tuberculosis genome rarely occur independently at exactly the same chromosomal locus, they can be considered unique and irreversible genetic events. The number and distribution of deletions provide a genomic pattern that can be used to construct phylogenetic relationships and to determine whether the loss of specific genes is related to the phenotype of a strain, such as its transmissibility or antigenicity (Salamon et al. 2000; Kato-Maeda et al. 2001).

5.3.6 Whole Genome and Microarray Technologies

Lessons Learned Using Molecular Epidemiology

Until recently, the main purpose of developing typing techniques using molecular markers was to differentiate between strains of M. tuberculosis and to identify chains of transmission. There is rising interest in identifying the genotype of specific strains and their correlations with specific clinical manifestations and other phenotypes, such as increased infectivity, virulence, or hyper-mutability. DNA sequencing is still too expensive and too complex to be applied to make direct comparisons of genomic sequences of multiple strains of M. tuberculosis, and it is likely that all strains vary to some degree at the nucleic-acid base level. However, it is now possible to analyze short segments of DNA for sequence similarities and differences. For example, genomic fragments can be amplified using PCR, and an automated DNA-sequencing procedure can be used to directly sequence the PCR-amplified DNA fragment. This approach allows a DNA fragment of 300- to 500-bp to be sequenced in 24 h (Tracy and Mulcahy 1991). In the future, improved automation of target amplification and direct sequence analysis may make this a practical approach for laboratories. Strains of M. tuberculosis can also be compared based on the whole genome sequence using DNA microarrays and DNA chip technology. These techniques allow simultaneous detection of the variation at many different genomic sites by analysis of the amount and specific location of mycobacterial DNA (Gingeras et al. 1998). Briefly, the genome of a strain of M. tuberculosis is compared with that of a known, sequenced reference strain by hybridization on an array containing thousands of oligonucleotides on a limited surface. Differences in gene expression and

Molecular genotyping has revolutionized our ability to track strains of M. tuberculosis as they spread through a community. Studies of the molecular epidemiology of tuberculosis have elucidated both suspected and unsuspected transmission, identified weaknesses in conventional contact investigations, identified risk factors for recent infection with rapid progression to disease, demonstrated exogenous reinfection with different strains, and documented the existence of laboratory cross-contamination as a cause for false-positive cultures.

5.4

5.4.1 Contact and Outbreak Investigations Conventional tuberculosis contact investigations use the"stone-in-the-pond" or concentric circle approach to collect information and to screen household contacts, coworkers, and increasingly distant contacts for tuberculosis infection and disease (Veen 1992). However, the concentric circle method may not be adequate in many out-of-household settings and there are considerable limitations with this approach. Molecular epidemiology has helped to identify some of these limitations. In low-incidence areas such as San Francisco (Small et al. 1994), Zurich (Pyffer et al. 1998), and Amsterdam (van Deutekom et al. 1997), a relatively small proportion (5-10%) of the cases that had identical IS6110-based genotyping patterns were actually identified as a contact by the source case. This suggests that unsuspected transmission of tuberculosis occurs and is not easily traced by con-

The Molecular Epidemiology of Tuberculosis

ventional contact tracing investigations. In a 5-year, population-based study in the Netherlands, contact investigations of persons in five of the largest clusters identified epidemiological links between them based on time, place, and risk factors. However, tuberculosis transmission also occurred through only short-term, casual contact that was not easily detected in routine contact investigations (van Soolingen et al. 1999). In a high-incidence area in Barcelona, Spain (163 tuberculosis cases/lOO,OOO population), there was 61.5% concordance between the genotype pattern and conventional contact tracing (Solsona et al. 2001). In that study, the authors concluded that conventional contact tracing was useful for identifying new tuberculosis cases, but it did not provide much information about the chains of transmission and how to block or prevent them. In a population-based molecular epidemiological study in an urban community in the San Francisco Bay area, 75 (33%) of 221 cases had the same strain of M. tuberculosis. Thirty-nine (53%) of the seventythree patients developed tuberculosis because they were not identified as contacts ofsource case-patients; twenty case-patients (27%) developed tuberculosis because of delayed diagnosis of their sources; and thirteen case-patients (18%) developed tuberculosis because of problems associated with the evaluation or treatment of contacts; and one case-patient (1 %) developed tuberculosis because of delay in being elicited as a contact. This study suggested that even in a community that has implemented the essential elements of tuberculosis control, ongoing transmission of M. tuberculosis will continue to produce cases unless patients are diagnosed earlier and contacts are more completely identified (Chin et al. 2000). Some populations, such as the urban homeless, present unique challenges for those conducting conventional contact investigations. However, studies that incorporate genotyping are able to provide information about the chains of transmission in these groups (Barnes et al. 1996; Gutierrez et al. 1998; Kimerling et al. 1998; Lemaitre et al. 1998; Dobbs et al. 2001). A prospective study of tuberculosis transmission in Los Angeles, California, USA identified 162 patients who had culture-positive tuberculosis, and interviewed the patients to identify their contacts and whereabouts (Barnes et al. 1997). Genotyping using 156110 RFLP typing and PGRS analysis were performed on all isolates of M. tuberculosis from these patients. Patients whose isolates had identical or closely related genotyping patterns were considered clustered, and the degree of homelessness and the instance of a patient using daytime services at

63

three shelters were independently associated with clustering. Ninety-six (59%) of one hundred and sixty-two patients were in eight clusters, but only two of the ninety-six clustered persons named others in the cluster as contacts. Thus, traditional contact investigations did not reliably identify patients infected with the same strain of M. tuberculosis. This study demonstrated that locations at which the homeless congregate are important sites of tuberculosis transmission for both homeless persons, who sleep at the shelters, and non-homeless persons, who use the facilities during the day. The authors recommended that strategies to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Additional studies support the idea that specific locations can be associated with recent or ongoing tuberculosis transmission. A study among predominantly HIV-seropositive gay men in Houston, Texas, found that barhopping, or visiting many bars in the same neighborhood each night, was associated with being in an IS6110 RFLP cluster, representing ongoing tuberculosis transmission (Yaganehdoost et al.1999).A population-based molecular epidemiologic study of tuberculosis transmission in Orizaba, southeastern Mexico, identified clandestine bars as the main site of ongoing tuberculosis transmission and bar patrons as the main sources of tuberculosis transmission in the community (Garda-Garda et al. 2000). In a case-control study of tuberculosis among non-institutionalized HIV-infected patients in King County, Washington, USA, six HIV-infected persons with the same strain of M. tuberculosis had contact at one or more of three bars and this was the only identifiable epidemiologic link among them (Tabet et al. 1994). Additional tools, such as geographic information systems (GIS) data, can add to molecular epidemiologic studies. In a 30-month prospective, city-wide study of all tuberculosis cases in Baltimore, Maryland, USA, using traditional contact investigations and IS6110-based genotyping, 46% (84/182) of isolates were clustered and 32% (58/182) were defined as being recently transmitted. Only 24% (20/84) of clustered cases had an identifiable epidemiologic link of recent contact. Using GIS data, there was significant spatial aggregation of the 20 clustered cases with epidemiologic links in geographic areas of the city with low socioeconomic status and high drug use (Bishai et al.1998). These findings and those described previously suggest that location-based control efforts may be more effective than contact tracing for early identification of cases, at least in some populations.

64

When a second case of tuberculosis is recognized as part of a contact investigation, it is generally assumed that the two cases harbor the same strain of M. tuberculosis, with one as the source-case and the other as the secondary case. Several studies have used genotyping to determine whether the contacts that had tuberculosis were, in fact, secondary cases or whether they were unrelated to the source case. Of 11,211 contacts evaluated in San Francisco, USA, there were 66 pairs of culture-positive index and contact cases (Behr et al. 1998). In the 54 pairs for whom genotyping was available, the index and contact cases were infected with a different strain of M. tuberculosis 30% of the time. Unrelated infections were more common among foreign-born, particularly Asian, contacts. The drug susceptibility pattern was more likely to be discordant in those pairs who harbored different strains. Similar findings have been reported from Denmark and the Netherlands (Sebek 2000). These studies highlight the fact that secondary cases are not always related to the presumed index case and that in areas with high rates of drug resistance, providers should be careful when assuming that the drug susceptibility patterns between the pairs are the same. Genotyping has been particularly useful in defining the presence and scope of tuberculosis outbreaks by identifying otherwise unsuspected and undetected transmission in the community. Traditionally, outbreak investigations depended on contact tracing in the field. This strategy can be ineffective in tuberculosis outbreaks if patients do not live in stable settings and either do not know or are unwilling to reveal the names of contacts. By identifying tuberculosis patients whose isolates have identical strains of M. tuberculosis, genotyping can infer epidemiologic links or connections between individuals and can highlight locations or settings where tuberculosis transmission is occurring. An outbreak of tuberculosis among the residents of a housing facility for HIV-positive persons in San Francisco, California, USA, was one of the first molecular epidemiologic studies that demonstrated the usefulness of genotyping techniques and helped to validate the IS611 a-based typing method (Fig. 5.3) (Daley et al. 1992). The isolates from persons who were part of the institutional outbreak had identical IS611 a RFLP banding patterns, but tuberculosis patients who were unrelated to the outbreak had different strain typing patterns. This study was timportant because it validated the IS6110 RFLP strain typing method in a well-characterized outbreak setting prior to its application in other epidemiologic studies and public health settings. The study also demonstrated the speed

K. DeRiemer and C. L. Daley

with which tuberculosis can spread through an HIVinfected population and the importance of specific locations as sites of transmission. Molecular epidemiology studies have demonstrated the impact that a single patient can have on the epidemiology of a community. A single patient was directly or indirectly responsible for 6% of the tuberculosis cases in San Francisco, including those in the HIV residential care facility noted above (Daley et al. 1992). In another report, IS6110 RFLP analysis showed that a single homeless tuberculosis patient with highly infectious pulmonary tuberculosis likely infected 42% (41197) of contacts who were regular customers and employees of a neighborhood bar that he patronized and caused disease in 14 (34%) of these contacts. Isolates of M. tuberculosis were available for 12/14 of the tuberculosis cases; all had identical IS611 aRFLP band patterns (Kline et al. 1995). In many instances, outbreak investigations are able to detect and document unsuspected transmission. For example, an early study using genotyping ofM. tuberculosis documented the spread of infection and the development of disease to specific individuals who mixed with marginalized drug users, mainly restaurant workers (Genewein et al. 1993). Transmission links between individuals with only casual contact were detected. The study elucidated the details and dynamics of a community sub-epidemic of tuberculosis in a way that went beyond traditional contact tracing and routine surveillance, and facilitated public health interventions designed to limit the tuberculosis transmission. Molecular epidemiologic techniques have confirmed suspected and unsuspected tuberculosis transmission in places such as crack houses (Leonhardt et al. 1994), sites of illegal floating card games (Bock et al. 1998), schools (Kim et al. 2001; Bauer et al. 2000), hospitals (Edlin et al. 1992; Haas et al. 1998), and clinics. Genotyping has been used in several tuberculosis outbreaks in prisons to identify the magnitude of transmission among jail inmates and prisoners, among inmates and guards, and from persons in the jail or prison to members of the community (Ferreira et al. 1996; Chaves et al. 1997; March et al. 2000; Hanau-Bercot et al. 2000; Mohle-Boetani et al. 2002). Tuberculosis transmission has also been demonstrated among groups such as church choirs (Mangura et al. 1998), interstate transgender social networks (Anonymous 2000b), and renal transplant patients (Jereb et al. 1993), as well as from patient to health care provider (Wilkinson et al. 1997) and from health care providers to patients (Frieden et al. 1996a,b; Ikeda et al.1995). Some quite unusual sources of tuberculosis transmission have been confirmed with molecular epidemiologic methods. For example,

The Molecular Epidemiology of Tuberculosis

65

processing contaminated medical waste resulted in transmission of M. tuberculosis to at least one medical waste treatment facility worker (Johnson et al. 2000). Genotyping was also used to document unsuspected bronchoscopy-related transmission and the cross-contamination ofpatients (Agerton et al.1997; Michele et al. 1997; Ramsey et al. 2002). In some instances, molecular typing may reveal outbreaks caused by more than one strain of M. tuberculosis and therefore, more than one likely source of infection. In other situations, molecular typing can effectively rule out an outbreak and thus avoid an expensive epidemiologic investigation and inappropriate interventions. Because of its usefulness, genotyping of M. tuberculosis has become a routine tool for outbreak investigations in many hospitals and health departments.

5.4.2 Community Epidemiology and Tuberculosis Control Tuberculosis results from rapid progression from a recently acquired infection, reactivation of LIBI, or

occasionally from exogenous reinfection (Small and Fujiwara 2001). Most molecular epidemiology studies have assumed that the proportion of clustered isolates in a population reflects the amount of recent or ongoing transmission of M. tuberculosis. Some studies established epidemiological links between clustered tuberculosis cases, inferring that the tuberculosis cases are part of a chain of transmission from a single common source or from several common sources (Genewein et al. 1993; Small et al. 1994). The number and proportion of tuberculosis cases that are in clusters, representing recent or ongoing tuberculosis transmission, have varied from study to study (Table 5.1). The frequency of clustering has varied from 17-18% in low incidence areas such as Vancouver, Canada and Norway to 30-40% in urban areas in the United States. Among gold miners in South Africa, 50% of tuberculosis patients were found to be in clusters. However, it is difficult to make meaningful cross-study comparisons because the studies differ in a number of important ways, including the population studied, the proportion of all tuberculosis cases studied, the duration of the study, the definition of clustering, and the method of secondary typing used.

Table 5.1. Frequency of clustering and risk factors for clustering in selected studies Study (period)

Location

Study population

N

Ever Risk factors for clustering clustered

Small et al. 1994 (1991-1992) Alland et al. 1994 (1989-1992)

San Francisco, USA

Community-based

473

40%

New York City, USA Hospital-based

104

38%

Bishai et al. 1998 (1994-1996) Bauer 1998 (1992-1996) van Soolingen et al. 1999 (1993-1997)

Baltimore, USA

182

46%

Acquired immune deficiency syndrome US-born HIV-seropositive Hispanic ethnicity Younger age Drug-resistant tuberculosis Low income Intravenous drug use

Community-based

Denmark, including Country-based Greenland Netherlands Country-based

1549

49%

Analysis not performed

4266

46%

Godfrey-Faussett et al. 2000 (1995) Dahle et al. 2001 (1994-1998) Hernandez-Garduno et al. 2002 (1995-1999)

South Africa

Gold miners

419

50%

Norway

Country-based

698

18%

Male gender Urban residence Dutch and Surinamese nationality Long term residence in Netherlands Treatment failure Time spent working in mines Analysis not performed

Vancouver, Canada

Community-based

793

17%

Diel et al. 2002 (1997-1999)

Hamburg, Germany Community-based

423

34%

Lockman et al. 2001 (1997-1998)

Botswana

301

42%

Community-based

Canadian-born aboriginals Canadian-born non-aboriginals Injection drug users Alcohol abuse History of contact tracing Unemployment Imprisonment

66

K. DeRiemer and C. L. Daley

The proportion and rate of clustering has been interaction between years of residence in the United used as an indicator to assess the performance of States and HIV infection; tuberculosis among foreigntuberculosis control programs. In an evaluation of born persons was more likely to result from recent tuberculosis transmission over a seven-year period in transmission among those who were HIV-infected San Francisco, the number and proportion of clustered and more likely to result from the reactivation of tuberculosis cases declined, particularly among the LTBI among those who were not infected with HIV US-born population. This was attributed to the imple- (Geng et al. 2002). The investigators recommended mentation of targeted tuberculosis prevention and that tuberculosis prevention and control strategies control programs such as screening high-risk popu- need to be targeted to the large number of foreignlations and the implementation of directly observed born persons in New York City who have LTB!. therapy to ensure high cure rates (Jasmer et al. 1999). The factors associated with clustering may be A recent study in New York City showed that as tuber- complex. For example, in a study of South Africa gold culosis case rates fell from recent high levels, the pro- miners, tuberculosis patients who had failed treatportion of cases caused by clustered isolates (i.e., cases ment at entry to the study were more likely to be in of tuberculosis due to recent transmission) decreased. clusters (adjusted OR = 3.41,95% confidence interval The proportion of tuberculosis cases caused by recent CI 1.25-9.27), and patients with multidrug-resistant transmission dropped from 63.2% in 1993 to 31.4% in tuberculosis were more likely to have failed tuberculo1999 (Geng et al. 2002). They also reported that tuber- sis treatment but less likely to be clustered than those culosis was unlikely to result from recent transmission with a drug-susceptible strain (OR = 0.27, 95% CI in persons born outside the United States. By contrast, 0.09-0.83). HIV seropositivity was common (53.6%) an 8-year study in Greenland showed that the annual but was not associated with clustering. Apparently, incidence of tuberculosis increased from 85/100,000 persistently infectious individuals who had previously in 1990 to 172/100,000 in 1997 and the percentage of failed treatment were responsible for one third of the culture positive tuberculosis cases in RFLP clusters tuberculosis cases in this population. In addition, the increased to 85%, reflecting micro-epidemics among World Health Organization's targets of70% case detecadults and young children in small, isolated settle- tion and 85% cure (World Health Organization 2002) ments (Soborg et al. 2001). were not sufficient to interrupt tuberculosis transmisConventional epidemiological methods can be used sion; indicators that are more closely linked to the in combination with molecular typing techniques to rate of ongoing tuberculosis transmission, are needed identify the risk factors associated with recent infec- (Godfrey-Faussett et al. 2000). tion and rapid progression to disease (Table 5.1). Some of the risk factors that are associated with recent infection are specific to a particular community, 5.4.3 whereas others are common to tuberculosis patients Exogenous Reinfection with M. tuberculosis in different geographical areas. For example, young age, being in an ethnic minority group, homelessness, Molecular typing can determine whether a patient and substance use have been identified in more than with a recurrent episode of tuberculosis has a relapse one study as being associated with recent infection with the previous strain of M. tuberculosis or exog(Small et al. 1994; Alland et al. 1994; Friedman et al. enous reinfection with a new strain (Table 5.2). In one 1995b; Barnes et al.1997; van Deutekom et al.1997). In of the earliest studies using genotyping to examine San Francisco, for example, among persons less than reinfection, Godfrey-Faussett and colleagues reported 60 years of age, Hispanic ethnicity (odds ratio OR = that the IS6110 RFLP band patterns from patients in 3.3, P = 0.02), black race (OR = 2.3, P = 0.02), birth in Malawi were different in pairs of isolates from four the United States (OR=5.8, P <0.001) and a diagnosis patients. The isolates were from different body sites of acquired immune deficiency syndrome (OR = 1.8,P in two patients, and from different episodes in the = 0.04) were independently associated with being in a other patients (Godfrey-Fausset and Stoker 1992). Since then, several groups have shown that exogcluster (Small et al.1994). In a recent study in New York City (Geng et al. enous reinfection can occur in both immunocom2002), birth outside the United States, age of 60 years promised and immunocompetent persons. More or older, and diagnosis after 1993 were factors inde- recently, researchers have tried to quantify the rate pendently associated with having a unique strain, at which exogenous reinfection occurs and assess while homelessness was associated with clustering its potential impact on tuberculosis transmission or recent transmission. There was also a significant dynamics. In Cape Town, South Africa, where there

67

The Molecular Epidemiology of Tuberculosis

Table 5.2. The frequency of exogenous reinfection in selected studies. NA not available, HIV+ Seropositive for the human immunodeficiency virus (HIV), HIV- Seronegative for HIV, AIDS Acquired immune deficiency syndrome, TB Tuberculosis Study (period)

Study site

Study population

Small et aI. 1993a (1987-1991)

Kings County Hospital, New York City

6

0

Godfrey-Faussett et aI. 1994 (1989-1991) Das et aI. 1995

Nairobi, Kenya

AIDS patients with positive culture 17 for> 1 year or Increasing drug resistance 31 TB recurrences NA

11 5

4 (36%) 1 (20%)

Gilks et aI. 1997 (1989-1992) Sudre et aI. 1999 (1988-1996) Van Rie et aI. 1999 (1992-1998) Chaves et aI. 1999 (1993-1994)

Nairobi, Kenya

Recurrence or isolated positive culture HIV + and HIV- women

30 32 4

30 32 4

11 (37%) 29 (91%) 2 (50%)

Switzerland

HIV cohort with two isolates

20

20

2 (10%)

Cape Town, South Africa Madrid, Spain

Recurrent TB

48

16

12 (75%)

11

9

2 (22%)

Sonnenberg et aI. 2000 (1995) Lourenc;o et aI. 2000 (1990-1994) Caminero et aI. 2001 (1991-1996) Fitzpatrick et aI. 2002 Bandera et aI. 2001 (1995-1999)

Gauteng Province, South Africa Rio de Janeiro, Brazil

HIV infected Spanish inmates who remained culture positive for >4 months HIV + and HIV- gold miners

57

48

2 (4%)

12

12

3 (25%)

23

18

8 (44)

de Viedma et aI. 2002 (1988-1999) Kriiiiner et aI. 2002 (1994-1999)

Madras, India

Patients with two episodes of tuberculosis or two isolates

Patients Patients with with reinfection genotyping

Gran Canaria Island, Spain Kampala, Uganda;

HIV + patients with multiple isolates Two positive cultures> 12 months apart HIV + and HIV - TB recurrences

NA

40

9 (23)

Lombardy, Italy

Recurrent TB separated >6 months NA

32

5 (16)

Madrid, Spain

HIV + and HIV - cases with two isolates> 100 days apart Treatment failures

172

43

14 (33)

35

11

11 (100)

Tartu, Estonia

is a high incidence of tuberculosis and ongoing transmission, 16 of 698 patients had more than one episode of tuberculosis of whom 75% (12/16) had pairs of isolates of M. tuberculosis with different IS6110 -based genotyping patterns (van Rie et al. 1999). Episodes of tuberculosis reinfection in areas with a low incidence of tuberculosis, such as the Netherlands (de Boer and van Soolingen 2000), the Canary Islands (Caminero et al. 2001), and Northern Italy (Bandera et al. 2001), are uncommon compared with those in high-incidence regions. In the study in Northern Italy, a four-fold increased risk for reinfection was observed among immigrant patients compared with Italian subjects. In contrast, a higher risk of relapse rather than reinfection was evidenced in HIY-positive subjects and in patients infected with multidrug-resistant tuberculosis. A person can be simultaneously infected with more than one strain of M. tuberculosis. Multiple infections were demonstrated in a patient in San Francisco, California (Yeh et al. 1999), in two patients who worked in

a medical-waste processing plant in Washington State (Braden et al. 2001) and among prisoners in Spain (Chaves et al. 1999). These observations indicate that simultaneous infections with multiple strains of M. tuberculosis occur in immunocompetent hosts and may be responsible for conflicting drug-susceptibility results. Knowing whether mixed infection is occurring is important because the susceptibility patterns of the two strains may be different and different treatment regimens may be needed (Niemann et al. 2000). In addition, some cases of suspected exogenous reinfection may be due to initial infections that include more than one strain.

5.4.4 Geographical Distribution and Dissemination

of M. tuberculosis

Surveillance networks incorporating conventional and molecular epidemiologic techniques were estab-

68

lished during the 1990s by the Centers for Disease Control and Prevention (CDC) and the European community to track long-distance dissemination of tuberculosis and to help suggest rational tuberculosis prevention and control strategies to interrupt such transmission (Glynn et al. 1999a; Castro and Jaffe 2002; Crawford et al. 2002). Several of the strains identified in outbreaks have been associated with large clusters that are widely dispersed both geographically and temporally, possibly because they are either more transmissible or they are more likely to cause disease once transmitted than are other strains (Murray and Nardell 2002b). In an early comparison of the strains present in New York City and San Francisco, California, USA, the most widely prevalent strain of M. tuberculosis from New York City was isolated from only one of 755 patients in San Francisco-a traveling salesman (Casper et al. 1996). A comparison of the strains of M. tuberculosis from San Francisco and the East Bay, two distinct regions separated by the San Francisco Bay, only 53 of 724 (7.3%) had genotype patterns that matched at least one isolate from the other region. Of 375 isolates from San Francisco with unique band patterns, only nine (2.4%) matched patterns of East Bay isolates. These population-based data suggest that in the San Francisco Bay Area, M. tuberculosis does not rapidly spread across geographic boundaries and tuberculosis control should focus on transmission within defined areas (Bradford et al. 1998). The most widely reported example of the geographical dissemination of a particular strain of M. tuberculosis is that of the "Beijing" and "W strains" (Bifani et al. 2002; Glynn et al. 2002) A multidrugresistant strain of M. tuberculosis called the "Wstrain" caused many cases of tuberculosis and deaths attributable to tuberculosis among patients and healthcare workers in nosocomial outbreaks and in other institutional settings in New York City during the 1990s (Anonymous 1993; Bifani et at. 1996). This same strain was later found in other parts of the United States (Agerton et al. 1999). By the late1990s, the "w strain" was recognized as a member of the "Beijing family" of strains. A study performed in Beijing, China in 1995 reported that 85% of the isolates were strains with greater than 66% similarity among their IS6110 RFLP patterns (van Soolingen et al. 1995). This "Beijing family" of strains was also detected in high proportions among strains in other parts of Asia (Anh et al. 2000), the former Russian Federation (Kurepina et al. 2000), Estonia (Kriiiiner et al. 2001), Latin America (Diaz et al. 1998), and Europe (Niemann et al. 2000).

K. DeRiemer and C. 1. Daley

Beijing strains, including the "w strain" and its variants, have an insertion of IS611 0 in the genomic dnaA-dnaN locus (Kurepina et al. 1998). Based on several early technical studies and a review of 16 studies of the Beijing or W strains that gave results on their spoligotyping, the W family and Beijing family strains have an identical, characteristic spoligotype based on DNA polymorphism in the direct repeat region that only contain spacers 35-43 (van Soolingen et al. 1995; Bifani et al. 1999; Sola et al. 1999; Soini et al. 2000; van Crevel et al. 2001; Glynn et al. 2002). Because this family of strains is so widespread and is often drug-resistant, there is concern that these strains are biologically hyper-mutable, hyper-virulent, and have a predilection for acquiring drug resistance. But as of this writing, comparable data are not available for other, Widespread strains. There may be a link between geographic location and the number of IS6110 copies. In low incidence areas with a high proportion of tuberculosis cases attributable to reactivation of LTBI and immigration (e.g. the Netherlands, San Francisco, Switzerland, and Norway), the IS6110 RFLP patterns are highly polymorphic. But in some areas of Asia, a high proportion of the isolates of M. tuberculosis contain no copies or very few copies ofIS611O. Specific clones or families of clones may be predominant in a specific geographical area. For example, two distinct populations of isolates were detected in Guinea-Bissau, a former Portuguese colony, one West African and the other European (Kallenius et al.1999). In the western Pacific region, the isolates from eight countries could be divided into three groups based on the mode of the copy number (Park et al. 2000). In Tunisia, 62% of the isolates belong to three genotype families that share greater or equal to 65% similarity in the IS611 0 RFLP patterns and in Ethiopia, 52% of the isolates belong to four genotype families (Hermans et al. 1995). As noted in the studies described above, there can be significant geographic variation in IS611 O-based genotype patterns. Therefore, it will be important to evaluate the geographic variation in new typing systems to determine their utility in different regions and populations.

5.4.5 Evaluation of Laboratory Cross-contamination Although laboratory cross-contamination of M. tuberculosis isolates was demonstrated during the 1980s using bacteriophage typing (Jones 1988), genotyping has become the most widely used tool for determin-

The Molecular Epidemiology of Tuberculosis

69

ing whether or not laboratory cross-contamination sary, and potentially toxic treatment for the patient, and has occurred. Careless specimen processing, contami- increased costs and burdens for both patients and local nated reagents (de C Ramos et al.1999), and contami- health care providers (Burman et al.1997a; Burman and nation with H37Ra, an avirulent reference strain of M. Reeves 2000; Northrup et al. 2002). tuberculosis that is used for standardization of laboratory procedures, can cause laboratory cross-contamination. A small but important proportion of cases with laboratory cross-contamination were detected 5.5 in every institution that looked for it (Anonymous The Future of Molecular Epidemiology 2000a; Perfecto et al. 2000; Breese et al. 2001; Chang et al. 2001; Jasmer et al. 2002). As a result, some settings Molecular genotyping, in combination with convenroutinely use genotyping to evaluate specimens for tional epidemiologic investigations, will continue to be possible laboratory cross-contamination. used to detect laboratory contamination, to describe Early investigations focused on the isolates of disease outbreaks, and to identify previously unsusM. tuberculosis that were processed together in the pected chains and locations of tuberculosis transmislaboratory and had identical IS6110 RFLP patterns, sion in the community. In addition, molecular epidebut were from at least one otherwise asymptomatic miologic methods will continue to play an important patient (Small et al. 1993b; Dunlap et al. 1995). In role identifying appropriate public health intervenNew York City, an isolate was collected from every tions and measuring their impact. The development of patient with a positive culture for M. tuberculosis real-time amplification based genotyping techniques during a I-month period, including both incident will likely improve our ability to do effective, timely and prevalent cases, and IS61 10 RFLP analyses were contact and outbreak investigations. performed. Of the 441 patients, 3% had clinical, laboRecent sequencing of the genome of M. tuberratory, and RFLP evidence of falsely positive cultures culosis has demonstrated that the IS6110 element for M. tuberculosis (Frieden et al. 1996b). The geno- is not distributed at random in the genome of the typing of all M. tuberculosis isolates from a I-year tubercle bacillus (McHugh and Gillespie 1998). The period in a 700-bed urban hospital in Chicago, USA, insertion sequence, and thus the number of RFLP revealed only one possible instance of nosocomial bands, is significantly more likely to be present at transmission and five (2.7%) false-positive M. tuber- some positions than would be expected by chance culosis cultures from 183 patients (French et al.1998). alone. This may interfere with the epidemiological Among isolates collected prospectively over 5 years interpretation of relatedness of strains. Improved, from a municipal health department laboratory, four more precise molecular typing techniques based on percent (8/199) of the culture-positive patients were specific genomic phenomenon such as point mutations, polymorphisms, and deletions will likely to be identified as probable or definite false-positives. Laboratory cross-contamination should be sus- developed in the near future. pected when there is a single positive culture in a cliniCurrent molecular epidemiologic approaches will cally well patient with negative acid fast bacillus (AFB) soon intersect with developments in mycobactesmears and no other evidence of tuberculosis. If the iso- rial genomics and other disciplines in many excitlate was cultured within one week of another specimen ing ways. The availability of the complete genome and has an identical genotype pattern, then laboratory sequence of M. tuberculosis now makes it possible cross contamination is likely. Laboratory cross-contam- to use a variety of typing techniques that can help ination should also be suspected when there is a sudden distinguish between different species and clonal increase in culture positive isolates without an epidemi- groupings of strains with specific phenotypic charological or clinical explanation. Published studies have acteristics such as transmissibility, pathogenicity, used IS6110 RFLP typing, VNTR typing (Gascoyne- or resistance to antimicrobial agents. Genome-wide Binzi et al. 2001), or spoligotyping (Nivin et al. 2000) analyses using techniques such as microarrays for to detect and evaluate laboratory cross-contamination. gene expression profiling or the detection of genomic Regardless of the typing method used, investigations deletions can be used to evaluate specific strains and of possible cross-contamination are based on good to determine the genetic basis of important phenocommunication between the laboratory and clinicians. typic traits. As molecular typing techniques improve, When laboratory or inter-patient cross-contamination molecular epidemiology will become an increasing occurs, it can result in an incorrect diagnosis, unneces- important tool in the prevention and control of sary clinic visits and consultations, lengthy, unneces- tuberculosis, globally.

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6

Molecular Epidemiology of Mycobacterium bovis ROBIN A. SKUCE and SYDNEY D. NEILL

CONTENTS 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11

Introduction 75 Bovine Tuberculosis 75 Epidemiology in Developed Countries 76 Epidemiology in Developing Countries 77 Epidemiological Typing 78 The Genetics of the M. tuberculosis Complex Bacteria 78 Strain Typing Methodology 79 Standardisation and Method Performance Evaluation 84 M. tuberculosis Molecular Epidemiology: Lessons Learned 85 M. bovis Molecular Epidemiology: Studies and Trends to Date 85 Conclusions 88 References 89

that includes farmed and feral animals, wildlife and also humans (O'Reilly and Daborn 1995). Its epidemiological pattern in developing and developed countries can be very complex. It is classified as a List B disease by the Office International des Epizootiques (OlE) and considered of socio-economic importance as well as public health significance, as it impacts significantly on the international trade of animals and animal products and hence locallivelihoods (Cousins 2001; Cousins and Roberts 2001).

6.2 Bovine Tuberculosis

Bovine tuberculosis is a chronic infection of animals, particularly cattle, and is caused by the tubercle bacillus Mycobacterium bovis. It is usually characterised by formation of nodular granulomas or tubercles, 6.1 particularly in the lungs and in the lymph nodes of Introduction the respiratory tract but lesions also may occur in In 1998 the World Health Organisation (Cosivi et al. the mesenteric lymph nodes, liver, spleen and other 1998) estimated that the incidence of human tuber- organs. Presentation of the disease is variable with culosis would be 88 million, resulting in 30 million an absence of clinical signs in less advanced infecdeaths for the period 1990-1999 and the majority tion, often the case in the majority of infected cattle of cases would be in developing countries. In these in developed countries with regular testing strategies countries, tuberculosis, caused by Mycobacterium (Neill 1994). It may also present non-specifically, with bovis, is present in animals. However, surveillance clinical signs such as weakness, anorexia, emaciation and control programmes for animal tuberculosis in and coughing in advanced cases. In most countries these countries are often inadequate or non-existent. diagnosis of tuberculosis in cattle is usually by tuberConsequently, the epidemiology of M. bovis in public culin skin testing, measuring delayed-type hypersensihealth issues remains largely unknown (Cosivi et al. tivity. However, in countries where prevalence of infec1998). M. bovis has an exceptionally broad host range tion is extremely low or freedom from infection has been declared, meat inspection alone is employed for diagnosis and surveillance. More recently, application of assays for bovine interferon-y has been employed R.A.SKUCE,BSc,PhD to indicate infection (Neill et al. 1994). Supporting Veterinary Sciences Division, Stoney Road, Stormont, Belfast, laboratory confirmation using bacteriological culture BT4 3SD, Northern Ireland techniques is also often employed. S. D. NEILL, BSc, PhD Bovine tuberculosis still can have serious, adverse, Veterinary Sciences Division, Department of Agriculture and financial consequences in agricultural regions of the Rural Development, Stoney Road, Stormont, Bellfast BT4 3SD, UK world (Bennett et al.1999; Cousins 2001; Cousins and M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

76

Roberts 2001; Goodchild and Clifton-Hadley 2001) and is considered a potential human health risk in some countries (Cosivi et al. 1998). Farmers incur financial losses from decreases in meat and milk production, carcass condemnations and restrictions on animal movement. Bovine tuberculosis presents a serious barrier to cattle trade within and between countries and there is considerable expenditure, usually by governments, on testing and compensation. Most European countries, through the introduction of voluntary and subsequently compulsory eradication programmes, experienced dramatic success in reducing the incidence of bovine tuberculosis. However, despite initial progress, eradication has not yet been achieved in all countries (European Commission Report 1998). Some European countries, e.g., the United Kingdom, have considerable regional variability with sporadic outbreaks, particularly in South-West England and Northern Ireland. Mycobacterium bovis infection in humans may result from ingestion of contaminated milk, inhalation via aerosols or through contact with infected clinical material. Pulmonary disease in humans, due to M. bovis is clinically, radiologically and pathologically indistinguishable from pulmonary tuberculosis caused by M. tuberculosis (O'Reilly and Daborn 1995). However, transmission of M. bovis from human to human is considered much less efficient than such transmission of M. tuberculosis (van Soolingen 2001). Evidence for transmission of M. bovis between humans is considered rare and largely anecdotal and equally rare are reports of humans infecting cattle (O'Reilly and Daborn 1995). Mycobacterium bovis tuberculosis was once common in children, affecting the cervical lymph nodes, the intestinal tract or the meninges, as a result of feeding infected cows milk. By 1937 up to 25% of tuberculosis cases in the USA and UK were due to M. bovis infections (Cousins 2001). The introduction of pasteurisation for milk and milk products, together with abattoir meat inspections significantly reduced this incidence and bovine tuberculosis in children is currently negligible. It was the recognition of the public health risk that prompted the introduction of control and eradication programmes for bovine tuberculosis in many countries in the early part of the last century (Cousins 2001). The success of such schemes has resulted in a low prevalence, or absence, of M. bovis infection in most national cattle herds, particularly in countries with advanced agriculture practices. This was reflected in a dramatic drop in the incidence of human tuberculosis due to M. bovis. However, the zoonotic nature of M. bovis still places

R. A. Skuce and S. D. Neill

farmers, abattoir workers and veterinarians at risk (Grange 2001).

6.3

Epidemiology in Developed Countries

In developed regions of the world, several countries have been unable to eradicate this disease, despite rigorously implementing comprehensive and costly strategies. The primary route of infection for cattle is respiratory but alimentary infection, cutaneous, congenital and genital routes have been recorded (Neill 1994). Social behaviour of cattle in an environment of intensive farming and a high degree of live cattle trade, together with the potential of M. bovis survival on pasture and inanimate objects contribute to exposure and facilitate subsequent spread of infection (Morris et al. 1994). Many countries, previously endemic for bovine tuberculosis, have now been declared tuberculosis-free, following intensive test and slaughter policies (Cousins and Roberts 2001). However, where there is interplay between infected wildlife and domestic animals, control has proven problematic. It is now evident that better understanding of this disease and its epidemiology is essential to overcome the impeded progress in some countries. Postulations on the residue of infection have included deficiencies in skin test specificity and sensitivity, poor cattle testing and the presence of reservoirs of M. bovis in cattle and wildlife. Traditional farming practices with significant cattle movement, intensive farming with high stocking densities and poor boundary fencing between farms allowing cattle 'nosing' between neighbouring farm herds have also been cited (Neill 1994; O'Reilly and Daborn 1995). In Ireland, inter-bovine spread of tuberculosis was identified as the single most important source of infection (Neill 1994).However,wildlife reservoirs are increasingly being considered a significant risk and responsible for protracted eradication programmes (Cheeseman et al. 1989; Livingstone 1992; Gallagher and Clifton-Hadley 2001). In the United Kingdom and Ireland, badgers (Meles meles) and feral deer (Cervus elaphus) are recognised wildlife reservoirs of M. bovis (Cheeseman et al. 1989; Hughes and Rogers 1994; Gallagher and Clifton-Hadley 2001). Badgers are indigenous and a protected species in the United Kingdom and Ireland with estimates in Great Britain of approximately 250,000 badgers, in Northern Ireland 30,000 (Hughes and Rogers 1994)

Molecular Epidemiology of Mycobacterium bovis

and 250,000 in the Republic of Ireland. The contribution of wildlife to tuberculosis in cattle in the United Kingdom and Ireland is not known precisely and this can be controversial with regard to the badger. One epidemiological study of risk factors for tuberculosis in dairy herds in Northern Ireland suggests that, although infected cattle may playa significant role in the transmission of tuberculosis, the importance of badgers may have been underestimated (Denny and Wilesmith 1999). In New Zealand, in marked contrast to Australia, tuberculosis in cattle is also exacerbated by a wildlife reservoir and maintenance host, the possum (Trichosurus vulpecula). Endemic infection in possums constitutes a major obstacle to disease control and eradication in some areas (Morris et al. 1994; de Lisle et al. 2001). Globally,M. bovis has been estimated to contribute 3.1 % of all human tuberculosis cases (2.1 % of pulmonary tuberculosis and 9.4% of extra-pulmonary tuberculosis) (Cosivi et al. 1998). In developed countries, such cases are now relatively rare and estimated at less than 1% of all tuberculosis cases, and are probably due to reactivation of dormant lesions amongst the elderly (Grange 2001). M. bovis has been isolated from HIV-infected individuals in some developed countries, with an additional serious complication of high primary resistance to isoniazid, streptomycin and pyrazinamide (Guerrero et al. 1997). Although M. bovis is thought to be less virulent and also less transmissible in humans than animals, human-to-human transmission of multi-drug resistant (MDR) M. bovis strains, over a relatively short interval, was demonstrated in Paris. HIV-induced immunosuppression may lower host immune responses and lead to overt disease more rapidly than under normal conditions. The true incidence of M. bovis infection in humans appears to be underreported, probably owing to the similar clinical presentation to M. tuberculosis infection but also to the fact that many diagnostic laboratories often would not have differentiated M. tuberculosis and M. bovis isolates.

6.4 Epidemiology in Developing Countries On the whole, there is very limited epidemiological information on bovine tuberculosis in cattle in the developing world (Cosivi et al.1998). Most cattle are in regions where no formal control measures are applied and implementation of a recognisable testand-slaughter policy would be considered excep-

77

tional. Information on M. bovis prevalence in other species is also very limited. In a study of 56 African countries, 33 provided information about M. bovis infection in animals. However, few reported M. bovis infection in humans (Kazwala et al. 2001a,b). The M. bovis cases reported in some developing countries may just be the tip of the iceberg, as, for example in Tanzania, medical authorities do not recognise M. bovis as a risk to human health. There has also been a lack of interest amongst medical practitioners and microbiologists in requesting speciation after primary isolation of mycobacteria from patients. In many developing countries there is little or no pasteurisation of milk or milk products and many people live a pastoral lifestyle, in close contact with their cattle, with resultant exposure to tuberculosis where present. Milk-borne infection is the main cause of non-pulmonary tuberculosis in areas where bovine tuberculosis is common and uncontrolled (Daborn et al. 1996). In Africa, approximately 85% of cattle and 82% of humans live in areas where bovine tuberculosis is only partially controlled (Cosivi et al. 1998). Approximately 90% of the total milk produced in Africa is consumed fresh or soured. Therefore, the consumption of unpasteurised milk, contaminated by M. bovis, is a likely route of zoonotic tuberculosis transmission to humans. In the remaining regions of these countries, control programmes operate only partially. It is evident that in Africa, Asia and Latin America/ Caribbean, significant human populations work in close physical contact with animals, and therefore zoonotic tuberculosis represents a significant, but in reality, unquantified risk. The global HIV epidemic adds a further complication. Tuberculosis is a major opportunistic infection, if not the most common infection, for HIV-infected individuals, the majority of whom live in developing countries. In those countries in which M. bovis infection is present in animals and where favourable conditions exist for zoonotic transmission, zoonotic tuberculosis is a serious public health threat. Zoonotic tuberculosis in HIV-positive individuals also resembles M. tuberculosis infection. WHO estimate that 70% of people (6.6 million) co-infected with tuberculosis and HIV live in sub-Saharan Africa (Cosivi et al.1998). In African countries, M. bovis infects a higher proportion of exotic dairy breeds (Bos taurus) than crossbred beef cattle and indigenous zebu cattle (Bos indicus) (Cosivi et al. 1998). In intensive conditions, a death rate from tuberculosis of up to 60% in zebu cattle has been recorded, with animal crowding con-

78

tributing to disease transmission. Tuberculosis control and eradication strategies, although understood, are inconsistently applied and usually not sustained in developing countries, largely due to lack of finance and trained professionals. Therefore there is obviously an increased likelihood of underestimation of zoonotic tuberculosis, the full economic implications of which is often overlooked.

R. A. Skuce and S. D. Neill

6.6 The Genetics of the Mycobacterium tuberculosis Complex Bacteria

The M. tuberculosis complex (Rastogi et al. 2001) comprises M. tuberculosis, M. africanum, M. bovis, M. microti and M. bovis BCG (Behr 2001), as well as newly characterised bacteria M. canetti (van Soolingen et al. 1997b) and M. caprae (Aranaz et al. 1999). Isolates with characteristics intermediate between M. tuberculosis and M. bovis have also been reported (Kallenius et al. 1999). The bacteria in the 6.5 Epidemiological Typing complex are usually regarded as subspecies and are characteristically 99.9% similar at the nucleotide Advances in molecular technologies have enabled level, with identical16S rRNA sequences (Brosch et specific identification and subsequent recognition al. 2002). However, there are distinct phenotypic difof strains of many pathogens involved in animal and ferences between the 'subspecies', not least their host human infections (van Belkum et al. 2001; van Belkum range and pathogenicity. M. bovis has the largest host 2002). 'Molecular epidemiology' is the integration of range within the M. tuberculosis complex, infecting these techniques with conventional epidemiological many mammalian species, including man (O'Reilly approaches for understanding better disease distri- and Daborn 1995), whereas M. tuberculosis is almost bution in populations (Small and van Embden 1994). exclusively a human pathogen. The M. tuberculosis complex genome sequencing Such studies are often designed to allow inference of patterns of disease transmission from similarities projects represent significant landmarks (Cole et al. between genetic patterns generated for infectious 1998). The sequencing projects,M. tuberculosis strain pathogens (Salamon et al. 2000). Using such genetic H37Rv and M. bovis strain AF2122/97 at the Sanger marker-assisted molecular epidemiology, it is now Centre (http://www.sanger.ac.uk. Cambridge, UK) possible to differentiate reproducibly between strains and M. tuberculosis strain CSU93 at The Institute of micro-organisms and to show spatial and temporal for Genome Research (http://www.tigr.org) have aspects of transmission, for example, between ani- provided a hugely valuable research resource and a mals. Epidemiological typing systems are generally unique insight into the biology of these major pathoused to study population dynamics and the spread gens. This should lead to major advances in diagnosof bacteria that undergo clonal reproduction both in tic, vaccination and chemotherapeutic options. The nature and in the clinical setting and often include M. tuberculosis H37Rv genome is relatively large for studies in bacterial population genetics and patho- an obligate pathogen, reflecting the complex biogenesis, providing 'early warning' through local, chemistry of its intracellular lifestyle and providing regional or national surveillance and in outbreak an insight into its origins. The genome is GC rich and it contains several different mobile genetic elements, investigations (Streulens 1996). Typing systems are applied primarily to assist epi- such as insertion sequences and prophages, as well demiological studies and in generating hypotheses as different classes of repetitive DNA (Gordon et al. 2001). about: Until very recently it was thought that M. tuber• the extent of epidemic spread of microbial culosis evolved from M. bovis, by adaptation of an clone(s) in an exposed population; • the number of clones involved in transmission animal pathogen to the human host. Recent studies have provided compelling genetic evidence to sugand infection; gest that the members of the M. tuberculosis complex it the identity of the source(s) of contamination and shared a common ancestor, which was likely to have vehicles of transmission; • the identification and monitoring of reservoirs of resembled M. tuberculosis or M. canetti and was epidemic clone(s) in the population and/or the probably already a human pathogen (Brosch et al. 2002). Comparative genomics has shown that the environment; M. bovis genome is smaller than M. tuberculosis, G the evaluation of the efficacy of control measures aimed at containing or interrupting the spread of which is intriguing due to its wider host range. This comparative approach has also identified a series of epidemic clone(s).

Molecular Epidemiology of Mycobacterium bovis

insertion and deletion events between members of the complex (Gordon et al. 1999a), whose distribution points to a common ancestor and has been used to derive an entirely new evolutionary scenario. M. bovis now appears to be the final member of a separate lineage that branched from the progenitor of M. tuberculosis and is represented by M. africanum, M. microti and M. bovis isolates having undergone successive loss of DNA segments. This may have contributed to clonal expansion and the selection of more successful pathogens in some hosts. This new data is inconsistent with the view that the proposed M. africanum - M. microti - M. bovis lineage could have spread and adapted to their wide host range within the suggested 15,000-20,000 years of speciation of the M. tuberculosis complex.

6.7

Strain Typing Methodology Phenotypic procedures based on physiological or biochemical characteristics and antibiotic susceptibility are often capable of discriminating between strains of micro-organism. However, this has not been the case for the M. tuberculosis complex (Saunders 1995) and in 1965 a WHO working group was established to facilitate development and standardisation of methods for typing mycobacteria. Phage susceptibility typing rapidly became a valuable epidemiological tool, which yielded data that contributed to the understanding of tuberculosis transmission and pathogenesis and provided the basis for current approaches to human tuberculosis control. However, the method is relatively crude and much greater discrimination can be achieved using molecular methods, which now supersede phage typing. The molecular epidemiology of M. tuberculosis in humans is much more advanced than that of M. bovis in cattle, largely because a robust and standardised strain typing method has been applied widely (van Soolingen 2001). Most M. bovis isolates are not amenable to this technique. Therefore, alternative methods have had to be developed and evaluated. The availability of genome sequences has led to the development of a generation of new molecular markers, which have the potential to offer high-resolution genotyping and high-throughput analysis. Collins and colleagues at AgResearch, Wallaceville, New Zealand pioneered bacterial DNA restriction enzyme fragment analysis, or restriction enzyme analysis (REA), as the earliest typing method applied

79

successfully to the organisms of the M. tuberculosis complex (Collins and de Lisle 1984, 1985). In REA, DNA extracted from batch cultures, using detergent and protease digestion followed by solvent extraction, is digested with each of three restriction enzymes (BstEII, Pvull and Bell), prior to electrophoretic separation (Fig. 6.1). Ethidium bromide staining of DNA fragments generates a complex DNA banding pattern, similar to a bar-code fingerprint, with observable differences in the fragment pattern, especially in the higher molecular weight fragments. This produced a highly discriminating typing system for the M. tuberculosis complex, most notably for M. bovis. Approximately 2000 M. bovis isolates have been REA typed over the last 20 years, and more than 165 stable REA types have been identified (Collins 1998). However, the REA technique is not widely used, due to difficulties in accurately reproducing the complex fingerprint patterns and in naming, recording and databasing them. It is a relatively cumbersome technique and technology transfer and inter-laboratory comparisons have proven particularly difficult. A logical modification of the REA technique is pulsed field gel electrophoresis (PFGE), where rare-cutting restriction enzymes are used to digest genomic DNA immobilised in agarose blocks (Hughes et al. 2001). The resulting large DNA fragments are then separated by electrophoresis in a dedicated apparatus (PFGE Fig.6.1). The smaller number of larger DNA fragments generated significantly simplifies the electrophoretic patterns produced, although the discrimination is considerably less than with REA. PFGE of M. bovis isolates has produced moderate discrimination and has been useful in epidemiological studies (Feizabadi et al. 1996). Significant improvements have been made recently to the culture and preparation of samples for PFGE and this should allow better evaluation of method performance (Hughes et al. 2001). Researchers have also exploited the finding ofrepetitive DNA (Tanaka et al. 2000) within the genomes of M. tuberculosis complex bacteria to develop an array of genotyping techniques, based either on DNA amplification using the PCR or Southern blotting, or a combination of the two. There are basically two types of repetitive DNA in bacterial genomes: dispersed repeats and tandem repeats. The former can include mobile genetic elements, including insertion sequences (ISs) and prophages (Gordon et al. 1999b). M. tuberculosis H37Rv contains 56 such IS elements, belonging to well-defined families. Mobile genetic elements contribute significantly to genetic diversity. Whilst most of these ISs are located in the same genome position

80

R. A. Skuce and S. D. Neill

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Fig.6.1. Schematic representation of the main restriction enzyme digestbased genotyping methods applicable to the M. tuberculosis complex. Actual gel images for M. bovis field isolates are shown for PFGEl, REA2 and RFLP. (PFGE gel reproduced with permission from VM Hughes. REA gel reproduced with permission from DM Collins. PFLP gel images reproduced with permission from M Rogers)

(Heersma et al.1998). The strain typing of M. tuberculosis isolates by IS611O-RFLP is now an internationally accepted part of standard control policies, a tool for detecting point source outbreaks and a method for monitoring the efficacy of human tuberculosis control programmes (van Soolingen 2001). Unfortunately, for M. bovis isolates, the IS6110 copy number is much lower, often occurring singly, especially in isolates from cattle (Collins et al. 1993; Skuce et al. 1996; Cousins et al. 1998b). Interestingly, however, the IS6110 copy number is often higher in M. bovis strains isolated from more 'exotic' animal species (van Soolingen et al. 1994). Therefore additional probes are often required to improve discrimination for low IS611 0 copy number strains. One such probe is the 'polymorphic GC-rich repeat' sequence

Molecular Epidemiology of Mycobacterium bovis

(PGRS), which is found in multiple copies within the genomes of M. tuberculosis complex mycobacteria, although it is not considered complex-specific, unlike IS611O. PGRS-RFLP, using the 'Doran repeat' oligonucleotide probe (Doran et al.1993) to hybridise to AluI-digested genomic DNA, produces relatively complex patterns. Most of the visible band differences are in the higher molecular weight fragments. The M. tuberculosis complex specific Direct Repeat (DR) locus (see below) can also be used as a probe in RFLP analysis (Groenen et al. 1993). DR-RFLP, using a DR oligonucleotide probe to hybridise Aluldigested genomic DNA produces a relatively simple banding pattern. Several studies have used the combined results of IS6110-RFLP, PGRS-RFLP and DRRFLP to produce an overall RFLP typing scheme (Skuce et al. 1996; Costello et al. 1999). Genomic libraries have revealed promising new probes for RFLP analysis, such as pUCD (O'Brien et al. 2000a,b). A bioinformatics search shows that pUCD contains several polymorphic loci, some of which have been found by other investigators using entirely different approaches (Supply et al. 2000). It is widely accepted that the RFLP techniques, although highly discriminating, are again relatively cumbersome, inconvenient and not amenable to high throughput genotyping and inter-laboratory comparisons. Hence, effort has been directed at exploiting the various classes of DNA repeat using DNA amplification procedures. For example, the spacer-oligotyping technique, (spoligotyping) (Kamerbeek et al. 1997) uses the PCR to amplify a region of DNA, the socalled Direct Repeat or DR region, the arrangement and sequence of which is highly specific to members of the M. tuberculosis complex (Fig. 6.2). The order of these spacer sequences is remarkably well conserved, so much so that a phylogeny and common ancestor to the M. tuberculosis complex bacteria can be inferred, with a much larger DR locus containing many more spacer sequences than is presently seen in modern members of the complex (van der Zanden et al. 2002). The DR locus belongs to a recently identified class of repeat sequences found in bacteria, known as 'spacer interspersed direct repeats' (SPIDRs) or 'clustered regularly interspaced short palindromic repeats' (CRISPRs), which are reminiscent of centromere-like structures with a possible role in replication partitioning. CRISPR-mediated variation may provide a bacterial population with the diversity required to be adaptable in changing environments. The DR locus is comprised of multiple 36 bp direct repeats interspersed by non-repetitive 34-41 bp DNA spacer sequences. Spoligotyping involves amplification of the

81

entire CRISPR locus using the DR as a target for PCR, followed by hybridisation of the amplified DNA to a set of spacer oligonucleotides derived from M. tuberculosis and M. bovis BCG strain P3. This reverse-cross blot hybridisation (Kaufhold et al. 1994) records the presence, or absence, of spacer DNA sequences and produces a simple digital pattern, which is readily named and databased. Spoligotyping is particularly attractive because the patterns generated may be a presumptive indicator of the M. tuberculosis complex species amplified. For example, M. bovis isolates generally lack spacers 39-43 inclusive, as well as spacers 6,9 and 16 (van Soolingen 200l). Spoligotype patterns are also useful for identifying M. microti (van Soolingen et al. 1998) and have proved extremely valuable for typing some emerging epidemic strains, such as M. tuberculosis WBeijing (Glynn et al. 2002). Differences in spoligotypes are due to deletions of DRs in the CRISPR locus,mediated by transposition ofIS611O, homologous recombination or replication slippage. Strain-specific differences in spoligotype are the result of a sequential loss of single spacers or blocks of contiguous spacers within the CRISPR locus. It has been possible, therefore, to infer a sequence of genetic events separating M. bovis of different spoligotypes. It appears that ST140 is a recent progenitor of several different M. bovis spoligotypes, all of which are represented in current field isolates. The molecular clock for the CRISPR locus appears to be very slow. For example, all M. bovis BCG daughter strains share the same spoligotype, a spoligotype still seen in French M. bovis field strains (Haddad et al. 2001). Current phylogenetic clustering tools consider the gain and loss of individual or contiguous spacers as an equal event. Due to the proposed mechanism of CRISPR evolution, this may misrepresent the relationship between strains. Spoligotyping is relatively easy to perform and can be used to detect and type M. tuberculosis complex bacteria directly in a range of clinical samples (Kamerbeek et al. 1997). However, a major problem with the current configuration of spoligotyping is its relatively limited discriminatory power. Even for M. bovis isolates, spoligotyping is only moderately discriminating, compared to REA and RFLP techniques (Aranaz et al. 1996; Cousins et al. 1998a). More extensive sequencing of M. tuberculosis complex isolates has disclosed additional spacer sequences (van Embden et al. 2000; Caimi et al. 200l), which have been incorporated into the second generation spoligotyping technique. The discrimination and reproducibility of spoligotyping using these new spacer sequences is currently being evaluated on

82

R. A. Skuce and S. D. Neill

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representative panels of M. tuberculosis complex bacteria (van der Zanden et al. 2002). Attempts have also been made to unify and simplify spoligotype pattern nomenclature in order to facilitate inter-laboratory studies of the transmission of strains between and within different countries (Dale et al. 2000) and relatively large databases of spoligotypes are being curated (Sola et al. 2000, 2001). Genomics and bioinformatics have revealed several different classes of repetitive DNA within the genomes of M. tuberculosis H37Rv and M. bovis AF2122/97, which can now be exploited to develop convenient and

discriminating strain typing tools (Brosch et al. 2000). Many tuberculosis genes were shown to be comprised of tandem repeats. The Tandem Repeats Finder software programme (Benson 1999) has been used to predict a number of repeats varying from 9-351 bp. Fifty-three PE_PGRS genes, 27 PPE proteins and 59 unrelated proteins contained such tandem repeats. This could represent a major source of genetic diversity and antige'lic v?riation in a pathogen with a striking lack of neutral, single nucleotide polymorphisms in key house-keeping genes and antigens (Sreevatsan t al. 1997; Musser et al. 2000). One such class of repeat,

Molecular Epidemiology of Mycobacterium bovis

83

which is generating considerable interest at present, was described, although clinical isolates were not is the so-called mycobacterial interspersed repetitive characterised in the study. Analysis of larger numbers unit (MIRU) (Supply et al. 2000). MIRUs range in size of M. tuberculosis complex bacteria allowed the comfrom 46-101 bp and fall into three classes depending pilation of allele naming tables, which form the basis on their length, sequence and organisation. MIRUs of the intuitive MIRU-VNTR nomenclature. Further, occur in 41 loci, mostly intergenic, in M. tuberculosis novel VNTR-type loci have been described recently in H37Rv. Most MIRUs contain an ORF and could poten- the M. tuberculosis H37Rv genome (Skuce et al. 2002) tially encode small peptides. MIRUs often occur as and were evaluated on a reference panel of M. bovis tandem repeats and can be exploited as strain typing isolates compared to existing VNTRs (Frothingham targets. Specific PCR primers are designed to flank and Meeker-O'Connell 1998) and spoligotyping data. such loci and PCR products consequently differ in size Different VNTR-PCRs had different discriminatory capacity and combining VNTR-PCRs produced a relative to the tandem repeat copy number (Fig. 6.3). Other loci, equivalent to MIRUs, known as variable more discriminatory technique. The majority of novel number of tandem repeat loci (VNTR), have also been VNTRs were located within ORFs including several described (Frothingham and Meeker-O'Connell 1998). representatives of the PPE protein family. For examThese MIRU-VNTR loci resemble eukaryotic mini- ple, there were 29 allelic variants of the PPE protein satellite loci, being comprised of tandem repeat units Rv1917c in the test panel of 100 M. bovis isolates. of varying size and copy number (Supply et al. 1997; Further, novel MIRU-VNTR loci have been Frothingham and Meeker-O'Connell 1998; Magdalena subsequently identified in M. tuberculosis genome et al. 1998a,b). Systematic bioinformatic searches of sequences (Roring et al. 2002). Again, their perforavailable genome sequence databases have disclosed mance has been systematically evaluated on a test more such loci within the M. tuberculosis complex panel of M. tuberculosis complex isolates, leading mycobacteria (Smittipat and Palittapongarnpim 2000) to the recommendation that MIRU-VNTR loci be and a simple scheme for naming the MIRU-VNTR loci selected, for genotyping studies, based on their proven

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---IIQUB11a

13451 QUB3232

Fig. 6.3. Schematic representation of PCR-based MIRU-VNTR genotyping of M. tuberculosis complex isolates. a Representation of tandem repeat copy number variation between four hypothetical strains at one MIRV-VNTR locus and sizing of PCR products to deduce copy number. b Hypothetical MIRU-VNTR profiles derived for four strains at five loci. c Gel images for the same six M. bovis field isolates MIRUVNTR typed at three loci (ETR-A, QUBI la and QVB3232)

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performance with a representative test panel of isolates and not just on the convenience of using predetermined sets of markers. For example, the 12 MIRU panel is at least as discriminatory as IS611O-RFLP for M. tuberculosis isolates (Mazars et a1. 2001). However, not all the MIRU loci are informative with the M. bovis isolates comprising the test panel (Roring et a1. 2002). There are now upwards of 30 MIRU-VNTR loci described within the M. tuberculosis complex. Their discriminatory ability will depend on the test panel used. The reproducibility of the MIRU-VNTR PCRs and allele naming is exceptionally high. These MIRU-VNTR loci detect differences between M. bovis BCG daughter strains, entirely consistent with the proposed genealogy (Magdelena et al. 1998a; Behr 2000; Roring et a1. 2002). A major future objective will be to determine the pace of change for each of these loci and to match their properties to specific epidemiological studies. It can be envisaged that a panel of MIRU-VNTR loci, with varying molecular clock speeds, will be used in future epidemiological studies of the M. tuberculosis complex mycobacteria. These MIRU-VNTR loci have many of the most desirable features required to progress rapidly the molecular epidemiology of the M. tuberculosis complex. Several other PCR-based techniques have been developed, based on amplification of DNA fragments between repetitive DNA elements. Some difficulties have been experienced with the reproducibility of such methods on inter-laboratory evaluation (Kremer et a1. 1999).

6.8 Standardisation and Method Performance Evaluation In order that strain-typing results are directly comparable between laboratories, it is important that standard procedures and analyses are performed. Novel strain typing methods are often applied without critical evaluation of their performance characteristics. They often lack standardisation in technical procedures, reference materials and quality assurance, as well as in the criteria used for the interpretation of results. Basic terminology is sometimes used ambiguously by different investigators, compounding the confusion created by variations in methodology (Struelens 1996). Consequently, progress towards performance comparison, or standardisation, has only been achieved for a limited number of bacterial and fungal pathogens (Struelens 1996).

R. A. Skuce and S. D. Neill

Different techniques will have different performance characteristics, e.g. discrimination, reproducibility, epidemiological concordance etc. (see Struelens 1996 for definitions). The most discriminating and the least practical strain-typing technique would be to produce the entire nucleotide sequence for every isolate. Several investigators have recognised the need for a unified approach to the strain typing of members of the M. tuberculosis complex mycobacteria and M. bovis in particular (Kremer et a1. 1999). This would comprise standardisation of procedures, reagents, reference strains, image analysis and nomenclature. Under the auspices of the International Union Against Tuberculosis and Lung Disease (IUATLD), one such study (Cousins et a1. 1998a) recommended IS611O-RFLP to define the copy number in the test population and that IS611O-RFLP was usually adequate for M. bovis strains possessing more than three copies of IS6110. For strains with less than three copies of IS6110, additional typing was often warranted and PGRS-RFLP was the method of choice. Spoligotyping was recommended to replace DR-RFLP for additional typing. The DNA typing of M. bovis specifically was also considered by a multi-centre evaluation of method performance (Skuce et a1. 1998). The candidate methods of IS6110-RFLP, PGRS-RFLP, DR-RFLP, PFGE, spoligotyping, ampliprinting and VNTR typing (Frothingham and Meeker-O'Connell 1998) were evaluated. Methods were optimised by working groups and standard protocols were developed, which detailed procedures, reference strains and reagents, as well as making recommendations for appropriate nomenclature schemes and databasing. The optimised methods were applied to a coded test population of M. bovis isolates and the various performance criteria evaluated were: typability, reproducibility, discrimination, concordance and convenience (Struelens 1996). Methods differed in their performance characteristics. For example, the combined IS611O, PGRS, DR-RFLP approach was found to be highly discriminating and, with care, was adequately reproducible. However, it was by far the least convenient. Spoligotyping was highly reproducible and convenient, but only moderately discriminating in its current configuration with this reference panel. Spoligotyping was recommended as a relatively simple and highly reproducible screening method with moderate discrimination and high concordance. Where further discrimination is required the highly reproducible, convenient and concordant VNTR technique (Frothingham and MeekerO'Connell 1998), or the RFLP and PFGE methods

Molecular Epidemiology of Mycobacterium bovis

were recommended. Further development and evaluation was merited for several methods, most notably the variations on VNTR typing, especially with the imminent release of the genome sequence for the M. bovis isolate AF2122/97. In another study, the discriminatory power and reproducibility of the main candidate DNA fingerprinting methods was assessed recently for M. tuberculosis complex bacteria, including a small number of M. bovis isolates (Kremer et al. 1999). Again the various RFLP-based methods, using IS611 0, IS1081, PGRS, DR and (GTGh as probes were found to be reproducible, although not very convenient. The PCR-based methods of spoligotyping, VNTR typing (Frothingham and Meeker-O'Connell 1998) and mixed-linker PCR were found to be highly reproducible. Subsequently, semi-automated versions ofVNTR-typing (Hughes et al. 2000) and MIRU-typing (Mazars et al. 2001) were developed and have been applied to the same test panel. They were found to be highly reproducible and discriminatory, with MIRU-typing as discriminating as the accepted standard IS611O-RFLP method. Additionally, IS611O-RFLP and MIRU-typing clustered the test panel isolates in a highly concordant manner, making MIRU-typing and possible developments thereof, an exciting, convenient and portable new tool with which to tackle molecular epidemiology of the M. tuberculosis complex. It is anticipated that the best DNA fingerprinting technique will be that which is based on genetic events occurring in synchrony with the time scale of the epidemiological situation. Thus, different epidemiological questions will be best addressed by different techniques. If the genetic events are occurring considerably faster than the epidemiological events, epidemiologically related strains will appear to be unrelated, whereas if the genetic events are occurring more slowly than the epidemiological events, then unrelated strains will appear to be related. A major future challenge of DNA fingerprinting of M. tuberculosis complex bacteria will be to determine the paces of change of the various markers and to match these to study specific epidemiological questions.

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(Kulaga et al. 2002). Epidemiological studies have generally relied on demonstrating plausible opportunities for infection in order to infer transmission. Strain typing of M. tuberculosis isolates by IS611ORFLP analysis is now well developed (van Soolingen 2001) and is being integrated into control policies, as a tool for detecting point source outbreaks and for monitoring the efficacy of tuberculosis control programmes (Small et al. 1994; Alland et al. 1994; van Soolingen et al. 1997aj Bauer et al. 1998). An internationally accepted standard procedure (van Embden et al. 1993) has been devised to facilitate inter-laboratory comparisons (van Embden et al. 1996; Heersma et al. 1998). For M. tuberculosis, strain typing is becoming a standard tool of public health investigators. If there is sufficient genetic diversity in a population, finding a group with the same, or highly related, pattern (cluster) infers epidemiological association, where individuals are either infected from each other or from a common source. Such cluster analysis must be interpreted with caution, especially where the survey has not been sufficiently comprehensive (Glynn et al. 1999a,b). Strain typing has been used to identify clonal transmission, e.g. between neighbours and within prisons, bars, clinics and hospitals. It has also been used to identify epidemiologically unsuspected transmission within groups, or unsuspected ongoing transmission in certain segments of the population, where risk factors, environments and common sources were missed by conventional contact tracing (van HeIden 1998). IS611O-RFLP has surprisingly and repeatedly demonstrated that tuberculosis transmission can occur without close personal contact (Kline et al. 1995). Prompt recognition of such events has helped to arrest such micro-epidemics. Strain typing has also played a role in identifying problematic procedures and laboratory cross-contamination in reference laboratories, where isolation of the organism is often the only basis for diagnosis and intervention.

6.10

Mycobacterium bovis Molecular

6.9

Mycobacterium tuberculosis Molecular Epidemiology: Lessons Learned

The ability to identify transmission events accurately and to intervene in order to prevent further spread is critical in effective tuberculosis control programmes

Epidemiology: Studies and Trends to Date

The REA technique has been used in several epidemiological studies, most notably in New Zealand (Collins 1998). It has provided a new insight into disease dynamics, demonstrating clustering of REA types in defined geographical areas of New Zealand. Possums, other wildlife and farmed animals in the

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same area were often infected with the same REA type (Collins et al. 1986, 1988, 1994). REA typing was used to include or exclude possible sources of infection in specific herds and it has clearly demonstrated whether infection in a farmed animal has come from the infected local wildlife reservoir or from infected cattle or deer brought onto those premises (de Lisle et al. 1995). REA data has been used to influence the level of herd testing or wildlife control in specific areas, e.g. REA and IS6110-RFLP were used to study the M. bovis strain types present in the MacKenzie basin in the South Island of New Zealand, a region that had been free of bovine tuberculosis prior to 1980. The identification of two main groups led to the suggestion that infection was introduced from two distinct, as yet unidentified, sources. Strain typing has, therefore, contributed to determining the extent and spread of infected wildlife in defined areas (de Lisle et al. 1995; Collins et al. 1999) and is now considered an integral component in local bovine tuberculosis control schemes (Collins 1998). Restriction enzyme analysis (REA) was used to type M. bovis isolates from a longitudinal field study of endemically infected possum, cattle, feral pigs and feral ferrets sharing the same habitat (Pfeiffer et al. 1994). A Geographical Information System (GIS) was used to manage the data, which was mapped on a terrain model of the study area. Statistical analysis was used to display the association between such variables as REA type, sex, age, season of detection and terrain covered. Four REA types were described, with two major types each dominating at different times. The same major types were evident in cattle when dominant in possums. Some important inferences regarding potential transmission routes for the wild possum population were possible. Shared REA types between mother and young identified pseudovertical transmission as an effective transmission route. Two successive, major cycles of transmission, showing only limited spatial overlap, were described within the possum population. REA was also used to infer that infected possum, not environmental survival of M. bovis, were responsible for reintroducing tuberculosis to an area following extensive possum depopulation (Corner et al., unpublished). Strain typing has also been used in a targeted fashion to show that domestic cats were usually infected with the same REA types as predominant in other local animal species and that they posed an unsuspected zoonotic risk (de Lisle et al. 1990). The application of molecular epidemiology to bovine tuberculosis is most advanced in New Zealand. However, several other countries have initiated stud-

R. A. Skuce and S. D. Neill

ies on methodology and preliminary epidemiological investigations (Collins 1998; Durr et al. 2000a,b; Skuce and Neill 2001). Despite some similarities, the epidemiology of bovine tuberculosis in other countries may well be very different from the New Zealand situation. For example, in Northern Ireland investigators have favoured the RFLP and spoligotyping techniques to disclose a surprisingly large number of M. bovis strain types in a panel of isolates from different animal species and from different geographical and temporal origins (Skuce et al. 1996; Roring et al. 1998). The RFLP types and spoligotypes identified did not appear to be host restricted, being isolated from cattle, badgers, deer and humans. One strain was over-represented in the panel of isolates, making up approximately 45% of RFLP types and approximately 70% of spoligotypes. Clonal expansion of such a strain is indicative of'success' and this strain is likely to be host-adapted to cattle, although not host-restricted. As with possum and cattle isolates in New Zealand, badger and cattle isolates in Ireland (Costello et al.1999) from the same locality or premises often share the same strain type, suggesting that a common pool of M. bovis strains is circulating between domesticated, wild and feral animals. Without more precise epidemiological data and statistically valid experimental design it is not possible yet to demonstrate, or quantify, the relative extent, if any, of transmission between cattle and wildlife, and vice versa. Mycobacterium bovis genotyping, using spoligotyping, has also shown that, in a significant number of herds for which M. bovis isolates are available for individual animals, multiple spoligotypes exist within an outbreak, usually one predominant spoligotype plus a few isolates of different spoligotypes (Skuce et al. 1996). This suggests that either the DR locus is not sufficiently stable within an outbreak or that such bovine tuberculosis outbreaks are due to multiple sources and that some types may be more transmissible than others. The balance of evidence suggests that such outbreaks are the result of infection from multiple sources, a phenomenon which may be under-reported due to the predominance of some spoligotypes, e.g. ST140. In a spoligotyping study of more than 600 M. bovis isolates in Northern Ireland, ST140 accounted for 70% of isolates, with ST263 accounting for a further 20%. The remaining five spoligotypes accounted for the remaining 10% of isolates (Skuce et al., unpublished). The clonal expansion of M. bovis isolates with the ST140 genotype is indicative of success. It may be speculated that ST140 spoligotype M. bovis possess some selective advantage.

Molecular Epidemiology of Mycobacterium bovis

IS611O-RFLP, PGRS-RFLP, DR-RFLP and spoligotyping were evaluated on a panel of 452 M. bovis isolates recovered from cattle, deer, pigs, sheep and goat in the Republic of Ireland (Costello et al. 1999). RFLP identified 85 strains with one type, the same type as identified in Northern Ireland, accounting for 20%. Spoligotyping identified 20 strains, with ST140 accounting for 52% of isolates. This major type was recovered from all animal species tested and was widely distributed geographically, suggesting that transmission between these animal species was a factor in the epidemiology of bovine tuberculosis in Ireland. However, some strain types were geographically more clustered. Subsequently, IS611O- RFLP, PGRS-RFLP, DR-RFLP and spoligotyping were compared with pUCD-RFLP on a panel of 299 M. bovis isolates (O'Brien et al. 2000a). Using a derived statistical function for discrimination, pUCD-RFLP was found to be more discriminating than the combined RFLP method and spoligotyping. pUCD-RFLP and DR-RFLP combined produced a more discriminating and epidemiologically concordant method. In Great Britain, the majority of isolates from recent outbreaks in cattle have been spoligotyped and mapped using the grid reference of the farm location (Clifton-Hadley et al.I998). Thirty-four spoligotypes were identified, 25 in cattle amongst 2668 isolates collected mainly during 1996 and 1997. In agreement with the Northern Ireland study, the same two spoligotypes (ST140 and ST263) accounted for 70% of all isolates. One spoligotype was detected in 623 herds and 2-5 spoligotypes were detected in the remaining 86 herds, again suggesting multiple source outbreaks. Fifty-three herds had isolates spoligotyped on repeat occasions and in all but five instances, at least one spoligotype was the same as the spoligotype initially detected. This suggests that new sources were responsible for introducing the different spoligotypes to retested herds. Through epidemiological trace-back a source for 426 of the outbreaks was proposed. In situations where badger M. bovis isolates were recovered following an outbreak in cattle, 90 of 99 (91%) isolates were of the same spoligotype. It was concluded that spoligotyping was a useful genotyping method but further discriminatory ability was desirable. A study of M. bovis recovered from a well-characterised badger population suggested that spoligotypes were relatively stable within a location and over a length of time, within a defined population and within individual animals. Using Geographical Information Systems (GISs) to manage the molecular typing data, a marked degree of geographical clustering was demonstrated for several spoligotypes, which can be

87

interpreted as evidence for a stable source of infection for cattle, such as wildlife. In marked contrast to the spoligotyping studies in Northern Ireland, the Republic of Ireland, Great Britain and several other countries, an extensive spoligotyping study of 1,349 M. bovis isolates collected mainly from cattle in France over a 20-year period (1979-2000) demonstrated that the technique had very acceptable and useful discriminatory capacity (Haddad et al' 2001). A large number (161) of spoligotypes were found, relatively more than were found in Great Britain, with two types responsible for 26 and 12% of isolates, respectively. The main spoligotype pattern was described as BCG-like. This may not be surprising, since M. bovis BCG was originally derived from a French M. bovis isolate and was proposed as the progenitor of further spoligotypes, which were linked through the unidirectional loss of CRISPR spacers. Spoligotypes were again mapped and there was marked geographical clustering for several of them. The remarkable diversity in spoligotypes was interpreted as reflecting the very low disease prevalence. Additional factors, such as traditional trading in a large number of different cattle breeds were also cited. It is interesting to note that the 'classical European' M. bovis BCG-like spoligotype pattern now dominates in northern Cameroon and that the introduction of Charolais cattle from France in 1913 is an alleged source. Although a small sample number, the spoligotype patterns in Cameroon appear relatively homogeneous. France is still the main exporter of cattle to Cameroon (Njanpop-Lafourcade et al' 2001). Molecular typing of M. bovis isolates in Tanzania (Kazwala et al' 1998) led to the hypothesis that there were two main categories of strains: autochthonous strains (atypical cultural properties) and strains imported from Europe (with classical cultural properties). Bovine tuberculosis has also become a very significant disease problem of wildlife in the major game reserves of South Africa, severely affecting nature conservation and eco-tourism. The African buffalo (Syncerus caffer) is a valuable big-game species which is susceptible to M. bovis infection, making them a risk for domestic cattle. Tuberculosis-free buffalo are also more valuable, a major driving force in illegal trade. The buffalo populations in Hluluwe-Umfolozi Game Reserve (HUP) and the Kruger National Park (KNP) are infected with M. bovis and act as a maintenance host, responsible for spill-over of infection into local wildlife. IS611 0- RFLP has been used to trace the original source of infection of buffalo populations in the KNP and in tracing the origin of infected animals that were moved to other parts of South Africa. Therefore,

88

R. A. Skuce and S. D. Neill

the identification of the probable origin of animals ogy, evolution and pathogenesis of a major pathogen may be possible by studying the strain types har- such as M. bovis. When integrated with developments boured by animals, where more conventional animal in animal traceability (Houston 2001) and technoloidentification methods have either failed or not been gies such as Global Positioning Systems (GPSs) and applied (Vosloo et al. 2001). Geographical Information Systems (GISs), which IS6110-RFLP and DR-RFLP were used to type are available in developed countries, they should M. bovis isolates from cattle in Mexico and Texas provide powerful and challenging new informa(Perumaalla et al. 1996, 1999). The disclosure of tion with which to inform control strategies better. multi-copy IS6110 strains implicated wildlife res- It is probable however that, as with M. tuberculosis, ervoirs in disease transmission, a theory which has this equivalent of contact tracing may not be very not been proven yet. In Argentina, the disclosure effective at detecting casual contacts and that strain of 'cattle' strain types infecting humans identified typing data will provide alternative explanations for an occupational zoonotic risk (van Soolingen et al. proposed sources and transmission routes. 1994). This contrasts with the current situation in These new tools should also prove valuable for the Netherlands, where bovine tuberculosis has been identifying sources and routes of M. bovis transsuccessfully eradicated. Strain typing of M. bovis mission, thus providing baseline information on isolates from humans showed that they were infected prevalence and distribution of M. bovis strain types with 'multi-copy' IS611 0 strains, which were likely to in animal species. Integration into imaginative and have been due to exposure to infected 'exotic' animals focussed epidemiological studies should help clarify specific issues such as the contributions from interin zoos or animal parks. Australia has succeeded in eradicating bovine bovine transmission and from wildlife. Availability tuberculosis. However, prior to this, M. bovis strain of more precise strain recording should be employed typing was used to propose potential sources and also to investigate: 'persistence' of infection within transmission routes for infection on specific farm a herd; the issue of recrudescence of infection and premises (Cousins et al. 1993, 1998b). Additionally, strain reintroduction, and therefore may ultimately strain typing has been used to investigate other zoo- help to elucidate further the issue of latency in cattle notic risks such as tuberculous captive seals, which referred to by Pollock and Neill (2002) were shown to have infected a seal trainer having the Additionally, it would be of significant value to same strain (Cousins et al. 1990). REA was used in know if particular M. bovis strains may be associated Sweden to show that M. bovis was independently, and with particular epidemiological features observed inadvertently, introduced into Sweden and New Zea- in outbreaks of this disease and if specific strains unusually influence the pathogenesis. Correlation land from Great Britain (Bolske et al. 1995). studies could be devised to compare biological properties of M. bovis isolates from, e.g., non-lesioned cattle, from weak tuberculin-reacting or tuberculinnegative animals exhibiting disease and from large 6.11 multi-reactor outbreaks of tuberculosis. IdentificaConclusions tion of key biological properties that influence strain Obviously, the medical and veterinary pnonties behaviour in such ways could have significant imporbetween developing and developed countries can tance in informing control strategies, particularly at differ significantly and hence their attitudes and the local level. For developing countries, molecular technologies responses to animal tuberculosis and any potential resulting zoonosis. Molecular epidemiology is a could be of equal value in the longer term. Howrelatively new discipline and particularly so when ever, in many instances, their use in surveillance is considering M. bovis. There is now a need to inte- likely to be prohibitively expensive for general and grate these new developments, which until recently widespread application. Nonetheless, collection and have been largely 'technology-driven', with classical collation of standard data could inform on matters of animal and human health, which would be epidemiological studies. Genetic tools to facilitate accurate diagnosis and a significant advance. The findings from molecular high-resolution identification of species and strains epidemiological studies in developed countries will of the M. tuberculosis complex now exist and are eventually yield significant information, which could amenable to high-throughput studies. These should have an impact if extrapolated to developing regions provide important new information about the biol- of the globe.

Molecular Epidemiology of Mycobacterium bovis

Acknowledgements. The authors would like to acknowledge the contributions of colleagues within the bovine tuberculosis statutory and research programme at Veterinary Sciences Division (VSD), Department of Agriculture and Rural Development (Northern Ireland) and the Department of Veterinary Science at the Queen's University of Belfast to some of the work discussed in this chapter. This work was funded by the Department of Agriculture and Rural Development (DARD, Northern Ireland), the Department of Environment, Food and Rural Affairs (DEFRA, Great Britain) and the European Union. The authors wish to thank Malcolm Taylor (VSD, DARD) for his assistance with figures and graphics.

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89 Cheeseman CL, Wilesmith JW, Stuart FA (1989) Tuberculosis: the disease and its epidemiology in the badger, a review. Epidemiol Infect 103:113-125 Clifton-Hadley RS, Inwald J, Archer J, Hughes S, Palmer N, Sayers AR, Sweeney K, van Embden JDA (1998) Recent advances in DNA fingerprinting using spoligotyping: epidemiological applications in bovine TB. Cattle Pract 6:79-82 Cole ST, Brosch R, Parkhill J et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544 Collins DM (1998) Molecular epidemiology: Mycobacterium bovis. In: Rutledge C, Dale J (eds) Mycobacteria-molecular biology and virulence. Blackwell Science, Boston Collins DM, de Lisle GW (1984) DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG. J Gen Microbiol 130:1019-1021 Collins DM, de Lisle GW (1985) DNA restriction endonuclease analysis of Mycobacterium bovis and other members of the tuberculosis complex. J Clin Microbiol 21:562-564 Collins DM, de Lisle GW, Gabric DM (1986) Geographic distribution of restriction types of Mycobacterium bovis isolates from brush-tailed possums (Trichosurus vulpecula) in New Zealand. J Hyg 96:431-438 Collins DM, Gabric DM, de Lisle GW (1988) Typing of Mycobacterium bovis isolates from cattle and other animals in the same locality. NZ Vet J 36:45-46 Collins DM, Erasmuson SK, Stephens DM, Yates GF, de Lisle GW (1993) DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridisation with the insertion elements IS1081 and IS6110. J Clin MicrobioI31:1143-1147 Collins DM, Radford AJ, de Lisle GW, Billman-Jacobe H (1994) Diagnosis and epidemiology of bovine tuberculosis using molecular biological approaches. Vet Microbiol 40:83-94 Collins DM et al. (1999) DNA typing of Mycobacterium bovis isolates from the Castlepoint area in New Zealand Cosivi 0, Grange JM,Daborn CJ et al. (1998) Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 4(1) CDC URL: http://www.cdc.gov/ ncidodleid/vol4no 11 cosivi.htm Costello E, O'Grady D, Flynn E et ai. (1999) Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection. J Clin MicrobioI37:3217-3222 Cousins DV (2001) Mycobacterium bovis infection and control in domestic livestock. Rev Sci Tech Off Int Epiz 20:71-85 Cousins DV, Roberts JL (2001) Australia's campaign to eradicate bovine tuberculosis: the battle for freedom and beyond. Tuberculosis 81:5-15 Cousins DY, Francis BR, Gow BL et al' (1990) Tuberculosis in captive seals. Bacteriological studies on an isolate belonging to the Mycobacterium tuberculosis complex. Res Vet Sci 48:196-200 Cousins DV, Williams SN, Ross BC, Ellis TM (1993) Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridization studies with Mycobacterium bovis as a new tool for epidemiological studies of bovine tuberculosis. Vet MicrobioI37:1-17 Cousins DV, Skuce RA, Kazwala RR, van Embden JD (1998a) Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. Int J Tuberc Lung Dis 2: 471-478

90 Cousins DV, Williams S, Liebana E et al. (1998b) Evaluation of four DNA typing techniques in epidemiological investigations of bovine tuberculosis. 1Clin Microbiol 36: 168-174 Daborn q, Grange 1M and Kazwala RR (1996) The bovine tuberculosis cycle: and African perspective. 1 Appl Bacteriol81 [Suppl):S27-S32 Dale IW, Brittain D, Cataldi AA et al. (2001) Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised nomenclature. Int 1Tuberc Lung Dis 5:216-219 De Lisle GW, Collins DM, Loveday AS, Young WA, Iulian AF (1990) A report of tuberculosis in cats in New Zealand, and the examination of strains of Mycobacterium bovis by DNA restriction endonuclease analysis. NZ Vet 1 38: 10-13 De Lisle GW, Yates GF, Collins DM, MacKenzie RW, Crews KB, Walker R (1995) A study of bovine tuberculosis in domestic animals and wildlife in the MacKenzie Basin and surrounding areas using DNA fingerprinting. NZ Vet 143:266-271 De Lisle GW, Mackintosh CG and Bengis RG (2001) Mycobacterium bovis in free-living and captive wildlife, including farmed deer. Rev Sci Tech Off Int Epiz 20:86-111 Denny GO, Wilesmith IW (1999) Bovine tuberculosis in Northern Ireland: a case-control study of herd risk factors. Vet Rec 144:305-310 Doran TI, Hodgson AL, Davies IK, Radford AI (1993) Characterisation of a highly repeated DNA sequence from Mycobacterium bovis. FEMS Microbiol Lett 111:147-152 Durr PA, Clifton-Hadley RS, Hewinson RG (2000a) Molecular epidemiology of bovine tuberculosis II: applications of genotyping. Rev Sci Tech Off Int Epiz 19:689-701 Durr PA, Hewinson RG, Clifton-Hadley RS (2000b) Molecular epidemiology of bovine tuberculosis I: Mycobacterium bovis genotyping. Rev Sci Tech Off Int Epiz 19:675-688 European Commission Report (1998) Trends and sources of zoonotic agents in animals, feedstuffs, food and man in the European Union in 1997. Document no VI/8495/98-Rev 2 Feizabadi MM, Robertson ID, Cousins DV, Hampson DI (1996) Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis. 1 Clin MicrobioI34:1136-1142 Frothingham R, Meeker-O'Connell WA (1998) Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144:1189-1196 Gallagher 1, Clifton-Hadley RS (2001) Tuberculosis in badgers: a review of the disease and its significance for other animals. Res Vet Sci 69:203-217 Glynn IR, Bauer 1, de Boer S, Borgdorff MW, Fine PEM, Godfrey-Faussett P, Vynnycky E (1999a ) Interpreting DNA fingerprint clusaters of Mycobacterium tuberculosis. Int 1 Tuberc Lung Dis 3:1055-1060 Glynn IR, Vynnycky E, Fine PEM (1999b ) Influence of sampling on estimates of clustering and recent transmission of Mycobacterium tuberculosis derived from DNA fingerprint techniques. Am 1 EpidemioI149:366-371 Glynn IR, Whiteley 1, Bifani PI et al. (2002) CDC-worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. CDC URL:http://www.cdc.gov/ ncidod/EID/voI8n08/02-0002-G.htm Goodchild AV, Clifton-Hadley RS (2001) Cattle-to-cattle transmission of Mycobacterium bovis. Tuberculosis 81:23-41

R. A. Skuce and S. D. Neill Gordon SV, Brosch R, Billault A, Garnier T, Eiglmeier K, Cole ST (1999a) Identification of variable regions in the genome of tubercle bacilli using artificial chromosome arrays. Mol Microbiol 32:643-655 Gordon SV, Heym B, Parkhill J, Barrell B, Cole ST (1999b) New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37 Rv. Microbiology 145:881-892 Gordon SV, Eiglmeier K, Garnier T, Brosch R, Parkhill 1, Barrell B, Cole ST, Hewinson RG (2001) Genomics of Mycobacterium bovis. Tuberculosis 81:157-163 Grange 1M (2001) Mycobacterium bovis infection in human beings. Tuberculosis 81:71-77 Groenen PMA, Bunschoten AE, van Soolingen D and van Embden IDA (1993) Nature of DNA polymorphisms in the direct repeat cluster of Mycobacterium tuberculosis: application for strain differentiation by a novel typing method. Mol Microbioll0:1057-1085 Guerrero A, Cobo 1, Fortun 1et al. (1997) Nosocomial transmission of Mycobacterium bovis resistant to 11 drugs in people with advanced HIV-l infection. Lancet 350:1738-1742 Haddad N, Ostyn A, Karoni C, Masselot M, Thorel MF, Hughes SL, Inwald 1, Hewinson RG, Durand B (2001) Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979-2000.1 Clin MicrobioI39:3623-3632 Heersma HF, Kremer K, van Embden ID (1998) Computer analysis of IS611 0 RFLP patterns of Mycobacterium tuberculosis. Methods Mol Bioi 101:395-422 Houston R (2001) A computerised database system for bovine traceability Hughes MS and Rogers M (1994) Vaccination of the badger (Meles meles) against Mycobacterium bovis. Vet Microbiol 51:363-379 Hughes SL, Frothingham R, Inwald 1 et al. (2000) The development of semi-automated fluorescent techniques for improved molecular typing of Mycobacterium bovis. Abstract of the 5th international meeting on bacterial epidemiological markers (IMBEM), Noordwijkerhout, Netherlands, Sept 2000 Hughes VM, Stevenson K, Sharp 1M (2001) Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria. 1 Microbiol Methods 44:209-215 Kallenius G, Koivula T, Ghebremichael S et al. (1999) Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau. 1Clin MicrobioI37:3872-3878 Kamerbeek 1, Schouls LM, Kolk A et al. (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbioi 35:907-914 Kaufhold A, Podbielski A, Baumgarten G, Blokpoel M, Top 1, Schouls L (1994) Rapid typing of group A Streptococci by the use of DNA amplification and non-radioactive allele specific oligonucleotide probes. FEMS Microbiol Lett 119:19-26 Kazwala RR, Daborn q, Kusiluka LIM, Iiwa SFH, Sharp 1M, Kambarage DM (1998) Isolation of Mycobacterium species from raw milk of pastoral cattle of the Southern HigWands of Tanzania. Trop Anim Health Prod 30:233-239 Kazwala RR, Daborn q, Sharp 1M et al. (2001a) Isolation of Mycobacterium bovis from human cases of cervical adenitis in Tanzania: a cause for concern? lnt J Tuberc Lung Dis 5:87-91 Kazwala RR, Kambarage DM, Daborn q et al. (2001b) Risk factors associated with the occurrence of bovine tubercu-

Molecular Epidemiology of Mycobacterium bovis losis in cattle in the Southern Highlands of Tanzania. Vet Res Commun 25:609-614 Kline SE, Hedemark LL, Davies SF (1995) Outbreak of tuberculosis among regular patrons of a neighbourhood bar. N Engl J Med 333:222-227 Kremer K, van Soolingen D, Frothingham R et al. (1999) Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J Clin MicrobioI37:2607-2618 Kulaga S, Behr M, Musana K et al. (2002) Molecular epidemiology of tuberculosis in Montreal. Can Med Assoc J 167:353-354 Livingstone PG (1992) Tuberculosis in New Zealand-current status and control policies. Surveillance 19:14-18 Magdalena J, Vachee A, Supply P, Locht C (1998a) Identification of a new DNA region specific for members of Mycobacterium tuberculosis complex. J Clin MicrobioI36:937-943 Magdalena J, Supply P, Locht C (1998b) Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex. J Clin Microbiol 36:2471-2476 Mazars E, Lesjean S, Banuls A-L, Gilbert M, Vincent V, Gicquel B, Tibayrenc M, Locht C, Supply P (2001) High-resolution minisatellit-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci USA 98:1901-1906 Morris RS, Pfeiffer DU, Jackson R (1994) The epidemiology of Mycobacterium bovis infections. Vet MicrobioI40:153-177 Musser JM, Amin A, Ramaswamy S (2000) Negligible genetic diversity of Mycobacterium tuberculosis host immune system protein targets: evidence of limited selective pressure. Genetics 155:7-16 Neill SD (1994) Pathogenesis of Mycobacterium bovis infection in cattle. Vet MicrobioI40:41-52 Neill SD, Cassidy J,Hanna J, Mackie DP, Pollock JM, Clements A, Walton E, Bryson DG (1994) Detection of Mycobacterium bovis infection in skin test-negative cattle with an assay for bovine interferon-gamma. Vet Rec 50:229-232 Njanpop-Lafourcade BM, Inwald J, Ostyn A, Durand B, Hughes S, Thorel M-F, Hewinson G, Haddad N (2001) Molecular typing of Mycobacterium bovis isolates from Cameroon. J Clin Microbiol 39:222-227 O'Brien R, Flynn 0, Costello E, O'Grady D, Rogers M (2000a) Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis. J Clin MicrobioI38:1723-1730 O'Brien R, Danilowiez BS, Bailey L et al. (2000b) Characterization of the Mycobacterium bovis restriction frament length polymorphism DNA probe pUCD and performance comparison with standard methods. J Clin Microbiol 38: 3362-3369 O'Reilly LM, Daborn CJ (1995) The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuberc Lung Dis 76 [SuppI1]:1-46 Perumaalla VS,Adams LG,Payeur J et al. (1996) Molecular epidemiology of Mycobacterium bovis in Texas and mexico. J Clin Microbiol 34:2066-2071 Perumaalla VS, Adams LG, Payeur J et al. (1999) Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain. Vet Microbiol 70:269-276 Pollock JM, Neill SD (2002) Review: Mycobacterium bovis infection and tuberculosis in cattle. Vet J 163:115-127

91 Pfeiffer DU, Jackson R, de Lisle GW, Collins DM, Morris RS (1994) The use of genetic markers to clarify tuberculosis epidemiology. Kenya Vet 18:293-295 Rastogi N, Legrand E, Sola C (2001) The mycobacteria: an introduction to nomenclature and pathogenesis. Rev Sci Tech OffInt Epi 20:21-54 Roring S, Brittain D, Bunschoten AE et al. (1998) Spacer oligonucleotide typing of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism analysis using PGRS, DR and IS611 0 probes. Vet MicrobioI61:111-120 Roring S, Scott A, Brittain D, Walker I, Hewinson G, Neill S, Skuce R (2002) Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping. J Clin MicrobioI40:2126-2133 Salamon H, Behr MA, Rhee JT, Small PM (2000) Genetic distances for the study of infectious disease epidemiology. Am J EpidemioI151:324-334 Saunders NA (1995) State of the art typing of Mycobacterium tuberculosis. J Hosp Inf 29:169-176 Skuce RA, Neill SD (2001) Molecular epidemiology of Mycobacterium bovis: exploiting molecular data. Tuberculosis 81:169-175 Skuce RA, Brittain D, Hughes MS, Neill SD (1996) Differentiation of Mycobacterium bovis isolates from animals by DNA typing. J Clin MicrobioI34:2469-2474 Skuce RA, Brittain D, van Embden JD, Smith T, Sharp M, Hewinson G, Rogers M, Garcia Marin J-F, Neill SD (1998) Development of novel standardised methodology and nomenclature for the identification of Mycobacterium bovis strains. EU Contract SMT4 CT96 2097 Final Report (EU Commission) Skuce RA, McCorry TP, McCarroll JF, Roring SM, Scott AN, Brittain D, Hughes SL, Hewinson RG and Neill SD (2002) Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. MicrobioI148:519-528 Small PM, van Embden JD (1994) Molecular epidemiology of tuberculosis. In: Bloom BR (ed) Tuberculosis: pathogenesis, protection and control. American Society for Microbiology Small PM, Hopewell PC, Singh SP et al. (1994) The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N Engl J Med 330:1703-1709 Smittipat N, Palittapongarnpim P (2000) Identification of possible loci of variable number of tandem repeats in Mycobacterium tuberculosis. Tubercle Lung Dis 80:69-74 Sola C, Filliol I, Legrand E, Rastogi N (2000) Recent developments of spoligotyping as applied to the study of epidemiology, biodiversity and molecular phylogeny of the Mycobacterium tuberculosis complex. Path Bioi 48:921-932 Sola C, Filliol I, Gutierrez M C et al. (2001) Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerg Inf Dis http://www.cdc.gov?ncidod/ eid.vol7n03/solaGl.htm Sreevatsan S, Pan X, Musser JM (1997) Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 94:9869-9874 Struelens MJ (1996) Consensus guidelines for the appropriate use and evaluation of microbial epidemiologic typing systems. Clin Microbiol Infect 2:2-11

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Supply P, Magdalena J, Himpens S, Locht C (1997) Identification of novel intergenic repetitive units in a mycobacterial twocomponent system operon. Mol MicrobioI26:991-1003 Supply P, Mazars E, Lesjean S, Vincent V, Gicquel Band Locht C (2000) Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol MicrobioI36:762-771 Tanaka MM, Small PM, Salamon H, Feldman MW (2000) The dynamics of repeated elements: applications to the epidemiology of tuberculosis. Proc Natl Acad Sci USA 97:3532-3537 Van Belkum A (2002) Molecular typing of micro-organisms: at the centre of diagnostics, genomics and pathogenesis of infectious diseases? J Med MicrobioI51:7-10 Van Belkum A, Struelens M, de Visser A, Verbrugh H, Tibayrenc M (2001) Role of genomic typing in taxonomy, evolutionary genetics and microbial epidemiology. Clin Microbiol Rev 14:547 Van der Zanden AG, Kremer K, Schouls LM, Caimi K, Cataldi A, Hulteman A, Nageikerke NJ, van Soolingen D (2002) Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides. J Clin Microbiol 40:4628-4639 Van Embden JD, Cave MD, Crawford JT et al. (1993) Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin MicrobioI31:406-409 Van Embden JD, van Soolingen D, Heersma HF et al. (1996) Establishment of a European Network for the surveillance of Mycobacterium tuberculosis, MRSA and penicillin-

R. A. Skuce and S. D. Neill resistant pneumococci (letter; comment). J Antimicrob Chemother 38:905-907 Van Embden JDA, van Gorkom T, Kremer K et al. (2000) Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J BacterioI182:2393-2401 Van Heiden P (1998) Molecular epidemiology: human tuberculosis. In: Rutledge C, Dale J (eds) Mycobacteria-molecular biology and virulence. Blackwell Science, Boston Van Soolingen D (2001) Review: molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements. J Intern Med 249:1-26 Van Soolingen D, de Haas PEW, Haagsma J et al. (1994) Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis. J Clin Microbiol 32:2425-2433 Van Soolingen D, Borgdorff MW, de Haas PEW et al. (1997a) Molecular epidemiology of tuberculosis in The Netherlands: a nationwide study from 1993 through 1997. J Infect Dis 180:726-736 Van Soolingen D, Hoogenboezem T, de Haas PE et al. (1997b) A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterisation of an exceptional isolate from Africa. Int J Syst BacterioI47:1236-1245 Van Soolingen D, van der Zanden AG, de Haas PW et al. (1998) Daignosis of Mycobacterium microti infections among humans by using novel genetic markers. J Clin Microbiol 36: 1840-1845

7 Tuberculosis in Special Groups and Occupational Hazards JAIME ESTEBAN

CONTENTS 7.1 7.2 7.3 7.4 7.5 7.6

7.7 7.8 7.9

7.10 7.11 7.12

Special Groups (1). Diabetics 93 Special Groups (2). Transplantation 94 Special Groups (3). Malignant Diseases 96 Special Groups (4). Drug Abusers 97 Special Groups (5). Correctional Facilities 98 Special Groups (6). Shelters 100 Special Groups (7). Immigrants 100 Occupational Hazards (1). Health Care Workers 101 Occupational Hazards (2). Laboratory Workers 104 Occupational Hazards (3). Silicosis 106 Occupational Hazards (4). Other Workers 106 Conclusion 107 References 108

host against the pathogen, some population groups, such as closed communities (correctional facilities, shelters, jails, hospitals, miners and others) seem to present unique characteristics. This is also the case in patients with underlying conditions that make the risk of active tuberculosis higher, either by immunosuppression or by local factors (silicosis, transplantation, diabetes, malignancies, drug abuse). The following pages describe the special characteristics of tuberculous diseases in these groups, with a special emphasis on protective measures.

7.1 Special Groups (1). Diabetics At the beginning of the last quarter of the twentieth century, tuberculosis seemed to be a disease in decay in the developed world. The image was that of a slow but progressive decrease in the prevalence of the disease due to the availability of a very effective therapy and the increasing development of hygienic measures. However, some years later, an increase in the number of cases of tuberculosis was detected in the USA. This fact, together with the appearance of cases of multidrug-resistant tuberculosis, which were very difficult to treat, increased the interest of the scientific community in this half-forgotten disease. In 1993, the WHO declared tuberculosis a global emergency because of the increasing number of cases throughout the world, and more measures were taken in the fight against the disease. The fact that tuberculosis is mainly a respiratory disease that can be transmitted airborne or by droplets makes any person in contact with a patient who suffers from active respiratory tuberculosis susceptible to infection. However, because of the nature of transmission and the immune response of the

J. ESTEBAN, MD Department of Medical Microbiology, Fundaci6n Jimenez Diaz, Av. Reyes Cat6licos 2, 28040 Madrid, Spain

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Diabetes is a well-known risk factor for tuberculosis. Diabetics have higher rates of tuberculosis than nondiabetics (2-5 times higher) (Opsald et al.1961; Small and Fujiwara 2001; Turner 1951). Although the reason for this is not well known, there are several hypotheses which try to explain this phenomenon. They include alterations in pulmonary immune cell function (including altered polymorphonuclear cell function, reduced activity of monocyte-macrophages, in particular impaired phagocytosis of intracellular organisms and depressed lymphocyte function) and the effect of diabetic microangiopathy in the capillaries of the alveolar septa and alveolar arterioles. The latter may affect local response to infection (Koziel and Koziel 1995). Tuberculous diabetics have the same symptoms and signs as non-diabetic patients, and their evolution seems to be similar. However, several reports describe atypical radiographic findings in diabetics, such as a higher prevalence of cavitations and tuberculous lesions in lower lobes. These data have been reported in several studies, which have in common small sample sizes, the results being statistically non conclusive (Morris et al. 1992). Two other reports with a higher number of patients gave different results. One of them supports the idea of a higher

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incidence of lower lobe disease in diabetics of all age groups (Perez-Guzman et al. 2000). The other report showed no statistical difference between all the groups, although a more detailed statistical analysis suggested an association between lower lobe disease and female gender. Another finding was that lower lobe disease in patients older than 40 years was more frequent in diabetics, whereas the frequency of cavitations seemed to be higher in patients with insulin-dependent diabetes (Bacakoglu et al. 2001). This report also showed a lower frequency of smear-positive patients in diabetics compared to non-diabetics. Given the higher incidence of tuberculosis in diabetics, tuberculin testing should be carried out in these patients. However, because of the risk of anergy, appropriate controls must be done in poorly controlled diabetics for a proper evaluation of the results. Prophylactic isoniazid is indicated in patients with a positive PPD skin test because of the high risk of reactivation of the disease. Treatment regimens are otherwise similar to those of non-diabetics. As peripheral neuropathy is a secondary effect of isoniazid, pyridoxine should be given with the antibiotic and all these patients should be carefully monitored for clinical symptoms of both neuropathy and hepatic disease (Koziel and Koziel 1995). Therapy of tuberculosis is the same as for nondiabetic patients. However, there are data suggesting higher relapse rates among diabetics, so longer courses of therapy may be indicated in order to minimise relapse incidence (Kameda et al. 1990). Moreover, a recent report establishes a higher incidence of multidrug-resistant tuberculosis among diabetics (Bashar et al. 2001). However, no other reports have confirmed this finding, so changes in therapeutic protocols are not currently indicated unless specific epidemiological data suggests a high rate of multidrug-resistant tuberculosis in the area. In this case, specific measures for proper management of the cases must be implemented and, probably, antimicrobial susceptibility testing would be indicated for all the isolates of M. tuberculosis from these patients.

7.2 Special Groups (2). Transplantation Transplantation is a therapeutic procedure that is increasingly being employed worldwide for numerous diseases. However, it implies several procedures to minimise the risk of rejection and most of them produce an alteration in the immunity of the patient.

J. Esteban The impairment of immune function is a circumstance that must be taken into account in all these patients because it increases the risk of infections, caused by both common and rare organisms. The degree of immunosuppression and other complications is quite different between bone marrow transplants and solid organ transplants and so is the risk of infection, which is higher for bone marrow transplantation. This is so because immunosuppressive therapy is given before and after the transplant; the risk is higher in allogeneic transplants than in autotransplants. In the pretransplantation period the presence of neutropenia and altered anatomical barriers are the main risk factors for development of infection. In the pre-engraftment period, aplasia due to the conditioning regimen employed, represents the major immune defect. In the third phase, neutropenia abates and the anatomic barriers are restored, although immune alterations persist. The presence of graft-versus-host disease and its therapy in this period represents further complications that increase the risk of infection. Later, in a fourth period, chronic graft-versus-host disease represents almost the only risk factor for the development of infection (Sable and Donowitz 1994). Tuberculosis (a disease that is closely related to immune function) was expected to be more frequent among transplanted patients due to the broad impairment of host immunity that they suffer. However, initial reports showed lower rates than those expected, although they were higher than those for the general population (Kurzrock et al.1984). An initial explanation for this finding was that these studies were done in countries with low global rates for tuberculosis (Roy and Weisdorf 1997). However, recent reports from countries with higher prevalence rates suggest that the initial findings should be reviewed and that tuberculosis rates in this group of patients are in fact similar to the rates of the general population (Aljurf et al. 1999; Budak-Alpdogan et al. 2000; George et al. 2001; Ip et al. 1998). Despite these data on incidence, tuberculosis in patients that underwent bone marrow transplantation had special characteristics compared to those of general population, perhaps due to the immune impairment that these patients suffer. There is a high number of cases of extrapulmonary or disseminated disease in many reports (Aljurf et al. 1999; George et al. 2001), although in others respiratory disease is the most frequent form of the disease (Ip et al. 1998; Kurzrock et al. 1984). However, even respiratory cases can have atypical presentations (Kurzrock et al. 1984). These facts, together with the relatively low

Tuberculosis in Special Groups and Occupational Hazards

incidence of the disease can often mislead the physician, and in many cases therapy is delayed. Some cases are diagnosed after the patient's death. Because of the severity of the disease in these cases, an aggressive approach should be used to diagnose the disease as soon as possible. Common therapeutic regimens can be used for these patients. These regimens, however, must be started as soon as possible, as diagnostic tests are not always available on time. A recent official report establishes the measures for prevention of tuberculosis in this group (Centers for Disease Control 2000). Measures include screening of candidates by careful medical history and tuberculin skin testing by the Mantoux method. For immunocompromised patients, a positive test is defined as 5 mm or more of induration. Persons with a positive test or a history of positive test must be evaluated for detection of active tuberculosis. If no disease is detected, a full 9month course of isoniazid chemoprophylaxis must be performed. This regimen is also indicated for patients who have been in close contact with an active tuberculosis case, regardless of skin test status. Routine anergy testing is not indicated in these patients. An alternative for isoniazid therapy is a 2-month course of daily pyrazinamide/rifampin, although there are only limited data about the safety and efficacy of this. It must also be taken into account that rifampin interacts with many drugs, such as cyclosporine or corticosteroids, so routine use of this regimen is not recommended. BCG vaccination is also contraindicated in these patients, because the BCG strain might cause severe (even fatal) disease in immunocompromised patients (Abramowsky et al.1993). Solid organ transplantation includes a broad range of procedures with variable degrees of immunosuppression that depends on the organ to be transplanted. Prevalence of tuberculosis among transplanted patients in developed countries is as high as 1% (Patel and Paya 1997), which is higher than that for the general population in these countries (Singh and Paterson 1998). Most cases of tuberculosis have been reported in renal transplant recipients, although cases have also been reported in liver, heart and lung transplanted patients (Singh and Paterson 1998). Unlike other infections, tuberculosis in these patients is not related to the time since the transplantation procedure (Patel and Paya 1997). However, the disease appeared earlier in heart, liver and lung transplant recipients than in renal ones. Although transmission of tuberculosis through the graft has been reported (Graham et al. 2001;

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Mourad et al. 1985), this mechanism of infection is rare, and reactivation and primary infection are the most common mechanisms of acquisition of the disease. There have also been reports of outbreaks of tuberculosis in transplantation programmes (Sundbery et al.199l). The clinical aspects of the disease include a high number of cases of extrapulmonary or disseminated tuberculosis (49%) (Singh and Paterson 1998). Transplantation of organs that imply a high level of immunosuppression, such as heart transplant, show an even higher proportion of extrapulmonary cases (Munoz et al. 1995). Among extrapulmonary cases, presentations include gastrointestinal, hepatic, skin, muscle, osteoarticular, genitourinary, ganglial, central nervous system and other more uncommon sites of disease such as larynx, tonsils or eye (Patel and Paya 1997; Singh and Paterson 1998). Clinical data from these patients were similar to those from the general population. Mortality of tuberculosis in transplanted patients is 30%. Disseminated disease, prior rejection and treatment with OKT3 or anti-T cell antibodies are significant predictors of mortality in these patients. Clinicians should suspect tuberculosis in patients who present with fever and radiographic infiltrates or symptoms of extrapulmonary disease. Smears for acid-fast stains and mycobacterial cultures should be performed on appropriate specimens. Pulmonary cases require an aggressive approach if the results of sputum testing are inconclusive because these patients have a high mortality risk. Procedures to be performed include bronchoscopy, including bronchoalveolar lavage or transbronchial biopsy, or even open lung biopsy (Patel and Paya 1997). Other techniques (bone marrow biopsy, liver or other organ biopsies, blood cultures for mycobacteria) are also useful when extrapulmonary or disseminated tuberculosis is suspected, although the results of some of these (for example, blood cultures) can take a long time (Esteban et al. 200lb). Samples should also be sent for pathological evaluation, looking for suggestive lesions such as granuloma formation. The treatment of transplanted patients with tuberculosis must be performed with at least two bactericidal drugs and during a longer period of time (l2 months) than that for common regimens (Patel and Paya 1997). Hepatotoxicity is a side effect of great importance in the context of liver transplantation, so these patients must be monitored carefully, including the performance of biopsies to detect hepatitis or graft rejection. Published data has shown that hepatotoxicity requiring discontinuation

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of isoniazid therapy occurs in 41 % of liver transplant patients. The percentages of hepatotoxicity for other patients were lower (Singh and Paterson 1998). It is also of interest to note that antimycobacterial therapy has important drug interactions. Rifampin can induce hepatic enzymes and accelerate metabolism of drugs such as cyclosporine or corticoids, so close monitoring of drug levels must be performed (Patel and Paya 1997). Tuberculin skin testing is indicated both in transplant recipients and, if possible, in donors. A history of tuberculosis exposure or past disease must also be recorded. Although no study has demonstrated the efficacy of isoniazid prophylaxis in tuberculin skin test positive transplanted patients, this therapy has been reported to minimise the risk of developing tuberculosis in renal patients (John et al. 1994). However, the indication of chemoprophylaxis must be balanced against the risk of hepatotoxicity. If possible, isoniazid should be given several months prior to transplantation. Other patients that should be considered as candidates for chemoprophylaxis include those with skin test conversion, a history of inadequately treated tuberculosis, close contacts with active tuberculosis cases, patients with chest radiograph abnormalities or the immunocompromised host. Prophylaxis should also be considered for patients who receive grafts from donors with a history of tuberculous infection or disease (Lichtenstein and MacGregor 1983; Qunibi et al. 1991).

J. Esteban

Patients with leukaemia have a higher risk of developing disseminated or extrapulmonary disease compared to those with other haematological tumours (Libshitz et al. 1997; Morii and Narita 1998). Another report mentions a high incidence of tuberculosis in hairy cell leukaemia (Advani and Banavali 1989), with incidences as high as 50% for patients suffering from this disease. The incidence of these manifestations is also higher in patients with lymphomas than in the general population, especially in Hodgkin lymphoma. However, in these diseases extrapulmonary tuberculosis is usually a focal disease and not a disseminated one (Melero et al. 1992). In bone marrow transplanted patients (a group of patients that often suffer from malignancies as well) the disease has unique characteristics that make diagnosis very difficult, such as involvement of extrapulmonary organs or atypical signs and symptoms. Atypical radiographic signs can also appear in pneumonia (Libshitz et al. 1997). Some of these presentations can go unrecognised until the death of the patient (Libshitz et al. 1997; Morii and Narita 1998; Muller et al. 1980; Rosenthal et al. 1975). An aggressive approach must be taken for an early diagnosis of these patients, including the taking of biopsies or the performance of bronchoscopic procedures. Proper therapy must then be performed using common schemes. Screening using the Mantoux technique must be done in patients who are going to receive aggressive chemotherapy, as the latter can induce severe immunosuppression (Morii and Narita 1998). In patients with a positive skin test, chemotherapy with isoniazid is indicated. 7.3 In solid organ cancers, tuberculosis is mainly pulmonary, and extrapulmonary or disseminated Special Groups (3). Malignant Diseases disease is less common (Libshitz et al. 1997). The Tuberculosis in cancer patients does not seem to have highest incidence is seen in head and neck cancer, very different characteristics to that in the general lung cancer and gastrointestinal cancer (Kumar et population, despite an increased incidence of the dis- al. 1999; Libshitz et al.I997). Pulmonary tuberculosis ease (Libshitz et al. 1997). However, some neoplastic and lung cancer can appear simultaneously, and in diseases have special characteristics that can affect some series the relation between these diagnoses is more frequent than other associations (Libshitz et the course of the disease. Haematological malignancies have a higher al. 1997). Nevertheless, in other series the incidence incidence of tuberculosis than solid organ cancers of simultaneous diseases and sequential diseases is (Libshitz et al. 1997; Melero et al. 1992). Among similar (Tamura et al. 1999). The location of tubercuthese malignancies, acute leukaemia and Hodgkin losis and lung cancer can be the same (in one series lymphoma have an even higher incidence than other M. tuberculosis was cultured from collapsed lobes in diseases (Advani and Banavali 1989; Libshitz et al. patients with lung cancer (Libshitz et al. 1997» or 1997; Melero et al.I992). The patients with these hae- different (Tamura et al. 1999). A normal radiographic matological disorders have impaired immunity. This pattern can appear if HIV infection is associated with is due to the disease itself and to cancer treatments, cancer and tuberculosis (Libshitz et al.I997). Because which can include radiotherapy, chemotherapy, of the risk of simultaneous infection and neoplastic corticosteroids and even bone marrow transplant. disease, samples for diagnosis should be sent to both

Tuberculosis in Special Groups and Occupational Hazards

microbiology and pathology laboratories. If cancer and tuberculosis appear in a sequential pattern, the disease seems to be more severe. This could be due to a greater debilitation of the patients during cancer therapy (Libshitz et al. 1997).

7.4 Special Groups (4). Drug Abusers Drug abusers have distinct characteristics that affect the course and management of concurrent tuberculosis. Many of them are outcasts and have antisocial behaviours, such as criminality or erratic behaviour (including auto-medication or even trading with the pills to obtain money or drugs), which are a serious hazard to the correct treatment of any disease. An important number of them also have other important risk factors, such as HIV infection, malnutrition or bad hygienic practices. The close association between HIV infection and intravenous drug abuse makes it difficult to differentiate between the role of the two factors in the development of the disease. Relatively little is known about the immune system in drug abusers. In the case of HIV-infected patients, the effects on the immune system of the HIV are so important that they minimise other possible defects. However, in HIV-non-infected patients, immune defects can also be found. In this group of intravenous drug abusers serum IgM and IgG levels are frequently elevated (Brown et al. 1974), and this elevation is usually accompanied by autoantibodies. The repeated injection of antigens is a possible explanation for the hypergammaglobulinaemia. Tcell function is also altered, as several in vitro studies with T-Iymphocytes have shown (Levine and Brown 2001). Delayed hypersensitivity skin test reactions are sometimes diminished or absent. Both increases and decreases in the T-Iymphocyte cell count have been reported. Morphine depresses several monocyte functions affecting antiviral defence and it also inhibits the monocyte response to activating stimuli (Stoll-Keller et al. 1997). Some studies describe the effects of morphine and, to a lesser degree, methadone on the neutrophil-monocyte system. The clinical implications of these in vitro findings are, however, unknown (Levine and Brown 2001). The relation between tuberculosis and intravenous drug abuse has been well known for many years (Perlman et al. 1995). Recent reports show rates of 44% positive Mantoux test for HIV-negative intravenous drug users (Daley et al.1998) and a 6.8% rate for

97

tuberculous disease among intravenous drug abusers (Friedman et al. 1996). Other studies have described drug abuse as a statistically significant risk factor for tuberculosis in the general population (Iftigo-Martinez et al. 2000; Moro et al. 2002). Drug abuse is linked to unemployment and low socio-economic status. In these population groups tuberculosis is a prevalent disease. Many intravenous drug users also have a previous history of imprisonment or homelessness. These circumstances increase the risk of tuberculosis, as will be explained further in the chapter. The AIDS epidemic has also affected the relation between drugs and tuberculosis. AIDS patients have a high risk for development of tuberculosis, both when they are positive for the tuberculin skin test and when they are anergic (Selwyn et al. 1992). In these patients, "atypical" presentations of the disease are also found, including a higher percentage of extrapulmonary or disseminated disease (Havlir and Barnes 1999; Hill et al. 1991; Small and Fujiwara 2001). In recent years, outbreaks of multidrug-resistant tuberculosis with extremely high mortality have been reported (Perlman et al. 1995). In these outbreaks, primary progressive disease has been described and many of the patients in these outbreaks were also intravenous drug abusers. Diagnosis of tuberculosis in intravenous drug abusers is affected by the presence of concomitant HIV infection and also by the association of other infections at the moment of diagnosis. This can mask clinical data, so clinicians should rule out tuberculosis in any drug abuser who presents with fever (Hamburg and Frieden 1994). Screening of tuberculous infection is also difficult in these patients as HIVinfected patients are generally anergic. In this setting a single skin test does not predict the development of tuberculosis (Friedman et al. 1996; Perlman et al. 1995; Selwyn et al. 1992). Moreover, recent data suggest that anergy tests are not reproducible, so it is not recommended to perform these tests in HIV patients anymore (Havlir and Barnes 1999). The use of chemoprophylaxis in infected patients has been proved to be effective in preventing the development of active disease (Gourevitch et al.1998; Selwyn et al.1992; Snyder et al.I999). However, recent studies have shown that chemoprophylaxis does not reduce the incidence of disease in HIV anergic patients, so in these cases it is not currently indicated (Daley et al.1998; Havlir and Barnes 1999). Treatment of the active disease has also been shown to be effective (Small and Fujiwara 2001), although in HIV patients some modifications should be made. In these patients, therapy with protease

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98

inhibitors and reverse-transcriptase inhibitors for the treatment of HIV infection complicates the therapy of tuberculosis because they interact with rifampin. The administration of this antibiotic can result in sub-therapeutic levels of antiretroviral drugs. In these cases, rifabutin, a rifampin derivative, can be used with efficacy instead of rifampin in the common regimens (Havlir and Barnes 1999; Small and Fujiwara 2001). However, the main problem in performing a proper anti-tuberculous therapy remains in the low compliance of these patients. A great number of intravenous drug abusers abandon therapy, and the sequels of this include therapeutic failure, prolonged infectivity, increased rates of transmission and the development of drug-resistant strains (Perlman et al. 1995). Several mechanisms have been developed in order to increase compliance among these patients. Directly observed therapy (DOT) is a strategy that has proved useful for the treatment of low compliance patients (Gasner et al. 1999; Perlman et al. 1995). However, some patients refuse any therapy. Legal measures have been developed for these cases, including the arrest of these patients during the treatment period (Gasner et al. 1999). These measures, however, must be restricted to only a few "impossible" patients who refuse any therapeutic measure (including DOTs), so arrest should remain only as the last resort to cure patients in tuberculous programmes.

7.5

Special Groups (5). Correctional Facilities

Prisons and other correctional facilities, such as jails, penal colonies and prisoner of war camps, represent a very special community for different reasons. Prisoners are often young males from socio-economically disadvantaged groups, poor educational backgrounds and ethnic minorities, including illegal immigrants. Prison conditions frequently include overcrowding, high incidence of HIV-infected individuals and low standards of hygiene. In addition, several behaviours, such as drug abuse and sex between men, often promote illness. Many of these circumstances, for example overcrowding, increase the risk of transmission of airborne infectious diseases, and HIV infection is considered the strongest known risk factor for the development of tuberculosis (Centers for Disease Control 1996). Moreover, correctional facilities are not closed environments; so many people circulate between the prison and the open society (Fig. 7.1).

These include liberated prisoners, but also workers and visitors. A history of previous imprisonment is one of the statistically significant risk factors for tuberculosis in some reports (Inigo-Martinez et al. 2000; Moro et al. 2002). Prisons are among the communities with the highest incidence of tuberculosis. Many reports have published annual case notification rates as high as 7200 cases/l00,000 (Coninx et al. 2000; Koffi et al. 1997; Wares and Clowes 1997) and even in developed countries, rates are several times higher than those of the general populations (Jones and Schaffner 2001; Fernandez-Martin et al. 2000; Centers for Disease Control 1996) (Table 7.1). An obvious consequence of this is the high risk of becoming infected for all the people who dwell in these institutions, especially when the period of imprisonment is long. Molecular epidemiology analysis confirmed internal transmission of tuberculosis inside correctional facilities, with more then 25% of the patients appearing in clusters (Fernandez-Martin et al. 2000; Laniado-Laborin 2001). This is true even if the time that the prisoner spends in the correctional facility is shorter than in prisons (Jones et aI.1999). Other problems include delay in the diagnosis of sputum smear-positive cases, failures in medical services, transfer of infectious patients inside and between prisons, and patients with low compliance for therapy. The diagnosis of these patients can be difficult because of the concomitant existence of other factors, such as HIV infection, that can increase the possibility of extrapulmonary, disseminated or even atypical forms of the disease. Other factors that could affect the diagnosis are of another nature: in some prisons, patients with disease sell smear-positive sputa to other prisoners so that they can avoid hard labour (Drobniewski 1995) or have access to better living conditions Non-infected Prisoners

i

__________

Infected"Prisoners ----------. Prisoners with active tuberculosis

Non-infeited prison workers

t Infected prison workers

~

Prison workers with active disease _

/

Outer Society

: Risk of contagion

-+ : Possible evolution of the patients

Fig.7.1. Relation between different kinds of patients in the correctional setting

Tuberculosis in Special Groups and Occupational Hazards Table 7.1. Incidence of tuberculosis in correctional facilities Location of correctional facility (year)

Annual rate (per 100,000)

Tomsk, Russia (1996) Azerbaijan (1994) Bouake, Cote d'ivoire (1990-1995) Madrid, Spain (1997-1998) Memphis, USA (1995-1997) New York, USA (1993)

7,000 4,667 7,200 693 274 139.3

in the prison hospital. Power structures in prisons also have direct implications in the management of tuberculosis, affecting detection and treatment of patients (Reyes and Coninx 1997). The therapy of diagnosed cases is also problematic. Some patients use the pills for trading with other prisoners or even correctional workers. Other prisoners manage to obtain the pills and take the drugs without proper medical and control. This inadequate therapy can lead to the development of resistant strains. This risk must be added to the bad compliance of many patients that implies the abandonment of therapy after a few weeks, especially if the prisoner is liberated and no specific programme exists to co-ordinate the correctional medical structure efforts with the open society structures. Sometimes, when anti-tuberculous drugs are scarce, a black market for these drugs can appear, especially if tuberculosis is an important problem in the facility (Coninx et al. 2000). This fact can be of extreme importance in the prisons of developing countries, where drug are not always available. Mobility of prisoners is another factor that can extend the disease between prisons, as prisoners can be transferred when they are still infectious (Reyes and Coninx 1997). This contributes to the transmission of multidrug-resistant strains, as in New York in 1991 (Drobniewski 1995). Proper measures must be implemented to minimise the problem of tuberculosis inside correctional facilities. These measures are based in a few clear points: rapid detection of patients with tuberculous disease, isolation and therapy for these cases, detection of infected patients and the institution of chemoprophylaxis when indicated. The pivotal measure for tuberculosis control is the active detection of infectious cases. This detection must be done using screening tests, such as symptom screening, tuberculin skin test or chest radiograph screening (Centers for Disease Control 1996). The selection of the initial screening test must be done according to the individual characteristics of each centre.

99

Symptom screening is useful in areas where tuberculosis prevalence is high. The presence of prolonged productive cough, fever, haemoptysis and other systemic symptoms (malaise, weight loss, etc.) suggest the possibility of active tuberculosis. In these cases, proper diagnostic measures must be performed, including radiological and microbiological tests, and also a careful medical history taken about previous episodes or treatments. The performance of chest radiographs for all symptomatic patients is always indicated. There are also studies showing cost-effective use of chest radiographs for all the prisoners in areas with high prevalence (Jones and Schaffner 2001). The results of chest radiography must be interpreted together with a careful medical history, and other diagnostic tests are indicated if there is suspicion of active tuberculosis. The Mantoux test is the preferred tuberculin skin test. However, its interpretation must be cautious in this setting, because there are areas where BCG vaccination is currently used. The high prevalence of HIV-infected patients can also affect the results, as severely immunocompromised patients have negative test results. All patients with positive skin test should be medically evaluated and other tests (like radiographs or sputum analysis) must be performed if indicated. Negative skin test individuals without anergy should be tested again periodically. Screening tests should be done both for correctional inmates and for correctional workers, because both groups have a high risk of infection. The earlier the detection of these patients, the less is the risk of infection for other people, as diagnosis must lead to proper isolation measures and initiation of therapy. Isolation measures are recommended for patients with respiratory tuberculosis. They will need transport to hospital if the prison lacks proper isolation measures. Therapy must be performed according to common protocols. It must be directed observed therapy (DOT), as there is a low level of compliance in many patients. DOT assures therapy of the patients and minimises the risk of developing resistances. Preventive therapy with isoniazid (6-12 months) is recommended also for infected individuals without active disease. Anergic HIV-infected patients should also receive prophylaxis because of the high risk of development of active tuberculosis in these patients (Selwyn et al. 1992). Co-ordination between correctional administrations and health programmes outside correctional facilities is essential, as many patients abandon therapy

J. Esteban

100

when they are liberated. Active searching and followup of these patients by tuberculosis programmes in the different communities should ensure the proper therapy of these patients and minimise the risk of relapse, therapeutic failures or development or resistances (Maher 1998; Centers for Disease Control 2000).

7.6 Special Groups (6). Shelters Homelessness is one of the risk factors for the development of tuberculosis, with estimated prevalences from 1.6% to 6.8% (Centers for Disease Control 1992a). Among these individuals, other risk factors for tuberculosis are found, such as malnutrition, poor hygiene, immigration from countries with high prevalence of the disease, substance abuse (both alcohol and/or intravenous drugs) and, in recent years, HIV infection (Centers for Disease Control 1992a; Moss et al. 2000). It is difficult to determine the true role of each of these factors, as they all playa role. However, an important fact is the recent finding of a high proportion of clustering when molecular epidemiology studies are applied to this population (Gutierrez et al. 1998; Moss et al. 2000). These data suggest a high proportion of transmission of tuberculosis in this population. Shelters and other provisional dwellings are considered as foci where transmission of the disease occurs and shelter-associated tuberculosis outbreaks have been described (Nardell et al. 1986). Because of the difficulties of establishing an adequate prevention programme in shelters, measures should be directed at rapid diagnosis and treatment of infectious cases (Stead 1989). Clinical characteristics of tuberculosis in this group are similar to the general population and variations in them are due to the presence of other concomitant factors, such as HIV infection. Thus, people with symptoms and signs of pulmonary tuberculosis should be studied with chest radiographs and, if available, proper microbiological studies. When the diagnosis of tuberculosis is made, therapy should be started using common therapeutic regimens (Centers for Disease Control 1992a). The most important variable in the outcome of these patients is the low compliance that many of them have. Directly observed therapy is indicated for these difficult cases, and even hospitalisation throughout all the treatment is indicated in individuals that have mental diseases that incapacitate them to follow the therapy. Several incentives have been recommended to increase the adherence to therapy, such as travel

tickets, food, clothes or even money. These incentives have proven to be effective in increasing the compliance of these patients (Centers for Disease Control 1992a; Moss et al. 2000). Contact study of the infective cases is more difficult. The mobility of this population makes them difficult to study. Other troublesome factors are the use of aliases by the patient or the potentially infected persons. Tuberculin skin test positive patients who were treated with isoniazid are not entirely free of risk, as shown by one of the first studies to describe an outbreak among these populations (Nardell et al. 1986). The frequent description of clustering supports this finding, even in populations with high prevalence of latent infection (Moss et al. 2000). However, if tuberculin skin testing is performed, the concomitant presence of factors such as HIV infection among these patients must be taken into account (Centers for Disease Control 1992a). Structural measures in the shelters have also been considered as useful to prevent transmission of the disease. The use of ultraviolet lamps has been recommended to disinfect the air. These lamps, however, must be installed appropriately to minimise the risk of eye and skin lesions. Enhanced ventilation systems could also help to reduce the airborne transmission of the disease (Centers for Disease Control 1992a).

7.7 Special Groups (7). Immigrants In the early years of the twenty-first century, immigration from many countries to more developed ones has become a problem of great importance. The migratory movements of populations between countries have become more frequent, and this represents an easy route for transmission of infectious diseases from one place to another. Tuberculosis prevalence is higher in countries from Africa and Asia, according to the WHO data (World Health Organization 2001). Movements affecting people from these high prevalence areas are of great interest because they can affect the control programmes of other countries. As a matter of fact, they have been considered the cause of resurgence of the disease in Western countries (Rieder et al. 1994). In our hospital, the relative percentage of immigrants with tuberculosis has gone from 5.05% in 1995 to 21.8% in 2000. This data probably explains the maintenance of tuberculosis rates in our area, despite the decreasing incidence in the local population (Dr. M.C. Alvarez-Castillo, personal

101

Tuberculosis in Special Groups and Occupational Hazards

communication, and Epidemiological Bulletins from grants and transmission between them (Lillebaek et al. 2001).A possible explanation is the low social conthe Comunidad de Madrid) (Figs. 7.2 and 7.3). Illegal immigration adds more problems to the tact that exists between the two communities. Other control of the potentially infected population. A reports suggest that tuberculosis in immigrants is the study from Spain, a country frequently crossed by result of reactivation and not of primary disease due immigrants en route to other European countries, to recent infection (Chin et al. 1998; Moro et al. 2002; detected that 78% of the immigrants with active Samper et al. 1998). tuberculosis were illegal ones (Huerga et al. 2000). Drug resistance among these patients is also a risk This population involves more factors that compli- to be taken into account. Resistance rates in many cate the management of patients, such as overcrowd- countries from which immigrants go to Western states ing, language difficulties, poverty, fear to go to health are high (The WHO/IUATLD Global Project on Anticare facilities and frequent imprisonment. All these tuberculosis Drug Resistance Surveillance 1994-1997, factors probably affect the therapy compliance in 1997). When these patients develop tuberculous disthese patients, and compliance is even lower for che- ease this data is of great importance in the planning moprophylaxis of infected patients without active of proper therapeutic regimens. Multidrug-resistance disease (Duran et al. 1996). However, strict follow-up is also important in these patients (Fig. 7.4). Antimiand other complementary measures, such as the use crobial susceptibility testing is indicated in patients of the patient's language for examinations, made it who come from world areas with high resistance rates possible to increase the compliance of tuberculous against anti-tuberculous drugs, as these rates are carried on by this group of patients (Huerga et al. 2000). patients to 78% (Huerga et al. 2000). The potential transmission of tuberculosis from immigrants to local population is a theoretical risk. However, a Danish study showed only minor transmission between these populations. This study 7.8 performed a molecular epidemiology analysis Occupational Hazards (1). using both RFLP and spoligotyping, and found only Health Care Workers nine minor clusters in both Danish and immigrant patients. Many other clusters were found, suggesting Among the different professions, health care workers the existence of predominant strains between immi- are at this time almost unanimously considered a spe-

50

45 40

35 30 25

20

15 10

District 7

5

o

1996

1997

1998

1999

Fig. 7.2. Incidence of tuberculosis (No. cases/lOO,OOO inhabitants). Comunidad de Madrid and District 7. 1996-1999

Fundaci6n Jimenez Diaz 5L--~-­

o

1996

1997

1998

1999

Fig. 7.3. Percentage of tuberculosis in immigrants. Fundaci6n Jimenez Diaz, Comunidad de Madrid and District 7Madrid (District 7 includes Fundaci6n Jimenez Diaz) 1996-1999

102

Fig.7.4. Multidrug-resistant tuberculosis in an immigrant patient

cial kind of worker with a higher risk of tuberculous infection than other workers or the general population. However, this common idea has not always been considered an unquestionable fact. It was not until the decade of 1950s that the increased risk for health care workers was recognised, in the years before that there were even several authors that considered the health care worker a profession with a lower risk of being infected than other people (Sepkowitz 1994). Nowadays, at least in developed countries, health care facilities are institutions where contact between workers and tuberculous patients is a frequent matter, chiefly in those hospitals where the number of tuberculous patients is high. In our experience, the majority of diagnoses of tuberculosis were made in the hospitals, where patients go when they are acutely (or even moderate chronically) sick. These are also the institutions where tuberculous patients spent at least the first days of their therapy. In some cases, the patients could spend several days undiagnosed, until a proper diagnosis was made after performing several tests of different kinds in different areas of the hospital. Even in a few cases death occurred and the diagnosis was delayed until autopsy or cultures gave the final results (Esteban et al. 2001bj Flora et al. 1990; Kramer et al.1990; Rosenthal et al.I975). Delay in diagnosis is a problem common in those hospitals or institutions where tuberculosis prevalence is low

J. Esteban and it affects only a minority among all the patients. However, this delay could have very important consequences. Health care workers could be infected because no proper preventive measures were taken before the diagnosis, as has been demonstrated in epidemic outbreaks (Frampton 1992; Griffith et al. 1995; Pearson et al. 1992) and, recently, in an environment where there was no outbreak (Greenaway et al. 2002). However, it is important to discriminate between the risk of infection and the risk of developing the active disease. Historical data concerning the last topic showed an increased incidence in nurses that could be as high as 500 times higher than in the general population (Sepkowitz 1994). Recent reports, however, have shown incidences analogous to that of the overall population (Capewell et al. 1988) or even lower (Raitio and Tala 2000). The low prevalence of tuberculosis and other risk factors for developing disease (such as HIV infection), together with BCG vaccination, has been considered in one of these reports as the explanation for this phenomenon (Raitio and Tala 2000). Speed in the detection of infectious cases and, again, BCG vaccination, are the reasons shown in the other article (Capewell et al.1988). Moreover, it must be taken into account that health care workers are usually healthy young people, without clear risk factors for developing tuberculosis, except for a few individuals (the healthy worker effect; Menzies et al. 1995). Probably the reasons for these data are difficult to find and possibly they are multifactorial. However, the risk of developing tuberculosis has been clearly recognised in several reports of outbreaks, including some due to multidrug-resistant strains (Griffith et al. 1995; Menzies et al.1995), so it must still be considered, mainly in some special circumstances. The risk of infection has also been considered another hazard of work in health care workers since the same time that the risk of tuberculous disease was acknowledged. Historical data documented conversions as high as 100% among health care workers (Sepkowitz 1994). More recent data indicate a high risk of infection among these workers (Louther et al. 1997; Menzies et al. 1995; Schwartzman et al. 1996), although clearly lower than the previous reports. Global annual infection risk has been reported between 0.2-10% (Menzies et al.1995). This risk could be even higher in outbreaks (Griffith et al.1995; Pearson et al. 1992; Wenger et al. 1995). However, not all health care workers, or even all health care facilities, had identical risks. More detailed studies indicated that the risk of infection is related to the number of tuberculous patients admitted by the hospital,

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Tuberculosis in Special Groups and Occupational Hazards

the contact between these patients and the different workers and other factors such as inadequate ventilation or delay in the diagnosis (Boudreau et al. 1997; Greenaway et al. 2002; Louther et al. 1997; Menzies et al. 1995,2000; Schwartzman et al. 1996). Hospitals with fewer than ten tuberculous patients/year of less than one patient with active disease/l00 workers per year had fewer Mantoux conversions than those with a higher number of admissions due to tuberculosis (Menzies et al.1995; Schwartzman et al.I996). Nurses are among those categories of workers with high conversion rates in many studies (Boudreau et al. 1997; Griffith et al. 1995; Ktsanes et al. 1986; Louther et al. 1997; Schwartzman et al. 1996), even when they are still students (Esteban et al. 2001a; Lainez et al. 1999; Sepkowitz 1994; Weiss 1973). However, other conditions such as housekeeping, ward clerks and workers in a bronchoscopy department have been documented as having high risk of infection (Boudreau et al. 1997; Louther et al. 1997; Schwartzman et al. 1996). Preventive measures has also been described for health care workers since the decade of 1930 (Sepkowitz 1994). Nowadays, several guidelines have been published where the measures for protecting the workers against infection are clearly specified (Centers for Disease Control 1994; Joint Tuberculosis Committee of the British Thoracic Society 2000; Moreno-Guillen et al. 1997), although these are not always adequately implemented in daily routine (Sutton et al. 2000). These measures have obviously been developed for respiratory disease, which is considered as contagious because tuberculosis is transmitted through respiratory secretions in most cases, although there are reports of infection due to extrapulmonary tuberculosis (Frampton 1992; Hutton et al.I990). Two important groups of measures could be defined: proper isolation measures and a programme of tuberculin screening for the workers. Isolation measures (Table 7.2) include both structural and personal protective measures. Structural interventions include the isolation of infectious patients in individual rooms with proper ventilation. Several reports of outbreaks defined poor ventilation as the cause, at least in part, of the contagion (Menzies et al. 1995). Another report also described poor ventilation as an important risk factor for infection in a non-outbreak setting (Menzies et al. 2000). Rooms with negative pressure are therefore recommended for isolation (Centers for Disease Control 1994). These rooms must have the door closed and at least six changes of air per hour (Centers for Disease ControI1994). Other structural measures, such as the use

Table 7.2. Recommended isolation measures for patients with tuberculosis Subject

Specific measure

Patient lodgings

Private room with negative pressure. Door must be closed Biosafety respirators (N95) Limited to strictly necessary. The patient wears a surgical mask during transportation Hand washing Gloves when necessary Face protection when necessary Clean, non-sterile gown Proper management of sharp devices Minimise aerosols or other exposures to samples

Respiratory protection Patient transport

Standard precautions

of ultraviolet lamps, have also been recommended as useful and cheaper than the negative pressure isolation rooms (Hannan et al. 2000; Menzies et al.I995). Protective measures for individuals are directed at blocking the access of infectious particles to the lower respiratory tract. For this purpose, wearing a mask is the measure recommended. However, not all the masks offer similar protection. Classical surgical masks are designed to avoid contamination from the person who wears it. However, they are not designed to protect that person, and there are reports where failure of these masks to protect health care workers has been documented (Sokolove et al.I994). Because of this fact, surgical masks are only recommended to be worn by the infectious patients when they are outside the isolation room (Centers for Disease Control 1994; Moreno-Guillen et al. 1997). Dust and HEPA respirators are designed to minimise the risk of a particle of a minimal size (l fl) reaching the alveolus. Because there are differences in the percentage of filtration efficacy, Centers for Disease Control and other protocols recommend the wearing of respirators with at least 95% of efficiency and the capacity to filter particles of 1 fl. These recommendations have been adopted by other guidelines (Centers for Disease Control 1994; Moreno-Guillen et al. 1997). However, there are reports where the efficacy of the different kinds of respiratory protection is similar (Catanzaro 1995). Another difficulty is how to evaluate one single measure if other additional isolation measures have been taken, and it is impossible to estimate the degree of protection that is due to each single measure. Because of all these data, it seems wise to use the recommended respirators if possible, but when they are not available, wearing a surgical mask probably offers a certain degree of protection that is better than nothing.

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The other great pillar in the measures for protection of health care workers is the programme for detection of recent infections and treating them through tuberculin skin testing and proper isoniazid chemotherapy (Bolyard et al. 1998; Centers for Disease Control 1994; Joint Tuberculosis Committee of the British Thoracic Society 2000; Moreno-Guillen et al. 1997). The programme is based on the performance of a two-step initial test for those workers who are negative in the first test to minimise the risk of a booster reaction that could be considered as a conversion, and the periodical re-testing of those workers that were negative in the previous test (Bolyard et al. 1998). However, in our experience and also the experience of others (Clague et al. 1991), it could be very difficult to develop a tuberculin programme in hospital. The tuberculin skin test is different from other tests performed in a protocol of health control because it must be performed on one day and read on another. This fact seems to be of great importance, and there are many workers that do not go to the reading of the test because they estimate that the result "is negative". Moreover, if it is difficult to convince the workers to perform one test, the two-step test for negative ones is even more difficult to perform, so, in some guidelines, this two-step test is not recommended for all the workers and must be individually evaluated (Moreno-Guillen et al. 1997). The periodicity to perform the test is another problem, because if it is performed too frequently (according to the worker's idea), the worker will not go to have the test the next time. BCG vaccination is recommended in some guidelines (Joint Tuberculosis Committee of the British Thoracic Society 2000), although many others do not recommend it for all workers (Centers for Disease Control 1994). However, the use of BCG vaccination could be indicated for some health care workers in some special conditions, such as outbreaks of multidrug-resistant tuberculosis (Bolyard et al. 1998). As in the case of the use of respiratory protections, in these cases a certain degree of protection is better than nothing.

7.9 Occupational Hazards (2). Laboratory Workers Laboratory workers are a special category among health workers because their risk has special characteristics. Laboratory workers are not usually exposed

J. Esteban to contagion by the usual route (contact with a patient with active respiratory tuberculosis), but they are exposed to infection through manipulation of clinical samples or cultures. Because tuberculosis is a disease mainly airborne transmitted, procedures that imply aerosol formation have infection risk. In a laboratory, aerosol-producing activities include subculturing and streaking cultures, cooling and flaming loops, pipetting for mixing microbial suspensions, needle and syringe manipulations, centrifugation of samples and cultures, mixing and homogenising procedures (use of shakers, homogenisers, blenders, sonicators and other mixing instruments such as vortex) pouring fluids, open culture containers (including plates or tubers), spillage of infectious material, lyophilisation and filtration under vacuum and even egg inoculation and harvesting (Sewell 1995). Other risk procedures include manipulation of samples or tissues from infected patients for processing, as done in microbiology and pathology departments. Even direct inoculation of cultures has also been reported as the cause of tuberculosis in a laboratory and must be taken into account when working with pure cultures of the organism (Peerbooms et al. 1995). The recent introduction of automated liquid cultures for rapid detection of mycobacteria introduces another potential risk, because inoculation of samples and extraction of liquid from positive cultures are done using syringes and this manipulation has the risk of creating aerosols (Richmond et al. 1996). Several reports have stated that laboratory workers have a risk of infection two to nine times higher than the general population (Grist and Emslie 1994; Harrington and Shannon 1976; Sewell 1995) There is a report in which incidence of infection was as high as 100 times the frequency observed in the general population (MUller 1988). The progressive lowering of the incidence of tuberculosis has been claimed as the cause of the decrease in the number of these infections. This is seen in pathology laboratories rather than in microbiology ones (Grist and Emslie 1994). In a recent report, laboratory personnel did not have the highest rates of infection among health care workers, and their rate was even lower than the global rate (4.4 conversions per 100 person/year versus 5.2 conversions per 100 person/year) (Louther et al. 1997). Probably, all these data suggest the efficacy of the safety measures implemented in the laboratories. Mycobacterium tuberculosis is an organism included in the biosafety level III in most guidelines for laboratory safety when manipulation of cultures is performed (Anonymous 1997; Richmond et al. 1996; Richmond and McKinney 1999), although procedures such as

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preparation of smears for acid-fast stains only require biosafety level II measures (Richmond and McKinney 1999). The organism is considered as an agent with potential for aerosol transmission and the disease that it produces may have serious or even lethal consequences, but the risk of infection is only individual, and the population as a whole has only a minimal risk (Richmond and McKinney 1999; World Health Organization 1994). Measures to minimise this risk include a broad spectrum that goes from rooms with negative pressure that must be away from the common laboratory to the use of biosafety cabinets, protective masks, gloves and other protective clothes (Table 7.3). The biosafety level III laboratory must have special design features. A double door access to the laboratory is recommended. The mycobacteria laboratory must also be separated from the laboratory and the ventilation must be balanced to provide directional airflow into the working room (negative pressure), although in some legislation this measure is only recommended and not obligatory (Anonymous 1997). In others this measure is always considered as necessary (Richmond and McKinney 1999; World Health Organization 1994). In the era of multidrug-resistant tuberculosis, and because of the risk of dissemination of these organisms, negative pressure is a desirable objective to be achieved for all the laboratories that perform manipulation of mycobacteria cultures. However, this measure is far from being available in many laboratories. In a recent survey performed by the Sociedad Madrileiia de Microbiologia Clinica (Society for Clinical Microbiology of Madrid), in the year 2001 only 4 out of 15 mycobacteriology laboratories from the Madrid Com-

munity had negative pressure (data reported in the 2002 congress of the SMMC). Biosafety cabinets (BSCs) are a measure included as necessary in all the legislation and recommendations (Anonymous 1997; Richmond et al. 1996; Richmond and McKinney 1999; World Health Organization 1994). The design of these containment devices differs in significant ways, although both BSC classes II and I protect the laboratory worker and the environment (Richmond et al. 1996). Class II devices are also designed for protecting samples and cultures from contamination. In the case of mycobacterial laboratories, both BSC classes I and II are adequate for biosafety level II measures, but when biosafety level III measures are required (manipulation of cultures for identification or susceptibility testing, for example) a BSC class II or even class III is recommended as necessary (Richmond and McKinney 1999). Protective clothing for working is another important protective measure for workers. Wearing gloves, solid-front gowns, scrub suits or coveralls is recommended, and this equipment should not be used outside the biosafety level III laboratory. Reusable equipment must be disinfected before been laundered. Frequent hand washing is also recommended (Richmond and McKinney 1999) and in some guidelines foot-operated lavatories are also suggested as necessary (World Health Organization 1994). Respiratory protection is recommended only when BSCs are not available (Richmond and McKinney 1999). However, because of the risk of accidents due to compromise of the containment capacity of BSCs or other potential accidents (Richmond et al. 1996),

Table 7.3. Recommended laboratory facilities for working with Mycobacterium tuberculosis complex organisms in laboratories (Richmond and McKinney 1999; Anonymous 1997; World Health Organization 1994) Measures

USA

European Union

W.H.O.

Physical separation from other working areas or corridors Filtration of air through HEPA filters

Yes

Desirable

Yes

Yes, for exhausted air from cabinets Yes Yes Yes

Yes, for exhausted air

Recommended Yes Yes Yes

Yes Yes Not stated Not stated Not stated Yes

Yes Desirable Yes, for workbench, floor, walls and ceilings Desirable Yes Yes Yes Yes Yes

Yes Yes

Yes Not stated

Yes Yes

Restricted access Negative airflow into laboratory Surfaces easily disinfected The working area should be sealed for disinfection Surfaces resistant to chemicals Security storage of biological agents Window or other system for control of the workers Laboratory with full exclusive equipment Use of biosafety cabinets for manipulation of specimens and cultures Autoclave or incinerator available Self-closing, double-door access

Yes Yes Not stated Not stated Not stated Yes

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respiratory protection for working at biosafety level III is recommended, even if a BSC is currently used. Together with the biosafety level II or III measures, periodic tuberculin skin testing is recommended for laboratory workers (Richmond and McKinney 1999). This procedure should be repeated at least annually for all the workers who are Mantoux negative, and probably the frequency of re-testing should be higher for those who work in high risk areas. If an accident with a high level of exposure potential occurs, all negative tuberculin skin test workers should be retested, and then re-tested again at 3 month intervals until no new conversions are detected (Richmond et al. 1996). Chemotherapy for infected workers should be performed according to the general criteria.

7.10 Occupational Hazards (3). Silicosis The relation between tuberculosis and silicosis has been known for many centuries. In the past century many reports established high rates of tuberculosis for silicotic patients throughout the world (Rosenman et al. 1997; Small and Fujiwara 2001; Snider 1978). Local alterations of the lung, the favourable conditions for contagion in the mines, the difficulties for a rapid diagnosis and the high prevalence of tuberculosis in the population may explain the high rates of tuberculous disease among these patients. Important local alterations include the impairment of macrophage function, a cell that plays a significant role in the pathogenesis of the disease. Small particles of silica dust are ingested by macrophages. Then, the ingested particles disrupt the phagosomal membranes and their enzymes are released, causing the death of the affected cells. The lysed cells stimulate the migration of more macrophages, the proliferation of fibroblasts and the production of collagen. Sublethal doses of silica dust also affect the ability of macrophages to inhibit the growth of Mycobacterium tuberculosis (Snider 1978). The diagnosis of tuberculosis in silicotic patients may be a difficult matter. Symptoms are usually non-specific and could be due to other conditions or diseases. Acid-fast stains from the sputa of these patients are negative in many cases, probably because the tuberculous bacilli hardly reach the bronchial tree due to silicotic fibrosis. Chest roentgenogram is considered of great value in the diagnosis (Barras 1970). The appearance of new infiltrates, cavities, pleural effusion, coalescence

J. Esteban of nodules and the development of bronchial stenosis are radiographic criteria that strongly suggest the diagnosis of tuberculosis (Snider 1978), even in the presence of negative microbiological diagnosis techniques results. Accordingly, clinical and radiological data should be used to establish a diagnosis and begin adequate therapeutic regimens in these patients. Therapy of tuberculosis among these patients includes longer therapeutic regimens with the same drugs as for non-silicotic patients. Longer regimens are necessary due to the lower penetration of the drugs in the silicotic nodules. A study in Hong-Kong showed that the standard 6-month short course therapy is inadequate for these patients, as 22% of patients had bacteriological relapses in the 6-month therapy group compared to 7% of bacteriological relapses in the 8-month therapy group (Hong Kong Chest Service/Tuberculosis Research Centre and Madras/British Medical Research Council 1991). As a general recommendation, in the silicotic patient the sterilising phase must be prolonged and patients should be treated for at least 9 months. If pyrazinamide is not included in the regimen, therapy with isoniazid and rifampin must be prolonged for a minimum of 12 months (Snider 1978). Retreatment regimens must also be prolonged as much as 12 months and the follow-up of these patients should also be prolonged for 5 years. Preventive chemoprophylaxis with isoniazid has also been considered of great value in diminishing the rates of active disease. A study that compared the treatment of 825 silicotic patients with 2,408 untreated silicotic patients showed that preventive therapy with isoniazid decreases the risk of developing active disease from 2.66% to 0.32% (annual rates) (Snider 1978). Shorter courses of chemoprophylaxis with several drugs have also achieved lower rates of tuberculosis than in non-treated patients, despite its efficacy being unacceptably lower (Cowie 1996). Other studies have shown the efficacy of isoniazid chemotherapy. However, the difficulties are the same as for the therapy of tuberculous disease and longer regimens are indicated for these patients.

7.11 Occupational Hazards (4). Other Workers Other groups of workers also have high rates of tuberculous diseases. Farm workers and other workers involved in animal management are considered a

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group with high risk for tuberculosis (McKenna et al. 1996). Specific measures for the prevention of the disease in these groups have been established. Among these, it has been reported that disease rates are even higher in migrant farm workers (Centers for Disease Control 1992b; Ciesielski et al. 1991; Garcia et al. 1996; Hibbs et al. 1989; McCurdy et al. 1997; Schulte et al. 2001). This population has unique characteristics, such as overcrowding, close contact between individuals and high rates of immigrants from countries with a high prevalence of tuberculosis (Centers for Disease Control 1992b; World Health Organization 2001). Prevention and control of tuberculosis among these workers includes the following measures: rapid diagnosis and therapy of tuberculous patients, contact investigation (Mantoux test) and chemoprophylaxis of infected persons, and screening and proper therapy in immunosuppressed workers, including HIV-infected ones. Diagnosis causes no difficulties, as these patients suffer from a similar disease to that suffered among the general population. Therapy should not be performed with short-course standard schemes and, if needed, should include directly observed therapy (Centers for Disease Control 1992b). In some cases, the disease can be caused by Mycobacterium bovis rather than Mycobacterium tuberculosis in workers in contact with animals (Dalovisio et al. 1992; Fanning and Edwards 1991; Pillai et al. 2000; Thompson et al.1993). In these cases, therapy schemes should consider that M. bovis strains are intrinsically pyrazinamide-resistant, so this drug cannot be used. Proper veterinary control measures, such as detection and management of tuberculous animals, could also be useful in the control of the infection. Workers with pneumoconiosis other than silicosis also have high rates of tuberculous disease (Mosquera et al. 1994; Taguchi et al. 2000). However, in these patients the characteristics differ from those of silicotic patients, probably because of the implications of silica particles in the pathogenesis of the disease, as has been previously explained (Snider 1978). Differences in the incidence of tuberculosis can also be found between the different kinds of pneumoconiosis, depending on the material inhaled (Starzynski et al. 1996). The rates of tuberculosis are higher in workers of the metallurgical industry than in those from refractory material manufacturing plants (such as ceramics). Nine-month therapeutic regimens (2 months of daily streptomycin, isoniazid, rifampin and pyrazinamide followed by 7 months of daily isoniazid and rifampin) have proven effective in these patients with only a 5% relapse rate and an identical amount of therapeutic failures (Lin et al. 1987).

Another profession that has been reported to have unusually high rates of tuberculosis is that of funeral director (McKenna et al.1996). Tuberculous infection has also been reported to be very frequent in other funeral home employees (Gershon et al.1998).A possible explanation is the contact with infected corpses, in which the tuberculous bacilli can be cultured even if they have been embalmed (Weed and Boggenstoss 1951). This theory has been recently demonstrated in a case of tuberculosis acquired by a funeral director, which was confirmed by both conventional and molecular epidemiology (Lauzardo et al. 2001). Periodic tuberculin skin testing and the use of respiratory protection has been advised for these workers because of the high incidence rates of disease and infection (Gershon et al. 1998). Another study has reported a higher risk of tuberculosis in unemployed people (McKenna et al. 1996). This is probably linked to neglect and other risk factors that are found among unemployed people and that are different to the mere occupational ones.

7.12 Conclusion Tuberculosis is perhaps one of the oldest known human diseases. However, in the beginning of the twenty-first century, and despite the existence of a very effective therapy, this disease is still affecting humans as one of the most frequent and lethal ones. Airborne transmission favours infection in those circumstances where a concentration of tuberculous patients occurs especially if there is overcrowding, poor hygiene and bad ventilation. Moreover, recently developed aggressive therapies that are used for other diseases favour the development of active disease through alterations in the normal defences of the organism. The knowledge of these facts must lead to the development of effective measures to control the expansion of the disease. However, we must not forget that the most effective measure for the control of the infection is early diagnosis and therapy of patients with active disease. Continuous efforts must be made in this direction in order to minimise the risk of healthy people acquiring this disease that has been called "captain among these men of Death" (Daniel 1997).

Acknowledgements. I want to acknowledge Dr. Francisco G. Santos-O'Connor for his help with the English language of this document.

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110 comial de la tuberculosis. Programa regional de prevenci6n y control de la tuberculosis en la Comunidad de Madrid. Direcci6n General de Prevenci6n y Promoci6n de la Salud. Consejeria de Sanidad y Servicios Sociales, Comunidad de Madrid, Madrid, pp 65-79 Morii T, Narita N (1998) Mycobacterial infection in patients with hematologic disorders. Nippon Rinsho 56:3209-3211 Moro ML et al. (2002) Two-year population-based molecular epidemiological study of tuberculosis transmission in the Metropolitan Area of Milan, Italy. Eur J Clin Microbiol Infect Dis 21:114-122 Morris JT, Seaworth BJ, McAllister CK (1992) Pulmonary tuberculosis in diabetics. Chest 102:539-541 Mosquera JA, Rodrigo L, Gonzalvez F (1994) The evolution of pulmonary tuberculosis in coal miners in Asturias, northern Spain. An attempt to reduce the rate over a 15-year period, 1971-1985. Eur J Epidemioll0:291-297 Moss AR et al. (2000) Tuberculosis in the homeless. A prospective study. Am J Respir Crit Care Med 162:460-464 Mourad G et al. (1985) Transmission of Mycobacterium tuberculosis with renal allografts. Nephron 41:82-85 Muller HE (1988) Laboratory-acquired mycobacterial infection. Lancet ii:331 Muller K, Fopp M, Senn HJ (1980) Unrecognized atypical tuberculosepsis in generalized hematologic neoplasms. Schweiz Med Wochenschr 110:1815-1817 Munoz P et al. (1995) Tuberculosis in heart transplant recipients. Clin Infect Dis 21:398-402 Nardell E, McInnis B, Thomas B, Weidhaas S (1986) Exogenous reinfection with tuberculosis in a shelter for the homeless. N Engl J Med 315:1570-1575 Opsald R, Riddervald HO, Aas TW (1961) Pulmonary tuberculosis in mitral stenosis and diabetes. Acta Tuberc Scand 4:290 Patel R, Paya CV (1997) Infections in solid-organ transplant recipients. Clin Microbiol Rev 10:86-124 Pearson ML et al. (1992) Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis. A risk to patients and health care workers. Ann Intern Med 117: 191-196 Peerbooms PGH et al. (1995) Laboratory-acquired tuberculosis. Lancet 345:1311-1312 Perez-Guzman C, Torres-Cruz A, Villarreal-Velarde H, Vargas MH (2000) Progressive age-related changes in pulmonary tuberculosis images and the effect of diabetes. Am J Respir Crit Care Med 162:1738-1740 Perlman DC et al. (1995) Tuberculosis in drug users. Clin Infect Dis 21:1253-1264 Pillai SD et al. (2000) Failure to identify non-bovine reservoirs of Mycobacterium bovis in a region with a history of infected dairy-cattle herds. Prev Vet Med 43:53-62 Qunibi WY et al. (1991) Mycobacterial infection after renal transplantation-report of 14 cases and review of the literature. Quart J Med 77:1039-1060 Raitio M, Tala E (2000) Tuberculosis among health care workers during three recent decades. Eur Respir J 15:304-307 Reyes H, Coninx R (1997) Pitfalls of tuberculosis programmes in prisons. Br Med J 315:1447-1450 Richmond JY, McKinney RW (1999) Biosafety in Microbiological and Biomedical Laboratories. US Government Printing Office, Washington DC Richmond JY, Knudsen RC, Good RC (1996) Biosafety in the clinical mycobacteriology laboratory. Clin Lab Med 16:527-550

J. Esteban Rieder HL et al. (1994) Tuberculosis control in Europe and international migration. Eur Respir J 7:1545-1553 Rosenman KD, Reilly MJ, Kalinowski DJ, Watt FC (1997) Silicosis in the 1990 s. Chest 111 :779-786 Rosenthal T, Pitlik S, Michaeli D (1975) Fatal undiagnosed tuberculosis in hospitalized patients. J Infect Dis 131 [Suppl]:S51-S56 Roy V, Weisdorf D (1997) Mycobacterial infections following bone marrow transplantation: a 20 year retrospective review. Bone Marrow Transplant 19:467-470 Sable CA, Donowitz GR (1994) Infections in bone marrow transplant recipients. Clin Infect Dis 18:273-284 Samper S et al. (1998) The molecular epidemiology of tuberculosis in Zaragoza, Spain: a retrospective epidemiological study in 1993. Int J Tuberc Lung Dis 2:281-287 Schulte JM, Valway SE, McCray E, Onorato 1M (2001) Tuberculosis cases reported among migrant farm workers in the United States, 1993-1997. J Health Care Poor Underserved 12:311-322 Schwartzman K, Loo V, Pasztor 1, Menzies D (1996) Tuberculosis infection among Health Care Workers in Montreal. Am J Respir Crit Care Med 154:1006-1012 Selwyn PA et al. (1992) High risk of active tuberculosis in HIV-infected drug users with cutaneous anergy. JAMA 268:504-509 Sepkowitz KA (1994) Tuberculosis and the health care worker: a historical perspective. Ann Intern Med 120:71-79 Sewell DL (1995) Laboratory-associated infections and biosafety. Clin Microbiol Rev 8:389-405 Singh N, Paterson DL (1998) Mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management. Clin Infect Dis 27: 1266-1277 Small PM, Fujiwara PI (2001) Management of tuberculosis in the United States. N Engl J Med 345:189-200 Snider DE (1978) The relationship between tuberculosis and silicosis. Am Rev Respir Dis 118:455-460 Snyder DC et al. (1999) Tuberculosis prevention in methadone maintenance dinics. Effectiveness and cost-effectiveness. Am J Respir Crit Care Med 160:178-185 Sokolove PE, Mackey D, Wiles J, Lewis RJ (1994) Exposure of emergency department personnel to tuberculosis: PPD testing during an epidemic in the community. Ann Emerg Med 24(3):418-421 Starzynski Z, Marek K, Kujawska A, Szymczak W (1996) Mortality among different occupational groups of workers with pneumoconiosis: results from a register-based cohort study. Am J Indust Med 30:718-725 Stead WW (1989) Special problems in tuberculosis. Clin Chest Med 10:397-405 Stoll-Keller F et al. (1997) Effects of morphine on purified human blood monocytes: modifications of properties involved in antiviral defenses. Int J Immunopharmacol 19:95-100 Sundbery R et al. (1991) A tuberculosis outbreak in a renal transplant program. Transplant Proc 23:3091-3092 Sutton PM, Nicas M, Harrison RJ (2000) Tuberculosis isolation: comparison of written procedures and actual practices in three California hospitals. Infect Contr Hosp EpidemioI21:28-32 Taguchi 0 et al. (2000) Mixed dust fibrosis and tuberculosis in comparison with silicosis and macular pneumoconiosis. Am J Indust Med 37:260-264

Tuberculosis in Special Groups and Occupational Hazards Tamura A et al. (1999) Active pulmonary tuberculosis in patients with lung cancer. Kekkaku 74:797-802 The WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance 1994-1997 (1997) Anti-tuberculosis drug resistance in the world. World Health Organization, Geneva Thompson PJ et al. (1993) Seals, seal trainers, and mycobacterial infecion. Am Rev Respir Dis 147:164-167 Turner WM (1951) Pulmonary tuberculosis and diabetes mellitus. Quart J Med 26:31 Wares DF, Clowes CI (1997) Tuberculosis in Russia. Lancet 350:957

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Weiss W (1973) Tuberculosis in student nurses at Philadelphia General Hospital. Am Rev Respir Dis 107:136-139 Wenger PN et al. (1995) Control of nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis among healthcare workers and HIV-infected patients. Lancet 345:235-240 World Health Organization (1994) Manual de bioseguridad en ellaboratorio. World Health Organization, Geneve World Health Organization (2001) Global tuberculosis control. WHO report 2001. WHO/CDS/TBI2001.287. World Health Organization, Geneva

Microbiology, Immunology, Pathogenesis and Pathology

8

Microbiology of Tuberculosis A. Ow OSOBA

CONTENTS 8.1 8.2 8.3 8.3.1 8.3.2 8.3.2.1 8.3.2.2 8.3.2.3 8.3.3 8.3.3.1 8.3.3.2 8.3.3.3 8.3.4 8.4 8.4.1 8.4.2 8.4.3 8.4.4 8.4.5 8.4.6 8.4.7 8.5 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.6.1 8.5.7

Aetiology and Morphology 115 Characteristics of Mycobacteria 116 Laboratory Diagnosis of Mycobacteria 117 Specimen Collection 117 Microscopic Examination 118 New AFB Detection Methods 119 Specimen Preparation and Culture 119 Liquefaction and Decontamination 119 Culture Media for the Isolation of Mycobacteria 120 Solid Media 120 Liquid Automated Culture Systems 122 Inoculation and Incubation Procedure 123 Identification of Mycobacteria Species 123 Molecular Techniques for Identification of Mycobacteria 123 Nucleic Acid Probes for Actively Growing Mycobacteria 123 The Accuprobe System 124 The BDProbe Tec ET 124 The Amplified Mycobacterium Tuberculosis Direct (MTD) Test 124 Amplicor M. Tuberculosis (PCR) Test 125 Ligase Chain Reaction (LCR) 125 Other Molecular Techniques 125 Antimicrobial Susceptibility Testing 126 Conventional Methods of Susceptibility Testing 126 Radiometric Method of Drug Susceptibility Testing 126 Non-Radiometric Methods of Drug Susceptibility Testing 127 The Mycobacterial Growth Indicator Tube (MGIT) System 127 The MB/BacT Microbial Detection System 127 Genetic Approach to the Detection of Drug Resistance by M. tuberculosis 127 Line Probe Assay (LIPA) 128 Newer Techniques of Drug Susceptibility Testing 128

A. O. OSOBA, MD, FRCPath (UK), FFPath (Ireland), FWACP Consultant & Head of Microbiology, King Khalid National Guard Hospital, P.O. Box 9515, Jeddah 2143, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

8.5.7.1 8.5.7.2 8.5.7.3 8.6 8.6.1 8.6.1.1 8.6.1.2 8.6.2 8.6.2.1 8.6.2.2 8.6.2.3

Luciferase-based Reporter Phage 128 Epsilometer (E-) test 128 Hybridisation Protection Assay (HPA) 128 Serological Diagnosis of Tuberculosis 129 Immunochromatographic Tests 130 ICT Tuberculosis Test 130 Rapid Test TB 130 Enzyme-linked Immunosorbent Assays 130 Tuberculosis IgA EIA 130 The Pathozyme TB Complex Test 130 PATHOZYME-MYCO IgG, IgA, and IgM Tests 130 References 131

8.1

Aetiology and Morphology

Tuberculosis is caused by Mycobacterium tuberculosis. The word tuberculosis is derived from the word tubercle, meaning a small lump or nodule (Wolinsky 1988). The disease was first described by Jean-Antoine Villemin, a French military doctor in 1868, when he successfully transmitted the infection to rabbits by inoculating them with material from man and cattle. He later showed that scrofula (tuberculous cervical lymphadenitis) and pulmonary tuberculosis were different manifestations of the same disease (Grange 1984). The organism was first isolated in pure culture by Robert Koch in 1882. The characterisation of the isolate led to the famous Koch's postulates, providing concrete evidence of M. tuberculosis as the causative agent of tuberculosis. In 1896, Lehmann and Neumann described the genus of Mycobacterium, which included M. tuberculosis and M. leprae, the leprosy bacillus. Mycobacterium tuberculosis has been classified in the genus of Mycobacterium, which belongs to the family of Mycobacteriaceae of the order of Actinomycetales. In this order are the families of Actinomycetaceae and Streptomycetaceae. In the order of Actinomycetaceae are the genera of Actinomyces and Nocardia, while the order of Streptomycetaceae includes the genus of Streptomyces.

116

A.O.Osoha

8.2 Characteristics of Mycobacteria Mycobacteria are primarily slow-growing intracellular curved bacilli. Their main characteristic feature is their distinctive staining properties. When stained with carbolfuchsin or other fluorochrome dyes, they resist decolorisation with acid and alcohol. Hence they are sometimes referred to as "acid-fast bacilli" (AFB). Robert Koch not only cultured M. tuberculosis but was also able to stain the organism with alkaline solution of methylene blue for 24 hours. The technique was later improved by Ehrlich using a hot solution of aryl ethylene dye fuchsin. This procedure was later modified by Ziehl and Neelsen (ZN), whose names are still today used to describe the staining technique used for staining Mycobacteria (Grange

Superflcialliplds (Myoosldes. cord lactor and 5ulpholipids)

Peptidoglycan (Murein layer) "'~--f-- Upoarabinomannan

1984).

Another characteristic feature of the Mycobacteria is their unique and complex cell wall. The cell walls are rich in lipid complexes such as peptidoglycolipids (mycosides), cord factor and sulpholipids, which give the organism its shape, rigidity and colonial characteristics (Fig. 8.1). These mycosides are structurally and functionally similar to the 0 antigens of Gram-negative bacteria and determine the seroagglutination and bacteriophage susceptibility of the organism. The cord factor (trehalose dimycolate) and sulpholipids have toxic properties but their contribution to virulence has not been elucidated (Davies et al. 1996). Recently, some workers have provided evidence to suggest that lipoarabinomannan plays a significant role in virulence (Moulding 1999). The unique, lipid-rich outer membrane confers on the organism an effective barrier to prevent entry of antimicrobials into the cell and helps to resist phagocytosis as well as the immune system of the host. (Fig. 8.2 + 8.3) The Mycobacteria are aerobic, non-sporing and non-motile organisms. They appear as thin slightly curved rods and, when stained by the ZN stain, may appear beaded. They measure 0.2-0.6 by 1.0-10.0 11m in size. They are only lightly stained or weakly Grampositive by Gram's stain, which is used to stain most bacteria, due to the high lipid content of their cell wall. They are characteristically slow growing on laboratory media and have a generation time of 12-36 hours. Mammalian tuberculosis is caused by M. tuberculosis (human tubercle bacillus), M. bovis (the bovine tubercle bacillus), M. africanum, found in equatorial Africa and intermediate between the above two species, and M. microti, a rare pathogen of voles and

Fig. 8.1. A diagrammatic representation of the mycobacterial cell wall

Fig. 8.2. False electron micrograph of the surface of Mycobacterium tuberculosis (After Medicine Digest, February, 1996, Cover page)

lower mammals. They are collectively referred to as M. tuberculosis complex. M. tuberculosis grows very slowly at 35°C and may take 3-6 weeks to produce visible colonies, which are buff-coloured, rough and friable.

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Microbiology of Tuberculosis

Human infections may also be produced by Mycobacteria other than the M. tuberculosis complex. These have been referred to as atypical, anonymous or MOTT (Mycobacterium other than typical tuberculosis). However the preferred terminology nowadays is non-tuberculous Mycobacteria (NTM) (Grange 1984). The most widely used classification of Mycobacteria is that of Runyon, which is based on clinical and microbiological features of the organisms, such as rate of growth, ability to grow and produce pigment in the presence or absence of light. These features provide an important method of differentiating between the various species in a clinical laboratory (Table 8.1) (Hopewell and Bloom 1994). The characteristic features of the NTM are shown in Table 8.1.

8.3 Laboratory Diagnosis of Mycobacteria The confirmation of the clinical diagnosis of mycobacterial infection depends on the identification of the mycobacteria in smears, isolation on laboratory media, identification of the species and susceptibility testing of the isolate.

8.3.1 Specimen Collection In order to make an accurate diagnosis of mycobacterial infection, adequate and proper collection of specimens submitted for examination must be made.

Table 8.1. Runyon's Classification of Non-tuberculous Mycobacteria Runyon's group and pigment production

Species

Rate of growth (days)

Clinical features and site of lesion

M. kansasii

10-21 7-14 7-14 7-14

Rare contaminant; lung disease Rare contaminant; skin and soft tissue Rare human pathogen; lung Rarely pathogenic

M.szulgae M.gordonae

10-28 12-28 10-28

M. flavescens

7-10

Rare human pathogen; lymphadenitis Rare human pathogen; lung disease Rarely pathogenic; environmental contaminant Rarely pathogenic; environmental contaminant

Group I Photochromogens Yellow/orange pigment after exposure to light

M. marinum M.simiae M. asiaticum

Group II Scotochromogens Orange/red pigment in the dark

M. scrofulaceum

Group III Nonphotochromogens

M. avium-

(Smooth and cream coloured colonies)

M. ulcerans

10-21

Lung disease in immunocompromised patients

28-60 14-28

Buruli ulcer (Africa) Optimal growth at 42°C; lung disease Environmental contaminant Rare human pathogen; lung disease

intracellulare (MAC) M. xenopi M. malmoense

M. haemophilum

}

18-84

10-21

Optimal growth at 20-32°C; skin and soft tissue disease in immunocompromised host Rarely pathogenic; environmental contaminant Rarely pathogenic; environmental contaminant Rarely pathogenic Environmental contaminant

3-7 3-7 3-7 3-7

Environmental contaminants; ulcers Environmental contaminant; ulcers Rarely pathogenic Rarely pathogenic

M. terrae-complex

10-21

M. gastri

10-21

M. flavescens M. triviale

Group IV M. fortuitum

Rapid growers (Growth in few days)

M. chelonae M. smegmatis M.phlei

118

In addition, the specimens must be submitted in the correct containers, held in the appropriate conditions and sent to the laboratory for processing as soon as possible, in leak-proof universal containers. If there is going to be a delay in culture, the sample must be refrigerated. Swabs are not recommended for isolation of mycobacteria. a) Pulmonary specimens - 5-10 ml of early morning sample of sputum collected on three consecutive days in a sterile container will be adequate. Deep coughing must be encouraged so that exudative material is obtained from the lungs. If the patient is unable to expectorate a satisfactory sample, deep coughing may be induced by inhalation of an aerosol of warm, hypertonic (5-10%) saline. The specimen should be properly labelled as ,induced sputum' so that it will not be rejected by the laboratory as saliva. Saliva and nasal drainage are unsatisfactory samples. Similarly 5-10 ml bronchoalveolar lavage submitted in a sterile container will be adequate for processing. b) Extrapulmonary specimens - For urine samples at least 40 ml of early morning samples collected on three consecutive days in a sterile container is recommended. Two millilitres of cerebrospinal fluid and 5-10 ml of gastric aspirates are usually required. Gastric lavage is particularly useful in children who do not produce adequate sputum specimens and who usually swallow coughed secretions. Hospitalisation of the child may be necessary so that the lavage can be collected very early in the morning. Synovial and pleural fluid require at least 5 ml of the sample in a sterile container. Tissues, fine needle aspirates and bone marrow samples can also be processed for mycobacteria, if submitted in sterile containers or inoculated at the bedside in liquid culture media (Shinnick and Good 1999).

8.3.2 Microscopic Examination After decontamination and concentration of the samples, two staining procedures are commonly used for the identification of mycobacteria in specimens. These are the ZieW-Neelsen stain and the aurarninerhodamine stain. Ziehl-Neelsen Stain. The smears are heat-fixed

and then flooded with carbolfuchsin and heated intermittently for 1 minute. The smears are then rinsed with tap water and then decolourised with

A. O. Osoba

Fig. 8.3. ZieW Neelsen stain of sputum sample showing slender acid-fast bacilli stained red against a blue background

acid alcohol, rinsed again with tap water and then counter-stained with methylene blue or malachite green for 2 minutes. The Mycobacteria are stained red against a blue background. This so-called "acid-fast" staining procedure is based on the fact that Mycobacteria are capable of retaining the red colour of the carbolfuchsin despite treatment with acid alcohol (Fig. 8.3). The smears are then examined under a light microscope with a 100x objective oil immersion. At the higher magnification, the smear should be read in three parallel sweeps of the long axis, with a minimum of 300 fields being examined or for a duration of 15 minutes. Usually, 5,000-10,000 organisms per millilitre of sample are required before they can be detected under the microscope. This makes the ZN stain less sensitive than cultures, although a properly performed smear has a positive predictive value of over 90% and specificity of over 90% in pulmonary tuberculosis (Shinnick and Good 1999; Marshall and Shaw 1997). Usually, 40 -70% of patients with M. tuberculosis isolated in culture will give a positive ZN stain reaction, while the rest will be ZN-negative. The advantage of the ZN stain is that it is cheap, the result can be made available in

119

Microbiology of Tuberculosis

a few hours and, more importantly, it can be used to determine the infectivity of a patient suspected of pulmonary open tuberculosis, based on clinical findings or chest radiography. (Fig. 8.6).

Auramine-rhodamine Stain. This is a fluorochrome stain employing auramine-rhodamine fluorescent dyes. The smear is flooded with auramine stain for 10 minutes and then rinsed with tap water. It is then decolourised with 5% acid alcohol for 2 minutes, rinsed again with tap water and then counter-stained for 5 minutes with potassium permanganate. The slide is allowed to air-dry and is then examined under fluorescent microscopy with 25x and 40x objectives. At lower magnification, the smear should be read in three parallel sweeps of the long axis, with a minimum of 30 fields of view. Acidfast bacilli appear as bright luminous yellow bacilli approximately 1-10 flm long, most typically in the form of slender rods but may appear curved or bent. Individual bacteria may display beaded and uneven staining. Some mycobacteria other than M. tuberculosis may appear pleomorphic, ranging from long rods to coccoid forms, with more uniform distribution of staining properties. A positive fluorochrome stain requires confirmation by over-staining the same slide with ZN stain. The fluorochrome stain is more sensitive and rapid than the ZN stain since the examiner is looking for yellowish-green fluorescing bacteria against a dark background and scanning of the smears is usually at a lower magnification.

8.3.2.1 New Acid-Fast Bacilli (AFBJ Detection Methods

Improved AFB detection methods in clinical specimens have been described, in order to increase the efficiency of standard microscopic procedures. One of these tests is the immunomagnetic separation strategy for capturing and concentrating mycobacteria from processed specimens. Magnetic polystyrene beads are coated with antibody to mycobacteria and then mixed with the sputum sample. The beads now covered with organisms are then recovered using a magnet. The organisms are washed off and stained and examined by fluorescent microscopy. The detection of M. tuberculosis by the immunocapture technique has been improved by the use of magnetic beads coated with rabbit IgG polyclonal antibody to lipoarabinomannan, a constituent of the mycobacterial cell wall (Fig. 8.1). The preliminary evaluation is very impressive but more clinical laboratory evaluation is still required (Shinnick and Good 1999).

B.3.2.2 Specimen Preparation and Culture

The definitive diagnosis of mycobacterial infection depends on the detection and recovery of AFB in clinical specimens. Since mycobacteria are often present in situations where there is resident bacterial flora (e.g. sputum) or are present in small numbers (e.g. CSF), specimens submitted for examination must be specially prepared for culture. Three main steps are usually required for processing specimens: a) Bacterial flora present in the specimens must be removed or reduced in numbers before inoculation in special media, since these are faster growing than mycobacteria; b) Mycobacteria trapped in mucin must be released by a liquefaction process without killing the mycobacteria, for easy identification and culture; c) Specimens with low bacterial load must be concentrated to enable detection by microscopy and culture; d) Suitable media (solid and/or liquid media) and appropriate incubation environment must be chosen to ensure optimum recovery of mycobacteria.

8.3.2.3 Liquefaction and Decontamination

The selection of a liquefaction and decontamination process that maintains the viability of the mycobacteria, eliminates the present bacterial flora and liquefies the mucin in the sample is desirable (Roberts et al. 1991). The three most widely used digestion methods are NaOH, the Zephiran-trisodium phosphate method and the NALC-NaOH method (Roberts et al. 1991). The NALC-NaOH method utilises a mucolytic agent, NALC for digestion and NaOH for decontamination and it is the most commonly used method in clinical laboratories. The advantage of this method is that a large number of specimens can be processed in a short time; concentrated smears can be ready for staining immediately and it has been shown to give a high rate of recovery of mycobacteria (Roberts et al. 1991) (Figs. 8.4, 8.5). Specimens collected aseptically from sterile body sites such as CSF, ascitic fluid and synovial fluids may be inoculated directly into appropriate media. Specimens other than sputum do not usually require digestion and decontamination. Urine specimens can be inoculated directly or treated with 10% CaCl and then centrifuged for 30 minutes at 2,500xg and the

A. O. Osoba

120

Fig. 8.4. Mycobacterium tuberculosis showing serpentine cords (Ziehl Neelsen stain. (After Meylan, 1993)

Fig. 8.6. Ziehl Neelsen stain of sputum showing a mass of acidfast bacilli in a patient with open tuberculosis

SMEAR Staining: Microscopy Ziehl-Neelsen Auramine-Rhodanine

deposit inoculated into appropriate media. For stool samples, the specimen can be processed by the NaOH sputum digestion method and then inoculated into appropriate media. After suitable treatment of the specimens, the suspension should be centrifuged for at least 15 minutes at 2,200-2,500xg or higher and the deposit inoculated into appropriate media. All centrifugation should be carried out in sealed safety cups.

CULTURE/ Inoculation of Media

SOLID

U Middlebrook

TB confirmation of +vesmears by PCR,LCR

LIQUID 12B Septi-Check BacT/Alert BAaEC MGIT 960

8.3.3 Culture Media for the Isolation of Mycobacteria 8.3.3.1 Solid Media

Three types of solid media have been described for the isolation of Mycobacteria and each has its advantages (Table 8.2).

* AST =Antimicrobial

susceptibility Testing

Fig. 8.5. Processing of clinical specimens for mycobacteria identification and susceptibility testing

a) Egg-based media - The most widely known media is the Lowenstein-Jensen (LJ) medium, which is an egg-based non-selective medium. LJ medium has a long shelf life, up to 1 year when refrigerated and is very cheap to prepare; it is used widely in developing countries. Its disadvantages are that it may be difficult to distinguish artefacts from true colonies on this medium and the fact that heat is required for solidification, which along with

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the presence of albumin inactivates some antituberculous drugs. On the whole it is good for the recovery of M. tuberculosis but not so much for the non-tuberculous mycobacteria (Table 8.2). It is generally regarded as the ,gold standard' as it results in a very high recovery rate of mycobacteria (Hopewell and Bloom 1994; Garcia et al. 1998; Palacios et al. 1999) (Fig. 8.7). The other types of egg-based media are the American Thoracic Society medium and a modified LJ medium by Petragnani, which contains a different concentration of malachite green used in the medium. Conventional culture on solid media, although inexpensive but specific, requires 1-5 x 102 bacilli per millilitre for reliable positive results and may take 6-8 weeks, since the growth of M. tuberculosis species is rarely visible to the naked eye until after 18 days of incubation. (Fig. 8.7) (Garcia et al. 1998; Palacios et al. 1999). b) Agar-based media - the agar-based selective media are the Middlebrook 7H1O and 7Hll media, which contain defined salts, vitamins, cofactors, oleic acid, glycerol, glucose as enrichment, catalase and biotin, to stimulate the regrowth of damaged bacilli, and albumin to promote growth by combining with the toxic products in the media (Table 8.2).

Fig. 8.7. Mycobacterium tuberculosis growing on LowensteinJensen's medium. Note buff-coloured colonies produced after 4 weeks incubation

c) Biphasic media - Another media used for the culture of Mycobacteria in some laboratories is the BBL-Septic-Chek AFB system, which is a biphasic system, containing both agar and broth. It uses the Middlebrook 7H9 broth in its lower chamber and agar slope of Middlebrook 7H 11 in the upper part. Mycobacteria grow readily in this medium

Table 8.2. Different Media used for the Isolation of Mycobacteria Medium

Composition

Malachite green present/absent (gllOO ml)

Inhibitory agents for non-mycobacterial contaminants

Egg-based (non-selective) Lowenstein-Jensen

Fresh whole eggs, defined salts, glycerol, potato flour

0.025

None

Egg-Based (liquid -selective) Middlebrook 7H12

7H9 broth base, bovine serum albumin, casein hydrolysate, catalase, 14C-labelled palmitic acid

None

Polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocillin

Agar-based medium Defined salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, glucose (selective) Middlebrook 7H10

0.00025

Cycloheximide, lincomycin, nalidixic acid

Agar-based medium Defined salts, vitamins, cofactors, oleic (selective) acid, albumin, catalase, glycerol, glucose, Middlebrook 7H11 casein hydrolysate

0.0025

Carbenicillin, amphotericin B, polymyxin B, trimethoprim lactate

Liquid culture Bactec (Middlebrook 7H12)

Same as Middlebrook 7H12 above

None

Same as in 7H12 above

Liquid culture MB/BacT (Middlebrook 7H9 broth)

Middlebrook 7H9 broth supplemented with growth factors

None

Amphotericin B, azlocillin, nalidixic acid, polymyxin B, trimethoprim

* Modified from Roberts et al' (1991)

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and at 2 or 3 days intervals during incubation the broth is made to run over the agar. Colonies are formed on the agar if they contain mycobacteria. The incorporation of NAP in the agar in a second tube will suppress the growth of M. tuberculosis and allow quick identification of MBT (Jenkins 1998). Agar-based media are expensive and have a relatively short shelf life, hence are not widely used in developing countries. However, they have the advantage of easy identification of microcolonies and accurate drug concentrations can be achieved for susceptibility testing. 8.3.3.2 Liquid Automated Culture Systems

1) The Bactec 460 TB instrument was the first automated instrument developed by Becton Dickinson, Sparks, Md. for the recovery of Mycobacteria from clinical specimens, using a radiometric detection system. The liquid medium is an enriched Middlebrook 7H9 base supplemented with bovine serum, albumin (fraction V) catalase, casein hydrolysate and 14 C-labelled substrate. The medium is used for the isolation, differentiation of M. tuberculosis complex from non-tuberculous mycobacteria and drug susceptibility testing. Although it is capable of recovering Mycobacteria in 7-14 days, it has several disadvantages, such as the inability to observe colonial morphology and mixed cultures and high costs. It is labour-intensive and uses radioactive reagents and needles, which could lead to cross-contamination. In view of these limitations, it is now less commonly used. 2) The MB/BacT system (Organon Teknika, Turnhout, Belgium) is the first fully automated nonradiometric system for the culture of Mycobacteria, approved by the FDA in 1996 (Fig. 8.8). It relies on a continuous colorimetric CO 2 detection to indicate Mycobacterial growth in a closed system. The measured values are transmitted continuouslyevery 10 minutes to a computer, which indicates vials with Mycobacterial growth based on a sophisticated algorithm. The system will recover Mycobacteria in 10-14 days. In the MB/BacT system the process bottles contain Middlebrook 7H9 broth, Tween 80, amaranth and an antibiotic supplement containing amphotericin B, azlocillin nalidixic acid, polymyxin Band trimethoprim. The MB/BacT cabinet holds 240 process bottles, which are continuously monitored for up to

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8 weeks (Garcia et al. 1998; Palacios et al. 1999). 3) BACTEC MGIT 960 - The MGIT (Mycobacterium growth indicator tube) system (Becton Dickinson, Sparks, Md.) uses tubes in which a fluorescent compound is embedded in silicone on the bottom of tubes. This fluorescent compound is sensitive to the presence of oxygen dissolved in the broth. Initially, the large amount of dissolved oxygen quenches emissions from the compound and little fluorescence can be detected. Later, actively respiring Mycobacteria consume the oxygen and allow the fluorescence to be detected. The mean time to detection (TTD) is about 15.4 days (range 4-47 days (Flanagan et al. 1999). It has the advantage of accommodating 960 tubes, which is suitable for large volume laboratories (Fig. 8.9). Both the MB/BacT and MGIT systems have the same limitations of failure to detect mixed cultures, the inability to identify colonial morphology and high cost of media and equipment. These preclude their Widespread use in developing countries. These liquid culture systems increase the recovery of mycobacteria while at the same time decreasing the time of recovery, since the mycobacterial growth is detected more rapidly in liquid media. Many clinicallaboratories now use a combination of solid media and liquid selective media in primary isolation of mycobacteria since most species grow in 7-14 days in liquid media as opposed to 6-8 weeks with solid media.

Fig.8.8. MB/BacT instrument for automated mycobacterial culture, identification and susceptibility testing (Organon Teknika)

Microbiology of Tuberculosis

Fig. 8.9. BACTEC MGIT 960 instrument for automated culture and susceptibility testing (Becton Dickinson)

8.3.3.3 Inoculation and Incubation Procedure

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niacin test, nitrate reduction, susceptibility to T2H, catalase test, urease test, arylsulphatase test, iron uptake test, growth on MacConkey agar without crystal violet, sodium chloride tolerance test, deamination of pyrazinamide, tel1urite reduction and Tween 80 hydrolysis. Mycobacterium tuberculosis can be identified by its slow growth on solid media,non-photochromogenicity, strongly positive niacin test, negative catalase test and positive nitrate reduction test. The p-nitro- -acetylamino-beta-hydroxypropiophenone (NAP) test is an intermediate compound in the synthesis of chloramphenicol and inhibits species of the Mycobacterium complex (i.e. M. tuberculosis, M. bovis, M. africanum, M. microti and bacilli Calmette-Guerin). This compound does not inhibit other mycobacteria. This test can be used to identify M. tuberculosis in positive cultures in the liquid automated culture systems in 4-6 days (Siddiqi 1999). However, although the NAP test has been recommended by the Centers for Disease Control and Prevention and widely used for the identification of M. tuberculosis, it is not to be used alone as a confirmatory test because of some reports of false-positive results (Rau and Libman 1999) (Fig. 8.5).

Solid Media

Egg-based Medium. After the decontamination and digestion of the samples, the sediment is inoculated into Lowenstein-Jensen slope and incubated in the dark at 35-37°C for 8 weeks, in an atmosphere of 5-10% CO 2 for the first 7 days, with the screw caps left loose. The cultures are visually inspected each week for evidence of growth (Fig. 8.7).

Liquid Automated Culture Systems. The supplements are reconstituted and inoculated into culture process bottles. The processed sample from the specimen is inoculated into the culture bottles. They are loaded into the incubator cabinet, which monitors the growth for 8 weeks. Bottles flagged positive are checked for purity and presence of acid-fast bacilli and then subjected to identification tests and susceptibility testing (Figs. 8.8, 8.9).

8.3.4 Identification of Mycobacteria Species

Conventional methods for the identification of Mycobacteria are (1) growth rate at different temperatures, (2) colonial morphology, (3) pigmentation in dark or light, and (4) biochemical tests. The biochemical tests commonly used to differentiate Mycobacteria are

8.4 Molecular Techniques for Identification of Mycobacteria Since conventional methods for the identification of Mycobacteria take 2-3 weeks, there has been increased effort to develop rapid identification techniques, especially in the wake of the resurgence of tuberculosis as a result of the HIV/AIDS pandemic. Molecular techniques have been developed in the last decade with the aim of achieving faster commencement of appropriate treatment, shortening time to detection of anti-mycobacterial drug resistance, and instituting appropriate control measures.

8.4.1 Nucleic Acid Probes for Actively Growing Mycobacteria

Actively growing mycobacteria may be identified by using commercial DNA probes, which detect specific DNA or ribonucleic acid (RNA) fragments by nucleic acid hybridisation. These tests require some amplification of the bacterial DNA. These methods include target, probe and signal amplification tech-

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niques, where ribosomal RNA or different parts of genomic DNA are selected as targets. The specificity of the tests is nearly 100%, but their sensitivity is still relatively lower compared with culture, especially in smear-negative specimens. The tests are cumbersome and expensive, which preclude their use in many developing countries. Probes are now available for the identification of M. tuberculosis complex, M. avium complex, M kansasii and M. gordonae isolated either on solid or liquid media.

8.4.2 The Accuprobe System In the Accuprobe system (Gen-Probe, San Diego, Calif.) the bacterial culture is sonicated to break up the bacterial cells, the lysate is incubated with an acridinium-Iabelled oligonucleotide probe. This hybridises to mycobacterial ribosomal RNA. The unbound oligonucleotide is removed and the bound probe is measured using a luminescence detection system. The Accuprobe can identify M. tuberculosis is under 2 hours. The test has been reported to be specific and when combined with the Bactec system can provide speciation of an isolate from a clinical sample in 14 days. However, false positives have been reported with M. avium-intercellulare complex strains (Marshall and Shaw 1997).

8.4.3 The BDProbe Tec ET The BDProbe Tec (Becton Dickinson, Sparks, Md.) is a nucleic amplification technique, which is based on homogeneous strand displacement amplification (SDA) and fluorescent energy transfer detection in an instrument system. SDA is an isothermal enzymatic process that amplifies nucleic acid exponentially. The process is based on the nicking of a modified recognition sequence by the restriction endonuclease BsoBl and the extension and repair of that site by the DNA polymerase Bst, which synthesises a new strand of DNA while displacing the existing strand. The displaced strand can then serve as a template for further amplification. The process takes place at a temperature of 52.5°C. An internal amplification control system is run with each sample and is

complex, M. avium complex and M. kansasii, performed on isolates grown in liquid or solid media. The test consists of three main steps: sample processing, priming and warming, and simultaneous amplification and detection. The whole procedure takes about 2 hours. While the sensitivity and specificity of the test is about 100% for smear-positive samples, however for smear-negative specimens the sensitivity and specificity are about 85% and 97%, respectively (Bergmann et al. 2000). Current probe technology, however, cannot distinguish live from dead mycobacteria, which indicates that while molecular techniques will achieve great usefulness in the primary diagnosis of mycobacterial infections, cultural methods will be required for monitoring the response to therapy. Most clinical laboratories now combine both molecular techniques and cultural methods in the laboratory diagnosis and susceptibility testing of Mycobacteria and this practice is likely to continue in the foreseeable future.

8.4.4 The Amplified Mycobacterium Tuberculosis Direct (MTD) Test The MTD test uses a transcription-mediated amplification (TMA) principle to detect M. tuberculosis complex directly from respiratory specimens and provides a billion-fold amplification of the rRNA targets (MTD, Gen-Probe, San Diego, Calif.). The amplification process uses a single temperature throughout the test. The target is 16s RNA. The mycobacterial cells are sonicated and the nucleic acids are released. The specimens are then heated to 42°C and multiple copies of the mycobacterial RNA are generated. In the test system, ampIicons are created through DNA intermediates by a reverse transcriptase. M. tuberculosis complex-specific sequences are then detected by the Gen-probe chemiluminescentlabelled DNA probes and the results are measured in a luminometer. The test is recommended for use only with smear-positive concentrated specimens such as bronchoalveolar lavages, bronchial aspirates and tracheal aspirates. The specificity of the test is 97-99%, while the sensitivity is 82-97%. The advantage of the test is that it uses a single temperature and a single tube format. The detection of rRNA accounts for its high sensitivity. The high reliability of the test makes

designed to verify the SDA reaction. The assay con-

it an ideal tool for diagnosing tuberculosis with low

sists of direct M. tuberculosis complex (DTB) assay performed on clinical respiratory specimens and the culture identification (ID) assays for M. tuberculosis

bacterial count. Since the test has a negative predictive value close to almost 100%, it can quickly help in ruling out tuberculosis from the differential diag-

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Microbiology of Tuberculosis

nosis, alternatively, if the smear is ZN positive, it suggests the presence of non-tuberculous mycobacteria if MTD negative (Pfyffer 1994).

working hours of a clinical diagnostic laboratory (Beavis et al. 1995).

8.4.6 8.4.5

Ligase Chain Reaction (LCR)

Amplicor M. Tuberculosis (PCR) Test

The Amplicor M. tuberculosis test by Roche Diagnostic Systems (Branchburg, N.J.) is based on a polymerase chain reaction (PCR), nucleic acid hybridisation for the detection of M. tuberculosis complex in digested, decontaminated sputum and bronchial alveolar lavage samples. The test is based on three major processes: PCR, target amplification, hybridisation of the amplified product to a specific nucleic acid probe and the detection of the amplified product by colour formation. In the test, gene-specific primers located in a highly conserved region of the 14S ribosomal RNA (rRNA) gene of M. tuberculosis are used to amplify a 584 base-pair sequence. The DNA-containing sample and reagent mixture are heated to separate the double-stranded helix and expose the primer target sequences. As the mixture cools, the biotinylated primers anneal to their targets. The thermostable DNA polymerase, in the presence of excess of deoxynucleoside triphosphates (dNTPs), extends the annealed primers along the target templates to produce amplicons. This process is repeated for a number of cycles, each cycle effectively doubling the amount of target DNA. After 37 cycles, over a billion copies can be produced from a single copy of DNA. The ampIicons are then chemically denatured to form single strands that are added to a microwell plate containing a bound, amplicon-specific oligonucleotide probe. The biotin-labelled amplicons will then bind (hybridise) to the amplicon specific probe and thus be captured on to the plate. The detection of the amplicons is carried out by an enzyme immunoassay technique by the addition of Av-HRP conjugate and the optical density of the mixture is measured in an automated microwell plate reader. In smearpositive samples the sensitivity was found to be 95% and the specificity to be 98% in one report, while in smear-negative samples the sensitivity was 75% and the specificity was 99% (Rau and Libman 1999; Gilpin et al. 1999; Carpenter et al. 1995). However there are reports of some current commercial kits having a sensitivity range of up to 70% for smearnegative M. tuberculosis samples (Rau and Libman 1999). The assay is rapid and easy to perform, taking about 8 hours and can be fitted into the normal

Ligase chain reaction is a probe amplification technique incorporating a DNA polymerase and it is based on sequential rounds of template-dependent ligation of two adjacent oligonucleotide probes. When a pair of probes has hybridised to the target sequence on a single strand of DNA, there is a gap of a few nucleotides between the probes. The addition of a DNA polymerase into the reaction acts to fill in this gap by incorporating nucleotides. Once the gap is filled ligase can covalently join the pair of probes to form an amplification product that is complimentary to the original target sequence and can itself serve as a target in subsequent cycles of amplification. The target nucleic acid sequence for LCR assay for M. tuberculosis (Abbott LCX probe system, Abbott Diagnostics, Chicago, Ill.) is found within the single copy chromosomal gene of M. tuberculosis which encodes for protein antigen b. This gene sequence is specific to the M. tuberculosis complex and has been detected in all M. tuberculosis complex strains. In the assay sediments from a clinical sample or culture material are sonicated and amplified in a thermocycler. The amplicons were detected in an LCX analyser (LCX probe system). In smear-positive samples the sensitivity was found to be 100% and the specificity to be 98% in one report, while in smearnegative samples the sensitivity was 89% and the specificity was 98% (Gilpin et al. 1999).

8.4.7 Other Molecular Techniques

A number ofmolecular techniques have been described for the rapid identification of mycobacteria growing on solid media. These are (1) thin-layer chromatography, (2) high-performance liquid chromatography, (3) gasliquid chromatography, (4) analysis with DNA probes. Although these procedures can produce rapid identification of mycobacteria growing on solid media, they are too insensitive for the identification of isolates in liquid media, due to the relatively low cell mass often produced, or they are only capable of identifying a limited number of species (Taylor et al. 1997). In summary, these newer techniques will have to be used to supplement the conventional methods and need to be interpreted in conjunction with the

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patient's clinical features, such as medical history, physical examination, chest radiograph, tuberculin test and therapeutic response to anti-tuberculous agents (Pfyffer 1994).

8.5 Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing (AST) of isolated Mycobacteria is very important not only for appropriate therapy but also for control of the disease and the identification of resistant strains. Conventional methods are still the method of choice but these take 3-8 weeks, while the Bactec 460 TB and the liquid culture systems take 8-12 days (Siddiqi 1999). The newer methods of susceptibility testing have distinct advantages and improvements over the older methods (Inderlied 1994).

8.5.1 Conventional Methods of Susceptibility Testing There are three methods of conventional susceptibility testing of M. tuberculosis and these are based on its growth on solid medium containing a fixed amount of the drug. (Hawkins IE et al.1991; Vareldzis et al. 1994). a) Absolute concentration method: medium containing known concentrations of antimicrobial agents and control medium without drugs are inoculated with standardised suspension of M. tuberculosis. A preliminary reading is made at 2 weeks and a final reading at 4 weeks. The strain is regarded as resistant if there is growth greater than 20 colonyforming units (CPU) at a specific drug concentration. The method is highly sensitive to variations in inoculum size. b) Resistance ratio method: this method is similar to the absolute concentration method in that both the drug-free media and the drug-containing media are inoculated with both the test strain and a standard strain of M. tuberculosis ( H37Rv). Resistance is expressed as the ratio of the MIC of the test strain divided by the MIC of the control organism used in the test. If the test strain has a ratio of 8 or more it is considered as resistant, while a resistance ratio of 2 or less is defined as sensitive. A resistance ratio of 2 or less is equivalent to the presence of less than 20

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colonies growing on 0.2 mgtl of isoniazid in the absolute concentration method. In this method, it is important that the inoculum be properly standardised. Variations in the inoculum size are difficult to prevent (Vareldzis et al. 1994). c) Proportion method: this is the most widely used method and it involves the inoculation of both drug-free media and drug-containing media in duplicate with various dilutions of the test organism and the plates incubated. The media used are the Middlebrook 7HlO or 7H11. The dilutions are made to produce between 50 and 100 colonies on at least one of the control media. The CPU on the drug-containing media is then compared with that of the drug-free media. Resistance is defined as Mycobacteria with greater than 1% of the population exhibiting growth in the presence of the lowest concentration of the drug tested. Results can be available in 14-21 days (Hawkins et al. 1991). The proportion method is the most widely used method in the USA, as it provides fairly accurate and reproducible results for primary and secondary anti-mycobacterial drugs. However the drawback of the method is the difficulty of standardisation of the inoculum and quality control. In addition, it has not been validated for testing new agents such as ciprofloxacin, ofloxacin and rifabutin (Hawkins et al. 1991; Vareldzis et al. 1994).

8.5.2 Radiometric Method of Drug Susceptibility Testing

Bactec 460 TB System. In 1980, Gardner Middlebrook introduced the Middlebrook 7H12 liquid medium, an automatable radiometric detection system for the growth of Mycobacteria in a liquid selective medium using the Bactec 460 TB system (Becton Dickinson Diagnostic Instruments Systems, Towson, Md.). The system revolutionised the isolation and susceptibility testing of Mycobacteria. The system is far superior to the conventional solid media in that it recovers more positive cultures in a significantly shorter period (2 weeks instead of 6-8 weeks) (Marshall and Shaw 1997; Siddiqi 1999). The medium contains 14 C_ labelled palmitic acid and the growth of Mycobacteria is detected by metabolic release of radio-labelled carbon dioxide from the palmitic acid. Radioactivity of the labelled CO 2 is determined quantitatively and is a measure of the rate and amount of growth in the vial. These numbers are designated the "growth index»

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(GI). By daily monitoring of the culture vials, detection 8.5.5 times have been shortened in this system to 7-14 days. The MB/BacT Microbial Detection System By the addition of anti-mycobacterial drugs into the vials, inhibition of growth results in a reduction in This is also a non-radiometric, automated colorithe amount of 14C produced and a reduction of the metric system consisting of a culture bottle with a GI, compared with the drug-free vial (control). The growth sensor for detecting mycobacterial growth. Bactec system allows the drug susceptibility testing to The instrument incubates and scans the bottles for be performed in 12-14 days after isolation and identi- positivity and a computerised data management fication, with excellent correlation with conventionally system records and reports results. The MB/BacT determined susceptibility testing (Marshall and Shaw susceptibility test is performed using the MB/BacT 1997). It can be used to test all primary drugs, i.e. process bottles, MB/BacT antibiotic supplement isoniazid, rifampicin, streptomycin, ethambutol and (MAS) reconstitution fluid and antimicrobial drugs. pyrazinamide as well as secondary drugs including The drugs are added to the new MB/BacT process quinolones and rifabutin. The main drawback of the bottles (one drug per bottle and one concentration method is the reporting of false susceptibility or resis- per drug). These bottles with drugs are inoculated tance due to testing of mixed populations of M. tuber- with broth from a positive MB/BacT bottle. Next to culosis and non-tuberculous mycobacteria. However, these bottles with drugs a process bottle without the critical concentrations have not been established drugs is inoculated with broth from the positive MB/BacT bottle. This bottle is considered the growth for the newer agents (Inderlied 1994). control. Growth of the M. tuberculosis isolate in the bottles with drugs is compared to the growth control 8.5.3 to decide if the isolate is resistant or susceptible. The Non-Radiometric Methods of Drug Susceptibility M. tuberculosis isolate is considered resistant if the Testing bottle containing the drug is flagged positive before or at the same time as the growth control. An isolate The worldwide resurgence of tuberculosis has under- is considered susceptible if the bottle containing the scored the necessity to develop rapid methods for drug remains negative during the test period or when isolation and susceptibility testing of mycobacteria. flagged positive after the growth control. The conBecause of the concerns for radioactive materials, centration at which the drugs are tested are streptosome governments placed restrictions on the Bactec mycin 1 Ilg/ml, isoniazid 1 Ilg/ml, rifampicin 1 Ilg/ml, 460 system culture bottles. This led to the develop- ethambutol 2 Ilg/ml and pyrazinamide 50 Ilg/ml. The ment ofnon-radiometric culture systems such as the test is concluded in 12 days. The MB/BacT cabinet Mycobacterial Growth Indicator Tube (MGIT) Bactec holds 240 process bottles, which are continuously MGIT 960 (Becton Dickinson) and the MB/BacT monitored for up to 8 weeks (Fig. 8.8). (Organon Teknika, Durham, N.C.).

8.5.6 8.5.4 The Mycobacterial Growth Indicator Tube (MGIT) System

It is an automated non-radiometric culture system using Middlebrook 7H9 broth and an oxygen sensitive fluorescence sensor to indicate microbial growth in a vial. It is similar to the Bactec 460 system. By the addition of anti-mycobacterial drugs, the actively respiring mycobacteria consume the oxygen and allow fluorescence to be detected by the instrument, indicating resistance. Control bottle without any drug is included with each run. The cabinet has a capacity to accommodate 960 bottles (Fig. 8.9). The mean time to determination of susceptibility or resistance is 10-12 days (Flanagan et al. 1999).

Genetic Approach to the Detection of Drug Resistance by Mycobacterium tuberculosis

Recent major advances have been made in uncovering the molecular basis of the resistance of M. tuberculosis to various antimicrobials, viz. isoniazid (Zhang et al. 1992; Banerjee et al. 1994), streptomycin (Finken et al.1993), quinolones (Takiff et al.1994), rifampicin (Telenti 1993a,b, 1997). For example, the mechanism of rifampicin resistance by M. tuberculosis has been associated with point mutations and small insertions or deletions in a limited region of the gene encoding for the B-subunit of the RNA polymerase (rpoB). In the case of isoniazid, mutations in the kat G, inh AI mab A, ahp C and oxy R genes confer resistance on M. tuberculosis. Based on this approach, commercial

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128

tests have become available for use in clinical diagnostic laboratories.

8.5.6.1 Line Probe Assay (UPA)

This test is based on the detection of mutations by hybridisation in the rpo B gene, which confers resistance on M. tuberculosis. Telenti (1993a,b; Cole and Telenti 1995) developed a screening method using PCR and single strand conformation polymorphism analysis and Williams (1994) used the PCR and heteroduplex formation to detect rifampicin resistance in M. tuberculosis cultures. In the LIPA test, oligonucleotide probes are immobilised as parallel lines at known locations on a nitrocellulose strip (Beenhower et al. 1995). The amplified material is denatured and incubated with a LIPA strip in a shaking water bath for 1 hour at 50°C in 1 ml of hybridisation mixture. The hybrids formed are revealed by an immunoenzymatic procedure, resulting in a coloured precipitate that enables visual reading of the results. If a mutation is present in one of the probe target regions, the mismatch created will prevent the corresponding probe from hybridising. The test is fast and easy to perform and allows the simultaneous detection of M. tuberculosis and the discrimination between rifampicin-sensitive strains and rifampicin-resistant strains of M. tuberculosis in clinical samples. The test can be completed in 2 days and when performed on cultures it takes about 8 hours. In one report, (Beenhower et al. 1995) the susceptibility of M. tuberculosis strains can be correctly determined in up to 97% of the strains tested. However, the test will fail to determine the susceptibility of strains utilising a different mechanism of resistance from the rpo B gene (Beenhower et al. 1995; Drobniewski and Pozniak 1996).

8.5.7 Newer Techniques of Drug Susceptibility Testing

8.5.7.1 Luciferase-based Reporter Phage

This is a molecular technique which has recently been described to provide a rapid drug susceptibility testing of M. tuberculosis. In this test, the gene encoding a luciferase enzyme from fireflies was inserted into a bacteriophage. When the bacteriophage infects M. tuberculosis, the luciferase enzyme is synthesised and emits light. Following the exposure of the organism to anti-mycobacterial agent, the organism dies and no light is emitted, indicating susceptibility to the drug.

Between 500 and 5,000 mycobacteria infected by this bacteriophage will produce a clear positive signal and the test can be completed in 3 hours (Marshall and Shaw 1997; Roth et al. 1997; Jacobs et al. 1993). This is a promising test, which will find useful application in clinical laboratories in the future.

8.5.7.2 Epsilometer (E-) test

The E-test has been used very recently to determine the Minimum Inhibitory Concentration (MIC) of many organisms. More recently the technique has been modified to determine the susceptibility of M. tuberculosis and M. avium-intercellulare to both first-line and second-line anti-mycobacterial agents (Flynn et al. 1997; Koontz et al. 1994; Wanger and Mills 1994). The E-test ( AB Biodisk, Solna, Sweden) is based on a stable-gradient strip impregnated with an antimicrobial drug placed on Middlebrook 7Hll media which has been inoculated with the test organism, and then incubated for 5 days. The results are interpreted by applying National Committee for Clinical Laboratory Standards (NCCLS) criteria. Four strips can be placed on a 150-mm diameter plate. The method produces MICs within 1 week of primary isolation, thus allowing early modification of therapy.

8.5.7.3 Hybridisation Protection Assay (HPA)

This is a simple and fast M. tuberculosis drug susceptibility test, which is now commercially available. After isolation of the organism from clinical specimen, it is grown on Middlebrook 7H9 broth and incubated at 37°C for 5-7 days. The inocula are standardised to McFarland no. 0.5 standard and then mixed with an anti-mycobacterial drug solution to give a final concentration of 0.1 and 1.0 Ilg/ml for isoniazid and 1.0 and 10.0 Ilg/ml for rifampicin. The resulting bacterial suspension with or without the test drug, is cultured on an agitating plate at 37°C for up to 5 days. At 0-, 1-,3- and 5-day intervals each sample is removed and subjected to the HPA test with acridinium ester (AE)labelled DNA probe{ AccuProbe; Gen-Probe, Inc. San Diego, Calif.). The assay results are read in a luminometer and expressed as relative light units (RLU). The statistical significance of the RLU between tubes with and without drugs is calculated (Miyamoto et al. 1996). In this test, drug-resistant strains of M. tuberculosis can be accurately detected after 1 day of incubation with the drug. The HPA test is safe, simple and fast. It can be incorporated into the routine of

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a clinical diagnostic laboratory. However, it is more serological diagnosis of tuberculosis have been expensive than the radiometric or conventional agar handicapped by the fact that the pattern of antibody methods. It may have useful application in some situ- response after M. tuberculosis infection varies from ations such as testing immunocompromised patients one lesion to another and from patient to patient. In many patients the antibody response is weak and is infected with multidrug-resistant M. tuberculosis. In general, a handicap of both genetic and phe- frequently directed predominantly against non-spenotypic tests for susceptibility of M. tuberculosis is cific mycobacterial antigens (Woodhead 1992). Antigen preparations have contained moieties that they cannot precisely distinguish between mixed cultures that contain 1% to 10% of resistant cells and present in many microbial species that lead to serocultures that contain only susceptible organisms. This logical cross-reactivity. Assays with crude antigens distinction is of clinical importance since the patient have had poor specificity because of the presence with a mixed culture would be regarded as having of cross-reacting epitopes between M. tuberculosis a drug-resistant M. tuberculosis and would be given and other mycobacteria. By means of chemical and immunological methods, several purified antigens, appropriate treatment (Shinnick and Good 1999). In conclusion, conventional methods based on such as A60 antigen (Amicosante et al. 1993; Alifano ZN microscopy and culture on LJ medium with et al. 1994; Charpin et al. 1990) and 38 kDa antigen biochemical tests to identify the isolates are the (Simonney et al. 1996; Young et al. 1986; Bothamley et most widely used for the diagnosis of tuberculosis al.1992; Harboe and Wiker 1992) have been prepared and are still considered the "gold standard" . These which have improved the enzyme-linked immunomethods are cost-effective but are rather slow for sorbent assay (ELISA) test chatacteristics (Wilkins the effective control of tuberculosis. The new tech- and Ivanyi 1990; Jackett et al. 1988). Recently, mouse niques of nucleic acid amplification offer the pros- monoclonal antibodies (Mabs) to specific epitopes pect of more rapid diagnosis, susceptibility testing on antigens of M. tuberculosis have been used in a and improved and accurate epidemiological infor- solid-phase antibody competition test to measure mation (Marshall and Shaw 1997). The resurgence antibody titres to these determinants in human sera of tuberculosis has necessitated the development (Jackett et al. 1988; Cocito 1991; Bothamley et al. of more rapid methods of diagnosis. The objec- 1992). The application of Mabs into the serological tive of clinical laboratories should be to report the tests for the diagnosis of tuberculosis is a significant isolation of M. tuberculosis within 10-14 days of the advance because they can be readily prepared in a receipt of the sample and the result of drug suscep- standard form and are usually free from problems tibility tests within the next 14 days (Shinnick and of cross-reactivity. They also allow the estimation of Good 1999; Rau and Libman 1999). both IgG and IgM in a single assay, thereby obviating the need to purify antigens from M. tuberculosis. The interest to develop serological tests for the diagnosis of tuberculosis stems from the fact that 8.6 blood is readily available in any form of the disease for serological testing. Therefore the measurement Serological Diagnosis of Tuberculosis of titres of antibody to M. tuberculosis specific Since 40-60% of patients with pulmonary disease antigens may be of value in the diagnosis of both and up to 75% of patients with extrapulmonary smear-positive and smear-negative pulmonary diseases are smear-negative, a number of alternative tuberculosis. Antibody titres may also help to diagnostic methods using molecular, chromato- identify those patients who should be treated in a graphic and immunological methods have been hospital, those who require observed chemotherdeveloped. While these tests are very specific and apy as well as those who might require additional only moderately sensitive, they rely on the provision drugs to the standard regimen for the therapy of of adequate material from the site of the lesion. This their infection. is often difficult, especially in children, and in most In order to meet these objectives, a number of cases of extrapulmonary tuberculosis, such as cere- serological tests developed for the diagnosis of bral tuberculoma or tuberculous osteomyelitis and tuberculosis have now become commercially availabdominal tuberculosis (Wilkins and Ivanyi 1990; able. These tests can be divided into two groups, viz.: Pottumarthy et al. 2000). Previous investigations into (a) Immunochromatographic tests and (b) Enzymetuberculous serology have been faced with problems linked immunosorbent Assay. Some of these will be of both sensitivity and specificity. These studies on briefly described.

A.O.Osoba

130

8.6.1 Immunochromatographic Tests 8.6.1.1

8.6.2.3 PATHOZYME-MYCO IgG, IgA, and IgM Tests

The three assays measure the levels in serum of IgG, IgA and IgM antibodies, respectively, to two antigens lipoarabinomannan (LAM) and recombinant 38 kDa This is a immunochromatographic test in which five antigen. These kits detect infection due to Mycobachighly purified antigens (including one of 38 kDa) terium species. secreted by M. tuberculosis during active infection In a study evaluating the performances of these are immobilised in four lines on the test strip (ICT tests in various categories of patients (Pottumarthy Diagnostics, Balgowish, New South Wales, Australia). et al. 2000), it was found that the ICT Tuberculosis The test detects the presence of immunoglobulin G test followed by PATHOZYME-MYCO IgG had the best performance characteristics with sensitivi(IgG) antibodies to these antigens. ties of 41 % and 55%, respectively, with sera from patients with acute tuberculosis and specificities 8.6.1.2 of 96% and 89%, respectively, with sera from the Rapid Test TB Mantoux test controls, and 88% and 90%, respecIt is a one-step coloured immunochromatographic tively, with sera from anonymous controls. In a assay (Quorum Diagnostics, Vancouver, British study in the United Arab Emirates (Nsanze et al. Columbia, Canada). It detects antibodies to the recom- 1997) on a population of patients with tuberculosis binant 38 kDa antigen from M. tuberculosis expressed and leprosy, using a combination of PATHOZYME TB-Complex test and PATHOZYME-MYCO IgG and purified in E coli (Pottumarthy et al. 2000). test, it was found that the tests showed a significant difference in antibody levels between the patients with active pulmonary disease, extrapulmonary 8.6.2 tuberculosis and leprosy in comparison with the Enzyme-linked Immunosorbent Assays control group. The sensitivity of the two tests combined for proven pulmonary tuberculosis was 8.6.2.1 about 95% using a higher EIA cut-off of 1.6, while Tuberculosis IgA EIA the specificities were 100%. The sensitivity for This test detects the IgA antibody to a mycobacterial extrapulmonary tuberculosis at the higher cutKp90 immuno-cross-reactive antigenic compound off was 51 %. The authors conclude that while the (ImCRAC). tests may be of some value in decision making in extrapulmonary tuberculosis, they may be useful in serological screening of populations at high risk in 8.6.2.2 The Pathozyme TB Complex Test endemic areas (Nsanze et al. 1997). Although a negative result by any of these tests This test detects serum IgG antibody to a recombi- would be useful in helping to exclude disease in a nant 38 kDa antigen from M. tuberculosis expressed population with a low prevalence of tuberculosis, a and purified from E coli, permitting the isolation of positive result may aid in clinical decision making significant quantities of protein (OMEGA DIAG- when applied to symptomatic patients being evaluNOSTICS LTD, Alloa, Scotland). This antigen has ated for active tuberculosis (Pottumarthy et al. 2000). been reported as the single most important antigen The tests now commercially available confirm a more for the serological diagnosis of tuberculosis. It is a limited antibody response in smear-negative pulmounique disease-associated protein, which appears to nary disease, but suggest that a battery of monoclobe completely specific to the M. tuberculosis complex, nal antibodies, with differing specificities, produces i.e. M. tuberculosis, M. bovis and M africanum. The better results than single monoclonal antibody kit is specific for the diagnosis of disease due to M. (Woodhead 1992). However they have the advantage tuberculosis complex. In this test the test sera are that they are rapid and relatively inexpensive. Overdiluted 1:50 and standards were provided to gener- all, more research is still required to produce better ate a semi logarithmic reference curve. The results tests with increased sensitivities in extrapulmonary tuberculosis which can be readily incorporated into are expressed as sero-units. clinical diagnostic laboratories. ICT Tuberculosis Test

Microbiology of Tuberculosis

References Alifano M et al. (1994) ELISA method for evaluation of anti-A 60 IgG in patients with pulmonary and extrapulmonary tuberculosis. Microbiologica 17:37-44 Amicosante M et al. (1993) Antibody repertoire against the A60 antigen complex during the course of pulmonary tuberculosis. Eur Respir J 6:816-822 Banerjee A et al. (1994) InhA, a gene encoding a target for Isoniazid and Ethionamide in Mycobacterium tuberculosis. Science 263:227-230 Beavis KG et al. (1995) Evaluation of amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens. J Clin MicrobioI33:2582-2586 Beenhower HD et al. (1995) Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tuberc Lung Dis 76:425-430 Bergmann JS, Keating WE, Woods GL (2000) Clinical evaluation of the BDProbeTec ET system for rapid detection of Mycobacterium tuberculosis. J Clin Microbiol 38:863-865 Bothamiey GH et al. (1992a) Clinical value of the measurement of M. tuberculosis-specific antibody in pulmonary tuberculosis. Thorax 47:9-14 Bothamley GH et al. (1992b) Clinical value of the measurement of Mycobacterium tuberculosis specific antibody in pulmonary tuberculosis. Thorax 47:270-275 Carpenter E et al. (1995) Diagnosis of tuberculosis by amplicor Mycobacterium tuberculosis test: a multicenter study. J Clin MicrobioI33:3106-3110 Charpin D et al. (1990) Value of ELISA using A60 antigen in the diagnosis of active pulmonary tuberculosis. Am Rev Respir Dis 142:380-384 Cocito CG (1991) Properties of the mycobacterial antigen complex A60 and its applications to the diagnosis and prognosis of tuberculosis. Chest 100:1687-1693 Cole ST, Telenti A (1995) Drug resistance in Mycobacterium tuberculosis. Eur Respir J B SuppI20:701s-713s Davies PDO, Girling DJ, Grange JM (1996) Tuberculosis. In: Weatherall DJ, Ledingham JGG, Warrell DA (eds) Oxford textbook of medicine. Oxford University Press, Oxford, pp 638-655 Drobniewski FA, Pozniak AL (1996) Molecular diagnosis, detection of drug resistance and epidemiology of tuberculosis. Br J Hosp Med 56:204-208 Finken M et al. (1993) Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: Alteration of the ribosomal protein S 12 gene and point mutations within a functional16S ribosomal RNA pseudoknot. Mol Microbiol 9: 1239-1246 Flanagan PG, Williams R, Paull A (1999) Comparison of two automated systems for the isolation of mycobacteria from clinical specimens. Eur J Clin Microbiol Infect Dis 18: 912-914 Flynn et al. (1997) Application of the E test to the antimicrobial susceptibility testing of Mycobacterium marinum clinical isolates. J Clin 35:2083-2086 Garcia FG et al. (1998) Evaluation of the MB/BACT automated mycobacteria culture system versus culture on Lowenstein medium. Clin Microbiol Infect 4:339-343 Gilpin CM et al. (1999) Comparative study of Amplicor polymerase chain reaction and Ligase chain reaction for direct

131 detection of M. tuberculosis in clinical specimens. Saudi Med J 20:79-84 Grange JM (1984) Tuberculosis. In: Wilson G, Miles A, Parker MT (eds) Topley and Wison's principles of bacteriology, virology and immunity, 7th edn. Arnold, London, pp 32-35 Harboe M, Wiker HG (1992) The 38-kDa Protein of Mycobacterium tuberculosis.: a review. J Infect Dis 166:874-884 Hawkins JE, Wallace RJ, Brown BA (1991) Antibacterial susceptibility tests: Mycobacteria. In: Balows A, Hausler WJ, Herrmann KL, Shadomy HJ (eds) Manual of clinical microbiology, 5th edn, chap 114. American Society for Microbiology, Washington DC, pp 1138-1152 Hopewell PC, Bloom BR (1994) Tuberculosis and other mycobacterial diseases, 2nd edn. In: Murray JF, Nadel JA (eds) Textbook of respiratory medicine. Saunders, Philadelphia, pp 1094-1117 Inderlied CB (1994) Antimycobacterial susceptibility testing: present practices and future trends. Eur J Clin Microbiol Infect Dis 13:980-993 Jackett PS et al. (1988) Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. J Clin MicrobioI26(11):2313-2318 Jacobs WR et al. (1993) Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260:819-822 Jenkins PA (1998) The microbiology of tuberculosis. In: Davies PO (ed) Clinical tuberculosis. Chapman and Hall, London, pp 69-79 Koontz FP et al. (1994) Etest for routine clinical antimicrobial susceptibility testing of rapid growing mycobacteria isolates. Diagn Microbiol Infect Dis 19:183-186 Marshall BG, Shaw RJ (1997) Diagnostic techniques: old and new. Eur Respir Mon 4:30-50 Meylan PR ASM News vol 59(12), 1993 (Cover page) Miyamoto J et al. (1996) New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay. J. Clin Microb. 34:1323-1326 Moulding T (1999) Tuberculosis and non nontuberculous mycobacterial infections, 4th edn. In: Schlossberg D (ed) Pathogenesis, pathophysiology, and immunology: clinical orientations. Saunders, Philiadelphia, pp 48-64 Nsanze H et al. (1997) Serodiagnosis of tuberculosis and leprosy by enzyme immunoassay. Clin Microb and Infection. 3:236-239 Palacios JJ et al. (1999) Fully automated liquid culture system compared with lowenstein-Jensen solid medium for rapid recovery of mycobacteria from clinical samples. Eur J Clin Microbiol Infect Dis 18:265-273 Pfyffer GE (1994) Amplification techniques: hope or illusion in the direct detection of tuberculosis. Med Microbiol Lett 3:335-347 Pottumarthy S, Wells VC, Morris AJ (2000) A comparison of seven tests for serological diagnosis of tuberculosis. J Clin MicrobioI38:2227-2231 Rau NV, Libman (1999) Laboratory implementation of the polymerase chain reaction for confirmation of pulmonary tuberculosis. Eur J Clin Microbiol Infect Dis 18:35-41 Roberts GD, Koneman EW, Kim YK (1991) Mycobacterium. In: Balows A, Hausler WJ, Herrmann KL, Shadomy HJ (eds) Manual of clinical microbiology, 5th edn, chap 34. American Society for Microbiology, Washington DC, pp 304-318 Roth A, Schaberg T, Mauch H (1997) Molecular diagnosis of

132 tuberculosis: current clinical validity and future perspectives. Eur Respir J 10:1877-1891 Shinnick TM, Good RC (1999) Diagnostic mycobacteriology laboratory practices. Clin Infect Dis 21 :291-299 Siddiqi SH (1999) Recent advances in the diagnosis of tuberculosis: an overview. J King Abdulaziz Univ Med Sci 7:31-35 Simonney N et al. (1996) Comparison of A60 and three glycolipid antigens in an ELISA test for tuberculosis. Clin Microbiol Infect 2:214-222 Takiff HE et al. (1994) Cloning and nucleotide sequence of the Mycobacterium tuberculosis gyrA and gyrB genes and characterization of quinolone resistance mutations. Antimicrobial Agents Chemother 38:773-780 Taylor TB et al. (1997) Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media. J Clin Microbiol 35(1):79-85 Telenti A (1993a) Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341: 647-650 Telenti A (1993b) Direct automated detection of Rifampicin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother 37:2054-2058 Telenti A (1997) Genetics of drug resistance in tuberculosis. Clin Chest Med 18:55-64

A. O. Osoba Tuberculosis. In: Wyngaarden JB, Smith LH (eds) Cecil textbook of medicine. Saunders I Harcourt Brace Jovanovich, Philadelphia, pp 1682-1696 Vareldzis BP et al. (1994) Drug-resistant tuberculosis: laboratory issues. World Health Organization recommendations. Tubercle Lung Dis 75:1-7 Wanger A, Mills K (1994) Etest for susceptibility testing of Mycobacterium tuberculosis and Mycobacterium aviumintracellulare. Diagn Microbiol Infect Dis 19:179-181 Wilkins EGL, Ivanyi J (1990) Potential value of serology for diagnosis of extrapulmonary tuberculosis. Lancet 336: 641-644 Williams DL (1994) Characterization of rifampicin resistance in pathogenic mycobacteria. Antimicrob Agents Chemother 38:2380-2386 Wolinsky E (1988) Tuberculosis. In: Wyngaarden JB, Smith LH (Eds.) Cecil Textbook of Medicine. Pp 1682-1696. WB Sounders Co., Harcourt Brace Javonovich, Inc., Philadelphia Woodhead M (1992) New approaches to the rapid diagnosis of tuberculosis. Thorax 47:264 Young D et al. (1986) Immunological activity of a 38-Kilodalton protein purified from Mycobacterium tuberculosis. Infect Immun 54(1):177-183 Zhang Y et al. (1992) The catalase-peroxidase gene and isoniazid reisstance of Mycobacterium tuberculosis. Nature 358:591-593

9

The Immunology and Pathogenesis of Tuberculosis GRAHAM

A. W.

ROOK

CONTENTS 9.1 9.1.1 9.1.2 9.1.3

9.2 9.2.1 9.2.2 9.2.2.1 9.2.2.2 9.2.3 9.2.4 9.2.4.1 9.2.4.2 9.2.5 9.2.5.1 9.2.5.2 9.2.5.3 9.2.5.4 9.2.6 9.2.7 9.2.8 9.2.8.1 9.2.9 9.2.9.1 9.2.9.2 9.2.9.3 9.2.9.4

Introduction 133 Latent Tuberculosis and Its Relationship to Persisters After Chemotherapy 134 Protection Versus Immunopathology 134 The Objectives of the Immunologist 135 Immunity to Tuberculosis 135 The Crucial Role of the Type 1 Response for Immunity to Tuberculosis in Mice 135 The Possible Role of Other Types of T Lymphocyte 135 The Detrimental Role of Type 2 Responses 135 Variability of Tuberculosis in Different Mouse Strains 135 The Protective Role of Tumour Necrosis Factor (TNF-a) 136 Immunity to Tuberculosis in Man 136 Genetics of Susceptibility to Tuberculosis 136 Natural Human Gene Knockouts; Proof of the Role of IFN-y and IL-12 136 Other T Cell Types That May Be Involved in Immunity 136 CD8+ IFN-y-secreting T Cells 136 CD-I-restricted T Cells 137 Gammaldelta (y/'6) T Cells 137 Regulatory T Cells 138 The Role of TNF-a in Human Tuberculosis 138 Type 2 Responses in Human Tuberculosis 138 The Causes of the Controversy over Expression ofIL-4 in Human Tuberculosis 139 The Mechanisms of the Shift Towards a Type 2 Cytokine Profile 139 Macrophage Function and M. tuberculosis 139 Uptake of Mycobacteria 139 Toll-like Receptors 140 Mycobactericidal Mechanisms Within Macrophages 140 Reactive Oxygen and Nitrogen Intermediates 140

9.2.9.5 Macrophage Apoptosis and the Death of Bacteria 141 9.2.10 Evasion of the Antimicrobial Functions of Macrophages 141 9.2.11 Bacterial Genetics and Mechanisms of Avoidance of Destruction by the Type 1 Response 142 Immunopathology 142 9.3 The Koch Phenomenon; the Response 9.3.1 Characteristic of Disease 142 9.3.2 Tuberculin Response and Protection 142 9.3.3 The Relationship Between the Koch Phenomenon and the Shwartzman Reaction 143 9.3.4 A Hypothesis to Explain the Koch Phenomenon 143 9.3.5 Detrimental Roles of Tumour Necrosis Factor-a in Human Tuberculosis 144 9.3.6 Fibrosis 144 9.4 Endocrinology 144 9.4.1 Vitamin D3 Metabolism in Tuberculosis Lesions 144 9.4.2 Adrenal Steroids in Tuberculosis 145 9.4.2.1 The Effects of Stress 145 9.4.2.2 The Hypothalamo-Pituitary-Adrenal Axis in Human Tuberculosis 145 9.4.2.3 Dysregulation of the Cortisol-Cortisone Shuttle 145 Vaccination 145 9.5 9.5.1 Interference by Exposure to Environmental Mycobacteria 145 9.5.2 Concurrent Parasite Infections 146 9.5.3 Vaccine Dose 146 9.5.4 A Possible Novel Approach to Vaccine Design 146 9.5.6 Immunotherapy 146 References 147

9.1

Introduction

G. A. W. ROOK, BA, MB Bchir, MD Professor, Department Medical Microbiology, Royal Free and University College Medical School, Windeyer Institute of Medical Sciences, 46 Cleveland Street, London WIT 4JF, UK

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Tuberculosis is a global emergency, currently aggravated by HIV, the break-down of health care systems and increases in drug resistance (Espinal et al. 2001). There are now more cases of tuberculosis than ever before in the history of mankind. The current failure to control tuberculosis is attributable to several factors that must be taken into consideration in any account of the immunology of the disease. These factors are:

134

1. The inadequacy of the current vaccine, Bacillus

2.

3.

4.

5.

Calmette-Guerin. The protection given varies from zero to about 80% (Fine 1995). Inadequate understanding of the biology of the organism itself. Mycobacterium tuberculosis can remain viable within the tissues without causing symptoms for the life of the individual (Wayne 1994) and, at least in countries with low or moderate tuberculosis endemicity, many cases of tuberculosis result from reactivation of latent infection (van Rie et al. 1999). The best available chemotherapy must be continued for at least 6 months. This treatment regimen is not a realistic proposition in most developing countries, or even in the inner cities of rich ones, because the patients feel well after a few weeks and stop taking the drugs. WHO now admits that directly observed therapy short-course (DOTS), in which the patient is supervised while taking every dose of therapy, helps but does not solve the problem (Butler 2000). Our inadequate understanding of the mechanism of protective immunity, which hampers efforts to design better vaccines, and the extraordinary problem of knowing how to select and test the best vaccine candidates. Inadequate understanding of how to implement immunotherapy. Immunological manipulations that correct the patients' failing immune responses may be the only way to shorten treatment regimens.

G.A,W.Rook

sites of fibrosis or calcification, where oxygen availability may be low. However, in a forgotten paper published in 1927, Opie and Aronson reported that 80% of tuberculous lesions were already sterile (as determined by culture in guinea-pigs) 5 years after the primary infection, whereas live bacilli could be found in macroscopically normal lung tissue (Opie and Aronson 1927). A major recent advance is the detection, by in situ polymerase chain reaction of DNA, of M. tuberculosis in lung tissue from victims of accidental death with no history or post mortem signs of tuberculosis (Hernandez-Pando et al. 2000a). When such samples were taken from countries with endemic tuberculosis (Ethiopia and Mexico), 30-40% of samples were positive. In contrast, no DNA of M. tuberculosis was found in samples from Norway, where transmission within the community ceased several decades ago. In the positive Ethiopian and Mexican samples, the DNA was seen in histopathologically normal tissue and was not confined to macrophages. Cell types containing M. tuberculosis DNA included type II pneumocytes, endothelial cells and fibroblasts (Hernandez-Pando et al. 2000a). These observations raise the possibility that by entering cells that are not "professional antigen-presenting cells",M. tuberculosis is able to escape detection and perhaps subvert the immune response.

9.1.2 Protection Versus Immunopathology 9.1.1 Latent Tuberculosis and Its Relationship to Persisters After Chemotherapy There are two interrelated reasons for the requirement for 6-month regimens. The first is obvious and often discussed. The chemotherapy kills the vast majority of the bacteria within a few days, but there are subpopulations of "persisters" (Grange 1992). It is not clear whether these organisms are in true stationary phase (Siegele and Kolter 1992) or merely replicating extremely slowly. Nor is it clear whether these persisters, defined as organisms that survive chemotherapy, are the same as "latent" or "dormant" bacilli that remain in the tissues of the approximately 90% of individuals who show no clinical disease after infection. These bacteria are evidently controlled by the immune response, but not killed by it. Most research has addressed dormant rather than persister bacilli, though they are likely to be similar populations. It was assumed that latent bacilli are in old lesions or

The second reason for the need for prolonged treatment is usually overlooked. Most tuberculosis patients have a necrotising pattern of response to M. tuberculosis, analogous to the phenomenon first noted by Koch in guinea-pigs (Koch 1891). There is overwhelming evidence that the Koch phenomenon is not a correlate of optimal protective immunity to tuberculosis. Indeed, pre-immunisation of animals so that they have a Koch phenomenon before they are challenged with virulent M. tuberculosis results in a clear and reproducible increase in susceptibility to the disease, compared to unimmunised controls (Wilson et al. 1940). This and other aspects of the Koch phenomenon are discussed in detail later. Its relevance at this point is that this inappropriate pattern of response may not correct itself rapidly during treatment. Therefore, if chemotherapy is stopped at 3 months, relapse rates are high even when the chemotherapy was an optimal rifampicin-containing one that achieved sputum negativity well before

135

The Immunology and Pathogenesis of Tuberculosis

3 months, and in spite of the fact that there are very few live organisms in the patients' tissues at this time (Balasubramanian et al. 1990).

9.2.2 The Possible Role of Other Types ofT Lymphocyte

In addition to conventional CD4+ a/~, Class II MHC9.1.3 The Objectives of the Immunologist The immunologist, therefore, needs to understand the differences between protective immunity and the Koch phenomenon, and the factors that determine which response pattern is present. The ultimate objectives are better prophylactic vaccines, and also therapeutic vaccines that can replace the pathological response with a protective one very early during treatment, using immunotherapeutic strategies that also cause recognition and clearance of persisters and dormant bacilli.

9.2 Immunity to Tuberculosis 9.2.1 The Crucial Role of the Type 1 Response for Immunity to Tuberculosis in Mice The ability to manipulate the immune system of mice with neutralising antibodies or gene knockout (KO) has provided strong evidence that, in this species, immunity to tuberculosis correlates with a Type 1 response. In vivo CD4+ lymphocytes act in concert with CD8+ cells and with numerous other cell types including macrophages, B cells and some stromal cells. Collectively these give rise to two patterns of cytokine release known as Type I (dominated by IL-12 from antigen-presenting cells, and by IL2, and IFN-y from Thl lymphocytes) and Type 2 (dominated by IL-4, 5 and 13 from Th2lymphocytes) (Clerici and Shearer 1994; Salgame et al. 1991). Disruption of the major histocompatibility (MHC) class II genes or of the gene for the ~ chain of the a/~ T cell receptor (Ladel et al. 1995), resulting in a deficiency of CD4+ a/~ T cells, render mice susceptible even to the avirulent Bacillus Calmette Guerin (BCG). Similarly, disruption of the gene for IFN-y makes mice very susceptible to M. tuberculosis (death within 3 weeks) and such mice may even die after many weeks from challenge with BCG (Flynn et al. 1993). A major inducer of the Type 1 pathway is IL-12, and IL-12 KO mice are more susceptible to tuberculosis (Wakeham et al. 1998).

restricted T cells, several other T cell types are also involved in the response to mycobacteria, including CD8+ cells, gamma/delta (y/o) T cells, CDl-restricted T cells and regulatory T cells. These are discussed later in relation to the immunology of the disease in man.

9.2.2.1 The Detrimental Role of Type 2 Responses

These data emphasise the crucial role of the Type 1 response. In agreement with this, other data indicate that the Type 2 response is not only unable to protect mice but can seriously undermine the efficacy of the Type 1 response. If a weak Type 2 response to the shared mycobacterial antigens is deliberately induced before challenge, mice are found to be strikingly more susceptible to tuberculosis than are unimmunised control animals (Hernandez-Pando et al.1997b; Lindblad et al. 1997). Similarly, in the Balb/c mouse model of pulmonary TB infection, the appearance of IL-4 in the lung lesions (as seen by immunohistochemistry and RT-PCR) coincides temporally and spatially with the appearance of areas of pneumonia and necrosis, leading to rapid clinical deterioration and death (Hernandez-Pando et al. 1996). These observations are not contradicted by a claim that, in IL-4 gene-disrupted mice, there is no evidence of increased resistance to the infection (North 1998). The animals used were resistant anyway and had controlled growth of the bacteria by 21 days, which is the optimum that can be achieved (North 1998). In contrast, Balb/c mice control the infection briefly at 21 days, and then relapse with progressive disease. This late phase is accompanied by a switch to Type 2 cytokine production (HernandezPando et al. 1996, 1997b), but progression is greatly attenuated if the IL-4 gene is knocked out (HernandezPando et al., in preparation).

9.2.2.2 Variability of Tuberculosis in Different Mouse Strains

As outlined above, the nature of the disease caused by M. tuberculosis in mice depends on the mouse strain used. For instance,A/J mice develop progressive interstitial pneumonitis, while C57BL/6 mice can develop massive granulomas mediated by a Type 1 response and so "drown" in Thl lymphocytes (Watson et al. 2000). Balb/c mice start with a Type 1 response and

G.A. W. Rook

136

control the infection at 21 days, but then switch to a Type 2 response and die with an increasing bacterial load and immunopathology (Hernandez-Pando et al. 1996, 1997b). Therefore, we cannot be sure which model is most similar to human tuberculosis without first studying the human disease.

9.2.3 The Protective Role ofTumour Necrosis Factor (TNF-a) This pro-inflammatory cytokine can have either protective or detrimental effects in murine disease, and the same is likely to be true in man, as discussed below in relation to immunopathology. Its effects appear to depend upon the other cytokines present. In the mouse, TNF-a is protective early in infection. TNF-a levels are elevated early (day 3) in mice infected via the intratracheal route and reach a second peak in the third week, coinciding with mature granuloma formation (Hernandez-Pando et al. 1997a). TNF-a receptor KO mice succumb faster to M. tuberculosis infection than control mice (Flynn et al.1995), and a disruption of the granulomatous response and increase in mycobacterial load is noted in M. tuberculosis-infected mice when TNF-a bioactivity is blocked (Adams et al. 1995).

culosis in man has come from the study of children with genetic defects of the Type 1 cytokine system. Vaccination with Bacille Calmette-Guerin (BCG), an avirulent derivative of the organism responsible for bovine tuberculosis, occasionally causes disseminated infection. The gene for the IFN-yRI gene in such a child had a single nucleotide deletion that resulted in the creation of a premature stop codon near the N-terminus (Jouanguy et al. 1996). Another study (Newport et al. 1996) involved four children from the same small town in Malta who presented with disseminated mycobacterial infections. The mycobacterial species isolated were M. fortuitum, M. avium (two strains) and M. chelonei. One child also had prolonged salmonellosis. These children had a single nucleotide substitution (A for C) rather than a deletion (Newport et al. 1996). It allowed normal levels of expression of the mRNA, but again introduced a premature stop codon. Interleukin-12 (IL-12) receptor deficiency has also been found in otherwise healthy individuals with mycobacterial infections. Unlike the children with IFNyR deficiency these patients are able to form mature granulomata, but their NK cells and T cells secrete little IFN-y. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas (Altare et al.1998; de Jong et al. 1998).

9.2.4 Immunity to Tuberculosis in Man

9.2.5

9.2.4.1

Other T Cell Types That May Be Involved in Immunity

Genetics of Susceptibility to Tuberculosis

Conventional genetic studies of tuberculosis patients showed that polymorphisms in genes encoding naturalresistance-associated macrophage protein (NRAMP1), the IL-l gene cluster, the vitamin D receptor and mannose-binding lectin were associated with susceptibility (Bellamy 2000). The relevance of vitamin D receptor polymorphisms is increased when there is also vitamin D deficiency as in the Gujarati population in London (Wilkinson et al. 2000). However, the effects on disease susceptibility are small, and so far studies of this type have cast little light on the mechanisms of immunity.

9.2.4.2 Natural Human Gene Knockouts; Proof of the Role of IFN-y and IL-12

On the other hand definitive evidence that, as in mice, the Type 1 response is crucial for immunity to tuber-

In addition to conventional CD4+ aJ~, Class II MHCrestricted T cells, several other T cell types are also involved in the response to mycobacteria. These are particularly fascinating because many of them recognise non-peptide antigens. Fig. 9.1 summarises the lymphocyte types involved, and some of their functions.

9.2.5.1 CD8+ IFN-y-secreting T Cells

There may be a high frequency of IFN-y-secreting CD8+ cytotoxic T cells in the peripheral blood of healthy individuals who have been exposed to tuberculosis (Pathan et al. 2000). This response was maintained in an asymptomatic contact over 2 years, so such cytotoxic T cells can clearly persist in individuals without active tuberculosis. These CD8 cells recognised a peptide from ESAT-6 (an immunogenic peptide released very early into cultures of M. tubercu-

137

The Immunology and Pathogenesis of Tuberculosis Various cell types thai recognise lipids, glycolipids, Isoprenoids

CD·1

1-1.

IL-12 Class I MHC Class II

COB

:-J

04

=

IFN'r

Activation - - - - - - ,

ltl- ' - - - - - - - - - - Suppression

Antigen· presenting cells

~IL-10 T re TGF·~

IL-10 Class II MHC Class I

Tc1 11----+----+ Cytotoxicity - - - - - - ,

fr'L~

IL·402

-

Tc

COB

' - - - - Fibrosis

Fig.9.1. Multiple lymphocyte types are involved in immunity to mycobacteria. IFN -r, the major activator of macrophages, may be released by CD4+ Thl cells, but also by CD8+ cytotoxic T cells and a variety of CD-I-restricted T cells that recognise unconventional antigens such as lipids and glycolipids (see main text). Macrophage activation and macrophage killing by cytotoxic cells can both lead to bacterial death. If the antigen-presenting cells are secreting IL-IO, rather than IL-12, the T cells generated will tend to be regulatory T cells (Treg ) or IL-4 (IL-402) secreting CD8+ cells and CD4+ Th2 cells. The presence of all of these in TB patients is well documented and, together with TNF·a (not shown), they will impair immunity and cause suppression, macrophage deactivation, fibrosis and immunopathology. The reality is still more complex, because TB patients also have T cells that make both IFN and lL-lO

-r

losis), and other peptides recognised by this cell type have been defined using cells from patients recovering from tuberculosis (Cho et al. 2000). These observations are important since both in mice (Silva and Lowrie 2000) and in man (Cho et al. 2000) CD8+ cytotoxic T cells that use the granule-mediated pathway of killing (as opposed to the Fas-FasL pathway) lead to killing of the contained mycobacteria, too. The role of the granule contents, particularly granulysin, has been reviewed (Krensky 2000). 9.2.5.2 CD-l-restricted T Cells

In humans and other large long-lived animals there are several types of T cell (including a~ T lymphocytes negative for both CD4 and CD8 molecules (socalled double-negative T cells), rb T cells and certain CD4+, CD8+, CD8a/a+ and NK lymphocytes) that recognise mycobacteriallipoarabinomannan (LAM) or mycolic acids presented by the MHC class I-like molecules of the CDI family (Beckman et al. 1994; Moody et al. 1997; Sieling et al. 1995). Similar T cells have been detected that recognise an evolutionarily conserved family of isoprenoid glycolipids that include essential components of protein glycosyl-

ation and cell wall synthesis pathways (Moody et al. 2000). The role of the CD-l restricted T cells remains unclear because rodents have a homologue of CDId, but have lost the genes for CD-la, b or c which are the forms that present mycobacterial lipids and glycolipids. Thus, their protective role will need to be tested in guinea-pigs or in large long-lived animals that have these genes, though it is reported that some types of human CD-I-restricted T cell not only produce significant amounts of IFN -y but also appear able to lyse infected cells and directly kill intracellular mycobacteria (Sieling et al. 1995; Stenger et al. 1998). It is fascinating that the different forms of CD-l "survey" different intracellular compartments, and so will encounter products of M. tuberculosis in different ways (Schaible et al. 2000; Sugita et al. 2000).

9.2.5.3 Gamma/delta (r/o) TCells

rio

Like CD8+ T cells, T cells can also kill infected cells by the granule pathway, and can reduce the viability of the contained bacteria (Dieli et al. 2000b). These cells are activated during pulmonary tuberculosis and

138

increase in frequency (Behr Perst et al.1999), but they decline after treatment (Dieli et al. 2000).

G.A.W.Rook

IL-12 provide definitive evidence of the importance of Type 1 cytokines, and suggest a close parallel with the mouse models. Recently the negative role 9.2.5.4 of Type 2 cytokines in human TB, again paralleling Regulatory T Cells the murine models, has been established (Marchant et al. 2001; Seah et al. 2000; van Crevel et al. 2000). A dominant theme in other domains of immunol- Expression of IL-4, whether measured by flow ogy is the regulatory T cell or "T reg" though they cytometry or by sensitive quantitative RT-PCR have received little attention in the context of on non-stimulated peripheral blood T cells (Seah tuberculosis. Treg characteristically release IL-lO and Rook 1999), is increased and correlates with and TGF-p, and suppress inflammation in models severity of disease and with cavitation (Seah et al. of allergy, inflammatory bowel disease and auto- 2000; van Crevel et al. 2000). The IL-4 mRNA copy immunity (Maloy and Powrie 2001). It has been number also correlates with total IgE (Seah et al. claimed that T cells cloned from the bronchoalveo- 2000) and with levels of soluble CD30 (Seah and lar lavage of human TB patients make both IFN-y Rook 2000). Thus, although it is true that actual and IL-l 0, and so may be a form of Treg (Gerosa et al. cytokine levels and mRNA copy numbers are 1999). Other authors have also noted the increased higher for Th-l than for Th-2 cytokines in tubersecretion of TGFp and IL-I0, though some of the culosis, the major change in cytokine expression IL-lO may come from macrophages rather than Treg compared to healthy donors is not, as previously (Ellner 1997). Inappropriate premature activation of stated, the small decrease in expression of Thl Treg is clearly a possible mechanism of failure of the cytokines, but rather a massive (80-100x) increase in expression of Th2 cytokines (Seah et al. 2000). immune response. Similarly, M. tuberculosis-responding T cells cloned from the peripheral blood of untreated tuberculosis patients, whether from Africa or Italy, secrete 9.2.6 The Role ofTNF-a. in Human Tuberculosis abundant IL-4 in response to M. tuberculosis. After treatment, the cytokine profile returns to Type 1 The other cytokine that is clearly implicated in man, (Marchant et al. 2001). Interestingly, many of the which again shows a parallel with data from the IL-4-secreting cells in TB patients are, in fact, mouse models discussed above, is tumour necrosis CD8+ (Gerosa et al. 1999; van Crevel et al. 2000). factor alpha (TNF-a.). Absolute proof of its role in Actually, it is likely that they are in reality secreting man is available only in relation to latent tubercu- IL-402, the splice variant of IL-4 that drives fibrosis losis. An antibody that neutralises TNF-a. has been (Atamas et al. 1999; Seah and Rook 1999). Finally, achieving some success as a treatment for rheu- it has emerged that M. tuberculosis contains commatoid arthritis, but a clear consequence in some ponents that drive striking expression of IL-4 in individuals is the reactivation of latent tuberculosis normal peripheral blood mononuclear cell cultures (Keane et al. 2001). It is therefore interesting that the in vitro (Seah and Rook 2001). DNA from M. tuberculosis detected by in situ PCR is These data have resolved a long-running controfrequently found in cells situated in tissues devoid versy which deserves explanation. That there is an of lymphocyte infiltration (Hernandez-Pando et al. IL-4 component in the response of human tubercu2000a), so that lymphocyte-derived cytokines may losis patients to M. tuberculosis ought to have been be at very low levels. On the other hand, numerous accepted 10 years ago, because there is no other components of M. tuberculosis may directly trigger known explanation for the presence of specific IgE TNF-a. release from macrophages and other non- antibody (Yong et al. 1989). Interestingly, the other lymphoid cell types, and this local release of TNF-a. largely Type 2 cytokine-dependent antibody, IgG4, is also reported to be increased in patients (Wilsher may maintain latency. et al. 1999). Similarly, immunohistochemistry reveals IL-4-expressing cells in the lymphoid tissue of tuberculosis patients (but not in tissue from patients with 9.2.7 sarcoidosis) (Bergeron et al. 1997). Type 2 Responses in Human Tuberculosis These observations and, above all, the susceptibility of children with defective receptors for IFN-y or

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139

9.2.8

Increasing antigen load is likely to be one factor, since the Thl/Th2 balance is strikingly linked to dose when immunising with particulate antigens such as mycobacteria (Hernandez-Pando and Rook 1994), or Why, then, has the matter been controversial? Firstly, leishmania (Bretscher et al. 1992). IL-4 is biologically active at much lower concentraThere are now conclusive data showing that M. tions than IFN-y and has a correspondingly lower tuberculosis contains components that drive IL-4 mRNA copy number, so methods that reliably pick expression and secretion in vitro when cultured with up IFN-yor its messenger RNA fail to detect biologi- human peripheral blood mononuclear cells (Seah cally significant levels of IL-4. Moreover the mRNA and Rook 2001). The nature and importance of these for IL-4 has a very short half-life, so it has often components is currently under investigation. disappeared from clinical specimens collected in In some populations excessive or inappropriate the field before they reach the laboratory. Workers contact with environmental mycobacteria may have who failed to detect IL-4 in either normal donors primed a Type 2 response to the crucial common or TB patients assumed, with no logical justifica- antigens. This mechanism is clearly demonstrable tion, that it was not increased in the latter. Secondly, in mice, in which it can massively increase susceptiattempts to increase cytokine expression by stimu- bility (Hernandez-Pando et al. 1997b) and has been lation of the cells in vitro do not yield an accurate suggested, but not conclusively proven, in man (Fine reflection of the cytokine balance present in vivo, 1995; Stanford et al.I981). and rapid early production of IFN-y can suppress During active infection, selective apoptosis of ThlTh2 cytokine release, as repeatedly demonstrated like T cells may be a factor (Das et al. 1999;Varadhachary by workers in the field of allergy. Finally, previous and Salgame 1998), as may prostaglandin release studies failed to take into account the presence of (Hilkens et al.1995; van-der-Pouw-Kraan et al.1995). the IL-4 splice variant (IL-482). This variant of lL-4 Finally there are now strong reasons for suggesting may be an inhibitor of IL-4 activity and is always that endocrine interactions with the immune system co-expressed with IL-4 at about the same level are important. Vitamin D3, cortisol metabolism and (Arinobu et al. 1999). In lung cells it may be dehydroepiandrosterone levels are discussed in the expressed at higher levels than IL-4 itself. However, endocrinology section below. almost every study of IL-4 mRNA levels used primers that would amplify mRNA for both lL-4 and the splice variant (Seah and Rook 1999). 9.2.9 Macrophage Function and M. tuberculosis 9.2.8.1 The Mechanisms of the Shift Towards a Type 2 9.2.9.1 Cytokine Profile Uptake of Mycobacteria

The Causes of the Controversy over Expression of IL-4 in Human Tuberculosis

What are the likely causes of this shift in cytokine profile? Several possibilities are shown in Fig. 9.2.

Mycobacteria are taken up by multiple pathways including complement receptors and mannose recep-

Fig. 9.2. Mechanisms that may lead to excessive activation of Type 2 cytokines and immunoregulation in human tuberculosis. These can impair mycobactericidal functions of macrophages and, in concert with TNF-a, contribute to immunopathology. Additional factors not shown are prostaglandins, and also prior induction of Th2 response to cross-reactive components of environmental mycobacteria

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tors (Kang and Schlesinger 1998; Schlesinger 1998). However, this is clearly not the whole story because in vitro M. tuberculosis can enter a variety of cell types that do not express these receptors (Filley and Rook 1991; Shepard 1958). The exact mode of uptake must affect the subsequent fate within the cell (AstarieDequeker et al. 1999).

9.2.9.2 Toll-like Receptors

Much of the initial activation and cytokine response of macrophages to mycobacteria may be mediated by interaction with the Toll-like receptors (TLR). These are members of the interleukin 1 (IL-1) receptor family, related to Toll, a molecule involved in innate microbial resistance mechanisms in Drosophila. Both virulent and attenuated strains of M. tuberculosis can activate in a TLR-dependent manner. TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Bacillus Calmette-Guerin failed to induce TLR-dependent activation. However, other soluble and cell wall-associated components of M. tuberculosis are involved. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4dependent activation (Means et al. 1999). Lipoproteins can activate via TLR, and several from M. tuberculosis will drive IL-12 production in this way (Brightbill et al. 1999), perhaps explaining the latter result. 9.2.9.3

Mycobactericidal Mechanisms Within Macrophages

Inhibition or killing ofM. tuberculosis is easily induced in murine macrophages by exposure to IFN-y but such

effects are extremely difficult to demonstrate convincingly in human cells (Rook et al. 1985, 1986). Success has been reported using human alveolar lavage macrophages exposed to TNF-a in vitro (Hirsch et al.1994). It is possible that the major killing mechanism is not a direct effect of activated macrophages, but rather an event that occurs during certain types of apoptosis (see below) or during killing of the macrophage by cytotoxic T cells that "inject" granulysin and perforin (see section on CD8+ effector cells above) (Fig. 9.3).

9.2.9.4 Reactive Oxygen and Nitrogen Intermediates

If macrophages do themselves kill M. tuberculosis, these are likely candidate killing mechanisms (Fig. 9.3) (Chan et a1.1992, 1995). The mechanism of action of the nitric oxide (NO) is uncertain, because it has important signalling and second messenger functions that may be as important as direct toxicity for the organisms (Roach et a1. 1994). However, KO mice unable to make NO or other reactive nitrogen intermediates (RNI) showed no increase in proliferation of M. tuberculosis in the lungs until very late in the infection, but there was increased growth in the spleen. In contrast, KO mice unable to make reactive oxygen intermediates (ROI) had increased growth of bacilli in the lungs. Interestingly, activation of macrophages by IFN-y in vitro to control proliferation of M. tuberculosis was dependent upon RNI rather than ROI, and so appeared to parallel immunity in the spleen rather than in the lungs (Adams et a1. 1997). The role of NO in man remains unclear. It probably is made by appropriately activated human macrophages (Nicholson et a1. 1996), but never in the very large quantities that murine macrophages can release. There is not universal agreement about the antimycobacterial relevance

MACROPHAGE

1) Activation

Acidification of phagosomes Maturation to hydrolytic vesicle

IFN-y lNF-a

Reactive oxygen and nitrogen derivatives ------l

2) Cytotoxic T cells

~ 'YJ 3) Induction of apoptosis

Perlorin Granulysin - - - - - +

Fas

-----A--------"

ATP ~ Death of some bacteria and H20 2 uptake of apoptotic bodies Fas Ligand by fresh macrophages

Multiple mechanisms attempt to kill M. tuberculosis within macrophages

Fig. 9.3. It is still not clear which are the most important mechanisms leading to killing of M. tuberculosis, particularly in human cells where killing is very difficult to achieve in vitro. Candidate pathways are shown and all are referenced and discussed in the text. Note that only some inducers of apoptosis result in reduced viability of the contained bacteria. Further macrophage activation must occur via the Toll-like receptors but their role in killing is not yet known

141

The Immunology and Pathogenesis of Tuberculosis

9.2.10

of this mechanism (Aston et al. 1998). There is some evidence that l,25(OHhD3 may be involved in the activation of iNOS in a human monocyte cell line (Rockett et al. 1998), so this could explain the antimycobacterial effect of this material in vitro (Rook et al. 1986).

Evasion of the Antimicrobial Functions of Macrophages

Apoptosis (programmed cell death) is a major regulator ofthe immune system,byproviding differential removal of different lymphocyte and macrophage subsets. Some human peripheral blood T cells undergo apoptosis in 9.2.9.5 the presence of antigen from M. tuberculosis (Das et Macrophage Apoptosis and the Death of Bacteria al. 1999; Hirsch et al. 1999). In contrast, several groups Large numbers of apoptotic T cells and macrophages report that M. tuberculosis actively inhibits apoptosis are seen in caseous foci. Immunohistochemistry and of macrophages (Fig. 9.4). Mannose-capped lipoarabiin situ hybridisation showed that the macrophages nomannan (Man-LAM) stimulates phosphorylation of surrounding caseous foci are negative for the anti- Bad, a pro-apoptotic protein, and so inhibits apoptoapoptotic protein bel2, but positive for the pro-apop- sis (Maiti et al. 2000). Another group has shown that totic protein bax, while the associated T cells express Man-LAM inhibits apoptosis by a mechanism involvIFN-y and FasL (Fayyazi et al. 2000). Macrophage ing calcium-dependent signalling (Rojas et al. 2000). apoptosis may be beneficial to the host, because Moreover cells containing M. tuberculosis are markedly death of the infected macrophage can be associated more sensitive to killing by TNF-a (Filley and Rook with death of the contained mycobacteria (Fig. 9.3). 1991), but infected cells secrete soluble TNF receptors Moreover, it has been suggested that reduction in (sTNF-R2) that can block their destruction by TNF-a bacillary numbers is only achieved by apoptosis of (Fratazzi et al. 1999). Thus it might be suggested that infected monocytes, not by the necrotic mode of M. tuberculosis causes death of a subset of T cells, while death (Molloy et al. 1994; Oddo et al. 1998). Apopto- preserving some macrophages as hiding places with sis induced by ATP promotes the killing of virulent reduced microbicidal and antigen-presenting funcM. tuberculosis within human macrophages (Kusner tion. A point against this hypothesis is that infected and Adams 2000; Lammas et al. 1997) as does apop- macrophages may also have down-regulated mRNA tosis induced by Fas ligand (Oddo et al. 1998), and expression of bcl-2, an inhibitor of apoptosis (Klingler hydrogen peroxide-induced apoptosis also causes et al. 1997). It may be that the organism is preferentially mycobactericidal effects (Lauchumroonvorapong et inducing forms of apoptosis that leave the organisms al.1996). Thus, perhaps M. tuberculosis has strategies unharmed. In addition to opposing apoptosis, mycobacteria that oppose macrophage apoptosis in order to limit have numerous other strategies for avoiding being the bacterial killing that can accompany this event.

M. tuberculosis avoids being killed by macrophages 1) Mechanisms during uptake Uptake via Mannose receptor Block Ca++ signalling by complement receptor Mannose receptor

Macrophage

Cell not activated

I Blocked function

2) Resistance to killing Tough cell w

SuperoXJde dismu1aSe

Free radlClll scavengers

H~"C?~ 8

3) Alterations to vesicles Failed maluratJon Allered fUSlOtl panern Blocked proton pump

4) Control cell signalling .....

S) Increased release soluble TNFf11 IL·l0

TGF·p IL-6

6) Decreased release or expression

Faa SHP-l

TNF-C

Actrvate phosphotyroslne phosphalase Block signalling via IFN-y receplOfS Illock an1Jgetl presa
Peptide + Class II MHC

9

Fig. 9.4. This figure should be compared with Fig. 3. Each of the killing pathways in Fig. 3 is opposed by some mechanisms in Fig. 4. Short thick lines indicate blocked pathways. Most of the figure is self-explanatory. Uptake via mannose receptors may avoid triggering killing mechanisms. The vacuoles containing mycobacteria lie within the sorting/recycling endosomal machinery of the macrophage and fail to acidify and fuse with acidic lysosomes. But they do fuse with LAMP-I-containing endosomes and they release vesicles containing LAM. Thus their fusion pattern is altered by the pathogen. (LAM lipoarabinomannan, LAMP-I lysosome-associated protein 1) SHP-I is a phosphotyrosine phosphatase

142

killed by phagocytes (Fig. 9.4) (Chan et al. 1991), M. tuberculosis may be taken up via mannose receptors that fail to trigger killing events (Astarie-Dequeker et al. 1999). It also inhibits complement-receptormediated Ca++ signalling, which may contribute to the failure of killing mechanisms (Malik et al. 2000). Mycobacteria can inhibit acidification of the phagosome (Sturgill-Koszycki et al. 1994) and modify intracellular trafficking of vacuoles so they behave like part of the endosomal recycling compartment rather than as toxic phagolysosomes (Xu et al. 1994). These vacuoles release quantities of lipoarabinomannan (LAM) which insert into glycosylphosphatidylinositol (GPI)-rich domains in the cell membrane (Ilangumaran et al. 1995). LAM is itself a GPI of unusual glycan structure, with the ability to modify numerous macrophage functions including the ability to respond to IFN -y and the ability to present antigen (reviewed in Ilangumaran et al. 1995). The last point may be relevant to the apparent inability of long-term mycobacterium-infected macrophages to present antigen to CD4+ T cells (Pancholi et al.1993). Thus the organisms may hide, undetected, in modified macrophages. The pattern of cytokine release from infected macrophages changes so that macrophage activation is diminished and T cell recruitment impaired (Fig. 9.4). Recruitment of Thl lymphocytes requires IL-12 production, which is inhibited by increased production of TGF-~ and IL-IO (Ellner 1997; Fulton et al.1998; Toossi et al.1997) and IL-6 release may also be a factor. TGF-~ and IL-I0 also impair macrophage microbicidal function and the IL-I0 contributes to increased release of TNF receptor-2, which blocks the activating role of TNF-a (Balcewicz-Sablinska et aI.1998).

9.2.11 Bacterial Genetics and Mechanisms of Avoidance of Destruction by the Type 1 Response There is intense interest in the bacterial factors that allow M. tuberculosis to resist destruction by the immune system in mice after the development of the full Type 1 response at about 21 days. The recent publication of the entire sequence of the M. tuberculosis genome has allowed a variety of genetic techniques to be applied. Mutations that inactivate mycolic acid cyclopropane synthase (Glickman et al. 2000) or isocitrate lyase, which is required for the metabolism of fatty acids (McKinney et al. 2000), both result in decreased ability to persist. Others have expressed candidate virulence genes,

G.A. W. Rook

or plasmid libraries of M. tuberculosis DNA in the avirulent organism M. smegmatis and then looked for increased ability to survive inside macrophages (Miller and Shinnick 2000; Wei et al. 2000). A review of the comparative genomics of mycobacteria gives an overview of the techniques used and the results obtained (Brosch et al. 2000).

9.3 Immunopathology 9.3.1 The Koch Phenomenon; the Response Characteristic of Disease As outlined in the introduction, Koch noted that 4-6 weeks after establishment of infection in guineapigs, intradermal challenge with whole organisms or culture filtrate resulted in necrosis locally and in the original tuberculous lesion (Koch 1891). Similar phenomena occur in humans. The tuberculin test is frequently necrotic in subjects who are, or have been, tuberculous, whereas necrosis does not occur when positive skin-tests to tuberculin are elicited in normal BCG recipients or in tuberculoid leprosy patients. Koch sought to exploit this phenomenon for the treatment of tuberculosis and found that injection of larger quantities of culture filtrate (Old Tuberculin) subcutaneously into tuberculosis patients would evoke necrosis in established tuberculous lesions at distant sites (Anderson 1891). This resulted in necrosis, sloughing and "cure" of the lesions of skin tuberculosis (Lupus vulgaris, usually caused by bovine strains), but when similar necrosis was evoked in deep lesions in the spine or lungs, the results were disastrous and merely provided further necrotic tissue in which the bacteria could proliferate (Fig. 9.5). This treatment was therefore abandoned, but it provided crucial clues as to the nature of the immunopathology of tuberculosis.

9.3.2 Tuberculin Response and Protection The error in Koch's thinking was highlighted in the 1940s. When guinea-pigs were pre-immunised so that they had powerful Koch phenomena in response to small doses of tuberculin, they became more susceptible to tuberculosis than were unimmunised control animals (Wilson et al.1940). Obviously this was seen

The Immunology and Pathogenesis of Tuberculosis

143

Fever, rigors. necrosis and sloug/ling 01 the skln lesion, and dangerous necrosis of other lesions In lUngs or spine.

24-48hrs

~

.~

only if the challenge infection was into the lungs or by deep intramuscular injection, so that necrosis could not result in shedding of the infected tissue. Optimal protection was seen in animals with moderate positive responses. This experiment is illustrated in Fig. 9.6 (Wilson et al.I940),and makes the point, also apparent from human epidemiology (Fine 1993), that the most protective response correlates with a small positive induration in the Mantoux test (delayed type hypersensitivity or DTH) rather than with the massive necrotic Koch phenomenon.

9.3.3 The Relationship Between the Koch Phenomenon and the Shwartzman Reaction Koch's discovery that soluble bacterial material could trigger necrosis in a distant tuberculous site (in addition to the site of injection) has some parallels with the Shwartzman reaction (Shwartzman 1937), and subsequent experiments strengthen the parallel still further. For instance injections of endotoxin-rich material into a distant site (instead of the tuberculin used by Koch) will also trigger necrosis in tuberculous lesions (Bordet 1931; Debonera et al.1932; Shands and Senterfitt 1972). These observations are compatible with the view that tuberculous lesions are susceptible to superimposed cytokine-mediated damage.

Fig. 9.5. Koch's failed immunotherapeutic treatment for tuberculosis, similar to the phenomenon he had previously described in guinea-pigs. It achieved necrosis and sloughing of the chronic lesions of cutaneous tuberculosis (Lupus vulgaris), but the simultaneous necrosis and liquefaction of cryptic lesions in deep tissues was disastrous and the treatment was abandoned

9.3.4 A Hypothesis to Explain the Koch Phenomenon There is increasing evidence for increased susceptibility to cytokine-mediated tissue necrosis of tissues undergoing inflammation mediated simultaneously by Type 1 and Type 2 cytokines. This is easily demonstrated in Balblc mice with pulmonary tuberculosis (HernandezPando et al. 1995). During the first 3 weeks, DTH sites were not sensitive to local injection of as much as 1 ~g of recombinant TNF-a. This is the period of Type 1 response (Orme et al.1993). After 50 days, the animals enter a phase of slowly progressive disease accompanied by increasing Th2 cytokine production, seen as IL-4 positive cells in the lesions. In these animals DTH sites become TNF-a-sensitive (Hernandez-Pando et al. 1997b; Hernandez-Pando and Rook 1994). Recently we have found that this state of TNF-a-sensitivity of the DTH sites does not occur in tuberculous IL-4 KO mice, but is restored if the IL-4 KO mice receive an intravenous injection of a persistent form of IL-4 (Hernandez-Pando et al., in preparation). This propensity for TNF-a toxicity in the presence of IL-4 is supported by several examples in non-mycobacterial models. Lawrence and co-workers, studying Trichinella spiralis infection in mice, have found that the enteropathy caused by TNF-a is dependent upon IL-4 (Lawrence et al. 1998). Other murine studies have also revealed that, in certain mouse strains, DTH caused by Th2 cells cor-

Result of inlramuscular infecllon with 18

~

~

I

Immunise with killed M. tuberculosis by various protocols \

W

•

, . . . . . - - - - - - - . . . , MORE susceptible ... 'Koch" than unimmunised

r-

ooMro~ Rank according to ~ Positive tuberculin ~

~ response

.. Negative Unimmunised controls

R' t t es,s an

~+----_Negative

Susceptible Susceptible

Fig.9.6. Large numbers of outbred guinea-pigs were immunised with killed M. tuberculosis by a variety of protocols an d then tested with a range of dilutions of tuberculin, before they were challenged by the intramuscular route with a very small does of M. tuberculosis. The most protected animals were those with small positive tuberculin responses, whereas animals with exquisite tuberculin sensitivity (Koch phenomenon) were more susceptible to the infection that were unimmunised controls (Wilson et al. 1940)

G.A. W. Rook

144

relates with TNF-a production by the cells (Muller et al. 1994). Finally, if an important component of the Koch phenomenon is cytokine-mediated damage in a site of mixed Type l/Type 2 inflammation, then the toxicity of Koch's "treatment" for tuberculosis is easily explained (Figs. 9.5, 9.2) (Anderson 1891).

9.3.5 Detrimental Roles ofTNF-a in Human Tuberculosis The previous section suggests that a component of the immunopathological state in human tuberculosis may result from the simultaneous presence of Type 1 and Type 2 cytokines and TNF-a. An increase in plasma TNF-a levels has been associated with clinical deterioration in patients with severe tuberculosis (Bekker et al. 1998). Both thalidomide and pentoxifylline, as inhibitors of TNF-a release, have been tried as treatments for the cachexia of chronic disease (Haslett 1998). Thalidomide, which reduces IL-6, IL-lO as well as TNF-a levels, and reduced lung pathology in a murine model (Moreira et al. 1993), was clearly clinically beneficial in the human disease (Tramontana et al. 1995).

9.3.6 Fibrosis The fibrosis is another aspect of the immunopathology of human tuberculosis that requires explanation. The dominant cytokine in tuberculosis, IFN-y, opposes fibrosis, so why does fibrosis occur? In fact, in all human diseases characterised by marked pulmonary fibrosis there is expression of Type 2 cytokines (systemic sclerosis, idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, chronic lung allograft rejection [reviewed in Lee et al. 2001]) and the same is true in the mouse in the bleomycin model (Lee et al. 2001) and in schistosomiasis, where fibrosis and tissue remodelling do not occur if induction of Type 2 cytokines by the ova is blocked by preimmunisation with ova+IL-12. These observations have led to the "Type 2 cytokine hypothesis offibrosis" (Lee et al. 2001). Type 2 cytokines such as IL-4, IL-482 and IL-13 activate fibroblasts and promote collagen formation. IL-13 also drives formation of TGF-~ and, by increasing release of the enzymes that activate TGF-~ (such as MMP-9) while down-regulating latent TGF-~-binding protein (LTBP), it also activates the TGF-~ (Lee et al. 2001). Significantly, the increase in expression of Type 2 cytokines in human TB extends to IL-13 which, like IL-4 and IL-482, is increased 80-100

fold compared to controls matched for age, race and gender (Seah et al. 2000).

9.4 Endocrinology There are several endocrine and metabolic changes in tuberculosis that may contribute to the failure of the Type 1 immune response to control the infection, and to the increasing level of Type 2 cytokine expression.

9.4.1 Vitamin D3 Metabolism in Tuberculosis Lesions The macrophages of tuberculosis patients, following activation by IFN-y, express an active la-hydroxylase, and rapidly convert 25(OH)-vitamin-D3 to calcitriol (Rook 1988; Rook et al. 1986). This is a potent phenomenon, leading occasionally to leakage of calcitriol into the periphery and to hypercalcaemia, though it has in the past been difficult to understand its role in the disease (Rook 1988). It now seems possible that this is a feed-back mechanism that tends to down-regulate Th1 and enhance Th2 responses, because calcitriol inhibits production of IFN-y and IL-2, and increases production ofIL-4 and IL-5 (Daynes et al. 1991; Rigby et al. 1987). This may well be related to the ability of calcitriol to inhibit release of IL-12 (Lemire 1995). In the 1940s attempts were made to treat tuberculosis with vitamin D. When patients with skin tuberculosis (Lupus vulgaris, often due to M. bovis) were treated with this vitamin, the chronic non-healing granulomatous lesions often underwent necrosis followed by resolution (Macrae 1947). However necrosis and liquefaction also occurred in deep lesions in the spine and lungs (Brincourt 1967), so the result was as disastrous as the use of Koch's immunotherapy described above (Anderson 1891). The mechanism of this effect remains unknown, but an increase in Type 2 cytokine expression in granulomata rich in Type 1 cytokines and TNF-a would be expected to cause necrotising immunopathology as discussed earlier.

The Immunology and Pathogenesis of Tuberculosis

9.4.2 Adrenal Steroids in Tuberculosis

9.4.2.1 The Effects of Stress

Glucocorticoids cause a switch to Type 2 cytokine production (Brinkmann and Kristofic 1995; Ramirez et al. 1996), probably because of effects on dendritic cells, which secrete less IL-12 and more IL-1O in their presence (Vieira et al. 1998; Visser et al.1998), but glucocorticoids also down-regulate the antimycobacterial effects of macrophages (Rook et al. 1987). It is therefore not surprising that reactivation or progression of infection with tuberculosis is sensitive to glucocorticoid therapy and to activation of the hypothalamopituitary-adrenal axis. Exposing humans to the stress of war or poverty (Spence et al. 1993), or cattle to the stress of transportation are enough to cause reactivation of disease. The disease-promoting effect of stress (Brown et al.1993; Tobach and Bloch 1956) and excessive activation of the hypothalamo-pituitary-adrenal axis (Hernandez-Pando et al.1998a,b) have been demonstrated under more controlled conditions in mice.

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that the site of abnormal conversion of inactive cortisone to active cortisol in the patients is the infected lung itself (Baker et al. 2000; Rook et al. 2000). This may be explained by the observation that TNF-a and IL1~ both increased the expression levels and reductase activity of ll~-HSD-l in a cell line in vitro (Escher et al. 1997). The result is a local increase in cortisol levels that is not apparent from measurements of serum cortisol. This cortisol excess will cause a shift towards Type 2 cytokine expression, deactivation of the antimycobacterial effects of macrophages, increased IL10 and increased TGF-~, so it may contribute to the changes seen in the human disease.

9.5

Vaccination The protective efficacy of BCG vaccination varies from 80% protection to no protection at all in different populations (reviewed in Fine 1993). The need to understand this variability before undertaking long and expensive trials of novel vaccines in man is obvious. Several hypotheses are currently under investigation.

9.4.2.2 The Hypothalamo-Pituitary-Adrenal Axis in Human Tuberculosis

In tuberculosis patients who are rested and acclimatised to hospital, the cortisol diurnal rhythm is normal and so are the responses of the adrenals to CRH and to very low doses (i.e. physiological) of ACTH (Rook et al. 2000). The total 24-hour cortisol output may be normal or modestly raised. However, there is a change in the pattern of metabolism of cortisol indicating a large alteration in the equilibrium point of the cortisol-cortisone shuttle, as discussed below (Rook et al' 1996).

9.4.2.3 Dysregulation of the Cortisol-Cortisone Shuttle

A major mechanism for the regulation of local tissue cortisol levels is the inter-conversion of active cortisol (II-hydroxy) and inactive cortisone (II-keto). Thus, effective cortisol concentrations in different organs can be very different from the values found in the serum. Gas chromatography and mass spectrometry revealed a striking excess of metabolites of cortisol relative to metabolites of cortisone in 24-hour urine collections from tuberculosis patients (Rook et al. 1996). Subsequent analysis of alveolar lavage samples from tuberculosis patients and controls has revealed

9.5.1 Interference by Exposure to Environmental Mycobacteria This hypothesis assumes that the common antigens, shared by all mycobacterial species, are relevant to protection. We have known for many years that the Bacillus Calmette-Guerin (BCG) is as effective a vaccine against leprosy as it is against tuberculosis, although M. leprae is an entirely different species (Fine et aI. 1986). BeG must therefore be able to work through common epitopes. Similarly, there is evidence that contact with an environmental organism leading to mycobacterial skin-test positivity is protecting the population of Malawi from both tuberculosis and leprosy (Fine et aI. 1994). In mice powerful protective or "anti-protective" effects can be induced with an environmental mycobacterial saprophyte, simply by altering the immunisation protocol so as to induce Type 1or Type 2 response (Hernandez-Pando et al. 1997b). These effects are obviously due to the common antigens. It is also significant that tuberculosis patients who still maintain necrotising skin-test positivity to antigens of M. tuberculosis itself, have diminished or absent skin-test responses to environmental saprophytes (Kardjito et al. 1986), whereas these do evoke responses in protected

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populations. The heat shock proteins (hsp), which are very highly conserved across species, are important examples of this concept. Indeed, the 65 kDa heat shock protein of M. leprae can protect mice against M. tuberculosis (Silva and Lowrie 1994), as can DNA vaccines based on its sequence. It is, therefore, logical to suggest that an apparent reduction in the efficacy of BCG could occur either because the environmental saprophytes were themselves protecting, or because they were priming deleterious patterns of response (e.g. Type 2) to the common antigens and so blocking the efficacy of the BCG. These two effects were suggested to be occurring in different countries (Stanford et al. 1981) and both effects are easily demonstrated experimentally (Hernandez-Pando et al. 1997b).

9.5.2 Concurrent Parasite Infections Some authors suggest that the presence of concomitant parasite infections may cause a systemic bias towards Th2 responses that undermines the ability of BCG to induce a Thl response to mycobacterial antigens.

9.5.3

then used as recombinant vaccinia or salmonella or as the purified protein plus adjuvant, or as DNA vaccines. The problem is that almost all such vaccines show some efficacy in mouse models, but rarely work as well as BCG, a vaccine already known to fail in man. This approach might be flawed. Virtually all patients develop a strong Type 1 response, so that a vaccine that merely accelerates the establishment of this Type 1 response provides marginal benefit. It may be that the solution is to identify the antigens and epitopes of M. tuberculosis shown to drive the "subversive"Type 2 component (Seah and Rook 2001), by screening for IL-4 induction, and then incorporate these in a vaccine with a potent Type 1 adjuvant, such as IL-12, or with an adjuvant capable of activating regulatory T cells that suppress Type 2 responses (Zuany-Amorim et al. 2002). The aim would be to suppress the Type 2 response to these antigens, or to force the establishment of a Type 1 response to them, even if they prove to be common mycobacterial antigens to which there is a pre-existing Type 2 response evoked by environmental species. Then if the individual were subsequently exposed to M. tuberculosis, the organism would be unable to activate the subversive IL-4secreting component. This novel approach is under investigation and should, moreover, provide a vaccine suitable for use as an immunotherapeutic.

Vaccine Dose Another suggestion is that BCG would be more reliable if used at a very low dose, because the dose at which the vaccine starts to evoke a deleterious Type 2 component may be much lower in some individuals than in others. This idea is derived from vaccination studies with Leishmania in different mouse strains, where a dose that evokes a Type 1 response in some strains is too high, and so Type 2-inducing, in others (Bretscher et al. 1992). Because BCG is a live vaccine it should still be effective at very low doses. In each individual it should proliferate to the level at which a mycobactericidal Thl response is induced (Power et al. 1998). In deer 5XI04 or 5xl07 is protective but 5x108 is less effective, so there might be some truth in this idea (Griffin et al. 1999).

9.5.4 A Possible Novel Approach to Vaccine Design Enormous effort has gone into testing purified or recombinant antigens and seeking those that evoke the most potent Type 1 response, with maximal output of IFN-yfrom human or murine cells. These antigens are

9.5.6 Immunotherapy This is perhaps the most important issue facing workers in the field of tuberculosis. As outlined in the introduction, DOTS helps but fails to solve the problem, and multi-drug-resistant disease is an increasing threat. Immunotherapy is the only solution, and the need for this approach has been recognised since the time of Robert Koch (Stanford et al. 1993). One potential approach to immunotherapy is the direct use of cytokines in patients, either systemically or given by aerosol. IL-2, IFN-y, IL-12 and GM-CSF have all been investigated (reviewed in Holland 2000). Their potential roles in therapy, other than as a potential adjunct to drug treatment in multi-resistant cases, have yet to be elucidated. Another approach that gives striking results in mice is the use of dehydroepiandrosterone or of the very similar androstenediol. These compounds oppose a subset of the effects of glucocorticoids, and can reverse the switch towards Type 2 cytokine profile in Balblc mice, but this has not been tested in man (Hernandez-Pando et al.I998ab).

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Immunotherapy is unlikely to be achieved by References simple antigen preparations, because there needs to be an immunoregulatory component with Abou-Zeid C et al. (1997) Induction of a Type 1 immune responses to a recombinant antigen from Mycobacterium down-regulation of pre-existing Type 2 cytokine tuberculosis expressed in Mycobacterium vaccae. Infect production. This may explain why antigen prepaImmun 65:1856-1862 rations that can protect against tuberculosis when Adams LB et al. (1995) Exacerbation of acute and chronic murine used as vaccines before challenge can fail to act tuberculosis by administration of a tumour necrosis factor receptor-expressing adenovirus. J Infect Dis 171:400-405 as therapeutics in mouse models (Turner et al. 2000). Interestingly, the only preparations shown Adams LB et al. (1997) Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to be effective immunotherapeutics in tuberculous to Mycobacterium tuberculosis using transgenic mice. mice are killed Mycobacterium vaccae (HernandezTubercle Lung Dis 78:237-246 Pando et al. 2000b; Rook and Hernandez-Pando Altare F et al. (1998) Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 280: 1996; Zhang et al. 2000) and more recently DNA 1432-1435 vaccines encoding common mycobacterial antigens (Lowrie et al. 1999). Attempts at immunotherapy Anderson MC (1891) On Koch's treatment. Lancet i:651-652 Arinobu Y et al. (1999) Antagonistic effects of an alternative using the environmental saprophyte, M. vaccae, splice variant of human IL-4, IL-4 delta 2, on IL-4 activifollowed studies of the influence of such organisms ties in human monocytes and B cells. Cell Immunol 191: 161-167 on disease susceptibility and on the efficacy of BCG vaccination (Stanford et al. 1981), a concept that Arkwright PD, David TJ (2001) Intradermal administration of a killed Mycobacterium vaccae suspension (SRL 172) is now widely accepted (Fine 1995). Two studies is associated with improvement in atopic dermatitis in of single injections of heat-killed M. vaccae have children with moderate-to-severe disease. J Allergy Clin been carried out to Good Clinical Practice (GCP) in Immunol107:531-534 human tuberculosis patients also receiving DOTS. Astarie-Dequeker C et al. (1999) The mannose receptor mediates uptake of pathogenic and nonpathogenic mycobacteThe results are conflicting (Group 1999; Johnson ria and bypasses bactericidal responses in human macroet al. 2000), though the striking success of recent phages. Infect Immun 67:469-477 studies in retreatment cases (National Cooperation Aston C et al. (1998) Early inhibition of mycobacterial growth Group on Clinical Study of Mycobacterium vaccae by human alveolar macrophages is not due to nitric oxide. Am J Respir Crit Care Med 157:1943-1950 and Luo 2001) and multi-drug-resistant (MDR) TB (Luo et al. 2000) in China suggest that further trials Atamas SP et al. (1999) Production of Type 2 cytokines by CD8+ lung cells is associated with greater decline in of multiple doses should be undertaken. In view of pulmonary function in patients with systemic sclerosis. the inevitable difficulty of demonstrating an effect Arthritis Rheum 42: 1168-1179 when a single dose is superimposed upon DOTS Baker RW et al. (2000) Increased cortisol-cortisone ratio in acute pulmonary tuberculosis. Am J Respir Crit Care Med under clinical trial conditions in patients with 162:1641-1647 drug-sensitive infections, there is a clear need for Balasubramanian R et al. 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(1999) Phenotypically activated gammadelta T lymphocytes in the peripheral blood of patients with and in clinical trials in human patients with asthma tuberculosis. J Infect Dis 180: 141-149 or eczema (Arkwright and David 2001; Camporota Bekker L et al. (1998) Selective increase in plasma tumour et al. 2000). Interestingly, it is now clear that the necrosis factor-a and concomitant clinical deterioration mode of action of M. vaccae is mainly the induction after initiating therapy in patients with severe tuberculosis. J Infect Dis 178:580-584 of regulatory T cells that suppress Type 2 responses Bellamy R (2000) Identifying genetic susceptibility factors (Zuany-Amorim et al. 2002). It can also induce Thl for tuberculosis in africans: a combined approach using a recognition of common mycobacterial antigens in candidate gene study and a genome-wide screen. 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terium tuberculosis in a human macrophge-like cell line. Infect Immun 66:5314-5321 Rojas M et al. (2000) Mannosylated lipoarabinomannan antagonizes Mycobacterium tuberculosis-induced macrophage apoptosis by altering Ca+2-dependent cell signaling. I Infect Dis 182:240-25 I Rook G (1988) The role of vitamin D in tuberculosis (editorial). Am Rev Respir Dis 138:768-770 Rook G, Hernandez-Pando R (1996) The pathogenesis of tuberculosis. Annu Rev Microbiol 50:259-284 Rook G et al. (1985) I-A restricted activation by T cell lines of anti-tuberculosis activity in murine macrophages. Clin Exp ImmunoI59:414-420 Rook G et al. (1986) Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology 57:159-163 Rook G et al. (1987) A direct effect of glucocorticoid hormones on the ability of human and murine macrophages to control the growth of M. tuberculosis. Eur I Respir Dis 71:286-291 Rook G et al. (1996) Urinary steroid metabolites in tuberculosis; a new clue to pathogenesis. Quart I Med 89:333-341 Rook Get al. (2000) Local regulation of glucocorticoid activity in sites of inflammation; insights from the study of tuberculosis. Ann NY Acad Sci 917:913-922 Salgame PR et al. (1991) Differing Iymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254:279-282 Schaible UE et al. (2000) Intersection of group I CDI molecules and mycobacteria in different intracellular compartments of dendritic cells. I ImmunoI164:4843-4852 Schlesinger LS (1998) Mycobacterium tuberculosis and the complement system. Trends MicrobioI6:47-49; discussion 49-50 Seah GT, Rook GA (1999) A sensitive, non-radioactive quantitative method for measuring IL-4 and IL-4delta2 mRNA in unstimulated cells from multiple clinical samples, using nested RT-PCR. J Immunol Methods 228:139-149 Seah GT, Rook GAW (2000) High levels of mRNA encoding IL-4 in unstimulated peripheral blood mononuclear cells from tuberculosis patients revealed by quantitative nested RT-PCR; correlations with serum IgE levels. Scand J Infect Dis 33:106-109 Seah GT, Rook GAW (2001) IL-4 influences apoptosis of mycobacterium-reactive lymphocytes in the presence of TNF-a. J ImmunoI167:1230-1237 Seah GT, Scott GM, Rook GA (2000) Type 2 cytokine gene activation and its relationship to extent of disease in patients with tuberculosis. J Infect Dis 181:385-389 Shands IW, Senterfitt VC (1972) Endotoxin-induced hepatic damage in BCG-infected mice. Am J Pathol 67:23-40 Shepard CC (1958) A comparison of the growth of selected mycobacteria in HeLa, monkey kidney, and human amnion cells in tissue culture. I Exp Med 107:237-246 Shwartzman G (1937) Phenomenon of local tissue reactivity and its immunological, pathological, and clinical significance. Hoeber, New York Siegele DA, Kolter R (1992) Life after log. I Bacteriol 174: 345-348 Sieling PA et al. (1995) CD I-restricted T cell recognition of microbial Iipoglycan antigens. Science 269:227-230 Silva CL, Lowrie DB (1994) Asingle mycobacterial protein (hsp65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis. Immunology 82:244-248

The Immunology and Pathogenesis of Tuberculosis Silva CL, Lowrie DB (2000) Identification and characterization of murine cytotoxic T cells that kill Mycobacterium tuberculosis. Infect Immun 68:3269-3274 Skinner MA et al. (1997) Immunization with heat-killed Mycobacterium vaccae stimulates CD8+ cytotoxic T cells specific for macrophages infected with Mycobacterium tuberculosis. Infect Immun 65:4525-4530 Spence DP et al. (1993) Tuberculosis and poverty. Br Med J 307:759-761 Stanford JL, Shield MJ, Rook GAW (198l) How environmental mycobacteria may predetermine the protective efficacy of BCG. Tubercle 62:55-62 Stanford JL et al. (1993) Old plague, new plague and a treatment for both? AIDS 7:1275-1277 Stenger S et al. (1998) An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282:121-125 Sturgill-Koszycki S et al. (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678-681 Sugita M et al. (2000) CDlc molecules broadly survey the endocytic system. Proc Natl Acad Sci USA 97:8445-8450 Tobach E, Bloch H (1956) Effect of stress by crowding prior to and following tuberculous infection. Am J Physiol 187: 399-402 Toossi Z et al. (1997) Modulation of IL-12 by transforming growth factor-beta (TGF-beta) in Mycobacterium tuberculosis-infected mononuclear phagocytes and in patients with active tuberculosis. J Clin Lab Immunol 49:59-75 Tramontana JM et al. (1995) Thalidomide treatment reduces tumor necrosis factor alpha production and enhances weight gain in patients with pulmonary tuberculosis. Mol Med 1:384-397 Turner J et al. (2000) Effective preexposure tuberculosis vaccines fail to protect when they are given in an immunotherapeutic mode. Infect Immun 68:1706-1709 Van Crevel R et al. (2000) Increased production of interleukin 4 by CD4+ and CD8+ T cells from patients with tuberculosis is related to the presence of pulmonary cavities. J Infect Dis 181:1194-1197 Van Rie A et al. (1999) Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N Engl J Med 341:1174-1179 Van-der-Pouw-Kraan TC et al. (1995) Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 181:775-779 Varadhachary AS, Salgame P (1998) CD95 mediated T cell apoptosis and its relevance to immune deviation. Oncogene 17:3271-3276 Vieira PL et al. (1998) Glucocorticoids inhibit bioactive IL-

151 12p70 production by in vitro- generated human dendritic cells without affecting their T cell stimulatory potential. J ImmunoI161:5245-5251 Visser J et al. (1998) Differential regulation of interleukin-1O (IL-I0) and IL-12 by glucocorticoids in vitro. Blood 91: 4255-4264 Waddell RD et al. (2000) Safety and immunogenicity of a fivedose series of inactivated Mycobacterium vaccae vaccine for the prevention of HIV-associated tuberculosis. Clin Infect Dis 30 [SuppI3]:S309-S315 Wakeham J et al. (1998) Lack of both types 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by Mycobacterium bovis bacille Calmette-Guerin in IL-12-deficient mice. J Immunol 160:6101-6111 Wang CC, Rook GAW (1998) Inhibition of an established allergic response to ovalbumin in Balblc mice by killed Mycobacterium vaccae. Immunology 93:307-313 Watson VE et al. (2000) Apoptosis in Mycobacterium tuberculosis infection in mice exhibiting varied immunopathology. Journal of Pathology 190,211-20 Wayne LG (1994) Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13: 908-914 Wei J et al. (2000) Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol 182:377-384 Wilkinson RJ et al. (2000) Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study. Lancet 355:618-621 Wilsher ML et al. (1999) Human in vitro immune responses to Mycobacterium tuberculosis. Tubercle Lung Dis 79: 371-377 Wilson GS, Schwabacher H, Maier I (1940) The effect of the desensitisation of tuberculous guinea-pigs. J Pathol Bacteriol 50:89-109 Xu S et al. (1994) Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol 153:2568-2578 Yong AJ et al. (1989) Total and antimycobacterial IgE levels in serum from patients with tuberculosis and leprosy. Tubercle 70:273-279 Zhang L, Ma W, Da J (2000) Mycobacterium vaccae influences the kinetics of Thl/Th2 cells and expression of iNOS in a marine model of experimental tuberculosis. Zhonghua Jie He He Hu Xi Za Zhi 23:43-46 Zuany-Amorim C et al' (2002) Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergenspecific regulatory T-cells. Nat Medicine 8:625-629

10 Pathology of Tuberculosis MOHAMMED AKHTAR

and

HADEEL AL MANA

CONTENTS 10.1 10.2 10.3

Pathobiology of Tuberculosis 153 Microscopic Pathology 154 Identification of Mycobacteria in Tissue Section 155 Macroscopic Pathology 156 Lung 156 Lymph Nodes 157 Intestine 157 Urinary Tract 157 Male Genital Tract 158 Female Genital Tract 158 Central Nervous System 159 Miliary Tuberculosis 159 Disseminated Bacille Calmette-Guerin (BCG) 160 Role of Fine-Needle Aspiration Biopsy in the Diagnosis of Tuberculosis 160 References 161

10.1

Pathobiology of Tuberculosis

Following inhalation, the droplet nuclei usually implant in the bronchioles and alveoli. The tubercu10.4 lous bacilli first elicit a polymorphonuclear leukocyte 10.4.1 reaction. Later the bacilli are ingested by pulmonary 10.4.2 macrophages. If the organisms are virulent or if the 10.4.3 inhaled dose is large, the bacilli may multiply within 10.4.4 the phagocytic vacuoles of the macrophages. The 10.4.5 10.4.6 resulting lesion may grow due to influx of additional 10.4.7 monocytes/ macrophages from the blood stream. At 10.5 this stage, this immune response is mainly due to a 10.6 tissue-damaging delayed hypersensitivity response, which kills the bacilli-laden macrophages thus pro10.7 ducing a solid caseating center containing extracellular bacilli. The next stage of the lesion depends upon the quality and intensity of cell mediated immunity. If only poor cell mediated immunity response develops, Tuberculosis is caused by Mycobacterium tuberculo- the bacilli escape from the periphery of the caseatsis, a slender, acid-fast aerobic organism which can ing center and are ingested by macrophages. Again be transmitted by inhalation, by ingestion or, rarely, the delayed hypersensitivity response kills these by direct implantation. A fetus can be infected in macrophages causing enlargement of the caseous utero transplacentally. Patients with active pulmo- center and progression of the disease. If, on the other nary tuberculosis are the major source of infection hand, an effective cell-mediated immunity response and inhalation is overwhelmingly the most frequent is mounted, the lesion is surrounded by highly actiroute. Droplet nuclei containing a few organisms vated macrophages, i.e. epithelioid cells, which ingest become airborne during coughing, speaking or even and destroy the bacilli often arresting the lesion at singing and may remain suspended in the room air the subclinical stage. for hours. In many cases, however, the bacilli continue to multiply extracellularly. Additional delayed hypersensitivity reaction may cause local tissue damage, thus resulting in progressive enlargement of the lesion with extension into surrounding tissues. With the passage of time, the areas of caseation undergo liquefaction. The cause of liquefaction is not known. Possibly hydrolytic enzymes released from the dead M. AKHTAR, MD, FCAP, FRCPA, FRCPath Chairman, Department of Pathology and Laboratory Medi- inflammatory cells and products of cell wall of the cine, King Faisal Specialist Hospital and Research Centre, P.O. bacilli may playa role. Liquefaction plays an imporBox 3354, MBCIO, Riyadh 11211, Saudi Arabia tant role in the progression of the disease in two H. AL MANA, MD important ways. First, the liquidized caseous mateFellow, Department of Pathology ad Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O. Box rial is frequently, but not always, an excellent growth 3354, Riyadh 11211, Saudi Arabia medium for tubercle bacilli. Second, the liquidized M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

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material causes further tissue damage and may easily erode into adjacent structures, such as blood vessels and bronchi, thus spreading the disease to other locations. Excessive localized tissue damage may also result in cavitation (Allison et al. 1962; Shima et al. 1972; Dannenberg 1993,1999; Dannenberg and Rook 1994; Fayyazi et al. 2000).

10.2 Microscopic Pathology

M. Akhtar and H. Al Mana

cells are believed to be formed by the fusion of several epithelioid cells. The granulomas are surrounded by a zone of lymphocytic cells, which are an integral part of the cell-mediated immunity against mycobacterial infection. Several subtypes of lymphocytes have been identified including natural killer cells, cytotoxic T cells and type I and type II T helper cells among others (Dannenberg 1999). Most of the granulomas in mycobacterial infections are characterized by the presence of central areas of caseation necrosis and are termed as caseating granulomas. (Figs. 10.2-1004) In these granulomas, the epithelioid cells form a palisading arrangement around the caseating center. Other granulomas may lack central caseation and are called non-caseating granulomas. (Fig. 10.1) Histologically, caseation necrosis consists of amorphous, coarsely granular, acellular debris. Liquefaction of the caseat-

The morphologic hallmark of tissue response to mycobacterial infection is inflammation which is composed of nodular microscopic lesions called granulomas. Granulomas are usually small «2 mm) and are composed of aggregates of epithelioid cells surrounded by a rim of lymphocytes (Fig. 10.1). The epithelioid cells are highly activated macrophages which are characterized by abundant slightly eosinophilic granular cytoplasm and a relatively small, usually eccentric, nucleus. The epithelioid cells may superficially mimic epithelial cells. At ultrastructural level, these cells lack junctional complexes and their cytoplasm is rich in lysosomes and abundant rough endoplasmic reticulum. These cells are less actively phagocytic than the typical macrophages, but have potent bactericidal function due to the abundant lysosomal content. A striking morphologic feature of granulomas is the presence of multinucleated giant cells. Some of the multinucleated cells, which have nuclei evenly dispersed through the cytoplasm, are called foreign body giant cells. Others have nuclei disposed in a horse-shoe arrangement and are termed Langhans giant cells. Both types of giant

Fig. 10.2. Granulomas in a lymph node composed of epithelioid cells and a few giant cells. The granulomas are noncaseating, except for one on the left which reveals extensive caseation. Hematoxylin and eosin stain magnification IOOx

Fig. IO.I. Photomicrograph featuring a lymph node with granulomas composed of epithelioid cells. No caseation is present. Hematoxylin and eosin stain magnification 200x

Fig. 10.3. A large caseating granuloma with palisading epithelioid cells and multinucleated giant cells. Hematoxylin and eosin stain magnification 200x

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ing centers may result in cavitation. As the granuloma matures, it becomes surrounded by a layer of fibrosis which may gradually extend towards the center of the granuloma. (Fig.IO.S) Healed granulomas are virtually completely replaced by fibrous tissue although occasional epithelioid cells or giant cells may be preserved. These fibrosed granulomas usually lack any organisms. Cavitary lesions heal by fibrosis and calcification, both of which may be quite extensive in longstanding lesions.

Fig. lOA. A caseating granuloma with a Langhans giant cell. Hematoxylin and eosin stain magnification 300x

Fig. 10.5. A non-caseating granuloma undergoing fibrosis

Fig. 10.6. Acid-fast bacilli stained by Ziehl-Neelsen stain. DiffQuik stain 400x

10.3 Identification of Mycobacteria in Tissue Section Histopathologic diagnosis of tuberculosis depends on the demonstration of granulomatous inflammation with caseation. However, similar morphologic changes may also be encountered in association with other infections, especially fungal organisms. Therefore, demonstration of the offending organism may be crucial for arriving at a definitive diagnosis of tuberculosis. If the tissue is received in a fresh state, part of the material may be submitted for microbiologic cultures. In addition, histologic sections of the tissue may be stained using special staining procedures for the demonstration of tubercle bacilli. Ziehl-Neelsen stain is the most commonly used stain for identification of tubercle bacilli in tissue sections. Owing to the waxy nature of the cell wall component, mycobacteria stain with the red dye carbolfuchsin and retain the stain after washing with acid alcohol (Stevens and Frances 1996). This indicates that the mycobacteria are acid-fast as compared to other bacilli, which are usually not acid-fast. When acidfast bacilli (AFB) are detected in the granulomatous area, it is highly specific for mycobacterial infection. In Ziehl-Neelsen stain, tubercle bacilli are identified as a straight or curved rod 0.2-0.sx2-S 11m, that stain uniformly bright red but often have a beaded appearance. (Fig. 10.6) The organisms are usually located at the junction of viable tissue with the necrotic zone. Ziehl-Neelsen stain, however, is relatively insensitive. It has been estimated that between 105_10 6 bacteria per milliliter of tissue is required before they can be visualized in a section stain by Ziehl-Neelsen stain (Jagirdar and Zagzag 1996). Auramine-rhodamine stain is a more sensitive stain for mycobacteria, but it may not be so specific. Furthermore, it requires a fluorescent microscope for detection of the organisms (Stevens and Frances 1996).

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Monoclonal antibodies directed to different proteins of M. tuberculosis have been employed by some workers for identification of these organisms. However, several technical problems remain to be addressed (Barbolini et al. 1989). Recently polymerase chain reaction (PCR) for identification of mycobacterium tuberculosis complex DNA has been employed successfully on formalin-fixed paraffin-embedded material as well as lesion material obtained by fine needle aspiration biopsy. These studies have provided evidence for higher sensitivity and specificity of PCR amplification for identification of M. tuberculosis in biopsy material (Vago et al. 2000). Ligase chain reaction (LCR) is another diagnostic alternative for detection of tubercle bacilli. This technique uses a combination of polymerase and ligase for amplification of target DNA sequences in the chromosome of M. tuberculosis. This technique may be applied in body fluids as well as material from tissue biopsies. It is commercially available as a semi-automatic assay which is rapid, sensitive and is claimed to be quite specific. However, additional studies are required to determine the usefulness of this technique for identification of tuberculous bacilli in material derived from paraffin embedded tissue sections (Ruiz-Serrano et al. 1998).

M. Akhtar and H. Al Mana

node component. The parenchymal lesion can occur anywhere in the lung but is usually seen in the middle and lower lobes. On gross examination, it is a well circumscribed, often subpleural, nodule. (Fig. 1O.7) Initially, these lesions are centrally necrotic but later they became fibrotic and calcified. The lymph node component of Ghon complex is found in the draining hilar or intrapulmonary nodes (Allen 1995). Post primary tuberculosis represents recurrence of pulmonary tuberculosis after previous sensitization to M. tuberculosis during primary infection. These lesions usually involve apical and posterior segments of the lungs and are grossly characterized by fibrotic and caseous areas, frequently with cavitation. (Figs. 10.8, 1O.9) These cavities may be quite extensive and fibrosis and calcification of their walls may prevent collapse. In severe cases, cavities may replace the entire lobe and may extend to the other parts of the lung. In

10.4 Macroscopic Pathology The macroscopic appearance of an organ or tissue involved by tuberculosis has several important clues which raise suspicion of tuberculosis. The presence of caseation necrosis, cavitation and enlarged regional lymph nodes with similar areas of caseation necrosis should strongly suggest tuberculosis. However, the macroscopic appearance may be somewhat modified according to the location and the organ involved.

Fig. 10.7. A localized subpleural nodule in the lower lobe of right lung, probably representing primary tuberculosis in a 20-year-old man

10.4.1 Lung The gross appearance of the lung involved by tuberculosis depends upon whether it is primary tuberculosis, which is encountered in patients who have no previous exposure to M. tuberculosis infection, or whether it is post primary tuberculosis in a patient who has had a previous encounter with tuberculosis infection. The clinical and pathologic manifestation of primary tuberculosis is the Ghon complex, which consists of a parenchymal component and a prominent lymph

Fig. 10.8. Wedge resection of the upper lobe of the right lung featuring multiple cavitary lesions extensively replacing the lung parenchyma

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volume of the diseased tissue in regional lymph nodes than at the original site of infection. Infected lymph nodes commonly become greatly enlarged. On sectioning, the nodes are found to be replaced by light yellow areas of caseation necrosis. (Fig. 10.10) Some of the nodes may be virtually completely replaced by caseating necrosis, which may also extend to adjacent extranodal tissues. Several lymph nodes may be matted together to form a large mass superficially mimicking a neoplasm. Overlying skin may be stretched or ulcerated, resulting in sinus tracts.

Fig. 10.9. Cut surface of left lung showing congestion of the lung parenchyma. Several yellowish areas in the upper part of the lung represent caseating lesions of tuberculous bronchopneumonia

10.4.3 Intestine

The ileocecal region is the most common site for gastrointestinal tuberculosis. The earliest changes occur most frequently in the terminal ileum or cecum in the form of small nodules which project into the intestinal lumen and may show evidence of ulceration. As the inflammatory process progresses, characteristic morphologic changes of ulcerative, ulceratohypertrophic or hypertrophic disease occur. In the ulcerative type, the disease is mostly limited to mucosa and submucosa with transverse ulcers which may involve the intestine circumferentially. Mesenteric lymph nodes are frequently involved by the disease and may show massive enlargement. The hypertrophic form (seen in 10% of patients) is characterized by extensive inflammation and fibrosis of all layers of the bowel wall. Fibrous adhesions involving bowel, mesentery and lymph nodes may result in a mass closely resembling Fig. 10.10. Multiple matted lymph nodes extensively replaced a neoplasm. Stricture of the bowel wall may lead to by caseating tuberculous lesions obstruction. The ulceratohypertrophic type of disease may have features of both ulcerative and hypertrophic the higWy susceptible, highly sensitized individual, the disease (Yangking and Chandrasama 1999). Colonic tuberculous infection may spread rapidly throughout tuberculosis may involve any part of the colon. In the large areas of the lung and produce a diffuse bron- acute phase, localized ulceration of the mucosa occur. chopneumonia or lobe-exudative consolidation. The ulcers tend to be transverse. In the chronic phase, Longstanding cavities may be secondarily infected by extensive caseation and fibrosis may be seen. Anobacteria or fungi (Scully et al. 1990; Anonymous 1970). rectal tuberculosis may be an ulcerative type similar Some of the fungal infections maybe in the form of to that seen in the colon or it may be characterized fungal balls. Rarely, tuberculous lesions may co-exist by complex fistulae. The latter type of involvement with carcinoma (Ting et al. 1976). may be difficult to distinguish from Crohn's disease (Kobayashi and Chandrasama 1999).

10.4.2 Lymph Nodes A nearly uniform event following infection by M. tuberculosis is spread from the primary focus to regional lymph nodes. This often results in a greater

10.4.4 Urinary Tract The renal parenchyma initially becomes infected by hematogenous spread, which may involve both the

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kidneys. The kidney may be enlarged or decreased in size with irregular scarring on the surface (Farrow 1997). The lesions usually start in the medullary region, later leading to papillary necrosis and extension of the disease into the renal pelvis. A cut surface reveals dilation of the renal pelvis and calyxes, which are filled by light yellow caseous material. (Fig.lO.11) Distinction between renal tuberculosis and xanthogranulomatous pyelonephritis may be difficult. In advanced cases, the renal parenchyma is replaced by caseous material leaving a thin rim of atrophic renal tissue at the periphery. (Fig. 10.12) Localized lesions may heal by calcification. In the urinary bladder, early macroscopic changes are typically disposed around the ureteral orifices. There is marked edema and hyperemia, later changing to fine mucosal nodulation and ulceration. The ulcers are sharply demarcated and have undermined edges and a layer of gray soft material on the floor. The ureteral orifice may be deformed by the disease; it may be distorted and nodular or remain rigidly open: the characteristic 'golf hole' ureter. The lesions may extend to the trigone or the rest of the bladder. In the late stages, there is fibrosis and shrinkage of the bladder with reduction of capacity (McClure and Young 1989). Occasionally, there is formation of exuberant granulation tissue forming polypoid masses which may mimic neoplasm. The lesions in the ureter and urinary bladder are always secondary to tuberculosis of the kidney. The most common site of involvement in the ureter is ureterovesical orifice or ureteropelvic junction.

10.4.5 Male Genital Tract

M. Akhtar and H. AI Mana

Fig. 10.11. Cut surface of nephrectomy specimen with marked dilatation of calyxes and the renal pelvis. Some of the calyxes are lined by light yellow caseous material. Renal parenchyma reveals variable atrophy

Fig. 10.12. A nephrectomy specimen showing extensive caseous material filling the dilated calyxes

Testis and epididymis are involved by metastatic spread through the blood stream and are usually associated with tuberculosis in other areas in the genitourinary system. On gross examination, there is enlargement, thickening and fibrosis of the testis and epididymis and there are variable numbers of necrotic and caseous lesions with discharging sinuses. (Figs. 10.13, 1O.14) Tuberculosis of the prostatic urethra and penis have rarely been reported.

10.4.6 Female Genital Tract The most common site for involvement by tuberculosis in the genital tract is the fallopian tubes. In the

Fig.IO.B. Epididymo-orchiectomy specimen in which epididymitis is involved by several caseating lesions. The testis is somewhat atrophic but is not involved

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Fig. 10.14. Cut surface of a testis virtually completely replaced by amorphous light yellow caseous material

Fig. 10.15. A space-occupying lesion (tuberculoma) resected from the posterior fossa in a 30-year-old man. There are cystic areas filled with necrotic caseating material

adhesive ulcerative form of tuberculous salpingitis, there is little distension and enlargement of the tube is mainly interstitial. The surface may show minute gray to yellow nodules and there may be adhesions to adjacent organs and development of fistulae. In the inflammatory exudative form, the tubes are markedly distended by exudates, which may be purulent rather than caseous. The wall is thin and serosa relatively smooth. There is eversion of the fimbria and the abdominal ostium is usually patent. Occasionally, the tube may appear normal at gross inspection. In the uterine cavity the disease is limited to the endometrium, which may be difficult to recognize at gross examination. Vulvovaginal disease takes the form of small shallow ulcers with sinus tracts and fibrosis. Involvement of the ovaries is uncommon but ovarian tissues may show adhesions and inflammatory exudates due to granulomatous inflammation.

reported throughout the cerebral hemispheres, basal ganglia, the brain stem and cerebellum. Tuberculoma is usually a well defined mass separated from the surrounding brain tissue by a zone of fibrosis. The mass is usually solid with areas of necrosis and may reveal focal cystic change. (Fig. 10.15) Distinction from a glioma may not always be possible and correct diagnosis usually requires microscopic examination.

10.4.7 Central Nervous System Involvement of the central nervous system is usually through the blood stream, although the spinal cord may be involved by extension of the disease from adjacent lesions in the vertebral bones. Leptomeningitis due to tuberculosis is characterized by a covering of the surface by a diffuse exudate, which later develops into small nodules. These changes may be more pronounced at the base of the brain. Tuberculous infection may also be present in the form of a space-occupying lesion in the brain or, rarely, in the spinal cord. These masses, called tuberculomas, may be single or multiple and mayor may not be associated with concomitant meningitis. They have been

10.5 Miliary Tuberculosis Miliary tuberculosis results from a massive hematogenous spread of tubercle bacilli, which may occur either at the time of primary infection or at a time remote from primary infection. Lymphohematogenous dissemination following primary infection is the usual mechanism causing miliary tuberculosis in infants and children. Miliary dissemination may also occur when an older tuberculous lesion in the lung or an extrapulmonary site reactivates and discharges massive numbers of tubercle bacilli into the blood stream. Miliary tuberculosis may involve many organs including lung, liver, spleen, bone marrow, kidney, adrenals, eyes and thyroid (Divinagracia and Harris 1999). The involved organs have numerous small, gray to reddish brown, punctate rounded lesions of more or less uniform size. Microscopically, miliary foci reveal tuberculous granulomas with or without central caseation. With appropriate stains, acid-fast bacilli may be found within the macrophages or epithelioid cell of the granulomas or in the areas of caseation.

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10.6 Disseminated Bacille Calmette-Guerin (BCG)

10.7 Role of Fine-Needle Aspiration Biopsy in the Diagnosis of Tuberculosis

Vaccination of the newborn and babies with Bacille Calmette-Guerin has been used widely throughout the world and has been shown to be a safe method for providing some protection against tuberculosis. However, in some cases local ulceration or regional lymph node enlargement due to granulomatous disease may be seen. In patients with congenital or acquired immune deficiency, a more progressive and disseminated BCG infection may develop, which may prove fatal unless treated by anti-tuberculous chemotherapy. In these cases, a biopsy usually reveals diffuse proliferation of histiocytes intermixed with variable numbers of neutrophil without distinct granulomas. (Fig. 10.16) The histiocytes on Ziehl-Neelsen staining usually reveal large numbers of intracytoplasmic acid-fast bacilli (Fig.10.1?) (AI BhlaI2000).

Fine-needle aspiration biopsy is an extremelyeffective technique for the pathologic diagnosis of tuberculosis. The morphologic pattern encountered in smears parallels closely with that seen in histologic preparations. Thus all the components of granulomatous inflammation including lymphocytes, macrophages, epithelioid giant cells and other inflammatory cells are easily recognized. (Fig. 10.18) Caseating necrosis can also be recognized by the presence of amorphous material with scattered polymorphonuclear cells and a few epithelioid cells (Das and Pant 1994; Prasoon 2000; Radhika et al. 1993; Bhattacharya et al. 1998) (Fig. 10.19). Aspirated material may also be used successfully for culturing the tubercle bacilli. In addition, aspiration smears may be stained by special stains to identify tubercle bacilli.

Fig. 10.16. Photomicrograph of a lymph node involved by disseminated BeG infection. The node is replaced by numerous histiocytes with abundant granular cytoplasm. No granulomas are present. Hematoxylin and eosin stain 300x

Fig.IO.IS. Fine-needle aspiration biopsy smear showing an epithelioid granuloma without caseation. Diff-Quik stain 300x

Fig. 10.17. Numerous intracytoplasmic acid-fast bacilli present within the macrophages. Ziehl-Neelsen stain 400x

Fig. 10.19. Aspiration smear featuring an epithelioid granuloma associated with a background of necrosis and inflammatory cells indicating caseation. Diff-Quik stain 200x

Pathology of Tuberculosis

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References AI Bhlal LA (2000) Pathologic findings for Bacille CalmetteGuerin infections in immunocompetent and immunocompromised patients. Am J Clin Pathol 113:703-708 Allen EA (1995) Tuberculosis and other mycobacterial infections of the lung. In: Thurlbeck WM, Churg AM (eds) Pathology of the lung, 2nd edn, pp 299-265 Allison MJ, Zappasodi P, Lurie MB (1962) Host-parasite relationships in natively resistant and susceptible rabbits on quantitative inhalation of tubercle bacilli. Am Rev Respir Dis 85:553-569 Anonymous (1970) Aspergilloma and residual tuberculous cavities - the results of a resurvey. Tubercle 51:227-245 Barbolini G, Bisetti A, Colizzi V, Damiani G, Migaldi M, Vismara D (1989) Immunohistologic analysis of mycobacterial antigens by monoclonal antibodies in tuberculosis and mycobacteriosis. Hum Pathol 20:1078-1083 Bhattacharya S, Raghuveer CV, Adhikari P (1998) FNAC diagnosis of tuberculosis - an eight years study at Mangalore. Indian J Med Sci 52:498-506 Dannenberg AM Jr (1993) Immunopathogenesis of pulmonary tuberculosis. Hosp Pract 28:33-40, 51-58 Dannenberg A (1999) Pathogenesis oftuberculosis. In: Schlossberg D (ed) Tuberculosis and non-tuberculous mycobacterial infections. Saunders, Philadelphia, pp 17-47 Dannenberg AM Jr, Rook GAW (1994) Pathogenesis of pulmonary tuberculosis: an interplay of tissue-damaging and macrophage-activating immune responses - dual mechanisms that control bacillary multiplication. In: Bloom BR (ed) Tuberculosis: pathogenesis, protection, and control. American Society for Microbiology, Washington DC, pp 459-483 Das DK, Pant CS (1994) Fine needle aspiration cytologic diagnosis of gastrointestinal tract lesions. A study of 78 cases. Acta CytoI38:723-729 Divinagracia R, Harris HW (1999) Miliary tuberculosis. In: Schlossberg D (ed) Tuberculosis and non tuberculous mycobacterial infection Saunders, Philadelphia, pp 271-284 Farrow GM (1997) Diseases of the kidney in the urologic pathology (Murphy WM ed). Saunders, Philadelphia, pp 440-442 Fayyazi A, Eichmeyer B, Soruri A, Schweyer S, Herms J, Schwarz P, Radzun HJ (2000) Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis. J Pathol 191:417-425 Jagirdar J, Zagzag D (1996) Pathology and insight into patho-

genesis of tuberculosis. In: Rom WN, Gary S (eds) Tuberculosis. Little Brown, Boston, pp 467-491 Kobayashi G, Chandrasama P (1999) Non neoplastic lesions of the colon. In: Chandrasama P (ed) Gastrointestinal pathology. Appleton and Lange, Stanford, pp 263-264 McClure J, Young RH (1989) Infectious disease of the urinary bladder, including malakoplakia. In: Young RH (ed) Pathology of urinary bladder Churchill Livingston, New York, pp 351-375 Prasoon D (2000) Acid-fast bacilli in fine needle aspiration smears from tuberculous lymph nodes. Acta Cytol44:297-300 Radhika S, Rajwanshi A, Kochhar R, Kochhar S, Dey P, Roy P (1993) Abdominal tuberculosis. Diagnosis by fine needle aspiration cytology. Acta Cytologica 37:673-678 Ruiz-Serrano MJ, Albadelejo J, Martinez-Sanchez L, Bouza E (1998) LCx: a diagnostic alternative for the early detection of mycobacterium tuberculosis complex. Diagn Microbiol Infect Dis 32:259-264 Scully RE, Mark EJ, Menealy WF, Menealy BU (eds) (1990) Case 45-1990 Case records of Massachusetts General Hospital. N Engl J Med 323:329-336 Shima K, Dannenberg AM Jr, Ando M et al. (1972) Macrophage accumulation, division, maturation, and digestive and microbicidal capacities in tuberculous lesions. 1. Studies involving their incorporation of tritiated thymidine and their content of lysosomal enzymes and bacilli. Am J PathoI67:159-180 Stevens A, Frances RJ (1996) Micro organisms. In: Bancroft JD, Stevens A (eds) Theory and practice of histopathologic techniques, 4th edn. Churchill Livingston, New York, pp 291-308 Thomas JO, Adeyi D, Amanguno H (1999) Fine needle aspiration in the management of peripheral lymphadenopathy in a developing country. Diagn Cytopathol 21:159-162 Ting YM, Church WR, Ravikrishnan KP (1976) Lung carcinoma superimposed on pulmonary tuberculosis 199: 307-312 Vago L, Zervi P, Caldarelli-Stefano R, Cannone M, D'Amico M, Bonetto S, Barberis M (2000) Polymerase chain reaction for mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples. Acta Cytol 44:1023-1028 Yanking M, Chandrasama P (1999) Pathology of small intestine. In: Chandrasama P (ed) Gastrointestinal pathology. Appleton and Lange, Stanford, pp 199-201

11 Nontuberculous Mycobacteria JAE-JOON YIM and STEVEN M. HOLLAND

CONTENTS 11.1 11.2 11.3 11.4 11.5 11.6 11.6.1 11.6.2 11.6.3 11.6.4 11.7

1l.8 11.9 11.10 11.11 11.11.1 11.11.2 11.11.3 11.11.4 11.12 11.12.1 11.12.2 11.13

Introduction 163 Classification of Nontuberculous Mycobacteriosis 163 Epidemiology 164 Host Susceptibility Factors 166 Host Defense Against Mycobacteria 168 Human Immune Defects 169 IFNy Receptor 1 Deficiency 169 IFNy Receptor 2 Deficiency 170 1L-12 Receptor ~ 1 Deficiency 170 IL-12 p40 Deficiency 170 NFKB Essential Modulator (NEMO) or IKB Kinase Defects 170 Familial Syndrome 171 Polymorphisms 171 Body Morphotype 171 Clinical Presentation and Diagnosis 172 Pulmonary Disease 172 Lymphadenitis 173 Skin and Soft-Tissue Infection 173 Disseminated NTM Infection 175 Treatment 175 Chemotherapy 175 Immunotherapy 177 Summary 178 References 178

11.1 Introduction The Norwegian physician G. H. Armauer Hansen described an organism subsequently known as Mycobacterium leprae in the skin of leprosy patients for the first time in 1874 (Hansen 1874). Eight years later, the German physician Robert Koch identified Mycobacterium tuberculosis. In 1885, 3 years after Koch's discovery of M. tuberculosis, a smegma bacillus, erroneously thought to be connected to syphilis but now

J.-J. YIM, MD Respiratory and Critical Care Medicine, Seoul National University College of Medicine, Seoul, South Korea 110744 S. M. HOLLAND, MD Immunopathogenesis Section, Building 10, Room 11N103, 10 Center Dr. MSC 1886, Bethesda, MD 20892 1886, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

known as one of the nontuberculous mycobacteria, M. smegma tis, was identified. Currently, 71 species of Mycobacterium are recognized or have been proposed (Shinnick and Good 1994). Despite the fact that these mycobacteria have occasionally been identified from clinical specimens (Wolinsky 1979), it is only relatively recently that serious attention has been paid to them. A variety of terms have been applied to these nontuberculous mycobacteria, including anonymous, unclassified, unknown, tuberculoid, environmental, opportunist, nyrocine, and mycobacteria other than tuberculosis (MOTT) (Collins et al. 1984; Debrunner et aI.1992). The word 'atypical' has been popular in the USA, while 'anonymous' and 'opportunist' have been preferred in the UK (Collins et al.1984). However, each term has its own limitations: These mycobacteria are certainly not 'anonymous', there is no reason to regard them as 'atypical', and the term 'opportunist mycobacteria' could be equally applied to the more virulent Mycobacterium tuberculosis, especially in the setting of AIDS (Grange 1989). Although the term nontuberculous mycobacteria (NTM) has been widely used and accepted after the American Thoracic Society adopted this term in its official statement about these mycobacteria (ATS 1990), NTM itself is a kind of misnomer. These mycobacteria usually do result in the formation of tubercles in infected tissue; in other words, they are 'tubercle-ous' (Grange 1989). Furthermore, the British Thoracic Society still uses the term 'opportunist mycobacteria'in its official guidelines (BTS 2000). In this chapter, we will review NTM disease in terms of classification, epidemiology, pathogenesis, diagnosis, and treatment. In addition, the state of knowledge of host susceptibility and related cytokine therapy will be underscored.

11.2 Classification of Nontuberculous Mycobacteriosis Growth rates, colony morphologies, and biochemical tests have traditionally been used to differentiate

J.-J. Yim and S. M. Holland

164

NTM from M. tuberculosis. M. tuberculosis takes 3-6 weeks to grow, has white colonies, and gives a positive niacin test result; most NTM are niacinnegative (Wayne 1985). In 1954, Timpe and Runyon classified NTM for the first time (Timpe and Runyon 1954). Their classification was based on NTM growth rates, morphology, and colony pigmentation. According to their scheme, if a mycobacterium took?.7 days to grow, it was a 'slow grower' and fell into groups I, II, or III. If it took <7 days, it was a 'rapid grower' and was classified as group IV. Among the slow growers, if colonies showed yellow pigmentation when grown in the light, they were photochromogens, type I. If they were strongly pigmented, yellow to orange when grown in the dark, they were scotochromogens, type II. If they showed no or shallow pigmentation when grown in the dark, they were classified as type III, nonphotochromogens. In fact, group IV, the rapid growers, shares many similarities with group III, the nonphotochromogens, except that the individual colonies grow in <7 days.

Although this Runyon classification system provided the first basis for a taxonomic understanding of mycobacteria, it has limitations. It is not particularly relevant to the clinical manifestations of NTM diseases, and some bacteria cannot be clearly classified. In fact, Runyon himself proposed that some mycobacterial species from different categories could share similar clinical manifestations (Runyon 1974). Consequently, the classification of NTM according to their predisposition to involve various organs has gained popularity (Table ILl). Over the last decade, the number of new species added to the NTM has increased dramatically (Wayne and Sramek 1992). Correct identification of these newly added NTM species by traditional culture-based and biochemical methods has become more challenging and is sometimes inadequate (Springer et al.I993). The development of new techniques, such as 16S rRNA gene sequencing (Sansila et al. 1998), PCR-RFLP of a 439 bp segment of the heat shock protein 65 (hsp-65) gene (Eriks et al. 1996), or high-performance liquid

Table 11.1. Classification of the nontuberculous mycobacteria recovered from humans Features of the common species Clinical disease species

Common etiologic Geography

Morphologic features species

Unusual etiologic

Pulmonary disease

M. avium complex Worldwide

Usually not pigmented Slow growth (>7 days) Pigmented; often large and beaded on acid-fast stain Rapid growth «7d) not pigmented Slow growth; pigmented Slow growth; not pigmented

M.simiae M.szulgai M. fortuitum M. celatum M. asiaticum M.shimodii M. haemophilum M. smegmatis

Usually not pigmented Pigmented Slow growth

M. fortuitum M. chelonae M. abscessus

M. kansasii

M. abscessus

M. xenopi M.

malmoense

USA, coal mining regions, Europe Worldwide but mostly USA Europe, Canada UK, northern Europe

Lymphadenitis

M. avium complex Worldwide M. scrofulaceum Worldwide M. malmoense UK, northern Europe (especially Scandinavia)

Cutaneous disease

M. marinum

M. fortuitum M. chelonae

M. abscessus M. ulcerans

Worldwide

Photochromogen; requires low temperatures (28-30°C) For isolation Worldwide, mostly USA Rapid growth; not pigmented

From ATS (1997)

M. kansasii M. nonchromogenicum M. smegmatis

M. haemophilum Australia, tropics, Africa, Grows slowly, pigmented SE Asia

Disseminated disease M. avium complex Worldwide

M. kansasii M. chelonae M. haemophilum

M. avium complex

USA USA USA, Australia

Isolates from patients with AIDS usually pigmented (80%) Photochromogen Not pigmented Not pigmented; requires hemin, often low temperatures, and CO 2 to grow

M. abscessus

M.xenopi M. malmoense M. genavense

M.simiae

M. conspicuum

M. marinum M. fortuitum

Nontuberculous Mycobacteria

chromatography (HPLC) mycolic acid fingerprinting (Butler and Kilburn 1990; Butler et al. 1992) have provided identifications and are in part responsible for the recent explosion of new species of NTM. Newly described or emerging human species of NTM are listed in Table 11.2.

11.3

Epidemiology

165

Table 11.2. Newly described or emerging human species of nontuberculous mycobacteria Newly described clinically significant human pathogens Rapid growing mycobacteria: M. mucogenicum M. goodii M. immunogenum M. wolinskyi M. fortuitum third biovariant complex, including M. septicum Slowly growing mycobacteria: M. genavense

M. triplex M. lentiflavum After the first report of disseminated NTM infection M. celatum in an AIDS patient in 1982 (Zakowski et al.1982), the M. interjeetum number of patients with NTM disease dramatically M. intermedium increased in developed countries (Selik et al. 1987; M. conspicuum Horsburgh and Selik 1989), even though the prevaM. branderi M. geidelbergense lence of NTM disease in AIDS patients in Africa is M. heckeshornense still low (Okello et al.1990; Morrissey et al.I992). The M. bohemicum epidemic of AIDS not only increased the number of M. tusciae cases of NTM disease, it also changed its characteris- Newly described potential human pathogens: tics. Before the AIDS epidemic, NTM disease was usuM. confluentis M. mageritense ally confined to the lungs, lymph nodes, or skin; only M. alvei in rare cases was it disseminated (Wolinsky 1979). M. hassiacum However, NTM disease in AIDS patients is usually M. kubicae disseminated. In addition, the most common dissemM. novocastrense inated NTM in AIDS patients is M. avium, (Zakowski M. brumae et al. 1982), whereas infection by M. kansasii and M. Old but emerging pathogens: M. ulcerans intracellulare was more frequent in the era before the M. haemophilum AIDS epidemic (Ahn et al. 1979; Falkinham 1996). M. xenopi Irrespective of the change related to the AIDS M. malmoense epidemic, the NTM which cause lymphadenitis in Adapted from Brown-Elliott et al. (2002) with permission children have also changed: M. scrofulaceum was the most frequent organism causing cervicallymphadenitis in children (Wolinsky 1979), but M. avium has now become the most common (Colville 1993; Wolinsky infection of AIDS patients from the hot water system 1995). This observation raises the possibility that the of hospitals (von Reyn et al. 1994). Although MAC is natural or environmental population of M. avium and one of the major causes of mycobacteriosis in poultry M. scrofulaceum is changing, that there is a change in and swine, recent serologic and molecular studies sugchildren's behavior, or that other factors (e.g., inciden- gest a low possibility of animal-to-human transmission tal antibiotic use) may be involved (Falkinham 1996). (Meissner et al. 1977; Ahrens et al. 1995; Guerrero et al. Water is the major source of infection for numerous 1995). The waterborne M. marinum has been reported NTM species including M. avium complex (MAC), M. to infect persons through aquarium exposure (Adams marinum, M. kansasii, M. xenopi, M. simiae, M. fortui- et al. 1970; Barrow and Hewitt 1971), and M. xenopi has tum, M. chelonei, M. abscessus, and others. MAC grows been suggested to infect humans through an aerosol well in the natural waters ofGermany (Beerwerth 1973), route (Collins and Yates 1984). In addition, the rapidly Japan (Ichiyama et al. 1988), Finland (von Reyn et al. growing mycobacteria M. fortuitum, M. chelonae, and 1993), Uganda (von Reyn et al. 1993), Zaire (von Reyn M. abscessus have been reported to cause nosocomial et al. 1993), the USA (Gruft et al. 1981), and probably outbreaks and pseudo-outbreaks from tap water many other untested regions. MAC can be aerosolized (Lowry et al. 1988; Maloney et al. 1994), ice prepared (Meissner and Falkinham 1986), raising the probability from tap water (Laussucq et al. 1988), processed tap of airborne infection for these mycobacteria. In 1994, water for dialysis (Bolan et al. 1985), and the whirlpool von Reyn et al. suggested the transmission of MAC footbaths of nail salons (Winthrop et al. 2002).

166

J.-J. Yim and S. M. Holland

Even though many mycobacteria including MAC the rapidly growing NTM M. fortuitum, M. chelonae, (Paull 1973; Reznikov and Leggo 1974; Reznikov and and M. abscessus (Wolinsky 1979; Griffith et al. 1993; Dawson 1980; Brooks et al. 1984; Eaton et al. 1995), Hadjiliadis et al. 1999). M. malmoense (Saito et al. 1994), and M. fortuitum Although disseminated M. avium infection has (Wolinsky and Rynearson 1968; Paull 1973) have been been common among patients with advanced HIV isolated from soil, there is no evidence that mycobacte- disease, pulmonary MAC disease has occurred infrequently in these patients. The frequency of MAC lung ria from soil can directly infect human beings. Although the prevalence of NTM disease cannot be disease in patients with disseminated MAC infection estimated easily, it has been suggested that the preva- is low, between 2.5% andlO% (Kalayjian et al. 1995). lence of NTM disease is now increasing in the USA This may reflect differences in organism tropism and (O'Brien et al.1987; Pae et al.1997), Switzerland (Deb- virulence, as bacteremic strains in AIDS patients runner et al. 1992), and Japan (Sakatani 1994). The tend to be M. avium, whereas M. intracellulare is reasons for this increase ofNTM disease are unknown, the more common pulmonary pathogen. Horsburgh but better clinical recognition and improved, increased et al. studied advanced AIDS patients with CD4+ Tamounts of culturing for NTM are felt to play impor- lymphocyte counts <25/mm 3 and found that the contant roles (ATS 1997). MAC is the most prevalent sumption of hard cheese and pate were associated species followed by M. kansasii (O'Brien et al. 1987; with recovery of MAC from stool, whereas consumption of raw shellfish was associated with recovery of Debrunner et al.1992; Sakatani 1994). The NTM are widely thought to be opportunistic. MAC from sputum (Horsburgh et al. 1994). Those patients most commonly afflicted are ones with structural lung disease (e.g., bronchiectasis, pneumoconiosis, chronic obstructive pulmonary disease [COPDj, prior tuberculosis) (Wolinsky 1979; O'Brien et al. 1987; Pae et al. 1999). Bronchiectasis 11.4 and MAC infection often coexist, making causality Host Susceptibility Factors difficult to determine in some cases. Specific morphotypes have been associated with pulmonary NTM In 1964, Engbaek reported the first familial outbreak infection, particularly in women with pectus excava- of fatal MAC infection (Engbaek 1964), suggesting a tum, thoracic scoliosis, and mitral valve prolapse genetic basis for the development of severe disease (Iseman et al. 1991). Pulmonary NTM disease has with these relatively nonpathogenic organisms. In been shown to have a growing female predominance, the 1980s, advanced HIV infection showed the critiincluding a significant number of older women with- cal role of CD4+ T-Iymphocytes as effectors against out clearly recognized predisposing factors (Prince et mycobacteria: NTM infections typically occurred al. 1989; Griffith et al. 1993). The majority of elderly only after the CD4+ T-Iymphocyte number fell below 3 male patients have risk factors such as smoking or 50/mm (Horsburgh and Selik 1989; ATS 1997), sugCOPD (Iseman 1996). In contrast to male smokers gesting that specific T-cell products or activities were with upper lobe cavitary disease who tend to carry required for mycobacterial resistance. The genetic the same single strain of MAC indefinitely, female basis of susceptibility to NTM has been characterized nonsmokers with nodular bronchiectasis tend to through identification of specific mutations involvcarry 3 or more strains over time. This suggests either ing the interferon-y (IFNy) synthesis and response polyclonal infection or recurrent infection with dis- pathways (Table 11.3). These include mutations in tinct strains (Wallace et al.1998). Patients with cystic interferon-y receptor 1 (IFNyR1), interferon-y recepfibrosis, in whom there are both structural lung dis- tor 2 (IFNyR2), interleukin-12 receptor ~1 subunit orders (Olivier et al. 1996) and abnormalities in the (IL12R~1), interleukin-12 p40 (IL12p40), the signal function of defensins, small antibacterial peptides transducer and activator of transcription 1 (STATl), secreted by bronchial epithelium (Goldman et al. and NFKb essential modulator (NEMO), an X-linked 1997), form a special risk group for pulmonary NTM trait that affects IL-12 production (Dorman and Holinfection. Patients with pulmonary alveolar pro- land 2000; Doffinger et al. 2001; Dupuis et al. 2001). teinosis are also at higher risk for pulmonary NTM However, despite the growing number of genetic infections as well as infection with the closely related defects identified in patients with disseminated NTM Nocardia (Witty et al. 1994). Esophageal motility dis- infections, only about one-third of cases without HIV orders such as achalasia have been found to be sig- infection have a genetic diagnosis. Therefore, almost nificantly associated with pulmonary disease due to two-thirds of all disseminated cases to date are still

167

Nontuberculous Mycobacteria

Table 11.3. Human identified immune defects Defect

Location

Zygosity Infections (disseminated mycobacterial disease)

c-IFNJ'RI

Sl16X

Homozygous

131delC 22delC 107ins4; 200+ 1 G~A 561de14; 373+1 G~T

Homozygous Homozygous Homozygous Heterozygous Heterozygous

561 del4

Homozygous

M. avium, Salmonella, M. chelonei, M. fortuitum BCG M. avium M. avium M. smegmatis, BCG? BCG, M. avium, M. kansasii, Listeria monocytogenes BCG

c-IFNJ'R2

278delA,G 791deiG

Homozygous Homozygous

M. avium, M. fortuitum BCG? M. abscessus

AR p-IFNJ'Rl

1871

Homozygous

BCG, S. enteritidis, 1. pneumophilia?, M. tuberculosis?

AR p-IFNJ'R2

R1l4C

Homozygous

BCG, M. abscessus

AD p-IFNJ'RI

818del4

Heterozygous

818delT 817insA

Heterozygous Heterozygous

BCG, M. avium, M. kansas ii, M. spp., H. capsulatum BCG, M. avium M. avium, M. kansasii, M. chelonei

c-IL-12 p40

p40de14.4

Homozygous

BCG, S. enteritidis

c-IL-12RPI

de1409-549 Q32X Q376X K305X 783+1 G~C Q214R

Homozygous Homozygous Homozygous Homozygous Homozygous Homozygous

BCG, S. typhimurium M. avium, S. paratyphi M. avium, S. group B BCG, S. enteritidis BCG M. avium, S. enteritidis

STATl

L706S

Homozygous

BCG, M. avium

NEMO

X420W

X-linked

M. kansasii, M. sp., S. enteritidis, S. pneumoniae, P. carinii M. avium, P. aeruginosa, CMV

201-2A~G

1167insC 1218insA D3l1N nd

H. influenzae, S. pneumoniae M.avium S. pneumoniae S. pneumoniae

R175P L227P C417F C417R A288G Polymorphism: NRAMPI 5'(CA)n 199/other INT4G~C D543NG~A

3'UTR TGTG+/del

S. pneumoniae H. influenzae S. pneumoniae

Heterozygous Heterozygous Heterozygous Heterozygous

Pulmonary tuberculosis Pulmonary tuberculosis Pulmonary tuberculosis Pulmonary tuberculosis

Abbreviations: c-IFNJ'Rl, complete interferon-y receptor 1 deficiency; c-IFNJ'R2, complete IFNJ'R2 deficiency; AR p-IFNJ'Rl, autosomal recessive partial interferon-y receptor 1 deficiency; AR p-IFNJ'R2, autosomal recessive partial IFNJ'R2; AD p-IFNJ'Rl, autosomal dominant partial interferon-y receptor 1; c-IL-12 p40, complete interleukin-12 p40 deficiency; c-IL-12RPl, complete interleukin-12 receptor ~ 1 deficiency; STATl, signal transduction and activation oftranscription factor 1; NEMO, NFKB essential modulator; NRAMPl, natural resistance-associated macrophage protein 1; BCG, bacille Calmette-Guerin. Adapted from Guide and Holland (2002) with permission.

168

syndromic (e.g., sarcoid, CD4+ T-Iymphocytopenia) or idiopathic. Interestingly, an association has been noted between pulmonary NTM infections and body habitus, predominantly in postmenopausal Caucasian women (pectus excavatum, scoliosis, mitral valve prolapse) (Iseman et al. 1991; Pomerantz et al. 1996).

J.-J. Yim and S. M. Holland

and Scott 1999). Binding results in phosphorylation of the associated cytoplasmic kinases, tyrosine kinase 2 (Tyk2) and Janus kinase 2 (Jak2), which in turn phosphorylate STAT4, leading to transcription of IL-12-responsive genes, in particular IFNy. IFNy activates neutrophils and macrophages to produce superoxide and nitric oxide, to increase the surface display of MHC molecules and Fc receptors, to decrease lysosomal pH, and to increase the intracellular concentration of certain antibiotics (Bermudez and Young 1989; Boehm et al. 1997). IFNy signals 11.5 through a membrane-bound receptor composed Host Defense Against Mycobacteria of two chains, IFNyRI and IFNyR2, which are conIn normal hosts, mycobacteria are initially phago- stitutively bound to their respective Janus kinases, cytosed by macrophages, which respond with the Jakl and Jak2. IFNy binding initiates aggregation of production of interleukin-12 (lL-12; see Fig. ILl) IFNyRI and IFNyR2, phosphorylation of Jakl and (Fulton et al. 1996). IL-12 is a complex heterodimer Jak2, and phosphorylation of the IFNyRI cytoplascomposed of p35 (chromosome 3) and p40 (chro- mic domain, which enables docking of STATl. Phosmosome 5) moieties, which together comprise p70. phorylated STATl (STATl-P) then homodimerizes IL-12p70 activates T-Iymphocytes and NK cells and translocates as a complex to the nucleus, where through binding to the IL-12 receptor, composed of it upregulates IFNy-responsive genes such as IL-12, IL-12R~1 and IL-12R~2 (Gately et al. 1998; Trinchieri major histocompatibility complex genes, and TNF . The positive feedback loop between IFNyand IL-12 is pivotal in the immune response to mycobacteria and other intracellular infections. Defects in any of these receptors or cytokine genes negatively affect the production of IFNy and/or IL-12, and consequently enhance mycobacterial susceptibility (Fig. 11.1) IL-12 (Dorman and Holland 2000). In mouse studies, IFNy has been shown to exert a mycobacteriostatic effect, possibly through acidification of mycobacteria-containing vesicles (Appelberg and Orme 1993). Toxic oxygen radicals do not control NTM infection in mice (Doherty and Sher 1997), but they may be important in early tuberculosis infection, TNFa especially in the lungs (Adams et aI. 1997; Cooper et al. 2000). Although in vitro data for IFNy control of NTM using macrophages has been inconsistent (Douvas et al. 1985; Bermudez and Young 1989; Denis et al. 1990), the in vivo data have been persuasive. Fig. ILL Cytokine pathways involved in mycobacterial infecDisruption of IFNy production in mice resulted in tion and host response. Mycobacteria infect macrophages and Widespread tuberculosis with poor granuloma forstimulate production of IL-12. Subsequently, IL-12 leads to T or NK cell production of IFNy. IFNy binds to a specific site on mation and rapid death (Flynn et al. 1993; Cooper IFNyRl, leading to aggregation of IFNyRl and IFNyRZ. Follow- et aI. 2000). Exogenous administration of IFNy did ing this aggregation, the associated cytoplasmic Janus kinases, not restore normal resistance in these animals, sugJakl and Jak2, transphosphorylate each other, leading to phosgesting that IFNy may playa critical developmental phorylation of the intracellular domain of IFNyRl. This phosphorylation creates a binding site for cytosolic STAT!, which is role, or that injecting systemic IFNy does not activate in turn phosphorylated. Phospho-STAT! then homodimerizes local defenses in the same manner as local producand is transported to the nucleus, where it upregulates IFNy- tion. Similar results were seen in IFNyR-deficient responsive genes. IFNy also upregulates TNFa production in mice (Kamijo et al. 1993) and in mice lacking the the setting of endotoxin (lipopolysaccharide). The mechanisms IFNy-induced interferon regulatory factor-l (IRF-l) by which IFNy mediates the killing of mycobacteria are not yet well characterized. *, denotes genes known to predispose to (Kamijo et al. 1994). These models define a critical role for IFNy in tuberculosis resistance and control in mycobacterial infection when mutated

Nontuberculous Mycobacteria

the mouse. Similar findings have been reported with MAC infection in IFNy-deficient mice (Doherty and Sher 1997). In normal mice infected with MAC, exogenously administered IFNy successfully augmented mycobacterial clearance, in part by suppressing the production of prostaglandin E2, an inhibitor of macrophage function (Edwards et al. 1986).

11.6

Human Immune Defects 11.6.1 IFNy Receptor1 Deficiency

In 1994, four children of an extended Maltese family were described with an autosomal recessive pattern of susceptibility to NTM (Levin et al. 1995). These related children had severe disseminated disease with a variety of NTM including M. chelonae, M. fortuitum, and MAC, in addition to recurrent infections with Salmonella. Affected children had impaired TNFa production in response to stimulation by IFNy, while their parents were intermediate between the patients and normals, suggesting a gene dosage effect in heterozygotes. Antigen presentation was also impaired (D'Souza et al.1996). Newport et al. identified a homozygous point mutation in the extracellular domain of IFNyRI in these patients, resulting in a premature stop codon (Newport et al. 1996). Simultaneously, a Tunisian infant was reported with disseminated BCG infection and a more proximal extracellular chain-terminating mutation (131delC) in the IFNyRI gene (Jouanguy et al. 1996). Since then, numerous autosomal recessive mutations in the extracellular domain of IFNyRI have been identified in patients with disseminated mycobacterial infections. Identification of compound heterozygotes in outbred populations indicates that mutations in IFNyRI are not simply the result of remote founder effects (Dorman and Holland 2000). Extracellular mutations leading to disruption of IFNyRI function lead to failure to activate STATl in response to IFNy. This leads to an inability to upregulate IFNy-responsive genes including TNFa. Since IFNy and IL-12 production are co-dependent, both show an impaired response to phytohemagglutinin (PHA) stimulation (Holland et al. 1998). However, complete IFNyRI or IFNyR2 deficiency leads to loss of IFNy removal from the plasma and elevated circulating levels of IFNy, despite low levels of IFNy generation in vitro (Fieschi et al. 2001).

169

Complete IFNyRI deficiency typically presents as disseminated BCG or NTM disease in infancy or childhood. Salmonella infections have occurred in cases from outside North America, probably due to the greater exposure to that organism in those parts of the world. Complete IFNyRI deficiency is associated with a poor prognosis and high mortality due to incomplete clearance of infection despite aggressive antibiotic therapy, as well as continued susceptibility to new infections. Other infections reported in these patients include Listeria monocytogenes, cytomegalovirus (CMV), severe respiratory syncytial virus (RSV), herpes simplex virus (HSV), and varicella zoster virus (VZV). Although these are common viruses prevalent in the general population, these infections are often much more severe in IFNyRI-deficient patients, suggesting that this may be an aspect of the clinical phenotype (Dorman et al. 1999). Two Portuguese siblings with disseminated BCG and presumed tuberculosis were identified with a homozygous mutation in the extracellular domain of IFNyRI (I87T) leading to an alteration in a Nlinked glycosylation site (Jouanguy et al. 1997). They were both cured with antibiotics alone and showed partial IFNy responsiveness in vitro. The formation of intact, tuberculoid granulomas by one child with disseminated BCG further demonstrated residual IFNyactivity. Autosomal dominant (AD) mutations in the IFNyRI gene have been shown to confer only a partialloss of IFNy responsiveness. Jouanguy et al. identified a four-base deletion (818deI4) in 12 unrelated people from around the world (Jouanguy et al. 1999), and another 2 Japanese patients with the same mutation were reported recently (Sasaki et al. 2002). This latter group also identified less common autosomal dominant mutations, 818delT and 818insA. These mutations result in deletions of the Jakl and STATl intracellular binding sites as well as deletion of the receptor recycling domain. These mutations impede signal transduction and receptor removal from the cell surface while preserving the receptor binding capacity. Autosomal dominant IFNyRI disease has a milder phenotype, lower mortality rate (<5%), and is associated with a later onset of infection and less likelihood of dissemination than complete ARIFNyRI mutations. These ADIFNyRl patients often develop mycobacterial osteomyelitis; other identified infections include histoplasmosis, herpesvirus infections, and Salmonella.

I.-I. Yim and S. M. Holland

170

11.6.2 IFNy Receptor 2 Deficiency

11.6.4 IL-12 p40 Deficiency

Complete IFNyR2 deficiency shows similar clinical features, histopathology, and in vitro responses to complete IFNyRI defects. Dorman and Holland (l998, 2000) identified a frameshift mutation in the IFNyR2 gene of a child with disseminated M. fortuitum and M. avium complex (MAC) infections resulting in a premature stop codon which caused IFNy unresponsiveness. A child with an extracellular domain mutation of IFNyR2 causing a partial defect leading to reduced IFNy responsiveness but normal surface IFNyR2 expression was also reported (Doffinger et al. 2000).

IL-12p40 is the first identified cytokine gene defect affecting mycobacterial susceptibility (Altare et al. 1998b). Autosomal recessive inheritance of the IL12p40 defect was demonstrated in a child with consanguineous Pakistani parents. The patient developed postvaccine disseminated BCG infection followed by disseminated Salmonella enteritidis. Both IFNy receptors 1 and 2 were intact along with the IL-12R~1 chain and a functional IL-12R. Similar to patients with IL12R~1 deficiency, this child's cells produced about 10% of normal IFNy but demonstrated normal TNFa and IL-8 production. Aggressive antibiotic therapy and two courses of IFNy (currently still on IFNy) were required for successful control of the Salmonella infection. The father had also had recurrent, severe Salmonella infections in his youth, raising the possibility of haploinsufficiency in heterozygous carriers.

11.6.3 IL-12 Receptor ~1 Deficiency

In 1998, mutations in the IL-12R~1 receptor were identified in several unrelated cohorts (Altare et al. 1998a; de Tong et al. 1998). These patients were infected with BCG, NTM, or Salmonella. In contrast to the patients with disabling mutations in IFNyRI or IFNyR2, patients with mutations in IL-12R~1 showed intact and mature-appearing granulomas, later onset of infections, and much higher rates of survival than did ARIFNyRI-deficient patients. Most of these patients were treated successfully with antibiotics and IFNy. However, despite aggressive therapy, several fatal cases of disseminated BCG in IL-12R~I-deficient patients have been reported from Turkey (Sanal et al. 2000). In vitro, despite a complete lack of expression of IL-12R~I, the cells maintained a normal proliferation to mitogen or antigen (PPD) and normal IFNy responsiveness. However, defective IL-12 signaling was exhibited in terms of NK activity, proliferation, and poor IFNy production (1-10% of normal). To date, most of the reported cases have been homozygous autosomal recessives, even in the absence of reported consanguinity, presumably reflecting founder effects in some populations. No mutations in IL-12R~2 have been found yet, although they are predicted to be similar in severity and theoretically as likely as mutations in IL-12R~1. However, IL-12R~1 is also one of the critical chains for the IL23 receptor, suggesting that IL-12R~1 deficiency also confers IL-23 unresponsiveness.

11.7 NFKB Essential Modulator (NEMO) or IKB Kinase Defects Recently, mutations in one of the proteins required for nuclear factor kappa B (NF-KB) activation have been shown to cause immunodeficiency associated with hypohidrotic ectodermal dysplasia (HED-ID) (Zonana et al. 2000; Doffinger et al. 2001; Orange et al. 2002). The NFKB essential modulator (NEMO) or IKKy encodes the regulatory subunit of the IKB kinase (IKK) complex, which is required for the activation of the NF-KB signaling pathways and immune defense. NF-KB regulates the transcription of genes encoding inflammatory proteins and antiapoptotic factors. Disruptions in NEMO that result in truncation of the protein or alteration of the zinc finger domain result in impaired NF-KB activation and a distinct physical phenotype of conical teeth, poor sweat gland formation, and abnormal hair growth (hypohidrotic ectodermal dysplasia) along with recurrent infections. Gram-positive, gram-negative and mycobacterial infections, bronchiectasis, and poor febrile responses are characteristic features of this disease. The defect in NEMO affects not only the signaling through the ectodysplasin/dysplasin pathway, explaining the ectodermal dysplasia phenotype, but also signaling through the TNFR, IL-IR, IL-18R, CD40, and Toll-like receptors (TLR), thereby explaining the infection susceptibility and lack of fever.

Nontuberculous Mycobacteria

11.8 Familial Syndrome A familial syndrome has been identified in which two generations of previously healthy women developed CD4+ T-Iymphocytopenia, disseminated NTM infection, and subsequent myeloid leukemia (Holland et al. 1994). Affected individuals displayed a similar clinical phenotype consisting of marked monocytopenia, indolent mycobacterial disease, and progressive lymphocytopenia prior to the development of myeloproliferative disease. This suggests an autosomal dominant common genetic defect that controls mycobacterial susceptibility and myeloid regulation. Holland et al. (1994) also reported a family with disseminated MAC infection in males from two generations. This family had abnormal regulation of IL-12, poor IFNy production, and clinical improvement with IFNytreatment (Frucht and Holland 1996; Frucht et al. 1999). These patients have recently been shown to have a novel mutation in NEMO (E315A).

11.9 Polymorphisms Aside from the specific defects described above, there is a growing interest in the role for genetic polymorphisms in mycobacterial susceptibility. In mice, polymorphisms in the natural resistance-associated macrophage protein (NRAMPl) gene were found to influence early, but not late mycobacterial susceptibility (Vidal et al. 1995). NRAMPI is expressed in activated macrophages and is clearly responsible for the higher bacterial burdens found 3 weeks after infection in BCG-susceptible mouse strains. However, by 6 weeks post infection, both susceptible and resistant strains have cleared the infection. The natural occurrence of this dichotomous trait in mice is due to a single amino acid change (Vidal et al. 1995). NRAMPI encodes an ion transporter that localizes to the lysosomal membrane during phagocytosis of mycobacteria as well as other pathogens. It is thought that NRAMPI may restrict organism replication through alteration of the phagolysosomal environment by regulating iron or other divalent cation transport (Supek et al. 1996; Gruenheid et al. 1997). The NRAMPI gene clearly plays a critical role in the early inflammatory response and early control of mycobacteria but may not influence the course of the disease later on, as demonstrated by the similar time to clearance of infection regardless of the NRAMPI

171 status of the strain. A study of the human orthologue of NRAMPI in a West African population with high rates of tuberculosis found significant associations of several polymorphisms with the development of tuberculosis (Bellamy et al.I998). However, in a small study of eight older women with pulmonary MAC infection, none of the described defects or polymorphisms in NRAMPI was found (Huang et al. 1998). A functional polymorphism in an intron of the IFNy gene has been identified that is correlated with decreased IFNy production, but this has not yet been linked to any infection susceptibility (Pravica et al. 1999). However, almost all patients with disseminated MAC infection produce low levels of IFNy in response to in vitro cell stimulation by the nonspecific T-cell mitogen PHA (Holland et al. 1994). No cases of genetic IFNy deficiency have been identified to date. Many more mutations and polymorphisms have been identified in cytokine receptors than in the cytokines themselves, although the reason for this is unclear.

11.10 Body Morphotype Many patients with pulmonary NTM infections appear to have similar clinical characteristics and body type, including scoliosis, pectus excavatum, mitral valve prolapse (MVP), and joint hypermobility (Iseman et al. 1991; Huang et al. 1999). Many of these physical characteristics are reminiscent of individuals with heritable collagen-vascular diseases (Bruno et al. 1984; Pyeritz and McKusick 1979; Grahame and Child 1984; Glesby and Pyeritz 1989; Roman et al.1989; Sponseller et al. 1995) or the hyper-IgE recurrent infection syndrome (Job's syndrome) (Grimbacher et al. 1999). Therefore, patients with pulmonary NTM infections may possess a subtle connective tissue defect. In this context, phenotypic abnormalities may be the consequence of a genetic defect that affects both body morphotype and infection susceptibility. The relationship between immunity and body morphotype has not been well characterized, but certain associations have been noted. Pulmonary abnormalities including spontaneous pneumothoraces, generalized emphysema, cystic lung disease, bronchiectasis, and increased susceptibility to pneumonia have long been noted in patients with Marfan syndrome (Guide and Holland 2002). Patients with cystic fibrosis (CF), who have been noted to develop NTM infections later in life (Olivier et al. 1996), showed an increased incidence of spinal curvature as well (Erkkila et al. 1978).

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J.-J. Yim and S. M. Holland

Patients with Job's syndrome suffer from recurrent more important for the diagnosis of a mycobacterial pulmonary infections and have recently been shown to disease than for routine bacterial infection, since labohave a characteristic phenotype that includes scoliosis, ratories typically do not look for mycobacteria withjoint hypermobility, and a high arched palate (Grim- out a specific request. In untreated cavitary disease, bacher et al. 1999). In a comprehensive phenotype sputum smears and cultures are usually positive. study of Job's patients, Grimbacher et al. found that The placement of mycobacterial skin tests usually 76% of patients had scoliosis, 68% had hyperextensible implies anticipation of M. tuberculosis infection, not joints, and 57% had recurrent fractures (Grimbacher et nontuberculous infection. In studies from the early al. 1999). The paradigm of Job's syndrome, in which a 1980s, about 50% of patients with pulmonary NTM distinct and somewhat similarbody morphotype is rec- infection had positive skin tests with purified protein ognized in the setting of recurrent infections, suggests derivative (PPD), the antigen filtrate derived from that an association between phenotype and immunity MTB (O'Brien et al. 1987). Recently, von Reyn and may also exist in patients with NTM infections. colleagues have revived the use of an antigen derived from M. avium, the M. avium sensitin (MAS), and compared it with testing with PPD in patients with pulmonary MAC or MTB. They looked for dominance of one skin test over the other when both MAS and 11.11 Clinical Presentation and Diagnosis PPD are placed simultaneously (>5 mm reaction and >3 mm larger than the heterologous skin test reac11.11.1 tion). MAS-dominant skin tests were 97% specific for Pulmonary Disease discriminating MAC from MTB disease (von Reyn et al. 1998). MAS is under trial but not yet available in Clinically, pulmonary NTM infection is usually milder the USA; it is used extensively in Scandinavia. than active pulmonary TB, although the destructive The radiographic appearance of NTM disease in the pulmonary changes and chest radiographs can be lung can be similar to TB, with extensive cavity formaindistinguishable. Symptoms can persist for months tion, nodules, and airspace disease (Fig.ll.2). Discrimito years before radiographs are taken or mycobacterial nating aspects of pulmonary MAC disease compared disease emerges in the differential diagnosis. Fatigue with TB are disease limited to the right middle lobe, is usually the most prominent symptom and one that disease limited to either lower lobe, or combined simulis quite troublesome to the patient. Cough productive taneous disease in the right middle lobe and lingula of mildly purulent sputum may be severe, but it is (Table 1104). Diffuse bronchiectasis involving four or usually simply annoying, worse in the morning or on more lobes of the lung is also more common for pulmorecumbency, and tolerated fairly well. Hemoptysis is nary MAC disease, whereas pleural calcification is more not usually serious but may occur in the course of dis- suggestive of MTB (Lynch et al.1995). The combination ease in up to one-third of patients (Griffith et al.1993). of bronchiectasis and lung nodules seen by chest CT Night sweats and fever eventually occur in the major- scanning had a sensitivity of80% for predicting positive ity; weight loss is common but not severe. Concurrent sputum cultures for MAC (Swensen et al.1994). The American Thoracic Society has proposed bacterial infection, especially with Pseudomonas aeruginosa, is not uncommon and may greatly exacerbate formal guidelines for the diagnosis of NTM disease, cough, sputum production, and fatigue. Various sapro- initially in 1990 (ATS 1990) and then revised in 1997 phytic fungi are frequently cultured from the sputum, (Table U.5) (ATS 1997). Although these are valuable but their influence on the pace or severity of disease is guidelines for the diagnosis of NTM disease, cautious undefined. The tempo of the infection is rarely rapid, interpretation of the radiologic or bacteriologic findand changes may be seen on radiographs evolving ings in the context of the clinical setting and follow-up over years if left untreated. Dyspnea is typically seen observation should be underscored. As we recognize only late in disease when extensive destruction has more cases of pulmonary NTM infection, and the relaoccurred. The physical examination is not very infor- tive frequency of pulmonary TB in the USA declines, mative, usually does not reflect the activity or quies- these guidelines may be unnecessarily restrictive in cence of mycobacterial disease, and is often relatively terms of the criteria required for the diagnosis of an active NTM infection. In most cases, if a patient has a normal, despite extensive involvement. The first step toward the diagnosis of pulmonary positive culture for NTM in the context of a compatible nontuberculous mycobacterial infection is to include history and radiograph, it is likely to be pulmonary that entity in the differential diagnosis. This is much NTM infection and not simple colonization.

Nontuberculous Mycobacteria

173

Table 11.4. Radiographic appearance of mycobacterial disease

a

Feature

Nontuberculous mycobacteria

Diffuse bronchiectasis' Focal bronchiectasis b RUL bronchiectasis RML bronchiectasis RML and lingular bronchiectasis RLL bronchiectasis Either RLL or LLL Nodules Diffuse bronchiectasis and well-defined nodules Cavities Airspace disease Pleural calcification

+++

+

+++ +++ +++ ++ +++ +++

Tuberculosis

+++ +++

+

+ +

+++

++ +++ +++

+

+++ +++ ++

RUL, right upper lobe; RML, right middle lobe; RLL, right lower lobe; LLL, left lower lobe; + indicates relative prevalence of radiographic feature in NTM compared with TB, with three plusses (+++) signifying a very common finding and one plus (+) a very uncommon one; - indicates absence of that feature • Diffuse bronchiectasis defined as involving ~4 lobes b Focal bronchiectasis defined as involving only one lobe. Adapted from Guide and Holland (2002) with permission

b Fig. 11.2a,b. Radiographic findings of pulmonary NTM disease. a Chest CT of a 69-year-old woman with pulmonary MAC disease shows characteristic bronchiectasis in the right middle lobe and lingula. b Chest CT of a 56-year-old woman with long-standing M. abscessus pulmonary infection showing extensive left lung destruction with bronchiectasis and consequent chest wall deformity

11.11.2 Lymphadenitis Lymphadenitis caused by NTM most commonly involves the submandibular, submaxillary, cervical, or preauricular lymph nodes of children between the ages of 1 and 5 years (Margileth et al. 1984; Wolinsky 1995). A unilateral, rapidly enlarging lymph node or group of nodes is usually the only presenting complaint, with occasional low-grade fever and malaise. In the USA currently, the majority of infections are due to MAC (80%) (Lai et al. 1984). This predominance of MAC is a change from 30 years ago, when M. scrofulaceum was the most common etiologic mycobacteria (Lincoln and Gilbert 1972). The presumptive diagnosis of NTM lymphadenitis can be made on the basis of caseating granulomata in the pathologic

specimen, with or without positive acid-fast bacillus smears and cultures, in the absence of a positive PPD reaction. Culturing NTM from the involved lymph node can provide a definitive diagnosis, but the yield of positive culture in cervical lymph nodes is only 50%-85% (Schaad et al.I979).Although the tuberculin skin test is helpful in making a diagnosis, it does not distinguish tuberculosis from NTM infection in one-third of cases (Wolinsky 1995). Excisional biopsy of the involved lymph node is recommend both for diagnosis and treatment. The role of fine needle aspiration in the diagnosis of NTM lymphadenitis has been controversial (Bailey et al. 1985; Lau et al. 1991; Gupta et al. 1993), and incisional biopsy may cause fistula formation with chronic drainage (Schaad et aI. 1979). In contrast to other forms of NTM infection, antibiotic therapy is often not needed; excision of the involved lymph node without chemotherapy is currently the best treatment option for isolated NTM lymphadenitis of childhood (Stewart et al. 1994).

11.11.3 Skin and Soft-Tissue Infection Although almost all species have been described as occasional pathogens in cutaneous disease (Wolinsky 1979; Falkinham 1996), M. jortuitum, M. abscessus,

c. Same

For a diagnosis of pulmonary disease, all three criteria - (1) clinical, (2) radiographic, and (3) bacteriologic - must be satisfied From ATS (1997)

c. Tissue biopsy • Any growth from bronchopulmonary tissue biopsy • Granuloma and/or AFB on lung biopsy with one or more positive cultures from sputum/bronchial wash • Any growth from usually sterile extrapulmonary site

I. Local immune suppression II. General severe immune suppression Alcoholism (M. avium complex) Leukemia Bronchiectasis Lymphoma Organ transplantation Cyanotic heart disease Other immunosuppressive therapy Cystic fibrosis Prior mycobacterial disease Pulmonary fibrosis Smokinglchronic obstructive lung disease None 1. Clinical criteria: a. Same a. Compatible signs/symptoms (cough, fatigue most common; fever, weight loss, hemoptysis, dyspnea may be present, particularly in advanced disease) with documented deterioration in clinical status if an underlying condition is present and b. Same b. Reasonable exclusion of other disease (e.g., tuberculosis, cancer, histoplasmosis) to explain condition, or adequate treatment of other condition with increasing signs/symptoms 2. Radiographic criteria: a. Same a. Any of the following chest X-ray abnormalities; if baseline films are more thanl year old, should be evidence of progression • Infiltrates with or without nodules (persistent >2 months or progressive) • Cavitation • Nodules alone (multiple) b. Same b. Any of these HRCT abnormalities • Multiple small nodules • Multifocal bronchiectasis with or without small lung nodules 3. Bacteriologic criteria: a. At least three available sputum/bronchial wash samples within I year a. Same • Three positive cultures with negative AFB smears or • Two positive cultures and one positive AFB smear or b. Same except b. Single available bronchial wash and inability to obtain sputum samples • culture positive with I + • Positive culture with 2+,3+, or 4+ growth or or greater growth • Positive culture with a 2+, 3+, or 4+ AFB smear or

Presumed or confirmed HIV-seropositive potential risk factors

Table 11.5. Criteria for the diagnosis of pulmonary nontuberculous mycobacterial disease

b. Same except • culture positive with I + or greater growth (excludes M. avium complex) c. Same

a. Same

b. Same

a. Same

b. Same

a. Same

CD4 count <200

Presumed or confirmed HIV-seronegative potential risk factors

0..

~

~

~

~

~

~

~ '"0..

,

--

...... ~

175

Nontuberculous Mycobacteria

M. marinum, and M. ulcerans are the most common NTM causes of skin and soft-tissue infection (Wolinsky and Rynearson 1968). Abscess formation and/or drainage at the site of puncture wounds, open traumatic injuries, or fractures are typical manifestations of cutaneous NTM infection. Furunculosis in the lower extremities by M. fortuitum through nail salon pedicure baths (Winthrop et al. 2002) and nosocomial infections involving long-term intravenous or peritoneal catheters have been reported (Wallace et al. 1983; Hoy et al. 1987). The diagnosis can be made by culture of NTM from the drainage material or on tissue biopsy. M. marinum can cause so-called 'swimming pool granuloma' or 'fish tank granuloma'. This bacillus infects abraded skin during the cleaning of freshwater fish tanks or from scratches from salt water fish, shrimp, fins, etc. (Wolinsky 1979). Skin infection by M. marinum commonly presents as a nodular or ulcerative sporotrichoid lesion on the elbows, fingers, hands, or knees. These lesions usually resolve spontaneously within 36 months (Catinella 1978). However, extensive tendonitis can occur. It is imperative to consider M. marinum early in the differential diagnosis of any necrotizing tendonitis, as the antibiotics effective against it are quite distinct, and failure to identify this organism often leads to recurrent operations and procedures with associated tendon destruction and loss of use. M. ulcerans usually causes a painless nodule on the leg without other signs or symptoms in young adults and children (Marston et al. 1995). It can progress to a shallow ulcer with a necrotic base. Spontaneous resolution may occur in 6-9 months, but sometimes the lesion will spread and cause serious deformity (Marston et al. 1995). NTM can cause chronic granulomatous infections of tendon sheaths, bursae, joints, bones, and prostheses inserted during orthopedic procedures (Kelly et al. 1967). M. marinum and MAC are prone to causing tenosynovitis of the hands (Wolinsky 1979; Hellinger et al. 1995; Falkinham 1996). On rare occasions, NTM can infect prosthetic heart valves (M. chelonae) (Repath et al. 1976) or the genitourinary tract (MAC, M. chelonae) (Clark et al. 1989).

11.11.4 Disseminated NTM Infection

Disseminated infection due to NTM usually occurs in the setting of immunosuppression such as AIDS (Horsburgh and Selik 1989), after solid organ transplantation (Lichtenstein and MacGregor 1983),

leukemia (Ingram et al. 1993), lymphoma (Ingram et al. 1993), collagen vascular disease (Ingram et al. 1993), severe combined immunodeficiency syndrome (Stone et al. 1992), and others. Patients with disseminated NTM infection without any apparent underlying disease have also been reported (Lincoln and Gilbert 1972). MAC has been the most common NTM infecting patients with HIV infection (Horsburgh and Selik 1989). However, in the other settings mentioned above, although MAC (Wolinsky 1979; Horsburgh et al. 1985) is most common, M. kansasii (Lichtenstein and MacGregor 1983), M. chelonae (Wallace et al. 1983; Cooper et al. 1989; Ingram et al. 1993),M. scrofulaceum (Wolinsky 1979),M. abscessus (Wolinsky 1979), and M. haemophilum (Kiehn and White 1994) also cause disease. In patients with disseminated NTM infection without AIDS, disease can manifest as a fever of unknown origin (Horsburgh et al.1985) or multiple subcutaneous nodules or abscesses (Wolinsky 1979; Cooper et al.1989; Ingram et al.1993; Kiehn and White 1994). In AIDS patients, prolonged fever accompanied by night sweats and weight loss in the setting of <50 CD4+ Tlymphocytes suggests disseminated NTM infection (ATS 1997). Diagnosis in both settings is confirmed by culture of NTM from bone marrow or blood. A case of disseminated MAC infection without known risk factors is illustrated in Fig. 11.3.

11.12

Treatment 11.12.1 Chemotherapy

Management of these infections is very much still evolving and remains challenging even in the most straightforward cases. Many of the NTM, such as MAC, are typically resistant to many antimycobacterial agents and frequently relapse after therapy. The newer macrolides, azithromycin and clarithromycin, have shown remarkable activity against these infections. Clear and defined roles for immunotherapy and surgery remain to be determined. In the pre-macrolide era, long-term success rates for the treatment of pulmonary MAC infection ranged from 44% to 80% and were not aided by the determination of in vitro susceptibilities. Dutt and Stead reported on 85 cases of pulmonary and 5 cases of extrapulmonary MAC infection from Arkansas in the era before macrolides. Some 65% of their popu-

176

J.-J. Yim and S. M. Holland

Fig. 11.3. a A 7-year-old-boy with extreme splenomegaly had a draining inguinal lymph node (b). Blood and drainage cultures grew MAC. c Bone marrow aspirate showed no granulomatous response, and staining of his bone marrow showed numerous acid-fast bacilli

lation had underlying pulmonary disease, and 31% were women. Although they found a high rate of drug resistance in vitro, they were still successful in treating patients with multiple agents including isoniazid, ethambutol, and streptomycin at least for 12 months. Some 63% of their patients cleared their sputa within 3 months, with an overall medical failure rate of 32%. They found no added benefit to treatment with more than four drugs (Dutt and Stead 1979). Engbaek et al. reported a retrospective study of 37 Danish patients (30% women) with pulmonary MAC disease treated with combinations of isoniazid, ethambutol, PAS, streptomycin, cycloserine, and rifampin. There was a 44% medical success rate despite very high levels of in vitro resistance of the organisms (Engbaek et al. 1981). Ahn et al. treated 46 patients with pulmonary MAC, 67% of whom had predisposing lung disease, and 33% of whom were women. Using a 9-week inpatient induction regimen of isoniazid, ethambutol, rifampin, and twice weekly streptomycin for 6 months, they achieved a 91 % sputum conversion rate. Subsequently, after 18-24 months of therapy, 4 patients relapsed or had recurrent disease (Ahn et al. 1986). Horsburgh et al. reported on 75 patients, 53% of whom were women, and 77% of whom had been treated previously. They started patients on an initial regimen of isoniazid, ethambutol, rifampin, capreomycin, ethionamide, and cycloserine. The overall response rate in 4 months was 66%, with more responders having received more drugs to which their organism was susceptible in vitro (Horsburgh et al. 1987). The existence of multiple case

series in the pre-macrolide era successfully treated without the benefit of in vitro susceptibility testing suggests that we are still fairly ignorant of the critical aspects of therapy and may be relying on inessential components of management currently. However, at least in the USA, the clinical epidemiology of MAC lung disease has changed and is now more female predominant, so the response rates may be changing for reasons other than treatment. Macrolides are now the mainstay of NTM therapy. Wallace et al. gave clarithromycin 500 mg twice daily for 4 months, followed by multidrug therapy, in an effort to determine the activity of the macrolide alone. They showed a 58% apparent cure rate in 20 adults, most of whom were previously untreated, and 40% of whom were women. The drug was fairly well tolerated; 16% of isolates developed clarithromycin resistance (Wallace et al. 1994). Azithromycin 600 mg/ day as monotherapy was studied by the same group in 23 patients. They found similar results except that toxicities such as gastrointestinal effects and altered hearing were more common; there was no development of macrolide resistance (Griffith et al. 1996). The combination of high-dose rifabutin (600 mg/day) with macrolides caused multiple toxicities including leukopenia, gastrointestinal symptoms, abnormal liver functions, diffuse polyarthralgias, and anterior uveitis (Griffith et al. 1995). Huang et al. have treated 27 patients with pulmonary MAC infection (93% were female, 90% Caucasian). After 12 months of multidrug therapy including azithromycin/clarithromycin, rifampin/rifabutin, and ethambutol, they had a 50%

Nontuberculous Mycobacteria

177

treatment failure rate (Huang et al. 1999). Whether counts rebounded. These results were seen in patients these changing rates of success with drug therapy with low CD4+ T-lymphocyte counts, suggesting that reflect changes in the demographics of the patient the IFNyeffect in this setting was not T-cell mediated. group (increasing numbers of Caucasian women) or We have identified a heterogeneous cohort of the organism or loss of potency of the multidrug regi- patients with abnormalities in IFNy generation mens remains to be determined in larger studies. and disseminated MAC infection without HIY. This The rapidly growing mycobacteria, especially has been associated with abnormal IL-12 producM. abscessus, present a special problem. Pulmonary tion (Frucht and Holland 1996) or idiopathic CD4+ disease due to M. abscessus is more likely to occur T-Iymphocytopenia (Holland et al. 1994) We have in Caucasian women who are nonsmokers without used subcutaneous IFNy in a total of 23 patients cavitary disease, more likely to be associated with with disseminated MAC and other NTM infections previous mycobacterial infection, less likely to be with favorable results (Fig. 11.4, unpublished obsermedically curable, and more likely to lead to death vations). Treatment has been used for up to 1 year due to lung destruction than other mycobacterial or longer. These patients have persistent in vitro lung diseases. This infection is a distinct entity that hypoproduction of IFNy, even long after cure. Three requires more intravenous therapy than does MAC patients have relapsed after discontinuation of their infection and may be less responsive (Griffith et al. IFNy, despite continuation of their antimycobacterial 1993). Although surgery is frequently attempted and therapy. may improve the pulmonary symptoms, M. abscessus may cause severe fistulizing disease if there has been contamination of the operative bed. Treatment guidelines from the American Thoracic Society and the British Thoracic Society from 1997 and 1999, respectively, are briefly summarized in Table 11.6 (ATS 1997; BTS 2000).

11.12.2 Immunotherapy It was not long after Koch's discovery of M. tuber-

culosis that he developed an extract of the organism, tuberculin, the forerunner of PPD, as a cure for tuberculosis (Koch 1891). The use of tuberculin and other extracts as a treatment for tuberculosis continued well into the middle part of this century. However, the lack of controlled studies, the requirement for individualization of treatment regimens to avoid complications, and relatively slow results prevented this approach from gaining wide application. Effective multidrug antituberculous therapy made tuberculin therapy obsolete. Animal models and human studies show that IFNy is a critical factor in the control of mycobacteria. Squires et al. (1989, 1992) treated 6 MAC-bacteremic AIDS patients with IFNy with or without concurrent antimycobacterials (Squires et al.1989, 1992). In those patients who received IFNy plus antimycobacterials, there was a clear decline in mycobacteremia. In contrast, when IFNywas used without concurrent antimycobacterials there was no change in bacterial counts. The treatment trial lasted no more than 6 weeks, at the end of which time IFNy was stopped, and bacterial

a

b Fig.l1.4a,b. Abdominal CT scan before (a) and after (a) treatment with subcutaneous IFNg in a 42-year-old man with disseminated MAC infection. Chylous ascites caused by MAC decreased dramatically after 4 months of IFNy treatment (Holland et al. 1994)

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178

Systemic IFNy has been tried in patients with refractory pulmonary MAC infections. These cases usually have severe parenchymal destruction and are difficult to clear, since penetration of antimycobacterials into diseased lung spaces is compromised. The best results have been in patients with noncavitary disease, but still in the 20% range (S.M. Holland, unpublished observations). Aerosolized IFNy has been proposed as a method of delivering organ-specific cytokine therapy, which may reach high levels in diseased airways (Jaffe et al. 1991). Chatte et al. used aerosolized IFNy 500 flg by nebulizer 3 days/week in a 38-year-old man with silicosis and severe refractory cavitary MAC disease (Chatte et al. 1995). His smears converted to negative, but the cultures remained positive; with the cessation of aerosolized IFNy, his smears reverted to positive, his disease progressed, and he died. This was the first report of aerosolized IFNy in the treatment of mycobacterial disease, and rapid conversion of sputum smear in such a long-term refractory patient was remarkable. However, the failure to convert his cultures is a concern for future applications. Similar results of smear but not culture conversion were seen with aerosolized IFNy therapy for multidrug-resistant tuberculosis (Condos et al. 1997). Placebo-controlled, prospective trials are necessary to determine the most appropriate delivery system and the patient groups that are most likely to benefit. To date, the adverse experience profile for IFNy is essentially the same as that seen in chronic granulomatous disease (The International Chronic Granulomatous Disease Cooperative Study Group 1991). The therapeutic use of IL-12 in human infectious diseases is still quite immature. Low dose IL-12 (60 ng/kg subcutaneously twice weekly) in a patient with pulmonary M. abscessus led to sputum conversion and cure despite failure to improve on IFNy (Holland and Dorman 1998). IL-12 has been shown to induce antimycobacterial activity in vitro (Bermudez et a1.1995) and in vivo (Holland and Dorman 1998) beyond the simple increase of IFNy. However, whether IL-12 has important therapeutic activity beyond that of IFNy is not yet clear, and whether there is any role for IL-12 in the setting of IFNy receptor deficiency is unknown. IL-12 toxicities so far have been greater than those of IFNy but can be controlled by careful dosing (Leonard et a1. 1997). Granulocyte macrophage colony-stimulating factor (GM-CSF) is produced by T-cells, NK cells, activated macrophages, and neutrophils. It stimulates myelopoiesis and activates phagocytes for superoxide production and intracellular killing of bacteria, including mycobacteria (Bermudez and

Young 1990). Monocytes and NK cell infection with MAC in vitro led to GM-CSF detection (Blanchard et a1. 1991). Exogenous administration of GM-CSF to MAC-infected beige mice resulted in increased killing of mycobacteria and an enhanced antimycobacterial effect (Bermudez et al. 1994). Kemper et a1. treated 8 MAC bacteremic AIDS patients with high-dose azithromycin alone or azithromycin plus subcutaneous GM-CSF daily for 6 weeks (Kemper et a1. 1998). Monocyte activation was enhanced in the GM-CSF group, and mycobacterial counts in the blood decreased more in the azithromycin plus GMCSF group than in the azithromycin alone group. The drug was well tolerated. Given the broad experience with GM-CSF in cancer patients and in vitro data supporting its efficacy, further evaluation in clinical trials are warranted. Steroids have been used extensively in the treatment of tuberculous meningitis, pleurisy, and pericarditis in an effort to reduce inflammation and subsequent fibrosis. Controlled studies in pleuritis have failed to show much benefit with steroids, although they reduced symptoms faster than medication alone (Wyser et a1. 1996). Wormser et a1. reported that 5 AIDS patients with disseminated MAC who received dexamethasone 1-4 mg daily in addition to antimycobacterial therapy noted improvement in symptoms, nutritional state, and laboratory markers over baseline (Wormser et a1.1994).

11.13 Summary Precisely because of their low virulence, the NTM have taught us much about both innate and acquired immune pathways. They highlight a critical relationship between the physical morphotype and immunity and have been unusually valuable in leading to the discovery of cytokine and receptor defects predisposing to infection. It is hoped that the observations about cytokine therapy developed in the treatment of the NTM will find broader application in the treatment of infectious diseases.

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Diagnosis

12 Serologic Testing for Tuberculosis CHAKRADHAR KOTARU and EDWARD D. CHAN

CONTENTS 12.1 12.2 12.2.1 12.2.2 12.2.3 12.2.4 12.2.5 12.2.6 12.2.7 12.2.8 12.2.9 12.2.10 12.2.11 12.2.12 12.2.13 12.2.14 12.2.15 12.3 12.3.1 12.3.2 12.3.3 12.3.4 12.3.5 12.4

Introduction 187 Specific Serological Tests for TB 187 Antigen 5 (38 kDa Antigen) 188 A60 Antigen 189 30 kDa Antigen 189 Antigen p90 189 19 kDa Antigen 189 16 kDa Antigen 189 45/47 kDa Antigen Complex 190 P32 Antigen 190 Cord Factor 190 Glycolipid Antigens 190 Lipoarabinomannan 191 ICT Tuberculosis Test 192 Dermal Response to MPB64 Antigen 192 Integral Membrane Antigens of M. habana TMC 5135 192 Lipopolysaccharide Antigen 192 Specific Targeted Populations 192 Childhood TB 193 TB in the Elderly 193 HIV-Positive Individuals 193 Extrapulmonary TB 194 Smear-Negative vs Smear-Positive TB 194 Conclusion 194 References 195

12.1 Introduction Tuberculosis (TB) is a leading cause of morbidity and mortality in the world. The World Health Organization (WHO) estimates that one-third of the world's population is latently infected with Mycobacterium tuberculosis. From this pool, roughly 10 million active TB cases emerge annually, resulting in 2 to 3 million deaths. Early identification and proper treatment of individuals with active TB have a great impact on public health. The bacteriologic methods are either not sufficiently efficient and specific (acidfast smear) or require an extended turnaround time from the laboratory (culture isolation). The development of a rapid nonbacteriologic diagnostic test that is both sensitive and specific for active TB continues to be formidable. This difficulty is due, in part, to the inability of many tests to distinguish latent infection from active disease. This chapter focuses on the current serological methods to diagnose TB.

12.2 Specific Serological Tests for T8 Edward D. Chan is supported by the NHLBIHL-66-112, the Parke-Davis Atorvastatin Research Award, and the American Lung Association Career Investigator Award.

C. KOTARU, MD Department of Medicine and Program in Cell Biology, National Jewish Medical and Research Center, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and Denver Veteran Administration Medical Center, K613e, Goodman Building, 1400 Jackson Street, Denver, CO 80206, USA E. D. CHAN, MD Associate Professor of Medicine, K613e Goodman Bldg., National Jewish Medical and Research Center, Denver, CO, 1400 Jackson St, Denver, CO 80206, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Although serological assays may not add to the diagnostic yield in those patients in whom sputa examination is possible an accurate serological test to diagnose TB would have considerable advantages in those patients who are unable to provide adequate sputum samples (e.g., in children and the very elderly), who are smear-negative and/or culture-negative, or have suspected extrapulmonary TB (Steele and Daniel 1991). In populations where the prevalence of tuberculin skin test positivity is high, earlier detection and treatment of active TB by blood screening may help prevent further transmission of TB. However, serological testing has been confounded by crossreactivity associated with bacillus Calmette-Guerin (BCG) vaccination or infection with nontuberculous mycobacteria. For example, using the purified protein

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derivative (PPD) status as a gold standard, the ability of immunoblot for the A60 antigen complex to discriminate between TB infection and noninfection was nearly 100% (Rovatti et al. 1996). However, 90% of the study group (n=140) tested positive for antiA60 antibody after BCG vaccination compared with only 1.4% prior to vaccination (Rovatti et al. 1996). Previous seroassays for TB have utilized either a mixture of M. tuberculosis antigens, such as purified extracted glycolipids, adsorbed mycobacterial sonicates, and PPD, or more distinct mycobacterial antigens (Table 12.1) (Chan et al. 2000). Measurement of tuberculostearic acid in clinical specimens was shown to have a high degree of sensitivity and specificity, but the assay required considerable expertise to perform (Brooks et al. 1987). Using mycobacterial sonicates in an enzyme-linked immunosorbent assay (ELISA) on samples of 31 children with clinical TB, Rosen (l990) found a sensitivity of 26% and specificity of 40%. Khomenko et al. (l996) evaluated an ELISA for M. tuberculosis H37Rv antigenic preparation and found the sensitivity to be lower at 72% and specificity to be 100%. Conversely, using affinity-purified H37Rv antibody to detect the antigen, they found the sensitivity and specificity to be similar, 77% and 90%-93%, respectively. Franco et al. (2001) assessed the serological response to four antigenic fractions of H37Rv sonicate by Western blot analysis. Patients with smear-positive pulmonary infection and tuberculous pleurisy had a positivity rate of 50%-60% against all fractions. Interestingly, of the 16 contacts, 3 had a conversion of their tuberculin skin tests, and the same 3 individuals also had a positive serologic response to the 19 kDa antigenic fractions, making it potentially

Table 12.1. Antigens used in the serological diagnosis of tuberculosis Mycobacterial sonicates Extracted glycolipids Purified protein derivative Antigen 5 (38 kDa antigen) A60 antigen 45/47 kDa antigen complex Antigen Kp90 30 kDa antigen P32 antigen Cord factor (trehalose dimycolate) Lipoarabinomannan 16 kDa antigen Integral membrane antigens of M. habana TMC 5135 Lipopolysaccharide antigen

useful in identifying patients who are candidates for the treatment of latent infection. In the following sections, we reviewed studies that examined the presence of specific antibodies directed against mycobacterial antigens.

12.2.1 Antigen 5 (38 kDa Antigen) The use of antigen 5, also known as the 38 kDa antigen, in a microtiter plate ELISA has been evaluated in a number of countries with reasonable sensitivity and high specificity (AIde et al. 1989; Daniel et al. 1985, 1986; Ma et al. 1986). In one of these studies from China, the test was 89% sensitive and 94%100% specific for patients with active pulmonary TB (Ma et al. 1986). Generally, the antibody titer correlated with the extent of pulmonary disease. In another study, detection of IgG against this 38 kDa revealed a sensitivity of 64% and a specificity of 81 % (Chiang et al. 1997). Recently, Cole and coworkers (l996) also used antigen 5 in a serological test but modified it by using a rapid membranebased antibody assay and found the sensitivity to be 89% (54 of 61) in patients with smear-positive, culture-positive TB. In a group of 91 patients who were both smear- and culture-negative, the sensitivity was 74% even though these patients had been on antituberculous therapy from 1 to 6 months at the time of the test. The overall specificity was 93%. In an ELISA using the monoclonal antibody TBn to the 38 kDa antigen, the sensitivity was about 80% for patients with either smear-positive/ culture-positive or smear-negative/culture-positive TB (Bothamley and Rudd 1994). Interestingly, the sensitivity rate was even higher (-90%) for patients successfully treated for presumed TB but without bacteriologic confirmation of disease. Combining an ELISA to detect antibody to both 38 kDa and a 34 kDa M. tuberculosis protein, Amicosante and colleagues (l995) found the overall sensitivity to be 86%. Importantly, the specificity in PPD-negative or PPD-positive control patients (n=30 each) and in 20 patients with nontuberculous bacterial pneumonia was 100%. Nsanze et al. (l997) reported the utility of the enzyme immunoassay (EIA) kit developed by Omega laboratories using the 38 kDa antigen. Depending on the cut-off points used, the sensitivities were >95%, while the specificities were 86%-100%. The test was much less useful for extrapulmonary TB, with sensitivities ranging from 51% to 71%.

Serologic Testing for Tuberculosis

12.2.2 A60Antigen

The A60 antigen, a thermostable component of PPD, has also been used in the serodiagnosis ofTB (Charpin et al. 1990; Cocito 1991; Fadda et al. 1992; Luh et al. 1996; Yu et al.1992; Zou et al.1994). Unfortunately, this molecule is not specific for mycobacteria because it is also present in Nocardia and Corynebacterium species. In 83 patients with smear-negative but culture-positive pulmonary TB, measurement of both IgM and IgG in an ELISA revealed a sensitivity of 68% and specificity of 100% (Charpin et al. 1990). Similarly, an ELISA revealed a sensitivity of 76.2% for patients with active pulmonary TB and 59% for patients with extrapulmonary TB (Luh et al. 1996). In 153 individuals with inactive tuberculous infection, the specificity was only 81.7%, and in over 500 patients with nontuberculous infection, the specificity was 91.3%. Thus, the positive predictive value was only 67.9% (Luh et al. 1996). In a study of 560 Chinese patients with pulmonary and extrapulmonary TB and over 700 controls, the measurement of IgM appeared to be sensitive (80%) for active primary TB and specific (100%) for latent TB (Zou et al. 1994). On the other hand, IgG against the A60 antigen was more predictive of active postprimary TB (sensitivity of 89%). Among 529 healthy persons most of whom were vaccinated with BCG, including 287 who were PPD positive, there was less than 1% false-positives. Anuradha et al. (2001) evaluated the utility of detecting antibodies to A60 for the diagnosis of extrapulmonary TB. In 72 patients with neuro-TB, antibodies were detected in the serum and/ or CSF in almost 80%. The diagnosis of other forms of extrapulmonary TB by anti-A60 antibodies could be made in about 60% of the cases.

12.2.3 30 kDa Antigen

Using a 30 kDa antigen purified from culture filtrates of M. tuberculosis, McDonough and colleagues (1992) compared a dot EIA with the standard ELISA assay on patients with active TB using control patients who had no clinical evidence of TB but whose PPD status was not known. The specificity for dot EIA and plate ELISA were 92% and 97%, respectively; the sensitivity rate for hospitalized patients with TB was 69% and 78%, respectively. Interestingly, the sensitivity rate was -48% for both methods in ambulatory patients with active TB, while in 26 HIV-positive patients with TB, both immunoassays showed extremely poor sensitiv-

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ity (12% for the plate ELISA and 0% for the dot EIA). In assessing the lymphocyte proliferative and humoral responses to patients with active pulmonary TB and of healthy household contacts, the group with active disease had a strong humoral response to the 30 kDa antigen but a poor proliferative response (Torres et al. 1994); the opposite pattern was seen in the exposed control patients. Thus, a humoral response to tuberculous antigen may not only signify active infection but may also indicate a nonprotective immune response. In a study that measured the presence of the 30 kDa antigen in 25 African patients with smear-positive TB using a monoclonal antibody, 20 of them tested positive (sensitivity 80%) (Ng et al. 1995).

12.2.4 Antigen p90

Arikan and co-workers (1997) showed that in 51 patients with active TB, the sensitivity of anti-Kp90 IgA in either sera or body fluids was 82%, while in 71 control patients, the specificity was 90%. In another study of 88 TB patients, comprised of 32 smear-positive and 56 smear-negative individuals, the overall sensitivity was 70% (Alifano et al. 1997). In a control group of 87 individuals with either nontuberculous lung disease, healed TB, or healthy volunteers, the specificity was 92%.

12.2.5 19 kDa Antigen

Although previously promlsmg, antibody titers to the 19 kDa antigen were found to have poor sensitivity (8%) in Indian patients with active TB, but it was higher in patients from the UK (55%-57%) (Bothamley et al. 1992).

12.2.6 16 kDa Antigen

Using a recombinant 16 kDa antigen of M. tuberculosis, Imaz et al. (2001) compared the seroresponse using EIA to detect IgG, IgM, and IgA in 74 children with active TB, in 149 with nonmycobacterial disease, and in 49 healthy contacts. The average levels of antibodies were higher in the healthy contacts when compared with children with nonmycobacterial disease, making it a potentially useful test for predicting patients who require prophylaxis against latent infection; however,

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the low overall sensitivity (34%, 19%,3%, and 43% for IgG, IgA, IgM, and combined IgG IlgM, respectively) prevented the test being useful in the diagnosis of active childhood TB.

controls who were all skin test-positive (Maekura et al. 1993). Interestingly, the cord factor antibody titers declined to normal levels with antituberculous chemotherapy.

12.2.7 45/47 kDa Antigen Complex

12.2.10 Glycolipid Antigens

In a study that evaluated the IgG response to the 451 47 kDa secreted protein ofM. tuberculosis, the specificity of the test was >98% in control subjects, comprised of healthy volunteers who were either PPD-positive or PPD-negative (Diagbouga et al. 1997). However, the sensitivity of the test was only 40% even in smearpositive TB patients.

Detection of IgG against M. tuberculosis glycolipid antigen in 57 patients (27 smear-positive and 30 smear-negative) revealed that the overall sensitivity was 96% and specificity 91 % (Dogan and Aksu 1997). Escamilla and colleagues (1996) used glycolipids from M. fortuitum (di- and tri-O-acylated trehaloses) to detect an antibody response and found the sensitivity for active TB was >80% and the specificity 98%. In a large multicenter study from Japan, Maekura et al. (2001) described the utility of an ELISA for antituberculous glycolipid (anti-TBGL) in the serodiagnosis of TB. Using a cut-off of anti-TBGL antibody titer >2 Ulml, the assay had a sensitivity of 81 % and specificity of 96% in 318 patients with active pulmonary TB (216 smear-positive andlor culture-positive and 102 smear- and culture-negative, but clinically diagnosed). Subgroup analysis revealed greater sensitivity (90%) in patients with smear- and culture-positive TB, as opposed to culture-positive only (70%) or smear- and culture-negative, clinically diagnosed patients (73%). This suggests a correlation between the positivity of the test and the mycobacterial burden. Another interesting observation that has potential implications in judging the response to therapy was the decline in titers in a subgroup of patients with initially high titers (> 10 U/ml) at the beginning of treatment. The test performance characteristics of ELISA to detect antibodies against M. tuberculosis antigenic glycolipids was evaluated in 142 patients with extrapulmonary TB compared with 578 patients with nontuberculous disease (Niculescu et al.1999). The sensitivity and specificity were 82 and 92%, respectively. Simmoney and co-workers (1996) compared the diagnostic utility of ELISA in detecting antibodies against glycolipid to those against A60 antigen in 46 HIV-positive and 50 HIV-negative patients with documented TB. Overall, antibody to A60 was detected in only 37% of the cases, while 85% of the patients had a positive antibody response to glycolipid antigens. When the data were analyzed in the context of HIV status and the associated level of immunosuppression, HIV-positive patients with lower CD4 counts were more likely to have negative antibody responses to A60 than to glycolipids.

12.2.8 P32Antigen

The serological response to purified protein of M. bovis BCG (known as P32 antigen) was also evaluated in patients with active TB (Turneer et al. 1988). Although there was a statistically significant difference in mean IgG and IgA antibody levels between active TB patients and healthy control subjects, the combined sensitivity rate for both immunoglobulin classes was only 47%. Interestingly, neither naturally acquired tuberculin hypersensitivity nor BCG vaccination affected the positive frequencies in healthy subjects. This same group of investigators also utilized a dot-blot assay with BCG cellular extract and found a good correlation between their ELISA assay and the dot-blot assay (van Vooren et al. 1988).

12.2.9 Cord Factor

The IgG antibody response to M. tuberculosis cord factor (trehalose-6,6'-dimycolate) was examined in a group of 99 patients with mycobacterial infection (42 with culture-positive TB, 46 with a clinical diagnosis of TB, and 11 patients with atypical mycobacterial infection), 5 patients with lung cancer, and 100 healthy controls. The overall sensitivity of patients with any bacterial or clinical diagnosis of a mycobacterial infection was 83%, and the specificity was 100% (He et al. 1991). The antibody response to M. tuberculosis cord factor was tested in an ELISA for patients with active TB and produced a sensitivity of 81 % and a specificity of 96% compared with healthy

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that of M. avium complex (the predominant atypical mycobacteria), the fine structural differences between the LAMs (e.g., degree and configuration The cell walls of mycobacterial organisms contain of mannose capping) have not been fully elucidated. complex macromolecules such as lipoarabinoman- Therefore, despite the relatively good specificity rate nan (LAM) (Fig. 12.1). LAM is a lipoglycan known to reported, the degree of antibody cross-reactivity have a number of immunomodulatory effects. LAM is against LAM of M. tuberculosis vs that of atypical comprised of a linear series of ringed mannose sugar mycobacteria remains to be determined. Second, there was a wide range of sensitivity of residues, with occasional branches of single mannose residues. At the proximal end of LAM, a phosphati- anti-LAM IgG among HIV-negative patients with dylinositol group anchors it to the plasma membrane. active TB, while the sensitivity was generally poor Distal to the mannose residues, a series of arabinose in HIV-positive patients. For example, in HIV-negasugar residues is attached. In M. tuberculosis, these tive patients, the sensitivity ranged from 21.5% to arabinose residues are further 'capped' with mannose 89% (del Prete et al. 1998; Julian et al. 1997; Lawn residues. Thus, the LAM of M. tuberculosis is called et al. 1997; Ratanasuwan et al. 1997; Sada et al. 1992, 'ManLAM'. In relatively recent reports, the detection 1999) but was between 7% and 40% in HIV-positive of anti-LAM antibodies has shown great promise in patients (Boggian et al. 1996; Julian et al. 1997; Lawn the serological diagnosis of active TB (Boggian et al. et al. 1997; Ratanasuwan et al. 1997; Sada et al. 1992, 1996; del Prete et al. 1998; Julian et al. 1997; Lawn et 1999). In HIV-negative patients, the sensitivity of the al. 1997; Ratanasuwan et al. 1997; Sada et al. 1990, MycoDot antiLAM IgG test in Tanzania, Ghana, and 1992; Somi et al. 1999). Thailand was 33%,56%, and 63%, respectively (Lawn In general, three important points can be taken et al. 1997; Ratanasuwan et al. 1997; Somi et al.I999). Third, as with most other types of serological tests, from these studies. First, the specificity of the test was excellent, ranging from 84% to 100%. Moreover, the sensitivity of the test fell with negative smears, Julian and co-workers (1997) tested 14 patients with a finding generally attributed to the lower burden atypical mycobacteria, and anti-LAM IgG could not of organisms in smear-negative cases and/or to the be detected in any of them although it is not clear how greater incidence of smear-negativity in patients comany of these patients were co-infected with HIV. infected with HIV. We recently tested the accuracy of Boggian et al. (1996) tested 104 patients with atypical a simplified, visually detectable, colloidal gold-based mycobacteria for anti-LAM IgG, and only 2 patients serological assay to qualitatively detect IgG directed were weakly positive; however, all the patients tested against LAM in a population of individuals largely were HIV-positive. Although the basic structure of comprised of immigrants to the USA from areas with LAM associated with M. tuberculosis is similar to a high prevalence of TB infection (Chan et al. 2000).

12.2.11

Lipoarabinomannan

ManLAM

Fig. 12.1. Cell wall of M. tuberculosis showing the various macromolecules that comprise the complex structure, including the plasma membrane, peptidoglycan, lipoprotein ,and various Iipoglycans such as mannose-capped Iipoarabinomannan, mycolic acid-arabinogaJactan peptidoglycan complexes (mAGP), Iipomannan (LM), and phosphatidylinositol dimannose (PIM z). The fatty acid chains (acyl groups) are attached to the plasma membrane

lipoproteins

mycolic acids

• 0- mannose-p • D-arabinose-f • galaetose-f '

phosphatidylinositol (palmitate, tuberculostearate)

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In patients with active TB, the sensitivity of anti-LAM IgG was 85%-93%. Importantly, in five patients with active TB who were smear-negative, all tested positive for anti-LAM IgG. The specificity of the test depended on the presence of tuberculous infection. In a group of US citizens comprised of young healthy adults and rheumatology patients, the specificity was 100%. In a population at risk for TB infection who were either tuberculin skin test-negative or -positive, the specificity of the test for active TB was 89%. The negative and positive predictive values of the test were 98% and 52%, respectively. The overall accuracy of the test was 81 %. The high negative predictive value of the test in a population at risk for tuberculous infection makes it a potentially valuable screening test for active TE.

12.2.12 ICT Tuberculosis Test

The ICT tuberculosis test is a card test based on the detection of IgG antibodies directed against five M. tuberculosis secreted antigens, using an anti-human IgG labeled with colloidal gold (Rasolofo et al. 2000), a method similar to the one we employed in the antiLAM IgG dot-blot assay (Chan et al. 2000). Rasolofo and colleagues (2000) showed that the ICT test had a sensitivity of only 68.2% for smear-positive TB and 65.2% for extrapulmonary TB in a group of patients from Madagascar. The specificity was 83.3%. Overall, the ICT assay is not sufficiently predictive for clinical application. The choice of a diagnostic test depends on the pretest probability of a positive response, and therefore the prevalence of disease in the population being tested. Such factors are especially important when decisions regarding health care policy and screening of large populations are being considered. McConkey et al. (2002) illustrated this point well by testing the diagnostic yield of a rapid antibody card test in areas of high prevalence of the disease (Cairo, Egypt) and of low prevalence(St. Louis, Missouri, USA). In Egypt, the antibody test was highly sensitive (87%) for smear-positive and -negative pulmonary TB; the specificity was 82%. The sensitivity and specificity in St. Louis were 29% and 79%, respectively.

12.2.13 Dermal Response to MPB64 Antigen

Although not a serological assay, Nakamura and colleagues (1998) recently reported that the MPB64

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mycobacterial antigen delivered transdermally by the patch test method was highly accurate for diagnosing active TB. In this promising study, 52 of 53 patients (98%) with active TB showed a positive reaction to MPB64, while none of the 43 PPD-positive controls were positive. This resulted in an overall sensitivity of the test for active TB of 98.1%, a specificity of 100%, and an accuracy of 98.9%.

12.2.14 Integral Membrane Antigens of M. habana TMC 5135

Chaturvedi and Gupta (2002) evaluated the usefulness of detecting antimycobacterial antibodies to antigens that belong to the integral compartment of the plasma membrane of M. habana in serum and body fluids of patients with mainly extrapulmonary TB (only 4 of 42 patients had exclusively pulmonary TB). The presence of antibodies against M. habana integral membrane antigens (IMAs) was detected in 36 patients, for an overall positivity rate of 86% (36/42). Anti-M. tuberculosis H37RA antibodies were also found in 29 out of 36 anti-M. habana IMA antibody-positive patients.

12.2.15 Lipopolysaccharide Antigen

Meena and co-workers (2002) recently reported the isolation of an immunodominant lipopolysaccharide (LPS) antigen from M. tuberculosis H37Rv for the serodiagnosis of TB. Serum samples obtained from 59 Indian patients (19 patients with active pulmonary TB, 20 with extrapulmonary TB, and 20 with nontuberculous pulmonary disease) and 20 healthy adults were tested for LPS and three other commercially available antigen assays. The presence of IgG against LPS was highly sensitive and specific (84 and 97%, respectively), similar to the test characteristics of A60 IgG and superior to the assays for antibodies to 38 kDa or p90 antigens.

12.3 Specific Targeted Populations In certain individuals, the diagnosis of active TB is particularly challenging. Such persons include young children, the elderly, HIV-positive individuals, and patients with extrapulmonary TE. This is due to the

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greater difficulty in obtaining a sputum sample, the lower incidence of acid-fast bacilli in respiratory samples, and/or the indolent or atypical presentation of TB in such groups of people.

12.3.1 Childhood TB Although childhood TB represents a small percentage of all cases, infected children are a reservoir for many adult cases (Khan and Starke 1995). The bacteriologic diagnosis of TB is particularly difficult in young children because of the greater incidence of disseminated disease and because children often do not produce an adequate amount of sputum. Thus, serological methods to diagnose childhood TB have great potential and relevance. For children with clinical TB, the detection of IgG and IgM against A60 antigen is comparable to that seen in adults. In a study that examined the utility of detecting specific IgM, IgA, and IgG antibodies to A60 antigen in childhood TB on the Indian subcontinent, the overall sensitivity and specificity of the test with combined IgA and IgM testing were 76% and 92%, respectively (Gupta et al. 1997). In children who were acid-fast positive, the sensitivity rose to 95%. For 150 children with extrapulmonary TB, the overall sensitivity was 77%. In a study to detect IgG against an autoclaved suspension of H37Rv in 132 clinical cases of childhood TB, the sensitivity of the test in the culture-positive group (n=35) was 69% (Hussey et al. 1991). Interestingly, there was a positive correlation between the titer level and increasing age. Prior BCG did not affect the ELISA result. However, in another study that examined IgM and/or IgG against A60 antigen in primary TB or TB adenitis in children, the authors concluded that the test was not diagnostically useful (Turneer et al.1994). Levels of antiglycolipid IgG were found to be higher in children with active TB than in nontuberculous controls, leading the authors to conclude that the detection of IgG antibodies against glycolipids was useful as a complementary technique in the serodiagnosis of pulmonary TB in children (Simonneyet al. 2000).

patterns of primary infection in the elderly compared with young adults may lower the health care provider's suspicion ofTB (Morris 1989). In adults, the incidence of active TB cases is higher in the elderly (Dorken et al. 1987). Miliary TB, often presenting without localizing signs or symptoms, is also more common in the elderly person than in young adults, although the diagnosis is frequently not made until autopsy (Korzeniewska et al. 1994). Reactivation is associated with co-morbid diseases that result in waning cell-mediated immunity, disclosed by reduced skin test positivity to PPD with increasing age. For example, in men, PPD reactivity drops from 50% at age 65-74 years to 10% at age 95+, and in women from 40% to about 5%, respectively (Dorken et al. 1987). Because of the high rate of conversion in the absence of symptoms in nursing home residents, a two-step skin test is recommended for residents on admission to the nursing home: an initial test and, if negative or equivocal, a repeat test 1-2 weeks later. A positive booster effect is defined as an increase of 6 mm or more from an induration <10 mm on the first test to 10 mm with the second test. Thus, this information at baseline can help to distinguish newly acquired disease from a prior infection. The bacteriologic diagnosis of TB in the elderly is generally more problematic due to the greater difficulty in obtaining sputum and the greater incidence of disseminated disease. Unfortunately, there has not been a systematic evaluation of the serologic diagnosis of active TB in the elderly. Whether age per se impairs the immune system and thus may affect the antibody response to bacterial antigens is controversial. For example, although a decline in the adaptive immunity has been observed in the elderly, in many cases it is not clear whether the defect is primary or secondary to an underlying systemic disorder such as cancer or diabetes.An age-associated decline in humoral immunity has been observed, as evinced by a lower rate ofseroconversion and decreased antibody titer after vaccination; this effect may largely be due to loss ofT-helper cell effector function (Miller 1996). We believe a prospective study is needed to determine the accuracy of serologic assays in the diagnosis of pulmonary and extrapulmonary TB in the elderly. This issue is especially relevant in the context of nursing home-associated TB, where transmission is not an uncommon occurrence.

12.3.2 TB in the Elderly Despite the fact that the clinical presentation of TB in the elderly shares features with disease in younger people, TB in the aged frequently goes unrecognized (Fulton and McCallioin 1987). Differing radiographic

12.3.3 HIV-Positive Individuals The ability to detect specific antibodies against M. tuberculosis antigens is substantially diminished in

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HIV-positive individuals. As noted above, the sensitivity of anti-LAM IgG in HIV-positive patients was uniformly lower than in HIV-negative patients. In Ugandan patients with TB who were also HIV-positive, the sensitivity of IgG antibody (ELISA) to the 30 kDa antigen of M. tuberculosis was only 28% compared with 62% for patients who were HIV-negative (Daniel et al. 1994). An ELISA and EIA to detect the 30 kDa antigen revealed an extremely low sensitivity (l1 % and 0%, respectively) in HIV-positive patients (McDonough et al. 1992). The level of immunosuppression may also affect the antibody response as evidenced by an earlier study, conducted prior to the inclusion of TB as an AIDS-defining criteria, which showed that anti-PPD IgG was positive in 8 of 22 patients (36%) co-infected with HIV and TB but without another criteria for AIDS vs 1 of 20 patients (5%) with AIDS (Barrera et al.1992). Thus, the generally poor negative predictive value of serologic assays for TB in HIV-positive individuals makes them unreliable and not cost-effective.

12.3.4 Extrapulmonary 18 An ELISA to detect IgG to a 43 kDa antigen of M. tuberculosis in various body fluids found it to be very sensitive (-100%) and highly specific [95.7% for cerebrospinal fluid (CSF) and 98.1% for pleural and ascitic fluid] (Wadee et al.1990).An antibody response to 38 kDa antigen was noted in 73% of patients with extrapulmonary TB, comparable to the 70% sensitivity rate for smear-negative pulmonary TB (Wilkins and Ivanyi 1990). In four groups of patients with various degrees of confirmed neurologic TB (postmortem proved, culture proved, clinically suspected, and tuberculoma), antibody to the 38 kDa antigen was detected in the CSF in 60%,80%,62.5%, and 0% of the patients, respectively (Kadival et al. 1994). Detection of the 30 kDa antigen using a monoclonal antibody was done on the sera of 51 Mrican patients with clinically diagnosed tuberculous pericardial effusion, of whom 25 had confirmation by pericardial fluid cultures. The sensitivity was 61% and specificity 96% (Ng et al. 1995). Zou et al. (l994) showed that IgG against A60 antigen was present in 88.5% with reactivation TB, and 69%-86% of patients with extrapulmonary TB. Similarly, Lub and co-workers (l996) found the sensitivity rate for anti-A60 IgG to be 76% for pulmonary TB and 59% for patients with extrapulmonary TB. Compared with children with pulmonary TB, the IgG antibody response to glycolipid was significantly

lower in patients with extrapulmonary TB (Simonney et al. 2000). Gupta and co-workers (l997) found that the sensitivity of IgA and IgM directed against A60 antigen was 72.4% for patients with pulmonary TB and 76.6% for patients with extrapulmonaryTB. In Indian patients with pulmonary and extrapulmonary TB, the sensitivity rate for IgA and IgG antibody directed against A60 was 98.3% for pulmonary TB, 88% for pleural TB, and 86% for extrapulmonary TB (Gupta et al. 1995). Overall, an assay for antibody against mycobacterial antigens in either serum or body fluids in patients with extrapulmonary TB holds great potential.

12.3.5 Smear-Negative vs Smear-Positive 18 If acid-fast smear of sputum or body fluids is positive, the diagnosis of TB or atypical mycobacteria is rarely questioned. However, -50% of sputum smear in patients with bacteriologic-confirmed pulmonary TB are smear-negative. Thus, an accurate seroassay may be particularly useful in this group of patients. Charpin and colleagues (l990) specifically examined patients with smear-negative, culture-positive TB using a combined IgG and IgM seroassay for the A60 antigen and demonstrated the sensitivity to be 68%, specificity 100%, and positive predictive value 100%. We had previously shown in our immunoblot assay for anti-LAM IgG that five patients with smear-negative, culture-positive TB had a positive seroassay (Chan et al. 2000). Few studies have directly compared the sensitivity rate of TB patients who are smear-negative and culture-positive vs those who are smear- and culture-positive. As shown in Table 12.2, the sensitivity rate for smear-negative patients is either similar or lower than for smear-positive cases. The sensitivity for the MycoDot assay for LAM was especially low for both groups, likely due to the fact that over 50% of the subjects were HIV-positive (Somi et al. 1999). Unfortunately, in those studies that directly compared smear-negative vs smear-positive cases, the majority (Table 12.2) showed suboptimal sensitivity and thus sufficiently low negative predictive value to be unreliable at present in smear-negative cases.

12.4

Conclusion In summary, the appropriate setting for the use of serological tests to diagnose M. tuberculosis dis-

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Serologic Testing for Tuberculosis Table 12.2. Sensitivity of smear-negative vs smear-positive tuberculosis Antibody detection Smear-positive, Smear-negative, Reference to antigen 'X' culture-positive culture-positive Glycolipid antigen 19kDa LAM (MycoDot)" LAM (ELISA) P32 antigen of M. bovis BCG 38kDa

96% 57% 26% 88% IgA 33% IgG 52% 45-89%

97% 55% 7% 67% IgA 24% IgG 35% 16-82%

P90 Glycolipid

75% 90%

68% 70%

a 51%

Dogan and Aksu (1997) Botharnley et al. (1992) Somi et al. (1999) Sada et al. (1992) Turneer et al. (1988) Cole et al. (1996); Botharnley and Rudd (1994); Chan et al. (1990) Alifano et al. (1997) Maekura et al. (2001)

of the patients were HIV-positive

ease remains to be definitively established. Obvious advantages to serologic diagnostic tests are a greater sensitivity compared with acid-fast smear and a more rapid turnaround time than culturing. However, in our opinion, these tests at the current state of their development cannot be considered as an alternative to the conventional methods for the following reasons: • An overall concern is whether any of these tests offers any significant advantage in the clinical setting over the gold standard of cultivation, which is still necessary to obtain the drug-susceptibility pattern. Importantly, whether the application of these tests improves the patient outcome has yet to be determined. • Suboptimal sensitivity of the serologic assays for patients with smear-negative TB diminishes the utility of these tests because this is the group of patients that is considered potentially to derive the most benefit from these nonconventional tests. Similarly, the poor negative predictive value of serologic assays for TB in HIV-positive individuals should, in its current state, preclude its use in AIDS patients. • The tests are associated with relatively high costs and greater personnel training requirements, issues that are of paramount concern especially in developing countries. • The relatively low positive predictive value due to the decreased ability to distinguish between active and latent TB infection. • The difficulty serological tests have distinguishing between M. tuberculosis vs atypical mycobacteria infection.

References Aide SLM et al (1989) Evaluation of an enzyme-linked immunosorbent assay (ELISA) using an IgG antibody to Mycobacterium tuberculosis antigen 5 in the diagnosis of active tuberculosis in children. Am Rev Respir Dis 139:748-751 Alifano M et al (1997) Evaluation of IgA-mediated humoral immune response against the mycobacterial antigen P90 in diagnosis of pulmonary tuberculosis. Chest Ill: 601-605 Amicosante M et al (1995) Evaluation of a novel tuberculosis complex-specific 34 kDa protein in the serological diagnosis of tuberculosis. Eur Respir J 8:2008-2014 Anuradha S et al (2001) Serodiagnosis of extra-pulmonary tuberculosis using A-60 antigen. J Commun Dis 33:12-16 Arikan S et al (1998) Anti-Kp 90 IgA antibodies in the diagnosis of active tuberculosis. Chest 114:1253-1257 Barrera L et al (1992) Humoral response to Mycobacterium tuberculosis in patients with human immunodeficiency virus infection. Tuberc Lung Dis 73:187-191 Boggian K, Fierz W, Vernazza PL (1996) Infrequent detection of lipoarabinomannan antibodies in human immunodeficiency virus-associated mycobacterial disease. J Clin MicrobioI34:1854-1855 Bothamley GH, Rudd RM (1994) Clinical evaluation of a serological assay using a monoclonal antibody (TBn) to the 38 kDa antigen of Mycobacterium tuberculosis. Eur Respir J 7:240-246 Bothamley G et al (1992) Serodiagnostic value of the 19 kilodalton antigen of Mycobacterium tuberculosis in Indian patients. Eur J Clin Microbiol Infect Dis 11:912-915 Brooks JB et al (1987) Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatograph. J Clin MicrobioI25:1201-1206 Chan ED et al (2000) Diagnosis of tuberculosis by a visually detectable immunoassay for lipoarabinomannan. Am J Respir Crit Care Med 161:1713-1719 Chan SL et al (1990) Serodiagnosis of tuberculosis using an ELISA with antigen 5 and a hemagglutination assay with glycolipid antigens. Am Rev Respir Dis 142:385-390 Charpin D et al (1990) Value of ELISA using A60 antigen in the diagnosis of active pulmonary tuberculosis. Am Rev Respir Dis 142:380-384

196 Chaturvedi V, Gupta HP (2002) Evaluation of integral membrane antigens of Mycobacterium habana for serodiagnosis of extrapulmonary tuberculosis: association between levels of antibodies and Mycobacterium tuberculosis antigens. FEMS Immunol Med MicrobioI33:1-7 Chiang 1-H et al (1997) Serodiagnosis of tuberculosis: A study comparing three specific mycobacterial antigens. Am J Respir Crit Care Med 156:906-911 Cocito CG (1991) Properties of the mycobacterial antigen complex A60 and its applications to the diagnosis and prognosis of tuberculosis. Chest 100:1687-1693 Cole RA et al (1996) Clinical evaluation of a rapid immunochromatographic assay based on the 38 kDa antigen of Mycobacterium tuberculosis on patients with pulmonary tuberculosis in China. Tuberc Lung Dis 77:363-368 Daniel TM, Debanne SM, van der Kuyp F (1985) Enzymelinked immunosorbent assay using Mycobacterium tuberculosis antigen 5 and PPD for the serodiagnosis of tuberculosis. Chest 88:388-392 Daniel TM et al (1986) Field evaluation of enzyme-linked immunosorbent assay for the serodiagnosis of tuberculosis. Am Rev Respir Dis 134:662-665 Daniel TM et al (1994) Reduced sensitivity of tuberculosis serodiagnosis in patients with AIDS in Uganda. Tuberc Lung Dis 75:33-37 Del Prete R et al (1998) Detection of anti-lipoarabinomannan antibodies for the diagnosis of active tuberculosis. Int J Tuberc Lung Dis 2:160-63 Diagbouga S et al (1997) Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis. Clin Diag Lab ImmunoI4:334-338 Dogan UB, Aksu HSZ (1997) Serodiagnostic value of ELISA in pulmonary tuberculosis in Turkey where tuberculosis is highly prevalent. Respiration 64:73-75 Dorken E, Grzybowski S, Allen EA (1987) Significance of the tuberculin test in the elderly. Chest 92:237-40 Escamilla L et al (1996) Mycobacterium fortuitum glycolipids for the serodiagnosis of pulmonary tuberculosis. Am J Respir Crit Care Med 154:1864-1867 Fadda G et al (1992) Serodiagnosis and follow up of patients with pulmonary tuberculosis by enzyme-linked immunosorbent assay. Eur J Epidemiol 8:81-87 Franco J et al (2001) Serological response (Western blot) to fractions of Mycobacterium tuberculosis sonicate antigen in tuberculosis patients and contacts. Int J Tuberc Lung Dis 5:958-962 Fulton JD, McCallioin J (1987) Tuberculosis - diagnostic difficulty in the elderly. J Clin Exp Gerontol 9:303-11 Gupta S, Bhatia R, Datta KK (1997) Serological diagnosis of childhood tuberculosis by estimation of mycobacterial antigen 6O-specific immunoglobulins in the serum. Tuberc Lung Dis 78:21-27 Gupta S et al (1995) Diagnostic utility of the estimation of mycobacterial antigen A60 specific immunoglobulins IgM, IgA, and IgG in the sera of cases of adult human tuberculosis. Tuberc Lung Dis 76:418-24 He H et al (1991) Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trahalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen. FEMS Microbiol ImmunoI3:201-204 Hussey G, Kibei M, and Dempster W (1991) The serodiagnosis of tuberculosis in children: an evaluation of an ELISA test

C. Kotaru and E. D. Chan using IgG antibodies to M. tuberculosis, strain H37Rv. Ann Trop Paediatr 11:113-118 Imaz MS et al (2001) Evaluation of the diagnostic value of measuring IgG, IgM and IgA antibodies to the recombinant 16-kilodalton antigen of Mycobacterium tuberculosis in childhood tuberculosis. Int J Tuberc Lung Dis 5: 1036-1043 Julian E et al (1997) Detection of lipoarabinomannan antibodies in patients with newly acquired tuberculosis and patients with relapse tuberculosis. J Clin Microbiol 35: 2663-2664 Kadival GV et al (1994) Detection of antibodies to defined M. tuberculosis antigen (38 kDa) in cerebrospinal fluids of patients with tuberculous meningitis. Zentralblatt BakterioI281:95-101 Khan EA, Starke JR (1995) Diagnosis of tuberculosis in children: Increased need for better methods. Emerg Infect Dis 1:115-123 Khomenko AG et al (1996) Serodiagnosis of tuberculosis: detection of mycobacterial antibodies and antigens. Tuberc Lung Dis 77:510-515 Korzeniewska M et al (1994) Tuberculosis in young adults and the elderly. Chest 106:28-32 Lawn SD, Frimpong EH, Nyarko E (1997) Evaluation of a commercial immunodiagnostic kit incorporating lipoarabinomannan in the serodiagnosis of pulmonary tuberculosis in Ghana. Trop Med Int Health 2:978-981 Luh KT et al (1996) Tuberculosis antigen A60 serodiagnosis in tuberculous infection: application in extrapulmonary and smear-negative pulmonary tuberculosis. Respirology 1:145-151 Ma Y, Wang YM, Daniel TM (1986) Enzyme-linked immunosorbent assay using Mycobacterium tuberculosis antigen 5 for the diagnosis of pulmonary tuberculosis in China. Am Rev Respir Dis 134:1273-1275 Maekura R et al (1993) Clinical evaluation of rapid serodiagnosis of pulmonary tuberculosis by ELISA with cord factor (trehalose-6,6'-dimycolate) as antigen purified from Mycobacterium tuberculosis. Am Rev Respir Dis 148:997-1001 Maekura R et al (2001) Clinical evaluation of anti-tuberculous glycolipid immunoglobulin G antibody assay for rapid serodiagnosis of pulmonary tuberculosis. J Clin Microbiol 39:3603-3608 McConkey SJ et al (2002) Evaluation of a rapid-format antibody test and the tuberculin skin test for diagnosis of tuberculosis in two contrasting endemic settings. Int J Tuberc Lung Dis 6:246-252 McDonough JA et al (1992) Microplate and dot immunoassays for the serodiagnosis of tuberculosis. J Lab Clin Med 120: 318-322 Meena LS et al (2002) Comparative study of three different mycobacterial antigens with a novel lipopolysaccharide antigen for the serodiagnosis of tuberculosis. J Clin Lab Anal 16:151-155 Miller RA (1996) The aging immune system: primer and prospectus. Science 273:70-74 Morris CDW (1989) The radiology, haematology, and biochemistry of pulmonary tuberculosis in the aged. Q J Med 71:529-535 Nakamura RM et al (1998) MPB64 mycobacterial antigen: a new skin-test reagent through patch method for rapid diagnosis of active tuberculosis. Int J Tuberc Lung Dis 2: 541-546

Serologic Testing for Tuberculosis Ng TT, Strang JI, Wilkins EG (1995) Serodiagnosis of pericardial tuberculosis. Quart J Med 88:317-320 Niculescu D et al (1999) Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA). Roum Arch Microbiol Immunol 58: 111-119 Nsanze H et al (1997) Serodiagnosis of tuberculosis and leprosy by enzyme immunoassay. Clin Microbiol Infect 3:236-239 Rasolofo V et al (2000) Predictive values of the ICT Tuberculosis test for the routine diagnosis of tuberculosis in Madagascar. Int J Tuberc Lung Dis 4:184-185 Ratanasuwan W et al (1997) Evaluation of the MycoDot test for the diagnosis of tuberculosis in HIV seropositive and seronegative patients. Int J Tuberc Lung Dis 1:259-264 Rosen EU (1990) The diagnostic value of an enzyme-linked immunosorbent assay using adsorbed mycobacterial sonicates in children. Tubercle 71:127-130 Rovatti E et al (1996) Evaluation of a western blot serum test for the diagnosis of Mycobacterium tuberculosis infection. Eur Respir J 9:288-292 Sada E et al (1990) Evaluation of lipoarabinomannan for the serological diagnosis of tuberculosis. J Clin Microbiol 28: 2587-2590 Sada E et al (1992) Detection oflipoarabinomannan as a diagnostic test for tuberculosis. J Clin Microbiol 30:2415-2418 Simonney N, Bourrillon A, Lagrange PH (2000) Analysis of circulating immune complexes (CICs) in childhood tuberculosis: levels of specific antibodies to glycolipid antigens and relationship with serum antibodies. Int J Tuberc Lung Dis 4:152-160 Simonney N et al (1996) Comparison of A60 and three glycolipid antigens in an ELISA test for tuberculosis. Clin Microbiol Infect 2:214-222 Somi GR et al (1999) Evaluation of the MycoDot test in

197 patients with suspected tuberculosis in a field setting in Tanzania. Int J Tuberc Lung Dis 3:231-238 Steele BA, Daniel TM (1991) Evaluation of the potential role of serodiagnosis of tuberculosis in a clinic in Bolivia by decision analysis. Am Rev Respir Dis 143:713-716 Torres M et al (1994) Comparison of the immune response against Mycobacterium tuberculosis antigens between a group of patients with active pulmonary tuberculosis and healthy household contacts. Clin Exp ImmunoI96:75-78 Turneer M et al (1988) Humoral immune response in human tuberculosis: Immunoglobulins G, A, and M directed against the purified P32 protein antigen of Mycobacterium bovis bacillus Calmette-Guerin. J Clin Microbiol 26: 1714-1719 Turneer M et al (1994) Determination of humoral immunoglobulins M and G directed against mycobacterial antigen 60 failed to diagnose primary tuberculosis and mycobacterial adenitis in children. Am J Respir Crit Care Med 150: 1508-1512 Van Vooren JP et al (1988) A multiblot immunobinding assay for the serodiagnosis of tuberculosis. J Immunological Methods 113:45-49 Wadee AA, Boling L, Reddy SG (1990) Antigen capture assay for detection of a 43-kilodalton Mycobacterium tuberculosis antigen. J Clin MicrobioI28:2786-2791 Wilkins EGL, Ivanyi J (1990) Potential value of serology for diagnosis of extrapulmonary tuberculosis. Lancet 336: 641-644 Yu CJ et al (1992) Evaluation of the A-60 IgG ELISA serodiagnostic test for tuberculosis in Taiwan. J Formosan Med Assoc 91:614-619 Zou YL et al (1994) Serological analysis of pulmonary and extrapulmonary tuberculosis with enzyme-linked immunosorbent assays for anti-A60 immunoglobulins. Clin Infect Dis 19:1084-1091

13 peR and Diagnosis of Tuberculosis DIANA

L. WILLIAMS

CONTENTS 13.1 13.2 13.3 13.4 13.5 13.6

Polymerase Chain Reaction 199 PCR of M. tuberculosis 200 Drug Resistance in M. tuberculosis 202 PCR Detection of Drug Resistance 204 Impact of PCR Diagnosis on Patient Care Summary 207 References 208

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The current 'gold standard' for the identification of M. tuberculosis in clinical specimens is based on acid-fast microscopy of sputum sediments or other specimens (AFB smears), followed by culture confirmation using definitive biochemical or DNA probe analyses. Although AFB smear results can be available within 1 day, they are only 50%-70% sensitive and do not distinguish between M. tuberculosis and other nontuberculosis mycobacteria that may be present in specimens. Traditional culture-based methodologies for the detection of M. tuberculosis require between 1 and 8 weeks to perform and often have low sensitivity when small numbers of organisms are analyzed (AFB smear-negative). In addition, culture-based drug-susceptibility testing requires several additional weeks to identify drug-resistant M. tuberculosis. These factors make patient diagnosis extremely difficult, and therefore, many patients are routinely started on treatment with minimal diagnostic information. This potentially results in postponing appropriate treatment and the failure to identify rapidly those patients who need to be isolated from the general population because they are harboring drug-resistant organisms. Therefore, the rapid and specific diagnosis of tuberculosis is one of the most pressing needs in the effort to control and eventually eradicate this disease. D. 1. WILLIAMS, PhD Molecular Biology Research Department, Laboratory Research Branch, National Hansen's Disease Programs at LSU-SVM, Rm 3517W, Skip Bertman Dr., Baton Rouge, LA 70803, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Molecular approaches to aid the diagnosis of human disease have begun to have a major influence on the current clinical management of various diseases. A major goal of tuberculosis research over the last decade has been to apply molecular approaches to develop rapid, reliable procedures that can detect M. tuberculosis directly from clinical specimens and thereby avoid the many weeks required for culture amplification. The result has been the development of numerous nucleic acid-based assays, including ones based on strand displacement amplification (Walker et al. 1992), transcription-mediated amplification (Jonas et al. 1993), oligonucleotide ligation amplification (Iovannisci and Winn-Deen 1993), Q-beta replicase amplification (Shah et al. 1995), and polymerase chain reaction (PCR) (Mullis and Faloona 1987) methodologies. Among them, PCRbased assays have been tested on thousands of clinical specimens and isolates in laboratories around the world. The purpose of this chapter is to summarize the progress that has been made in the past few years in the development and characterization of PCRbased assays for the rapid diagnosis of tuberculosis and drug-resistant tuberculosis, to highlight several critical differences between PCR-based testing and conventional microbiology, and to discuss the impact that PCR analysis can potentially have on patient care and the control of tuberculosis.

13.1 Polymerase Chain Reaction PCR is a nucleic acid-based methodology, originally described in 1987, that uses short oligonucleotide primers to direct the amplification of specific segments of target DNA sequences (template) by repeated cycles of denaturation of the template DNA, primer annealing to this DNA, and primer extension (Mullis and Faloona 1987). This allows exponential multiplication of the specified template, so that beginning with as few as one copy of this sequence,

D. L. Williams

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as many as one billion copies (amplicons) can be produced within 2-4 h of specimen processing. In addition, because of the designed specificity, PCR is a powerful technique for the detection of small numbers of bacterial DNA in crude biological specimens without the necessity of culture. PCR requires: (1) specifically selected primers; (2) an appropriately prepared sample; (3) a thermostable DNA polymerase, buffer, and dNTPs; (4) appropriate positive and negative controls; (5) a thermocycler; and (6) a detection assay. The specificity of PCR lies in the selection of oligonucleotide primers used in this assay. Therefore, primers are typically selected from specific regions of the chromosome not found in other bacterial or eukaryote DNA. Once identified, primers can be synthetically manufactured and used specifically to amplify template from crude biological specimens. In addition, since the primers are incorporated in the amplicon during the PCR reaction, primers can be labeled with biotin or specific Buors for subsequent amplicon detection as discussed below.

13.2 peR of M. tuberculosis Numerous PCR-based assays have been developed for the detection of M. tuberculosis, and several of them are summarized in Table 13.1. All of these assays use specific procedures to recover DNA efficiently from low numbers of M. tuberculosis from clinical specimens and to remove potential contaminants that may inhibit PCR. Sputum specimens are typically used for the PCR detection of M. tuberculosis, but bronchial alveolar washes, skin biopsies, cerebrospinal fluid, blood, gastric lavage fluid, fecal specimens, and tissue biopsy specimens have also been used. The standard protocol for preparing sputum for PCR uses N-acetyl-L-cysteine sodium hydroxide (NALC-NaOH) treatment to digest and decontaminate specimens (Kent and Kubica 1985). The next step is the efficient lysis of the bacteria. This can be accomplished by sonication, treatment with sodium dodecyl sulfate (SDS) plus lysozyme plus heat, protease treatment, and chaeotropic salts followed by a nucleic acid purification step. This insures that the majority of potential inhibitors of the PCR are removed. However, one easy and commonly used procedure for the preparation of sputum sediments for PCR is the fixation of a portion (100-250 Ill) of these sediments in a final concentration of 70% ethanol for at least 2 h (Williams et al. 1995). This

procedure kills M. tuberculosis and preserves bacterial DNA. The fixed sediments can be stored at room temperature indefinitely or can be pelleted, resuspended in sterile H20, and placed in a boiling water bath for 10 min. These crude celllysates serve as an excellent template for PCR or can be stored at -20°C for later use. PCR cycling parameters vary primarily because of the melting temperature of the primers selected for amplification. However, a standard protocol involves an initial denaturation step of 94°C for 5 min followed by a 30-45 cycle program consisting of: 30 s at 94°C; 30 s at the optimal primer annealing temperature; and 30 s at noc, the optimal extension temperature. This program is followed by a final 5-10 min hold at n°e. Once the template is amplified, many methods are available for the detection of PCR amplicons. One of the most commonly used and inexpensive methods is the electrophoretic separation of amplicons on agarose or polyacrylamide slab gels with subsequent visualization of ampIicons in ethidium

Table 13.1. PCR-based assays for detection of M. tuberculosis in clinical specimens Assay

Target

Reference(s)

'In-house' Eisenach et aI. (1990, 1991) Brisson-Noel et aI. (1991) Kent et aI. (1995) Mulcahy et aI. (1996) DesJardin et aI. (1998) Almeda et aI. (2000) Borun et aI. (2001) Williams et aI. (1 998b) rpoB Brisson-Noel et aI. (1991) hsp65 Shinnick and Jonas (1994) MPB70 Cousins et aI. (1992) MTB40 DelPortilio et aI. (1991) 32 kDa antigen Soini et aI. (1992) 65 kDa/IS611O Yeboah-Manu et aI. (2001) (multiplex) 16S rRNA gene Bottger et aI. (1989) Boddinghaus et aI. (1990) IS6110

Commercial: Roche Amplicor 16S rRNA gene www.roche-diagnostics.com Bergmann and Woods (1996) MTB Bennedsen et aI. (1996) Dalovisio et aI. (1996) Soini et aI. (1996) Forbes (1997) Mitarai et aI. (2001) Gomez-Pastrana et aI. (2001) RocheCOBAS 16S rRNA gene www.roche-diagnostics.com Eing et aI. (1998) Amplicor MTB Bogard et aI. (2001) Oh et aI. (2001)

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Because of the large number of copies of this elebromide-stained gels using UV-light transillumination. Documentation of results can be accomplished ment in most strains (1-25) and the random inserusing a standard or Polaroid camera fitted with the tion of this element in different strains of M. tuberappropriate orange filter or by using a computerized culosis, the IS6110 PCR assay has been modified to image acquisition and analysis instrument, such as a produce a DNA fingerprint of a strain (Thierry et al. GelDoc 2000 (BioRad Laboratories, Hercules, CA). 1990; Cave et al.1991). This analysis and others based To increase the sensitivity of amplicon detection, on repeated insertion elements in the genome of M. amplicons can be transferred from gels to nitrocellu- tuberculosis have been used to type clinical isolates lose or nylon membranes using a simple capillary dif- for epidemiological studies (Thierry et al.1990; Cave fusion-based nucleic acid transfer technique or South- et al. 1991; Plikaytis et al. 1993; van Embden et al. ern blotting (Southern 1975). PCR products can then 1993; van Soolingen et al. 1994; Otal et al. 1997). be visualized using one of many hybridization-based Other 'in-house' PCR assays target many different detection techniques with radiolabeled, chemilumines- genes and repeated sequences within the M. tubercucent, or fluorescent-labeled probes. Some protocols use losis genome (Table 13.1). Even though these assays reverse hybridization of amplicons to probes bound on rely on the amplification of different targets, they nitrocellulose filters (de Beenhouwer et al. 1995) or to all have similar specimen preparation and specificthe wells of microtiter plates (Amplicor MTB, Roche ity for smear-positive specimens. However, because Molecular Systems, Emery, CA, USA). In addition, one the majority of these assays are based on single copy commercially available assay provides amplification genes, they are not as sensitive as the IS6110 assay. and detection in a semiautomated system (COBAS In addition to these M. tuberculosis-specific PCR Amplicor MTB, Roche Molecular Systems). assays, another 'in-house' PCR assay has been develThe majority of the assays developed for the detec- oped which identifies the presence of M. tuberculosis tion of M. tuberculosis are referred to as 'in-house' or other mycobacteria in clinical specimens. This PCRs because they have been developed and charac- assay uses mycobacterial genus-specific primers terized by individual laboratories. While these assays which flank a hypervariable region of the 16S rRNA offer the potential for diagnosing tuberculosis with gene in which species-specific differences have been a high degree of sensitivity and specificity in a few characterized (Bottger et al. 1989). The PCR amplihours, only a few have been successfully transferred cons are then subjected to DNA sequencing to disto the clinical laboratory. The IS611 0 'in-house' PCR criminate between mycobacterial species (Bottger et assay, originally developed by Eisenach et al. in 1990, al. 1989; Boddinghaus et al. 1990). This analysis has targets the multicopy IS611 0 insertion element of M. proven to be a rapid and reliable method for the identuberculosis. This assay, or variations of it, have been tification of mycobacteria in the clinical laboratory successfully used in many clinical laboratories to (Kirschner et al. 1993). detect M. tuberculosis (Table 13.1). Presently, there are only a few commercially availWhen the IS6110 PCR assay was evaluated for its able nucleic acid amplification assays for the direct ability to detect M. tuberculosis in sputum specimens detection of tuberculosis from clinical specimens routinely processed in several clinical laboratories, the (Table 13.1). An example of one of these assays is the following conclusions were made: (1) this PCR was Amplicor MTB assay, which is based on PCR amplieasy to fit into the daily routine of a clinicallabora- fication of the 16S rRNA gene and a colorimetric tory with a turnaround time of 24-36 h from time of detection system. This assay is recommended for the specimen collection; (2) existing laboratory personnel detection of M. tuberculosis in liquefied, decontamicould perform these assays; and (3) false-positives, due nated, and concentrated human respiratory specito amplicon contamination of specimens, were rare mens, including expectorated and induced sputum, when personnel strictly adhered to specific laboratory bronchial washings, and bronchial alveolar lavages procedures (Clarridge et al. 1993; Shalwar et al. 1993; (www.roche-diagnostics.com). Nolte et al. 1993). In addition, when the results of this Clinical trials to evaluate the reliability of this study were compared with culture-based detection for assay for the diagnosis of pulmonary tuberculosis the same specimens, there was 100% specificity for M. have concluded that this assay has excellent sensitivtuberculosis and 95% sensitivity with smear-positive, ity, specificity, and positive and negative predictive culture-positive specimens. However, the PCR-based values for smear-positive, culture-positive specimens assay had a lower sensitivity for smear-negative, cul- when compared with other 'in-house' assays (Bergture-positive specimens, thus being of lesser value for mann and Woods 1996; Eing et al. 1998). One such the early detection of disease development. trial evaluated 956 respiratory specimens from 502

202

patients and compared the results of the Amplicor MTB assay with those of culture. For 135 specimens that were culture-positive for mycobacteria, the sensitivity, specificity, and positive and negative predictive values of the Amplicor MTB assay were 79.4%,99.6%, 92.6%, and 98.6%, respectively. For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of Amplicor MTB were 97.6%, 100%, 100%, and 90.9%, respectively. For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values were 40.0%,99.5%,69.2%, and 98.7%, respectively. In another study, the sensitivity was ~95% and specificity 100% in smear-positive specimens (Woods 2001). Another commercially available PCR assay is the Roche COBAS Amplicor MTB assay. This assay is a semiautomated version of the manually performed Roche Amplicor MTB test (www.rochediagnostics.com). When compared with culture and an IS611O-based 'in-house' PCR protocol, the sensitivity, specificity, and positive and negative predictive values for this assay were 66.33%, 99.71 %, 94.36%, and 97.66%, respectively (Eing et al. 1998). The corresponding values for the 'in-house' PCR were 91.08%, 99.85%, 97.87%, and 99.37%, respectively. Another study showed similar results, giving an overall specificity of 99.7% and sensitivities ranging from 85.7% to 90.9% (Bogard et al. 2001). Therefore, it appears that these commercially available assays have good sensitivity, specificity, and predictive values for the detection of M. tuberculosis in smear-positive and culture-positive specimens (EI-Hajj et al. 2001). However, many laboratories, particularly in developing countries, continue to use methods developed 'in house'. This is primarily due to a lack of funding required for the expensive reagents and equipment used to perform these assays. In this environment, great care should be taken in the validation of these 'in-house' assays and in maintaining proper quality control. In summary, many PCR-based assays have been developed for the detection ofM. tuberculosis directly from clinical specimens. Several of these assays have been shown to be extremely sensitive for the direct detection of M. tuberculosis from clinical specimens.

13.3 Drug Resistance in M. tuberculosis Treating tuberculosis with combinations of effective antibiotics is a proven method for limiting the

D. L. Williams

emergence and spread of existing antibiotic-resistant strains of M. tuberculosis. In most areas of the world, current short-course multidrug therapy formulated for tuberculosis (MDT-TB) includes a 2-month daily combination of isoniazid (INH), rifampin (RMP), and pyrazinamide (PZA) and either streptomycin (STM) or ethambutol (EMB) followed by daily therapy with INH and one of the other primary drugs for the next 4 months (Stratton and Reed 1986). However, because of the length of MDT-TB, patient noncompliance often occurs, particularly when direct observation of therapy is not instituted (Frieden et al. 1993; Bradford et al. 1996). Since drug-resistant strains often emerge during intermittent or inadequate chemotherapy (Frieden et al. 1993; Bradford et al. 1996), clusters of disease caused by drug-resistant strains are still reported globally (Cohn et al. 1997; Moore et al. 1997). Therefore, there is a great need for rapid drug-susceptibility testing to monitor the evolution of drug resistance and track the spread of multidrug-resistant tuberculosis (MDR-TB). Conventional drug-susceptibility testing for tuberculosis relies on the cumbersome and time-consuming culture-based assays which can take up to 2 months to obtain results (Kent and Kubica 1985; Heifets 1991). Accordingly, drug-susceptibility testing is not a routine component of tuberculosis control programs in many parts of the world. This limits the ability to monitor the evolution of drug resistance and to establish the success of a treatment strategy like MDT-TB. Recently, substantial progress has been made in our understanding of the molecular basis of M. tuberculosis drug resistance as summarized by Musser (1995), Blanchard (1996), Ramaswamy and Musser (1998) and Cockerill (1999). Drug resistance in M. tuberculosis is primarily attributed to chromosomal mutations in genes encoding drug targets. These mutations occur spontaneously as a result of errors in DNA replication. For example, the frequency of drug-resistant mutants in a population of M. tuberculosis is estimated at 10-6 for INHR mutants and 10-7 for RMp R mutants. These frequencies suggest that one organism in a population of 106 is resistant to INH and one organism in 107 is resistant to RMP. Since it has been estimated that some untreated, smear-positive tuberculosis patients can harbor >109 viable M. tuberculosis/gram of lung tissue, the population of M. tuberculosis in such a patient could contain thousands of drug-resistant organisms. Inappropriate therapy for patients with such large bacterial loads selects for these subpopulations of drugresistant bacteria, resulting in the development of drug-resistant tuberculosis and leading to the spread of one or more resistant phenotypes.

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Isoniazid. INH is an effective, synthetic bactericidal agent for M. tuberculosis that requires the bacterial catalase peroxidase to convert it to its active form. Resistance to INH in the majority of isolates correlates with changes in the catalase enzyme as a result of missense mutations, deletions, or insertions within the katG which encodes the catalase of M. tuberculosis (Zhang et al. 1992; Musser 1995). In addition, INH and ethionamide (ETH), a structural analogue of INH, interfere with new cell wall synthesis in M. tuberculosis (Winder et al. 1971). Resistance to these drugs also develops as a result of defined mutations within the inhA locus encoding the enoyl-ACP reductase and the 3-ketoacyl-ACP reductase involved with mycolic acid biosynthesis (Banerjee et al. 1994; Musser et al. 1996; Ramaswamy and Musser 1998). Additional mechanisms have been described for the development of INH resistance in M. tuberculosis, but all targets and modes of action of this drug in M. tuberculosis are still unknown (Table 13.2).

Rifampin. RMP [3,4-(methylpiperazinyl-iminomethylidene-rifamycin)J, a semisynthetic derivative of rifamycin, has been a key bactericidal component in most antituberculosis chemotherapeutic regimens since the early 1970s. RMP appears to exert its potent

bactericidal activity by binding to the DNA-dependent RNA polymerase complex and uncoupling transcription (Winder 1982). The target for RMP in M. tuberculosis is the ~-subunit of the DNA-dependent RNA polymerase encoded by rpoB (Telenti et al. 1993a; Williams et al. 1994, 1998a). Resistance to RMP in virtually all resistant isolates of M. tuberculosis correlates with changes in the structure of the ~-subunit of the DNA-dependent RNA polymerase as a result of missense mutations, small deletions, and insertions within an 81-base pair region of the rpoB referred to as the RMP resistance-determining region (RRDR) (Telenti et al. 1993a; Williams et al. 1994; Musser 1995; Ramaswamy and Musser 1998). The resultant ~-subunit is still functional, but it is refractory to the effects of RMP or requires much higher levels of the drug to produce bacterial cell death (Williams et al. 1998a).

Pyrazinamide. PZA is a structural analogue of nicotinamide that is used as a first-line tuberculosis drug. The mode of action of this drug is not understood, but it is thought that PZA is converted to the bioactive form of the drug by pyrazinamidase, an enzyme produced by M. tuberculosis (Konno et al. 1967). PZAR isolates lose some or all pyrazinamidase

Table 13.2. PCR-based assays for detection of drug-resistant M. tuberculosis Reference(s)

Assay

Drug

Target

'In-house': PCR-DNA sequencing

INH

EMB FQs RMP INH RMP

Marttila et aI. (1996); Musser et aI. (1996) Musser et aI. (1996) Sreevatsan et aI. (1997c) Telenti et aI. (1993b); Kapur et aI. (1994) Williams et aI. (1994); Nachamkin et aI. (1997) Rossau et aI. (1997); Bartfai et aI. (2001) pncA Sreevatsan et aI. (1997a) Sreevatsan et aI. (1 997b) embB Takiff et aI. (1994) gyrA rrs, rpls Sreevatsan et aI. (1996); Nachamkin et al. (1997) katG Heym et aI. (1995); Rouse et aI. (1995) Victor et aI. (1996); Telenti et aI. (1993a, 1997b) Temesgen et aI. (1997) inhA Rouse et aI. (1995); Telenti et aI. (1997) Telenti et aI. (1997a) ahpC rpoB Telenti et al. (1993a,b, 1997a,b) Felmlee et aI. (1995); Whelen et aI. (1995) Selvakumar et aI. (1997); Kim et aI. (2001) embB Sreevatsan et aI. (1 997b) gyrA Takiff et aI. (1994); Delgado and Telenti (1996) rpoB Williams et aI. (1996, 1998b); Thomas et aI. (2001) katG Piatek et aI. (2000) rpoB Piatek et aI. (1998,2000); EI-Hajj et aI. (2001)

RMP

rpoB

RMP

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katG inhA ahpC rpoB

de Beenhouwer et aI. (1995); Rossau et aI. (1997) Bartfai et aI. (2001)

204

activity (Butler and Kilburn 1983). This occurs as a result of defined missense mutations within pncA which encodes this enzyme (Sreevatsan et al. 1997a; Ramaswamy and Musser 1998; Cockerill 1999).

Streptomycin. STR is an aminocyclitol glycoside antibiotic that appears to function by binding to the 16S rRNA of M. tuberculosis to inhibit translational initiation and detrimentally affects translation fidelity, which results in protein production termination (Douglass and Steyn 1993; Finken et al. 1993; Ramaswamy and Musser 1998; Cockerill 1999). Specific mutations within the 16S rRNA gene (rrs) and rpsi gene encoding the ribosomal protein S12 in M. tuberculosis are responsible for the development of resistance to this drug (Douglass and Steyn 1993; Finken et al. 1993; Sreevatsan et al. 1996; Ramaswamy and Musser 1998; Cockerill 1999). However, not all resistant isolates have mutations in these genes. Ethambutol. EMB ([ S,S']- 2,2'-[ethylenedimino]di-lbutanol) is a bactericidal drug that targets M. tuberculosis cell wall synthesis by inhibiting incorporation of mycolic acids into the cell wall (Takayama et al. 1979; Takayama and Kilburn 1989). This appears to be a function of three genes organized into an operon designated as embCAB (Telenti et al.1997b). Mutations in a single codon of emb are associated with the development of EMB resistance in M. tuberculosis (Telenti et al. 1997; Ramaswamy and Musser 1998; Cockerill 1999). Fluoroquinolones. Ciprofloxacin, ofloxacin, and levofloxacin are fluorinated derivatives of quinolones (FQs) (Hooper and Wolfson 1989) used primarily as second-line drugs in the treatment of drug-resistant tuberculosis (Cambau et al. 1994; Sullivan et al. 1995; Takiff et al.1994; Ramaswamy and Musser 1998). The target for this drug class is the DNA gyrase, an enzyme that catalyzes negative supercoiling of DNA (Hooper and Wolfson 1989). Specific missense mutations within the quinolone resistance-determining region (QRDR) of the gyrA, encoding the GyrA subunit of the gyrase, are responsible for the development of resistance in M. tuberculosis (Cambau et al. 1994; Sullivan et al. 1995; Takiff et al. 1994; Ramaswamy and Musser 1998).

D. L. Williams

has been used to develop a variety of rapid, PCR-based assays for the detection of drug-resistant M. tuberculosis directly from clinical specimens (Table 13.2). These assays are based on the specific genotypic identification of mutations associated with the drug-resistant phenotype within amplicons derived from drug target genes. Some of these assays include: direct PCR-DNA sequencing, PCR single-strand conformation polymorphism (SSCP), PCR heteroduplex formation analysis (PCR-HDA), PCR line probe assay (LiPA), and molecular beacon sequence analysis (Table 13.2).All of these assays can be performed on sputum specimens or clinical isolates within 24 h of sample acquisition, are highly specific and sensitive, and, therefore, have the capability to significantly reduce the time needed for determining drug resistance.

PCR-DNA Sequencing. Direct DNA sequencing of PCR amplicons is the most definitive method to detect mutations associated with drug resistance. This analysis can be performed in a well-equipped diagnostic laboratory and requires approximately 1-2 days to obtain results from clinical specimens. As a result, numerous PCR-DNA sequencing assays have been developed to identify M. tuberculosis (Kapur et al. 1995; Kirschner et al. 1993) and the presence of drug-resistant mutant strains (Table 13.2). These assays are based on PCR amplification of the respective target gene directly from crude celllysates of processed sputum specimens using oligonucleotide primers which flank these regions of the chromosome of M. tuberculosis, purification of the amplicons using commercial reagents, such as QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, www.qiagen.com). and dideoxy DNA sequencing of the amplicons using either manual or automated DNA sequencing strategies. Figure 13.1 shows representative DNA electropherograms of a small fragment of the RRDR of RMp s and RMp R strains of M. tuberculosis obtained by automated DNA sequencing. In the resistant strain, a single missense mutation (TCG-"7TTG) in codon 531 is identified. This results in a substitution of a serine amino acid residue with a leucine residue in the ~-subunit of the RNA polymerase of this mutant and results in the development of highlevel RMP resistance (MIC >64 Ilg/ml) (Williams et al. 1998a).

13.4

PCR-SSCP. PCR single-strand conformation polymor-

Knowledge of the specific mechanisms that result in the development of antituberculosis drug resistance

phism analysis assays have been developed and used to detect drug-resistant mutants of M. tuberculosis directly from patient specimens or clinical isolates (Telenti et al. 1993a,b; Kim et al. 2001) (Table 13.2).

peR Detection of Drug Resistance

205

PCR and Diagnosis of Tuberculosis

This is accomplished using the same basic approach taken to produce PCR arnplicons for DNA sequencing for gene targets. These double-stranded amplicons are then dissociated into single strands by heat denaturation and separated very slowly by gel electrophoresis under stringently controlled temperature conditions. Gels are stained to observe ssDNA fragment mobility patterns called SSCP profiles. A schematic representation of the SSCP assay is shown in Fig. 13.2. DNA strands from a RMp R strain demonstrate a unique mobility pattern compared with RMPS, reflecting the change in nucleotide composition. Numerous studies have used this analysis to study drug resistance in M. tuberculosis (Table 13.2).

affect the mobility of the resultant DNA fragments (Fig. 13.3). Therefore, when the heteroduplexes are separated by gel electrophoresis on polyacrylamide gels and stained with ethidium bromide, unique heteroduplex profiles are observed for susceptible and resistant genotypes (Williams et al. 1998b). The PCR-UHG requires approximately 6 h to complete and uses precast minigels and a nonradioactive detection format.

RRDR rpoB PCR amplieon RMP,Strain

111111111111111 j --u'--

PCR-HDA. PCR heteroduplex assay is another gel electrophoresis-based assay which has been developed to detect simultaneously the presence of M. tuberculosis and its susceptibility to RMP directly from clinical isolates (Williams et al.1994). This assay has been modified to reduce the time taken and technical complexity and can be used directly on clinical specimens with the use of a universal heteroduplex generator (UHG) (Williams et al. 1996, 1998a). The UHG contains several base pair mismatches, insertions, and deletions within the RRDR region of M. tuberculosis rpoB (Williams et al. 1996). When the UHG is heat-denatured and slowly annealed to these denatured amplicons, it provides enhanced mutation detection. Enhanced mutation detection occurs because large areas of unmatched nucleotides (bubbles) in the newly formed duplexes greatly

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Fig. 13.2. Schematic representation of the PCR single-strand conformation polymorphism (PCR-SSCP) assay for detection of rifampin susceptibility of M. tuberculosis

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Fig. 13.3. Schematic representation of the PCR universal heteroduplex generator (PCR-UHG-Rif) assay for detection of rifampin susceptibility of M. tuberculosis

206

D. L. Williams

PCR-LiPA. The line probe assay is a PeR-based reverse hybridization assay that has been developed for the rapid detection of RMP susceptibility of M. tuberculosis (de Beenhouwer et al. 1995; Rossau et al. 1997). LiPA uses PCR to amplify the RRDR region of rpoB and, at the same time, label the fragment with biotin. Denatured PCR amplicons are hybridized with a set of DNA probes immobilized on a membrane strip at specific sites (Fig. 13.4). The immobilized probes are small DNA fragments that are either homologous with short segments of the RRDR of rpoB from a RMp s strain or contain mutations in the RRDR associated with RMp R M. tuberculosis mutants. The stringency of the hybridization reaction is designed so that the single-stranded PCR amplicons will bind only to probes with 100% sequence homology. The resultant hybrids are detected by immunoenzymatic staining, and the results are read visually. An example of LiPA for RMP susceptibility testing is shown in Fig. 13.4. The genotype of the test organism is determined by which lines test positive. Therefore, the presence or absence of a RMp R strain of M. tuberculosis can be determined from the pattern obtained. This assay is commercially available in certain areas of the world (Rossau et al. 1997).

probes contain homologous sequences to either the susceptible or resistant genotype. In addition, they contain a fluorophore on one end and a molecule that quenches fluorescence when it is in close proximity to a fluorophore on the other end. When no PCR amplicons are present, the probe remains in its hairpin shape, and the fluorescence is quenched. However, after these probes bind to their intended targets, they undergo a conformational change that restores thefiuorescence of the internally quenched fluorophore. Therefore, the more amplicons produced, the more fluorescence. The specificity for drug resistance detection is in the short sequence that binds to the amplicon. In summary, there are several PCR-based mutational detection assays to identify drug-resistant mutants of M. tuberculosis. These assays can be performed within days of specimen acquisition or isolation of strains from specimens and, therefore, can be useful for the early detection of drug-resistant tuberculosis. Early detection of drug-resistant tuberculosis can potentially improve patient care and reduce the transmission of drug-resistant tuberculosis by effective isolation of infected individuals.

Molecular Beacons. PCR molecular beacon sequence

13.5 Impact of PCR Diagnosis on Patient Care

analysis has been developed to detect mutations in the rpoB that are associated with RMP resistance (Piatek et al. 1998,2000; EI-Hajj et al. 2001) and INH resistance (Piatek et al. 2000) in M. tuberculosis. Molecular beacons are small hairpin-shaped DNA probes that report the presence of specific nucleic acids (Fig. 13.5). These

BiotioylatedRRDR rpoD PCR Amplicoo RMP.

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Because of the specificity of PCR assays for the detection of M. tuberculosis, a smear-positive, PCR-positive specimen would indicate that the acid-fast bacilli in the sputum of a particular patient are M. tuber-

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Fig. 13.4. Schematic representation of the PCR line probe assay (PCR-LiPA) for detection of rifampin susceptibility of M. tuberculosis

207

peR and Diagnosis of Tuberculosis

~+ Qumched Targd DNA Molecular Beaooo (ss AqJIicoo)

Fig. 13.5. Schematic representation of molecular beacon sequence analysis. The black circle represent the quenching molecule. The open circle represents the fluorophore

developed to monitor drug therapy (DesJardin et al. 1996; Hellyer et al. 1999a) and has been shown to be very useful for rapidly determining the efficacy of therapeutic regimens directly from sputum specimens (DesJardin et al.1996; Hellyer et al. 1999a,b).

13.6

Summary

culosis (Kaul 2001). Therefore, the patient should be appropriately retained in isolation and treated until their sputum becomes smear-negative. A PCR-negative specimen would indicate the presence of a nontuberculosis mycobacterial infection, and the patient could be released from isolation and treated more appropriately. In addition, because of the availability of PCR-based assays for the detection of mutations associated with antituberculosis drug resistance, a smear-positive, PCR-resistant specimen would indicate that this patient is also harboring drug-resistant tuberculosis. Therefore, the patient should be appropriately retained in isolation and treated with additional drug therapy until their sputum becomes smear-negative. The immediate impact on patient care, the reduction of transmission of tuberculosis, and cost of providing care is very apparent. Because of the high specificity of these assays, they cannot identify the presence of other mycobacteria in mixed infections. It is, therefore, recommended that PCR should be augmented by culture to determine if other nontuberculosis mycobacteria are present in specimens. This is especially important in areas of the world where HIV infection is highly endemic and other nontuberculosis mycobacteria present problems for the differential diagnosis of tuberculosis. Recently, newer multiplex PCR assays have been developed which can be used for the detection of M. tuberculosis complex and nontuberculous mycobacteria. These assays are currently being characterized for the identification of mixed infections (Ahmed 1999; Yeboah-Manu et al. 2001). Most M. tuberculosis PCR assays are based on the detection of DNA or ribosomal RNA targets. Although beneficial for the initial diagnosis, such assays have proven unsuitable for rapidly monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after the conversion of smears and cultures to negative (DesJardin et al. 1998; Hellyer et al. 1999a). It has recently been ascertained that the presence of mRNA is a good indicator for determining the viability of M. tuberculosis from sputum specimens. Using mRNA, an assay has been

Methodologies developed over 30 years ago for the detection of tuberculosis and drug susceptibility testing are cumbersome and require protracted periods of time to obtain results. The resurgence of tuberculosis and drug-resistant tuberculosis has promoted the development of a number of new options for the rapid laboratory diagnosis of M. tuberculosis. One of the most promising and exciting methodologies has been the introduction of assays employing PCR amplification technology to detect M. tuberculosis directly from clinical specimens. PCR-based assays for the diagnosis of tuberculosis and identification of drug-resistant strains are configured to yield results in a few hours to days because these assays can detect the presence of M. tuberculosis and/or mutations present in its genome that are associated with the development of drug resistance directly from crude biological specimens. This represents a major step forward in the ability to rapidly identify tuberculosis patients and patients harboring drug-resistant tuberculosis. Therefore, the implementation of these assays has the potential to improve patient care and provide substantial savings in the overall cost of patient care compared with conventional smear, culture, and speciation alone. In addition, since rapid detection allows for the rapid isolation of tuberculosis patients, the nosocomial or environmental spread of tuberculosis and drug-resistant-tuberculosis to susceptible individuals will most likely be reduced. However, the major drawbacks for the implementation of many of these assays in clinical laboratories in developing countries are the cost of the reagents used and the need for expensive equipment and trained individuals to perform the analysis. This review has attempted to describe many of the current molecular aspects of the detection of tuberculosis using PCR, the modes of action, the resistance mechanisms of the major drugs used to treat tuberculosis, and the current molecular assays for detecting drug resistance in M. tuberculosis. Although amplification assays hold significant promise to improve the laboratory diagnosis of tuberculosis,

208

the decision to perform these assays is complicated because most assays have decreased sensitivity with specimens that are AFB smear-negative. In addition, many other factors, such as cost, assay performance, and technical difficulty must be considered in order to facilitate the decision-making process about whether an amplification assay would be appropriate in a particular laboratory setting. As nucleic acid-based tests evolve, it is anticipated that they will become cheaper and simpler to perform. This is extremely important in developing countries, where resources are limited, and tuberculosis is highly endemic and often complicated by HIV infection. Therefore, research continues to be conducted to automate and simplify sample preparation, amplification, and amplicon detection procedures, thus making them easier to implement in clinical laboratories within both developed and developing countries. This will assist tuberculosis control programs with their overall goals of providing better patient care, reducing the transmission of tuberculosis, and providing the capability to monitor trends in drug resistance at the regional and local levels.

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losis by automated DNA sequencing. Arch Pathol Lab Med 119:131-138 Kent L et al (1996) Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp.? J Clin Microbiol 33:2290-2293 Kent P, Kubica G (1985) Public health mycobacteriology: a guide for the Level III laboratory. US Department of Health and Human Services, Public Heath Service, CDC, Atlanta, pp 31-56 Kim BJ et al (2001) Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked singlestrand conformation polymorphism and DNA sequencing. J Clin MicrobioI39:2610-2617 Kirschner P et al (1993) Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31:2882-2889 Konno K, Feldmann FM, McDermott W (l967) Pyrazinamide susceptibility and amidase activity of tubercle bacilli. Am Rev Respir Dis 95:461-469 Marttila HJ et al (1996) katG mutations in isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Finnish patients. Antimicrob Agents Chemother 40:2187-2189 Mitarai S et al (2001) Potential use of Amplicor PCR kit in diagnosing pulmonary tuberculosis from gastric aspirate. J Microbiol Methods 47:339-344 Moore M et al (1997) Trends in drug-resistant tuberculosis in the United States. J Am Med Assoc 278:833-837 Mulcahy GM et al (1996) IS611O-based PCR methods for the detection of Mycobacterium tuberculosis. J Clin Microbiol 34:1348-1349 Mullis KB, Faloona FA (l987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335-350 Musser JM (l995) Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 8:496-514 Musser JM et al (1996) Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis 173: 196-202 Nachamkin I, Kang C, Weinstein MP (l997) Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of Mycobacterium tuberculosis by molecular methods. Clin Infect Dis 24:894-900 Nolte FS et al (1993) Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. J Clin MicrobioI31:1777-1782 Oh EJ et al (2001) Improved detection and differentiation of mycobacteria with combination of Mycobacterium Growth Indicator Tube and Roche COBAS AMPLICORiZ> System in conjunction with Duplex PCR. J Microbiol Methods 46: 29-36 Otal I et al (1997) Use of a PCR method based on IS611 0 polymorphism for typing Mycobacterium tuberculosis strains from BACTEC cultures. J Clin Microbiol 35:273-277 Piatek AS et al (1998) Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat BiotechnoI16:359-363 Piatek AS et al (2000) Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular

210 beacons: implications for rapid susceptibility testing. Antimicrob Agents Chemother 44:103-110 Plikaytis BB et al (1993) Rapid, amplification-based fingerprinting of Mycobacterium tuberculosis. J Gen Microbiol 139:1537-1542 Poa CC et al (1990) Direct detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin MicrobioI28:1877-1880 Ramaswamy S, Musser JM (1998) Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuberc Lung Dis 79:3-29 Rossau R et al (1997) Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrob Agents Chemother 41:2093-2098 Rouse DA et al (1995) Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 39: 2472-2477 Selvakumar N et al (1997) Single strand conformation polymorphism profiles with biotinylated PCR products to detect mutation in rpoB gene of Mycobacterium tuberculosis. Curr Sci 73:774-777 Shalwar R et al (1993) Detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin MicrobioI29:712-717 Shah J et al (1995) Q-beta replicase-amplified assay for detection of Mycobacterium tuberculosis directly from clinical specimens. J Clin MicrobioI33:1435-1441 Shinnick TM, Jonas V (1994) Molecular approaches to the diagnosis of tuberculosis. In: Bloom B (ed) Tuberculosis: pathogenesis, protection and control. American Society for Microbiology Press, Washington DC, pp 517-530 Soini H et al (1992) Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. J Clin MicrobioI30:2025-2028 Soini H et al (1996) Comparison of AMPLICOR® and 32-kilodalton PCR for detection of Mycobacterium tuberculosis from sputum specimens. J Clin MicrobioI34:1829-1830 Sougakoff W et al (1997) Nonradioactive single-strand conformation polymorphism analysis for detection of fluoroquinolone resistance in mycobacteria. Eur J Clin Microbiol Infect Dis 16:395-398 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bioi 98:503-517 Sreevatsan S et al (1996) Characterization of rpsL and rrs mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from diverse geographical localities. Antimicrob Agents Chemother 40:1024-1 026 Sreevatsan S et al (1997a) Mutations associated by pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms. Antimicrob Agents Chemother 41:636-640 Sreevatsan S et al (1997b) Ethambutol resistance in mycobacterium tuberculosis: critical role of embB mutations. Antimicrob Agents Chemother 41:1677-1681 Sreevatsan S et al. (1997c) Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Chemother 41:600-606 Stratton MA, Reed MT (1986) Short-course drug therapy for tuberculosis. Clin Pharm 5:977-987

D. 1. Williams Sullivan EA et al (1995) Emergence of fluoroquinolone-resistant tuberculosis in New York City. Lancet 345:1148-1150 Takayama K, Kilburn JO (1989) Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 33:1493-1499 Takayama K et al (1979) Inhibition by ethambutol of mycolic acid transfer into the cell wall of Mycobacterium smegmatis. Antimicrob Agents Chemother 16:240-242 Takiff HE et al (1994) Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 38:773-780 Telenti A et al (1993a) Detection of rifampin-resistance mutations in Mycobacterium tuberculosis. Lancet 341:647-650 Telenti Aet al (1993b) Direct, automated detection of rifampinresistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother 37:2054-2058 Telenti A et al (1997a) Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J Clin Microbiol 35:719-723 Telenti A et al (1997b) The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat Med 3:567-570 Temesgen Z et al (1997) Use of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis to detect a point mutation in the catalase-peroxidase gene (katG) of Mycobacterium tuberculosis. Mol Cell Probes 11 :59-63 Thierry D et al (1990) IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 18:188 Thomas GA, Williams DL, Soper SA (2001) Capillary electrophoresis-based heteroduplex analysis with a universal heteroduplex generator for detection of point mutations associated with rifampin resistance in tuberculosis. Clin Chern 47:1195-1203 van Embden JD et al (1993) Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31: 406-409 van Soolingen D et al (1994) DNA fingerprinting of Mycobacterium tuberculosis. Methods EnzymoI235:196-205 Victor TC et al (1996) katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis are not infrequent. Antimicrob Agents Chemother 40:1572 Walker GT et al (1992) Strand displacement amplification-an isothermal, in vitro DNA amplification technique. Nucleic Acids Res 20:1691-1696 Whelen A et al (1995) Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR. J Clin Microbiol 33:556-561 Williams DL et al (1994) Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob Agents Chemother 38:2380-2386 Williams DL, Gillis TP, Dupree WG (1995) Ethanol fixation of sputum sediments for DNA-based detection of Mycobacterium tuberculosis. J Clin MicrobioI33:1558-1561 Williams DL et al (1996) PCR-heteroduplex detection of rifampin-resistant Mycobacterium tuberculosis. In: Persing D (ed) PCR protocols for emerging infectious diseases. ASM Press, Washington DC, pp 122-129

PCR and Diagnosis of Tuberculosis Williams DL et al (l998a) Contribution of rpoR mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 42:1853-1857 Williams DL et al (l998b) Evaluation of a polymerase chain reaction-based universal heteroduplex generator assay for direct detection of rifampin-susceptibility of Mycobacterium tuberculosis directly from sputum specimens. Clin Infect Dis 26:446-450 Winder FG, Collins PB, Whelan D (1971) Effects of ethionamide and isoxyl on mycolic acid synthesis in Mycobacterium tuberculosis BCG. J Gen MicrobioI66:379-380 Winder FG (1982) Mode of action of the antimycobacterial

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agents and associated aspects of the molecular biology of the mycobacteria. In: Ratledge C, Stanford J (eds) The biology of the mycobacteria, vol 1. Academic Press, London, pp 353-438 Woods GL (2001) Molecular techniques in mycobacterial detection. Arch Pathol Lab Med 125:122-126 Yeboah-Manu D, Yates MD, Wilson SM (2001) Application of a simple multiplex PCR to aid in routine work of the mycobacterium reference laboratory. J Clin MicrobioI39:4166-4168 Zhang Y et al (1992) The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358:591-593

14 Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients GORGUN AKPEK

CONTENTS 14.1

Introduction

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14.1.1 Mycobacterium Tuberculosis 214 14.2 14.2.1 14.2.2 14.2.3 14.2.4 14.2.5 14.2.6 14.2.7 14.3

14.3.1

14.3.2 14.3.3 14.3.4 14.3.5 14.3.6 14.3.7 14.3.8 14.3.9 14.4 14.4.1 14.4.2 14.4.3 14.5

Hematologic Findings in Tuberculosis 215 Anemia 216 White Blood Cell Abnormalities 216 Thrombocytopenia 216 Pancytopenia 216 Possible Mechanisms of Hematologic Findings in Tuberculosis 217 Prognostic Significance of Hematologic Abnormalities. 219 Tuberculosis-Induced Lymphopenia and Immunosuppression 219 Hematologic Abnormalities Associated with Mycobacterial Infections in Imrnunocompromised Hosts 220 Hematologic Complications of HIV Infection and AIDS 220 Mycobacterium avium Complex in HIV-Infected Patients 220 Anemia Associated with MAC in HIV-Infected Patients 220 Other Cytopenias Associated with MAC in HIV-Infected Patients 222 MAC Infections in Other Immunocompromised Hosts 222 Hemophagocytic Syndrome Associated with MAC 223 Miliary/Disseminated Tuberculosis in AIDS 223 Diagnostic Yield of Bone Marrow Aspiration/Biopsy for Mycobacterial Infections in Patients with HIV Infection 224 Other Mycobacterial Infections Associated with Hematologic Disorders 226 Coagulation Abnormalities Associated with Mycobacterial Infections and Clinical Presentations 226 Disseminated Intravascular Coagulation 226 Deep Vein Thrombosis Associated with Tuberculosis 226 Thrombotic Thrombocytopenic Purpura Associated with Mycobacterial Infections 227 Conclusion 227 References 227

G.AKPEK,MD Greenebaum Cancer Center at University of Maryland School of Medicine, 22 South Greene Street, Baltimore MD 21201, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

14.1 Introduction The relationship between hematologic abnormalities and mycobacterial infections dates back to the beginning of the 20th century. Flinn and his colleagues suggested at that time that the change in hematologic parameters is the earliest finding of tuberculosis (TB) (Flinn and Finn 1929). The authors said,'In connection with the above studies we have formed the impression that an extension of the pathologic process in a tuberculosis lung usually manifests itself first in the blood picture, later by physical signs and x-ray, and often last of all by symptoms: Before embarking upon a general discussion of hematologic manifestations of mycobacterial infections, I would like to present two cases that were previously discussed at the Medical Staff Conference in the University of San Francisco, California, USA, in 1967. These cases have certain features of the history and clinical findings of TB, in particular, the striking difference in the hematological manifestations between the two cases. A brief summary about the demographics and clinical presentation of Mycobacterium TB infection is included thereafter.

Case 1. The first patient is a 50-year-old white man who came in for evaluation of a fever of about 7 months' duration. His symptoms began with multiple episodes of severe pain in the left upper quadrant of the abdomen and nausea. These symptoms were followed in a few days by fever and generalized malaise. According to the patient's description, the febrile episodes would last for approximately 1 week and recurred at approximately 4-week intervals with temperatures in the range of 38.3°C-40.6°C (lOI°F105°F). He was noted to have axillary and inguinal adenopathy 2 months later when seen by a physician because of persistent symptoms. Laboratory data included hemoglobin of 12.2 gm/dl, markedly elevated erythrocyte sedimentation rate, increased concentration of serum gammaglobulin, and a positive tuberculin test. Two months later, the patient underwent surgery for the repair of an incarcer-

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ated incisional hernia. An exploratory laparotomy performed at that time revealed no intraabdominal pathology. Two months later, the patient's fever recurred and was associated with night sweats and 13 pounds (6 kg) of weight loss since the onset of his symptoms. At that time no history could be obtained of cough, pleuritic chest pain, hemoptysis, or production of sputum. The family history was negative. The patient had been a heavy smoker for 20 years. On physical examination, the only abnormalities were slight adenopathy in the inguinal and axillary regions, hepatomegaly, and questionable splenomegaly. Body temperature was 38.3°C (101°F). Hemoglobin content was 8.7 g/dl, WBC of 3400/mm3, with 30% neutrophils, 65% lymphocytes, 3% monocytes, and 2% basophils. The platelet count was 301,000/mm 3, with reticulocytes forming 2.1 % of the total. Additional hematologic data included normal haptoglobulin, leukocyte alkaline phosphatase, coagulation parameters, and serum iron and iron-binding capacity. The bone marrow at that time showed generalized hyperplasia, predominantly of the erythroid series. Skin test was positive with PPD. A chest X-ray showed fibrocalcific densities. There was no active pulmonary disease, but there was evidence of old granulomatous disease. There were granulomas in the right upper lung field and some scattered granulomas in the right mid-lung field. All other work-up for fever of unknown origin was negative. A scalene lymph node biopsy showed multiple confluent and epithelioid caseous granulomas. The patient improved on an anti-TB regimen. Cultures of the scalene lymph node specimen grew acid-fast organisms.

Case 2. The second patient is a 62-year-old man with a chief complaint of tenderness in the left upper quadrant of the abdomen, with weight loss and weakness of 5 months' duration. A mass in the left upper quadrant was noted on routine physical examination. Four months later, the patient first noticed anorexia, weight loss, and weakness. Three months later, he developed anemia and was admitted to the hospital. The patient has had regular chest X-rays taken every 6 months for the past 2 years. These films have shown stable apical infiltrates. He was also known to have a positive PPD skin test. He had been a heavy smoker for the past 45 years; he has had chronic nonproductive cough and a history of acute upper gastrointestinal bleeding, which responded to medical management. On physical examination, the patient was pale and cachectic. Body temperature was 38°C (101 OF). He had several small lymphadenopathies. Lung expansion was decreased bilaterally, and the anteroposterior diam-

G.Akpek

eter of the chest was increased. There was dullness and increased fremitus on the right. Peristaltic movement was visible beneath a thin abdominal wall. The spleen, which was large and rock-hard, extended 8 cm below the left costal margin, and there was a tender nodule, 2x3 cm, at the anterior margin. The liver edge was felt 4 cm below the right costal margin. It was not tender and firm. Early clubbing of the fingers and severe muscular wasting of the extremities were noted. Leukocyte numbered 16,000/mm3 , with 60% neutrophils, many immature myeloid cells, 10% basophils, and 10% eosinophils. Hematocrit was 19%, reticulocyte count 2.5% of the total, and platelet count 80,000/mm 3• Normoblasts were present in the peripheral blood. Xray examination revealed dense bones. Patchy apical infiltrates, which had increased since the previous examination, were noted on chest X-ray. There was a large right pleural effusion. A skin test with PPD was positive. Thoracentesis and pleural biopsy were performed. The latter revealed caseating granulomas. No intrabronchiallesions were seen at bronchoscopy. A bone biopsy was performed since three previous marrow taps had been dry. The bone biopsy revealed sclerotic bone and myelofibrosis. Triple anti-TB therapy was begun. Subsequently, cultures of both sputum and pleural fluid grew acid-fast bacilli.

14.1.1

Mycobacterium Tuberculosis Currently, more than one-third of the world's population is infected with Mycobacterium TB: 8 million new cases and approximately 2 million deaths are reported each year (Dye et al. 1999). Although the lung is the primary site of disease in 80%-84% of cases of TB in the USA (Center for Disease Control and Prevention 2000), extrapulmonary TB has become more common with the advent of HIV infection, and the risk of TB increases as the immunosuppression progresses (Moore et al.1997; Jones et al.1993; Iseman 2000). The most common extrapulmonary sites of disease are the lymph nodes, pleura, and bones or joints (Center for Disease Control and Prevention 2000). Lymphatic TB is usually seen in children and young adults; more commonly in women (especially Asian and Indian women). It presents with unilateral, painless, cervical adenopathy, which is connected to the skin by sinus tracts late in the course of disease. Excisional biopsy with culture yields the diagnosis of TB lymphadenitis. PPD is usually positive. It may respond slowly to medication and rarely may require excision (Mandel et al. 2000).

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

Clinical presentations ofTB have changed dramatically since the introduction of anti-TB agents. In the pre-antibiotic era, late generalized TB was often the primary disease, occurring mainly in young adults and frequently associated with pulmonary symptoms. In the antibiotic era, TB commonly occurs together with and is frequently obscured by other diseases. It often afflicts the elderly and is much less frequently accompanied by pulmonary symptoms (Slavin et al. 1980). Disseminated TB involving the lung, liver, spleen, bone marrow, and lymph nodes can occur occasionally in an immunocompetent host and is usually associated with hematologic abnormalities and poor outcome (Charfi et al.1998). Splenomegaly associated with fever is another clinical sign of disseminated TB (O'Reilly 1998). The histologic appearance in this rare form of disseminated hematogenous TB (nonreactive TB) shows nonspecific necrosis containing disintegrating polymorphonuclear leukocytes and enormous numbers of tubercle bacilli (O'Brien 1954). In a typical case, granulomas and epithelioid cells are lacking, although intermediate cases have areas more typical for T8. The gross pathologic findings are minute soft abscesses up to 1 cm, which always involve the liver and spleen, usually the marrow, commonly the lungs and kidneys, but almost never the meninges. The clinical picture may be overwhelming sepsis, with splenomegaly and often an inconspicuous diffuse mottling on the chest X-ray. Major hematologic abnormalities are common. Whether the lesions are typical or not, it is necessary to have bacteriologic confirmation of TB. Near these lesions, the bone marrow cellularity is often raised or lowered, reticular fibers are often increased, and sometimes reticulin fibrosis is marked (Tulliez 1976). In the following sections, common types of mycobacterial infections that are associated with various hematologic findings are discussed.

14.2 Hematologic Findings in Tuberculosis While most patients with TB do not manifest major hematologic abnormalities, some patients with late generalized or chronic hematogenous TB and most with nonreactive TB have serious hematologic abnormalities, including leukopenia, thrombocytopenia, anemia, leukemoid reactions, myelofibrosis, and polycythemia (Cameron 1974). Leukemoid reactions may suggest acute leukemia, although most patients in whom hematogenous TB coexists with the clinical picture ofleukemia have both diseases. Slavin and his

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colleagues reviewed the medical records of a community-based university teaching hospital over a lO-year period (1978 to 1987) to determine the clinical and laboratory characteristics, diagnostic methods, and prognostic variables in adults treated for miliary TB in the rifampicin era. They identified a total of 109 patients including 12 who did not have miliary nodules on the chest X-ray (all of whom were shown to have hematogenous dissemination). Hematologic abnormalities were common: leukopenia (less than 4xlO(9)/I) was present in 16 of 107 patients (15%), thrombocytopenia (less than 150x10(9)/L) in 24 of 104 (23%), and lymphopenia (less than 1.5X10(9)/L) in 82 of 94 (87%). Pancytopenia was found in 6 patients, 3 of whom recovered. Disseminated intravascular coagulation occurred in 4 patients, all of whom died (Slavin et al. 1980). In a recent study, investigators from India compared peripheral blood and bone marrow findings in patients with disseminated/miliary TB (DTB/MTB) as well as pulmonary TB (PTB) (Singh et al. 2001). They also assessed the effect of anti-TB therapy on the occurrence of hematologic abnormalities. Thirty-two patients with DTB/MTB and 23 patients with PTB were prospectively studied. All patients received standard anti-TB treatment. They were subjected to a hemogram including a peripheral blood examination, which was repeated on completion of the anti-TB therapy. Bone marrow aspiration and biopsy were also done in all patients before starting the treatment. Normocytic normochromic anemia was the most common abnormality observed in all groups and subgroups (DTB/MTB 84%, PTB 86%). Other hematological abnormalities of the white blood cells include leukopenia (DTB/MTB 25%, PTB 0%; p<0.02), neutropenia (DTB/MTB 22%, PTB 0%; p<0.04), lymphocytopenia, monocytopenia, leukocytosis, neutrophilia, lymphocytosis, and monocytosis. Pancytopenia was observed only in patients with DTB/MTB (p<0.05). Thrombocytopenia was more common in patients with DTB/MTB (p<0.007), whereas thrombocytosis was more common in patients with PTB (p
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G.Akpek

14.2.1 Anemia

even be the presenting feature of the latter form (Perez et al. 1998; Ghobrial and Albornoz 2001). When thrombocytopenia occurs in TB, it does so Severe anemia was quite rare in a large series of most commonly via nonimmunologic mechanisms, patients with pulmonary TB, occurring in less typically manifesting in the context of pancytopenia than 20%. It is, however, quite common and occurs that develops secondary to granulomatous infiltrain 60%-80% of patients with disseminated TB. tion of the bone marrow. However, thrombocytopeTherefore, severe anemia is the rule in advanced TB nia and possibly other hematologic complications (Mandell 2000). Normocytic, normochromic anemia associated with TB can be immune-mediated. In a (hemoglobin<10 gm/dl) and unexplained fever recent case report, a 49-year-old man who presented should raise the suspicion of disseminated TB. with immune thrombocytopenic purpura (ITP) and disseminated TB was described. In that particular case, the hematological and infectious abnormali14.2.2 ties, which did not respond to high-dose intravenous White Blood Cell Abnormalities corticosteroids and immunoglobulin, resolved after anti-TB treatment (Ghobrial and Albornoz 2001). An Leukopenia, leukocytosis, and lymphocytosis are immune basis for TB-induced ITP was also supported relatively uncommon in TB. The white blood cell by the presence of either platelet antigen-specific count is usually normal but may be between 10,000 antibodies or platelet surface membrane IgG in two and 15,000 cells/mm3 in miliary TB (Mandell 2000). studies (Jurak et al. 1983; Boots et al. 1992). In the Monocytosis is seen in less than 10% of patients. previous report, thrombocytopenia associated with The presence or absence of monocytosis used to be TB developing concurrently in a mother and son considered a prognostic marker for the clinical course was described. Antiplatelet antibodies were demonand outcome of patients with tuberculosis (Medical strated in the serum of both mother and son (Jurak Staff Conference 1967). A rising lymphocyte count et al. 1983). Thrombocytopenia and hyponatremia and falling monocyte count could be signs of a poor associated with miliary TB have also been described prognosis, and a rising monocyte count a sign of a (Cockcroft et al. 1976). good prognosis. However, this concept has never been There are other case reports in which patients with proved. Eosinophilia occurs in about 15% of patients disseminated TB presented with clinical and laboraand is more common in pulmonary TB than in extra- tory features of ITP (Talbot et al. 1998; Hernandezpulmonary TB. Maraver et al. 1996; al-Majed et al. 1995; Yasuda et al. Leukemoid reactions are extremely rare, occur- 1994; Pavithran and Vijayalakeshmi 1993; Singh et al. ring in less than 3% of patients with disseminated 1986; Chia and Machin 1979; Levy and Cooper 1964). TB. The peripheral blood findings and the clinical The majority of reported patients with TB and ITP features may be exactly the same as those of chronic were women of Middle Eastern and Asian descent. myelogenous leukemia or, rarely, acute myeloblastic Although TB-related ITP was distributed across all leukemia. Tuberculous leukemoid reactions and leu- age groups, it occurred most commonly between kemia may be differentiated by cytogenetic studies, the 3rd and 8th decades of life. Pulmonary TB repwhich in the case ofleukemia will reveal an abnormal resented the most common clinical presentation, karyotype pattern. In patients in whom TB is associ- having occurred in 33% of patients, followed by disated with the hematologic picture of acute leukemia, seminated TB and lymphadenitis at 19% each. Some it is likely that both diseases are present and that TB 81 % and 35% of patients presented with a platelet is complicating acute leukemia. The documented count less than 20x109II and 5X109/l, respectively. instances of TB producing the entire disease picture The highest platelet counts at presentation were seen of acute leukemia are extremely rare. in 3 of the 4 patients with TB lymphadenitis.

14.2.3 Thrombocytopenia

14.2.4 Pancytopenia

Thrombocytopenia is very rare in pulmonary TB. However, it is a relatively common feature of disseminated or miliary TB. Thrombocytopenia can

TB may be associated with pancytopenia and aplastic anemia. Disseminated TB should be considered when pancytopenia is associated with fever and weight loss

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

or as a cause of other obscure hematologic disorders. Medd and Hayhoe described a disease called TB miliary necrosis with pancytopenia (Medd and Hayhoe 1955). This disorder had rather distinctive pathologic features with widespread miliary necrosis and very little tissue reaction. Clinical symptoms include a high, spiking fever which is called the 'typhoidal variant' of TB. Leukopenia and splenomegaly are present. This is a rare disease, but it is important to be aware of its existence and to differentiate it from aplastic anemia of other causes. The clue to the diagnosis is splenomegaly, which is not consistent with typical aplastic anemia. Pancytopenia can be due to hemophagocytic syndrome, an unusual hematologic manifestation of TB (Basu et al. 2000). TB-associated hemophagocytic syndrome was also reported in a hemodialysis patient who presented with fever of unknown origin, anorexia, weight loss, hepatomegaly, and pancytopenia (Yang et al. 1996). Bone marrow biopsy revealed marked hemophagocytosis and granuloma formation with positive staining for acid-fast bacilli. The patient expired shortly after making the diagnosis. Despite its high mortality, disseminated TB in association with pancytopenia may occasionally respond to anti-TB chemotherapy with bacteriologic cure of TB and concomitant resolution of the pancytopenia. Disseminated intravascular coagulopathy can be accompanied by pancytopenia in some of these patients (Rosenberg and Rumans 1978).

14.2.5 Possible Mechanisms of Hematologic Findings in Tuberculosis There are five possible explanations for hematologic abnormalities seen during the course of TB: 1. The first possibility is that the association of a blood disorder and TB may be purely coincidental. 2. The second possibility is that the blood disorder favors the development of TB, although this is relatively rare. TB is one of the infections commonly found in patients with hematologic disorders, particularly in those whose immunologic defense mechanisms have been further compromised by chemotherapy. 3. The third explanation is that treatment of blood disorders results in increased susceptibility to TB. Patients are treated with corticosteroids and alkylating agents, which may suppress the normal immune responses and impair the host defense. In patients with hematologic disorders, cellular

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immunity may be suppressed by the disease itself or by treatment with corticosteroids, immunosuppressants, or anticancer agents. Miliary TB developing in such compromised hosts is cryptic, and thus its diagnosis is difficult to make. Missing the diagnosis of miliary TB is a growing problem and demands special attention. In the years 1964 to 1974,37 patients with active TB were misdiagnosed in the Chaim Sheba Medical Center. The diagnosis was made only after death. Twentyone patients were over 60 years old. Eleven had hematological disorders (Rosenthal et al. 1975). Miliary TB is fatal if it cannot be detected in time. Therefore, the possibility of mycobacterial infection should always be kept in mind when treating patients with hematologic disorders. Consequently, early suspicion and diagnosis of systemic TB may be important. Unexplained pyrexia is the most common symptom of systemic TB (Uetake et al. 1990). Because of the poor prognosis of patients with mycobacterial infection and primary hematologic disorders, prophylactic antiTB medication may be considered when treating patients with hematologic disorders in areas endemic for TB (Morii and Narita 1998). 4. The fourth possibility is that TB is associated with the blood disease. The most common hematologic abnormality that is associated with TB is cytopenia. There are three possible ways in which TB could cause a reduction in circulating blood cells: a. As with any chronic infection, there may be a suppression of normal hematopoiesis. Chronic infections are the major causes of anemia of chronic disease. In association with the latter there is usually low serum iron and iron-binding capacity. The plasma iron turnover rate is rapid, but there is poor utilization of iron for erythropoiesis. It is likely that suppression of erythropoiesis in TB is partly due to increased cytokine release (TNF-a, tumor necrosis factoralpha) that is the major mechanism in anemia of chronic disease (Dallalio et al. 1999). b. TB can produce hypersplenism, and this might be a second cause of cytopenia. It essentially involves increased sequestration because of the large spleen. c. There is actual replacement of the marrow with tubercles and the associated fibrotic reaction. Occasionally, TB may produce changes in the blood that are difficult to distinguish from the underlying hematologic disease. In several large series of patients with TB, approximately 8%-10%

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were found to have associated primary blood disorders (Oswald 1963; Glasser et al.1970). Corr and his colleagues, in reviewing 364 patients with all types of TB, found that 17% had anemia, 3% had leukocytosis, and 1% had leukopenia. They found only one patient with immature granulocytes in the blood, a patient with metastatic carcinoma. They also described 6 patients who had primary hematologic disorders with superimposed TB (Corr et al. 1964). In the old days, patients who had leukemic blasts in the blood and bone marrow were given antiTB drugs, and all of them died during the initial acute illness without improvement of the hematologic picture. But since autopsy failed to reveal widespread leukemic infiltrates, leukemia was not believed to be present, and the blasts in the blood were considered to be a response to TB. However, subsequent epidemiologic studies failed to prove this concept. Some of these findings are undoubtedly secondary to the TB. In some cases, however, there is an underlying hematologic disease such as leukemia and aplastic anemia. Glasser et al. in their review suggest that anemia, leukopenia, monocytosis, and granulocytic leukocytosis certainly represent hematologic responses to TB, for they disappear following successful treatment with anti-TB drugs. But the 'leukemic' blood picture or pancytopenia does not resolve, and these patients almost invariably die after very brief illnesses (Glasser et al. 1970). Based on these observations, patients with TB who have blast cells in the peripheral blood and bone marrow also have leukemia and, therefore, merit consideration of anti-leukemic therapy in addition to treatment of TB. Similarly, there is no strong evidence that TB causes a reversible pancytopenia. Such patients, even with anti-TB therapy, invariably die in a relatively short time. However, several case reports suggest that treatment of TB rarely restores the blood picture to normal. This is important in the prognosis of these diseases. For example, the diagnosis of refractory anemia with excess blasts, a subtype of myelodysplastic syndrome, was made based upon morphological and cytogenetic criteria (del 20q) in a patient with TB lymphadenitis. Hematological parameters remained stable without any specific treatment for several months, cell counts even normalized under anti-TB therapy (Ugo et al. 1996). In another case report, large granular lymphocytosis associated with pulmonary TB was described. Anti-TB therapy was started, and

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soon after the clinical symptoms and pancytopenia improved, and 1 year later, the hematological abnormalities had disappeared (Otsuji et al. 1990). TB may occur in the setting of all myeloproliferative disorders. More than 100 patients with pulmonary TB and polycythemia vera have been reported. The association was sufficiently frequent that in the old days, the physicians considered that the lipids of the tubercle bacilli might have erythropoietic activity. Of all the myeloproliferative syndromes, it is myelofibrosis and myeloid metaplasia that have the most intriguing association with TB. There are a number of reasonably documented cases in which the myeloid metaplasia followed the onset of TB. The second case presented at the beginning of this chapter is an example of this. However, the mechanism of this association between the two diseases remained speculative. Karel Pelger described an abnormality of granulocyte nuclear segmentation in the context of advanced TB. It is now recognized that the Pelger-Huet nuclear anomaly (PHNA) can be either hereditary or acquired with systemic diseases, commonly hematologic dysplasias. A male patient presenting with cachexia, high fever, severe hypoproliferative anemia, and acquired PHNA was described. At autopsy, an overwhelming TB was discovered in the absence of any other underlying disease (Shenkenberg et al. 1982). Based on the above observations, the following guidelines in the approach to the association of TB and hematological disorders can be used. First, that the possibility of TB exists in any case of myelofibrosis and myeloid metaplasia, aplastic anemia, or atypical or poorly characterized blood disorder. In such a setting, one must make a rigorous search for acid-fast organisms by cultures of gastric aspirates, sputum, BAL, bone marrow, blood, and urine. In addition, the physicians may be forced to consider biopsy of an enlarged lymph node or, failing this, a 'blind' biopsy of the scalenus anticus node. This last procedure yielded the diagnosis in the first case presented at the beginning of this chapter. Molecular diagnostic methods such as polymerase chain reaction analysis should be utilized to diagnose TB in clinically suspicious cases when the standard methods fail to identify TB. 5. Finally, anti-TB drugs can cause hematologic abnormalities. These drugs may all individually or collectively produce pancytopenia or decrease individual blood elements. Anti-TB medications can cause hematologic side-effects, and the inci-

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

dence of hematologic abnormality appears to be associated with the degree of immunosuppression (Kavesh et al. 1989). Leukopenia is the most common side-effect of anti-TB drugs. Anti-TB drugs may cause idiosyncratic reactions, malabsorption, interference with iron metabolism, and hemolysis in patients with red blood cell enzyme deficiencies. Idiosyncratic reactions manifested by depression of any or all of the three cellular blood elements may be caused by any of the anti-TB drugs. Para-aminosalicylic acid (PAS) and streptomycin are the drugs most often responsible (Whitfield 1970). In patients who have red blood cells deficient in enzymes such as glucose 6-phosphate dehydrogenase, oxidizing agents such as PAS may cause hemolytic anemia (Whitfield 1970). Evidence of small-bowel intestinal malabsorption was found in 36% of 14 patients studied who were taking PAS for TB (Akhtar et al. 1968). In another study of 68 patients taking PAS, approximately 90% had subnormal blood folic acid levels, in contrast to 35% of a control group of similar patients (Roberts et al. 1966). Both serum and stainable bone marrow iron levels are commonly elevated in patients taking isoniazid (INH) for 6 months. Sideroblastic anemia has also been reported in patients receiving INH therapy. This is likely to be related to interference with pyridoxine metabolism by INH (Whitfield 1970). Hematologic abnormalities developed in 4 of 814 patients (0.5%) with pulmonary TB who were treated for 9 months with INH and rifampicin, daily for 1 month and twice weekly for the other 8 months, between January 1976 and June 1981. Given the infrequency of hematologic side-effects, only clinical surveillance for toxicity was recommended (Dutt et al. 1983) for patients receiving anti-TB medication. Perhaps a complete blood count should be added to the liver function studies that are evaluated during the course of TB therapy.

14.2.6 Prognostic Significance of Hematologic Abnormalities Some of the hematologic manifestations of TB are predictive for the prognosis. In a recent study, the extent and severity of hematological abnormalities in 380 patients with pulmonary TB were evaluated. Full blood count, bone marrow aspiration smears, and bone marrow biopsy were performed. There was a

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close correlation between the hematologic abnormalities and the severity of clinical findings of pulmonary TB. This survey revealed that hematologic abnormalities are relatively common in severe pulmonary TB. Body weight loss, white blood cell count, hemoglobin level, and erythrocyte sedimentation rate appeared to be useful indices for the severity of TB. The return of these indices to a normal level may indicate disease control correlating with sputum conversion to acidfast bacilli negative status (Bozoky et al. 1997). In a retrospective cohort study, 26 patients (24%) died of miliary TB a median of 6 days after starting treatment. Survivors were followed up for a median of 51 weeks. Stepwise logistic regression identified age greater than 60 years, lymphopenia, thrombocytopenia, hypoalbuminemia, elevated transaminase levels, and treatment delay as independent predictors of mortality (Maartens et al. 1990).

14.2.7 Tuberculosis-Induced Lymphopenia and Immunosuppression TB is clearly responsible in some patients for CD4 cell lymphocytopenia that reverses with treatment (Onwubalili et al. 1987; Turett and Telzak 1994). Many HIV-negative patients with active TB have CD4 cell counts much lower than 500 cells/I, which return toward normal with treatment of TB. Recent clinical data on 85 HIV-negative patients with TB indicated that there is a close relationship between the CD4+ T-cell count and the severity of TB. The disease severity was associated with greater depression of the total lymphocyte and CD4 cell counts. CD4 cell counts returned to normal levels in most patients after 1 month of therapy (Jones et al. 1997). In TB, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani TB patients to both mycobacterial and candidal antigens were suppressed by approximately 50% compared with healthy purified protein derivative (PPD)-positive household contacts. Production of interferon-gamma (IFN-y) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30 kDa alpha antigen of MTB (30 kDa antigen) induced greater secretion of transforming growth factor-beta (TGF-~), but not of interleukin 10 (IL-lO) or TNF-a, by PBMCs from TB patients compared with healthy contacts. The degree of suppression correlated with the duration of treatment:

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patients treated for less than 1 month had significantly lower T-cell blastogenesis and IFN-y production and higher levels of TGF-~ than did patients treated for longer than 1 month. Neutralizing antibody to TGF-~ normalized the lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production ofIFN-yin TB patients but not in contacts. Neutralizing antibody to IL-lO augmented, but did not normalize, T-cell responses to both PPD and Candida in TB patients and candidal antigen in contacts. TGF-~, produced in response to MTB antigens, may therefore playa prominent role in down-regulating potentially protective host effector mechanisms and can be an important mediator of immunosuppression in TB (Hirsch et al. 1996).

14.3 Hematologic Abnormalities Associated with Mycobacterial Infections in Immunocompromised Hosts 14.3.1 Hematologic Complications of HIV Infection and AIDS

The hematologic manifestations of HIV infection and AIDS are common and may cause symptoms that are life-threatening and impair the quality of life of these patients. These manifestations include morphologic abnormalities of peripheral blood and bone marrow changes (Aboulafia and Mitsuyasu 1991). Cytopenias are the most common hematologic findings in HIV patients. Anemia and neutropenia are generally caused by inadequate production because ofsuppression ofthe bone marrow by the HIV infection through abnormal cytokine expression and alteration of the bone marrow microenvironment. Thrombocytopenia is caused by immune-mediated destruction of the platelets in addition to inadequate platelet production. The incidence and severity of cytopenia are generally correlated to the stage of the HIV infection. Other causes of cytopenia in these patients include adverse effects of drug therapy, the secondary effects of opportunistic infections, especially mycobacterial infections, malignancies or other preexisting or coexisting medical problems that may be prevalent in the HIV-infected population. Diagnosis of

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effects of various infectious pathogens or malignancies. Infectious agents such as Mycobacterium avium complex (MAC) or TB may cause direct bone marrow suppression or reticuloendothelial system dysfunction, resulting in depressed blood cell counts (Castella et al. 1985; O'Hara 1989).

14.3.2

Mycobacter;um av;um Complex in HIV-Infected Patients Disseminated MAC infections are common in patients with HIVI AIDS, occurring in up to 50% of patients (Ellner et al.1991; Nightingale et al.1992). In contrast to the immunocompetent host, in which MAC disease is usually limited to the lungs, bacteremia is by far the most common syndrome in patients with AIDS. Frequently, there is widespread dissemination, often to the liver, bone marrow, lymph nodes (Fig. 14.1), and spleen. In one study, lymphadenopathy, hepatosplenomegaly, and severe anemia «8.5 g/dl) were seen in 37%, 24%, and 85% of patients evaluated, respectively (Benson and Ellner 1993). MAC infection is one of the most common complications seen during the course of HIV infection and is associated with significant suppression of blood counts (Castella et al. 1985). Bone marrow aspiration and biopsy may be useful in making a specific diagnosis of several complications of AIDS that result in cytopenia. In histologic sections of bone marrow, the diagnosis of MAC infection is suggested by the finding of granulomas and aggregates of foamy histiocytes (Figs. 14.1b and 14.2a,b) in which the organisms can be seen by acid-fast staining (Fig. 14.2c). Granuloma formation is frequently lacking or scarce (Fig. 14.2b), so the pathologist should be alerted that disseminated MAC is suspected to ensure that acidfast staining is performed (French et al. 1997).

14.3.3 Anemia Associated with MAC in HIV-Infected Patients Severe anemia (hematocrit <26%) has been reported to be present in 76% of patients with disseminated MAC disease (Horsburgh et al. 1994). The presence of anemia has been correlated with the presence of

the mechanism and cause of the cytopenia may allow

disseminated MAC disease in HIV-infected patients

for specific management (Coyle 1997). In evaluating HIV-infected patients with abnormal blood counts, one must consider the hematologic

who have absolute CD4+ lymphocyte counts below 100lml (Havlick et al. 1992). The survival of patients with MAC disease was negatively correlated to the

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

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a

a

b

b

c Fig.14.la-c. Lymph node histology in an HIV-infected patient with co-existing MAC infection. a Poorly formed granulomas (-100). b Poorly formed granulomas composed of numerous foamy histiocytes (-400). c AFB stain shows numerous intracellular acid-fast mycobacteria (M. avium intracellulare) (-1000). Courtesy of Metin Ozdemirli, MD, PhD, Assistant Professor in Hematopathology, Georgetown University, Washington, DC, USA

Fig.14.2a-c. Bone marrow histology in an HIV-infected patient with co-existing MAC infection. a Numerous, well formed granulomas (-100). b A few paratrabecular poorly formed granulomas (-100). c AFB stains show scattered acid-fast mycobacteria (M. avium intracellulare) (-1000). Courtesy of Metin Ozdemirli, MD, PhD, Assistant Professor in Hematopathology, Georgetown University, Washington, DC, USA

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presence of anemia (Horsburgh et al. 1994). The presence of anemia, fever, and weight loss in patients with advanced immunodeficiency should prompt an evaluation for MAC disease (Coyle 1997). The anemia in MAC disease is thought to be due to high levels of inflammatory cytokines as well as a direct disturbance of the bone marrow microenvironment by mycobacteria (Coyle 1997). Serum TNF levels were demonstrated to be markedly elevated in children with anemia and MAC diseases. In a recent study, the hematologic manifestations of MAC in 37 HIV-infected infants and children were reviewed. Anemia was the predominant feature in all patients, with severe anemia (hemoglobin <6 g/dl) occurring in 7 of 34 (21 %) patients. This was followed by leukopenia (79%), monocytosis (82%), thrombocytopenia (59%), leukoerythroblastic changes (68%), and neutropenia (41 %). Serum TNF-a was markedly elevated in all patients with MAC, and there appeared to be an association between elevated TNF-a and anemia in these patients (Ellaurie and Rubinstein 1995). MAC infection may disrupt erythropoiesis in HIV patients by direct invasion of the bone marrow. Patients with disseminated MAC appear to have greater impairment of erythropoiesis than those without MAC. This impairment appears to be mediated by selective suppression of hematopoietic progenitors (Gascon et al. 1993) and production of inhibitory TNF-a (Ellaurie and Rubinstein 1995). To elucidate the mechanisms of anemia and other cytopenias in HIV patients, various cytokine and cytokine receptor concentrations were measured by ELISA in bone marrow aspirate supernatants from 19 HIV patients undergoing diagnostic evaluation and 14 healthy paid volunteer controls. IL-l ~ and IFN-y were rarely detectable. All cytokines/ receptors detectable in marrow supernatant, except RANTES, showed mean concentrations 1.6- to 6.2-fold higher in patients with HIV compared with healthy controls. Elevated TNF-a and MIP-l ~ was associated with marrow involvement by lymphoma, Hodgkin disease, or mycobacterial infection. Concentrations of all cytokines/receptors measured correlated with the severity of the anemia. CD8+ lymphocytes were inversely correlated with concentrations of all cytokines measured other than MIP-la (Dallalio et al.1999).

14.3.4 Other Cytopenias Associated with MAC in HIV-Infected Patients

Bone marrow infiltration with MAC may cause thrombocytopenia or increase the severity of exist-

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ing HIV-associated thrombocytopenia. However, thrombocytopenia due to bone marrow infiltration is not nearly as common as anemia of MAC infection (Coyle 1997). The absence or presence of megakaryocytes on bone marrow examination is often useful in distinguishing thrombocytopenia caused by decreased production due to marrow infiltration of mycobacteria from thrombocytopenia caused by increased destruction during the course of HIV infection or due to drug side-effect (Coyle 1997). Bone marrow infiltration with disseminated MAC infection may cause neutropenia by suppression of bone marrow progenitors (Coyle 1997).

14.3.5 MAC Infections in Other Immunocompromised Hosts

Infection with atypical mycobacteria occurs mainly in patients with a compromised cellular immune system, in particular in those with a defective Tcell or monocyte function. The specific immune response of an adolescent HIV-negative patient with disseminated M. avium infection and fatal varicella zoster virus infection was studied in a patient who presented with dysplastic hematopoesis of all cell lineages and anemia plus leukopenia. No hematologic malignancy was found. Peripheral lymphopenia and monocytopenia as well as a lack of natural killer (NK)-cells and B-cells were noted. Lymphocytes consisted of T-cells (95%), which contained up to 40% of TCR y8+CD4-CD8- T-cells, few monocytes and B-cells. Approximately 50% of CD3+ T-cells showed a CD57+ NK-like phenotype. Functional analysis of mononuclear cells revealed a good antigen-specific T-cell function if the antigen-presenting cells were supplemented from a HLA-matched donor. Moreover, a strong M. avium specific cytotoxicity mediated by TCR a~+ T-cells could be found in vitro and even ex vivo. In contrast, NK-killing was absent. No evidence for a defect in IL-12 or IFN-g production and signaling was found (Wendland et al. 2000). In a patient with pulmonary MAC disease associated with idiopathic CD4+T lymphocytopenia, hematologic studies showed a low CD4+ cell count in the absence of any identifiable immunodeficiency, including HIV infection. With the combination of chemotherapy and surgery, he had a good clinical outcome (Ishida et al. 1998). Disseminated atypical MAC infection in a patient receiving interferon treatment for hairy-cell leukemia was also described (Maurice et al. 1988).

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

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14.3.6 Hemophagocytic Syndrome Associated with MAC This rare manifestation of MAC was recently described. A 37-year-old man with insulin-dependent diabetes mellitus complicated by end-stage renal disease who had received a second cadaveric kidney transplant 4 years earlier presented with symptomatic splenomegaly, progressive anemia, prolonged fever, dysphagia, and weight loss. The first kidney transplant, as well as a pancreatic transplant, had been lost because of acute rejection 7 and 3 years previously, respectively. Owing to chronic rejection of his kidney transplant, he required intensive immunosuppressive therapy consisting of tacrolimus, mycophenolate mofetil, and prednisone. Abdominal CT performed on admission revealed splenomegaly with a subcapsular hematoma. Analysis of a bone marrow aspirate revealed numerous large histiocytes with striated cytoplasm (onion-skin appearance), resembling Gaucherie's cells. Acid-fast staining of a bone marrow biopsy specimen showed that the histiocytic inclusions were actually innumerable acidfast bacilli. PCR analysis of the bone marrow identified the microorganism as MAC. Despite appropriate antibiotic therapy with azithromycin and ethambutol, respiratory, renal, and liver failure developed, and the patient ultimately died of profound lactic acidosis and septic shock due to disseminated MAC infection (Argiris et al. 1999).

a

14.3.7 Miliary/Disseminated Tuberculosis in AIDS HIV-infected patients are predisposed not only to reactivation of remote TB but also to rapid progression of recently acquired infection (Daley et al.1992; Di Perri et al.1989). It is also probable that AIDS increases the susceptibility to acquisition of new infection, and the diagnosis of TB among hospitalized adults aged 15-44 years is also very commonly associated with concurrent HIV infection (40% of patients) (Rosenblum et al. 1994). With advanced HIV infection, TB often has an atypical presentation, with extrapulmonary disease a prominent feature. The most common forms of extrapulmonary TB are lymphadenitis and disseminated disease (Fig. 14.3). In patients with advanced HIV, TB may present as progressive primary infection or disseminated disease. In AIDS patients, 10% with TB and 38% with extrapulmonary TB have miliary disease (Shafer et al. 1991).

Fig. 14.3a-c. Lymph node histology in an HIV-infected patient with co-existing tuberculosis. a Granulomas with central necrosis (-25). b Granulomas with central necrosis and occasional Langerhans giant cells (-100). c AFB stain shows acidfast mycobacteria (M. tuberculosis) (-1000). Courtesy of Metin Ozdemirli, MD, PhD, Assistant Professor in Hematopathology, Georgetown University, Washington, DC, USA.

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Major constitutional symptoms and hectic fevers are characteristic. The chest X-ray is abnormal in 80% and may include typical miliary mottling. Only 10% are tuberculin-positive (Salzman et al. 1992). The sputum smear is positive in only 25% (Shafer et al. 1991), but cultures of many materials are positive, including blood in 50%-60%. Biopsies show a typical tuberculous histologic appearance but with more stainable organisms than in non-HIV miliary TB (Fig. 14.3). In contrast, the histologic picture is often nonreactive TB in fatal cases (Salzman et aI. 1992). Miliary TB in HIV-infected persons may also cause acute respiratory distress syndrome or tuberculous papular skin lesions. Smears of respiratory secretions are positive in 80%. Fulminant miliary TB may be associated with severe refractory hypoxemia (adult respiratory distress syndrome) and disseminated intravascular coagulation (DIC). In such cases, adjunctive corticosteroids (60-80 mg of prednisone daily) are indicated.

14.3.8 Diagnostic Yield of Bone Marrow Aspiration! Biopsy for Mycobacterial Infections in Patients with HIV Infection Hematology consultants are frequently involved in the care of patients with HIV1AIDS through requests for diagnostic bone marrow (BM) aspiration and biopsy, particularly when opportunistic mycobacteria or histoplasma infections are suspected. MAC and TB infections in particular continue to be important causes of HIV-related deaths (Selik et aI. 1995; Sehonanda et aI.1996; Chin et aI.1994). Consequently, a determination of optimal strategies for the diagnosis of these infections continues to be an important clinical issue. While multiple retrospective studies have been reported (Barreto et aI. 1993; Northfelt et aI. 1991; Ciaudo et aI. 1994; Riley et aI.1995; Benito et aI. 1997; Kilby et aI. 1998), no consensus has emerged with respect to the specific value of BM examinations in the diagnosis of MAC or TB infections in immunocompromised HIV-infected patients. BM examination can be a useful method of diagnosing opportunistic mycobacterial and fungal infections in patients with fever, anemia, or neutropenia, and underlying HIV infection. Detection of granuloma with or without positive AFB staining is a common and valuable histologic clue to opportunistic infection. The diagnostic yield of BM granuloma formation was evaluated in 6988 BM biopsies taken from 1973 to 1986. Granulomas were identified in

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72 specimens. The granulomas were associated with infectious disease (30%), hematologic disorders (25%), sarcoidosis (11 %), nonhematologic malignancies (10%), drug reaction (5%), other diseases (6%), and no final diagnosis (6%). Patients with fever of unknown origin were 15 times more likely to have marrow granulomas than patients biopsied for other reasons (Bhargava and Farhi 1988). In another series of 342 BM examinations from 314 patients with HIV infection, 70 examinations (20%) detected opportunistic mycobacterial or fungal infections. Of the 314 patients, III had such infections, and, hence,63% (70/111) were detected by BM examination. Special stains for microorganisms detected 16 (32%) of 50 MAC, 10 (22%) of 45 TB infections, 8 (73%) of 11 Histoplasma capsulatum (HC) infections, and 5 (83%) of 6 Cryptococcus neoformans infections. BM cultures detected 36 (72%) of the 50 MAC infections, 13 (29%) of the 45 TB infections, and 63% of the fungal infections. Marrow examination revealed infection in only 1 of the 70 specimens (1 %) collected to evaluate thrombocytopenia alone or hematologic malignancy, but in 69 (25%) of 274 with fever, neutropenia, anemia, or miscellaneous other indications for marrow examination. Granulomas were detected in 102 (30%) of the biopsy specimens, including 71 (64%) of those in patients with mycobacterial or fungal infection. The granulomas showed caseous necrosis in 9, all in patients with TB, and the 27 patients with TBassociated granulomas tended to show large, tightly cohesive granulomas. The presence of granulomas correlated with opportunistic infection in 82 (80%) of 102 cases. Without granulomas, special stains were positive in only 8 (3%) of 240 specimens, suggesting that special stains alone may not be a cost-efficient way to diagnose such infections (Nichols et al. 1991). Although these pathogens are not associated with specific BM findings, disseminated MAC or TB may cause increased reticulum, occasional granulomas, and positive smears for AFB (more abundant in MAC cases and relatively sparse in TB cases), and rarely pseudo-Gaucher cells in the BM (Solis et al. 1986; Argiris et al. 1999). The most common manifestation of fungal and mycobacterial infection is a diffuse marrow infiltrate of loose aggregates and clusters of macrophages (Fig. 14.2b) (Castella et al. 1985; O'Hara 1989). The configuration of acid-fast bacilli in or around the granuloma also differs between these two Mycobacterium species. In TB,AFBs resemble a bunch of cigarettes with a single cigarette dropped next to the bunch. In MAC, the AFBs tend to be seen as clumps of bacilli in loose aggregates (Carl J. O'Hara, personal communication). Histiocytic erythrophagocytosis has

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Table 14.1. Sensitivity, specificity, and positive and negative predictive values of studies for identification of mycobacterial infection and histoplasma (Akpek et al. 2001) Predictive value Study

Sensitivity"

Specificitt

PositiveC

Negatived

BM culture Blood culture (+) AFB in BM (+) Granuloma

63% 63% 19% 34%

100% (24/24) 100% (24124) 100% (24/24) 96% (23124)

100% (20/20) 100% (20/20) 100% (616) 91% (11112)

67% 86% 48% 52%

(20/32) (20132) (6132) (11132)

(24/36) (31/36) (24/50) (23/44)

Sensitivity: The percent (or proportion) of patients ultimately diagnosed with MAC/TB or HC who had positive studies. b Specificity: The percent (or proportion) of patients who had negative studies who ultimately did not have MAC/TB or He. C Positive predictive value: The percent (or proportion) of patients who had positive studies who were ultimately diagnosed with MAC/TB or He. d Negative predictive value: The percent (or proportion) of patients who had negative studies who were ultimately not diagnosed with MAC/TB or HC a

also been observed and is a nonspecific finding often seen in association with infections, including mycobacterial infections and lymphomas. Successful detection of organisms in the BM of AIDS patients requires the use of special stains and sensitive culture techniques (O'Hara 1989). The highest yield from special stains is obtained when there is a plentiful infiltrate of macrophages, especially in aggregates or in poorly formed granulomas (Castella et al. 1985). Even in the absence of noticeable granulomas or macrophage infiltration, acid-fast organisms and fungi can be diagnosed when present in high numbers (Cohen et al.1983). In order to determine the value of BM culture and BM histology in the diagnosis of opportunistic MAC/ TB and HC infections in immunosuppressed patients with HIV, we retrospectively reviewed the records of 56 adult patients with HIV who underwent a single BM aspiration, biopsy, and culture because of unexplained fever and/or other clinical features suggestive of MAC/TB or HC infection (Akpek et al. 2001). Thirty-two patients (57%) were ultimately diagnosed with MAC/TB (n=30) or HC (n=2) infection by positive cultures of BM, blood, sputum, or bronchoalveolar lavage (BAL) fluid or by the histologic detection of organisms in biopsies of BM or other tissues. The diagnostic sensitivity of blood cultures was equivalent to that of BM culture (63%) in the diagnosis of MAC/TB and HC infections, as reported by others (Northfelt et al.1991; Kilby et al.1998) and was greater than that of BM histopathology alone (34%) (Table 14.1). Blood cultures were found to be negative in 5 of 20 patients for which BM samples were culture-positive. Granuloma and/or histologically apparent organisms were seen in BM biopsy specimens in 11 of 32

individuals (34%) ultimately diagnosed with MAC/TB or HC infections. Among these 11 patients, both granuloma and acid-fast staining organisms were found in the BM biopsy specimens of 2 individuals for whom both BM and blood cultures were negative. The diagnosis would have been missed in 6 patients (19%) ifthe BM had not been examined. The 34% diagnostic sensitivity of BM histopathology observed in our study is similar to the 20%-30% sensitivities reported in other studies (Riley et al. 1995; Kilby et al. 1998; Nichols et al. 1991). One-fourth of BM biopsy specimens showing granuloma were not associated with positive blood or BM cultures, and this finding suggested a 9% falsepositive rate for this histopathologic feature by itself. The low diagnostic sensitivity of BM histopathology alone is not surprising. An autopsy study of 44 AIDS patients with MAC bacteremia found no histologic evidence of MAC in 30% (Torriani et al. 1994). Other studies have also indicated that some individuals suffer mycobacterial bacteremia without detectable organ or tissue invasion (Torriani et al. 1996). A detailed evaluation of clinical and laboratory parameters observed in our study population indicates that the specific clinical features of high peak temperature, longer duration of febrile days, and elevated direct bilirubin at least greater than 1 mg! dl of the upper limit are predictive of MAC/TB or HC infections and are therefore useful in selecting patients most likely to derive diagnostic benefit from BM examination (Akpek et al. 2001). With recent evidence supporting the value of MAC prophylaxis in immunosuppressed HIV-infected patients (Pierce et al. 1996), it is likely that in the future an increasing proportion of patients may receive antimycobacterial prophylaxis at the time of diagnostic evaluation for infection. This change in practice

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could affect the diagnostic yield of both cultures and BM histopathology. Furthermore, with the development of new molecular methods for the detection of mycobacterial DNA or ribosomal RNA in clinical specimens within 24 h, with sensitivities ranging from 40% to 77% and specificities greater than 95% (Havlir and Barnes 1999; Barnes 1997; Condos et al. 1996), it is likely that a reluctance to carry out diagnostic BM examinations will continue, particularly given the additional considerations of cost, patient discomfort, risk of needle-stick exposures for individuals involved in BM sampling, and evidence for responses to empiric anti-MAC therapy in some patients. While the diagnostic sensitivity of BM cultures may not be greater than that of blood cultures in detecting MAC/TB or HC infections in immunosuppressed HIV + patients, histopathologic examination of BM specimens often results in the relatively rapid identification of nearly one-third of infected patients who underwent BM examination and can also identify infections in some patients who are culture-negative. Based on the above findings, we should continue to use BM aspiration, biopsy, and culture for the diagnosis of opportunistic MAC/TB or HC infections in immunosuppressed HIV + patients, particularly when selected clinical features are present.

14.3.9 Other Mycobacterial Infections Associated with Hematologic Disorders Mycobacterial disease, especially atypical mycobacteria, is relatively often seen in hairy-cell leukemia (HCL). The clinical picture is usually dominated by persistent fever. Sweet's syndrome manifested by fever, skin rash, and HCL has been described (Kramers et al. 1992). Blood cultures grew M. kansasii. The patient recovered after treatment with recombinant IFN-a and tuberculostatic drugs. The skin lesions completely regressed within 1 week after the start of r-IFN-a. Leukopenia secondary to M.leprae was also described (Brooks et al. 1990).

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14.4 Coagulation Abnormalities Associated with Mycobacterial Infections and Clinical Presentations 14.4.1 Disseminated Intravascular Coagulation DIC is a very rare complication of pulmonary TB. However, there have been numerous case reports published about the association between DIC and TB (Krishnaswamy 1969; Goldfine et al. 1969; Mavligit et al. 1972; Jenss and Ostendorf 1980). Clotting factors might be locally consumed, thus producing the syndrome of 'multifocal vasculopathic coagulation' (Krauss and Walker 1979).A patient with cavitary TB complicated with DIC has been described (Fujita et aI.1997). Rifampin was considered to treat the clinical course of DIe. Another patient developed subclinical DIC due to anti-TB drugs, probably rifampicin. The patient also developed marked leucocytosis, a flu-like illness, intravascular hemolysis, and acute renal failure as part of the drug reaction (Ip et al. 1991). A review of all 13 patients reported to date shows that the patients are generally black, middle-aged, male, alcoholic, and febrile. The form of TB is generally miliary and is associated with a high mortality. Eight of these patients had associated ARDS. Only one patient had an acute tuberculous peritonitis. In 6 patients the coagulopathy began after the start of therapy; steroids did not appear to affect survival. The exact pathophysiologic mechanisms involved in the development of DIC are unknown (Stein and Libertin 1990). TB-associated hemophagocytic syndrome (HPS) has recently been recognized as a benign reactive histiocytic proliferation with marrow hemophagocytosis. A patient with TB complicated by severe BM failure and DIC was reported. The immunological disturbances usually occurring in miliary TB may playa role in the pathogenesis of HPS (Eliopoulos et al. 1992).

14.4.2 Deep Vein Thrombosis Associated with Tuberculosis Deep venous thrombosis (DVT) associated with TB infection has rarely been reported (Gogna et al. 1999). In a pediatric patient who developed DVT of the left leg in association with pulmonary TB, transient protein S deficiency and anticardiolipin

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients

IgG and IgM antibodies were identified (CasanovaRoman et al. 2002). In a retrospective analysis of clinically diagnosed and lower limb DVT proven by contrast venography, DVT complicated admissions in 46 (3.4%) of 1366 adult patients treated in a TB hospital during 1986. Analysis of 7542 admissions during 1978-1986 showed a relative risk of about 5 in patients treated with regimens including rifampicin compared with other regimens. DVT was significantly more common in the winter months and usually occurred within 2 weeks of treatment being started (White 1989). This probable association between rifampicin and DVT does not contraindicate use of this drug, but measures to prevent DVT should be taken for patients receiving rifampicin.

14.4.3 Thrombotic Thrombocytopenic Purpura Associated with Mycobacterial Infections

Thrombotic thrombocytopenic purpura (TTP) during infectious diseases is a known but rare event (Pene et al. 2001). Various therapeutic approaches were used with the patient who developed TTP during the course of primary TB infection: fresh frozen plasma infusions and plasma exchange, specific anti-TB therapy, antiplatelet drugs, and steroids. A complete remission occurred 3 months after the onset of the acute disease (Toscano et al. 1995). One of the hypotheses about the pathogenesis of TTP might be an increased procoagulant activity of IL-l on endothelial cells. Rifampicin therapy was reported to be associated with the development of TTP in a patient with TB (Fahal et al. 1992). A patient with disseminated MAC infection also developed TTP (Skopinski et al. 2001). Excessive bleeding with unclear etiology can occur rarely in patients with TE. A fatal case of pulmonary TB masquerading as diffuse alveolar hemorrhage after autologous stem-cell transplant was recently reported (Keung et al. 1999).

14.5 Conclusion The types and mechanisms of hematologic findings seen during the course of mycobacterial infections were covered in this chapter. Hematologic findings in patients with active mycobacterial

227

infections are usually considered by clinicians a part of the chronic inflammatory condition, and thus they are usually overlooked. However, it is extremely important to recognize the diagnostic and prognostic value of these hematologic findings because of the possible impact on the outcome of these patients. Severe hematologic abnormalities indicate an advanced mycobacterial infection that requires immediate attention regarding diagnosis and treatment, which may alter the course in these deadly diseases.

References Aboulafia DM, Mitsuyasu RT (1991) Hematologic abnormalities in AIDS. Hematol Oncol Clin North Am 5:195-214 Akhtar AJ, Crompton GK, Schonell ME (1968) Para-aminosalicylic acid as a cause of intestinal malabsorption. Tubercle 49:328-331 Akpek G et al (2001) Bone marrow aspiration, biopsy, and culture in the evaluation of HIV-infected patients for invasive mycobacteria and histoplasma infections. Am J HematoI67:100-106 Al-Majed SA et al (1995) Tuberculosis presenting as immune thrombocytopenic purpura. Acta HaematoI94:135-138 Argiris A, Maun N, Berliner N (1999) Mycobacterium avium complex inclusions mimicking Gaucher's cells. N Engl J Med 340:1372 Barnes PF (1997) Rapid diagnostic tests for tuberculosis: progress but no gold standard. Am J Respir Crit Care Med 155:1497-1498 Barreto JA et al (1993) Isolation of Mycobacterium avium complex from bone marrow aspirates of AIDS patients in Brazil. J Infect Dis 168:777-779 Basu S, Mohan H, Malhotra H (2000) Pancytopenia due to hemophagocytic syndrome as the presenting manifestation of tuberculosis. J Assoc Physicians India 48:845-846 Benito N et al (1997) Bone Marrow Biopsy in the diagnosis of fever of unknown origin in patients with acquired immunodeficiency syndrome. Arch Intern Med 57:1577-1580 Benson CA, Ellner JJ (1993) Mycobacterium avium complex infection and AIDS: advances in theory and practice. Clin Infect Dis 17:7 Bhargava V, Farhi DC (1988) Bone marrow granulomas: clinicopathologic findings in 72 cases and review of the literature. Hematol PathoI2:43-50 Boots RJ, Roberts AW, McEvoy D (1992) Immune thrombocytopenia complicating pulmonary tuberculosis. Case report and investigation of mechanism. Thorax 47:396-397 Bozoky G et al (1997) Hematologic abnormalities in pulmonary tuberculosis. Orv HetilI38:1053-1056 Brooks BJ Jr,Alvarez S, Yoder L (1990) Leukopenia secondary to Mycobacterium leprae. J La State Med Soc 142:35-36 Cameron SJ (1974) Tuberculosis and the blood: a special relationship. Tubercle 5:65-72 Casanova-Roman M et al (2002) Deep venous thrombosis associated with pulmonary tuberculosis and transient protein S deficiency. Scand J Infect Dis 34:393-394

228 Castella A et al (1985) The bone marrow in AIDS: a histologic, hematologic and microbiologic study. Am J Clin Pathol 84: 425 Centers for Disease Control and Prevention (2000) Reported tuberculosis in the United States, 1999. Centers for Disease Control and Prevention, Atlanta Charfi MR et al (1998) Disseminated tuberculosis in nonimmunocompromised host: three case reports. Rev Med Interne 19:917-920 Chia YC, Machin SJ (1979) Tuberculosis and severe thrombocytopenia. Br J Clin Pract 33:55-56 Chin DP et al (1994) The impact of Mycobacterium avium complex bacteremia and its treatment on survival of AIDS patients - a prospective study. J Infect Dis 170:578-584 Ciaudo M et al (1994) Revisited indications for bone marrow examinations in HIV-infected patients. Eur J Haematol 53: 168-174 Cockcroft DW et al (1976) Miliary tuberculosis presenting with hyponatremia and thrombocytopenia. Can Med Assoc J 115:871-873 Cohen RJ et al (1983) Occult infections with M. intracellulare in bone marrow biopsy specimens from patients with AIDS. N Engl J Med 308:1475 Condos R et al (1996) Peripheral-blood-based PCR assay to identify patients with active pulmonary tuberculosis. Lancet 347:1082-1085 Corr WP, Kyle RA, Bowie EJW (1964) Hematologic changes in tuberculosis. Am J Med Sci 248:709-714 Coyle TE (1997) Hematologic complications of human immunodeficiency virus infection and AIDS. In: Gold JWM, Telzak EE, White DA (eds) Management of the HIV-infected patient, part II. Infections and malignancies associated with HIV infection. Saunders, Philadelphia, pp 449-470 (Medical clinics of North America, vol 81) Daley CL, Small PM, Schecter GF (1992) An outbreak of tuberculosis with accelerated progression among persons infected with the human immunodeficiency virus. N Engl J Med 36:231-235 Dallalio G et al (1999) Cytokine and cytokine receptor concentrations in bone marrow supernatant from patients with HIV: correlation with hematologic parameters. J Invest Med 47:477-483 Di Perri G, Danzi MC, DeChecchi G (1989) Nosocomial epidemic of active tuberculosis among HIV-infected patients. Lancet 2:1502-1504 Dutt AK, Moers D, Stead WW (1983) Undesirable side effects of isoniazid and rifampin in largely twice-weekly shortcourse chemotherapy for tuberculosis. Am Rev Respir Dis 128:419-424 Dye C et al (1999) Consensus statement: global burden of tuberculosis: estimated incidence, prevalence, and mortality by country: WHO Global Surveillance and Monitoring Project. JAMA 282:677-686 Eliopoulos G et al (1992) Tuberculosis associated hemophagocytic syndrome complicated with severe bone marrow failure and disseminated intravascular coagulation. Nouv Rev Fr HematoI34:273-276 Ellaurie M, Rubinstein A (1995) Elevated tumor necrosis factor alpha in association with severe anemia in human immunodeficiency virus infection and mycobacterium avium intracellulare infection. Pediatr Hematol Oncol 12: 221-230 Ellner n, Goldberger MJ, Parenti DM (1991) Mycobacterium

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avium infection and AIDS: a therapeutic dilemma in rapid evolution. J Infect Dis 163:1326-1335 Fahal IH et al (1992) Thrombotic thrombocytopenic purpura due to rifampicin. Br Med J 304:882 Flinn JW, Finn RS (1929) Annu Rev Tuberculosis 20:347 French AL, Benator DA, Gordin FM (1997) Nontuberculous mycobacterial infections. In: God JWM, Telzak EE, White DA (eds) The management of the HIV-infected patient, part II. Infections and malignancies associated with HIV infection. Saunders, Philadelphia, pp 361-379 (Medical clinics of North America, vol 81) Fujita M et al (1997) Disseminated intravascular coagulation associated with pulmonary tuberculosis. Intern Med 36: 218-220 Gascon P, Sathe SS, Rameshwar P (1993) Impaired erythropoiesis in the acquired immunodeficiency syndrome with disseminated Mycobacterium avium complex. Am J Med 94:41-48 Ghobrial MW, Albornoz MA (2001) Immune thrombocytopenia: a rare presenting manifestation of tuberculosis. Am J HematoI67:139-143 Glasser RM, Walker RI, Herion JC (1970) The significance of hematologic abnormalities in patients with tuberculosis. Arch Intern Med 125:691-695 Gogna A et al (1999) Tuberculosis presenting as deep vein thrombosis. Postgrad Med J 75:104-105 Goldfine ID et al (1969) Consumption coagulopathy in miliary tuberculosis. Ann Intern Med 71:775-777 Havlick JA et al (1992) Disseminated mycobacterium avium complex infection: clinical identifiers and epidemiologic trends. J Infect Dis 165:577-580 Havlir DV, Barnes PF (1999) Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med 340:367-373 Hernandez-Maraver D et al (1996) Immune thrombocytopenic purpura due to disseminated tuberculosis. Acta Haematol 96:266 Hirsch CS et al (1996) Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production. Proc Natl Acad Sci USA 93:3193-3198 Horsburgh CR et al (1994) Predictors of survival in patients with AIDS and disseminated mycobacterium avium complex disease. J Infect Dis 170:573-577 Ip M, Cheng KP, Cheung WC (1991) Disseminated intravascular coagulopathy associated with rifampicin. Tubercle 72:291-293 Iseman MD (2000) Tuberculosis in relation to human immunodeficiency virus and acquired immunodeficiency syndrome. A clinician's guide to tuberculosis. Lippincott Williams and Wilkins, Philadelphia, pp 199-252 Ishida T et al (1998) Pulmonary Mycobacterium avium disease in a young patient with idiopathic CD4+ T lymphocytopenia. Intern Med 37:622-624 Jenss H, Ostendorf P (1980) Leukopenia and consumption coagulopathy in miliary tuberculosis. Schweiz Med Wochenschr 110:1208-1213 Jones BE et al (1993) Relationship of the manifestations of tuberculosis to CD4 cell counts in patients with human immunodeficiency virus infection. Am Rev Respir Dis 148: 1292-1297 Jones BE et al (1997) CD4 cell counts in human immunodeficiency virus-negative patients with tuberculosis. Clin Infect Dis 24:988-991

Hematologic Findings in Mycobacterial Infections Among Immunosuppressed and Immunocompetent Patients Jurak SS, Aster R, Sawaf H (1983) Immune thrombocytopenia associated with tuberculosis. Clin Pediatr (Phila) 22: 318-319 Kavesh NG, Holzman RS, Seidlin M (1989) The combined toxicity of azidothymidine and antimycobacterial agents. A retrospective study. Am Rev Respir Dis 139:1094-1097 Keung YK et al (1999) Mycobacterium tuberculosis infection masquerading as diffuse alveolar hemorrhage after autologous stem cell transplant. Bone Marrow Transplant 23:737-738 Kilby JM et al (1998) The yield of bone marrow biopsy and culture compared with blood culture in the evaluation of HIV-infected patients for mycobacterial and fungal infections. Am J Med 104:123-128 Kramers C et al (1992) Sweet's syndrome as the presenting symptom of hairy cell leukemia with concomitant infection by Mycobacterium kansasii. Ann Hematol 65:55-58 Krauss JS, Walker DH (1979) Miliary tuberculosis and consumption of clotting factors by multifocal vasculopathic coagulation. South Med J 72:1479-1481 Krishnaswamy V (1969) Disseminated tuberculosis presenting as a bleeding disorder. J Indian Med Assoc 53:85-87 Levy M, Cooper BA (1964) Thrombocytopenic purpura associated with tuberculous lymphadenitis. Can Med Assoc J 90:373-374 Maartens G, Willcox PA, Benatar SR (1990) Miliary tuberculosis: rapid diagnosis, hematologic abnormalities, and outcome in 109 treated adults. Am J Med 89:291-296 Mandell GL et al (2000) Principles and practice of infectious diseases. Mycobacterial infections Maurice PD et al (1988) Mycobacterium avium-intracellulare infection associated with hairy-cell leukemia. Arch Dermatol 124:1545-1549 Mavligit GM, Binder RA, Crosby WH (1972) Disseminated intravascular coagulation in miliary tuberculosis. Arch Intern Med 130:388-389 Medd WE, Hayhoe FGJ (1955) Tuberculous miliary necrosis with pancytopenia. Quart J Med 24:351 Medical Staff Conference (1967) Hematologic manifestations of tuberculosis. Calif Med 106:215-221 Moore M, McCray E, Onorato 1M (1999) TB-AIDS versus nonAIDS TB cases, United States, 1993-1997.1nt J Tuberc Lung Dis S20-S20 abstract Morii T, Narita N (1998) Mycobacterial infection in patients with hematologic disorders. Nippon Rinsho 56:3209-3211 Nichols L et al (1991) Bone marrow examination for the diagnosis of mycobacterial and fungal infections in the acquired immunodeficiency syndrome. Arch Pathol Lab Med 115:1125-1132 Nightingale SD et al (1992) Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J Infect Dis 165:1082-1085 Northfelt DW et al (1991) The usefulness of diagnostic bone marrow examination in patients with human immunodeficiency virus (HIV) infection. J Acquir Immune Delic Syndr 4:659-666 O'Brien JR (1954) Nonreactive tuberculosis. J Clin Pathol 7: 216-225 O'Hara CJ (1989) The lymphoid and hematopoietic systems. In: Harawi SJ, O'Hara CJ (eds) Pathology and pathophysiology of AIDS and HIV-related diseases. Mosby, St Louis, pp 150-164

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Onwubalili JK, Edwards EJ, Palmer L (1987) T4lymphophenia in human tuberculosis. Tubercle 68:195 O'Reilly RA (1998) Splenomegaly in 2,505 patients in a large university medical center from 1913 to 1995. West J Med 169:78-87 Oswald NC (1963) Acute tuberculosis and granulocytic disorders. Br Med J 2:1489-1496 Otsuji A et al (1990) Transient large granular lymphocytosis associated with pulmonary tuberculosis: a case report. Rinsho Ketsueki 31:1955-1959 Pavithran K, Vijayalekshmi N (1993) Thrombocytopenic purpura with tuberculous adenitis. Indian J Med Sci 47: 239-240 Pene F et al (2001) Septic shock and thrombotic microangiopathy due to Mycobacterium tuberculosis in a nonimmunocompromised patient. Arch Intern Med 161: 1347-1348 Perez de Llano LA, Soilan del Cerro JL, Garcia Pais MJ (1998) Imune thrombocytopenic purpura as presenting form of miliary tuberculosis. Arch Broncho PneumoI34:411-412 Pierce M et al (1996) A randomized trial of clarithromycin as prophylaxis as prophylaxis against Mycobacterium avium complex infection in patients with advanced acquired immunodeficiency syndrome. N Engl J Med 335:384-391 Riley UB et al (1995) Detection of mycobacteria in bone marrow biopsy specimens taken to investigate pyrexia of unknown origin. J Clin PathoI48:706-709 Roberts PD, Hoffbrand AV, Mollin DL (1966) Iron and folate metabolism in tuberculosis. Br Med J 5507:198-202 Rosenberg MJ, Rumans LW (1978) Survival of a patient with pancytopenia and disseminated coagulation associated with miliary tuberculosis. Chest 73:536-539 Rosenblum LS et al (1994) Effect of HIV infection and tuberculosis on hospitalizations and cost of care for young adults in the United States, 1985 to 1990. Ann Intern Med 121:786-792 Rosenthal T, Pitlik S, Michaeli D (1975) Fatal undiagnosed tuberculosis in hospitalized patients. J Infect Dis 131 [Suppl):S51-S56 Salzman SH et al (1992) The role of bronchoscopy in the diagnosis of pulmonary tuberculosis in patients at risk for HIV infection. Chest 102:143-146 Sehonanda A, Choi YJ, Blum S (1996) Changing patterns of autopsy findings among persons with acquired immunodeficiency syndrome in an inner-city population. Arch Pathol Lab Med 120:459-464 Selik RM, Chu SY, Ward JW (1995) Trends in infectious disease and cancers among persons dying of HIV infection in the United States from 1987 to 1992. Ann Intern Med 123:933-936 Shafer RW et al (1991) Extrapulmonary tuberculosis in patients with human immunodeficiency virus infection. Medicine 70:384-397 Shenkenberg TD, Rice L, Waddell CC (1982) Acquired PelgerHuet nuclear anomaly with tuberculosis. Arch Intern Med 142:153-154 Singh KJ et al (2001) Significance ofhaematological manifestations in patients with tuberculosis. J Assoc Physicians India 49:790-794 Singh SP et al (1986) Tubercular lymphadenitis with purpura. J Indian Med Assoc 84:247-249 Skopinski S et al (2001) Mycobacterium avium complex sep-

230 ticemia: a new cause of thrombocytopenic thrombotic purpura. Rev Med Interne 22:400-401 Slavin RE, Walsh TJ, Pollack AD (1980) Late generalized tuberculosis: a clinical pathologic analysis and comparison of 100 cases in the preantibiotic and antibiotic eras. Medicine (Baltimore) 59:352-366 Solis DC, Belmonte AH, Ramaswamy G (1986) Pseudo-Gaucher cells in Mycobacterium avium-intracellulare infection in the acquired immunodeficiency syndrome (AIDS). Am J Clin Pathol 85:233 Stein DS, Libertin CR (1990) Disseminated intravascular coagulation in association with cavitary tuberculosis. South Med J 83:60-63 Talbot S et al (1998) Mediastinal nodal tuberculosis presenting as immune thrombocytopenia. Aust NZ J Med 28:465-466 Torriani PJ et al (1994) Autopsy findings in AIDS patients with mycobacterium avium complex bacteremia. J Infect Dis 170:1601-1605 Torriani PJ et al (1996) Disseminated Mycobacterium avium complex: correlation between blood and tissue burden. J Infect Dis 173:942-949 Toscano Vet al (1995) Thrombotic thrombocytopenic purpura associated with primary tuberculosis. Infection 23:58-59 Tulliez M (1976) Bone marrow's histopathology in tuberculosis

G.Akpek of the hematopoietic organs. Pathol Bioi (Paris) 24:699-711 Turett GS, Telzak EE (1994) Normalization of CD4+ T-Iymphocyte depletion in persons without HIV infection treated for tuberculosis. Chest 105:1335 Uetake T et al (1990) Clinicopathological study of miliary tuberculosis in patients with hematologic disease. Kekkaku 65:273-283 Ugo V et al (1996) Myelodysplasia and tuberculosis. Bull Cancer 83:548-552 Wendland T et al (2000) Strong alpha beta and gamma delta TCR response in a patient with disseminated Mycobacterium avium infection and lack of NK cells and monocytopenia. Immunol Lett 72:75-82 White NW (1989) Venous thrombosis and rifampicin. Lancet 2:434-435 Whitfield CL (1970) Hematologic abnormalities in tuberculous patients. Arch Intern Med 126:698 Yang CW et al (1996) Tuberculosis-associated hemophagocytic syndrome in a hemodialysis patient: case report and review of the literature. Nephron 72:690-692 Yasuda Y et al (1994) A case of intractable pulmonary tuberculosis complicated by idiopathic thrombocytopenic purpura. Nippon Kyobu Geka Gakkai Zasshi 42: 2301-2305

15 Immunodiagnostics for Latent Tuberculosis Infection ROHIT

K.

KATIAL

CONTENTS 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 15.10

Introduction 231 History of TB Skin Testing 231 TB Immune Response 232 PPD Strength 232 PPD Testing Methodology 233 Mantoux Testing Method of Measuring Induration 233 TB Skin Test Interpretation 234 Booster 235 Adverse Reactions to PPD Testing 235 New Trends in Diagnosing Latent TB Infection 235 References 237

The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

15.1 Introduction Historical accounts date mycobacterial infections back thousands of years, but it was not until the late 19th century that the causative, agent responsible for tuberculosis (TB) was discovered. The veterinary industry in its attempt to eradicate bovine TB led the effort to diagnose both latent and active TB infections. The skin testing method successfully used in animals was soon optimized for human diagnostics. Screening methods have changed little in the last 100 years, and the skin test is fraught with limitations including poor specificity due to crossR. K. KATIAL, MD Associate Professor of Medicine, National Jewish Medical and Research Center Denver, 1400 Jackson Street, Denver, CO 80206, USA

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

reactivity with nontuberculous strains, difficulty in interpretation, and test interference by underlying co-morbid conditions. Because of the resurgence of TB infection associated with the AIDS epidemic and with increasing urban crowding, more specific and sensitive tests that will detect exposure to Mycobacterium tuberculosis (MTB) are needed. New in vitro diagnostics are being evaluated as potential replacements for the old TB skin test.

15.2 History of T8 Skin Testing TB was an epidemic that first appeared in animals in the Paleolithic period long before the disease occurred in man some 8000 years ago (Bates and Stead 1993). Four thousand years later, one finds written reference to TB in the Vedas (Kiple 1993). In the Indian subcontinent, the Hindus referred to TB as consumption (Davis 2000), a term used until quite recently. As urban development increased and city centers were formed in the early 1600s, the incidence of TB increased sharply. Over the subsequent 200 years, the epidemic spread throughout Western Europe. At the end of the 18th century, TB was rampant, but the causes were speculative and treatment ineffective. In 1882, Robert Koch isolated the tubercle bacillus and thus began the rapid advancement in understanding both the disease pathophysiology and diagnostics (Koch 1890, 1891). Robert Koch developed a MTB filtrate known as old tuberculin (OT) from heat-sterilized cultures, which he mistakenly promoted as a therapeutic agent for TB. Soon thereafter, OT began to be evaluated for use in early diagnosis, initially in cattle by comparing skin testing reactivity with autopsy data. The association of subclinical infection with a reactive skin test was made when asymptomatic cattle with positive skin tests demonstrated tuberculi on autopsies (Pearson 1892). Any skin test reactivity at this point was considered positive for TB because nonspecific

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reactivity from nontuberculous strains had not been recognized. After widespread testing in animals, the occasional discrepancy between skin test reactivity and the absence of organisms on autopsy became apparent. By the 1930s, it was widely accepted in the veterinary community that skin test conversion could occur after exposure to nontuberculous strains of mycobacteria (Hagan 1931). Similar nonspecific reactivity was seen in humans when large-scale testing was done through the 1930s and 1940s. In addition, it was noted that OT showed batch-to-batch variability and led to inconsistent skin test results (Snider 1982). With this observation, work began on developing a more consistent and standardized product. In 1934, Florence Seibert prepared the purified protein derivative (PPD), and in 1941, Seibert and Glenn prepared lot no,49608, the international skin test standard reagent PPD-S (Seibert and Glenn 1941). All PPD preparations from that point forward had to be bioequivalent to PPD-S.

15.3 18 Immune Response The injurious effects from MTB are a result of the defensive responses exhibited by the host. The infectious process begins with inhalation of particles in the respirable range of 1-5 flm. These particles settle in the alveolus where they are engulfed by alveolar macrophages. The immune response to TB is a complex interaction between antigen-presenting cells (APC) and a variety of T-Iymphocytes, with a humoral response being much less significant. The MTB bacillus is taken up by a phagosome within the APC that attempts to fuse with a lysosome and ultimately create a mature phagolysosome. However, one of the mechanisms of immune evasion by TB is the prevention of the formation of the mature phagolysosome (Kaufmann 2001). The intracellular presence of the bacillus results in complexing of TB antigens with MHC class II molecules. This leads to activation of CD4+ T-cells, which produce IFN-y among other cytokines and ultimately recruit more macrophages to the site of infection, thus perpetuating the cycle (Iseman 1999). Antigen presentation also occurs through nonpolymorphic CD1 and polymorphic MHC class I molecules, thus leading to activation of CD8+ and double-negative a~T-cells. Evidence is also mounting that yoT-cells may contribute to the tuberculin response (Kaufmann 2001). All these T-Iymphocytes have the ability to produce IFN-y, with CD4+ cells being the primary producer

R. K. Katial

as well as the cell central to the recall delayed-type hypersensitivity (DTH) response seen after injection of PPD in individuals previously exposed to MTB. The contribution of other cytokines such as IL-12, IL-18, and TNF-~ to the pathogenesis and protective response of TB infection has been nicely elaborated in other reviews (Collins and Kaufmann 2001; Iseman 1999; Daniel 1980; Daniel et al.2000; Tsuyuguchi 1996). IFN-yis the primary cytokine involved with governing immune protection to TB and has recently been used in the development of immunodiagnostics. TB-infected individuals present tuberculin antigens by various ways and generate effector T-cells, which then continue to circulate in the blood. The nonsterilizing quality of the initial exposure may serve to continually stimulate the immune system, thus maintaining a reservoir of MTB-specific T-cells. The response to PPD is a classic DTH reaction, involving vasodilation, edema, and infiltration initially of antigen-specific T-cells which then secrete cytokines leading to recruitment of other nonspecific inft.ammatory cells (Ahmed and Blose 1983). The result of these inft.ammatory events is induration beneath the skin and some degree of erythema. The maximum induration is generally seen 48 h after injection of PPD, and this is not correlated with the erythema. DTH responses can persist for years after exposure to the bacillus. The immune mechanism of the DTH response differs from the IFN-yresponse mounted for protection from the disease. Orem and Copper (1999) postulated that immune protection is governed by cytokine production such as IFN-y, while the DTH response may be a primary result of chemokine production. The clinical importance of this is that in actively infected individuals, the DTH reaction may be depressed initially but returns within a month of therapy, whereas IFN-y may remain suppressed for at least 12 months (Ellner 1997; Hirsch et al. 1996, 1999). This clearly suggests that in vitro measurement of IFN-y production is not a direct correlate of the less expensive PPD and recall antigen (e.g., measles, mumps, tetanus) skin tests but rather that the two reactions are governed by different immune mechanisms.

15.4 PPD Strength In the USA, two types of tuberculin are available: OT and PPD. OT is only used with multipuncture devices, whereas PPD is used for both multipuncture and the intracutaneous test. PPD is manufactured by protein

Immunodiagnostics for Latent Tuberculosis Infection

233

precipitation of culture titrate using either tricWoro- Induration at the site of injection is usually read at 48acetic acid or ammonium sulfate. Although both prep- 72 h. Howard and Solomon (1988) evaluated readings arations are standardized by bioequivalency using a at 24, 48, and 72 h and reported 71 % sensitivity and skin testing methodology (Villarino et al. 1999), the 9% false-positive rate at 24 h. Thus, the authors recomantigen content of the two may differ due to the dif- mended reading the test at the standard 48-72 h, while ferences in the precipitation methods. PPD contains being aware that some reactions may occur earlier and primarily small proteins (less than 10,000 Da) but also others may persist longer than 72 h. has polysaccharides and lipids. The small proteins and lack of adjuvant prevent PPD from being sensitizing (American Thoracic Society 2000). The PPD used for intracutaneous testing comes in 15.6 three concentrations: 1 tuberculin unit (TU), 5 TU, Mantoux Testing Method of Measuring and 250 TV. PPD 5 TU is the only strength that is Induration bioassayed (standardized in vivo to the PPD-S). PPD 1 TU was shown to have only 50% sensitivity in The CDC standard for reading PPD induration is children, while PPD 250 TU identified all specific and palpating along the transverse axis of the forearm nonspecific reactors (Murtagh 1980; Gryzbowski et (Core Curriculum on Tuberculosis, Centers for al. 1969). Palmer (1945) studied student nurses who Disease Control 1994). However, several investigawere tested with PPD 5 TU and 250 TV. PPD 5 TU tors have compared this palpation method with a proved to be highly sensitive in identifying students ballpoint method first introduced by Sokal (1975), with pulmonary calcifications and/or lung infiltrates. who claimed a reduction in the observer variability On the other hand, reactivity with PPD 250 TU did with the pen method. Correlation between these two not correlate with pulmonary findings or contact methodologies is high, and differences between readhistory. Therefore, the 1 and 250 TU concentrations ings are small and often not clinically relevant. When should not be used for testing because of lack of Pouchot et al. (1997) compared PPD readings by palstandardization. pation versus the ballpoint method, they found a high correlation between the two techniques but reported a 38% broader area of imprecision with palpation. The skin test readers in the study were not blinded 15.5 to the patients, and only two very experienced readPPD Testing Methodology ers performed all the tests. A study by Howard and Solomon (1988) evaluating 806 volunteers found The two primary skin-testing methods used are the discordance between the two methodologies in the multipuncture device and the Mantoux test. Multiple 5-14 mm range. The readers in their study were also puncture devices such as the tine test are inexpen- not blinded. The authors suggested that the readings sive, simple to apply, and much more rapid. They obtained with the pen method should be interpreted introduce antigen through tuberculin-coated prongs with caution in the 5-14 mm range. Jordan et al. on the applicator device, or by puncturing through (1987) and Bouros et al. (1991) conducted doublea liquid film placed on the forearm; however, such blind studies comparing the two methods, and both devices have numerous drawbacks including unre- showed that the pen method is comparable to palpaliable dose delivery (Lunn 1980), inconsistent tech- tion in the hands of experienced readers. Bouros and niques of administration, and significant false-posi- colleagues (1992) later studied the two methods with tive reactions. A study by the Research Committee of readings from both experienced and inexperienced the British Thoracic Association (1982) showed up to readers. No significant differences were found in any 23% variability in subjects with duplicate tine tests. range of measurement by the two groups using both Because of these shortcomings, the current recom- methods. The authors concluded that inexperienced mendation is that positive tine tests need validation readers can use either methodology accurately. Longby the commonly adopted Mantoux method, which field and colleagues (1984) studied interobserver has become the standard recommended by the variability in 101 patients and found no significant United States Centers for Disease Control (CDC). difference between the two methods. They found The Mantoux test is performed by injecting 0.1 ml comparable variability between the two techniques; of PPD 5 TU intradermally with a 26-27 gauge needle using a 10 mm cut-off, less than 7% of readings would with bevel upward, creating a pale bleb above the skin. have changed the clinical interpretation.

234

R. K. Katial

At our institution we use the ballpoint method, measuring along two axes of the forearm. We retrospectively evaluated 5680 skin test records and analyzed only the positive records (380), comparing the transverse to the longitudinal axis as well as the average of the two. We found no statistically significant difference (p=0.135) between the transverse and longitudinal readings; in fact, the total number of subjects classified positive and negative were the same in each group, using both a 10 mm and 15 mm cut-off, although the specific individuals varied (Table 15.1) (Hershey et al. 2001). This suggests that the two readings are equivalent. However, when the larger of the two readings was used to determine the classification, a slightly higher number of people (24/380 with a 15 mm cut-off) were classified as positive than by the single transverse reading. When the average of the two readings was used, the results were similar to the standard transverse method but may reflect enhanced precision because two readings have been made. The intraobserver variability has been reported to be 3.0 mm greater or less than the first measurement (Pouchot et al. 1997). In our data, the variability between the two readings was

3 mm or less in 81 % of the subjects. However, choosing the larger of the two measurements provided slightly greater sensitivity. It appears that adherence to one technique and proper application and training are more important than which technique or axis is used.

15.7 T8 Skin Test Interpretation In determining the cut-off for a clinically positive result indicating exposure to MTB, one must understand the reactivity patterns in various populations as well as the factors contributing to false-positive and false-negative results. In the 1950s, the World Health Organization (WHO) collated data of skin test induration for patients in TB hospitals throughout the USA, Europe, and Southeast Asia. The highest number of reactors had indurations of approximately 16-17 mm with a normal distribution. In every country, the frequency curves overlapped. In some regions, a bimodal distribution was described

Table 15.1. Comparison of skin test readings by reading direction (transverse vs longitudinal) No. of subjects

Percentage

(n=380)

Transverse classification at ~1O mm cut-off: Negative Positive Longitudinal classification at ~10 mm cut-off: Negative Positive Transverse classification at ~15 mm cut-off: Negative Positive Longitudinal classification at ~15 mm cut-off: Negative Positive Identical readings in mm Transverse reading larger than longitudinal No. of readings with difference >3 mm Longitudinal reading larger than transverse No. of readings with difference >3 mm No. of readings reclassified" at 15 mm cut-off No. reclassified" at 15 mm cut-off where reading difference >3 mm No. of readings reclassified" at 10 mm cut-off No. reclassified" at 10 mm cut-off where reading difference >3 mm

59 321

15.5% 84.5%

59 321

15.5% 84.5%

167 213

43.9% 56.1%

167 213 108 106 34 166 39 24

43.9% 56.1% 28.4% 27.9% 8.9% 43.7% 10% 6.3%

9 7

2.4% 1.8%

3

0.7%

" Standard classification is based on transverse reading. Reclassification reflects change to positive classification when larger longitudinal direction is used

235

Immunodiagnostics for Latent Tuberculosis Infection

with one peak at 5-9 mm and another at 16-17 mm (Snider 1982). Based on previous animal data, it was felt that the smaller peak reflected reactivity to nontuberculous (NTM) strains, such as M. aviumintracellulare (MAl). Between 1958 and 1970, a study evaluating 275,558 Navy recruits tested all subjects with both PPD-S and PPD-B (MAl strains). The individuals with large reactions to nontuberculous PPD tended to have smaller reactions (6-11 mm) with PPD-S (Edwards et al. 1969). The incidence of active TB in 0-5 mm PPD reactors was 36/100,000, in 6-11 mm PPD reactors it was 110/100,000, and with indurations with PPD >12 mm the incidence of disease was 380/100,000. In another study, Palmer et al. (1959) evaluated skin-test reactivity in Pakistani children and again demonstrated a bimodal distribution with peaks at 2-5 mm and 17 mm. They concluded that the first peak was due to NTM, and the larger size peak represented true TB reactors. The reactions from NTM tend to be smaller than from MTB. Thus, increasing the cut-off for a positive reaction increases the specificity but reduces the sensitivity. In the USA, this has been the rationale for a 15 mm cut-off, since the prevalence of NTM is high (particularly in the southern USA) and of MTB low. A value of 15 mm is also close to the peak of reactivity described by the WHO in true reactors. In groups at high risk for TB, the cut-off is lowered in order to improve the sensitivity; immunocompromised groups, in particular, may have smaller size reactions or become anergic due to immune dysregulation, and thus the cut-off has been reduced to 5 mm.

15.8 Booster Although the PPD skin-test reactivity can persist throughout life, occasionally there is a diminution or absence in the DTH response to PPD over time. In such individuals, a return in response may be seen if tested sequentially over several weeks. This phenomenon of accentuation of response after repeat testing is commonly known as the booster effect and must not be interpreted as a new skin test conversion. Boostering most often occurs in persons older than age 55 years in whom cellular immunity may wane. Health care institutions where repeat TB testing is performed use an initial screening two-step test (two PPD skin tests about 3 weeks apart) to establish a baseline reactivity, in order to avoid falsely classifying a repeat test at a later time as a new conversion.

15.9 Adverse Reactions to PPD Testing Generally, adverse reactions to TB skin testing are rare; however, immediate hypersensitivity reactions have been reported and are in no way correlated to underlying TB infection (Tarlo et al. 1977; Wright et al. 1989). Individuals may also develop vesicular or ulcerating lesions at the site of PPD injection. These severe reactions occur in a small percentage ofpositive reactors; however, a previous positive does not predict an increased likelihood of developing a severe cutaneous reaction (Reichman and O'Day 1977). Health care providers use topical corticosteroid creams to treat these local reactions, but one controlled study using hydrocortisone did not demonstrate efficacy, although the use of a more potent steroid may have shown benefit (Hanson and Comstock 1968).

15.10 New Trends in Diagnosing Latent TB Infection The immune response to mycobacterial infection is predominantly cellular (Kaufmann 2001). The PPD skin test has been a convenient, cost-effective method for assessing cell-mediated immune responses to mycobacterial-derived proteins. Although the test is reasonably priced, there continue to be multiple factors challenging the accuracy of the PPD skin test (ST) in different settings. These factors include (but are not limited to) variability in operator placement and reading, cross-reactivity among mycobacterial species (including M. avium and BeG), the need for the patient to return in 48-72 h for a reading, and the modulation of the skin response due to underlying illness or immunosuppression. Clearly, there is a need for another more sensitive and specific test that would overcome the limitations of the skin test. In addition, the tools used to diagnose active disease are currently different from the skin test, which is used to detect exposure. The major immunodiagnostic advances to date have centered on more sensitive and rapid testing to determine the presence of active disease, but progress in the area of detecting exposure in healthy individuals has been slower. Recent work has shifted to identifying recombinant and purified antigens that may be used for skin testing and in immunoassays with greater sensitivity. One of the first tuberculosis complex-specific antigens discovered was MPT64, a

R. K. Katial

236

24 kDa protein. More recently, other antigens have been purified, including 38 kDa (antigen b), 10 kDa, 18 kDa, MPT59 (Ag85B), and ESAT-6 (Gennaro 2000). MPT64 has produced disappointing results when used as a skin-test reagent. Only 6% of PPD-reactive TB-positive patients responded to MPT64 in comparison with a 50% reactivity with MPT59 (Wilcke et al. 1966). Antigen b has been shown to be very antigenic and, when studied in guinea pigs, elicited a positive skin test in MTB-vaccinated animals but not in those exposed to other strains. The antigen appears to be more specific than PPD (Haslov et al. 1990). Vordermeier et al. (1992), using an epitope located near the 38 kDa antigen of MTB, elicited a DTH reaction in PPD-positive individuals but not in negative ones. These antigens may find a role in immunoassays and possibly in skin testing by providing the ability to detect newly exposed individuals with greater specificity as well as to differentiate those vaccinated from those infected. The immune response to MTB is highly dependent upon IFN-y production by macrophages and antigen-specific T-cells; over the past decade, there has been an increasing interest in the development and application of in vitro culture assays measuring IFN-y production in response to tuberculin antigen stimulation as a substitute diagnostic screening test for the classic PPD skin test (Lein and von Reyn 1997). Initially using peripheral blood mononuclear cells, the methodology evolved to a whole blood culture technique that was first validated in Australian cattle. These studies demonstrated that the IFN-y test had greater diagnostic sensitivity, lower cost, and rapid results for cattle TB screening (Rothel et al. 1992; Wood et al. 1991; Wood and RotheI1994). This method was developed for human TB testing using human PPD, avian PPD, and the mitogen phytohemagglutinin (PHA). A standardized diagnostic kit with a specifically defined data analysis procedure has been marketed by CSL in Australia (now Cellestis, Australia): QuantiFERON-TB or Q-IFN. In various studies comparing the Q-IFN assay with PPD-ST, the agreement ranges from 40% to 100% when looking at subjects without active disease or a history of active disease (Table 15.2). Agreement has been lower in those with active disease. Streeton et al. (1998) reported a sensitivity and specificity of 90.5% and 98%, respectively, for diagnosing MTB exposure in human subjects. In their study, the gold standard for diagnosis was the PPD-ST, and the study subjects were stratified according to their skin test induration and risk of exposure to tuberculosis. Kimura et al. (1998) and

Table 15.2. Agreement between Q-1FN and SKT for latent TB infection in various studies Reference

Skin test classification

Agreement between Q-1FN test and SKT

(Q-1FN/SKT)a N (HIV-) 68% P (HIV-) 70% 700/0 Overall, H1VN (H1V+) 85% 40% P (H1V+) 61% Overall, H1V+ 40% N (H1V -) Converse et aI. (1997) P (H1V - ) 100% Overall, H1V72% N (H1V+) 73% P(H1V+) 92% Overall, H1V+ 79% 100% Desem and Jones N 90% (1998) P 95% Overall 80% KatiaI et aI. N (2001) 80% P Overall 80% 500/0 N (H1V-) Kimura et aI. (1998) P (H1V -) 89% 59% Overall, HIV 85% N (H1V+) 56% P (H1V +) 82% OveraIl, H1V + 900/0 Mazurek et aI. N 65% (2001) P 85% Overall Pottumarthy et aI. N (high-risk country) 89% (1999) P (high-risk country) 64% N(HCW) 81% P (HCW) 67% 79% OveraIl 88% N Streeton et aI. (1998) 900/0 P 88% Overall Bellete et aI. (2002)

(43/63) (921131) (135/194) (22126) (ll/28) (33/54) (6115) (17117) (23/32) (16122) (ll/12) (27/34) (10/10) (9/10) (19/20) (16/20) (16/20) (32/40) (115/229) (63171) (178/300) (128/151) (9/16) (137/167) (738/818) (1471227) (885/1045) (135/151) (55/86) (64179) (32/48) (286/364) (480/545) (163/182) (643/727)

With ~15% = positive Q-1FN, excluding those with active disease or past history of active disease N =negative skin test defined at various cut-offs depending on risk; P =positive skin test defined at various cut-offs depending on risk; Hew = health care worker

a

Converse et al.(1997) used Q-IFN in studies comparing the IFN release assay with PPD-ST in populations at risk for MTB exposure (intravenous drug users with or without HIV infection). They found that the Q-IFN assay detected more reactors than PPD-ST. Agreement between the two tests was weak. We studied 40 patients and found good agreement between PPD-ST and the Q-IFN kit (kappa=0.73). We evaluated the agreement between in vivo and in vitro tests and did not refer to sensitivity and specificity because they assume comparison to an adequate gold standard, which we feel does not exist

237

Immunodiagnostics for Latent Tuberculosis Infection

(Katial et al. 2001). Recently, Mazurek and colleagues (2001) evaluated 1226 volunteers and found overall 83% agreement between the IFN-y assay and skin test in individuals at both low and high risk for latent TB infection. However, only 68% agreement was noted in the positive PPD-ST group. A similar study by Bellete et al. (2002) reported an overall agreement in a USA cohort of 79% (in Baltimore), but lower agreement (68%) was seen in Ethiopia, an endemic TB area. In addition, the authors found poor reproducibility in the in vitro results from one test to another. One significant drawback of both the skin test and the Q-IFN test is the nonspecific response to PPD because of cross-reactivity between tuberculin and other mycobacterial species. Although the Q-IFN test does distinguish M. avium from MTB reactivity, it does not differentiate between responses from MTB and other mycobacterial species such as M. kansasii, M. marum, and M. africanum. The false-positivity in the PPD skin testing due to BCG vaccination is well documented. However, the in vitro IFN-yproduction by BCG-vaccinated individuals in response to PPD may also be influenced by cross-reacting mycobacterial antigens. Streeton et al. (1998) included BCG vaccinees in their study but were unable to discern the effects of BCG on the assay results. Therefore, additional studies are needed to delineate the diagnostic value of the Q-IFN kit in this population. The in vitro diagnostic system affords a distinct advantage over PPD-ST in that one can test for tuberculin-specific proteins without unnecessarily exposing the patient. The low-molecular-weight antigen ESAT-6 (6 kDa) has been shown to differentiate between MTB and BCG strains and thus may serve as an additional stimulant to determine the effect of BCG. Recently, ESAT-6 was evaluated in the Q-IFN assay and found to differentiate those infected with TB from controls, with high sensitivity and specificity (Johnson et al. 1999). We studied low-molecular-weight culture filtrate proteins, culture filtrate proteins minus lipoarabinomannan, soluble cell wall proteins, and cytosolic proteins in whole blood culture, and none of the subfractions performed any better than the whole PPD (Katial et al. 2001). However, the crude preparations used in this study did stimulate IFN-y production in sensitized individuals and should be further purified and studied in the whole blood system. The immune response to TB is complex and directed to a heterogeneous mixture of antigens rather than anyone protein. Therefore, studying a cocktail of native or recombinant antigens may prove to be more specific and sensitive in diagnosing TB than anyone immunodominant protein. The advantages of the

blood test include the absence of any reading or placement variability and the need for only one office visit. The current disadvantages are the need for stringent laboratory requirements dealing with blood handling, cell culture, and ELISA testing. The areas needing clarification include defining the performance characteristics and lower cut-off limit for the enzyme immunoassay. Data are needed on temporal measurements to determine if the time of day or different days have any impact on the whole blood stimulation response. Finally, the cut-offs differentiating a positive from a negative response suggested in the Q-IFN kit need to be verified based on the prevalence of disease in different populations as has been done with PPD-ST. These issues should be addressed and followed up by large-scale trials to assess the true sensitivity, specificity, and positive predictive value of the Q-IFN kit, prior to Widespread use for clinical MTB testing. The assessment of IFN-y production by TB-specific lymphocytes is also being studied by more sensitive techniques such as flow cytometry and Elispot assays. Tilley and Menon (2000) demonstrated the correlation between intracellular staining of IFN-y in TB-specific lymphocytes with the extracellular secretion of IFN-y as measured by the Elispot assay. The authors were also able to conclude that the IFNy response as measured by the intracellular staining correlated directly with the Mantoux skin test. As the data in TB patients using these techniques increase, the understanding of the underlying TB immune response will become clear, and some combination of tests may become the new standard of care for the diagnosis of latent TB infection.

References Ahmed AR, Blose DA (1983) Delayed type hypersensitivity skin testing: a review. Arch DermatoI119:934-945 American Thoracic Society and Centers for Disease Control and Prevention (2000) Diagnostic standards and classification of tuberculosis in adults and children. Am J Crit Care Med 161:1376-1395 Bates JH, Stead WW (1993) The history of tuberculosis as a global epidemic. Med Clin North Am 77:1205-1217 Betiete B et al (2002) Evaluation of a whole-blood interferon-y release assay for the detection of Mycobacterium tuberculosis infection in 2 study populations. Clin Infect Dis 34: 1449-1456 Bouros D et al (1991) Palpation vs pen method for the measurement of skin tuberculin reaction (Mantoux test). Chest 99:416 Bouros D et al (1992) The role of inexperience in measuring tuberculin skin reaction (Mantoux test) by the pen or palpation technique. Respir Med 86:219-223

238 Centers for Disease Control (1994) Core curriculum on tuberculosis, 3rd edn. Centers for Disease Control, Atlanta, p 9 Collins H, Kaufmann S (2001) The many faces of host responses to tuberculosis. Immunology 103:1-9 Converse PJ et al (1997) Comparison of a tuberculin skin test in high-risk adults of human immunodeficiency virus infection. J Infect Dis 176:144-150 Daniel T (1980) The immunology tuberculosis. Clin Chest Med 1:189-201 Daniel TM, Boom WH, Ellner JJ (2000) Immunology of tuberculosis. In: Reichman L, Hershfield ES (eds) Tuberculosis: a comprehensive international approach, 2nd edn. Dekker, New York, pp 187-214 Davis AL (2000) A historical perspective on tuberculosis and its control. In: Reichman L, Hershfield ES (eds) Tuberculosis: a comprehensive international approach, 2nd edn. Dekker, New York, pp 3-54 Desem N, Jones SL (1998) Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection. Clin Diagn Lab Immunol 5: 531-536 Edwards LB et al (1969) An atlas of sensitivity to tuberculin, PPD-B and histoplasmin in the United States. Am Rev Respir Dis 99:1-132 Ellner JJ (1997) The immune response in human tuberculosis-implications for tuberculosis control. JID 176: 1351-1359 Gennaro ML (2000) Immunologic diagnosis of tuberculosis. Clin Infect Dis 30:s243-s246 Gryzbowski S, Brown MT, Stothard D (1969) Infections with atypical mycobacteria in British Columbia. CMAJ 100: 896-900 Hagan WA (1931) The no lesion case problem in the tuberculosis eradication campaign. Cornell Vet 21:163-171 Hanson ML, Comstock GW (1968) Efficacy of hydrocortisone ointment in the treatment of local reactions to tuberculin. Am Rev Respir Dis 97:472-473 Haslov K et al (1990) Comparison of the immunological activity of five defined antigens from mycobacterium tuberculosis in seven inbred guinea pig strains. The 38 kDa antigen is immunodominant. Scand J ImmunoI31:503-514 Hershey J, Engler RJ, Katial RK (2001) Tuberculin skin tests: to measure or not to measure in two directions? JACI 107: s254 Hirsch CS et al (1996) Cross-modulation by transforming growth factor b in human tuberculosis: suppression of antigen-driven blastogenesis and interferon g production. Proc Natl Acad Sci USA 93:3193-3198 Hirsch CS et al (1999) Depressed T-cell interferon-y responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. JID 180:2069-2073 Howard T, Solomon DA (1988) Reading the tuberculin skin test: Who, when and how. Arch Intern Med 148:2457-2459 Iseman M (1999) Immunity and pathogenesis. In: A clinician's guide to tuberculosis. Lippincott Williams and Wilkins, Philadelphia, pp 63-96 Johnson PDR et al (1999) Tuberculin-purified protein derivative, MPT-64, and ESAT-6 stimulated gamma interferon responses in medical students before and after mycobacterium bovis BCG vaccination and in patients with tuberculosis. Clin Diagn Lab Immunol 6:934-937 Jordon T et al (1987) Tuberculin reaction size measurement by

R. K. Katial the pen method compared to traditional palpation. Chest 92:234-236 Katial RK et al (2001) Cell mediated immune response to tuberculosis purified protein derivative: comparison of TB skin testing to in-vitro g-interferon production in whole blood culture. Clin Diagn Lab Immunol 8:339-345 Kaufmann SH (2001) How can immunology contribute to the control of tuberculosis? Nature Rev Immunol1:20-30 Kimura M et al(1998) Comparison between a whole blood interferon-gamma release assay and tuberculin skin testing for the detection of tuberculosis infection among patients at risk for tuberculosis exposure. J Infect Dis 179: 1297-1300 Kiple KF (ed) (1993) The Cambridge world history of human disease. Cambridge University Press, Cambridge Koch R (1890) Ober bacterilogische Forschung. Dtsch Med Wochenschr 16:756 Koch R (1891) Fortsetzung der Mitteilungen tiber ein Heilmittel gegen Tuberculose. Dtsch Med Wochenschr 891:17-101 Lein D, von Reyn F (1997) In vitro cellular and cytokine responses to mycobacterial antigens: application to diagnosis of tuberculosis infection and assessment of response to mycobacterial vaccines. Am J Med Sci 313:364-371 Longfield J et al (1984) Interobserver and method variability in tuberculin testing. Pediatr Infect Dis 3:323-326 Lunn JA (1980) Reasons for variable response to tine test. Br Med J 280:223 Mazurek GH et al (2001) Comparison of a whole-blood interferon gamma assay with tuberculin skin testing for detecting latent Mycobacterium tuberculosis infection. JAMA 286:1740-1747 Murtagh K (1980) Unreliability of the Mantoux test using 1 TU PPD in excluding childhood tuberculosis in Papua New Guinea. Arch Dis Child 55:795-799 Grme I, Cooper A (1999) Cytokine/chemokine cascades in immunity to tuberculosis. Immunol Today 20:307-312 Palmer CE (1945) Nontuberculous pulmonary calcification and sensitivity to histoplasmin. Public Health Rep 60,513-520 Palmer CE et al (1959) Experimental and epidemiologic basis for the interpretation of tuberculin sensitivity. J Pediatr 55:413-428 Pearson L (1892) Tuberculin as a diagnostic agent. Med News 60:358-359 Pouchot J et al (1997) Reliability of tuberculin skin test measurement. Ann Intern Med 126:210-214 Pottumarthy S et al (1999) Evaluation of the tuberculin gamma interferon assay: potential to replace the Mantoux skin test. J Clin Microbiol 37:3229-3232 Reichman LB, G'Day R (1977) The influence of a history of a previous test on the prevalence ands size of reactions to tuberculin. Am Rev Respir Dis 115:737-741 Research Committee of the British Thoracic Association (1982) Reproducibility of the tine tuberculin test. Br J Dis Chest 76:75-78 Rothel JS et al (1992) The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma-interferon in whole blood culture. Aust Vet J 69: 1-4 Seibert FB, Glenn JT (1941) Tuberculin purified protein derivative: preparation and analysis of a large quantity for standard. Am Rev Tuberc 44:9-25 Snider D (1982) The tuberculin skin test. Am Rev Respir Dis 125:s108-s118

Immunodiagnostics for Latent Tuberculosis Infection Sokal JE (1975) Measurement of delayed skin test responses. N Engl J Med 293:501-502 Streeton JA,Desem N, Jones SL (1998) Sensitivity and specificity of a gamma interferon blood test for tuberculosis infection. Int J Tuberc Lung Dis 2:443-445 Tarlo SM et al (1977) Immediate hypersensitivity to tuberculin in in-vivo and in-vitro studies. Chest 71:33-37 Tilley PAG, Menon IN (2000) Detection of Mycobacteriumspecific interferon-gamma-producing human T lymphocytes by flow cytometry. APMIS 108:57-66 Tsuyuguchi I (1996) Regulation of the human immune response in tuberculosis. Infect Agents Dis 5:82-97 Villarino ML et al (1999) Comparable specificity of 2 commercial tuberculin reagents in persons at low risk for tuberculous infection. JAMA 281:169-171

239 Vordermeier HM et al (1992) M. tuberculosis-complex specific T-cell stimulation and DTH reactions induced with a peptide from the 38-kDa protein. Scand J Immunol 35: 11-718 Wilcke JT et al (1966) Clinical evaluation of MPT-64 and MPT59, two proteins secreted from mycobacterium tuberculosis, for skin test reagents. Tuber Lung Dis 77:250-256 Wood PR, Rothel JS (1994) In vitro immunodiagnostic assay for bovine tuberculosis. Vet MicrobioI40:125-135 Wood PR et al (1991) Field comparison of the interferongamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis. Aust Vet J 68:286-290 Wright DN, Ledford DK, Lockey RF (1989) Systemic and local allergic reactions to the tine test purified protein derivative. JAMA 262:2999-3000

Organ-related Chapters

16 Childhood Tuberculosis PETER R.

DONALD

CONTENTS Introduction 243 Epidemiology of Childhood Tuberculosis 243 Pathogenesis and Clinical Manifestations of Childhood Tuberculosis 245 Central Nervous System Tuberculosis 248 16.4 Intracranial Tuberculomata 250 16.5 16.6 Childhood Tuberculosis and HIV/AIDS 251 16.7 Diagnosis of Tuberculosis in Childhood 251 Tuberculosis in Infancy 254 16.8 Tuberculosis During Adolescence 255 16.9 16.10 Treatment of Tuberculosis in Childhood 256 16.11 Drug-resistant Tuberculosis in Childhood 257 16.12 Prevention and Control of Tuberculosis in Childhood 258 References 261

16.1 16.2 16.3

tion. It is of considerable interest that the frequency with which these various features are seen varies with age and therefore probably reflects changes in the immune response to tuberculosis infection. Perhaps because of this wide spectrum of pathology, childhood tuberculosis will be looked upon by some as a relatively benign manifestation of tuberculosis infection and regarded by others as a major child health problem, particularly in developing countries. This chapter will describe the epidemiology of tuberculosis in childhood, the most important clinical features of childhood tuberculosis, the interaction of childhood tuberculosis and HIV/AIDS, and the diagnosis and management of childhood tuberculosis.

16.1 Introduction

16.2 Epidemiology of Childhood Tuberculosis

Childhood tuberculosis refers to a wide spectrum of manifestations of tuberculosis seen in children from birth to adolescence. At one extreme, particularly in the very young, are life-threatening disseminated forms of tuberculosis, such as tuberculous meningitis and miliary tuberculosis. At the other extreme, enlargement of the mediastinal lymph nodes may pass entirely unnoticed, and the development of a positive tuberculin test will be the only sign that tuberculosis infection has occurred. As the children enter adolescence, "adult-type" pulmonary tuberculosis will be seen with increasing frequency, characterized by the development of cavitation involving mainly the apices of the lungs. Also at this age, large pleural effusions with straw-coloured fluid become more common as a manifestation of primary infec-

The incidence of childhood tuberculosis will be determined by the frequency of exposure to a source of infection, which will nearly always be an adult with sputum microscopy smear-positive, cavitating pulmonary tuberculosis. In developed communities, tuberculosis has become mainly a disease of the elderly, with a male predominance, occurring amongst the economically deprived, living on the fringes of society. Small foci of disease may also be found amongst disadvantaged groups such as immigrants. Children will thus not often be exposed to the risk of infection and disease. In developing communities, tuberculosis will be a disease of young adults, often with a female predominance. Children aged 15 years or less may constitute 40% or more of the population, and it is therefore not surprising that the incidence of childhood tuberculosis is far higher in a developing community than a developed community and that children make up a far greater portion of the tuberculosis case load. Thus, not only are there more young children in these communities, but they will be exposed to tuberculosis infection at a much

P. R. DONALD, MD, FRCP (Edin), FCP (SA), DCH (Glasgow), DTM&H (London) Department of Paediatrics and Child Health, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, 7505, Tygerberg, South Africa M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

244

younger age than would be the case in a developed community. Because more serious forms of tuberculosis such as miliary tuberculosis and tuberculous meningitis tend to occur in younger children, these potentially deadly forms of tuberculosis will occur more frequently in developing communities. During the last decade tuberculosis has been acknowledged as a worldwide threat to health, and in 1993 the World Health Organization declared the tuberculosis situation in the world a global emergency. Although considerable effort has since been expended in promoting the diagnosis of adult pulmonary tuberculosis by sputum smear microscopy and curing patients with directly observed short course treatment (DOTS), the burden of childhood tuberculosis frequently goes unrecognized. In developed countries, this is understandable as children may constitute less than 5% of the tuberculosis case load; in developing countries, however, this percentage may rise to 20% or even 40% in certain high-incidence countries and communities. Because of the difficulty of diagnosing tuberculosis in childhood, estimates of the global burden of childhood tuberculosis are at best an educated guess. In an often quoted estimate, the number of cases of childhood tuberculosis occurring in 1990 was put at 1.3 million, leading to 450,000 deaths (Kochi 1991). In other estimates, an arbitrary figure of 10% appears to have been used to calculate the portion of the tuberculosis burden attributable to children. Thus, it was predicted that during 2000 just over 1,000,000 cases of childhood tuberculosis would occur in the world. In the African region, however, under the influence of HIV/AIDS, 447,000 children would make up 20% of the tuberculosis burden, in contrast to the 1990 estimate of 90,000 cases of childhood tuberculosis for the region (Dolin et al. 1994). That such a dramatic increase has in fact occurred is suggested by data from several sources. In Malawi, for example, the number of children 0-14 years of age with tuberculosis managed at the Queen Elizabeth Central Hospital increased from 64 in 1986 to 507 in 1995. In 1995 and 1996,64% of children being treated for tuberculosis were mY-seropositive (Harries et al. 1997). In contrast to the above estimates, which must be open to considerable doubt, the risk of infection to which children are exposed can be objectively quantified by determining the annual risk of tuberculosis infection (ART!). ART! is determined by tuberculin testing a representative group of children at regular intervals to detect the incidence of infection and is considered one of the most objective ways to evaluate the tuberculosis situation in any community (Bleiker

P. R. Donald

1991). While ART! in most developed countries will be in the region of 0.01 %, it may rise as high as 2% or 3% in developing countries, implying that between a third and a half of the population will be infected by 15 years of age. From this figure it then becomes possible to predict more accurately the number of cases of childhood tuberculosis that are likely to arise in a particular community. It is disturbing that several recent studies from the developing world have shown no fall in ART! over long periods. In the Chingleput district of south India, for example, ART! has remained at 2% since observations started in 1968 (Tuberculosis Research Centre 2001). In Tanzania, ART! was 1.0% during 1988-1992 and 0.9% during 1993-1998. In this instance, the relatively constant ART! is reason for a certain amount of optimism, in that adult tuberculosis notifications have doubled under the influence of mY/AIDS during this same period (Tanzania Tuberculin Survey Collaboration 2001). This "success" is ascribed to the early adoption and implementation of the DOTS strategy by Tanzania. Figure 16.1 illustrates the age- and sex-related incidence of tuberculosis up to the age of 20 years in a developing community. There are several noteworthy features. Firstly, there is a very high incidence of tuberculosis in a developing community in early childhood, with a slight but consistent male predominance in the first year of life. This susceptibility of the young infant to tuberculosis is even more striking when considered as a proportion of the infected population, and very high rates of disease following infection of 5,000 to 6,000/100,000 population have been calculated for infected infants (Rich 1951; Bentley et al. 1954). Not only are the very young more susceptible to tuberculosis disease, but the disease is c:

o

~::>

250

Co

8. 8o 200 o o....

g 150

Tuberculous meningitis Miliary tuberculosis Lymphobronchial tuberculosis 'Adult type' tuberculosis

C1l

"t:l

'0

.5

'" 100

.~

~ 2 ~

501....&........- -......- _ - -.....-__.--_

Fig. 16.1. Age- and sex-related incidence of tuberculosis up to the age of 20 years in a typical developing community

245

Childhood Tuberculosis

likely to be more serious, disseminated and to take the form of tuberculous meningitis or miliary tuberculosis. With increasing age both the frequency and severity of disease decline until a nadir is reached between 5 and 10 years of age, the so-called "safe school age" of the old German authors. As adolescence is entered, the incidence of disease rises again, "adult-type" pulmonary tuberculosis with apical cavitation becomes the norm, and large pleural effusions with a straw-coloured exudate are frequently encountered.

16.3 Pathogenesis and Clinical Manifestations of Childhood Tuberculosis Following infection, the clinical manifestations of tuberculosis will develop in a certain pattern, which was described as the "time-table of tuberculosis" by the great Swedish paediatrician, Arvid Wallgren (1948). As a generalization it is usually stated that disease will occur in approximately 10% of individuals following infection and that the greatest risk for most manifestations of disease is in the 3-12 months following upon infection, although a lifetime risk of disease does remain. In some communities disease may follow infection in as many as 15% or 20% of individuals. The "timetable of tuberculosis" is summarized in Fig. 16.2. Within 3-8 weeks of infection, the majority of infected individuals will become tuberculin-positive

Time after primary infection

and experience an unobtrusive ,fever of onset' (Wallgren 1928). By 1 month later, the features of primary tuberculosis will start to be seen, and by the end of one year after infection, most cases of tuberculous meningitis, miliary tuberculosis and pleural effusion will have occurred. In contrast, although many cases of osteoarticular tuberculosis will develop within a year ofinfection, they will continue to occur for several years after infection. Interestingly' although haematuria will often be detected shortly after infection and M. tuberculosis can be cultured from the urine, renal tuberculosis will seldom be seen sooner than 5 years after infection (Munro 1944). Following upon infection, two events playa major role in determining the clinical manifestations of childhood tuberculosis. Firstly, before the establishment of cellular immunity to M. tuberculosis, lymphohaematogenous dissemination of organisms occurs and will distribute small numbers of organisms throughout the body, but particularly to sites with a good blood supply. In addition to reaching the bone ends, meninges and kidneys, bacilli also again reach the lungs and in the form of Simon foci may later give rise to adult tuberculosis. Should exceptionally large numbers of organisms be liberated into the blood by erosion into a blood vessel within a lymph node or following the establishment of tubercles within a blood vessel, a tuberculous septicaemia ensues, and small tubercles will develop throughout the body. In the lungs, these give rise to small, uniformly sized nodules the size of millet seeds on chest radiography, and hence the name miliary tuberculosis (Fig. 16.3). The second important factor in the pathogenesis of childhood tuberculosis is the role of the medias-

Clinical Manifestation Fever ofOnset

2 - 3 months Tuberculin Test Positive

Primary puhnonary TB TB Meningitis Miliary TB TB Pleural effusion

3 - 12 months

I

I

ttl

oS

is ~

isc.. 0

§'"

f I ()

0

.3.

~

~:

r;;'

6 - 24 months

II

Osteo-articular TB

> 5 years

II

RenalTB

Fig. 16.2. The timetable of tuberculosis

Fig.16.3. Massive haematogenous dissemination of bacilli leading to multiple small nodules less than 2 mm in diameter evenly distributed throughout the lungs

246

tinal nodes which form part of the primary or Ghon complex in the lungs. Following inhalation of a small droplet 5-10 flm in diameter containing a very small number of M. tuberculosis organisms, a tuberculous focus becomes established in the lungs. From the primary or Ghon focus organisms spread via the lymph ducts to the regional lymph nodes. Most often, only the nodes, either hilar (Fig.16.4) or paratracheal (Fig. 16.5} or both, are seen on a chest radiograph. Occasionally, the primary focus is visible or more commonly it may become visible some 6-9 months later as calcification develops. In a majority of cases, there is only a single primary focus, but in approxi-

P. R. Donald

mately 20% of individuals two or more primary foci may be seen, as illustrated in Fig. 16.6. Enlargement of the hilar nodes with or without ulceration gives rise to a spectrum of pathological changes including segmental or lobar collapse or hyperinflation, collapse consolidation, and segmental or lobar expansile consolidation with or without cavitation. With complete obstruction, collapse of a lobe or segment will develop (Fig. 16.7}. With partial obstruction and a ball-valve effect, hyperinflation of a lobe or segment or a whole lung may develop (Fig. 16.8). Ulceration of a node and discharge of its contents into the relevant bronchus may lead to collapse consolidation or in the absence of obstruction to a lobar or segmental opacification or the appearance of an expansile pneumonia. The histology of these lesions may vary according to the immune response and the numbers of organisms present in the lesion (Seal and Thomas 1956). At one extreme, a benign ,tuberculin' response may be seen with a good prognosis and clearing within 2-3 months. At the other extreme, caseation with cavity formation may develop and lead to bronchiectasis and permanent lung damage (Fig. 16.9}. Should partial obstruction also be present, a grossly enlarged tension cyst may be formed. This course of events is summarized in Fig. 16.10. In the past, these usually well defined lesions were referred to as epituberculosis, segmental tuberculosis, or endobronchial tuberculosis. They are perhaps best grouped together

Fig. 16.4. Enlargement of the hilar nodes in a child 2 years of age with a Mantoux test giving an induration of 20 mm and the gastric aspirate positive on culture for M. tuberculosis

Fig. 16.5. Enlargement of the right paratracheal nodes in a 5year-old child. Sputum positive on culture for M. tuberculosis

Fig. 16.6. Right-sided paratracheal enlargement with calcification appearing in the nodes and in three right-sided primary foci in a 3-year-old child. Gastric aspirate positive on culture for M. tuberculosis

Childhood Tuberculosis

247

Fig. 16.7. Collapse of the right middle lobe in a 2-year-old child who presented with persistent cough and wheeze. Gastric aspirate positive on culture for M. tuberculosis. A Mantoux test gave an induration of 18 mm

b

Fig. 16.8. Enlargement of the paratracheal nodes with hyperinflation of the right lung in a 2-year-old child with gastric aspirate positive on culture for M. tuberculosis

Fig. 16.9. Consolidation of the left upper lobe with several cavities enlarged by pressure inflation. A fine nodularity is also present in the right lung, which may indicate bronchogenic dissemination and a small left-sided pleural reaction. Gastric aspirate positive on culture for M. tuberculosis

as lymphobronchial tuberculosis, thus emphasizing the role of both lymph node enlargement and bronchial disease in determining the varied features of these lesions which, none the less, have a common origin. Any primary tuberculosis infection will be accompanied by a greater or lesser degree of lympho-hae-

matogenous dissemination of organisms throughout the body. These have a predilection for certain organ systems where they may establish themselves and give rise to disease if not controlled by the individual's immune response. The most important site where extrapulmonary disease may arise in childhood is the central nervous system.

P. R. Donald

248

Benign "Tuberculin Response"

Fig.16.10. Lymphobronchial tuberculosis: the spectrum of possible consequences of tuberculous involvement of the mediastinal nodes during primary tuberculosis

16.4

Central Nervous System Tuberculosis Tuberculous meningitis (TBM) is the most serious complication of childhood tuberculosis and has its highest incidence between the ages of 2 and 3 years. At this age it is often associated with miliary tuberculosis, and if untreated a majority of children with miliary tuberculosis will die of TBM. In both developed and developing countries the diagnosis of TBM is frequently delayed, with disastrous consequences for the patient as the prognosis is closely linked to the stage of the disease at the time of diagnosis. A classification of the stage of TBM developed by the British Medical Research Council in 1948 is still useful. Patients with early signs of meningeal irritation but no other focal signs are at stage I TBM. Patients diagnosed at this point should recover fully. When focal neurological signs appear or the patient is mentally confused, the disease is considered to be at stage II. Although the majority of patients presenting at stage II will survive, they are likely to do so with a physicalor intellectual deficit. By stage III, the patient is comatose, unable to localise pain, and may have a complete hemiplegia or rarely a quadriplegia. A third of these patients may die, and the great majority of survivors will have severe physical and mental handicaps (Medical Research Council 1948). Although TBM may occur at any time following primary infection, it occurs most often approximately 3 months after primary infection as result of the rupture of a parenchymal focus, or less often a

meningeal focus into the cerebrospinal fluid (CSF). This release of antigens into the CSF elicits a severe inflammatory response, which gives rise to a thick gelatinous exudate. This exudate envelops the base of the brain and the adjacent structures. This in turn leads to cranial nerve palsies, vasculitis, and raised intracranial pressure due to obstruction to the flow of CSF. The vasculitis affects the larger blood vessels such as the middle cerebral artery and the smaller blood vessels of the basal ganglia and will often lead to infarctions and irreversible brain damage. TBM usually has an insidious onset lasting a week or more marked by increasing lassitude, loss of appetite and irritability. Occasionally, a more acute onset may be seen resembling that of other forms of bacterial meningitis. Many children will experience a preceding loss of weight. Common presenting complaints include vomiting, cough, fever, apathy and headache; this last complaint being seen mainly in children older than 3 years. Because of the non-specific nature of these features, a high index of suspicion is necessary when these complaints persist. A tuberculin test, a chest radiograph or enquiry as to household contact with an adult with ,smear-positive' pulmonary tuberculosis may lead to the early diagnosis of TBM or to the prevention of TBM in some instances by the early prescription of antituberculosis treatment. In any case where doubt is present, a lumbar puncture should be done. The CSF in TBM will typically be clear with a mean cell count of approximately 100Xl06/L, predominantly lymphocytes, a mean protein of 1.5 giL, and a mean glucose of 1.8 mmollL. It is very important to note that this is a typical picture. In 5%-10% of cases, a cell count of >500x106 /L with a predominance of polymorphonuclear leucocytes may be found; CSF protein levels may be normal or between 0.45 giL and 0.8 giL in close to 20% of cases; the CSF glucose may be >2.2 mmollL in as many as 30%-40% of cases (Donald et al. 1991). There is thus ample room for mistakes to be made. In any case of doubt, it is better to initiate antituberculosis treatment and review the diagnostic evidence later. The diagnosis of TBM is confirmed by a culture of M. tuberculosis from the CSF. This is, however, positive in only a minority of cases. This is probably due to the relatively small amounts of CSF that are submitted for culture from children. Support for the diagnosis is provided by culture of M. tuberculosis from another source such as gastric aspirate or lymph node biopsy, a chest radiograph suggestive of tuberculosis, a positive Mantoux test, or a history of close contact with an adult with sputum "smear-positive" tuberculosis.

Childhood Tuberculosis

249

A chest radiograph may show evidence of respira- raised CSF white cell count or protein concentration tory tuberculosis in up to 70% of children with in such cases would provide sound support for a deciTBM, while a Mantoux test should be positive in a sion to continue antituberculosis treatment. The management of TBM involves three aspects: majority of children. It must be emphasized, however, that a negative Mantoux test does not exclude TBM. the elimination of viable M. tuberculosis from the Approximately 50% of children with TBM will have lesions, the control of raised intracranial pressure a history of contact with an adult with pulmonary and the prevention of further brain damage related tuberculosis, and in many cases this will be a family to the presence of the basal exudate, and the associated hypersensitivity and vasculitis. member, often the mother or a grandparent. There are almost no controlled trials evaluating Cranial computed tomography or magnetic resonance imaging, if available, may also assist and show different regimens for treating TBM. Consequently, hydrocephalus associated with the basal enhance- a variety of recommendations can be found in the literature and various official documents. Standard ment and possibly cerebral infarctions (Fig. 16.11). Other diagnostic aids include CSF polymerase short course regimens given for 6 months and all chain reaction, the detection of tuberculostearic acid containing rifampicin for the full 6 months have been in the CSF, CSF adenosine deaminase concentrations, reported to be successful by several groups (Biddulph CSF lactate or lactate dehydrogenase concentrations, 1990; Jacobs et al. 1992). Our own preference is for a and the bromide partition test. None of these tests at 6-month regimen of isoniazid, rifampicin, pyrazinpresent has a totally satisfactory sensitivity or speci- amide and ethionamide, with isoniazid and rifampificity, and while they may be valuable in individual cin given in a dose of 20 mg/kg body weight. In our patients, the final decision as to whether to treat a region there is an overall incidence of isoniazid resispatient remains a clinical one assisted mainly by the tance of 10%. Approximately half of these isolates are resistant at isoniazid concentrations of <5 fig/ml, a clinical features and the CSF results. In cases of doubt it can sometimes be valuable concentration easily exceeded even in fast acetylto repeat the lumber puncture a week or two after ators by giving a higher dose of isoniazid (Donald treatment has been started. It would be most unusual et al. 1992). Rifampicin is a critical sterilizing agent for the CSF findings to normalize in such a relatively in tuberculosis regimens. It is, however, 80% protein short space of time in the case of TBM. A persistently bound, and relatively low concentrations enter the CSF when given in a dosage of 10 mg/kg (Humphries 1992). Ethionamide is notorious for causing gastric irritation and nausea but obtains a satisfactory entry into the CSF (Donald and Seifart 1992) and is much better tolerated by children than adults. We have used this regimen in over 200 patients to date and have experienced relatively few problems with drug toxicity; we documented relapse in only one patient in whom the dosages of the drugs were reduced after 2 months of treatment. If the intracranial pressure is monitored, it will be found to be raised in nearly all children presenting with stage II or III TBM. In those children who have a communicating hydrocephalus and the absence of other intracranial pathology has been confirmed by computed tomography or magnetic resonance imaging, raised intracranial pressure can be successfully managed by using furosemide (1 mg/kg/day) and acetazolamide (100 mglkg/day). The presence of communication between the ventricles and the spinal Fig. 16.11. Cranial computed tomography with contrast in a canal can be demonstrated by injecting air at the time 2-year-old child with tuberculous meningitis showing acute of lumbar puncture and taking an X-ray of the skull, hydrocephalus and periventricular oedema. Diffuse enhancewhich should show air in the ventricles (Fig. 16.12). If ment in the territory of the right middle cerebral artery is present indicating ischaemia. A mass effect also indicating air is seen at the base of the brain and not in the ventricles, a non-communicating hydrocephalus is probably ischaemic changes is seen in the right caudate nucleus

250

Fig. 16.12. Skull radiograph of a child with tuberculous meningitis. Air was injected at the time of lumbar puncture and is seen at the base of the brain and in the ventricular system, indicating that a communicating hydrocephalus is present

present, and a ventriculo-peritoneal shunt is urgently required to relieve the raised intracranial pressure. As tuberculin hypersensitivity is thought to play an important role in the formation of the basal exudate in TBM and its associated vasculitis, there is a long tradition of using corticosteroids to manage this problem. At first, it was reported that corticosteroids decreased mortality but did not affect the outcome amongst survivors (Freiman and Geefhuysen 1970; Shao et al. 1980). In a later study corticosteroids improved not only morbidity but also outcome (Girgis et al.1991). In a more recent, randomised trial, prednisone was also shown to improve morbidity and outcome. Interestingly, it did not appear to have any effect upon raised intracranial pressure (Schoeman et al. 1996). Tuberculomata, when present, resolved more quickly, and fewer new tuberculomata developed in those patients receiving corticosteroids.

P. R. Donald

In a review of 202 cases of TBM seen at our hospital, associated granulomas were present in 34 (17%) children (Ravenscroft et al. 2001). Two necrotic processes occur in these lesions. In gummatous necrosis the inflammatory granulation tissue itself becomes necrotic, and the fibrovascular stroma can be identified on silver reticulin staining. The process can repeat itself intermittently, giving rise to a lamellated appearance. In the second process there is no fibrovascular component, and the consistency of the contents can vary from caseous to purulent liquid. Gummatous necrosis is isodense or hyperdense on cranial CT, while caseous necrosis is hypodense or isodense, reflecting the more fluid character of the contents (Rutherfoord and Hewlett 1994). Intracranial tuberculomata may be asymptomatic and discovered by chance or present with convulsions or signs or symptoms of raised intracranial pressure. Unless the lesion is situated at some critical point in the central nervous system, medical treatment as for TBM and augmented by corticosteroids will usually be sufficient. As is the case with tuberculous cervical lymphadenopathy, it is not unknown for these lesions to enlarge paradoxically despite successful antituberculosis treatment. Should the lesion give rise to symptoms, either due to its location or due to raised intracranial pressure, a course of corticosteroids may expedite the shrinkage of the lesion.

16.5 Intracranial Tuberculomata Tuberculomata are common intracranial tumours in those communities where tuberculosis is common. They may be single or multiple in 15%-20% of cases (Jamieson 1995). They may occur in isolation or in association with tuberculous meningitis (Fig. 16.13).

Fig. 16.13. Computed cranial tomography with contrast in a child with stage II tuberculous meningitis. There is meningeal enhancement in the left Sylvian fissure and multiple, homogeneously enhancing granulomata

251

Childhood Tuberculosis

16.6 Childhood Tuberculosis and HIVI AIDS The last decade has witnessed the continued spread of HIV/AIDS throughout the world and its pernicious effect upon the incidence of tuberculosis. Nowhere has the deleterious interaction of these two formidable diseases been more dramatic than in the countries of sub-Saharan Africa where the tide of HIV/AIDS and tuberculosis is threatening to overwhelm the already struggling health services. Not surprisingly, this dual epidemic is also affecting childhood tuberculosis, and very high rates of HIV seropositivity have been recorded amongst children with tuberculosis (Chintu et al. 1993; Harries et al. 1997). Because of the difficulty ofconfirming the diagnosis of tuberculosis in children, it is uncertain how many of these children really do have tuberculosis. In addition to tuberculosis, these HIV-infected children are also susceptible to a variety of other bacterial pathogens including S. pneumonia, H. influenza and S. aureus as well as pathogens such as Pneumocystis carinii, respiratory syncytial virus and cytomegalovirus (Graham et al. 2001). Lymphocytic interstitial pneumonitis frequently presents with a reticulonodular appearance and hilar adenopathy on chest radiographs and can easily be confused with miliary tuberculosis. In a study from Johannesburg, M. tuberculosis was isolated from 8.5% of 423 HIVinfected children and 7.6% of 434 uninfected children (Mahdi et al. 2000). In Cape Town M. tuberculosis was similarly isolated from 10% of HIV-infected children and from 14% ofa group of non-HIV-infected children (Zar et al. 2000). Amongst children with lung disease persisting more than a month, M. tuberculosis was isolated from 29% of both HIV-infected and non-HIVinfected children (Jeena et al. 1998). A post-mortem study of 32 HIV-infected children from Botswana confirmed the presence of tuberculosis in 4 (l2.5%) (Ansari et al. 1999). These findings all suggest that tuberculosis is definitely a problem in HIV-infected children and that a high index of suspicion is always required. In an endemic area, however, it is but one of a number of other infections to which these children are subject. HIV-infected children may present with all of the features of childhood tuberculosis seen in immunocompetent children. Several studies from the developed world have found an increased incidence of extrapulmonary tuberculosis, as is the case in HIVinfected adults (Chan et al. 1996; Khouri et al. 1987; Thomas et al. 2000). This has, however, not been the case in studies from Africa (Chintu and Zumla 1995). Despite this, it is necessary to consider tuberculosis in

the differential diagnosis of any lesion not responding to conventional treatment in these children.

16.7 Diagnosis of Tuberculosis in Childhood The diagnosis of tuberculosis in childhood requires a high degree of suspicion but at the same time a balanced clinical perspective. Suspicion is required, as the features of childhood tuberculosis may be subtle and non-specific. Failure to gain weight properly may be the only clinical sign that a child is in the active phase of primary tuberculosis infection. Balance is required, as the urgency of making a diagnosis of tuberculosis must be weighed against factors such as the child's age and the severity of the child's illness. In a child 8 years of age with a subtle picture of hilar enlargement on chest radiography and a vague history of indisposition, one may safely choose to await further developments and the results of investigations before making a final diagnosis. In a young infant with an enlarged liver and spleen and an illdefined nodular opacification on chest radiography, it is a matter of urgency to commence antituberculosis treatment as soon as possible and to dispute later as to the accuracy of the diagnosis. In an adult it is unwise to accept a diagnosis of tuberculosis without microbiological confirmation either by way of culture of M. tuberculosis or by visualization of acid-fast bacilli on microscopy of a clinical specimen. Childhood tuberculosis is paucibacillary, and cavitation of the lung tissue is relatively unusual. Consequently, as few organisms are excreted, it would be equally unwise to await . microbiological confirmation of the diagnosis of childhood tuberculosis before commencing treatment. Even with intensive investigation of children with probable tuberculosis in specialist institutions, microbiological confirmation of the diagnosis will seldom be obtained in more than 50% of cases (Schaaf et al. 1995; Smith et al. 1996; Lobato et al. 1998). In the community, where most cases of childhood tuberculosis must be diagnosed and managed, fewer than 10% of cases are likely to be confirmed by culture or microscopy (Jacobs et al. 1987). In view of the lack of sensitivity of gastric aspirate culture and microscopy to detect M. tuberculosis in children, attempts have been made to use PCR in the detection of mycobacterial DNA. While satisfactory sensitivity and specificity have been claimed by some (Pierre et al. 1993; Delacourt et al. 1995), other

252

P. R. Donald

investigators consider the technique to be unreliable at present (Noordhoek et al. 1994; Smith et al. 1996). Unfortunately, whatever one's views of this technology, it is unlikely to be available in precisely those communities where most cases of childhood tuberculosis occur. The diagnosis of childhood tuberculosis consequently remains a clinical process drawing on the patient's history, clinical examination, chest radiography, tuberculin testing and lastly culture of gastric aspirate or sputum in older children. Making use of this process, the World Health Organization has recommended a hierarchical approach leading to children being classified as suffering from suspect, probable or confirmed tuberculosis. This approach is summarized in Table 16.1 (World Health Organization 1989). A history of close household contact with an adult with microscopy smear-positive pulmonary tuberculosis should immediately raise the suspicion that infection may have occurred. From one-third to twothirds of children so exposed may be infected (Brailey 1940; Shaw and Wynn-Williams 1963; van Geuns et al. 1975). It is also recommended that a cough and wheeze not responding to appropriate treatment, failure to recover from a preceding illness, or loss of weight or overt malnutrition should also raise the suspicion of tuberculosis in a child. The sensitivity and specificity of these criteria have, however, seldom been determined. Amongst a group of 627 children <5 years of age attending an outpatient department in an area with a particularly high incidence of tuberculosis, 206 (33%) were found to have one or more of the above criteria for suspect tuberculosis (Houwert et al. 1998). The children were evaluated by gastric aspi-

rate, Mantoux testing and chest radiography. After reviewing the diagnostic criteria 2 months later, 23 children (11 %) were considered to have probable tuberculosis, and a further 10 (5%) had confirmed tuberculosis. The most rewarding criterion was close household tuberculosis contact, and 23 (35%) of such children had either confirmed or probable tuberculosis compared with 13 (22%) of 46 children with persistent cough and 24 (15%) of 157 children with loss of weight. Of 11 children who had all 3 criteria, 7 (63%) had probable (4) or confirmed (3) tuberculosis. Since this study the spread of HIV/AIDS has complicated the interpretation of these diagnostic criteria considerably. Many children with HIV/AIDS will have lost weight, many will be exposed to household tuberculosis contact and will be suffering from recurrent or persistent respiratory infections. As the resources of many developing countries to carry out further diagnostic investigations in children are limited, it seems inevitable that many HIV-infected children who do not have tuberculosis will be placed on antituberculosis treatment. In another study children who presented with probable tuberculosis were investigated. There was no difference between the proportion of children later thought to have probable or confirmed tuberculosis and those found not to have tuberculosis in the presence of clinical features such as weight loss, cough or wheezing lasting more than 2 weeks, hepatosplenomegaly or peripheral lymphadenopathy. Furthermore, in this high tuberculosis incidence population, although 43% and 56%, respectively, of the children with confirmed and probable tuberculosis had an adult household contact with tuberculosis, so did 21 % of those later thought not to have tuberculosis. To add further diagnostic confusion, 10% of

Table 16.1. World Health Organization (1989) provisional guidelines for the diagnosis of pulmonary tuberculosis in children A. Suspect tuberculosis

B. Probable tuberculosis

C. Confirmed tuberculosis

1. An ill child with a history of contact with a confirmed case of pulmonary tuberculosis

A suspect case and any of the following:

2. Any child:

1. Positive (~10 mm) induration on tuberculin testing

1. Detection by microscopy or culture of tubercle bacilli from secretions or tissues or 2. The identification of the tubercle bacilli as Mycobacterium tuberculosis by culture characteristics

2.1 Not regaining normal health after measles or whooping cough 2.2 With loss of weight, cough and wheeze not responding to antibiotic therapy for respiratory disease 2.3 With painless swelling in a group of superficial nodes

2. Suggestive appearances on chest radiograph 3. Suggestive histologic appearance of biopsy material 4. Favourable response to specific antituberculous therapy

Childhood Tuberculosis

253

Tuberculin testing is one of the most important the children with tuberculosis confirmed by culture had a chest radiograph which was considered normal investigations supporting the diagnosis of tubercuon blind reading by an independent panel (Schaaf et losis in a child. It must, however, be emphasized that al. 1995). This last finding is not unexpected, as it a positive tuberculin test, however defined, means is known that when such children are evaluated by only that a child has been infected by M. tuberculosis. chest computed tomography, hilar nodal enlarge- The younger the child, the more significant a positive ment will frequently be visible (Delacourt et al.1993). tuberculin test is as infection is likely to have occurred This finding does emphasize the uncertain division recently, and infection in a young child is more likely between what clinicians regard as infection only and to proceed to disease than in an older child. disease in childhood tuberculosis. The tuberculin test must be interpreted in the light In any area where childhood tuberculosis is of the child's clinical condition, the epidemiological common, children will also suffer from a multitude of background and whether or not BCG vaccination has other infectious, allergic and congenital conditions, been administered. There is also a small number of and will often be malnourished to a greater or lesser individuals who appear unable to respond to tubercudegree. It is not surprising that many of these chil- lin, perhaps on a genetic basis (van Eden et al. 1983). dren will be inappropriately treated for tuberculosis. Recent childhood viral disease, live virus vaccines Table 16.2 lists some of these conditions which in our such as measles and rubella vaccines, severe malnuexperience have caused diagnostic problems. When trition, overwhelming tuberculosis disease, corticotuberculosis treatment of a child is commenced on steroid therapy and HIV/AIDS may all be responsible uncertain grounds, it is essential for the clinician to for a false-negative tuberculin test. Nearly two-thirds keep the case under review and to consider other of HIV-infected children with tuberculosis may have a negative tuberculin test (Chan et al. 1996; Mukadi diagnostic options. Culture and microscopy of gastric aspirate are et al. 1997). Nonetheless, even in communities with a the means by which tuberculosis will most often be particularly high incidence of tuberculosis and HIV confirmed in childhood. Although a relatively simple infection, the tuberculin test remains a valuable diagprocedure, attention to detail will improve the results nostic aid. The majority of children with tuberculo(Pomputius et al. 1997). Although hospitalization is sis in these communities will not be HIV-infected, usually considered necessary for gastric aspirate, this and even amongst those who are HIV-infected, the is not necessarily so (Lobato et al. 1998). Alternative suppression of tuberculin hypersensitivity will be means of collecting specimens for culture from chil- related to the stage of AIDS in each child (Schaaf et dren are nasopharyngeal aspirate, which could also al. 1998). be used in community work (Franchi et al.1998), and In individuals who have received BCG vaccine, a sputum induction (Zar et al. 2000), both of which will transverse induration of ~15 mm should be taken give a comparable yield of positive cultures. When- to indicate M. tuberculosis infection, where BCG has ever sufficient resources are available, every attempt not been given ~10 mm, and in those who are HIVshould be made to confirm the diagnosis of tuber- infected ~5 mm (Harries and Maher 1996). culosis in a child. A successful culture may reveal Chest radiography may provide valuable supportunexpected drug resistance (Steiner et al. 1985), ing evidence of the presence of unsuspected tubercuwhile RFLP strain typing may explain unexpected losis in a child and is often the means by which the transmission patterns (Barnes et al. 1997). extent of disease will be evaluated. It is therefore nec-

Table 16.2. Differential diagnosis of childhood tuberculosis Segmental or bronchopneumonic opacification with or without cavitation on chest radiograph Hilar or paratracheal adenopathy Asthma with a mucous plug Foreign body Bronchial compression due to tumours, congenital abnormalities Lobar emphysema Bronchiectasis Recurrent aspiration

Cystic fibrosis Hypersensitivity syndromes Loeffler syndrome

Lymphoma Teratoma Neurofibroma

Tropical eosinophilia Necrotizing bacterial pneumonias Lung abscess Diaphragmatic hernia Echinococcus cysts

Other tumours and malignancies Fungal infections Sarcoidosis Bronchogenic or dermoid cyst

254

P. R. Donald

b

a Fig. 16.14a,b. Chest radiographs of a 3-year-old child presenting with recurrent cough and wheeze. a Chest radiograph taken in poor inspiration showing possible hilar enlargement. b A chest radiograph of the same child taken 30 min later showing the shadow to be the descending branch of the right pulmonary artery

essary that the clinician be aware of the importance of a good quality chest radiograph. Common technical faults in children's chest radiographs include inadequate inspiration, excessive lordosis, rotation of the chest, and faulty exposure or processing, leading to a film that is overexposed or underexposed. One of the most common problems is poor inspiration, leading to excessive prominence of the descending branch of the right pulmonary artery (Grzybowski 1954). To the inexperienced this can easily assume the appearance ofhilar adenopathy (Fig. 16.14). In babies up to the age of 18 months, the thymus will frequently be visible and give rise to confusion, particularly if the shadow is atypical or the radiograph rotated or taken in poor inspiration (Fig. 16.15).

of the neonate (Pillay et al. 2001; Ahmed et al. 1999; Adhikari et al. 1997). Infection of the very young infant or neonate may be the result of transplacental infection in utero or infection may take place during or shortly after birth. If infection is antenatal and transplacental, the primary focus of infection will be in the liver, and there will be enlargement of the regional nodes in the porta hepatis. These infants are often symptomatic, with loss of weight, tachypnoea and wheezing or bronchial breathing audible on auscultation (Schaaf et al.I993).

16.8 Tuberculosis in Infancy If untreated, tuberculosis has its highest mortality and morbidity during infancy, and the consequences of a delay in diagnosis and treatment are potentially more serious at this age than in older children. In the experience of Wallgren (1938), 36% of infants less than 1 year of age at the time of diagnosis died, while Edith Lincoln (1950) reported that 55% of infants diagnosed when less than 6 months of age died. With the spread of HIVI AIDS, tuberculosis has become a common cause of obstetric mortality and morbidity, and this has inevitably increased the risk of antenatal or postnatal tuberculosis infection

Fig. 16.15. A typical thymus shadow projecting into the upper right lung field in a 15-month-old child

Childhood Tuberculosis

Hepato-splenomegaly may be present, and tuberculosis may be a cause of prolonged neonatal jaundice (Schaaf and NelI992). Chest radiography will often show a miliary pattern. Airway narrowing is frequently present and will sometimes be more clearly seen on a high kilo-volt radiograph. Cavitation may be present as early as 5 weeks of age (Cunningham 1982). Gastric aspirate or tracheal aspirate will give a positive culture in a higher percentage of cases than in older children, and as many as 70%-80% of infants may have a positive culture for M. tuberculosis (Schaaf et al.1993; Vallejo et al.1994). Unfortunately, the tuberculin test will frequently be negative during the acute phase of the illness but should not be ignored. Extrathoracic lymph node enlargement may be palpated as early as 4 weeks of age, and hilar node enlargement may be seen on chest radiograph even earlier. Chest radiography and sputum culture of the mother may be rewarding procedures (Schaaf et al. 1991), and the possibility of maternal urogenital tuberculosis should be considered. Other family members and even hospital personnel should not be forgotten as the possible source of infection. Infants born to a mother with tuberculosis are at considerable risk of infection and disease. Even if the mother is sputum microscopy ,smear-negative' but culture positive, a significant risk of infection still exists because of the closeness of the contact between the mother and her newborn infant. A number of studies have shown convincingly that isoniazid will protect such exposed infants against the risk of infection and disease (Dormer et al. 1959; Kendig 1960; Light et al. 1974). If the diagnosis of maternal tuberculosis is made shortly before or shortly after birth, an alternative approach would be to give the infant isoniazid and rifampicin for as long as the mother is herself being treated.

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Tuberculin testing of large groups of adolescents has found similar rates of tuberculosis infection in both genders or a slight male predominance in some cases (Hall 1957). Nonetheless, the risk of developing tuberculosis during adolescence has been found to be 2 to 6 times higher in girls (Smith 1967). The development of tuberculosis is also often associated with menarche (Comstock et al. 1974). It is therefore attractive to ascribe this susceptibility to ,adult-type' tuberculosis to the hormonal and metabolic perturbations of puberty, and Arvid Wallgren (1938) commented, "It is quite likely that the liability to pulmonary tuberculosis (in adolescence) is intimately linked up with endocrine activity". It is now known that the balance between the Thl and Th2 families of cytokines may be regulated by the adrenal steroids. Glucocorticoids may promote a Th2-type response, which would be disadvantageous to the course of tuberculosis, whereas dehydroepiandosterone will tend to promote a Thl-type response (Rook et al. 1993). This is also the age at which large strawcoloured pleural effusions are more often seen following primary infection (Fig. 16.16). It is of interest that despite the higher incidence of adult-type tuberculosis in girls at this age, these large effusions are much more frequent in boys (Bentley et al. 1954; Enarson et al.1982; Weber et al. 2000). If infection occurs after 7 years of age, the chance of developing disease during puberty has been put at 8% in American studies (Lincoln et al. 1960). When tuberculin conversion occurs during adolescence, it

16.9 Tuberculosis During Adolescence Following the onset of puberty there is a rapid rise in the incidence of tuberculosis, although presumably the risk of tuberculosis infection remains similar. The nature of tuberculous disease also undergoes a marked change and now takes the form of,adult-type' disease. This is characterized by the development of apical lesions and the propensity to develop cavitation, which not only contributes to lung destruction but also leads to the expectoration of microscopy smear-positive sputum, so completing the cycle of infection.

Fig. 16.16. A large, right-sided, pleural effusion in a 12-yearold adolescent who presented with fever and right-sided chest pain. The Mantoux test gave an induration of 19 mm, and the sputum was positive on culture for M. tuberculosis, but negative for acid-fast bacilli on microscopy

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is far more likely to be followed by disease. As many as 15% of such recently infected adolescents may develop the disease, and disease is most likely to occur within 1-3 years of infection (Smith 1967). In view of the various hormonal and social adaptations occurring during adolescence, it is not surprising that non-compliance has been identified as a problem in these children (Nemir 1986; Weber et al. 2000). Careful attention to potential problems and community networking is necessary to assist these patients to successfully complete therapy (Wilcox and Laufer 1994). The combination of HIV infection and tuberculosis in adolescence makes compliance even more difficult to achieve (Hoffman et al. 1996).

16.10

Treatment of Tuberculosis in Childhood The principles of the treatment of childhood tuberculosis are the same as those that apply to adults. A 2-month intensive bactericidal phase of isoniazid, rifampicin, pyrazinamide and ethambutol or streptomycin is followed by a 4-month sterilizing continuation phase with isoniazid and rifampicin.. The aim of the intensive phase is to reduce the number of rapidly metabolizing active organisms as quickly as possible. This leads to the symptomatic improvement, a reduction in the risk of infection to others, while the use of a multidrug regimen will prevent the development of drug resistance. The most effective bactericidal drugs are isoniazid, which kills 90% of active organisms within 48 h (Donald et al. 1997), ofloxacin and rifampicin (Sirgel et al. 1993). Most cases of tuberculosis in childhood are paucibacillary and "smear-negative", as children do not often develop cavitation. Consequently, the risk of developing drug resistance is probably not as great as in adults, and the intensive phase can safely be undertaken with isoniazid, rifampicin and pyrazinamide, unless there is a history of contact with a case of drug-resistant tuberculosis. In that case, the regimen should be appropriately adjusted to take into account the resistance pattern of the contact's isolates. Where cavitation is present, it is probably advisable to assume the presence of a reasonably large bacterial population even if the smear is negative, and to use four drugs in the intensive phase as in adults. The 4month continuation phase is also undertaken with isoniazid and rifampicin. The aim of the 4-month continuation phase is the elimination of all remaining residual forms of M.

P. R. Donald

tuberculosis to prevent relapse occurring. The most valuable sterilizing agents are rifampicin and pyrazinamide and to a lesser extent isoniazid (Mitchison 2000). The loss of rifampicin with the development of multidrug resistance necessitates the use of much longer, complicated and potentially toxic regimens. In the case of serious forms of extrapulmonary tuberculosis or disseminated tuberculosis, official documents often recommend the same treatment regimens as for new ,smear-positive' adult pulmonary tuberculosis. More conservative clinicians might prefer to extend the continuation phase to 7 months of isoniazid and rifampicin (Harries and Maher 1996). Our own practice for children with miliary tuberculosis, tuberculous meningitis or other cases where sequestered lesions might be present is to use a combination of isoniazid, rifampicin, pyrazinamide and ethionamide, all given for 6 months (Donald et al. 1998). Isoniazid has excellent pharmacokinetics and a very large therapeutic margin between the highest serum concentration that can be achieved without undue toxicity and the lowest concentration at which a bactericidal effect can still be measured (Donald et al. 1997). Pyrazinamide also has good pharmacokinetics and enters the cerebrospinal fluid with ease (Donald and Seifart 1988), as does ethionamide (Donald and Seifart 1989). Rifampicin, however, is highly protein bound and at the usual dose of 12 mg/kg body weight is probably operating at the lower border of its efficacy. Any further reduction in dose will increase relapse rates (Long et al. 1979) and decrease bactericidal activity (Sirgel et al. 1993). Rifampicin has also been shown to have poor penetration into chronic tuberculous empyema (Elliot et al. 1995) and cerebrospinal fluid, as is the case with ethambutol (Ellard et al. 1993). As ethionamide is better tolerated by children than adults, we advocate its use in preference to ethambutol in those situations where isoniazid resistance is a potential threat or where other drugs, such as rifampicin, may be at a pharmacokinetic disadvantage. The dosages of the more commonly used drugs for the management of tuberculosis in children are given in Table 16.3. In Table 16.4 the dosages of the alternative drugs are given. The use of these agents may be necessitated by the detection or suspicion of drug resistance or by the development of drug toxicity. These agents are as a generalization less efficient in the treatment of tuberculosis and tend to be associated with greater toxicity. Before embarking on their use, it is wise to consult those experienced in their usage. There is good evidence that adult smear-negative pulmonary tuberculosis can be treated for 4 months

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Childhood Tuberculosis Table 16.3. Treatment of tuberculosis in children. Dosage and action of antituberculosis drugs Drug Isoniazid Rifampicin Pyrazinamide Ethambutol Ofioxacin a Streptomycin

Action Bactericidal Sterilizing

Twice weekly intermittent Daily dosage (maximum mg) mglkg (maximum mg) mglkg

+++ ++

10-20 10-20 25-35 15-20 15 20

-

++ ++ +

+ +++ +++ ?a

(300) (600) (2000) (1250) (800) (1000)

20-40 10-20 50-70 50 25-30

(900) (600) (4000) (1250) ? (1500)

aThere is as yet relatively little experience with the use of ofioxacin. It has been shown to have a high early bactericidal activity and is a valuable drug to have in reserve when hepatotoxicity or drug resistance precludes the use of other established drugs. There is, as yet, no information as to its possible sterilizing activity

Table 16.4. Treatment of tuberculosis in children. Dosage of ,second-line' drugs Drug

Daily dosage mg/kg

Maximum dosage

Ethionamide Kanamycin Amikacin Capreomycin Cycloserine Para-aminosalicylic acid Thiacetazonea

15-20 15-20 15-20 15-20 15 300 4

1g 750 mg 750 mg 750 mg 1g 10 g 150 mg

aCommence thiacetazone with 0.5 mglkg for the 1st week, 1 mglkg the 2nd week and 4 mglkg from the 3rd week

with very low relapse rates (Hong Kong Chest Service et al. 1989; Dutt et al. 1989). As most childhood tuberculosis is of limited extent and smear-negative, it seems probable that the same 4-month regimens might also apply to children. However, as there are, as yet, no clinical trials to support this approach, most official documents still recommend 6 months of treatment for all forms of childhood tuberculosis. In contrast to the prospect of, perhaps, shortening the length of treatment for children with tuberculosis, there is increasing evidence that in the presence of HIV infection, treatment should be prolonged to 9 months. Of 14 HIV-infected children with cultureproven tuberculosis, 4 were still culture-positive 4-14 months after commencing treatment (Schaaf et al. 1998). Recently, it was reported that HIV-infected adults with pulmonary tuberculosis treated for less than 9 months had an increased incidence of relapse within 30 days of stopping treatment as compared with those treated for longer than 9 months (Driver et al. 2001). The reasons for this finding are uncertain, but the poor absorption of antituberculosis agents in adults with HIVI AIDS has been documented (Peloquin et al. 1996). Others have not found any associa-

tion between tuberculosis recurrence and antituberculosis drug concentrations in HIV-infected patients (Narita et al. 2001). No similar pharmacokinetic data exist for children. The management of childhood tuberculosis is not a priority in most developing communities, where hard-pressed health service staff have difficulty in ensuring the compliance of smear-positive adult pulmonary tuberculosis patients. Under these circumstances treatment is often given by family members, and there must be some doubt about the extent of compliance that is achieved. Intermittent treatment of children, given two or three times weekly, is an acceptable alternative to daily treatment and can lead to equivalent rates of adherence (te Water Naude et al. 2000). This would also make supervision of treatment easier for the health services. A further problem encountered in the management of childhood tuberculosis is the lack of formulations specifically designed for children. Several commercial fixed dose combination preparations are now available, but in their absence ad hoc preparations must often be constituted by clinic personnel. It is very important that such ad hoc preparations should not be made up before administration and that they should not be mixed with vitamin solutions as this can lead to a rapid decline in the drug concentrations (Seifart et al. 1991).

16.11

Drug-resistant Tuberculosis in Childhood Drug-resistant tuberculosis in children is important from two points of view. Firstly, it is important for the individual patient that it should be diagnosed expeditiously and managed appropriately. This is particularly important in HIV-infected individuals,

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who may deteriorate rapidly and die if appropriate treatment is not started as soon as possible (Small et al. 1993). Secondly, it is important as drug resistance in children will nearly always be primary resistance, and the incidence and patterns of resistance in children will probably reflect those circulating amongst adults in a community (Rieder 1993). Despite early evidence that INH-resistant organisms were less pathogenic for laboratory animals, there can be no doubt now that INH-resistant organisms can be transmitted and can cause disease (Snider et al. 1985). In an evaluation of 128 children who were in close household contact with an adult with multidrug-resistant tuberculosis, 66 (52%) were found to be infected and a further 15 (12%) to have tuberculosis disease. RFLP strain typing of M. tuberculosis cultured from these children found identical drug susceptibility patterns and strains amongst the children and their contacts in a majority of cases (Schaaf et al. 2000). In nearly 80% of cases, the index case was a family member. As only a minority of diseased children are likely to produce a culture of M. tuberculosis for drug susceptibility testing, it is important that an accurate history always be obtained of possible drugresistant contacts. This will enable the construction of the most appropriate treatment regimen as soon as possible. This is particularly important in those children with HIV-infection and life-threatening forms of disease such as tuberculous meningitis or miliary tuberculosis. In the Western Cape Province of South Africa, the rates of initial isoniazid resistance and multidrug resistance among adults were 3.9% and 1.1 %, respectively, between 1992 and 1993 (Weyer et al. 1995). From 1995 to 1998, 306 cultures of M. tuberculosis obtained from children aged 0-13 years of age were evaluated for drug susceptibility. Among children 0-5 years of age, the incidence of isoniazid resistance and multidrug resistance was 5.6% and 1%, respectively, which did not differ significantly from that found earlier in adults. This indicates that the incidence of resistance in children probably reflects the transmission of drug-resistant strains that is occurring amongst adults. Where cultures can be obtained from children and tested for drug susceptibility, this will offer a relatively simple means of monitoring drug resistance in a community. In children with a culture of M. tuberculosis resistant to isoniazid only, the use of a standard WHO retreatment regimen is considered adequate. This consists of 2 months of isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin followed by 1 month of isoniazid, rifampicin, pyrazinamide and

P.R. Donald

ethambutol and a 6-month continuation phase of isoniazid, rifampicin and ethambutol (Crofton et al. 1997). The use of isoniazid in the face of resistance is controversial. In primary isoniazid resistance, low-level isoniazid resistance (MIC<5Ilg/ml) may be found in more than half of the patients (Canetti 1965; Tripathy et al. 1969; Schaaf et al. 2000). These concentrations are easily attainable with a dose of 10-20 mg/kg isoniazid, which will usually be well tolerated by young children. In the case of resistance to at least isoniazid and rifampicin (multidrug resistance), a 5-drug intensive phase is considered mandatory (Crofton et al. 1997) and should be given for 3 months or until smear conversion. The continuation phase should last for 18 months after smear conversion and should contain at least three drugs to which the organism is sensitive. In most childhood disease a culture of M. tuberculosis may not be obtained. In these cases it is advisable to treat the child according to the susceptibility pattern of the adult contact and to use at least three drugs to which the child has not been exposed before.

16.12 Prevention and Control of Tuberculosis in Childhood Tuberculosis infection in childhood is the consequence of the inhalation of small aerosol droplets 5-10 flm in diameter containing 1-3 M. tuberculosis bacilli. These are expectorated by adults, who will usually be suffering from cavitating pulmonary tuberculosis and whose sputum will be positive on microscopy for acid-fast bacilli. There is therefore little doubt that the control of tuberculosis in adults would rapidly eliminate childhood tuberculosis as a serious health problem. It is equally clear that there is little prospect that tuberculosis will be controlled in developing countries in the foreseeable future and that the main focus of tuberculosis control programmes will continue to be the supervision of directly observed short course treatment for sputum ,smear-positive' adults. Nevertheless, it should be emphasized that even within the constraints imposed by financial restrictions and the lack of resources, opportunities exist to reduce the impact of tuberculosis on children. In order to make use of these opportunities, we need to continually remind ourselves that tuberculosis is an infectious disease and that in a high incidence community those most

Childhood Tuberculosis

often affected will be young adults in their reproductive years. In many developing communities it is also women who are bearing the brunt of the HIV/AIDS epidemic and, as indicated above, HIV/AIDS-associated tuberculosis has become an important cause of obstetric mortality and morbidity. These women will be in constant close contact with their own children and those of their extended families. In addition to the more obvious measures of chemoprophylaxis and BCG vaccination, there are several other ways by which the force of the infectious process as it affects children, but also adults, can be reduced. It is often forgotten that paediatric outpatient facilities and in particular the overnight holding wards which are commonly encountered in the developing world can serve as a focal point for infectious disease transmission. Young children attending these facilities will often be accompanied by parents or caregivers, who may themselves be sick. In an evaluation of young children less than 18 months of age with suspected tuberculosis attending one such facility, a policy of taking a chest radiograph of the accompanying parent or caregiver revealed pulmonary tuberculosis in 18% of cases. Amongst the adults accompanying 17 children less than 6 months of age, this was an even more rewarding procedure, and tuberculosis was found in 6 adults (35%). The identification of these adults not only contributed to strengthening the diagnosis of tuberculosis in their children, but also helped prevent the further spread of tuberculosis in their families and in the outpatient department. Two other measures, which can be considered in busy health care facilities where children and their caregivers congregate, are attention to ventilation and the use of ultraviolet light. Good ventilation can significantly reduce the density of bacilli in the air. Merely renewing the air 6 times in an hour has been calculated to reduce the density of bacilli to 100th of what it was beforehand (Riley and O'Grady 1961). Ultraviolet light can also contribute to the killing of bacilli (Riley et al. 1976). If the provision of artificial ultraviolet light is thought too expensive, the provision of large windows directed appropriately to make maximum use of available sunlight can contribute further to reducing the risk of tuberculosis infection.

259

In the very young, disease is particularly likely to take the form of disseminated forms of tuberculosis such as tuberculous meningitis or miliary tuberculosis. Mortality rates for those infected at an age of under 1 year and untreated have been calculated to be between 5000 and 6000 per 100,000 population (Rich 1951; Bentley et al. 1954). The identification of young children in close contact with an adult with sputum microscopy "smear-positive" pulmonary tuberculosis therefore offers an opportunity for the prevention of disease in these children and the early diagnosis and treatment of disease in those already infected. Depending upon the resources available, such children should have a Mantoux test to identify those infected and a chest radiograph to detect subclinical disease. As it may take up to 10 -12 weeks for tuberculin hypersensitivity to develop, it is prudent to commence chemoprophylaxis with isoniazid in these children exposed to infection. After 3 months the Mantoux test can be repeated. If this is positive, chemoprophylaxis should be continued to 6 months, and if negative, BCG vaccination can be considered. A number of controlled clinical trials have demonstrated the efficacy of isoniazid chemoprophylaxis when given to children and adults with asymptomatic primary infection (Ferebee 1970). Not only is the incidence of disease reduced, but more serious forms of disseminated tuberculosis are prevented. Isoniazid chemoprophylaxis for 3 months, 6 months and 12 months reduced the risk of tuberculosis by 21%, 65% and 75%, respectively. (International Union against Tuberculosis Committee on Prophylaxis 1982). In an analysis of the cost effectiveness of different durations of prophylaxis, it was then claimed that 6 months of chemoprophylaxis offered the best compromise (Snider et al.1986), and this is currently the duration of isoniazid chemoprophylaxis recommended in many official documents. A more recent analysis of data from several studies has however shown that 9 months of chemoprophylaxis probably offers the optimal protection against the development of tuberculosis disease (Comstock 1999). A major impediment to the successful implementation of chemoprophylaxis is non-compliance, and relatively low percentages of adults and children in both developing and developed countries who complete the prescribed course of treatment (Woebeser et al. 1989; Kopanoff et al. 1978). In developing comChemoprophylaxis. Between 50% and 60% of young munities health care staff are already hard pressed children in close household contact with an adult to ensure that their adult ,smear-positive' patients with sputum microscopy ,smear-positive' pulmo- complete their therapy, and it is hardly surprising nary tuberculosis will become infected. Approxi- that chemoprophylaxis is not accorded a very high mately 10% of those infected may become diseased. priority. In our own experience of 193 children pre-

260

senting with tuberculous meningitis in the Western Cape Province of South Africa, 77 (40%) had a close household contact who had been treated for pulmonary tuberculosis within the previous 2 years. Only 17 (22%) of these children had, however, been prescribed isoniazid chemoprophylaxis, and a mere 7 completed 3 months of prophylaxis (Donald et al. 1995).

In the face of these dismal figures, it is important to prioritise and to give our attention to those at greatest risk. These are undoubtedly the children under 2 years of age in household contact with an adult with sputum ,smear-positive' pulmonary tuberculosis. These adults will frequently be a parent or grandparent or another caretaker. As these individuals must themselves receive supervised treatment, it should add relatively little to the burden of the health care personnel to ensure that their children receive prophylactic treatment at the same time. Although isoniazid for 6 months remains the most frequently recommended prophylaxis regimen, there has been considerable interest in shorter regimens containing rifampicin and pyrazinamide. This interest has been sharpened by our inability to halt the ongoing spread of HIV-associated tuberculosis and evidence from Peru that even a model national tuberculosis control programme may not be able to reduce the incidence of tuberculosis successfully even in the absence of HIV/AIDS (Chaisson 2000). In experiments with M. tuberculosis-infected mice, rifampicin for 3 months or rifampicin and pyrazinamide for 2 months were more effective than 6 months of isoniazid in reducing the proportion of mice with positive spleen cultures (Lecoeur et al. 1989). Several controlled trials have now found rifampicin and pyrazinamide for 2 months to give results equivalent to those of 6 months of isoniazid (Halsey et al. 1998; Mwinga et al. 1998; Gordin et al. 2000). There are at present few data relating to children regarding this approach, but there seems no reason to expect different results.

Bacille

Calmette-Guerin Vaccination. Bacille Calmette-Guerin (BCG) vaccination has been practised for more than 80 years, but there is still no consensus as to its value in preventing tuberculosis in childhood. Analysis of a number of clinical trials in which BCG was evaluated has shown effects ranging from significant protection to an increased incidence of disease. Case-control studies have consistently shown a protective effect against disseminated forms of tuberculosis such as TBM and miliary tuberculosis. It should be pointed out that some case-control stud-

P. R. Donald

ies made use of controls that were not in contact with a patient with tuberculosis and so would automatically tend to show some degree of protection. Nevertheless, the current consensus is that BCG does offer a degree of protection of between 60%-80% against disseminated forms of tuberculosis, and BCG forms part of the World Health Organization Expanded Programme of Immunization. BCG will, however, have only a limited effect upon the epidemiological situation in a community. It does not break the chain of infection and protects the individual only against disease dissemination, but not infection (Styblo and Meijer 1976). BCG has also been evaluated in a variety of populations in different parts of the world. In the light of the above variations in strains, methods of administration and populations in which BCG has been evaluated, it is perhaps not surprising that conflicting results have been obtained. BCG is derived from a virulent bovine tubercle bacillus which was attenuated over a 13-year period of subculturing and 230 transfers from one subculture to another. Following this, a number of culture samples were distributed all over the world, and these were in turn subjected to further subculturing on a variety of different media. In this manner a number of daughter strains have been created, possibly by encouraging the emergence of minority strains within each population (Osborn 1983). BCG has been administered to a diversity of populations in a number of different countries orally, by scarification, by percutaneous multiple puncture and by intradermal injection. It is therefore perhaps not surprising that widely differing results have been obtained and that there is no consensus as to the value of BCG. Amidst the uncertainty of its action with regard to tuberculosis, it is of interest to note a number of other instances in which BCG appears to have had an undoubted effect upon the immune system. Thus, BCG has been documented to be instrumental in helping to control leprosy in Malawi (Karonga Prevention Trial Group 1996) and has a well substantiated role in the management of bladder cancer (Alexandroff et al. 1999). It is also significant to note that following the discontinuation of BCG immunization in Sweden, the incidence of atypical mycobacterial lymphadenitis increased dramatically (Katila et al. 1987). It has also been speculated that BCG immunization might be the reason why HIV/AIDS patients in Sweden have a much lower incidence of Mycobacterium avium complex infections than patients in the USA and that it may therefore be of importance in reducing the impact of HIV infection (Kallenius et al. 1989). There is also some evidence that BeG vaccina-

261

Childhood Tuberculosis

tion may have a role in preventing certain forms of malignancy (Grange and Stanford 1990). Intradermal BCG vaccination will usually lead to the formation of a small superficial ulcer approximately 4 weeks after vaccination. This heals over 8-10 weeks and leaves a flat scar some 50 mm in diameter. Complications may be seen in 3%-4% of individuals, particularly if care is not taken with regard to the technique. The importance of staff training has been emphasized (Jeena et al. 2001). For this reason complications tend to be more common following intradermal vaccination than after percutaneous vaccination. Complications encountered include axillary lymphadenopathy with suppuration in some cases, abscess formation at the injection site, especially if the vaccine is delivered subcutaneously, and disseminated forms of disease such as osteoarticular involvement or, more rarely, tuberculous meningitis. Disseminated BCG disease will usually be seen exclusively in those with severe immunosuppression. Although disseminated BCG was in the past mainly a disease of infants, it is now increasingly seen in adults and older children and often in association with HIV/AIDS (Talbot et al. 1997). At present, HIV infection is not considered a contraindication to BCG vaccination, and BCG should continue to be given to all infants even if HIV-infected provided they have no symptoms of AIDS. Other factors which playa role in the occurrence of complications include the type of vaccine used and the dose of vaccine given. In Sweden between 1972 and 1974, 29 patients with osteo-articular disease were reported per 100,000 children vaccinated. When the BCG strain was changed, no more cases occurred (Romanus 1987). In several countries the Pasteur strain has been noted to be more reactogenic than other strains, and several outbreaks of lymphadenitis followed a change to the Pasteur strain. In another instance an outbreak of lymphadenitis followed a change of vaccine from the Pasteur strain to Tokyo 172 and was found to be due to the injection of an inappropriately high dose of vaccine (Kabra et al. 1993). The management of BCG lymphadenitis is controversial. Most lesions will ultimately heal spontaneously. In the case of suppurative lesions, needle aspiration from a distance of 2-3 cm has been shown to bring about a more rapid regression of adenopathy and to prevent sinus formation (Banani and Alborzi 1994). It has also been claimed that streptomycin administration into the nodes during the non-suppurative phase prevents suppuration and causes earlier healing than the previously advised use of erythromycin (Kuyucu et al. 1998). Surgery has been advo-

cated for large lesions, but it is uncertain whether the long-term results are indeed cosmetically better than a more conservative approach. In the case of disseminated forms of BCG disease, chemotherapy must be given, and it should be remembered that BCG is resistant to pyrazinamide. With regard to HIV infection it is currently recommended that BCG vaccine should not be given to those who are HIV-infected and have signs and symptoms of AIDS. In the case of those who are HIVinfected only, the benefits of BCG vaccination appear to outweigh the potential risks (O'Brien et al. 1995). In this context it should be noted that a recent study amongst adult volunteers demonstrated viable BCG mycobacteria draining from the vaccination ulcer 2 months after vaccination. These organisms could theoretically pose a risk to other immunosuppressed individuals who might be in close contact with the vaccinated infant (Hoft et al. 1999).

References Adhikari M, Pillay T, Pillay DV (1997) Tuberculosis in the newborn: an emerging disease. Pediatr Infect Dis J 16: 1108-1112 Ahmed Y, Mwaba P, Chintu C, Grange J, Ustianowski A, Zurnla A (1999) A study of maternal mortality at the University Teaching Hospital, Lusaka, Zambia: the emergence of tuberculosis as a major non-obstetric cause of maternal death. Int J Tuberc Lung Dis 3:675-680 Alexandroff AB, Jackson AM, O'Donnel et al (1999) BCG immunotherapy of bladder cancer: 20 years on. Lancet 353:1689-1694 Ansari NA, Kombe AH, Kenyon TA et al (1999) Mortality and pulmonary pathology of children with HIV infection in Francis town, Botswana. Int J Tuberc Lung Dis 3 [Suppl]:S201 Bentley FJ, Grzybowski S, Benjamin R (1954) Pleural effusion. Tuberculosis in childhood and adolescence. National Association for the Prevention of Tuberculosis, London, pp 50-54 Bleiker MA (1991) The annual risk of infection rate, the tuberculin survey and the tuberculin test. Bull Int Union Against Tuberc 66:53-56 Brailey M (1940) A study of tuberculous infection and mortality in children of tuberculous households. Am J Hyg 31:1-43 Canetti G (1965) Present aspects of bacterial resistance in tuberculosis. Am Rev Respir Dis 92:687-703 Cauthen GM, Pio A, ten Darn HG (1988) Annual risk of infection. World Health Organization WHO/TB/88 154:1-34 Chaisson RE (2000) New developments in the treatment of latent tuberculosis. Int J Tuberc Lung Dis 4:S176-S181 Chan SP, Birnbaum J, Rao M, Steiner P (1996) Clinical manifestations and outcome of tuberculosis in children with acquired immunodeficiency syndrome. Pediatr Infect Dis J 15:443-447 Chintu C, Zumla A (1995) Childhood tuberculosis and infection with the human immunodeficiency virus. J R Coli Phys Lond 29:92-95

262 Chintu C, Bhat G, Luo C et al (1993) Sero-prevalence of human immunodeficiency virus type 1 in Zambian children with tuberculosis. Pediatr Infect Dis J 12:499-504 Comstock GW (1999) How much isoniazid is needed for the prevention of tuberculosis among immunocompetent adults? Int J Tuberc Lung Dis 3:847-850 Comstock GW, Livesay VT, Woolpert SF (1974) The prognosis of a positive tuberculin reaction in childhood and adolescence. Am J EpidemioI99:131-138 Crofton J, Chaulet P, Maher D (1997) Guidelines for the management of drug-resistant tuberculosis. World Health Organization, Geneva, WHO/TB/96.210 (Rev 1) Delacourt C, Mani TM, Bonnerot V et al (1993) Computed tomography with normal chest radiograph in tuberculous infection. Arch Dis Child 69:430-432 Delacourt C, Poveda J-D, Chureau C, Beydon N et al (1995) Use of polymerase chain reaction for improved diagnosis of tuberculosis in children. J Pediatr 126:703-709 Dolin PJ, Raviglione MC, Kochi A (1994) Global tuberculosis incidence and mortality during 1990-2000. Bull WHO 72: 213-220 Donald PR, Seifart HI (1988) Cerebrospinal fluid pyrazinamide concentrations in children with tuberculous meningitis. Pediatr Infect Dis J 7:469-471 Donald PR, Seifart HI (1989) Cerebrospinal,fluid concentrations of ethionamide in children with tuberculous meningitis. J Pediatr 115:483-486 Donald PR, van Zyl LE, de Villiers J (1995) BCG vaccination status of children with tuberculous meningitis and the use of unsupervised isoniazid prophylaxis. S Afr Med J 85:167-170 Donald PR, Schoeman JF, van Zyl LE, de Villiers IN, Pretorius M, Springer P (1998) Intensive short course chemotherapy in the management of tuberculous meningitis. Int J Tuberc Lung Dis 2:704-711 Dormer BA, Swart JA, Harrison I (1959) Prophylactic isoniazid protection of infants in a tuberculosis hospital. Lancet 2: 902-904 Driver CR, Munsiff SS, Li J et al (2001) Relapse in persons treated for drug-susceptible tuberculosis in a population with a high coinfection with human immunodeficiency virus in New York City. Clin Infect Dis 33:1762-1769 Dutt AK, Moers D, Stead WW (1989) Smear- and culture-negative pulmonary tuberculosis: four-month short course chemotherapy. Am Rev Respir Dis 139:867-870 Ellard GA, Humphries MJ, Allen BW (1993) Cerebrospinal fluid drug concentrations and the treatment of tuberculous meningitis. Am Rev Respir Dis 148:650-655 Elliot AM, Berning SE. Iseman MD, Peloquin CA (1995) Failure of drug penetration and acquisition of drug resistance in chronic tuberculous empyema. Tubercle Lung Dis 76: 463-467 Franchi LM, Cama RI, Gilman RH, Montenegro-James S, Sheen P (1998) Detection of Mycobacterium tuberculosis in nasopharyngeal aspirate samples in children. Lancet 352:1681-1682 Gordin FM, Chaisson RE, Matts JP et al (2000) An international randomized trial of rifampin and pyrazinamide versus isoniazid for prevention of tuberculosis in HIV-infected persons. J Am Med Assoc 283:1445-1450 Graham SM, Coulter JBS, Gilks CF (2001) Pulmonary disease in HIV-infected African children. Int J Tuberc Lung Dis 5:12-23 Grange JM, Stanford JL (1990) BCG vaccination and cancer. Tubercle 71:61-64

P. R. Donald Grzybowski S (1954) Radiology in childhood tuberculosis. Tubercle 35:113-117 Halsey NA, Coberly JS, Desormeaux J et al (1998) Rifampicin and pyrazinamide vs. isoniazid for prevention of tuberculosis in HIV-I infected persons. Lancet 351:786-792 Harries AD, Maher D (1996) TB/HIV a clinical manual. World Health Organization, Geneva, WHO/TB/96.200 Harries AD, Parry C, Mbewe LN et al (1997) The pattern of tuberculosis in Queen Elizabeth Central Hospital, Blantyre, Malawi: 1986-1995. Int J Tuberc Lung Dis 1:346-351 Hoffman ND, Kelly C, Futterman D (1996) Tuberculosis infection in human immunodeficiency virus-positive adolescents and young adults: a New York City cohort. Pediatrics 97:198-203 Hoft DF, Leonardi C, Milligan T et al (1999) Clinical reactogeincity of intradermal Bacille Calmette-Gutfrin vaccination. Clin Infect Dis 28:785-790 Hong Kong Chest Service/ Tuberculosis Research Centre, Madras/British Medical Research Council (1989) A controlled trial of 3-month, 4-month and 6-month regimens of chemotherapy for sputum-smear-negative pulmonary tuberculosis. Am Rev Respir Dis 139:871-876 Houwert KAF, Borggreven PA, Schaaf HS, Nel E, Donald PR, Stolk J (1998) Prospective evaluation of World Health Organization criteria to assist diagnosis of tuberculosis in children. Eur Respir J 11:1116-1120 Humphries MJ (1992) The management of tuberculous meningitis. Thorax 47:577-581 International Union Against Tuberculosis (1982) Efficacy of various durations of isoniazid preventive therapy for tuberculosis: five years of follow up in the IUAT trial. Bull WHO 60:555-564 Jacobs M, Yach D, Fisher S, Kibel M, Hattingh, Coetzee G (1987) Mangement of children with tuberculosis in a local authority of Cape Town. Southern Afr J Epidemiol Infect 2:15-18 Jamieson DH (1995) Imaging intracranial tuberculosis in childhood. Pediatr Radiol 25: 165-170 Jeena PM, Mitha T, Bamber S, Wesley A, Coutsoudis A, Coovadia HM (1996) Effects of human immunodeficiency virus on tuberculosis in children. Tuberc Lung Dis 77:437-443 Jeena PM, Coovadia HM, Thula SA et al (1998) Persistent and chronic lung disease in HIV-l-infected African children. AIDS 12:1185-1193 Jeena PM, Chhagan MK, Topley J et al (2001) Safety of the intradermal Copenhagen BeG vaccine in neonates in Durban, South Africa. Bull WHO 79:337-343 Kabra SK, Jain Y, Seth MV (1993) BCG associated adenitis. Lancet 341:970 KaIlenius G, Hoffner SE, Svenson SB (1989) Does vaccination with Bacille Calmette-Guerin protect against AIDS? Rev Infect Dis 11:349-351 Karonga Prevention Group (1996) Randomised controlled trial of single BCG, repeated BCG, or combined BCG and killed Mycobacterium leprae vaccine for prevenyion of leprosy and tuberculosis in Malawi. Lancet 348:17-24 Katila ML, Brander E, Backman A (1987) Neonatal BCG vaccination and mycobacterial cervical adenitis in childhood. Tubercle 68:291-296 Kendig EL (1969) Prognosis of infants born to tuberculous mothers. Pediatrics 26:97-100 Khouri YF, Mastrucci MT, Hutto C, Mitchell CD, Scott GB (1987) Mycobacterium tuberculosis in children with immu-

Childhood Tuberculosis nodeficiency virus and routine childhood immunisations. Lancet 2:669-672 Kochi A (1991) The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 72: 1-6 Kuyucu N, Kuyucu S, Ocal B et al (1998) Comparison of oral erythromycin, local administration of streptomycin and placebo therapy for non-suppurating Bacillus CalmetteGuerin lymphadenitis. Pediatr Infect Dis J 6:524-525 Lecoeur HF, Truffot-Pernot, Grosset JH (1989) Experimental short-course preventive therapy of tuberculosis with rifampin and pyrazinamide. Am Rev Respir Dis 140: 1189-1193 Light IT, Saidleman M, Sutherland JM (1974) Management of newborns after nursery exposure to tuberculosis. Am Rev Respir Dis 109:415-418 Lincoln EM, Gilbert LA, Morales SM (1960) Chronic pulmonary tuberculosis in individuals with known previous primary tuberculosis. Dis Chest 5:473-482 Lobato MN, Loeffler AM, Furst K, Cole B, Hopewell PC (1998) Detection of Mycobacterium tuberculosis in gastric aspirates collected from children: hospitalization is not necessary. Pediatrics 102, URL: http://www.pediatrics.orglcgi/ content/full/l02/4/e40 Long MW, Snider DE, Farer LS (1979) US Public Health Service co-operative trial of three rifampicin -isoniazid regimens in treatment of pulmonary tuberculosis. Am Rev Respir Dis 119:879-894 Madhi SA, Petersen K, Madhi A, Khoosal M, Klugman KP (2000) Increased disease burden and antibiotic resistance of bacteria causing severe community-acquired lower espiratory tract infections in human immunodeficiency virus type-1 infected children. Clin Infect Dis 31:170-176 Medical Research Council (1948) Streptomycin treatment of tuberculous meningitis. Lancet 1:582-596 Mitchison DA (2000) Role of individual drugs in the chemotherapy of tuberculosis. 1nt J Tuberc Lung Dis 4:796-806 Munro WT (1944) Tubercle bacilluria in the child. Edinb Med J 51:101-106 Mukadi YD, Wiktor SZ, Coulibaly I-M et al (1997) Impact of HIV infection on the development, clinical presentation and outcome of tuberculosis among children in Abidjan., Cote d'Ivoire. AIDS 11:1151-1158 Mwinga A, Hosp M, Godfrey-Faussett P et al (1998) Twice weekly tuberculosis preventive therapy in HIV infection in Zambia. AIDS 12:2447-2457 Narita M, Hisada M, Thimmappa B et al (2001) Tuberculosis recurrence:multivariate analysis of serum levels of tuberculosis drugs, human immunodeficiency virus status, and other risk factors. Clin Infect Dis 32:515-517 Nemir RL (1986) Perspectives in adolescent tuberculosis: three decades of experience. Pediatrics 78:399-405 Noordhoek GT, Kolk AH, Bjune G et al (1994) Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin MicrobioI32:277-284 O'Brien KL, Ruff AJ, Louis MA et al (1995) Bacillus CalmetteGuerin complications in children born to HIV-1-infected women with a review of the literature. Pediatrics 95: 414-418 Peloquin CA, Nitta AT, Burman WJ et al (1996) Low antituberculosis drug concentrations in patients with AIDS. Ann Pharmacother 30:919-925

263 Pillay Y Kahn M, Moodley Jet al (200!) The increasing burden of tuberculosis in pregnant women, newborns and infants under 6 months age in Durban, Kwazulu-Natal. S Afr Med J 91:983-987 Ravenscroft A, Schoeman JF, Donald PR (2001) Tuberculous granulomas in childhood tuberculous meningitis: radiological features and course. J Trop Pediatr 47:5-12 Rieder HL (1993) Drug-resistant tuberculosis: issues in epidemiology and challenges for public health. Tuberc Lung Dis 75:321-323 Riley RL, O'Grady F (1961) Airborne infection: transmission and control. Macmillan, New York Riley RL, Knight M, Middlebrook G (1976)Ultraviolet susceptibility of BCG and virulent tubercle bacilli. Am Rev Respir Dis 113:413-418 Rook GAW, Onyebujoh P, Stanford JL (1993) Th1->Th2 switch and loss of CD4 cells in chronic infections: an immunoendocrinological hypothesis not exclusive to HIV, Immunol Today 14:568-569 Romanus V (1987) Bacillus Calmette-Guerin-immunized and unimmunized children in Sweden: a ten-year evaluation following the cessation of general Bacillus Calmette Guerin immunization of the newborn in 1975. Pediatr Infect Dis J 6:272-280 Rutherfoord GS, Hewlett RH (1994) Atlas of correlative surgical and neuropathology and imaging (Gresham GA, ed). Kluwer Academic, Dordrecht, pp 47-74 Schaaf HS, Nel ED (1992) Tuberculosis presenting as cholestatic jaundice in early infancy. J Pediatr Gastroenterol Nutr 15:437-439 Schaaf HS, Smith J, Donald PR, Stockland B (1989) Tuberculosis presenting in the neonatal period. Clin Pediatr 28: 474-475 SchaafHS, Donald PR, Scott F (1991) Maternal chest radiography as supporting evidence for the diagnosis of tuberculosis in childhood. J Trop Pediatr 37:223-225 Schaaf HS, Gie RP, Beyers N, Smuts, Donald PR (1993) Tuberculosis in infants less than 3 months of age. Arch Dis Child 69:371-374 Schaaf HS, Geldenhuys A, Gie RP, Cotton MF (1998) Culturepositive tuberculosis in human immunodeficiency virus type I-infected children. Pediatr Infect Dis J 17:599-604 Schaaf HS, Vermeulen HAS, Gie RP, Beyers N, Donald PR (1999) Evaluation of young children in household contact with adult multidrug-resistant pulmonary tuberculosis cases. Pediatr Infect Dis J 18:494-500 Schaaf HS, Gie RP, Beyers N, Sirgel FA, de Klerk, Donald PR. (2000a) Primary drug-resistant tuberculosis in children. Int J Tuberc Lung Dis 4:1149-1155 SchaafHS,Van Rie A, Gie RP et al (2000b) Transmission of multidrug-resistant tuberculosis. Pediatr Infect Dis J 19:695-699 Seifart HI, Parkin DP, Donald PR (1991) The stability of isoniazid, rifampicin and pyrazinamide suspensions used for the treatment of tuberculosis in children. Pediatr Infect Dis J 10:827-831 Shaw JB, Wynn-Williams N (1954) Infectivity of pulmonary tuberculosis in relation to sputum status. Am Rev Tuberc 69:724-732 Sirgel FA, Botha FJH, Parkin DP et al (1993) The early bactericidal activity of rifabutin in patients with pulmonary tuberculosis measured by sputum viable counts - a new method of drug assessment. J Antimicrob Chemother 32: 867-875

264 Small PM, Schafer RW, Hopewell PC et al (1993) Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection. N Engl J Med 328:1137-1144 Smith KC, Starke JR, Eisenach K, Ong LT, Denby M (1996) Detection of Mycobacterium tuberculosis in clinical specimens from children using a polymerase chain reaction. Pediatrics 97:155-160 Smith MHD (1967) Tuberculosis in adolescence. Characteristics, recognition, management. Clin Pediatr 6:9-15 Snider DE, Kelly GD, Cauthen GM, Thompson NJ, Kilburn JO (1985) Infection and disease among contacts of tuberculosis cases with drug-resistant and drug susceptible bacilli. Am Rev Respir Dis 132:125-132 Snider DE, Caras GJ, Koplan JP (1986) Preventive therapy with isoniazid. Cost effectiveness of different durations of therapy. J Am Med Assoc 255:1579-1583 Steiner P, Rao M, Mitchell M, Steiner M (1985) Primary drug resistant tuberculosis in children. Am J Dis Child 139:780-782 Tanzania Tuberculin Survey Collaboration (2001) Tuberculosis control in the era of the HIV epidemic: risk of tuberculosis infection in Tanzania, 1983- I998. Int J Tuberc Lung Dis 5:103-112 Talbot EA, Perkins MD, Fagundes S et al (1997) Disseminated Bacille Calmette-Guerin disease after vaccination: case report and review. Clin Infect Dis 24:1139-1146 Te Water Naude JM, Donald PR, Hussey GD et al (2000) Twice weekly vs.daily chemotherapy for childhood tuberculosis. Pediatr Infect Dis J 19:405-410 Thomas P, Bornschlegel K, Singh TP et al (2000) Tuberculosis in human immunodeficiency virus-infected and human immunodeficiency virus-exposed children in New York City. Pediatr Infect Dis J 19:700-706 Tripathy SP,Menon NK, Mitchison DA et al (1969) Response to

P. R. Donald treatment with isoniazid plus PAS of tuberculosis patients with primary isoniazid resistance. Tubercle 50:257-268 Udani PM (1983) Tuberculosis on children in India. A major health hazard! Pediatr Clin India 18: 11-42 Vallejo JG, Ong LT, Starke JR (1994) Clinical features, diagnosis, and treatment of tuberculosis in infants. Pediatrics 94: 1-7 Van Eden W, De Vries RR, Stanford JL et al (1983) HLA-DR3 associated genetic control of response to multiple skin tests with new tuberculins. J Immunogenet 10:107-114 Van Geuns HA, Meijer J, Styblo K (1975) Results of contact investigation in Rotterdam, 1967-1969. Bull Int Union Against Tuberc 50:107-121 Wallgren A (1928) Initial fever in tuberculosis. Am J Dis Child 36:1341-1345 Wallgren A (1938) Pulmonary tuberculosis. Relation of childhood infection to disease in adults. Lancet 1:417-420 Wallgren A (1948) The ,time-table' of tuberculosis. Tubercle 29:245-251 Weber HC, Beyers N, Gie RP, Schaaf HS, Fish T, Donald PR (2000) The clinical and radiological features of tuberculosis in adolescents. Ann Trop Paediatr 20:5-10 Weyer K, Groenewald P, Zwarenstein M, Lombard CJ (1995) Tuberculosis drug resistance in the Western Cape. South Afr Med J 85:499-504 Wilcox WD, Laufer S (1994) Tuberculosis in adolescents. Clin Pediatr 33:258-262 World Health Organization (1989) Provisional guidelines for the diagnosis and classification of the EPI target diseases for primary health care, surveillance and special studies. EPIIGEN/83/4 Zar HJ, Tannenbaum E, Appoles P, Roux P, Hanslo D, Hussey G (2000) Sputum induction for the diagnosis of pulmonary tuberculosis in infants and young children in an urban setting in South Africa. Arch Dis Child 82:305-308

17 Primary Tuberculosis in Adults M. MONIR MADKOUR

CONTENTS 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 17.10 17.11 17.12

Epidemiology of Primary Tuberculosis in Adults 265 Pathogenesis of Primary Pulmonary Tuberculosis 266 Culture Yield of Bacilli from Primary Lesion and Primary Complex 267 Groups at Risk of Primary Tuberculosis 267 Clinical Features and Diagnosis of Primary Tuberculosis in Adults 268 Lung Infiltrates and Consolidation 268 Lymphadenopathy 269 Pleural Effusion 269 Miliary Tuberculosis 269 Microbiological Confirmation 269 Treatment and Possible Paradoxical Transient Worsening 270 Conclusion 270 References 270

Traditionally, primary tuberculosis is a disease of childhood (Beyers 1979). This remains true in poor, developing countries where tuberculosis is endemic, and infections commonly start during childhood (see Chap. 16). This tradition, however, has been modified in rich, developed and industrialized countries that adopted successful tuberculosis control programs starting in the nineteenth century. Developed industrialized countries at present harbor only 5% of the world burden of tuberculosis, and the population group most infected is over 65 years of age (Alexander et al. 1979) (see Chap. 3). In these developed countries, exposure to the infection occurs mostly in the elderly population, leading to primary pulmonary tuberculosis. During the current epidemic of HIV, co-infection of adults with primary tuberculosis has been increasingly reported, leading to its dissemination (Pitchenik and Rubinson 1985; Wasser et al. 1988). This shift towards the presentation of primary tuberculosis in the elderly and among HIV-infected patients appears M. M. MADKOUR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

to be related to decreasing childhood exposure in the developed, industrialized countries (Khan et al. 1977; Miller and MacGregor 1978; Woodring et al. 1986; Choyke et al. 1983; Hulnick et al.I983). As a consequence of this shift of primary tuberculosis to the adult population, some authors from developed countries suggested that the term 'childhood tuberculosis' and 'adult tuberculosis' be abandoned (Berger and Granada 1974; Choyke et al.I983). We reviewed the medical records of 10 adult patients with primary tuberculosis who attended our hospital from 1990 through 2000. Their clinical features will be discussed in this chapter. The imaging features of these patients, including the follow-up radiograph findings during treatment and after its completion, are clearly described in our separate chapter specially devoted to the radiology of pulmonary tuberculosis. Imaging figures on primary pulmonary tuberculosis are numbers: 23.3,23.4,23.8,23.9,23.10,23.12,23.16, 23.17 and 23.18.

17.1 Epidemiology of Primary Tuberculosis in Adults Since the shift of patient care from long-term sanatorium management into general hospitals, the clinicians' awareness of primary tuberculosis has decreased. This is because tuberculous patients are no longer managed by clinicians with a special interest in the disease, along with the decline in the incidence of the disease in the populations of developed countries (Ellersten 1959; Khan et al. 1977; Varkey and Politis 1981; Choyke et al. 1983). The incidence of primary tuberculosis has been reported to range between 10% and 34% (Stead et al. 1968; Khan et al. 1977; Miller and MacGregor 1978; Woodring et al. 1986; Buckner et al. 1990; Miller et al. 1993; Miller 1994; McAdams et al. 1995). Most of the epidemiological data on primary tuberculosis in adults are reported from the USA and Europe.

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The criteria adopted to determine the incidence of adult primary tuberculosis in various reported series vary slightly among different authors. The criteria for diagnosis basically rested upon the conversion of PPD skin testing as well as imaging features (Chiba and Kurihara 1979; Choyke et al. 1983). Choyke and colleagues from Duke and Yale Universities reviewed 103 patients with adult-onset primary pulmonary tuberculosis seen over a period of 6 years. Their criteria included recent PPD skin test conversion with adenopathy or pleural effusion in a patient with no history of tuberculosis and previously normal chest radiographs. Black male predominance was noted in approximately 60%, and 73.8% was aged 40 years or more. These authors reported that the incidence of active primary tuberculosis in adult is approximately 10%-20%. Miller and Miller (1993) considered a recent conversion of the PPD skin test with isolated pleural effusion, lymphadenopathy, isolated middle and lower lobe infiltration, and most cases of miliary tuberculosis as diagnostic of primary tuberculosis. These authors estimated the incidence of primary tuberculosis in adults as ranging from 23% to 34%. In Norway, Heldal and colleagues (2000) reviewed the cases of Norwegian-born patients with pulmonary tuberculosis. They found that most cases in their series were due to endogenous reactivation of the disease. The incidence of adult primary pulmonary tuberculosis in these patients was reported to be 10% in 1975,19% in 1985, and 16% in 1995.

17.2 Pathogenesis of Primary Pulmonary Tuberculosis Primary pulmonary tuberculosis is transmitted by inhalation of air-borne Mycobacterium tuberculosis present in the cough particles from the sputum of a smear-positive patient. The inhaled bacilli usually deposit in the middle or lower lung field. The exact mechanisms of the protective immunity response against the development of the disease in humans have not been totally clarified (Ellner 1997). M. tuberculosis lipoproteins and glycolipids induce activation of macrophage, T-cell, and cytokine expression (Wallis and Johnson 2001). Alveolar macrophages phagocytose the bacilli, and activation of the cell-mediated immune response (CMI) plays an essential role in combating infection (Reddy and Hayworth 2002; Garcia et al. 2002). The bacilli may

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survive, especially within nonactivated monocytel macrophages that enter the alveoli from the bloodstream (Dannenberg 2001). Early in the primary infection (within a few hours), the bacilli are transported by macrophages to the hilar and/or paratracheal lymph nodes (Milburn 2001). Tissue-damaging delayed hypersensitivity (DTH) develops, and the bacilli-laden macrophages are killed. Key factors in this process include monocyte-derived interleukin (IL)-12 and tumor necrosis factor (TNF)-oc as well as T-cell-derived IL-2 and interferon (IFN)-y. These cytokines playa crucial role in the induction of macrophage-mediated elimination of mycobacteria (Vanham et al. 1997; Garcia et al. 2002). During active pulmonary tuberculosis, signs of both immune depression and immune activation are concomitantly present (Vanham et al.1997). The balance of cytokines produced by lymphocytes in response to infection is believed to have a profound effect on the clinical outcome. Depression of peripheral blood T-cell and cytokine production has been demonstrated during active tuberculosis disease (Garcia et al. 2002). As the infection progresses, local areas of inflammation with granuloma formation and cellular infiltrates occur in the middle or lower zones of the lung. This is the pulmonary component of the primary Ghon focus and, in combination with hilar and/or paratracheallymphadenitis, forms the primary complex. Tuberculin conversion may occur 3-6 weeks after the initial infection (Milburn 2001). The outcome of the infection depends on the interaction between the host immune system response and the bacilli. In patients with strong and competent cellular immunity, the Ghon focus and regional lymphadenitis heal with fibrosis and calcifications within weeks or months. However, the primary complex lesion may progress into lung parenchymal or lymph node infection or both in what is known as progressive primary tuberculosis. Many tuberculosis specialists adopted the use of the term 'progressive primary tuberculosis' if it occurs within 3-8 months after tuberculin conversion. About 10% of all cases of primary tuberculosis progress to a chronic form indistinguishable from reactivation disease (Goppert and Left 1979). Within the first year of primary pulmonary infection, the incidence of clinically significant disease is approximately 1.5%, and the cumulative risk during the first 5 years is 5%-10% (Chiba and Kurihara 1979). Balasubramanian and colleagues (1994) from Wisconsin, USA, extensively reviewed the published work of other investigators which dealt with the pathogenesis of tuberculosis. Particular emphasis was laid on the infective dose, the yield of bacilli from the pri-

267

Primary Tuberculosis in Adults

mary lesion, predominant locations, and the argument regarding the endogenous versus exogenous pathway leading to post-primary tuberculosis. The following paragraphs include some personal abstracts as well as the investigators' views and schools of thought on the issues of the pathogenesis of primary pulmonary tuberculosis. The size of the infective dose has been debated among investigators in a review article. On the one hand, some investigators indicated that a large infectious dose occurred more frequently in household contacts of sputum-positive cases. The infective dose includes the number of bacilli per infectious particles inhaled during anyone period of exposure. Other investigators indicated that the infectious dose in tuberculosis was very small. The size of a droplet nucleus disseminated during a cough by a sputum-positive tuberculosis patient is 5.0 ~m in diameter. It is estimated that the number of bacilli in a droplet nucleus is 1-10. Accordingly, these observations suggest that the infectious dose in tuberculosis is very low. The location of the primary complex was noted by Medlar (1948) in his series of 105 individuals found during autopsies performed on 1225 bodies. The single primary complex was located within 1 cm of the pleural surface in 85%, and in the lower half of the lung field in 66%, while only 12% were supraclavicular; he ascribed this pattern of lung lesion distribution to the direction of the airflow. The primary focus is the initial lesion produced by tubercle bacilli in any tissue. The primary or Ghon complex includes both the primary focus and homologous lesions in the draining lymph nodes. The primary complex most commonly occurs in the lung, but may also develop in the skin, intestines, genital tract, and tonsils. A mucous membrane primary focus may not incite a typical tuberculous healing reaction, so that only fibrocalcific lymph nodes may remain as indicators of a primary infection in these tissues. Therefore, fibrocalcific lesions in the lung or lymph nodes of a patient with organ and miliary tuberculosis may be taken to represent evidence of a prior primary infection (Slavin et al. 1980).

17.3 Culture Yield of Bacilli from Primary Lesion and Primary Complex Canetti (1972) suggested 'that tubercle bacilli do not survive indefinitely in tuberculous foci'. Work on guinea-pigs with inoculations of the component

of the primary complex (before the era of chemotherapy) indicated that no live bacilli could be cultured in 85% of calcified lesions and in 50% in the encapsulated stage. This review explored as well the work and school of thought of other investigators on how a calcified primary focus can be a source of reactivation of the disease when live bacilli no longer exist. Theories of alternative locations created during the primary disease by the progeny of bacilli from the primary lesion which had been disseminated hematogenously have been postulated. Other aspects of pathogenesis including the postprimary tuberculosis and the specified pathways (endogenous versus exogenous) by which the disease may occur later in life have already been elaborated.

17.4 Groups at Risk of Primary Tuberculosis Adults and the elderly in developed countries with diseases that affect the integrity of their immune system will increase the chances for the development of primary tuberculosis infection (Alexander et al. 1979; Ikejoe et al. 1992). Young adults with preexisting HIV infection are particularly susceptible to co-infection with primary pulmonary tuberculosis. Patients on chemotherapy or steroids for cancer and other diseases are particularly vulnerable to the development of primary tuberculosis. Drug abusers, the homeless, and the elderly residing in nursing homes are susceptible to the development of adult primary pulmonary tuberculosis. Traveling to endemic areas or a long airplane flight has been reported as risk factors for the transmission of tuberculosis. Kenyon and colleagues (1996) from the USA reported the transmission of tuberculosis during a long flight (8.75 hours) from a 32-year-old Korean woman tourist to four passengers on the same flight in April 1994. The patient was traveling from Chicago to Honolulu, and she had been on antituberculous treatment for the past 2 years. She was coughing during the flight and had fever. Eventually, she became very sick and was admitted to hospital and died 1 week later with massive hemoptysis. The sputum smear was highly positive, and culture was positive for M. tuberculosis. The CDC was involved in the investigation that included notifications to the passengers of that flight. Careful and thorough epidemiological screening was conducted including all risk factors and previous PPD skin test history. Four patients who were known

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tuberculin skin test-negative had initial testing after exposure which was negative and had a final test repeated 12 weeks later that showed conversion to positive results. In the Netherlands, Cobelens et al. (2000) investigated Dutch long-term travelers to countries of high tuberculosis endemicity. PPD skin testing was done before traveling and 2-4 months after their return. The results of 656 individuals were reported: there were 12 with M. tuberculosis infection, and 2 had active disease (1.8%). The overall incidence rate was estimated as 3.5 per 1000 person-months of travel and 2.8 per 1000 person-months of travel after the exclusion of health-care workers. The inclusion criteria for this study were over 15 years of age, not yet received BCG vaccination, and intended to travel for 3-12 months to these highly endemic countries. Exclusion criteria were history of tuberculosis or positive skin test, diabetes mellitus, HIV, or history of immunosuppressive therapy.

17.5 Clinical Features and Diagnosis of Primary Tuberculosis in Adults Primary pulmonary tuberculosis in adults may present with constitutional symptoms as well as cough, fever, hemoptysis, or weight loss that are often misdiagnosed initially as bacterial pneumonia. It is only because of the atypical clinical features in association with the so-called 'unusual' radiographic findings (Segarra et al. 1963; Parmar 1967; Dodd et al. 1978; Palmer 1979; Hadlock et al. 1980; Stead 1981; Lee et al. 1993) or the adoption of 'routine' microbiological examination of the sputum by smear staining and culture for acid-fast bacilli that an accurate diagnosis can be achieved (Choyke et al. 1983). A high index of suspicion by the attending clinician is essential for an early diagnosis. Failure in considering tuberculosis as a possible cause in these patients will delay the initiation of appropriate chemotherapy. The disease may progress, and serious dissemination to other body organs or systems may occur. Guidelines and criteria were used by several authors to identify and classify patients as having adult primary pulmonary tuberculosis (Stead et al. 1968; Khan et al. 1977; Choyke et al. 1983). Khan and colleagues (1977) classified adult patients with tuberculosis in the lower lung field as having primary pulmonary tuberculosis if one or more of the following criteria were also present: (1) known recent tuberculin conversion, (2) tuberculous pleural effusions

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without roentgenographic evidence of post-primary disease, (3) hilar adenopathy with or without parenchymal infiltration. These authors found 12 patients with adult primary pulmonary tuberculosis in their large series of 88 adult patients with newly proven diagnosis of active pulmonary tuberculosis who fulfilled these criteria. They were seen over a 12-month period and attended two hospitals in Boston, USA. Ten patients were young adults (19-38 years old), and 2 were in their 60s. Choyke and colleagues (1983) from the USA adopted the same criteria and reported their own series of 103 adult patients with primary pulmonary tuberculosis with positive M. tuberculosis cultures from at least one source. In our own series of 10 adult patients with primary pulmonary tuberculosis, the criteria for the classification depended mainly on radiographic findings. Recent PPD skin test conversion or the status of a previous tuberculin test is unknown in many of our patients. Choyke and colleagues (1983) from the USA stated: 'The diagnosis of primary tuberculosis rests upon tuberculin skin test (PPD) conversion.' However, they reported that there was inadequate documentation of the PPD skin test in their own series. Symptoms lasting a few days or weeks at the time of presentation are commonly reported in 84% by Khan et al. (1977) and Choyke et al. (1983). These included constitutional symptoms, fever, cough, and hemoptysis. The initial diagnosis of tuberculosis on admission was only made in 10%, and 70% were initially diagnosed to have 'bacterial pneumonia', and diagnosis was delayed for up to 6 weeks as reported in Choyke's series of 103 patients.

17.6 Lung Infiltrates and Consolidation Plain radiographs of the chest may look normal, and lung infiltrates may only be depicted on CT. Segmental or lobar consolidation of the right middle or lower lobe is the most common pattern of distribution in primary tuberculosis in adults as we noted in 6 of our patients (60%) (Figs. 23.3,23.9,23.16, and 23.18). Consolidation in the upper lobe may also occur, but it is rare. Associated hilar or right paratracheal lymphadenopathy with consolidation was noted in these 6 patients as well. Cavitation (Dahl 1952) in primary tuberculous consolidation was noted in 1 of our patients (Fig. 23.4). In the series of 12 patients reported by Khan et al. (1977), consolidations in the lower and middle lobes

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were noted in 6 patients (50%) and in the upper lobe, in 1 patient. In Choyke and colleagues' (1983) series of 103 patients, they reported consolidations in the lower and middle lobes in 77 patients (75%) and in the upper lobe in 36 patients (35%), bilateral in 13 (13%), cavitations in 8 (8%), and normal radiographs in 10 patients (10%). In Belgium, van den Brande and colleagues (1998) reported the incidence of adult pulmonary tuberculosis in a low prevalence area. They reviewed the chest radiographs of patients who had bacteriologically proven pulmonary tuberculosis over two periods (1981-1985 and 1986-1990). They found 114 and 105 patients, respectively. Approximately 50% of these patients were aged 65 years and over. Radiological features were classified as 'usual' or 'unusual' presentation.'Unusual' radiological features were defined as solitary pleural effusion, isolated hilar or mediastinal lymphadenopathies, normal chest xray, lower lung field tuberculosis, nodular lesions, diffuse infiltration, and atelectasis. The usual imaging features of pulmonary tuberculosis were present in 76% and 68%, respectively. Unusual imaging features of pulmonary tuberculosis were present in 24% and 35%, respectively. The authors noted the shift in the incidence of tuberculosis to elderly subjects who had never been infected before. They also noted that their 'unusual' imaging features resemble those observed in childhood primary pulmonary tuberculosis. They concluded that there is a recent trend towards a more 'unusual' presentation of primary pulmonary tuberculosis in the elderly.

17.7 Lymphadenopathy Unilateral or bilateral hilar lymphadenopathy and/or right paratracheal lymph node enlargement are the hallmarks of primary tuberculosis. They were noted in 9 patients with adult primary pulmonary tuberculosis (90%) in our own series, and their imaging features are illustrated in our chapter on radiography of pulmonary tuberculosis (Figs. 23.3, 23.4, 23.6, 23.7, 23.8,23.9,23.12,23.16, and 23.18). Hilar and/or paratracheal tuberculous lymphadenopathy in primary tuberculosis is more commonly reported in children than adults (McAdams et al. 1995; Woodring et al. 1986; Leung et al. 1992). Caseation and necrosis of tuberculous hilar or paratracheal lymph nodes may occur (Fig.23.8). Enlarged lymph nodes may obstruct the airway and may lead to atelectasis. It may invade the surrounding structures, particularly

the esophagus, phrenic nerve, recurrent laryngeal nerve, superior vena cava, or pericardium (Figs. 23.10 and 23.11). Other authors reported the incidence of tuberculous hilar lymphadenopathy as 15%-50% in their series (Khan et al. 1977; Choyke et al. 1983).

17.8 Pleural Effusion Pleural effusion in primary tuberculosis in an adult may occur in 29%-70% of patients (Roper and Waring 1955; Stead et al. 1968; Miller and MacGregor 1978; Khan et al. 1977; Choyke et al. 1983; Epstein et al. 1987). It is often unilateral, but bilateral effusion may rarely occur (Fig. 23.12). Associated parenchymal lesions and hilar lymphadenopathy may be present but not depicted on plain radiographs. Computed tomography is more sensitive in depicting such an association. Pleural effusion due to primary tuberculosis in adults was noted in one patient from our series (Fig. 23.12).

17.9 Miliary Tuberculosis Miliary tuberculosis may occur in both primary and reactivation tuberculosis (Munt 1972; Gelb et al.1973; Sahn and Neff 1974; Grieco and Chmel 1974). It is increasingly reported in immunosuppressed adults, particularly with HIV co-infection, in up to 13% (Choyke et al. 1983; Lee and 1m 1995). The plain radiograph may initially look normal, and CT is more sensitive in depicting the miliary lung shadowing.

17.10 Microbiological Confirmation Microbiological confirmation of the diagnosis in adult primary pulmonary tuberculosis may be difficult. The sputum smear and culture with positive yield were reported in as many as 60%-70% (Khan et al. 1977; Choyke et al. 1983). Bronchoscopy to obtain samples was performed in 34 patients, and in 27, the cultures were positive as reported by Choyke et al. (1983). Pleural fluid and biopsies were positive with yield of the bacilli in 10 out of 26 cases reported by the

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same authors. Other sources reported about pathological specimens obtained by open lung biopsies and thoracotomy in difficult-to-diagnose cases.

17.11

Treatment and Possible Paradoxical Transient Worsening The treatment of primary tuberculosis in adults does not differ from the treatment of reactivation disease. Directly observed therapy (DOT) is the most appropriate therapeutic regimen for primary pulmonary tuberculosis in adults. It is usually given on an outpatient basis for 6 months. In the initia12-month phase, four drugs are used (rifampicin, isoniazid, pyrazinamide, and either ethambutol or streptomycin) followed by a 4-month continuation phase of rifampicin and isoniazid. Treatment is usually given daily but can be given thrice weekly (see Chap. 44). During the initial 3 months of chemotherapy, paradoxical transient worsening of the original radiographic findings or development of new lesions may occur, as reported by many authors (Weber et al. 1968; Matthay et al. 1974; Campbell and Dyson 1977; Amodio et al. 1986; Lamont et al. 1986; Akira et al. 2000). The mechanisms and pathogenesis of this paradoxical response remain unclear. Enhanced immune responses to a sudden destruction and release of tubercular antigens or even a drug reaction has been postulated by several authors (Onwubalili et al. 1986; Marshall and Chambers 1988; Akira et al. 2000). Paradoxical transient worsening should be differentiated from true worsening of tuberculous lesions due to co-infection with HIY, other bacteria, or fungi. In our series, worsening of the original parenchymal shadowing as well as the development of new ipsilateral or contralateral consolidations were noted in two patients (Fig. 23.16 and 23.17). Radiological evaluation should be done at 2-3 months after initiation of chemotherapy (Bass et al. 1986).

17.12

Conclusion Primary pulmonary tuberculosis in adults is rarely reported in endemic developing countries, where it is mostly reported in children. The recent PPD skin test conversions, considered as important criteria for the diagnosis in developed countries, are difficult to

M. M. Madkour

document in most patients in endemic, resourcepoor countries. In developed, industrialized countries where the tuberculosis control program has been applied for many years, with a low incidence of the disease in the population, primary pulmonary tuberculosis has shifted to adults and the elderly population. The criteria for classification of primary pulmonary tuberculosis in adults, particularly, the recent PPD skin test conversions, can be applicable. The clinical and imaging features of adult primary pulmonary tuberculosis are no longer considered as 'usual' because of the shift of the disease to adults in developed countries. The clinical, imaging, and other investigations to confirm the diagnosis are discussed. Our own experiences with 10 patients with adult primary pulmonary tuberculosis have been discussed, and the imaging features of these patients can be seen in our separate chapter, Imaging of Pulmonary Tuberculosis, Figs. 23.3, 23.4, 23.8, 23.9, 23.10, 23.12, 23.16,23.17, and 23.18. Paradoxical transient worsening of radiology features during the early phase of chemotherapy is also documented.

References Akira M, Sakatani M, Ishikawa H (2000) Transient radiographic progression during initial treatment of pulmonary tuberculosis: CT findings. J Comput Assist Tomogr 24:426-43 I Alexander WJ, Avent CK et al (1979) Simple primary tuberculosis in an elderly woman. J Am Geriatr Soc 27:123-125 Amodio J, Abramson S, Berdon W (1986) Primary pulmonary tuberculosis in infancy: a resurgent disease in urban United States. Paediatr RadioI16:185-189 Balasubramanian V et al (1994) Pathogenesis of tuberculosis: pathway to apical localization. Tuberc Lung Dis 75: 168-178 Bass JB, Farer LS, Hopewell PC et al (1986) Treatment of tuberculosis and tuberculosis infection in adults and children. Am Rev Respir Dis 134:355-363 Berger HW, Granada MG (1974) Lower lung field tuberculosis. Chest 65:522-526 Beyers JA (1979) The radiological features of primary pulmonary tuberculosis. S Afr Med J 55:994-997 Buckner CB, Walker CW et al (1990) Radiologic manifestations of adult tuberculosis. J Thorac Imaging 5:28-37 Campbell lA, Dyson AJ (1977) Lymphnode tuberculosis: a comparison of various methods of treatment. Tuberculosis 58:171-179 Canetti G (1972) Endogenous reactivation and exogenous reinfection. Their relative importance with regard to the development of non-primary tuberculosis. Bull Int Union Tuberc 47:116-122 Chiba Y, Kurihara T (1979) Development of pulmonary tuberculosis with special reference to the time interval after tuberculin conversion. Bull Int Union Tuberc 54:263-264

Primary Tuberculosis in Adults Choyke PL,Sostman HD et al (1983) Adult onset of pulmonary tuberculosis. Radiology 148:357-362 Cobelens FGJ, Deutekom HV et al (2000) Risk of infection with Mycobacterium tuberculosis in travelers to areas of high tuberculosis endemicity. Lancet 356:461-465 Dahl RV (1952) The first appearance of a pulmonary cavity after primary infection with relation to time and age. Acta Tuberc Scand 27:140 Dannenberg Am Jr (2001) Pathogenesis of pulmonary Mycobacterium bovis infection: basic principles established by the rabbit model. Tuberculosis 81:87-96 Dodd GD, Capitanio MA et al (1978) Case 11 (primary tuberculosis). In: Film interpretation session. Radiological Society of North America, 64th scientific assembly and annual meeting, Nov 1978. Summary of discussion and diagnoses. Radiology 129:831 Ellertsen E (1959) Epidemics of primary tuberculosis and their significance. Acta Tuberc Scand 37:203 Ellner JJ (1997) Regulation of the human immune response during tuberculosis. J Lab Clin Med 130:469-475 Epstein DM, Kline LR et al (1987) Tuberculous pleural effusions. Chest 91: 106 Garcia M, Vargas A, Castej6n R et al (2002) Flow-cytometric assessment of lymphocyte cytokine production in tuberculosis. Tuberculosis 82:37-41 Gelb AF, Lefler C et al (1973) Miliary tuberculosis. Am Rev Respir Dis 108:1327 Goppert EF, Left A (1979) The pathogenesis of pulmonary and military tuberculosis. Arch Intern Med 139: 1381-1383 Grieco MH, Chmel H (1974) Acute disseminated tuberculosis as a diagnostic problem. Am Rev Respir Dis 109:554 Hadlock FP, Park SK et al (1980) Unusual radiographic findings in adult pulmonary tuberculosis. AJR 134:1015-1018 Heldal E et al (2000) Pulmonary tuberculosis in Norwegian patients. The role of reactivation, re-infection and primary infection is assessed by previous mass screening data and restriction fragment length polymprphism analysis. Int J Tuberc Lung Dis 4:300-307 Hulnick DH, Naidich DP, McCauley Dr (1983) Pleural tuberculosis evaluated by computed tomography. Radiology 149: 759-765 Ikejoe J, Kakeuchi N et al (1992) CT appearance of pulmonary tuberculosis in diabetic and immunocompromised patients: comparison with patients who had no underlying disease. AJR 159:1175-1179 Kenyon TA, Valway SE et al (1996) Transmission of multidrug-resistant Mycobacterium tuberculosis during a long airplane flight. N Engl J Med 334:933-938 Khan MA, Kovnat DM et al (1977) Clinical and roentgenographic spectrum of pulmonary tuberculosis in the adult. Am J Med 62:31-38 Lamont AC, Cremin BJ, Pelleret RM (1986) Radiological patterns of pulmonary tuberculosis in paediatric age group. Pediatr RadioI16:2-7 Lee KS, 1m JG (1995) CT in adults with tuberculosis of the chest: characteristic findings and role in management AJR 164:1361-1367 Lee KS, Song KS et al (1993) Adult-onset pulmonary tuberculosis: findings on chest radiographs and CT scans. AJR 160:753-758 Leung AN, Muller NL, Pineda PR et al (1992) Primary tuberculosis in childhood. Radiographic manifestations. Radiology 182:87-91

271 Marshall BG, Chambers MA (1988) Central nervous system tuberculosis: the paradox of host immune response. J Infect 36:3-4 Matthay RA, Neff TA, Iseman MD (1974) Tuberculous pleural effusion developing during chemotherapy for pulmonary tuberculosis. Am Rev Respir Dis 109:469-472 McAdams HP, Erasmus J, Winter JA (1995) Radiologic manifestations of pulmonary tuberculosis. Radiol Clin North Am 33:655-678 Medlar EM (1948) The pathogenesis of minimal pulmonary tuberculosis: a study of 1225 necropsies in cases of sudden and unexpected death. Am Rev Tuberc 58:583-611 Milburn HJ (2001) Primary tuberculosis. Curr Opin Pul Med 7:133-141 Miller WT (1994) Tuberculosis in the 1990s. Radiol Clin North Am 32:649-661 Miller WT, MacGregor RR (1978) Tuberculosis: the frequency of unusual radiographic findings. AJR Am J Roentgenol 130:867-875 Miller WT, Miller WT Jr (1993) Tuberculosis in the normal host: Radiological findings. Semin RoentgenoI28:109-118 Munt RW (1972) Miliary tuberculosis in the chemotherapy era: with a clinical review in 69 American adults. Medicine (Baltimore) 51:139 Onwubalili JK, Scott GM, Smith H (1986) Acute respiratory distress related to chemotherapy of advanced pulmonary tuberculosis. A study of two cases and review of the literature. OJ Med 59:599-610 Palmer PES (1979) Pulmonary tuberculosis - usual and unusual radiographic presentations. Semin RoentgenoI14:204-243 Parmar MS (1967) Lower lung field tuberculosis. Am Rev Respir Dis 96:310 Pitchenik AE, Rubinson HA (1985) The radiographic appearance of tuberculosis in patients with the acquired immunodeficiency syndrome (AIDS) and pre-AIDS. Am Rev Respir Dis 131:393-396 Reddy VM, Hayworth DA (2002) Interaction of Mycobacterium tuberculosis with human respiratory epithelial cells (Hep-2). Tuberculosis 82:31-36 Roper WH, Waring JJ (1955) Primary serofibrinous pleural effusion in military personnel. Am Rev Tuberc 71:616 Sahn S, Neff TA (1974) Miliary tuberculosis. Am J Med 56:495 Segarra F, Sherman DS et al (1963) Lower lung field tuberculosis. Am Rev Respir Dis 87:37-40 Slavin RE, Walsh TJ et al (1980) Late generalized tuberculosis: a clinical pathologic analysis and comparison of 100 cases in the preantibiotic and antibiotic eras. Medicine 59: 352-366 Stead WW, Kerby GR et al (1968) The clinical spectrum of primary tuberculosis in adults. Confusion with reinfection in the pathogenesis of chronic tuberculosis. Ann Intern Med 68:731-745 Stead WW (1981) Tuberculosis among elderly persons: an outbreak in a nursing horne. Ann Intern Med 94:606 Van den Brande PV, Dockx S et al (1998) Pulmonary tuberculosis in the adult in a low prevalence area: is the radiological presentation changing? Int J Tuberc Lung Dis 2:904-908 Vanham G, Toossi Z, Hirsch CS et al (1997) Examining a paradox in the pathogenesis of human pulmonary tuberculosis: immune activation and suppression/anergy. Tuberc Lung Dis 78:145-158 Varkey B, Politis J (1981) Pulmonary tuberculosis. A multifaceted disease. Postgrad Med 69:117-121, 124-126, 129

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M. M. Madkour Weber AL, Bird KT, Janower ML (1968). Primary tuberculosis in children with particular emphasis on changes affecting the tracheobronchial tree: AJR 103:123-132 Woodring JH, Vandiviere H et al (1986) The radiographic features of pulmonary tuberculosis. AJR 146:497-506

18 Miliary/Disseminated Tuberculosis M. MONIR MADKOUR

mary tuberculosis, immediately after the post-primary period, or at a time far remote from the post-primary 18.1 Introduction 273 period as late generalized tuberculosis (LGT) (Slavin 18.2 Epidemiology of MiliarylDisseminated et al. 1980). A wide spectrum of pathological features Tuberculosis 274 occurs as a result of hematogenous dissemination of 18.3 Pathology and Pathogenesis the bacilli. This spectrum of pathology is determined of Miliary/Disseminated Tuberculosis 275 18.4 Clinical Features of Miliary/Disseminated by the size of the bacillary inoculum load, the viruTuberculosis 277 lence of the bacilli, and the status of the host immune 18.5 Meningitis and Tuberculomas response. It ranges from a rare but acute fulminating in Miliary/Disseminated Tuberculosis 277 form due to the release of massive myriads of caseous 18.5.1 Case Illustration 1 279 18.5.2 Case Illustration 2 283 and necrotic tubercles into the blood with a nonreac18.5.3 Case Illustration 3 288 tive and an anergic response, very low count of CD4+ 18.5.4 Case Illustration 4 290 T-cells with scanty or absent granuloma formation; 18.5.5 Case Illustration 5 292 that form of the disease is only diagnosed at autopsy. 18.6 Adult Respiratory Distress Syndrome If, on the other hand, tuberculous bacteremia is slight and Disseminated Intravascular Coagulation in Miliary Tuberculosis 292 with the formation of few tubercles, this discrete type of 18.7 Pneumothorax in Miliary/Disseminated generalized dissemination is usually without immediTuberculosis 293 ate clinical significance, although these tubercles serve 18.8 Diagnosis of MiliarylDisseminated as 'seed beds' for the later development of organ tuberTuberculosis 293 culosis or LGT (Slavin et al. 1980). Between these two 18.9 High Resolution CT 296 18.10 Laboratory Diagnosis of Miliary pathological and clinical extremes lies a spectrum of Tuberculosis 296 pathology that varies in severity. The more common 18.10.1 Polymerase Chain Reaction 297 classic miliary tuberculosis resembles the fulminating 18.11 Treatment of MiliarylDisseminated form (Prout et al. 1980). Miliary tuberculosis is defined Tuberculosis 297 as a hematogenous dissemination of the bacilli resultReferences 298 ing in widespread, active, visceral, caseous tubercle formations measuring 1-3 mm in diameter (the size of millet seeds) with radiologic or pathologic evidence of pulmonary micronodules (Sahn and Neff 1974; Slavin et al. 1980; Penner et al. 1995). Disseminated tubercu18.1 losis is defined as a hematogenous transmission of the Introduction bacilli with active caseous tubercle formation in two Miliary/disseminated tuberculosis signifies the wide- or more extrapulmonary sites and with no pulmonary spread occurrence of caseating visceral tuberculosis miliary nodular shadowing on chest radiography that occurs by hematogenous dissemination of the (Penner et al. 1995; Sahn and Neff 1974). Proudfoot bacilli from an active caseous focus or foci located in the et al. (1969) described the fulminating dissemination lung or extrapulmonary sites. Hematogenous dissemi- form of tuberculosis in patients with other co-existing nation of the bacilli may occur during the course of pri- underlying disease that may lead to a clinical presentation with atypical features due to an impaired cellmediated immune response and the absence of miliary lung shadowing as 'cryptic disseminated tuberculosis', M. M. MADKOUR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces which is usually diagnosed at autopsy. Disseminated tuberculosis is a diagnostic challenge even in endemic Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia

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M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

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areas when the attending clinician has a high index of suspicion. Chest radiography is usually the initial diagnostic investigation for miliary tuberculosis. The sensitivity of chest radiography was found to range from 59% to 69% in a study based on population and group control subject (Kwong et al. 1996). ARDS may occur as a complication of miliary tuberculosis, and chest radiography may be difficult to interpret since the miliary nodules are superimposed on a more diffuse ground (Armstrong et al. 1995). Fatal consequences if undiagnosed and untreated at an early stage may affect 21 %-64% of patients with disseminated tuberculosis (Monie et al. 1983; Al-Jahdali et al. 2000; Prout et al. 1980; Bobrowitz 1982). Miliary/disseminated tuberculosis has increased in incidence in the USA, particularly among HIV co-infected patients (Rieder et al. 1991; Hill 1991; FitzGerald et al. 1991; Korzeniewska-Kosela et al. 1992). Iatrogenic miliary/disseminated tuberculosis due to Mycobacterium bovis, induced by intravesical BCG immunotherapy for the treatment of urinary bladder cancer, has been increasingly reported (Foster 1997; McParland et al. 1992; Palayew et al. 1993).

18.2 Epidemiology of Miliary/Disseminated Tuberculosis The incidence of miliary/disseminated tuberculosis is difficult to determine in either developing or developed countries (Vijayan 2000). Many patients were undiagnosed in life, and it is only at autopsy that the disease may be found (JuuI1977; Bobrowitz 1982; Slavin et al. 1980; Prout et al. 1980). In the USA, Slavin et al. (1980) from Johns Hopkins reviewed the records of 120 autopsies performed between May 1937 and December 1959. They excluded 20 cases due to missed records. The remaining 100 autopsies had microbiologically proven TB, histopathological features, and evidence of hematogenous spread involving the spleen, liver, lung, bone marrow, kidney, adrenal glands, and other organs. The total number of autopsies performed during these 22 years was 14,224, and LGT was found in 120 (0.8%). Sixty of the 100 patients lived in the pre-antibiotic era (mid-1948) and 40 in the antibiotic period. Male predominance was noted in 61 (61 %), of whom 44% were black. There were 39 women, 28% of whom were black. The total number of black patients was 72 and white patients, 28. The authors estimated that 1 case of LGT was found in every 142 autopsies.

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In Canada, Long et al. (1997) reviewed the records of 2013 patients with active tuberculosis between January 1, 1979, and December 31,1993, to determine the clinical-pathologic-radiologic correlation in disseminated tuberculosis with and without a miliary pattern on radiography. All patients came from Manitoba, with a low seroprevalence of HIV in that area. They adopted a rigorously defined; radiographic or histopathologic evidence of hematogenous dissemination and found 56 patients (3%) (42 miliary and 14 nonmiliary). Women were 2.4 times more likely to be affected than men in this series. The mean age for both sexes was 50±26 years. Fourteen patients died, of whom 5 (12%) had miliary and 9 (64%) had nonmiliary disseminated tuberculosis. In Glasgow, Scotland, Monie et al. (1983) reviewed the records of 1000 patients with tuberculosis reported during a 3-year period (1976-1978) and found 28 (2.8%) patients with miliary tuberculosis. Seventeen (61%) had miliary shadowing, 7 (25%) had other radiological changes consistent with active or previous pulmonary tuberculosis, and 4 (14%) had no radiological changes suggesting pulmonary tuberculosis. About 50% of patients were over 60 years of age, and 23% were non-European in origin. These authors reported fatalities in 9 (32%) of the 28 patients with miliary tuberculosis. Ormerod and Horsfield (1995) from Blackburn, UK, reviewed notified cases of tuberculosis in the Blackburn, Hyndburn and Ribble Valley District Health Authority (DHA), an area of high prevalence for tuberculosis. They found 39 cases of miliary tuberculosis over 16 years (1978-1993 inclusive). Fourteen patients had miliary TB, and 25 had cryptic miliary TB. The authors noted ethnic differences with regard to the number of cases and the age at onset. The mean age among Caucasian patients was 51.3 years, while among Indian subcontinent (ISC) patients, it was 37 years. The incidence of miliary tuberculosis in Blackburn was approximately 0.25/100,000 in the Caucasian ethnic group and 7.99/100,000 in the ISC ethnic group. These authors reported the death rate due to miliary tuberculosis as 10%. In Denmark, Juul (1977) reported on clinically undiagnosed, active tuberculosis in 895 autopsies. They found active tuberculosis undiagnosed in life in 86 (0.1 %) patients. Miliary tuberculosis found in 38 (44.2%). The mean age of these patients was 70.7 years, with a female predominance. In NewYork,Bobrowitz (1982) reported 21 patients in whom tuberculosis was not diagnosed in life. At autopsy, 10 had suffered miliary tuberculosis and 11, pulmonary tuberculosis. Fifteen were male and

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6 female, and 13 were white, 6 black, and 2 Hispanic. Over 70% of the patients were more than 60 years of age. The main diagnosis of the 10 patients with miliary tuberculosis on admission was pneumonia, malignancy, or sepsis. The authors explained the failure to discover tuberculosis in these patients as due to combinations of other diseases and conditions. The incidence of miliary tuberculosis in developing countries is more difficult to determine (Prout et al. 1980). Lack of notification of miliary tuberculosis is due to failure to make the diagnosis while alive in countries like South Africa, which has a high incidence of tuberculosis (Prout et al. 1980). These authors reviewed the computerized records of medical diagnoses over a 6year period and found 64 cases of miliary or disseminated tuberculosis. Thirty-seven cases fulfilled the inclusion criteria: miliary lung shadowing, detection of the bacilli by stain or culture, caseating granulomas from liver or bone marrow biopsies, and involvement of two or more organs. These authors also reviewed the autopsies of 2,200 patients who died between 1973 and 1978 and found a further 34 cases (of them 9 fell into both groups and were excluded). A total number of 62 cases (live and autopsies) was reviewed. Eight (12.9%) were white, 20 (32.3%) colored, and 34 (54.8%) black. Thirty-six (58.1%) were male and 26 (41.9%) female. Over 72% of men and 42% of women were over 40 years of age. Alcoholism, malnutrition, diabetes, cytotoxicity or radiotherapy for malignancies were concomitantly present in 25 patients (40.3%). Forty patients (64%) died, and 31 deaths were directly attributed to disseminated tuberculosis. In Saudi Arabia, AI-Jahdali et al. (2000) reported 780 cases of tuberculosis seen over a period of 7 years. Miliary tuberculosis was found in 47 (6%) patients, and 68% were over 60 years of age. A male predominance was noted (n=30), and the mean age was 61 years for both sexes. Risk factors such as diabetes, prior tuberculosis, chronic liver disease, renal failure, immunosuppressive drugs, and malignancies were present in over 50% of these patients. Ten patients died (21 %), all were due to miliary tuberculosis. In our own hospital in Riyadh, computerized records of medical diagnoses for patients admitted during the period from 1983-2000 inclusive (using ICD-9 code description) revealed a total number of 2,484 tuberculosis patients, with miliary tuberculosis reported in 79 patients (3.18%). Since the HIV epidemic in 1980s, the incidence of miliary/disseminated tuberculosis in HIV co-infected patients in the USA has increased. The incidence of miliary/disseminated tuberculosis has therefore increased from 1.3% of all reported cases of tuber-

culosis to 8%-10% among HIV co-infected patients (Rieder et al. 1991; Hill 1991; Schaefer et alI991).

18.3 Pathology and Pathogenesis of Miliary/Disseminated Tuberculosis Acute miliary/cryptic disseminated tuberculosis is part of a broad spectrum of hematogenous spread of Mycobacterium tuberculosis with various and variable degrees of clinical manifestation and severity. It is most commonly encountered in children and young adults in developing countries. It may occur during primary disease or within 6-8 months of primary disease (progressive primary). In endemic areas, hematogenous spread may occur in neonates, infants, and children or in immunocompromised patients, leading to primary miliary or disseminated tuberculosis. In developed countries, adults particularly those co-infected with HIV may develop primary miliary/disseminated tuberculosis via hematogenous transmission of the infection. Miliary/disseminated tuberculosis may occur in post-primary disease, either due to endogenous reactivation or exogenous re-infection (Auerbach 1944,1959). Hematogenous spread of the bacilli from a focus or foci located in the lung or extrapulmonary sites is the source of infection. Transmission of the organisms via the lymphatics to the blood circulation or direct invasion of the blood vessels by liquefied caseous material may occur due to a rupture or erosion of a contiguous focus or foci (Geppert and Leff 1979; Yu et al. 1986; Murray et al. 1978; Prout et al. 1980). Slavin et al. (1980) used the term late generalized tuberculosis (LGT) to describe generalized hematogenous spread occurring at a time far remote from the post-primary period. If tuberculous bacteremia is slight, a discrete type of generalization is usually without immediate clinical significance, although these 'seed beds' may serve later in the development of organ tuberculosis and LGT. However, if the generalization of the tuberculous bacteremia is massive, necrotizing tubercles are formed in various organs, and this is known as acute miliary tuberculosis. In postprimary disease, it is often believed that a single focus at the pulmonary or extrapulmonary site is considered the source of hematogenous spread of the bacilli. Clinical and pathological studies reported by several authors indicated that more than one focus located at different unrelated anatomical sites in various body organs may be reactivated simultaneously, discharg-

276

ing an enormous quantity of mycobacterial inoculum into the bloodstream. Slavin et al. (1980) stated that 'Late Generalized Tuberculosis (LGT) derived from a single extrapulmonary focus was very infrequent (7%); whereas in more than half of the cases, the origin of dissemination appears to have been derived from large foci present in a variety of organs'. This may occur intermittently, episodically, or continuously in 54% of cases (Pagel et al. 1964; Saye 1936; Slavin et al. 1980; Jacques and Sloan 1970). Evidence of intermittent or episodic hematogenous spread of infection was noted at autopsy. Soft exudative tuberculous lesions were mixed with other lesions showing partial or complete healing with fibrosis, calcifications, and ossifications (Slavin et al. 1980; Saye 1936; Jacques and Sloan 1970). It has been debated by some authors whether the lung is the main source of focal active caseous spread via the hematogenous route to other body organs; they believe that an extrapulmonary focus or foci are the most common source of hematogenous spread of infection. Slavin et al. (1980) reported that 'in the antibiotic era, chronic pulmonary tuberculosis and late generalized tuberculosis (LGT) coexisted far less commonly than they did prior to the advent of chemotherapy'. They reviewed the clinical and autopsy findings of 100 patients with LGT in two eras (pre-antibiotic and antibiotic periods) and concluded,'thus, both clinical and autopsy findings in the antibiotic period support Biehl's statement - that only a minority of cases of miliary tuberculosis arise from patients with pulmonary tuberculosis'. Hematogenous release of large quantities of necrotic caseous material of uniform size spread via the blood will cause embolization of the capillaries of most organs, particularly those with the richest vascular supply such as the liver, spleen, bone marrow, brain, and lungs. It is generally fatal if undetected and not treated early. At the other end of the spectrum of the hematogenous bacteremia, few bacilli may be discretely seeded into these body organs without immediate clinical manifestation. At a later stage, these hematogenous 'seed beds' may be reactivated in an organ or in all organs, with the development of miliary tuberculosis. They may also remain nonprogressive and well localized and cause few or no clinical symptoms. The course of the disease may take the form of 'chronic miliary tuberculosis', which is slowly progressive and not fatal (Pagel et al. 1964; Saye 1936). Fatal cases due to late generalized miliary tuberculosis do occur, but some clinicians consider these as representative of the terminal event of long-standing pulmonary or extrapulmonary disease (Yu et al.1986; Slavin et al. 1980). Factors determining the pattern of

M. M. Madkour

the disease presentation will depend on the size of the bacilli inoculum, its virulence, and the host immune response. In its severe acute fulminating form, septicemia may occur with extensive tissue damage and necrosis with very little evidence of a cell-mediated immune response. The diagnosis of this form is mostly missed in life because of the absence of the typical miliary lung shadowing seen on chest radiography, and the diagnosis is only made at autopsy (Chapman and Whorton 1946; Biehl 1958; Treip and Meyers 1959; Proudfoot et al.1969; Munt 1972; Prout et al.1980). Proudfoot et al. (1969) described this form of the disease as 'cryptic disseminated tuberculosis', Patients with cryptic disseminated tuberculosis may have a co-existing underlying disease that may present with atypical features and also cause impairment of the cell-mediated immunity such as alcoholism, cirrhosis, malnutrition, diabetes mellitus, connective tissue diseases, HIV, or may be taking immunosuppressive chemotherapy, and the diagnosis is then missed in life, with an absence of miliary lung shadowing, and is usually made at autopsy (Millar and Horn 1979; Bobrowitz 1982; Yu et al. 1986; Crump 1998). Patients in the late stage of AIDS with disseminated tuberculosis have a very low count of CD4+ T-cells and an anergic response with scanty or absent granuloma formation as reported by Zumla and Grange (see Chap. 29). In immunocompetent patients, the cell-mediated immune response (CM!) and tissue-damaging delayed hypersensitivity (DTH) responses will be activated. The bacilli cell wall antigens will activate the macrophages, CD4+ T cell, increased production of cytokines, TNF-a, IFN -y, IL-12 to combat the infection. The bacilli may survive, and during these responses concomitant immune depression and immune activation occur at the same time (Dannenbergh 2001; Vanham et al. 1997; Ellner 1997). The infection may overcome the immune system responses, and the disease progresses. At sites of the capillary bed seeded with bacilli, granuloma formation with central caseation and necrosis may occur simultaneously in all affected body organs. Adult respiratory distress syndrome (ARDS) is known as a complication of miliary/disseminated tuberculosis; it requires mechanical ventilation and has a high mortality rate ranging from 40% to 60% (Mohan et al. 1996; Dyer and Potgieter 1984; Murray et al. 1978; Dyer et al. 1985; Dee et al. 1980). The exact pathogenetic mechanisms by which miliary tuberculosis causes ARDS is not clearly understood. Penner et aI. (1995) suggested that the immunological aspects and pathogenesis of ARDS caused by miliary tuberculosis are similar to those caused by Gram-negative sepsis. In

MiliarylDisseminated Tuberculosis

277

miliary tuberculosis, a massive release of mycobacteria including fever, sweating, loss of appetite, weight lipoarabino-mannan cell wall antigens causes wide- loss, and weakness. Respiratory symptoms may spread perifocal inflammatory responses, injury to the include dry or productive cough, hemoptysis, shortalveolocapillary membrane, interstitial granulomatous ness of breath, and pleuritic chest pain. Neurological infiltration, obliterative endarteritis, increased capillary symptoms such as headaches, confusion, disorientaendothelial cell susceptibility to the toxic effect of the tion, cranial nerve palsies, or even coma may be the released TNF-a and ICAM-l (Penner et al.1995; Sutton presenting features. A history of tuberculosis may be et al. 1974; Petty and Ashburgh 1971; Massaro and Katz present in 10%-20% of patients (Slavin et al. 1980). 1964; Ashburgh et al.1967; McClement et al.1951). Fever is the most common symptom and may be The alveoli will be filled with inflammatory exu- the only presenting feature (pyrexia of unknown dative edema due to increased local vascularity, with origin). Other symptoms of tuberculosis related to vasculitis leading to pulmonary edema which is the other body systems or organs such as musculoskhallmark of ARDS (So and Yu 1981; Penner et al. eletal, genitourinary tract, or the abdomen are less 1995; Dee et al. 1980). The alveolar exudate mate- frequently encountered at the time of presentation. rial includes mostly polymorphonuclear leukocytes, Concomitant chronic illnesses such as diabetes melerythrocytes, fibrin, as well as caseating granulomas. litus, alcoholism, connective tissue diseases, chronic This will lead to mismatching of ventilation and per- renal failure, silicosis, HIV, or immunosuppressive fusion and an impaired diffusion capacity (Mohan et chemotherapy may be found in patients with miliary/ al.1996; William and Yoo 1973). disseminated tuberculosis. The physical signs may be Acute miliary/disseminated tuberculosis may few, including tachypnea, wasting, lymphadenopathy, also be caused by Mycobacterium bovis. Miliary M. hepatic and splenic enlargement, cutaneous lesions, bovis induced by intravesical BCG immunotherapy or features of meningitis (Ray et al. 2002; Sharma used for the treatment of bladder cancer has been et al. 1981; Pasculle et al. 1980; Bateman et al. 1980; reported (Deresiewicz et al. 1990; Rawls et al. 1990; K6ylii et al.1997; Shibolet et al.1979; Prout et al.1980; Kesten et al. 1990; Gupta et al. 1988; McParland et al. Evans et al. 1998; Long et al. 1997). The frequency of 1992; Iantorno et al. 1998; Palayew et al. 1993; Foster symptoms and signs of miliary/disseminated tuber1997; Jasmer et al. 1996; Rabe et al. 1999; Soloway culosis as reported by some authors are shown in 1988). The mechanism by which BCG induces mili- Tables 18.1 and 18.2. ary tuberculosis has been debated among authors (Lotte et al. 1984; de Hertogh et al. 1989; Orihvela et al. 1987). Some authors suggested that it is a hypersensitivity reaction in response to BCG, particularly 18.5 when they failed to grow the organisms. Other Meningitis and Tuberculomas authors explained the widespread lung shadowing in Miliary/Disseminated Tuberculosis as due to hematogenous dissemination of the bacilli which were isolated from various pathological fluid Miliary/disseminated tuberculosis may involve the and tissue biopsy specimens. Other nontuberculous central nervous system with evidence of meningitis Mycobacterium species may cause miliary and dis- or tuberculomas in up to 30% of patients (Slavin et seminated diseases similar to those caused by M. al.1980; Biehl 1958; Maartens et al.1990). Headaches, tuberculosis. These nontuberculous mycobacteria are hemiplegia, seizures, and coma may indicate involveparticularly found in patients with HIV. Mycobacte- ment by meningitis and/or tuberculoma, as noted in rium avium-intracellulare, M. kansasii, and M. fortui- our case no. 3 (Fig. 18.3f,g). However, the patients may tum are the ones most frequently reported (Bone and have no symptoms that are related to the central nerStableforth 1981; Longdale et al. 1992). vous system, and cerebral tuberculoma may only be depicted by MRI. The cerebrospinal fluid (CSF) may be either clear or turbid with lymphocytic pheocytosis, raised protein concentration, with a low level of 18.4 glucose (Gelb et al. 1973). Cerebrospinal fluid smear Clinical Features of Miliary/Disseminated is rarely positive for acid-fast bacilli, and culture may Tuberculosis yield the bacilli in up to 20% of patients (Hill 1991). The prognosis may be poor if undiagnosed and not The clinical symptoms at the time of presentation of treated early, with the development of hydrocephalus, miliary/disseminated TB may only be constitutional coma, and death.

54

109

21 26

59

67

62

63

Long et al. (1997)

Maartens et al. (1990)

Bobrowitz (1982) Stenius-Aarniala (1979)

Prout et al. (1980)

Munt (1972)

Biehl (1958)

Chapman and Whorton (1946)

79

62

84

44

95 88

92

82

72

Fever (%)

82

63

65

63

43 15

72

59

60

Cough (%)

-

-

-

37

-

19

72

26

30

Expectorant (%)

64

23

1

37

33 31

72

30

34

Dyspnea (%)

-

-

-

15

-

72

11

-

84

69

63

Munt (1972)

Biehl (1958)

62

72 82 96 44

60

-

33

-

62

67

-

-

62

6

6

8.5

15

-

13

Pleural effusion 8 46 36 57

30

-

12

15 12

52 62

72 53

35

13 36

-

4 20 21 28

9

14

-

-

85

61

95

59

33 38

92

62

30

Hemoptysis Weight loss (%) (%)

-

-

38 16

Epididymo-orchitis, subcutaneous abscess arthritis 13

Others

Fatigue - 49 Sweating - 45 Sweating - 37 Weakness - 70 Abdominal pain - 21 Musculoskeletal - 13 Disorientation and confusion - 52 Abdominal pain - 35 Fatigue - 35 Nausea & vomiting - 23 Anorexia - 51 Weakness - 42 Abdominal pain - 30 Confusion - 17 Abdominal pain - 7 Weakness - 93 Anorexia - 91 Weakness - 65 Anorexia - 42 Abdominal pain - 10 Anorexia - 90

Others

Neck stiffness 26 Pericardial effusion 8 Choroidal TB 7 -

20 -

7

18

10

17

12

25

-

-

Headaches (%)

Hepato- Spleno- Ascites CNS megaly megaly

Chest pain (%)

No. of Fever Tachypnea Wasting Lympha- Chest signs denopathy cases

Al-Jahdali et al. (2000) 47 Long et al. (1997) 54 Maartens et al. (1990) 109 Prout et al. (1980) 62

Reference

Table 18.2. Most common signs of miliary/disseminated tuberculosis

47

No. of cases

Al-Jahdali et al. (2000)

Reference

Table 18.1. Most common symptoms of miliary/disseminated tuberculosis

s:: l:

~ ...

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~ ~

I~

MiliarylDisseminated Tuberculosis

18.5.1 Case Illustration 1

A 39-year-old male clerk working in our hospital presented to me on 8 September 1999 with a I-month history of right knee pain which was mild and associated with swelling. Symptoms were progressive. He was in good health before presentation and denied a history of trauma. He was always overweight and did not notice any recent weight loss, and had no fever or sweating. Musculoskeletal system investigation was otherwise normal. He was using analgesics as self-medication, which were controlling his knee pain. There was no low-back pain or other systemic symptoms that could be related to connective tissue diseases. He was treated in the ophthalmology clinic for dryness of the eyes but that lasted only for a few months before resolving. He denied a history of drinking raw milk or consumption of raw liver or meat or contact with animals. Other systemic enquiries were normal. On physical examination he looked well, apyrexial, not in pain and not pale. The right knee was swollen with effusion but not tender or warm and had a full range of movement. There was no thigh or leg muscle wasting. Other joint examinations were normal. Other systematic physical examination showed no lymphadenopathy, and other systems were normal. Synovial fluid aspiration was obtained, and other investigations included hemogram, ESR, biochemical profiles including serum urate, blood culture, and Brucella agglutinins, plain radiography of the knees and chest. He was referred to our orthopedic surgeon for arthroscopy, but he refused this investigation. The hemoglobin was 13.2 gldl with normal white cell count, but the ESR was raised 50 mm/h. Biochemical parameters were normal apart from slightly raised serum urate at 467 f.lmolll (normal range 210-430 f.lmolll). Chest and knee radiography results were normal (Fig. 18.1a,b). Synovial fluid white cell count was normal, and culture was negative. Plain radiography and MRI of the right knee showed two small defects in the joint's surface on the medial condyle of the femur, one measured 7 mm and the second 3-4 mm in diameter. This was interpreted as osteonecrosis but could also be due to an inflammatory process (Fig. 18.1c-f, j). Arthroscopy was refused again by the patient. He was taking Diclofenac retard 100 mg once daily orally and was controlling his mild knee pain, and the swelling resolved. Four months later he presented to the emergency room with a 1O-day history of fever, rigor, night sweating, and 4-day history of cough with whitish sputum, left pleuritic chest pain, and shortness of breath. He also had a history of diarrhea for 3 days before presentation. He was anorexic

279

with occasional vomiting, and although he was on a weight-reducing diet, he did not notice faster weight reduction. On physical examination in the emergency room, he looked unwell, tachypnea, feverish 40°C, pale, and had lost 5 kg of weight since last weighed 4 months earlier. There was no lymphadenopathy, no positive abnormal sounds heard over the lung, and no pleural rub. Abdominal examination revealed an enlarged, tender liver extending 4 cm below the costal margin and enlarged spleen extending 3 cm below the costal margin, but there was no ascites or other palpable masses. The right knee was swollen with mild knee effusion. Chest radiograph looked normal, but the hemoglobin decreased to ILl gldl with normal blood film, and there was evidence of malaria, while the white cell count was normal. Blood culture was done, and the blood biochemical parameters were normal. When I reviewed the patient the following day, a softer chest radiograph was done which showed faint reticular lung shadowing at both lung bases (Fig. 18.lg). Miliary tuberculosis was the presumptive admission diagnosis, and he was started on four-drug chemotherapy. Other investigations were carried out during admission. The sedimentation rate (ESR) was 90 mm/h, negative blood and sputum cultures. Brucella serology was repeated, autoimmune screenings were all negative. Liver enzymes were abnormal; ALT 79 U/l (normal range 2-40 U/I); alkaline phosphatase 1151 U/I (normal range 98-279 U/I), albumin 33 gil (normal range 38-51 gil), urate 467 f.lmolll (normal range 210-430 Ilmolll), and total bilirubin was normal 12 f.lmolll (normal range 2-22 f.lillolll). The tuberculin test was negative; it had been done twice, once at the initial visit 4 months earlier and during the current admission. MRI of the chest showed diffuse confluent miliary lung shadowing widely spread over both lung fields with small areas of nonhomogeneous consolidation in the lateral segment of the right middle lobe (Fig. 18.1h,i). Ultrasound of the right knee showed significant synovial thickening but no effusion. Ultrasound of the abdomen showed an enlarged spleen with a span of 16 cm but without focal lesions in it; the liver was enlarged, but no focal lesions were depicted; and there was no definite para-aortic lymphadenopathy. The other abdominal organs were normal. The nonsteroidal anti-inflammatory medication was discontinued, and the patient remained on antituberculous medication. Bronchoscopy was done which showed an inflamed bronchial mucosa but no endobronchial lesions, and bronchoalveolar lavage (BAL) was performed. BAL was sent for cytology, AFB smear, and culture - post-bronchoscopy sputum specimens were sent for microbiological examination.

280

M. M. Madkour

a

b

[>

c Fig. 18.1 a Frontal radiograph of the right knee at the initial presentation with monoarthritis. This was interpreted as showing no bony abnormalities. b Chest radiograph at the initial presentation with right knee monoarthritis, showing no abnormal imaging features. c, d MRI of the right knee: sagittal Tl-weighted images demonstrate presence of effusion (arrow) and two osteochondral lesions (arrowheads) at the medial femoral condyle appearing as central areas of increased signal intensity surrounded by signal void rim. Appearances are typical of osteochondritis dissecans, but an infective pathology is also possible. e, f MRI of the right knee: sagittal T2-weighted images at the same level demonstrates joint effusion better (arrow) and the osteochondral defect as high signal intensity indicating activity (arrowheads). g Chest radiograph during acute presentation demonstrates numerous fine discrete nodules bilaterally (arrowheads) consistent with miliary tuberculosis. h Axial CT scan with lung windowing demonstrates generalized increase in parenchymal density with ill-defined fine nodular pattern in a random distribution (arrowheads). i Axial CT scan at a caudal level demonstrates air-space consolidation in the left lower lobe (arrowheads). j Frontal radiograph of the right knee during the acute miliary tuberculosis presentation demonstrates evidence of periarticular osteoporosis (arrowheads) and subchondral erosions (arrows). Note preservation of joint space (*). k Isotope bone scan (99mTc) demonstrates marked tracer uptake in the right knee (arrowheads). I Isotope bone scan demonstrates tracer uptake in 010 vertebral body (arrowheads). m Axial CT of the lung with mediastinal windowing demonstrates destruction of the lower dorsal vertebral body with associated paravertebral abscess (arrows). n Sagittal gadolinium-enhanced Tl-weighted MR image of the spine demonstrates destruction of the vertebral body (arrows), paraspinal abscess (large arrowheads) with subligamentous spread (small arrowheads) causing compression of the spinal cord (*).0 Axial gadolinium-enhanced Tl-weighted MR image with fat saturation demonstrates paraspinal abscess with wall enhancement (arrows)

281

Miliary/Disseminated Tuberculosis

e

f

[>

282

M. M. Madkour

k

n

m

o

Fig. 18.1 (Continued) k-o

MiliarylDisseminated Tuberculosis

283

The patient agreed to arthroscopy, which showed a cough, expectoration, loss of appetite, and weight hemorrhagic hypertrophic synoviurn with grade II loss. The swelling in the forehead was painless, and degenerative changes at the medial femoral condyle. she denied any history of head trauma. She had low Debridement, synovial tissue biopsies, and wash-out back pain and amenorrhea for the same duration. She were sent for histopathological and microbiological was in perfect health before the present illness. Other examination. Bone marrow biopsy was performed. systemic reviews were noncontributory, but she gave The patient responded to the antituberculous treat- a history of contact with a housemaid who had pulment, and the fever and dyspnea began to improve monary tuberculosis 6 years before the onset of the within 1 week of hospitalization. However, he com- current illness. She had no family history of tubercuplained of back pain, and there was tenderness over losis. She was a high school student and single. She T9-1O. He later admitted that he had had occasional was initially diagnosed to have tuberculosis a few back pain before, but it was so mild that it was relieved days before her presentation to our hospital. She initially presented to our satellite military hoswith the nonsteroidal anti-inflammatory treatment he used for his knee. Isotope bone scan and MRI of the pital in Al-Kharj (80 km south of Riyadh) and was spine and other joints were performed. MRI showed diagnosed as having tuberculosis and was referred to destruction of T9 with collapse of the vertebral body, our hospital a few days after initiating her on antitugibbus deformity, and para-spinal abscess with exten- berculous antibiotics. On physical examination, the sion into the epidural space (Fig. 18.1m-o). Isotope patient was unwell, pale, underweight (38 kg) with bone scan showed increased uptake at T9-1O and in forehead cystic swelling measuring 3.0x2.0 cm in the right knee (Fig. 18.1k,1). Histopathological results diameter. It was not tender or hot or red, and there of the bone marrow showed multiple areas of granulo- was no discharge from it. She had fever with a temmatous lesions with central caseation, but bacilli were perature of 39SC and tachycardia of llO/min. She not seen, and the culture was later reported as negative. had no peripheral lymphadenopathy. Cardiovascular, The histopathological findings of the synovial tissue respiratory, abdominal, and central nervous system and debridement specimen showed multiple granulo- examinations showed no abnormalities. Joint examimatous synovitis with central caseation. BAL culture nations were normal; she had localized tenderness and post-bronchoscopy sputum grew M. tuberculosis on percussion over multiple areas of the spine, but that was sensitive to isoniazid, rifampicin, pyrazin- there were no deformities or kyphosis. The hemoamide, and ethambutol. The temperature settled after gram showed WBC 5.3X109, hemoglobin 8.1 g/dl, and 9 days of antituberculous medication. The difficulties ESR 90 mm/h. Clinical chemistry parameters were in the diagnosis of this patient were due to several normal. Chest radiograph showed normal cardiac factors: the mild nature of the right knee symptoms size and clear lung fields. There were two ill-defined, with absence of constitutional symptoms, the refusal soft-tissue mediastinal masses on the right lateral of exploratory arthroscopy as an important diagnos- border and upper left border of the cardiac shadow tic means of investigation, the use of nonsteroidal with no calcification. Frontal and lateral dorsolumbar anti-inflammatory medication that masked back pain spine radiographs showed loss of height of L2 and despite the enquiry into spinal symptoms, the initially reduced bone density ofL! and L3 (Fig. 18.2a). Sagitnormal-appearing chest radiograph was done in the tal CT showed destruction of L2 (Fig. 18.2b). MRI of emergency room when he presented with acute chest the lumbar spine showed destruction of the body of and constitutional symptoms, the multiplicity of skel- L2 with compression of the dural space by a paraetal foci that were the most likely source of hematog- spinal abscess (Fig. 18.2c-e). Postcontrast CT of the enous dissemination rather than a single focus. The abdomen showed anterior subligamentous abscess patient responded well and continued antituberculous with a large right psoas muscle abscess (Fig. 18.2f,g). medication for 12 months. He has been followed up MRI of the abdomen at the same level showed a for over 2 years with no evidence of recurrence or any large right psoas abscess displacing the right kidney neurological or other complications. (Fig. 18.2h). Drainage of the right psoas abscess was performed in the prone position (Fig. 18.2i). The dorsal spine MRI showed another lesion 18.5.2 affecting the mid-dorsal region (Fig. 18.2j,k). Chest Case Illustration 2 CT with contrast of lung and mediastinal windows depicted a small lung parenchymal granuloma A 16-year-old Kuwaiti girl presented on 29 July 2000 and caseating lymph nodes in the retrosternal with a 6-month history of forehead swelling, fever, carina, subcarinal region, and prevascular groups

284

M. M. Madkour

a

b

c

d[> Fig. IS.2a-s. A 16-year-old girl presented with painless, soft-tissue swelling of the forehead, fever, cough, and low-back pain. a Frontal and lateral radiographs of the dorsolumbar spine demonstrate loss of height of L2 vertebral body (arrow) due to destructive lesion demonstrated better on the lateral view (arrowheads). There is also reduced bone density of the vertebral body of L1 and L3 and narrowing of disc spaces (D). b Sagittal reformatted CT image demonstrates destruction of the body of L2 with sclerosis of the remaining part (arrowhead). c MRI of the lumbar spine: sagittal post-enhanced Tl-weighted image demonstrates destruction of the body of L2 with large tuberculous cavity (arrow). Note posterior comparison on the dural space (open arrow). d Right para-sagittal image demonstrates anterior subligamentous tuberculous abscess (arrows) which had a tract to form right paraspinal abscess (A) in a. e Sagittal T2-weighted image with fat suppression demonstrates the paraspinal abscess as high signal intensity mass (A), the subligamentous abscess (long arrows), diffused increased signal intensity of the body of L2 and to lesser extent the body of L1 (open arrowheads). Note abscess cavity in L2 (arrow). f Post-enhanced CT of the abdomen at the level of L2 demonstrates destruction of the vertebral body (black arrow), anterior subligamentous abscess (arrowhead), and a large right psoas muscle abscess. g Post-enhanced axial CT at a caudal level demonstrates further destruction of L2 vertebral body (black arrow), tracking of the anterior subligamentous abscess (open black arrow), into a large right psoas muscle abscess (A). h MRI of the abdomen at same level as f: post-enhanced Tl-weighted image demonstrates destruction of

MiliarylDisseminated Tuberculosis

285

e

f

g

h the body of L2 (black arrow) and a large psoas muscle abscess (A), note the enhancing thick wall of the abscess (small arrows). The abscess is displacing the right kidney (thick black arrow). i Axial CT of the abdomen demonstrates percutaneous insertion of drainage catheter at the site of psoas abscess (arrow) (the examination is performed in the prone position). j Sagittal MRI of the dorsal spine: T2-weighted image with fat suppression demonstrates large anterolateral abscess appearing as diffuse high signal intensity anterior and to the right of mid-dorsal region (arrows). No associated bone or disc lesion is seen. k, I MRI of the same patient. Post-enhanced II-weighted sagittal (k) and axial (I) images demonstrate the abscess (A) as low signal intensity surrounded by thick enhancing wall (arrows). m, n CT of the chest: post-enhanced CT at the level of aortic arch. m Lung window demonstrates narrow lung parenchyma separate from small granuloma (arrow). n Mediastinal window demonstrates caseating lymph node with characteristic ring enhancement in the retrosternal and prevascular group (arrows). 0, p Post-enhanced axial CT at the level above the carina (0) and subcarinal (p) demonstrates appearance characteristic of subcarinal region. Note ring enhancement of the wall (arrow). q, r Frontal and lateral skull radiographs demonstrate focal area of bone destruction in the frontal bone. Note loss of the outer table of the skull bone on the lateral radiograph (white arrows) and focal ill-defined radiolucency due to bone destruction (black arrows). s Axial CT of the brain demonstrates bone destruction of the frontal bone (curved arrow) and associated soft-tissue mass due to abscess formation (white arrow)

l>

286

Fig. 18.2 (Continued) i-m

M. M. Madkour

m[>

MiliarylDisseminated Tuberculosis

287

n

o

q

p

s r

Fig. 18.2 (Continued) n-s

288

(Fig. 18.2m-p). Plain radiograph of the skull depicted a focal area of bone destruction in the frontal bone (Fig. 18.2q,r). Axial CT of the brain showed a softtissue abscess associated with frontal bone destruction (Fig. 18.2s). Ultrasound of the abdomen showed no evidence of hepatic or splenic involvement, and CT showed no intra-abdominal free fluid or other abdominal organ involvement. Culture of psoas abscess drainage yielded M. tuberculosis after 14 days incubation and was sensitive to all antituberculous agents. The forehead abscess was drained by our neurosurgeon, and tissue biopsies were obtained. Histopathological examination of the forehead abscess showed caseating granulomas, and culture yielded M. tuberculosis. The patient was treated with isoniazid 300 mg, rifampicin 450 mg, pyrazinamide IG, ethambutol 800 mg, and pyridoxine 25 mg as single oral daily dosing for 2 months and continued on isoniazid and rifampicin for a further 10 months. She responded well to treatment while in hospital, and her temperature settled with weight gain. She remained in hospital for 2 months and was discharged in September 2000. She stayed in Riyadh with members of her family and was followed up in the outpatient clinic regularly. She made a full recovery with no sequelae. This patient had disseminated tuberculosis with spinal disease at multiple levels, mediastinallymph node abscesses, psoas abscess, and forehead abscess. Although there was no lung parenchymal active disease depicted on plain chest radiography, CT showed small parenchymal lung granuloma. This patient did not have HIV or other diseases and was not on any medication that could have contributed to such severe disseminated tuberculosis.

M. M. Madkour

18.5.3 Case Illustration 3

A 27-year-old man transferred to our infectious disease unit isolation room with advanced AIDS, multidrug-resistant pulmonary tuberculosis, left hemiplegia due to tuberculous abscess of the brain, and recurrent seizures. This patient was diagnosed in another hospital in Riyadh with pulmonary tuberculosis and HIV co-infection in 1997. His pulmonary tuberculosis relapsed in 1999 due to poor compliance with medication. He was moved from one hospital to another and finally came to our hospital on 29 April 2001 until his death on 3 November 2001 due to disseminated multidrug-resistant tuberculosis. On arrival at our hospital, he was cachectic, pale, dyspneic, had oral thrush, and looked very ill. The hemogram showed low WBC 2.8109, and CD4 count ranged between 14 and 30/111, hemoglobin was 8.2 g/dl, and ESR was 84 mm/h. The HIV viral load was 75,000 COP/ml in 1999. Sputum culture yielded M. tuberculosis resistant to rifampicin and isoniazid. He was already on AZT, lamuvidine, rifampicin, clarithromycin, co-trimoxazole, fluconazole, phenytoin, and pyridoxine. Chest radiograph showed paratracheal and hilar lymphadenopathy and fibronodular infiltrative left upper lobe lesions with thin-walled cavities (Fig. 18.3a). Axial CT of the chest showed evidence of endobronchial tuberculosis with caseating hilar lymphadenopathy (Fig. 18.3b-d). In June 2001, the patient developed abdominal pain, vomiting, abdominal distension, and constipation for 2 days. The abdomen was tender all over with absent bowel sounds. Abdominal radiograph and axial CT showed

Fig. 18.3a. Plain chest radiograph demonstrates paratracheal and hilar adenopathy (arrows) and fibronodular infiltrative changes in the left upper lobe (curved arrow) with thin-walled cavities (arrowheads). b Axial CT of the chest with lung windowing demonstrates nodular and branching opacities of endobronchial tuberculosis (arrowheads) in both upper lobes, dilated bronchi (arrows). c Axial CT at caudal level, lung windowing demonstrates multiple thin cavities (arrows). d Axial post-enhanced CT of the chest at the level of carina demonstrates enlarged lymph nodes with typical ring enhancement and caseating center (arrows). e Axial post-enhanced CT of the abdomen demonstrates significantly dilated small-bowel loops (B) and caseating mesenteric lymph node (curved arrow). There was no ascites (dry type). At surgery, 20 cc of pus were aspirated from the mesenteric abscess. f MRI of the brain: post-enhanced TI-weighted image demonstrates tuberculoma and tuberculous abscess in the left parietal lobe. Note the enhancement of the tuberculoma (arrowhead) and the irregular enhancing wall of the tuberculous abscess (arrow). g MRI post-enhanced fast spin-echo inversion recovery image (FSEIR) at the same level demonstrates surrounding edema (arrowheads)

a

l>

Miliary/Disseminated Tuberculosis

dilated small-bowel loops and caseating mesenteric lymphadenopathy. Exploratory laparotomy after taking all precautionary measures in the theatre was performed. A massively distended small bowel was noted with mild changes in color. There was a large inflammatory mass in the root of the mesentery of

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the small bowel. Thick pus was freely present in the abdominal cavity, and 20 cc was aspirated and sent to the Microbiology Department. Bowel adhesions were noted and released. It was decided to close with interrupted sutures, and the patient tolerated the procedure. In October 2001, he went into a coma,

b

c

d

e

f

g

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and brain MRI showed enlargement of the tuberculoma of the brain with development of left parietal lobe brain abscess (Fig. 18.3f,g). The patient died 1 month later.

18.5.4 Case Illustration 4 A 21-year-old married female patient presented to the surgical department with a right iliopsoas mass. She had a history of abdominal pain lasting 6 months associated with fever, profuse night sweating, loss of appetite, and weight loss. She was well before that time and had no previous illnesses. She noted a swelling in the right inguinal region which was associated with a gradual but progressive pain that increased with flexion of the right hip and was relieved by extension of the leg. The pain became worse 2 months before presentation, and the swelling became larger, and she noted a loss of 13 kg in weight. She had an associated history of dysuria and dribbling of urine during the 2 months before presentation. She had no hematuria but had loin pain on both sides. Other systematic reviews were noncontributory. She was using pain-killers to relieve her pain during that time which had been provided by a private clinic. She was also given several courses of antibiotics for a period

M. M. Madkour

of 2-3 months before presentation to our hospital. There was no past history of any illnesses. She had married at the age of 18 years and had one healthy boy. She had a history of vaginal discharge which was yellowish after IUCD insertion 1 year before presentation, and it was subsequently removed. On physical examination, she was underweight, and blood pressure was 92/66 mmHg. There was a cystic swelling at the right inguinal area. Chest examination showed dextrocardia and situs inversus. There were positive clinical findings in the lungs including crepitations and bronchial breathing in the left upper zone. The heart was otherwise normal. Abdominal examination revealed soft lax abdomen with no masses or organomegaly. The right iliac fossa was tender with vague fullness but no definite masses felt. She was pale and feverish, with a temperature of 38.5°C. The patient felt pain during examinations of her hip joint and resisted manipulation. Other systemic examinations were normal. The hemogram showed normal white cell count, hemoglobin 8.8 gldl, and ESR 90 mml h. Biochemical parameters were normal. Chest radiograph showed dextrocardia and pneumonic consolidation in the left mid-zone (Fig. 18.4a). Axial CT of the chest showed airspace consolidation with air bronchogram (Fig. 18.4b). Axial CT of the chest postcontrast showed an abscess in the left erector spinae muscle (Fig. 18.4c,d).

a Fig. 18.4a-h. A 21-year-old woman with situs inversus and dextrocardia presented with rigor, fever, night sweating, loss of weight, and right hip pain. She was found to have a right iliopsoas mass. a Chest radiograph demonstrates pneumonic consolidation in the left mid-zone (arrow) (figure should read situs inversus). b Axial CT of the chest with lung windowing demonstrates air space consolidation with air bronchogram (arrowheads). There is minimal para-pneumonic effusion (open arrow). c, d Axial post-enhanced CT of the patient at the thoraco-abdominal region demonstrates abscess cavity in the left erector spinae muscle (arrows). Note the situs inversus with the liver on the left (arrowhead) and dextrocardia. e, fAxial CT of the pelvis: nonenhanced (e) and post-contrast-enhanced (0 axial images at the same level demonstrate multiple pelvic abscesses (A). Note enhancement of the walls of the abscesses (arrows; B). g, h Axial CT at the level of the hip joints: nonenhanced (g) and post-contrast-enhanced (h) axial images demonstrate right inguinal abscess

Miliary/Disseminated Tuberculosis

Axial CT of the pelvis pre- and post-enhancement showed large multiple pelvic abscesses with wall enhancement (Fig. 18.4e,f). Axial CT at the level of the hip joints showed right inguinal abscess with ring enhancement after contrast (Fig. 18.4g,h). Percutaneous drainage of the right inguinal abscess and other pelvic abscesses was performed under CT guidance.

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Other sputum cultures and cultures of the abscess contents yielded M. tuberculosis that was sensitive to all antituberculous drugs. Histopathology of the inguinal abscess wall showed granulomas. The patient had negative testing for HIV and no evidence of any underlying diseases. She responded well to antituberculous treatment without any sequelae.

c

e

f

g

h

292 18.5.5 Case Illustration 5

A 26-year-old man was referred from Al-Kharj (80 kIn south of Riyadh) with a 6-month history of fever, sweating, dry cough, left-sided chest pain, dyspnea on mild exertion, weakness, loss of appetite, weight loss, and back pain. He had a strong family history of tuberculosis. He was initially treated with antibiotics and analgesics, but his symptoms gradually progressed. He was healthy previously and was not on any medication prior to the presenting illness. Other systemic reviews were normal. On physical examination, he looked unwell, pale, underweight, and his temperature was 38.5°C. There was no palpable peripherallymphadenopathy. Chest examination revealed features of leftsided pleural effusion. Cardiovascular system examination was normal. The abdomen was soft, but there was a splenomegaly with tenderness on palpation. There was no other organ enlargement and no ascites. The spine showed no deformities, but tenderness was elicited at different areas on percussion, mostly at the mid-thoracic and lumbar spine. There was no evidence of neurological deficit, and the fundi were normal. The hemogram showed a normal white cell count, hemoglobin 8.3 g/dl, and ESR 94 mm/h. All biochemical parameters were normal. Chest radiograph showed left-sided, encysted pleural effusion, and a paraspinal shadow was noted, consistent with a paraspinal abscess (Fig. 18.5a). Plain radiograph of the thoracolumbar spine depicted a paraspinal abscess with narrowing of the disc space at TlO-ll {Fig. 18.5d). Ultrasound of the abdomen showed a poorly defined, hypoechoic lesion in the spleen consistent with a splenic abscess (Fig. 18.5j). Axial CT of the chest showed a large, encysted, left pleural effusion with pleural thickening {Fig. 18.5b). MRI of the dorsolumbar spine showed several lesions affecting the vertebral bodies at multiple levels in the thoracic and lumbar vertebrae with paraspinal abscess formation {Fig. 18.5c,f,h,i). Pleural aspiration and biopsy were performed for histopathological and microbiological investigations. Pleural biopsy showed multiple areas of granulomas consistent with tuberculosis. The patient was seen by the spinal surgeon, and surgery was planned after initiating antituberculous treatment. Paraspinal abscess drainage and bone fusion for stabilization of the spine were performed {Fig. 18.5e). Culture of the granulation tissue and abscess content were positive for M. tuberculosis which was sensitive to all antituberculous drugs. Histopathology of spinal granulation tissue showed caseating granulomas. Serological testing

M. M. Madkour

for HIV was negative. The patient responded well to treatment. His antituberculous treatment was extended to 16 months because of the multiplicity of spinal lesions. He made a full recovery and had no spinal deformities or late neurological deficits.

18.6 Adult Respiratory Distress Syndrome and Disseminated Intravascular Coagulation in Miliary Tuberculosis ARDS is an uncommon but serious complication of miliary tuberculosis with a high hospital mortality rate of up to 69%, similar to that of other etiologies such as Gram-negative sepsis (Penner et al. 1995). It has no pathognomonic clinical or imaging features that could differentiate a tuberculous from a nontuberculous etiology. Goldfine et al. (1969) were the first to describe respiratory failure in patients with miliary tuberculosis, and Agarwal et al. (1977) were the first to use mechanical ventilation for such patients. The criteria for diagnosing ARDS is based on the American-European Consensus Conference (Bernard et al. 1994) which includes: 1. The ratio of partial pressure of arterial oxygen (Pa02) to the fraction of inspired oxygen (Fi0 2) is ::;200. 2. Chest radiographic features of bilateral lung infiltrates. 3. A pulmonary artery occlusion pressure ::;18 mmHg. 4. Or absence of clinical or imaging evidence of raised left atrial pressure. The onset of ARDS due to miliary tuberculosis is often preceded by fever and chest symptoms for a few weeks, but may occur acutely over a few days with intense dyspnea (Mohan et al.1996; Murray et al. 1978; So and Yu 1981; Dyer et al. 1985). A high index of suspicion by the treating clinician and willingness to consider tuberculosis as a possible cause of ARDS of unknown origin are essential and may reduce the mortality. Tachypnea and bilateral inspiratory crackles over both lungs may be found. Other physical examination results may detect splenomegaly, hepatomegaly, and signs of meningitis. Some authors have suggested that ARDS due to miliary tuberculosis may have a longer duration of symptoms than 1 week, while Gram-negative septicemia and viral pneumonia produce symptoms much faster. Hypotension is uncommon (7%) in ARDS due to miliary tuberculosis, while it is more frequent in ARDS due

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to sepsis (Dyer et al. 1985). Disseminated intravascu- TB is not clear, but disseminated emphysematous lar coagulation (DIC), acute renal failure, hepatitis, tuberculosis, rupture of a pleural bleb, or rupture of and intercurrent infections are common features a subpleural caseous miliary nodule may be the cause (up to 87.5%) in ARDS due to miliary tuberculosis (Mert et al. 2001). There are only a few single case and Gram-negative septicemia (Murray et al. 1978; reports on such complications in the literature. Mert Piqueras et al. 1987; Dyer et al. 1985; Mohan et al. el al. (2001) found eight cases in the English literature 1996). Death may occur with 1-7 days of the onset between 1974 and 1999 and reported one case of their of ARDS (Murray et al. 1978), and early diagnosis own. These authors reported their own series of 38 and initiation of antituberculous chemotherapy may patients with miliary tuberculosis, only 1 of whom reduce the mortality (Mohan et al.1996). had pneumothorax while on antituberculous treatThe etiological diagnosis of ARDS due to miliary ment. The size of the pneumothorax as noted by other tuberculosis may be achieved in up to 35% of patients authors ranged from small up to 80%. Pneumothorax by sputum smear and in 20% by urine sediment smear in these rare cases was mostly unilateral. Graf-Deuel showing the bacilli. The chest radiograph may be and Knoblauch (1994) described 12 patients with bilatstrongly suggestive of miliary tuberculosis in 15% eral pneumothorax due to various causes and reviewed of patients (Dyer et al. 1985). Other investigations the literature and found 56 published cases including in smear-negative patients should include bron- 3 patients due to miliary tuberculosis. Chandra et al. choscopy with brushing, transbronchial lung biopsy, (1988) described an 18-year-old woman with miliary bone marrow trephine, and liver biopsies as well as tuberculosis. She was given antituberculous chemolumbar puncture if features of meningitis are present. therapy, but on the 20th day of admission, she develThe hemogram often shows normal white cell count, oped bilateral simultaneous pneumothorax that was anemia, or pancytopenia. Patients with ARDS due to aspirated by needle and tube drainage and improved. miliary tuberculosis may develop disseminated intra- Two weeks later, recurrent right-sided pneumothorax vascular coagulation (Die). The pathogenesis and was followed by left-sided pneumothorax and a bronfactors contributing to DIC occurrence are not clear. chopleural fistula that required surgery and responded Tuberculous vasculitis may possibly be a contributing well to treatment. Severe chest pain and breathlessness factor to the initiation oflocal consumption of clotting may occur at the onset of pneumothorax, even while factors leading to Die. Subclinical coagulopathy may the patient is on chemotherapy. It may respond to possibly occur as indicated by the presence of micro- needle aspiration, tube drainage, or may require thothrombi in the biopsy specimen from these patients. racotomy if a bronchopleural fistula develops. The development of DIC may be noted with clinically overt bleeding or the results of laboratory tests consistent with its occurrence (Murray et al. 1980; Dyers et al. 1985; Maartens et al. 1990). The development 18.8 of DIC in such cases is often fatal (Rosenberg and Diagnosis of Miliary/Disseminated . Rumans 1978). In endemic areas or when clinical and Tuberculosis imaging features are suggestive of miliary tuberculosis as a possible cause of ARDS, empirical antituberculous The chest radiograph is the most important initial treatment should be commenced while the diagnosis diagnostic tool for patients with miliary tuberculosis is actively pursued. (Berger and Samortin 1970). However, most authors reported a wide range in the incidence of depicting miliary lung nodularity: from 30% to 93% of patients. The diagnostic accuracy of the chest radiograph was 18.7 determined with a high specificity and good interobPneumothorax in Miliary/Disseminated server agreement by Kwong and colleagues (1996) from Vancouver, Canada. These authors reviewed all Tuberculosis cases with miliary TB diagnosed over a lO-year period Pneumothorax as a complication of miliary tuberculo- (1982-1992), with inclusion of a group of control subsis is extremely rare (Mert et al. 2001; Graf-Deuel and jects and interpretation by three independent blinded Knoblauch 1994; Chandra et al. 1988). Pneumothorax observers. They identified miliary nodular lung shadin cavitary pulmonary tuberculosis may occur due to owing with a sensitivity ranging from 59% to 69%, rupture of the subpleural cavity into the pleural space. higher in those with HIV (71 %-85%), and lower when The possible mechanism of pneumothorax in miliary the diagnosis was made only at autopsy (30%-60%).

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b a

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h

g

Fig.18.5a-j. A 26-year-old male patient presented with chest pain, fever, rigor, abdominal and back pain. a Chest X-ray demonstrates large, left-sided, encysted, pleural effusion (arrowhead). Note paraspinal shadow consistent with abscess formation (open arrows). b Axial post-contrast-enhanced CT demonstrates collapsed left upper lobe (black arrowhead) and a large pleural effusion (open arrow). Note thickening of the parietal pleura (small arrows). c MRI of the spine: post-enhanced II-weighted image demonstrating lesion in the vertebral body of Dll (large white arrow); note dural enhancement (small arrowheads). Note also enhancement of the thickened visceral and parietal pleura (small white arrows) around the pleural effusion (large arrowhead). d Frontal X-ray of the dorsolumbar spine demonstrates paraspinal abscess (curved arrow). Narrowing of the disc at DlO-ll level (open arrows). e Postsurgical frontal radiograph of dorsolumbar spine demonstrates resolution of the paraspinal abscess (drained surgically). Bone fusion for stabilization has been performed (long arrows). f Sagittal post-enhanced II-weighted image demonstrates multiple spinal involvement by tuberculous foci (arrows). Note large intraspinal abscess involving two adjacent vertebral bodies and crossing the intervening disc space (open arrow). Abscess is also tracking anteriorly along the anterior spinal ligament (long arrows). Note enhancement of the dura (arrowheads). g MRI of the spine after surgery: sagittal proton-density and T2-weighted image demonstrate marked improvement with resolution of the osteomyelitis and paraspinal abscess. Bone graft (arrow) has been performed for stabilization. h Sagittal post-enhanced II-weighted image demonstrates enhancement of the last two lumbar vertebral bodies (arrows) and a small intravertebral abscess (arrowhead). i Sagittal proton and T2-weighted images at the same level demonstrates the abscess cavity at L5 (arrows). j Same patient, ultrasound examination of the spleen demonstrates poorly defined, hypoechoic lesion in the spleen consistent with splenic abscess (arrows)

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The interobserver agreement of the three independent radiologists was 90%. The chest radiography features of miliary tuberculosis characteristically consist of widespread, bilateral, nodular lung opacities in the upper and lower zones often measuring 1-3 mm in diameter in 90% of patients and greater than 3 mm in 10% (Kwong et al. 1996). Areas of consolidation may be noted in up to 30% of patients (Fig. 18.1i) and are depicted better by CT of the chest. Parenchymal lung cavities may be seen in 3%-11.9% of patients with miliary tuberculosis (Kwong et al. 1996; Long et al. 1997; Hong et al. 1998). An associated mediastinal and/or hilar lymphadenopathy may be noted in about 15% of patients (Kwong et al. 1996). Other additional radiographic findings may include pleural effusion and calcified granulomas (Fig. 23.14a, b).

18.9

High Resolution (T HRCT of the chest findings in patients with miliary tuberculosis has been reported in only a few articles with a relatively small number of patients (Oh et al. 1994; Optican et al. 1992; McGuinness et al.1992). These reports suggested that HRCT is more specific and observed the following imaging findings in patients with miliary tuberculosis, miliary nodules, groundglass opacities (GGOs), reticulation, and interlobular septal thickening. Oh et al. (1994) described the GGOs as multiple microscopic granulomas with acid-fast bacilli after obtaining transbronchiallung biopsies of these areas. The GGOs appear as random patchy opacities and are the second most common (miliary nodules being the first) HRCT feature of miliary tuberculosis. A large retrospective study on HRCT findings of miliary tuberculosis was reported by Hong et al. (1998) from Seoul, Korea. They reviewed 25 patients with microbiologically andlor histopathologically proven miliary tuberculosis who had undergone HRCT. Miliary nodules that are uniformly distributed throughout both lung fields, mostly ranging from 1-3 mm in diameter and sharply defined, were the most common findings in 24 of the 25 patients (96%). Larger nodules up to 5 mm in diameter due to coalescence or enlargement of granulomas were noted in 5%-10% of all nodules. GGOs occurred in 23 (92%) of these patients and are considered the second most common CT finding. These authors observed that in two of their patients with severe dyspnea and impending ARDS due to miliary TB, extensive GGOs were depicted by HRCT. Interlobar reticulation and interlobar septal thicken-

M. M. Madkour

ing were found in 44% each. The authors also reported other HRCT findings including pre-existing TB lesions in 44%, lymphadenopathy in 32%, pleural effusion in 16%, and bronchogenic spread in 16%. Diverse diseases may cause a similar miliary lung shadowing including hematogenous dissemination of infectious diseases, sarcoidosis, metastatic malignancy, histoplasmosis, pneumoconiosis, and interstitial fibrosis (Curull et al.1985; Willcox et al.1986). Centrilobular (core) and paraseptal (peripheral) nodules may be identified with HRCT. Similar additional findings in chest radiography such as pleural effusion, lung cavities, and lymphadenopathy may also be noted (Hong et al. 1998). The imaging features of miliary tuberculosis are not pathognomonic of the disease (McGuinness et al.1992; Chugh and Agarwal 1997; Miyake et al. 1997).

18.10

Laboratory Diagnosis of Miliary Tuberculosis A rapid and specific laboratory diagnosis is the most pressing need in confirming the diagnosis of miliary tuberculosis. The many weeks that are required for culture yield in patients with miliary tuberculosis cannot be used as a means of confirming the diagnosis before starting chemotherapy. A rapid confirmatory laboratory investigation may not always be easy to achieve. A chest radiograph showing miliary nodular lung shadowing in the presence of clinical Table 18.3. Rapid diagnosis by Ziehl-Neelsen stain: number of patients and percentage of positive AFB Body fluid

Prout et aI. (1980)

39 (31%) Sputum 17 (18%) Urine 31 (3%) CSF Bronchial lavage 3 (100%)

Maartens et aI. (1990) 64 22 24 51

(33%) (14%) (8%) (27%)

Table 18.4. Rapid diagnosis by tissue biopsies: number of patients and percentage of positive granulomata Biopsy site

Al-Jahdali et al. (2000)

Maartens et aI. (1990)

Prout et al. (1980)

Liver Bone marrow TransbronchiaI Lymph nodes

16 (88%) 11 (73%) 10 (70%) 2 (100%)

11 (100%) 22 (82%) 48 (63%) 9 (100%)

12 (92%) 15 (71%) 2 (100%)

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features that suggest miliary tuberculosis may be the only available clues for the attending clinician. Maartens et al. (1990) reported on 109 patients with miliary tuberculosis, and in 7 patients, the diagnosis was based on clinical grounds and classical miliary nodules on chest radiography; they were treated with chemotherapy with a good response. Rapid confirmation of the diagnosis of miliary tuberculosis may be achieved in up to 83% of patients by means of ZieW-Neelsen stain and by the finding of granulomata in tissue biopsies or by fine-needle aspiration (AI BWa12001; Maartens et al. 1990; Prout et al. 1980; Al-Jahdali et al. 2000). The most common body fluids used for Ziehl-Neelsen stain include sputum, gastric fluid aspirate, urine, CSF, bronchial lavage and brushings, and pleural fluid (Sharma et al. 1988) (Table 18.3). Common sites of tissue biopsies to detect granulomata in patients with miliary TB include transbronchial lung tissue, bone marrow trephine, liver, pleura and lymph nodes (Stallworth et al. 1980; Steiner et al.1976) (Table 18.4). Sputum culture in patients with miliary tuberculosis may be positive in 30%-60% (Prout et al. 1980; Monie et al. 1983; Maartens et al. 1990; Al-Jahdali et al. 2000).

18.10.1 Polymerase Chain Reaction PCR assays are a rapid, specific, and noninvasive method of confirming the diagnosis of tuberculosis. PCR using the IS6110 method can be used in identifying the DNA fingerprinting of M. tuberculosis in the sputum, urine, bronchial lavage, gastric aspirate, blood, and tissue biopsies. It has a specificity of 100% and sensitivity of 95% with a turnaround time of 2436 h (Clarridge et al. 1993; Shalwar et al. 1993; Nolte et al. 1993). This rapid and specific diagnostic tool is very useful in confirming the diagnosis of miliary tuberculosis when it is available. Penner et al. (1995) used PCR to determine the strain of Mycobacterium. Tuberculosis was found in 6 of their 13 patients (46%) with miliary tuberculosis, pulmonary and disseminated disease with respiratory failure that required mechanical ventilation. In disseminated tuberculosis, extrapulmonary systems and organs affected can be diagnosed with other investigations appropriate to the site of involvement. These may include various imaging modalities with guided tissue biopsies, sur- . gical drainage of abscesses as demonstrated in the five case illustrations from our own patients. Other investigations should include serological testing for

HIV and PPD skin test. The hemogram may show pancytopenia or 10wWBC (Cassim et al.1993). Tuberculin skin test may be positive in 25%-75% of patients with miliary/disseminated tuberculosis, as reported in various series, but is low among those co-infected with HIV (Hill 1991; Maartens et al. 1990; Gelb et al. 1973; Grieco and Chmel1974; Munt 1972).

18.11 Treatment of Miliary/Disseminated Tuberculosis The treatment of miliary/disseminated tuberculosis is similar to that used for pulmonary and extrapulmonary disease. In miliary tuberculosis, the four-drug regimen includes isoniazid, rifampicin, pyrazinamide, and ethambutol which is used for 2 months and is followed by isoniazid and rifampicin for a further 4-6 months. Drug susceptibility and resistance should be determined. In patients co-infected with HIY, the duration of treatment should be extended to at least 9 months. In disseminated tuberculosis, the duration of treatment may be extended to at least 12 months, particularly when the bone is involved. Surgical intervention may be required to stabilize the spine, release abdominal adhesions if intestinal obstruction arises, drain abdominal, paraspinal, inguinal abscesses, or treat other pulmonary or extrapulmonary complication (see case illustrations). The use of corticosteroids as a modulator of the inflammatory process in miliary tuberculosis with ARDS, meningitis, or pericarditis has been reported by many authors and debated by others (Dyer and Potgieter 1984; So and Yu 1981). Penner et al. (1995) used steroids in 8 of their 13 patients (61.5%) with miliary and pulmonary tuberculosis with ARDS and respiratory failure; they required mechanical ventilation. The median time interval between hospitalization and commencement of treatment was 2 days (range 1-36 days) (Maartens et al.1990). Fever resolved in approximately 7 days (range 1-55 days). Death occurred in 25% of patients within 1-2 weeks of receiving treatment. The most important single factor in the mortality of patients with miliary tuberculosis is a delay in the diagnosis (Maartens et al. 1990). Other factors that contribute to mortality include old age, lymphopenia, hypoalbuminemia, elevated liver transaminases, and delay in early commencement of treatment. Other underlying chronic illnesses may contribute to mortality. The development of complications such as ARDS, DIC, Addison's

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disease, acute renal or multiorgan failure also contributes to the raised rate of mortality. The mortality rate among patients with miliary tuberculosis is approximately 70%, and 77% among those requiring mechanical ventilation due to ARDS, DIe leading to respiratory failure. Mortality rates reported in large series ranged between 20% and 80% of patients (Maartens et al. 1990; Prout et al. 1980; AI-Jahdali et a1.2000; Monie et al.1983; Stenius-Aarniala and Tukiainen 1979; Dahmash et al. 1995; Evans et al. 1998; Mohan et al.1996; Ormerod and Horsfield 1995).

References Agarwal MK, Muthuswamy PP et al (1977) Respiratory failure in pulmonary tuberculosis. Chest 72:605-609 Al Bhlal LA (2001) Fine-needle aspiration cytology of postvaccinial disseminated bacillus Calmette-Guerin infection. Diagn CytopathoI24:333-335 Al-Jahdali H,Al-Zahrani K et al (2000) Clinical aspects of miliary tuberculosis in Saudi adults. Int J Tuberc 4:252-255 Armstrong P, Wilson AG, Dee P et al (1995) Imaging of disease of the chest, 2nd edn. Mosby Year Book, Chicago, p 183 Ashburg DG, Bigelow DB et al (1967) Acute respiratory distress in adults. Lancet 2:319 Auerbach 0 (1944) Acute generalized miliary tuberculosis. Am J Pathol 20: 121-136 Auerbach 0 (1959) The natural history of the tuberculous pulmonary lesion. Med Clin North Am 43:239-251 Bateman DE, Makepeace W et al (1980) Miliary tuberculosis in association with chronic cutaneous tuberculosis. Br J Dermatol103:557-560 Berger HW, Samortin TG (1970) Miliary tuberculosis: diagnostic methods with emphasis on the chest roentgenogram. Chest 58:586-589 Bernard GR, Artigas A et al (1994) The American-European Consensus conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 149:818-824 BieW JP (1958) Miliary tuberculosis: a review of 68 adult patients admitted to a municipal general hospital. Am Rev Tuberc Pul Dis 77:605-622 Bobrowitz ID (1982) Active tuberculosis undiagnosed until autopsy. Am J Med 72:650-658 Bone M, Stableforth D (1981) Miliary infection to Mycobacterium aYium-intracellulare. Tubercle 62:211-213 Cassim KM, Gathiram Vet al (1993) Pancytopenia associated with disseminated tuberculosis, reactive histiocytic haemophagocytic syndrome and tuberculous hypersplenism. Tubercle Lung Dis 74:208-210 Chandra KS, Prasad AS et al (1988) Recurrent pneumothoraces in miliary tuberculosis. Trop Geogr Med 40:347-349 Chapman CB, Whorton CM (1946) Acute generalized miliary tuberculosis in adults. A clinicopathological study based on 63 cases diagnosed at autopsy. N Engl J Med 235:239-248 Chugh 1M, Agarwal AK (1997) Bilateral miliary pattern in sarcoidosis. Indian J Chest Dis Allied Sci 39:245-249

M. M. Madkour Clarridge J et al (1993) Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J Clin Microbiol 31: 2049-2056 Crump JA (1998) Miliary tuberculosis with paradoxical expansion of intracranial tuberculomas complicating human immunodeficiency virus infection in a patient receiving highly active antiretroviral therapy. Clin Infect Dis 26: 1008-1009 Curull V, Morell F et al (1985) Dyspnea, fever and miliary pattern. Chest 88:285-286 Dahmash NS, Fayed DF et al (1995) Diagnostic challenge of tuberculosis of the elderly in hospital: experience at a university hospital in Saudi Arabia. J Infect 31:93-97 Dannenberg AM Jr (2001) Pathogenesis of pulmonary Mycobacterium boYis infection: basic principles established by the rabbit model. Tuberculosis 81:87-96 Dee P, Teja K et al (1980) Miliary tuberculosis resulting in adult respiratory distress syndrome: a surviving case. Am J Roentgenol134:569-572 De Hertogh Fierer E et al (1989) Hypersensitivity reaction to bacillus Calmette Guerin treated with plasmapheresis. Am J Med 86:343-344 Deresiewicz RL, Stone RM, Aster JC et al (1990) Fatal disseminated mycobacterial infection following intravesical bacillus Calmette Guerin. J UroI144:1331-1334 Dyer RA, Potgieter PD (1984) The adult respiratory distress syndrome and bronchogenic pulmonary tuberculosis. Thorax 39:383-387 Dyer RA, Chappell WA et al (1985) Adult respiratory distress syndrome associated with miliary tuberculosis. Crit Care Med 13:12-15 Ellner JJ (1997) Regulation of the human immune response during tuberculosis. J Lab Clin Med 130:469-475 Evans RH, Evans M et al (1998) Massive hepatosplenomegaly, jaundice and pancytopenia in miliary tuberculosis. J Infect 36:236-239 FitzGerald JM, Grzyrowski S et al (1991) The impact of immunodeficiency virus infection on tuberculosis and its control. Chest 100: 191-200 Foster DR (1997) Miliary tuberculosis following intravesical BCG treatment. Br J Radiol 70:429-431 Gelb AF, Leffler C et al (1973) Miliary tuberculosis. Am Rev Respir Dis 108:1327-1333 Geppert EF, Leff A (1979) The pathogenesis of pulmonary and miliary tuberculosis. Arch Intern Med 139:1381-1383 Goldfine ID,Schachter H et al (1969) Consumption coagulopathy in miliary tuberculosis. Ann Intern Med 71:775-777 Graf-Deuel E, Knoblauch A (1994) Simultaneous bilateral spontaneous pneumothorax. Chest 105:1142-1146 Grieco MH, Chmel H (1974) Acute disseminated tuberculosis as a diagnostic problem. A clinical study based on 28 cases. Am Rev Respir Dis 109:554-560 Gupta RC, Lavengood R et al (1988) Miliary tuberculosis due to intravesical bacillus Calmette-Guerin therapy. Chest 94: 1296-1298 Hill AR (1991) Disseminated tuberculosis in the acquired immunodeficiency syndrome era. Am Rev Respir Dis 144: 1164-1170 Hong SH, 1m JG et al (1998) High resolution CT findings of miliary tuberculosis. J Comput Assist Tomogr 22: 220-224 Iantorno R, Nicolai M et al (1998) Miliary tuberculosis of the

Miliary/Disseminated Tuberculosis lung in a patient treated with bacillus Calmette-Guerin for superficial bladder cancer. J Urol 159: 1639-1640 Jacques J, Sloan JM (1970) The changing pattern of miliary tuberculosis. Thorax 25:237-240 Jasmer RM, McCowin MJ et al (1996) Miliary lung disease after intravesical bacillus Calmette-Guerin immunotherapy. Radiology 201:43-44 Juul A (1977) Clinically undiagnosed active tuberculosis. Acta Med Scand 202:225-229 Kesten S, Title L et al (1990) Pulmonary disease following intravesical BCG treatment. Thorax 45:709-710 Korzeniewska-Kosela M, FitzGerald JM et al (1992) Spectrum of tuberculosis in HIV infected patients in British Columbia: a report of 40 cases. Can Med Assoc J 146: 1927-1934 Koylii R, Tozkoparan E et al (1997) Unusual miliary tuberculosis presenting with generalized lymphadenopathy and abdominal involvement. Int J Tuberc Lung Dis 1:474-476 Kwong JS, Carignan S et al (1996) Diagnostic accuracy of chest radiography. Chest 110:339-342 Langdale LA, Meissner M et al (1992) Tuberculosis and the surgeon. Am J Surg 163:505-509 Long R, O'Connor R et al (1997) Disseminated tuberculosis with and without a miliary pattern on chest radiograph: a clinical-pathologic-radiologic correlation. Int J Tuberc Lung Dis 1:52-58 Lotte A, Wasz-Hockert 0 et al (1984) BCG complications. Estimates of the risks among vaccinated subjects and statistical analysis of their main characteristics. Adv Tuberc Res 21: 107-193 Maartens G, Willcox PA et al (1990) Miliary tuberculosis: rapid diagnosis, haematologic abnormalities, and outcome in 109 treated adults. Am J Med 89:291-296 Massaro D, Katz S (1964) Rapid clearing in hematogenous pulmonary tuberculosis. Arch Intern Med 113:573 McClement JH, Renzetti AD, Carroll D et al (1951) Cardiopulmonary function in hematogenous pulmonary tuberculosis in patients receiving streptomycin therapy. Am Rev Tuberc 64:583 McGuinness G, Naidich DP et al (1992) High resolution CT findings in miliary lung disease. J Comput Assist Tomogr 16:384-390 McParland C, Cotton DJ et al (1992) Miliary Mycobacterium bovis induced by intravesical bacilli Calmette-Guerin immunotherapy. Am Rev Respir Dis 146:1330-1333 Mert A, Bilir M et al (2001) Spontaneous pneumothorax: a rare complication of miliary tuberculosis. Ann Thorac Cardiovasc Surg 7:45-48 Millar JW, Horne NW (1979) Tuberculosis in immunosuppressed patients. Lancet 1:1176-1178 Miyake S, Yoshizawa Yet al (1997) Non-Hodgkin's lymphoma with pulmonary infiltrates mimicking miliary tuberculosis. Intern Med 36:420-423 Mohan A, Sharma SK et al (1996) Acute respiratory distress syndrome (ARDS) in miliary tuberculosis: a twelve year experience. Indian J Chest Dis Allied Sci 38:157-162 Monie RDH, Hunter AM et al (1983) Retrospective survey of the management of miliary tuberculosis in South and West Wales, 1976-8. Thorax 38:369-372 Munt PW (1972) Miliary tuberculosis in the chemotherapy era: with a clinical review in 69 American patients. Medicine 51:139-155 Murray HW, Tuazon CU et al (1978) The adult respiratory

299 distress syndrome associated with miliary tuberculosis. Chest 73:37-43 Myers JA (1970) Miliary tuberculosis in the elderly. Br Med J 1:565 Nolte FS et al (1993) Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. J Clin MicrobioI31:1777-1782 Oh YU, Kim YH et al (1994) High-resolution CT appearance of miliary tuberculosis. J Comput Assist Tomogr 18:862-866 Optican RJ, Ost A et al (1992) High-resolution computed tomography in the diagnosis of miliary tuberculosis. Chest 102:941-943 Orihuela E, Herr HW, Pinsky CM et al (1987) Toxicity of intravesical BCG and its management in patients with superficial bladder tumors. Cancer 60:326-333 Ormerod LP, Horsfield N (1995) Miliary tuberculosis in a high prevalence area of the UK: Blackburn 1978-1993. Resp Med 89:555-557 Pagel W, Simmonds FAH et al (1964) Pulmonary tuberculosis, 4th edn. Oxford University Press, London . Palayew M, Briedis D et al (1993) Disseminated infection after intravesical BCG immunotherapy. Chest 104:307-309 Pasculle AW, Kapadia SB et al (1980) Tuberculous bacillemia, hyperpyrexia, and rapid death. Arch Intern Med 140:426-427 Penner C, Roberts D et al (1995) Tuberculosis as a primary cause of respiratory failure requiring mechanical ventilation. Am J Respir Crit Care Med 151:867-872 Petty TL, Ashburg DG (1971) The adult respiratory distress syndrome: clinical features, factors influencing prognosis and principles of management. Chest 60:233-239 Piqueras AR, Marruecos L et al (1987) Miliary tuberculosis and adult respiratory distress syndrome. Intensive Care Med 13:175-182 Pitchenik AE, Fertel D, Bloch AB (1988) Mycobacterial disease: epidemiology, diagnosis, treatment,and prevention. Clin Chest Med 9:425-441 Proudfoot AT, Asktan A, Douglas AC et al (1969) Miliary tuberculosis in adults. Br Med J 2:273-276 Prout S, Benatar SR et al (1980) Disseminated tuberculosis. S Afr Med J 58:835-842 Rabe J, Neff KW et al (1999) Miliary tuberculosis after intravesical bacilli Calmette-Guerin immunotherapy for carcinoma of the bladder. Am J Roentgenoll72:748-750 Rawls WH, Lamm DL et al (1990) Fatal sepsis following intravesical bacillus Calmette Guerin administration for bladder cancer. J UroI144:1328-1330 Ray M, Kataria S et al (2002) Unusual presentation of disseminated tuberculosis. Indian Pediatr 39:88-91 Rieder HL, Kelly GD, Bloch AB et al (1991) Tuberculosis diagnosed at death in the United States. Chest 100:678-681 Rosenberg MJ, Rumans LW (1978) Survival of a patient with pancytopenia and disseminated coagulation associated with miliary tuberculosis. Chest 73:536-539 Sahn SA, Neff TA (1974) Miliary tuberculosis. Am J Med 56: 495-505 Saye L (1936) Tuberculosis miliar cronica. Rev Med Barcelona 25:387-423 Shafer RW, Kim DS et al (1991) Extrapulmonary tuberculosis in patients with human immunodeficiency virus infection. Medicine 70:384-397 Shalwar R et al (1993) Detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin MicrobioI29:712-717

300 Sharma SK, Shamim SQ et al (1981) Disseminated tuberculosis presenting as massive hepatosplenomegaly and hepatic failure. Am J Gastroenterol 76:153-156 Sharma SK,Pande IN et al (1988) Bronchoalveolar lavage (Bal) in miliary tuberculosis. Tubercle 69:175-178 Sharma SK, Pande IN et al (1992) Pulmonary function and immunologic abnormalities in miliary tuberculosis. Am Rev Respir Dis 145:1167-1171 Shibolet A, Dan M et al (1979) Recurrent miliary tuberculosis secondary to infected ventriculoatrial shunt. Chest 76:328-330 Slavin RE, Walsh TJ et al (1980) Late generalized tuberculosis: a clinical pathologic analysis and comparison of 100 cases in the preantibiotic and antibiotic eras. Medicine 59:352-366 So SY, Yu D (1981) The adult respiratory distress syndrome associated with miliary tuberculosis. Tubercle 62:49-53 Soloway MS (1988) Intravesical therapy for bladder cancer. Urol Clin North Am 15:661-669 Stallworth JR, Brasfield DM et al (1980) Congenital miliary tuberculosis proved by open lung biopsy specimen and successfully treated. Am J Dis Child 134:320-321 Steiner P,Rao M et al (1976) Miliary tuberculosis in two infants

M. M. Madkour after nursery exposure: epidemiologic, clinical, and laboratory findings. Am Rev Respir Dis 113:267-271 Stenius-Aarniala BS, Tukiainen P (1979) Miliary tuberculosis. Acta Med Scand 206:417-422 Sutton FD, Hudson LD et al (1974) Recognition and management of the adult respiratory distress syndrome. Chest 66:34S-36S Treip C, Meyers D (1959) Fatal tuberculosis in a general hospital. A diagnostic problem. Lancet 1:164-1'67 Vanham G, Toossi Z, Hirsch CS et al (1997) Examining a paradox in the pathogenesis of human pulmonary tuberculosis: immune activation and suppression/anergy. Tubercle Lung Dis 78:145-158 Vijayan VK (2000) Disseminated tuberculosis. J Indian Med Assoc 98:107-109 Willcox PA, Potgieter PD et al (1986) Rapid diagnosis of sputum negative miliary tuberculosis using the flexible fibreoptic bronchoscope. Thorax 41:681-684 Williams MH, Yoo OH (1973) Pulmonary function in miliary tuberculosis. Am Rev Respir Dis 107:858 Yu YL, Chow WH et al (1986) Cryptic miliary tuberculosis. Q J Med 59:421-428

19 Tuberculosis and Pregnancy M. MONIR MADKOUR

risk factor for the development of tuberculosis has never been proven, as suggested in the past (Snider 19.1 Introduction 301 1984; Espinal et al. 1996). Pregnancy has no effect 19.2 Epidemiology 301 on the incidence or prognosis of tuberculosis. The 19.3 Pathogenesis 302 effect of tuberculosis on the outcome of pregnancy, 19.4 Pregnancy-Tuberculosis: Interrelations 303 the fetus, and the neonate has lost most of its 19.5 Clinical Features 303 19.6 Personal Series 303 importance due to the existence of effective anti19.6.1 Case Illustration 1 304 tuberculous agents. However, a delay in diagnosing 19.6.2 Case Illustration 2 304 active tuberculosis poses a real risk to the pregnant 19.6.3 Case Illustration 3 306 woman, fetus, and infant. Tuberculosis in pregnant 19.7 Diagnosis 306 19.8 PPD Skin Test 306 women may be asymptomatic, particularly when 19.9 Chest Radiography 307 it invades extrapulmonary sites in the body, and 19.10 Microbiology and Other therefore presents a great risk of neonatal morbidAdvanced Laboratory Tests 307 ity and mortality. Symptoms related to tuberculosis 19.11 Breast-Feeding and Its Contraindications 307 may be mild and mimic those caused by normal 19.12 Treatment of Active TB During Pregnancy 308 19.13 Treatment of Multidrug-Resistant TB pregnancy such as dyspnea and fatigue (Gogus et al. During Pregnancy 309 1993; Machin et al. 1992). The disease may first pres19.14 Preventive Treatment During Pregnancy 309 ent in the neonate, and acute tuberculosis may later References 311 be discovered in the mother. Hageman et al. (1980) reported that tuberculosis was initially presented and diagnosed in neonates in 15 of 26 cases, and subsequent investigations of their mothers detected 19.1 active disease. Congenital and neonatal tuberculosis Introduction associated with undiagnosed and untreated mothers carries an infant mortality rate of 50% (Nemir The prevalence of tuberculosis in women of child- and O'Hare 1985). Antenatal screening of pregnant bearing age and during pregnancy is increasing women in endemic areas should include plain chest both in developing countries and among ethnic radiography if pulmonary tuberculosis is suspected, immigrants in urban areas of developed countries with proper shielding of the abdomen. (Centers for Disease Control and Prevention 1994, The outcome of pregnancy for the tuberculous 1995; Ahmed et al. 1999; Hageman 1998). The mor- woman, fetus, and neonate who are treated promptly bidity and mortality among pregnant women with with antituberculous drugs is similar to that for pregtuberculosis, the fetus, and the neonate have also nant women without tuberculosis. increased, particularly among those co-infected with HIV (Adhikari et al.1997).A high index of suspicion by attending clinicians and effective measures during the prenatal period are essential for reducing these 19.2 risks (Starke 1997). Pregnancy as a predisposing or Epidemiology

CONTENTS

M. M. MADKOUR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Specific epidemiological data on the incidence of tuberculosis during pregnancy at present is unknown even in developed countries such as the USA. Available data from the 1970s, based on hospitalized

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pregnant women with tuberculosis in a New York hospital, was estimated by Schaefer et al. (l975) as ranging from 0.6% to 1% and was higher (3.2%) among those with pulmonary disease. These data were equivalent to those from nonpregnant women with tuberculosis of comparable age. In the late 1980s and 1990s, the incidence of tuberculosis in the USA has increased in young adults and children, which implies that tuberculosis during pregnancy may be more prevalent. Such a rise was attributed to the HIV epidemic as the most important risk factor, immigrants of different ethic origin coming from countries where tuberculosis is endemic, decline in public health services, and increased transmission in congregate settings. Tuberculosis among foreign-born persons has increased from 22% in 1986 to 35% in 1995 in the USA (McKenna et al.1995). The epidemiology of tuberculosis in England and Wales has changed its pattern since 1988 (Ormerod 2001). During the 1980s, white ethnic women over the age of 50 contributed 50% of the cases, and only a minority of patients were of child-bearing age (Medical Research Council 1992). In 1998, over 56% of cases were foreign-born immigrants from the Indian subcontinent and of African ethnic origin. The median age of these immigrants was under 30 years, while among the white ethnic women, tuberculosis declined to 37% with a minority of child-bearing age (Rose et al. 2001). In developing countries, poverty compounded by HIV infection form closely associated risk factors and contributed to an estimated 70% mortality due to tuberculosis in the age group 15-40 years including women of child-bearing age (Connolly and Nunn 1996). In Africa, there are no data on the number of women of child-bearing age co-infected with HIV and tuberculosis. The World Health Organization (WHO) estimated that about 50% of women of childbearing age living in sub-Saharan Africa has been infected with M. tuberculosis (Connolly and Nunn 1996). In South Africa, the incidence of tuberculosis is 311 per 100,000 population, and the prevalence rate of HIV infection at an antenatal clinic was reported as 7.6% (Epidemiology Comments, Department of Health, Pretoria 1996). In developing countries, the maternal mortality attributed to tuberculosis is high. Ahmed and colleagues (l999) from Zambia reported hospital-based, nonobstetric maternal mortality among pregnant women with tuberculosis. These authors analyzed 251 maternal deaths over a 2-year period (1996-1997) in Lusaka University Hospital.

A total of 106 (42%) maternal deaths was due to direct obstetric causes, and 145 (58%) were due to nonobstetric causes. Tuberculosis, a nonobstetric cause of maternal death, was associated with 25% of pregnant women. Other reports from Zambia indicated that 25% of antenatal women were infected with HIV, and a rate of 1 woman in 8 was presumed to be co-infected with HIV and TB. The risk of developing overt tuberculosis is expected at 8% annually (Fylkesnes et al. 1997; Dolin et al. 1994). Neonatal tuberculosis is rare, and there are no reports of a recent increase in incidence from countries with a high incidence of HIV infection. Adhikari et al. (l997) from South Africa reported a hospitalbased incidence of neonatal tuberculosis. Eleven neonates with culture-confirmed tuberculosis were reported from King Edward VIII Hospital in Natal, seen in a I-year period (l996-1997). The mothers of six of these neonates were co-infected with TB and HIV. One neonate and two mothers died within the first 3 months. These data suggest that the increase in the co-infections of pregnant women in endemic areas for both diseases was associated with an increased incidence of neonatal TB.

19.3 Pathogenesis The pathogenesis of tuberculosis during pregnancy is similar to that in nonpregnant women. Mycobacterium tuberculosis enters the body commonly by inhalation. The bacilli are ingested by the alveolar macrophages. A few weeks later two responses take place: tissuedamage response (delayed-type hypersensitivity-specific humoral response) and macrophages-activating response (cell-mediated immunity). Cytokines produced by alveolar macrophages and T-Iymphocytes (CD4+) take an active role in the host defense. Subsequently, granuloma formation occurs in the lungs. Granulomas may heal with fibrosis and scarring, but some bacilli may remain viable and reactivate if the patient's immunity is suppressed. Hematogenous spread of the bacilli to the placenta from pulmonary or extrapulmonary disease is the most common route of transmission. Rarely, transmission by direct spread and extension of the disease from pre-existing genital tuberculosis may occur (Hallum and Thomas 1995; Kaplan et al. 1980; BazazMalik et al. 1983). The organisms are transmitted to the placenta and may infect the chorionic villi, amniotic fluid, and decidua. The bacilli may be transmit-

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ted to the fetus via the umbilical vein, infecting the liver first, and may pass through the main circulation, leading to lung disease. Fetal ingestion or aspiration of infected amniotic fluid may also be another mode of transmission of tuberculosis to the fetus in utero or the time of birth (Vallejo and Starke 1992). Transmission of infection in the neonatal period may also occur via inhalation if the mother is not diagnosed or treated.

19.4 Pregnancy-Tuberculosis: Interrelations "For the virgin, no marriage; For the married, no pregnancy; For the pregnant, no confinement; For the mother, no suckling." In an excellent comprehensive review on the interrelationships between tuberculosis and pregnancy, Miller and Miller (1996) abstracted these statements from archaic reports reflecting the opinions and advice of scientists and clinicians in various medical eras. The influence of pregnancy as a risk factor for the development of tuberculosis, altering the course of the disease and prognosis, has intrigued clinicians for centuries since the days of Hippocrates through the early 1920s. The pendulum of opinion swung widely from the beneficial value of pregnancy on tuberculosis to the detrimental effect on the patient as it worsened the course of the disease, and abortion was even advised. Even before the discovery of antituberculous treatment, pregnancy was reported not to worsen the course of the disease (Hill 1928, cited by Miller and Miller 1996). Shortly after the introduction of antituberculous drugs, many reports in the early 1950s, 1960s, and 1970s confirmed that there was no significant difference between the patients who improved and those whose tuberculosis progressed during pregnancy or after delivery (Cohen et al.1952; Hedvalll953; Schaefer et al.1975; Espinal et al.1996). There is no evidence that pregnancy increases the risk of tuberculosis in the postpartum period in HIVinfected or uninfected women (Espinal et al. 1996). The effect of tuberculosis on the course of pregnancy, fetus, and neonate is similar to that in nontuberculous pregnant women. Untreated active tuberculosis poses a real great risk to the pregnant woman, fetus, and neonate. Women with active tuberculosis have a significantly

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higher incidence of spontaneous abortion, congenital malformations of the fetus, and neonatal disease (Bjerkedal et al. 1975).

19.5 Clinical Features Tuberculosis may present initially with symptoms mimicking those physiologically related to normal pregnancy such as dyspnea, fatigue, and lack of energy. The attending clinician has to have a high index of suspicion and make diligent efforts to diagnose the disease in order to prevent fetal and neonatal infection, particularly in endemic areas and among groups at risk (HIV, alcoholics, drug addicts, the homeless). Pregnancy does not alter the mode of onset or the presentation of initial symptoms. However, one-half to one-third of pregnant women with tuberculosis are asymptomatic and unaware of the disease, and the diagnosis may be missed (Wilson et al. 1973; Schaefer et al. 1975; Carter and Mates 1994). However, the clinical features of tuberculosis during pregnancy are similar to those in nonpregnant women.

19.6 Personal Series In our series, review of the medical records of pregnant women with active tuberculosis who attended the Military Hospital in Riyadh were analyzed. Between 1985 and 2000, there were 39 patients with active tuberculosis during their pregnancy. Their age ranged between 16 and 42 years (average 19.7 years). We found a past history of tuberculosis (treated or partially treated) in 13 patients (33.3%). History of contact and a family history of tuberculosis were found in 9 patients (23%). Pulmonary tuberculosis was the most common site of infection and found in 23 of these pregnant women (59%). Extrapulmonary localizations of active tuberculosis were found in 16 (41 %). Cough, fever, easy fatigability, shortness of breath, and weight loss were the most common presenting features and found in 23 patients (59%). Accidental discovery of cervical lymph adenopathy during antenatal screening was noted in 9 patients (23%). Four women were aware of the cervical lymphadenopathy, while the other 5 women were unaware of

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its presence. None of the pregnant women with tuberculous lymphadenitis were symptomatic at the time of the antenatal screening. The 4 women who were aware of lymph node enlargement did not notice any recent changes in size. Two of these women with TB lymphadenitis presented with pancytopenia or thrombocytopenic purpura (see case illustrations). The mean gestational age at the time of clinical presentation or suspicion during antenatal screening was 24.6 weeks, with a range of 1-35 weeks. Tuberculous pleural effusion was found in 3 patients (7.7%). One patient had a vulval swelling that was biopsied and found to be tuberculous by culture and histopathology findings (2.6%). One patient had military tuberculosis (2.6%). One patient had tuberculosis of the knee (2.6%). One patient had cold abscess in the right thigh (see case illustration) (2.6%). All 39 patients had positive tuberculin skin tests. Pulmonary tuberculosis was diagnosed in 23 pregnant women by plain chest radiography findings and confirmed by cultures from sputum, early morning gastric aspiration, and urine, identifying M. tuberculosis. None of the 39 patients in our series had HIV co-infection, drug-resistant tuberculosis, or nontuberculous mycobacterial disease. In New York, the incidence of tuberculosis during pregnancy has increased in association with the HIV epidemic. Margono et al. (1994) reviewed the computerized records of 16 pregnant women with active tuberculosis attending two hospitals. In a 6year period (1985-1991) they found 5 cases, while they found 11 cases in the following 2-year period (1991-1992). A HIV test was positive in 7 among 11 tested pregnant women with tuberculosis. Pulmonary tuberculosis was the most common site (10 cases, 62.5%), and extrapulmonary sites were found in 6 cases (37.5). Tuberculous lymphadenitis was the least common site of extrapulmonary localization reported by these authors in contrast to our own series where the lymph node was the most common location. Early reports on tuberculosis during pregnancy in the USA mostly involved pulmonary localizations (Good et al. 1981). In Great Britain, pregnant women with tuberculosis mostly had an extrapulmonary site of the disease. In a prospective study carried out in Northwick Park District General Hospital, Llewelyn et al. (2000) reported 13 pregnant women with active tuberculosis seen prospectively over a 30-month period (1995-1998). Extrapulmonary sites of localization of tuberculosis were present in 9 patients (69.2%). All patients in this recent study were immigrants to Britain.

M. M. Madkour

19.6.1 Case Illustration 1 A 36-year-old Saudi woman in the 24th week of pregnancy presented with a swelling in the middle third of the right thigh. The swelling started as a small area but increased in size over the following 3 months before presentation, when it became painful. There was no history of trauma or discharge. The patient had fever, sweating, and loss of appetite over the same period. She had never been ill before, and other systematic enquiries were negative. The patient was not acutely ill or in distress. A large soft-tissue swelling extending through the anterolateral aspect of the proximal third of the right thigh was seen. The skin over it was normal, and it was slightly tender. The uterus size corresponded with 24 weeks' gestation. The hemogram, coagulation profile, and biochemical values were normal. ESR was 40 mm/h. Ultrasound of the right thigh depicted a large, soft-tissue, cystic swelling with thick mobile fluid, which could be an abscess or hematoma. It measured 16 cm in length and contained about 800 ml of fluid (Fig. 19.1a). MRI of the right thigh was done, axial T1 and T2 slices obtained through both upper femora and the lower pelvis. The post-gadolinium images were not diagnostic due to the patient's movement. MRI depicted a huge, well-defined, elliptical mass in the right rectus femoris muscle extending from its attachment down to the distal thigh (Fig.1b,c). Similar masses were also noted in the right iliopsoas muscle and right pectineus muscle. These masses showed low T1 and high T2 signal intensity, most probably indicating pus caused by tuberculous abscesses. No bone involvement was seen, and MRI of the thoracolumbar spine showed no evidence of spinal involvement (Fig. 19.1b,c). Aspiration of 200 ml of yellowish purulent pus was obtained and sent for microbiological examination and culture for tuberculosis. The culture yielded M. tuberculosis that was sensitive to all antituberculous drugs. Rifampicin, INH, and ethambutol were given to this patient, and she responded well to treatment. A normal, spontaneous, full-term delivery of a healthy infant was the result in this patient.

19.6.2 Case Illustration 2 A 33-year-old woman presented to my clinic with a 2-month history of fever, sweating, and drug cough.

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a

c Fig.19.1a-d. A 36-year-old pregnant woman presented with a swelling of her right thigh. a Ultrasound shows a well-defined cystic mass with relatively thick wall (curved arrow) consistent with localized abscess. TI-weighted (b) and T2-weighted (c) MRI of the same patient show the abscess in the right vastus lateralis muscle as low signal intensity lesion on TI and as homogeneous high signal intensity on T2 (arrowheads). d MRI of lower pelvis demonstrated a gravid uterus with gestational sac (arrowheads). This was done in an attempt to trace the source of this tuberculous abscess and avoid missing abdominal or pelvic pathology (the safety of MRI in pregnancy is not yet clear). Patient completed the treatment and delivered a normal healthy boy

She had lost 21 kg in weight over that period. She cough, and weight loss. She looked pale, feverish, but was 95 kg in weight before the illness and 74 kg at there was no change in the size of the left supraclathe time of presentation. She had a past history of vicular lymph nodes. There were no other clinical two swellings on the left side of the neck for the past findings. There were purpura and petechiae over the 17 years with no recent changes in their size. She had lower limbs. Repeat chest radiograph, with shield five healthy children. The patient looked pale, her tem- over the abdomen, remained normal. The hemoperature was 38.3°C. There were two enlarged lymph gram showed pancytopenia with Hb 10.9 G/dl, WBC nodes in the left supraclavicular region but no other 2.400/mm3, platelets 31,OOO/mm 3, and the blood film clinical findings. The hemogram showed Hb 1O.9G/dl, showed microcytic hypochromic features. Her serum WBC 9000/mm 3 , platelets 211,OOO/mm 3, and the blood iron and other biochemical parameters were otherfilm showed hypochromic microcytic anemia. wise normal. Coombs test was negative. Bone marrow Biochemical parameters were normal, but the aspiration and biopsy showed hypercellularity in the Mantoux test was positive. The patient refused lymph myeloid and erythroid series with normal maturanode biopsy and discharged herself against medical tion and increased number of megakaryocytes. advice. Two months later she presented to me with a The marrow iron store was normal. Ultrasound of 3-day history of hematuria and skin petechiae over the abdomen was normal. Lymph node biopsy was her legs. She was pregnant with gestational age of performed and showed granulomas with caseation. 9 weeks. She continued to have fever, sweating, dry The biopsy culture yielded M. tuberculosis 4 weeks

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later. She responded well to antituberculous treatment, and the hemogram returned to normal after 3 months. She subsequently had a normal, spontaneous delivery of a healthy baby.

19.6.3 Case Illustration 3 A 30-year-old woman from AI-Qurayyat, northern Saudi Arabia, was referred to our hematologist because of thrombocytopenia, anemia, and convulsions 5 days after a normal delivery. The patient was well during her antenatal follow-up until the last month of her pregnancy when she presented with epistaxis and melena. At that time, her hemoglobin was only 5.8 Gldl, low platelet of 12,OOO/mm3 with normal white cell count. The bleeding time was prolonged, and bone marrow aspiration could not be done at the referring hospital. She was suspected to have idiopathic thrombocytopenic purpura and was started on prednisolone 60 mg oral daily. She was given 5 units of blood and 4 units of platelets and vitamin K. Five days prior to her transfer to our hospital, she delivered a healthy girl. After delivery, she developed headache and recurrent attacks of convulsions. Brain CT scan with contrast was normal. Her fits were controlled with phenytoin. She was admitted on arrival to our general intensive care unit (GICU). She gave a history of easy bruising for the past few years and had not been investigated for this complaint. She had delivered a baby boy 4 years ago with fetal abnormalities, the exact nature of which was not known, who died 3 months after birth. Her history included pulmonary tuberculosis while pregnant with that congenitally deformed boy 4 years ago. She discontinued her antituberculous medication after 2 months when she discovered that she was pregnant. She decided herself to discontinue taking the antituberculous drugs for fear of taking medication during pregnancy. The patient looked ill, emaciated, with bruises all over her body, particularly in the upper limbs. She was not jaundiced and had no lymphadenopathy. Fine apical crepitations were heard over the right lung. Other systemic examinations were normal. The hemogram showed features of severe iron deficiency anemia with low platelets of 18,OOO/mm3 and normal white cell count. Biochemical parameters were normal. Plain chest radiography showed multiple cavitating lesions in the right upper lobe compatible with active tuberculosis. Abdominal cT showed no abnormalities. Bone

M. M. Madkour

marrow aspiration and trephine showed marked hypercellular marrow with hyperactive myeloid and erythroid series with normal maturation and an increased number of megakaryocytes. The iron store was normal. EEG and MRI of the brain were normal. Lumber puncture showed normal CSF. She was seen by our pulmonologist and started on four antituberculous antibiotics. The prednisolone dosage was gradually reduced and stopped. The patient's platelets and hemoglobin returned to normal. She had no further convulsions or bruises. Her follow-up at the chest and hematology clinic was satisfactory, and the patient had a full recovery.

19.7 Diagnosis Prenatal health care screening may help in identifying pregnant women with active pulmonary or extrapulmonary tuberculosis. Early detection of active disease may help in preventing serious consequences to the mother, fetus, and neonate. A high index of suspicion by the attending clinician is essential' particularly in endemic areas and among women at high risk for tuberculosis infection. Detailed history and clinical examination and screening investigations are required to achieve this goal. Investigations may include microbiological methods, plain chest radiography, tuberculin skin testing, and other laboratory tests as appropriate to each patient.

19.8 PPD Skin Test Earlier concepts suggested that a false-negative tuberculin skin testing might occur as a result of the suppression of cell-mediated immunity due to pregnancy (Finn et al. 1972). Pregnancy is found by controlled studies to have no demonstrable effect on tuberculin skin testing, and the test is valid at any time during pregnancy (Snider 1984; Present and Comstock 1975; Huebner et al. 1993). In endemic areas, a positive tuberculin skin test identifies individuals with previous infection by M. tuberculosis but does not indicate the state of disease activity, and a negative response may be noted in patients with active disease. HIV-infected pregnant women react to tuberculin skin testing in a similar way to nonpregnant women with HIV infection with

Tuberculosis and Pregnancy

similar rates of anergy. Anergy is not more frequent as a result of pregnancy among women with HIV infection (Mofenson et al.1995). In the USA, the recommendations of the Advisory Committee at the Center for Disease Control (CDC 1990) with regard to performing the tuberculin test for screening individuals at high risk are as follows: (a) pregnant woman with clinical features suggestive of tuberculosis, (b) women with known or suspected exposure to tuberculosis, (c) women at high risk of developing tuberculosis. A skin reaction of 15 mm in low-risk patient and 5-10 mm in immunocompromised patients is considered positive and should lead to the inclusion of chest radiography as part of the screening (Division of Tuberculosis Elimination, CDC 1991). The interpretation of the tuberculin skin test and the rationale of using various induration size measurements as a positive indication among different populations at variable risk have been the subject of debates in many recent publications. In populations at high risk of developing tuberculosis, contact with active tuberculosis patients, those with clinical or imaging evidence of tuberculosis, or patients with HIV or immunocompromised individuals, induration of at least 5 mm is considered positive and indicating infection with M. tuberculosis. For children in contact with high-risk adults, immunesuppressed patients due to other medical illness, and foreign-born persons, an induration of at least 10 mm is considered positive. In populations at low risk for tuberculosis, an induration of at least 15 mm is considered positive. There has been no classification scheme study of tuberculin induration measurements for pregnant women with or without HIV infection, nevertheless, the tuberculin skin test reaction during pregnancy is not different from that for nonpregnant women (American Academy of Pediatrics 1992; American Thoracic Society 1993; Centers for Disease Control and Prevention 1995).

19.9 Chest Radiography In the past, routine chest radiography was performed as part of screening investigations for all pregnant women (Freth 1952). This routine was found to be potentially hazardous due to irradiation of the fetus, particularly during the first trimester (Bonebrake et al. 1978). However, chest radiography can be per-

307

formed for pregnant women if active pulmonary tuberculosis is suspected particularly in endemic areas and among women at high risk. In nonendemic areas, a positive tuberculin skin test in pregnant women is an indication for chest radiography. Appropriate shielding of the abdomen will reduce irradiation to 50 millirads, which is a less hazardous dose for the fetus, and chest radiography is preferably done after the first trimester. Women with chest symptoms even with a negative tuberculin skin test should have a chest radiograph done (Swartz and Reichling 1978; Brent 1989).

19.10 Microbiology and Other Advanced Laboratory Tests The diagnosis of active tuberculosis during pregnancy may be difficult, particularly when conventional sputum smear staining is negative. Specimens from sputum, gastric or bronchial aspirates, urine and other body fluids, or tissue biopsies should be sent for culturing. Recent advances in automated culture systems such as BACTEC (Becton Dickson Diagnostic Instrument Systems, Towson, MD, USA) are now widely used in many laboratories. Immunological tests and PCR are useful tools that can be used, when available, for the diagnosis of tuberculosis during pregnancy.

19.11 Breast-Feeding and Its Contraindications Breast-feeding by mothers with newly discovered active tuberculosis, before initiating treatment, has been a subject of debate among authors in developed and developing countries. Authors from developed countries, where alternative feeding of infants is readily available, have their own views. Authors from poor, developing countries where feeding substitutes are not easily available have different views as well. To make matters worse, co-infection with HIV complicates the issue of breast-feeding in these poor countries. Transmission of HIV via breast milk to infants is a real threat, and starvation and infant death follow if breast-feeding is contraindicated in such circumstances. This issue is clearly defined in developed countries as a contraindication to breastfeeding (Oxtoby 1988).

308

Nursing mothers with active tuberculosis who are receiving antituberculous treatment should be encouraged to breast-feed their infants. Concerns about the side-effects of antituberculous drugs secreted in the mother's milk have been investigated by many authors. Rifampicin, INH, and streptomycin concentrations in breast milk have been measured after the administration of 600 mg, 300 mg, and 1 g of these drugs, respectively. Peak milk concentrations ranged between 10 and 30 mg!l when rifampicin was given in a dose of 600 mg. A concentration level of 16.6 mg!l was found 3 h after INH injection at a dose of 300 mg, while streptomycin 30 min after intramuscular injection of 1 g dose reached a concentration of 1.3 mg!I. These concentrations of antituberculous drugs in breast milk have no toxic effect on the infant (Lawrence and Lawrence 2001; Berlin and Lee 1979; Vorherr 1974; Fugimoritt and Imais 1957; Snider and Powell 1984). These antituberculous medications have been used directly and safely in infants (Snider 1984). Tuberculosis infection and active tuberculosis are considered two separate issues with regard to breastfeeding. Breast-feeding is not contraindicated in women with previous tuberculosis infection. In women with active tuberculosis, the American Academy Committee has indicated that respiratory contact puts these infants in jeopardy. Breast milk in such patients does not contain tubercle bacilli and can be pumped into bottles and fed to these infants until medication has commenced, and the mother is no longer considered infective. The committee also recommended that active tuberculosis is an indication for temporary isolation of the mother until she has received about 2 weeks of antituberculous therapy. Mothers with TB mastitis should have the milk pumped and discarded until the breast lesion has completely healed (American Academy of Pediatrics 1992). The presence of concomitant HIV and active tuberculosis in mothers living in developed countries is an absolute contraindication to breast-feeding. The rate of HIV transmission to infants via breast milk is estimated to range between 5% and 20% or higher (Lawrence 1997; Lawrence and Lawrence 2001; Oxtoby 1988). Data on freezing or heating breast milk to destroy HIV type I are sparse and insufficient (Ando et aI. 1989). In developing countries where alternative affordable infant feeding, sanitary measures, and medical resources are not available to prevent the transmission of HIV to infants, the World Health Organization, UNICEF, and the United Nations AIDS program recommend the following measures: (l) If

M. M. Madkour

an adequate and safe milk substitute is available, then breast-feeding should not be used. (2) If substitutes are not available, then women who are HIV-positive should be offered the choice of what is appropriate for their circumstances and supported in their choice (WHO 1998).

19.12 Treatment of Active T8 During Pregnancy Pregnant women with active pulmonary or extrapulmonary tuberculosis should be treated without delay. Delay in treatment poses a real threat to the pregnant women, her fetus, and the contact. Treatment should not be interrupted in women with active tuberculosis if pregnancy is discovered while she is on antituberculous medication. The only exception is streptomycin, which may cause fetal ototoxicity and should be replaced unless no alternative drugs are available (Hamadeh and Glassroth 1992). Concerns about the teratogenic effect of antituberculous drugs have been extensively studied by many authors in recent years. Isoniazid, rifampicin, ethambutol, and pyrazinamide are the most widely used and carefully evaluated drugs with no evidence of increased teratogenicity (Steen and Seainton-Ellis 1977; Snider et al. 1980; Starke 1997; Czeizel et aI. 2001). The human teratogenic potential of isoniazid, rifampicin, ethambutol, pyrazinamide, ethionamide, prothionamide, and cycloserine during pregnancy was carefully studied by Czeizel from Hungary and his colleagues from Denmark (Czeizel et al. 2001). These authors surveyed case-controlled, populationbased, congenital abnormalities from the Hungarian National Birth Registry between 1980 and 1996, regarding the safety of 7 oral antituberculous drugs used during pregnancy. These drugs were used during the second and third months of gestation to treat pregnant women with active tuberculosis (critical period for most major congenital abnormalities). The authors reported their findings as shown in their table (Table 19.1). They found no detectable teratogenic risk to the fetus; however, the number of pregnant women who were treated with these drugs during the critical period was limited (6 cases vs 21 controls). The guidelines and recommendations with regard to the choice of the antituberculous drug regimen are subject to revisions and updating depending on drug sensitivity and resistance tests. The most commonly used regimen at present involves the use of two to four or more oral drugs including isoniazid, rifam-

309

Tuberculosis and Pregnancy Table 19.1 Characteristics of the antituberculosis drugs studied, in addition to the number of cases and controls during the study period between 1980 and 1996 (reproduced with permission from Czeizel et al. 2001) Antituberculosis drugs Isoniazid Rifampicin Ethambutol Pyrazinamide Ethionamide Prothionamide Cycloserine Total

Table dose (mg) 50 150,300 250 500 250 250 250

Recommended daily treatment (mg) 50-300 450-600 15-15 mglbwkg 35 mglbw kg (1000-2000) 750-1000 500-1000 750-1000

Cases

Controls for all CAs

Crude POR

(n=22865) n (0/0)

(n=38151) n (0/0)

POR (950/0CI)

6 (0.03) 0(0.00) 4 (0.02) 0(0.00) 0(0.00) 0(0.00) 1 (0.00) 11 (0.05)

17 (0.04) 1 (0.00) 6 (0.02) 3 (0.01) 1 (0.00) 1 (0.00) 0(0.00) 29 (0.08)

0.6 (0.2-1.5) 0.6 (0.0-13.7) 1.1 (0.3-13.7) 0.2 (0.0-4.6) 0.6 (0/0-13.7) 0.6 (0.0-13.7) 5.0 (0.2-122.9) 0.6 (0.3-1.3)

POR, prevalence odds ratio; CA, congenital abnormality; CI, confidence interval; bw, body weight

picin, ethambutol, and pyrazinamide. The duration of treatment may range between 6 and 9 months. Ethambutol should be added in the first 2 months to INH and rifampicin. Alternatively, pyrazinamide can be used in the first 2 months with INH and rifampicin. Pyridoxine 50 mg daily should be added to prevent seizure in the newborn when INH is used. The dosages of these drugs are similar to those used in nonpregnant women. Streptomycin should be avoided unless no alternative antituberculous drug is available. Kanamycin and capreomycin share the same potential for producing ototoxicity as streptomycin. Successful treatment of active tuberculosis during pregnancy depends on the identification of the organisms by culture and sensitivity, the use of multiple drugs to which the organisms are sensitive, and close follow-up of the patient for treatment compliance.

19.13 Treatment of Multidrug-Resistant T8 During Pregnancy Single or multiple drug-resistant tuberculosis during pregnancy is increasing. Isoniazid-resistant tuberculosis during pregnancy can be treated with rifampicin and ethambutol for 18 months (Vallejo and Starke 1992). However, the management of pregnant women with multidrug-resistant tuberculosis is a real dilemma. Iseman recommended the use of 5-6 drugs in areas of known multidrug-resistant tuberculosis until drug sensitivity patterns are identified. This makes the treatment of pregnant women with multidrug-resistant tuberculosis extremely difficult, because some of these medications may

be contraindicated or of unknown teratogenic effect (Iseman 1993). There are no clear recommendations or guidelines to this problem because of possible serious consequences to the fetus. Good and colleagues reported their views and stated: 'it is reasonable to consider therapeutic abortion in this setting' (Good et al. 1981). Nitta and Milligan (1999) from the MDR tuberculosis unit in Los Angeles reported their experience of management of four pregnant women with multidrug-resistant pulmonary tuberculosis. MDR tuberculosis was acquired due to their nonadherence to previous regimens of antituberculous treatment. One of them chose termination of her pregnancy. In one, treatment was withheld, and only rifampicin and INH were given to prevent TB progression; another patient was allowed to continue self-administered medication during her pregnancy. Yet another patient was not treated during pregnancy. All three women who carried their gestation to full term had healthy infants. Retreatment of all four women was done using 5-6 drugs including ethambutol, cycloserine, para-aminosalicylic acid (PAS), capreomycin, ofloxacin, and clofazimine given as daily DOT for 17-24 months. The authors concluded that individualized treatment is considered according to each patient's medical and psychosocial needs. They also demonstrated that MDR tuberculosis during pregnancy can be managed safely and successfully.

19.14 Preventive Treatment During Pregnancy Pregnant women infected with tuberculosis with a positive tuberculin skin test but who are asymptomatic form

M. M. Madkour

310

a controversial issue (Vallejo and Starke 1992). Should they receive preventive treatment, and what are the guidelines in developed and developing countries? Authors from developed countries do not adopt the same policy or guidelines with regard to preventative therapy during pregnancy. Not all infected, pregnant women in developed countries have the same chance of developing active tuberculosis. In the USA, the incidence of tuberculosis infection (not disease) in the general population is approximately 7% (MedchillI999). Individuals who have a positive tuberculin test have a 5% chance of developing active tuberculosis within the first 1-2 years after exposure and can gain an additional 5% chance over the rest of their lives (Barnes and Barrows 1993). As pregnancy has no effect on the development of active tuberculosis, some clinicians may delay initiating preventive therapy until after delivery (Hamadeh et al. 1992). Starke (1997) has debated the value of initiating isoniazid treatment during pregnancy for such individuals particularly those with poor compliance to treatment. However, he structured an algorithm to serve as a guideline for clinicians in the USA to help identify those who will need treatment and when to start it (Fig. 19.2).

Starke recommended the initiation of preventive therapy to those co-infected with HIV. The cost-effectiveness of preventive therapy was reported in the USA by Medchill from Phoenix in 1999. His study was based on the data about the chance of development of active TB among those with a positive tuberculin test and found preventive therapy to be cost-effective. Isoniazid is given as a single daily dose of 300 mg for 6-12 months. Hepatotoxicity should be monitored at least monthly (Hamadeh and Glassroth 1992). In Great Britain, preventive therapy is less widely used in asymptomatic women with a positive tuberculin test but may be recommended for refugees and new immigrants (Ormerod 2001). In developing countries, preventive treatment during pregnancy is not an adopted policy as tuberculosis is highly endemic, and the prevalence of a positive tuberculin test is high. In a large study from Chile, 840 pregnant women in the third trimester were assessed by tuberculin test. Over 50% of them had a positive tuberculin test, none developed symptoms of tuberculosis during pregnancy, and 93% were followed up for 1 year with no evidence of active disease. The Chilean Ministry of Health policy is 'not to perform tuberculin testing as a means of

Positive Mantoux Sj Test During Pregnancy Chest Radiograph (abdominal shield) I Abnonnal

Nonnal

Recent High risk Exposure for progression to disease

y

Start isoniazid and monitor carefully

Consistent with "Older" infection Calcified or likely fibrotic lesion active (not active) and tuberculosis no symptoms or symptoms

I.

'd . Start Isom8Z1 After delivery

Start isoniazid after delivery if repeat chest radiograph is nonnal

I

Three sputum AFB smears and cultures

I

Start multidrug therapy (isoniazid, rifampin, ethambutol, usually pyrazinamide)

Fig. 19.2. Evaluation and treatment of a pregnant woman with a positive Mantoux skin test. AFB, acid-fast bacilli (Starke's algorithm, Starke 1997, from Clinics in Perinatology; with permission from Dr. Stark)

Tuberculosis and Pregnancy

identifying infected women and not to treat asymptomatic tuberculin-positive individuals' (Sepulveda et al. 1995).

References Adhikari M et al (1997) Tuberculosis in the newborn: an emerging disease. Pediatr Infect Dis J 16:1108-1112 Ahmed Yet al (1999) A study of maternal mortality at the university teaching hospital, Lusaka, Zambia: the emergence of tuberculosis as a major non-obstetric cause of maternal death. Int J Tuberc Lung Dis 3:675-680 American Academy of Paediatrics (1992) Committee on infectious diseases: screening for tuberculosis in infants and children. Pediatrics 93:313 American Thoracic Society (1993) Control of tuberculosis in the United States. Am Rev Respir Dis 146:1623 Ando Y et al (1989) Effect of freeze thawing breast milk on vertical HTLV-l transmission from seropositive mothers to children. Jpn J Cancer Res 80:405 Barnes PF, Barrows SA (1993) Tuberculosis in the 1990's. Ann Intern Med 19:400-410 Bazaz-Malik G et al (1983) Tuberculosis endometritis. A clinicopathological study of 1000 cases. Br J Obstet Gynaecol 90:84-88 Berlin C, Lee C (1979) Isonizid and acetylisoniazid disposition in human milk, saliva and plasma. Fed Proc 38:426 Bjerkedal T et al (1975) Course and outcome of pregnancy in women with pulmonary tuberculosis. Scand J Respir Dis 56:245-250 Bonebrake CR et al (1978) Routine chest roentgenography in pregnancy. JAMA 240:2747-2748 Brent RL (1989) The effect of embryonic and fetal exposure to x-ray, microwaves and ultrasound: counseling the pregnant and non-pregnant patient about these risks. Semin Oncol 16:346-368 Carter EJ, Mates S (1994) Tuberculosis during pregnancy: the Rhode Island experience, 1987 to 1991. Chest 106: 1466-1470 Centers for Disease Control (1990) Screening for tuberculosis and tuberculosis infection in high-risk populations. Recommendations of the Advisory Committee for Elimination of Tuberculosis. MMWR Morb Mortal Wkly Rep 39:1-7 Centers for Disease Control and Prevention (1994) COR Curriculum on tuberculosis, what the clinician should know, 3rd edn. Department of Health and Human Services, Atlanta, pp 6-7 Centers for Disease Control and Prevention (1995) Screening for tuberculosis and tuberculosis infection in high-risk populations. MMWR Morb Mortal Weekly Rep 44:19 Cohen JD et al (1952) The tuberculous mother. Am Rev Respir Dis 65:1-23 Connolly M, Nunn P (1996) Women and tuberculosis. World Health Statist Quart 49:115-119 Czeizel AE et al (2001) A population-based case-control study of the safety of oral anti-tuberculous drug treatment during pregnancy. Int J Tuberc Lung Dis 5:564-568 Division of Tuberculosis Elimination, Center for Prevention Services (1991) Center for Disease Control and American

311 Thoracic Society: core corriculum on tuberculosis. US Public Health Services, New York Dolin PJ et al (1994) Global tuberculosis incidence and mortality during 1990-2000. Bull WHO 72:213-220 Epidemiology Comments (1996) Report of the review of the tuberculosis control programme of South Africa, June 10 to 25, 1996: Department of Health, Pretoria, South Africa, vol 23, pp 2-20 Espinal MA et al (1996) The effect of pregnancy on the risk of developing active tuberculosis. J Infect Dis 173:488-491 Finn R, Hill CA et al (1972) Immunological responses in pregnancy and survival of foetal hemograft. Br Med J 3: 150-152 Freth A (1952) Routine x-ray examination of the chest at an antenatal clinic. Lancet 1:287-288 Fujimori H, Imais (1957) Studies on dihydrostreptomycin administered to the pregnant and transferred to their fetuses. Jpn Obstet Gynecol Soc 4:133-149 Fylkesnes K et al (1997) The HIV epidemic in Zambia: sociodemographic prevalence patterns and indications of trends among childbearing women. AIDS 11:339-345 Gogus S et al (1993) Neonatal tuberculosis. Pediatr Pathol13: 299-304 Good JT et al (1981) Tuberculosis in association with pregnancy. Am J Obstet GynecoI140:492-498 Hageman J (1998) Congenital and perinatal tuberculosis: discussion of difficult issues in diagnosis of difficult in diagnosis and management. J Perinatal 18:389-394 Hageman J et al (1980) Congenital tuberculosis: critical reappraisal of clinical findings and diagnostic procedures. Paediatrics 66:980-984 Hallum JL, Thomas HE (1995) Full-term pregnancy after proved endometrial tuberculosis. J Obstet Gyaecol Br Emp 62:548 Hamadeh MA, Glassroth J (1992) Tuberculosis and pregnancy. Chest 101:1114-1120 Hedvall E (1953) Pregnancy and tuberculosis. Acta Med Scand 147:1-101 Huebner RE et al (1993) The tuberculin skin test. Clin Infect Dis 17:968-975 Iseman MD (1993) Treatment of multi-drug-resistant tuberculosis. N Engl JMed 329:784-791 Kaplan C et al (1980) Placental tuberculosis in early and late pregnancy. Am J Obstet Gynecol137:858-860 Lawrence RA (1997) A review of the medical benefits and contraindications to breast-feeding in the United States. Maternal and Child Health Technical Information Bulletin. National Center for Education in Maternal and Child Health, Arlington Lawrence RM, Lawrence RA (2001) Given the benefits of breast-feeding, what contraindications exist? Pediatr Clin North Am 48:235-251 Llewelyn M et al (2000) Tuberculosis diagnosed during pregnancy: a prospective study from London. Thorax 55: 129-132 Machin GA et al (1992) Perinatally acquired neonatal tuberculosis: report of two cases. Peadiatr PathoI12:707-716 Margono F, Mroueh J, Garely A et al (1994) Resurgence of active tuberculosis among pregnant women. Obstet Gynecol 83:911-914 McKenna MT et al (1995) The epidemiology of tuberculosis among foreign-born persons in the United States, 1986 to 1993. N Engl JMed 332:1071

312 Medchill MT (1999) Prenatal purified protein derivative skin testing in a teaching clinic with a large Hispanic population. Am J Obstet GynecoI180:1579-1583 Medical Research Council Cardiothoracic Epidemiology Group (1992) National Survey of Notifications of Tuberculosis in England and Wales in 1988. Thorax 47:770-775 Miller KS, Miller JM Jr (1996) Tuberculosis in pregnancy: Interactions, diagnosis and management. Clin Obstet GynecoI39:120-142 Mofenson et al (1995) Mycobacterium tuberculosis infection in pregnant and non-pregnant women infected with HIV in the women and infants transmission study. Arch Intern Med 155:1066-1072 Nemir RL, O'Hare D (1985) Congenital tuberculosis. Am J Dis Child 139:284-287 Nitta AT, Milligan D (1999) Management of four pregnant women with multi-drug-resistant tuberculosis. Clin Infect Dis 28:1298-1304 Ormerod P (2001) Tuberculosis in pregnancy and puerperium. Thorax 56:494-499 Oxtoby MJ (1988) Human immunodeficiency virus and other viroses in human milk: placing the issue in broader perspective. Pediatr Infect Dis J 7:825-835 Present PA, Comstock GW (1975) Tuberculosis sensitivity in pregnancy. Am Rev Respir Dis 112:413-416 Rose AMC et al (2001) Tuberculosis at the end of the 20th century in England and Wales: results of a national survey in1998. Thorax 56:173-179

M. M. Madkour Schaefer G et al (1975) Pregnancy and pulmonary tuberculosis. Obstet GynaecoI46:706-715 Sepulveda RL et al (1995) The influence of BCG immunization on tuberculin reactivity in healthy Chilean women in the third trimester of pregnancy. Tuberc Lung Dis 76:28-34 Snider D (1984) Pregnancy and tuberculosis. Chest 86: 105-135 Snider DE, Powell K (1984) Should women taking anti-tuberculosis drugs breastfeed? Arch Intern Med 144:589-590 Snider DE et al (1980) Treatment of tuberculosis during pregnancy. Am Rev Respir Dis 122:65-79 Starke JR (1997) Tuberculosis, an old disease but a new threat to the mother, fetus and neonate. Clin Perinatol 24: 107-127 Steen JSM, Seainton-Ellis DM (1977) Rafampicin in pregnancy. Lancet ii:604-605 Swartz HM, Reichling BA (1978) Hazards of radiation exposure for pregnant women. JAMA 239:1907-1908 Vallejo JG, Starke JR (1992) Tuberculosis and pregnancy. Clin Chest Med 13:693-707 Vorherr H (1974). Drug excretion in breast milk. Postgrad Med J 56:97-104 Wilson EA et al (1973) Tuberculosis complicated by pregnancy. Am J Obstet GynecoI115:526-529 World Health Organization (1998) HIV and infant feeding: guidelines for healthcare managers and supervisors. World Health Organization, Geneva. Publication WHO/FRH/NUT 98.2, UNAIDS/98.4, UNICEF/PD/NUT/(J) 98.2

20 Post-primary Pulmonary Tuberculosis M. MONIR MADKOUR, Y. ABusABAAH, ALI BEN MOUSA, ALI AL MASOUD

CONTENTS 20.1 20.2 20.2.1 20.2.2 20.2.3 20.2.4 20.2.5 20.2.6 20.2.7 20.2.8 20.2.9 20.2.10 20.2.11 20.2.12 20.3 20.3.1 20.3.2 20.3.3 20.3.4 20.3.5 20.4 20.4.1

Pathogenesis and Pathology 313 Clinical Features and Complications 315 Hemoptysis in Pulmonary Tuberculosis 316 Massive Hemoptysis in Tuberculosis 316 Rasmussen Aneurysm 317 Endobronchial Tuberculosis 318 Bronchiectasis as a Complication of Post-primary Pulmonary TB 318 Pneumothorax as Complication of Post-primary Pulmonary TB 318 ARDS and Tuberculosis 319 Tuberculous Bronchopleural Fistula 319 Aspergilloma (Mycetoma) 319 Tuberculomas 320 Tuberculous Pulmonary Gangrene 320 Tuberculosis, Lung Cancer and Other Neoplasia 320 Diagnosis of Post-primary Pulmonary TB 320 Imaging Features of Post-primary Pulmonary TB 320 Microbiology of the Sputum and Bronchial Aspirate 321 Bronchoscopic Diagnosis 321 Tuberculin Skin Test 322 Other Diagnostic Investigations 322 Treatment of Post-primary Pulmonary TB 322 Response to Treatment 324 References 324

M. M. MADKouR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia Y. ABusABAAH, ABIM Fellow, Respiratory Medicine, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia A. BEN MousA, MD Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia A. AL MAsouD, MD Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Post-primary pulmonary tuberculosis is a chronic disease commonly caused by either endogenous reactivation of a latent infection or exogenous re-infection by Mycobacterium tuberculosis. It has other synonyms derived mostly from the route of transmission of the infection or from the age of the patient at the onset of the disease, including endogenous reactivation primary tuberculosis, exogenous re-infection pulmonary tuberculosis, or adult-onset pulmonary tuberculosis. The term being used here is 'post-primary pulmonary tuberculosis'to include both re-infection and reactivation forms. The clinical features of the disease are not specific, and the imaging features are suggestive but can simulate other diseases. The definitive diagnosis depends on the identification of M. tuberculosis bacilli, using conventional microbiological methods of sputum smear and culture or radiometric culture methods such as BACTEC or DNA probe PCR-based assays which can identify drug-resistant strains as well. Sputum smear and culture remain the most important investigative methods. Smear-negative sputum may delay the diagnosis for 4-8 weeks or longer if the culture is also negative. A presumptive diagnosis based on the clinical and radiographic features should be made with initiation of treatment after the exclusion of other possible causes of the radiographic findings. Infection with HIV is a great risk for the development of either endogenous reactivation or exogenous re-infection pulmonary disease (Millar and Horne 1979; Barnes et al. 1991; Heyderman et al. 1998). The development of drug-resistant pulmonary tuberculosis is a real global concern that indicates a failure of the tuberculosis control program. The current treatment is both toxic and expensive, and new drug development is sparse at present.

20.1 Pathogenesis and Pathology The development of post-primary pulmonary tuberculosis as a result of endogenous reactivation or exog-

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enous re-infection in low-risk and high-risk areas has been debated for many years among authors (Glynn et al. 2001). Balasubramanian and colleagues reviewed the world literature on these issues and added their own views (Balasubramanian et al. 1994). Schools of thought adopted by those researchers based on microbiological studies of tissue specimens from the lung and lymph nodes of the primary complex. Some authors quoted by reviewers suggested that the primary complex is sterile within 5 years, while other authors suggest that virulent bacilli lie dormant in a metastatic site seeded hematogenously within the vulnerable region. Cultures of apical lung lesions yielded viable bacilli in 25%-76% as reported by some of the quoted authors, and transmission via the bloodstream was suggested. Other researchers examined the sputum and urine cultures in patients living in Bangalore, India, and compared the bacilli virulence, INH sensitivity, and phage typing of the isolates and drew conclusions about the biological evidence of exogenous re-infection in that study. Epidemiologists have noted that high- or lowrisk incidence rates of infection in different areas of the world played an important role (in exogenous reinfection or endogenous reactivation). In areas with a risk greater than 1% of developing countries, with the likelihood of repeated episodes of droplet infection via the airway, the disease is more likely to be due to exogenous re-infection. In areas of low-risk (less than 0.05%), as in developed countries, endogenous reactivation is more likely to be the cause of the disease. Some other authors gave evidence of the contributions of both exogenous re-infection as well as endogenous reactivation. Balasubramanian et al. hypothesized that the implantation of the vulnerable region is directly transmitted via the airway (exogenous re-infection). They also concluded that more studies using genetic fingerprinting of Mycobacterium bacilli isolates would help in the future in disease control in endemic areas and in the development of new vaccines. Immune suppression resulting from HIV infection leads to higher rates of co-infection with tuberculosis. Recent molecular epidemiological studies have indicated that up to 40% of newly diagnosed tuberculous patients and over 70% of recurrences may be due to exogenous re-infection (Stead and Bates 2000; Bates et al. 2001). Among the immunosuppressed patients due to HIV, exogenous re-infection was the leading mechanism. Endogenous reactivation contributed to only 16% of patients in a low-risk area in developed countries and more than 70% in a high-risk area in developing countries (Alland et al. 1994; Brande et al. 1998; Small et

M. M. Madkour et aI.

al. 1994; van Rie et al. 1999; McDonough et al. 2000; Rook and Zumla 2001; Caminero et al. 2001; Bandera et al. 2001; Wallis and Johnson 2001). Caminero and colleagues (2001) reported 912 patients with culture-proved pulmonary tuberculosis between 1991 and 1996 on a Spanish island with a moderate risk rate of incidence. They were treated and followed up for at least 12 months after completing chemotherapy and were culture-negative. Twentythree patients (2.5%) became culture positive again. DNA fingerprinting results were available from 18 patients with recurrence of the disease. DNA fingerprinting was available of pretreatment and recurrent isolates and was reviewed. In 8 patients (44%), the genotype of the recurrent isolate showed a different pattern to the pretreatment isolate. These authors concluded that in 8 patients, 2 of them HIV-positive, exogenous re-infection was the cause of the recurrent pulmonary tuberculosis. Immune suppression or reduction of immune responses may also occur in certain diseases such as silicosis, diabetes mellitus, or with corticosteroid and other immunosuppressive drugs used for malignancies or connective tissue diseases. Regardless of the mode of infection (exogenous or endogenous), the apex of the lung (apical-posterior segments) is the most common site of post-primary pulmonary tuberculosis. Specific factors permitting the progression of tuberculosis in most cases are not yet known (Wallis and Johnson 2001). The host immune response and the role of the cellmediated immunity of activated macrophages and T-cells and the expression of cytokines (Garcia et al. 2002) in response to M. tuberculosis glycolipids and lipoproteins have been discussed in the pathogenesis of primary pulmonary tuberculosis in Chap. 17. Post-primary pulmonary tuberculosis commonly affects the apical and posterior segments of the upper lobe or the superior segment of a lower lobe. After its localization, inflammatory granulomatous nodular formations with cellular infiltrates, fibrosis, central necrosis, and caseation may take place. Hilar and paratracheal lymphadenitis (Woodring 1986) in post-primary tuberculosis is a rare occurrence and reported in approximately 5% of patients (see Figs. 23.18, 23.20, 23.21, 23.22, 23.39, 23.40b). Healing with fibrosis and calcification (Fig. z.25) of lung parenchymal lesion may occur, leading to traction of the trachea (Fig. 23.21, 23.22, 23.23), and in late advanced stages when the lung is destroyed and replaced by extensive fibrosis, displacement of the mediastinum may occur (Fig. 23.24). The initial upper lobe infiltration may form a pneumonic consolidation, and cavitation often

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occurs in 40%-80% of patients (Fig. 23.20, 23.26a, b) sheep with udder tuberculosis. It can also be transmit(Rohenberg and Shaw 1996). Tuberculous consolida- ted to humans from cows as an airborne infection tion with cavity formation may expand and form a (Geppert and Leff 1979; Liesegang and Cameron 1980; lung abscess discharging large numbers of the bacilli Sauret et al. 1992). The incidence ofM. bovis in humans in the sputum as was noted in one of our patients is higher in rural areas with infected herds (Moda et al. (Fig. 23.26d). Tuberculous cavities with areas of 1996). Ingestion of contaminated milk, particularly by necrosis and caseation may rupture into the pleura, young children living in developing countries, leads to leading to empyema (Fig. 23.34b) or a bronchopleu- establishment in the cervical and less frequently in the raj fistula (Fig. 23.36). Rupture of a tuberculous cavity axillary lymph nodes (scrofula). The bacilli may also into the trachea or bronchi is a common complica- affect the intestine, kidney, bones, and central nervous tion and occurs in up to 40% of patients (Rohenberg system in endemic areas with a similar pathogenicity and Shaw 1996). Bronchogenic spread of necrotic and to M. tuberculosis (Moda et al. 1996). HIV patients caseous tissue loaded with the bacilli to other parts of co-infected with M. bovis have been reported in San the lung fields on the same side or opposite lung may Diego, USA, near the border with Mexico (Dankner occur (Figs. 23.19,23.20, 23.27b,c, 23.28b,c). Infection et al.1993). Nontuberculous mycobacterial organisms starts at the newly seeded sites with nodular infiltrate may also cause a similar pulmonary infection (see bronchopneumonia or similar tissue destruction and Chap. 11). fibrosis to the lung parenchyma (Figs. 23.25, 23.26a, z.27b, c, 23.28b). A severe form of transbronchial spread of infection takes the form of dissemination in both lungs (Fig. 23.28c). 20.2 Endobronchial involvement of the bronchial wall is Clinical Features and Complications common, leading to scarring and luminal narrowing and post-stenotic emphysema (Figs. 23.22b, 23.30a, b, Post-primary tuberculosis often presents with a 23.25, 23.31b), atelectasis, and cystic or tubular bron- gradual onset of symptoms that may be tolerated by chiectasis due to traction or endobronchial fibrosis as the patient. The duration of symptoms before prea common complication of the tuberculous disease sentation may vary widely, from 3 days to 23 months process. It is often located in the upper lobes but can (Dahmash et al. 1995; Maartens and Beyers 2002). occur at any other site (Figs. 23.30a, b, 23.31a, b). Post- The presenting features may initially be related to the tuberculous bronchiectasis, although often asymp- respiratory system or present as constitutional symptomatic, may cause hemoptysis in these patients. toms or both. Cough is the most frequently reported A tuberculous cavity larger than 25 mm in diameter presenting feature. Initially, it is dry but later becomes may persist long after successful antibiotic treatment. productive. The sputum may be mucoid, muco-puruColonization with fungi forming a ball of mycetoma lent, blood-stained, or with massive hemoptysis. Chest may rarely occur (Fig. 23.26b, 23.37a,b), and hemopty- pain due to associated pleurisy or pneumothorax may sis may rarely be a presenting complaint. be a presenting symptom. Dyspnea due to tuberculous Tuberculous granulomatous tissue nodules may pneumonia or bilateral fibrocavitary disease may be be encapsulated with connective tissue during vari- the presenting symptom. Fever with sweating and able stages of disease healing and activity, leading chills are common, particularly at night. Other constito tuberculomas found in 3%-6% of patients. It may tutional symptoms including weakness, anorexia, and be single or multiple with a central area of necrosis weight loss, which are nonspecific, may also be present or calcification (Figs. 23.32a-d). Tuberculomas may (Dunlap et al. 2000; Johnson and Ellner 2000). occur in both primary and post-primary pulmonary Symptoms related to extrapulmonary tuberculotuberculosis. sis such as tuberculous monoarthritis, Pott's disease, In bovine tuberculosis, the M. bovis bacilli are genitourinary symptoms, or other organ involvebacteriologically distinctive from M. tuberculosis but ment might accompany the respiratory symptoms at otherwise cause identical pathogenesis, lung lesions, the time of presentation. We retrospectively reviewed and clinical disease. In humans, M. bovis is a zoonotic the records of 176 adult patients with post-primary disease that has been virtually eliminated in developed pulmonary tuberculosis who had a positive sputum countries and other developing countries that practice culture for M. tuberculosis. They attended our hospipasteurization of milk and immunization of dairy tal between 1998 and 2000. The frequency of history/ herds (Grange et al. 1994; Dannenberg 2001). The symptoms, physical findings, and imaging features of organism is excreted in the milk from cows, goats, and these patients are presented in Table 20.1.

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Table 20.1. Clinical features of 176 patients with culture-positive post-primary pubnonary tuberculosis No. of patients

Percentage

166 128 40 135 80 98 76 58

94.3 72.7 23.0 76.7 45.4 55.7 43.2 32.9

105 Crackles Localized wheezes 23 Bronchial sound 55 Diminished sound 66 20 Clubbing 16 Extrapulmonary 28 Bronchoscopy

59.6 13.0 31.2 37.5 11.4 9.0 16.0

Clinical features

Symptoms: Cough Expectoration Hemoptysis Fever Sweating Weight loss Dyspnea Chest pain

Signs:

The frequency of these symptoms may vary, being more intense among those co-infected with HIV (Corbett et al. 2000). The frequency of symptoms was also reported from a large tuberculosis center in Riyadh for 1566 hospitalized patients with pulmonary tuberculosis during 1983-1987. Fever and constitutional symptoms were reported in 77.7%, cough with or without expectoration in 94.3%, and hemoptysis in 40.7% (AlHajjaj et al. 1991). In another series from the south of Saudi Arabia, Al Wabel et al. (1995) reported on 190 patients with post-primary pulmonary tuberculosis who were hospitalized over a 2.5-year period. Cough was noted in 84%, expectoration in 65%, hemoptysis in 23%, fever and constitutional symptoms in over 40% of patients. Pulmonary tuberculosis in elderly patients with underlying and concomitant other chronic illnesses in 80 patients were reported from Riyadh (Dahmash et al. 1995). These patients had diabetes mellitus or malignancies, and some were on steroids, and these illnesses occurred in 86% of them, but none had HIY. Cough was noted in 85%, expectoration in 60%, fever in 66%, hemoptysis in 17.5%, anorexia, weight loss, and other constitutional symptoms in over 50%. Choyke and colleagues (1983) reported on 103 patients with adult-onset pulmonary tuberculosis: 85% were symptomatic, with fever in 40%, cough in 37%, weight loss in 23.6%, and hemoptysis in 8%. Physical examination of the chest may be normal despite the presence of pulmonary infiltrations depicted by chest radiography. Displacement of the trachea due to lung fibrosis and collapse may

be found (Fig. 23.21). Chest wall retraction due to fibrosis or prominence due to associated pleural effusion may be found. Features of consolidations with crepitation and bronchial breathing may be detected. Localized wheezes may be present, indicating endobronchial disease. Other systemic features including pallor, rarely clubbing of the fingers and toes in chronic cases, weight loss, dyspnea, and other extrapulmonary involvement may be found on physical examination. Patients with post-primary pulmonary tuberculosis may present with clinical features similar to those of community-acquired pneumonia. Al-Zeer and colleagues (1998) reported a series of 64 patients admitted to hospital during the pilgrimage season to Mecca in 1994 with an initial diagnosis of community-acquired pneumonia. All patients came from developing countries. Microbiologically proven M. tuberculosis was found in 13 patients (20.3%) and was the most common cause of pneumonia among this group.

20.2.1 Hemoptysis in Pulmonary Tuberculosis Hemoptysis is often an alarming presenting symptom in patients with tuberculosis. It may vary in severity from a slight tinge of blood mixed with the sputum, to mild, moderate, or severe, massive, lifethreatening hemoptysis. In our series of 176 patients with microbiologically proved pulmonary tuberculosis, 28 patients (16%) had slight to mild hemoptysis that was managed with chemotherapy and conservative treatment. Active pulmonary tuberculosis with or without cavitation or post-tuberculous bronchiectasis is the most common cause among these patients. Rarely, the development of bronchial carcinoma in these patients as well or mycetoma or other unrelated illnesses could be the cause of hemoptysis in tuberculous patients (Stebbings and Lim 1999; Hirshberg et al. 1997).

20.2.2 Massive Hemoptysis in Tuberculosis Life-threatening massive hemoptysis due to active tuberculosis or post-tuberculous bronchiectasis has been reported as 'common in comparison to other causes listed in many publications', as reported by Hsiao et al. (2001) from the USA. Life-threatening, massive hemoptysis is defined by most authors as

Post-primary Pulmonary Tuberculosis

expectoration of at least 200 ml of blood in 24 h, significant drop of hemoglobin requiring blood transfusion, or failure to respond to conservative treatment such as oxygen supplement, morphine, and antibiotics (Wong et al. 2002; Abal et al. 2001; Lee et al. 2000; Hsiao et al. 2001; Conlan et al. 1983). In South Africa, Conlan et al. (1983) reviewed 123 patients with massive hemoptysis due to different causes. Tuberculosis was by far the most common cause, either due to culture-proved active disease or bronchiectasis as a sequela to tuberculosis. These authors reported that active pulmonary tuberculosis as a cause of massive hemoptysis was found in 47 patients (38%),24 men and 23 women aged between 19 and 60 years. Thirty-seven patients had bronchiectasis, and 17 (45.9%) had bilateral upper lobe bronchiectasis secondary to former tuberculosis. The total number of patients with massive hemoptysis with or who had tuberculosis was 64 (52%). The remaining causes were chronic narcotizing pneumonitis in II, lung abscess in 6, lung cancer in 6, primary fungal pneumonia in 4, bronchovascular fistula in 5, and miscellaneous causes in 7 patients. In France, Mal and colleagues (1999) reported the intermediate and long-term outcome of bronchial artery embolization (BAE) performed on 46 patients with massive hemoptysis. Tuberculosis (active or sequela) was found in 23 patients (50%) as a cause of the massive hemoptysis. Idiopathic in 10, bronchiectasis in 4, lung cancer in 2, and 1 patient each due to other various causes. The outcome of BAE was favorable with an immediately successful result in stopping the bleeding, but complications were also frequent. Revascularization may occur with recurrence of hemoptysis, technical failure in BAE, spinal cord injury related to invisible anastomotic connections between the bronchial circulation and the anterior spinal artery. The authors recommended that BAE should be avoided in patients with minor hemoptysis. In the USA, Hsiao et al. (2001) from Stanford reported the assessment of modalities of investigations used for the localization of the site of bleeding in patients with massive hemoptysis. They reviewed the records of 28 patients seen between 1988 and 2000 who presented with massive hemoptysis. They found 16 patients with tuberculosis (57%), 14 with tuberculous bronchiectasis, and 2 with active disease, bronchogenic carcinoma in 2, and other causes had 1 patient each. These authors noted, 'Contrary to the statistics reported in many recent series, tuberculous bronchiectasis is the most common underlying etiology for massive hemoptysis in our patients'.

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The authors also indicated that the overall mortality of patients with massive hemoptysis was 7%-80% and operative mortality was 30%-40% as they found from reviewing the literature on the subject. These authors reported, 'The angiographic signs in hemoptysis include hyperplasia of the bronchial artery trunk and branches, bronchopulmonary anastomoses, and bronchial arterial aneurysms', which were seen on angiography of these patients. They concluded that fibroptic bronchoscopy before BAE is unnecessary in patients with hemoptysis of known origin. In Kuwait, Abal et al. (2001) prospectively studied 52 hospitalized patients with hemoptysis of variable degrees of severity over a period of 1 year. Twenty had blood-stained sputum, and 32 had frank hemoptysis including 16 with massive blood expectoration. They found that pulmonary tuberculosis (active or old) was the most common cause, found in 17 patients (32.7%). Other causes included carcinoma in 5, bronchitis in 3, 1 patient each due to other causes, and unknown cause in 13 patients. These authors managed 80.8% of their patients conservatively, and only 19% required BAE or surgery. Recurrent hemoptysis occurred in 12% at the I-year follow-up in this series. In a retrospective study of BAE for massive hemoptysis, Wong et al. (2002) from South Africa reported on 165 patients, and bilateral post-tuberculous bronchiectasis was the most common cause (75%). The short-term outcome of BAE was satisfactory in controlling hemoptysis in all patients. Thoracic aortography was done during the initial assessment, and they noted pathologic enlargement of the bronchial arteries and the presence of nonbronchial systemic collaterals with arteriovenous shunting including intercostal arteries. One patient had transient paraparesis, and the authors noted that the presence of spinal arteries was not considered a contraindication to embolization.

20.2.3 Rasmussen Aneurysm This is a rare phenomenon that can cause massive, life-threatening hemoptysis. It involves invasion of a peripheral pulmonary artery located within a tuberculous cavity often in the upper lobe. Invasion of the arterial wall by tuberculous granulation tissue leads to granulomatous vasculitis with replacement of the adventitia and media with fibrin during the process of healing. This will lead to weakening of the arterial wall with mycotic pseudoaneurysmal formation in one or more locations (see Chapter 43). Rupture of

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the aneurysm will lead to massive, life-threatening hemoptysis as reported in 5% of postmortem cases (Winer-Muram and Rubin 1990; Kim et al. 2001).

20.2.4 Endobronchial Tuberculosis Endobronchial tuberculosis is a common complication of post-primary pulmonary tuberculosis. It may occur in up to 40% of patients, and the most common source of bronchial wall infection is a contiguous tuberculous cavity (Rohenberg and Shaw 1996). Rarely, the bronchial wall may be affected by a hilar or paratracheal tuberculous caseating lymphadenitis. Hematogenous or lymphatic spread of infection to the bronchial wall may occur (Buckner and Walker 1990). The role of the bronchial tree in the spread of tuberculosis to other parenchymal segments or lobes on the same or opposite side by transbronchial spread of caseous material leading to bronchopneumonia is well recognized (1m et al.1993). Hoarseness ofvoice due to laryngeal involvement and localized wheezes due to bronchial disease in the chest are common clinical features. The imaging features of plain radiography and CT may show multiple nodular opacities, bronchial wall thickening, post-stenotic dilatation, lobar hyperinflation, pulmonary collapse, and atelectasis (Figs. 23.27 and 23.28). Endobronchial tuberculosis, its definition, epidemiology, pathogenesis and pathology, classification, clinical features, diagnosis, the role of bronchoscopy in the diagnosis and management, and the treatment are considered in a separate chapter (see Chap. 21).

20.2.5 Bronchiectasis as a Complication of Post-primary PulmonaryTB Bronchiectasis is a common sequela of pulmonary tuberculosis (primary and post-primary). It may occur primarily as a result of endobronchial tuberculosis with irreversible bronchial wall dilatation (Lee et al. 1991). Secondary tuberculous bronchiectasis occurs as a result of lung parenchymal destruction with fibrosis (traction bronchiectasis). Based on high-resolution CT, bronchiectasis was found in 27% of patients with pulmonary tuberculosis, and the upper lobes were the most common site (Cartier et al. 1999). In our series of 176 patients with pulmonary tuberculosis, 17 (9.6%, see Table 20.2) had imaging features of tuberculous bronchiectasis (Figs. 23.9, 23.27c, 23.28c, 23.29, 23.30, and 23.31). Typically, the

M. M. Madkour et al. Table 20.2. Imaging features of 176 patients with culture-positive post-primary pulmonary tuberculosis Imaging features

No. of patients Percentage

Unilateral-parenchymal 119 Bilateral-parenchymal 57 Infiltrates and consolidation 144 Cavities 61 Bronchiectasis 17 Destroyed lungs 2 Calcifications 22 (LN, parenchymal and pleural) Pneumothorax 8 Lymphadenopathy 19 (hilar and paratracheal) Lung infiltrates with pleural 46 effusion Mycetoma 2 Tuberculomas 3 Bronchopleural fistula 1

67.6 32.3 81.8 34.6 9.6 1.1

12.5 4.5 10.8 26.1 1.1 1.7 0.5

apical and posterior segments of the upper lobes are the most common sites of tuberculous bronchiectasis (Fig. 23.27c). Chest radiography may show ring shadows with occasional fluid levels (Figs. 23.9 and 23.29). Features on CT and HRCT will show the details of the bronchial wall changes (Figs. 23.28c, 23.30, and 23.31) (McAdams et al. 1995). Tuberculous bronchiectasis may also occur in the lower lobes of the lung. It is usually asymptomatic, but secondary bacterial infection or hemoptysis may be a presenting feature.

20.2.6 Pneumothorax as Complication of Post-primary PulmonaryTB Pneumothorax may occur during active post-primary pulmonary tuberculosis. Rupture of a tuberculous cavity contiguous to the pleura may result in pneumothorax. The incidence has been reported as low, ranging from 0.6% to 1% in hospitalized tuberculous patients (Wilder et al.1962; Ihm et al.I972). In our own series of 176 patients (see Table 20.2), pneumothoraxoccurred in 8 (4.5%). In 1 patient,pneumothorax failed to respond to antituberculous treatment and chest tube insertion, and a bronchopleural fistula was suspected. A fistulogram confirmed the presence of a bronchopleural fistula and active tuberculosis in the apical segment of the left upper lobe (Fig. 23.36). Pneumothorax may also occur in treated and healed pulmonary tuberculosis (Lambert 1956).

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20.2.7 ARDS and Tuberculosis

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20.2.8 Tuberculous Bronchopleural Fistula

Acute respiratory distress may occur in patients with Tuberculous bronchopleural fistula is rarely reported bilateral chronic cavitary or bronchogenic pulmo- nowadays with the recent advances in chemotherapy. nary tuberculosis with a high hospital mortality rate Most recent reports are on patients with late complicaof up to 47%. Dyer and Potgieter (1984) described tions of collapse therapy for pulmonary tuberculosis three adult patients from South Africa with adult that was done in the past (Johnson et al. 1973; Iseman respiratory distress syndrome (ARDS) due to pul- and Madsen 1991; Uchida et al. 1999; Weissberg and monary tuberculosis (nonmiliary). Weissberg 2001). It may develop after lung resection A 31-year-old woman with cough, fever, and for pulmonary tuberculosis. The lung parenchymal dyspnea lasting 2 months had bilateral pulmonary tuberculous cavity may rupture into the pleural space tuberculosis, and because of the severe dyspnea and with pneumothorax, and the diagnosis can be made the abnormal arterial blood gases, she was mechani- by fistulography (Fig. 23.36 radiology of pulmonary cally ventilated. The diagnosis of tuberculosis was TB chapter). A tract may form between the bronchus made by sputum-positive direct smear. Antituber- contiguous to the tuberculous cavity and the pleura, culous treatment was started, but the patient died. producing a bronchopleural fistula. It is estimated that Postmortem examination showed left upper and a bronchopleural fistula can occur in patients with right lower lobe cavitary tuberculosis. The second active extensive pulmonary parenchymal tuberculosis patient was a 22-year-old woman with a similar pre- in less than 1% (Miller 1981; Woodring 1986; Winersentation who died in the ICU with postmortem evi- Muram and Rubin 1990). Patients are usually sympdence of bilateral tuberculous bronchopneumonia tomatic, and sputum production may increase. Plain with a left bronchopleural fistula. The third patient chest radiography may show air in the pleural space, had a similar presentation but did not require a changing air-fluid level, and contralateral spread of ventilation and responded well to antituberculous tuberculous infiltration. CT may depict the sites of the treatment. bronchopleural fistula (Kim et al. 2001). Levy and colleagues (1987) from South Africa The management of these patients is usually reported a retrospective study of 15 patients admit- difficult. Patients with a susceptible organism may ted to the ICU between January 1982 and June 1985 respond to antituberculous treatment and intercoswith a confirmed diagnosis of pulmonary tuberculo- tal tube drainage. Patients with multidrug-resistant sis. These patients required ICU admission for respi- tuberculosis are treated with chemotherapy and ratory failure. They comprised 1.5% of 933 patients lobectomy or pneumonectomy and pleural decortiwith pulmonary tuberculosis hospitalized in Hillbow cation unless found to have respiratory insufficiency Hospital. Eleven patients required ventilation, and 5 (Iseman and Madsen 1991). Endobronchial occlusion died. Miliary tuberculosis was found in 6 patients, by coils has been found to be an effective method tuberculous bronchopneumonia in 5 patients, lobar (Uchida et al. 1999). pneumonia in 3 patients, bilateral lung parenchymal destructive disease in 2. Penner and colleagues (1995) from Canada 20.2.9 reviewed the records of 13 patients with confirmed Aspergilloma (Mycetoma) pulmonary tuberculosis (7 women and 6 men) from 1984 to 1993, and all had respiratory failure requir- A chronic tuberculous cavity may be colonized by ing mechanical ventilation. Seven patients had mili- the spores of Aspergillus fumigatus to form a fungal ary or disseminated tuberculosis and 6, tuberculous ball (Aderaye and Jajaw 1996). It may occur in 11% of pneumonia. Nine patients died, and only 4 survived. patients with a chronic tuberculous cavity, and approxM. tuberculosis was isolated in all patients from imately 25%-55% of patients with aspergilloma have respiratory or nonrespiratory sites. These authors a history of the disease (Kim et al. 2001). Patients with also quoted the work of Agarwal and colleagues aspergilloma are often asymptomatic but may present (1977) regarding 16 patients with respiratory fail- with hemoptysis (Kaestel et al. 1999). Plain radiography ure due to tuberculous pneumonia who required of the chest may depict a mobile, rounded mass with mechanical ventilation. air-crescent ring (Fig.23.26b and 23.37a,b). Surgical resection is essential if associated with hemoptysis and systemic antifungal treatment is ineffective.

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20.2.10 Tuberculomas

Solitary or multiple, round or oval, tuberculous pulmonary mass lesions may be noted on the chest radiographs in approximately 5% of patients (Kim et al. 2001; Winer-Muram and Rubin 1990). They may be the only radiographic manifestation of primary or post-primary pulmonary tuberculosis. Their diameter may range between 0.5 and 4.0 cm or larger with a smooth or sharply defined margin. Central caseation or calcifications may be found in 20%-30% of tuberculomas. Satellite lesions may be seen in up to 80% of these lesions.

20.2.11 Tuberculous Pulmonary Gangrene

An extremely rare but fatal complication of tuberculous cavitary lesion is the involvement of adjacent vessels and the development of arterial and venous vasculitis with thrombosis, leading to pulmonary gangrene (Reich 1993). Khan and colleagues (1980) from New York reported on 4 patients with pulmonary gangrene due to pulmonary tuberculosis, and only 1 survived. The first patient was a 55-year-old man who presented with fever, cough, and hemoptysis lasting 3 months. Chest radiography showed bilateral upper lobe infiltrate with cavitation and intracavitary mass. The sputum smear was positive for the bacilli. Chemotherapy was started, but the patient developed pneumothorax and died. Autopsy showed tuberculous pneumonia with extensive tuberculous arteritis and occlusion of the lumen by thrombosis. The second patient died before establishing the diagnosis, and subsequent sputum culture taken on admission grew M. tuberculosis. Autopsy showed pulmonary vasculitis in arteries and veins contiguous with the tuberculous cavity. The third patient was known to be tuberculous before and had undergone lobectomy, and now presented with fever and a large pulmonary cavity with an intracavitary mass in the right upper lobe. The sputum smear was positive, and culture grew M. tuberculosis. She responded well to chemotherapy. The fourth patient had a similar chest radiographic cavity with intracavitary mass and died. The bacilli were found at autopsy as well as lung parenchymal cavities, tuberculous granulomatous vasculitis with thrombosis of the pulmonary arteries and veins. We reviewed the world literature and found 18 previously reported patients with pulmonary gangrene mostly due to Klebsiella pneumonia and other

organisms but not due to M. tuberculosis. Lopez-Contreras et al. (1994) reported a 61-year-old alcoholic man with a 4-month history of cough, fever, and weight loss. Sputum smear and culture were positive for M. tuberculosis. Chest radiography showed a large cavity with air-fluid level and a free-floating mass. The patient died, and autopsy was denied.

20.2.12 Tuberculosis, Lung Cancer and Other Neoplasia

The relationship between pulmonary tuberculous and lung cancer has been frequently debated. Such a relationship was raised by Greenberg and colleagues (1964), indicating the co-existence of carcinoma and tuberculosis of the lung. The co-existence between bronchogenic carcinoma and tuberculosis creates a difficult diagnostic problem for radiologists as the radiographic changes may be misinterpreted as progression of tuberculosis (Kim et al. 2001; WinerMuram and Rubin 1990). Brown and Almenoff (1992) reviewed the literature of various retrospective studies on this co-existence. They reviewed other malignancies including leukemia, lymphoma, myelofibrosis, head and neck malignancies, as well as the use of immunosuppressive chemotherapy, and their relationship with the development of tuberculosis. They noted that tuberculosis was 6-9 times more common among patients with Hodgkin's disease, lung cancer, and non-Hodgkin's lymphoma than those with other malignancies. These authors noted, 'Tuberculosis was more likely to be diagnosed at the time of tumor diagnosis in patients with lung, head and neck malignancies, and disease in these patients was predominantly confined to the lungs'. Profound suppression of the cell-mediated immune response caused by malignant diseases or as a result of severe immunosuppressive chemotherapy is the most likely cause of the high risk of developing tuberculosis among these patients (Brown and Almenoff 1992).

20.3 Diagnosis of Post-primary Pulmonary T8 20.3.1 Imaging Features of Post-primary PulmonaryTB Various imaging modalities are used for the depiction of features of post-primary pulmonary

321

Post-primary Pulmonary Tuberculosis

tuberculosis. The imaging features of post-primary pulmonary tuberculosis can be broadly classified as lung parenchymal disease with cavitation, endobronchial tuberculosis, pleural extension of the disease, and other complications such as tuberculoma and mycetoma. Conventional plain chest radiography is the mainstay imaging modality in depicting pulmonary features of the disease. However, a normal chest radiograph does not exclude pulmonary tuberculosis and has been reported in approximately 10%-20% of immunocompetent and immunocompromised patients, respectively (Fitzgerald et a1.1991; Miller and Miller 1993; Greenberg et al.1994; Lee and 1m 1995). Computed tomography (CT) is useful in depicting cavitation and in patients with pleural effusion that may be masking lung parenchymal involvement on the same side (Kuhlman et aI.1990). CT is more sensitive than chest radiography (Hulnick et al. 1983; Lee et a1.1996) in depicting mediastinal and paratracheal lymphadenopathy, endobronchial tuberculosis, and dissemination to the lung parenchyma and other rare complications (Hatipoglu et al. 1996). Bronchography, now replaced by CT, is still used as the investigation of choice for the detection of bronchiectasis in poor-recourse countries of endemic areas (Fig. 23.29). Arteriography for diagnostic or therapeutic methods of bronchial artery embolization (BAE) is used for these rare but life-threatening massive hemoptysis cases. Imaging features of post-primary pulmonary tuberculosis, although suggestive, are not characteristic as they can simulate other diseases (Lee and 1m 1995). Upper lobe infiltration or consolidation should always be suspected as tuberculous. Cavitation may occur in approximately 40%, and the diagnosis of tuberculosis is usually not difficult if it is present in the upper lobe and associated with bronchogenic spread to other parts of the lung (Miller and Miller 1993). In some instances, upper lobe consolidation should always be considered to be tuberculosis until proven otherwise. The presence of lung parenchymal scarring, fibronodular or calcific changes should not be assumed as inactive, and follow-up is essential. We retrospectively reviewed the radiological records of 176 adult patients with post-primary pulmonary tuberculosis who had positive sputum culture for M. tuberculosis. They attended our hospital between 1998 and 2000, and their imaging features and frequencies are presented in Table 20.2. The imaging features of post-primary pulmonary tuberculosis have been reported by us in a separate chapter (Chap. 23).

20.3.2 Microbiology of the Sputum and Bronchial Aspirate Sputum microscopy for the detection of acid-fast bacilli by the Ziehl-Nielsen stain remains the cornerstone of rapid diagnosis of pulmonary tuberculosis (Maartens 2002). In HIV patient's sputum, microscopy is positive less often. At least 3 single specimens of sputum should be initially collected from patients with productive cough. In those who have difficulty in providing sputum, an aerosol inhalation of sterile hypertonic saline can be used to stimulate sputum production. Morning gastric aspiration, bronchoalveolar lavage, or transbronchoscopic brush or biopsies may be required. At least 5,000 to 10,000 bacilli per milliliter of sputum must be present to enable the detection of the organism by stained smear (Hobby et al. 1973). Sputum culture will require 10 to 100 bacilli to yield a positive result (Yeager et al. 1967). The American Thoracic Society (2000) reported on the diagnostic standards and classifications of tuberculosis in adult and children, and indicated that 50%-80% of patients with pulmonary tuberculosis will have positive sputum smears. Traditional culture media required 4-8 weeks to yield the bacilli, while radiometric culture methods (BACTEC) combined with a DNA probe allow identification of M. tuberculosis in 1-3 weeks. Currently, PCR-based assays for the diagnosis of tuberculosis and identification of drug-resistant strains are configured to yield results in a few hours to days (see Chapter PCR and Diagnosis of Tuberculosis by Dr. Diana L. Williams).

20.3.3 Bronchoscopic Diagnosis The use of bronchoscopy to obtain diagnostic specimens for patients with a chest radiograph suggesting tuberculosis but with negative sputum specimen has been reported (Willcox et al. 1982). Willcox and colleagues reported on 275 patients seen from 1976 to 1980, with imaging features of suspected tuberculosis and negative sputum smear. Specimens collected by bronchoscopy included bronchial brushings in 83, transbronchiallung biopsies in 18,and post-bronchoscopy sputum. Positive brushing yield was 67.5%, and transbronchial biopsies were positive in 50%. They also reported the co-existence of tuberculosis and bronchial carcinoma in 4 of their patients (4%).

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In our series of 176 patients, bronchoscopy was done on 27 patients with a chest radiograph suggesting pulmonary tuberculosis but with negative direct smear. Tuberculosis was confirmed by positive culture of bronchoscopic specimens, and no bronchial carcinoma was found. Tuberculous infection after bronchoscopy may be transmitted from patient to patient. Molecular epidemiological studies by molecular typing of DNA supported the transmission of M. tuberculosis isolates to other patients via bronchoscopic contamination (Michele et al. 1997; Argeton et al. 1997). It is recommended that the instrument be cleaned prior to its immersion in 2% aqueous solutions of glutaraldehyde for 45-min exposure times (Food and Drug Administration 1992).

20.3.4 Tuberculin Skin Test The tuberculin skin test is still widely used to identify infection with M. tuberculosis. Antigenic extracts of culture 'PPD' produces a delayed-type hypersensitivity reaction. Intradermal injection of 0.1 mg/0.1 ml of the standard 5-tuberculin unit (TU) dose (Mantoux method) is done into the volar or dorsal surface of the forearm (American Thoracic Society 2000). The test should be read between 48 and 72 h after injection. Three cut-off points have been recommended for defining a positive PPD test. A cut-off point of 2:5 mm of induration using the ballpoint pen method of Sokal is considered positive in a person with recent contact or in the presence of abnormal chest radiographs consistent with tuberculosis. A cut-off point of 2:10 mm is suggested for individuals who have normal or mildly impaired immunity and a high likelihood of being infected with the disease but without other risk factors. A cut-off point of2:15 mm for individuals with no risk factors for tuberculosis is considered positive (American Thoracic Society 2000). Tuberculin tests have several limitations including difficult administration, anergy, poor specificity, and the need for repeated testing to detect boosting (Maartens 2002).A comparative study between skin tests with PPD and measurement of the response by in-vitro culture assays measuring IFN-y production in response to tuberculin antigen stimulation is described in detail in a separate chapter. Dr. Rohit Katial has demonstrated the superiority of these assays and compared it with the PPD skin test (see Chap. 15).

20.3.5 Other Diagnostic Investigations Ultrasound or CT-guided, transthoracic, percutaneous, fine-needle aspiration (FNA) cytology may be useful in diagnosing pulmonary tuberculosis in patients initially suspected of having malignancies. Das et al. (1995) reported the use of FNA to diagnose tuberculosis in 29 patients in their series of 190 patients with malignancies and other causes. Serological tests for antimycobacterial antibodies in the serum using an ELISA immunoassay may be positive in up to 88% (Barnes et al. 1993). DNA fingerprinting PCR-based assays provide a noninvasive method of diagnosing M. tuberculosis as well as identifying drug-resistant strains, with results ready in a few hours or days (see Chap. 13). Pulmonary function tests may be required in tuberculous patients, particularly when lung surgery is required. Radionuclide studies in pulmonary tuberculosis are useful imaging tools in assessment of the disease (see Chap. 24 and 26).

20.4 Treatment of Post-primary Pulmonary T8 The history of the management of tuberculosis has been called 'the story of medical failure' (Holme 1998). 'The patients have been blamed for non-compliance with the therapeutic regimen, but sociologic studies have shown that, in most cases, the providers of health care are at fault' (Grange and Zumla 2000). The infectivity of tuberculous patients for close contacts after starting chemotherapy has been studied by several authors. Riley et al. (1962) reported that the effluent air from the rooms of patients receiving chemotherapy became noninfectious for guinea-pigs within 2 weeks. Other authors found the rapid reduction in the number of viable bacilli by 1-2 logarithmic counts within 2 weeks (Yeager et a1.1967; Hobby et al. 1973; Jindani et al. 1980). An editorial (1980) reviewed the issue of isolation of infectious patients with pulmonary tuberculosis, indicating that admission to a sanatorium was justified for supervised treatment, considering its toxicity and to safeguard their close contacts. It also referred to the work of Jenkinson et al. (1979) who found viable bacilli in the sputum of 15 patients who had received 6 weeks of treatment, and active disease was produced when injected into guinea-pigs. Studies on the duration of antituberculous treatment for patients with a positive

Post-primary Pulmonary Tuberculosis

sputum smear to achieve three consecutive negative results has been carefully studied prospectively by Telzak and colleagues (1997) from the South Bronx in New York. The study started from April 1993 to March 1995 of all patients with culture-confirmed tuberculosis. Data included the results of smears, cultures, and drug susceptibility, HIV status, CD4 cell count (for HIV-positive patients). The main objective of the study was to identify the time duration between initiating antituberculous treatment to the first of three consecutive negative sputum smears and the first of three consecutive negative sputum cultures. During the period of the study, 199 patients with culture-positive tuberculosis were diagnosed, 75% had lung parenchymal disease alone, 2% had lung and pleural abscess, 14% had pulmonary and extrapulmonary TB, 3% had only pleural disease, and 7% had extrapulmonary disease alone. They had complete information on 100 of the sputum smearpositive patients (85%). They found that the mean number of days before the first of three consecutive negative sputum smears was 33, and the median was 23 days. The mean number of days until the first of three consecutive negative sputum cultures was 32, and the median was 26 days. These authors noted that the following factors were associated with an increased number of days to achieve their objective: the high number of AFB on initial smear, the presence of cavitary disease, and no previous history of tuberculosis. They noted that HIV had no effect on the duration of treatment before achieving the study objectives. HIV-positive patients are less likely to have cavitary disease, and therefore more likely to produce few AFB. In conclusion, patients with smearpositive sputum require hospitalization and must be isolated for a mean duration of 32 days after initiating appropriate treatment or longer if the initial AFB count was high in the presence ofcavitary disease and if the patient had no prior history of tuberculosis. The aim of the appropriate chemotherapy is to use drugs to which the bacilli are susceptible, with bactericidal activity to cure the patient, reduce infectivity to contacts, and prevent the emergence of drug resistance (Centers for Disease Control 1994). Clinicians are often required to take the decision of either to initiate the treatment of smear-negative patients with a presumptive diagnosis of pulmonary tuberculosis based on the presence of clinical and imaging features, or to wait for the sputum culture results that may require 4-8 weeks to obtain and may not yield the bacilli. In a prospective study of 139 patients with a presumptive diagnosis of pulmonary tuberculosis, Gordin et al. (1989) reported their findings. The

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study was carried out in San Francisco between mid1981 to the end of 1982. The presumptive diagnosis in this series was based on the presence of clinical and radiological features suggestive of pulmonary tuberculosis with negative sputum smear. Treatment with isoniazid, rifampicin, and ethambutol was given to all patients. Positive culture was reported later in 16 patients. Among the culture-negative patients, 43 showed radiographic improvement after 3 months of initiating the treatment, clinical improvement in 7, and bronchoscopic confirmation of the diagnosis in 1 patient. There were 72 individuals with radiological stability who were considered as having inactive previous tuberculosis. The authors concluded that the treatment was appropriate in 66 of 139 (48%) of patients. The British Medical Research Council (BMRC) recommended the initiation of antituberculous treatment for such patients after other causes for abnormal chest radiography findings have been excluded (Dutt and Stead 1994; Fox et al. 1999). The modern short course of antituberculous chemotherapy, Directly Observed Therapy (DOT), is the best strategy for the treatment of post-primary pulmonary tuberculosis to achieve the three goals, that is, cure the patient, reduce infectivity to contacts, and prevent the emergence of drug resistance if appropriately adhered to. DOT is usually given as an outpatient treatment and supervised by nurses. Such visits to the outpatient facility may be difficult as it involves traveling and may be costly for patients in developing countries. Training of a family member or a community lay person to supervise the administration of DOT may be more practical in developing countries (Wilkinson 1994). WHO indicated that for the DOT strategy to be effective, it requires government commitment, the availability of microscopic and other diagnostic facilities, a continuous supply of high-quality medication, direct observation of treatment administration, and recording the response to treatment (Netto et al. 1999). DOT consists of an initial2-month phase of intensive treatment using four drugs (rifampicin, isoniazid, pyrazinamide, and either ethambutol or streptomycin) followed by a 4-month continuation phase of rifampicin and isoniazid. These drugs are usually given as daily treatment but can be given thrice weekly, either throughout or during the continuation phase, to facilitate the supervision of treatment. The isoniazid daily dose is 5 mg/kg orally or intramuscularly (maximum 300 mg), rifampicin daily dose is 10 mg/kg orally (maximum 600 mg), pyrazinamide daily dose is 15-30 mg/kg orally (maximum 2 g),

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streptomycin daily dose is 15 mg/kg intramuscularly (maximum 1 g) for persons below the age of 60 years and 10 mg/kg intramuscularly (maximum 750 mg) for persons above 60 years, ethambutol daily dose is 15-25 mg/kg orally (maximum 2-5 g). The cure rate of drug-susceptible disease when the patient completes the course of treatment is up to 98%. HIV co-infected patients have a similar outcome of tuberculosis treatment success rate (Grange and Zumla 2000). Although DOT has been successful in some countries, endemic areas with HIV and MDR tuberculosis have not achieved the WHO targets for disease control (Netto et al. 1999). Multidrug-resistant tuberculosis is a much more serious problem to treat. Drugs used for treatment are more toxic, more expensive, and require a prolonged period of up to 24 months, and surgery may be considered to decrease the load of the organisms during chemotherapy (Iseman 1993; Iseman et al.1990).

20.4.1 Response to Treatment Appropriate chemotherapy is usually associated with clinical and radiological improvements. Clinical improvement with resolution of fever occurred in 92% of patients 4 weeks after starting treatment (Teklu and AI-Wabel 1994; Vanham et al. 1997). Radiological improvement is noticed after 3 months (Gordin et al. 1989). Paradoxical worsening of the clinical and imaging responses to treatment may occur particularly in the first few weeks or months. HIV co-infected patients are more prone to paradoxical worsening after starting antituberculous treatment, and this is reported to occur in 36% (Wendel et al. 2001). In tuberculous patients without HIV, paradoxical worsening may occur in 16% (AI-Majed 1996). Paradoxical worsening after antituberculous treatment is defined as worsening of fever, cough, shortness of breath or even development of ARDS, enlargement of lymph nodes or other extrapulmonary site of the disease during appropriate treatment (Smith 1987; AI-Majed 1996; Rodriguez-Bano et al. 1997; Hung and Chang 1999; Wendel et al. 2001). Radiological worsening of the pre-existing pulmonary infiltrates, development of new lesions on the same or apposite lung, enlargement of hilar or paratracheal lymph nodes were noted in our series (Figs.23.16a-c and 23.17a-c). The pathogenesis of paradoxical worsening is not yet clear. It is believed that it may be due to enhanced immune responses (immunological rebound) with increased proliferation of

M. M. Madkour et al.

mononuclear cells and IFN-y production in response to M. tuberculosis antigens (Wendel et al. 2001). Corticosteroid therapy has been used to modify the intensity of symptoms (Rodriguez-Bano et al. 1997). There have been no controlled trials on the management of these paradoxical worsening responses.

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326 Johnson JL, Ellner 11 (2000) Adult tuberculosis overview: African versus Western perspectives. Curr Opin Pulm Med 6:180-186 Johnson TM, McCann et al (l973) Tuberculous bronchopleural fistula. Am Rev Respir Dis 107:30-41 Kaestel M, Meyer W, Mittelmeier HO et al (1999) Pulmonary aspergilloma - clinical findings and surgical treatment. Thorac Cardiovasc Surg 47:340-345 Khan FA, Rehman M et al (l980) Pulmonary gangrene occurring as a complication of pulmonary tuberculosis. Chest 77:76-80 Kim HY,Song KS et al (2001) Thoracic sequelae and complications of tuberculosis. Radiographics 21:839-860 Kuhlman JE et al (1990) CT features of thoracic mycobacterial disease. Radiographics 10:413-431 Lambert HP (1956) Spontaneous pneumothorax and pulmonary tuberculosis. Tubercle 37:207-209 Lee KS,Im JG (l995) CT in adult with tuberculosis of the chest: Characteristic findings and role of management. AJR 164: 1361-1367 Lee KS, Kim YH, Kim WS et al (l99l) Endobronchial tuberculosis: CT features. J Comput Assist Tomogr 15:424-428 Lee KS, Hwang JW, Chung MP et al (l996) Utility of CT in the evaluation of pulmonary tuberculosis in patients without AIDS. Chest 110:977-984 Lee TW, Wan S, Choy DK et al (2000) Management of massive hemoptysis: a single institution experience. Ann Thorac Cardiovasc Surg 6:232-235 Levy H, Kallenbach JM, Feldman C et al (l987) Acute respiratory failure in active tuberculosis. Crit Care Med 15: 221-225 Liesegang TJ, Cameron Douglas (l980) Mycobacterium bovis infection of the conjunctiva. Arch Ophtalmol 98:1764-1766 Lopez-Contreras JL, Ris J et al (1994) Tuberculous pulmonary gangrene: report of a case and review. Clin Infect Dis 18: 243-245 Maartens G (2002) Advances in adult pulmonary tuberculosis. Curr Opin Pulm Med 8:172-177 Maartens G, Beyers N (2002) Tuberculosis in the tropics. Clin Chest Med 23:341-350 Mal H, Rullon I, Mellot F (1999) Immediate and long-term results of bronchial artery embolization for life-threatening hemoptysis. Chest 115:996-1001 McAdams HP, Erasmus J et al (l995) Radiologic manifestations of pulmonary tuberculosis. Radiol Clin North Am 33:655-678 McDonough KA, Florczyk MA, Kress Y (2000) Intracellular passage within macrophages affects the trafficking of virulent tubercle bacilli upon reinfection of other macrophages in a serum-dependent manner. Tuberc Lung Dis 80:259-271 Michele TM, Cronin WA, Graham NMH et al (l997) Transmission of Mycobacterium tuberculosis by a fiberoptic bronchoscope: identification by DNA fingerprinting. JAMA 278: 1093-1095 Millar JW, Horne NW (l979) Tuberculosis in immunosuppressed patients. Lancet 1:1176-1178 Miller WT (l981) Tuberculosis in the adult. Postgrad Radiol 1:147-167 Miller WT, Miller WT (l993) Tuberculosis in the normal host: radiological findings. Semin Roentgenol 28:109-118 Moda G, Daborn q, Grange JM et al (l996) The zoonotic importance of Mycobacterium bovis. Tuberc Lung Dis 77:103-108

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21 Endobronchial Tuberculosis HEE SOON CHUNG

21.15.1.1 Interventional Management 343 21.15.1.2 Tracheobronchial Stent 344 21.15.1.3 Endobronchial Electrosurgery vs Laser Photoresection 346 21.15.1.4 Surgical Management 346 21.15.2 Bronchiectasis 346 21.15.3 Broncholith 346 References 347

CONTENTS 21.1 21.2 21.3 21.4 21.5 21.6 21.7

Introduction 329 Definition 330 Epidemiology 330 Pathogenesis 330 Pathology 331 Classification 332 Characteristics of the Each Subtype of Endobronchial Tuberculosis 333 21.7.1 Actively Caseating Type 333 21.7.2 Edematous-Hyperemic Type 333 21.7.3 Fibrostenotic Type 334 21.7.4 Tumorous Type 334 21.7.5 Granular Type 335 21.7.6 Ulcerative Type 335 21.7.7 Nonspecific Bronchitic Type 336 21.8 Location of Bronchial Involvement 336 21.9 Natural Course of Endobronchial Tuberculosis 21.10 Endobronchial Tuberculosis in HIV-Infected Patients 337 21.11 Clinical Features 338 21.12 Diagnosis 338 21.12.1 Clinical History 338 21.12.2 Physical Examination 339 21.12.3 Laboratory Testing 339 21.12.4 Bacteriology 339 21.12.5 Tuberculin Testing 340 21.12.6 Pulmonary Function Test 340 21.12.7 Radiology 340 21.12.7.1 Simple Roentgenograms 340 21.12.7.2 Computerized Tomography 340 21.12.8 Bronchoscopy 341 21.13 Differential Diagnosis 341 21.13.1 Pneumonia 341 21.13.2 Fungal Infection and Actinomycosis 342 21.13.3 Lung Cancer 342 21.13.4 Bronchial Asthma 342 21.14 Treatment 342 21.14.1 Anti-Tuberculosis Chemotherapy 342 21.14.2 Corticosteroid Treatment 343 21.15 Complications and Their Management 343 21.15.1 Bronchostenosis 343

21.1 Introduction

337

H. S. CHUNG, MD, FCCP Associate Professor of Medicine, Seoul National University College of Medicine and Seoul Municipal Boramae Hospital affiliated to Seoul National University Hospital, # 395 Shindaebang-2-Dong, Dongjak-Gu, Seoul, 156-707, Korea

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Endobronchial tuberculosis is a specific form or a significant complication of pulmonary tuberculosis. It is important to remember that endobronchial tuberculosis has the following pitfalls: (1) its diagnosis is frequently delayed, since the decreased incidence itself diminishes the suspicion of tuberculosis (Greenbaum et al. 1980; Chung et al. 1991a); (2) bronchostenosis may develop as a serious complication despite efficacious antituberculosis chemotherapy (Albert and Petty 1976; Hoheisel et al. 1994; Park et al. 1997); and (3) it is often misdiagnosed as bronchial asthma (Watson and Ayres 1988; Williams et al.1988; Park et al.1995) or lung cancer (Matthews et al.1984; Smith et al. 1987). Recently there has been an unprecedented resurgence of tuberculosis, which is related to the human immunodeficiency virus (HIV) epidemic, multidrugresistant strains, poverty and homelessness, immigration, and failures in the treatment system (Millard et al. 1994; Glynn 1998; Lerner 1999). The HIV epidemic may be associated with a higher incidence of endobronchial tuberculosis (Judson and Sahn 1994; Calpe et al.1995). Therefore, endobronchial tuberculosis continues to be a health problem, though the incidence of tuberculosis affecting respiratory organs including the trachea and bronchi has been greatly reduced (Shulutko et al. 1979). Endobronchial tuberculosis should be borne in mind when patients are young female adults or adolescents who present with symptoms suggestive of asthma and/or with unusual roentgenographic patterns, or in patients with HlV infection.

330

21.2 Definition Endobronchial tuberculosis was first described by Richard Morton in 1694 (Jenks 1940), and is defined as a specific inflammation of the trachea or major bronchi caused by tubercle bacilli. Active disease can be diagnosed when certain endobronchial lesions, such as whitish gelatinous material, ulcer, tumor, stenosis or inflammation, exist on bronchoscopy and tuberculosis is proven by bronchoscopic biopsy of these lesions. Bronchial anthracofibrosis is defined as a luminal narrowing associated with anthracotic pigmentation on bronchoscopy, without a relevant history of pneumoconiosis or smoking, and may be a sign of active endobronchial tuberculosis (Chung et al. 1998; Kim et al. 2000), but histologic confirmation is necessary for definitive diagnosis (Garimella 2001). Bronchial fibrostenosis is sometimes a surprise finding in patients with a previous history of tuberculosis, which must be the presumed cause of stenosis. Since the sequela of endobronchial tuberculosis and reactivated pulmonary tuberculosis can be present coincidently, fibrotic stenosis of bronchi may be inactive lesions resulting from prior endobronchial tuberculosis even though sputum examination for acid-fast bacilli is positive. Therefore, it is necessary to obtain histologic proof of tuberculosis for a definite diagnosis of endobronchial tuberculosis. Localized tuberculous bronchitis in segmental bronchi communicating with diseased portions of lung is common in pulmonary tuberculosis (Daniel 1994). Resected lung specimens frequently show either ulceration or stenosis of the draining bronchioles or bronchi, and the same endobronchial processes may result in bronchiectasis due to destruction of the bronchial wall (Rossman and Oner-Eyuboglu 1998). Since these endobronchial lesions distal to lobar bronchi do not have clinical significance, they should be included in the disease entity of pulmonary tuberculosis rather than that of endobronchial tuberculosis.

21.3 Epidemiology Tuberculosis is still a common disease despite its great decline in recent years. In high-prevalence areas, tuberculosis afflicts chiefly young adults. In countries where HIV infection is endemic, tuberculosis is one of the most important causes of morbid-

H. S. Chung

ity and mortality in acquired immune deficiency syndrome (AIDS) patients (Daniel 1994). The incidence of endobronchial tuberculosis among tuberculous postmortem specimens was as high as 40% prior to the chemotherapy era (Wolinsky 1989). With modern treatment, endobronchial tuberculosis has been reported in about 10% of cases of pulmonary tuberculosis, when fiberoptic bronchoscopy is routinely performed (Jokinen et al. 1977). In my previous study, the incidence of endobronchial tuberculosis in pulmonary tuberculosis was 5.88 percent. However, it should be borne in mind that the actual incidence is somewhat higher than this, because bronchoscopy was not performed routinely in all tuberculosis patients but only when an endobronchial lesion was highly suspected. Endobronchial tuberculosis shows a marked preference for female patients, and the male-to-female ratio is approximately 1:5. The disease is usually detected in the third decade. The reason why endobronchial tuberculosis is more common in young females is not clear. One of the possible reasons is that organism implantation from infected sputum may occur more easily in females, especially in teenagers and those in their twenties, because they do not expectorate sputum, for sociocultural and cosmetic reasons (Chung and Lee 2000). In addition, it is well recognized that endobronchial tuberculosis frequently occurs as a part of primary pulmonary tuberculosis in young adults (Smith et al. 1987).

21.4

Pathogenesis

First infection with the tubercle bacillus is known as primary tuberculosis, and usually includes the involvement of the draining lymph nodes in addition to the initial lesion. The combination of the primary or Ghon focus and draining lymph nodes is termed the primary complex. Although primary tuberculosis was formerly relatively common in intestines or tonsils, due to infection from milk, and may occur in various other unusual sites, in the vast majority of cases the route of infection is by inhalation and, consequently, the primary lesion is pulmonary. All other tuberculous lesions are regarded as post-primary. Multiple terms have been used to describe this stage of tuberculosis - for example, chronic tuberculous postprimary disease, reinfection or recrudescent tuberculosis, adult-type progressive tuberculosis, endogenous reinfection and reactivation tubercu-

331

Endobronchial Tuberculosis

losis (Garay 1996). Regardless of the nomenclature, these tuberculous lesions are not accompanied by major involvement of the draining lymph nodes. In primary pulmonary tuberculosis, the first focus of exudative inflammation may occur in any bronchopulmonary segment, more often near the periphery in the middle or lower lung, i.e. that portion of the lung receiving the greatest ventilation, and this is where bacilli within droplet nuclei tend to be implanted. Tubercle bacilli multiply in the exudates and may be carried into some of the very abundant pulmonary lymphatics, and then to the regional lymph nodes. Hematogenous seeding to the distant organs then probably occurs with considerable frequency, either by way of lymphatic channels or more directly from smaller pulmonary veins within the exudate. However, lesions of clinical magnitude do not develop by hematogenous seeding unless native resistance is low or the number of bacilli is great. Regional lymph node involvement is a fairly consistent characteristic of primary pulmonary tuberculosis. The hilar lymph node is most commonly involved, but the paratracheal node is also frequently enlarged, and a substantial minority of cases show enlargement of both the hilar and paratracheal nodes. These nodes develop an intense cellular inflammatory response and may become so large that they compress the major bronchi, with obstructive atelectasis or obstructive pneumonitis. This phenomenon was formerly called epituberculosis. Caseation often follows in the nodes, but healing by partial resorption of the caseum, and the calcification of the lesion in the lung and lymph nodes, is the rule. However, such nodes occasionally rupture or protrude into bronchi. In children especially, rupture of the caseous glands into the trachea or major bronchi causes collapse of the lung or even sudden death by suffocation in young children. In adults, the lung component of the primary complex is usually more obvious and the nodal component may not be seen, whereas in children often only an enlarged hilar or paratracheal node is apparent (Seaton et al.1989). In post-primary tuberculosis, lymph node involvement is seldom extensive and pulmonary lesions on chest roentgenograms are usually located in the apical or subapical areas of the upper lobe. The predilection for the upper lung probably relates to the high oxygen tensions at the lung apex (Riley 1960). Even though the pathogenesis of endobronchial tuberculosis is not yet fully established, sources of endobronchial tuberculosis may include direct implantation of tubercle bacilli into the bronchus from an adjacent pulmonary parenchymal lesion,

direct airway infiltration from an adjacent tuberculous mediastinal lymph node, erosion and protrusion of an intrathoracic tuberculous lymph node into the bronchus, hematogenous spread and extension to the peribronchial region by lymphatic drainage (Myerson 1944; Smart 1951; Matthews et al. 1984; Smith et al.1987; Lee et al. 1992; Kim et al.1993). Endobronchial tuberculosis, before the era of chemotherapy, was considered a complication of advanced post-primary disease. Effective antituberculosis chemotherapy and preventive measures have reduced childhood tuberculosis exposure, which has resulted in an increase in adult primary tuberculosis with unusual clinical and roentgenographic presentations. In the 1970s and early 1980s, a number of reports concerning the so-called unusual radiographic manifestations of adult tuberculosis were published. Up to one-third of adult cases were reported to have atypical findings, such as mediastinal adenopathy, lower lung consolidation and miliary disease. However, these "unusual manifestations" are the usual manifestations of primary tuberculosis. The only unusual aspect is the increasing incidence of primary tuberculosis in adults (McAdams et al.1995). In the modern drug era, endobronchial tuberculosis is more likely to be discovered in adults with primary tuberculosis (Smith et al. 1987), whereas post-primary disease is by far the most common type of pulmonary tuberculosis.

21.5

Pathology

The pathologic changes that occur in the involved bronchi differ in primary tuberculosis from those of post-primary tuberculosis. The involvement of the trachea and bronchi in primary tuberculosis usually results from the pressure created by the enlarged lymph nodes, which may cause partial or complete bronchial obstruction, necrosis and ulceration of the bronchial wall, often in association with the rupture of caseous nodes into the bronchi. In post-primary tuberculosis, the pathogenesis differs, and bronchial lesions are usually secondary to repeated implantation of tubercle bacilli from a sloughing parenchymal source distal to the lesion (Medlar 1955). The earliest lesions often are lymphocytic infiltrations of the mucosa with or without congestion or edema. These usually clear when the parenchymal focus ceases to slough. However, bronchial lesions may progress to tubercle formation in the mucosa and submucosa. Rarely, necrosis with ulceration and sloughing may involve

332

portions of the bronchial wall, and it is this type of lesion that causes bronchial narrowing and stenosis, distal to which further bronchial inflammation may lead to obstructive pneumonitis or bronchiectasis. The healing of more extensive lesions is frequently accompanied by cicatricial stenosis (Wolinsky 1989).

21.6

Classification The clinical course of endobronchial tuberculosis is variable, not only because there are several possible pathogenetic mechanisms, but also because the interactions between the effects of mycobacteria, host immunity and antituberculous drugs is complex, and any variation in these three factors may result in an altered course (Chan and Pang 1989). Therefore, it is an oversimplification to view cases of endobronchial tuberculosis as a homogeneous group, and endobronchial tuberculosis is probably better divided into subtypes. Over the past five decades, many classifications of endobronchial tuberculosis have been published. Judd (l947) classified endobronchial tuberculosis into intrabronchial and extrabronchial types on the basis of their pathogenetic mechanisms. In 1957, the Sixth All-Union Congress of Phthisiologists adopted a classification which categorized endobronchial tuberculosis into four forms: infiltrative, ulcerative, cicatricial and fistulous (or glandular). After the introduction of the flexible bronchoscope, Oho and Amemiya (l984) suggested that endobronchial tuberculosis be classified as edematous-hyperemic, infiltrative-proliferative, ulcerative-granulative and fibrostenotic. In 1991, my colleagues and I suggested a classification of endobronchial tuberculosis which considered the above-mentioned classifications and bronchoscopic features. This classification categorized endobronchial tuberculosis into seven forms: actively caseating, stenotic without fibrosis, stenotic with fibrosis, tumorous, granular, ulcerative and nonspecific bronchitic (Chung et al. 1991a), and was partially reported at the Sixth World Congress for Bronchology (Han et al. 1989). However, the terms "stenotic without fibrosis" and "stenotic with fibrosis" were later renamed "edematous-hyperemic" and "fibrostenotic:' respectively, due to the fact that gross bronchoscopic findings cannot reflect the presence of fibrosis. As a result, the forms of endobronchial tuberculosis were classified into seven subtypes by bronchoscopic findings: actively caseating, edematous-hyperemic,

H. S. Chung

fibrostenotic, tumorous, granular, ulcerative and nonspecific bronchitic (Chung and Lee 2000). The actively caseating, edematous-hyperemic, fibrostenotic and tumorous forms of endobronchial tuberculosis have varying degrees of bronchostenosis proximal to the segmental bronchi, whereas the granular, ulcerative and nonspecific bronchitic forms do not have luminal narrowing of the bronchi. This new classification is valuable for predicting the therapeutic outcome of endobronchial tuberculosis, because it is closely related to the extent of disease progression (Chung and Lee 2000). Pathologically, the initial lesion, which presents as simple erythema and edema of the mucosa with lymphocytic infiltration of the submucosa (Medlar 1955; Shulutko et al.1979),corresponds to nonspecific bronchitic endobronchial tuberculosis. This lesion is followed by submucosal tubercle formation, which produces the erythema and granularity seen at bronchoscopy (Myerson 1944), and partial bronchial stenosis, which is caused by considerable congestion and edema of the mucosa (Medlar 1955). These are the granular and edematous-hyperemic types, respectively. At this point, the development of caseous necrosis, with the formation of tuberculous granuloma, can be found at the mucosal surface (Medlar 1955), which constitutes actively caseating endobronchial tuberculosis. However, when the inflammation erupts through the mucosa, an ulcer is seen, which may be covered by caseous material (Myerson 1944), and the disease is considered to be of the ulcerative type. Finally, the bronchial mucosal ulcer evolves into hyperplastic inflammatory polyps, and the endobronchial tuberculous lesion heals by fibrostenosis (Salkin et al. 1943; Smith et al. 1987). During this process, the lesion has moved through either the tumorous or fibrostenotic type. In addition, tumorous endobronchial tuberculosis can develop via a pathway involving erosion and later protrusion of an intrathoracic tuberculous lymph node into the bronchus (Shulutko et al.1979; Judson and Sahn 1994). This may be the principal mechanism oftumorous endobronchial tuberculosis, given that computed tomography usually reveals an endobronchial mass, as well as enlarged lymph nodes adjacent to the bronchus, and anthracotic pigment is frequently seen in biopsy specimens (Yee et al. 1985; Smith et al.1987). Interestingly, endobronchial tuberculosis in patients with HIV infection shows such bronchoscopic and computed tomography findings (Judson and Sahn 1994; Alame et al. 1995; Calpe et al. 1995; Saadoun et al.1998). In my previous study (Chung and Lee 2000), the actively caseating type was the most common form, the edematous-hyperemic, fibrostenotic, tumorous and granular types were relatively common, the non-

333

Endobronchial Tuberculosis

specific bronchitic type occurred less frequently and the ulcerative type was the rarest form (Table 21.1).

21.7 Characteristics of the Each Subtype of Endobronchial Tuberculosis

In my previous study, about a third of the cases of the edematous-hyperemic type became of the nonspecific bronchitic type and healed within three months of treatment. The remaining two-thirds became fibrostenotic within three months of drug treatment, and complete obstruction of the bronchial lumen often occurred. The prognosis of edematoushyperemic endobronchial tuberculosis is poor.

21.7.1 Actively Caseating Type Actively caseating endobronchial tuberculosis is diagnosed when the bronchial mucosa is swollen, hyperemic and diffusely covered with a whitish cheese-like material. This form is usually accompanied by luminal narrowing at diagnosis, whether granulation tissue is present or not (Fig. 21.1). In my previous study, approximately one-third of the cases of actively caseating endobronchial tuberculosis healed via the edematous-hyperemic, granular or nonspecific bronchitic types without complication. The remaining two-thirds transformed into the fibrostenotic type within three months of antituberculosis and corticosteroid treatment. The formation of granulation tissue is a poor prognostic factor, because all cases that showed granulation tissue on follow-up bronchoscopy changed into the fibrostenotic type. The prognosis of actively caseating endobronchial tuberculosis is poor.

Fig.21.1. Bronchoscopic finding of actively caseating endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

21.7.2 Edematous-Hyperemic Type In cases of edematous-hyperemic endobronchial tuberculosis, the bronchial lumen is narrowed due to severe mucosal swelling with surrounding hyperemia. However, neither caseous material nor fibrous contracture is found at diagnosis (Fig. 21.2). Table 21.1. Classification of 114 endobronchial tuberculoses by Bronchoscopic Features Type

Cases

%

Actively caseating type Edematous-Hyperemic type Fibrostenotic type Tumorous type Granular type Nonspecific bronchitic type Ulcerative type

49 16 12 12 13 9 3

43.0 14.0 10.5 10.5 11.4 7.9 2.7

From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

Fig. 21.2. Bronchoscopic finding of edematous-hyperemic endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

334

21.7.3 Fibrostenotic Type

H. S. Chung

Tumorous endobronchial tuberculosis is characterized by an endobronchial mass that has a surface often cov-

ered with caseous material, and which nearly totally occludes the bronchial lumen (Fig. 21.4). This form of endobronchial tuberculosis is frequently mistaken for lung cancer because of its bronchoscopic appearance and the fact that the computerized tomography findings mimic those of lung cancer. Anthracotic pigmentation or anthracofibrosis can appear after the endobronchial lesion has been successfully treated. If the lymph nodes caseate, they may point towards and discharge intrabronchially. Following discharge, the resultant fistulae usually heal slowly, leaving small, sometimes pigmented, pitted scars in the bronchial wall. These can be visualized as hard, depressed, black plaques by bronchoscopy, and have been observed to follow the intrabronchial perforations of tuberculous lymph nodes. The black pigment derives from the anthracotic material present in the tuberculous nodes, which is subsequently incorporated into scarred areas. In my previous study, the evolution of tumorous endobronchial tuberculosis was very complicated and unpredictable. Approximately 70% of cases eventually changed into the fibrostenotic type, which included the complete obstruction of the bronchial lumen with fibrosis. Six months after treatment, another 20% were found to exhibit impending obstruction of the left main bronchus due to a re-growing mass, although this were successfully corrected by bronchoscopic electrocautery. In 20% of cases, new tumorous lesions appeared five or six months after treatment, and anthracofibrosis or anthracotic pigmentation was

Fig.21.3. Bronchoscopic finding of fibrostenotic endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

Fig.21.4. Bronchoscopic finding of tumorous endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

Fibrostenotic endobronchial tuberculosis presents as a marked narrowing of the bronchial lumen with fibrosis. Usually) endobronchial tuberculous lesions do not encircle the bronchial mucosa and normal mucosa is partly spared, and the stenotic bronchial lumen becomes a crushed waterdrop-shape, as shown in Fig. 21.3. In some cases, the bronchial lumen is completely occluded. The edges of this stricture are avascular and pale because of dense fibrosis, and it is impossible to biopsy due to its very hard consistency. However, active tuberculosis can be diagnosed by bronchoscopic biopsy of the chronic inflamed mucosa at the periphery of the lesion, which is usually swollen and reddened. In my previous study, all cases remained in a fibrostenotic state inspite of drug therapy. In addition, roughly half of the cases showed progressive fibrostenosis and this resulted in complete obstruction of the bronchial lumen two or three months after treatment. The prognosis of fibrostenotic endobronchial tuberculosis is very poor.

21.7.4 Tumorous Type

335

Endobronchial Tuberculosis

observed in 30% of cases. The prognosis of tumorous endobronchial tuberculosis is grave. It is very difficult to analyze the evolution of tumorous endobronchial tuberculosis; this is due to the fact that the disease exhibits diverse progress and unexpected changes. However, the observations of Shulutko and coworkers (l979) are extremely valuable. In bronchoglandular tuberculosis, which is equivalent to tumorous endobronchial tuberculosis, bronchoglandular fistulas occur when the necrotic foci of tuberculous lymph nodes rupture into the bronchial lumen, extruding the lymph node contents. During this stage, bronchial stenosis is often temporary, and the bronchoglandular fistula usually heals, leaving a thin tender scar that neither deforms the wall nor narrows the lumen of the bronchus, provided endobronchial treatment (removal of caseous masses and granulations, cauterization of the fistulous opening or peribronchial blockades) is performed. However, if scarring of the bronchoglandular fistula continues, the bronchial lumen may be permanently narrowed by persistent cicatricial stenosis, or it may even be completely obliterated. Most cicatricial stenoses of endobronchial tuberculosis appear to be the sequelae of tumorous bronchadenitis, and occasionally more lymphoglandular fistulas arise near the first fistula. When the insights of Shulutko et al. were applied to my previous series, unexpected changes were no longer unexpected, but probable. In order to maintain bronchial patency, early diagnosis and efficacious treatment, including interventional

modalities (Smith et al.1987; Watson and Ayres 1988; Chung et al. 1991a), are very important in tumorous endobronchial tuberculosis.

Fig.21.5. Bronchoscopic finding of granular endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

Fig.21.6. Bronchoscopic finding of ulcerative endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

21.7.5 Granular Type Granular endobronchial tuberculosis appears macroscopically like scattered grains of boiled rice, and the underlying bronchial mucosa shows severe inflammatory change (Fig. 21.5). In my previous study, roughly 20% of the cases showed fibrostenosis of the bronchial lumen two months after treatment, and the other 80% of cases healed without endobronchial sequelae three or four months after treatment. The prognosis of granular endobronchial tuberculosis is good.

21.7.6 Ulcerative Type The appearance of the bronchial ulcer in ulcerative endobronchial tuberculosis is very similar to that of a peptic ulcer (Fig. 21.6), and it can result from the submucosal lymphatic spread of organisms from adjacent parenchymal disease or implantation. In my previous study, the prognosis of ulcerative endobronchial tuberculosis was excellent, as all cases were completely resolved within three months of

336

H. S. Chung

treatment commencement. However, the number of cases involved was too small to serve as a basis for evaluating prognosis.

21.7.7 Nonspecific Bronchitic Type In nonspecific bronchitic endobronchial tuberculosis, only mild mucosal swelling and/or hyperemia are evident by bronchoscopy (Fig. 21.7). Tuberculosis is proven by bronchoscopic biopsy of the lesions. As my previous investigation indicated, all cases of nonspecific bronchitic endobronchial tuberculosis that were studied healed within two months of treatment, suggesting that the prognosis for this disease is excellent.

Fig. 21.7. Bronchoscopic finding of nonspecific bronchitic endobronchial tuberculosis. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

21.8 Location of Bronchial Involvement As pulmonary tuberculosis shows a right-sided predominance in most series (McAdams et al. 1995), so endobronchial tuberculosis may occur more frequently in the right side (Garay 1996). In my previous study (Chung et al. 1991a), endobronchial tuberculosis showed a mild right-sided predominance, whereas luminal narrowing of the bronchi was more frequently observed in the left. Moreover, tracheal involvement was not rare. Interestingly, the

Trachea: 17.3

Rt main B: 6.1

Cas' 48.6 E-H . 37.1 Fs : 8.6 NsB. 5.7

Cas: 56.2 E-H :18.7 Fs : 12.5 Tum: 6.3 Gra . 6.3

Lt Main B: 18.4 E-H : 35.6 Cas: 26.7 Fs : 24.4 Tum: 8.9 Gra : 4.4

Rt upper B: 17.9 Cas : 40.0 E-H : 29.3 Fs . 17.3 NsB : 6.7 Ulcer: 6.7 Rt lower B : 15.1 NsB : 55.6 E-H : 18.5 Fs : 18.5 Cas. 7.4

most common site of endobronchial tuberculous lesions was found to be the left mainstem bronchus. Lowet al. (2001) confirmed this finding. Endobronchial tuberculosis of both lower lobe bronchi and the right middle lobe bronchus was found to be mainly of the nonspecific bronchitic type, and tumorous endobronchial tuberculosis was found to occur mainly in the left mainstern bronchus. The distributions of involved sites and their types are illustrated in Fig. 21.8.

Lt upper B: 4.8 Fs : 37.5 Cas: 37.5 E-H : 25.0 Upper div: 5.8

E-H : 50.0 Cas: 25.0 Tum: 25.0 Rt middle B : 3.7 NsB Fs E-H Tum

42.8 28.6 14.3 14.3

Cas: E-H : Fs : Tum:

30.8 30.8 23.0 15.4

Lingular div: 1.2 Fs: 100.0

Lt lower B: 7.6 NsB : 84.6 Tum: 15.4

Fig. 21.8. Distributions of involved sites and their types versus their relative frequencies (%) in endobronchial tuberculosis. *Rt right, Lt left, Interm intermediate, B bronchus, div division. Cas actively caseating type, E-H edematous-hyperemic type, Fs fibrostenotic type, Tum tumorous type, era granular type, Ulcer ulcerative type, NsB nonspecific bronchitic type

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Endobronchial Tuberculosis

21.9 Natural Course of Endobronchial Tuberculosis

21.10 Endobronchial Tuberculosis in HIV-Infected Patients

The presumptive natural course of endobronchial tuberculous lesions (Fig. 21.9, dashed arrow) can be deduced by observing the healing process (Fig. 21.9, solid arrow). The desired end result of endobronchial tuberculosis is healing without significant sequelae; the other possibility is fibrostenosis. All subtypes of endobronchial tuberculosis are situated between these two conclusions, and they can transform into other subtypes during treatment. However, there is a critical point between these two extremes, the position of which is mainly determined by the extent of disease progression (Kim et al. 1993) and the formation of granulation tissue (Shulutko et al. 1979; Smith et al. 1987). Bronchial stenosis is inevitable (Albert and Petty 1976; Caligiuri et al. 1984) if the disease progresses beyond this critical point. Therefore, prompt diagnosis and efficacious treatment are of paramount importance in cases of endobronchial tuberculosis in order to minimize the resultant bronchial stenosis (Park et al. 1997). To alleviate bronchostenosis that has already developed, aggressive therapeutic modalities should be considered before dense fibrosis progresses or complete obstruction of the bronchus occurs.

In a normal host the immunologic response to infection by the tubercle bacillus provides a degree of protection against additional tubercle bacilli that may be subsequently inhaled in droplet nuclei. The likelihood of reinfection is a function of the risk of re-exposure, the intensity of such exposure, and the integrity of the host's immune system. In developed countries the risk of re-exposure to an infectious case is low. Furthermore, in the otherwise healthy, but previously infected person, any organisms deposited in the alveoli are likely to be killed by cell-mediated immune response. Mycobacterial infections have been commonly observed in patients infected with HIV. These individuals are 200 times more likely to contract tuberculosis than HIV-negative individuals (Broughton and Bass 1999). In contrast to tuberculosis infections in normal adults, tuberculosis in AIDS patients cannot be readily identified by clinical or radiological criteria. It has become apparent that infection with HIV, because of its profound suppression of normal immune response, predisposes the individual to much more severe forms of tuberculosis. In HIVinfected persons with tuberculosis, dissemination of the tubercle bacilli and a variety of extrapulmonary manifestations are common. Thus, unusual clinical

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Fig. 21.9. A scheme summarizing the observed healing process (solid arrow) and the presumptive natural course (dashed arrow) of endobronchial tuberculous lesions. From Chung and Lee (2000). Reproduced, with permission, from Chest 117:385

338

H. S. Chung

presentations of tuberculosis in HIV-infected persons present a special diagnostic challenge (American Thoracic Society 1990). Tuberculosis in HIV-infected patients may have the radiographic characteristics of primary disease. Infiltrates may appear in any lung zone, cavitation is uncommon, and mediastinal or hilar lymphadenopathy is often present (Mangura and Reichman 1989). The tumorous type of endobronchial tuberculosis predominates in patients with HIV infection, and the condition may be the result of the erosion and protrusion of a tuberculous lymph node into the bronchus, a pathogenetic mechanism of primary tuberculosis. The diagnosis of endobronchial tuberculosis in HIV patients should always be considered, since it may be more frequent than suspected (Calpe et al. 1995). Consequently, a more liberal indication of bronchoscopy helps in the early detection of endobronchial tuberculosis in patients with AIDS (Alame et al. 1995). Even if no endobronchial lesion is apparent by bronchoscopy, bronchoalveolar lavage and transbronchial biopsy are particularly useful for diagnosing HIV-related pulmonary tuberculosis, since only two-thirds to three-quarters of the patients have a positive acid-fast smear, and the tuberculin skin test is less sensitive in these patients (Rossman and Oner-Eyuboglu 1998).

A cough is the most common symptom, which has an almost imperceptible onset. Coughs slowly progress over weeks or months and become more frequent; they may be nonproductive at first, but occasionally may be associated from the onset with mucopurulent sputum or blood, and later, the expectoration of sputum usually appears. Shortness of breath frequently occurs, and may be associated with localized wheezes, stridors or the absence of a breathing sound. Fever is frequently associated with the disease, and though low-grade at the onset it may become marked as the disease progresses. Characteristically, the fever develops in the late afternoon and may not be accompanied by pronounced symptoms. With defervescence, usually during sleep, sweating occurs - the classic «night sweats." In more extensive disease, hemoptysis may occur but is seldom massive. The clinical features of endobronchial tuberculosis as detailed in several reports are summarized in Table 21.2. Symptoms are usually nonspecific and are caused mainly by the coexisting pulmonary tuberculosis. In contrast to uncomplicated pulmonary tuberculosis, hemoptysis was less frequent and dyspnea was much more common in my previous study.

21.12 Diagnosis 21.11 Clinical Features

21.12.1 Clinical History

The clinical features of endobronchial tuberculosis vary widely, depending on the site(s) and the extent of involvement, and endobronchial tuberculosis may occur in the absence of recognized symptoms.

When endobronchial tuberculosis is suspected, inquiries concerning patient exposure to a person with an open case of pulmonary tuberculosis may be helpful, especially if this contact is long-standing and close. A

Table 21.2. Clinical features of endobronchial tuberculosis as detailed in several reports Symptom and sign (%)

Song et al. (1985)

Chung et al. (1991)

Ip et al. (1986)

Hoheisel et al. (1994)

Low et al. (2001)

Cough Sputum Dyspnea Hemoptysis Chest pain Fever Absence of a BS Wheezing/Stridor Rhonchi Weight loss None

87.5 75 24 17.5 45 35 27.5 27.5

75.3 68.7 33.1 1.2

100 95 35 25 15 50

87 89

86

BS breathing sound

26.5 28.3 27.7 14.5

15

57 8 15 87 19 9 12

24 19 29

35

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past diagnosis of pneumonia that has recurred from time to time should always arouse suspicion regarding endobronchial tuberculosis. All previous chest films should be obtained. Medical conditions that increase the risk of tuberculosis (i.e. diabetes mellitus, gastrectomy, drug abuse, etc) should also be reported.

21.12.2 Physical Examination Acomplete physical examination should be performed, although an examination of the chest will usually furnish the main clues. Wheezing is frequently heard on auscultation. Persistent unilateral wheezing is more indicative of endobronchial tuberculosis, while transient wheezing due to bronchial secretions usually clears with cough. Stridor may occur with an ulceration and cicatrix of the trachea or larynx. It is nearly impossible to differentiate wheezing and stridor heard in endobronchial tuberculosis from that heard in bronchial asthma. Decreased breathing or the absence of a breathing sound is another frequent finding. When tuberculosis involves the larynx, hoarseness is usually present and is often accompanied by severe pain.

21.12.3 Laboratory Testing Routine laboratory examinations are rarely helpful in establishing or suggesting a diagnosis. A broad range of hematologic manifestations has been reported in endobronchial tuberculosis. The most common of these are increases in peripheral blood leukocyte counts and modest anemia, each of which occurs in approximately 10% of patients. WBC counts of over 20,000/f.lL suggest another infectious process, and the erythrocyte sedimentation rate is usually elevated. With endobronchial tuberculosis, HIV testing is recommended in patients who have known or suspected risk factors for the acquisition of HIV infection.

21.12.4 Bacteriology Endobronchial tuberculosis, especially the extensive form, is usually higWy infectious. In my previous study, the sputum smear for acid-fast bacilli was positive in about half of the patients, and the sputum culture for tubercle bacilli was positive in 70% or more.

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Conversely, some recent studies have found a low yield on sputum acid-fast bacilli smears. The explanation given was that the expectoration of sputum is difficult because of mucus entrapment by proximal bronchial granulation tissue (Ip et al. 1986; Lee et al. 1992; van den Brande et al. 1990). It should not be surprising that large differences are observed in the positive rates of acid-fast bacilli, because culture yield, like microscopic examination, is affected by the clinical status of patients. Sputum examination for acid-fast bacilli is much more important, since patients with endobronchial tuberculosis occasionally present with apparent acute pneumonia. In fact, the diagnosis of tuberculosis is made only on routine sputum examination or because of a failure of clinical or radiological resolution using broad-spectrum antibiotics. However, it should be noted that a positive acid-fast smear is not specific for Mycobacterium tuberculosis. Other mycobacteria, both saprophytes and potential pathogens, can be acid-fast. Thus, the only absolute way of confirming a diagnosis of M tuberculosis is by culture. FresWy expectorated sputum is the best sample to stain and culture for M tuberculosis. Twenty-four hour old sputum samples are frequently overgrown with mouth flora and are much less useful. If a patient is not producing sputum spontaneously, induced sputum is the next best specimen for study. When a patient cannot provide a spontaneous sputum sample, a gastric aspirate to obtain swallowed sputum may be useful. This sample must be obtained in the morning before the patient arises or eats. Bronchoscopy is preferable to gastric aspiration, since bronchial secretions are readily available for acid-fast smear and culture, as well as for cytologic study, and a careful evaluation of the tracheobronchial tree can be carried out at the same time. Specimens for culture should be obtained using a minimal amount of anesthesia, because the local anesthetics used for fiberoptic bronchoscopy may be lethal to M tuberculosis. The portion of the biopsy specimen to be used for culture should not be placed in formalin. Post-bronchoscopy sputum can be another valuable source of diagnostic material, and bronchoscopy may cause the patient to continue producing sputum for several days. These later specimens should also be collected and examined, as they may reveal tubercle bacilli absent from the sample taken on the day of the bronchoscopy. Newer technologies, such as radiometric technology (the BACTEC system), polymerase chain reaction and genetic probes, immunoassay of mycobacterial antigens, and the detection of biologic compounds,

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can allow an early diagnosis or improve our ability to isolate mycobacteria from clinical specimens (Glassroth 1993).

21.12.5 Tuberculin Testing The tuberculin skin test is used as an indicator of M. tuberculosis infection, and relies on a cell-mediated immune response. However, a positive delayed hypersensitivity reaction to tuberculin indicates only the occurrence of a prior primary infection and not the presence of clinically active disease. Very large reactions (greater than 25 mm of induration) are more frequently associated with active tuberculosis. However, a negative reaction to tuberculin does not rule out the diagnosis, because the patient may be anergic or have a specific anergy to tuberculin (Rossman and Oner-Eyuboglu 1998). Nevertheless, the absence of skin reactivity to tuberculin makes the possibility of tuberculosis unlikely in most situations.

21.12.6 Pulmonary Function Test Bronchial disease and pulmonary parenchymal lesions coexist in endobronchial tuberculosis. Although this may have an influence on pulmonary function, no pathognomonic pattern of physiologic disturbance exists in endobronchial tuberculosis. Nevertheless, the usual pattern is that of a predominantly restrictive ventilatory defect. The reason why endobronchial tuberculosis shows this restrictive pattern may be due to organic obstruction of the bronchial tree and chronic inflammatory or bronchiectatic changes of the lung parenchyme. The pulmonary function test may be helpful in the differential diagnosis of endobronchial tuberculosis and bronchial asthma. The results of pulmonary function testing in my previous study are illustrated in Fig. 21.10.

Pulmonary function testing is useful for the followup of endobronchial tuberculosis. Changes in pulmonary function during treatment in endobronchial tuberculosis are significantly correlated with changed bronchoscopic findings, although the indices of pulmonary function testing at diagnosis do not correlate significantly with the gross bronchoscopic findings of endobronchial tuberculosis (Chung and Lee 1996).

21.12.7 Radiology 21.12.7.1 Simple Roentgenograms

Despite the fact that roentgenographic examination itself is not diagnostic of endobronchial tuberculosis, it is a necessary procedure. A lateral chest film should be a part of every roentgenographic inquiry, since approximately 25 percent of the lung fields cannot be visualized on the conventional posteroanterior film. Endobronchial tuberculosis, as well as pulmonary tuberculosis, may produce almost any form of pulmonary radiographic abnormality. However, endobronchial tuberculosis frequently presents with unusual roentgenographic findings, such as pneumonic consolidation, lobar or segmental collapse, a mass-like lesion or hilar or paratracheal adenopathy. A normal chest radiograph cannot completely exclude endobronchial tuberculosis, because roughly 10% of patients show normal chest roentgenograms. Radiologic findings of endobronchial tuberculosis in my previous study are shown in Table 21.3. 21.12.7.2 Computerized Tomography

Special imaging techniques, such as computerized tomography and magnetic resonance imaging, may be of particular value in defining nodules, cavities, cysts, Table 21.3. Radiologic findings of 166 endobronchial tuberculoses Characteristics

Fig.21.10. The results of pulmonary function testing in 85 cases of endobronchial tuberculosis

Cases

Pneumonic patchy infiltration 42 Atelectasis or Collapse 33 Fibrostreaky densities 27 Cavitary lesion 23 Mass-like lesion 12 Bronchiectatic change 7 18 Mediastinal widening No active lesion 21

%

25.3 19.9

16.3 13.9

7.2 4.2 10.8 12.7

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calcifications, the contours oflarge bronchi and vascular details in lung parenchyma. Moreover, since the length of bronchial involvement, the thickness of bronchial wall and mediastinal lymph node enlargement can be evaluated noninvasively, computerized tomography is a useful adjunct to direct endoscopic visualization, particularly when performed at a 5-mm intervals with 5-mm slice collimation through the hila. The technique accurately depicts bronchial abnormality in 93 to 100% of all cases (McAdams et al. 1995), and it can also reconstruct three-dimensional images of the trachea and of the major bronchi (Choi et al. 2002). Computerized tomography findings of endobronchial tuberculosis include: isolated long segment bronchial narrowing with concentric wall thickening, complete endobronchial obstruction, extrinsic compression by adjacent adenopathy and even direct bronchial invasion of a caseous node. The erosion of calcified lymph nodes into adjacent bronchi, known as broncholithiasis, can also be observed, along with the resultant segmental collapse or overinflation. Computerized tomographic scans of the central airways usually show active endobronchial tuberculosis as an irregular narrowing of the airways with marked wall thickening; the scans also frequently show mediastinal lymph node enlargement, whereas fibrotic tuberculosis shows as a rather smooth narrowing of the airways with minimal wall thickening (Kim et al.1997; Moon et al.1997).

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essential that aggressive therapy be performed before the disease progresses too far and bronchostenosis becomes inevitable. Therefore, the bronchoscopic approach is mandatory not only for the prompt diagnosis of endobronchial tuberculosis, but also for the prevention of further bronchostenosis. The suspicion of endobronchial tuberculosis provides an important indication for bronchoscopy. The presence of a wheeze or of a persistent and uncontrollable cough, or the presence of tubercle bacilli in the sputum without an obvious source in the lung parenchyma, provides sufficient reason for the procedure. Roentgenographic findings of atelectasis or selective segmental or lobar collapse, unexplained shadows near the hilum, the sudden or gradual appearance of localized obstructive over-distention or ballooning cavities should also be investigated by bronchoscopy. A ballooning cavity is often referred to as the tension cavity, regardless of whether or not the intracavitary pressure is known. Ballooning cavities are frequently secondary to the involvement of bronchi in tuberculosis (Wolinsky 1989). Tuberculosis produces two main bronchoscopically visible changes: endobronchial inflammation and distortion due to extrabronchial lymph-node enlargement. One may see inflamed, swollen mucosa and purulent secretions or blood; alternatively, one may sometimes see fibrous masses ofwhite-pink color resembling cauliflower and simulating a cancerous tumor, or even ulceration, in the bronchial draining 21.12.8 lobes or segments afflicted with active tuberculosis. Bronchoscopy Acute inflammatory changes can respond rapidly to chemotherapeutic treatment, leaving normal bronBronchoscopy is the single most useful modality chi, but healing may lead to bronchial scarring and in the diagnosis of endobronchial tuberculosis, sometimes to marked contractive stenosis. whereas the chest radiograph is the most important In summary, the diagnostic use of bronchoscopy for suggesting a diagnosis of pulmonary tuberculo- in obtaining a biopsy from an area of bronchial sis. Bronchoscopy is necessary not only to make the ulceration or obstruction can clinch the diagnosis of diagnosis of endobronchial tuberculosis, but also endobronchial tuberculosis. to exclude bronchogenic carcinoma. Bronchoscopy has a unique value in the management of endobronchial tuberculosis. When the different forms of endobronchial tuberculosis are classified into seven 21.13 subtypes (actively caseating, edematous-hyperemic, Differential Diagnosis fibrostenotic, tumorous, granular, ulcerative and nonspecific bronchitic), the therapeutic outcome of 21.13.1 each subtype, except the tumorous type, can be pre- Pneumonia dicted by follow-up bronchoscopy during the initial two to three months of treatment. However, in the In contrast to endobronchial tuberculosis, in which case of tumorous endobronchial tuberculosis, close symptoms are nonspecific, the acute pneumonic and long-term follow-up is advisable, given that the patient usually has typical symptoms of fairly recent evolution of lesions during treatment is very compli- onset. If the patient is symptomatic, an oral antibicated and bronchial stenosis may develop later. It is otic should be prescribed, and if the radiographic

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342

opacities do not clear or improve in 2 to 3 weeks, then tuberculosis is a possibility. If the pneumonia is refractory to standard treatment, especially if the white count is normal, then repeated sputum specimens should be examined for acid-fast bacilli.

21.13.2 Fungal Infection and Actinomycosis Polesky et al. (1999) presented data on 38 cases of coccidioidomycosis of the airways, which included 6 cases detailed from their own experience and 32 from the literature. In histoplasmosis, regional lymph node involvement is invariable during the initial infection. The extrinsic pressure of enlarged nodes on the airways may cause obstruction and distal infection or atelectasis. Complications of histoplasmosis are similar to those of primary pulmonary tuberculosis (Fraser et al. 1994). In 1999, Lee et al. reported a case of biopsy-proven endobronchial actinomycosis. The bronchoscopic findings of this disease are very similar to those of endobronchial tuberculosis.

21.13.3 Lung Cancer This differential diagnosis occurs primarily in the middle-aged and elderly age groups. Consolidation distal to a proximal carcinoma may be cavitated and closely mimic tuberculosis. Moreover, carcinoma of the lung and tuberculosis may be present simultaneously; in fact, the frequency of coexistent lung cancer and tuberculosis is as high as 5% (McAdams et al. 1995). In cases with a simultaneous presentation of carcinoma and tuberculosis, the diagnosis of tuberculosis is frequently made first, and the carcinoma diagnosis is often delayed for several months. Thus, if the radiographic and clinical findings suggest carcinoma, but the sputum has acid-fast bacilli, further procedures to diagnose carcinoma may still be indicated (Rossman and Mayock 1988). On bronchoscopy, tuberculous granulation tissue may be observed to erupt through the bronchial mucosa to form a tumor-like mass. The caseous tracheobronchial lymph nodes can produce ominous, irregular swellings into the main bronchi or trachea that suggest the malignant invasion of lymph nodes (Stradling 1981). Bronchoscopic biopsy will reveal the true situation.

21.13.4 Bronchial Asthma Some patients with endobronchial tuberculosis show wheezing on physical examination but have normal chest films, which mimics bronchial asthma. One should always consider the possibility of endobronchial tuberculosis during the differential diagnosis of bronchial asthma if patients with wheezing show normal airway responsiveness (Park et al. 1995) or a poor response to bronchodilation (Williams et al. 1988).

21.14 Treatment 21.14.1 Anti-Tuberculosis Chemotherapy In principle, the short-course regimens are the regimens of choice for patients with endobronchial tuberculosis. Many short-course regimens have been investigated and proposed for pulmonary tuberculosis, and these can also be applied in endobronchial tuberculosis. A 6-month regimen consisting of isoniazid, rifampin and pyrazinamide for 2 months, followed by isoniazid and rifampin for 4 months, is the preferred treatment for patients with fully susceptible organisms and who can adhere to treatment. Ethambutol (or streptomycin in children too young to be monitored for visual acuity) should be included in the initial regimen until the results ofdrug susceptibility studies are available, unless there is only a small possibility of drug resistance (i.e. if there is less than 4% primary resistance to isoniazid in the community, and the patient has had no previous treatment with antituberculosis medications, is not from a country with a high prevalence of drug resistance and has no known exposure to a drug-resistant case). The four-drug,6-month regimen is effective even when the infecting organism is resistant to isoniazid. This recommendation applies for both HIV-infected and uninfected persons. However, in the presence of HIV infection, it is critically important to comprehensively assess the clinical and bacteriologic response. Any problem with the response to treatment indicates that the usual evaluation should be undertaken and the therapy possibly prolonged. Consideration should be given to treating all patients with directly observed therapy, and the duration of treatment should total at least 6 months and 3 months beyond culture conversion. Children should be managed in essentially the same ways as adults by using appropriately adjusted

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doses of the drugs. In multidrug-resistant tuberculosis (i.e. resistance to at least isoniazid and rifampin), treatment must be based on susceptibility studies (American Thoracic Society 1994). With endobronchial tuberculosis, therapy needs to be prolonged as decided on a case-by-case basis and should be individualized after considering the clinical, bacteriologic and bronchoscopic response, especially if the dominant form is of the tumorous type or if interventional management is performed.

loon dilatation, which tends to be only a temporary measure, Nd-YAG laser resection and surgical reconstructive procedures, which are generally considered to be the gold standard, although some patients will have contraindications to surgery. Stent placement, in contrast, is a new therapeutic modality. For select patients, a multidisciplinary approach, whereby pulmonologists, otolaryngologists and thoracic surgeons decide on the most appropriate use of laser resection, stent placement and surgical techniques has been advocated (Dineen et al. 2002).

21.14.2 Corticosteroid Treatment

21.15.1.1 Interventional Management

The use of corticosteroids has been and remains controversial. Nevertheless, under certain conditions corticosteroids are of benefit in patients with endobronchial tuberculosis. It appears that steroids are effective in rapidly reducing the mass effects of mediastinallymphadenopathy in patients with primary tuberculosis. Steroids may, therefore, decrease the severity of local obstructive complications (Nemir et al. 1963). Corticosteroids are more likely to be beneficial in the earlier stages of endobronchial tuberculosis, when hypersensitivity is the predominant mechanism (Dooleyet al. 1997), and they are unlikely to be helpful in more advanced cases when extensive fibrosis is present. Therefore, corticosteroids can be cautiously prescribed for actively caseating and edematous-hyperemic endobronchial tuberculosis (which occur in the earlier stages of the disease) and for tumorous endobronchial tuberculosis, which may be derived from primary tuberculosi~. The usual dose required is 40-60 mg of prednisone (or approximately 1 mg/kg ofbody weight) orally daily for 4-6 weeks, with gradual tapering over the next few weeks. It should be emphasized that before corticosteroids are prescribed, one must be confident that adequate antituberculosis chemotherapy is being given. Prompt treatment upon early diagnosis, before the formation of dense fibrosis, is a prerequisite for the prevention and amelioration of bronchostenosis (Park et al.1997).

Since bronchostenosis develops despite adequate antituberculosis therapy and/or corticosteroid treatment, more aggressive interventional management may be indicated to restore the patency of the involved bronchus in selected cases of endobronchial tuberculosis. In tumorous endobronchial tuberculosis, because the prognosis is grave if the condition is not treated aggressively, the endobronchial tumorous lesion itself should be removed by laser resection or electrosurgery to prevent further bronchostenosis. The fibrous membrane can simply be resected by laser photoresection or electrocautery (in the fibrostenotic type of endobronchial tuberculosis), if the membrane has a concentric web-like stricture (Becker et al. 1991). In cases where the fibrostenosis is relatively long, an endobronchial stent can be placed after dilatation with a high-pressure balloon catheter. Some, but not all, patients with tuberculous bronchostenosis may be provided with long-term control of their disease process by stent placement. Restenosis by granulation tissue formation can develop, but it may be successfully treated by laser ablation or electrosurgery. The therapeutic results of stent placement have been very poor in actively caseating, edematoushyperemic and tumorous endobronchial tuberculosis, which have active severe inflammation of the involved bronchus. Thus, appropriate antituberculosis chemotherapy and/or corticosteroid treatment should be administered prior to stent placement, until active inflammation disappears. This may take up to 5 months, after which, the endobronchial tuberculosis is usually of the fibrostenotic type, which is the main indication of stent placement. During drug therapy, repeated balloon dilatation may be needed to prevent complete obstruction and to maintain the patency of the narrowed bronchus (an example is shown in Figs. 21.11 and 21.12). Once active inflammation has been ameliorated by drug treatment, a

21.15 Complications and Their Management 21.15.1 Bronchostenosis Traditional strategies for repairing benign tracheobronchial stenosis include repeated endoscopic bal-

344

tracheobronchial stent can be placed, if necessary (Figs. 21.13-15}. Further chemotherapy should be given for a minimum of 3 months after the procedure to prevent recurrence. The stent may be successfully removed one year after placement, and the success rate of stenting is roughly 50% (Kim, personal communication). Granular, ulcerative and nonspecific bronchitic endobronchial tuberculosis do not indicate stent placement, because they do not involve significant bronchostenosis. 27.75.7.2

TracheobronchialStent The ideal tracheobronchial stent should possess several vital characteristics. It should be capable of establishing and maintaining airway patency; it should be easy and safe to place and (if necessary) remove;

H. S. Chung

it should be biocompatible; and, it should be flexible enough to fit appropriately in irregular anatomy. In addition, it should not cause mucosal injury or granulation tissue formation, hamper the ability to clear secretions, migrate from its desired position or obstruct otherwise patent lumens (Jantz and Silvestri 2000). As of yet, no such stent has been developed. Essentially, five types of stent have been developed and used for tracheobronchial obstructions. The earliest types were of silicone, most notably the Dumon or Endoxane stent. The next stents used were of uncovered stainless steel, such as the Gianturco Z and the Palmaz stents, which were the first stents capable of being deployed by flexible bronchoscopy. The third type were the second generation of metal stents as represented by the Wallstent and the Ultraflex. These stents are alloy-based mesh or interwoven loop stents and are also available with a polyurethane covering.

Fig. 21.11a-d. Repeated balloon dilatation of the left mainstem bronchus in a case with endobronchial tuberculosis. a At diagnosis. b At 1 month of treatment. c At 2 months of treatment; d At 3 months of treatment

Fig. 21.12a, b. Three-dimensional images of central airways in the case illustrated in Fig. 21.11. a At diagnosis. b At 2 months of treatment

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Fig. 21.13a-d. Stent placement for endotracheal tuberculosis. a Before stenting. b After stenting. c Just after stent-removal. d At 4 months after stent-removal

Fig. 21.14a, b. Three-dimensional images of the trachea in the case illustrated in Fig. 21.13. a Before stenting. b After stenting

Fig.21.15a, b. Stent placement for mainstem endobronchial tuberculosis. a Before stenting. b Just after stenting

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The polymer coating prevents the ingrowth of tumor or granulation tissue through the stent mesh framework. The fourth type of stent, the bifurcated Y-stent, was designed specifically to deal with the anatomic complexities of the central airways and the carina, and the fifth type of stent was constructed from a variety of different materials, such as polyester/silicone and nitinollsilicone. Generally, the most common complications associated with stents are stent migration, secretion retention and granulation tissue formation. Refinements in stent designs are required to improve biocompatibility and reduce migration and granulation tissue formation (Dineen et al. 2002). Overall, silicone stents have been very successful in treating benign airway obstructing lesions and may well be the gold standard for years to come. Recently, animal model trials have been reported with a bioabsorbable stent (Korpela et al. 1999). In the future, bioabsorbable stents may have a role in the management of benign airway stenoses. 21.15.1.3 Endobronchial Electrosurgery vs Laser Photoresection

Nd-YAG laser photoresection is the most effective therapy for treating tracheobronchial obstructions from benign or malignant lesions. However, the widespread use of this technique is limited by the perceived need for rigid bronchoscopy, expensive equipment and special training and fear of major complications. Advances in flexible bronchoscopy have allowed developments in other techniques directed at alleviating airway obstruction, including cryotherapy, brachytherapy and photodynamic therapy. Although costeffective, their effects are delayed and they may require repeated treatments (Coulter and Mehta 2000). Labeled "the poor man's laser:' electrosurgery offers equivalent laser-like tissue effects at a fraction of the cost. The ability to perform endobronchial electrosurgery in the outpatient setting is more accommodating for patients and time-saving for physicians, and it offers Significant cost savings. The lesions found to be most amenable to endobronchial electrosurgery are polypoid in morphology and attached to the airway by a stalk. Flat or sessile lesions can be treated by fulguration with the coagulation probe (Coulter and Mehta 2000). Endobronchial lesions in the tumorous type of endobronchial tuberculosis are good candidates for electrosurgery, because they usually present as polypoid masses or sessile lesions. Although the number of cases was small, my previous study found that electrosurgery produced successful results in this

H. S. Chung

type of endobronchial tuberculosis (Chung and Lee 2000). Granulation tissue caused by metallic stents can also be removed by endobronchial electrosurgery. Special care must be taken not to touch the wires when ablating these lesions, since electrical conduction may occur through the wire mesh. The circumferential treatment of endobronchial lesions should be avoided in endobronchial electrosurgery, as this may lead to cartilaginous damage, fibrosis and subsequent stenosis. 21.15.1.4 Surgical Management

Severe bronchostenosis with poor response to medical treatment and intervention usually requires later resection. Bronchoscopy and computed tomography are the treatments of choice in the accurate diagnosis of bronchial involvement and the assessment of surgical indications (Watanabe et al. 1997). To preserve lung function, bronchoplastic surgery is essential, especially for bronchial stricture of the trachea or larger bronchi, and appropriate antituberculosis chemotherapy should be given for 9 to 12 months perioperatively to prevent recurrence and restenosis (Hsu et al. 1997).

21.15.2 Bronchiectasis Distention by mucus, caseous tissue or secondary infection beyond a bronchial stenosis may result in bronchiectasis, especially following lobar or segmental lesions. (Seaton et al. 1989). The incidence of bronchiectasis is reduced by prompt antituberculosis chemotherapy.

21.15.3 Broncholith In primary tuberculosis, gross tracheobronchial lymph-node enlargement may take place. In many cases, these caseous nodes do not rupture but instead inspissate, contract and calcify, and occasionally such calcifications may later begin to ulcerate through the bronchial wall to produce repeated hemoptysis and eventually be extruded into a bronchus as a "broncholith:' A partially protruded broncholith can sometimes be removed bronchoscopically if not expectorated by the patient. However, attempts to remove broncholiths bronchoscopically can lead to profuse hemorrhage and, therefore, are best avoided (Stradling 1981).

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Acknowledgements. I am very grateful to Dr. Ho Joong Kim, and Miss Jin Ha Park for their generous contribution.

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347 ticosteroid therapy for tuberculosis: a critical reappraisal of the literature. Clin Infect Dis 25:872-887 Fraser RS et al (1994) Infectious disease of the lungs, chap 7. In: Fraser RS, Pare JAP, Fraser RG, Pare PD (eds) Synopsis of diseases of the chest, 2nd edn. Saunders, Philadelphia, pp 287-391 Garay SM (1996) Pulmonary tuberculosis, chap 32. In: Rom WN, Garay SM (eds) Tuberculosis. Little Brown, Boston, pp 373-412 Garimella PS (2001) Anthracofibrosis. Chest 120 [Suppl): 327s

Glassroth J (1993) Diagnosis of tuberculosis. In: Reichman LB, Hershfield ES (eds) Tuberculosis: a comprehensive international approach. Dekker, New York, pp 149-166 Glynn JR (1998) Resurgence of tuberculosis and the impact of HIV infection. Br Med Bull 54:579-593 Greenbaun M, Beyt BE, Murray PR (1980) The accuracy of diagnosing pulmonary tuberculosis at a teaching hospital. Am Rev Respir Dis 21:447-481 Han SK et al (1989) Balloon dilatation of bronchial stenosis in endobronchial tuberculosis. Program and book of abstracts. The 6th world congress for bronchology, Tokyo/ Kyoto,p 130 Hoheisel G et al (1994) Endobronchial tuberculosis: diagnostic features and therapeutic outcome. Respir Med 88:593-597 Hsu HS et al (1997) Surgical treatment of endobronchial tuberculosis. Scand Cardiovasc J 31:79-82 Ip MSM et al (1986) Endobronchial tuberculosis revisited. Chest 89:727-730 Jantz M, Silvestri GA (2000) Silicone stents versus metal stents for management of benign tracheobronchial disease-pro: metal stents. J Bronchol 7:177-183 Jenks RS (1940) Tuberculous tracheobronchitis: a review. Am Rev Tuberc 37:315-326 Jokinen K, Palva T, Nuutinen J (1977) Bronchial findings in pulmonary tuberculosis. Clin Otolaryngol 2: 139-148 Judd AR (1947) Tuberculos tracheobronchitis. J Thoracic Surg 16:512-521 Judson MA, Sahn SA (1994) Endobronchial lesion in HIVinfected individuals. Chest 105:1314-1323 Kim HY et al (2000) Bronchial anthracofibrosis (inflammatory bronchial stenosis with anthracotic pigmentation): CT findings. Am J Roentgenol 174:523-527 Kim Y et al (1997) Tuberculosis of the trachea and main bronchi: CT findings in 17 patients. Am J Roentgenol168: 1051-1056 Kim YH et al (1993) Serial fiberoptic bronchoscopic observations of endobronchial tuberculosis before and early after antituberculosis chemotherapy. Chest 103,673-77 Korpela A et al (1999) Bioabsorbable self-reinforced poly-Llactide, metallic, and silicone stents in the management of experimental tracheal stenosis. Chest 115:490-495 Lee JH et al (1992) Endobronchial tuberculosis: clinical and bronchoscopic features in 121 cases. Chest 102:990-994 Lee SH et al (1999) Endobronchial actinomycosis simulating endobronchial tuberculosis: a case report. J Korean Med Sci 14:315-318 Lerner BH (1999) Catching patients: tuberculosis and detention in the 1990 s. Chest 115:236-241 Low SY, Eng P, Hsu A (200l) Interventional bronchoscopy in tuberculous tracheobronchial stenosis. Chest 120 [Suppl): 160s Mangura BT, Reichman LB (1989) Pulmonary tuberculosis. In:

348 Pennington JE (ed) Respiratory infections: diagnosis and management, 2nd edn. Raven, New York, pp 528-543 Matthews JI, Matarese SL, Carpenter JL (1984) Endobronchial tuberculosis simulating lung cancer. Chest 86:642-644 McAdams HP, Erasmus J, Winter JA (1995) Radiologic manifestation of pulmonary tuberculosis. Radiol Clin North Am 33:655-678 Medlar EM (1955) The behavior of pulmonary tuberculous lesion: a pathological study. Am Rev Tuberc 71:132-145 Millard PS et al (1994) Rurality and tuberculosis incidence trends in North and South Carolina, 1982 to 1992. J Rural Health 10:226-236 Moon WK et al (1997) Tuberculosis of the central airways: CT findings of active and fibrotic disease. Am J Roentgenol 169:649-653 Myerson MC (1994) Tuberculosis of the trachea and bronchus. Thomas, Springfield, pp 250-275 Nemir RL et al (1963) Prednisone therapy as an adjunct in the treatment of lymph node-bronchial tuberculosis in childhood: a double-blind study. Am Rev Respir Dis 88:189-198 Oho K, Amemiya R (1984) Pathological bronchoscopic findings, chap 4. In: Oho K, Amemiya R (eds) Practical fiberoptic bronchoscopy, 2nd edn. Igaku-Shoin, Tokyo, pp 94-150 Park CS et al (1995) Bronchial hyperreactivity in patients with endobronchial tuberculosis. Respir Med 89:419-422 Park IW, Choi BW, Hue SH (1997) Prospective study of corticosteroid as an adjunct in the treatment of endobronchial tuberculosis in adults. Respirology 2:275-281 Polesky A et al (1999) Airway coccidioidomycosis: report of cases and review. Clin Infect Dis 28:1273-1280 Riley RL (1960) Apical localization of pulmonary tuberculosis. Bull Johns Hopkins Hosp 106:232-239 Rossman MD, Mayock RL (1988) Pulmonary tuberculosis, chap 7. In: Schlossberg D (ed) Tuberculosis, 2nd edn. Springer, Berlin Heidelberg New York, pp 71-76 Rossman MD, Oner-Eyuboglu AF (1998) Clinical presentation and treatment of tuberculosis, chap 162. In: Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, Senior RM

H. S. Chung (eds) Fishman's pulmonary diseases and disorders, 3rd edn. McGraw-Hill, New York, pp 2483-2503 Saadoun R et al (1998) Endobronchial tuberculosis presenting as an obstructive tumor in an HIV-l-positive patient: apropos of a case and review of the literature. Rev Med Interne 19:344-347 Salkin D, Cadden V, Edson RC (1943) The natural history of tuberculous tracheobronchitis. Am Rev Tuberc 47:351-359 Seaton A, Seaton D, Leitch AG (1989) Clinical features of tuberculosis, chap 14. In: Seaton A, Seaton D, Leitch AG (eds) Crofton and Douglas's respiratory diseases, 4th edn. Blackwell, Oxford, pp 395-422 Shulutko et al (1979) Tuberculosis. In: Lukomsky GI (ed) Bronchology. Mosby, St Louis, pp 287-305 Smart J (1951) Endobronchial tuberculosis. Br J Dis Chest 45: 61-68 Smith LS, Schillaci RF, Sarlin RF (1987) Endobronchial tuberculosis: serial fiberoptic bronchoscopy and natural history. Chest 91:644-647 Song JH, Han SK, Heo 1M (1985) Clinical observation of endobronchial tuberculosis(in Korean). Tuberc Respir Dis 4:276-284 Stradling P (1981) Inflammatory and associated changes, chap 5. In: Stradling P (ed) Diagnostic bronchoscopy, 4th edn. Churchill Livingstone, New York, pp 61-81 Van den Brande PM et al (1990) Clinical spectrum of endobronchial tuberculosis in elderly patients. Arch Intern Med 150:2105-2108 Watanabe Y et al (1997) Treatment of bronchial stricture due to endobronchial tuberculosis. World J Surg 21:480-487 Watson JM, Ayres JG (1988) Tuberculous stenosis of the trachea. Tubercle 69:223-226 Williams DJ et al (1988) Endobronchial tuberculosis presenting as asthma. Chest 93:836-838 Wolinsky E (1989) Tuberculosis, chap 26. In: Baum GL, Wolinsky E (eds) Textbook of pulmonary diseases, 4th edn. Little Brown, Boston, pp 465-520 Yee A et al (1985) Pigmented polypoid obstructive endobronchial tuberculosis. Chest 87:702-703

22 Pleural Tuberculosis M. MONIR MADKOUR, MAJDY IDREES, MONA

CONTENTS 22.1 22.2 22.3 22.4 22.5 22.5.1 22.5.2 22.5.3 22.5.4 22.5.5 22.5.6 22.5.7

Introduction 349 Epidemiology 349 Pathogenesis of Tuberculous Pleural Effusion 350 Clinical Features 351 Diagnosis of Tuberculous Pleural Effusion 352 Diagnosis 352 Pleural Fluid Examination 352 Pleural Biopsy 353 Thoracoscopy in Tuberculous Patients 354 Tuberculin Skin Test (PPD) 354 Imaging Features of Pleural Tuberculosis 355 Treatment 355 References 356

22.1 Introduction Tuberculous pleurisy is a common disease in developing countries and its incidence is increasing in developed countries. It is considered an extrapulmonary manifestation of tuberculosis despite its intimate anatomical relationship with the lung parenchyma. In the United States, because of the recent increase in the incidence of tuberculosis due to the HIV epidemic and immigrants from developing endemic countries, the incidence of pleural tuberculosis has increased in parallel with, and has been found in 20% of, AIDS patients (Ankobiah et al. 1990). Although tuberculous pleural effusion is associated with parenchymal infiltrates and cavitation in approximately 50% of patients, isolated pleural effusion without lung parenchymal disease can present M. M. MADKOUR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia M. IDREES, MD, FRCP (C), FCCP Head, Pulmonary Function Laboratory, Division of Pulmonary Medicine, Department of Medicine, Riyadh Armed Forces Hospital, ClIO, P.O. Box 7897, Riyadh 11159, Saudi Arabia M. AL SHAHED, MBBS, FRCR Consultant Radiologist, Department of Radiology, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

AL SHAH ED

a diagnostic challenge; malignancy is of particular concern (Hunlnick et al. 1983; Hirsch et al. 1979). It is estimated that between 15 to 20% of patients with exudative pleural effusion in general are difficult to diagnose. In developing countries with poor resources, conventional methods of diagnosis by pleural fluid culture may only be positive in 25-50% of patients (Idell 1994). Presumptive diagnoses of tuberculous origin in exudative pleural effusion, based on clinical features, radiography and response to antituberculous chemotherapy in endemic countries are still adopted by many clinicians. Patients who are not treated for tuberculous pleural effusion often develop reactivation pulmonary disease (Palmer 1979). In developed countries, due to recent advances in investigative facilities, particularly in the field of molecular biology assays, the diagnosis can be made in few hours or days if other investigations are not helpful (Takagi et al.1998; Querol et al.1995; Lassence et al. 1992).

22.2 Epidemiology Pleural tuberculosis is the second commonest form of extra pulmonary parenchymal disease, after the tuberculous lymphadenitis. Pleural tuberculosis is also more common in patients that are co-infected with AIDS (11%) than among those without AIDS (6%). Other reports indicating a higher incidence of pleural tuberculosis (20%) in AIDS patients have found it to be the most common cause of pleural effusion in this patient population (Modilevsky et al. 1989; Shivaram et al.1989;Ankobiah et aI.1990). In developed countries, pleural tuberculosis is more commonly caused by post-primary disease than by primary infection. Reactivation of the disease was the cause of tuberculous pleural effusion in 64% of patients seen in Scotland and reported by Moudgil et al. (1994). Primary pleural tuberculosis was noted in 7% of patients with active pulmonary disease (Aktogu

350

et al. 1996). In young adults with HIV who are coinfected with tuberculosis, the primary disease as a cause of pleural tuberculosis was more common than reactivation (post-primary) (Sudre et al.1992; Batungwanayo et al.1993; Richter et al.1994). Seibert et al. (1991) from Alabama, USA, reviewed the medical records of 1,738 patients with tuberculosis seen between 1968 and 1988, to determine the age distribution, pleural fluid culture and frequency of pleural effusion among those with parenchymal disease, as well as the prevalence of positive tuberculin test. Pleural tuberculosis was found in 70 patients (4.0%) of all forms of the disease. The mean age was 47±17.7 years, with male predominance and pulmonary parenchymal involvement noted in 50% of patients. In Spain, two large studies of patients with pleural effusion were undertaken to determine the causes. The first series was a retrospective study of 414 patients with pleural effusion of unknown cause, who were seen between 1979 and 1986. Tuberculous cause was identified in 107 patients (28.5%) (Bueno et al. 1990). Male predominance was noted and the average age was 55 years. In the second series, 642 patients with pleural effusion were seen between 1989 and 1993. Tuberculous pleural effusion was found in 25% of these patients, similar to the earlier series, and the average age was also similar (57.1±21.1 years) with male predominance as well (Valdes et al.I996b). In Australia, Christopher et al. (1998) reported a prospective study of 27 patients with plural effusion that were hospitalized for the purpose of etiological diagnosis. Tuberculosis was found in 16 patients (59%) and was considered as the single most common cause of exudative pleural effusion in that series. In the United States, the incidence of tuberculosis as a cause of pleural effusion in AIDS patients was found to be increased. In New York, Relkin and colleagues (1994) retrospectively studied pleural tuberculosis in HIV-positive patients and found it to occur at a younger age. They found 70 patients with tuberculosis pleural effusion, including 43 HIV-positive (mean age 38±1 years) and 27 HIV-negative (mean age 52±3 years). They also confirmed previous observations that pleural tuberculosis in HIV-negative patients occurs more frequently in the older age group. In developing countries, such as some sub-Saharan African countries, the prevalence of tuberculosis has increased as a result of the HIV epidemic and the incidence of pleural tuberculosis has also increased in parallel. Richter and colleagues from Tanzania (Richter et al. 1991) prospectively studied 127 patients with pleural effusion. Tuberculosis was the

M. M. Madkour et al.

most common cause (70%) in this series. The authors also found that among the 89 patients with tuberculous pleurisy 57 (64%) were HIV-positive. In Zimbabwe where HIV is endemic, tuberculosis has become the single most likely cause of opportunistic infection (Heyderman et al. 1998). The prevalence of HIV among male factory workers was 19.4% and in females was over 30% (Mbizvo et al. 1996). In 1995, Harare provided an annual report of the incidence of tuberculosis, which was 195 cases per 100,000 population, with over 40% of the TB cases co-infected with HIV. In our series of 176 patients with culture positive sputum for M. tuberculosis, 46 patients had pleural effusion (26.1 %) - see Table 2 in the chapter on postprimary tuberculosis. The average age group was 40 years, with male predominance (75%). The majority of the patients were from the central (57%) and southern (29%) provinces of Saudi Arabia. Unlike the situation in the sub-Saharan African countries, HIV was not found to be a contributing factor for the development of tuberculous pleural effusion.

22.3 Pathogenesis of Tuberculous Pleural Effusion Tuberculous pleurisy has been described by an immunologist as an excellent model for studying the immune response in vivo. The mode of transmission of M. tuberculosis to the pleura may be hematogenous spread, as in primary tuberculosis, or it may be secondary to the direct invasion from adjacent tuberculous lymphadenitis or the rupture of subpleural tuberculous lung parenchymal cavity; it may also result from extrapulmonary disease such as spinal tuberculosis (Sahn 1988; Stevenson 1955; Mohammed et al. 1998; Maeda et al. 1993; Wallis 1996; Zhang et al. 1994; Morehead 1998). The pleura respond to the mycobacterial antigens with an extensive infiltration of mononuclear phagocytic cells and increased production of cytokines; however, the mechanisms by which these cells are recruited to the pleural space remains unclear. Several recent immunological studies have suggested that activated macrophages and CD4 lymphocytes play the most important role in cell-mediated immunity with granuloma formation against M. tuberculosis. Antigens from the wall of the bacilli have a potent stimulant effect and lead to the excessive production of cytokines (TNF-a and IFN-y) from these inflammatory cells, which in turn contribute to

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the formation of granuloma and to the defense mechanism against infection (Epstein et al. 1987; Shimokata et al. 1986; Barnes et al. 1989, 1990; Lorgat et al. 1992; Kunkel et al. 1989; Willis et al. 1990; Zhang et al. 1994; Wallis 1996). Several observations have suggested that a vigorous in-situ or "compartmentalized" intrapleural immune response with activation of cell-mediated immunity (CMI) and delayed type hypersensitivity (DTH) with cytokine production in response to the bacilli cell-wall antigens take place (Barnes et al. 1989). Mycobacterium tuberculosis bacilli contain multiple antigens, including polysaccharides (arabinogalactan and arabinomannan) and proteins (mainly glycolipids and lipoproteins). Polysaccharides from the bacilli generally do not elicit delayed type hypersensitivity (DTH) (Chaparas et al. 1971; Yamamura et al. 1968; Barnes et al. 1993). Lipoprotein and glycolipids antigens elicit DTH and stimulate T lymphocyte and cytokine production, which can be detected in high concentration in the pleural fluid. Pleural mesothelial cells are the first cells to respond to bacilli invading the pleural space. These cells are metabolically active and likely initiate and propagate an inflammatory reaction (Mohammed et al. 1998). Mesothelial cells have been found to initiate inflammation in response to various agonists by the production and release of chemokines (Boylan et al. 1992; Goodman et al. 1992; Jonjic et al. 1992).lnterleukin 12 (IL-12) cytokine has been found in high concentration in the pleural fluid of patients with tuberculous pleurisy (Zhang et al. 1994). Zhang and colleagues from California found that IL-12 play an important role in the immune response by enhancing the production of interferon-y, facilitating development of Thl cells and augmenting cytotoxicity of antigen-specific T-cells and natural killer cells. IL-12 enhances natural killer cell-mediated cytotoxicity and augments antigen-dependant proliferation by CD8+ cytotoxic T lymphocytes (Zhang et al. 1994). It has also been suggested that IL12 enhances cytotoxicity by activating CD4 + T cells against the bacilli loaded phagocytic macrophages, leading to its apoptosis and the destruction of the bacilli within it. Evidence for the excessive production ofantigen-reactive T lymphocytes,IFN-y, TNF-u and IL-12 is provided by the elevated levels of these cytokines in the pleural fluid of tuberculous patients. There is evidence that IL-12 is produced locally in the pleura in response to M. tuberculosis antigens, and that when IL-12 is experimentally suppressed, the bacilli proliferate again. These findings suggest that IL-12 contributes to lymphocyte recognition of M. tuberculosis antigens, probably by enhancing proliferation of activated T cells. The high concentration

of IFN-y and TNF-u (both have anti-mycobacterial activity) in the pleural fluid are a likely indication that they playa role in immune resistance. TNF-u augments the macrophages' capacity to phagocytose and kill the bacilli (Zhang et al. 1994). The authors conclude that IL-12 contribute to the immune response against M. tuberculosis by enhancing production of IFN-y, the T cells and orchestrating antigen-specific cytolytic mechanisms. CD4 T cells also playa critical role in the immune response to M. tuberculosis antigens. In 1996, Wallis (from Ohio), studied the response of T-cells to alpha antigen, a major protein from the wall of the bacilli, and reported the increased IFN-yproduction. As a result of the immune response and the outcome of the infection, inflammatory cellular infiltrates and granulomata formations may occur. As the infection progresses, inflammatory responses and cytokines release will lead to increased vascular permeability of the local capillaries. Subsequently, plasma protein exude fluid into the pleural space forming pleural effusion (Ellner et al. 1988). In postprimary pleural effusion, the disease may be complicated by empyema, lung parenchymal spread or bronchopleural fistula. Pleural fibrosis and calcification may occur in chronic tuberculous empyema.

22.4 Clinical Features Pleural effusion due to tuberculosis most commonly occurs 3 to 12 months, or even more, after the primary infection in adolescents and young adults, but it may occur at any time during the course of the disease (Gedde-Dhal 1952; Berger and Mejia 1973; Sahn 1988; Ansari and Idell 1998). Pleural tuberculosis, due to reactivation of the disease, may be noted in up to 50% of patients with post-primary lung parenchymal involvement, particularly with cavitations (Seibert et al. 1991). The onset of symptoms is acute in about 70% of patients with cough, fever and chest pain. Gradual onset may be noted in about 30% with dyspnea, weight-loss and weakness. The frequency of these symptoms may vary from one series to another. The presence or absence of co-infection with HIV does not change the frequency of symptoms, but non-HIV patients are more symptomatic (See Table 22.1). Non-symptomatic pleural effusion may occur in HIV co-infected patients and was reported in 4 of 21

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Table 22.1. Symptoms

Our Own Hong Kong USA Zimbabwe series (Chang et al. 1991) (Ankobiah et al. 1990) (Heyderman et al. 1998) No HIV No HIV ±HIV ±HIV

Cough 0/0 Dyspnea 0/0 Fever 0/0 Weight loss 0/0 Chest pain 0/0

63

77 86.5 90 44

71 48 71 55 53

60 38 72 59 44

HIV patients (20%) by Ankobiah et al. (1990) from New York. Clinically, sharp pain with pleural rub during deep inspiration on the affected side may be noted. The clinical features of pleural effusion may depend on its size, and are manifested as decreased chest movement, dullness of percussion, decreased breath sound or retraction of chest wall due to extensive pleural fibrosis. These features are not characteristic of tuberculous pleurisy. Pleural tuberculosis is most commonly unilateral but may be bilateral in 5% of patients (Chan et al.1991; Heyderman et al.1998). In our experience with 46 patients with proven pleural tuberculosis, 44 (96%) had unilateral pleural effusion. One patient (2%) had transudative effusion based on Light's criteria. Thirty patients (65%) had moderate effusions and 11 (24%) had massive effusions. Loculated effusion was reported in 9 patients (20%) and pleural calcification in 7 (15%).

97 82 73 51 73

cell count and bloodstained exudative effusion were the best discriminating functions in the screening for pleural tuberculosis. The diagnosis, however, can only be confirmed by the presence of M. tuberculosis determined by direct staining or by culture. The diagnosis of pleural tuberculosis has been defined for the purpose of clinical studies by several authors when one or more of the following were confirmed: (1) positive microbiological findings (smear or culture) in pleural fluid or biopsy; (2) histopathological evidence of granulomatous pleuritis with clinical andlor radiological response to anti-tuberculous treatment; (3) positive skin testing, recent conversion in young patient with a lymphocytic, exudative effusion (Ankobiah et al. 1990; Seibert et al. 1991; Morehead 1998).

22.5.2 Pleural Fluid Examination

22.5

Diagnosis of Tuberculous Pleural Effusion

22.5.1 Diagnosis The diagnosis of pleural tuberculosis begins with a high index of suspicion, clinical features, PPD skin test, imaging features and a culture of all potentially diagnostic specimens (body fluids or tissue biopsies). Screening methods used to differentiate tuberculous from non-tuberculous pleural effusion have been evaluated to identify the discriminate power of each investigative parameter. In a recent report from Spain, Carrion-Valero and Perpina-Tordera (2001) screened tuberculous pleural effusion by discriminant analysis of 189 patients. In this retrospective study, the discriminating power of routine imagining features and laboratory parameters was reviewed. Using the backward elimination method, the authors found that age, the tuberculin skin test, pleural white

Pleural fluid examination remains an important tool in the of investigation of tuberculous pleurisy. Tuberculous pleural fluid is an exudate which may be cloudy (turbid), yellow, serosanguineous or hemorrhagic in appearance. The PH usually ranges between 7.30 and 7.40 or lower, particularly among those co-infected with HIV (George et al. 1985; Pablo et al. 1997). Total WBC may range from 5,000 to 10,000/mm3, with more than 50% lymphocytes; however, polymorphs may be seen early after the onset of symptoms. Red cell count may be raised to a variable degree in the pleural fluid. Mesothelial cell count is usually less than 5% of the pleural fluid white cell count. Raised mesothelial count above 5% is considered by many authors to argue against tuberculous pleurisy (Spriggs and Boddington 1960). However, some authors have reported mesothelial cell counts above 5% in patients with tuberculous pleurisy (Hirsch et al. 1979; Lau 1989; Santos-Santre et al. 1990). Pleural fluid protein is usually greater than 3.0 Gldl, and glucose may be decreased. Pleural fluid

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lactic dehydrogenase (LDH) is usually above 200 LU.I L. Pleural fluid adenosine deaminase (ADA) measurement has been used as a biochemical parameter in an attempt to differentiate between tuberculous and other exudative non-tuberculous pleural effusions (Valdes et al. 1996). In a prospective study from India, Sharma et al. (2001) reported the diagnostic value of ADA as a marker for tuberculous pleurisy. Seventy-five patients with exudative pleural effusion were assessed. Pleural fluid ADA, as well as serum ADA, levels were significantly higher in tuberculous pleurisy compared with non-tuberculous effusions. The authors reported the sensitivity and specificity of pleural ADA at two cut-off points, 35 lUlL and 100 lUlL. At 100 lUlL, the sensitivity of ADA was 40% and the specificity was 100%. The authors suggested that using 100 lUlL pleural ADA level may spare as many as 40% of patients from having a pleural biopsy. However, ADA level estimation is complicated by false-positive and false-negative results (Yamada et al. 2001). Maartens and Bateman (1990) from South Africa found in their prospective study of 111 patients with pleural effusion that ADA did not provide a valuable diagnostic test of pleural tuberculosis as has been suggested. In recent years, the use of pleural fluid cytokines as parameters for the diagnosis of tuberculous pleurisy has been reported (Yamada et al. 2001; Pablo et al. 1997; Xirouchaki et al. 2002). In a comparative study between raised pleural levels of ADA and cytokines, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-y), Yamada et al. reported their findings. The study involved samples of pleural fluid obtained from 21 tuberculous, 21 inflammatoryand 18 malignancy cases. The authors indicated that IFN-y was a "very reliable marker" of tuberculous pleurisy. Xirouchaki and colleagues (2002) indicated that the measurement of pleural fluid cytokines might be helpful in differentiating malignancy from tubercu10sis in exudative pleural effusion. Methods for measuring antigens and antibodies to M. tuberculosis in the pleural fluid have been reported by many authors. It has further been reported that the sensitivity of these tests are only about 50% (Murante et al.1990; Hara et al. 1992; Caminero et al.1993). See chapter on Immunological Tests for Tuberculosis. Molecular biology techniques utilizing various PCR protocols have been developed for detecting the M. tuberculosis genome in pleural and other body fluid samples, as well as in tissue biopsies. The reported sensitivity and specificity has varied from one technique to another. Querol and colleagues (1995) from Spain reported 107 patients with pleural effusion

between 1991 and 1993. Twenty-one patients had proven tuberculosis and 86 non-tuberculous (cancer, parapneumonia, cirrhosis, heart failure, emphysema, lymphoma, SLE and non-specific pleuritis). The PCR assay was based on the detection of a 123-bp DNA segment belonging to the insertion sequence IS6100, specific for M. tuberculosis. The diagnoses of twentyone tuberculous patients were confirmed by clinical, PPD skin test, pleural ADA, cytology, microbiology and histopathology of pleural biopsies. Positive pleural biopsy with granuloma formation was found in 72%, positive culture in 67% and raised ADA activity in 86%. PCR sensitivity and specificity were found to be 81% and 100%, respectively. Other authors applied PCR to detect DNA (IS 6110) in pleural biopsy specimens (Takagi et al.1998). These authors reported PCR sensitivity and specificity of 89% and 100%, respectively, of patients, which was similar to the results of Querol and colleagues. These two studies demonstrate the excellent sensitivity and specificity of PCR using either pleural fluid or pleural tissue for rapidly diagnosing pleural tuberculosis. Pleural fluid microbiological examination (smear and culture) for M. tuberculosis may detect only 25% to 50% of patients (Idell 1994). In patients coinfected with HIV, pleural fluid cultures are more often positive than in non-HIV patients. Parenchymal lung infiltrates are more severe and depicted by various imaging modalities in HIV positive patients, suggesting more mycobacterial extension from the lung into the pleural space, in contrast to HIV negative individuals (Luzze et al. 2001). In a prospective study of 111 patients with pleural effusion, and from an area with a high prevalence of tuberculosis in South Africa, radiometric cultures using BACTEC and conventional cultures were compared (Maartens and Bateman 1990). Tuberculosis was confirmed by histopathology, microbiology or both in 62 patients (56%). Positive pleural fluid culture was found in 47%, while histology and tissue culture were positive in 84% and 71%, respectively. BACTEC was faster than conventional mycobacterial culturing, and the yield appeared after 18 versus 33 days. The incidence of a positive pleural fluid culture as reported in some series is shown in Table 22.2.

22.5.3 Pleural Biopsy Closed needle biopsy of the pleura should be considered for further evaluation if clinical features, PPD skin test, imaging findings and pleural fluid

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Table 22.2. Pleural tuberculosis in some recent reports: diagnostic procedures, % yield Series Year

Fluid culture % positive

Biopsy culture % positive

Biopsy histology % positive

PPD % positive

Maartens and Bateman (l990) Ankobiah et al. (l990) Seibert et al. (1991) Chang et al. (l991) Relkin et al. (1994) Kitinya et al. (1994) Heyderman et al. (1998) Our Own Series

47 57 58 23 83.3 28 14b (smear) 23

71 45.6 66.7 40 65.5 45.6 40 65

84 80.7 84.6 97 80 100 60 80

78 60 93 77 56 74 30' 76

'Had problem with PPD reading compliance bCulture was not reported

biochemical parameters and culture do not provide a definitive diagnosis. It is an invasive procedure with significant discomfort and potential complications (Ansari and IdellI998). Good quality single pleural biopsy sampling is sufficient to diagnose pleural tuberculosis, in contrast to the increased diagnostic yield with multiple biopsies for malignancy (Jimenez et al. 2002). Positive pleural biopsy culture was found to be higher than pleural fluid (39% versus 13%) by Bueno and colleagues (1990). Histopathological findings of granuloma in pleural biopsy may be found in up to 80% of cases. PCR of pleural tissue biopsy for M. tuberculosis was found to be rapid, sensitive and specific in 89-100% of cases for diagnosing pleural tuberculosis (Takagi et al. 1998). An audit to value the routine practice of pleural fluid aspiration and biopsy for the investigation of pleural effusion has been reported by Walshe and colleagues (1992). The authors reviewed the investigative results of pleural fluid and biopsy procedures of 112 patients. They reported that the protein content of pleural fluid was of little value as a diagnostic indicator, as it overlapped substantially between various other diagnostic groups. A low positive yield for microbiological findings was of significance. Pleural biopsy was performed only in 30% in this series. The authors, however, indicated that the practical difficulties regarding pleural biopsy included the lack of experience among junior staff, lack of pleural biopsy needles and inadequate or poor quality samples. The authors reported the rate of complications (pneumothorax and emphysema) as 2% for aspiration alone and 4% for aspiration combined with biopsy. The incidence of pleural tissue biopsies, positive cultures and the presence of granulomata on histopathological examination in different series are shown in Table 22.2.

22.5.4 Thoracoscopy in Tuberculous Patients

Thoracoscopic examination was first described by Professor Hans Christian Jabobaeus from Sweden in 1910. He inserted a cystoscope into the pleural space of two patients with exudative pleuritis (Thomas 1994). He reported his observations of patients with pulmonary tuberculosis that had been treated with pneumothorax in which there was no pleuritis. He also explored the therapeutic application of thoracoscopy in breaking adhesions in patients with pulmonary tuberculosis that were selected for lung collapse therapy. He noted the complications of thoracoscopy, including haemorrhage, pleural effusion and subcutaneous emphysema. At present, the role of thoracoscopy in obtaining pleural tissue biopsies in the diagnosis of pleural tuberculosis is restricted, as a closed-needle pleural biopsy is the most reasonable method of initially obtaining a diagnostic tissue specimen.

22.5.5 Tuberculin Skin Test (PPD)

The use ofPPD skin testing is valuable in the diagnosis of patients with pleural tuberculosis. In patients with recent conversion, pleural tuberculosis will be classified as primary tuberculous pleurisy. Patients with a past history of positive PPD or active tuberculosis will be classified as secondary (Ankobiah et al. 1990). The PPD skin test is considered positive when the induration measurement is over 9 mm, and it is found in 60 to 90% of patients with pleural tuberculosis as reported in several series (See Table 22.2). A negative PPD skin test is more frequently reported in patients with tuberculous pleurisy co-infected with

Pleural Tuberculosis

HIV than in those with negative HIV. Ankobiah and colleagues (1990) from New York found positive PPD reaction in only 12% of AIDS patients and 80% in non-AIDS patients. Relkin et al. (1994) found a similar low incidence of positive PPD in their patients with tuberculous pleurisy co-infected with HIV. compared with HIV negative ones (41 versus 76%). The high frequency of negative PPD skin tests among those co-infected with HIV reflects the alternation of delayed hypersensitivity due to HIV.

355

sion (Fig.22b in chapter 23). Bronchopleural fistula may be depicted by CT or fistulograrn (see Fig. 36 in chapter 23). Fibrothorax with diffuse pleural thickening but without effusion may be seen on CT. Ultrasonography (US) may play a role in the investigation of tuberculous pleural effusions. It may help in detecting pleural thickening, nodularity and in obtaining US-guided closed-needle pleural biopsy (Chang et al. 1991; Akhan et al. 1992). The imaging features of various modalities for pleural tuberculosis are non-specific, but may help in distinguishing tuberculous from non-tuberculous pleural disease.

22.5.6 Imaging Features of Pleural Tuberculosis 22.5.7 The imaging modalities most commonly used in Treatment tuberculous pleural disease are plain chest radiography, CT and ultrasonography. Rarely, a fistulogram Treatment of pleural tuberculosis is similar to treatfor bronchopleural fistula is used when CT localiza- ment of pulmonary disease. Full assessment is essention of the tract is not clear. CT is more sensitive tial, particularly in regard to prior treatment for TB, and useful than plain radiography in the evaluation drug susceptibility or resistance. The co-infection of pleural disease. It may show unrecognized small with HIV has to be determined. Detection of comsubpleural cavities, parenchymal lung involvement, plicated pleural effusion is essential, as it may lead lymphadenitis or rib involvement (see Figs. 36a, bin to therapeutic failure if medical treatment is used chapter on "Radiology of Pulmonary Tuberculosis") alone without surgical intervention. If left untreated, (Moon et al. 1999; Yilmaz et al. 1998; Winer-Muram pleural tuberculosis will develop into active pulmonary or extra-pulmonary disease within 5 years in at and Rubin 1990; Hunlnick et al. 1983). Unilateral, moderate or large pleural effusion can least 65% of immunocompetent patients (Roper and easily be depicted by plain radiography (see Figs. 12a, Waring 1955). Uncomplicated, HIV negative tuberculous pleub, 34b in the chapter on "Radiology of Pulmonary Tuberculosis"). Bilateral small or moderate pleural risy can be treated with a short course of 6-9 months effusions may be depicted by plain radiography and of anti-tuberculous chemotherapy. CT (see Fig. 12c in chapter 23). Imaging features of Six months of anti-tuberculous chemotherapy contiguous subpleural tuberculous cavities in the lung using two drugs for pleural tuberculosis was reported parenchyma as localized empyema may be seen on by Dutt et al. (1992). The authors used isoniazid 300 mg plain radiography or CT (see Figs. 22b, 26 in chapter 23) and rifampicin 600 mg daily for one month, followed (Seibert et al. 1991). Other imaging features of lung by twice weekly treatment with isoniazid 900 mg and parenchymal disease with bronchiectasis may be rifampicin 600 mg for five more months. This regimen noted in patients with tuberculous pleural effusion used by Dutt and colleagues on 161 patients with TB (see Fig. 30b in chapter 23). Features of large empy- pleurisy and follow-up for about 4-6 months revealed ema with increased thickness of the pleura may be no relapses. In areas with high risk for HIV infection found on plain radiography and CT (see Fig.34b in or with high incidence of drug-resistant mycobacteria, chapter 23). Tuberculous pleural empyema may be initial treatment should include at least four drugs localized with thick, calcified wall better depicted on until drug susceptibility testing is available. CT than plain chest radiography (see Figs. 13, 22b, 23, Immunosuppressed patients with pleural tuber34a, 35a in chapter 23). CT may depict focal pleural culosis should be treated for a much longer period, thickness with calcifications in the absence of fluid at least 12 months. Patients with multi-drug resistant collection (see Figs. 23, 32d in chapter 23). An asso- tuberculosis should be treated with 5-6 drugs. ciated destruction of adjacent ribs with cold abscess Complicated tuberculous pleural effusion may formation and empyema may be depicted better by CT be due to the development of loculated chronic (35a, b in chapter 23). Other calcifications in the lung empyema, drug resistance or bronchopleural fistula. parenchyma or mediastinal lymph nodes may also be Medical treatment alone will not be sufficient in such depicted in association with tuberculous pleural effu- patients and surgical intervention will be required

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(Brown and Pomerantz 1995; Iseman et al. 1991). Surgical treatment is discussed in detail in a separate chapter (see chapter XX). In a recent report from India, Lahiri et al. (1998) analyzed the surgical procedures for thoracic tuberculosis in 1655 cases seen over a 20-year period. These procedures were needed in only 2.2% of the patients, including tuberculous empyema with or without broncho-pleural fistula. Intercostal drainage with irrigation, thoracostoma and chest wall tube drainage were the most frequent minor procedures. Major procedures were less frequently required and included decortication and thoracoplasty.

References Akhan 0 et al (1992) Tuberculous pleural effusions: ultrasonic diagnosis. J Clin Ultrasound 20:461-465 Aktogu S et al (1996) Clinical spectrum of pulmonary and pleural tuberculosis: a report of 4, 480 cases. Eur Respir J 9:2031-2035 Ankobiah WA, Finch P Powell S et al (1990) Pleural tuberculosis in patients with and without AIDS. J Assoc Acad Minor Phy 1:20-23 Ansari T, Idell S (1998) Management of undiagnosed persistent pleural effusions. Clin Chest Med 19:407-417 Barnes PF, Mistry SD, Cooper CL et al (1989) Compartmentalization of a CD4+ T lymphocyte subpopulation in tuberculous pleurisy. J ImmunoI142:1114-1119 Barnes PF, Fong S-J, Brennan PJ et al (1990) Local production of tumor necrosis factor and IFN-'Yin tuberculous pleurisy. J ImmunoI145:149-154 Barnes P et al (1993) Cytokine production at the site of disease in human tuberculosis. Infect Immun 61:3482-3489 Batungwanayo J et al (1993) Pleural effusion, tuberculosis and HIV-l infection in Kigali, Rwanda. AIDS 7:73-79 Berger H, Mejia E (1973) Tuberculous pleurisy. Chest 63:88-92 Boylan AM, Ruegg C, Kim KJ et al (1992) Evidence of a role for mesothelial cell derived interleukin-8 in the pathogenesis of asbestos-induced pleurisy in rabbits. J Clin Invest 89: 1257-1267 Brown J, Pomerantz M (1995) Extrapleural pneumonectomy for tuberculosis. Chest Surg Clin North Am 5:289-296 Bueno CE et al (1990) Cytologic and bacteriologic analysis of fluid and pleural biopsy specimens with Cope's needle. Arch Intern Med 150:1190-1994 Caminero JA et al (1993) Diagnosis of tuberculosis by detection of specific IgG antigen 60 in serum and pleural fluid. Respiration 60:58-62 Carrion-Valero F, Perpina-Tordera M (2001) Screening tuberculous pleural effusion by discriminant analysis. Int J Tuberc Lung Dis 5:673-679 Chang DB et al (1991) Ultrasound-guided pleural biopsy with tru-cut needle. Chest 100:132-133 Chaparas SD, Thor DE, Hedrick SR (1971) Comparison oflymphocyte transformation, inhibition of macrophage migration and skin tests using dialyzable and nondialyzable tuberculin fractions from Mycobacterium bovis (BCG). J

M. M. Madkour et al. Immunol107:149-153 Christopher DJ, Peter JV, Cherian AM (1998) Blind pleural biop~y using a tru-cut needle in moderate to large pleural effUSIOn - an experience. Singapore Med J 39:196-199 Dutt AK et al (1992) Tuberculous pleural effusion: 6-month therapy with isoniazid and rifampicin. Am Rev Respir Dis 145:1429-1432 Ellner JJ et al (1988) The immunology of tuberculous pleurisy. Semin Respir Infect 3:335-342 Epstein DM et al (1987) Tuberculosis pleural effusions. Chest 91:106-109 Gedde-Dahl T (1952) Tuberculosis infection in the light of tuberculin maticuolation. Am J Hyg 56:139-214 George RB et al (1985) Mycobacterial, fungal, actinomycotic and nocardial infection of the pleura. Clin Chest Med 6:63-75 Goodman RB, Wood RG, Martin TR et al (1992) Cytokinestimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-lIlL-8. J Immunol 148:457-465 Hara N et al (1992) Pleural SC56-9 in differential diagnosis of tuberculous, malignant and other effusions. Chest 102: 1060-1064 Heyderman RS,Makunike R,Muza T et al (1998) Pleural tuberculosis in Harare, Zimbabwe: the relationship between human immunodeficiency virus, CD4 lymphocyte count, granuloma formation and disseminated disease. Trop Med Int Health 3:14-20 Hirsch A et al (1979) Pleural effusion: laboratory test in 300 cases. Thorax 34:106-112 Hunlnick D et al (1983) Pleural tuberculosis evaluated by computed tomography. RadioI149:759-765 Idell S (1994) Evaluation of perplexing pleural effusion. Contemp Intern Med 6:31-39 Iseman MD et al (1991) Chronic tuberculous empyema with bronchopleural fistula resulting in treatment failure and progressive drug resistance. Thorax 100:124-127 Jimenez D et al (2002) Determining the optimal number of specimens to obtain with needle biopsy of the pleura. Respir Med 96:14-17 Jonjic N, Peri G, Bernasconi S et al (1992) Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med 176:1165-1174 Kitinya IN, Richter C, Perenboom R et al (1994) Influence pf HIV status on pathological changes in tuberculous pleuritis. Tuberc Lung Dis 75:195-198 Kunkel SL, Chensue SW, Strieter RM et al (1989) Cellular and molecular aspects of granulomatous inflammation. Am J Respir Cell Mol Bioi 1:439-447 Lassence AD et al (1992) Detection of mycobacterial DNA in pleural fluid from patients with tuberculous pleurisy by means of the polymerase chain reaction: comparison of two protocols. Thorax 47:265-269 Lau KY (1989) Numerous mesothelial cells in tuberculous pleural effusion. Chest 96:438-439 Lorgat F et al (1992) Evidence for in vitro generation of cytotoxic T-cells. PPD-stimulated lymphocytes from tuberculous effusion demonstrate enhanced cytotoxicity with accelerated kinetics of induction. Am Rev Respir Dis 145:418-423 Luzze H etal (2001) Evaluation ofsuspected tuberculous pleurisy: clinical and diagnostic findings in HIV-positive and HIV-negative adults in Uganda. Int J Tuberc Lung Dis 5:745-755 Maartens G, Bateman ED (1990) Tuberculous pleural effusion: increased culture yield with bedside inoculation of pleural

Pleural Tuberculosis fluid and poor diagnostic value of adenosine deaminase. Thorax 46:96-99 Maeda J, Ueki N, Ohkawa T et al (1993) Local production and localization of transforming growth factor-beta in tuberculous pleurisy. Clin Exp Immunol 92:32-38 Mbizvo MT et al (1996) HIV seroincidence and correlates of seroconversion in a cohort of male factory-workers in Harare, Zimbabwe. AIDS 10:895-901 Modilevsky T et al (1989) Mycobacterial disease in patients with human immunodeficiency virus infection. Arch Intern Med 147:2201-2205 Mohammed KA, Nasreen N, Ward MJ et al (1998) Mycobacterium-mediated chemokine expression in pleural mesothelial cells: role of C-C chemokines in tuberculous pleurisy. J Infect Dis 178:1450-1456 Moon WK et al (1999) Complicated pleural tuberculosis in children: CT evaluation. Pediatr RadioI29:153-157 Morehead RS (1998) Tuberculosis of the pleura. South Med J 91:630-636 Moudgil H et al (1994) Reactivation disease: the commonest form of tuberculous pleural effusion in Edinburgh. Respir Med 88:301-304 Murante T et al (1990) Antipurified protein-derivative antibody in tuberculous pleural effusion. Chest 97:670-673 Pablo AD et al (1997) Are pleural fluid parameters related to the development of residual pleural thickening in tuberculosis? Chest 112:1293-1297 Palmer PES (1979) Thoracic tuberculosis. Semin Roentegenol 14:204-243 Querol JM et al (1995) Rapid diagnosis of pleural tuberculosis by polymerase chain reaction. Am J Respir Crit Care Med 152:1977-1981 Relkin F,Aranda CP,Garay SM et al (1994) Pleural tuberculosis and HIV infection. Chest 105:1338-1341 Richter C et al (1991a) Extrapulmonary tuberculosis - a simple diagnosis? A retrospective study in Dar es Salam. Trop Geog Med 43:375-378 Richter C et al (1991b) Pleural effusion aetiology, diagnosis and clinical picture in HIV-positive and negative patients. A prospective study in Dar es Salam. Proceedings of the VIth international congress on AIDS in Africa, Dakar, Senegal, Dec 1991; WA.220 Richter C et al (1994) Clinical features ofHIV-seropositive and HIV-seronegative patients with tuberculous pleural effusion in Dar es Salam, Tanzania. Chest 106:1471-1476 Roper WH, Waring JJ (1955) Primary serofibrinous pleural effusion in military personnel. Am Rev Tuberc 71:616-635 Sahn SA (1988) State of the art - the pleura. Am Rev Respir Dis 138:184-234 Santos-Sastre et al (1990) Mesothelial cells and tuberculous pleuritis. Chest 98:518 Seibert AF et al (1991) Tuberculous pleural effusion. Twentyfive year experience. Chest 99:883-887

357 Sharma SK et al (2001) A prospective study of sensitivity and specificity of adenosine deaminase estimation in the diagnosis of tuberculous pleural effusion. Indian J Chest Dis Allied Sci 43:149-155 Shimokata K, Kishimoto H, Takagi E et al (1986) Determination of the T-cell subunit producing g-interferon in tuberculous pleural effusion. Microbiol Immunol 30:353-361 Shivaram I et al (1989) Pleural effusion in AIDS/ARC. Chest 96:2215 Spriggs AL, Boddington MM (1960) Absence of Mesothelial cells from tuberculous pleural effusions. Thorax 15:169-171 Stevenson F (1955) The natural history of pleural effusion and orthopaedic tuberculosis. J Bone Joint Surg Br 37:80-91 Sudre P et al (1992) Tuberculosis: a global overview of the situation today. Bull WHO 70:149-159 Takagi N et al (1998) Polymerase chain reaction of pleural biopsy specimens for rapid diagnosis of tuberculous pleuritis. Int J Tuberc Lung Dis 2:338-341 Takagi N, Hasegawa Y,Ichiyama S et al (1998) Polymerase chain reaction of pleural biopsy specimens for rapid diagnosis of tuberculous pleuritis. Int J Tuberc Lung Dis 2:338-341 Thomas PA (1994) A thoracoscopic peak: what did Jacobaeus see? Ann Thorac Surg 57:770-771 Valdes L et al (1996a) Adinosine deaminase (ADA) isoenzyme analysis in pleural effusion: diagnostic role and relevance to the origin of increased ADA in tuberculous pleurisy. Eur Respir J 9:747-751 Valdes L et al (1996b) The etiology of pleural effusion in an area with high incidence of tuberculosis. Chest 109:158-162 Wallis RS (1996) New approaches to identification of antigens of Mycobacterium tuberculosis. Infect Agents Dis 5: 119-125 Walshe AD, Douglas JG, Kerr KM et al (1992) An audit of the clinical investigation of pleural effusion. Thorax 47:734-737 Willis RS, Amiy-Tahmasseb M, Ellner JJ (1990) Induction of interleukin-l and tumor necrosis factor by mycobacterial proteins: the monocyte Western blot. Proc Nat! Acad Sci USA 87:3348-3352 Winer-Muram HT, Rubin SA (1990) Thoracic complications of tuberculosis. J Thorac Imag 5:46-63 Xirouchaki N et al (2002) Diagnostic value of interleukin-lalpha, interleukin-6 and tumor necrosis factor in pleural effusion. Chest 121:815-820 Yamada Yet al (2001) Cytokines in pleural liquid for diagnosis of tuberculous pleurisy. Respir Med 95:577-581 Yamamura Y, Onoue K, Azuma I (1968) Biology of the mycobacterioses. Chemical and immunological studies on peptides and polysaccharides from tubercle bacilli. Ann NY Acad Sci 154:88-97 Yilmaz MU et al (1998) Computed tomography findings of tuberculous pleurisy. Int J Tuberc Lung Dis 2:164-167 Zhang M, Gately MK, Wang E et al (1994) Interleukin 12 at the site of disease in tuberculosis. J Clin Invest 93: 1733-1739

23 Radiology of Pulmonary Tuberculosis MONA AL SHAHED, MOHAMMED ABD EL BAGI, M. MONIR MADKOUR

CONTENTS 23.1 23.2 23.2.1 23.2.2 23.2.3 23.2.4 23.2.5 23.3 23.3.1 23.3.1.1 23.3.1.2 23.3.1.3 23.3.1.4 23.3.2 23.3.3 23.3.3.1 23.3.3.2 23.3.3.3 23.3.3.4 23.3.3.5 23.3.3.6 23.4 23.5 23.6 23.6.1 23.6.2 23.6.3 23.6.3.1 23.6.3.2 23.6.3.3 23.6.3.4

Introduction 359 Imaging 359 Conventional Radiography 359 Computed Tomography (CT) 360 High Resolution Computed Tomography 360 Bronchography 360 Arteriography 360 Classification and Radiological Presentation 360 Primary Tuberculosis 360 Parenchymal Disease 361 Lymphadenopathy 362 Pleural Disease in Primary Tuberculosis 364 Miliary TB 365 Paradoxical Transient Worsening Phenomenon 366 Post Primary Tuberculosis 368 Parenchymal Disease 369 Cavitation 370 Endobronchial Disease 371 Tuberculoma 374 Pleural Disease in Post-primary Tuberculosis 375 Mycetoma Formation 378 Assessment of Activity of Pulmonary TB 379 Non-tuberculous Mycobacteria 379 Mycobaqerial Infection and Human Immunocompromised Virus (HIV) 379 Background 379 Mycobacterial Tuberculosis 380 Non-Tuberculous Infections 380 Mycobacterial Avium-intercellular Complex (MAl) 380 Mycobacterial Kansasii 381 Other Non-Tuberculous Infections 381 Non-Tuberculous Bacteria in Immunodeficiency States Other than HIV 382 References 382

M. AL SHAHED, MBBS, FRCR Senior Consultant Radiologist, Department of Radiology, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia M. ABD EL BAGI, MB BCh, DMRD, FSRRCSI Senior Consultant Radiologist, Department of Radiology, Riyadh Armed Forces Hospital, P.O. Box 7897, Riyadh 11159, Saudi Arabia M. M. MADKOUR, MD, DM, FRCP Consultant, Department of Medicine, Riyadh Armed Forces Hospital, P.O. Box 7897, C-119, Riyadh 11159, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

23.1

Introduction

Tuberculosis (TB) is a disease caused by infection with Mycobacterium tuberculosis and accounts for more than 95% of pulmonary mycobacterial infection. Other non-tuberculous mycobacteria, e.g. M. Kansasii, M. avium-intercellular complex and others, account for the remainder. Tuberculosis (TB) can affect virtually any organ system in the body. It is a topic of universal concern due to the recent resurgence of (TB) in both immunocompetent and immunocompromised individuals (Davis et al. 1993). Much of this increase has been ascribed to human immunocompromised virus infection (HIV), poverty and homelessness. The radiology of pulmonary (TB) reflects, and is influenced by, the immune status of the host, the virulence of the organisms, the presence of delayed hypersensitivity due to mycobacterium tuberculosis and the method of spread. Pulmonary (TB) is classically divided into primary and post primary (reactivation) disease. There is considerable overlap in the radiological manifestation of these two entities. Should primary (TB) pass into the post primary form without a break, the term progressive primary tuberculosis is used.

23.2

Imaging

23.2.1 Conventional Radiography Conventional chest radiography is the mainstay in the detection and follow up examination of patients with pulmonary TB. Good quality chest radiographs are essential and remain the first line of investigation. Normal radiographs do not exclude tuberculosis and have been reported in up to 10% of immunocompetent patients and in up to 20% of immunocompromised patients (Fitzgerald et al. 1991; Greenberg et al. 1994).

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23.2.2

23.2.4

Computed Tomography (CT)

Bronchography

Recently, conventional Computerized Tomography (CT) has been used in selected cases. It is required in many circumstances such as in the detection of cavitation and in the evaluation of the route of spread (Kuhlman et al. 1990). CT is most useful in assessing pleural disease and in patients with extensive opacification on the conventional radiographs because of the marked destruction of the lung by the disease process. CT has proven to be more sensitive than conventional radiography in detecting adenopathy, parenchymal shadowing, particularly in miliary disease, and in detecting bronchogenic dissemination. CT is used in the evaluation of complications that might occur in the process of the disease, including bronchopleural fistula formation and extension into chest wall. CT is also used to guide percutaneous intervention procedures and pre-surgical planning.

Bronchography was once the tool of choice for the detection of bronchiectasis, but it has now been replaced by HRCT.

23.2.3 High Resolution Computed Tomography High resolution CT (HRCT) is extremely useful in understanding the pathological process of the disease and route of spread; it is also useful in the evaluation of disease activity (1m et al. 1993).

23.2.5 Arteriography

Bronchial artery angiography is currently performed for therapeutic reasons in the treatment of lifethreatening haemoptysis, which is not a common presentation of bronchiectasis due to tuberculosis.

23.3 Classification and Radiological Presentation 23.3.1 Primary Tuberculosis Primary (TB) is a term used when the infected individual has not been previously exposed to M. tuberculous and lacks hypersensitivity to tuberculoprotein. Initially, primary TB was described in infants and children, in whom it remains the most common

b

a

Fig. 23.1. a Primary TB. Chest x-ray of 7 year old showing ill-defined air space consolidation (arrowheads). b CT the same patient showing dense air space consolidation with air bronchogram (arrowhead)

Radiology of Pulmonary Tuberculosis

form (Miller and Miller 1993; Lamont et al. 1986). Increasingly, however, it has been encountered in adult population, and it is now estimated to account for 23-34% of all adult cases (Buckner et al. 1990; McAdams et al. 1995; Miller and Miller 1993; Miller 1994; Woodring et al. 1986). Radiologically, primary pulmonary TB typically manifests in four major ways, singly or in aggregate: (l) parenchymal disease; (2) lymphadenopathy; (3) pleural effusion; and (4) miliary disease (Agrons et al. 1993; 1m et al. 1995; Leung et al. 1992; Palmer 1979; Stansberry 1990). Ten percent (lO%) of all cases of primary TB progress directly into the chronic form, which is indistinguishable from reactivation (Gepport and Left 1979). Moreover, the result of a chest radiograph may be normal in 15-50% of cases (McAdams et al. 1995; Miller and Miller 1993; Woodring et al.1986). 23.3.7.7

ParenchYn1alDisease By radiological examination, parenchymal shadowing is commonly manifested as homogenously dense air space consolidation with ill-defined margins, frequently segmental or lobar in distribution (Fig. 23.1a). Expansion of a consolidated lobe may occur (Fig. 23.2). The focal parenchymal lesion may be mass-like, and in adults it may be confused with neoplasia (Fig. 23.3). It is usually unilateral and single, but multilobar consolidation can be seen in up to 25% of cases (Leung et al. 1992; Woodring et al. 1986). A tendency to favor the right lung has

Fig. 23.2. Chest x-ray demonstrating dense air space consolidation with expansion of right upper lobe in 10 years old child with primary tuberculosis.

361

emerged in several series (Lamont et al. 1986; Leung et al. 1992; Weber et al. 1968). This was also noted in our patients. No consensus has been reached as to regional preference within the lung. Upper lobe predominance (Weber et al. 1968; Nagakura 1960; Joffe 1960; Choyke et al. 1983), mid and lower lobes predominance (Woodring et al. 1986; Choyke et al. 1983) and no anatomic bias (Lamont et al.1986) have each been reported. Cavitation may occur in primary TB (Fig. 23.4). The incidence varies from 10% (Choyke et al. 1983) to 30% (Woodring et al. 1986). It is more common in infants and children raised in communities in which TB has been introduced comparatively recently (Weizman et al. 1980). At Computed Tomography (CT), primary TB typically manifests as dense hemogenous air space consolidation with well-defined margins (Harishigani et al. 2000) (Fig. 23.1b). Parenchymal shadowing usually resolves with no radiological sequelae over 6-24 months. Some may be left with residual scarring or a calcified focus (Ghon focus). This is seen in up to 20% of patient (Fig. 23.5). Occasionally during the first three months of treatment, worsening of parenchymal shadowing occurs despite appropriate therapy, a phenomenon referred to as paradoxical transient worsening (see below). Single or multiple tuberculomas may develop in primary TB, but they are seen much less frequently than in post primary TB.

Fig.23.3. Chest x-ray of a 35-year old female with primary tuberculosis presenting with a focal lung lesion (arrow), right paratracheal and left hilar lymph adenopathy (arrowhead)

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b

a Fig. 23.4. a Chest x-ray showing cavitation within tuberculous consolidation in the right upper lobe. b The lesion has resolved with minimal residual scaring after the completion of antituberculous treatment

Fig.23.5. Chest x-ray showing foci of the calcification in the right perihilar region (arrowhead) a stigma or previous tuberculous infection

23.3.1.2 Lymphadenopathy

Lymphadenopathy is the hallmark of primary TB, with or without concomitant parenchymal abnormality. Age stratification qas been identified in many series (Lamont et al. 1986; Leung et al. 1992; Weber et al. 1968), with a higher prevalence of lymphadenopathy in children under 3 years of age than in

older children and adults with primary tuberculosis (McAdams et al.1995; Woodring et al.1986). It can be the only manifestation in young children (Campbell and Dyson 1977). Any lymph node group may be involved, but the patterns commonly seen are unilateral hilar, unilateral hilar plus right paratracheal or isolated right paratracheal adenopathy (Fig. 23.6). Right paratracheal nodal involvement predominates in our experience, similar to the experience of others (Amorosa et al. 1978; Rottenberg and Shaw 1996; Weber et al.1968). Bilateral adenopathy has also been described in up to 32% of cases (Leung et al. 1992); when present, it is almost always asymmetrical and can be strikingly extensive, resembling lymphoma, metastatic disease and sarcoidosis (Fig. 23.7). CT demonstrates lymphadenopathymore accuratelythan chest radiographs. Characteristically, on post-contrast studies the nodes larger than 2 cm in diameter consistently display a low density center, 40-50 HU, with enhancing peripheral rims (Harishigani et al. 2000) (Fig. 23.8), and hence citing dissimilarity to lymphoma, sarcoidosis or histoplasmosis. Lymphadenopathy usually resolves at a slower rate than parenchymal lesions. Nodal calcification occurs 6 months or more after infection and is more frequently seen than parenchymal calcification; it is encountered in up to 35% of all cases (Weber et al. 1968; Leung et al. 1992). There are significant differences in the distribution and pattern of calcification of lymph nodes in TB and sarcoidosis, which can be explained by the route of lymphatic drainage and the

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a Fig.23.6. Primary tuberculous presenting with isolated right paratracheallymphadenopathy in 12-year old male patient

b

Fig. 23.8. Post contrast CT of the chest demonstrating characteristic appearance of tuberculous lymphadenopathy with low density centre and enhancing peripheral rim (arrowheads)

Fig. 23.7. a Frontal and lateral. Chest x-ray of primary tuberculosis presenting with extensive lymphadenopathy resembling lymphoma. Note large hilar (arrow) and bilateral paratracheal (arrowhead) lymph node involvement. b Lateral x-ray of the same patient demonstrating hilar adenopathy (arrows)

caseating nature of tuberculous granulomas. When hilar node calcification is present, it is more likely to be unilateral in TB and bilateral in sarcoidosis. A focal pattern of calcification is more common in sarcoidosis, while complete nodal calcification is common in TB (Fig. 23.9). Nodal pressure and/or erosion into adjacent structures may lead to the following complications: (1) Obstruction of airways leading to over-inflation and/or segmental/lobar collapse, usually in the anterior segment of the right middle lobe; (2) Perforation into an airway, leading to Widespread bronchogenic dissemination. The healing of these

lesions, though occasionally without sequelae, often results in various combinations of bronchostenosis, bronchiectasis, parenchymal fibrosis and loss of lung volume; (3) Haematogenous spread from infected nodes, which can lead to "metastatic" lesions such as soft tissue abscesses. These "metastatic" foci may lie dormant for years before they become active, manifesting as bone, joint or renal TB; (4) Erosion of the node into the pericardium, leading to tuberculous pericarditis. Pericarditis can also be a complication of miliary TB. Involvement of the pericardium can present in the form of pericardial effusion (Fig. 23.10) or

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Fig. 23.10. Chest x-ray demonstrating cardiac enlargement due to pericardial effusion caused by tuberculous pericarditis Fig.23.9. Chest x-ray demonstrating complete lymph node calcification typical of tuberculosis (arrowhead). Note extensive cystic bronchiectasis changes in the right middle and lower lobes (arrows), some showing air fluid level (small arrows). Also node calcined granuloma in the left mid zone (short arrow)

in the form of constrictive pericarditis, with or without calcification (Fig. 23.11). Other complications of nodal involvement include erosion into oesophagus, phrenic and recurrent laryngeal nerve paresis, superior vena cava obstruction and fistula formation. 23.3.1.3 Pleural Disease in Primary Tuberculosis

Pleural effusion is fairly common, occurring in up to 25% of cases. Unlike lymphadenopathy, it is seen more in adults than in children (Weber et al. 1968; Stead et al. 1968; Derham 1956). The effusions are generally unilateral, the exception being when the disease is a complication of miliary TB. Associated pulmonary parenchymal lesions or adenopathy may be radiologically occult. Tuberculosis should be considered in any young patient with moderate size or large unilateral pleural effusion in the absence of demonstrable pulmonary disease (Fig. 23.12a, b). Pleural effusion in primary tuberculosis can be self-limiting, or it can lead to serious disease years later. The effusions are usually painless and can be very large at presentation. The majority of these effusions are reactive in nature, rather than due to direct pleural involvement

Fig. 23.11. Lateral chest x-ray showing pericardial calcification as sequelae to tuberculous pericarditis (arrowheads)

by tuberculosis. Therefore, mycobacterium bacilli are rarely isolated from culture of the pleural fluid. The diagnosis is best established by pleural biopsy. CT is more sensitive than plain radiographs in depicting associated pulmonary infiltrate and adenopathy in addition to pleural fluid (Fig. 23.12c, d). CT will also demonstrate evenly thickened parietal pleura, which will enhance when contrast material is used. Com-

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a

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d Fig. 23.12. a Chest x-ray demonstrating left sided pleural effusion with no visible parenchymal lesion or adenopathy in a young patient presenting with fever, night sweat, loss of weight and high ESR. Pleural biopsy confirms the diagnosis. b Post enhanced CT of the chest of the same patient demonstrating right sided simple effusion (arrow). c Thoracic post-enhanced CT of another patient presented with fever and weight loss showing thin bilateral pleural effusion (arrowheads) and lymph nodes (arrows). d CT scan of the same patient at lower level showing posterior mediastinal adenopathy. No parenchymal lesions were seen Bronchial aspirate was positive for tuberculosis

plete resolution is the usual sequelae to antituberculous treatment. Incompletely resolved effusions will commonly develop into secondary disease (Palmer 1979). Tuberculous empyema is a less common form of pleural disease in primary tuberculosis, resulting from discharge of mycobacterium bacilli into pleural space (Fig. 23.13). It is usually seen in post primary cases (see below). 23.3.7.4 Miliary 18

Miliary TB results from haematogenous dissemination of the organisms and is seen in both primary and reactivation tuberculosis. It is increasingly encountered in adults, particularly the immunosuppressed, in whom it has been reported in up to 13% of cases

(Lee and 1m 1995). It is associated with a very high mortality if untreated, respiratory failure being the major cause of death. The characteristic radiologiCal appearance consists of Widespread nodular shadows measuring 2-3 mm in diameter and randomly distributed throughout the parenchyma (Fig. 23.14). In approximately 50% of cases the initial radiographs appear normal. Ill-defined haze can be an early manifestation before the characteristic miliary pattern became discernable. High resolution CT (HRCT) can detect diffuse lung changes where the initial radiographic appearance is normal or undetermined (Fraser et al. 1989). Miliary TB at HRCT manifests as numerous fine, discrete nodular or reticulonodular shadows. An associated nodular appearance of the interlobular septa and vessels may also be seen (McGuinness et al. 1992) (Fig. 23.15). Miliary tuber-

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culosis differs from bronchogenic spread in its even distribution throughout the lung and its uniform size. Diseases which have a similar appearances to that of miliary TB are varicella pneumonia, sarcoidosis, histoplasmosis, metastasis, pneumoconiosis and haemosiderosis. The resolution of these nodules is

Fig. 23.13. Tuberculous empyema as a complication of pleural disease. Note the presence of irregular pleural calcification (arrowheads)

a

usually slow and complete, with no residual calcification after treatment. Radiological improvement can be seen within 3 weeks of starting therapy.

23.3.2 Paradoxical Transient Worsening Phenomenon Paradoxical transient worsening of the radiographic findings, both parenchymal and extraparenchymal, is common in the first three months following the initiation of therapy (Akira et al. 2000; Amodio et al. 1986; Bobrowitz 1980; Campbell and Dyson 1977; Lamont et al. 1986; Matthay et al. 1974; Weber et al. 1968). Paradoxical response refers to progression of the original lesions or development of new ones during apparently adequate antituberculous treatment (Bobrowitz 1980), usually regresses without a change in the initial drug regimen. As we and others have observed (Akira et al. 2000), progression often manifests as enlargement of the original parenchymal shadowing. Areas of ground glass opacities, new areas of consolidations ipsilateral or contralateral to the initial lesion and new macronodules in the ipsilateral site were also encountered; none were with cavitations (Figs. 23.16, 23.17). It is important to recognize this transient benign phenomenon to avoid unnecessary invasive procedures or changes of appropriate therapy. The mechanisms of these clinically and radiologically impressive changes remain unclear. Several hypotheses have been considered,

Fig. 23.14. a Chest x-ray of a child presenting with miliary tubeculosis. Note widespread nodular shadowing involving both lung fields. b Miliary tuberculosis in another child presenting with massive mediastinal adenopathy (arrowheads) and superadded consolidation in the right middle lobe (arrow)

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a Fig. 23.15. a CT scan of the chest demonstrating numerous fine discrete nodular shadow. Note the nodular appearance of the interlobular septa (arrows). b CT scan of same patient more caudally showing the miliary pattern of tuberculosis. Note the nodular irregular outline of the blood vessels (arrows)

b

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Fig. 23.16. a Paradoxical transient worsening: A young female patient presented with malaise, fever, weight loss and night sweats. Chest x-ray showed right paratracheal adenopathy (arrow) and ill-defined consolidation in the right perihilar region (arrowhead). Tuberculosis was diagnosed based on positive bronchial aspirates and was started on antituberculous treatment. b Chest x-ray 8 weeks after initiation of antituberculous treatment showed progression of the parenchymal lesion with development of new macronodular lesions (arrowheads). Note significant regression of the right paratracheal adenopathy. c Chest x-ray demonstrates complete resolution after completion of antituberculous treatment

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Fig.23.17. a Paradoxical transient worsening: A middle aged male patient with positive bronchial aspirate. Initial chest x-ray showed patchy infiltrates in the right lower lobe. b Six weeks after initiation of antituberculous treatment a new lesion with thick wall developed in the right upper lobe (arrowhead). Note resolution of the initial changes. c Chest x-ray 6 months after initiation of treatment showing almost complete resolution

a

b

including enhanced focal immune responses (Iwai et al. 1979; Marshall and Chambers 1988), local hypersensitivity to sudden destruction of the bacilli and tuberculoprotein (Onwubalili et al. 1986) and local hypersensitivity to drug reaction (Akira et al. 2000). Interestingly, new pulmonary lesions during therapy for extrapulmonary TB have been reported by Sze-chunhung et al. (Hung and Chang 1999). Transient paradoxical worsening should be differentiated from true worsening of tuberculous lesions, complication by bacterial and fungal infection and drug reaction. Thin-cut CT in this aspect may playa role. In a study by (Akira et al. 2000), the dominant CT findings of transient progressions were ground glass opacities and/or consolidation, whereas the dominant CT findings of true worsening of TB were macronodules and centrilobular nodules, often with cavitations. Currently, the American Thoracic Society

and Centers for Disease Control recommend a radiographic evaluation at 2-3 months after initiation of therapy (Bass et al. 1986). Parenchymal abnormalities can then be evaluated every 2-3 months until they clear, and lymphadenopathy at yearly intervals until radiographically stable (Abernathy 1989).

23.3.3 Post Primary Tuberculosis The term post-primary TB is used to describe tuberculosis in patients who have acquired tubercula protein hypersensitivity from a previous infection or BCG vaccination. Other terminology used include: reactivation, secondary, reinfection or adult tuberculosis. Postprimary disease results from reactivation of a previously dormant primary infection in 90% of

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cases, and as extension of primary TB in 10% of cases (Woodring et al. 1986). It is a disease of adolescence and adulthood characterized by its chronicity, strong site preference, cavitation and fibrosis. Radiologic features of postprimary TB can be broadly classified as lung parenchymal disease with cavitation, endobronchial airway disease, tuberculoma, pleural extension and other complications. 23.3.3.1

ParenchYlnalDisease The earliest radiological manifestation is the development of exudative lesions of patchy ill-defined segmental or subsegmental infiltrations in typical anatomic regions: apical and posterior segments of an upper lobe or a superior segment of a lower lobe in 95% of patients. This site of preference has been attributed to the oxygen tension being relatively high, and to lymphatic drainage being relatively ineffective in these areas. The lesions consist of peripheral consolidation, often patchy and frequently associated with accentuated bronchovascular markings, extending to the ipsilateral hilar region (Fig. 23.18). Widespread bronchopneumonia is usually a result of endobronchial seeding (Spencer et al. 1990). Despite this strong site preference, no portion of the lung is immune; isolated anterior and basal involvement have been reported (Woodring et al. 1986). Pulmonary TB presenting as segmental consolidation has similar radiological appearances as non-tuberculous segmental pneumonia. Park et al. (1999) have shown

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that CT findings of fluid-bronchogram, in an area of consolidation, bronchial dilatation and proximal bronchial wall thickening is significantly more prominent in tuberculous pneumonia than in nontuberculous, non obstructive pneumonia (Fig. 23.19, 23.24b). Hilar and mediastinal lymph node involvement is veryrare,being reported in only5% ofcases (Woodring et al. 1986) (Fig. 23.20). The initial exudative infiltrates will be replaced by a more sharply defined nodular pattern (acinar nodules), interspersed with reticular opacities of fibroproductive lesions (Fig. 23.21). The

Fig. 23.19. CT chest demonstrating dense consolidation with fluid-bronchogram (arrowheads), and evidence of endobronchial spread (small arrows). The posterior mass (large arrows) is due to enlarged lymph nodes

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b Fig. 23.18. a Chest x-ray of an adult female showing patchy consolidation with element of collapse in the right upper lobe. Note bilateral hilar calcification as evidence of previous tuberculous infection (arrowheads). b Chest x-ray of an adult male showing consolidation in the apical segment of both upper lobes. Note calcification of the hilar lymph nodes (arrows)

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Fig.23.20. Chest x-ray of 40 year old male presented with fever and malaise, chest x-ray showed upper lobe and lower lobe consolidation with associated paratracheal adenopathy

(arrowheads)

b

Fig.23.21. Post primary TB. Chest x-ray demonstrating noduloreticular opacities in the apical segment of the right upper lobe with element of fibrosis. Note shifting of the trachea to the ipsilateral site

majority of patients show both patterns. Marked tissue destruction may occur at this stage, with cavitation and necrosis. Healing may occur by fibrosis, resulting in cicatrization, considerable loss of lung volume, traction bronchiectasis and atelectasis. Scattered calcification may also be seen (Fig.23.22) (Lee et al. 1991). Secondarily, compensatory signs include elevation of ipsilateral hilum, over-infiltration of adjacent lung tissue and bullate formation (Figs. 23.23, 23.24). Extensive fibrosis and bullae formation with scattered

Fig. 23.22. a Post primary TB. Chest x-ray demonstrating fibrotic changes with cicatrization, deviation of trachea to the right indicating loss of lung volume in the right upper lobe (black arrowheads). Bronchial distortion is also present (white arrowhead). b CT of different patient with post primary TB showing evidence of fibrosis and thickened septi in the left upper lobe and scattered calcific densities (arrows). Patchy emphysematous changes (white arrowhead) and thickened pleura (black arrowheads). Note complete calcification of lymph nodes (large black

arrowhead)

macronodular shadows are seen in end-stage pulmonary tuberculosis (Fig. 23.25). 23.3.3.2 Cavitation Cavitation is a distinct feature of postprimary TB and is of considerable diagnostic significance, since it indicates the likelihood of activity (1m et al. 1993). It is seen in up to 40-80% of patients with postpri-

Radiology of Pulmonary Tuberculosis

Fig. 23.23. Post primary TB. Chest x-ray demonstrating fibrotic changes with considerable loss of lung volume of the right upper lobe. Note deviation of trachea to the ipsilateral site, elevation of right hilum, (small arrow) and compensatory hyperinflation of right lower lobe (large arrow). Pleural thickening and calcification (arrowheads)

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Fig. 23.24. Post primary TB chest x-ray demonstrate end stage tuberculous changes involving the right lung with extensive fibrosis, loss of lung volume, deviation of the mediastinum, bullae formation (arrowheads) and hyperinflation of the left lung

mary TB (Rottenberg and Shaw 1996). Cavities tend to occur in the areas of consolidation. They have no diagnostic features and may be single or multiple, small or large, thin or thick-walled (McAdams et al. 1995). Air fluid level may be present, but it is uncommon and usually indicates superadded infection (Fig. 23.26). Spontaneous pneumothorax may be seen (Woodring et al. 1986). A residual tuberculous cavity may lead to endobronchial spread or become a site for the development of mycetoma. Rasmussen aneurysm is a rare but a life-threatening complication of cavitary lesion involving the wall of the pulmonary artery, and may lead to massive haemoptysis if ruptured. 23.3.3.3

Endobronchial Disease Endobronchial disease is a common complication seen in up to 40% of patients with active TB. The source of bronchogenic spread is usually from an adjacent tuberculous cavity or tuberculous lymph node containing liquefied materials rich in tuberculous bacilli (Smith and Schillaci 1987; McLoud and Naidich 1992). The spread through peribronchial lymphatic channels or directly via infected sputum are less common routes (Collins et al. 1998; Rottenberg and Shaw 1996). Rarely, the airways can be involved through a haematogenous route via bronchial arteries (Auerbacho 1949; Buckner and Walker

Fig.23.25. Post primary TB. Chest x-ray showing end stage tuberculous changes with extensive fibrosis, macro noduloreticular shadows, hyperinflation and bullae formation involving both lung fields. Note scattered calcifications

(arrowheads)

1990). Bronchogenic spread can be ipsilateral or contralateral, and bilateral dissemination can also occur. (see chapter 21, Endobronchial Tuberculosis) The radiological manifestations of bronchogenic spread include multiple small nodular opacities that

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a

d

c Fig. 23.26. a Post-primary TE. Chest x-ray demonstrating a large thick wall cavity in a consolidated left upper lobe with an air fluid levels (arrow). Patchy infiltrate with macronodular shadows in the right lower lobe (small arrowheads) indicating bronchogenic spread. Note two nodular opacities in the left midzone representing tuberculous granuloma (open arrows). b CT chest of a different patient demonstrating thick-walled subpleural tuberculous cavity with air fluid level. Note localized empyema as a complication (arrowheads). c Tomography of the chest demonstrates a thin-wall tuberculous cavity (arrow). d Tuberculous abscess. Chest x-ray of a female patient with bilateral thin wall cavities demonstrating air fluid level (arrowheads)

may later become confluent (Fig. 23.27). Bronchial wall thickening, post-stenotic dilatation, lobar hyperinflation, persistent pulmonary collapse, obstructive pneumonia and mucoid impaction are other radiological features of bronchogenic disease. HRCT is more sensitive than conventional radiographs in demonstrating early endobronchial spread. The most common findings are the formation of 2-4 mm centrilobular nodules and branching linear structures ((tree-on bud" appearance), which represent caseation necrosis within and around the bronchioles. The centrilobular nodules are well defined and of high attenuation, a finding which is unusual in other

causes of bronchopneumonias (1m et al. 1993, 1995) (Fig. 23.28). Other findings include ill defined 8 mm centrilobular nodules, lobular consolidation, thickened interlobular septa and long segment narrowing with acentric wall thickening of the bronchi (1m et al. 1993; Lee et al.I993). Bronchiectasis is a common complication of endobronchial TB (Lee et al. 1991). It is defined as the irreversible dilatation of a bronchial tree. It is typically secondary to pulmonary destruction and fibrosis (traction bronchiectasis). It may also result from central bronchostenosis (Fig. 23.27c). Since the vast majority of post primary TB affects apical and

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b

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Fig. 23.27. a Endobronchial tuberculosis: chest x-ray showing multiple small nodular opacities (arrows). b Endobronchial TE. Chest x-ray showing consolidation of the left upper lobe with large cavity with irregular outline and an intracavitary mass, mycetoma (arrowheads). Multiple macronodular shadows are seen bilaterally (arrows) indicating bronchogenic spread. Note hyperinflation of the right upper lobe and the paraspinal tuberculous abscess (curved arrows). c Endobronchial tuberculosis: chest x-ray demonstrates extensive bronchiectasis and cavitation in the right upper lobe with multiple bilateral basal nodular and bronchial linear shadows of indicating bronchogenic spread (arrows)

posterior segments of the upper lobes, facilitating bronchial drainage, bronchiectasis is usually asymptomatic. Chest radiographic findings include tram line opacities (dilated thickened wall bronchi with loss of normal tapering) and ring shadows, occasionally with air-fluid levels (Fig. 23.9). Bronchography was until recently the investigation of choice for the diagnosis of bronchiectasis (Fig. 23.29). CT, particularly HRCT, has replaced bronchography in the diagnosis of bronchiectasis in the recent years (Park et al.1999), with sensitivity up to 82-97% (Munroe et al. 1990; Grenier et al. 1986). Features on HRCT include uniform bronchial dilatation extending to the lung periphery without tapering (Fig. 23.28a), bronchial wall thickening and ring shadows representing markedly dilated bronchi (Fig. 23.30). HRCT is able to demonstrate the lobular distribution of airway abnormalities in early bronchiectasis

not seen using conventional x-ray or conventional CT. Features include subpleural nodules and branching linear structures ("tree-on-bud" appearance) corresponding to impacted bronchioles (Fig. 23.28c) (Davis et al. 1993; Moon et al. 1997). Hypertrophy of bronchial arteries, secondary to bronchiectasis, is demonstrated on contrast enhanced CT. With effective treatment, most of the changes of endobronchial spread resolve over time; residual fibrosis, bronchiectasis and emphysematous changes are invariably seen. The latter is believed to result from traction by adjacent fibrosis, from paracicatricial emphysema, and from bronchial stricture (1m et al. 1993). CT after treatment will demonstrate the residual changes of fibrosis, bronchiectasis and emphysema. Lobular emphysematous changes will give rise to a mosaic pattern (Fig. 23.31) (Eber et al. 1993; Martin et al. 1986).

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Fig.23.28. a Endobronchial tuberculosis: HRCT scan showing irregular bronchial wall thickening, loss of tapering (long arrow), multiple scattered linear branching densities, tree-on-bud (small arrow), and macronodular shadows. b CT of the same patient at lower level showing widespread macronodular and linear branching shadows of endobronchial spread (arrowheads). Note solid consolidation in the left lower lobe and massive adenopathy (arrow). c CT scan of the same patient at lung bases demonstrating impaction of the peripheral bronchioles with infective material resulting in extensive linear and nodular shadows (arrowheads)

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23.3.3.4 Tuberculoma

Fig.23.29. Bronchogram demonstrating both cystic (arrowheads) and tubular (arrows) dilatation of the lower lobe bronchial tree

A tuberculoma is a focus of acid-fast bacilli that is encapsulated by connective tissue. It can be a manifestation of either primary or secondary TB, and it is believed to represent localized parenchymal disease that alternately activates and heals. Tuberculoma always carry a potential risk of cavitation and dissemination. They are seen in 3-6 % of cases, can be multiple and may be associated with satellite nodules (Leung 1999). Tuberculoma, by radiographic investigation, appears as a well-defined, round or oval masslike lesion (Fig. 23.32a). Most tuberculoma are <3 cm in size (range 0.5-5 cm) (Lee et al. 1991). The majority of these lesions remains stable for a long time; 20-30% may calcify. CT shows them as low attenuation masses that do not enhance with contrast media (Gawne-Cain and Hansell 1996) (Fig. 23.32b-d).

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a b Fig. 23.30. a CT chest showing multiple ring shadows of cystic bronchiectasis in the superior segment of the right upper lobe. b CT scan of patient presented with post-primary TB showing extensive cystic bronchiectatic changes as complication of endobronchial disease. Note fluid levels (arrows). Pleural effusion was due to cardiac failure

b Fig. 23.31. a CT scan demonstrating change of fibrosis with residual bronchiectatic changes m the left upper lobe. Note the mosaic pattern in the lung fields due to lobular emphysematous changes (arrowheads). b CT demonstrating end result of endobronchial disease. There is evidence of traction bronchiectasis (arrowheads), bullae (open arrow) and tubular bronchial dilatation (arrow). Note pleural thickening in the right apical zone. Note mosaic appearance in the left upper lobe from lobular emphysema

23.3.3.5

Pleural Disease in Post-primary Tuberculosis The pleural effusion accompanying post primary TB is usually an empyema, and it carries a worse prognosis than does effusion in primary TB. When present, the condition is invariably associated with parenchymal disease. Radiologically, it appears as lobulated pleural collections associated with pleural calcification (Figs. 23.13, 23.33). Presence of air fluid level (hydropneumothorax) usually indicates presence of

bronchopleural fistula. CT demonstrates the pleural disease to better advantage than ordinary radiography (Fig. 23.34) (Hulnick et al.1983). CT is also more sensitive in demonstrating associated parenchymal lesions and peripheral subpleural cavitary lesions, which could be the cause of empyema formation (Fig. 23.26b); CT is also more sensitive in demonstrating associated complications such as bronchopleural fistula, rib destruction and cold abscess (empyema necessitatis) (Fig. 23.35). Fistula can also be demonstrated by fistulogram (Fig. 23.36).

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Fig.23.32. a Chest x-ray of an adult showing well-defined round lesion with central lucency representing tuberculoma (arrow). Two other nodular opacities are seen in the right lower lobes representing multiple tuberculomas. b CT of the same patient in prone position showing the tuberculoma of soft tissue density and central lucency (arrow). c CT at lower level demonstrating two other tuberculomas presenting as homogenous round tissue opacities (arrowheads). d CT chest of another patient showing single tuberculoma in the left lower lobe with central focal calcification (small arrowhead) Note the fibrotic band extending to an area of focal pleural thickening (large arrowhead)

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a

Fig. 23.33. Chest x-ray demonstrates extensive pleural disease caused by previous tuberculous infection. There is considerable pleural thickening and calcification causing fibrothorax (arrowhead). Note compensatory emphysema of the right upper and middle lobe b

Fig. 23.34. a CT of the chest demonstrating tuberculous empyema (arrow) manifesting as localized collection of high density fluid associated with pleural thickening (arrowheads). b CT of the chest showing a large empyema with associated thickening of parietal pleura (large arrowheads). Note fluid bronchogram in collapsed consolidated apical segment of the left upper lobe (small arrowheads)

b

a Fig. 23.35. a CT chest showing pleural thickening (short arrows), localized collection with irregular calcification (large arrow) associated with rib destruction and localized cold abscess appearing as low density area within the intercostal muscles (arrowheads). Note localized pleural thickening with calcification on the left. b Chest x-ray of the same patient later after completion of antituberculous treatment showing resolution of the pleural pathology demonstrated on a and residual changes in the affected ribs (arrowheads)

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Fig.23.36. Fistulogram demonstrate pleuro bronchial fistula caused by tuberculous changes in the apical segment of the left upper lobe (arrowheads)

23.3.3.6 Mycetoma Formation

Pulmonary mycetomas are masses of Aspergillus hyphae matted together to form a fungus ball. The fungi colonize a pre-existing cavity in the lung, usu-

ally tuberculous in origin, but can also develop in sarcoidosis, emphysematous bullae, histoplasmosis and in cavitary bronchial carcinoma. Approximately 15% of tuberculous cavitary lesions larger than 25 mm in diameter become colonized by Aspergillus Fumigatus (Research Committee 1970). Fungal ball develops in both tuberculous cavities as well as in areas of cystic bronchiectasis. The majority of mycetomas are found in the upper lobes and superior segments of the lower lobes. The majority are asymptomatic. Haemoptysis, although uncommon, being the most important complication that may warrant surgical resection or bronchial artery embolization. When haemoptysis is a presenting symptom, differentiation between mycetoma formation, reactivation of tuberculosis and development of bronchial carcinoma becomes difficult. The radiographic findings include demonstration of a mass within a cavity. Air crescentic sign, an air rim between the wall of the cavity and the fungal ball, is highly suggestive but not specific (Fig. 23.37). The wall of the cyst is usually thickened, and an associated adjacent pleural reaction is not uncommon; osteomyelitis of the adjacent rib can also occur. Right and left lateral decubitus films may show the mobile nature of the fungal ball. Immature mycetoma is seen as an irregular spongework of soft tissue containing air spaces and obliterating the cavity (Rottenberg and Shaw 1996). HRCT aids in the diagnosis of mycetoma as well as in pre-operative assessment (Kuhlman et al. 1990).

a Fig.23.37. a Mycetoma. Chest x-ray demonstrates a large cavity in the left upper lobe with a fungal ball (arrow). (Courtesy of Dr. M. Mutairi) b Localized view of this region shows the characteristic appearance of crescentic sign (arrow). (Courtesy of Dr. M. Mutiari)

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23.4 Assessment of Activity of Pulmonary T8 Chest radiography plays an important role in the diagnosis of pulmonary TB, both assessing the activity and monitoring the response to treatment. Inactivity cannot be reliably diagnosed on a single radiograph, and serial radiographs are required. Tuberculous foci, which appear inactive over an extended period, such as extensive calcification and pulmonary fibrosis, can harbour active disease with the potential to break down under certain favorable circumstances. Features that suggest active disease are ill-defined air space shadowing, miliary nodulation and cavitation. CT is more sensitive in the detection of miliary disease, cavitation and lymphadenopathy. HRCT has been proven to playa role in the early detection of endobronchial spread and in the determination of disease activity. The presence of branching centrilobular lesions without evidence of fibrotic bronchovascular distortion are highly suggestive of active disease (1m et al. 1995). Conventional radiographs are used to follow up patients once treatment has started. Radiographic resolution of pulmonary changes can take from 6 to 24 months. Complete resolution is seen in two-thirds of patient (Lee et al. 1991). Radiographic worsening can be seen despite appropriate therapy "transient radiographic progression" and should be differentiated from true worsening (see text). In patients with HIV, assessment of disease activity is less sensitive as the possibility of secondary infection and other pulmonary complications are common and should always be considered (Barnes et al. 1991).

23.5 Non-tuberculous Mycobacteria One to three percent of mycobacteria other than M. tuberculosis have been identified as causes of pulmonary infection (Woodring and Vandiviere 1990). These include M. kansasii, M. avium-intercellular complex, M. fortuitum and M. gordonae. These are free-living sporocyte. Infection is acquired by inhalation or ingestion from the environment. Non-tuberculous mycobacterial infections have a predilection for individuals with pre-existing chronic obstructive pulmonary disease, underlying debilitating disease, a previous tuberculous infection and patients who are immunocompromised. Males are more commonly affected than females, and tuberculin skin tests are usually negative or weakly positive. Radiological fea-

tures of non-tuberculous mycobacterial infections are in general indistinguishable from post primary TB and have similar site preferences: posterior segments of the upper lobes and the superior segments of the lower lobes. Cavitation is a distinct feature, occurring in up to 96% of cases. Cavities tend to be multiple (Abelda et al. 1985; Erasmus et al. 1999). CT findings of non-tuberculous mycobacteria ranges from small to large nodular infiltrates, consolidation, cavitations and bronchiectasis (Hartman et al. 1993).

23.6 Mycobacterial Infection and Human Immunocompromised Virus (HIV) 23.6.1 Background Mycobacterial infection is an important cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIY. The association between HIV and tuberculosis has been observed since 1984 (Barnes et al. 1991; Greenberg et al. 1994). Pulmonary TB occurs in 4-21 % of patients with acquired immunocompromised syndrome (AIDS) (Davis et al.1993), and it was declared to be an AIDS-defining disease in 1993. The increased susceptibility of these patients to tuberculosis is best explained by their altered immunity function. The T-helper lymphocyte (CD 4 Tcell) and to lesser extent macrophage depletion and dysfunction process are central to the HIV disease rendering these patients particularly susceptible to tuberculous infection. Both M. tuberculosis and non-tuberculous mycobacteria have become a serious problem in patients infected with HIY. Noninvasive diagnostic methods, such as sputum stain and culture, are less effective in the immunocompromised host. Moreover, other infections and non-infectious disease processes are common in these patients and there is considerable overlap in radiological manifestation, making accurate diagnosis more difficult. Clinicians are often forced to choose between the use of invasive techniques to determine the exact cause of the pulmonary disease or an empirically chosen therapy (McLoud 1989; McLoud and Naidich 1992). The role of radiology in this setting is important and involves not only detection of abnormalities on chest radiographs or CT, but also analysis of these changes with regard to the possible diagnosis and choice of an appropriate interventional technique, e.g. percutaneous needle biopsy and aspiration. The role of radiology

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disease and in the identification and characterization of lymphadenopathy. HRCT is also helpful in demonstrating parenchymal lesions in patients whose chest radiographs are normal (Leung etal. 1996).

23.6.2 Mycobacterial Tuberculosis Reactivation is the primary mechanism of infection. Rapid progression of newly acquired infection has also been described (Small et al. 1994). Clinical and radiological features depend on the degree of cellular immune compromise, especially CD 4 T-cell count. In early stages of HIV disease (CD 4 count>200 cell/mm\ radiological findings are typical of post primary TB, especially apical cavitary disease and bronchogenic spread. Skin testing to tuberculin is usually positive at this stage. With a greater degree of immunocompromise (CD 4 count <200 cell/mm\radiographs typically show a pattern more consistent with primary TB, including hilar and mediastinal adenopathy, non-cavitary infiltrates equally distributed in both upper and lower lobes, multiple nodules, miliary disease and unilateral and bilateral pleural effusion (Figs. 23.38,23.39). However, normal radiographs have been reported in up to 15% of cases (Greenberg et al. 1994). Skin testing at this stage is positive in only 20-40% of patients, similarly bronchoalveolar lavage is positive in only 20% of cases. In contradistinction, chest radiographic abnormalities can be identified in up to 85% of cases, making accurate interpretation imperative, as therapy with antituberculous drugs are usually effective (Barnes et al.1991; Leung 1999; Greenberg et al. 1994). HRCT is helpful, especially in the evaluation of miliary

Fig.23.38. Atypical mycobacteria. Chest x-ray of immunocompromised patient presented with miliary pattern. Note the diffuse fine nodular opacities in both lung fields from MAl infection

23.6.3 Non-Tuberculous Infections 23.6.3.1 Mycobacterial Avium-intercellular Complex (MAl) The incidence of (MAl) in patients with AIDS has risen since pneumocystis prophylaxis has been widely instituted (Hoover et al. 1993). Unlike infection with M. tuberculosis, pulmonary disease is relatively uncommon in patients infected with MAL Significant radiographic abnormalities occur in approximately 5% of cases and then only late in the course of HIV disease (Horsburgh 1991). Infection with MAl typically causes disseminated extrapulmonary disease, presumably due to the fact that the main route of entry is the gastrointestinal tract. There are no distinctive pulmonary radiographic abnormalities in patients with MAl infection and AIDS; moreover, other associated opportunistic diseases are common. The radiographic pulmonary findings described in the literature range from normal chest x-rays to mediastinal adenopathy, pulmonary infiltrates, diffuse nodules, miliary spread and pleural effusion (Fig. 23.40). Localized pulmonary disease is uncommon, occurring in less than 5% of patients (Horsburgh 1991), and cavitation is rare. MAl is very resistant to antituberculous drugs, and no treat-

Fig.23.39. Post renal transplant patient presented with respiratory failure. Chest x-ray show extensive consolidation, macronodular pattern and massive mediastinal adenopathy (arrowheads). Lung biopsy confirmed MAl infection

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b

a

Fig.23.40. a Chest x-ray of an adult patient with atypical mycobacterium MAl infection demonstrating consolidation, patchy infiltration and bronchiectatic changes in the right middle lobe (straight arrow). Note cavitation (curved arrow) and pleural effusion (arrowhead). (Courtesy of Dr. M. Enani). b CT scan of the same patient showing macro-nodular opacities in the right lower lobe (arrows), consolidation in the middle lobe c pleural thickening and effusion (small arrowhead), and adenopathy (large arrowhead). (Courtesy of Dr. M. Enani). c CT scan of the same patient at higher cuts showing bronchiectatic changes in the right middle lobe and medial basal segment of the right lower lobe (arrows)

ment regimen has proven entirely satisfactory. Because of the poor outcome of treatment, prophylaxis is recommended in patients with CD4 count <50 celI/mm 3• 23.6.3.2 Mycobacterial Kansasii

M. Kansasii is second only to M. tuberculosis in the frequency with which it causes pulmonary disease. Radiographic manifestation mimics post primary tuberculosis, including upper lobe cavitary infiltrates, endobronchial lesions and lymphadenopathy (Bamberger et al.1994; Christensen et al.1978, 1981; Connolly et al. 1993). Normal chest radiographs have also been reported (Fishman et al. 1997).

c

23.6.3.3 Other Non-Tuberculous Infections

Virtually all atypical mycobacterial infections that complicate HIV infection have been caused by MAl and M. Kansasii. M. gordonae, M. fortuitum, M. chelonei, M. haemophilum, M. malmoense and M. terrae are extremely rare causes and usually manifest as disseminated disease (Fakih et al. 1996; Saubolle et al. 1996; Carbonara et al. 2000). Radiological manifestations are nonspecific, ranging from normal radiographs to focal and diffuse infiltrates. Prognosis is generally poor, as most of these organisms are resistant to antituberculous treatment.

382

M. Al Shahed et al.

a Fig. 23.41. Post enhanced CT of the chest for a 67-year-old female patient with hairy cell leukemia. a Mediastinal setting at the level of aortic arch showing multiple enlarged lymph nodes (arrowheads). b Lung window setting at the same level demonstrates ill-defined increased parenchymal densities and macronodular shadows (arrows)

23.6.3.4 Non-Tuberculous Bacteria in Immunodeficiency States Other than HIV Malignancy and immunosuppressive therapy for organ transplantation (steroid and chemotherapy) can predispose patients to infection with non-tuberculous mycobacteria. Among malignancies, there is a particular strong association with hairy cell leukemia (Fig. 23.41) (Libshitz et al. 1981; Weinstein et al. 1981). A review of the literature points to the prevalence of MAl infection in these patients and to the fact that the disease is more common than was previously thought. Pulmonary involvement, according to multiple reports, is most often a manifestation of disseminated infection rather than a primary lung disorder. Reported radiographic manifestations include normal chest radiographs, single or multiple pulmonary nodules and masses, lymphadenopathy and patchy alveolar infiltrates with or without cavitation (Albelda et al. 1985; Bamberger et al. 1994; Long et al. 1991).

References Abernathy RS (1989) Tuberculosis in children and its management. Semin Respir Infect 4:232-242 Agrons G A, Markowitz RI, Kraner SS (1993) Pulmonary tuberculosis in children. Semin RoentgenoI2:158-172 Akira M. Sakatani M, Ishikawa H (2000) Transient radiographic progression during initial treatment of pulmonary tuberculosis: CT findings. J Comput Assist Tomogr 24:426-431

Albelda SM, Kern JA, Marinelli DL, Miller WT (1985) Expanding spectrum of pulmonary disease caused by nontuberculous mycobacteria. Radiology 157:289-296 Amodio J, Abramson S, Berdon W (1986) Primary pulmonary tuberculosis in infancy: a resurgent disease in urban United States. Paediatr RadioI16:185-189 Amorosa JK, Smith PR, Cohen JR (1978) Tuberculous mediastinallymphadenitis in the adult. Radiology 126:365-368 Auerbacho (1949) Tuberculosis of trachea and major bronchi. Am Rev Tuberc 60:604-606 Bamberger DM, Driks MR, Gupta MR et al. (1994) Mycobacterium kansasii among patients infected with human immunodeficiency virus in Kansas city. Kansas city AIDS Research Consortium. Clin Infect Dis 18:395-400 Barnes PF, Bloch AB, Davidson PT, Snider DE (1991) Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med 324:1644-1650 Bass JB, Farer LS, Hopewell PC et al (1986) Treatment of tuberculosis and tuberculosis infection in adults and children. Am Rev Respir Dis 134:355-363 Bobrowitz D (1980) Reversible roentgenographic progression in the initial treatment of pulmonary tuberculosis. Am Rev Respir Dis 121:735-742 Buckner CB, Walker CW (1990) Radiologic manifestation of adult tuberculosis. J Thorac Image 5:28-37 Campbell lA, Dyson AJ (1977) Lymphnode tuberculosis: a comparison of various methods of treatment. Tuberculosis 58:171-179 Carbonara S, Tortoili E, Costa D et al (2000) Disseminated Mycobacterium terrae infection in a patient with advanced human immunodeficiency virus disease. Clin Infect Dis 30: 831-835 Christensen EE, Dietz GW, Ahn CH et al (1978) Radiographic manifestations of pulmonary mycobacterium kansasii infections. AJR 131:985-993 Christensen EE, Dietz GW, Ahn CH et al (1981) Initial roentgenographic manifestation of pulmonary M. tuberculosis, M. kansassi, M. intercellularis infections. Chest 80: 132-136

Radiology of Pulmonary Tuberculosis Choyke PL, Soskman HD, Curtis AM et al (1983) Adult onset tuberculosis. Radiology 148:357-362 Collins J, Blankenbaker D, Stern E (1998) CT patterns ofbronchiolar disease: what is tree-in-bud? AJR 171:365-370 Connolly MG Jr, Baughman RP, Dohn MN (1993) Mycobactrium kansasii presenting as an endobronchial lesions. Am Rev Respir Dis 148:1405-1407 Davis SD, Yankelevits DF, Williams T et al (1993) Pulmonary tuberculosis in immunocompromized hosts: epidemiological, clinical and radiological assessment. Semin Roentgenol 28:119-130 Derham RJ (1956) Post primary intrathoracic tuberculosis in childhood with special reference to its sequelae. Taxes J Med 52:583-585 Eber CD, Stark P, Bertozzi P (1993) Bronchiolitis obliterans on high resolution CT: a pattern of mosaic oligemia. J Comput Assist Tomogr 17:853-856 Erasmus JJ, McAdams HP, Farrell MA et al (1999) Pulmonary non-tuberculous mycobacterial infection: Radiologic manifestations. Radiographics 19:1487-1503 Fakih M, Chapalamadugu S, Ricart A, Corriere N Amsterdam D (1996) Mycobactrium malmoense bacteremia in two AIDS. J Clin MicrobioI34:731-733 Fishman JE, Schwartz DS, Sais GJ (1997) Mycobacterium kansasii pulmonary infection in patients with AIDS: spectrum of chest radiographic findings. Radiology 204:171-175 Fitzgerald JM, Gizybouski S, Allen EA (1991) The impact of human immunodeficiency virus infection on tuberculosis and its control. Chest 100:191-200 Fraser RG, Pare JAP, Pare PD et al (1989) Infectious diseases of the lung. In: Diagnosis of diseases of the chest, vol 2, 3rd edn. Saunders, Philadelphia pp 882-940 Gawne-Cain ML, Hansell DM (1996) The pattern and distribution of calcified mediastinal lymph nodes in sarcoidosis and tuberculosis: A CT study. Clinical Radio 51:263-267 Gepport EF, Left A (1979) The pathogenesis of pulmonary and miliary tuberculosis. Arch Intern Med 139:1381-1383 Greenberg SD, Frager D, Suster B, Walker S, Stavropoulos C, Rothpeal A (1994) Active pulmonary tuberculosis in patients with AIDS: spectrum of radiographic findings (including a normal appearance). Radiology 193:115-119 Grenier P, Maurice F, Musset D et al (1986) Bronhiectasis: assessment by thin section CT. Radiology 161:95-99 Harishigani M, Mcloud T, Shepard J, Ko J et al (2000) Tuberculosis from head to toe. Radiographics 20:469-470 Hartmann TE, Swensen SJ, Williams DE (1993) Mycobactrium avium-intercellulare complex: evaluation with CT. Radiology 187:23-26 Hoover DR, Saah AJ, Bacellar H et al (1993) Clinical manifestation of AIDS in the era of pneumocytis prophylaxis. N Engl J Med 329:1922-1926 Horsburgh CR Jr (1991) Mycobacterium avium complex infection in the acquired immunodeficiency syndrome. N Engl J Med 324:1332-1338 Hulnick DH, Naidich PD, McCauley DL (1983) Pleural tuberculosis evaluated by computed tomography. Radiology 1489:759-765 Hung S, Chang S (1999) New pulmonary lesions during therapy for extrapulmonary tuberculosis. Chest 116: 1794-1797 1m JG, Hoh H, Shim YS, Lee JH,Ahn J, Han MC, Noma S (1993) Pulmonary tuberculosis: CT findings early active disease and sequential change in antituberculous therapy. Radiology 186:653-660

383 1m JG, Itoh H, Han MC (1995) CT of pulmonary tuberculosis. Semin Ultrasound CT/MR 16:420-434 Iwai K, Kiao C, Tsuchiya S, Kita R, Kinoshita T (1979) Histological observation on the tuberculous lesions aggravated during rifampicin treatment (in Japanese). Kekkaku 54: 473-478 Joffe N (1960) Cavitating primary pulmonary tuberculosis in infancy. Br J Radiol 33:430-433 Kuhlman JE, Deutsch JH, Fishman EK et al (1990) CT features of thoracic mycobacterial disease. Radiographics 10: 413-431 Lamont AC, Cremin BJ, Pelleret RM (1986) Radiological patterns of pulmonary tuberculosis in paediatric age group. Pediatr RadioI16:2-7 Lee KS, 1m JG (1995) CT in adults with tuberculosis of the chest: characteristic findings and role in management. AJR 164:1361-1367 Lee KS, Kim YH, Kim WS, Hwang SH et al (1991) Endobronchial tuberculosis: CT features. J Comput Assist Tomogr 15:424-428 Lee KS, Song KS, Lim TH, Kim PN et al (1993) Adult onset pulmonary tuberculosis; findings on chest radiographs and CT scans. AJR 160:753-758 Leung AN, Millet NL, Pired PR et al (1992) Primary tuberculosis in childhood. Radiographic manifestations. Radiology 182:87-91 Leung NA (1999) Pulmonary tuberculosis: the essentials. Radiology 210:307-322 Leung NA, Brauner MW, Gamus G et al (1996) Pulmonary tuberculosis: comparison of CT findings in HIV-seropositive and HIV-seronegative patients. Radiology 198: 687-691 Libshitz HI, Shuman LS, Gresik MV (1981) Pneumonia in hairly cell leukemia Radiology 139:19-24 Long R, Maycher B,Scaicini M et al (1991) The chest roentgenogram in pulmonary tuberculosis patients seropositive for human immunodeficiency virus type 1. Chest 99:123-127 Marshall BG, Chambers MA (1988) Central nervous system tuberculosis: the paradox of host immune response. J Infect 36:3-4 Martin KW, Sagel SS, Siegel BA (1986) Mosiac oligemia stimulation pulmonary infiltrates on CT. AJR 147:670-673 Matthay RA, Neff TA, Iseman MD (1974) Tuberculous pleural effusion developing during chemotherapy for pulmonary tuberculosis. Am Rev Respir Dis 109:469-472 McAdams HP, Erasmus J, Winter JA (1995) Radiologic manifestations of pulmonary tuberculosis. Radiol Clin North Am 33:655-678 McGuinness F, Naidich DP, Jagirdar J, Leitman B, Mc Cauley DL (1992) High resolution CT findings in miliary lung disease. J Comput Assist Tomogr 16:384-390 McLoud TC (1989) Pulmonary infections in the immunocompromised host. Radiol Clin North Am 27:1059-1062 McLoud TC, Naidich DP (1992) Thoracic disease in the immunocompromised patient. Radiol Clin North Am 30: 525-554 Miller WT (1994) Tuberculosis in the 1990s. Radio Clin North Am 32:649-661 Miller WT, Miller WT (1993) Tuberculosis in the normal host: radiological findings. Semin Roentgenol 28:109-118 Moon Wk, 1m JG, Yeon KM et al (1997) Tuberculosis of the central airways: CT findings of active and fibroxic disease. AJR 169:649-653

384 Munroe NC, Cooke JC, Currie DC et al (1990) Comparison of thin section computed tomography with bronchography for identifying bronchiectatic segments in patients with chronic sputum production. Thorax 45:135-139 Nagakura Y (1960) Pathologic-anatomic studies or resected lungs of younger patients with pulmonary tuberculosis. Clin Resp Organs (Tokyo) 15:462 Onwubalili JK, Scott GM, Smith H (1986) Acute respiratory distress related to chemotherapy of advanced pulmonary tuberculosis; a study of two cases and review of the literature. Q J Med 59:599-610 Palmer PES (1979) Pulmonary tuberculosis usual and unusual radiographic presentations. Semin RoentgenoI14:204-242 Park S, Hong YK, Joo SH, Choe KO, Cho SH (1999) CT findings of pulmonary tuberculosis presenting as segmental consolidation. JComput Assist Romogr 23:736-742 Research committee of the British Thoracic and Tuberculosis Association (1970) Aspergilloma and residual tuberculous cavities: the result of survey. Tubercle 51 :227- 245 Rottenberg GT, Shaw P (1996) Radiology of pulmonary tuberculosis. Br J Hosp Med 56: 195-199 Saubolle MA, Kiehn TE, White MH, Rudinsky MF, Armstrong D (1996) Mycobacterium haemophilum: microbiology and expanding clinical and geographic spectra of disease in humans. Clin Microbiol Rev 9:435-447 Small PM, Hopewell PC, Singh SP et al. (1994) The epidemiology of tuberculosis in San Francisco. A population based

M. Al Shahed et al. survey using conventional and molecular methods. New Engl J Med 330:1703-1709 Smith LS, Schillaci RF (1987) Endobronchial tuberculosis: serial fibroptic bronchoscopy and natural history. Chest 91:644-647 Spencer D, Yagan R, Blinkhorn R, Spagnuolo PJ (1990) Anterior segment upper lobe tuberculosis in the adult. Occurrence in primary and reactivation disease. Chest 97:384-388 Stansberry SD (1990) Tuberculosis in infants and children. J Thorac Imag 5:17-27 Stead WW, Kerby GR, Schuleter DP et al (1968) The clinical spectrum of primary tuberculosis in adults: confusion with reinfection in the pathogenesis of chronic tuberculosis. Ann Intern Med 68:731-737 Weber AI, Bird KT, Janower ML (1968) Primary tuberculosis in children with particular emphasis on changes affecting the tracheobronchial tree. AJR 103:123-132 Weinstein RA, Golomb HM, Grumet G et al (1981) Hairy cell leukemia: association with disseminated atypical mycobacterial infection. Cancer 48:380 Weizman Z, Shvi Y, Ayalon A (1980) Primary cavitary tuberculosis in an infant chest 77:578-581 Woodring JH, Vandiviere HM (1990) Pulmonary disease caused by non tuberculous mycobacteria. J Thorac Imag 5:64-76 Woodring JH, Vandiviere HM, Fried AM et al (1986) Update: the radiographic feature of pulmonary tuberculosis. AJR 146:497-506

24 Pulmonary Function Test and Tuberculosis MAJDY M. IDREEs, SIRAJ

0 WALl, ABDULLA AL-AMOUDI

CONTENTS 24.1 24.1.1 24.2 24.2.1 24.2.2 24.2.3 24.2.4 24.3 24.3.1 24.3.2 24.4 24.5 24.6 24.7 24.8

Pulmonary Function Abnormalities in Tuberculosis 385 Historical Aspect 385 TB and Restrictive Lung Disease 385 Parenchymal Lung Disease 385 Pleural Disease and Fibrothorax 386 Endobronchial Tuberculosis 386 Surgical and Collapse Procedures 386 TB and Obstructive Lung Disease 387 Endobronchial Tuberculosis 387 Bronchiectasis 388 TB and Mixed Pattern of Obstructive and Restrictive Abnormality 389 TB and Respiratory Failure/Hemodynamics Preoperative Evaluation 390 Case Illustration 391 Discussion 392 References 392

389

24.1 Pulmonary Function Abnormalities in Tuberculosis 24.1.1 Historical Aspect Tuberculosis (TB) is an ancient disease. It has been found in the spines of Egyptian mummies. Drawings of persons with Potts's disease have been found on the walls of Egyptian tombs. Hippocrates (460-370 B.C.) called the disease "phthisis," and Varro (116-28 B.C.) conceived the notion that organisms too small to be seen by the naked eye might be causing the disease. M. M. IDREEs, MD, FRCP (C), FCCP Head, Pulmonary Function Laboratory, Division of Pulmonary Medicine, Department of Medicine, Riyadh Armed Forces Hospital, ClIO, P.O. Box 7897, Riyadh 11159, Saudi Arabia S. O. WAll, MD, FRCP (C), FCCP Head, Division of PulmonaryMedicine,Department of Medicine, King Khalid Hospital, National Guard, Jeddah, Saudi Arabia A. AL-AMOUDI, MD, FRCP (C) Deputy Head, Department of Medicine, King Faisal Specialist, Hospital & Research Center, Jeddah, Saudi Arabia

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

Before the availability of chemotherapy, pulmonary function assessment was frequently performed in patients with pulmonary tuberculosis. In 1846, Hutchinson suggested a correlation between the severity of the reduction in vital capacity and the extent of the parenchymal disease. There are no characteristic pulmonary function abnormalities in patients with pulmonary tuberculosis. Restrictive, obstructive or mixed pattern can present, depending on the extent, chronicity and the location of the disease (see below). However, in most patients with mild to moderate disease, the pathological abnormality is transient and complete recovery in pulmonary function tests takes place after completion of therapy. As an infection control measure, in our lab a pulmonary function test is not performed until three sputum samples have stained negative for acid-fast bacilli (AFB).

24.2 T8 and Restrictive Lung Disease Both complications and therapeutic measures for controlling tuberculosis can result in restrictive lung disease.

24.2.1 Parenchymal Lung Disease The pulmonary tissue response to the inhalation of Mycobacterium tuberculosis is characterized by hypersensitivity leading to necrosis that is surrounded by inflammatory cells. These cells form a fibrotic tissue encompassing the area involved in the inflammatory process, isolating it from the remaining lung parenchyma. Limitation of the infection to this stage has no significant effect on pulmonary function or gas exchange abnormality. Decreased static and dynamic lung volume, reduced forced expiratory volume in one second (FEV!) propor-

M. M. Idrees et aI.

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tionally to forced vital capacity (FVC) and reduction in the diffusion capacity (DLco) can result from necrotizing tuberculous pneumonitis. In massive pneumonia, severe hypoxemia and widening of the alveolar-arterial oxygen gradient (A-a 02) take place. This is related to the low V/Q ratio and the development of venous admixture. In the absence of coexisting significant airway obstructions, hypercapnia is rarely observed. Tuberculosis is an uncommon, but treatable, cause of Acute Lung Injury I Acute Respiratory Distress Syndrome ALIIARDS that might develop as a complication of tuberculous pneumonitis. The exact pathogenesis of ALII ARDS in TB is unclear. Massive release of mycobacteria into pulmonary circulation with secondary hyperimmune and inflammatory reaction leading to infiltrative and obliterative endarteritis, basal membrane thickness and vascular and endothelial leakage is the postulated mechanism. Whether partial or complete resolution of the lung function abnormalities will take place depend on the extent of scarring and fibrous tissue deposition after the acute injury. Most patients who survive the acute attack will have a mild degree of parallel reduction in both FEVI and FVC three months after hospital discharge that will continue to improve during the first year. A subset of survivors might end with permanent residual abnormality manifested by low diffusion capacity. In a group of 73 patients with cavitary disease and 27 patients with non-cavitary disease, Marcus and colleagues reported an average vital capacity of 67% and a diffusing capacity of 63% of that predicted at baseline (Marcus et al. 1963). In contrast, Malik and Martin reported an average arterial oxygen tension (Pa02) of 76 mmHg at baseline in 104 patients with tuberculosis, but the number of cavities was not specified (Malik and Martin 1969). More recently, Long and coworkers reported milder changes in pulmonary function in these patients. They prospectively studied 25 patients with pulmonary tuberculosis. Pulmonary function tests were performed at baseline, 1 and 6 month intervals. Patients with non-cavitary disease (15 cases) had virtually normal lung function, while those with cavitary disease (10 cases) had very mild restrictive changes (vital capacity and diffusing capacity averaging 88 and 85%, respectively, of that predicted) and trivial hypoxemia with Pa02 averaging 85 mmHg. In the same study, some correlation of lung function with structure (number of cavities) was found (Long et al. 1998). This may explain why patients in earlier studies had inferior values, i.e. they had more disease or pre-existing lung function abnormalities.

24.2.2 Pleural Disease and Fibrothorax Pleural involvement is very common in tuberculous infection. It usually occurs through direct extension from a subpleural focus. Tuberculous pleural effusion and residual pleural thickness may occur in up to 50% of patients (Barbas et al.I991). The exact pathogenesis behind the development of pleural thickness is probably related to a delayed hypersensitivity reaction, rather than to an inflammatory response to the infection. The role of corticosteroids in reducing the risk of pleural thickness is controversial. Recent reports suggest no beneficial effect (Lee et al. 1988; Senderovitz and Viskum 1994). The coexistence of empyema thoracis may complicate the clinical picture, leading to grossly thickened pleura with loculated pleural fluid. Once extensive, this process can result in trapped lung syndrome and a variable degree of volume loss leading to significant restrictive lung disease. This is manifested by low TLC and a proportional reduction in both FEVI and FVC. DLco may also be reduced, resulting in a variable degree of gas exchange abnormalities. Shunting and venous admixture may take place at the atelectatic lobes, leading to a significant degree of widening of A-a 02 gradient and hypoxemia.

24.2.3 Endobronchial Tuberculosis Endobronchial tuberculosis will be discussed in detail as a cause of obstructive lung disease (see below). It should be noted, however, that a restrictive pattern is not uncommon in patients with endobronchial TB. Post obstructive pneumonitis, atelectasis or coexisting parenchymal or pleural disease are likely causes for restrictive lung disease in this patient population.

24.2.4 Surgical and Collapse Procedures The need for collapse procedures such as thoracoplasty, pneumothorax, pneumoperitoneum and phrenic nerve crush has been virtually eliminated by the great success of anti-TB chemotherapy. However, excisional surgery can still be called upon to deal with complications, such as bronchopleural fistula, persistent empyema, significant hemoptysis from bronchiectasis or aspergilloma or excision of a lobe

Pulmonary Function Test and Tuberculosis

or segment infected by multi-drug resistant organisms. These "volume reduction" procedures result in a variable degree of restriction. In addition, a mixed obstructive-restrictive pattern has been observed. The obstructive element in these patients may be related to the development of compensatory hyperinflation of the lung postoperatively. It has also been suggested that airway obstruction may be related to the alteration of the bronchial caliber, coexisting cigarette smoking or endobronchial location of specific inflammation (Marino and Giuliano 1998). DLco may be normal when compared to residual alveolar volume. The net effect on gas exchange and pulmonary circulation hemodynamics depend upon the integrity of the residual pulmonary tissues.

24.3 18 and Obstructive Lung Disease Obstructive airway disease came to be recognized as a complication of advanced pulmonary tuberculosis within a few years after the advent of effective chemotherapy.Anno and Tomaschefskyin 1955 reported pulmonary function studies in 25 selected patients with proven pulmonary tuberculosis. They demonstrated that airway obstruction and pulmonary hyperinflation were more common in far advanced than in less advanced disease (Anno and Tomaschefsky 1955). Hallet and Martin (1961) reported on 710 patients with pulmonary tuberculosis admitted to the Firland Sanitarium. The obstructive pattern was found in 34% of these patients and occurred more frequently in men than in women, although the difference was not statistically significant. The incidence of the obstructive abnormality increased with advanced age. The duration of smoking appeared to influence the incidence of obstructive disease; however, when the age factor was controlled, the smoking relationship was no longer significant (Hallet and Martin 1961; Martin and Hallet 1961). The failure to show a very strong association between smoking and airway obstruction in patients with pulmonary tuberculosis is an interesting observation that is difficult to explain. It may be related to the predominant effect of tuberculosis on the airways and the subsequent damage, irrespective of the smoking status of the patient. However, current data suggest an additive effect of smoking with tuberculosis in producing airway obstruction. The mechanisms of airway obstruction in pulmonary tuberculosis remain largely speculative.

387

Obstructive lung disease might be due to tuberculous involvement of the bronchial tree. Sub-mucosal tuberculous involvement and nonspecific inflammation are frequently observed in resected specimens of bronchi from tuberculous patients (Thompson and Kent 1958). Bronchial obstruction and dilatation can complicate lymph node involvement of the primary infection. In chronic post primary tuberculosis, severe bronchiectasis may result from the involvement of the tuberculous lesion in specific segments of the bronchial tree (see below). Chronic bronchitis, emphysema and bronchial asthma may develop as purely coincidental phenomena, especially in patients with atopy or a history of heavy smoking. The specific pathological entities involved in pulmonary tuberculosis presenting with obstructive pulmonary insufficiency will be discussed in detail below.

24.3.1 Endobronchial Tuberculosis Endobronchial tuberculosis (EBTB) is most often a complication of primary pulmonary tuberculosis in children, although it may also occur in adults. The prevalence of EBTB varies among studies, ranging from 10-50%, probably reflecting the heterogeneity of the studies' population and design. In the authors' experience of 98 adults with proven pulmonary TB, 12 (12%) were found to have endobronchial disease (unpublished data); 10 (10%) of them had submucosal infiltration and hyperemia and 2 (2%) had a polypoid tumor. Bronchoscopically, EBTB may present as an endobronchial mass lesion (either polypoid or ulcerative granuloma), submucosal infiltration, hyperemia with edema and fibrostenosis. In one study, 50 EBTB cases were diagnosed by bronchial biopsy, bronchial fine needle aspiration and washing. Mass lesions were found in 31 cases (62%),21 of which were ulcerous granulomas and 10 polypoid masses, while 11 cases (22%) were of submucosal infiltrative, 16 (32%) fibrostenosis and 5 (10%) hyperemia and edema (Altin et al.1997). The pathogenesis of EBTB is not yet completely understood, and the course of EBTB differs according to the type. The pathogenesis, presentation, treatment and prognosis of EBTB are discussed in detail in the chapter on Endobronchial TB. The prognosis of the actively caseating and edematous-hyperemic types is poor, resulting in fibrostenosis in two-thirds of patients. Granular and non-specific bronchitic types have a more favorable prognosis (Shim 1996).

388

Clinically,cough, mostly nonproductive,is invariably present and usually persistent. General symptoms, such as fever, anorexia and weight loss, usually predominate over specific pulmonary symptoms. However, dyspnea, wheeze and strider may be the prominent features and mimic other conditions such as asthma. A variety of lung function abnormality patterns are recognized by PFT in patients with EBTB. They include: iP Pure, restrictive defect as manifested by a proportional reduction of FVC and FEV1, with normal FEVlIFVC % ratio. (Post obstructive pneumonitis, atelectasis or coexisting parenchymal or pleural disease are likely causes for restrictive lung disease in EBTB.) lIIil Pure obstructive pattern defined as FEV1/FVC % ratio is less than 70%, with a low FEVI value. (This is usually caused by the generalized spread of the disease endobronchially to large and small airways.) (I Possible increased airway hyperresponsiveness. One study, however, looked at this particular issue by comparing the provocation concentrations of histamine required to reduce FEVI by 20% (PC 20) of the pre-challenge baseline between patients with EBTB, symptomatic asthmatics and normal healthy controls. PC 20 in EBTB patients and normal controls was significantly higher than in asthmatics. Furthermore, PC 20 was not affected by disease location within the bronchial tree and did not correlate with FEVI or FVC (Park et al. 1995). .. Abnormal inspiratory curve of the flow volume loop, indicating a variable upper airway (Le. extrathoracic) obstruction (Fig. 24.1) leading to inspiratory flow reduction. This might occur secondary to the involvement of the extrathoracic upper airway, typically found in extensive laryngeal tuberculosis (LTB). LTB can result from the direct spread of contaminated sputum to the larynx, or it can be a secondary manifestation of hematological spread. .. Fixed large airway obstruction, characterized by equally reduced inspiratory and expiratory flow (Fig.24.2). This can be a direct result of large airway stenosis secondary to EBTB. Hoheisel and coworkers found that 7% of their patients had a total occlusion of their airways secondary to EBTB, 13% had less than total, 13% had significant,45% minor and 22% had no residual stenosis (Hoheisel et al. 1994).

M. M. Idrees et al. FIoWI~J

FNex

6

8

10

Fig. 24. I. FIV loop: variable extrathoracic obstruction

AawIVa)

FNex

10

5 10 FN~

Fig. 24.2. FIV loop: fixed obstruction

24.3.2

Bronchiectasis

The pathogenesis of bronchiectasis in tuberculosis is multifactorial. Bronchial stenosis secondary to EBTB can result in post obstructive pneumonitis with a secondary bacterial infection. Exogenous compression of the bronchial lumen by enlarged lymph node produces results similar to endobronchial obstruction. In both situations, the inflammatory destruction of the bronchial lumen and the subsequent dilatation of the bronchi is mainly caused by a mixed bacterial infection and purulent bacterial secretion, with an exaggerated local immune reaction, rather than by the local spread of M. tuberculosis. Pulmonary function abnormalities in this situation can show pure obstruction, pure restriction or a combination of both abnormalities, depending on the type and extent of the involvement and the degree of residual damage. In extensive disease, both DLco and lung compliance are reduced, indicating a significant degree of gas exchange abnormality, with venous admixture and increased lung stiffness. The

Pulmonary Function Test and Tuberculosis

A-a 02 gradient is widened, and, at a more advanced stage, chronic hypoxemia and respiratory failure can develop. Chronic corpulmonale may develop secondarily to a persistent increase in pulmonary artery pressure. The physical destruction of the pulmonary vessels by the inflammatory process may play an important role in the development of pulmonary hypertension, but this is unlikely to be the major cause. Hypoxemia, active immune modulators and cytokines release are more likely to be the most important factors in the development of full-blown pulmonary hypertension and corpulmonale.

389

13 patients with pulmonary tuberculosis requiring mechanical ventilation. Seven patients had miliary tuberculosis and six had tuberculosis pneumonia. Eight had ARDS and another two had probable ARDS. The mortality rate of these patients was 69%, which is almost twice that of the hospital mortality for non-tuberculosis pneumonia requiring mechanical ventilation (36%) (Penner et al. 1995). Circulatory disturbance characterized by septic shock is a rare syndrome which has been reported in patients with disseminated tuberculosis (Gachot et al. 1990; Piqueras et al. 1987; Sandoval et al. 1991). In recent years, the occurrence of extra-pulmonary and disseminated tuberculosis has been increasing, especially among HIV patients and intravenous drug 24.4 users, and the incidence of septic shock complicating T8 and Mixed Pattern of Obstructive disseminated tuberculosis is expected to be on the and Restrictive Abnormality rise as well. Pulmonary hypertension (PHT) is a known sequela In miliary TB, the reported physiological changes are of complicated pulmonary tuberculosis. The exact of an obstructive-restrictive pattern. In a group of 33 pathophysiology for PHT is not clear. However, various patients with miliary TB, pulmonary function test- mechanisms have been attributed to the development ing was performed in 31 patients and arterial blood of PHT and corpulmonale in tuberculosis, including gases were analyzed in 13 patients (Shama et al.1992). chronic hypoxia and a reduced pulmonary capillary The physiologic abnormalities observed suggested a bed, caused either by the disease itself or as a result mild to moderate restrictive defect (vital capacity of surgical therapy (Ershov et al. 1996). Sandoval and of 56% of that predicted, ±14), with impairment of colleagues studied the hemodynamic behavior of pulpulmonary diffusing capacity (68±2l) and arterial monary circulation in both anatomical and functional hypoxemia (Pa02 of 75 mmHg ±12), and widening settings. Six patients with miliary tuberculosis were of the A-a 02 gradient. In addition, there was a mild studied. Cardiac catheterization, with subsequent reduction in the flow rates, based on reduced mid- hemodynamic measurements, was performed at rest expiratory flow and increased residual volume-to- and during exercise. The mean values of the baseline total lung capacity. This suggests peripheral airway pulmonary function testing, expressed as percentage involvement and subsequent gas trapping. These of the predicted value, showed a vital capacity of 62%, changes are consistent with the diagnosis of inter- FEVI of 50%, FEVlIFVC 76% and TLC of 79.5%. The stitial lung disease and peripheral airway dysfunc- resting pulmonary artery pressure, cardiac index, tion. With treatment, however, there was significant pulmonary vascular resistance and the PAd-PWP improvement in gas exchange (mean Pa02 increased (pulmonary diastolic minus pulmonary wedge pressignificantly, P value of <0.03), but no change in pul- sure) were all within normal limits. As a group, the monary function abnormality (Shama et al. 1992). hemodynamic response to exercise was characterized Other pathological entities causing mixed obstruc- by an increase in both the mean pulmonary artery tive-restrictive abnormalities are EBTB and post pressure and cardiac index, while pulmonary vascular resectional surgeries (see above). resistance remained within the normal range. At rest, hypoxemia (Pa02 59±3 mmHg) without hypercapnia and normal pH were found in the group as a whole. The alveolar arterial oxygen gradient and the dead 24.5 space (VDIVT) ratio increased. During exercise, no T8 and Respiratory Failure/Hemodynamics significant changes occurred in the gas exchange variables, except for oxygen consumption, respiratory rate Tuberculosis is an uncommon primary cause of and minute ventilation (Sandoval et al.199l). respiratory failure requiring mechanical ventilaThe correlation between survival and pulmonary tion. In a retrospective study that extended over a hypertension in patients with sequelae of pulmonary lO-year period, Penner and colleagues reported only tuberculosis was also studied. Twenty-one patients

390

with a past history of treated pulmonary tuberculosis and a mean Pa02 of 59 mmHg and PaC02 of 51 mmHg were included in the study. Spirometry showed a mean vital capacity of 44% of that predicted and a FEVI of 66%. Twenty patients had an mPAP greater than 30 mmHg, and 18 received home oxygen therapy. Among those patients who required home oxygen therapy, blood gases and pulmonary hemodynamics did not vary significantly between those who died within 2 years (short-term survivors) and those who lived for more than 5 years (long-term survivors). However, lung function parameters (FVC and FEV!) were significantly lower in the short-term vs.long-term survivors, suggesting that the principle prognostic determinant for long-term survival in patients with post tuberculosis sequelae is the degree of restrictive pulmonary abnormality, and not PHT or the severity of gas exchange abnormalities (Sasaki et a1.1998).

24.6 Preoperative Evaluation Preoperative pulmonary function testing emerged in the 1950s as an appealing approach for predicting complications after thoracotomy. The goals of preoperative evaluation are to: • Identify patients at risk • Assess the degree of risk for post operative complications • Identify factors associated with increased risk • Keep both morbidity and mortality low In a retrospective analysis of 454 patients who underwent thoracic surgery, age (greater than 65 years), obesity and hypocapnia (PaC02 less than 31 mm Hg) were found to be associated with increased morbidity and mortality (Beaufils et al. 1992). In patients undergoing lung resection for pulmonary tuberculosis, mortality was as high as 40% among those with maximum voluntary ventilation (MVV) less than 50% of the predicted and FVC less than 70%. Other parameters associated with poor outcome included age older than 70 and FEVI less than 21 (Gaensler et aI.1995). More recent studies confirmed the correlation between MVV and poor postoperative outcome. Olsen and coworkers (1975) reported that 5 out of

M. M. Idrees et aI.

6 patients deemed inoperable by pulmonary artery pressure and perfusion scan-predicted FEVI measurement had MVV less than 50% of the predicted outcome, whereas only 2 out of 23 patients who were deemed operable had low MVV. FEV1 is also frequently measured to assess the risk of resection surgery for multi-drug resistant tuberculosis or other indications, such as lung cancer. Preoperative FEVI has been used in conjunction with perfusion lung scan to estimate postresectional FEVl. Based on data extrapolated from COPD patients, estimated postoperative FEVI (FEVI-PPO) less than 0.8 I, or 30% of the predicted, has been considered the threshold below which resection is considered prohibitive. When this criterion was used to determine operability in high-risk lung resection patients, denying surgery to patients with an FEVI-PPO less than 0.81 defined a group having a 20% postoperative mortality rate (Olsen et aI, 1975). More recently, Melendez and Fischer divided the preoperative tests into three predictive value groups: lljl Spirometry, blood gas analysis and stair climbing tolerance are helpful for screening but not very discriminating. This group had a poor outcome predictive value. " An intermediate predictive value was achieved using diffusion capacity, exercise-induced decrease in oxygen saturation and exercise pulmonary vascular resistance. G A high predictive value was achieved by the full cardiopulmonary exercise test for the measurement of the oxygen consumption (V02) at 40 watts of exercise, or V02 max (Melendez and Fischer 1997). Richter and coworkers also evaluated the best predictor of postoperative morbidity and mortality for thoracic resection surgery. In all, they prospectively evaluated 97 patients. All patients had preoperative maximal exercise testing and dynamic spirometry. Logistic regression showed maximum preoperative workload to be the only predictor of cardiopulmonary complications. Maximum oxygen consumption (V02 max) was predictive of cardiopulmonary death. V02 max of less than 50% of the predicted value was associated with a high risk of death from cardiopulmonary causes. However, both FEV 1 and V02 max were predictive of postoperative complications. Finally, V02 max was correlated with long-term survival, while spirometric variables were not (Richter et al. 1997).

Pulmonary Function Test and Tuberculosis

391

Recently, the role of cardiopulmonary exercise testing in the preoperative evaluation for thoracic resection surgery has become the subject of great interest. It has been recommended that patients with FEV1-PPO of less than 40% of predicted values should be exercised to assess oxygen transport and consumption. Patients with FEVI-PPO greater than 30% of predicted values and peak exercise V02 greater than 15 mllkg/min should be offered surgery with the goal of resecting the smallest volume of tissue that would be compatible with a cure (Gilbreth and Weisman 1994).

monary illness and no contact with sick people. She has no pets, nor had she recently traveled. She was given a three-day course of azithromycin by her GP with no significant response. Clinically, she looked ill and mildly distressed. Temperature was 37.8 0c. She had very audible wheezes all over the lung fields. Chest-x-ray showed mild degree hyperinflation, but was otherwise unremarkable.

24.7 Case Illustration N.S.S is a 54-year-old woman, with no previous illness aside from mild rhinitis. She presented with a 3-week history of low-grade fever, cough, shortness of breath and wheeze. She is a non-smoker and had no family history of atopy. There was no occupational risk factor for pul-

FNa

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Arterial blood gases on room air were: iii

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24.8 Discussion Pulmonary function testing showed a moderate degree of obstruction. The borderline FEVl/FVC ratio was related to incomplete exhalation and underestimation of FVC. Total lung capacity was normal, but both residual volume (RV) and RV% TLC were high, indicating gas trapping. There was a significant response to bronchodilator. Arterial blood gases showed a mild degree of hypoxemia and widening of the A-a 02 gradient. The patient was treated initially as a case of bronchial asthma/bronchitis. Steroids, bronchodilator and antibiotics were started with partial initial response followed by deterioration. Three sputum samples for AFB were negative. Bronchoscopy was done and an endobronchial mass lesion in the left main stem bronchus was found. This was associated with generalized airway hyperemia. Anti-TB chemotherapy was started and the patient subsequently improved.

References Altin S, Cikrikcioglu S, Morgul M et al (1997) 50 endobronchial tuberculosis cases based on bronchoscopic diagnosis. Respiration 64:162-164 Anno H, Tomaschefsky JF (1955) Studies on the impairment of respiratory function in pulmonary tuberculosis. Am Rev Tuberc 71:333 Barbas CSV, Cukier A, de Varvalho CRR et al (1991) The relationship between pleural fluid finding and the development thickening in patients with pleural tuberculosis. Chest 100:1264-1267 Beaufils LC, Brachet A, Manuelian M et al (1992) Preoperative evaluation of respiratory function in thoracic surgery. Do reliable predictive criteria exists in that type of surgery. Agressologie 33:15

M. M. Idrees et al. Ershov AL, Evstaf'ev IuA, Sobkin AL et al (1996) The significance of exacerbations of lung diseases in the development of chronic corpulmonale and their treatment. Probl Tuberk 4:14-16

Richter LK, Svendsen UG, Milman N et al (1997) Exercise testing in the preoperative evaluation of patients with bronchogenic carcinoma. Eur Respir J 10:1559 Gachot B, Wolff M, Clair B (1990) Severe tuberculosis in patients with human immunodeficiency virus infection. Intensive Care Med 16:491-493 Gaensler EA, Cugell DW, Lindgren I et al (1995) The role of pulmonary insufficiency in mortality and invalidism following surgery for pulmonary tuberculosis. J Thorac Cardiovasc Surg 29:163 Gilbreth EM, Weisman 1M (1994) Role of exercise stress testing in preoperative evaluation of patients for lung resection. Clin Chest Med 15:389 Hallet WY, Martin q (1961) The diffuse obstructive pulmonary syndrome in a tuberculosis sanatorium. Etiological factors. Ann Intern Med 54:1146 Hoheisel G, Chan BK, Chan CH et al (1994) Endobronchial tuberculosis: diagnostic features and therapeutic outcome. Respir Med 88:593-597 Lee CH, Wang WJ, Lan RS et al (1988) Corticosteroids in the treatment of tuberculous pleurisy: a double blind, placebocontrolled, randomized study. Chest 94:1256-1259 Long R, Maycher B, Dhar A et al (1998) Pulmonary tuberculosis treated with directly observed therapy. Chest 113: 933-943

Malik SK, Martin q (1969) Tuberculosis, corticosteroid therapy, and pulmonary function. Am Rev Respir Dis 100:13-18 Marcus H, Yoo OH,Akyol T et al (1963) A randomized study of the effect of corticosteroid therapy on healing of pulmonary tuberculosis as judged by clinical, roentgenographic and physiologic measurements. Am Rev Respir Dis 88:55-64 Marino de Rosa, Giuliano C (1998) Respiratory function impairment in pulmonary tuberculosis. Rays 23:87-92 Martin q, Hallet WY (1961) The diffuse obstructive pulmonary syndrome in a tuberculosis sanatorium. Incidence and symptoms. Ann Intern Med (Chic) 54:1156 Melendez JA, Fischer ME (1997) Preoperative pulmonary evaluation of the thoracic surgical patient. Chest Surg Clin North Am 7:641 Olsen GN, BlockAJ, Swenson EW et al (1975) Pulmonary function evaluation of the lung resection candidate: a prospective study. Am Rev Respir Dis 111:379 Park CS, Kim KU, Lee SM et al (1995) Bronchial hyperreactivity in patients with endobronchial tuberculosis. Respir Med 89:419-422 Penner C, Robert D, Kunimoto D et al (1995) Tuberculosis as a primary cause of respiratory failure requiring mechanical ventilation. AJRCCM 151:867-872 Piqueras AR, Marrueces L, Artugas A (1987) Miliary tuberculosis and adult respiratory distress syndrome. Intensive Care Med 13:175-182 Sandoval J, Cicero R, Seoane M et al (199l) Behavior of the pulmonary circulation at rest and during exercise in miliary tuberculosis. Chest 99:152-154 Sasaki Y, Yamagishi F, Suzuki K et al (1998) Survival and

Pulmonary Function Test and Tuberculosis pulmonary hemodynamics in patients with sequelae of pulmonary tuberculosis who received anti-tuberculosis chemotherapy and home oxygen therapy. Nihon Kokyuki Gakkai Zasshi 36:934-938 Senderovitz T, Viskum K (1994) Corticosteroids and tuberculosis. Respir Med 88:561-565 Shama SK, Pande JN, Singh YN et al (1992) Pulmonary func-

393

tion and immunologic abnormalities in miliary tuberculosis. Am Rev Respir Dis 145:1167-1171 Shim YS (1996) Endobronchial tuberculosis. Respirology 1: 95-106 Thompson JR, Kent G (1958) Occult tuberculous endobronchitis in surgically resected lung specimens. Am Rev Tuberc 77:931

25 Thoracic Surgery for Tuberculosis YUJI SHIRAISHI

CONTENTS 25.1 History of Thoracic Surgery for Tuberculosis 395 25.1.1 Role of Surgery in the Pre-Rifampin Era 395 25.1.2 Post-Rifampin Era and Emergence of Drug-resistant Tuberculosis 396 25.2 Surgery for Multidrug-Resistant Pulmonary Tuberculosis 396 25.2.1 Background 396 25.2.2 Preoperative Medical Treatment 396 25.2.3 Indications for Surgery 399 25.2.4 Preoperative Evaluation 399 25.2.5 Surgical Technique 399 25.2.6 Postoperative Care 400 25.2.7 Types of Operative Procedures 400 25.2.8 Operative and Late Mortality 401 25.2.9 Postoperative Complications 401 25.2.10 Cure Rate of Surgical Treatment 402 25.2.11 Surgical Treatment Other than Pulmonary Resection 402 25.3 Surgery for Tuberculous Infection of the Pleural Space 403 25.3.1 Clinical Manifestations and Diagnosis 403 25.3.2 Medical Therapy 403 25.3.3 Surgical Treatment 405 References 407

25.1

History of Thoracic Surgery for Tuberculosis

25.1.1 Role of Surgery in the Pre-Rifampin Era Thoracic surgery began when the specialty was "chest" surgery for treating tuberculosis. Our hospital was established as a sanatorium owned by the Japan Anti-Tuberculosis Association. The Section of Chest Surgery began to operate on patients with pulmonary tuberculosis in 1948. At first, most patients underY. SHIRAISHI, MD Head, Section of Chest Surgery, Fukujuji Hospital, 3-1-24 Matsuyama Kiyose, Tokyo 204-8522, Japan

M. Monir Madkour et al. (eds.), Tuberculosis © Springer-Verlag Berlin Heidelberg 2004

went thoracoplasty. However, pulmonary resection, such as lobectomy, pneumonectomy or segmentectomy, soon became the predominant operation. The number of pulmonary resections performed annually reached around 240 in 1954. On the other hand, the number of cases treated with collapse therapy decreased (Fig. 25.1). Until the advent of antituberculous chemotherapy in the 1940s, pulmonary resection frequently resulted in high mortality because of bronchial failure or poor wound healing due to uncontrolled tuberculous infection. Therefore, rather than resectional surgery, collapse therapy, especially thoracoplasty, was the primary operation for the management of tuberculosis (Pomerantz and Mault 2000). The concept behind collapse therapy involves compressing the cavities, which prevents oxygen from entering them, causing aerobic tubercle bacilli to die from suffocation (Pomerantz and Brown 1995). Other examples of collapse therapy, which are less frequently performed, include plombage, therapeutic pneumothorax, pneumoperitoneum and phrenic nerve crush. Under the auspices of adequate chemotherapy, pulmonary resection became safer and thus was performed more frequently in the 1950s and 1960s. Collapse therapy eventually yielded to pulmonary resection. However, with the introduction of rifampin, almost all patients with tuberculosis could be successfully treated with medication alone. Even the removal of cavities persisting after medical treatment proved unnecessary. The need for surgery, therefore, markedly diminished and sanatoriums in many countries were closed down. In our hospital, the number of operations performed for tuberculosis decreased year by year. Eventually, fewer than 20 operations were performed for tuberculosis in 1975 (Fig. 25.1). In the late 1970s and early 1980s, indications for the surgical treatment of tuberculosis were limited to the following: massive hemoptysis, bronchopleural fistula, bronchial stenosis, a trapped lung and the need to rule out cancer (Pomerantz and Mault 2000).

396

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(van Leuvan et al. 1997), South Korea (Sung et al. 1999), Turkey (Kir et al. 1997), and the United States (Treasure and Seaworth 1995; Pomerantz et al. 2001). Exceptions are France (Mouroux et al. 1996; Souilamas et al. 2001) and Hungary (Furak et al. 2001), where surgery for tuberculosis is seldom performed for multidrug-resistant strains.

-----"---------"'-------'-----'---.::....::...---" 1955

1960

1965

1970

1975

Year - - Pulmonary resection - ..-. Thoracoplasty

Fig. 25.1. The number of operations performed for pulmonary tuberculosis annually at Fukujuji Hospital between 1948 and 1975. At first, the number of thoracoplasty was higher than that of pulmonary resection, but soon the situation was reversed. Pulmonary resections were most frequently performed in the late 1950s and early 1960s

25.1.2 Post-Rifampin Era and Emergence of Drug-resistant Tuberculosis Unfortunately, during the last five decades of the chemotherapy era, certain strains of Mycobacterium tuberculosis have acquired resistance to various antituberculous drugs. Particularly, multidrug-resistant strains, which are defined as resistant to isoniazid and rifampin, have emerged. These organisms are often resistant to other first-line drugs (ethambutol, streptomycin, and pyrazinamide) and also to some of the other less efficacious drugs. The emergence of multidrug-resistant tuberculosis has posed a major problem in antituberculous therapy (Iseman 1993). Medical therapy alone has been associated with high rates of relapse or treatment failure and a mortality rate of approximately 50% (Goble et al. 1993). Patients with such resistant strains invariably have either a thick-walled cavitary disease or a destroyed lobe or lung (Figs. 25.2-25.4). Cavities contain from 107 to 4 109 organisms compared with 10 2 to 10 in nodules (Canetti 1965). Thus, the removal of this heavy bacterial burden by pulmonary resection may enhance the efficacy of medical treatment. A destroyed lobe or lung acts similar to a cavitary disease. Therefore, the surgeon's role has recently come back to the armamentarium in the treatment of tuberculosis (Pomerantz and Brown 1995). Currently, multidrug-resistant tuberculosis is the most common indication for surgery for tuberculosis in many countries. Examples are Italy (Rizzi et al. 1995, 1997), Japan (Nakajima 1997), South Africa

25.2 Surgery for Multidrug-Resistant Pulmonary Tuberculosis 25.2.1 Background A team at the National Jewish Center for Immunology and Respiratory Medicine in the United States at Denver, Colorado, is a pioneer of the surgical treatment for multidrug-resistant tuberculosis. Between the 1970s and early 1980s, pulmonary resection was still infrequently performed for multidrug-resistant tuberculosis at the National Jewish Center. Only nine (5.3%) of 171 patients with multidrug-resistant tuberculosis underwent surgery from 1973 to 1983 (Goble et aI.1993). However, the center has employed resectional surgery aggressively over the last two decades, due to the fact that even the best medical regimens available have been associated with unacceptably high rates of treatment failure, whereas surgical treatment has proved safe and efficacious (Iseman et al. 1990). Beginning with lobectomies in patients having abundant respiratory reserve, the National Jewish Center group has progressed to pneumonectomies or bilateral resections in patients with very marginal cardiopulmonary reserve. In contrast with the earlier series (Goble et al. 1993), out of 109 patients treated between 1983 and 1993 and eligible for long-term follow-up, 62 (57%) underwent pulmonary resections (Iseman 2000).

25.2.2 Preoperative Medical Treatment Patients with multidrug-resistant tuberculosis initially should be administered the best antituberculous chemotherapy possible based on drug sensitivity tests. The recommended regimen consists of at least four drugs to which the organism is sensitive, two of which have not been used previously in the patient. Resectional surgery should be performed only after approximately

Thoracic Surgery for Tuberculosis

397

a

c Fig. 25.2a, b. A 59-year-old man had bilateral cavitary diseases in the both upper lobes (a, b). His tubercle bacilli were resistant to isoniazid, rifampin, and streptomycin. The patient was treated with chemotherapy employing ethambutol, pyrazinamide, cycloserine, kanamycin, ethionamide, and levofloxacin. After six months of chemotherapy, cavities shrunk, but still remained (c). He underwent staged bilateral upper lobectomies (d). Latissimus dorsi muscle flaps were used to cover the both bronchial stumps

a

b Fig. 25.3a, b. A 48-year-old man had multiple cavitary diseases in the upper and lower lobes of the right lung (a, b). The left lung was intact. His tubercle bacilli were resistant to seven drugs: isoniazid, rifampin, ethambutol, streptomycin, cycloserine, paraaminosalicylcclic acid, and ethionamide. After being treated with isoniazid, rifampin, kanamycin, ethionamide and levofloxacin, he underwent a right pneumonectomy. The bronchial stump was reinforced with the latissimus dorsi

398

Y. Shiraishi

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a

b

c

d Fig. 25.4a-d. A 59-year-old man having the destroyed left upper lobe (a, b) was infected with tuberculosis resistant to isoniazid, rifampin, ethambutol, streptomycin, cycloserine, ethionamide, and levofloxacin. He was treated with pyrazinamide, kanamycin, and para-aminosalicylcclic acid, and then underwent a left upper lobectomy with a superior segmentectomy of the lower lobe. The latissimus dorsi was used, but air leakage prolonged (c). Thoracoplasty removing from the second to the seventh ribs was performed on postoperative day 35, resulting in the obliteration of residual space (d)

three months of intensive medical treatment, and when the lowest bacterial count possible is achieved in the sputum smear and culture results. This may decrease the incidence of postoperative complications. Attempting resection without establishing antituberculous control poses an increased risk of bronchial stump or chest wall wound dehiscence. It is ideal if the sputum smear and culture of the patient becomes negative at the time of operation. This has been possible, however, in only about half

of the patients with multidrug-resistant tuberculosis, even those treated with appropriate antituberculous regimens (Pomerantz et al. 2001). The analysis of the 1973-1983 series at the National Jewish Center revealed that the median time for cultures to become negative was two months, with the great majority becoming negative at four months (Goble et al.I993). Therefore, if the patient remains culture positive after three or four months of chemotherapy, the patient is unlikely to be cured with medication alone

399

Thoracic Surgery for Tuberculosis

and should be considered for surgery if feasible. Improvement in pulmonary hygiene by pulmonary rehabilitation is essential during the preoperative period. Nutritional support is also important during both the medical treatment phase before surgery and the postoperative period.

25.2.3 Indications for Surgery

The following indications for surgery proposed by Iseman (2000) have been widely accepted. (A) The patient is persistently culture positive for multidrugresistant tuberculosis despite extended drug retreatment. (B) Patterns of drug resistance are so extensive as to be likely to be associated with the risk of treatment failure or relapse with additional resistance. (C) The patient has localized cavitary or necrotic/destructive disease in a lobe or region of the lung that is amenable to resection without producing respiratory insufficiency and/or severe pulmonary hypertension. Surgery is sometimes performed on patients who have achieved negative sputum cultures with chemotherapy but are at high risk of relapse because of cavitary diseases or a destroyed lung and extensive patterns of resistance. Pulmonary resection is also performed on patients with anticipated noncompliance and on those with little or no medical options if the current treatment fails. Contraindication to surgery is bilateral disease that is so extensive that resection is not feasible.

25.2.4 Preoperative Evaluation

Patients undergoing pulmonary resection for multidrug-resistant tuberculosis should be carefully evaluated in order to keep morbidity and mortality rates low. Preoperative evaluation should include standard chest x-rays, computed tomography (CT) scan of the chest, pulmonary function tests and a ventilation perfusion scan. These studies are necessary to assess the extent of the disease and to determine the acceptable resection based on anticipated postoperative pulmonary reserve. Bronchoscopy should also be performed if there is hemoptysis or a possibility of bronchostenosis. When pulmonary hypertension is suspected, right heart catheterization should be performed to determine whether the patient is suitable for resectional surgery. A mean pulmonary artery pressure of 25 mmHg or greater often precludes pneumonectomy (Pomerantz and Brown 1997).

The purpose of surgery for patients with multidrug-resistant tuberculosis is to remove all gross lesions. This requires the removal of all cavitary diseases as well as the destroyed lung. Scattered nodular lesions may remain because their mycobacterial burden is low. Indications for a pneumonectomy, in contrast to a lesser procedure, include destruction of an entire lung, multiple cavities in one lung (Fig. 25.3), stenosis of the main stem bronchus, infection involving more than one lobe of the lung and tuberculosis associated with a chronic empyema.

25.2.5 Surgical Technique

At the beginning of the surgery, bronchoscopy is performed to cleanse the airway in order to prevent spread to other portions of the lung. A double-lumen endobronchial tube, which prevents intraoperative contamination of the contralateral lung, is routinely used to achieve lung isolation. A posterolateral thoracotomy incision is routinely used, and the fifth or sixth rib is usually removed. When the chest cavity is entered, dense adhesions are usually found over the upper lobe and often over the superior segment of the lower lobe. Dissection of adhesions may be done intrapleurally or extrapleurally. However, extrapleural dissection has been recommended whenever the pleural space is obliterated to prevent the operative field from being contaminated with tubercle bacilli (Brown and Pomerantz 1995; Conlan et al. 1995). During extrapleural dissection, hemostasis should be achieved using electrocautery and/or bipolar scissors (PowerStar Bipolar Scissors; Ethicon, Somerville, NJ) to minimize blood loss. Special care must be taken to avoid spillage of the contents of cavities into the operative field. Frequently, once pneumolysis is completed, the hilar structures can be isolated without difficulty. However, in the case of a completion pneumonectomy, pulmonary arteries and veins may have to be dissected within the pericardium. The bronchial stump is covered with muscle flaps or omentum if indicated. Bronchial closure can then be done either by suture or with staples. The liberal use of muscle and omental flaps in combinationwith pulmonaryresection for multidrug-resistant tuberculosis has been advised in order to decrease the incidence of postoperative complications (Pomerantz et al. 1991; Treasure and Seaworth 1995). Indications for using these flaps include a positive sputum culture preoperatively, a preoperative bronchopleural fistula, polymicrobial contamination of the pleural space and

Y. Shiraishi

400

anticipated space problems after lobectomy (Pomerantz and Brown 1997). The choice of muscle flap is the latissimus dorsi. The technique for harvesting the latissimus dorsi has been well described previously (Pairolero et al.1983). The flap is sutured to the mainstem bronchial stump, in the case of a pneumonectomy, and used to cover the hilum. When a lobectomy is performed, the flap is sutured to the bronchial stump and also placed at the top of the remaining lung. Other flaps less frequently used include the pectoralis major muscle, the serratus anterior muscle and the intercostal muscle. An omental flap is used when a previous thoracotomy precludes the mobilization of a muscle flap (Pomerantz and Brown 1997). It is also used when a left or right pneumonectomy is performed in the presence of massive contamination. These flaps may be unnecessary despite positive sputum in middle lobe and lingula resections, lower lobectomies and segmental resections. Hemostasis then should be achieved carefully, occasionally with the argon beam coagulator. The chest cavity is thoroughly irrigated and drained. If a muscle flap has been used, a suction drain should be placed below the skin flap and left until drainage becomes less than 25 mllday. On completion of the surgery, bronchoscopy is usually done to cleanse the tracheobronchial tree of secretions that may result from manipulation of the lung during resection. Pomerantz (2000) has advocated that an Eloesser's procedure (Eloesser 1935) be done for patients with extensive contamination after completion of a pneumonectomy. The Eloesser is closed 4 to 6 weeks later with a Clagett procedure (Clagett and Geraci 1963).

25.2.6 Postoperative Care Postoperative care is usually the same as that for pulmonary resection for lung cancer. It should be emphasized that surgery is an adjunct to medical therapy.

Therefore, the patient should be kept on antituberculous medication for 12 to 24 months postoperatively, depending on the bacteriologic and radiographic findings. Stopping antituberculous drugs too soon leads to further resistance or mycobacterial spread.

25.2.7 Types of Operative Procedures Since it is crucial to resect all grossly diseased areas of the lung, the rates of pneumonectomy have been high in some reports (Table 25.1). Kir and colleagues (1997) reported an extremely high rate of pneumonectomy. Out of 27 operations, 20 (74%) were pneumonectomies. Van Leuvan and associates (1997), Pomerantz and coworkers (2001) and Treasure and Seaworth (1995) reported pneumonectomy rates of 56 to 42%. When a lobectomy or an operation that is more than a lobectomy, but less than a pneumonectomy, is performed, the right upper lobe is most commonly involved. In a report by Pomerantz and associates (2001), 55 of 93 patients undergoing lobectomy had the right upper lobe, or the right upper lobe in combination with another portion of the lung, resected. In patients with pulmonary tuberculosis, the destruction of the lung requiring a pneumonectomy occurs more frequently on the left than on the right side. This is revealed by the fact that 73% of all pneumonectomies were on the left in a series by Pomerantz and colleagues (2001). Van Leuven and associates (1997) reported a similar incidence (80%) ofleft-sided pneumonectomies. Similarly, in a report by Kir and colleagues (1997), 75% of pneumonectomies were performed on the left side. Ashour (1997) reported that 16 (80%) of 20 pneumonectomies were done on the left side when pneumonectomy was performed for patients with post-tuberculosis lung destruction. The reasons for this left-sided predominance remain unclear. Ashour and associates (1990) postulated that the predilection for left-lung destruction might be

Table 25.1. Types of pulmonary resections for multidrug-resistant tuberculosis Reference Treasure and Seaworth (1995) Kir et aI. (1997) Van Leuvan et aI. (1997) Nakajima (1997) Sung et aI. (1999) Pomerantz et al. (2001)

No. of Pneumonectomy operations 19 27 62 40 27 180

8 20 (right 5, left 15) 35 (right 7, left 28) 14 9 82 (right 22, left 60)

Lobectomy Segmental resection 9

2

7 26 24 16 93

1 2 2 5

401

Thoracic Surgery for Tuberculosis

related to the smaller caliber of the left main stem bronchus, a tight mediastinum through which the left main stem bronchus traverses and the more horizontal course of the left main stem bronchus.

25.2.8 Operative and Late Mortality In general, pulmonary resection for multidrugresistant tuberculosis can be performed with low mortality. Operative mortality rates ranging from zero to 3.3% have been reported (Table 25.2). Causes of operative deaths described in the reported series include postpneumonectomy pulmonary edema, pulmonary embolism, bronchopleural fistula with respiratory failure, myocardial infarction and a cerebral vascular accident. Operative survivors have seldom died from the progression of multidrug-resistant tuberculosis. Pomerantz and colleagues (2001) reported that 11 late deaths occurred but only three were caused by multidrug-resistant tuberculosis. Of the three patients, one patient had his antituberculous medication stopped inexplicably in the postoperative period, a second had cavitary disease on the other side and refused a second operation and a third died from progressive tuberculosis in spite of continued chemotherapy. Other causes included respiratory failure, a self-induced drug overdose, myocardial infarction, renal failure and unknown causes.

25.2.9 Postoperative Complications Although operative mortality has been acceptably low, postoperative complication rates have been high. Morbidity rates have ranged from 12 to 26% (Table 25.2). Complications described after pulmonary resection for multidrug-resistant tuberculosis are summarized in Table 25.3. These complications occur more com-

Table 25.2. Morbidity and mortality after pulmonary resections for multidrug-resistant tuberculosis Reference Treasure and Seaworth (1995) Kir et al. (1997) Van Leuvan et al. (1997) Sung et al. (1999) Pomerantz et al. (2001)

No. of Mortality Morbidity operations (No.) (No.) 19 27 62 27 180

0% (0)

21% (4)

0% (0) 1.6% (1) 0% (0) 3.3% (6)

19% (5) 23% (14) 26% (7) 12% (20)

Table 25.3. Complications after pulmonary resections for multidrug-resistant tuberculosis Reference

Complications (no. of patients)

Kir et al. (1997)

Revision because of hemorrhage (2); bronchopleural fistula (2); apical residual space (1) Van Leuvan et al. (1997) Postoperative hemorrhage (5); postpneumonectomyempyema (4); wound infection (2); pneumonectomy stump fistula (2); postpericardiotomy syndrome (1); respiratory failure (1); space problem (1) Sung et al. (1999) Prolonged air leakage (3); reoperation due to bleeding (2); bronchopleural fistula (1); reversible blindness (1) Pomerantz et al. (2001) Respiratory failure (6); bronchopleural fistula (5); wound infection (3); postoperative hemorrhage (3); recurrent nerve injury (2); intrathoracic bowel herniation (1)

monly after surgery for tuberculosis because of the adhesive nature of the disease process, as well as the debilitated catabolic condition of the patient. A bronchopleural fistula is a dreaded complication after surgery for mycobacterial infections. Factors adversely affecting the incidence of bronchopleural fistula include right pneumonectomy, positive sputum at the time of surgery, significant polymicrobial contamination, diabetes and prior chest wall irradiation (Pomerantz 2000). Although some investigators have not used muscle flaps to cover the bronchial stump (Kir et al. 1997; van Leuvan et al. 1997; Sung et al. 1999), the use of muscle flaps has been advocated to decrease the incidence of bronchopleural fistula (Treasure and Seaworth 1995; Pomerantz et al. 2001). Bronchopleural fistula is rarely encountered after resection of the middle lobe, lingula, lower lobe or segment. When resections less drastic than pneumonectomies are performed, the inability of the remaining lung to fill the pleural space has occasionally resulted in a chronic space problem (Fig. 25.4). Thoracoplasty has been used to treat space problems (Pomerantz and Mault 2000). However, Pomerantz and coworkers (1991) have recommended using muscle flaps to prevent this complication and to avoid thoracoplasty. While operative morbidity and mortality are the obvious disadvantages of pulmonary resection, overall mortality is reduced, compared with simple medical treatment, and the patient's opportunity to rejoin society outweighs the risk.

402

25.2.10 Cure Rate of Surgical Treatment

The combination of pre- and postoperative antituberculous medications and pulmonary resection has resulted in high cure rates in patients with multidrugresistant tuberculosis. Iseman and colleagues (l990) noted that 92% of patients remained sputum-culture-negative for tuberculosis a mean of 39 months after resection. Recent studies around the world have also demonstrated that cure can be obtained in more than 80% of the surgical cases (Table 25.4). Although no randomized study has been done to compare the cure rates of medical treatment plus surgical treatment with those of