Reichman and Hershfield's Tuberculosis: A Comprehensive, International Approach, Third Edition (Two-Volume Set) (Lung Biology in Health and Disease)

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Reichman and Hershfield's

Tuberculosis A Comprehensive, International Approach

Third Edition Part A

Edited by

Mario C. Raviglione World Health Organization Geneva, Switzerland

New York London

This volume is not an official publication of the World Health Organization, and the opinions expressed herein do not necessarily represent the views of the Organization.

Informa Healthcare USA, Inc. 270 Madison Avenue New York, NY 10016 © 2006 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9271-3 (Hardcover) International Standard Book Number-13: 978-0-8493-9271-9 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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Introduction

The year 2006 is an important year in the history of tuberculosis and in the fight to control it. As was declared during World TB Day in March 2006, the world showed a new resolve by announcing a ‘‘Global Plan to Stop TB, 2006–2015.’’ The goal of this worldwide effort, or global strategy, is to begin to reverse the incidence of tuberculosis by 2015. The TB Fact Sheet (1), published by the World Health Organization on the occasion of the 2006 World TB Day, provides staggering data from 2004 on the incidence, prevalence, and mortality from tuberculosis, indicating that there were as many as 1.7 million deaths. As we see such numbers, it becomes easy to also see the rationale for the Global Plan to Stop TB, 2006–2015. For this strategy to be successful, however, it will be necessary to have a solid foundation for the development of new actions, be they therapeutic or in the public health arena. However, just as well, it will require a strong and continuous international commitment to support and implement the program. Very likely, the cynics will say that this new program has a bit of ‘‘de´ja` vu.’’ Indeed, tuberculosis is not a new disease, and much work has been done over the years to combat it, but it is the first time that a strategic plan has iii

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Introduction

been formulated with the endorsement of health, business, and financial world leaders, not at the World Health Organization, but during the 2006 Economic Forum in Davos, Switzerland. The publication in 2006 of the third edition of Reichman and Hershfield’s Tuberculosis: A Comprehensive, International Approach, presented by the series of monographs Lung Biology in Health and Disease, may be a coincidence, but it is very opportune as it gives the reader not only a status report of where the field of tuberculosis is today, but also provides a panoramic review of the many new research avenues that are in need of investigation. The purpose of this volume, as stated by its editor, Dr. Mario C. Raviglione, is to show ‘‘what needs to be integrated in practice through effective collaboration between research scientists, physicians, public-health officials, epidemiologists, and policy makers’’ if indeed the Global Plan to Stop TB is to succeed. Truly, this volume gives us a road map to reach this goal! Dr. Raviglione, the chief officer of the World Health Organization tuberculosis program, has shown a remarkable vision in the preparation and development of this volume. Suffice it to review the list of contributors to see that they represent the ‘‘Who’s Who’’ of the tuberculosis field and that they have paved the way to reaching the Stop TB goal. As the editor of this series of monographs, Lung Biology in Health and Disease, I am immensely grateful to Dr. Raviglione and to all the experts who participated in the preparation of this volume for the opportunity to present this new edition of Reichman and Hershfield’s Tuberculosis: A Comprehensive, International Approach. Claude Lenfant, MD Gaithersburg, Maryland, U.S.A. Reference 1. World Health Organization Tuberculosis Fact Sheet #104. Revised March 2006.

Historical Background

Sixteen years ago, the first edition of Tuberculosis: A Comprehensive, International Approach was published; it was the first textbook on tuberculosis in several years. At that time, although very prevalent, tuberculosis was a forgotten disease. There had been no new drugs or diagnostic initiatives in several decades, the DOTS strategy had not been named, and the World Health Organization tuberculosis program was a small office with one professional and one support person. Advocacy and concerned groups of patients and caregivers relating to tuberculosis were not even a figment of one’s imagination. The relationship between tuberculosis and HIV was recognized but routinely ignored. Multidrug-resistant tuberculosis was scary but accepted without intervening or even treating patients so afflicted. Because of rapid advances in the field, a second edition was published in 2000. There was increasing interest and progress. Consequently, other textbooks devoted to this subject and its ramifications were published by many different publishers. Now we are pleased to see that the continuing vast progress and improvements in the field require a third edition of this text. There is nothing more fitting for us, then, to pass the editorship to an individual who is widely recognized for his tuberculosis expertise as well as his influence in v

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policy matters, Dr. Mario C. Raviglione. He has put together a table of contents that is a ‘‘Who’s Who’’ of the unquestionable world leaders in the field. He has also made provisions to distribute the book at a reduced cost to developing nations where the global burden of tuberculosis is most strongly reflected. It is with humility and pride that we hand over the editorship of this volume to our successor, and our gratitude goes to him and the publisher, Informa Healthcare, for extending and enhancing the franchise of Reichman and Hershfield’s Tuberculosis: A Comprehensive, International Approach. Lee B. Reichman, MD, MPH Newark, New Jersey, U.S.A. Earl S. Hershfield, MD Winnipeg, Manitoba, Canada

Preface

Tuberculosis, an ancient scourge of humanity known for several thousands of years, is still a major public health challenge in many countries today. Global targets for tuberculosis control by the year 2005 were set by the World Health Assembly to encourage a concerted worldwide effort to end this situation. It is therefore timely and appropriate for the international tuberculosis control and research community to assess what has been achieved in the past decade since the launch and promotion in 1995 of a modern tuberculosis control strategy, branded under the name of ‘‘DOTS’’a. It is also time to consider what more needs to be done to eliminate tuberculosis as a significant disease threat. Looking back, there have been remarkable achievements in recent years. They include: the rapid adoption of the DOTS strategy, which is now being applied in most countries; the high rate of cure demonstrated a The DOTS strategy has five essential components: government commitment to tuberculosis control; diagnosis via bacteriology through an effective laboratory network; standardized short-course chemotherapy with supervision and full patient support throughout treatment; uninterrupted supply of quality-assured anti-tuberculosis drugs; and recording and reporting to measure patient and program outcomes.

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among millions of infectious tuberculosis patients throughout the world; the efforts to tackle multi-drug resistant tuberculosis and human immunodeficiency virus–associated tuberculosis; the launch of successful initiatives, such as the Global Drug Facility and the Green Light Committee, to provide access to anti-tuberculosis drugs in resource-poor countries; the inclusion of tuberculosis among the targeted diseases for support by new international financial mechanisms, which are mobilizing unprecedented resources; and major public–private research initiatives to develop new tools to improve the diagnosis, treatment, and prevention of tuberculosis. This is undoubtedly an impressive track record that has been widely supported and facilitated by the establishment in 2001 of the global coalition to fight tuberculosis: the Stop TB Partnership. However, despite substantial progress in many countries, tuberculosis still kills 1.7 million people and affects 8.9 million new patients every year. Clearly, more detailed knowledge is necessary to strengthen research efforts toward development of new diagnostics, new anti-tuberculosis drugs, and an effective vaccine. Better understanding of pathogenesis, for instance, is crucial. At the same time, while awaiting the tools of the future, we cannot afford to relax our efforts to control tuberculosis using the current tools more effectively in the (often weak) health systems existing today in the world. On the contrary, the threat of resistance to anti-tuberculosis drugs, especially in the former USSR, and the overlap of the tuberculosis and human immunodeficiency virus/acquired immunodeficiency virus epidemics, especially in Africa, oblige us to reinforce DOTS programs everywhere by promoting public–private approaches and engaging communities to reach more patients, establish collaboration between tuberculosis and human immunodeficiency virus/acquired immunodeficiency virus programs, and contribute to health system strengthening to address, for instance, the scarcity of human resources capable of delivering high quality tuberculosis services. With this in mind, the new Stop TB Strategy recently announced by the World Health Organization was formulated to widen the focus of DOTS and address all modern challenges. All these issues, whether they are in the field of research or that of program implementation, deserve our full attention. Therefore, in preparing the third edition of this widely respected book, I sought to link the clinical ‘‘state of the art’’ to the principles of programmatic tuberculosis control and to current and upcoming research initiatives, thereby integrating on paper what needs to be integrated in practice through effective collaboration between research scientists, physicians, public health officials, epidemiologists, and policy makers. The book’s 50 chapters bring together our current knowledge of tuberculosis, its control, and related scientific discoveries. It is addressed to all who work in the field of tuberculosis control, whether in low-income, high-prevalence areas or in low-prevalence industrialized countries. As a compendium of information on all aspects of tuberculosis, it enables up-to-date knowledge to reach

Preface

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all parts of the world, especially those settings where access to modern scientific ideas may still be difficult. I am immensely grateful to Lee B. Reichman and Earl S. Hershfield for inviting me to continue the successful tradition of their book. Aspiring to match their previous standards, I have asked many of the world’s leading authorities on tuberculosis to contribute chapters to this book and I thank them for their generous and expert collaboration. Finally, I thank my family for patiently tolerating my enthusiasm for this important endeavour to share knowledge on the global fight against tuberculosis. Mario C. Raviglione

Acknowledgments

First of all, I would like to thank Dr. Kitty Lambregts-van Weezenbeek for her contributions in the initial phase of preparation of this new edition and for providing input for the preface, the structure, and the outline of the book. Her advice has been particularly important for the identification of the contributors and the organization of the table of contents. Second, I would like to thank the technical editor, Dr. Lindsay Martinez, for having worked with me very closely throughout the production of the book and for carefully reviewing the scientific language style used by the contributors and making it as consistent as possible for a multiauthor book. Third, I would like to recognize the work of Ms. Monika Tatranska and Ms. Michelle Lavergne for having helped in coordinating the production, especially the tasks of contacting peer-reviewers and compiling information for the contributors. Their support has been crucial and the book would not have been possible without them. Finally, I would like to thank all the peer reviewers who patiently provided their assistance to the contributors in ensuring that the chapters were of the highest standards: C. E. Barry, M. Behr, N. Binkin, A. Bone, M. W. Borgdorff, M. Bugiani, K. Caines, P. Caminero, P. Cegielski, P. Chaulet, xi

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Acknowledgments D. Chemtob, R. L. Cowie, M. R. Dal Poz, P. R. Donald, K. Duncan, N. Ezard, A. Fanning, J. I. Figueroa, T. Frieden, P. Fujiwara, J. Foulds, G. Gargioni, H. Getahun, J. Glynn, P. Gondrie, A. Gori, S. Graham, J. Grosset, E. Heldal, S. Hoffner, E. Jaramillo, S. H. E. Kaufmann, D. Kibuga, S. J. Kim, A. Laszlo, M. Levy, K. Lo¨nnroth, F. Luelmo, D. Maher, R. Matiru, J. Mazurek, A. Mwinga, E. Nathanson, C. Nolan, R. O’Brien, V. Pathania, T. Pennas, M. Pomerantz, R. Pray, S. Rangan, H. L. Rieder, J. Sbarbaro, J. C. Sadoff, H. Sawert, F. Scano, R. Scherpbier, L. Schlesinger, K. Siddiqi, G. Steenbergen, W. Stewart, E. A. Talbot, F. Varaine, A. Vernon, J. Walley, R. S. Wallis, C. Wells, B. G. Williams, V. G. Williams, J.-P. Zellweger.

Contributors

Francis Adatu-Engwau National Tuberculosis and Leprosy Programme, Wandegeya, Kampala, Uganda Chantelle Allen

ADRA Nepal, Kathmandu, Kingdom of Nepal

Virginia C. Arnold Office of the Director–General, World Health Organization, Geneva, Switzerland Mohamed Abdel Aziz Geneva, Switzerland

Stop TB Department, World Health Organization,

Marcel A. Behr Department of Medicine, Research Institute of the McGill University Health Centre, McGill University, and Division of Infectious Diseases and Medical Microbiology, Montreal General Hospital, Montreal, Quebec, Canada William R. Bishai Division of Infectious Diseases, Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Leopold Blanc Stop TB Department, World Health Organization, Geneva, Switzerland Kai Blo¨ndal-Vink The Netherlands

KNCV Tuberculosis Foundation, The Hague,

Fadila Boulahbal Mycobacteriology, National Reference TB Laboratory, Institut Pasteur d’Alge´rie, Alger, Alge´rie Roland Brosch Unite´ de Ge´ne´tique Mole´culaire Bacte´rienne, Institut Pasteur, Paris, France xiii

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Contributors

Kenneth G. Castro United States Public Health Service and Division of Tuberculosis Elimination, National Center for HIV, STD, and Tuberculosis Prevention, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Antonino Catanzaro Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, School of Medicine, UCSD Medical Center, San Diego, California, U.S.A. Peter Cegielski United States Public Health Service and Division of Tuberculosis Elimination, National Center for HIV, STD, and Tuberculosis Prevention, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Rosella Centis World Health Organization Collaborating Centre for Tuberculosis and Lung Diseases, Fondazione Salvatore Maugeri, Care and Research Institute, Tradate, Italy Richard E. Chaisson Johns Hopkins University Center for Tuberculosis Research, Baltimore, Maryland, U.S.A. Pierre Chaulet

Faculty of Medicine, University of Algiers, Algiers, Algeria

Gavin J. Churchyard Aurum Institute for Health Research, CAPRISA, University of Kwa-Zulu Natal, Marshalltown, Gauteng, South Africa Jacqueline S. Coberly Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland, U.S.A. David L. Cohn Denver Public Health and the Division of Infectious Diseases, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A. George W. Comstock Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland, U.S.A. Ma´ire A. Connolly Disease Control in Humanitarian Emergencies, Communicable Diseases, World Health Organization, Geneva, Switzerland Elizabeth L. Corbett Clinical Research Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine Funded by the Wellcome Trust, London, U.K. Peter D. O. Davies Tuberculosis Research, Cardiothoracic Centre and University Hospital Aintree (NHS) Trusts, Mercers, Liverpool, U.K.

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Isabel N. de Kantor Tuberculosis Consultants Panel, World Health Organization, Buenos Aires, Argentina T. Mark Doherty Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark Gilles Dussault Human Development, World Bank Institute, Washington, D.C., U.S.A. Christopher Dye Stop TB Department, World Health Organization, Geneva, Switzerland Jerrold J. Ellner Department of Medicine and Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, UMDNJ—New Jersey Medical School, Newark, New Jersey, U.S.A. Wafaa M. El-Sadr Division of Infectious Diseases, Harlem Hospital Center and International Center for AIDS Care and Treatment Programs (ICAP), Columbia University, College of Physicians and Surgeons, and Mailman School of Public Health, New York, New York, U.S.A. Gijs Elzinga National Institute of Public Health and the Environment, Bilthoven, Utrecht, The Netherlands Donald A. Enarson International Union Against Tuberculosis and Lung Disease, Paris, France Marcos A. Espinal Fuentes Stop Tuberculosis Partnership Secretariat, World Health Organization, Geneva, Switzerland Sue C. Etkind Division of Tuberculosis Prevention and Control, State Laboratory Institute, Massachusetts Department of Public Health, Boston, Massachusetts, U.S.A. Anne Fanning Faculty of Medicine and Dentistry, Walter McKenzie Health Sciences Center, University of Alberta, Edmonton, Alberta, Canada Kevin P. Fennelly Department of Medicine, Center for the Study of Emerging and Re-emerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, U.S.A. Jose´ I. Figueroa Public Health Improvement, City and Hackney Primary Care Trust, London, U.K.

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Contributors

Mark FitzGerald Centre for Clinical Epidemiology and Evaluation, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Katherine Floyd Stop TB Department, World Health Organization, Geneva, Switzerland Michelle Gayer Disease Control in Humanitarian Emergencies, Communicable Diseases, World Health Organization, Geneva, Switzerland Ann Ginsberg Clinical Department, Global Alliance for TB Drug Development, New York, New York, U.S.A. Peter Godfrey-Faussett Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, U.K. Ruth Griffin CMMI, Department of Infectious Diseases and Microbiology, Imperial College London, London, U.K. Malgorzata Grzemska Geneva, Switzerland

Stop TB Department, World Health Organization,

Maria-Cristina Gutie´rrez Laboratoire de Re´fe´rence des Mycobacte´ries, Institut Pasteur, Paris, France Christy L. Hanson Division of Infectious Disease, Bureau for Global Health, U.S. Agency for International Development, Washington, D.C., U.S.A. Anthony D. Harries HIV Unit, Ministry of Health, Lilongwe, Malawi, and Family Health International, Arlington, Virginia, U.S.A. Leonid Heifets Mycobacteriology Clinical Reference Laboratory, National Jewish Medical and Research Center, Denver, Colorado, U.S.A. Petra I. Heitkamp

World Health Organization, Jakarta, Indonesia

Philip C. Hopewell Division of Pulmonary and Critical Care Medicine, Medical Service, San Francisco General Hospital, Francis J. Curry National Tuberculosis Center, and Department of Medicine, University of California, San Francisco, California, U.S.A. Michael F. Iademarco United States Public Health Service and Division of Tuberculosis Elimination, National Center for HIV, STD, and Tuberculosis

Contributors

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Prevention, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Ernesto Jaramillo Stop TB Department, World Health Organization, Geneva, Switzerland Sirinapha Jittimanee TB Cluster (NTP), Bureau of AIDS, TB, and STIs, Ministry of Public Health, Bangkok, Thailand Michael E. Kimerling Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Phung K. Lam Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, School of Medicine, UCSD Medical Center, San Diego, California, U.S.A. Kitty Lambregts-van Weezenbeek Hague, The Netherlands

KNCV Tuberculosis Foundation, The

Adalbert Laszlo Mycobacteriology Laboratory Consultant, Ottawa, Ontario, Canada Philip A. LoBue Division of Tuberculosis Elimination, Field Services and Evaluation Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Knut Lo¨nnroth Stop TB Department, World Health Organization, Geneva, Switzerland Fabio Luelmo Tuberculosis Control Programmes Consultant, The Hague, The Netherlands Dermot Maher Stop TB Department, World Health Organization, Geneva, Switzerland Jaouad Mahjour Directorate of Epidemiology and Disease Control, Ministry of Health, Rabat, Morocco Dick Menzies Epidemiology, Biostatistics, and Occupational Health, Montreal Chest Institute, McGill University, Montreal, Quebec, Canada Giovanni Battista Migliori World Health Organization Collaborating Centre for Tuberculosis and Lung Diseases, Fondazione Salvatore Maugeri, Care and Research Institute, Tradate, Italy

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Contributors

John Moore-Gillon Department of Respiratory Medicine, St. Bartholomew’s and Royal London Hospitals, London, U.K. Flor M. Munoz Pediatrics Section of Infectious Diseases and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A. Edward A. Nardell Division of Social Medicine and Health Inequalities, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Paul Nunn Stop TB Department, World Health Organization, Geneva, Switzerland Richard J. O’Brien Foundation for Innovative New Diagnostics, Geneva, Switzerland Philip Onyebujoh Implementation Research and Methods, UNICEF/ UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland Salah-Eddine Ottmani Geneva, Switzerland

Stop TB Department, World Health Organization,

Kathleen R. Page Johns Hopkins University Center for Tuberculosis Research, Baltimore, Maryland, U.S.A. Mikhail I. Perelman Moscow, Russia

Sechenov Moscow Medical Academy,

Mark D. Perkins Foundation for Innovative New Diagnostics, Geneva, Switzerland Sharon Perry Division of Geographic Medicine and Infectious Diseases, Stanford University School of Medicine, Stanford, California, U.S.A. Antonio Pio Public Health and Respiratory Disease, Mar del Plata, Argentina Franc¸oise Portaels Department of Microbiology, Institute of Tropical Medicine, Antwerpen, Belgium Mario C. Raviglione Stop TB Department, World Health Organization, Geneva, Switzerland

Contributors

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Michael Leonard Rich Partners in Health, Division of Social Medicine and Health Inequalities, Brigham and Women’s Hospital, Boston, Massachusetts, U.S.A. Leen Rigouts Department of Microbiology, Institute of Tropical Medicine, Antwerpen, Belgium William Rodriguez Harvard Medical School Division of AIDS, The Landmark Center, Boston, Massachusetts, U.S.A. John A. Sbarbaro University of Colorado Health Sciences Center, University Physicians, Inc., Denver, Colorado, U.S.A. S. K. Schwander Department of Medicine and Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, UMDNJ—New Jersey Medical School, Newark, New Jersey, U.S.A. Kevin Schwartzman Respiratory Division and Respiratory Epidemiology Unit, Montreal Chest Institute, McGill University, Montreal, Quebec, Canada Isdore Chola Shamputa Department of Microbiology, Institute of Tropical Medicine, Antwerpen, Belgium Ian M. Smith Office of the Director–General, World Health Organization, Geneva, Switzerland Melvin Spigelman Research and Development, Global Alliance for TB Drug Development, New York, New York, U.S.A. Sergio Spinaci Global Malaria Programme, World Health Organization, HIV/AIDS, Tuberculosis and Malaria Cluster, Geneva, Switzerland Jeffrey R. Starke Pediatrics Section of Infectious Diseases, Baylor College of Medicine and Ben Taub General Hospital, Houston, Texas, U.S.A. Roberto Tapia-Conyer Subsecretariat of Prevention and Control of Diseases, Mexican Secretariat of Health, Mexico City, Mexico Mukund Uplekar Stop TB Department, World Health Organization, Geneva, Switzerland Richard Urbanczik Tuberculosis Consultants Panel, World Health Organization/International Union Against Tuberculosis and Lung Disease, Scho¨mberg, Germany

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Contributors

Armand Van Deun Department of Microbiology, Institute of Tropical Medicine, Antwerpen, Belgium Jeroen van Gorkom The Netherlands

KNCV Tuberculosis Foundation, The Hague,

Jaap Veen Head Unit Europe, KNCV Tuberculosis Foundation, The Hague, The Netherlands Suzanne Verver Head Unit Europe, KNCV Tuberculosis Foundation, The Hague, The Netherlands Ve´ronique Vincent Stop TB Department, World Health Organization, Geneva, Switzerland Diana E. C. Weil Stop TB Department, World Health Organization, Geneva, Switzerland Virginia G. Williams International Union Against Tuberculosis and Lung Disease, Paris, France Jean Woo Department of Community and Family Medicine, Division of Geriatrics, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, and School of Public Health, Prince of Wales Hospital, Shatin, Hong Kong, China Samuel C. Woolwine Division of Infectious Diseases, Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Douglas Young CMMI, Department of Infectious Diseases and Microbiology, Imperial College London, London, U.K. Richard Zaleskis Regional Office for Europe, TB Unit, World Health Organization, Copenhagen, Denmark Noureddine Zidouni Algiers, Algeria

Faculty of Medicine, University of Algiers,

Matteo Zignol Stop TB Department, World Health Organization, Geneva, Switzerland Alimuddin Zumla Windeyer Institute of Medical Sciences, Centre for Infectious Diseases and International Health, University College London, Royal Free and University College London Medical School, London, U.K.

Contents

Introduction Claude Lenfant . . . . iii Historical Background Lee B. Reichman and Earl S. Hershfield . . . . v Preface . . . . vii Acknowledgments . . . . xi Contributors . . . . xiii PART A SECTION I: BASIC ASPECTS OF TUBERCULOSIS 1. The Global Tuberculosis Epidemic: Scale, Dynamics, and Prospects for Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Christopher Dye I. Introduction . . . . 1 II. Global and Regional TB Epidemics: Scale and Dynamics . . . . 2 III. Tuberculosis Control . . . . 6 IV. Chemotherapy and the DOTS Strategy . . . . 8 V. Implementation and Impact of DOTS, 1991 to 2005 . . . . 10 VI. Prospects for Tuberculosis Control, 2006 to 2015 and Beyond . . . . 12 VII. Conclusion . . . . 21 References . . . . 25

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2. Bacteriology of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . Fadila Boulahbal and Leonid Heifets I. Mycobacterium tuberculosis Complex . . . . 29 II. Bacterial Populations in Patients . . . . 32 III. Bacteriological Diagnosis of Tuberculosis . . . . 33 IV. Methods for Mycobacterium tuberculosis Identification . . . . 35 V. Detection of Drug Resistance . . . . 38 References . . . . 42

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3. Genomics and Evolution of Tubercle Bacille . . . . . . . . . . . 47 Ve´ronique Vincent, Maria-Cristina Gutie´rrez, and Roland Brosch I. Introduction . . . . 47 II. The Genome Sequence and Biology of Mycobacterium tuberculosis . . . . 48 III. Comparative Genomics and Evolution Within the Mycobacterium tuberculosis Complex . . . . 51 IV. Evolution of Mycobacterium bovis BCG . . . . 52 V. Population Structure and Clonal Evolution of the Mycobacterium tuberculosis Complex . . . . 54 VI. Ecotypes Within the Mycobacterium tuberculosis Complex . . . . 56 VII. Mycobacterium prototuberculosis: The Progenitor of the Mycobacterium tuberculosis Complex Orphan Clone . . . . 57 VIII. Conclusion . . . . 59 References . . . . 60 4. Epidemiology of Tuberculosis . . . . . . . . . . . . . . . . . . . . . Jacqueline S. Coberly and George W. Comstock I. Introduction . . . . 65 II. Etiologic Epidemiology . . . . 66 III. Administrative Epidemiology . . . . 79 IV. Conclusion . . . . 92 References . . . . 92 5. Overview of the Pathogenesis of Tuberculosis from a Cellular and Molecular Perspective . . . . . . . . . . . . . . . Samuel C. Woolwine and William R. Bishai I. Introduction . . . . 101 II. Infection . . . . 103

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III. IV. V. VI.

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Host Response . . . . 106 Cavitary Tuberculosis . . . . 109 Latent Tuberculosis . . . . 110 Conclusion . . . . 111 References . . . . 112

6. The Human Host: Immunology and Susceptibility . . . . . . 117 S. K. Schwander and Jerrold J. Ellner I. Introduction . . . . 117 II. The Natural History of Mycobacterium tuberculosis Infection in Humans . . . . 118 III. Human Immunity to Mycobacterium tuberculosis . . . . 119 IV. Susceptibility to Mycobacterium tuberculosis Infection and Tuberculosis Development . . . . 133 V. Resistance to Mycobacterium tuberculosis Infection . . . . 138 References . . . . 140 SECTION II: CLINICAL TUBERCULOSIS 7. Diagnosis of Pulmonary and Extrapulmonary Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Phung K. Lam, Philip A. LoBue, Sharon Perry, and Antonino Catanzaro I. Introduction . . . . 155 II. Medical History and Physical Examination . . . . 156 III. The Tuberculin Skin Test . . . . 156 IV. Interferon Release Assays . . . . 157 V. Chest Radiography . . . . 158 VI. Respiratory Specimen Sampling: AFB Smear and Culture . . . . 159 VII. Culture-Negative Pulmonary Tuberculosis . . . . 160 VIII. Extrapulmonary Tuberculosis . . . . 160 IX. Clinical Use of Diagnostic Tests: Comparing Sensitivity and Specificity to PPV and NPV . . . . 160 X. Using Newer Diagnostic Tests: Incorporating Clinical Suspicion of Tuberculosis . . . . 161 XI. Newer Diagnostic Tests: NAA Assays . . . . 164

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XII. A Proposed Diagnostic Algorithm for the Diagnosis of TB Using Clinical Suspicion of TB with NAA Testing . . . . 166 XIII. Creating New Diagnostic Tests Based on Older Technology: Serodiagnosis by Immunoassays . . . . 168 XIV. Conclusion . . . . 171 References . . . . 172 8. Treatment of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . 183 Philip C. Hopewell I. Tuberculosis Treatment as a Public Health Measure . . . . 183 II. History of Antituberculosis Chemotherapy . . . . 184 III. Antituberculosis Drugs . . . . 185 IV. Promoting Adherence to Treatment . . . . 195 V. Current Treatment Regimens . . . . 196 VI. Treatment in Special Situations . . . . 201 VII. Adjunctive Treatments for Pulmonary Tuberculosis . . . . 204 VIII. Extrapulmonary Tuberculosis . . . . 205 IX. New Drugs for Tuberculosis . . . . 209 References . . . . 210 9. Diagnosis of Latent Tuberculosis Infection . . . . . . . . . . . Dick Menzies and T. Mark Doherty I. Tuberculin Skin Testing . . . . 215 II. Chest X-ray (for Diagnosis of Tuberculosis Infection) . . . . 242 III. Interferon-g Release Assays . . . . 243 IV. Conclusions . . . . 248 Glossary . . . . 250 References . . . . 251

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10. Treatment of Latent Tuberculosis Infection . . . . . . . . . . David L. Cohn and Wafaa M. El-Sadr I. Introduction . . . . 265 II. Efficacy of Treatment of LTBI . . . . 266

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III. Safety and Tolerability of Treatment of LTBI . . . . 279 IV. Treatment of LTBI in Special Populations . . . . 286 V. Recommendations for the Treatment of LTBI . . . . 288 VI. Programmatic and Other Issues Related to the Treatment of LTBI . . . . 292 VII. Future Directions . . . . 296 VIII. Conclusions . . . . 297 References . . . . 297 11. Childhood Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . 307 Flor M. Munoz and Jeffrey R. Starke I. Introduction . . . . 307 II. Epidemiology . . . . 309 III. Pathogenesis . . . . 314 IV. Clinical Forms of Pediatric Tuberculosis . . . . 316 V. Diagnosis of Tuberculosis in Children . . . . 325 VI. Treatment . . . . 329 VII. Summary . . . . 336 References . . . . 336 12. Tuberculosis in the Elderly . . . . . . . . . . . . . . . . . . . . . . 345 Peter D. O. Davies, Jean Woo, and John Moore-Gillon I. Introduction . . . . 345 II. The Aging Population . . . . 346 III. Epidemiology . . . . 346 IV. Decline in Immunocompetence with Increasing Age . . . . 348 V. Tuberculosis in Special Situations . . . . 351 VI. Clinical Presentation . . . . 353 VII. Mortality . . . . 354 VIII. Human Immunodeficiency Virus Infection . . . . 356 IX. Diagnosis . . . . 357 X. Treatment . . . . 358 XI. Preventive Therapy . . . . 361 XII. Case Reports . . . . 362 XIII. Conclusions . . . . 366 References . . . . 366

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13. Tuberculosis–HIV Coinfection: Epidemiology, Clinical Aspects, and Interventions . . . . . . . . . . . . . . . . Kathleen R. Page, Peter Godfrey-Faussett, and Richard E. Chaisson I. Introduction . . . . 371 II. Risk of Tuberculosis in Persons with HIV Infection . . . . 372 III. Prevalence of HIV Infection Among Patients with Tuberculosis . . . . 374 IV. Influence of HIV Infection on the Pathogenesis of Tuberculosis . . . . 375 V. Influence of Tuberculosis on the Course of HIV Infection . . . . 377 VI. Diagnosis of Tuberculosis Infection and Disease . . . . 378 VII. Treatment of Tuberculosis in Patients with HIV . . . . 385 VIII. Tuberculosis and HIV in Children . . . . 392 IX. Tuberculosis Caused by Multidrug-Resistant Organisms . . . . 394 X. Treatment of Latent Tuberculosis Infection . . . . 396 XI. Programs and Interventions . . . . 399 References . . . . 404 14. Diagnosis and Treatment of Multidrug-Resistant Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael Leonard Rich I. Introduction . . . . 417 II. History . . . . 417 III. Mechanisms of Resistance . . . . 418 IV. Cross-Resistance . . . . 419 V. Pathogenicity, Transmissibility, and Drug Resistance . . . . 419 VI. Preventing the Evolution and Transmission of Drug Resistance . . . . 422 VII. Multidrug-Resistant Tuberculosis Diagnosis . . . . 424 VIII. Multidrug-Resistant Tuberculosis Treatment . . . . 427

371

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IX. Drug-Resistant Tuberculosis and HIV (and Other Immunosuppressive States) . . . . 445 X. Factors Associated with Good Treatment Outcomes . . . . 447 XI. Summary . . . . 448 References . . . . 449 15. Surgical Treatment of Pulmonary Tuberculosis . . . . . . . . Mikhail I. Perelman I. Historical Background . . . . 459 II. Indications for Surgery . . . . 462 III. Contraindications . . . . 467 IV. Types of Operation . . . . 468 V. Conclusion . . . . 479 References . . . . 480

459

SECTION III: CONTROL OF TUBERCULOSIS—BASIC PRINCIPLES AND TOOLS 16. History of Tuberculosis Control . . . . . . . . . . . . . . . . . . 483 John A. Sbarbaro and Sergio Spinaci I. Introduction . . . . 483 II. Compulsory Isolation and the Beginnings of TB Control in the United States . . . . 484 III. The Origins of the Tuberculosis Clinic . . . . 484 IV. The Impact of Effective Chemotherapy . . . . 485 V. Nonadherence and the Introduction of Directly Observed Treatment . . . . 486 VI. Preventive Treatment: Expanding the Role of Chemotherapy in the United States . . . . 487 VII. The Tuberculin Skin Test . . . . 488 VIII. Tuberculosis Control in Europe . . . . 489 IX. Tuberculosis Control in Developing Countries . . . . 491 X. Advancing the Goals of Tuberculosis Control . . . . 493 XI. The Impact of Social Trends upon TB Control . . . . 495 XII. Conclusion . . . . 496 References . . . . 497

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17. Tuberculosis Control Interventions: A Stepwise Approach . . . . . . . . . . . . . . . . . . . . . . . . . . Antonio Pio I. Concepts of Tuberculosis Control, Elimination, and Eradication . . . . 501 II. Overview of Tuberculosis Control Interventions . . . . 502 III. The Global Stop TB Strategy . . . . 502 IV. Case Management Intervention . . . . 504 V. Case Management Impact on the Risk of Infection . . . . 504 VI. Case Management Impact on Morbidity Incidence . . . . 505 VII. Case Management Impact on Case Fatality and Mortality . . . . 506 VIII. Specific Tuberculosis-Control Interventions Other than Case Management . . . . 507 IX. Bacille Calmette–Gue´rin Immunization . . . . 507 X. Chemoprophylaxis . . . . 507 XI. Nonspecific Tuberculosis-Control Interventions . . . . 509 XII. A Stepwise Approach to Implementation of Tuberculosis-Control Interventions . . . . 510 XIII. Summary . . . . 517 References . . . . 517

501

18. The Laboratory Network in Tuberculosis Control in High-Prevalence Countries . . . . . . . . . . . . . . . . . . . . 521 Adalbert Laszlo, Isabel N. de Kantor, and Richard Urbanczik I. The Concept of Diagnosis in Tuberculosis Control . . . . 521 II. Diagnosis as a Strategy of the NTP . . . . 523 III. TB Laboratory Network Technical Profile . . . . 525 IV. TB Laboratory Network Organizational Profile . . . . 525 V. Resources of the NTP Laboratory Network . . . . 527 VI. Management of Laboratory Supplies . . . . 530 VII. Training and Human Resource Development . . . . 531 VIII. Quality Assurance in the Laboratory Network . . . . 534 IX. Evaluation . . . . 537 References . . . . 539

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19. BCG Vaccines: History, Efficacy, and Policies . . . . . . . . Anne Fanning and Mark FitzGerald I. Introduction . . . . 541 II. History of Bacille Calmette Gue´rin Development . . . . 541 III. Vaccine Efficacy Trials . . . . 542 IV. Global Immunization Practices . . . . 548 V. World Health Organization Bacille Calmette Gue´rin Policy . . . . 549 VI. Hope for Future Vaccines . . . . 551 References . . . . 552

541

20. The Role of Contact Tracing in Low- and High-Prevalence Countries . . . . . . . . . . . . . . . . . . . . . . Sue C. Etkind and Jaap Veen I. Introduction . . . . 555 II. Definitions . . . . 556 III. Contact-Tracing Objectives . . . . 557 IV. Contact Tracing in Low-Prevalence Countries . . . . 558 V. Contact Tracing in High-Prevalence Countries . . . . 568 VI. New Technologies . . . . 574 VII. Summary . . . . 576 References . . . . 577

555

21. Managing Tuberculosis Patients: The Centrality of Nurses . . . . . . . . . . . . . . . . . . . . . . . 583 Virginia G. Williams, Chantelle Allen, and Sirinapha Jittimanee I. Introduction . . . . 583 II. Role of Nurses in Tuberculosis Control . . . . 586 III. Conclusion . . . . 595 References . . . . 595 22. Involving Community Members in Tuberculosis Care and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597 Dermot Maher, Jeroen van Gorkom, and Francis Adatu-Engwau I. Introduction . . . . 597 II. Definition of Terms . . . . 598 III. Background . . . . 599

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IV. Review of Published Experience . . . . 601 V. Principles of Community Contribution to Tuberculosis Care as Part of NTP Activities . . . . 606 VI. The Future . . . . 611 References . . . . 613 23. Molecular Epidemiology: Its Role in the Control of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . 617 Marcel A. Behr and Kevin Schwartzman I. Introduction . . . . 617 II. IS6110-Based RFLP . . . . 618 III. Spoligotyping . . . . 627 IV. Mycobacterial Interspersed Repetitive Units—Variable Number of Tandem Repeats . . . . 629 V. Molecular Epidemiology in Suspected Outbreaks . . . . 631 VI. Community-Level Studies . . . . 633 VII. Concluding Thoughts and Future Directions . . . . 642 References . . . . 644 24. Economic and Financial Aspects of Global Tuberculosis Control . . . . . . . . . . . . . . . . . . . . . . . . . . 649 Katherine Floyd I. Introduction . . . . 649 II. Overview of Economic and Financial Analyses Related to TB Control Undertaken in Recent Years . . . . 654 III. Major Results from Two Recent Cost-Effectiveness Studies . . . . 665 IV. Recent Trends in Financing of TB Control and Projected Needs for the Decade 2006–2015 . . . . 671 V. What New Work Is Needed in the Next 5 to 10 Years? . . . . 677 References . . . . 680 25. Advancing and Advocating Tuberculosis Control Globally Through the Stop Tuberculosis Partnership . . . . . . . . . . . Petra I. Heitkamp and Marcos A. Espinal Fuentes I. Introduction . . . . 685 II. Role of Partnerships and Advocacy . . . . 685

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III. Advocacy, Communication, and Social Mobilization . . . . 687 IV. The Stop Tuberculosis Partnership . . . . 690 V. Characteristics of a Successful Partnership . . . . 694 VI. Conclusion . . . . 701 References . . . . 701 26. The Global Drug Facility: A Revolution in Tuberculosis Control . . . . . . . . . . . . . . . . . . . . . . . . Virginia C. Arnold and Ian M. Smith I. Introduction . . . . 705 II. History of the GDF . . . . 706 III. Operating Mechanisms of the GDF . . . . 707 IV. Achievements . . . . 708 V. Future Challenges and Opportunities . . . . 711 VI. Conclusion . . . . 713 References . . . . 714

705

Index . . . . I-1 PART B SECTION IV: CONTROL OF TUBERCULOSIS—TAILORING TUBERCULOSIS CONTROL 27. Fundamentals of Tuberculosis Control: The DOTS Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . 717 Fabio Luelmo, Leopold Blanc, and Donald A. Enarson I. Introduction . . . . 717 II. Principles of Tuberculosis Control . . . . 718 III. A Strategy Called DOTS . . . . 719 IV. How the DOTS Strategy Has Been Expanded: From the London Meeting to the Global DOTS Expansion Plan . . . . 720 V. The Expanded DOTS Framework for Effective Tuberculosis Control . . . . 721 VI. The Success of the DOTS Strategy Well Applied . . . . 724 VII. Threats to Progress in the Fight Against Tuberculosis and the Way Forward . . . . 726 References . . . . 727

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28. Tuberculosis Control in the Countries of Eastern Europe and the Former Soviet Union . . . . . . . . . . . . . . . . . . . . . . . . . . 731 Malgorzata Grzemska and Richard Zaleskis I. Introduction . . . . 731 II. Historical Review of TB Control in the Countries of Eastern Europe and the Former Soviet Union . . . . 732 III. Epidemiology . . . . 733 IV. Adaptation to the International Standards . . . . 736 V. Applying International Standards . . . . 737 VI. Policy Development with Examples from Countries . . . . 738 VII. Challenges . . . . 739 VIII. Conclusions . . . . 743 References . . . . 743 29. Tuberculosis Control in Low-Prevalence Countries of Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . Giovanni Battista Migliori and Rosella Centis I. The Framework for Tuberculosis Control: An Evolving Strategy . . . . 747 II. Definitions . . . . 749 III. Context: New Challenges for Tuberculosis Control in Low-Incidence Countries . . . . 750 IV. Aims of the Elimination Strategy . . . . 755 V. Approach to Control and Eliminate Tuberculosis . . . . 755 VI. Prerequisites to Implementation of the European Framework . . . . 758 VII. Conclusions . . . . 763 References . . . . 764

747

30. Tuberculosis in the United States: Toward Elimination? . . . . . . . . . . . . . . . . . . . . . . . . . . 767 Michael F. Iademarco and Kenneth G. Castro I. Introduction . . . . 767 II. Factors Associated with the Tuberculosis Epidemic in the United States . . . . 768 III. The Response to the Epidemic and Associated Reversal in Trend . . . . 769

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IV. The Choice Between Elimination and Stagnation: Another Cycle of Neglect? . . . . 775 V. The Way Forward . . . . 776 VI. Summary . . . . 783 References . . . . 784 31. Tuberculosis Transmission and Infection Control in Congregate Settings . . . . . . . . . . . . . . . . . . . . . . . . . Edward A. Nardell and Kevin P. Fennelly I. Introduction . . . . 793 II. Tuberculosis Transmission . . . . 796 III. TB Infection Control in Low-Prevalence, Resource-Rich Settings . . . . 803 IV. High-Risk, Resource-Limited Settings . . . . 811 V. TB Infection Control in Prisons . . . . 815 VI. Summary . . . . 816 References . . . . 817

793

32. Tuberculosis Drug Resistance in the World . . . . . . . . . . . 823 Franc¸oise Portaels, Leen Rigouts, Isdore Chola Shamputa, Armand Van Deun, and Mohamed Abdel Aziz I. Introduction . . . . 823 II. Extent of the Worldwide Drug Resistance Problem . . . . 825 III. Drug-Resistance Surveillance . . . . 832 IV. Causes of (Multi)drug Resistance and Risk Factors for Its Development . . . . 834 V. Role of the Laboratory Activities in Drug-Resistance Surveillance . . . . 838 References . . . . 840 33. Programmatic Control of Multidrug-Resistant Tuberculosis 845 Peter Cegielski, Kai Blo¨ndal-Vink, Kitty Lambregts-van Weezenbeek, and Ernesto Jaramillo I. Background and Introduction . . . . 845 II. Drug-Resistant TB: Definitions and Program Implications . . . . 847 III. Etiology of Drug Resistance and Program Implications . . . . 850 IV. Epidemiology of Drug-Resistant Tuberculosis . . . . 851 V. The Global Response to Multidrug-Resistant Tuberculosis . . . . 855

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VI. The Framework for Multidrug-Resistant Tuberculosis Control . . . . 858 VII. Emerging Issues in Multidrug-Resistant Tuberculosis Control Programs . . . . 861 VIII. Summary and Conclusions . . . . 864 References . . . . 865 34. Tuberculosis Control and Migration . . . . . . . . . . . . . . . . Suzanne Verver and Jaap Veen I. Introduction . . . . 869 II. History of Migration of TB . . . . 870 III. Epidemiology . . . . 872 IV. Contribution of Migration to Transmission in the Host Country . . . . 884 V. Interventions . . . . 885 VI. Legal Aspects of TB Control Among Immigrants . . . . 895 VII. Epilogue . . . . 895 References . . . . 896

869

35. Tuberculosis Control in Refugee and Displaced Populations . . . . . . . . . . . . . . . . . . . . . . 907 Michelle Gayer and Ma´ire A. Connolly I. Introduction . . . . 907 II. The Changing Context of Conflict Situations . . . . 908 III. Risk Factors . . . . 908 IV. Burden of TB . . . . 909 V. Constraints to Implementing TB Control Programs for Refugee and Displaced Populations . . . . 911 VI. Management of TB in Refugee and Displaced Populations . . . . 914 VII. TB Control Successes in Refugee and Displaced Populations . . . . 915 VIII. Challenges for the Future . . . . 916 IX. Conclusions . . . . 917 References . . . . 918 36. Tuberculosis Control in Prisons . . . . . . . . . . . . . . . . . . . Michael E. Kimerling I. Introduction: Two Sides of the Wall . . . . 921 II. Access to Adequate TB Care and Human Rights in Prisons . . . . 922

921

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III. Prisons as Special Communities . . . . 923 IV. Epidemiology of TB in Prisons: The Convergence of Risk Groups and a Disproportionate Burden of Disease . . . . 923 V. The Revolving Door of Prisons and Links to the General Community . . . . 928 VI. Establishing DOTS Programs in Prisons . . . . 930 VII. Conclusions and Challenges Ahead . . . . 944 References . . . . 945 37. Tuberculosis Control in Mines . . . . . . . . . . . . . . . . . . . Gavin J. Churchyard and Elizabeth L. Corbett I. Introduction . . . . 949 II. Epidemiology . . . . 950 III. Tuberculosis Control . . . . 957 IV. Conclusion . . . . 960 References . . . . 961

949

SECTION V: NEW CHALLENGES FOR A NEW CENTURY 38. Programmatic Management of Human Immunodeficiency Virus–Associated Tuberculosis . . . . . . . . . . . . . . . . . . . 967 Anthony D. Harries and Paul Nunn I. Introduction . . . . 967 II. Global Burden of TB and HIV Infection . . . . 967 III. Current Interaction Between Tuberculosis and AIDS Programs . . . . 968 IV. Strategic Work to Decrease the Burden of TB–HIV . . . . 972 V. Principles, Policies, and Guidelines for Implementing Collaborative TB–HIV Activities . . . . 972 VI. General Overview of Initiatives to Scale Up Antiretroviral Treatment in Resource-Poor Countries . . . . 977 VII. TB as an Entry Point to Antiretroviral Therapy: Benefits and Risks for TB Control . . . . 979 References . . . . 980 39. Engaging Private Providers in Tuberculosis Control: Public–Private Mix for DOTS . . . . . . . . . . . . . . . . . . . Mukund Uplekar and Knut Lo¨nnroth I. Introduction . . . . 985 II. Global Assessment . . . . 986

985

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III. What Makes Public–Private Mix for DOTS Work? . . . . 989 IV. Evidence Base . . . . 994 V. Economic Analysis . . . . 998 VI. Scaling Up Public–Private Mix for DOTS . . . . 999 VII. Public–Private Mix for DOTS and the Millennium Development Goals . . . . 1001 References . . . . 1002 40. Controlling Tuberculosis in Large Metropolitan Settings . . . . . . . . . . . . . . . . . . . . . . . . . 1005 Knut Lo¨nnroth, Matteo Zignol, and Mukund Uplekar I. Introduction . . . . 1005 II. Rapid Urbanization and Sprawling Slums . . . . 1006 III. Urban TB Epidemiology . . . . 1007 IV. Major Barriers to TB Control in Large Cities . . . . 1010 V. Two Examples of TB Control in Large Cities . . . . 1013 VI. A Provisional Framework for TB Control in Large Cities . . . . 1020 References . . . . 1024 41. Health Education and Social Mobilization in Tuberculosis Control . . . . . . . . . . . . . . . . . . . . . . . . . . 1029 Roberto Tapia-Conyer and Ernesto Jaramillo I. Introduction . . . . 1029 II. Health Education in TB Control . . . . 1030 III. Social Mobilization in TB Control . . . . 1032 IV. Promoting Social Mobilization: The Experience of the National TB Control Program of Mexico . . . . 1035 V. The Impact of Social Mobilization . . . . 1037 References . . . . 1038 42. Workforce Constraints in Tuberculosis Control . . . . . . . . 1041 Gijs Elzinga, Gilles Dussault, and Jose´ I. Figueroa I. Introduction . . . . 1041 II. Global Tuberculosis Control and Health Workforce Constraints . . . . 1041 III. Human Resources for Health Constraints and Tuberculosis Control Targets . . . . 1042

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IV. Positioning Tuberculosis Programs Within the Health System . . . . 1045 V. Strategies and Policies Required . . . . 1049 VI. Conclusions . . . . 1055 References . . . . 1057 43. The Practical Approach to Lung Health Strategy for Integrated Respiratory Care . . . . . . . . . . . . . . . . . . . . . 1059 Salah-Eddine Ottmani and Jaouad Mahjour I. Introduction . . . . 1059 II. Burden of Respiratory Illnesses in Populations . . . . 1060 III. Demand of Care for and Management of TB and Other Respiratory Illnesses in PHC Settings . . . . 1061 IV. Objectives of the PAL Strategy . . . . 1064 V. Components of the PAL Strategy . . . . 1065 VI. Adaptation of the PAL Strategy . . . . 1066 VII. Steps to Introduce the PAL Strategy in Countries . . . . 1067 VIII. Preliminary Results from Country Experiences . . . . 1075 IX. Perspectives of the PAL Strategy . . . . 1078 X. Conclusion . . . . 1079 References . . . . 1080 44. The Responsibilities of Medical and Nursing Schools in Tuberculosis Care and Control in Countries with Medium and High Tuberculosis Incidence . . . . . . . . . . . . 1083 Pierre Chaulet and Noureddine Zidouni I. Introduction . . . . 1083 II. The Social Responsibility of Training Institutes . . . . 1084 III. Limitations of the Traditional Approach to the Teaching of TB . . . . 1084 IV. Introducing Innovative Teaching Techniques in TB Control . . . . 1085 V. Basic Training for Health-Care Workers . . . . 1087 VI. Training of Trainers . . . . 1090 VII. Participation in Continuous Training . . . . 1093

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VIII. Building Partnerships to Assess the Impact of Training on the Performance of National Programs . . . . 1093 IX. Enhancing the Quality of Training and Stemming the Outflow of Trained Staff . . . . 1094 References . . . . 1094 45. Tuberculosis in the Poverty Alleviation Agenda . . . . . . . . 1097 Christy L. Hanson, Diana E. C. Weil, and Katherine Floyd I. Introduction . . . . 1097 II. Associations Between TB and Poverty . . . . 1099 III. TB Control and the Poverty-Reduction Agenda . . . . 1106 IV. Conclusions . . . . 1110 References . . . . 1111 SECTION VI: BUILDING THE FUTURE 46. New Diagnostics for Tuberculosis: An Essential Element for Global Control and Elimination . . . . . . . . . . . . . . . . . . . 1115 Mark D. Perkins and Richard J. O’Brien I. Introduction . . . . 1115 II. The Need for Improved Diagnostics . . . . 1116 III. Obstacles to TB Diagnostic Development . . . . 1118 IV. TB Diagnostic Priorities . . . . 1120 V. Characteristics of Needed Tests . . . . 1122 VI. TB Diagnostics Currently in the Development Pipeline . . . . 1122 VII. Public–Private Partnerships and a Development Strategy . . . . 1128 VIII. Conclusion . . . . 1130 References . . . . 1130 47. New Drugs for Tuberculosis . . . . . . . . . . . . . . . . . . . . . 1135 Ann Ginsberg and Melvin Spigelman I. Introduction . . . . 1135 II. Treatment of Active Tuberculosis . . . . 1135 III. Treatment for MDR-TB . . . . 1136 IV. Treatment of Active TB in Individuals Infected with HIV . . . . 1137

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V. Treatment of Latent TB . . . . 1137 VI. The Global Alliance for TB Drug Development . . . . 1137 VII. The Drug Development Process . . . . 1138 VIII. The Emerging Global Tuberculosis Drug Portfolio . . . . 1139 IX. Future Prospects in TB Drug Development . . . . 1148 References . . . . 1149 48. The Future of Tuberculosis Vaccinology . . . . . . . . . . . . . 1153 Ruth Griffin and Douglas Young I. The TB Vaccine Challenge: To Improve on Bacille Calmette–Gue´rin . . . . 1153 II. The Genome and New Vaccine Candidates . . . . 1155 III. Clinical Trials . . . . 1161 References . . . . 1164 49. Research Priorities in Tuberculosis . . . . . . . . . . . . . . . . 1169 Philip Onyebujoh, William Rodriguez, and Alimuddin Zumla I. Background and Introduction . . . . 1169 II. Specific TB Research Priority Areas . . . . 1176 References . . . . 1223 50. The New Stop TB Strategy of WHO: Reaching Global Targets . . . . . . . . . . . . . . . . . . . . . . . 1227 Mukund Uplekar, Diana E. C. Weil, and Mario C. Raviglione I. Introduction . . . . 1227 II. Challenges and Opportunities . . . . 1228 III. Goals and Targets . . . . 1230 IV. Components of the Stop TB Strategy . . . . 1232 V. Measuring Global Progress and Impact . . . . 1241 VI. Conclusion . . . . 1243 References . . . . 1244 Index . . . . I-1

SECTION I: BASIC ASPECTS OF TUBERCULOSIS

1 The Global Tuberculosis Epidemic: Scale, Dynamics, and Prospects for Control

CHRISTOPHER DYE Stop TB Department, World Health Organization, Geneva, Switzerland

I. Introduction Drugs that can cure most tuberculosis (TB) patients have been available since the 1950s, yet TB remains the world’s second most important cause of death from an infectious agent, after the human immunodeficiency virus (HIV) (1). TB control is high on the international public health agenda, not just because of the enormous burden of the disease, but also because short-course chemotherapy is recognized to be among the most cost effective of all health interventions (2–4). This evidence has been central to the global promotion of the DOTS strategy, the package of measures combining best practices in the diagnosis and treatment of patients with active TB, in which direct observation during treatment is a key element (5,6). This chapter provides an update on epidemiological burden and trends, and an overview of the documented and potential impact of DOTS as the principal method of control. But the scope of the discussion is intended to be broader in two respects. First, to contain the TB epidemic, and to show that it has been contained, requires strong, quantitative epidemiology. Epidemiological methods are needed, not only to measure the size and direction of the TB epidemic, but also to explain what is measured. 1

2

Dye

Second, the DOTS strategy as originally formulated may not be sufficient on its own to bring TB under control and drive the disease toward elimination. Numerous regional variations in TB epidemiology, and in the organization of health care, mean that the core DOTS strategy needs to be enhanced in different ways in different parts of the world. In sub-Saharan Africa, a huge proliferation of TB patients is associated with the spread of HIV infection. Countries of the former Soviet Union have especially high rates of drug resistance, a problem undoubtedly exacerbated by the deterioration of health and health care during the 1990s. In Asia, a relatively high proportion of TB patients first seek help from private practitioners rather than from the public health system, which underlines the importance of forming links between the two. On top of this, multimillion-dollar initiatives were launched around the turn of the millennium to develop better diagnostics, drugs, and vaccines, operating under the umbrella of the Stop TB Partnership (8). Some of the products of this new research, especially a new high-efficacy vaccine, could stimulate changes to the way in which TB control is done. II. Global and Regional TB Epidemics: Scale and Dynamics Based on surveys of the prevalence of infection and disease, on assessments of the performance of surveillance systems, and on death registrations, there were an estimated 8.9 million new cases of TB in 2004, fewer than half of which were reported to public health authorities and World Health Organization (WHO). Approximately 3.9 million cases were sputum-smear positive, the most infectious form of the disease (5,8,9). The WHO African region has the highest estimated incidence rate (356 per 100,000 population per year), but the majority of TB patients live in the most populous countries of Asia: Bangladesh, China, India, Indonesia, and Pakistan together account for half the new cases arising each year (Fig. 1). About 80% of new cases arising each year occur in the 22 top-ranking countries. TB is predominantly a disease of adult men. In regions where the transmission of Mycobacterium tuberculosis has been stable or increasing for many years, the incidence rate is relatively high among infants and young adults, and most cases are due to recent infection or reinfection. As transmission falls, the caseload shifts to older adults, and a higher proportion of cases come from the reactivation of latent infection. Therefore, in the countries of Western Europe and North America that now have low incidence rates, indigenous TB patients tend to be elderly, whereas patients who are immigrants from high-incidence countries tend to be young adults. Allowing for the difficulties of diagnosing childhood TB, estimation exercises indicate that there are relatively few cases among 0 to 14 year olds, even in areas of high transmission (18% of all new cases in Africa in 2004, but only 4% in the established market economies). In 2004, countries reported 1.4 million sputum-smear TB cases among men, but only 775,000 among women. In some instances, women have poorer

The Global Tuberculosis Epidemic

3

Figure 1 Distribution of TB in the world in 2004. Maps show (A) the estimated numbers of new TB cases (all forms) by country, and (B) the incidence per 100,000 population. Source: From Ref. 5.

access to diagnostic facilities (10), but the broader pattern also reflects real epidemiological differences between the sexes: although there is some evidence that young adult women (15–44 years) are more likely than men to develop active TB following infection, this effect is typically outweighed by the much higher exposure and infection rates among adult men (11–14). Although the TB incidence rate appears to be growing slowly in the world as a whole, incidence rates have been steady or falling for at least two decades in the Southeast Asia and Western Pacific Regions, and in Western and Central Europe, North and Latin America, and the Middle

4

Figure 2 (Caption on facing page )

Dye

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East (as judged from trends in case notifications; Fig. 2). The global increase is attributable to the striking proliferation of cases in countries of Eastern Europe (mainly the former Soviet Union) since 1990 and in subSaharan Africa (regions with high and low rates of HIV infection) since the mid-1980s. However, trends in case reports suggest that the rate of increase in both regions has slowed significantly since the mid-1990s, and the incidence in Eastern Europe may now be in decline (5,6). The downturn in case notifications in Eastern Europe is clear in data from Russia, Belarus, and the Baltic States of Estonia, Latvia, and Lithuania, whereas incidence rates appear still to be increasing in the central Asian republics of Kazakhstan, Tajikistan, and Uzbekistan (5). Summing up the figures obtained across the nine regions depicted in Figure 2 gives the global trend in incidence; the case rate per capita was increasing most quickly at 1.5% per year in 1995, but, because of the dynamics in Africa and Eastern Europe, has since been decelerating. The continued increase in TB incidence rate worldwide from 2004 onwards is entirely due to the increase in Africa. The resurgence of TB in Eastern European countries can be explained by economic decline and the failure of TB control and other health services since 1991 (15). Based on periodic surveys, more than 10% of new TB cases in Estonia, Latvia, and some parts of the Russia are multidrug-resistant (MDR) TB, i.e., resistant to at least isoniazid and rifampicin, the two most effective antituberculosis drugs (16,17). Drug resistance is likely to be a by-product of the events that led to TB resurgence in these countries, not the primary cause of it, for three reasons. First, resistance is generated initially by inadequate treatment due, for example, to interruption of the treatment schedule, the use of low-quality drugs, or the use of high-quality drugs at low dosage. Second, resistance tends to build up over many years, even though there was a sudden increase in TB incidence in Eastern European countries after 1991. And third, although formal calculations have not been done, resistance rates are probably too low to attribute all of the

Figure 2 (Figure on facing page) Trajectories of the tuberculosis epidemic for nine epidemiologically different regions of the world. Points mark trends in incidence rates, derived from case notifications for 1990–2004. The two panels separate regions with estimated incidence rates (A) above or (B) below the global average in 1990 (heavy line). Groupings of countries are based on the WHO regions of Africa (subdivided into two regions comprising those countries with high HIV-infection rates, more than or equal to 4% of patients aged 15 to 49 years, in 2004, and those with low rates of HIV infection, less then 4%), Central Europe, Eastern Europe (former Soviet countries plus Bulgaria and Romania), Eastern Mediterranean, Established Market Economies [all 30 Organization for Economic Cooperation and Development (OECD)] countries, except Mexico, Slovakia, and Turkey, plus Singapore, Latin America, Southeast Asia, and Western Pacific. The countries in each region are listed in full elsewhere. Source: Adapted from Ref. 6.

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increase in caseload to excess transmission from treatment failures. While parts of Eastern Europe are clearly hotspots for MDR-TB, only 4–6% of TB cases arising each year (new and previously treated) are estimated to be MDR (18,19) though the frequency among previously treated cases is higher. Much of the recent increase in global TB incidence can be explained by the spread of HIV in Africa (5,6,9). Globally, an estimated 13% of new adult tuberculosis cases were infected with HIV in 2004, but there was marked variation among regions—from 33% in sub-Saharan Africa to 1.4% in the Western Pacific Region. HIV infection rates in TB patients have so far remained below 1% in Bangladesh, China, Indonesia, and Pakistan. In African populations with higher rates of HIV infection, a higher proportion of TB patients are women 15 to 24 years (5,9). Across Africa, the rise in the number of TB cases is slowing (though this may not be true in every country), perhaps because HIV infection rates are also beginning to stabilize or fall (20), and perhaps also because TB surveillance systems have been overloaded by patients. HIV has probably had a smaller effect on TB prevalence than on incidence because the duration of TB among HIVinfected patients is relatively short, with a rapid onset of severe illness and a marked reduction in life expectancy (21). In places where HIV infection rates are high in the general population, they are even higher among TB patients; estimates for 2004 exceeded 50% in Botswana, Malawi, South Africa, Zambia, and Zimbabwe, among other countries. Approximately 1.7 million people died of tuberculosis in 2004, including 248,000 patients who were coinfected with HIV (6,9). Although these are usually reported as AIDS deaths under the International Classification of Diseases-10 and by WHO, TB control programs need to know the total number of TB deaths, whatever the underlying cause. Because few countries with high burdens of TB compile reliable statistics on the cause of death, the global and regional trends in TB deaths are uncertain. However, recent assessments based on modeling have suggested that the global TB mortality rate began to fall around year 2000, after growing during the 1990s (5,6).

III. Tuberculosis Control The methods of TB control are to prevent infection, to stop progression from infection to active disease, and to treat active disease. Roughly, 100 million infants (more than 80% of the annual cohort) are vaccinated each year with bacille Calmette–Gue´rin (BCG), and the effect of this vaccine is mainly to prevent serious forms of disease in children—meningitis and miliary TB. The most complete analysis of effectiveness to date suggests that BCG given to children worldwide in 2002 will have prevented approximately 30,000 cases of childhood meningitis and about 11,500 cases of miliary TB during their first five years, or 1 case for every 3400 and 9300 vaccinations, respectively (22). The protective efficacy against pulmonary tuberculosis in adults is highly variable, and often very low (23).

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Individuals at high risk of TB who have a positive tuberculin skin test but not active disease (e.g., associates of active cases, especially children, and immigrants to low-incidence countries) can be offered preventive therapy (also called treatment of latent TB infection), most commonly with the relatively safe and inexpensive drug, isoniazid (isoniazid preventive therapy, IPT). Randomized, controlled, clinical trials have shown that 12 months of daily isoniazid gives 25% to 92% protection against developing active TB (range of point estimates),but towards the upper end of this range when patients adhere fully to the treatment regimen (Chapter 10). However, IPT is not widely used, mainly because compliance with long-term daily treatment tends to be poor among healthy people—a relatively high risk of TB among people carrying latent infections is usually still a low risk in absolute terms. The exceptionally high risk of TB among people coinfected with M. tuberculosis and HIV is a reason for encouraging wider use of IPT, especially in Africa. Among the control methods that are possible with current technology, only the treatment of active disease has so far been implemented and shown to be effective on a large scale. The cornerstone of TB control at the start of the 21st century is the prompt treatment of active cases with short-course chemotherapy using first-line drugs that are administered via the DOTS strategy with targets framed by the United Nations Millennium Development Goals (MDGs) (Table 1) (5,6). Because of the importance of treating active TB, now and for the foreseeable future, the following discussion focuses on chemotherapy, delivered via the DOTS strategy and its extensions. Table 1 Goals, Targets, and Indicators for Tuberculosis Control Millennium Development Goal 6 Combat HIV/AIDS, malaria and other diseases Target 8: To have halted by 2015 and begun to reverse the incidence of malaria and other major diseases Indicator 23: Prevalence and death rates associated with TB Indicator 24: Proportion of TB cases detected and cured under DOTS (the basis of the WHO recommended Stop TB Strategy) Stop TB Partnership targets By 2005: At least 70% of people with sputum-smear positive TB will be diagnosed (i.e., under the DOTS strategy), and at least 85% cured. These are targets set by the World Health Assembly of WHO By 2015: The global burden of TB (prevalence and death rates) will be reduced by 50% relative to 1990 levels. This means reducing prevalence to approximately 150 per 100,000 or lower and deaths to approximately 15 per 100,000 per year or lower by 2015 (including TB cases coinfected with HIV). The number of people dying from TB in 2015 should be less than approximately one million, including those coinfected with HIV By 2050: The global incidence of TB will be less than one case per million population per year Source: From Ref. 24.

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The core DOTS strategy has five elements: political commitment, diagnosis primarily by sputum-smear microscopy among patients attending health facilities, short-course chemotherapy with effective case management (including direct observation), a regular supply of quality-assured anti-TB drugs, and systematic monitoring to evaluate the outcomes for every patient started on treatment. Standard short-course chemotherapy can cure more than 90% of new, drug-susceptible tuberculosis cases, and high cure rates are a prerequisite for expanding case finding (25,26). Although the DOTS strategy aims primarily to provide free treatment for smear-positive patients, most DOTS programs also treat smear-negative patients, often without a fee. DOTS is increasingly being used as the basis for more complex interventions, for example, where rates of drug resistance or HIV infection are high, as reflected in connection with the Stop TB Strategy (27) (Table 2). The internationally agreed targets for TB control, embraced by the United Nations MDGs, are to detect 70% of sputum-smear–positive cases and successfully treat 85% of such cases by the end of 2005 (Table 1). Mathematical modeling (based mainly on European and North American data) suggests that the incidence rate of TB will decline at 5% to 10% per year when these targets are met, even though this represents a treatment success among all infectious cases of only 60% (25,26,28). These expectations do not, however, allow for the complications associated with HIV coinfection and drug resistance. For example, the impact of DOTS will be less than that suggested above when HIV is spreading through a population. Nor do the anticipated reductions in TB incidence account for changes in risk attributable to factors such as air pollution (29,30), tobacco smoke (31,32), diabetes (33–35), malnutrition and undernutrition (36), and alcohol abuse (37). In principle, TB incidence could be forced down more quickly than seen in Europe and North America, by as much as 30% per year, if new cases could be found soon enough to eliminate transmission. In general, the decline is faster when a larger fraction of cases arises from recent infection (i.e., in areas where transmission rates have recently been high) and slower where there is a large backlog of asymptomatic (latent) infection, and where rates of reactivation are higher among latently infected people. These facts explain why it should be easier to control epidemic than endemic disease: during an outbreak in an area that previously had little TB, the reservoir of latent infection is small, and most new cases come from recent infection. In practice, the best results in the control of endemic TB by chemotherapy (largely) have been achieved in native communities (Inuit and others) of Alaska, Canada, and Greenland, where the incidence rate was reduced by 13% to 18% annually from the early 1950s (26). Over a much wider area in Western Europe, TB declined at 7% to 10% per year after drugs became available during the 1950s, though the incidence rate was already falling at 4% to 5% per year before chemotherapy.

Global Plan to Stop TB, 2006–2015 Budget for specific activities including Increased case detection and treatment success toward and beyond target levels Enhanced advocacy, communication, and social mobilization Strengthened laboratory services for microscopy, culture, and DST Improved case management including directly observed treatment and patient support to increase adherence and chance of cure, and to lower risk of acquiring drug resistance Regular supply of high-quality drugs; improvement of drug management capacity Technical assistance, including monitoring and evaluation Contributing to case finding, treatment success, and TB prevention TB/HIV control including active TB case finding among HIV-infected people, treatment of latent infection for HIV-infected people without active TB, antiretroviral therapy, and cotrimoxazole preventive therapy; MDR-TB management including expanded DST and treatment with second-line drug regimens Investment in staff, infrastructure, health information, and management, oriented to TB but more widely applicable; investment in syndromic management of adult respiratory disease Improved links between public, non-governmental, and private health care systems Community TB care; advocacy, communication, and social mobilization New diagnostics and drags (new vaccine not expected before 2015)

Abbreviations: DST, drug susceptibility testing; MDR-TB, multidrug-resistant TB.

Engage all care providers Empower patients and communities Enable and promote research

Contribute to health system strengthening

Drug supply Monitoring and impact evaluation Additional components Address TB/HIV, multidrug resistant TB (MDR-TB) and other challenges

Principal components DOTS expansion and enhancement Political commitment Case detection through bacteriology Standardized treatment, with supervision and patient support

Stop TB Strategy

Table 2 Elements of the Global Plan to Stop TB, 2006–2015, Underpinned by the Revised Stop TB Strategy of WHO

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The long-term aim of TB control is to eliminate all new cases [defined as an incidence less than one case per million population per year (24,38)], but cutting prevalence and death rates are arguably more important in the short term. About 86% of the burden of TB, as measured in terms of disabilityadjusted life years lost, is due to premature death rather than illness, and prevalence and mortality can be reduced faster than incidence in chemotherapy programs. For example, the TB death rate among the Alaskan Inuit population dropped by an average of 30% per year in the period 1950–1970. V. Implementation and Impact of DOTS, 1991 to 2005 A. Case Detection

More than 21 million TB patients were diagnosed and treated in DOTS programs between 1994 and 2004. Despite this mounting total, only 53% of all estimated new smear-positive cases were reported by DOTS programs to WHO in 2004, with much variation between regions of the world (Fig. 3A). The case detection rate in DOTS programs has been accelerating globally. The recent improvement in case finding has been due mostly to rapid implementation in India, where detection increased from 1.7% in 1998 to 57% in 2004, and in China, where detection increased from 30% in 2002 to 63% in 2004. In 2004, almost all (91%) TB cases reported to WHO were reported by DOTS programs. Because very few of the undetected TB patients were reported elsewhere in the public health system, DOTS programs must seek other sources of patients in order to reach the 70% target for detection. Among these sources are private practitioners, and public hospitals that have not traditionally been linked to TB clinics (39). B. Treatment Success

Many of the 183 national DOTS programs in existence by the end of 2004 have shown that they can successfully treat a high proportion of patients. The average treatment success among 1.7 million smear-positive patients in the 2003 DOTS cohort was 82%, not far below the 85% target (Fig. 4). Concealed by this high proportion of successful treatments are poorer results in Africa, Eastern Europe, and in the established market economies (Fig. 3). In the African countries most affected by HIV (Africa—high HIV), 7% of patients died during treatment, and 21% were lost to follow-up (defaulted, transferred to other treatment centers, or not evaluated). In the established market economies, the death rate was higher than in any other region (10%), because a large proportion of patients are elderly. In Eastern Europe, where rates of drug resistance are relatively high, 9% of patients failed to respond to treatment and 6% died during treatment. C. Incidence, Prevalence, and Mortality

Although the decline in TB has almost certainly been accelerated by good chemotherapy programs in countries such as Chile, Cuba, and Uruguay, there have been only few recent, unequivocal demonstrations of impact in

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Figure 3 Case detection and treatment failure rates reported by DOTS programs at the end of 2004. (A) Smear-positive case detection rates in 2004 for the nine regions defined in Figure 2. (B) Adverse outcomes of treatment in the 2003 DOTS cohort, for each of the nine regions defined in Figure 2. Source: From Ref. 5.

high-burden countries. Two persuasive examples come from Morocco and Peru. Between 1994 and 2000, the incidence of pulmonary TB among Moroccan children up to four years of age fell by more than 10% per year, suggesting that the risk of infection was falling at least as quickly (Ministry of Health Morocco, unpublished data). Moreover, average age of TB cases has been rising for over 20 years in Morocco. And yet the overall reduction in pulmonary TB was only 4% per year, in part because of the large reservoir of infection in adults. DOTS was launched in Peru in 1991, and high rates of case detection and cure appear to have pushed down the incidence rate of pulmonary TB by 6% per year (40). For epidemic TB, as a result of aggressive intervention following an outbreak in New York City, the number of MDRTB cases fell by over 40% per year (41).

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Figure 4 Progress toward the targets of 70% smear-positive case detection by DOTS programs (filled points, heavy lines) and 85% treatment success (open points, light lines). Points show the measured progress in smear-positive case detection from 1995 to 2004 and treatment success for the 1994 to 2003 cohorts. Lines project changes to 2015 according to Global Plan scenarios 2 (dashed) and 3 (continuous) for improved TB control (case detection by DOTS programs is zero in scenario 1, so treatment success does not apply).

Indirect assessments of the effect of DOTS suggest that 70% of the TB deaths expected in the absence of DOTS were averted in Peru between 1991 and 2000, and more than half the TB deaths expected in the absence of DOTS are prevented each year in DOTS-served provinces of China (40,42). There have been few direct measures of the reduction in TB prevalence over time, but surveys done in China in 1990 and 2000 showed a 32% [95% confidence limits, 9% to 51%] reduction in the prevalence per capita of all forms of TB in DOTS areas, as compared with the change in the prevalence in other parts of the country (43). Preliminary findings from a 2004 national survey in Indonesia indicate that the per capita prevalence of smear-positive TB was threefold lower than in surveys carried out around 1979 and 1982 (Ministry of Health, Indonesia; WHO, unpublished data) (44,45). But not all of this reduction can be attributed to the DOTS program, or even to the direct effects of chemotherapy. VI. Prospects for Tuberculosis Control, 2006 to 2015 and Beyond The prospects for TB control over the next 10 years, implementation and impact, have been set out in the Global Plan to Stop TB, 2006–2015 (46). The Global Plan imagines three scenarios: Scenario 1: No DOTS. This assumes that the strategy was never introduced in any region, so chemotherapy would continue in the same way as it was pre-DOTS, with variable rates of case detection and typically

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lower rates of cure. This gives a baseline against which to compare the gains that have already been made under DOTS, and which might be made in future. Scenario 2: Sustained DOTS. Case detection and treatment success both increase until 2005, and then remain steady until 2015 (Fig. 4). Approximately, 50 million patients would be treated under DOTS between 2006 and 2015, as compared with 21 million in the previous decade, 1996–2005. Scenario 3: Enhanced DOTS. Case detection and treatment success continue to increase beyond 2005, up to 2015 (Fig. 4). As in scenario 2, roughly 50 million patients would be treated between 2006 and 2015 (a higher proportion of patients treated sooner means that, as a result of reduced transmission, there are fewer patients later; Table 3). To reach high rates of case detection and cure requires various additions to the basic DOTS strategy, including community-based care, a syndromic approach to diagnosing and treating TB among other respiratory conditions, and improved collaboration between public and private health sectors. To improve the management of drug-resistant disease, more patients will be given drug sensitivity tests, and around 800,000 MDR-TB patients will be treated with regimens including second-line drugs (Table 3, Fig. 5A). HIV testing and counselling will be provided to 29 million TB patients, and antiretroviral therapy (ART) and cotrimoxazole preventive therapy offered to 3.2 million (Fig. 5B). Approximately 200 million people infected with HIV will be screened for TB, and 24 million will be offered IPT. Analyses carried out for the Global Plan investigate the impact of these additions to DOTS used in varying combinations, and implemented at different rates, in seven endemic regions of the world (i.e., excluding established market economies and Central Europe). The potential impact of scenario 3, as compared with scenarios 1 and 2, has been evaluated with a mathematical transmission model describing, as in previous models (27,47–49), how the planned interventions determine incidence, prevalence, and death rates through time. Model calculations show that scenarios 2 and 3 should both satisfy MDG target 8 ‘‘to have halted and begun to reverse incidence,’’ globally (Fig. 6) and in each of the seven regions (Fig. 7). In fact, the annual incidence of new cases is expected to be in decline well before 2015, even under scenario 2. Ambitious plans for the South-East Asia and Western Pacific regions are reflected in the relatively rapid declines in incidence expected by 2015 (7–9% per year). Even with these rates of decline in Asia, TB incidence would still exceed 10 per 100,000 globally in 2050, which is 100 times greater than the target for TB elimination. The targets of halving prevalence and death rates between 1990 and 2015 are more challenging. Projections suggest that these targets can be met globally with full implementation of the enhanced DOTS strategy (scenario 3, Fig. 6), but not in Africa or Eastern Europe (Fig. 8A). Based on the calculated rate of decline in mortality from 2006 to 2015 in the African countries most affected by HIV (Africa—high HIV), the target death rate (Text continues on page 18.)

DOTS expansion Total number of new ssþ patients treated in DOTS programs (millions) Case detection rate (%) Total number of new ssþ patients successfully treated in DOTS programs (millions) Treatment success rate (%) Total number of new ss-/extra-pulmonary patients treated in DOTS programs (millions) Percentage of new ss-/extra-pulmonary patients treated in DOTS programs Management of drug resistance Total number of detected MDR-TB patients that are treated in DOTS-Plus programs (millions) Percentage of detected MDR-TB patients that are treated in DOTS-Plus programs TB/HIV Total number of PLWHA attending HIV services screened for TB (millions) 2.2 (2.8) 79% 1.9 (2.2) 86% 3.0 (3.9) 77%

0.09 (0.14) 64%

23 (23)

64% 1.8 (2.1) 86% 3.0 (4.6) 67%

0.02 (0.12) 17%

11 (18)

2010

2.1 (3.3)

2006

26 (26)

100%

0.11(0.11)

84%

89% 2.7 (3.2)

82% 1.6 (1.8)

1.8 (2.2)

2015

209 (225)

62%

0.8 (1.3)

77%

81% 30 (39)

78% 17 (21)

21 (27)

2006–2015

Interventions with current technologya

Table 3 Interventions and New Technology to Be Introduced Under the Global Plan to Stop TB, 2006–2015

NA

0.01c

26%

80% 12 (46)b

32% 9 (11)

12 (37.5)b

1996–2005 comparison

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100% 2.6 (35) 7% 3.2 (3.8) 84% 0.3 (0.6) 50%

61% 1.2 (30) 4% 2.5 (3.4) 74% 0.2 (0.5) 40%

57%

0.4 (0.7)

85%

8% 2.9 (3.4)

100% 3.1 (40)

52%

3.2 (6.1)

81%

7% 29 (36)

93% 24 (354)

NA

NA

NA

Numbers in parentheses indicate the denominator. For DOTS expansion it is the number of new TB cases. For DOTS-Plus it is the total number of detected MDR-TB cases. For PLWHA screened for TB, it is the total number of PLWHA. For PLWHA-offered IPT, it is all PLWHA. For TB patients, HIV tested and counseled, it is TB patients treated under DOTS covered by TB/HIV activities. For TB patients enrolled in ART, it is TB/HIV–positive patients known to be eligible for ART. b Assumes values for 2004 and 2005 as for 2003 as published in the Global Plan. c Refers to number of patients approved by WHO’s Green Light Committee from 2000–2004. d HIV services include testing and counseling and HIV treatment and care services. Abbreviations: ART, antiretroviral therapy; IPT, izoniazid preventive therapy; PLWHA, persons living with HIV/AIDS; MDR-TB, multidrug-resistant TB; NA, data not available, but likely to be very low; SSþ, sputum smear-positive TB; SS
, sputum smear-negative TB. Source: From Ref. 42.

a

Percentage of PLWHA screened for TBd Total number of newly diagnosed and eligible PLWHA offered IPT (millions) Percentage of PLWHA offered IPT Total number of TB patients in DOTS programs, HIV tested, and counseled (millions) Percentage of TB patients treated in DOTS programs, HIV tested, and counseled Total number of TB patients (HIV positive and eligible) in DOTS programs enrolled on ART (millions) Percentage of TB patients (HIV positive and eligible) in DOTS programs enrolled an ART

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Figure 5 Expected numbers of people and patients to be treated in components of the Global Plan to Stop TB, 2006–2015, (scenario 3) related to the management of DOTS plus for the management of MDR-TB (A, B) and to the joint management of TB and HIV/AIDS (C, D, logarithmic scale). Numbers of people and patients are summed by region (A, C) and year (B, D). MDR-TB patients are detected by procedures including drug sensitivity testing. In the context of TB/HIV, testing and counseling is with respect to HIV infection. Abbreviations: PLWHA, persons living with HIV/AIDS; ART, antiretroviral therapy; IPT, izoniazid preventive therapy; MDR-TB, multidrug resistant tuberculosis.

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Figure 6 Expected changes in tuberculosis incidence, prevalence, and deaths from 1990 to 2015, assuming full implementation of the Global Plan to Stop TB, 2006–2015. Changes are expressed as rates (upper row) and numbers (lower row, millions), for scenarios 1 (dotted line), 2 (light continuous line), and 3 (heavy continuous line), and with reference to the targets of halving prevalence and death rates between 1990 and 2015 (horizontal dashed lines).

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Figure 7 Expected annual changes in TB incidence rate by 2015 (measured as the difference between 2014 and 2015), assuming full implementation of the Global Plan to Stop TB, 2006–2015 in seven regions of the world.

would not be reached before 2025. If the rate of decline in mortality slows, as it has in Europe and North America, then the target will be reached later than 2025. In Eastern Europe, but not in Africa, prevalence rates are also expected to remain high compared with 1990 levels. In Eastern Europe, a relatively high proportion of patients have chronic TB, which is commonly MDR. In Africa, patients who are infected with HIV do not suffer from TB for long; their illness typically progresses quickly, and they either are cured or die (21). This bleak outlook for TB control in Africa and Eastern Europe arises in large part from the choice of 1990 as the MDG reference year. In that year, TB incidence rates in these two regions were close to their lowest levels for at least half a century, and most of the recent rise in incidence happened during the 1990s. Although the epidemiology of the recent past is not ignored, the impact of Global Plan in the near future is more relevant here. The same projections also show that, over the 10 years from 2006 to 2015, the impact of the enhanced DOTS strategy, assuming full implementation, would be almost as great in Africa and Eastern Europe as in other regions of the world: a reasonable goal in all regions would be to halve prevalence and death rates between 2005 and 2015 (Fig. 8). To that end, the implementation of the enhanced DOTS strategy will be especially important in Africa and Eastern Europe, where the incremental benefits of enhanced DOTS (scenario 3) compared with sustained DOTS (scenario 2) are greatest. Indeed, the proportional reduction in TB cases under scenario 3 (as compared with scenario 2) would be greater in Eastern Europe than any other region (Fig. 9A). The proportional reduction in deaths would be greatest in Africa (high-HIV countries) and Eastern Europe (Fig. 9B). In other regions of the world, a higher proportion of the benefits to be obtained over the next 10 years come from sustaining what has been achieved over the past 10 years, and TB epidemiology in these other regions, notably

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Figure 8 Expected reductions in prevalence and death rates by 2015, as compared with the estimated rates in (A) Millennium Development Goals baseline year 1990, or (B) with the rates in 2005. Although targets for halving prevalence and death rates between 1990 and 2015 (bars < 0.5) are unlikely to be reached in Africa and Eastern Europe (A), the impact of the Global Plan to Stop TB, 2006–2015 is not expected to be much less in these two regions during the period 2006–2015 (B).

Asia, governs the global trend (Fig. 6). Thus, enhanced DOTS (scenario 3), as compared with sustained DOTS (scenario 2), would save only 2.7 million deaths globally over the next decade. But if scenario 3 is considered to be the logical extension of the program of global DOTS expansion that began in the early 1990s, then enhanced DOTS will save 13.7 million deaths between 2006 and 2015 (compared with scenario 1). In continuing this program of

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Figure 9 (A) TB cases and (B) deaths that would be saved through the implementation of the Global Plan to Stop TB, 2006–2015 (comparing scenarios 2 and 3), for seven regions separately and combined. Bars show the percentage of cases and deaths saved; numbers over the bars are millions of cases and deaths saved by scenario 3 in comparison with scenarios 1 (open bars) and 2 (solid bars).

DOTS expansion, most cases and deaths saved will be in the Southeast Asian and Western Pacific regions (Fig. 9). Two outcomes of the analyses for Africa and Eastern Europe are not visible in the summary statistics (Figs. 6–9). First, while expanding DOTS

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Figure 10 Expected changes in the incidence rate of TB in region ‘‘Africa—high HIV’’ under the Global Plan to Stop TB, 2006–2015 (scenario 3; heavy line), and the different trends in incidence among people with (light line) and without HIV infection (dotted line).

programs are not expected to bring down the overall incidence of TB in eastern and southern Africa (Africa—high HIV) before 2005, the incidence in the HIV-negative population has been, according to model calculations, in continuous decline since 1990 (Fig. 10). Data, rather than models, provide some evidence of a similar decline in the HIV-negative population in Malawi (50,51). Second, DOTS-plus programs using second-line drug regimens in Eastern Europe are expected to reduce deaths from MDR-TB more quickly than the TB death rate overall (so the percentage of TB deaths due to MDR-TB falls; Fig. 11). The incidence of MDR-TB cases has fallen faster than the incidence of all TB in Hong Kong (52), Republic of Korea (53), and Mexico (54), but it is not yet known whether treatment with second-line drugs can disproportionately reduce MDR-TB deaths on a large scale.

VII. Conclusion Although countries of the former Soviet Union suffered big increases in TB incidence during the 1990s, there are strong signs in some, notably Russia, that incidence began falling again at the turn of the millennium. The increase of TB in Africa appears to be slowing, although case reports to 2004 give no indication of when the incidence will peak. In other regions of the world, most importantly in Asia where the majority of new TB patients are found

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Figure 11 Expected changes in the TB death rate in Eastern Europe among all cases (heavy line) and among those with MDR-TB (light line), and the percentage of deaths due to MDR-TB (dotted line, right axis). Abbreviation: MDR, multidrug resistant.

each year, TB continued to decline through the 1990s, albeit slowly. TB incidence was probably still increasing slowly in 2005 throughout the world, whereas the number of deaths may already have begun to fall. These epidemiological trends have undoubtedly been influenced by chemotherapy, which has existed in most countries in some form since the 1950s. But drug treatment delivered via DOTS programs will have had a measurable impact only in countries that achieved high coverage by the mid-1990s. Although there is a great deal of circumstantial evidence that chemotherapy programs can drive TB incidence downwards, more direct, recent demonstrations of the impact of DOTS are restricted to a few countries such as Peru (reduced incidence) and China (reduced prevalence). DOTS programs are likely to have an impact in populations with high rates of HIV infection and MDR-TB, but the evidence remains equivocal. To evaluate the full impact of the DOTS strategy and its extensions, much more effort needs to be given to measurement. The effects of largescale public health programs, such as DOTS, can never be assessed under experimental conditions, but periodic population-based surveys of the prevalence of active disease and infection will show trends, which could be attributable to progress in TB control. TB deaths need to be counted more frequently and more accurately, either as a component of general cause-ofdeath surveys or through systems of routine death registration. The evidence from surveys of prevalence and mortality should be supplemented by fuller

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analyses of the vast body of surveillance data that is routinely collected by national control programs (following the example of Peru). The MDG framework has highlighted the importance of measuring epidemiological impact and, by including TB prevalence and deaths as indicators, has added momentum to the process of evaluation. Although data are being gathered from all these sources, planning for TB control over the next decade and beyond must be done with the evidence at hand. The Global Plan to Stop TB, 2006–2015 has been developed by asking what methods of TB control would be needed, and at what rate they must be implemented, in order to reverse the rise in incidence, and to halve TB prevalence and death rates between 1990 and 2015. The mathematical models used to assess the impact of different control scenarios are heavily influenced by the experiences of Europe and North America since the 1950s; they may omit some important but unknown features of TB epidemiology that now apply in Asia, Africa, or the Americas, such as the changing risks associated with tobacco smoking, air pollution, nutritional disorders, urban crowding, and other factors. With these qualifications, the calculations made for the Global Plan suggest that enhanced DOTS (scenario 3), if fully implemented, would bring down TB incidence long before 2015 (satisfying MDG target 8), although not fast enough to reach the 2050 target for elimination (less than one case per million population). Enhanced DOTS should also, by 2015, halve prevalence and death rates worldwide, and in all major endemic regions except Africa and Eastern Europe. Although Africa and Eastern Europe cannot quickly regain all the ground lost since 1990, the impact of enhanced DOTS over the period 2006–2015 is expected to be almost as big in these two regions as in other regions of the world. Scenario 3 would prevent 2.7 million deaths in comparison with scenario 2 (sustained DOTS), and fewer than one million people would die from TB in 2015. In the larger picture, considering enhanced DOTS as an extension of the project to expand DOTS worldwide since the 1990s, nearly 14 million deaths would be prevented between 2006 and 2015 (comparing scenarios 1 and 3). By 2015, enhanced DOTS should begin to make incremental gains over sustained DOTS that will grow larger in the years beyond. These forecasts are certainly not precise; rather they are intended to be broadly indicative of what can and cannot be achieved, regionally and globally, by implementing the Global Plan over the next 10 years. To do as much as planned, enhanced DOTS must successfully adapt the basic package of care so that it can move beyond the limits of public notification systems. This means forging better links between DOTS programs and other, nonparticipating public clinics and hospitals. It also means engaging medical services in prisons and the armed forces, private clinicians, nongovernmental organizations, mission hospitals, and clinics in the corporate sector. Besides encouraging all medical practitioners to adopt the basic package of care (55–57), DOTS programs must participate in the expansion of health services to serve populations where no

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professional health care is yet available. For example, half the population of Ethiopia has no access to health services and therefore no access to TB diagnosis and treatment. To do better than planned, especially in Africa and Eastern Europe, would require one or more of the following: higher rates of case detection and cure, for smear-positive and smear-negative patients and for those with MDR-TB, achieved in part through active case finding; more widespread use of drug sensitivity testing and second-line drug regimens; more rapid distribution of ART and cotrimoxazole for patients infected with HIV; greater use of preventive therapy for people with and without HIV coinfections; and faster reductions in HIV incidence. Some of these gains could be made more easily with new technology,as proposed in the Global Plan (Table 4). Improved TB diagnostics will probably be the first to reach field application (58). A new drug could improve cure rates by shortening the duration of treatment to one or two months, by increasing treatment success among patients with MDR-TB,

Table 4 Development of New Technology for Tuberculosis Control, as Described in the Global Plan to Stop TB, 2006–2015 By 2000 Vaccines

Drugs

Diagnostics

By 2010

By 2015

Four phase III efficacy Nine candidates in phase II trials; at least trials carried out. One safe, effective, two vaccines in phase licensed vaccine IIb or proof of concept trials by 2003; available by 2015 beginning of phase III trials 27 new compounds One to two new drugs Seven new drugs registered for TB in the TB pipeline registered for TB indication; regimen indication; treatment revolutionized: shortened to 3–4 mo clinical testing of drugs that can shorten treatment to 1–2 mo Predictive test for Rapid culture for case Point of care: rapid LTBI in culture; improved detection and DST demonstration microscopy; phage in demonstration phase detection (þDST); phase and simplified NAAT introduced

Three candidates in phase I trials by end 2005

Abbreviations: DST, drug susceptibility testing; NAAT, nucleic acid amplification test; LTBI, latent tuberculosis infection. Source: From Ref. 46.

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or by reducing the frequency of relapse among patients coinfected with HIV (59). There is no guarantee that a new vaccine will be available before 2015, or that it will have high efficacy against pulmonary TB (60). But if such a vaccine can be made, mass immunization could change the approach to TB control, shifting the emphasis from cure to prevention. Whatever the technological developments, they need to find field application urgently, not just to accelerate progress toward the MDGs, but to provide any hope that TB can be eliminated by 2050. Acknowledgments I thank Bernadette Bourdin, Katherine Floyd, Mehran Hosseini, Knut Lonnroth, Dermot Maher, Eva Nathanson, Andrea Pantoja, Alasdair Reid, and Catherine Watt who helped to compile information and data for this chapter. References 1. World Health Organization. The World Health Report 2004: Changing History. Geneva: World Health Organization, 2004. 2. Jamison DT, Mosley WH, Meashem AR, Bobadilla JL, eds. Disease Control Priorities in Developing Countries. New York: Oxford University Press for the World Bank, 1993. 3. de Jonghe E, Murray CJ, Chum HJ, Nyangulu DS, Salomao A, Styblo K. Costeffectiveness of chemotherapy for sputum smear-positive pulmonary tuberculosis in Malawi, Mozambique and Tanzania. Int J Health Plann Manage 1994; 9:151–181. 4. Murray CJL, De Jonghe E, Chum HJ, Nyangulu DS, Salomao A, Styblo K. Cost effectiveness of chemotherapy for pulmonary tuberculosis in three sub-Saharan African countries. Lancet 1991; 338:1305–1308. 5. World Health Organization. Global Tuberculosis Control: Surveillance, Planning, Financing. Geneva: World Health Organization, 2006:362. 6. Dye C, Watt CJ, Bleed DM, Hosseini SM, Raviglione MC. Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, and deaths globally. J Am Med Assoc 2005; 293:2767–2775. 7. www.stoptb.org. 8. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. J Am Med Assoc 1999; 282:677–686. 9. Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 2003; 163: 1009–1021. 10. Hudelson P. Gender differentials in tuberculosis: the role of socio-economic and cultural factors. Tuberc Lung Dis 1996; 77:391–400. 11. Borgdorff MW, Nagelkerke NJ, Dye C, Nunn P. Gender and tuberculosis: a comparison of prevalence surveys with notification data to explore sex differences in case detection. Int J Tuberc Lung Dis 2000; 4:123–132. 12. Hamid Salim A, Declercq E, Van Deun A, Saki KAR. Gender differences in tuberculosis: a prevalence survey done in Bangladesh. Int J Tuberc Lung Dis 2004; 8: 952–957. 13. Radhakrishna S, Frieden TR, Subramani R. Association of initial tuberculin sensitivity, age and sex with the incidence of tuberculosis in south India: a 15-year follow-up. Int J Tuberc Lung Dis 2003; 7:1083–1091.

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14. Rieder HL. Epidemiologic Basis of Tuberculosis Control. 1st ed. Paris: International Union Against Tuberculosis and Lung Disease, 1999:1–162. 15. Shilova MV, Dye C. The resurgence of tuberculosis in Russia. Philos Trans R Soc Lond B Biol Sci 2001; 356:1069–1075. 16. Espinal MA, Laszlo A, Simonsen L, et al. Global trends in resistance to antituberculosis drugs. N Engl J Med 2001; 344:1294–1303. 17. World Health Organization IUATALD. Anti-Tuberculosis Drug Resistance in the World: Third Global Report. WHO/IUATLD Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Geneva: World Health Organization, 2004:299. 18. Dye C, Espinal MA, Watt CJ, Mbiaga C, Williams BG. Worldwide incidence of multidrug-resistant tuberculosis. J Infect Dis 2002; 185:1197–1202. 19. Zignol M, Hosseini MH, Wright A, et al. Global Incidence of Multidrug-Resistant Tuberculosis. J Infect Dis. In Press. 20. Asamoah-Odei E, Garcia Calleja JM, Boerma JT. HIV prevalence and trends in subSaharan Africa: no decline and large subregional differences. Lancet 2004; 364:35–40. 21. Corbett EL, Charalambous S, Moloi VM, et al. Human immunodeficiency virus and the prevalence of undiagnosed tuberculosis in African gold miners. Am J Respir Crit Care Med 2004; 170:673–679. 22. Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 2006; 367:1173–1180. 23. Rieder HL. BCG vaccines. In: Davies PDO, ed. Clinical Tuberculosis. London: Arnold, 2003:337–353. 24. Dye C, Maher D, Weil D, Espinal M, Raviglione M. Targets for global tuberculosis control. Int J Tuberc Lung Dis 2006; 10:460–462. 25. Styblo K, Bumgarner JR. Tuberculosis can be controlled with existing technologies: evidence. Tuberc Surveill Res Unit Prog Rep 1991; 2:60–72. 26. Styblo K. Epidemiology of Tuberculosis. 2nd ed. The Hague: Royal Netherlands Tuberculosis Association (KNCV), 1991:1–136. 27. Raviglione MC, Uplekar MW. WHO’s new stop TB strategy. Lancet 2006; 367: 952–955. 28. Dye C, Garnett GP, Sleeman K, Williams BG. Prospects for worldwide tuberculosis control under the WHO DOTS strategy. Lancet 1998; 352:1886–1891. 29. Smith KR, Mehta S. The burden of disease from indoor air pollution in developing countries: comparison of estimates. Int J Hyg Environ Health 2003; 206:279–289. 30. Baris E, Ezzati M. Should interventions to reduce respirable pollutants be linked to tuberculosis control programmes? Br Med J 2004; 329:1090–1093. 31. Kolappan C, Gopi PG. Tobacco smoking and pulmonary tuberculosis. Thorax 2002; 57:964–966. 32. Gajalakshmi V, Peto R, Kanaka TS, Jha P. Smoking and mortality from tuberculosis and other diseases in India: retrospective study of 43000 adult male deaths and 35000 controls. Lancet 2003; 362:507–515. 33. Kim SJ, Hong YP, Lew WJ, Yang SC, Lee EG. Incidence of pulmonary tuberculosis among diabetics. Tuberc Lung Dis 1995; 76:529–533. 34. Ponce de Leon A. Tuberculosis and diabetes in southern Mexico. Diabetes Care 2004; 27:1584–1590. 35. Olmos P, Donoso J, Rojas N, et al. Tuberculosis and diabetes mellitus: a longitudinalretrospective study in a teaching hospital. Revista Med Chile 1989; 117:979–983. 36. Cegielski JP, McMurray DN. The relationship between malnutrition and tuberculosis: evidence from studies in humans and experimental animals. Int J Tuberc Lung Dis 2004; 8:286–298.

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37. Coetzee N, Yach D, Joubert G. Crowding and alcohol abuse as risk factors for tuberculosis in the Mamre population. Results of a case-control study. S Afr Med J 1988; 74:352–354. 38. Institute of Medicine. Ending Neglect: The Elimination of Tuberculosis in the United States. Washington, D.C.: Institute of Medicine, 2000. 39. World Health Organization. Public-Private Mix for DOTS. Global progress. Report of the second meeting of the PPM subgroup for DOTS Expansion. WHO/HTM/TB/ 2004.338. 40. Suarez PG, Watt CJ, Alarcon E, et al. The dynamics of tuberculosis in response to 10 years of intensive control effort in Peru. J Infect Dis 2001; 184:473–478. 41. Frieden TR, Fujiwara PI, Washko RM, Hamburg MA. Tuberculosis in New York City—turning the tide. N Engl J Med 1995; 333:229–233. 42. Stop TB partnership and World Health Organization. The Global Plan to Stop TB, 2006–2015. 2006 TB Partnership:Geneva. 43. China Tuberculosis Control Collaboration. The effect of tuberculosis control in China. Lancet 2004; 364:417–422. 44. Aditama TY. Prevalence of tuberculosis in Indonesia, Singapore, Brunei Darussalam, and the Philippines. Tubercle 1991; 72:255–260. 45. Ministry of Health-Republic of Indonesia. Tuberculosis survey in Indonesia, 2004. National Institute of Health Research and Development, Ministry of Health, Jakarta Indonesia 2005:99. 46. Stop TB Partnership and World Health Organization. The Global Plan to Stop TB, 2006–2015. 2006. Stop TB Partnership:Geneva. 47. Blower SM, McLean AR, Porco TC, et al. The intrinsic transmission dynamics of tuberculosis epidemics. Nat Med 1995; 1:815–821. 48. Dye C, Williams BG. Criteria for the control of drug-resistant tuberculosis. Proc Natl Acad Sci USA 2000; 97:8180–8185. 49. Dye C, Espinal MA. Will tuberculosis become resistant to all antibiotics? Proc R Soc London B 2001; 268:45–52. 50. Glynn JR, Crampin AC, Ngwira BM, et al. Trends in tuberculosis and the influence of HIV infection in northern Malawi, 1988–2001. AIDS 2004; 18:1459–1463. 51. Borgdorff MW, Corbett EL, DeCock KM. Trends in tuberculosis and the influence of HIV infection in northern Malawi, 1988–2001. AIDS 2004; 18:1465–1467. 52. Kam KM, Yip CW. Surveillance of Mycobacterium tuberculosis drug resistance in Hong Kong, 1986–1999, after the implementation of directly observed treatment. Int J Tuberc Lung Dis 2001; 5:815–823. 53. Hong YP, Kim SJ, Bai JY, Lew WJ, Lee EG. Twenty-year trend of chronic excretors of tubercle bacilli based on the nationwide tuberculosis prevalence surveys in Korea, 1975–1995. Int J Tuberc Lung Dis 2000; 4:911–919. 54. DeRiemer K, Garcia-Garcia L, Bobadilla-del-Valle M, et al. Does DOTS work in populations with drug-resistant tuberculosis? Lancet 2005; 365:1239–1245. 55. Elzinga G, Raviglione MC, Maher D. Scale up: meeting targets in global tuberculosis control. Lancet 2004; 363:814–819. 56. UN Millennium Project. Investing in strategies to reverse the global incidence of TB. Task Force on HIV/AIDS, Malaria, TB, and Access to Essential Medicines. London: Earthscan, 2005. 57. Stop TB Partnership. Report on the Meeting of the Second ad hoc Committee on the TB Epidemic. Geneva: World Health Organization, 2004:17. 58. www.finddiagnostics.org. 59. www.tballiance.org. 60. www.aeras.org.

2 Bacteriology of Tuberculosis

FADILA BOULAHBAL

LEONID HEIFETS

Mycobacteriology, National Reference TB Laboratory, Institut Pasteur d’Alge´rie, Alger, Alge´rie

Mycobacteriology Clinical Reference Laboratory, National Jewish Medical and Research Center, Denver, Colorado, U.S.A.

I. Mycobacterium tuberculosis Complex A. Taxonomy

The genus Mycobacterium is the only genus in the family of Mycobacteriaceae, which, along with six families of Actinomycetes, belongs to the order of Actinomycetales. The taxonomy and nomenclature of the genus Mycobacterium within this order has been addressed in special reviews (1–4). Hundreds of species of this genus, usually called nontuberculous mycobacteria (NTM) or mycobacteria other than tubercle bacilli, can be found in the environment (water, soil, etc.), and about 60 or 70 of them have been identified (or suspected) as potential human pathogens (5). The elucidation of new mycobacterial species is currently based on a molecular approach, which relies on the definition of species-specific nucleotide sequences, particularly within the hypervariable regions (termed A and B) of the 16S ribosomal DNA molecule. Mycobacterium tuberculosis (complex) and Mycobacterium leprae are known as the only mycobacteria that are not found in the environment, and the only pathogens in this genus transmissible from person to person. The generic term ‘‘tubercle bacilli’’ incorporates at least five species belonging to a group termed the M. tuberculosis complex: M. tuberculosis, Mycobacterium bovis, 29

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Mycobacterium africanum, Mycobacterium canettii, and Mycobacterium microti. The first four of these species are definite human pathogens that can cause tuberculosis (TB), and a few cases of disease in humans caused by M. microti have also been reported. Recently, two additional species have been suggested: Mycobacterium caprae (6) and Mycobacterium pinnipedii (7). bacille Calmette–Gue´rin (BCG), an attenuated M. bovis subspecies, can be found in diagnostic specimens in cases where BCG is used for bladder cancer therapy, or results after BCG vaccination in a disseminated infection (for example, in HIV-positive individuals) or in the so-called ‘‘cold abscesses.’’ BCG is not transmissible from person to person. B. Common Features of the Genus Mycobacterium

1.

2.

3.

4.

5.

6.

7.

Due to the high content of lipids in their cell wall, all mycobacteria are acid-fast bacteria (AFB), which means that special efforts are required to make the dyes penetrate through the bacterial cell wall, and it is difficult to decolorize them with acid–alcohol after they are stained. Mycobacteria are gram-positive rods 1 to 4 mm long and 0.3 to 0.6 mm in diameter; because of their acid fastness they may be seen as clear rod-shaped zones in smears stained by the Gram method. Mycobacteria have a high lipid content in the outer layer of the cell wall, which includes glycolipids and esters of fatty acids with fatty alcohols; one of the water-soluble glycolipids (mycosides), known as ‘‘cord factor,’’ is considered to be related to the virulence of tubercle bacilli. Mycolic acid of the cell wall is considered one of the specific features of this family of bacteria, although this fatty acid can also be found in cell walls of some genuses of aerobic Actinomycetes (Nocardia, Gordonia, Tsukamurella, Rhodococcus, etc.) and in most species of the genus Corynebacterium. Most of the pathogenic mycobacterial species are fastidious with regard to culture conditions. M. tuberculosis will only grow on specially designed culture media, with optimal temperature conditions limited to 35 C to 37 C, optimal pH of 6.4 to 7.4, and the presence of CO2 in the atmosphere for some media. Some species, such as Mycobacterium haemophilum, Mycobacterium ulcerans, Mycobacterium avium spp. paratuberculosis, and Mycobacterium genavense are particularly fastidious. Mycobacteria grow slowly in culture media (generation time for M. tuberculosis is from 12–20 hours), and the colonies do not become visible on solid media before two to three weeks of cultivation; however, a group of mycobacteria called ‘‘rapid-growers’’ may produce visible colonies after only a few days of cultivation. Most of the mycobacterial species are strict aerobes; some M. bovis isolates may grow in an oxygen-reduced atmosphere.

Bacteriology of Tuberculosis 8.

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Members of M. tuberculosis complex, M. avium complex, and a number of other pathogenic mycobacteria are considered facultative intracellular parasites (in macrophages); it is possible that long periods of persistence of the tubercle bacilli in individuals with latent TB may be related to their intracellular survival.

C. Genetics: Typing

The sequencing of the M. tuberculosis (strain H37Rv) genome has been completed (8), indicating that it contains about 4000 genes that encode proteins and about 50 genes that encode RNA other than mRNA. An unusual feature, when compared with other bacteria, is the large number of genes (250) that encode enzymes for the metabolism of fatty acids. Progress in the molecular biology of mycobacteria is essential not only for the current knowledge on the biology of these organisms, but also for practical solutions related to issues such as the development of new rapid methods for diagnosis and detection of drug resistance, as well as for better differentiation between various species (9). Molecular biology tools are also useful for application in modern ‘‘molecular epidemiology’’ (see Chapter 23). This new discipline is based on molecular typing methods of M. tuberculosis strains, or DNA fingerprinting. The DNA-based methods replaced various phenotype characteristics (such as drug susceptibility or resistance patterns and phage typing) used in the past to distinguish between different strains or clinical isolates and for cluster analysis of outbreaks. One of the DNAbased methods, restriction fragment length polymorphism (RFLP) pattern analysis, is based on analysis of strain divergence within the same species in restriction sites (identified by restriction enzymes), leading to changes in the length of fragments between genomes (10). Description of the repetitive insertion sequence IS6110 in M. tuberculosis (11) led to the establishment of the IS6110 RFLP technology, which became the most widely used genotyping method for investigating TB outbreaks (12–15). This technology has been standardized for broad application (16) and is now available in regional laboratories assigned by the Centers for Disease Control and Prevention (CDC) for the evaluation of M. tuberculosis isolates obtained from TB patients in the United States. Other methods of DNA fingerprinting usually employ polymerase chain reaction (PCR), such as spoligotyping, PCR for IS6110 and for other genetic loci, as well as whole-genome fingerprinting. These methods have certain advantages and disadvantages compared with the more traditional IS6110 RFLP analysis in regard to the level of sensitivity of the assay, turnaround time of the report, labor intensity, and cost (17–21). It is important to stress that all these methods require a high level of technical competency in both laboratory procedures and interpretation of results. Therefore, implementation of genotyping procedures should be considered only for large and well-equipped TB laboratories that already have all basic techniques in their arsenal and provide service to a large population of TB patients.

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For more details on the biology of mycobacteria, particularly on the genetics and physiology of M. tuberculosis, please refer to other chapters of this publication and to special reviews in the monograph recently published by the American Society for Microbiology (22). II. Bacterial Populations in Patients A. Latent Tuberculosis Infection

Various aspects of latent tuberculosis infection (LTBI) have been addressed in a recent comprehensive review (23). According to the information presented in this review, the probability of an individual becoming infected with tubercle bacilli depends on a number of factors such as closeness and duration of exposure, the degree of infectiousness of the source (number of bacteria excreted in sputum, involvement of larynx, type of inflammation, and ability to produce aerosols by coughing), actual size of the inhaled infectious dose, ability of the bacterial strain to survive and multiply within the host (so-called fitness of the organism), and the immunological status of the host. It is estimated that only about 10% of infected immunocompetent individuals will develop the active disease during their lifetime. The usual event after infection is the development of a LTBI, which is defined as a clinical condition without clinical or radiological signs of active disease, and is manifested only by a positive tuberculin skin test (23). Persons with LTBI may harbor viable tubercle bacilli for many years in a state of relative dormancy, or rather, as currently defined, in a state of nonreplicating persistence (NRP). This rather descriptive term indicates that the tubercle bacilli are not replicating, with no reference made to the mechanism behind this phenomenon (24). Factors that may induce NRP include depletion of nutrients, shifts in pH, accumulation of growth-inhibiting products, and depletion of oxygen. Extensive research using various in vitro and in vivo models indicates that NRP is associated with certain genetic changes in the physiology of tubercle bacilli. Annually, about one-third, or about 2.5 million, of all new cases of active TB in the world result from activation from LTBI, while the remaining 4 to 6 million cases result from new infection (23). Treatment of LTBI and the mechanism of the NRP status of tubercle bacilli remain among the most important topics of modern TB research. B. Bacterial Population in Patients with Active Tuberculosis

The bacterial population in patients with active TB is quite heterogeneous. It consists of at least four subpopulations: actively metabolizing and relatively rapidly growing bacteria, semidormant bacteria whose growth is partially inhibited by the low pH of the early acute inflammation sites, semidormant bacteria that have occasional short spurts of metabolism in locations other than those with low pH, and bacteria in a dormant state (25,26). Recognition of this diversity is essential to designing appropriate treatment regimens that include drugs active against these subpopulations. The relative sizes of these subpopulations change during the course of the

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disease, and their proportions alter under the effect of drugs implemented for therapy. In addition, the structure of the patient’s bacterial population may be modified by the development of drug resistance. III. Bacteriological Diagnosis of Tuberculosis A. Smear Examination

The bacteriological diagnosis of TB is most commonly based on examination of sputum specimens (smear examination, culture isolation, molecular methods, etc.). Depending on the site of infection, other specimens include various body fluids (pleural, cerebrospinal, synovial, etc.), blood, lymph nodes, and other biopsy specimens. Sputum smear examination for AFB detection is the method most widely used for the provisional diagnosis of pulmonary TB. There are several technical options for this diagnostic tool. The most traditional among them (especially in countries with low resources) is a direct sputum smear stained by the Ziehl–Neelsen method and examined under light microscopy. The sensitivity of this method is limited: the results can be positive if the specimen contains no less than 104 AFB/mL of sputum, with variations depending on the skill of the technologists. Higher sensitivity, perhaps 10-fold greater, compared with the direct method examined after Ziehl– Neelsen stain, can be achieved when a sputum specimen is concentrated and examined after a fluorescent acid-fast stain (for example, with auramine O). Unfortunately, the feasibility of this approach is limited in most laboratories of low-income countries by lack of resources, expertise, and maintenance capabilities. Patients with positive smear results are considered to be the most infectious, and, therefore, their detection in the community is one of the priorities set by World Health Organization (WHO) for TB programs. According to the WHO standards, a definitive TB case is defined as ‘‘a patient who is culture-positive for M. tuberculosis complex’’ (27). The same definition also applies to patients with two AFB-positive sputum smears in countries where the culture isolation procedure is not available (27). It is now well established that AFB smear examination detects no more than 50% of all culture-positive adult patients with pulmonary TB (28). According to a study employing molecular epidemiology methods, the remaining smear-negative (culture-positive) patients represented a source of infection for 17% of all new cases. This conclusion was reached in a situation (in San Francisco) where all the smear-negative patients were detected and treated. It is not clear what role such patients may play as a source of infection when they are undetected and untreated. It is recommended by WHO to expand the methods for bacteriological diagnosis of TB beyond smear examination where resources allow (29). B. Culture Isolation

Detailed protocols for culture isolation, including descriptions of various culture media, methods for processing of the specimens, their applicability

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in different settings, as well as the advantages and disadvantages of different methods, can be found in technical manuals, handbooks, and special reviews (29–32). According to these descriptions, culture isolation of tubercle bacilli from patients’ specimens with subsequent identification procedures is a ‘‘gold standard’’ for the definitive diagnosis of TB. Culture is not only more sensitive than smear examination, but also allows identification of the mycobacterial species and determination of the drug susceptibility pattern of the isolate. The major obstacles to the broad application of culture isolation are the necessity for decontamination of specimens from nonsterile sites, cost, need for appropriate biosafety conditions, and expensive modern technology for rapid detection of mycobacteria in cultures. Processing specimens for cultivation involves aerosol-generating procedures such as homogenization and centrifugation. Therefore, culture isolation should not be considered in laboratories that do not have aerosol-contained centrifuges and other safe equipment and supplies. Besides more complete and accurate diagnosis, culture isolation and subsequent or concurrent drug susceptibility testing (DST) are important for making decisions on suitable therapy, but only if the laboratory reports are timely enough to be useful for adjustment of the treatment regimen. Isolation of the tubercle bacilli on the egg-based culture media most often used around the world [such as Lo¨wenstein-Jensen (L-J)] usually takes four to five weeks, but may take up to eight weeks. Visible colonies of M. tuberculosis can be detected on agar-based media after only two to four weeks of cultivation for most of the isolates, but may take up to six weeks. Even faster detection of growth (one to two weeks for most isolates) can be achieved in automated or semiautomated systems employing liquid selective media, such as BACTEC-460 and -960 (Becton Dickinson, Sparks, Maryland, U.S.A.), BacT/Alert (Biomerieux, Inc., Durham, North Carolina, U.S.A.), and ESP (Trek Diagnostics, Westlake, Ohio, U.S.A.). Each of the liquid medium systems has certain advantages and disadvantages, and the decision for preference is usually made depending on the specific conditions of the laboratory. In most countries, mycobacterial isolation is based on the inoculation of an egg-based medium with homogenized or decontaminated and concentrated sputum specimens, and the methods used for decontamination may vary in different laboratories (29–31). In some countries, it is recommended to use more than one medium for culture isolation. In the United States, it is mandatory to use a combination of solid and liquid media, including an option of combining up to four units of the medium. Detailed protocols on these and other procedures can be found in special reviews and laboratory manuals (29–32). In conclusion, we should stress that culture isolation has obvious advantages over the direct smear examination in regard to the more complete detection of all new TB patients in the community and an opportunity, when combined with DST, for early identification of patients with drug resistance. Nevertheless, so far, direct smear examination remains the only affordable tool for detection of most of the infectious patients in

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low-income countries. Insufficient resources for proper equipment and a safe and efficient laboratory environment, lack of well-trained technical personnel, and general infrastructure problems, limit the use of culture isolation in these countries. Implementation of culture isolation, when resources become available, should be done in a stepwise manner, leaving to the end introduction of the most attractive (and most expensive) rapid methods, such as liquid media systems and molecular methods described below. Among the less expensive solid media, the egg-based (for example, L-J) media are more familiar to most laboratories in the world, and, therefore, the easiest to implement first. Subsequently or simultaneously, the agar-based medium (7H10/7H11) should be considered, because it has an advantage over L-J slants for earlier detection of colonies due to the medium transparency, especially when used in Petri dishes. The disadvantage of the 7H10/7H11 agar medium is that it contains an expensive and difficult to standardize nutrient supplement, oleate-albumin-dextrosecatalase (OADC), and cultivation is required in a 5% to 10% CO2 atmosphere. For future development, the use of a new formulation of agar medium can be considered (33): in this medium OADC is replaced with an animal serum, and CO2 incubators are not required. This medium can be even less expensive than an egg-based medium, especially when used for DST. IV. Methods for Mycobacterium tuberculosis Identification Differentiation of M. tuberculosis complex from other mycobacteria represents an important public health issue, particularly in countries with a high prevalence of diseases caused by NTM. The next step is the determination of the species among the members of M. tuberculosis complex, which in practice usually involves differentiation between M. tuberculosis and M. bovis. A. Conventional Identification Tests

Isolation of M. tuberculosis requires incubation at 35 C to 37 C, while no growth appears at 25 C, 32 C, or 42 C. The buff-colored colonies are always nonpigmented and have a rough, dry surface with irregular edges, and often have a wrinkled surface. It is typical to find serpentine cording in smears made from a broth culture. Results of four tests, with cultures from any medium, may be used for final identification of M. tuberculosis and its differentiation from M. bovis. Identification of M. tuberculosis is based on the following results: positive niacin production, positive nitrate reduction, resistance to 5 mg/mL of thiophen-2-carboxylic acid hydrazide (TCH), and positive pyrazinamidase test. The results of these four tests (30,32,34,35) are reversed for M. bovis. The niacin test can be negative for some M. tuberculosis strains, especially those resistant to isoniazid; this test is more reliable when performed with an L-J culture. The pyrazinamidase test with M. tuberculosis can be negative if the patient has been treated with

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pyrazinamide (PZA) and resistance to this agent has developed. In addition, false-negative results with any of these tests may appear due to (quite common) deviations from the prescribed technique. The Niacin Test

Formerly, this test was relied upon for identification of M. tuberculosis, but rapid methods described below have greatly reduced its value. Laboratories that still use this and other conventional identification methods should be aware that the niacin test can be positive with some mycobacterial species other than M. tuberculosis, such as Mycobacterium simiae, some BCG strains, and some rapidly growing mycobacteria. Many laboratories use three- to four-week-old cultures grown on Middlebrook 7H10 or 7H11 agar, and the test works well in most cases. However, some M. tuberculosis strains do not produce a sufficient amount of niacin on this medium for detection. The frequency of these false-negative results can be decreased if the agar medium is enriched with L-asparagine (0.25%) or its potassium salt (0.1%). False-negative results can be almost completely eliminated by using sixweek-old or older cultures on L-J medium. This can serve as a backup if results with the agar-grown culture after three weeks of cultivation are negative. The presence of some contaminants in the culture may cause false-positive results, so it is necessary to confirm the purity of the culture by examining a smear stained by the Ziehl–Neelsen method. The test is based on detection of niacin in the medium, not in the bacteria. In cases of confluent growth, it is therefore necessary to pierce through the bacterial growth with a pipette to expose the medium. Nitrate Reduction Test

Nitrate reduction test is the second most important conventional test for identifying M. tuberculosis, and particularly for differentiating it from M. bovis. Besides M. tuberculosis, other species (Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium terrae, Mycobacterium triviale, Mycobacterium phlei, Mycobacterium smegmatis, and Mycobacterium vaccae) produce nitrate reductase. The test is negative with M. bovis but it can be weakly positive with some BCG strains. The Pyrazinamidase Test

The pyrazinamidase test detects the presence of the enzyme that converts PZA to pyrazinoic acid. Pyrazinamidase can be found in cultures of M. tuberculosis strains susceptible to PZA, but PZA-resistant M. tuberculosis strains do not possess detectable amounts of the enzyme. All M. bovis strains, including BCG, are resistant to PZA, and the organisms show negative results in the pyrazinamidase test. Positive results of these tests confirm M. tuberculosis, while negative results suggest the presence of M. bovis. One must be alert to negative reactions with M. tuberculosis strains resistant to PZA that were isolated from patients previously treated with this drug. M. africanum is susceptible to PZA and is positive in the pyrazinamidase test.

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The Test for Susceptibility to Thiophen-2-Carboxylic Acid Hydrazide

The test for susceptibility to TCH is especially useful for differentiation between M. bovis and the multidrug-resistant strains of M. tuberculosis, because these strains can produce negative results in the three other differentiation tests described above. Only M. bovis strains (including BCG) are susceptible to 1.0 and 5.0 mg of TCH/mL incorporated into Middlebrook 7H10/7H11 agar medium, or in a similar test using L-J medium. B. Rapid Methods

The most widely used rapid identification procedures include cell-wall lipid (mycolic acid) analyses by high-performance liquid chromatography (HPLC), nucleic acid probes, and amplification procedures with raw specimens. In addition, a nucleic acid sequencing test, currently used in only a few laboratories, may have potential for wider use in the future. High-Performance Liquid Chromatography Procedure

This technique is now available in almost all TB laboratories in the United States (36–38). The mycolic acids extracted from mycobacterial cells are converted to esters and subjected to chromatographic separation to detect the characteristic patterns associated with various mycobacterial species. The analysis is based on a comparison of retention time of the peaks and their height ratios. The recent review on HPLC in mycobacteriology describes 63 chromatographic patterns representative of 73 known mycobacterial species (36). Introduction of fluorescence detection instead of ultraviolet spectrophotometry made the method much more sensitive, so that the AFB-positive broth cultures can be used instead of a large harvest from solid media (37), but the sensitivity of this method for detection of mycobacteria directly in sputum specimens remains insufficient to rely upon. More details on cell-wall lipid analysis methods can be found in the reviews (36–38). Nucleic Acid Probes

The AccuProbe technology (Gen-Probe, San Diego, California, U.S.A.) is commercially available for identification of the M. tuberculosis complex, M. avium complex, M. avium, M. intracellulare, M. kansasii, and M. gordonae. The test employs chemiluminescent acridinium ester–labeled single-stranded DNA probe complementary to the rDNA of the target bacteria. The association of the two strands forms a stable hybrid, and chemiluminescence is developed by the addition of hydrogen peroxide and is measured in a luminometer (39). The test can be performed with a culture grown either on solid or in liquid media. With most of the clinical isolates, it takes a minimum of three weeks on 7H11 agar plates and about 10 days in liquid media to achieve growth sufficient for this test. If the test with the M. tuberculosis complex probe is positive, the results are reported as M. tuberculosis complex, because this probe does not distinguish between

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M. tuberculosis and other members of the complex. This type of report is sufficient for timely public health actions and appropriate treatment decisions. Amplification Procedures

The concept of amplification of discrete fragments of bacterial DNA or RNA opened a new opportunity for making the nucleic acid probe hybridization method (described above) highly sensitive, leading to the idea of detection and identification of mycobacteria (particularly M. tuberculosis) directly in the patient’s specimen. A number of amplification systems have been developed recently, both ‘‘in-house’’ assays and commercial systems (40–43). The most popular among them are PCR and Gen-Probe amplified Mycobacterium tuberculosis Direct (MTD) test. The appeal of the amplification methods is their high sensitivity, allowing detection of only a few bacterial cells in a raw specimen, which means that diagnosis of TB can be completed in a few days or perhaps hours after arrival of a specimen in the laboratory. The techniques are very expensive and should be used in addition to other methods when their use is fully justified. To avoid false-positive results, these methods should not be used in patients who have had a history of TB. Other shortcomings are related mainly to the low sensitivity of these methods for smear-negative (culture-positive) sputum specimens—only 70% or less. The primary reason for using any of the amplification systems is to provide rapid differentiation between M. tuberculosis and NTM in a smear-positive sputum specimen, so that necessary actions, including a patient’s isolation and treatment, can be carried out without delay. The insufficient sensitivity and specificity of the new methods for smear-negative sputum specimens suggests the use of amplification techniques for broth cultures at the earliest possible detection of growth in addition to and/or instead of a test with raw specimens (41). C. Nucleic Acid Sequencing

Hypervariable regions of the 16S rRNA molecule have been found to be useful in the identification of mycobacterial species (44–46). The identification is based on comparison of the isolate with known sequences. Among the disadvantages of this procedure is the high cost, insufficient standardization of the method, and limited availability of libraries of mycobacterial species. Nevertheless, in the near future, this procedure could prove valuable to the reference laboratories dealing with a substantial number of NTM. At the same time, other less costly rapid methods may have advantages for the specific purpose of differentiating between M. tuberculosis and NTM. V. Detection of Drug Resistance A. General Concepts

Correct implementation of effectively supervised therapy programs is indispensable to the prevention of epidemics of drug-resistant TB. In addition, DST, especially in new patients, can be an effective tool in parts of the world

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where an increase in prevalence of multidrug-resistant TB (MDR-TB) is already taking place. Unfortunately, this measure can only be applied in laboratories that perform cultivation of tubercle bacilli, and have sufficient resources, expertise, and an appropriate biosafety environment. Therefore, in many countries, detection of patients with drug resistance is based on a rather empirical approach. Drug resistance, particularly multidrug resistance, is suspected in patients who fail to respond to the standard treatment regimen. In such cases, an alternative treatment regimen is administered on the assumption that the patient’s isolate is resistant to the first-line drugs. In countries that have TB laboratories with limited capabilities, specimens are often submitted to the laboratory for DST only if the patient fails to respond to the initial standard treatment regimen. Either of these options can be unsafe and costly: there is danger for individual patients who may receive inappropriate treatment, and extra cost for society because of the prolonged period during which a patient with MDR-TB is infectious. Management of patients with MDR-TB can be much more expensive than the cost of DST of the initial isolates from all new patients. Therefore, when resources become available, development of an appropriate laboratory system in countries with growing rates of drug resistance should be among the priorities of national programs (29). In the meantime, systematically conducted epidemiological surveys to evaluate the dynamics of drug resistance prevalence in different countries are recommended for identification of problematic areas and proper adjustment of the local TB control programs (47). The DST system is most likely to be affordable if it is based on direct delivery of raw specimens to a central mycobacteriology laboratory that has a large operational volume and well-trained personnel, and is properly equipped. Unfortunately, this option is currently difficult to implement in large lowincome countries with underdeveloped general infrastructure. The classical definition of a drug-resistant strain of M. tuberculosis is that it is significantly different by the degree of susceptibility from a wild strain that has never come into contact with the drug (48,49). The drug concentrations to distinguish susceptible and resistant strains (so-called ‘‘critical concentrations’’) should be somewhere between the highest minimal inhibitory concentration (MIC) found among the wild strains and the lowest MIC found among the isolates considered resistant. Progress in molecular biology may provide a new definition of drug resistance, but may also raise new questions to be addressed, especially considering that resistance to some drugs has more than one genetic mechanism responsible for low and high levels of resistance (50,51). Different methods for M. tuberculosis DST, as well as their advantages and disadvantages, have been previously discussed in detail (52–55). The main requirement for a DST is the ability to distinguish between susceptible and resistant M. tuberculosis strains, which can be reliably achieved by the traditional strategy based on cultivation. Newer molecular biology– based methods for the detection of mutations associated with drug resistance offer several advantages over the conventional phenotypic method,

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including: (i) possible availability of the results in a matter of hours or days, (ii) better reproducibility, especially in cases of low degree of resistance, and (iii) ability to work with poorly growing and/or mixed cultures. On the other hand, classical phenotypic methods, which involve culturing mycobacteria in the presence of drugs under standardized conditions to detect inhibition of growth, offer several advantages; these methods are less expensive, which makes them more affordable in resource-limited settings. B. Testing on Solid Media

Drug susceptibility methods based on mycobacterial cultivation on solid media, either egg or agar based, can be performed as a direct or indirect test. In the direct test, a set of drug-containing and drug-free media is inoculated directly with a concentrated specimen. An indirect test is the inoculation of the media with a pure culture, and it is classically performed with a bacterial suspension made from growth on solid media (L-J, or 7H10 or 7H11 agar). However, a 7H9 broth culture can also be used as an inoculum when it is grown within five to eight days up to the turbidity of a McFarland Standard No. 1 (about 108 CFU/mL). An advantage of the direct over the indirect test is that the results are available sooner and better represent the patient’s original bacterial population. If the results of the direct test are not valid because there is insufficient or excessive growth in drug-free controls, or there is heavy contamination, the test must be repeated with a pure culture, i.e., as an indirect test. Three methods were originally suggested by the WHO panel for starch-free L-J medium: (i) the proportion method, (ii) the resistance ratio method, and (iii) the absolute concentration method (48,49). The option of using agar medium instead of L-J is little known in laboratories outside the United States. More details on these and other techniques can be found in the CDC manual (30) and other publications (52–55). An advantage of performing tests on agar plates is that the final results can be reported within three weeks instead of four to six weeks or more, as is necessary when using the L-J medium. According to the practices of the TB laboratories in the United States, three weeks of total turnaround time for a direct test on agar plates provides an advantage over the indirect test, which requires initial isolation followed by a susceptibility procedure, with a total turnaround time of six weeks on agar medium and 10 to 12 weeks on L-J medium for most isolates. A significant disadvantage of this method is that cultivation on 7H10 or 7H11 agar requires an incubator that can provide 5% to 10% CO2. This problem can be addressed with use of the new agar medium (33). C. Indirect Test in Liquid Medium: BACTEC-460 Method

New automated liquid medium systems (MB/BacT, MGIT-960, ESP, etc.) were introduced recently, but most of the experience has been accumulated by using the radiometric BACTEC-460 system. Technology for liquid media systems other than BACTEC-460 is based on similar principles and can be found in appropriate manufacturer’s inserts. The 7H12 broth in 12B

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vials (4.0 mL in each) is composed of 7H9 broth base, casein hydrolysate, bovine serum albumin, catalase, and 14C-fatty acid. Consumption of the 14 C-substrate by the growing bacteria results in release of the 14CO2, the amount of which is expressed by the instrument as a growth index (GI) on a scale of 0 to 999. In the presence of an antimicrobial agent, susceptibility is detected by the inhibition of daily GI increases (56–58). The major advantage of this technique is the ability to detect growth, and its inhibition earlier than by other means. An indirect DST in this system usually required an average of 9.3 days (58). The overall mean time for primary isolation plus indirect testing was 18 days in a cooperative study by five institutions (56). The major disadvantage of the BACTEC-460 system is the problem of disposing large volumes of radioactive materials (12B vials), even though radioactivity is very low. Another disadvantage is the cost, which is much higher than for solid media, but is less expensive than the newer nonradioactive liquid medium systems mentioned above. Technical details on use of the BACTEC-460 system can be found in published reviews and in the manufacturer’s manuals. D. A Test in Mycobacterial Growth Indicator Tubes

The nonradioactive automated BACTEC-960 system with mycobacterial growth indicator tubes from Becton Dickinson is another option for performing an indirect test in a liquid medium with five first-line drugs. Comparison of this method with the BACTEC-460 system (59), as well as with the agar proportion method, indicates that this new method is quite promising. Further studies in different laboratory settings with a large number of clinical isolates will probably be required before this method is considered as reliable as the proportion method on solid media or the BACTEC-460 method. E. Molecular Approaches

Molecular biology–based methods can be divided into two categories: phenotypic and genotypic. In view of the current limitations for their worldwide applicability, only a very basic listing of these methods is provided here; readers may find more detailed descriptions in special reviews (54,60–63). Phenotypic Methods

New phenotypic methods, as well as the traditional phenotypic cultivation methods addressed above, target detection of drug resistance regardless of its genetic basis. Unlike the traditional techniques, new methods employ molecular indicators of changes in bacterial metabolic activity related to the development of drug resistance. The following procedures can be classified in this category: bioluminescence assay of adenosine triphosphate (64), some colorimetric techniques (65,66), flow cytometry (67,68), quantitation of mycobacterial antigens in agglutination with beads coated with polyclonal antibodies (69), luciferase reporter phages test (70,71), and phage-based assays (72,73). Among other suggestions is the so-called microscopic observation drug susceptibility (74–76).

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These methods are based on current knowledge regarding mutations in specific genes responsible for resistance to different antimicrobial agents (50,51): isoniazid—katG, inhA, kasA, cpM; rifampicin—rpoB; streptomycin—rpsL; ethambutol—group emb; PZA—pncA; and quinolones—gyrA and gyrB. The usual first step in most of the genotypic methods for detection of drug resistance is amplification of the corresponding DNA segments. With this approach, there is a tendency to perform the test with the raw specimen, which may provide the main advantage of such methods—minimizing the turnaround time. Development of these methods and their specifics has been addressed in a number of reviews (50,51,54,55,60,77,78). Most attention has been given in these reviews to the following methods: automated DNA sequencing, PCR single-strand confirmation polymorphism, PCR-heteroduplex formation, and Line Probe Assay. Oligonucleotide ligation assay using DNA microchips is a new, rapidly developing approach (79,80). Another new suggestion is based on the combined use of two microassays for detection of resistance to rifampicin and isoniazid (81). All these and other novel methods represent an obvious interest for future development of methods for rapid detection of drug resistance, but before any of them is introduced for routine use in a clinical laboratory, their justification should be based on sensitivity and specificity analyses in comparison with the most advanced conventional methods (for example, direct test on solid media), total turnaround time, total cost (including labor), cost-effectiveness analyses, and proper qualification of the laboratory. It is important that such methods, at least initially, are introduced in addition to, not instead of, the well-standardized conventional protocols. References 1. Goodfellow M, Wayne LG. Taxonomy and nomenclature. In: Ratledge C, Stanford J, eds. The Biology of the Mycobacteria. 1. 1. London, England: Academic Press Inc., Ltd., 1982:471–521. 2. Woods GL, Washington JA. Mycobacteria other than Mycobacterium tuberculosis: review of microbiologic and clinical aspects. Rev Infect Dis 1987; 9:275–294. 3. Goodfellow M, Magee JG. Taxonomy of mycobacteria. In: Gangadharam PRJ, Jenkins PA, eds. Mycobacteria: Basic Aspects. New York, New York: Chapman and Hall (International Thomson Publishing), 1998:1–71. 4. Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of 1990s. Clin Microbiol Rev 2003; 16:319–354. 5. Heifets L. Mycobacterial infections caused by nontuberculous mycobacteria (NTM). Semin Resp Crit Care Med 2004; 25(3):283–295. 6. Aranaz A, Cousins D, Mateos A, Domonguez L. Evaluation of M. tuberculosis subsp. caprae to species rank as M. caprae comp. nov. sp. Int J Syst Evol Microbiol 2003; 53:1785–1789. 7. Cousins DV, Bastida R, Cataldi A, et al. Tuberculosis in seals caused by a novel member of Mycobacterium tuberculosis complex: M. pinnipedii sp. nov. Int J Syst Evol Microbiol 2003; 53:1305–1314.

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8. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393:537–544. 9. Cole ST. Comparative mycobacterial genomics as a tool for drug target and antigen discovery. Eur Respir J 2002; 20:78S–86S. 10. Tenover TC, Arbet RD, Goering V, et al. Interpreting chromosomal DNA restriction pattern produced by pulsed-field gel electrophoresis criteria for bacterial strain typing. J Clin Microbiol 1995; 33:2233–2239. 11. Thierry D, Cave MD, Eisenach KD, et al. IS6110, an IS-like element of M. tuberculosis complex. Nucleic Acids Res 1990; 18:188. 12. Poulet S, Cole ST. Characterization of the highly abundant polymorphic GC-richrepetitive sequence (PGRS) present in M. tuberculosis. Arch Microbiol 1995; 163: 87–95. 13. van Soolingen D. Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements. J Intern Med 2001; 349:1–26. 14. Small PM, Hopewell PC, Singh S, et al. The epidemiology of tuberculosis in San Francisco: a population-based study using conventional and molecular methods. N Engl J Med 1994; 330:1703–1709. 15. DeRiemer K, Daley CL. Tuberculosis transmission based on molecular epidemiologic research. Sem Resp Crit Care Med 2004; 25(3):297–306. 16. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of M. tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993; 31:406–409. 17. Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD. Nature of DNA polymorphism in the direct repeat cluster of M. tuberculosis: application for strain differentiation by novel typing method. Mol Microbiol 1993; 10:1057–1065. 18. Haas WH, Butler WR, Woodley CL, Crawford JT. Mixed-linker polymerase chain reaction: a new method for fingerprinting of M. tuberculosis complex isolates. J Clin Microbiol 1993; 31:1293–1298. 19. Kato-Maeda M, Rhee JT, Gingeras TR, et al. Comparing genomes within the species of M. tuberculosis. Genome Res 2001; 11:547–554. 20. Kremer K, van Soolingen D, Frothingham R, et al. Comparison of methods based on different molecular epidemiological markers for typing complex strains: inter-laboratory study of discriminatory power and reproducibility. J Clin Microbiol 1999; 37:2607–2618. 21. Supply P, Lesjean S, Savine E, et al. Automated high throughout genotyping for study of global epidemiology of M. tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 2001; 39:3563–3571. 22. Cole ST, Eisenach KD, McMurray DN, Jackobs WR, eds. Tuberculosis and the Tubercle Bacilli. Washington, DC: ASM Press, 2005. 23. Nuermberger E, Bishai WR, Grosset JH. Latent tuberculosis infection. Sem Resp Crit Care Med 2004; 25(3):317–336. 24. Wayne LG, Sohaskay CD. Nonreplicating persistence of M. tuberculosis. Annu Rev Microbiol 2001; 55:139–163. 25. Mitchison DA. The action of antituberculosis drugs in shout-course chemotherapy. Tubercle 1985; 66:219–225. 26. Mitchison DA. Role of individual drugs in the chemotherapy of tuberculosis. Int J Tuberc Lung Dis 2000; 4:796–806. 27. Harries A, Maher D, Graham S. TB/HIV: a Clinical Manual. 2d ed. Geneva: World Health Organization, 2004. 28. Behr MA, Warren SA, Salamon H, et al. Transmission of M. tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 1999; 353:444–449.

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29. World Health Organization. An Expanded DOTS Framework for Effective Tuberculosis Control. Geneva: WHO/CDS/TB/2002.297, 2002. 30. Kent PT, Kubica JP. Public Health Mycobacteriology: a Guide for the level III Laboratory. Atlanta: Center for Disease Control, 1985. 31. Hall GS. Primary processing of specimens, and isolation and cultivation of mycobacteria. Clin Lab Med 1996; 16(3):551–568. 32. Heifets L, Desmond E. Clinical mycobacteriology (tuberculosis) laboratory: services and methods (Chapter 4). In: Cole ST, Eisenach K, McMurray DN, Jackobs WR, eds. Tuberculosis and Tubercle Bacilli. Washington, DC: ASM Press, 2005:49–69. 33. Heifets L, Sanchez T. New agar medium for mycobacteria (HSTB). US Patent 6,579,694 B2; June 17, 2003. 34. Witebsky FG, Kruczak-Filipov P. Identification of mycobacteria by conventional methods. Clin Lab Med 1996; 16:569–602. 35. Heifets L, Jenkins PA. Speciation of mycobacteria in clinical laboratories. In: Gangadharam PRJ, Jenkins PA, eds. Mycobacteria: Basic Aspects. Vol. 1. New York: Chapman & Hall, 1998:308–350. 36. Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin Microbiol Rev 2001; 14(4):704–726. 37. Jost KC Jr., Dunbar DF, Barth SS, Headley VL, Elliot LB. Identification of Mycobacterium tuberculosis and M. avium complex directly from smear-positive sputum specimens and Bactec 12B cultures by high-performance liquid chromatography with fluorescence detection and computer-driven pattern recognition models. J Clin Microbiol 1995; 33:1270–1277. 38. Roberts GD, Bo¨ttger EC, Stockman L. Rapid methods for the identification of mycobacterial species. Clin Lab Med 1996; 16:603–616. 39. Arnold LJ, Hammond PW, Wiese WA, Nelson NC. Assay formats involving acridinium-ester-labeled DNA probes. Clin Chem 1989; 35:1588–1594. 40. Eisenach KD, Sifford MD, Cave MD, Bates JH, Crawford JT. Detection of Mycobacterium tuberculosis in sputum samples using polymerase chain reaction. Am Rev Resp Dis 1991; 144:1160–1163. 41. Forbes BA, Hicks KES. Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction. J Clin Microbiol 1993; 31:1688–1694. 42. Shinnick TM, Jonas V. Molecular approach to the diagnosis of tuberculosis. In: Bloom BR, ed. Tuberculosis: Pathogenesis, Protection, and Control. Washington, DC: ASM Press, 1994:517–530. 43. Desmond E, Loretz K. Use of the Gen-Probe amplified Mycobacterium tuberculosis direct test for early detection of Mycobacterium tuberculosis in BACTEC 12B medium. J Clin Microbiol 2001; 39(5):1993–1995. 44. Rogall T, Flohr T, Bo¨ttger EC. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol 1990; 136:1915–1920. 45. Kirschner P, Springer B, Vogel U. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a two-year experience in a clinical laboratory. J Clin Microbiol 1993; 31:2882–2889. 46. Kirschner P, Meier A, Bo¨ttger EC. Genotypic identification and detection of mycobacteria facing novel and uncultured pathogens. In: Pershing DH, Tenover F, White TJ, eds. Diagnostic Molecular Microbiology. Washington, DC: American Society for Microbiology, 1993:173–190. 47. Espinal MA, Laszlo A, Simonsen L, et al. Global trends in resistance to tuberculosis drugs. N Engl J Med 2001; 344:1294–1303.

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48. Canetti G, Fox W, Khomenko A, et al. Advances in techniques of testing mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control programs. Bull World Health Org 1969; 41:21–43. 49. Canetti G, Froman S, Grosset J, et al. Mycobacteria: laboratory methods for testing drug sensitivity and resistance. Bull World Health Org 1963; 29:565–578. 50. Telenti A. Genetics of drug resistance in tuberculosis. Clin Chest Med 1997; 18: 55–64. 51. Takiff HE. The molecular mechanisms of drug resistance in M. tuberculosis (Chapter 6). In: Bastian I, Portaels F, eds. Multi-drug Resistant Tuberculosis. The Netherlands: Kluwer Academic Publishers, 2000:77–114. 52. Heifets L. Drug susceptibility tests in the management of chemotherapy of tuberculosis (Chapter 3). In: Heifets LB, ed. Drug Susceptibility in the Chemotherapy of Mycobacterial Infections. Boca Raton, FL: CRC Press, 1991:89–121. 53. Heifets L. Drug susceptibility testing in mycobacteriology. Clin Lab Med 1996; 16:641–656. 54. Heifets L, Cangelosi GA. Drug susceptibility testing of Mycobacterium tuberculosis—a neglected problem at the turn of the century. Int J Tuberc Lung Dis 1999; 3(7): 1–18. 55. Parsons LM, Somoskovi A, Urbanchzik R, Salfinger M. Laboratory diagnostic aspects of drug resistant tuberculosis. Front Biosci 2004; 9:2086–2115. 56. Roberts G, Goodman NL, Heifets L, et al. Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of M. tuberculosis from acid-fast smear-positive specimens. J Clin Microbiol 1983; 18:689–696. 57. Siddiqi SH, Libonati JP, Middlebrook G. Evaluation of rapid radiometric method for drug susceptibility testing of M. tuberculosis. J Clin Microbiol 1981; 13: 908–912. 58. Heifets L. Rapid automated method (BACTEC system) in clinical mycobacteriology. Sem Respir Med 1986; 1(4):242–249. 59. Bemer PF, Palicova S, Rusch-Gerdes H, et al. Multicenter evaluation of fully automated BACTEC Mycobacteria Growth Indicator Tube 960 system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2002; 40(1):150–154. 60. Palomino JC. Novel rapid antimicrobial susceptibility tests for M. tuberculosis (Chapter 9). In: Bastian I, Portaels F, eds. Multi-drug Resistant Tuberculosis. The Netherlands: Kluwer Academic Publishers, 2000:145–162. 61. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in M. tuberculosis. Tuberc Lung Dis 1998; 79:3–29. 62. Zhang A, Telenti A. Genetics of drug resistance in M. tuberculosis. In: Hatfull GF, Jacobs WR, eds. Molecular Genetics of Mycobacteria. Washington, DC: ASM Press, 2000:225–254. 63. Telenti A, Tenover FC. Genetic methods for detecting bacterial resistance genes. In: Lewis K, Salyers AA, Taber HW, Wax RG, eds. Bacterial Resistance to Antimicrobials. New York: Marcel Dekker, Inc., 2002:239–264. 64. Nillson LE, Hoffner SE, Ansehn S. Rapid susceptibility testing of M. tuberculosis by luminescence assay of mycobacterial ATP. Antimicrob Agents Chemother 1988; 32:1208–1212. 65. Franzblau SG, Witzig RS, McLaughlin JC, et al. Rapid, low-technology MIC determination with clinical M. tuberculosis isolates by using the microplate alamar blue assay. J Clin Microbiol 1998; 36:362–366. 66. Palomino JC, Portaels F. Simple procedure for drug susceptibility testing of M. tuberculosis using a commercial colorimetric assay. Eur J Clin Microbiol Infect Dis 1999; 18:380–383.

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67. Kirk SM, Schell RF, Moore AV, et al. Flow cytometric testing of susceptibility of M. tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours. J Clin Microbiol 1998; 36:1568–1573. 68. Moore AV, Kirk SM, Callister SM, et al. Safe determination of susceptibility of M. tuberculosis to antimicrobial agents by flow cytometry. J Clin Microbiol 1999; 37:479–483. 69. Drowart A, Cambasso CL, Huygen K, et al. Detection of rifampicin and isoniazid resistance of M. tuberculosis by particle counting immunoassay (PACIA). Int J Tuberc Lung Dis 1997; 1:284–288. 70. Jacobs W Jr., Barletta WR, Bloom BR Jr., et al. Rapid assessment of drug susceptibilities of M. tuberculosis by means of luciferase reporter phages. Science 1993; 260:819–822. 71. Riska PF, Su Y, Bardarov S, et al. Rapid film-based determination of antibiotic susceptibilities of M. tuberculosis strains by using a luciferase reporter phage and the Bronx box. J Clin Microbiol 1999; 37:1144–1149. 72. Wilson SM, al-Suwaidi Z, McNerney R, et al. Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis. Nat Med 1997; 3:465–468. 73. Albert H, Trollip AP, Mole RJ, et al. Rapid indication of multidrug-resistant tuberculosis from liquid cultures using FASTPlaque TB-RIFTM, a manual phage-based test. Int J Tuberc Lung Dis 2002; 6:523–528. 74. Caviedes L, Lee TS, Gilman RH, et al. Rapid, efficient detection and drug susceptibility testing of M. tuberculosis in sputum by microscopic observation if broth cultures. J Clin Microbiol 2000; 38:1203–1208. 75. Park WG, Bishai WR, Chaisson RE, Dorman SE. Performance of microscopic observation drug susceptibility assay in drug susceptibility for M. tuberculosis. J Clin Microbiol 2002; 40:4750–4752. 76. Moor DAJ, Mendoza D, Gilman RH, et al. Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis in resource-poor setting. J Clin Microbiol 2004; 42(10):4432–4437. 77. Alcaide F, Telenti A. Molecular techniques in the diagnosis of drug-resistant tuberculosis. Ann Acad Med 1997; 26:647–650. 78. Drobniewski FA, Wilson SM. The rapid diagnosis of isoniazid and rifampicin resistance in M. tuberculosis—a molecular story. J Med Microbiol 1997; 47:189–196. 79. Michailovich V, Lapa S, Gryadunov D, et al. Identification of rifampin-resistant M. tuberculosis strains by hybridization, PCR, and ligase detection reaction on oligonucleotide microchips. J Clin Microbiol 2001; 39:2531–2540. 80. Deng J-Y, Zhang X-E, Lu H-B, et al. Multiplex detection of mutations in clinical isolates of rifampin-resistant M. tuberculosis by short oligonucleotide ligation assay on DNA chips. J Clin Microbiol 2004; 42(10):4850–4852. 81. Nikolayevsky V, Brown T, Balabanova Y, et al. Detection of mutations associated with isoniazid and rifampin resistance in M. tuberculosis isolates from Samara region, Russian Federation. J Clin Microbiol 2004; 42(10):4498–4502.

3 Genomics and Evolution of Tubercle Bacille

VE´RONIQUE VINCENT

MARIA-CRISTINA GUTIE´RREZ

Stop TB Department, World Health Organization, Geneva, Switzerland

Laboratoire de Re´fe´rence des Mycobacte´ries, Institut Pasteur, Paris, France

ROLAND BROSCH Unite´ de Ge´ne´tique Mole´culaire Bacte´rienne, Institut Pasteur, Paris, France

I. Introduction Soon after the discovery of the bacterial agent of tuberculosis by Robert Koch in 1888, Lehmann and Neumann formally described Mycobacterium tuberculosis (1). By the early 1900s, culture techniques and bacteriological tests allowed the differentiation of M. tuberculosis from Mycobacterium bovis by growth rate, colonial morphology, biochemical characteristics, and behavior in experimental animals (2). According to the same criteria, Mycobacterium microti and Mycobacterium africanum were identified in the 1950s and in the 1970s, respectively. Moreover, serial passages of a M. bovis strain over 13 years on potato slices soaked in ox bile and glycerol led Calmette and Gue´rin to the selection of a strain that lost its virulence and was used for human vaccine purposes, beginning from 1921. DNA/DNA hybridization studies showed a high level of genomic relatedness among the above bacille, indicating that they all belong to a single species (3). Sequence analyses confirmed these data, showing that tubercle bacille share great genetic similarity, seen by homology at the DNA level of greater than 99% (4–6). However, some particular genetic and phenotypic characteristics based mainly on different host preferences have led researchers to maintain 47

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the traditional species names, and to create additional ones. The M. tuberculosis complex now encompasses M. tuberculosis, M. bovis, M. africanum (7), M. microti (8), Mycobacterium pinnipedii (9), and Mycobacterium caprae (10). In this chapter, the additional agent of tuberculosis, Mycobacterium canettii (11), is not included in the designation ‘‘M. tuberculosis complex’’ and will be discussed separately. II. The Genome Sequence and Biology of Mycobacterium tuberculosis Until a few years ago, M. tuberculosis was called the ‘‘genetically intractable organism’’ (12). With the development of genome sequencing, a growing body of information became available in the last 10 years. Genomics, the systematic analysis of the complete genome by means of DNA sequencing and bioinformatics, generates complete data sets of genes, proteins, and antigens, allows in vitro reconstruction of evolution, and provides rich biological, evolutionary, and medical knowledge. Five complete genome sequences of pathogenic mycobacteria have been published (two for M. tuberculosis, one for M. bovis, one for Mycobacterium leprae, and one for Mycobacterium paratuberculosis) (6,13–16), and several other mycobacterial sequencing projects [Mycobacterium abscessus, Mycobacterium avium, M. bovis bacille Calmette–Gue´rin (BCG), Mycobacterium marinum, M. microti, Mycobacterium smegmatis, and Mycobacterium ulcerans] are either in the finishing phase or have been completed, awaiting publication (17). The M. tuberculosis H37Rv genome consists of 4,411,532 base pairs (bp) containing approximately 4000 genes encoding proteins and 50 genes encoding RNAs (15,18,19). Similarly to other prokaryotes, a 91% potential coding capacity and gene density of one gene for 1.1 kb are found. The genome sequence highlighted several characteristics of the biology of M. tuberculosis. 1.

M. tuberculosis differs radically from other bacteria in that at least 8% of its coding capacity is dedicated to lipid metabolism. The genome reveals more genes encoding potential lipid biosynthetic activities than there are known products in in vitro–grown tubercle bacille, thus raising the intriguing possibility that many more novel lipids and polyketides remain to be found. Moreover, numerous genes and proteins associated with lipolytic functions seem to enable M. tuberculosis to degrade exogenous lipids and sterols from host tissues. Support for this proposal is available because Wheeler et al. have reported that mycobacteria obtained from infectedtissuescanavidlydegradeexogenouslipidsandcontribute to energy metabolism (20,21). In addition to the classical b-oxidation cycle catalyzed by the multifunctional FadA/FadB proteins, the genome sequence suggests that alternative lipid oxidation pathways may well exist. A striking level of redundancy in

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lipid metabolic genes was apparent in the M. tuberculosis H37Rv genome, with 36 fadD alleles encoding acyl-CoA synthase, 36 fadE genes encoding acyl-CoA dehydrogenase, and 21 echA genes encoding enoyl-CoA hydratase/isomerase (15). The apparent redundancy in lipid enzymes may, however, need to be readdressed in light of the recent work on the fadD genes. Gokhale and coworkers have shown that some of the fadD alleles do not encode fatty acyl-CoA ligases, but instead code for a new class of fatty acyl-AMP ligases that are linked to a proximal pks gene encoding a unique polyketide synthase (22). It is therefore possible that the apparent redundancy in lipid enzymes hides novel enzyme activities. As another example, the genome comparison of M. tuberculosis H37Rv and the more distantly related Corynebacterium glutamicum has greatly helped to identify the gene encoding the condensase, which is responsible for the final condensation step in mycolic acid biosynthesis. Although M. tuberculosis harbors several pks genes encoding various polyketide synthases, C. glutamicum only shows one pks gene ortholog of pks13. Because both mycobacteria and corynebacteria produce mycolic acids, Pks13 that contains the four catalytic domains theoretically required for the condensation reaction was selected as the candidate condensase. These predictions were then experimentally confirmed. Because mycobacteria, in contrast to corynebacteria, are not viable without mycolic acids, Pks13 is also a promising target for the development of new antimycobacterial drugs (23). In addition to electron transport chains using oxidative phosphorylation, components of several anaerobic electron-transport chains were identified. The dual presence of these energy-generation systems allows M. tuberculosis to adapt its metabolism to environmental changes, actively growing in an oxygen-rich lung cavity on the one hand or surviving in oxygen-poor tissues or granulomas on the other. An operon, narGHJI, is present for the formation of nitrate reductase, which allows utilization of nitrate as a terminal electron acceptor. Immunodeficient mice infected with a narG mutant of M. bovis BCG developed smaller granulomas than those infected with the wild BCG type, and presented no clinical signs of disease after more than 200 days (24). It therefore appears that the ability to respire anaerobically contributes to virulence. It is also noteworthy that one of the classical microbiological methods to differentiate M. bovis from M. tuberculosis is based on nitrate, reductase activity. M. tuberculosis reduces nitrate to nitrite, whereas M. bovis performs this reduction very poorly. Bange and coworkers have also shown that this defect in nitrate reductase activity is due to a point mutation in the promoter of the M. bovis narGHIJ cluster (24).

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4.

5.

Almost 3.4% of the genome is composed of insertion sequences (IS) and phages. Some of these IS elements are highly similar to those encountered in other actinomycetes, suggesting that horizontal transfers occurred in nature when the ancestor of M. tuberculosis lived in soil and shared its ecological niche with other bacteria. Moreover, IS6110, the most abundant element, which varies in copy number from 0 to 23 in M. tuberculosis, plays an important role in genome plasticity of individual strains, causing either inactivation of genes by insertion of the IS element or deletion of chromosomal fragments in the size range of several kilobases by homologous recombination of two adjacent IS6110 elements (25,26). The phospholipase encoding genes plcA-D genes are hotspots of IS6110 integration (27). The M. tuberculosis sequence presents large gene families, the PE and PPE families, encoding nearly 10% of the coding sequence and comprising 100 and 67 members, respectively. Members of these families share a conserved N-terminal domain with the characteristic motifs ProGlu (PE) or ProProGlu (PPE), whereas the C-terminal segment varies in length and sequence. These proteins are cell-wall associated and surface exposed. The two multigene families have a significant higher genetic variability compared with the genome as a whole (6). They may represent the principal source of antigenic variation and interfere with immune responses by inhibiting antigen processing (15). Disruption of the gene encoding a PE protein leads to a marked reduction of bacterial clumping, suggesting that this protein may mediate cell–cell adhesion and altered phagocytosis by macrophages (28). Another PE protein can bind fibronectin and could mediate bacterial interaction with host cells (29,30). Several characteristics can be considered to contribute to the slow growth rate of the tubercle bacille. (i) M. tuberculosis, like the other slow-growing mycobacteria, has only a single copy of the genes coding for the ribosomal RNAs, whereas most bacteria have several copies of these genes (31). (ii) This single ribosomal RNA operon is located unusually at 1500 kb from the origin of replication, unlike most bacteria which have several copies of this operon close to the origin of replication to exploit the gene-dosage effect obtained during replication. (iii) Genes are evenly distributed along the two DNA strands and 59% of the genes are transcribed with the same polarity as the replication fork. By contrast, a high percentage of genes located on the leading replication strand are thought to be associated with higher gene-expression levels, because replication and transcription act with the same polarity (15).

In addition to the H37Rv type strain, another M. tuberculosis strain has also been fully sequenced. The strain CDC1551 is a clinical, highly

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transmissible strain and was the cause of an outbreak in a rural area of the United States (32). The strain induces a strong immunologic response by the host and is less virulent than strain H37Rv in animal models (33). Its genome sequence is nearly identical to that of H37Rv but slightly smaller at 4,403,836 bp (6,34). The M. bovis strain AF2122/97, isolated from a cow in the United Kingdom, has a genome nearly 70 kb smaller than those of the M. tuberculosis strains at 4,435,492 bp (13,35). The main reason for this size difference is the absence of several previously identified regions from the genome of M. bovis, described in more detail below. The number of about 2400 single nucleotide polymorphisms (SNPs) between M. bovis and M. tuberculosis corresponds to approximately twice as many point mutations than the number of SNP between the two sequenced M. tuberculosis strains. Several of these SNPs may have important consequences for the biology of the organism as shown by a SNP causing one of the key in vitro differences between M. bovis and M. tuberculosis, which is a requirement for pyruvate when glycerol is the sole carbon source. Strikingly, the genome sequence of M. bovis revealed that the gene encoding pyruvate kinase activity, pykA, contains a SNP that affects binding of the Mg2þ cofactor. As such, M. bovis glycolytic intermediates are blocked from feeding into oxidative metabolism, meaning that in vivo M. bovis must rely on amino acids or fatty acids as a carbon source for energy metabolism. The same SNP is also responsible for the classical phenotypic difference between the ‘‘eugonic’’ growth of M. tuberculosis on media containing glycerol, compared to the small ‘‘dysgonic’’ colonies produced by M. bovis on the same media (36). III. Comparative Genomics and Evolution Within the Mycobacterium tuberculosis Complex The availability of several genomes from members of the M. tuberculosis complex now allows genome-wide comparisons to be employed at the nucleotide level. However, larger polymorphisms between tubercle bacille were first identified by subtractive hybridization, bacterial artificial chromosome (BAC) arrays, and microarrays (37–39). By these methods, regions of differences (RDs) have been identified as being absent from M. bovis BCG but present in M. tuberculosis. The presence or absence of these RD regions was analyzed in a large set of strains and gave rise to a scheme of the phylogenetic lineages within members of the M. tuberculosis complex (40). Sequencing of the flanking regions of the different RD regions—and particularly of RD9, which is present in full length in M. tuberculosis and absent from M. africanum, M. microti, M. bovis, M. pinnipedii, and M. caprae— revealed that most of the deletions had occurred within coding regions, exactly at the same sites, indicating firstly that the involved genetic events were deletions in the M. africanum–M. bovis lineage and not insertions into M. tuberculosis. Secondly, the identity of the junction sequences in various members of the M. tuberculosis complex from different hosts and geographical locations suggested that the deletions did not occur independently in the

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individual strains of the M. tuberculosis complex, but resulted from successive, irreversible genetic events in common progenitor strains, which were then inherited from daughter strains. M. bovis has lost these RD regions corresponding in total to approximately 70 kb, which are still present in M. tuberculosis. This logically excludes M. bovis as the ancestor of M. tuberculosis. In addition, except for one region (TbD1), which is present in M. bovis but absent from most tested M. tuberculosis strains, M. bovis did not contain any additional genomic material compared to M. tuberculosis. Thirdly, the members of the M. tuberculosis complex show a clonal population structure, with very little or no recent horizontal transfer as discussed below (41). Taken together, these results refute the previous hypothesis on the origin of human tuberculosis, suggesting that humans acquired tuberculosis through milk consumption in the era of domestication of cattle. This hypothesis was consistent with the large host range of M. bovis and the restricted human one of M. tuberculosis, and implied that M. tuberculosis evolved from the bovine bacille by adaptation from the bovine to the human host. The present hypothesis suggests that M. tuberculosis and M. bovis derived from a common ancestor, which resembled M. tuberculosis and could well have been a human pathogen already (40). It is also interesting that the DNA of tubercle bacille amplified from a North American Pleistocene extinct bison (dated 17,000 years) showed a genetic profile based on the DR region that resembled M. africanum or M. tuberculosis more closely than it resembeled M. bovis (42). For the M. tuberculosis lineage, presence or absence of the M. tuberculosis–specific TbD1 divides strains into ancestral M. tuberculosis strains, which still harbor the TbD1 region, and the modern strains, which have lost it. In the evolutionary pathway drawn by RDs, the loss of RD9 corresponds to a major divergence leading to a main lineage encompassing M. africanum and all the tubercle bacille that have animals as preferential hosts. Successive deletions, some species specific, led to M. microti, M. pinnipedii, M. caprae, M. bovis and eventually M. bovis BCG (Table 1). Thus, the RD analysis provides a useful molecular tool for the rapid and unambiguous identification of the various members of the M. tuberculosis complex (Table 1). These markers are in very good agreement with other genetic markers, such as for example, the pncA SNP, which is responsible for the natural resistance of M. bovis strains to the antituberculosis drug pyrazinamide. All tested M. bovis strains that showed the characteristic pncA SNP also lacked the RD4 region. Therefore, rapid polymerase chain reaction (PCR) screening for absence of RD4, using internal and flanking primers of the RD4 region, may be a useful additional procedure for identification of clinical isolates. IV. Evolution of Mycobacterium bovis BCG The use of genomic deletion analysis in strains of M. bovis BCG allowed the reconstruction of the BCG genealogy (45). It has to be remembered that early after the development of the vaccine, Calmette and Gue´rin distributed

Nitrate reductase þ – – V þ – – –

Niacin þ – – V – þ – –

– – þ þ þ

þ þ – – – – – – – –

þ þ

þ –

– –

PZA –

Pyruvate –

þ

TCH

þ þ þ þ þ

– –

þ

RD4

– þ – – –

– –

þ

RD9

þ þ þ – þ

– –

þ

RD12

RDpind

RDcanb RDmicc

RD1

TbD1a

RD specific

Note: The phenotypic tests include niacin production, presence of nitrate reductase, growth on TCH, growth stimulated by pyruvate, and growth in the presence of PZA. The genotypic characteristics consist of the presence (þ) or absence (
) of regions RD4, RD9, and RD12. a TbD1 region is absent from ‘‘modern’’ M. tuberculosis only and present in all members of the MTBC (40). b RDcan corresponds to a region specifically absent from M. canettii that only partially overlaps RD12 (40). c RDmic corresponds to a region specifically absent from M. microti that only partially overlaps RD1 (40). d RDpin corresponds to a region specifically absent from M. pinnipedii that only partially overlaps RD2 (40). Abbreviations: PZA, pyrazinamide; RD, regions of difference; V, variable; TCH, thiophen-2-carboxylic acid hydrazide. Source: From Refs. 8, 11, 37, 40, 43, and 44.

Mycobacterium tuberculosis Mycobacterium bovis Mycobacterium bovis bacille Calmette–Gue´rin Mycobacterium africanum Mycobacterium canettii Mycobacterium microti Mycobacterium caprae Mycobacterium pinnipedii

Strain

Table 1 Phenotypic and Genotypic Characteristics of the Mycobacterium tuberculosis Complex Members

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the BCG strain throughout the world for local vaccine production. Preparation of seed lots was performed only in the 1960s, and approximately 1000 passages were carried out in vaccine production centers. Local manufacturers also distributed cultures of the strain to other vaccine producers. This history explains the present diversity of BCG strains, which differ by phenotypic features, mycolic acid content, antigenic proteins as well as virulence and protective efficacy in mouse models (45,46). Because the vaccines never reverted to virulent strains, genetic changes that occurred during the attenuation process have permanently disabled BCG from causing tuberculosis in immunocompetent individuals. Comparative genomics of various BCG vaccines identified 10 genomic deletions that mark the molecular evolution of BCG daughter strains (39). Deletions specific for some BCG strains, which may contribute to the spectrum of protective immunity, as well as deletions shared by all BCG strains, which are likely to account for the attenuation of the vaccine, have been identified. The RD1 region is deleted in all BCG strains. This region encodes for two immunodominant, exported proteins in M. tuberculosis. Animal models infected with recombinant BCG vaccines expressing the two proteins, ESAT-6 and CFP10, showed that the presence of the RD1 region is simultaneously associated with increased virulence in immunocompromised hosts and with improved protection in immunocompetent hosts. The RD1 region is therefore an interesting target for second-generation vaccine candidates, combining the safety of the BCG vaccine with an improved protection against tuberculosis (47).

V. Population Structure and Clonal Evolution of the Mycobacterium tuberculosis Complex Even before genome sequences were available, Musser and coworkers stressed that SNPs were rare among tubercle bacille. Their pioneering studies on 26 structural genes totalling 2 Mb showed that the level of allelic variation in M. tuberculosis is 2000 times less than in Borrelia burgdorferi and 600 times less than in Neisseria meningitidis (48). The authors proposed the hypothesis that M. tuberculosis was evolutionarily young and had only recently spread globally. The availability of the three genome sequences confirmed these results and established that SNPs account for less than 0.2% of the differences between the two M. tuberculosis genomes and less than 0.05% of the differences between M. bovis and the two M. tuberculosis strains. Genomic studies were conducted on large number of strains to elucidate the structure of the M. tuberculosis population. Studies have investigated SNPs based on a set of structural genes (49,50) or at a genome-wide scale (51–53); large genomic deletions on microarrays or membranes (54–56); or the variability generated by intergenic sequences such as the mycobacterial interspersed repetitive units (MIRUs). MIRUs occur as tandem repeats between genes in operons and vary according to their copy number (41,57). Results showed significant linkage disequilibrium corresponding to nonrandom association

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of genomic polymorphism at different chromosomal loci, which were consistent with a highly clonal population structure of M. tuberculosis, where no lateral gene transfer, i.e., recombination of chromosomal sequences between strains was detectable (41). Based on the SNP of the katG and gyrA genes, Musser and coworkers identified three genetic groups: a large one (group 1) comprising all bacille of the M. tuberculosis complex and two other groups (groups 2 and 3) corresponding to M. tuberculosis sensu stricto (48). This first attempt to differentiate genetic groups among the tubercle bacille was confirmed and enriched by the RD classification (40). All strains of genetic groups 2 and 3 belong to ‘‘modern’’ M. tuberculosis strains as defined by the deletion of the TbD1 region. The polymorphism of a specific molecular marker identified only in the M. tuberculosis complex and not in other mycobacteria, the DR locus, has been used to define genotype families within the M. tuberculosis complex (58,59). The direct repeat (DR) region consists of a single locus characterized by 36-bp direct repeats interspersed by polymorphic spacer regions. It belongs to the family of Clustered Regularly Interspaced Short Palindromic Repeats identified in prokaryotic genomes (60). One of the most successful M. tuberculosis genotype families is the ‘‘Beijing’’ family first identified in China, which spread to other countries of the region and emerged in other parts of the world, specially in the former Soviet Union and in the West (61), and was recently found in Africa (62). The ‘‘Beijing’’ family was the focus of specific interest and studies because strains of this family show particular virulence and are often associated with frequent resistance to several antituberculosis drugs. The ‘‘W’’ strains, involved in an outbreak of more than 250 patients with multidrug-resistant M. tuberculosis in the United States, belong to the ‘‘Beijing’’ family (63). Molecular markers described above (DR locus, MIRUs, IS6110, RD, and/or SNP analysis) all contribute to identify genetic lineages within the two main agents of tuberculosis—M. tuberculosis and M. bovis. Because the population structure is clonal and the occurrence of horizontal gene transfer is rare, starting from a common ancestor, each phylogenetic lineage evolves independently from others (40). This peculiar population structure of the M. tuberculosis complex has important implications in public health because distinct lineages may differ in phenotypes such as virulence, transmissibility, ability to reactivate, and host-specific association. Interestingly, the role of lipids in the pathogenesis of M. tuberculosis has been recently revisited. Some M. tuberculosis strains, such as the 210 (Beijing type) strain, produce a particular phenolic glycolipid (PGL), whereas most M. tuberculosis strains do not. The absence of the PGL has been associated with a deletion of 7 bp that introduces a frameshift in the pks15/1 gene (64) found in strains belonging to the genetic group 2 or 3 (65), as defined by Sreevatsan et al. (48). The synthesis of this PGL molecule is correlated with a decrease in the production of proinflammatory cytokines by host immune cells (66). This finding could explain in part the

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hypervirulent phenotype observed for strains from the Beijing family (33,67) and the stability of the associated phenotype due to a specific deletion (64). VI. Ecotypes Within the Mycobacterium tuberculosis Complex For the M. bovis lineage, host preference has been demonstrated with the description of genetic clades prevalent in voles (M. microti), goats (M. caprae), sea lions, and fur seals (M. pinnipedii), and in artiodactyls (antelopes, oryx, etc.) (68). These observations led Smith et al. to propose the application of the ecotype concept to the M. tuberculosis complex. According to Cohan’s model of bacterial speciation (69), adaptation of a strain to a new niche corresponds to the emergence of a new ecotype that becomes immune to diversity. In other words, strains adapted to one host are less prone to infect another host. In the evolutionary scheme of Brosch et al. (Fig. 1), the lineage that encompasses animal-pathogen tubercle bacille may be seen as a series of host-adapted clades that follow the Cohan’s ecotype concept. M. microti, M. pinnipedii, and M. caprae have distinct host preference, and their evolution is related to specific SNPs and genomic deletions (40,71). They can be considered as ecotypes although further division within these groups is possible. The specific adaptation to a host and maladaptation to others is illustrated by the historical rabbit infection model used to differentiate M. tuberculosis

Figure 1 Scheme of the evolutionary pathway of the tubercle bacille. Source: Adapted from Refs. 40, 70.

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from M. bovis. In this animal, lesions due to M. tuberculosis regress over time and those due to M. bovis lead to pulmonary cavities. Whereas M. bovis has been regarded as a generalist and M. tuberculosis as a restricted human pathogen, Smith et al. stress that the ecotype concept applied to the M. tuberculosis complex should lead to new research insights in the identification of authentic ecotypes and the investigation of genetic differences associated with host specificity (68). This issue has already been addressed in a study led by P. Small, specifically designed to determine whether distinct strains of M. tuberculosis infect different human populations and whether associations between host and pathogen are stable despite global traffic in large cosmopolitan cities (55). The study supports the expected results from the theory of population genetics and shows highly stable association between host and pathogen populations. Recently, Gagneux et al. expanded these results and showed that M. tuberculosis lineages were more prone to spread in sympatric than in allopatric patient populations. These observations suggest that, similarly to M. bovis clades adapted to specific animal hosts, M. tuberculosis genetic lineages have adapted to specific human host populations and are maladapted to others (72). VII. Mycobacterium prototuberculosis: The Progenitor of the Mycobacterium tuberculosis Complex Orphan Clone The M. tuberculosis complex (as defined in this chapter, encompassing the species M. tuberculosis, M. africanum, M. microti, M. caprae, M. pinnipedii, and M. bovis) represents one of the most extreme examples of genetic homogeneity with about 0.01% to 0.03% synonymous nucleotide variations in their genomes. In comparison, the synonymous nucleotide variation is close to 6% and 12% in bacterial species such as N. meningitidis and Escherichia coli, respectively. No significant trace of horizontal genetic exchange has been detected among members of the complex. These data indicate that the M. tuberculosis complex is evolutionarily young and corresponds to a clone that had recently spread globally, roughly 15,000 to 20,000 years ago (48). However, the clone remained an orphan, with no identified progenitor species. Similarly, other main human pathogens such as Salmonella enteritica serotype Typhi and Yersinia pestis consist of single clones of recent emergence. These clones have evolved from well-known progenitor species, Yersinia pseudotuberculosis and S. enteritica, respectively. The progenitor of M. tuberculosis has been elucidated recently (70). In the evolutionary scheme based on deletion analysis (Fig. 1), Brosch et al. indicated that M. canetti presented all the genomic regions deleted in the members of the M. tuberculosis complex (40). M. canetti strains are rare strains that present an unusual smooth colony type. Although the first smooth colony of a tubercle bacille had been described in the 1970s by G. Canetti, only few isolates with this unusual phenotype had been isolated worldwide (11). Recently, a collection of 37 smooth strains was investigated

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(70). Strains were isolated from tuberculous patients, mostly immunocompetent patients who were living in or had lived in Djibouti, East Africa. Genetic data, based on the molecular markers usually used for the M. tuberculosis complex, on 16S rDNA sequencing and on the analysis of polymorphism within six housekeeping genes totalling 3387 nucleotides, showed that the smooth bacille do not all correspond to M. canetti but define eight distinct genetic clusters (Fig. 2). All these groups and the M. tuberculosis complex share the same 16S rRNA sequence and form a single mycobacterial species. However, the synonymous nucleotide variation within the smooth tubercle bacille was much higher than in the M. tuberculosis complex alone but interestingly in the range of other bacterial species. In addition, the housekeeping genes display mosaic structures, providing direct evidence of intragenic recombination within the smooth

Figure 2 Split decomposition analysis of the concatenated sequences of the six housekeeping genes of the eight genetic groups of smooth tubercle bacille (A to I) and of those of the Mycobacterium tuberculosis complex. Groups A and C/D correspond to the previous formal description of Mycobacterium canetti. The nodes represent strains and are depicted as small squares (smooth tubercle bacille) or small circles (MTBC members). The scale bar represents Hamming distance. Abbreviation: MTBC, Mycobacterium tuberculosis complex. Source: Adapted from Refs. 11, 70.

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strains. The sequences of these housekeeping genes in the M. tuberculosis complex appear as a patchwork of sequences from different smooth groups. The M. tuberculosis genome therefore appears to be a composite assembly of smooth bacille genomic fragments resulting from ancient horizontal DNA exchanges. In other words, the M. tuberculosis complex is only a clone of the larger tubercle bacille species defined by the smooth strains. Knowledge of this ancient history contributes to explain the extraordinary adaptation of the M. tuberculosis complex to mammals, because genetic exchange is known to play a crucial role in the adaptation of pathogens to their hosts (70). Assuming the same hypothesis to estimate the age of the M. tuberculosis clone of 35,000 years, the minimal time needed to accumulate the observed amount of synonymous divergence in the smooth strains is between 2.6 and 2.8 million years (70). Instead of having a recent origin as previously thought, tuberculosis could be much older than the plague, typhoid fever, or malaria, and might have affected early hominids. It is fascinating that the history of the tubercle bacille may mirror the history of the human species, with a common birthplace in East Africa, where hominids were present three million years ago. It is also striking that the diversity found in smooth strains, all originating from Djibouti, and the homogeneity retrieved in the globally spread M. tuberculosis complex, are reminiscent of the distribution of human populations with the highest genetic distance found in Africa. These findings suggest that tubercle bacille emerged in Africa where they diversified and a successful clone expanded and spread globally, possibly carried by the waves of human migration out of Africa. The long interaction lasting almost three million years between the tubercle bacille and hominids may explain the successful persistence of the bacille in the human host with the sophisticated protection conferred by granulomas, which both protect living bacteria and isolate them from their host for decades, as long as the immune system is not impaired. VIII. Conclusion The investigation of smooth bacille and the description of M. prototuberculosis, the progenitor of the M. tuberculosis complex clone, suggest that the tubercle bacille are much more ancient than previously thought. Tubercle bacille could have been present in early hominids as long as three million years ago. The human–chimpanzee divergence is estimated to have occurred between five and seven million years ago (73). It is noteworthy that comparative genomics of the chimpanzee and human genomes showed that among the most rapidly evolving genes is the gene for granulysin, a protein which directly kills extracellular M. tuberculosis, alters the membrane integrity of the bacille, and contributes to immunity against intracellular pathogens (74). The description of M. prototuberculosis opens new avenues of research for a better understanding of the successful expansion of M. tuberculosis. Comparative genomics and proteomics of smooth tubercle bacille, which disclose an extensive polymorphism and are confined to a limited geographical area, and

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classical tubercle bacille will contribute to elucidate the global expansion of the M. tuberculosis clone. Moreover, the identification of genetic families and the concept of ecotypes applied to the M. tuberculosis complex and its animal as well as human hosts have profound implications for tuberculosis control and specifically for the development of new vaccines. The variety of M. tuberculosis genotypes and host–pathogen adaptation should be considered in relation to the design and evaluation of vaccine candidates. References 1. Lehmann KB, Neumann R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik. 1st ed. Munchen, 1896. 2. Smith T. A comparative study of bovine tubercle bacilli and of human bacilli from sputum. J Exp Med 1898; 3:451–511. 3. Imaeda T. Deoxyribonucleic acid relatedness among selected strains of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium microti, and Mycobacterium africanum. Int J Syst Bacteriol 1985; 35:147–150. 4. Brosch R, Gordon SV, Pym A, Eiglmeier K, Garnier T, Cole ST. Comparative genomics of the mycobacteria. Int J Med Microbiol 2000; 290(2):143–152. 5. Gordon SV, Eiglmeier K, Garnier T, et al. Genomics of Mycobacterium bovis. Tuberculosis 2001; 81(1/2):157–163. 6. Fleischmann RD, Alland D, Eisen JA, et al. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 2002; 184(19): 5479–5490. 7. Niemann S, Kubica T, Bange FC, et al. The species Mycobacterium africanum in the light of new molecular markers. J Clin Microbiol 2004; 42(9):3958–3962. 8. van Soolingen D, van der Zanden AG, de Haas PE, et al. Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers. J Clin Microbiol 1998; 36(7):1840–1845. 9. Cousins DV, Bastida R, Cataldi A, et al. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov. Int J Syst Evol Microbiol 2003; 53(Pt 5):1305–1314. 10. Aranaz A, Cousins D, Mateos A, Dominguez L. Elevation of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to species rank as Mycobacterium caprae comb. nov., sp. nov. Int J Syst Evol Microbiol 2003; 53(Pt 6):1785–1789. 11. van Soolingen D, Hoogenboezem T, de Haas PE, et al. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int J Syst Bacteriol 1997; 47(4):1236–1245. 12. Jacobs WR. Mycobacterium tuberculosis: a once genetically intractable organism. In: Hatfall GF, Jacobs WR, eds. Molecular Genetics of Mycobacteria. Washington, DC: American Society for Microbiology, 2000:1–16. 13. Garnier T, Eiglmeier K, Camus JC, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U.S.A. 2003; 100(13):7877–7882. 14. Li L, Bannantine JP, Zhang Q, et al. The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci U.S.A. 2005; 102(35): 12344–12349. 15. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393:537–544. 16. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the leprosy bacillus. Nature 2001; 409(6823):1007–1011. 17. http://www.genomesonline.org/

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18. Camus JC, Pryor MJ, Medigue C, Cole ST. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology 2002; 148(Pt 10):2967–2973. 19. http://genolist.pasteur.fr/TubercuList/ 20. Wheeler PR, Bulmer K, Ratledge C. Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis? J Gen Microbiol 1990; 136(1):211–217. 21. Wheeler PR, Bulmer K, Ratledge C. Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens. J Gen Microbiol 1991; 137(4):885–893. 22. Trivedi OA, Arora P, Sridharan V, Tickoo R, Mohanty D, Gokhale RS. Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature 2004; 428(6981):441–445. 23. Portevin D, De Sousa-D’Auria C, Houssin C, et al. A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc Natl Acad Sci USA 2004; 101(1):314–319. 24. Stermann M, Sedlacek L, Maass S, Bange FC. A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis. J Bacteriol 2004; 186(9):2856–2861. 25. Brosch R, Gordon SV, Eiglmeier K, et al. Genomics, biology, and evolution of the Mycobacterium tuberculosis complex. In: Hatfull GF, Jacobs WR, eds. Molecular Genetics of Mycobacteria. Washington, DC: ASM Press, 2000:19–36. 26. Ho TB, Robertson BD, Taylor GM, Shaw RJ, Young DB. Comparison of Mycobacterium tuberculosis genomes reveals frequent deletions in a 20 kb variable region in clinical isolates. Yeast 2000; 17(4):272–282. 27. Vera-Cabrera L, Hernandez-Vera MA, Welsh O, Johnson WM, Castro-Garza J. Phospholipase region of Mycobacterium tuberculosis is a preferential locus for IS6110 transposition. J Clin Microbiol 2001; 39(10):3499–3504. 28. Brennan MJ, Delogu G, Chen Y, et al. Evidence that mycobacterial PE-PGRS proteins are cell surface constituents that influence interactions with other cells. Infect Immun 2001; 69(12):7326–7333. 29. Singh KK, Zhang X, Patibandla AS, Chien P Jr., Laal S. Antigens of Mycobacterium tuberculosis expressed during preclinical tuberculosis: serological immunodominance of proteins with repetitive amino acid sequences. Infect Immun 2001; 69(6):4185– 4191. 30. Espitia C, Laclette JP, Mondragon-Palomino M, et al. The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins? \questMicrobiology1999; 145ðPt12Þ : 3487 3495: 31. Bercovier H, Kafri O, Sela S. Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. Biochem Biophys Res Commun 1986; 136:1136–1141. 32. Valway SE, Sanchez MP, Shinnick TF, et al. An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N Engl J Med 1998; 338(10):633–639. 33. Manca C, Tsenova L, Bergtold A, et al. Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of INF-alpha/beta. Proc Natl Acad Sci USA 2001; 98:5752–5757. 34. http://www.tigr.org 35. http://genolist.pasteur.fr/BoviList/ 36. Keating LA, Wheeler PR, Mansoor H, et al. The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth. Mol Microbiol 2005; 56(1):163–174.

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37. Gordon SV, Brosch R, Billault A, Garnier T, Eiglmeier K, Cole ST. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 1999; 32(3):643–655. 38. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996; 178(5):1274–1282. 39. Behr MA, Wilson MA, Gill WP, et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 1999; 284(5419):1520–1523. 40. Brosch R, Gordon SV, Marmiesse M, et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 2002; 99(6): 3684–3689. 41. Supply P, Warren RM, Banuls AL, et al. Linkage disequilibrium between mini satellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area. Mol Microbiol 2003; 47(2):529–538. 42. Rothschild BM, Martin LD, Lev G, et al. Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present. Clin Infect Dis 2001; 33(3):305–311. 43. Aranaz A, Liebana E, Gomez-Mampaso E, et al. Mycobacterium tuberculosis subsp. caprae subsp. nov.: a taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Int J Syst Bacteriol 1999; 49: 1263–1273. 44. Espinosa de los Monteros LE, Galan JC, Gutierrez M, et al. Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism. J Clin Microbiol 1998; 36(1):239–242. 45. Brosch R, Behr MA. Comparative genomics and evolution of Mycobacterium bovis BCG. In: Cole ST, Eisenach KD, McMurray DN, Jacobs WR, eds. Tuberculosis and the Tubercle Bacilli. Washington, DC: American Society for Microbiology, 2005:155–164. 46. Behr MA, Small PM. A historical and molecular phylogeny of BCG strains. Vaccine 1999; 17(7–8):915–922. 47. Brodin P, de Jonge MI, Majlessi L, et al. Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity. J Biol Chem 2005; 280(40):33,953–33,959. 48. Sreevatsan S, Pan X, Stockbauer KE, et al. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 1997; 94(18):9869–9874. 49. Baker L, Brown T, Maiden MC, Drobniewski F. Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis. Emerg Infect Dis 2004; 10(9):1568–1577. 50. Filliol I, Motiwala AS, Cavatore M, et al. Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 2006; 188(2): 759–772. 51. Alland D, Whittam TS, Murray MB, et al. Modeling bacterial evolution with comparative-genome-based marker systems: application to Mycobacterium tuberculosis evolution and pathogenesis. J Bacteriol 2003; 185(11):3392–3399. 52. Gutacker MM, Mathema B, Soini H, et al. Single-nucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 2006; 193(1):121–128.

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53. Gutacker MM, Smoot JC, Migliaccio CA, et al. Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains. Genetics 2002; 162(4):1533–1543. 54. Goguet de la Salmoniere YO, Kim CC, Tsolaki AG, Pym AS, Siegrist MS, Small PM. High-throughput method for detecting genomic-deletion polymorphisms. J Clin Microbiol 2004; 42(7):2913–2918. 55. Hirsh AE, Tsolaki AG, DeRiemer K, Feldman MW, Small PM. Stable association between strains of Mycobacterium tuberculosis and their human host populations. Proc Natl Acad Sci USA 2004; 101(14):4871–4876. 56. Tsolaki AG, Hirsh AE, DeRiemer K, et al. Functional and evolutionary genomics of Mycobacterium tuberculosis: insights from genomic deletions in 100 strains. Proc Natl Acad Sci USA 2004; 101(14):4865–4870. 57. Supply P, Magdalena J, Himpens S, Locht C. Identification of novel intergenic repetitive units in a mycobacterial two-component system operon. Mol Microbiol 1997; 26(5):991–1003. 58. Kremer K, van Soolingen D, Frothingham R, et al. 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 Microbiol 1999; 37(8):2607–2618. 59. Sola C, Filliol I, Gutierrez MC, Mokrousov I, Vincent V, Rastogi N. Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerg Infect Dis 2001; 7(3): 390–396. 60. Gordon SV, Supply P. Repetitive DNA in the Mycobacterium tuberculosis complex. In: Cole ST, Eisenach K, McMurray D, Jacobs WR Jr., eds. Tuberculosis and the Tubercle Bacillus. Washington: American Society for Microbiology Press, 2005:191–202. 61. Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 2002; 8(8):843–849. 62. Glynn JR, Crampin AC, Traore H, et al. Mycobacterium tuberculosis Beijing genotype, northern Malawi. Emerg Infect Dis 2005; 11(1):150–153. 63. Bifani PJ, Plikaytis BB, Kapur V, et al. Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. JAMA 1996; 275(6):452–457. 64. Constant P, Perez E, Malaga W, et al. Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methly esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. J Biol Chem 2002; 277(41):38,148–38,158. 65. Marmiesse M, Brodin P, Buchrieser C, et al. Macro-array and bioinformatic analyses reveal mycobacterial ‘core’ genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex. Microbiology 2004; 150(Pt 2):483–496. 66. Reed MB, Domenech P, Manca C, et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 2004; 431(7004):84–87. 67. Lopez B, Aguilar D, Orozco H, et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 2003; 133(1):30–37. 68. Smith NH, Kremer K, Inwald J, et al. Ecotypes of the Mycobacterium tuberculosis complex. J Theor Biol 2006; 239:220–225. 69. Cohan FM. What are bacterial species? Annu Rev Microbiol 2002; 56:457–487.

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4 Epidemiology of Tuberculosis

JACQUELINE S. COBERLY and GEORGE W. COMSTOCK Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland, U.S.A.

I. Introduction Tuberculosis (TB) remains a leading contender for the dubious distinction of being the most important plague of mankind. The World Health Organization (WHO) estimated that in 2003, 8.8 million people developed TB, of whom 3.9 million had so many tubercle bacille in their sputum that the bacille could be identified by simple microscopy, and that there were 1.7 million deaths due to TB (1). In 2003, the incidence of TB was stable or falling in five of six WHO regions. The exception was the African region (AFR). Incidence was increasing in this region, especially in areas with high human immunodeficiency virus (HIV) prevalence rates (1), enough so that the global incidence of TB continued to increase at about 1% per year. Accentuating the impact of TB on the world’s well-being is its concentration among young adults throughout most of the developing world, and its airborne spread from person to person, especially to household members. TB has been exacting a toll for many centuries. Of particular interest from an epidemiologic point of view is the reported frequency of skeletal lesions suggestive of TB among pre-Columbian populations of North America (2). Although such lesions were occasionally noted in skeletons of 65

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the Late Woodland peoples (800–1050 A.D.), their successors, the Mississippians, had a much higher frequency of TB-like bony lesions, associated with their coming together in larger and relatively permanent settlements. That TB and crowding go together is now so generally accepted that the reason(s) for the association is (are) rarely considered. Is it solely because crowding increases the risk of becoming infected if infectious cases are present? Is it because there is something associated with crowding, which makes it more likely that an infected person will develop tuberculous disease? Is it some combination of these sets of risks? Answers to questions like these comprise the ‘‘etiologic epidemiology’’ of TB. This chapter will first address ‘‘etiologic epidemiology’’ by reviewing what is known about risk factors for becoming infected with tubercle bacille, then risk factors for developing disease given that infection has occurred, and finally risk factors for relapse following apparent cure or spontaneous healing of the disease. ‘‘Administrative epidemiology’’ will then be reviewed. This aspect of epidemiology deals with the occurrence of TB based on routine reporting or special surveys. These data are vital for public health administrators who must know the distribution of cases by time, place, and personal characteristics regardless of what caused these distributions. II. Etiologic Epidemiology A. Risk of Becoming Infected with Tubercle Bacille Causes of Tuberculous Infection

Three related organisms—Mycobacterium tuberculosis, M. africanum, and M. bovis—are the necessary causes of TB. M. tuberculosis is by far the most common. M. africanum is rarely found outside of northwestern Africa, and disease due to M. bovis is limited in developed countries by widespread pasteurization of milk and in the developing world by the low consumption of milk along with the practice of boiling much that is consumed. Mycobacterium canettii and M. microti have recently been found to be genetically related to the tubercle bacille and have been included in the group on this basis. M. microti generally causes disease in rodents, but has been linked retrospectively to infections in llamas, ferrets, and cats. It has also been implicated as the cause of pulmonary TB in a small number of humans (3,4). M. canettii appears to be strictly a human pathogen. The probability of having been infected with one of the tubercle bacille can be assessed by the size of induration caused by the tuberculin test and more recently by various blood tests. However, these new tests have not had the rigorous long-term evaluation accorded the tuberculin test (see Chapters 7 and 46). Risk of Infection by Time and Place

The reported incidence of TB in the United States declined at an average rate of 5.9% per year for several decades until 1985. The case rate then rose

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from 9.3 per 100,000 in 1985 to a high of 10.5 in 1992 when it began to decline steadily (5,6). The now familiar increase in the mid-1980s was caused by the advent of HIV/AIDS and was mirrored in populations worldwide; however, the initial decline in incidence seen until the 1980s has also been duplicated in many countries as they develop economically. The causes of this type of decline are undoubtedly similar around the world and examination of the well-documented decline in the United States is instructive. The best estimate of the decrease in the risk of becoming infected for residents of the United States still comes from the extensive and carefully standardized tuberculin testing of Navy recruits (7,8). Among white males aged 17 to 21 years, the proportion of positive reactors fell from 6.6% in 1949 to 1951 to 3.1% in 1967 to 1968. Subsequent testing on a routine basis showed the prevalence of positive reactors among all recruits to be 1.5% from 1980 to 1986 and to have risen to 2.5% in 1990 (9). Although the mean age of recruits had probably changed little since 1950, the two later study populations included sizeable proportions of nonwhites who, in the earlier study, had much higher proportions of infected people than the white recruits. In addition, the positive reactors throughout this entire period undoubtedly included some who were infected with nontuberculous mycobacteria (NTM) and not with M. tuberculosis. Correcting for this mixture of infections led to an estimate that only 1.4% of the white male recruits tested in 1968 had been infected with M. tuberculosis (8). Very little is known about the prevalence of positive tuberculin reactions among adults in the United States. The only data that might be considered representative of the total adult population comes from the first Health and Nutrition Examination Survey in 1971 to 1972. Among 1494 adults aged 25 to 74 years, 16.1% were classified as reactors (10). The likelihood of having been infected among household contacts of infectious cases of TB has also declined with time, at least in the United States (11). In Williamson County, Tennessee, U.S., in the period 1931 to 1955, 67% of household contacts aged five to nine years were positive tuberculin reactors. In a large study of contacts in 1958, this proportion was 48%. In 1996, only 17.7% of children under the age of 15 years, who were household contacts of pulmonary TB cases, were positive tuberculin reactors (12). In 2003, the risk of becoming infected was falling throughout the world except in Africa, where rates were increasing (1,13). Reasonably good estimates can be obtained in countries where there are enough children and young adults who have not been vaccinated with bacille Calmette– Gue´rin (BCG) to allow the risk to be estimated (14,15). For example, in the Netherlands, the risk of becoming infected was 0.5% per year in 1950 and only 0.02% in 1971. In contrast, several African countries had an estimated risk of becoming infected of 3.0% per year in 1950, with only a slight increase during the next 20 years. Recent findings have been reported from Cambodia and Orissa State of Eastern India (16,17). Among children five to nine years of age in Cambodia, the average risk of infection in 1995 was estimated to be 1.0% in Phnom Penh and 0.75% in the provinces. In

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Orissa State during 2002 to 2003, the average annual risk of infection was 2.5% in urban areas and 1.6% in rural areas. The most dramatic decrease in the risk of infection was documented among the Inuit residents of the Yukon and Kuskokwim River deltas in Alaska (18). In 1949 to 1951, 62% of children aged zero to six years were infected with tubercle bacille, equivalent to an average annual risk of becoming infected of approximately 25% per year. An intensive program of case-finding and treatment, supplemented by isoniazid preventive therapy, was instituted. By 1963 to 1964, only 2.4% were infected, and in 1969 to 1970, there were only two reactors among 1535 tested children in this age group. Personal Risk Factors for Acquiring Infections Degree of Contact and Intensity of Exposure

Because TB is a communicable disease primarily spread by the airborne route, it is not surprising that the risk of an uninfected person becoming infected is strongly associated with the probability of coming in contact with someone with infectious TB, the closeness or intimacy of that contact, its duration, and the degree of infectiousness of the case. Crowding increases both the likelihood of coming into contact with a case and the closeness of the contact. In the U.S. Navy recruit testing program, the prevalence of positive tuberculin reactions among white males aged 17 to 21 years who were lifetime residents of metropolitan areas was 4.2% compared to 2.8% among lifetime residents of farms (7). The associations of infection risk with closeness of contact, with factors related to race, and with the degree of infectiousness of the source case are shown in Table 1 (19). In the Canadian provinces of British Columbia and Saskatchewan, Indian contacts were more likely to have been infected than whites, probably because Indian households were more crowded. For both Indians and whites, infection risk was greater if the contact was Table 1 Age-Adjusteda Percentages of Positive Tuberculin Reactors Among White and Indian Children Aged 0–14 Years in British Columbia and Saskatchewan by Sputum Status of Source Case, 1966–1971 Race and closeness of tuberculosis contact Indian children (%) Sputum status of source case Positive smear Positive culture only Negative culture a

White children (%)

Intimate (n ¼ 1012)

Casual (n ¼ 619)

Intimate (n ¼ 1873)

Casual (n ¼ 3031)

44.7 27.7 25.7

37.4 15.6 18.7

34.7 8.9 7.2

10.1 2.4 3.3

Adjusted to age distribution of total study population aged 0–14 years. Source: From Ref. 19.

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intimate (household associates or partners) than if it was casual (other friends, fellow employees). If sputum of the source case contained so many tubercle bacille that they were demonstrable by microscopic examination of a stained sputum smear, the risk of infecting a contact was also greatly increased. In this population, there was only equivocal evidence that cases with positive sputum cultures were more infectious than those with negative cultures. In other populations, the infectiousness of cases with positive sputum cultures was appreciably greater than those with negative cultures (20). Other characteristics of the source case are related to the prevalence of positive tuberculin reactions among children who are household contacts (19). The extent of pulmonary involvement was strongly associated with infectivity: 62% of contacts of cases with far advanced disease were reactors compared to only 16% reactors among contacts to minimal cases. Also related to the risk of infection was cough frequency, which decreased appreciably during the first week of chemotherapy. Similar findings were noted in a study in Mysore State, India (21). Duration of Exposure

Duration of exposure is important in comparing the infectiousness of TB with that of other communicable diseases. Although an occasional tuberculous patient can be as infectious as a child with measles (22), in most instances, the proportion of exposed contacts who become infected with tubercle bacille is much lower than the risk of infection from cases of other acute communicable diseases. When the duration of exposure is taken into account, the average TB patient has a low degree of infectiousness per unit of time. Virulence of Organism

It has been known for some time that strains of M. tuberculosis from different parts of the world show considerable variation in their virulence in guinea pigs (23). Isoniazid-resistant organisms also have decreased virulence in guinea pigs (24). Until recently, however, the possibility of strain resistance has not been seriously considered in the pathogenesis or epidemiology of human TB. During 1994 to 1996, 21 cases of TB developed in a small rural community in the midwestern part of the United States (25). Investigation of the outbreak showed an unusually high rate of infection among contacts of the source case. Of the 42 identified and tested contacts of the source case, 37 (88.1%) were positive reactors, and 8 (21.6%) of the reactors had documented tuberculin conversions. High proportions of the contacts of the index case were also tuberculin reactors and converters. Environmental investigations revealed no explanation for these high infection rates. The strain of tubercle bacille responsible for the outbreak was initially reported to be more virulent than the standard virulent Erdman strain. However, subsequent laboratory investigations did not confirm this finding (26). The high proportion of infections could also have resulted from chance (27). Whether or not M. tuberculosis varies in its virulence for humans remains uncertain.

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Foreign Residence

There is little evidence that a period of foreign residence is associated with an important risk of infection for people born in the United States. Navy recruits who had lived abroad at a time when TB was common even in many developed countries were only slightly more likely to be tuberculin reactors than lifetime residents of the United States (7). At least some of the difference must have resulted from BCG vaccinations received in the foreign country. The fact that the excess risk was so low is probably attributable to the lifestyle of most expatriate U.S. citizens, most of whose exposures must have occurred in public places and have been very short in duration. Age

There is some evidence that the risk of acquiring infections increases with age during the period from infancy to early adult life (28), probably because of increasingly numerous contacts with other people. Although tuberculin sensitivity, once acquired as a result of infection with tubercle bacille, persists for many years, the prevalence of positive tuberculin reactions tends to level off around 50 to 60 years of age. In some populations, there is even a decreased prevalence in older ages, possibly because the infecting bacille in some people had died out at an early age. Sex and Race

In nearly all populations around the world, adult males are more likely to have been infected than females, again probably reflecting their opportunity for more and varied contacts in most societies (29). This sex difference was clearly illustrated in a large tuberculin-testing program among New York City school employees (30,31). The prevalence of positive reactors was also higher among nonwhites than whites. Socioeconomic Status

In the New York City study, the prevalence of positive tuberculin reactors decreased steadily with increasing socioeconomic status of their neighborhood. In the highest socioeconomic areas, the frequency of reactors was similar among whites and nonwhites (26,27). Among high school students in Washington County, Maryland, U.S.A., large tuberculin reactions typical of those resulting from tuberculous infection were much more common among students living in crowded, inadequate housing (32). Chemotherapy of Source Case

Effective chemotherapy of the source case appears to reduce infectiousness rapidly, perhaps even more rapidly than is indicated by results of sputum examinations (22,33,34). Although isoniazid-resistant organisms have reduced virulence for guinea pigs, there is no indication that drug resistance per se has any effect on infectiousness for humans (35). However, when source cases with drug-resistant organisms had a history of prior and probably ineffective treatment, their contacts were at increased risk of being

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infected. It is likely that this increased risk resulted from the long duration of exposure that is associated with multiple episodes of treatment. Institutionalization

Both voluntary and involuntary confinement in two types of institutions has been shown to be associated with an increased risk of becoming infected with tubercle bacille. In a survey of nursing homes in Arkansas, it was found that the risk of becoming a positive tuberculin reactor was 3.5% per year even if there had been no recognized TB cases in the home within the previous three years (36). Periodic tuberculin testing in an elderly population in poor health can be misleading in individuals because of the relatively high degree of instability of the tuberculin reaction in such people (37). Using the two-step procedure at the time of initial testing will help identify many of the conversions that are due to ‘‘boosting’’ (anamnestic reaction), which might otherwise be subsequently classified as new infections (37–39). Identification of new TB infections among people in long-term correctional institutions also faces the problem of differentiating new infections from boosted reactions. This problem can be minimized by the use of two-step testing at the initial examination (39,40). A conversion from a negative to a positive test within a ‘‘week or two’’ interval in two-step testing is highly likely to be a boosted reaction. A subsequent conversion at a semiannual or annual retest among people negative to the second of the two tests should be considered as evidence of a new infection. Repeated tuberculin testing in state prisons in two states showed conversion rates from a negative to a positive tuberculin test of 6.3% and 9% per year (41,42). Since then, TB has been recognized as a serious threat because of gross overcrowding in correctional institutions and the ease of airborne spread of infection (40,41). A growing problem concerns TB transmission in shelters for the homeless. The presence of an untreated infectious case of TB in these often crowded, poorly ventilated buildings confers a considerable risk of infection on the other clients and the shelter personnel (43). Intrinsic Susceptibility

A review of the foregoing shows that the known determinants of becoming infected are extrinsic to the exposed person or, in other words, environmental. Whether or not there is also an intrinsic risk factor is still uncertain. In one study, blacks were more likely to become positive tuberculin reactors than whites, when exposed similarly in nursing homes and prisons (44). However, a careful study of a primary school outbreak found no difference in infection rates among white and black children similarly exposed to an infectious physical education teacher (45). At present, the issue of intrinsic susceptibility to infection remains unsettled, kept alive by the fact that individuals do differ in almost all characteristics and by anecdotal reports of people who are still negative tuberculin reactors after a lifetime of caring for TB patients.

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Relatively few studies have been able to investigate the factors that influence whether or not an infected person will develop TB. Although most of them were done 25 or more years ago, the relative risks are still likely to be relevant. Reinfection

For the past 100 years, there have been many discussions and opinions about the relative importance of exogenous reinfection and endogenous reactivation in the development of clinical TB following the initial infection with M. tuberculosis (46). With the ability to genotype tubercle bacille, it is now clear that reinfection from a new source case can occur. However, it is still uncertain how often reinfection is responsible for the development of manifest disease. In any case, it is likely that the risks of being exposed to possible reinfection are similar to the risks of first becoming infected, as reviewed in the previous section. Time and Place

The change in risk of disease occurring after infection is not known with respect to calendar time. There are, however, some data showing that the risk of disease is highest shortly after receipt of infection and that it declines thereafter. Findings from a controlled trial of isoniazid prophylaxis among contacts of active TB cases and a trial among hospitalized psychiatric patients can be combined to yield a reasonable estimate (47). In these two trials, 1472 people allocated to the placebo regimen converted from a negative to a positive tuberculin reaction at some time within the first study year. Of the 29 new cases that developed during a seven-year follow-up period, 64% occurred during the first year, the year in which they became reactors, 22% developed during the next three years, and 13% during the last three years. In South India, the risk of developing TB was 2.6% within the first year after tuberculin conversion, and only 0.5% during the next three years (48). Incidence of TB among tuberculin reactors varies by place, probably related to the intensity of exposure. Among 265,488 tuberculin reactors with negative chest radiographs who participated in a mass campaign in 1950 to 1952 in Denmark (exclusive of Copenhagen), the average annual incidence over the next 12 years was 29 per 100,000 (49). At the other extreme was the Inuit population in the Yukon-Kuskokwim delta of Alaska, where the average annual incidence rate from 1957 to 1964 was over 500 per 100,000 people with initially negative chest radiographs, virtually all of whom were tuberculin reactors (50). In Denmark, in the 1960s, rural tuberculin reactors 15 to 44 years of age had a risk of subsequently developing TB, which was only 60% of the risk for their urban counterparts (49). Personal Characteristics Age

Among TB contacts in British Columbia and Saskatchewan, Canada, who had positive tuberculin reactions, the frequency of active TB discovered

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during a six-month period following diagnosis of the index case decreased with increasing age of the contacts (19). A similar pattern by age was observed in South India (48). The higher risk among younger contacts may have resulted in part from the fact that a higher proportion of infections among young people are likely to have been recent. The incidence of TB among tuberculin reactors by age was investigated as a by-product of a controlled trial of BCG vaccination in Puerto Rico (51). Among 82,269 tuberculin reactors aged 1 to 18 years, who were followed for 18 to 20 years, 1400 cases of TB were identified. As shown in Figure 1, there were two peaks of incidence. One occurred among children in the one- to four-year-old age group, probably reflecting the fact that these infections must have been recent. The second peak occurred during late adolescence and early adult life and was experienced by all birth cohorts as they passed through this period of life. A similar peak was noted among British adolescents, although at a slightly lower age (53). The cause of increased incidence at this age, even for people infected in early childhood, is unknown. The risk among older adults is not well established, but all evidence points to the persistence of at least a low risk of developing TB during the lifetime of infected people. For this reason, life expectancy is a major determinant of the lifetime risk of developing TB among tuberculin reactors. Sex

In seven studies that reported sex- and age-specific incidence rates among positive tuberculin reactors, rates for females were higher during their childbearing ages than the rates for males; at older ages, rates were higher in

Figure 1 Incidence of tuberculosis among Puerto Rican children who were reactors to tuberculin, by age when tuberculosis was first diagnosed. Source: From Ref. 52.

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males (29). An exception to this pattern occurred in the large BCG trial in the Chengleput area of South India (54). Among people with tuberculin reactions of 12 mm or larger, males had higher incidence rates than females at all ages. Findings from a directly observed treatment program in another area of South India confirm the excess risk among infected males (55). Prevalence of positive reactors was only 20% higher among males, whereas the attack rate of tuberculous disease was 6.5 times greater among males. Race

Race per se appears to have little influence on the risk of disease once infection has occurred. Case rates were not significantly different among black and white reactors in Georgia and Alabama (56), nor among Navy recruits (57). As can be seen in Table 2, Indian and white reactors in Canada also had similar rates when age, intimacy of contact, and infectiousness of source case had been controlled (19). Dosage of Infection

The findings shown in Table 2 also bear on the relationship of dosage of infection to the risk of developing TB (19). All the subjects in this study were tuberculin reactors and can be considered to have been infected. Because the risk of disease was greatest among those exposed to the most infectious cases and among those with the closest contact, the conclusion seems inescapable that people infected with larger numbers of tubercle bacille are at greater risk than those infected with smaller numbers of organisms. A study in Mysore State, India, also showed that among contacts who were strongly positive tuberculin reactors, development of pulmonary disease was most likely among those with the most intense exposure, i.e., the contacts most likely to have received larger doses of infection (58).

Table 2 Age-Adjusteda Prevalence of Active Tuberculosis Among Infected Tuberculosis Contacts in British Columbia and Saskatchewan by Race, Type of Contact, and Sputum Status of Source Case, 1966–1971 Prevalence (%) Indian contacts Sputum status of source case Positive smear Positive culture only Negative culture a

White contacts

Intimate (n ¼ 352)

Casual (n ¼ 169)

Intimate (n ¼ 412)

Casual (n ¼ 216)

14.4 5.1 3

10 3.9 0

14 5 2.3

8.2 6.2 0

Adjusted to age distribution of total study population aged 0–14 years. Source: From Ref. 19.

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Size of Tuberculin Reaction

It has been known for several decades that infections with NTM often cause tuberculin sensitivity but rarely result in disease, and also that crossreactions to tuberculin caused by these organisms are usually smaller than those caused by M. tuberculosis (59). Consequently, it is not surprising that where nontuberculous mycobacterial infections are present, small tuberculin reactions are less likely to be caused by infections with tubercle bacille, and hence are less likely to be associated with a risk of subsequent disease than larger reactions. The importance of this risk was illustrated by a study of Puerto Rican children (52). Children with reactions measuring 16 mm or more in diameter to one tuberculin unit of a purified tuberculin had a subsequent risk of tuberculous disease, more than five times greater than children with reactions of 6 to 10 mm, following a test with 10 tuberculin units of a purified tuberculin. The prognostic importance of this widely available risk factor has recently been recognized in recommended standards for TB control (60). Immunosuppression

The fact that the great majority of people do not develop TB after they have been infected indicates the ability of the normal immune system to hold the infecting organisms in abeyance or even, in some instances, eradicate them. Treatment with immunosuppressive agents can upset this balance, as can infection with HIV. TB is reported to be rampant in populations throughout the world who have dual infections with both the tubercle bacille and HIV. An early illustration of the enormous magnitude of this risk was afforded by a longitudinal study among intravenous drug users in New York City (61). No cases of TB were observed among 298 reactors who were HIV negative, compared to eight among 215 HIV-positive people. Seven of the eight TB cases occurred among 36 who were known to have been positive tuberculin reactors but who had not received isoniazid chemoprophylaxis, an average annual case rate on the order of approximately 8000 per 100,000. The temporary loss of tuberculin sensitivity following measles has also been equated with immunosuppression, and hence increased susceptibility to activation of a latent tuberculous infection. However, a careful review of the pertinent literature failed to substantiate this belief (62). Relative Weight

Among the few benefits of being overweight is its association with protection against TB. Among white male recruits with positive tuberculin reactions and negative chest radiographs on entry to the Navy, those who were 10% or more underweight were 3.4 times more likely to develop TB than those who were 10% or more overweight (63). Genetics

Genetic factors clearly play some role in the development of TB in humans, but the extent of the role is unclear. Lurie has shown that there is some

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genetic basis for resistance to TB in rabbits (64), and a variety of evidence suggests the same is true for humans. In a tragic accident in 1926, 249 infants in Lubeck, Germany were vaccinated with live M. tuberculosis instead of BCG vaccine. All 249 developed TB, although the severity of the illness varied widely; 76 infants died. None of the infants had been previously vaccinated with BCG, none were old enough to have developed natural acquired immunity, and all received the same dose of mycobacteria. The variation in their response to the infection may be partially due to nutritional status and other factors, but some must have been due to differences in innate susceptibility to TB (65,66). When TB was introduced for the first time in the Qu’Appelle Indians in the late 19th century, 10% of the population died annually from the disease. Half of the families in the community died out within two generations, but the annual TB death rate in the remaining population dropped to only 0.2% (67). A similar scenario occurred when TB was introduced to the Yanomami Indians in the Amazon (68). These scenarios appear to show the development of population immunity to TB where intrinsically more susceptible people die from infection, leaving only those genetically more capable of surviving infection. Perhaps the strongest evidence that genetics plays a role in development of disease comes from twin studies, which examine the incidence of disease in pairs of twins. When both twins in a pair develop a disease, they are called concordant; they are discordant when only one of the twins is diseased. If genetics plays no role in a disease, monozygotic and dizygotic twin pairs should have similar concordance rates. If genetics is important, then concordance should be higher in monozygotic twins because they have identical genomes. Several twin studies have shown that the concordance for TB is roughly two times higher in monozygotic twins even when data was adjusted for the effects of sex, age, infectivity of the index case, type of TB, and years of contact (69–71). Although environment must play a role in the development of disease in twin pairs, it seems clear that monozygotic twins are at greater risk of disease. There have been other types of studies suggesting a genetic component in TB. In a well-done study by Overfield and Klauber, the prevalence of a positive tuberculin response and active TB was correlated with ABO and MN blood types in a random sample of Alaskan Eskimos with a very high TB infection rate. The authors reported that people with blood type AB and B were three times more likely to have moderate to severe TB compared to people with blood type O and A (72). There also appears to be some correlation between development of TB and histocompatibility types (73). Several studies have also shown that specific genetic polymorphisms may be associated with TB. Allelic variation in the gene nramp-1 was associated with TB in a case–control study in the Gambia (74). Another study from Cambodia identified a specific HLADQ haplotype associated with TB (75). Interferon-cR1 deficiency has also been shown to be associated with increased susceptibility to TB in a study of French children who developed disseminated BCG after vaccination.

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A few children with no underlying immune defect had a specific autosomal recessive mutation in the gene coding for interferon c-R1 (65,76,77). Studies have also suggested that several other genes or chromosome regions may effect susceptibility to TB, and research continues in this area (65). Smoking

Although a link between smoking and TB has been long been postulated, the evidence supporting the association has been lacking until recently. A 2003 study in India showed that male smokers were three times more likely to report a history of TB than nonsmokers (78). A prospective study in Hong Kong also examined the incidence of TB in smokers and nonsmokers and found that the incidence was highest in current smokers (735/100,000), lowest in never smokers (174/100,000), and intermediate in ex-smokers (427/100,000). The trend was significant (p < 0.001) and persisted after adjustment for multiple factors. In addition, current smokers who developed TB smoked more cigarettes than those who did not (13.43 cigarettes per day vs. 7.87, p ¼ 0.01), and a significant dose response was observed (79). A study in Kuwait suggests that smoking may delay sputum conversion in some people receiving treatment (80). Although the mechanics of the smoking–TB association have not been explained, some have suggested that iron-loading of pulmonary macrophages secondary to smoking may damage the cells and make them more susceptible to infection with the tubercle bacille (81). Socioeconomic Status

There is almost no evidence about the relationship of social and economic factors to the development of tuberculous disease among tuberculin reactors. In Muscogee County, Georgia, U.S.A., the incidence of TB among reactors during the period 1950 to 1962 showed no association with the quality of their housing, as recorded in a private census in 1946 (56). This held true for both whites and blacks. There are no data on reactors living under conditions of extreme deprivation, although anecdotal evidence indicates a high risk. C. Risk of Reactivation of Disease

The third risk to be considered in etiologic epidemiology is relapse, namely the risk of developing active disease following spontaneous or therapeutically associated ‘‘cure.’’ Relatively little is known about these risks, except for those related to chemotherapy. Adherence to Chemotherapy

Chemotherapy has made an almost miraculous improvement in the prognosis of people who develop TB. Conscientious adherence to an appropriate regimen even in the earlier days of chemotherapy came close to guaranteeing a lasting cure (82). It is not surprising, therefore, that poor compliance with therapy is a major risk factor not only for treatment failure, but also for relapse after apparent cure (83–85). The presence of drug-resistant tubercle bacille is also an important risk factor for relapse. In 12 controlled trials of short-course chemotherapy, patients with bacille resistant to

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streptomycin or isoniazid were much more likely to relapse than patients with bacille sensitive to these drugs (86). Even though completion of an appropriate regimen comes close to guaranteeing a cure (60), close is not perfection. As one example, among 582 patients who completed a six-month regimen with isoniazid and rifampicin throughout and pyrazinamide plus either ethambutol or streptomycin for the first two months and who were followed for five years, the relapse rate during this period was 3.4% (87). Expressed as a percentage, this appears low, but it is equivalent to an average annual rate of 680 per 100,000. Life-table reanalysis of the data from the U.S. Public Health Service (USPHS) TB short-course chemotherapy trial 21 showed a relapse rate of approximately 600 per 100,000 per year for the two regimens combined (88). Time

The risk of relapse by calendar time has clearly been influenced by the markedly reduced risk following the introduction of chemotherapy. In Denmark, after the introduction of isoniazid into the therapeutic regimen, the relapse rate fell from nearly 13% to 6% (89). The risk of relapse by time following completion of therapy has also been influenced by the introduction of antibiotic chemotherapy. Prior to its introduction, relapse was most likely to occur shortly after treatment stopped (90,91); after chemotherapy was introduced, relapses were less likely during the year or two following adequate treatment (88,91). Longterm risk after adequate chemotherapy is not known. Age, Sex, and Race

Relapse rates by age do not show a consistent pattern. In untreated people whose disease was judged to be inactive or fibrotic at the time of diagnosis, reactivation was less likely with increasing age (91,92). Among people whose disease became inactive after treatment, relapse rates went up with age in Denmark (67), and showed no significant trend with age in India (93). There was a tendency for relapse rates to be somewhat higher in males than females (89,92,93), although not in all populations (94). Reactivation rates were more common among Canadian Indians than other Canadians (95) and, in the state of Georgia, more common among blacks than whites (90). Socioeconomic Status

Among blacks in Georgia, degree of skin pigmentation was not related to the risk of relapse, suggesting that socioeconomic factors might be more important than race per se (90). Another indication that socioeconomic factors might play a role came from a geographic comparison of relapse rates in Denmark (89). Relapse rates among residents of Copenhagen were higher than among people living in the more rural areas of Denmark. Extent of Disease

In Georgia and in Europe, reactivation in untreated people was much more likely among people with extensive fibrotic disease than among those with

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only minimal lesions (90,96). A similar finding was reported among previously treated patients in Wisconsin and South Africa (83,97). III. Administrative Epidemiology Information on TB morbidity and mortality is voluminous compared to that available for etiological epidemiology. Even so, most of it is based on official reports and can be related only to time, place, race, sex, and age. Hard data on other factors are sparse. The available information on many aspects of administrative epidemiology is included in other chapters of this volume. A. Time and Place

The risk of TB infection varies widely around the world. It is stable or falling throughout in five of the six WHO regions. Rates are lowest and continue to drop in developed and more affluent developing countries in Latin America and Eastern Europe. Incidence is stabilizing in Asia, although the sheer size of the population means there are millions of cases in the region. Rates in Africa continue to rise, however, particularly in areas with high HIV prevalence, although the rate of increase is slowing in some countries (1). In the United States, reported incidence declined at an average rate of 5.9% per year for several decades until 1985 (Fig. 2) (5). The case rate then rose from 9.3 per 100,000 in 1985 to a high of 10.5 in 1992. Since then, the case rate has fallen steadily to 5.1 in 2003 (6).

Figure 2 Number of tuberculosis cases and deaths, United States, 1953 to 2003, all ages, all types. Source: From Refs. 5, 6.

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The so-called resurgence of TB from 1985 to 1992 was far from uniform among U.S. states and cities, with marked variation in case rate changes both during and after the resurgence period (98). Seven states—California, Nevada, New Jersey, New York, Texas, Utah, and Washington—showed average annual increases of 4% to 17% during the period 1984 to 1991. Rates for the rest of the country showed a slight decline throughout this entire period. Most Western European countries and Canada showed a slowing of the previous decline in TB rates after 1985, and some even showed slight increases. A notable exception was Finland, where the decrease in case rates accelerated after 1985. Various factors have been suggested as the cause of the resurgence in the United States (99). These include HIV infection, poverty, homelessness, drug abuse, immigration, and, usually last, decreased funds for TB control. The only one of these factors to have changed in a favorable direction since 1985, however, was the considerable increase in TB control funds during the 1990s, which led to a revitalization of TB control activities in critical areas (100). The WHO collects and reports global TB incidence data annually. Although reporting to WHO is voluntary, nearly all countries in the world comply by reporting the number of new TB cases of all types, the number of new smear-positive cases, and age and sex information for smear-positive cases, as well as information on the status of their TB control program (101). The quality of the incidence data reported to WHO varies. To compensate for these differences, WHO reports both the number of cases reported by each country (case notifications) and an estimated number of TB cases, which is a standardized adjustment of the case notifications based on countryspecific information (102). There are considerable differences between the number of case notifications and number of estimated cases in some instances (Table 3), generally due to undercounting of incident disease. For comparison across countries or between WHO world regions, therefore, the estimated incidence is preferable. WHO presents data annually by geographic regions of the world (101). Although convenient and logical, in some of the six regions, there are countries with very different risks of TB. This confuses the epidemiologic picture somewhat, but the WHO regions are the only available source of standardized global information. Among the WHO regions, the absolute burden of TB as measured by the number of cases is greatest in the Southeast Asian region (SEAR), but the rate of disease is highest in the AFR. Either way, the SEAR and AFR together accounted for more than 60% of incident cases in the world in 2004 (1). The rates of disease in the East Mediterranean region (EMR) and the Western Pacific region (WPR) are also high, but only about onethird of the rate seen in AFR. The number of cases and rate of disease are lowest in the Americas and European region (EUR), which are dominated by the more developed countries of the world. Figures 3 and 4 show trends in case notification rates collected by WHO for 1982 to 2002 for different world populations. The notification

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Table 3 Number of Notified and Estimated Tuberculosis Cases (All Types), Cumulative Incidence, and Estimated Rate of Tuberculosis, All Types, Worldwide and by World Health Organization Region, 2002 Number of cases

Estimated annual rate (per 100,000)

WHO region

Notified

Estimated

Ratio of estimated to notified

AFR Americas EMR EUR SEAR WPR

992,054 233,648 188,458 373,497 1,487,985 806,112

2,354,000 370,000 622,000 472,000 2,890,000 2,090,000

2.37 1.58 3.30 1.26 1.94 2.59

350 43 124 54 182 122

Global

4,081,754

8,707,000

2.13

141

Abbreviations: AFR, African region; EMR, East Mediterranean region; EUR, European region; SEAR, Southeast Asian region; WPR, Western Pacific region. Source: From Ref. 101.

rates in the figure are expressed relative to an arbitrary standard of 100 in 1990 to emphasize time trends. The error bars show the 95% confidence interval of the standardized rates. Countries included in the graphs were considered representative of countries within the region for the time period specified. Increases observed in notification rates in the late 1990s in some areas may be due to improvements in TB control programs rather than true increases in disease rates, but, in general, the standardized notification rates shown accurately reflect changes in disease trends in the populations (101). In the established market economies and Central Europe, notification rates dropped fairly steadily over the time period, except for the 1985-to1992 period, mentioned earlier, which is visible here as a flattening of the trend line around 1990. Similarly, rates in the EMR and Latin America appear to have been dropping steadily. In the SEAR, the trend is less clear, but, since 1996, notification rates have been dropping steadily. Declines seen in the WPR between 1990 and 1996 have been lost, and the trend in notification rates has flattened. In Eastern European countries, case and death rates from TB have historically been higher than in Western Europe, but were declining in most of the countries through 1992 (103). After that, notification rates climbed steadily until 2001 when they were roughly double the rate seen 20 years earlier. Revised data from 2004 show that rates peaked in 2001 and have been falling since then (1). Notification trends in Africa vary by HIV prevalence. In high-prevalence areas, notification rates continue to increase as they have since the mid-1980s, although the increase in some countries has been mitigated by effective TB control activities (103,104). In low-prevalence countries, a previously flat trend began increasing at the turn of the 20th century.

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Figure 3 Trends in tuberculosis case notification rates, all types, for selected countries and world regions, 1981 to 2002. Established market economies: Australia, Austria, Belgium, Canada, Czech Rep., Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Japan, Luxembourg, Netherlands, New Zealand, Norway, Portugal, Singapore, Spain, Sweden, Switzerland, United Kingdom, United States. Central Europe: Albania, Croatia, Cyprus, Hungary, Poland, Serbia and Montenegro, Slovakia, Slovenia, Turkey. Latin America: Argentina, Bolivia, Brazil, Chile, Cuba, Dominican Republic, El Salvador, Guatemala, Guyana, Honduras, Jamaica, Nicaragua, Paraguay, Peru, Puerto Rico, Uruguay, Venezuela. Eastern Mediterranean: Iran, Jordan, Lebanon, Morocco, Oman, Qatar, Saudi Arabia, Syria, Tunisia. Pacific: China Hong Kong, SAR, China Macao SAR, Lao People’s Democratic Republic, Malaysia, Republic of Korea, Vietnam. SE Asia: Bhutan, India, Maldives. Source: From Ref. 101.

TB is more common in large cities than in rural areas (97,103). In the United States, in 2002, metropolitan statistical areas with populations greater than 500,000 had 77% of the new cases, for a case rate of 6.3/ 100,000. In the less populous areas, the rate was only 3.0/100,000. Forty-nine percent of the 3142 U.S. counties reported no cases in 1998; most were located in the northern plains and Rocky Mountain areas (84).

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Figure 4 Trends in tuberculosis case notification rates, all types, for Africa and Eastern Europe, 1981 to 2002. Africa–low HIV: Algeria, Benin, Comoros, Ghana, Guinea, Madagascar, Mali, Mauritania, Mauritius. Africa–high HIV: Botswana, Coˆte d’Ivoire, Democratic Republic of Congo, Kenya, Lesotho, Malawi, Uganda, UR Tanzania, Zambia, Zimbabwe. Eastern Europe: Armenia, Bulgaria, Estonia, Kazakhstan, Kyrgyzstan, Latvia, Lithuania, Republic of Moldova, Romania, Russian Federation, Tajikistan, Turkmenistan, Ukraine, Uzbekistan. Source: From Ref. 101.

The same situation is also seen in other parts of the world, even highly endemic areas. In Orissa State, India, the prevalence of TB in children residing in urban areas was 60% higher than the risk of those living in rural areas (16). Similarly, the estimated annual risk of TB infection from 1955 through 1995 was consistently higher in Phnom Penh, Cambodia, than it was in the surrounding provinces (17). Not all of the considerable geographic differences can be explained by stage of economic development, immigration, or prevalence of HIV infections. Case rates within the original European community varied from 7.4 to 31.9 per 100,000 in 1983; among six members of the Eastern Bloc, the range was 20.3 to 72.8 (105). In 2002, Iceland had the lowest estimated rate of TB in Europe, 3.0/100,000, with Sweden and Norway close behind at 3.0 and 5.0/100,000, respectively (81). Rates in England and Wales ranged from 3.1 in Anglia to 37.0 per 100,000 in some boroughs of London in 1983 (106), while among the 50 U.S. states, the case rate in 2003 ranged from 0.8/100,000 in Wyoming to 14.0/100,000 in Washington, DC (104). Unfortunately, interpretation of geographic variations is more difficult than generally recognized. Within the United States in 1992, the percentage of

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pulmonary cases not bacteriologically confirmed varied from 0% to 35.4% among states with 25 or more reported cases (107). Considerable variations between nations in both the extent and nature of cases of pulmonary TB have also been recorded (108). Influx of immigrants from an area where the prevalence of TB is high has also affected temporal trends adversely, especially in industrialized countries. An early experience was reported from British Columbia, Canada. TB case rates had been declining from 1970 to 1985, except for the city of Vancouver (109). On investigation, it was found that the failure of the rates to decline in Vancouver was selective immigration into the poorer areas of the city of a group of high risk, socially disadvantaged immigrants. In the United States, the proportion of cases from abroad increased from 29% in 1993 to 53% in 2003 (103). Perhaps the heaviest burden of immigration has fallen on Israel, where more than a million immigrants, mostly from high-prevalence countries, were admitted during 1990 to 2000 (110). By adapting TB control procedures to the problems and needs of immigrants, Israel has had considerable success in controlling TB among them, with no evidence to date that there has been significant spread of disease to the host population. Genotyping has cast considerable light on the risks of TB among immigrants. In New York City, genotyping indicated that reactivation of latent TB infection was the cause of most cases among the foreign-born, whereas among US-born people, many cases were due to recent transmission (111). A large study in Hamburg, Germany, also concluded on the basis of molecular and epidemiologic investigations that recent transmission was not important among immigrants (112). Spoligotyping of tubercle bacille from Swedish cases indicated that only 14% were due to recent transmission and that the strains of tubercle bacille among immigrants corresponded closely with the countries from which the immigrant cases had come (113). The results of genotyping 1100 isolates from 11 countries also showed that tubercle bacille tended to have characteristics that were unique to the country from which they were obtained (114). B. Age, Race, and Sex

The number of reported TB cases per 100,000 population for 1989 through 2002 by ethnic group for the United States is shown in Figure 5 (5,103,107). All ethnic groups are currently showing a downward trend in TB rates. The trend began about 1992 for Hispanics, blacks, and whites; 1995 for Asian/ Pacific Islanders; and in 1994 for American Indian/Alaskan Natives. In the United States, in 2002, case rates were low in infancy and decreased somewhat during early childhood (5). After puberty, they showed a generally steady increase with age (Fig. 6). For all ethnic groups, rates among females are lower than among males. TB case rates for whites are the lowest at all ages, and Asian/Pacific Islanders are the highest. Rates among blacks, Hispanics, and American Indian/Alaskan natives are intermediate.

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Figure 5 Reported tuberculosis rates per 100,000 by race/ethnicity group, United States, 1989 to 2002 (up to 1993, Hispanics are also included in one of the other groups). Source: From Ref. 103.

Figure 6 Tuberculosis case rates, United States, 1996, by race/ethnicity and age groups. Abbreviations: A, American Indian/Alaska native; A/P, Asian/Pacific Islander; H, Hispanic; B, black, non-Hispanic; W, white, non-Hispanic; M, male; F, female. Source: From Ref. 76.

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Figure 7 Number of smear-positive cases of tuberculosis per 100,000 population in the World Health Organization Americas, East Mediterranean, and European regions by age and sex, 2002. Abbreviations: AMR, American region; EMR, East Mediterranean region; EUR, European region; M, male; F, female. Source: From Ref. 81.

Smear-positive TB notification rates by age, sex, and WHO world region are shown in Figures 7 and 8. Among women in five of the six WHO regions of the world, rates start low in infancy, climb to a peak at 25 to 35 years of age (101), and then decline or flatten with increasing age. In the EMR, the rates also begin low but then continue to rise with increasing age. In high-prevalence areas such as the AFR and SEAR, the

Figure 8 Number of smear-positive cases of tuberculosis per 100,000 population in the World Health Organization Southeast Asia, Western Pacific, and African regions by age and sex, 2002. Abbreviations: AFR, African region; SEAR, Southeast Asian region; WPR, Western Pacific region; M, male; F, female. Source: From Ref. 101.

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increase between 14 and 35 years of age is much steeper than that seen in low incidence areas. The pattern is not quite so clear in men across the six regions. In all regions, rates start low and rise into early adulthood. In the Americas, EUR, and AFR, the rates increase to a peak at 25 to 45 years and then decline or remain roughly level with increasing age. The pattern is similar in the SEAR but the peak is delayed until 55 to 64 years of age. In contrast, in the WPR and EMR, the rates of smear-positive disease continue to increase throughout life. Figures 7 and 8 also show that age- and sex-specific curves of smearpositive notification rates throughout the world fit one of two patterns. In the most common one, the curves are similarly shaped and smear-positive TB rates are lower in women at all ages. This is the pattern seen in five of the six WHO regions. The pattern in the AFR is different, however. The curves for males and females still parallel each other in shape but the rates in females are higher from birth through 24 years of age and then lower than males throughout the rest of life. However, Figures 7 and 8 can be misleading. Examination of case-specific age/sex rates for individual countries shows that there are some countries in each region of the world, except the EUR, in which the rates are greater in women than men from birth through 24 years of age. This is obscured on a region-wide level because the first pattern predominates in all but the AFR. C. Socioeconomic Status and Nutrition

The association of TB with poor socioeconomic status has long been noted (7,99,115). Decades ago, homeless men in New York City were found to have high rates of TB (115) and a similar excess was noted among unmarried men living in central Copenhagen (116). The situation is no different today. In the United States, in 2002, 56% of new TB cases occurred in people who had been unemployed for more than 24 months and 6% in the homeless (103,117). The situation in the United States and some other developed countries is aggravated by an increase in the number of homeless people and the continued high frequency of TB among them (117,118). Further aggravation in more developed countries comes from the tendency of poor immigrants to crowd into large cities (119,120). Initially, their TB risk reflects the prevalence of the disease in their native countries and, although the risk decreases with their duration of stay in their adopted homes (109,121,122), they still produce foci of infection in areas with otherwise low rates of TB (119). Even in highly endemic areas such as Hong Kong, more TB cases reside in rooming houses or one-room apartments than in more luxurious accommodations (123,124). Interestingly, the reverse situation can be found in some developing countries. In Sabah, Malaysia, the rate of TB in native-born Malaysians is higher than in immigrants from the Philippines and Indonesia, even though overall rates in those countries are higher. In this case, it may be that immigrants are wealthier than native-born Malaysians and have had a lower risk of disease within their home country (125).

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Based on a survey from 29 states in 1984 to 1985, occupational status was strongly associated with TB case rates (126). Executives and professionals had the lowest rates, and laborers, farm workers, and household servants had the highest rates. Health-care workers had rates of TB about the same as that of the general population, except for higher rates among inhalation therapists, nursing aides, orderlies, and attendants. An interesting exception to the inverse association of TB with occupational status was the higher-than-expected rate among funeral directors. A recent study showed that funeral home employees who performed embalming were twice as likely to have been infected with tubercle bacille as other employees (127). Historically, moderate to severe malnutrition has also been linked to the development of TB, although the available supporting evidence in humans is limited. The best evidence suggesting a link comes from the first National Health and Nutrition Examination, in which a representative cross section of U.S. adults were followed longitudinally for more than 10 years. Individuals with body mass index or upper arm circumference in the lowest decile of the population had a 6- to 10-fold increased risk of TB after controlling for other known risk factors (128). A more recent study in Malawi also shows that the severe pulmonary TB is more common in people with lower body mass index and fat mass (129). There is also a suggestion that certain micronutrients may effect TB infection. In a study in India, people with TB had lower levels of serum vitamin A than did controls (130). D. Institutional Living

Because poverty is associated with both crime and TB, it is not surprising that TB is often a problem among inmates of correctional institutions. Various surveys have estimated that the frequency of TB is increasing among such populations and that it is three to six times higher than expected from rates in the general population (131). TB among people living and working in nursing homes and other facilities providing long-term medical care has only recently been recognized as a problem (36). A survey of 29 states suggested that the case rate for patients was approximately 50% higher and the rate among employees was three times higher than expected from the rates in similar age sex groups in the general population (36,131). E. Special Medical Situations

A variety of medical conditions are associated with TB. Although these risk factors are presumably limited to people already infected with M. tuberculosis, studies to substantiate this presumption are few and rarely definitive. By far, the most important is immunosuppression, particularly that resulting from infection with the HIV (129,132). Other causes of immunosuppression also accompanied by an increased TB risk include treatment with immunosuppressive drugs, including prolonged adrenocorticosteroid therapy, some hematologic and reticuloendothelial diseases such as leukemia and lymphoma, and end-stage renal disease and renal transplantation (133).

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Silicosis has long been linked with TB, so much so that silico TB is an accepted disease entity. Although the causal nature of this association is largely based on uncontrolled reports, silica dust has long been known to have an adverse effect on TB in animals (134). Diabetes, too, has long been accepted as a risk factor for TB (135). Two studies in the 1950s indicated that the prevalence among diabetics was approximately four times that in a comparable general population and that the risk was greatest among those with severe diabetes (136,137). Alcoholism and drug addiction are also associated with TB, although it is not clear whether these diseases increase susceptibility to TB or whether conditions conducive to substance abuse are similar to those leading to TB. In any case, alcoholism was well known to physicians in TB sanatoria, because 10% to 30% of patients were reported to be alcoholics (7). Current surveys also show a high prevalence of TB among alcoholics and drug addicts (138). Smoking has also been implicated as a risk factor for TB. In a historical cohort study among elderly people in Hong Kong, annual TB case rates per 100,000 were 174 among never smokers, 427 among ex-smokers, and 735 among current smokers (79). The trend persisted after adjustment for possible confounders, including alcohol intake. F. Infection with HIV

The immune deficiency characteristic of HIV infection increases both the risk of acquiring new TB infection and the risk of developing disease once infected. Add to that the fact that HIV infection is most prevalent in the areas of the world where TB incidence is highest and the outcome is a massive coepidemic of TB and HIV infection centered in sub-Saharan Africa and Asia. The greatly increased risk of clinical TB among people infected with M. tuberculosis and HIV has been clearly demonstrated in this country and abroad (61,132,139). Only in the study by Selwyn et al. (61) is it clear that new tuberculous infection was not the major contributor to the increased risk. In recent years, there have been frequent reports of localized outbreaks (clusters) of TB cases among groups at high risk of developing TB. Many of these people were known to be HIV-positive. In these clusters, a very high proportion of cases were found to have tubercle bacille with the same restriction fragment-length polymorphism pattern, strongly suggesting that all cases came from a single source, probably recent (140,141). In these cluster situations, it is reasonable to believe that there was a high risk of becoming infected and, consequently, a high attack rate of tuberculous disease. Only in populations with a low risk of becoming infected is it reasonable to assume that most HIV-associated TB is the result of reactivation of latent infection. Two features distinguish the epidemiology of HIV-associated TB. First is the speed with which clinical disease becomes manifest following exposure. In two outbreaks among HIV-infected people, attack rates of

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16.7% and 29.4% were observed within somewhat less than a two-year period (142,143). For comparison, during the first two years of observation of HIV-negative household contacts admitted to the placebo arm of an isoniazid prophylaxis trial, only 1.3% of 6608 initially positive reactors and 2.2% of 867 tuberculin converters developed TB (47). The likelihood of developing manifest TB depends on the stage of the HIV infection. Most cases of TB are recognized at about the same time that other AIDS-defining conditions occur; the majority of the remaining cases occur somewhat prior to that time (144). The other feature is that HIV-related TB under usual circumstances appears to be somewhat less infectious than TB not associated with HIV infection (145). Although this decreased infectiousness might appear to be related to the tendency for HIV-infected people to have noncavitary and extrapulmonary disease, the decreased risk of infection persisted after allowing for these and other conditions considered to be related to infectiousness. G. Genetic Susceptibility

In the 19th century, it was commonly believed that TB was a hereditary disease (146). In the early part of the 20th century, Karl Pearson and Raymond Pearl each attempted to disentangle the hereditary and environmental factors that led to the familial concentration of TB (135)—investigations that were continued in the Williamson County Tuberculosis Study by Puffer (147). Subsequent studies of monozygotic and dizygotic twins indicated that some degree of susceptibility was inherited (71). Because of these indications of genetic susceptibility, investigators have looked for associations of TB with various genetic markers. Among the Inuits in Alaska, TB was more prevalent among people with blood groups B and AB than among those with blood groups O or A (72). Although various human leukocyte antigen types have also been suspected of playing a role in TB susceptibility, no consistent associations have been found (148,149). H. Nontuberculous Mycobacterial Infections

It has long been known that NTM are common in the environment, residing in water, soil, and a variety of animals and plants (135). Their global distribution had not been systematically studied until recently, however. In 1996, members of the Bacteriology and Immunology Section of the International Union Against Tuberculosis and Lung Disease formed a working group to collect systematic data on NTM isolated in laboratories around the world and have confirmed a number of conjectures with previously limited support. The most basic point is that the number of NTMs isolated from people increased steadily from 1976 to 1996, probably because of improved laboratory techniques and the advent of the HIV pandemic. The working group also identified the five most commonly isolated NTMs and discovered that the geographic distribution of NTM species is constantly changing and that new species are constantly emerging. For example, Mycobacterium

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xenopi was first isolated in 1980 in Spain, but by 1996, it was the third most common NTM isolated from people, although the rate of isolation varies considerably in the countries studied. There is also considerable geographic variation of chromogenic and nonchromogenic NTM species (150). In a series of experiments unlikely to be rivaled in size and sophistication, Palmer and Long showed that infections with a variety of mycobacteria increased the resistance of guinea pigs against TB (151). Evidence of protection was also found among British adolescents who reacted only to the 100 TU (the standard measurement unit for PPD tuberculin) dose of tuberculin and U.S. Navy recruits who reacted to antigens prepared from Mycobacterium avium-intracellulare or Mycobacterium scrofulaceum but not to the intermediate dose of purified-protein derivative (PPD)-tuberculin (152,153). These findings were not confirmed in a large study in Puerto Rico (51), although it is possible that the nonreactors to the strong dose of tuberculin were like some of Palmer’s guinea pigs that showed some evidence of protection even after failing to develop hypersensitivity after two injections of NTM. Although the question is unsettled, there is a strong possibility that human infections with NTM do confer some protection against TB. I. Psychosocial Stress

Although medical scientists are often hesitant to study the possible effects of mind on the body, there have been persistent hints in the TB literature that psychological, social, and economic stresses have an adverse effect on TB (154–156). Stress is a common thread running throughout the risk factors of poverty, homelessness, marital disruption, institutionalization, and substance abuse. A study that controlled for many other risk factors involved Navy recruits (57). White, black, and Filipino recruits who were tuberculin reactors on entry to the Navy had very similar housing, diet, and income during the first four years of their enlistment. Case rates among white and black reactors decreased during this period; case rates among Filipino reactors increased, possibly because of stresses associated with separation from families and with being a small minority with few social supports. J. Multidrug-Resistant Tuberculosis

Multiple drug resistance, currently defined as resistance to at least isoniazid and rifampicin, has become a major problem in many areas throughout the world (157). Although multiple drug–resistant tubercle bacille have been involved in many outbreaks of TB during the past decade or two, there is at present no evidence that their virulence differs from that of susceptible organisms. Rather, their association with outbreaks appears to be due largely to the situations in which outbreaks occur, namely people at high risk because of immunosuppression, crowding, homelessness, drug abuse, and/or poverty, especially in circumstances where chemotherapeutic regimens are poorly administered or accepted, often because of inadequate TB control programs. Understanding of the global distribution of multidrug-resistant tuberculosis (MDR-TB) is limited by a lack of systematic information. WHO and

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the International Union Against Tuberculosis and Lung Disease founded the Global Project on Anti-tuberculosis Drug Resistance Surveillance in 1994 and have sought to collect representative global data. Unfortunately, the project covers only 60 regions of the world and may not include data from all parts of a given country (158,159). Thus, although their data suggest that MDR-TB rates are 1% or less of all cases in most industrialized nations, there are conflicting reports that suggest much higher rates in at least some parts of some industrialized nations such as the Baltic and some countries of the former Soviet Union (159). In addition, information is very limited from parts of Asia and Africa where TB incidence is highest. Modeling of available data suggests that 250,000 to 500,000 new cases of MDR-TB occur globally each year (159). There are also numerous reports that detail foci of MDR-TB infection in select cities and populations (160–164), but a clearer understanding of the global distribution of MDR-TB remains elusive. IV. Conclusion Although this review of risk factors seems lengthy, it should be noted that much of the information rests on relatively few studies and that some of the most important ones were performed 30 to 40 years ago. Of concern is the current risk of disease following tuberculous infection in a variety of populations. Some way of reliably identifying people who continue to harbor tubercle bacille after having been infected would allow TB control efforts to be much more sharply focused on the seedbed of disease. Even small and individually nondefinitive studies of these and other risk factors would be helpful if they all pointed to the same conclusion. Increased knowledge of current risks of infection and subsequent disease could help greatly in efforts to bring TB back under control and, in developed countries, could even lead to its elimination. References 1. WHO Stop TB Programme. Global Tuberculosis Control, WHO Report 2005. World Health Organization, Geneva, 2005. 2. Buikstra JE, Cook DC. Pre-Columbian tuberculosis in West-Central Illinois: prehistoric disease in biocultural perspective. In: Buikstra JE, ed. Prehistoric Tuberculosis in the Americas. Evanston, IL: Northwestern University Archeological Program, 1981:115–139. 3. Pfyffer G, Auckenthaler R, van Embden JDA, van Soolingen D. Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg Infect Dis 1998; 4(4):631–634. 4. Niemann S, Richter E, Dalugge-Tamm H, et al. Two cases of Mycobacterium microti-derived tuberculosis in HIV-negative immunocompetent patients. Emerg Infect Dis 2000; 6(5):539–542. 5. CDC. Reported Tuberculosis in the United States, 2002. Atlanta, GA: US Department of Health and Human Services, CDC, September 2003.

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94. Chan-Yeung M, Galbraith JD, Schulson N, Brown A, Grzybowski S. Reactivation of inactive tuberculosis in northern Canada. Am Rev Respir Dis 1971; 104: 861–865. 95. Nakielna EM, Cragg R, Grzybowski S. Lifelong follow-up of inactive tuberculosis: its value and limitations. Am Rev Respir Dis 1975; 112:765–772. 96. International Union against Tuberculosis Committee on Prophylaxis. Efficacy of various durations of isoniazid preventive therapy for tuberculosis: five years of follow-up in the IUAT trial. Bull WHO 1982; 60:555–564. 97. Cowie RL, Langton ME, Becklake MR. Pulmonary tuberculosis in South African gold miners. Am Rev Respir Dis 1989; 139:1086–1089. 98. Comstock GW. Variability of tuberculosis trends in a time of resurgence. Clin Infect Dis 1994; 19:1015–1022. 99. Brudney K, Dobkin J. Resurgent tuberculosis in New York City. Human immunodeficiency virus, homelessness, and the decline of tuberculosis control programs. Am Rev Respir Dis 1991; 144:745–749. 100. U.S. Congress, Office of Technology Assessment. The Continuing Challenge of Tuberculosis, OTA-H-574. Washington, DC: U.S. Government Printing Office, 1993. 101. World Health Organization. Global tuberculosis control: surveillance, planning, financing. WHO Report 2004, Geneva, Switzerland, ISBN 92 4 156264 1. 102. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Global burden of tuberculosis estimated incidence, prevalence and mortality by country. JAMA 1999; 282:677–686. 103. CDC. Reported Tuberculosis in the United States, 2003. Atlanta, GA: U.S. Department of Health and Human Services, CDC, September 2004. 104. CDC. Reported Tuberculosis in the United States, 1998. Atlanta, GA: U.S. Department of Health and Human Services, CDC, September 1999. 105. Tala E. Registration of tuberculosis in Europe. Bull Int Union Tuberc Lung Dis 1987; 62(1–2):74–76. 106. Medical Research Council Tuberculosis and Chest Diseases Unit. The geographical distribution of tuberculosis notifications in a national survey of England and Wales in 1983. Tubercle 1986; 67:163–178. 107. Centers for Disease Control and Prevention. Reported Tuberculosis in the United States, 1993. Atlanta: Public Health Service, 1994. 108. Horwitz O, Comstock GW. What is a case of tuberculosis? The tuberculosis case spectrum in eight countries evaluated from 1235 case histories and roentgenograms. Int J Epidemiol 1973; 2:145–152. 109. Enarson DA, Wang JS, Grzybowski S. Case-finding in the elimination phase of tuberculosis: tuberculosis in displaced people. Bull IUATLD 1990; 65(2–3):71–72. 110. Chemtob D, Leventhal A, Weller-Ravell D. Screening and management of tuberculosis in immigrants: the challenge beyond professional competence. Int J Tuberc Lung Dis 2003; 7:959–966. 111. Geng E, Kreiswirth B, Driver C, et al. Changes in the transmission of tuberculosis in New York City from 1990 to 1999. N Engl J Med 2002; 246:1453–1459. 112. Diel R, Rusch-Gerdes S, Nieman S. Molecular epidemiology of tuberculosis among immigrants in Hamburg, Germany. J Clin Microbiol 2004; 42:2952–2966. 113. Brudey K, Gordon M, Mostrom P, et al. Molecular epidemiology of Mycobacterium tuberculosis in Western Sweden. J Clin Microbiol 2004; 42:3046–3051. 114. Ahmed N, Alam M, Rao KR, et al. Molecular genotyping of a large, multicentric collection of tubercle bacilli indicates geographical partitioning of strain variation and has implications for global epidemiology of Mycobacterium tuberculosis. J Clin Microbiol 2004; 42:3240–3247.

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115. Chaves AD, Robins AB, Abeles H. Tuberculosis case finding among homeless men in New York City. Am Rev Respir Dis 1961; 84:900–901. 116. Horwitz O. Tuberculosis risk and marital status. Am Rev Respir Dis 1971; 104: 22–31. 117. Von Ville P, Holtzhauer F, Long T, et al. Tuberculosis among residents of shelters for the homeless—Ohio, 1990. Morb Mortal Wkly Rep 1991; 40:869–877. 118. Lukacs J, Tubak V, Mester J, et al. Conventional and molecular epidemiology of tuberculosis in homeless patients in Budapest, Hungary. J Clin Microbiol 2004; 42:5931–5934. 119. Faggiano F, Vigna-Taglianti FD, Versino E, Bugiani M. Tuberculosis incidence in Turin, Italy, 1973–1999. Int J Tuberc Lung Dis 2004; 8(2):171–179. 120. Froggatt K. Tuberculosis: spatial and demographic incidence in Bradford, 1980– 1982. J Epidemiol Commun Health 1985; 39:20–26. 121. Centers for Disease Control. Tuberculosis among Asians/Pacific Islanders— United States, 1985. Morb Mortal Wkly Rep 1987; 36:331–334. 122. Sutherland I, Springett VH, Nunn AJ. Changes in tuberculosis notification rates in ethnic groups in England between 1971 and 1978/79. Tubercle 1984; 65:83–91. 123. Noland CM, Elarth AM. Tuberculosis in a cohort of Southeast Asian refugees. A five-year surveillance study. Am Rev Respir Dis 1988; 137:805–809. 124. Leung CC, Yew WW, Tam CM, et al. Socio-economic factors and tuberculosis: a district-based ecological analysis in Hong Kong. Int J Tuberc Lung Dis 2004; 8: 958–964. 125. Dony JF, Ahmad J, Tiong YK. Epidemiology of tuberculosis and leprosy, Sabah, Malaysia. Tuberculosis 2004; 84:8–18. 126. McKenna MT, Hutton M, Cauthen G, Onorato IM. The association between occupation and tuberculosis. A population-based survey. Am J Respir Crit Care Med 1996; 154:587–593. 127. Gershon RRM, Vlahov D, Escamilla-Cejudo JA, et al. Tuberculosis risk in funeral home employees. J Occup Environ Med 1998; 40:497–503. 128. Cegielski JP, McMurray DN. The relationship between malnutrition and tuberculosis: evidence from studies in humans and experimental animals. Int J Tuberc Lung Dis 2004; 8:286–298. 129. Van Lettow M, Kumwenda JJ, Harries AD, et al. Malnutrition and severity of lung disease in adults with pulmonary tuberculosis in Malawi. Int J Tuberc Lung Dis 2004; 8(2):211–217. 130. Ramachandran G, Santha T, Garg R, et al. Vitamin A levels in sputum-positive pulmonary tuberculosis patients in comparison with household contacts and health ‘normals’. Int J Tuberc Lung Dis 2004; 8(9):1130–1133. 131. Hutton MD, Cauthen GM, Bloch AB. Results of a 29-state survey of tuberculosis in nursing homes and correctional facilities. Public Health Rep 1993; 108: 305–314. 132. Braun MM, Badi N, Ryder RW, et al. A retrospective cohort study of the risk of tuberculosis among women of childbearing age with HIV infection in Zaire. Am Rev Respir Dis 1991; 143:501–504. 133. Klote MM, Agodoa LY, Abbott K. Mycobacterium tuberculosis infection incidence in hospitalized renal transplant patients in the United States, 1998–2000. Am J Transplant 2004; 4:1523–1528. 134. Snider DE Jr. The relationship between tuberculosis and silicosis. Am Rev Respir Dis 1978; 118:455–460. 135. Pinner M. Pulmonary Tuberculosis in the Adult. In: Its Fundamental Aspects. Springfield, IL: Charles C Thomas, 1945:190.

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136. Boucot KR, Dillon ES, Cooper DA, Meier P, Richardson R. Tuberculosis among diabetics. The Philadelphia survey. Am Rev Respir Dis 1952; 65(No. 1, Part 2):1–50. 137. Oscarsson PN, Silwer H II. Incidence of pulmonary tuberculosis among diabetics. Search among diabetics in the county of Kristianstad. Acta Med Scand 1958; 161(suppl 335):23–48. 138. Friedman LN, Sullivan GM, Bevilaqua RP, Loscos R. Tuberculosis screening in alcoholics and drug addicts. Am Rev Respir Dis 1987; 136:1188–1192. 139. Allen S, Batungwanayo J, Kerlikowske K, et al. Two-year incidence of tuberculosis in cohorts of HIV-infected and uninfected urban Rwandan women. Am Rev Respir Dis 1992; 146:1439–1444. 140. Genewein A, Telenti A, Bernasconi C, et al. Molecular approach to identifying route of transmission of tuberculosis in the community. Lancet 1993; 342:841–844. 141. Small PM, Hopewell PC, Singh SP, et al. The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N Engl J Med 1994; 330:1703–1709. 142. Dooley SW, Villarino ME, Lawrence M, et al. Nosocomial transmission of tuberculosis in a hospital unit for HIV-infected patients. JAMA 1992; 267:2632–2635. 143. Daley CL, Schechter GF, Rutherford GW. Tuberculosis outbreak among people in a residential facility for HIV-infected people—San Francisco. Morb Mortal Wkly Rep 1991; 40:649–652. 144. Rieder HL, Cauthen GM, Bloch AB, et al. Tuberculosis and acquired immunodeficiency syndrome—Florida. Arch Intern Med 1989; 149:1268–1273. 145. Cauthen GM, Dooley SW, Onorato IM, et al. Transmission of Mycobacterium tuberculosis from tuberculosis patients with HIV infection or AIDS. Am J Epidemiol 1996; 144:69–77. 146. Daniel T. Captain of death. In: The Story of Tuberculosis. Rochester: University of Rochester Press, 1997:69–76. 147. Puffer RR. Familial Susceptibility to Tuberculosis. In: Its Importance as a Public Health Problem. Cambridge, MA: Harvard University Press, 1944. 148. Hwang C-H, Khan S, Ende N, Mangura BT, Reichman LB, Chou J. The HLA-A, -B, and -DR phenotypes and tuberculosis. Am Rev Respir Dis 1985; 132:382–385. 149. Hawkins BR, Higgins DA, Chan SL, Lowrie DB, Mitchison DA, Girling DJ. HLA typing in the Hong Kong Chest Service/British Medical Research Council study of factors associated with the breakdown to active tuberculosis of inactive pulmonary lesions. Am Rev Respir Dis 1988; 138:1616–1621. 150. Martin-Casabona N, Bahrmand AR, Bennedsen J, et al., Spanish Group for Non-tuberculosis Mycobacteria. Non-tuberculosis mycobacteria: patterns of isolation. A multi-country retrospective survey. Int J Tuberc Lung Dis 2004; 8(10): 1186–1193. 151. Palmer CE, Long MW. Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. Am Rev Respir Dis 1966; 94:553–568. 152. Hart PDA, Sutherland I. 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 1977; 2:293–295. 153. Edwards LB, Acquaviva FA, Livesay VT. Identification of tuberculous infected. Dual tests and density of reaction. Am Rev Respir Dis 1973; 108:1334–1339. 154. Downes J, Price CR. The importance of family problems in the control of tuberculosis. Milbank Memorial Fund Quart 1942; 20:7–22. 155. Hendricks CM. Psychosomatic aspects of tuberculosis. In: Hayes EW, ed. The Fundamentals of Pulmonary Tuberculosis and Its Complications. Springfield, IL: Charles C Thomas, 1949:233–244.

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156. Holmes TH. Multidiscipline studies of tuberculosis. In: Sparer PJ, ed. Personality, Stress and Tuberculosis. New York: International Universities Press, 1956. 157. Cohn DL, Bustreo F, Raviglione MC. Drug-resistant tuberculosis: review of the worldwide situation and the WHO/IUATLD Global Surveillance Project. International Union Against Tuberculosis and Lung Disease. Clin Infect Dis 1997; 24(suppl 1):S121–S130. 158. Drobniewski F, Balabanova Y, Coker R. Clinical features, diagnosis, and management of multiple drug-resistant tuberculosis since 2002. Curr Opin Pulmonary Med 2004; 10:211–217. 159. WHO: Anti-tuberculosis Drug Resistance in the World. Report No. 2: Prevalence and Trends. Geneva: WHO, 2000. 160. Kruuner A, Hoffner SE, Silastu H, et al. Spread of drug-resistant pulmonary tuberculosis in Estonia. J Clin Microbiol 2001, 39:3339–3345. 161. Toungoussova S, Caugant DA, Sandven P, et al. Drug resistance of M. tuberculosis strains isolated from patients with pulmonary tuberculosis in Archangels, Russia. Int J Tuberc Lung Dis 2002; 6:406–414. 162. Drobniewski F, Balabanova Y, Ruddy M, et al. Rifampin and multiple drug resistant tuberculosis in the Russian civilian and prison sectors-dominance of the Beijing strain family. Emerg Infect Dis 2002; 8:1320–1326. 163. Almeida D, Rodrigues C, Udwadia ZF, et al. Incidence of multidrug-resistant tuberculosis in urban and rural India and implications for prevention. Clin Infect Dis 2003; 36:e152–e154. 164. Shemyakin IG, Stepanshina VN, Ivanov IY, et al. Characterization of drugresistant isolates of Mycobacterium tuberculosis derived from Russian inmates. Int J Tuberc Lung Dis 2004; 8:1994–1203.

5 Overview of the Pathogenesis of Tuberculosis from a Cellular and Molecular Perspective

SAMUEL C. WOOLWINE and WILLIAM R. BISHAI Division of Infectious Diseases, Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

I. Introduction Mycobacterium tuberculosis, the cause of human tuberculosis (TB), has been a scourge of humanity throughout recorded history. Even today, this bacille claims 2 to 3 million lives per year, remains one of the leading causes of death among the infectious diseases (1), and is the leading killer of people with AIDS (2). Human TB is a multistage disease. Any rational approach to TB control must be based upon the pathogenic processes at work during these stages. The pathogenic process begins with the inhalation of infectious aerosols. Bacille lodging in the alveoli are engulfed by the alveolar macrophage (AM) and, if able to survive this initial encounter with the innate immune system, begin a period of logarithmic growth, doubling every 24 hours until the macrophage bursts to release the bacterial progeny. New macrophages attracted to the site engulf these bacille and the cycle continues. The bacille may spread from the initial lesion via the lymphatic and/or circulatory systems to other parts of the body. After three weeks, the host develops specific immunity to the bacille. The resulting M. tuberculosis–specific lymphocytes migrate to the site of 101

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infection, surrounding and activating the macrophages there. As the cellular infiltration continues, the center of the cell mass, or granuloma, becomes caseous and necrotic (Fig. 1). In the majority of cases, the immunocompetent human is able to arrest the growth of the bacille within the primary lesion with little or no signs of illness. The initial lesion, which eventually resolves or calcifies, may still harbor viable bacille, in which case the host is said to harbor latent TB infection (LTBI, see below). However, in about 10% of infected individuals (3–7), the disease progresses during the initial weeks or months after infection, and

Figure 1 The histopathology of caseous necrosis in rabbit pulmonary tubercles five weeks following infection with Mycobacterium bovis. (A) A caseous center (hematoxylin and eosin staining) revealing disintegrating epithelioid macrophages at the center and a rim of basophilic mononuclear cells and early capillary formation on the periphery. (B) Same caseous center (acid-fast stain) showing the relative paucity of acid-fast bacille in rabbit tubercles at five weeks. (C) Multinucleated (Langhans) giant cell in rabbit lung tissues at five weeks (hematoxylin and eosin staining). (D) Acid-fast staining and high power (400
) view of a peripheral region of a five-week-old caseous rabbit tubercle showing several acid-fast bacille and some weakly acid-fast debris from destroyed bacille. Source: Courtesy of M. Yoder, Johns Hopkins University, Baltimore, Maryland, U.S.A.

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Figure 2 (A) Whole lungs from a nonsensitized New Zealand White (outbred) rabbit infected transthoracically with 108 colony forming units of Mycobacterium bovis in paraffin–lanolin according to the method of Yamamura revealing a large tuberculous lesion 3 cm in diameter. (B) Incision reveals an early cavitary lesion containing thick partially liquefied caseous material. Source: Courtesy of M. Yoder, Johns Hopkins University, Baltimore, Maryland, U.S.A.

the patient develops the typical symptoms of active (or progressive) primary TB: cough, fever, lethargy, and weight loss. In some cases, the granuloma becomes quite large and the caseous material liquefies, a phenomenon referred to as cavitary TB (see below and Fig. 2). This phenomenon is more commonly seen in cases of reactivation of latent TB. If the wall of the cavity erodes into an airway, the patient may become highly infectious as the liquefied contents of the cavity are expelled by coughing. Both caseous granulomas and cavities are devoid of blood supply, impairing both the immune system’s ability to fight the infection as well as the clinician’s attempts to treat the disease chemotherapeutically. The progression of TB from infection to either containment of disease or demise of the host has been well characterized anatomically. Although the molecular mechanisms responsible for many aspects of TB pathogenesis remain unknown, progress has been made in recent years on a number of fronts. This chapter will follow the typical course of the disease through its various stages and describe what is known regarding the molecular processes involved. II. Infection Although M. tuberculosis can infect by atypical routes and manifest in a number of anatomic sites, this pathogen is acquired in the overwhelming majority of cases by aerosol inhalation. The classic experiments of Wells and Riley (8–11) investigating the mechanics of airborne TB transmission have been revisited after more than 40 years in a recent study by Fennelly et al. (12), demonstrating that a large proportion of viable tubercle bacille expelled by coughing are contained in droplets less than 5 mm in size,

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consistent with the predictions made by the earlier investigators. Such infectious droplets, being small enough to reach the alveolus, allow the bacille to avoid the mucociliary clearance mechanisms of the airways. Once in the alveolus, the bacille is engulfed by the AM. AMs are continually ingesting inhaled particulates and as a result are usually in a partially activated state, depending on the nature of the particulates and the mechanism by which the material is ingested (e.g., with or without opsonization, specific receptors involved, etc.) (13,14). Phagocytosis by an insufficiently activated AM allows the bacille to avoid being killed and to begin a phase of exponential replication. That the AM actually contributes to productive M. tuberculosis infection is suggested by the observation that selective depletion of AMs from mice by using liposomeencapsulated dichloromethylene diphosphonate, which induces apoptosis of AMs, prior to and shortly after infection with M. tuberculosis resulted in 100% survival of the mice at 150 days following infection, compared to 60% survival with liposome treatment alone (15). Thus, ironically, the unactivated macrophage is used by the pathogen as a site for intracellular multiplication, and apoptosis of infected macrophages may be an antibacterial host defense mechanism. A few in vitro studies have suggested that M. tuberculosis might be capable of invading respiratory epithelial cells (16–19). Sato et al. reported that approximately 10% of the bacille observed by electron microscopy in lung sections (from mice intravenously infected with 5
107 organisms) were located inside type II alveolar cells at two days postinfection, with the remaining 90% contained within macrophages or neutrophils (20). At 14 days postinfection, few if any mycobacteria were observed within type II alveolar cells. Because most natural infections result from the successful aerosol implantation of one or a few bacille, the relevance of bacillary uptake by respiratory epithelial cells remains to be substantiated. A. Macrophage Receptors

A great deal of effort has gone into investigating the molecular interactions leading to phagocytosis of M. tuberculosis by the macrophage. A number of macrophage cell–surface molecules have been shown to bind to and promote internalization of M. tuberculosis, including multiple complement receptors (CR1, CR3, and CR4), mannose receptor, CD14, immunoglobulin G Fcc receptor, and scavenger receptors (21–26). The mechanism by which phagocytosis occurs may influence subsequent cytoplasmic events, thus M. tuberculosis may have evolved mechanisms to promote its uptake via specific pathways to avoid intracellular killing. Selectively blocking individual phagocytic pathways with antibody or competitive ligands does not seem to have an appreciable effect on M. tuberculosis survival or growth in macrophages (26). However, opsonization of M. tuberculosis with specific antibody results in antibody receptor–mediated phagocytosis and subsequent killing of the bacille after phagosome–lysosome fusion (21), whereas the bacille is able to prevent phagosome–lysosome fusion otherwise (27). For example,

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one study found that coating the bacille with monoclonal antibody to arabinomannan prior to infection had a protective effect in mice (28). B. Survival Within the Macrophage

Perhaps more articles have been published on mycobacterial survival within the macrophage than on any other aspect of TB pathogenesis. Ever since the observation that macrophage phagosomes containing M. tuberculosis fail to fuse with lysosomes (the normal fate of ingested bacteria) (27), investigators have attempted to unravel the underlying mechanisms of M. tuberculosis intracellular survival. The mycobacterial phagosome has recently been reviewed in great depth by Vergne et al. (29). Whereas the interior of the phagosome ordinarily becomes more acidic as it progresses down the endosomal/lysosomal pathway, phagosomes containing M. tuberculosis and other pathogenic mycobacteria do not completely acidify (30,31). This reduced acidification has been attributed to failure of the M. tuberculosis phagosome to accumulate vesicular proton– adenosine triphosphatase (ATPase) (30). Consistent with the observation that M. tuberculosis–containing phagosomes do not fuse with lysosomes (27), such phagosomes are also lacking in mature lysosomal hydrolases and other lysosomal markers (32–36). C. Calcium Signaling and Phagosome Maturation

The mechanism responsible for phagosome maturation arrest has been the subject of intense investigation in recent years. Deretic and coworkers have performed extensive characterization of the molecules involved in vesicle trafficking and whether or not these molecules localize normally in M. tuberculosis–infected cells (37–42). Chief among their findings were the aberrant accumulation of Rab5 and failure to acquire Rab7 on the mycobacterial phagosome (41). Rab5 and Rab7 are small guanosine triphosphate (GTP)-binding proteins involved in vesicular trafficking, being markers of early and late endosomes, respectively. GTP-binding proteins bind to GTP, thereby becoming activated for interaction with and/or regulation of other specific proteins. The GTP-binding protein also hydrolyzes the GTP to guanylnucleoside diphosphate (GDP), thus reverting to an inactive form until the GDP is exchanged for GTP once again. In this way, GTP hydrolysis acts as a molecular timer for protein activation. Following up on the observed block at the Rab5/Rab7 stage, Fratti et al. discovered that early endosomal autoantigen 1 (EEA1), a Rab5regulated protein involved in endosome docking and fusion (43), failed to localize to M. tuberculosis phagosomes (39). EEA1 also interacts with phosphatidylinositol-3-phosphate (PI3P) (44), generated from membrane phosphatidylinositol (PI) by another protein, hVPS34, a PI kinase (45). This interaction helps to localize EEA1 to the phagosome membrane (44). Vergne et al. showed that hVPS34 interacts with the Ca2þ-binding protein calmodulin in a Ca2þ-dependent manner (46). These findings intersect with another line of study by Malik et al. demonstrating that M. tuberculosis

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blocks the intracellular rise in Ca2þ associated with phagocytosis and that the Ca2þ/calmodulin pathway contributes to phagosome–lysosome fusion (47,48). These observations have collectively led to a model (Fig. 3) in which M. tuberculosis prevents phagosome maturation by blocking the increase of intracellular Ca2þ. Lower intracellular Ca2þ, in turn, prevents the Ca2þ/calmodulin-dependent activation of the PI kinase hVPS34, and thus no PI3P is generated on the phagosome membrane to recruit and retain EEA1 (29). It has recently been shown that the M. tuberculosis cell wall glycolipid lipoarabinomannan (LAM) inhibits intracellular increases in Ca2þ (46). It appears that the M. tuberculosis–dependent blocking of Ca2þ elevation occurs via inhibition of host cell sphingosine kinase (49), which has been shown to link a number of cell surface receptors to a rise in cytosolic Ca2þ (50,51). One observation that is not explained by this model is the inability of heat- or radiation-killed M. tuberculosis to inhibit the Ca2þ spike (47). This result seems to be at odds with the aforementioned ability of purified LAM to inhibit Ca2þ increase in the macrophage (46), as dead M. tuberculosis would still contain LAM. This may suggest that the active secretion or release of LAM from the cell wall of M. tuberculosis requires bacterial viability. The failure to synthesize PI3P on the phagosomal membrane would explain many observations regarding the mycobacterial phagosome, including its failure to acquire the vesicular proton-ATPase and lysosomal hydrolases (cathepsins) normally delivered to the phagosome by vesicles of the trans-Golgi network (Fig. 3) (29,32,35). III. Host Response A. The Dendritic Cell

If successful in preventing phagosome–lysosome fusion, the bacille replicates within the macrophage, filling the cell with its progeny until the macrophage ruptures to release its microbial cache. These bacille are in turn engulfed by the more immature monocyte-derived macrophages recruited to the area by chemoattractants such as complement components, bacterial products, and cytokines released by the infected host cell (52). Some of these secondarily infected host cells are dendritic cells (DCs) that migrate to the draining lymph nodes to initiate the onset of the adaptive immune response (53,54). DCs are derived from the monocyte/macrophage lineage and function as effective scavengers that phagocytose, process, and present antigens to T-lymphocytes, a necessary event in the development of cellmediated as well as humoral immunity. A growing body of literature suggests that M. tuberculosis interferes with DC function. Infection of blood monocytes with M. tuberculosis prevents their subsequent interferon-a–induced differentiation into DCs (55). It has also been discovered that the DC-specific intercellular adhesion molecule–grabbing nonintegrin (DC-SIGN) binds to LAM, a prominent glycolipid on the surface of M. tuberculosis (56). DC-SIGN has been

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Figure 3 A model for phagosome maturation arrest caused by Mycobacterium tuberculosis. Lipoarabinomannan from the mycobacterial cell wall prevents the normal rise in intracellular Ca2þ associated with phagocytosis. This in turn prevents the calcium/calmodulin activation of hVPS34 and its subsequent generation of PI3P. PI3P synthesized from the phagosome membrane phosphatidylinositol ordinarily serves to recruit both Rab5 and early endosomal autoantigen 1, which interact with syntaxin-6 on the membrane of vesicles from the trans Golgi network carrying molecules such as cathepsins and the vesicular proton–ATPase. Abbreviations: LAM, lipoarabinomannan; LBC, phagosome containing latex bead; MPC, phagosome containing M. tuberculosis; PI3P, phosphatidylinositol-3-phosphate; CaM, calcium/ calmodulin; PI, phosphatidylinositol; TGN, trans Golgi network; ATPase, adenosine triphosphatase; EEA1, early endosomal autoantigen 1; GDI, guanosine nucleotide dissociation inhibitor. Source: From Ref. 29.

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found to bind to surface molecules of a number of bacterial, parasitic, and viral pathogens, notably HIV-1 and -2, Ebola virus, and Dengue virus (57). The binding of LAM to DC-SIGN has been shown to inhibit both lipopolysaccharide-induced secretion of interleukin (IL)-12 and Mycobacterium bovis bacille Calmette–Gue´rin (BCG)-mediated DC maturation (56). LAM–DC–SIGN interaction also stimulates secretion of the anti-inflammatory cytokine IL-10 (56). Whether or not the effects of M. tuberculosis on DC function play a role in TB pathogenesis, the fact remains that TB patients do develop specific cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH). Therefore, it stands to reason that either impairment of DC function by M. tuberculosis does not occur in vivo (or at least not to the extent of preventing an adaptive immune response), or that other antigen-presenting cells (e.g., macrophages) must play a role in T-cell activation and proliferation, or both. B. Granuloma Formation

The rabbit model of TB, pioneered by Lurie and Dannenberg, has provided a wealth of information regarding the histopathologic changes that occur following aerosol infection with M. tuberculosis. Following ingestion of M. tuberculosis by the AM and the initial replication within this cell, the resulting bacille are taken up by the newly arriving macrophages derived from blood monocytes. By two weeks postinfection, infected macrophages at the center of the lesion have acquired an epithelioid morphology. As these cells die, they provide the raw material for the process of caseation necrosis. The periphery of the lesion consists largely of activated macrophages and neutrophils. At four weeks postinfection, the number of mature macrophages seen at the periphery of the caseous lesion has increased. Multinucleated giant cells (Langhans cells, see Fig. 1) are often seen by this time, formed by the fusion of activated macrophages. In addition, M. tuberculosis–specific lymphocytes have appeared and surrounded the lesion, along with plasma cells and fibroblasts. The onslaught of the immune response has by this time destroyed a large proportion of the bacille, and by six weeks the rare remaining bacille are typically found at the edge of the caseous center of the lesion, surrounded by a zone of activated macrophages. Eight weeks following infection, the lesion consists of a caseous necrotic core, surrounded by lymphocytes and a few remaining macrophages. Few if any viable bacille may be present at this time. These lesions may range in size from 1 to 5 mm (58,59). C. Cell-Mediated Immunity vs. Delayed-Type Hypersensitivity

Dannenberg has made a distinction between mechanisms of macrophageactivating CMI and tissue-damaging DTH (60). In this dual mechanism model, CMI is described as an immunologic mechanism in which macrophages are activated by antigen-specific T-cells, thereby acquiring an enhanced ability to destroy bacille they have ingested. DTH, on the other hand, is viewed as a separate phenomenon whereby bacille-laden macrophages are themselves destroyed, along with some surrounding tissue, resulting in

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caseous necrosis. Both CMI and DTH occur within the environment of the granuloma after the onset of adaptive immunity. If the host produces effective CMI, the macrophages attracted to the site of infection become highly activated and surround the existing lesion, engulfing and destroying any bacille that have been released from dying cells or from the periphery of the caseous center of the granuloma. In this way, the infection is contained and the spread of caseous necrosis is prevented. However, in hosts that fail to mount effective CMI, the incoming macrophages are insufficiently activated and become parasitized by the bacille, serving as reservoirs for further bacterial replication until they are destroyed by the tissue-damaging DTH response. In this way, more lung tissue is destroyed as the caseous lesion grows larger. Although the operational distinction between CMI and DTH is useful in terms of understanding the pathology of the disease, it should be noted that experimental evidence for immunologically distinct mechanisms of CMI and DTH has yet to be conclusively demonstrated. Perhaps a more fundamental way of viewing CMI and DTH as defined above is that these processes represent opposing blades of a double-edged sword: the more effective the immune response at killing or halting replication of the bacille, the less collateral tissue damage occurs and vice versa. IV. Cavitary Tuberculosis In some cases, the caseous lesion becomes quite large and transforms into a liquid-filled cavity. Unfortunately, little is known regarding the process of liquefaction at the molecular level. As more tissue surrounding the cavity is destroyed by caseous necrosis, the cavity expands. The bacille multiply to high numbers within the liquefied cavity, and if the cavity erodes into the wall of an adjacent blood vessel, the patient may become seeded throughout the body with tubercle bacille. On the other hand, if the cavity ruptures into an airway, the fluid is expelled as an infectious aerosol when the patient coughs. Thus cavitary TB is one of the most important manifestations of the disease from the perspective of transmission. As with the process of granuloma formation, the rabbit has provided an excellent model for studying the development of cavitary TB. Whereas the rabbit is usually able to contain and even clear infection with M. tuberculosis, infection with M. bovis leads to progressive disease. Cavitation in the rabbit is often seen 8 to 12 weeks following infection or reinfection with M. bovis (61–63). Yamamura et al. developed a rabbit model of cavitary TB following direct injection of bacille or bacterial extracts into the lung through the chest wall (64,65). These investigators found that previous exposure of the rabbit to heat-killed M. bovis by subcutaneous injection greatly increased the frequency of cavity formation upon subsequent infection. In fact, Yamamura observed that transthoracic injection of even heat-killed bacille led to cavitation in 40% to 85% of sensitized rabbits, but failed to do so in na€
ve animals. Therefore, the process of cavitation seems to be purely a host response phenomenon. Immunosuppressive agents (6-mecaptopurine and

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azathioprine) were found to reduce the occurrence of cavity formation in sensitized rabbits (66). This finding is consistent with the more current observation that TB patients with advanced HIV infection are less likely to develop cavitary disease (67). V. Latent Tuberculosis Of those who become infected with M. tuberculosis, about 10% will progress to active primary disease. The roughly 90% of individuals (3–7) who contain the infection within the initial lesion may still harbor viable bacille, a condition referred to as LTBI (5). They are at risk for developing secondary (or reactivation) TB, which may manifest as pulmonary or extrapulmonary disease. In the immunocompetent host, the lifetime risk of developing reactivation disease is usually estimated to be 5% to 10%. There is good evidence that the use of various immunosuppressive drugs increases the risk of reactivation substantially. Tumor necrosis factor-a inhibitors such as infliximab, etanercept, and adalimumab are associated with an increased incidence of TB, usually within the first months of administration (68–71). Advanced HIV infection has been calculated to increase the relative risk of reactivation TB 10-fold compared to non–HIV-infected patients with LTBI (72). Given that M. tuberculosis can persist for years in the human host before recrudescence of active disease, it follows that either the replication of the bacille must drastically slow or even cease, or that host bactericidal mechanisms must keep pace with the growth of the bacille. In fact, this is typically observed in the murine model of TB: after a period of around three weeks of exponential growth of M. tuberculosis following inoculation of the mouse, the bacillary burden plateaus. This change in the rate of bacterial growth coincides with the onset of specific immunity. Although one might at first conclude this represents ‘‘latency,’’ there are important differences between the condition of the mice and that of humans with LTBI. First, the mice continue to harbor high titers of bacille in their lungs. Second, the mice eventually succumb to the disease, specifically to the loss of airspace in the lungs as a result of progressive inflammatory infiltration. Unfortunately, there are still no truly adequate animal models for LTBI, although the rabbit model shows great promise. Rabbits infected with M. tuberculosis form the caseous lesions typical of human TB pathology, and, like most humans, are often able to contain the disease such that no cultivable bacille are recovered from lung homogenates of these animals. If these animals harbor latent bacille, it may be possible to precipitate reactivation TB through the use of immunosuppressive agents. A. In Vitro Models of Latency

In vitro models in which M. tuberculosis halts replication, yet remains viable, have been developed to better study the bacterial physiology in LTBI. One of these models, developed by Wayne, is based on the gradual depletion of oxygen from exponentially growing cultures (73). Given the

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avascular nature of the caseous lesion, and the fact that hypoxia results in a nonreplicating yet viable state, the Wayne model seems to represent a logical hypothesis to explain the change in bacillary metabolism in LTBI. Wayne and coworkers found specific enzyme activities that were induced upon transition to the hypoxic state. Specifically, glycine dehydrogenase activity catalyzing the reductive amination of glyoxylate to glycine was significantly increased (74). The production of glycine in this reaction is coupled to the oxidation of NADH to NADþ, and it has been postulated that this regeneration of NADþ allows the bacille to complete the current cycle of DNA replication and achieve an orderly metabolic shutdown (74). Boon et al. subsequently used the Wayne model to study protein changes in M. bovis BCG and found that four proteins were significantly induced (75). Two of these are proteins of unknown function. The third protein, HspX (Acr), belongs to a family of proteins known as chaperones. Chaperones assist in the proper folding (or refolding) of other proteins either during synthesis or after they have become misfolded due to heat shock or chemical stress. The fourth protein, DosR, is a transcriptional regulator that activates the expression of many genes, including hspX, in response to hypoxic conditions (76). More recently, whole-genome expression profiling of M. tuberculosis identified a host of genes induced in response to hypoxic conditions, including dosR, hspX, and several genes involved in diverse metabolic pathways, many of which were subsequently found to be regulated by DosR (76). Several genes found to be induced by hypoxia were also found to be induced by low levels of nitric oxide, a potent antibacterial molecule implicated in the defense against M. tuberculosis (77). Among these genes were dosR, hspX, nrdZ (ribonucleotide reductase class II), narX (fused nitrate reductase), narK2 (nitrite extrusion protein), ctpF (cation transport ATPase), and fdxA (ferredoxin). Another in vitro model that maintains bacterial viability in a state of nonreplication is nutrient starvation. Using whole-genome expression profiling, Betts et al. identified several hundred genes that were induced during nutrient limitation. Among these were hspX, discussed above, and four genes (sigB, sigE, sigF, and sigD) belonging to the sigma factor family (78). Sigma factors are components of the bacterial RNA polymerase, which confer gene target specificity to the polymerase; i.e., it is the sigma factor that determines which genes a given molecule of RNA polymerase will recognize and express. Although these genomic studies have identified many genes and metabolic pathways involved in response to starvation or hypoxia, the actual molecular mechanisms by which tubercle bacille enter a latent state have yet to be elucidated. In fact, it is still not known whether these in vitro conditions truly represent the bacterial physiology at work in LTBI. VI. Conclusion What, then, does biomedical science have to offer to the fight against TB? One might argue that an effective vaccine would be a beneficial tool.

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However, roughly 90% of individuals infected with TB are able to contain the infection, and in spite of this successful immune response, many of these individuals are unable to completely eradicate the organism from the body. Effective vaccination schemes, hence, will need to protect newborns from infection, and ideally also protect adults with LTBI from the risk of reactivation. Because reactivation of LTBI most often occurs in the context of a weakened immune system, these goals remain daunting challenges. New drugs active against the tubercle bacille are perhaps the most desperately needed weapons in the war on TB. Our current arsenal of drugs contains both bactericidal and sterilizing antibiotics, which work both against active TB and as secondary prevention in patients with LTBI. It is likely that the new anti-TB drugs may also have dual applicability for these two therapeutic needs. Safe, short-course, effective therapy against latent infection is certainly a high priority for successful TB control. In fact, bacille with latent-state physiology may comprise a significant percentage of the bacillary population even during active disease, and may be one reason why such long treatment regimens are required. As more information is gathered on the survival mechanisms of M. tuberculosis within the macrophage and during latent infection, new targets for drug intervention may be realized. Clearly it will take a concerted effort on the part of scientists, physicians, and other health care workers, as well as political leaders, to continue making progress against one of the most successful bacterial killers the world has ever known. References 1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione M. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. J Am Med Assoc 1999; 282:677–686. 2. Hopewell P, Chaisson R. Tuberculosis and human immunodeficiency virus infection. In: Reichman L, Hershfield E, eds. Tuberculosis: A Comprehensive International Approach. 2nd ed. New York: Marcel Dekker, Inc., 2000:525–552. 3. Ferebee S. Controlled chemoprophylaxis trials in tuberculosis: a general review. Adv Tuberc Res 1969; 17:28–106. 4. Grzybowski S, Barnett G, Styblo K. Contacts of cases of active pulmonary tuberculosis. Bull Int Union Tuberc 1975; 50:90–106. 5. Nuermberger E, Bishai W, Grosset J. Latent tuberculosis infection. Semin Respir Crit Care Med 2004; 25:317–336. 6. Sutherland I. Recent studies in the epidemiology of tuberculosis, based on the risk of being infected with tubercle bacilli. Adv Tuberc Res 1976; 19:1–63. 7. Sutherland I. The Ten-Year Incidence of Clinical Tuberculosis Following ‘‘Conversion’’ in 2550 Individuals Aged 14 to 19 Years. TSTRU Progress Report. The Hague: KNCV, 1968. 8. Riley R, Mills C, Nyka W, et al. Aerial dissemination of pulmonary tuberculosis: a two year study of contagion in a tuberculosis ward. Am J Hyg 1959; 70:185–196. 9. Wells W. On air-borne infection. II: Droplets and droplet nuclei. Am J Hyg 1934; 20:611–618.

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10. Wells W, Lurie M. Experimental airborne disease: quantitative natural respiratory contagion of tuberculosis. Am J Hyg 1941; 34:21–40. 11. Wells W, Ratcliffe H, Crumb C. On the mechanics of droplet nuclei infection. II: Quantitative experimental air-borne tuberculosis in rabbits. Am J Hyg 1948; 47:11–28. 12. Fennelly K, Martyny J, Fulton K, Orme I, Cave D, Heifets L. Cough-generated aerosols of Mycobacterium tuberculosis. Am J Respir Crit Care Med 2004; 169: 604–609. 13. Brown D, Donaldson K, Borm P, et al. Calcium and ROS-mediated activation of transcription factors and TNF-a cytokine gene expression in macrophages exposed to ultrafine particles. Am J Physiol Lung Cell Mol Physiol 2004; 286:L344–L353. 14. Kobzik L, Huang S, Paulauskis J, Godleski J. Particle opsonization and lung macrophage cytokine response. J Immunol 1993; 151:2753–2759. 15. Leemans J, Juffermans N, Florquin S, et al. Depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice. J Immunol 2001; 166: 4604–4611. 16. Reddy V, Hayworth D. Interaction of Mycobacterium tuberculosis with human respiratory epithelial cells (HEp-2). Tuberculosis 2002; 82:31–36. 17. Mehta P, King C, White E, Murtagh J, Quinn F. Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication. Infect Immun 1996; 64:2673–2679. 18. Bermudez L, Goodman J. Mycobacterium tuberculosis invades and replicates within type II alveolar cells. Infect Immun 1996; 64:1400–1406. 19. Bermudez L, Sangari F, Kolonoski P, Petrofsky M, Goodman J. The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells. Infect Immun 2002; 70:140–146. 20. Sato K, Tomioka H, Shimizu T, Gonda T, Ota F, Sano C. Type II alveolar cells play roles in macrophage-mediated host innate resistance to pulmonary mycobacterial infections by producing proinflammatory cytokines. J Infect Dis 2002; 185:1139– 1147. 21. Armstrong J, Hart P. Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J Exp Med 1975; 142:1–16. 22. Ernst J. Macrophage receptors for Mycobacterium tuberculosis. Infect Immun 1998; 66:1277–1281. 23. Peterson P, Gekker G, Hu S, et al. CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Infect Immun 1995; 63: 1598–1602. 24. Schlesinger L. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. J Immunol 1993; 150:2920–2930. 25. Schlesinger L, Bellinger-Kawahara C, Payne N, Horwitz M. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990; 144:2771–2780. 26. Zimmerli S, Edwards S, Ernst J. Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages. Am J Respir Cell Mol Biol 1996; 15:760–770. 27. Armstrong J, Hart P. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med 1971; 134:713–740.

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28. Teitelbaum R, Glatman-Freedman A, Chen B, et al. A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proc Natl Acad Sci USA 1998; 95:15688–15693. 29. Vergne I, Chua J, Singh S, Deretic V. Cell biology of Mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 2004; 20:367–394. 30. Sturgill-Koszycki S, Schlesinger P, Chakraborty P, et al. Lack of acidification in mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 1994; 263:678–681. 31. Crowle A, Dahl R, Ross E, May M. Evidence that vesicles containing living virulent M. tuberculosis or M. avium in cultured human macrophages are not acidic. Infect Immun 1991; 59:1823–1831. 32. Sturgill-Koszycki S, Schaible U, Russell D. Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J 1996; 15:6960–6968. 33. Barker L, George K, Falkow S, Small P. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect Immun 1997; 65:1497–1504. 34. Clemens D, Horwitz M. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med 1995; 181:257–270. 35. Fratti R, Chua J, Vergne I, Deretic V. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proc Natl Acad Sci USA 2003; 100:5437–5442. 36. Xu S, Cooper A, Sturgill-Koszycki S, et al. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol 1994; 153:2568–2578. 37. Fratti RA, Chua J, Deretic V. Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes. J Biol Chem 2003; 278:46961–46967. 38. Fratti RA, Chua J, Deretic V. Cellubrevin alterations and Mycobacterium tuberculosis phagosome maturation arrest. J Biol Chem 2002; 277:17320–17326. 39. Fratti RA, Backer JM, Gruenberg J, Corvera S, Deretic V. Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J Cell Biol 2001; 154:631–644. 40. Fratti RA, Vergne I, Chua J, Skidmore J, Deretic V. Regulators of membrane trafficking and Mycobacterium tuberculosis phagosome maturation block. Electrophoresis 2000; 21:3378–3385. 41. Via LE, Deretic D, Ulmer RJ, Hibler NS, Huber LA, Deretic V. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem 1997; 272:13326–13331. 42. Deretic V, Via LE, Fratti RA, Deretic D. Mycobacterial phagosome maturation, rab proteins, and intracellular trafficking. Electrophoresis 1997; 18:2542–2547. 43. Christoforidis S, McBride HM, Burgoyne RD, Zerial M. The Rab5 effector EEA1 is a core component of endosome docking. Nature 1999; 397:621–625. 44. Simonsen A, Lippe R, Christoforidis S, et al. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 1998; 394:494–498. 45. Christoforidis S, Miaczynska M, Ashman K, et al. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat Cell Biol 1999; 1:249–252. 46. Vergne I, Chua J, Deretic V. Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2þ/Calmodulin-PI3K hVPS34 cascade. J Exp Med 2003; 198:653–659. 47. Malik ZA, Denning GM, Kusner DJ. Inhibition of Ca(2þ) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages. J Exp Med 2000; 191:287–302.

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48. Malik ZA, Iyer SS, Kusner DJ. Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages. J Immunol 2001; 166:3392–3401. 49. Malik ZA, Thompson CR, Hashimi S, Porter B, Iyer SS, Kusner DJ. Cutting edge: Mycobacterium tuberculosis blocks Ca2þ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase. J Immunol 2003; 170:2811–2815. 50. Spiegel S, Milstien S. Sphingosine 1-phosphate, a key cell signalling molecule. J Biol Chem 2002; 277:25851–25854. 51. Melendez A, Floto R, Gillooly D, Harnett M, Allen J. FccRI coupling to phospholipase D initiates sphingosine kinase-mediated calcium mobilization and vesicular trafficking. J Biol Chem 1998; 273:9393–9402. 52. Sadek M, Sada E, Toossi Z, Schwander S, Rich E. Chemokines induced by infection of mononuclear phagocytes with mycobacteria and present in lung alveoli during active pulmonary tuberculosis. Am J Respir Cell Mol Biol 1998; 19:513–521. 53. Jiao X, Lo-Man R, Guermonprez P, et al. Dendritic cells are host cells for mycobacteria in vivo that trigger innate and acquired immunity. J Immunol 2002; 168: 1294–1301. 54. Marino S, Pawar S, Fuller C, Reinhart T, Flynn J, Kirschner D. Dendritic cell trafficking and antigen presentation in the human immune response to Mycobacterium tuberculosis. J Immunol 2004; 173:494–506. 55. Mariotti S, Teloni R, Iona E, et al. Mycobacterium tuberculosis diverts alpha interferon-induced monocyte differentiation from dendritic cells into immunoprivileged macrophage-like host cells. Infect Immun 2004; 72:4385–4392. 56. Geijtenbeek T, van Vliet S, Koppel E, et al. Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med 2003; 197:7–17. 57. van Kyook Y, Geijtenbeek T. DC-SIGN: escape mechanism for pathogens. Nat Rev Immunol 2003; 3:697–709. 58. Lurie M. Resistance to Tuberculosis. Cambridge: Harvard University Press, 1964. 59. Lurie M. The correlation between the histological changes and the fate of living tubercle bacilli in the organs of tuberculous rabbits. J Exp Med 1932; 55: 31–54. 60. Dannenberg AM Jr. Roles of cytotoxic delayed-type hypersensitivity and macrophageactivating cell-mediated immunity in the pathogenesis of tuberculosis. Immunobiology 1994; 191:461–473. 61. Dannenberg AM Jr. Pathogenesis of pulmonary Mycobacterium bovis infection: basic principles established by the rabbit model. Tuberculosis 2001; 81:87–96. 62. Lurie M. The fate of tubercle bacilli in the organs of reinfected rabbits. J Exp Med 1929; 50:747. 63. Lurie M. A correlation between the histological changes and the fate of living tubercle bacilli in the organs of reinfected rabbits. J Exp Med 1933; 57:181. 64. Yamamura Y. The pathogenesis of tuberculous cavities. Adv Tuberc Res 1958; 9: 13–37. 65. Yamamura Y, Yasaka S, Yamaguchi M, et al. Studies of the experimental tuberculosis cavity: the experimental formulation of the tuberculous cavity in the rabbit lung. Med J Osaka Univ 1954; 5:187–197. 66. Yamamura Y, Ogawa Y, Yamagata H. Prevention of tuberculous cavity formation by immunosuppressive drugs. Am Rev Respir Dis 1968; 98:720. 67. Lucas S, Nelson A. Pathogenesis of tuberculosis in human immunodeficiency virusinfected people. In: Bloom B, ed. Tuberculosis: Pathogenesis, Protection and Control. Washington, DC: American Society for Microbiology, 1994:503–513.

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68. Keane J, Gershon S, Wise RP, et al. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001; 345:1098–1104. 69. Tuberculosis associated with blocking agents against tumor necrosis factor-alpha— California, 2002–2003. Morb Mortal Wkly Rep 2004; 53:683–686. 70. Wallis RS, Broder MS, Wong JY, Hanson ME, Beenhouwer DO. Granulomatous infectious diseases associated with tumor necrosis factor antagonists. Clin Infect Dis 2004; 38:1261–1265. 71. Wallis R, Broder M, Wong J, Beenhouwer D. Granulomatous infections due to tumor necrosis factor blockade: correction. Clin Infect Dis 2004; 39:1254–1255. 72. Horsburgh CR Jr. Priorities for the treatment of latent tuberculosis infection in the United States. N Engl J Med 2004; 350:2060–2067. 73. Wayne L, Sohaskey C. Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 2001; 55:139–163. 74. Wayne L, Lin K. Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions. Infect Immun 1982; 37:1042–1049. 75. Boon C, Li R, Qi R, Dick T. Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model. J Bacteriol 2001; 183:2672–2676. 76. Park H, Guinn K, Harrell M, et al. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 2003; 48:833–843. 77. Voskuil M, Schnappinger D, Visconti K, et al. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 2003; 198:705– 713. 78. Betts J, Lukey P, Robb L, McAdam R, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 2002; 43:717–731.

6 The Human Host: Immunology and Susceptibility

S. K. SCHWANDER and JERROLD J. ELLNER Department of Medicine and Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, UMDNJ—New Jersey Medical School, Newark, New Jersey, U.S.A.

I. Introduction Epidemiological evidence suggests that protective immunity against Mycobacterium tuberculosis exists in most exposed humans. The proportion of nonimmunocompromised individuals who undergo primary infection or reactivation disease following M. tuberculosis infection is low. Only 5% to 10% of M. tuberculosis–infected individuals develop tuberculosis (TB) disease during their lifetime (Fig. 1) (1,2). The study of human immunity to M. tuberculosis infection is critical to the understanding of the mechanisms that contain the initial tuberculous focus and maintain clinical latency. Such studies validate basic concepts established in animal models and are required because differences are apparent between experimental models in cell function, effector molecules, and disease expression. Immunological markers of protection in humans also serve as desirable end points for the study of new antituberculous vaccines and new vaccine delivery strategies and as readouts for adjuvant immunotherapies.

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Figure 1 Hypothetical model of the natural history of Mycobacterium tuberculosis infection in humans. Abbreviations: IFN-c, interferon gamma-c; M. tb, Mycobacterium tuberculosis; TST, tuberculin skin test.

II. The Natural History of Mycobacterium tuberculosis Infection in Humans Infection with M. tuberculosis in most instances occurs by inhalation of droplet nuclei (1–5 mm) that contain the infectious bacteria and are aerosolized from the lung tissue of TB patients (3–6) by respiratory maneuvers such as coughing or speaking. Droplet nuclei are deposited in the terminal airspaces and the initial site of exposure is most often in the lower and middle lobes due to the higher ventilation of these lung regions. Most latently, M. tuberculosis–infected individuals do not develop active TB. M. tuberculosis infection in such persons is typically accompanied only by the development of a positive tuberculin skin test (TST) or M. tuberculosis antigen–specific lymphocyte proliferation or interferon gamma (IFN-c) production in vitro. The risk of developing active TB is greatest in the first two years following infection and is associated with more intense exposure to M. tuberculosis. It is higher for persons infected through contact with an index sputum smear-positive TB case (7). The age at the time of M. tuberculosis infection is also important. Persons who become infected in infancy, adolescence, or old age are more likely to progress to active TB. Overall, 5% to 10% of immunocompetent persons develop active TB during their lifetime. A. Primary Tuberculosis

The vast majority of immunocompetent persons develop an effective immune response against M. tuberculosis and contain the primary infection,

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leaving only small, calcified parenchymal scars (Ghon complex). Following primary infection, immunocompetent persons develop specific acquired resistance to reinfection. This specific acquired resistance (also called adaptive or memory immunity) is long lasting, possibly due to low-level endogenous bacterial replication or, in high prevalence areas, repeated exogenous exposure. B. Progressive Primary Tuberculosis

Persons who fail to develop specific acquired immune responses following primary M. tuberculosis infection may develop progressive primary TB. This form of disease is most common in young children, the immunocompromised, and the elderly. Miliary or meningeal disease may result after widespread hematogenous dissemination of M. tuberculosis. The clinical presentation is frequently cryptic with nonspecific symptoms such as malaise and fatigue or fever of unknown origin (8). Progressive primary disease in young adults presents with fever, productive cough, night sweats, weight loss, and upper lobe cavitary lesions, which can be reliably distinguished from reactivation TB only when recent TST conversion has been documented (9). C. Reactivation (Postprimary) Tuberculosis

The risk of developing progressive primary or reactivation TB following infection with M. tuberculosis is increased in immunocompromised persons; the degree of risk varies with the underlying disease impairing host defenses. The lungs are the most common sites of reactivation TB. Chronic productive cough with mucopurulent sputum of greater than three weeks’ duration, night sweats, weight loss, and anorexia are the most frequent complaints. Forty percent to 60% of patients are afebrile at presentation. The onset of symptoms is usually insidious. About one-fifth of patients with reactivation TB have no pulmonary symptoms and the diagnosis is detected on a routine chest radiograph (10). In areas of high TB prevalence, exogenous reinfection may occur and progress to active disease. III. Human Immunity to Mycobacterium tuberculosis Progression of M. tuberculosis infection to primary or reactivation TB has in the past been interpreted as a function of the extent and efficiency of protective human immunity against M. tuberculosis. However, there is increasing evidence for a complex mutual interplay of host and pathogen biology and for a major role of M. tuberculosis isolates in triggering a variety of host responses. Much of the current knowledge of human immunity to M. tuberculosis is derived from studies of blood cells because they are most accessible. Blood provides the source of cells that are recruited to and compartmentalized at the inflammatory focus and form the building blocks of the granulomatous tissue reaction. Studies in healthy household contacts (HHC) of TB patients provide a model of protective immunity, and correlates of immunity established here may guide in the development of

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new vaccination approaches. Pulmonary TB in the adult represents a good model to delineate pathogenesis, immunopathology, and concomitants of reactivation disease and to test concepts concerning immunotherapy. With increasing attention to TB, a great deal now is known about the human immune response in infection and disease. The observations should be placed in the context of the natural history of M. tuberculosis infection (Fig. 1). Several themes emerge. In exposed household contacts and individuals with latent infection, innate and acquired immune mechanisms prevent the establishment of infection and progression to disease. Stimulation of Toll-like receptors (TLR) on monocytes (MN), alveolar macrophages (AM), and dendritic cells (DC) by M. tuberculosis and its constituents is a key component of innate immunity. Specific acquired resistance and ‘‘protective immunity’’ are characterized by a predominant T helper 1 (TH1) response (with production of interleukin [IL]-2), and IFN-c and suppression of T helper 2 (TH2) responses (with production of IL-4, IL-5, and IL-13). It has become clear that the initial interaction of M. tuberculosis with antigen-presenting cells (APC) may be the crucial event that determines the balance between TH1 and TH2 immunity and the effectiveness of the immune response. Further, genetic and acquired factors (e.g., helminthic infection and atopy) may modify this balance. The eventual ‘‘breakdown’’ of clinically latent foci of M. tuberculosis infection may result from immunosuppression, aging, comorbidities (diabetes mellitus), or unknown factors and culminates in reactivation disease. Deciphering the immune response in the presence of disease is complex and findings related to pathogenesis may be confounded by disease-related factors (fever, weight loss, etc.). The systemic immune response during TB is dominated by immunosuppression. This may avert the deleterious effects of systemic immune activation (i.e., septic shock). This specialized form of immunosuppression has as a downside: susceptibility to progression of exogenous reinfection to active disease. This, in fact, has been recognized recently in patients from high-prevalence areas, who were shown to frequently become exogenously reinfected. The local immune response in active TB shows unregulated immune activation with an abundance of inflammatory mediators demonstrable in the blood, bronchoalveolar lavage fluid, pleural fluid, and sputum. Some of these mediators are immunosuppressive or proapoptotic. They disappear early during the course of TB chemotherapy. Immune activation is a dominant finding in studies of bronchoalveolar lavage cells (BAC) from radiographically involved lungs of TB patients. The occurrence of high local levels of potentially protective cytokines such as IFN-c concomitant to active disease suggests that the response to these cytokines may be blocked. A. Cell-Mediated Immunity

There is general consensus that cell-mediated immunity (CMI) is the primary host defense mechanism against intracellular pathogens such as M. tuberculosis

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(Fig. 2). M. tuberculosis is taken up by phagocytic antigen–presenting MN, macrophages (MØ), DC, and epithelial cells. M. tuberculosis is taken up by MN and MØ through binding to complement and/or mannose receptors (11,12) and by DC through binding to a C-type lectin DC-specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) (13–17). In their role as APC, MN, MØ, and DC process mycobacterial antigens and present them bound to the major histocompatibility complex (MHC) class I or II molecules or to T-cells, primarily CD4 T-cells and CD8 T-cells, or through unconventional [CD1b, CD1c, CD1d (18–22)] or undefined receptors to double-negative (Valpha24þCD4
CD8
) T-cells and natural killer T (NKT) cells or to gamma delta (cd) T-cells, respectively. The relative contribution of the various T-cell or natural killer (NK) populations to the control of M. tuberculosis infection in humans may vary by infection site, involved organ, and natural history of the M. tuberculosis infection. Clearly, T-cells play a central role in host defense and mucosal immunity against M. tuberculosis. The balance of receptor expression, receptor signaling, and cytokine production by APC (MØ, MN and DC) determines

Figure 2 Active immune surveillance by Mycobacterium tuberculosis–specific CD4 and CD8 T cells and the presence of interferon gamma and tumor necrosis factor alpha are required to control latent M. tuberculosis infection.

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whether or not the T-cell will differentiate along the TH1 or TH2 pathway. Activated CD4þ T-cells can differentiate into TH1 or TH2 cells that secrete specific subsets of cytokines. The TH1 cell product of greatest relevance to M. tuberculosis immunity is IFN-c, the predominant activator of MØ and MN (23,24), whereas TH2 cells principally produce IL-4, IL-5, and IL-13. IL-10 from TH2 cells, naturally occurring CD4þ T regulatory cells (Tregs), CD8 T-cells, MØ and DC, and transforming growth factor beta (TGF-b) (from TH3 cells) provide immune regulation via inhibition of both TH1 and TH2 responses. Following M. tuberculosis infection, effector T-cells are stimulated. M. tuberculosis antigen–specific memory T-cells are subsequently generated. These memory T-cells can be distinguished into T effector memory (TEM) and T central memory (TCM) cells. TEM cells are found predominantly in peripheral tissue and at sites of inflammation where they exhibit rapid effector function. TCM cells reside predominantly in lymphoid organs and cannot be immediately activated. Upon exposure to M. tuberculosis, reinfection or vaccination TCM are thought to rapidly expand and differentiate to resupply the effector T-cell pool at peripheral sites. Specific surface markers distinguish the two memory cell types (TEM: CCR7lo, CD62Llo, CD69hi; TCM: CCR7hi, CD62Lhi, CD69lo, CD27hi) (25–27). IFN-c regulates a host of genes that are involved in direct bactericidal effects against M. tuberculosis, and antigen processing and generation of reactive nitrogen intermediates (RNI). The evidence for a central role of IFN-c in M. tuberculosis immunity derived from IFN-c knockout mice, which fail to produce RNI, and succumb rapidly to the experimental infection with M. tuberculosis (28,29). In children with hereditary IFN-c receptor deficiencies (30,31), there is progression and uncontrolled dissemination of mycobacterial infections. As the role of external addition of recombinant human (rh) IFN-c to the control of mycobacterial growth in in vitro studies remains controversial (32–34) and therapeutic administration of rhIFN-c (in patients with multidrug-resistant TB) resulted, at best, in transitory therapeutic benefits only (35), much remains uncertain about the expression of IFN-c–dependent protective immunity in humans. Monocytes and Macrophages

Nitric oxide is the primary product of inducible nitric oxide synthase (iNOS, NOS2 isoform) expressed by MN and MØ and can kill M. tuberculosis at low concentrations. The relevance of nitric oxide in human protective immunity against M. tuberculosis has accumulated slowly. Nitric oxide is released from M. tuberculosis–infected blood MN (36,37), AM (38–41), and respiratory epithelial cells (42,43) and is increased in exhaled air of TB patients (44). iNOS is present in TB lung granulomata and expressed in combination with eNOS in MØ and multinucleated cells in the inflammatory zone and the surrounding lung tissue and is significantly elevated in granulomatous tissue from TB patients relative to control tissue (45). Nitric oxide production as determined by expression of iNOS mRNA can be triggered in human AM

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by bacille Calmette–Gue´rin (BCG) (46) and confers antimycobacterial activity against M. tuberculosis (37,40) and BCG (46). However, there is a large interindividual variability in nitric oxide production from AM following M. tuberculosis infection, which correlates with intracellular growth inhibition of M. tuberculosis by AM (40). Increased generation of nitric oxide by AM (38,39) or MN (36) from TB patients amplifies the synthesis of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-c) (36,38) and IL-1 (38), in an autoregulatory manner. Conversely, the release of RNI in human cells is triggered by IFN-c, TNF-a, and IL-1R (37) and mycobacterial components such as lipoarabinomannan and M. tuberculosis 19 kDa lipoprotein (47). Studies in the murine system also suggest that RNI production may be mediated by the interaction of pathogens with TLR and the downstream cytoplasmic adapter protein myeloid differentiation primary response gene 88 (myD88) (48). MN and MØ [including AM (49)] can undergo apoptosis following intracellular infection with M. tuberculosis. Apoptosis of these phagocytes upon M. tuberculosis infection may present a protective immune response that averts the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies (50,51). More recently, transcriptional responses of MN and MØ to M. tuberculosis infection have been studied using high-density DNA microarrays. These comprehensive assessments of gene expression patterns provide the possibility of elucidating the complex cross talk between M. tuberculosis and host cells and to identify the genes involved in host pathogen responses that so far remained elusive. Initial studies of the MN transscriptome during M. tuberculosis infection indicate that M. tuberculosis interferes with the human MØ activation program (52). MØ infected with M. tuberculosis upregulate many nuclear factor kappa B (NF-jB) members, receptors for chemokines, and ILs and downregulate molecules necessary to respond to the immunosuppressive cytokine TGF-b. Induction of IL-12 also was significantly depressed after infection with M. tuberculosis, which may enhance the survival of M. tuberculosis (52). Dendritic Cells

The differentiation of na€
ve T-cells to effector TH1 and TH2 subtypes is greatly influenced by APC and thus also by DC. The understanding of the role of DC in the immune regulation, production of cytokines, and growth control of M. tuberculosis during M. tuberculosis infection is evolving. DC phagocytose particulate antigens including M. tuberculosis are the most potent APC for the activation of CD4 T-cells and CD8 T-cells in both primary and recall immune responses (53). DC maturation is induced by microbial lipopeptides that stimulate DC via TLR2 (54). DC are present in the respiratory tract (55), constitute 1% of epithelial cells in the airways, and are found peripherally in the human lung as far distally as the alveolar septa (56). Infection of human MN–derived DC with live M. tuberculosis

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induces expression on their surfaces of costimulatory signals (CD54, CD40, and B7.1) as well as of MHC class I molecules (57). Human MN–derived DC infected with M. tuberculosis secrete TNF-a, IL-1, IL-12 (57), IL-6, IL-10, IFN-a (58), and IFN-b (59); however, their capacity to control the growth of M. tuberculosis growth is inferior to that of MØ (60). The presence or function of DC in ex vivo pulmonary material from TB patients or HHC has not been assessed to date. CD4 T-Cells

CD4 T-cells are critical to adaptive CMI in adults (61–64) and children (32). MHC class II–restricted CD4 T-cells are the predominant source of IFN-c and IL-2 and are critical for the induction of delayed type hypersensitivity responses during M. tuberculosis infection and the development and maintenance of CD8 cytotoxic T-lymphocyte (CTL) responses (65). CD4 T-cells are directly involved in inhibiting M. tuberculosis growth in human MN (33). The predominant importance of CD4 T-cells in M. tuberculosis immunity is apparent during HIV-1 infection. The reduction of CD4 T-cell numbers and function confers the greatest known risk for the reactivation of latent and progression of primary M. tuberculosis infection to active TB disease. The increased risk that a clinically latent focus will reactivate during HIV-1 infecion (5–10% per year) indicates that active immunologic surveillance involving CD4 T-cells is required to maintain latency. It further indicates that ‘‘latency’’ is a misnomer because there is a dynamic balance between bacterial replication and host immune response at the infectious site(s). CD8 T-Cells

MHC class I–restricted CD8 T-cells were first shown in the murine model to be components of protective immunity against infection with Mycobacterium bovis BCG (66) and M. tuberculosis (67–69). Evidence is evolving that CD8 T-cells may have a role in human M. tuberculosis immunity (41,70–75) both as effector T-cells and as CTL. Human CD8 T-cells are reactive to several M. tuberculosis antigens including early secretory antigenic target 6, antigen (Ag) 85A, Ag85B, 38 kDa protein, heat shock protein 65, and 19 kDa lipoprotein (76–79). CTL that are primarily CD8 T-cells play an essential role in the lysis of M. tuberculosis–infected target cells (67–70). CD8 CTL lyse M. tuberculosis infected MØ via a Fas-independent granule exocytosis pathway (72,80–82) and via a Fas–FasL interaction (83) that results in the apoptotic death of M. tuberculosis–infected target cells. Alveolar CD4 T-cells and CD8 T-cells confer cytotoxic activity against M. tuberculosis–infected MØ from healthy individuals (71), suggesting that either or both of these cell types that are present in the alveoli during active TB (84) could act as CTL in the lung. Cytotoxic products of CTL, granulysin, perforin, and granzymes, are the effector molecules released by CD8 and CD4 CTL upon interaction with M. tuberculosis–infected target cells (22,72). Human peripheral CD8 effector T-cells also contribute to the production of IFN-c (41,85,86) and TNF-a upon stimulation with M. tuberculosis (41,86).

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The overlap in function of T-cell populations suggests that events that impact on their presence and differentiation at local sites of bacterial replication may determine their specific role in acquired resistance. Natural Killer Cells

Unconventional cells are of increasing interest in human M. tuberculosis immunity because of their potential roles in local innate immunity and the induction of adaptive immune responses. NK cells coregulate human CD8 T-cell effector function against M. tuberculosis (lysis of M. tuberculosis– infected target cells), which depends on the release of IFN-c by NK cells and of IL-15 and IL-18 by MN (87). Direct NK-mediated killing of M. tuberculosis in vitro occurs early (within 24 hours) and requires direct cell-to-cell contact (88). NK cells are activated by MN-derived immature DC in the presence of M. tuberculosis or IFN-c and reciprocally enhance DC maturation and IL-12 production (89). Human NKT cells are a unique subset of T-cells, are non–MHCrestricted, and recognize the nonclassical APC molecule, CD1d, which is expressed on human MN–derived cells (90). NKT cells secrete IFN-c and IL-13 and have bactericidal effects that appear to be mediated by granulysin (22,90). Gamma/Delta T-Cells (c/dT-Cells)

Human T-cells with a cd rather than the usual ab T-cell receptor are called cdT-cells and account for less than 10% of T-cells in blood and in the lung and respond to stimulation with unusual, nonpeptide phosphoantigens from M. tuberculosis (91,92). Non–peptide-reactive cd T-cells are only found in humans. cd T-cells proliferate in response to whole M. tuberculosis, are unrestricted by MHC class I or II molecules (93), are competent cytotoxic effector cells, and recognize the antigen that is presented by both AM (94) and MØ (91). cd T-cells contribute to M. tuberculosis immunity by producing IFN-c, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-3, and TNF-a, and thus by activating MØ to eliminate M. tuberculosis (92,95). A subgroup of human cd T-cells that is activated by M. tuberculosis and recognizes mycobacterial nonpeptide phosphoantigens is Vc9Vd2þ (91,92). Proportions of Vc9Vd2þ cells are decreased in blood and lung cells from TB patients (96), probably as a consequence of M. tuberculosis– triggered apoptotic death (97). BCG vaccination expands and activates this cell population to provide helper functions for M. tuberculosis–specific CD4 T-cells and CD8 T-cells (98). These findings suggest a role for cd T-cells in the protective immune response to M. tuberculosis infection and one that may be expressed in the respiratory epithelium. Regulatory T-Cells

Several control and regulatory immune mechanisms prevent and minimize tissue damage from autoreactive and overexuberant immune responses to pathogens (99,100). Naturally occurring Tregs have recently been shown to be crucial elements of the regulation of immune responses by suppressing

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self-reactive T-cells and preventing ‘‘collateral’’ damage from pathogendirected protective immune responses by antigen-reactive T-cells (99). The suppressive function of Tregs depends critically on cell–cell interaction (101) and can involve the action of IL-10 (Tr1 cells), cytotoxic T-lymphocyte– associated protein (CTLA-4), or TGF-b (TH3 cells) (100). Major Tregs subsets are naturally occurring CD4 T-cells that express high levels of the IL-2 receptor alpha chain CD25 (CD4þCD25þ Tregs) and produce IL-10 and TGF-b. Murine Tregs express TLR (102) and can be activated in vitro by lipopolysaccharide (LPS), suggesting that Tregs can be stimulated by components of M. tuberculosis. Increased IL-10 levels have been detected in patients with advanced TB. Patients with TB, who lack TST reactivity to purified protein derivative (PPD) and often have a poor clinical outcome, are characterized by IL-10 production (not IFN-c production) from constitutively IL-10–producing T-cells (103). IL-10–producing Tregs inhibit T-cell responses to M. tuberculosis antigens (103,104). Reciprocally, neutralizing antibodies to IL-10 in in vitro–stimulated peripheral blood mononuclear cells (PBMC) from HIV-1–infected and HIV-1–negative TB patients were shown to increase IFN-c production by enhancing IL-12 production from MN (105). IL-10–producing Tregs, thus, may play a major role in limiting human immune responses to M. tuberculosis. High-level expression of CD25 and expression of the DNA-binding transcription factor Foxp3 (a master control gene for Tregs development) are functional markers for the identification of natural CD4þ Tregs. However, as stable phenotypic markers for unequivocal identification of human Tregs are still unidentified, the exact role of Tregs in human immunity to M. tuberculosis is evolving. Tregs may turn out to be of major importance in human immunity against M. tuberculosis as the balance between effector T-cells and Tregs at sites of infection may determine the pathogen survival in the presence of protective host immunity. Suppression of immune responses by enhancing Tregs function or boosting of immunity by suppressing Tregs function may become a future immunomodulating means of great therapeutic promise (101,106). B. Humoral Immunity

The prevailing view has been that humoral immunity has little or no impact on the course of M. tuberculosis infection. This may not be accurate. There is evidence in the murine model that arabinomannan-specific antibodies alter the course of (M. tuberculosis) infection and increase survival (107). Further, the presence of antibodies against M. tuberculosis liparabinomannan (LAM) is associated with resistance to dissemination of disease in children (108). Antibodies also may play a pathogenic role during adult TB by enhancing proinflammatory and by blocking downregulatory cytokines. PPD-specific immunoglobulin (Ig) G1 antibodies augment secretion of TNF-a (109,110) and IL-6 and IL-10 (110) by PPD-stimulated MN from patients with TB. Absorption of IgG1 removed the augmenting activity for TNF-a and IL-6 and increased, rather than decreased, IL-10 secretion. The role

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of antibodies during the initial encounter between M. tuberculosis and host cells in the alveolar spaces, prevention of M. tuberculosis dissemination, and induction of anti–M. tuberculosis antibodies following vaccination are important areas of active research (111). C. Systemic Immunity in Tuberculosis

MN of TB patients (112) depress PPD-stimulated lymphocyte transformation (112–114) and production of TH1-type cytokines IL-2 and IFN-c (115–117) selectively. This, in part, accounts for the decreased responsiveness of blood T-cells during TB (112). TGF-b and IL-10 are important MN and MØ, as well as T-cell–derived mediators of cytokine-mediated suppression (116,118,119). In vitro depletion of adherent cells (120) and the use of neutralizing anti– TGF-b antibodies (116) normalize lymphocyte proliferation in response to PPD and significantly increase PPD-stimulated production of IFN-c in TB patients. IL-10 inhibition has a similar effect on IFN-c production. The recent description of Tregs that may produce IL-10 or TGF-b requires reexamination of the cellular source of these cytokines in active TB. Blood MN from patients with active TB are activated as determined by the release of cytokines (112,114) and expression of markers of activation such as Fcc receptor type I and III and human leukocyte antigen (HLA)-DR on their cell surface (121). The antigen specificity of suppression in TB is due to the fact that blood MN primed in situ are restimulated in vitro with M. tuberculosis constituents to overproduce immunosuppressive cytokines. For example, M. tuberculosis cell wall lipoglycans and culture filtrate proteins directly stimulate MN to produce cytokines including TNF-a (122) and TGF-b (123). MN-dependent T-cell suppression is associated with negative TST responses in patients with TB. Apoptosis (124) of M. tuberculosis antigen–specific T-cells and compartmentalization of antigen-specific T-cells to the sites of inflammation (typically the alveolar and pleural spaces) contribute to decreased immune responses in the peripheral blood. In longitudinal studies of immune responses during and following antituberculous therapy, TGF-b and IL-10 responses normalize by three months, whereas IFN-c production remained depressed for at least 12 months. The protracted primary T-cell defect in IFN-c production may be due to apoptosis that occurs in the course of active disease (119). The uncontrolled immune activation in the course of active TB is associated with high levels of cytokines in plasma, sputum, bronchoalveolar lavage (BAL), and pleural fluid. These inflammatory markers disappear rapidly during the course of treatment. They may well contribute to programmed cell death and the protracted primary defect in T-cell function in pulmonary TB. D. Lung Immunity in Tuberculosis

Studies of local lung immunity at the entry site of aerosolized M. tuberculosis provide a window into the fundamental biologic interactions at the interface of M. tuberculosis with the human host. The interplay of activating

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and suppressing immune mechanisms determines whether M. tuberculosis infection remains in latency and confinement or whether TB disease will develop. The epidemiological evidence of resistance to M. tuberculosis infection despite aerogenic exposure suggests that the study of local innate and adaptive immune mechanisms against M. tuberculosis may reveal correlates of human protective immunity. The Granulomatous Tissue Response

Granulomatous tissue reactions are the pathologic hallmark of TB. By immunohistochemistry, TB granulomas comprise MN, MØ, and T-cells. The latter mostly are CD4 T-cells and CD8 T-cells. Granuloma formation in human TB is associated with the expression of characteristic cytokine profiles. By reverse transcriptase PCR (RT-PCR) from biopsy material of granulomatous TB lymph nodes, IFN-c, IL-12 (p40), IL-1b, TNF-c, GMCSF, and lymphotoxin-b (LT-b) production are increased twofold (IL-1b) to 19-fold (IFN-c) compared to control biopsy material from patients with carcinomas or chronic organizing pneumonias (125). mRNA of TH2 cytokines (IL-4, IL-5) is detectable in only one-half of the studied TB granulomas (126). Interestingly, levels of GM-CSF expression are tightly linked to the intensity of the granulomatous response, and GM-CSF is present in epitheloid cells and lymphocytes surrounding granulomatous lesions (125). Levels of TNF-a and LT-b mRNA correlate negatively with the extent of caseous necrosis present in TB lesions (125). In a study of surgically removed lung granulomas from five TB patients, employing in situ hybridization with riboprobes, detectable levels of IFN-c and TNF-a mRNA were found in all and IL-4 mRNA in three of five subjects. Two patients with granulomas exhibited IFN-c but not IL-4 cytokine production, expressed low levels of TNF-a, and presented with more necrotic lesions than did the three TB patients positive for both IFN-c and IL-4. Lung granuloma MØ from TB patients also express TGF-b, which may interfere with antimycobacterial mechanisms and effective granuloma formation (127). Granulomas in TB are simultaneously present in multiple stages of maturity that correlate with different patterns of cytokine mRNA expression. Newer, less mature granulomas exhibit IFN-c and TNF-a but no IL-4; intermediate granulomas exhibit IFN-c, TNF-a, and IL-4 and mature granulomas show greater expression of TNF-a, intermediate amounts of IL-4, and little IFN-c. TNF-a appears to correlate positively with IL-4 gene expression and negatively with the presence of caseous necrosis. Cells stained with the myeloid marker CD68, probably MØ, show mRNA production for IFN-c and IL-4 (128). The latter observation coincides with the finding that AM from patients with TB (129) and in vitro M. tuberculosis–infected AM express IFN-c mRNA (130). The granulomatous tissue reactions depend on the regulation of apoptosis. Initiation of apoptosis requires death-inducing signals such as lack of survival factors, metabolic supplies, binding to death signal–transmitting receptors, etc. (131). Members of the TNF-a superfamilies of TNF-aR

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and TNF-a-ligands are involved in the control of such apoptotic processes during granuloma formation. TNF-a induces apoptosis of M. tuberculosis– infected AM (49,132), favoring formation of caseating granulomas. Pleural Fluid Cells

Tuberculous pleuritis is most common in primary TB and usually resolves spontaneously (133,134). Immune responses occurring in the pleural spaces therefore probably represent protective immunity that controls multiplication of M. tuberculosis. Compartmentalization of PPD-specific immune cells in TB was first described in pleural effusion cells on the basis of increased antigen-specific DNA synthesis (120,135–137) as compared to autologous blood cells. TH1 cytokine responses are increased and CD4 T-cells with a memory phenotype (CD45RA
) accumulate locally (124,138,139) with increased frequencies of cells responding to PPD (120,137). These cells express multiple homing receptors such as CD11a, CC chemokine receptor 5 (CCR5), and CXC chemokine receptor (CXCR) 3 (139). Pleural fluid cells also produce increased concentrations of cytokines in comparison with autologous blood cells. IFN-c (49,140–144) and TNF-a production by pleural fluid cells is increased both constitutively (124,141–143,145) and in response to stimulation with protein–peptidoglycan complex, lipoarabinomannan (138), and M. tuberculosis Erdman (141). The increases in IFN-c production are paralleled by increases in IL-6 (146), free IL-12p40, and heterodimeric IL-12, both constitutively and when stimulated with heat-killed M. tuberculosis (147). Pleural effusion cells from TB patients also showed increased production of IL-10 (141) and of TGF-b (148). IL-10 and TGF-b might play a role in limiting the inflammation in this compartment (149). Levels of IFN-c and the proapoptotic molecules TNF-a, FasL, and Fas are increased in pleural fluid from TB patients relative to plasma (124). Spontaneous apoptosis of CD4 T-cells and non-CD4 T-cells is increased in pleural TB, suggesting that immune activation and loss of antigen-responsive T-cells may occur concomitantly, favoring persistence of M. tuberculosis infection (124). In HIV-1– seronegative pleural TB, vigorous immune responses usually are associated with negative M. tuberculosis cultures. In HIV-1–seropositive pleural TB, M. tuberculosis cultures most often are positive. Therefore, the finding that levels of IFN-c (as well as the proapoptotic molecules and apoptosis) are increased further in HIV-1–seropositive pleural TB is suggestive of a block in the protective response to IFN-c (150). Bronchoalveolar Cells

Bronchoalveolar cells (BAC) obtained by BAL permit the study of localized immunoregulatory functions during TB and in M. tuberculosis–exposed but healthy subjects. BAC provide insights into immunologic compartmentalization and are thought to reflect processes in the granulomatous tissue that is adjacent to the bronchoalveolar spaces. The procedure generally samples approximately one million alveoli, the walls of which contain the granulomas. In health, BAC are composed of 90% to 95% AM and 5% to 10% alveolar lymphocytes (AL), with occasional neutrophils and eosinophils.

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The most prominent finding of BAL studies in TB patients is a compartmentalized pulmonary immune response. Unstimulated total BAC in TB contain increased numbers of cells expressing IFN-c (129,151,152) and IL-12 mRNA (152) but not IL-4 or IL-5 mRNA (129,152) and release increased amounts of IL-1b, IL-6, and TNF-a (153). Spontaneous TNF-a secretion in AM is associated with reduced in vitro M. tuberculosis growth in normal AM (154). BAC in pulmonary TB are also characterized by an alveolitis of ab T-cell receptor–bearing AL that are activated as manifested by HLA-DR and CD69 membrane expression (84). Upon challenge with PPD, these BAC produce a TH1 type cytokine host response (155). Compartmentalization of mycobacterial antigen-specific responses to the lung in TB is due, in part, to recruitment of T-cells from the blood to the lungs, as well as mycobacterial antigen–specific expansion in situ. Recruitment of M. tuberculosis–specific cells to the lung compartments is mediated by chemokines with lymphocyte chemotactic activity such as that conferred by Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES), IL-8, and MN chemoattractant protein-1 (MCP-1). These chemokines, among others, are increased in the BAL fluid of patients with pulmonary TB (156,157). Recent studies in a bronchoscopic challenge model of TST-positive healthy subjects showed induction of IFN-c–inducible protein 10 (IP-10) and monokine induced by IFN-c (Mig) and presence of resident CD45ROþ memory cells after PPD challenge in the lungs. The PPD-specific AL were predominantly CD4þ T-cells that produce IFN-c (158). There is evidence for a role of CTL in CMI against M. tuberculosis in the human lungs. Alveolar and blood CD4 T-cells and CD8 T-cells express M. tuberculosis antigen–specific CTL activity when stimulated by M. tuberculosis–infected AM from healthy TST-positive donors (71). Mycobacterial antigen-pulsed AM are targets of blood CTL activity but are significantly more resistant to cytotoxicity than antigen-pulsed autologous blood MN (71). The findings that both CD4 T-cells and CD8 T-cells are present and increased in number and activated in alveoli during active pulmonary TB (84) suggest that either or both of these cell types could function as antigen-specific CTL in the lung during disease. As mediators of innate and adaptive immune responses, AM contribute to the production of cytokines and of chemokines that facilitate the recruitment of lymphocytes and MN. About 20% of AM are immature and possibly recently recruited from the blood during active TB (84). M. tuberculosis–infected AM release TNF-a, IL-1, IL-6, IL-12, IL-15, IL-18 (153,159–161), GM-CSF, and deactivating cytokines IL-10 and TGF-b (161,162). Expression of IFN-c mRNA from AM of TB patients ex vivo (129) and production of IFN-c from AM upon in vitro infection with M. tuberculosis (130) have also been observed. Upon in vitro infection with M. tuberculosis, AM release RANTES, MCP-1, macrophage inflammatory protein (MIP)-1a, and IL-8 (157,163) in amounts that may vary depending on the virulence of the infecting M. tuberculosis strain (163).

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AM (as well as DC and MN) express the recently described TLR. Qualitative and quantitative differences in the protein expression of these TLR have profound implications on the modulation of both innate and adaptive immunity, e.g., by mediating TNF-a, IL-1, and IL-6 and killing M. tuberculosis (164) and probably RNI production (48). Innate resistance against M. tuberculosis is thought to be critically dependent on the engagement of three of the known 13 TLR on macrophages (TLR2, 4, and 9). TLR2 agonists include a variety of bacterial cell wall components, such as peptidoglycan as well as lipoarabinomannan, which is a major cell wall–associated glycolipid derived from M. tuberculosis. Arabinose-capped lipoarabinomannan, purified from rapidly growing mycobacteria, induces TNF-a production in macrophages in a TLR2-dependent manner (165,166). TLR4 agonists include gram-negative bacterial LPS, and unmethylated CpGcontaining mycobacterial DNA is a TLR9 agonist (165). Expression of TLR1–5 is present in almost all granulomas from TB patients. All TLR can utilize myD88 to propagate signals to target genes and generate rapid protective responses. Granulomas expressing IL-4 show low expression of TLR2 (126). In summary, the study of localized immunity has revealed prominent local TH1 responses that are characterized by enhanced DNA synthesis, activation of lung cells, large numbers of IFN-c–producing M. tuberculosis antigen–specific cells, and increased IL-12 and IL-18 production. This pattern of immunity that is considered to be protective is, however, observed consistently in affected TB lungs despite uncontrolled progression of M. tuberculosis infection. The final answers to this conundrum in M. tuberculosis immunity need to be found and may involve complex interactions between host cell responses and M. tuberculosis–induced immune evasion mechanisms. Probably, protective TH1 responses overlay synchronous deactivation events in the lungs or upregulated immune-mediated processes including apoptosis may facilitate extracellular bacterial replication. However, the evolving understanding of local and mucosal immunity and its initial interactions with inhaled M. tuberculosis suggest that research to maximize vaccine-based prevention of aerogenic M. tuberculosis infection needs to take into consideration the compartmentalization of immune responses and elicitation of local protective immunity. HIV-1 and Mycobacterium tuberculosis Infection in the Lung

HIV-1 replication is greatly enhanced at anatomical sites of active M. tuberculosis coinfection (167). The compartmentalization of antigenspecific cells at sites of infection in the local milieu of proinflammatory cytokines creates an environment that is conducive to HIV-1 replication with enhanced viral burden and conditions favoring dissemination of M. tuberculosis infection. The failure of effective immune response that permits development of opportunistic infections may in addition be due to continued exposure of immunoreactive cells to M. tuberculosis at sites of infection, with subsequent apoptosis of CD4 T-cells and non-CD4

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T-cells. This initiates a vicious cycle of simultaneous immune activation and loss of antigen-responsive T-cells, which permits persistence of M. tuberculosis infection (168). In BAC, cellular and immunocytological characteristics in HIV-1– coinfected TB patients compared with healthy controls portray an inflammatory cell profile with reduced CD4 T-cell (151) and increased CD8 T-cell numbers in affected lungs (151,169), increased AM and neutrophil numbers (169), and decreased constitutive IFN-c production (151). These findings suggest reduced enrichment and activation of immune cells in the lungs. In pleural TB, HIV-1 replication is enhanced both in the cellular (pleural compared with blood mononuclear cells) and acellular (pleural fluid compared with plasma) compartments of the pleural space (167,170). These observations are explained by augmented local TNF-a and HIV1–noninhibitory b-chemokine (MCP-1) concentrations, and low levels of HIV-1 inhibitory b-chemokines (MIP-1a), MIP-1b, and RANTES, with upregulation of the HIV-1 coreceptor, CCR5, by pleural fluid mononuclear cells (170). Source cells of HIV-1 in pleural fluid are, in part, HLA-DRþ cells, CD26þ lymphocytes, and CD14þ MØ (167). AM from HIV-infected subjects with CD4 T-cell counts lower than 200/mL and detectable HIV load produce HIV-1 (p24) upon stimulation with PPD and M. tuberculosis. This indicates that M. tuberculosis and its PPD can induce HIV replication in latently infected AM (171). AM in TB patients and upon in vitro M. tuberculosis infection express CXCR4, producing a permissive environment for replication of HIV-1 using CXCR4. Because progression to acquired immunodeficiency syndrome (AIDS) is associated with a shift in viral coreceptor use from CCR5 to CXCR4, these recent findings form a molecular basis for the observed acceleration of the course of HIV-1 infection to AIDS in TB (172). Intracellular M. tuberculosis growth is reduced in AM from HIV-1– infected subjects with CD4 T-cell counts greater than 200/mL compared with AM from healthy controls. As binding and internalization of M. tuberculosis are augmented in AM from HIV-1–infected subjects compared with healthy controls, this reduced intracellular M. tuberculosis growth is not due to impaired phagocytosis or a defect in innate immunity of AM (154). The Alveolar Epithelial Barrier

Epithelial cells and their products such as defensins and chemokines are important components of antibacterial mucosal innate immunity that are difficult to study in humans due to their inaccessibility. Because type II alveolar epithelial cells cover 5% to 8% of the alveolar surface, early interaction between M. tuberculosis and epithelial cells after entry of infectious microdroplets into the alveolar spaces is plausible. The mechanisms by which M. tuberculosis crosses the alveolar wall to establish infection in the human lung tissue or gain access to the lymphatic

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and circulatory system are not well defined. M. tuberculosis laboratory strains H37Ra (avirulent) and H37Rv (virulent) invade the human type II alveolar epithelial cell line (A549) in a microfilament- and microtubuledependent manner using antivitronectin receptor (CD51) and b1 integrin (CD29) as receptors (173) in vitro. Within A549 cells, H37Ra and H37Rv can replicate and survive (173–175). We have recently shown that infection of A549 cells with H37Ra induces production of human beta defensin 2 (176), a peptide with antimicrobial and chemokine activities. Earlier in vitro studies had provided evidence that M. tuberculosis (not BCG) can cross epithelial-endothelial barriers and that this passage triggers the release of IL-8 and MCP-1 (177) from both epithelial and endothelial cells. This data indicates that respiratory epithelium may create a gradient for infected mononuclear cells to migrate across the alveolar barrier following exposure to M. tuberculosis. Alveolar and other respiratory epithelial cells, thus, may be involved in initiating and linking innate and adaptive antimycobacterial immune responses. IV. Susceptibility to Mycobacterium tuberculosis Infection and Tuberculosis Development A. The Impact of HIV-1

Active immune surveillance is required to maintain the latency of quiescent M. tuberculosis foci. Concurrent HIV-1 infection profoundly alters the natural history of TB and increases the risk of active progressive primary TB and of reactivation TB (63,178). In nosocomial outbreaks, 30% to 40% of HIV-1–infected individuals develop TB within one to two months after diagnosis of the source TB index case and TB development is associated with skin test anergy and CD4 T-cell lymphopenia (63,179). The risk of developing reactivation TB among HIV-infected persons is 79-fold greater than in HIV-noninfected persons (178) and may be up to 170-fold higher in patients with AIDS (180). Risk of death from TB is increased in HIV-1–infected persons (181). In immunocompetent subjects, TB pathology is restricted to the lungs (85%) and rarely extrapulmonary or both pulmonary and extrapulmonary (15%) (182). With HIV-1 coinfection, TB is 38% pulmonary, 30% extrapulmonary, and a combination of both pulmonary and extrapulmonary disease in the remainder (183). Development of extrapulmonary TB is associated with compromised granulomatous tissue reactions (182–185) that result from HIV-1–induced alterations of cell-mediated immunity. With advanced immunosuppression, cavitary tissue damage and the number and size of cavitary lesions within the lung are reduced. M. tuberculosis granulomatous host responses are greatly impaired and correlate with the residual degree of immunocompetence and peripheral CD4 T-cell depletion. In early stages of HIV-1 infection, histological features of the TB granuloma are abundant epitheloid MØ, Langhans giant cells, and a peripheral rim of CD4 T-cells. M. tuberculosis numbers are low. With moderate immunodeficiency, Langhans giant cells

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are missing. Epitheloid differentiation and activation of MØ are absent. CD4 T-cells are depleted and M. tuberculosis numbers increased. Lastly, with advanced HIV-1–related immunosuppression and AIDS, granuloma formation is rare and there is little cellular recruitment and very few CD4 T-cells can be found. M. tuberculosis numbers are high (64). HIV-1 infection associated with immunosuppression leads to lowered resistance to exogenous reinfection (186). Perturbations in the cytokine expression, that is a reduced TH1 response, have been suggested as contributing to the susceptibility of HIVinfected patients to TB. Upon stimulation with M. tuberculosis in vitro, PBMC from HIV-infected TB patients show reduced proliferative and type 1 responses that are a direct result of CD4 T-cell depletion and related to IL10 production (187). Similarly, low CD4 cell counts and low IFN-c production are correlated with impaired ability to regulate growth of BCG in whole blood assays of HIV-1–infected children (32). TNF-a production is increased, suggesting that HIV-1–associated TB is accompanied by immune activation that triggers increased HIV-1 expression and accelerated progression to AIDS (188). In line with the observed clinical synergism between the two infections, blood MN from TB patients are highly permissive in vitro to productive infection with HIV-1 (189). M. tuberculosis and PPD increase in vitro HIV-1 replication in MN via transcriptional activation (190) and activation of NF-jB (191). Conversely, growth of M. tuberculosis is increased in HIV-1–infected human MN–derived MØ (192). In vivo viral load in HIV-1– and M. tuberculosis–coinfected persons is increased compared with single HIV-1–infected persons at CD4 T-cell counts greater than 500/mL but not at CD4 T-cell counts lower than 500/mL, suggesting that TB, as an early HIV-1 opportunistic infection, increases early viral replication and dissemination and progression of HIV-1 disease (193). IFN-c production is preserved in HIV-1–infected TB patients with CD4 T-cell counts of 200/mL to 500/mL and is low in patients with CD4 T-cell counts of 500/mL or more and in HIV-1–uninfected patients. TNFa levels are similar, regardless of the CD4 T-cell numbers (194). Interestingly, among TST-positive HIV-1–infected subjects, TB incidence is high and the development of TB is associated with an IL-10 response to PPD or with positive IL-5 responses when a BCG scar is present (195). Recent data in M. tuberculosis–stimulated PBMC from TST-nonreactive donors showed that IL-10 significantly decreases replication of T-cell–tropic HIV-1 isolates in M. tuberculosis recall antigen–stimulated cells (196). These effects likely result from the inhibition of TNF-a, which enhances HIV-1 replication. This and data showing increased numbers of IL-10– producing CD4 T-cells in TST-nonreactive TB patients (103) indicate that HIV-1 replication in M. tuberculosis–coinfected individuals is coregulated by Tregs. B. Immune Reconstitution Inflammatory Syndrome

Reappearance or paradoxical worsening of previous TB manifestations or appearance of new manifestations (fever, lymph node enlargement, cough,

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pulmonary infiltrates, etc.) despite administration of effective anti-TB therapy have been reported in HIV-1–infected patients after the initiation of antiretroviral therapy (ART) (78,79,197,198). Immune reconstitution inflammatory syndrome (IRIS) is a frequent and sometimes severe event that occurs in 30% to 45% of patients after initiation of ART (198). Exaggerated immune inflammatory responses have been observed upon reconstitution of immunity, specifically in individuals with strong increases in CD4 T-cell percentage and ratio of CD4 T-cells to CD8 T-cells, as well as TST conversion. Individuals starting treatment for disseminated TB and HIV-1 coinfection quickly increase frequencies of IFN-c–producing peripheral M. tuberculosis–specific blood CD4 T-cells (199). Further identification of immunological determinants of IRIS development could aid the diagnosis of IRIS and lead to preventive strategies. The development of IRIS in the context of ART may need to be considered as a relevant public health problem, because its incidence may sharply rise with wider availability of ART in TB-endemic countries with high rates of HIV-1 infection. C. Inhibitors of Tumor Necrosis Factor

TNF-a is essential for granuloma formation and maintenance. The importance and complexity of the role of TNF-a as a regulator of local antimycobacterial host defense has been clearly demonstrated in murine studies using anti–TNF-a antibodies and TNF-a receptor gene–disrupted or transgenic mice. These interventions result (i) in a decrease in number of granulomas and their delayed formation, (ii) decreased containment and elimination of infecting mycobacteria (BCG) (200), (iii) accelerated lethal course of M. tuberculosis infection, (iv) delay in iNOS production (201), and (v) widespread pulmonary inflammatory infiltrates, necrosis, and dysregulation of pulmonary cytokine and chemokine production (202). With the increased human use of TNF-a inhibitors to treat inflammatory conditions such as rheumatoid arthritis and Crohn’s disease (203), reports of simultaneous occurrence of granulomatous infectious diseases have increased in the past five years. Patients receiving TNF-a inhibitors have been shown to be at increased risk of TB reactivation disease (203–207). In a large retrospective study of the Food and Drug Administration adverse events reporting system, TB incidence rates were reported for between 35 and 144/100,000 treated patients (206). Currently under investigation are the relative risk of therapy with monoclonal anti–TNF-a antibodies and with TNF-a receptor antagonists and in vitro mechanisms of their interference with protective host immune mechanisms. D. Other Immunocompromising Conditions

Diabetes mellitus, chronic renal failure, carcinoma of head or neck, and iatrogenic immunosuppression, increase the relative risk for reactivation of latent M. tuberculosis infection and development of TB markedly by 2-, 3.6-, 10-, 15-, 16-, and 11.9-fold, respectively, relative to healthy individuals

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with no known risk factors (208). Persons with silicosis, end-stage renal disease, poorly controlled diabetes mellitus, chronic malnutrition, rapid weight loss, chronic treatment with corticosteroids and other immunosuppressive drugs, and tobacco smoking are also at increased risk of developing TB, although the relative risk is much less than that of HIV-infected persons. The duration and dose of corticosteroid use associated with an increased risk of TB are unknown; however, treatment for less than three to four weeks with doses of 15 mg of prednisone or less daily probably causes little increased risk.

E. T Helper 2 Cytokines and Helminth Infection

Whereas TH1 cytokines are considered protective in human M. tuberculosis infection, dominant TH2 cytokine immune profile and immune perturbation by chronic infections contribute to the development and alter the extent of human TB (209,210). Long-term control of M. tuberculosis infection is associated not just with elevated TH1 responses but also with inhibition of the TH2 response. Individuals progressing to TB tend to have decreased levels of TH1 cytokines and increased levels of IL-10 compared with healthy M. tuberculosis–infected and –uninfected community controls, whereas healthy M. tuberculosis–infected subjects have increased IL-4 antagonist and IL-4d2 message, compared with both TB patients or uninfected individuals (211,212). In a recent study of M. tuberculosis–exposed health-care workers, increased median percentages of IL-4–producing CD8 and cd T-cells were associated with progression to active TB. Individuals who remained healthy showed increased percentages of IFN-c–producing CD8 and cd cells and lower percentages of IL-4–producing CD8 and cd T-cells (197). PBMC from healthy donors respond to sonicated M. tuberculosis antigens with increased IL-4 gene activation, CD30 expression, and apoptosis, probably by sensitizing lymphocytes to TNF-a–mediated apoptosis. Interestingly, these changes are significantly greater than those observed when cells were stimulated with antigens from nonpathogenic Mycobacterium vaccae (210). IL-4 also modulates expression of TLR (126), thus interfering with the intracellular killing of M. tuberculosis and induction of TLR signaling–dependent immune mechanisms. TB without TST reactivity to PPD has been described in individuals with predominant TH2 type cytokine response patterns and occurs in geographic areas with high rates of Helminth infections. In individuals with Helminth infections, PPD-specific in vitro cellular proliferative responses are decreased and expression of costimulatory signaling molecules (CTLA-4) altered (213). Conversely, upon anthelmintic treatment, in vitro PPD-specific cell proliferation and IFN-c production, TST reactivity, and post-BCG vaccination PPD-specific immune responses are significantly increased (214). Lowered resistance to mycobacterial infections or failure of protection from anti-TB vaccines (214) may require special public health measures in Helminth-endemic areas.

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F. Mycobacterium tuberculosis Host Immune Evasion Mechanisms

The recent years have provided evidence that M. tuberculosis exploits and circumvents host immunoregulatory mechanisms to promote chronic infection, persistence, and the opportunity for transmission (215,216). For example, M. tuberculosis lipids arrest phagosome maturation and target host cell membrane–trafficking processes and organelle biogenesis (217), allowing escape from lysosomal bactericidal mechanisms and preventing efficient Ag presentation in phagocytic host cells. M. tuberculosis also interferes with the induction of IFN-c–regulated genes and the stimulation of MØ to kill M. tuberculosis (218). This has been shown in the human acute monocytic leukomia cell line named THP-1, in which induction of CD64 (FccR1) surface expression and transcription are impaired following M. tuberculosis infection despite normal activation of Signal Transducer and Activator of Transcription 1 (STAT1). An additional mechanism of M. tuberculosis interference with IFN-c–regulated genes has recently been described in human MN–derived MØ (219). IL-6 from M. tuberculosis–infected MØ was shown to decrease the transcription of class II transactivator and expression of IFN-c–induced MHC class II that affected directly the infected MØ and via a bystander effect also the uninfected MØ. The inflammatory cytokine microenvironment at the site of infection, where IL-6 is produced, may thus interfere with important protective IFN-c effects (219). In a murine model, M. tuberculosis 19-kDa lipoprotein and an M. tuberculosis cell wall peptidoglycan inhibit via a TLR2 and myD88–dependent mechanism macrophage responses to IFN-c at a transcriptional level and IFN-c activation of murine MØ to kill virulent M. tuberculosis (220). The 19-kDa lipoprotein also inhibits IFN-c–regulated expression of MØ HLA-DR protein and mRNA and of FccRI in a TLR2-dependent manner and thus Ag processing and presentation of soluble protein antigens to MHC-II–restricted CD4 T-cells (221). Another immunosuppressive mechanism of M. tuberculosis appears to be its inhibition of the phenotypic and functional maturation of human MN–derived DC (222). M. tuberculosis targets a DC-specific C-type lectin (DC-SIGN) (13,14) that is an important receptor of mycobacteria and of HIV-1 through the mycobacterial cell wall component manLAM, thus preventing mycobacteria- or LPS-induced DC maturation (14,223). This array of M. tuberculosis–induced alterations of host cell function and immune responses provides additional clues as to the lack of M. tuberculosis control and the unhindered M. tuberculosis infection at local sites of infection despite presence of protective immune mechanisms such as high IFN-c levels. G. The Impact of Mycobacterium tuberculosis Isolates on Human Immunity

There is increasing evidence that heterogeneity between M. tuberculosis isolates and variably expressed bacterial factors modify host CMI and epidemiological and clinical features. Strains of M. tuberculosis differ in their virulence and properties to induce host cytokines that modify the

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granulomatous tissue reaction as well as TST. For example, differences in M. tuberculosis phenotypes (H37Rv, CDC1551) result in differences in TST positivity in humans (224) and in TNF-a, IL-6, IL-10, and IL-12 production levels by human MN and of TNF-a in AM (225). Although not yet demonstrated in the human model, host immunomodulatory effects of an isolate of a subset of the W-Beijing family of M. tuberculosis (HN878) with hyperlethality in the murine model are correlated with a biologically active polyketide synthase–derived phenolic glycolipid (PGL) (226). Disruption of the synthesis of PGL in the HN878 strain was associated with an increase of TNF-a, IL-6, and IL-12 release from murine bone marrow MØ in vitro. Further, studies with H37Ra and H37Rv indicate that virulence of the infecting strain alters the release of chemokines such as RANTES, MCP-1, MIP-1a, and IL-8 (163), which affect recruitment of inflammatory cells capable of controlling infection and are involved directly in M. tuberculosis growth control. Taken together, these and other findings suggest that individual M. tuberculosis components confer immunomodulatory effects on host immune cells. M. tuberculosis manipulates its environment by interfering with the host immune response on multiple levels. This redundancy of interferences suggests their importance for the survival of M. tuberculosis during its coevolution with the human host. V. Resistance to Mycobacterium tuberculosis Infection A. Household Contact Studies

More than half of intensely exposed household contacts fail to manifest M. tuberculosis infection despite continued aerogenic M. tuberculosis exposure, presumably due to innate resistance. The initial interaction of M. tuberculosis with the alveolar environment controls growth of M. tuberculosis efficiently, because 50% to 75% of M. tuberculosis–exposed HHC of TB patients do not acquire M. tuberculosis infection as determined by TST (7,227). Because immunity in HHC reflects early immune responses that can be associated with protection and efficient M. tuberculosis growth control, study of HHC may permit identification of correlates of human protective immunity. Household contact studies offer the best possible approach to study human-to-human M. tuberculosis transmission and concurrent protective host immunity to M. tuberculosis under conditions of known exposure (228). Studies from our group showed increased immune responses against secreted M. tuberculosis Ag85 in PBMC from HHC (229) and increased frequencies of M. tuberculosis Ag85–specific IFN-c–producing BAC in HHC compared with healthy community controls (230). This presence of M. tuberculosis Ag85–reactive T-cells in BAC suggested induction of protective local memory immunity during concurrent M. tuberculosis exposure and probably containment of M. tuberculosis infection in the alveolar environment. This hypothesis was inferred from murine (231–233) and guinea

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pig (234) vaccine studies in which M. tuberculosis Ag85 conferred protection against M. tuberculosis. Recent data from our group show that autologous blood CD8 T-cells from concurrently aerogenically M. tuberculosis–exposed HHC, but not from unexposed healthy community controls, contribute to growth control of M. tuberculosis in AM (41). The findings also indicate that M. tuberculosis–specific effector CD8 T-cells expand in vivo in individuals who are exposed aerogenically to M. tuberculosis (41). Interestingly, there was a positive correlation between TNF-a—but not IFN-c or nitric oxide levels—and the growth-controlling activity of the AM/CD8 cocultures (41). Quantification of exposure to M. tuberculosis and respiratory uptake of M. tuberculosis in humans cannot be studied directly, making human resistance to M. tuberculosis difficult to study. Because the search for correlates of protective immunity is critical for the evaluation of rationally designed new antituberculous vaccines, new approaches to quantifying human M. tuberculosis exposure are under development (235). B. Host Immunogenetics

Genetic variation influences immune responses and may contribute to differential development of TB (236,237). There is evidence suggesting that innate resistance or susceptibility to TB disease or M. tuberculosis infection may be genetically determined. Understanding the essential host genes and alleles associated with susceptibility or resistance allows gaining insights into effector mechanisms of protection. Resistance to TB disease and M. tuberculosis infection is polygenic in nature. Associations with the development of mycobacterial infections and TB disease in population-based studies have been described for polymorphisms in multiple genes. A selection of these genes is listed subsequently: HLA (238–242), IFN-c gene (243,244), Natural Resistance Associated Macrophage Protein (NRAMP) (245–249), TGF-b and IL-10 (250,251), mannose-binding protein (252,253), IFN-c receptor (254), TLR2 (255,256), vitamin D receptor (257–259), and IL-1 (260,261). The identification of patients with mutations in single receptor genes that are involved in IL-12 and IFN-c binding and signaling provided evidence for the importance of TH1 cytokines in human resistance to mycobacteria. Defects in the IL-12R (262–264) and IL-12Rb1 (265,266) and complete or partial defects in the IFN-cR or IFN-cR1 genes (30,266–270) have been found worldwide in individuals with disseminated infections caused primarily by BCG (following vaccination) or nontuberculous poorly virulent mycobacteria. Studies with blood cells from such individuals often show an inability or reduced in vitro production of IFN-c (271) or TNF-a (269) upon stimulation. Interestingly, infections with M. tuberculosis or TB disease in association with the IL-12 or IFN-c receptor defects have rarely been reported (265,272,273). However, as the relative contribution of genes involved in resistance or susceptibilty to disease depends on the genetic background of the studied populations and the stage of infection, much remains to be understood.

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