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Foreword
I am honoured to be asked to write the Foreword for this comprehensive textbook. It is a compilation of the writings of more than 100 distinguished TB experts from all over the world. They cover almost every aspect of the disease. I am impressed at the concerted effort being made by scientists and health staff working at different levels, through programmes and in hospitals and clinics, to save lives and prevent morbidity. I applaud and commend them for their efforts to find solutions to curtail the devastating effects of a relentless pandemic. Tuberculosis is a very old disease which we are still unable to contain. Since early biblical times the two main mycobacterial diseases, tuberculosis (consumption) and leprosy have caused great suffering and death. Although leprosy is now largely controlled, TB continues its rampant course throughout the world. Despite the fact that the cause of TB was discovered nearly 120 years ago it continues to kill 1.8 million people every year and is responsible for 1 in 4 preventable deaths. Tuberculosis has escalated to the point where millions of people a year develop the disease and die. This pandemic is further complicated by the additional burden of drug-resistant TB, HIV and poor health services in many countries that experience the highest number of TB infections. Tuberculosis is primarily a disease of poverty—patients are often victims of their circumstances rather than being responsible for the problem themselves. The resurgence of TB reflects the failure of global, political and economic institutions to improve the lives of poor people. This textbook with excellent illustrations and photographs will assist the professional and lay reader to understand TB from a variety of perspectives. It provides the latest updates on progress in tackling the disease such as finding solutions for rapidly diagnosing TB, newer drugs and shorter treatment regimens, and more effective
vaccines. The chapters ominously illustrate the enormity of the problem: nearly two million deaths each year, the emerging problems of drug-resistant TB and the serious hindrances in diagnosing and managing the disease caused by coinfection with HIV. While the overall picture for achieving TB control is still gloomy and advance in TB scientific research is slow, there remains room for optimism. Current data from WHO shows that the number of cases of TB reported last year is levelling out in several countries. This surely can be repeated elsewhere. In the short term, TB is likely to continue killing millions each year in poorly resourced countries; sadly the international donor community’s response has been disappointing. This is a disease that can be overcome and I challenge donors to support the work of thousands of dedicated healthcare workers, volunteers, scientists and doctors who are doing their best at the coalface. I am encouraged that this book contains substantial contributions from authors from Africa, particularly South Africa, where TB is taking its toll. By uniting African authors with their colleagues in Europe, Asia and the USA the editors have demonstrated that the way forward in the fight against TB is to move forward together. A vast amount of knowledge has been generated on the subject of TB in the past decade. This book gives a very helpful overview of this material in a readable form. Its publication is timely. I remind the donor community of the urgent need to work towards controlling TB worldwide. We can do it— together. Archbishop Emeritus Desmond Tutu
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Preface
On March 24, 1882, Dr. Robert Koch announced the discovery of Mycobacterium tuberculosis, the bacterium causing tuberculosis (TB). Koch’s achievement was the first step towards developing tools to control TB. Despite initial success with the discovery of anti-tuberculosis chemotherapy 60 years ago, the tubercle bacillus has been a tenacious human enemy, hard to conquer, and today TB disease still kills 5,000 people daily; that is one person every 20 seconds. More than 80 percent of TB cases arise in only 22 countries, most of which are under-resourced. One-third of the world’s population is infected with M. tuberculosis, and 5 to 10 percent of infected people are likely to develop active TB; for those infected with human immunodeficiency virus (HIV) and very young children, the likelihood of developing and dying from TB is much higher. In 2005, an estimated 1.8 million people died of TB; 195,000 were HIV co-infected. Today, fuelled by the HIV epidemic, TB disproportionately affects young adults in their most productive years. It is pushing medical research to its limits with synergic efforts, interdisciplinary approaches, and translational research using the latest technology. M. tuberculosis and HIV co-infection produces a deadly partnership making both infections more destructive together than alone. This is most apparent in Africa where, according to World Health Organization (WHO), TB cases increase 10 percent annually in the wake of increasing levels of HIV infection and, directly and indirectly, involve increasing numbers of children. TB control programmes worldwide are increasingly faced with multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) unresponsive to treatment that may return control programmes to the pre-antibiotic era, unless care is taken of currently existing drugs and new classes of anti-tuberculosis drugs are developed. The huge mortality caused by the TB epidemic underscores the importance of continuing clinical, basic science and operational research to better understand how M. tuberculosis interacts with the host. Research findings translated into health care interventions could improve the diagnosis, treatment and prevention of TB. This textbook was developed from the understanding that advances in infectious disease research should be translated into applied clinical and management practice using newer guidelines. Over the past decade there has been an explosion of research into all aspects of TB (conventional and molecular epidemiology, organism and host interactions, clinical management schedules for TB and HIV co-infected individuals, new diagnostics, newer therapeutic regimens, newer vaccines, operational and implementation research). The revolution has generated new data that is influencing clinical practice in HIV-infected and uninfected adults and children. Research and funding agendas are better coordinated and serious moves to link funding of TB research and development
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work are emerging between a conglomerate of official and nongovernmental agencies. The WHO STOP TB Partnership has established a ‘research movement’ to assist in operational and implementation research. With so much activity in the TB field and growing knowledge being translated into clinical terms, a need to update the literature exists. Currently there are several TB textbooks available, but they are focused either exclusively on research developments or clinical management guidelines. Furthermore most textbooks focus on adult TB and ignore the growing problem of childhood TB as this was not considered important to public health a decade ago. An enormous amount of new data has now accumulated changing our understanding of transmission dynamics, epidemiology, clinical presentation in HIV-infected adults and children, diagnostics, management and infection control. These data need condensation in a practical clinical textbook incorporating management of the HIVinfected patient with active TB. It is apparent on reading several existing TB textbooks that these have been written by authors with limited experience of clinical practice in developing countries, where the majority of TB cases occur. Existing textbooks lack representation of authors from developing countries, particularly Africa. Currently new information on TB generated from clinical and basic science research is published in scientific and medical journals, not readily accessible health workers in poor countries where the brunt of the TB epidemic is borne. The practical management of TB in these countries is based on guidelines formulated several years ago. Most of the practitioners are too busy to catch up with, or do not have access to, the latest developments in the field. Three years ago an urgent need was recognized for an up-todate, affordable, comprehensive textbook on the clinical management of TB in adults and children, preferably by experts from all over the world, but particularly from TB and HIV/AIDS endemic areas. The challenge was taken up by Professor Alimuddin Zumla (editor of Manson’s Tropical Diseases 21st and 22nd editions; himself a survivor of near fatal TB meningitis as a junior doctor); extensive international work on TB and TB advocacy activities led him to pursue this task with Elsevier Publishing convincing them of the need for a new clinical TB textbook. A strong editorial team was assembled to assist with the challenging task. To balance the book between adult and childhood TB, Professor Zumla (an adult physician) chose Professor Simon Schaaf, a distinguished and very experienced paediatrician from the Department of Paediatrics & Child Health, Stellenbosch University, Cape Town, South Africa, as co-editor. For the book to be truly globally representative, Professors Zumla and Schaaf enlisted six international leading TB authorities as associate editors: Professor John M Grange (UK and Europe), Dr. Mario C Raviglione (WHO, Geneva), Dr. Wing
PREFACE
Wai Yew (Asia), Professor Jeffrey R Starke (USA), Professor Madhukar Pai (Canada), and Professor Peter Donald (Africa). This editorial team invited 158 eminent authors from several continents (53 authors from Africa, 32 from USA and Canada, 56 from Europe and 17 from Asia and Australia) to write 107 chapters and 4 appendices to create ‘Tuberculosis: A Comprehensive Clinical Reference’ – an up-to-date, globally relevant TB treatise. The chapters present state-of-the-art on nearly every aspect of clinical TB relevant to clinical practice bringing readers up to date with scientific developments. Most chapters are amply illustrated with figures, graphs, tables and a diversity of clinical, pathological and radiological pictures. The chapters are clearly written with a healthy overlap between some chapters. A wide range of approaches is presented, reviewing current knowledge and indicating gaps and challenges; focused on practical information, a thorough overview of the latest topics places a strong emphasis on clinical practice. This textbook arises from the world-wide need for a comprehensive clinical text with the latest developments in TB research incorporated into advice on day to day clinical practice. Current TB clinical dogma and changing clinical practice due to the ominous consequences of co-infection with HIV and TB are highlighted and the collective experience of 158 authors ensures a unique and practical approach with global appeal. There is information and knowledge for clinical practitioners from major city hospitals to the rural health worker in the TB clinic. Knowledge is provided in clear practical terms to aid a variety of audiences, from general physicians, pulmonologists, TB and HIV clinic staff, medical assistants, nurse practitioners, TB programme staff, medical students, postgraduates, technical staff, clinician scientists (as a clinical guide for scientists), policy makers, politicians and donor agencies.
The bulk of the book is on clinical presentation, diagnosis and management and principles are demonstrated by system based case histories of adults and children. Tuberculosis-a comprehensive reference contains eleven sections. As an introduction to clinical TB, the first three sections provide a brief history, background updates on new knowledge of epidemiology and transmission dynamics, basic science (immunology, pathogenesis of TB and HIV, development of new TB vaccines and pathology and pathogenesis of TB) and natural history of TB. The main clinical thrust is manifested in sections 4 to 8. In these sections clinical presentation and diagnosis of pulmonary and extrapulmonary TB in adults and children are discussed. Complicated tuberculosis such as HIV/TB co-infection, MDR and XDR-TB and TB in pregnancy is also covered. Wellillustrated chapters on diagnostics and imaging are included. Clinical management (treatment and drug doses) of TB follow on the clinical presentation. Illustrative case histories emphasize day to day problems of clinical practice. Section 9 presents clinical cases of TB in nearly all organ systems and convey the considerable experience of the authors. Section 10 covers Social Science and structural issues in TB and TB/HIV which are always important in the overall study of TB. The epilogue introduces perspective and emphasises that current advances allow room for optimism to achieve control, but not total eradication. Professor Alimuddin I Zumla and Professor H Simon Schaaf Chief Editors February, 2009
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Contributors
Miriam Adhikari MBChB, FCP (Paeds) Professor and Head Paediatrics Department of Paediatrics Nelson R Mandela School of Medicine University of KwaZulu Natal Durban, South Africa
SA, MD (Univ
of Natal)
Vineet Ahuja Department of Gastroenterology All India Institute of Medical Sciences New Delhi, India Savvas Andronikou MBBCh, Radiologist Diagnostic Working Group Medicins Sans Frontiers Amsterdam, The Netherlands
FC Rad (Diag), FRCR (Lond) PhD
Phillip S Barie MD, MBA, FCCM, FACS Professor of Surgery and Public Health Department of Surgery New York-Presbyterian Hospital and Weill Medical College of Cornell University New York, NY, USA DCH, MD(UCT)
David W Beatty MBChB, MD Emeritus Professor of Paediatrics Red Cross Children’s Hospital and the School of Child and Adolescent Health University of Cape Town Rondebosch, South Africa Marcel A Behr MD, MSc Associate Professor of Medicine McGill University Montreal, QC, Canada
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Nulda Beyers MBChB, FCP (SA), MSc (Med), PhD Professor of Paediatrics and Child Health Faculty of Health Sciences Desmond Tutu TB Centre and Department of Paediatrics and Child Health Stellenbosch University Tygerberg, South Africa Juanita Bezuidenhout MBChB, MMed, PhD Professor of Anatomical Pathology Department of Anatomical Pathology Stellenbosch University Tygerberg, Cape Town, South Africa
Ludwig Apers MD, MPH, PhD Clinician and Public Health Specialist Institute of Tropical Medicine Antwerpen, Belgium
Eric D Bateman MBChB (UCT), FRCP, Professor of Respiratory Medicine Division of Pulmonology University of Cape Town Cape Town, South Africa
Linda-Gail Bekker MBChB, DTMH, DCH, FCP (SA), PhD Associate Professor Deputy Director of the Desmond Tutu HIV Centre Institute of Infectious Diseases and Molecular Medicine University of Cape Town Cape Town, South Africa
Leopold Blanc MD, MPH Coordinator Tuberculosis Strategy and Operations Stop TB Department, HIV/AIDS, TB and Malaria Cluster World Health Organisation Geneva, Switzerland Bj rn Blomberg MD, PhD Consultant Physician Department of Medicine Haukeland University Hospital and Institute of Medicine Bergen, Norway Franc¸ois GE Bonnici MD Associate Director Global Health Initiative, World Economic Forum Cologny, Geneva, Switzerland Matthys H Botha MBChB, FCOG, MMed Senior Specialist, Obstetrics and Gynaecology Department of Obstetrics and Gynaecology Stellenbosch University and Tygerberg Academic Hospital Tygerberg, South Africa
CONTRIBUTORS
Guillaume Breton MD Department of Internal Medicine Pitie´-Salpe´trie´re Hospital Groupe Hospitalier Pitie´-Salpeˆtrie`re Paris, France
Catherine M Corbishley FRCPath Consultant Histopathologist Department of Cellular Pathology St George’s Hospital London, UK
Daniel Brodie MD Assistant Professor of Clinical Medicine Columbia University College of Physicians and Surgeons New York, NY, USA
Charles L Daley MD Head of Division of Mycobacterial and Respiratory Infections Professor of Medicine National Jewish Medicine and Research Center Denver, CO, USA
Ertan Bulbuloglu MD Associate Professor Department of Surgery and Pathology Kahramanmaras Sutcuimam University Kahramanmars, Republic of Turkey
Thomas M Daniel MD Professor Emeritus of Medicine and International Health Center for Global Health and Diseases Case Western Reserve University Cleveland, OH, USA
William J Burman MD Associate Professor of Medicine Infectious Diseases Clinic Denver Public Health Denver, CO, USA
Rodney Dawson MBChB, FCP (SA) Cert Pulm Consultant Pulmonologist and Head Centre for Tuberculosis Research Innovation University of Cape Town Lung Institute and Division of Pulmonary Medicine Mowbray, Groote Schuur, South Africa
Jose´ A Caminero MD Pneumology Department Hospital General de Gran Canaria Las Palmas, Spain
Adelard I De Backer MD, PhD Head Department of Radiology General Hospital Sint-Lucas Ghent, Belgium
Mete C ¸ ek MD Associate Professor of Urology Department of Urology Osmanaga Mah Istanbul, Turkey
Ashwin S Dharmadhikari MD Department of Medicine Division of Pulmonary and Critical Care Medicine Brigham and Women’s Hospital Boston, MA, USA
Jeremiah M Chakaya MD Chief Research Officer Kenya Medical Research Institute Nairobi, Kenya Lakhbir S Chauhan MD Deputy Director General of Health Services Ministry of Health and Family Welfare Government of India New Delhi, India Chiang Chen-Yuan MD, MPH Director Department of Lung Health International Union against Tuberculosis and Lung Disease Taipei, Taiwan Gavin J Churchyard MB BCh, FCP (SA), Chief Executive Aurum Institute for Health Research Marshalltown, South Africa Harun Ciralik MD Assistant Professor Department of Pathology Kahramanmaras Sutcuimam University Kahramanmaras, Republic of Turkey
MMed, PhD
Keertan Dheda MBBCh, FCP(SA), FCCP, PhD (Lond) Consultant Physician in Respiratory and General Internal Medicine Honorary Senior Lecturer Division of Infection and Immunity University College London London, UK T Mark Doherty MD Coordinator Research and Strategy, Infectious Disease Immunology Statens Serum Institute Copenhagen, Denmark Peter R Donald MBChB (Stellenbosch), DCH (Glasgow), DTM&H (London), FCP(SA), FRCP (Edin) MD (Stellenbosch) Emeritus Professor of Paediatrics and Child Health Department of Paediatrics and Child Health Stellenbosch University and Tygerberg Children’s Hospital Western Cape, South Africa
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CONTRIBUTORS
Francis Drobniewski MB BS, MA, MSc, PhD, Head, Clinical TB and HIV Group CID Institute of Molecular Sciences London, UK
DTM&H, FRCPath
Christopher Dye DPhil Scientist Stop Tuberculosis Department HIV/AIDS, TB and Malaria Cluster World Health Organisation Geneva, Switzerland Soumitra R Eachempati MD, FACS New York-Presbyterian Hospital New York, NY, USA John B Eastwood MD, FRCP Professor of Medicine Department of Renal Medicine St. George’s Hospital London, UK Mary E Edginton MB BCH, FFCH, PhD, MSc, DPH, DOH, DTM&H, DHSM Associate Professor School of Public Health University of the Witwatersrand Johannesburg, South Africa Asma I ElSony MD, PhD Director Epidemiological Laboratory (Epi-Lab) Khartoum Epidemiological Laboratory (Epi-Lab) for Public Health/Implementation Research Khartoum, Sudan
Adem Fazlioglu MD Department of Urology Taksim Teaching Hospital Istanbul Medical School Istanbul, Turkey Katherine Floyd Economist Tuberculosis Monitoring and Evaluation Team Stop TB Department World Health Organization Geneva, Switzerland Gerald Friedland MD Professor of Medicine Yale University School of Medicine Connecticut, USA Mathew Gandy BA, PhD Professor of Geography University College London London, UK Neel R Gandhi MD Assistant Professor of Medicine and Epidemiology Division of General Internal Medicine Montefiore Medical Center and Albert Einstein College of Medicine New York Deliana Garcia MA Director of International Research and Development Migrant Clinicians Network Inc Austin, Texas, USA
Brian S Eley MBChB, FCPaed(SA), BSc(Hons) Head of Paediatric Infectious Diseases Red Cross Children’s Hospital School of Child and Adolescent Health University of Cape Town Rondebosch, Cape Town, South Africa
Claudia Garcia-Moreno MD, MSc Coordinator Gender, HIV/AIDS and Violence Department of Reproductive Health and Research World Health Organization Geneva, Switzerland
Donald A Enarson MD Senior Advisor International Union Against Tuberculosis and Lung Disease (IUATLD) Paris, France
Giuliano Gargioni MD Medical Officer Stop TB Department World Health Organization Geneva, Switzerland
Marcos Espinal MD, PhD, MPH Executive Secretary Stop TB Partnership Secretariat Stop TB Department, HIV/AIDS, TB and Malaria Cluster World Health Organisation Geneva, Switzerland
Haileyesus Getahun MPh, PhD Stop TB Department; HIV/AIDS, TB and Malaria Cluster World Health Organization Switzerland
Elizabeth L Fair PhD Assistant Professor Division of Pulmonary and Critical Care Medicine San Francisco General Hospital University of California San Francisco, CA, USA
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Robert P Gie MMed (Paed), FCP (SA) Department of Paediatrics and Child Health Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa
CONTRIBUTORS
Ishwarprasad Gilada MBBS, DVD Consultant in HIV/AIDS Medicine Unison Medicare and Research Centre Mumbai, India Michael Gotway MD Clinical Associate Professor Professor of Radiology and Pulmonary Critical Care Medicine UCSF; Consulating Staff, Scottsdale Medical Imaging (an Affiliate of Southwest Diagnostic Imaging) Scottsdale, AZ, USA Pierre Goussard MBChB, Mmed(paed) Pediatric Pulmonologist Tygerberg Children’s Hospital Department of Paediatrics and Child Health Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa Stephen M Graham MB BS, FRACP, DTCH Associate Professor in International Child Health University of Melbourne Department of Paediatrics Royal Children’s Hospital Australia John M Grange MBBS(Lond), MSc(Lond), MD(Lond) Visiting Professor Centre for Infectious Diseases and International Health Windeyer Institute London, UK Malgorzata Grzemska MD, PhD Medical Officer Stop TB Department HIV/AIDS, TB and Malaria Cluster World Health Organization Geneva, Switzerland
Tony Hawkridge MSc, FCPHM (SA) Clinical Director South African TB Vaccine Initiative Institute of Infectious Disease and Molecular Medicine University of Cape Town Cape Town, South Africa Leonid Heifets MD, PhD Director, Microbiology Laboratory National Jewish Medical and Research Center Denver, CO, USA Anneke C Hesseling MD, MSc Senior Researcher Department of Paediatrics and Child Health Desmond Tutu TB Centre Stellenbosch University Tygerberg, South Africa Christopher J Hoffmann MD, MPH, Fellow Division of Infectious Diseases John Hopkins School of Medicine Baltimore, MD, USA
MSc
Philip C Hopewell MD Professor and Associate Dean Division of Pulmonary and Critical Care Medicine San Francisco General Hospital University of California, San Francisco San Francisco, CA, USA S Mehran Hosseini MD Epidemiologist Stop TB Department HIV/AIDS, TB and Malaria Cluster World Health Organization Geneva, Switzerland
Phillipe Guerin MD, MPH, PhD Scientific Director of Epicentre Me´decins Sans Frontie`res Paris, France
Gregory D Hussey MBChB, DTM&H, MSc, MMed, FFCH Director South African TB vaccine Initiative Institute of Infectious Diseases and Molecular Medicine Cape Town, South Africa
Willem A Hanekom MBChB, DCH(SA), FCPaed(SA) Laboratory Director South African Tuberculosis Vaccine Initiative Institute for Infectious Disease and Molecular Medicine University of Cape Town Cape Town, South Africa
Elvis Irusen MD Stellenbosch University Tygerberg Hospital Tygerberg, South Africa
Anthony D Harries MA, MD, FRCP, DTM&H Technical Advisor in HIV Care and Support Ministry of Health Malawi Country Office Lilongwe, Malawi
Ernesto Jaramillo MD, PhD Medical Officer Stop TB Department World Heath Organisation Geneva, Switzerland
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CONTRIBUTORS
Prakash M Jeena MD Department of Paediatrics and Child Health University of Kwazuku-Natal Durban, South Africa
Christian Lienhardt MD, DTM, MSc, PhD Head, Clinical Trial Division International Union Against Tuberculosis and Lung Diseases Paris, France
John L Johnson MD Project Investigator Department of Medicine Case Western Reserve University and University Hospitals Case Medical Center Cleveland, OH, USA
Knut Lo¨nnroth MD, MSc (Clinical Epidemiology), PhD (Social Medicine) Medical Officer TB Strategy and Health Systems Stop TB Department World Health Organisation Geneva, Switzerland
Nico E Jonas MBChB, FRCS (Glas), FCORL Consultant Division of Otolaryngology University of Cape Town Medical School Cape Town, South Africa
Gary Maartens MBChB, MMed, FCP(SA), Professor of Clinical Pharmacology Division of Clinical Pharmacology UCT Health Sciences Faculty Cape Town, South Africa
DTM&H
H Francois Jordaan MBChB MMed Associate Professor and Head Division of Dermatology Department of Medicine Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa
Dermot Maher MA, BM BCh, DM, MRCGP, FRCP, Senior Clinical Epidemiologist MRC/UVRI Uganda Research Unit on AIDS Entebbe, Uganda
Shaloo P Kamble MBBS, India Program Manager Global Health Initiative World Economic Forum Geneva, Switzerland
Ben J Marais MBChB, MRCP (Paed UK), FCP (Paed MMed, PhD Associate Professor of Paediatrics and Child Health Department of Paediatrics and Child Health Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa
DTCD
Priya Khanna Clinical TB and HIV Group CID Institute of Molecular Sciences London, UK Sharon Kling MMed (Cape Town) FC Paed (SA) Senior Specialist Department of Paediatrics and Child Health Stellenbosch University Tygerberg, South Africa Fatih O Kurtulus MD Department of Urology Taksim Teaching Hospital Istanbul Medical School Istanbul, Turkey Stephen D Lawn MD The Desmond Tutu HIV Centre Institute for Infectious Disease and Molecular Medicine Faculty of Health Sciences University of Cape Town Cape Town, South Africa Jonathan Levin PhD Statistician MRC/UVRI Uganda Research Unit on AIDS Uganda Research Unit on AIDS Entebbe, Uganda
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Bongani M Mayosi Dphil, Professor and Head Department of Medicine University of Cape Town Groote Schuur Hospital Cape Town, South Africa
FFPH
SA),
FCP (SA), FESC, MASSAf
Christopher RE McEvoy BSc (Hons), PhD Postdoctoral Research Fellow DST/NRF Centre of Excellence in Biomedical TB Research Department of Medical Biochemistry Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa Helen McIlleron MBChB, PhD Senior Medical Researcher Pharamcokinetic Research Unit Division of Clinical Pharmacology, UCT Groote Schuur Hospital Cape Town, South Africa Salil Mehta MS, DNB Consultant Ophthalmologist Department of Opthalmology Lilavati Hospital and Research Centre Mumbai, India
CONTRIBUTORS
Graeme Meintjes MD Department of Medicine Institute of Infectious Diseases and Molecular medicine University of Cape Town Cape Town, South Africa Dick Menzies MD, MSc Professor of Respirology and Epidemiology Director Respiratory Division of the MUHC Montreal Chest Institute Montreal, Canada Anthony P Moll BSc, MBChB Chief Medical Officer Church of Scotland Hospital Tugela Ferry, South Africa Joia Mukherjee MD Assistant Professor Division of Social Medicine and Health Inequalities Brigham and Women’s Hospital Harvard Medical School Boston, MA, USA W Mulwafu MBChB Senior Registrar, Division of Otolarygology University of Cape Town Medical School Groote Schuur Hospital Cape Town, South Africa Jean B Nachega MD, PhD Professor of Medicine and Epidemiology Director Centre for Infectious Diseases Stellenbosch University Cape Town, South Africa Nani Nair MD Regional Advisor - Tuberculosis Regional Office for South-East Asia World Health Organisation New Delhi, India Edward A Nardell MD Associate Professor FXB Centre for Health and Human Rights Harvard School of Public Health Boston, MA, USA Lisa J Nelson MD Country Director, Mozambique, Global AIDS Program Centers for Disease Control and Prevention (CDC) Maputo, Mozambique Andrew Nunn MSc Associate Director MRC-Clinical Trials Unit London, UK
Paul Nunn MD, FRCP Coordinator TB/HIV and Drug Resistance Stop TB Department HIV/AIDS, TB and Malaria Cluster World Health Organization Geneva, Switzerland Philip C Onyebujoh MD, PhD, FRCP (Lond) Medical Officer and TB/HIV Research Officer Implementation Research and Methods UNICEF/UNDP/ World Bank/WHO Special Programme for Research and Training in Tropic Geneva, Switzerland Madhukar Pai MD, PhD Assistant Professor of Epidemiology Department of Epidemiology, Biostatics and Occupational Health, McGill University Montreal, Canada Mark D Perkins MD Chief Scientific Officer Foundation for Innovative New Diagnostics (FIND) Cointrin, Switzerland Robert J Pratt BA, MSc, RN, RNT, DN(Lond) Director Richard Wells Research Centre Institute for Research in Health and Human Sciences Thames Valley University London, UK Chris AJ Prescott MBChB, MMed, FCS(SA) Professor Division of Otolaryngology University of Cape Town Medical School Cape Town, South Africa Mario C Raviglione MD Director Stop TB Department (STB) HIV/AIDS, TB and Malaria Cluster World Health Organisation Geneva, Switzerland Helmuth Reuter MBChB, FCP (SA), MMed (Int), FRCP (Edinb) PhD Professor of Internal Medicine Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa Etienne de la Rey Nel MBChB, MMed Consultant Department of Paediatrics and Child Health University of Stellenbosch Cape Town, South Africa
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CONTRIBUTORS
John C Ridderhof PhD, HCLD (ABB) Associate Director of Health Sciences National Center for Preparedness Detection and Control of Infectious Diseases CCID Centers for Disease Control and Prevention Atlanta, GA, USA Colebunders Robert MD Head, HIV/STD unit Department of Clinical Science Institute of Tropical Medicine Antwerp, Belgium Graham AW Rook BA, MB, BChair, MD Professor of Microbiology Centre for Infectious Diseases and International Health Windeyer Institute of Medical Sciences London, UK
Kevin Schwartzman MD, Assistant Professor Department of Medicine McGill University Montreal, QC, Canada
MPH, FRCPC
N Sarita Shah MD, MPH Assistant Professor of Medicine and Epidimiology Division of General Internal Medicine Montefiore Medical Center and Albert Einstein College of Medicine New York Mahesh P Sharma MD, DM, FACG, FAMS Head of Department of Gastroenterology Department of Gastroenterology Rockland Hospital New Delhi, India
ID Rusen MD, MSc Director Department of Tuberculosis Control and Prevention International Union Against Tuberculosis and Lung Disease Paris, France
Om P Sharma MD, FRCP Professor of Medicine Department of Pulmonology and Critical Care Medicine LAC and USC Medical Center Los Angeles, CA, USA
Fabio Scano MD Stop TB Department HIV/AIDS, TB and Malaria Cluster World Health Organisation Geneva, Switzerland
Surendra K Sharma MD, PhD Chief Division of Pulmonary and Critical Care Medicine All India Institute of Medical Sciences New Delhi, India
H Simon Schaaf MBChB (Stell), MMed Paed (Stell), DCM (Stell), MD Paed (Stell) Professor of Paediatrics, sub-specialist Infectious Diseases, Department of Paediatrics and Child Health and Desmond Tutu Tuberculosis Centre, Faculty of Health Sciences, Stellenbosch University; Consultant, Tygerberg Children’s Hospital and Brooklyn Hospital for Chest Diseases, Cape Town, South Africa
Hari S Shukla MS, PhD, FRCSEd Professor and Head Department of Surgical Oncology Institute of Medical Sciences Banaras Hindu University Lanka, Varanasi, India
Neil W Schluger MD Professor of Medicine, Epidemiology, and Environmental Health Sciences Chief Division of Pulmonary, Allergy, and Critical Care Medicine Columbia University College of Physicians and Surgeons New York, NY, USA Johann W Schneider MBChB, MMed (Anat Path) Professor of Anatomical Pathology Department of Pathology Faculty of Health Sciences University of Stellenbosch Cape Town, South Africa Johan F Schoeman MBChB, MMed (Paeds), Professor of Paediatrics Department of Paediatrics and Child Health Faculty of Health Sciences Stellenbosch University Tygerberg, South Africa
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S Bertel Squire BSc, MBChir, MD, FRCP Senior Lecturer in Clinical Tropical Medicine Consultant Physician Liverpool School of Tropical Medicine Liverpool, UK Jeffrey R Starke MD Professor and Vice-Chairman Department of Pediatrics Baylor College of Medicine Texas Children’s Hospital Houston, TX, USA Martin Storm BSC, MBChB, FCS(Orth), Consultant Orthopaedic Surgeon Vredenburg, South Africa
FCP (Paeds) SA, MD
Willem A Sturm MD, PhD Professor of Medical Microbiology Department of Medical Microbiology Nelson R Mandela School of Medicine Congella, South Africa
MMed(Orth)
CONTRIBUTORS
Mallika Tewari MS, MRCSEd Senior Resident Department of Surgical Oncology Institute of Medical Science Banaras Hindu University Varanasi, Uttar Pradesh, India Rachael Thomson Co-ordinator EQUS-TB Project Liverpool School of Tropical Medicine Liverpool, UK Anna Thorson MD, PhD Division of International Health (IHCAR) Department of Public Health Services Karolinska Institute Stockholm, Sweden Guy E Thwaites MD, MBBS, MRCP, MRCPath, PhD Wellcome Trust Clinical Research Fellow Centre for Molecular Microbiology and Immunity Imperial College London, UK Jacqueline P Tulsky MD Professor of Clinical Medicine Department of Medicine, Positive Health Program University of California, San Francisco San Francisco, CA, USA Mukund W Uplekar MD Medical Officer Stop TB Department, HIV/AIDS, TB & Malaria Cluster World Health Organization Geneva, Switzerland Mahnaz Vahedi MBBS, MIPH Medical Officer Implementation and Research methods UNICEF/UNDP/World Bank/WHO Special program for Research and Training in Tropical Diseases (TDR) World Health Organisation Geneva, Switzerland Armand Van Deun MD Research Assistant & Bacteriology Consultant The Union Mycobacteriology Unit Institute of Tropical Medicine Antwerp, Belgium Paul van den Brande MD Pulmonologist Department of Pulmonology University Hospital Gasthuisberg Catholic University of Belgium Leuven, Belgium
Frederick H Van der Merwe MBChB, MMed (O&G), FCOG Specialist Obstetrician and Gynaecologist Department of Obstetrics and Gynaecology Tygerberg Academic Hospital and Stellenbosch University Tygerberg, South Africa Paul D van Helden PhD Director DST/NRF Centre of Excellence in Biomedical Tuberculosis Research Division of Molecular Biology and Human Genetics Department of Biomedical Sciences Faculty of Health Sciences University of Stellenbosch Tygerberg, South Africa Jan van den Hombergh Country Director PharmAccess Foundation Dar-Es-Salaam, Tanzania
MD, MSc, MPH
Filip M Vanhoenacker MD, PhD Medical Faculty Department of Radiology University Hospital Antwerp Edegem, Belgium Johan van Wijgerden RGN, BSc, Public Health Manager Ealing Primary Health Care Trust Southall, Middlesex, UK
MSc
Gert J Vlok MBChB (Stell), MMed (Orth) Stell, Professor and Head Department of Orthopaedic Surgery Faculty of Health Sciences University of Stellenbosch Tygerberg, South Africa
FC Orth (SA)
Robert S Wallis MD Medical Director Pharmaceutical Product Developments Inc. Washington DC, USA Fraser Wares MBChB, DTM&H, MPH Medical Officer (Tuberculosis) Office of the WHO Representative to India New Delhi, India Robin M Warren PhD DST/NRF Centre of Excellence in Biomedical Tuberculosis Research Division of Molecular Biology and Human Genetics Department of Biomedical Sciences Faculty of Health Sciences University of Stellenbosch Tygerberg, South Africa
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CONTRIBUTORS
Catherine J Watt DPhil Epidemiologist Stop TB Department, HIV/AIDS, TB and Malaria Cluster World Health Organization Geneva, Switzerland Diana EC Weil MD Senior policy Advisor Stop TB Department, HIV/AIDS, TB and Malaria Cluster World Health Organization Geneva, Switzerland Mary C White RN, MPH, PhD Professor Community Health Systems University of California, San Francisco San Francisco, CA, USA Andrew C Whitelaw MBBch, MSc, FCPath (Micro) SA Senior Specialist, National Health Laboratory Service Senior Lecturer, University of Cape Town Groote Schuur Hospital Cape Town, South Africa Nicky Wieselthaler MBChB, FCRad(DiagSA) Consultant Department of Radiology University of Cape Town Red Cross War Memorial Children’s Hospital Cape Town, South Africa Brian G Williams PhD Epidemiologist Tuberculosis Monitoring and Evaluation Stop TB Department, HIV/AIDS, TB AND Malaria Cluster World Health Organisation Geneva, Switzerland Robin Wood BSc, BM, BCH, FCP (SA) Professor of Medicine Desmond Tutu HIV Research Centre Institute of Infectious Disease and Molecular Medicine University of Cape Town Medical School Cape Town, South Africa
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Colleen A Wright MD Professor and Head of Discipline Department of Anatomical Pathology Faculty of Health Sciences NHLS Tygerberg Hospital Tygerberg, South Africa Alimuddin I Zumla BSc, MBChB, MSc (Lond), PhD (Lond), FRCP(Lond), FRCP(Edin) Professor of Infectious Diseases and International Health, University College London Medical School; Director, Centre for Infectious Diseases and International Health, Windeyer Institute of Medical Sciences, University College London, UK; Honorary consultant infectious diseases physician, University College London Hospitals NHS Foundation Trust, London, UK; Honorary Professor, Liverpool School of Tropical Medicine, University of Liverpool, UK; Visiting Professor, Department of Medicine, University of Cape Town, South Africa; Honorary Professor, University of Zambia School of Medicine, Lusaka, Zambia; Vice President, Royal Society of Tropical Medicine and Hygiene (2004-2006); Member of the Court of Governors, London School of Hygiene & Tropical Medicine, London, UK; Member of the Steering Technical Advisory Group, WHO STOP TB Partnership, Geneva, Switzerland; Member TB Alert; and formerly Associate Professor, Centre for Infectious Diseases, University of Texas Health Science Centre at Houston, School of Medicine and Public Health, Houston, Texas, USA. Edward Zuroweste MD Chief Medical Officer Migrant Clinicians Network Austin, TX, USA
Dedication
This textbook is dedicated to: all those selfless, committed individuals and institutions who are engaged in the fight against the killer disease Tuberculosis; the millions of people who suffer from the disease each year; and those who have succumbed to it.
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Acknowledgements
At the start of this project we were aware of the massive undertaking that we had taken on board. We were strongly encouraged by many members of the global TB fraternity who saw a great need for this textbook and unflinchingly supported us during the entire period of book development. The end result has been absolutely superb. We are very grateful to many individuals who assisted us in this formidable task. Our gratitude goes to our six associate editors, Professor John M Grange, Dr. Mario C Raviglione, Dr. Wing Wai Yew, Professor Jeffrey R Starke, Professor Madhukar Pai, and Professor Peter R Donald who were always willing to assist and review chapters in their areas of expertise. This high powered combination of TB leaders enabled us to easily attract the 158 willing authors who enthusiastically contributed chapters of the highest quality. Their excellent contributions will go a long way to furthering knowledge on TB and will help in the management of millions of adults and children with TB in the world.
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The staff at the publishers Elsevier Science were very cooperative and sincere thanks to Ms Sarah Penny, Ms Louise Cook, Ms Shereen Jameel for the efficient way they handled the production schedule and liaised with us. To our wives and families a special thank you for their support, patience and encouragement. We are indebted to Professor John M Grange for his generous and voluntary contributions to editorial activities. To our research and clinical teams at University College London and Stellenbosch University, we are indebted for their support received during the time we spent on editing the text. It’s been a pleasure editing this volume and we hope that the hard work of the editorial team and authors will be of major benefit to the readers world wide, and help in the battle against TB worldwide. Chief Editors Professors Alimuddin I Zumla and H Simon Schaaf
Abbreviations
AAFB AAP ABC ACH ACSM ACTH ADA AE AFB Ag AIDS AII AMTD APC ARDS APHA ARI ART ARV ATS AUC BAE BAL BCG BM BMI BOF bp BSC BTS CAPD CDC CF CFP CFU/cfu CHW CI CL
acid-alcohol-fast bacilli American Academy of Pediatrics abacavir air changes per hour advocacy, communication and social mobilization adrenocorticotropic hormone (corticotropin) adenosine deaminase adverse event acid-fast bacilli antigen acquired immunodeficiency syndrome airborne infection isolation Amplified Mycobacterium Tuberculosis Direct antigen presenting cell adult respiratory distress syndrome American Public Health Association annual risk of (tuberculosis) infection antiretroviral treatment (therapy) antiretrovirals American Thoracic Society area under the concentration-time curve bronchial artery embolization bronchoalveolar lavage Bacillus Calmette-Gue´rin bacterial meningitis body mass index broncho-oesophageal fistulae base pair biological safety cabinet British Thoracic Society continuous ambulatory peritoneal dialysis Centers for Disease Control and Prevention cystic fibrosis culture filtrate protein colony forming units community health worker confidence interval confidence limit
CMI CMV CNS COPD CPT CRF CRISPA CRP CSF CSO CT CXR CYP450 DALY db DC DC-SIGN DMC DOT DOTS DR DRS DST DTH DVR DWI E EBA ECG EFV EIB ELISA ELISPOT EMB ENT EPI EQA ESAT ESR
cell mediated immunity (or immune response) cytomegalovirus central nervous system chronic obstructive pulmonary disease co-trimoxazole preventive therapy case-report form clustered regularly interspaced short palindromic repeats C-reactive protein cerebrospinal fluid civil society organization computed tomography (scan) chest radiograph cytochrome P450 disability-adjusted life-years decibel dendritic cell dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin Data Monitoring Committee directly observed treatment (therapy) directly observed treatment, shortcourse Direct Repeat drug resistance surveillance drug susceptibility test delayed type hypersensitivity direct variable repeat diffusion weighted imaging ethambutol early bactericidal activity electro cardiogram efavirenz erythema induratum of Bazin enzyme-linked immunosorbent assay enzyme-linked immunospot ethambutol ear, nose and throat Expanded Programme on Immunization external quality assessment Early Secreted Antigenic Target erythrocyte sedimentation rate
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ABBREVIATIONS
ESRF ETH EU EUS FAS FBO FDA FDC FDG-PET FEV1 FIND FLAIR FliP FNA FNAB FNAC FTT GATB GCP GDF GFATM GFR GLC GLP GM-CSF gp H HAART HAI HBC HCW HþE HEPA HHV HIV HLA HPA HPLC HR HRCT HSP IC ICH ICP ICSI IDPs IDSA IDU
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end-stage renal failure ethionamide European Union endoscopic ultrasound fatty acid synthase faith-based organisation Food and Drug Administration fixed-dose combination 18 F-fluoro-2-deoxy-D-glucose positron emission tomography forced expiratory volume-1 second Foundation for Innovative New Diagnostics fluid-attenuated inversionrecovery fast ligation-mediated PCR fine needle aspiration fine needle aspiration biopsy fine needle aspiration and cytology failure to thrive Global Alliance for TB drug development Good Clinical Practice Global Drug Facility Global Fund to fight AIDS, Tuberculosis and Malaria glommerular filtration rate Green Light Committee Good Laboratory Practice granulocyte-macrophage colonystimulating factor glycoprotein isoniazid highly active antiretroviral therapy healthcare-associated infections high-burden country healthcare worker Haematoxylin and Eosin (stain) high-efficiency particulate air (filters/filtration) human herpes virus human immunodeficiency virus human lymphocyte antigen hypothalamo-pituitary adrenal high-performance liquid chromatography hazard ratio high resolution computed tomography heat shock protein informed consent International Conference on Harmonisation intracranial pressure intracytoplasmic sperm injection internally displaced persons Infectious Diseases Society of America injection drug user
IEC IFN IGRA IL IM IMP IMR INGO INH iNOS INR IPT IRB IRD IRIS IS ISTC ITT IUATLD IVU IWGMT kb kDa KS kV LAM LAMP LDH LED LEV LIP LJ LP LPS LRP LSPs LTBI LTCF LTR MAC MAI MBL M. bovis MDG MDR MGIT MHC MIC MIRUs MOTT MRC
Independent Ethics Committees interferon interferon-gamma release assay interleukin intramuscular Interventional Medical Product Infant Mortality Rate international non-governmental organizations isoniazid inducible nitric oxide synthetase international normalized ratio isoniazid preventive therapy Institutional Review Board immune restoration disease immune reconstitution inflammatory syndrome insertion sequence International Standards for Tuberculosis Care intention to treat International Union against Tuberculosis and Lung Disease intravenous urogram International Working Group on Mycobacterial Taxonomy kilobase kiloDalton Kaposi sarcoma kiloVolt lipoarabinomannan loop-mediated isothermal amplification lactate dehydrogenase light emitting diode local exhaust ventilation lymphoid interstitial pneumonitis Lo¨wenstein-Jensen lumbar puncture liopolysaccharide Luciferase reporter phages large-sequence polymorphisms latent tuberculosis infection long-term care facility long terminal repeat Mycobacterium avium complex Mycobacterium avium intracellulare mannose-binding lectin Mycobacterium bovis Millennium Development Goal multidrug-resistant Mycobacterial Growth Indicator Tube major histocompatibility complex Minimal inhibitory concentration Mycobacterial Interspersed Repetitive Units mycobacteria other than (typical) tuberculosis Medical Research Council
ABBREVIATIONS
MRI MRS MTB MTBC MTD M. tuberculosis MVA NAA NAATs NALC NANDA-International NAT NGO NICE NIH NIOSH NK cells NNRTI NO NOD2 NPV NRL NRTI NSAID nsSNPs NTCA NTP NTCP NVP OR PAF PAL PAMPs PAS PAS (in Chapter 21 & 87) PBMC PCP PCR PET PFR PGL PGRS PHC PI PK
magnetic resonance imaging magnetic resonance spectroscopy Mycobacterium tuberculosis Mycobacterium tuberculosis complex Mycobacterium Tuberculosis Direct Mycobacterium tuberculosis Modified vaccinia Ankara nucleic acid amplification nucleic acid amplification tests N-acetyl-L-cysteine North American Nursing Diagnosis AssociationInternational N-acetyltransferase non-governmental organisation National Institute for Health and Clinical Excellence National Institutes of Health National Institute for Occupational Safety and Health natural killer cells non-nucleoside reverse transcriptase inhibitor nitric oxide nucleotide oligomerization binding domain 2 gene negative predictive value national reference laboratory nucleoside reverse transcriptase inhibitor non-steroid anti-inflammatory drug non-synonymous SNPs National Tuberculosis Controllers Association National Tuberculosis Programme National Tuberculosis Control Programme nevirapine odds ratio population attributable fraction Practical Approach to Lung Health pathogen-associated molecular patterns para-aminosalicylic acid Periodic acid-Schiff (stain) peripheral blood mononuclear cells Pneumocystis jiroveci pneumonia polymerase chain reaction positron emission tomography particulate filter respirator persistent generalized lymphadenopathy polymorphic GC-rich repetitive sequence Primary Health Care protease inhibitor pharmacokinetic
PLWHA PMTCT PNT PPAs PPD PPM PPV PRR PWBs PZA QFT-G R RCT RD R&D RES RFLP RIF RMP RNTCP S SAE SC SCC SCID SI SIADH SLD SLE SM SNPs sSNPs SOP SRL STAG STIR SUR SUV T TAG TB TBM TCH TCR TDF TGF-b Th1/Th2 The Union TIR
people living with HIV/AIDS prevention of mother-to-child transmission papulonecrotic tuberculid Participatory Poverty Assessments purified protein derivative public-private mix positive predictive value pattern recognition receptor patient-wise drug boxes pyrazinamide QuantiFERONW-TB Gold rifampicin randomized controlled trial Regions of Difference (genome) research and development reticulo-endothelial system restriction fragment length polymorphism rifampicin rifampicin Revised National Tuberculosis Control Programme (India) streptomycin serious adverse event subcutaneous short-course chemotherapy severe combined immunodeficiency Spreading Index syndrome of inappropriate antidiuretic hormone (secretion) second-line drugs systemic lupus erythematosus streptomycin single nucleotide polymorphisms synonymous SNPs standard operational procedure Supra-Regional Laboratory/ Supra-National Reference Laboratory Scientific and Technical Advisory Group short Tau inversion recovery standardized uptake ratio standardized uptake value thioacetazone Treatment Action Group tuberculosis tuberculous meningitis thiophen-2-carboxylic acid hydrazide T-cell receptor tenofovir disoproxil fumarate transforming growth factor-b T-helper type 1/T-helper type 2 International Union Against Tuberculosis and Lung Disease toll/interleukin-I receptor
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ABBREVIATIONS
TLR TNF TNF-a Treg TSH TSRU TST TU TUR-P UK UN UNHCR UNICEF UPJ US USA (or US)
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toll-like receptor tumor necrosis factor tumor necrosis factor-alpha regulatory T cells thyroid stimulating hormone Tuberculosis Surveillance and Research Unit tuberculin skin test tuberculin units transurethral resection of the prostate United Kingdom United Nations United Nations High Commission for Refugees United Nations Children Fund ureteropelvic junction ultrasound United States of America
USPHS UVGI VATS VCT VDR VNTR VOC WHA WHO WHO-TDR
XDR X-ray Z ZN
United States Public Health Service ultraviolet germicidal irradiation video-assisted thoracoscopic surgery voluntary counseling and testing VitD3 receptor variable nucleotide tandem repeats volatile organic compounds World Health Assembly World Health Organization World Health organization – Special Programme for Research and Training in Tropical Diseases extensive(ly) drug-resistant radiograph pyrazinamide Ziehl-Neelsen (stain)
SECTION 1
HISTORY AND EPIDEMIOLOGY OF TUBERCULOSIS
CHAPTER
The history of tuberculosis
1
Past, present, and challenges for the future Thomas M Daniel
Most of the technologically advanced world has seen more than a century of declining incidence of tuberculosis (TB). The less developed world has not been so fortunate, and in those populous regions TB incidence is increasing. In acquired immunodeficiency syndrome (AIDS)-stricken regions – sub-Saharan Africa, for example – TB case rates now are 50–100 times those in North America and Europe. Spurred by immunodeficiencies resulting from human immunodeficiency virus (HIV) infection, these case rates will continue to increase in coming decades. The history of TB reveals that this disease has swept across large regions of the globe in slowly moving epidemic waves with periods measured in centuries. The factors contributing to the rise and decline of these waves are only partly known and are difficult to control in the absence of massive social and political changes. There is, however, much that we might learn from considering the historical spread of TB and the ways in which those countries now favoured with low TB incidences managed their high-incidence problems in their pasts.
ORIGINS The genus Mycobacterium has a slow rate of mutation, and this fact has enabled the development of hypotheses concerning the origins and evolution of Mycobacterium tuberculosis. There is some inferential reason to suspect that the genus, as represented by Mycobacterium ulcerans, may have existed 150 million years ago in the Jurassic period.1 Gutierrez and her colleagues2 at the Pasteur Institute have concluded that the progenitor of M. tuberculosis emerged from an array of mycobacterial species about 3 million years ago, presumably infecting early hominids and other primates in prehistoric times. It seems likely that all modern members of the M. tuberculosis complex evolved from a common ancestor 15,000–20,000 years ago.3,4 Mycobaterium bovis and other species in the complex split off from the central line at later times. Figure 1.1 presents the phylogenetic tree developed by Gutierrez and her colleagues. The hypothesized genome of the common progenitor more closely resembles M. tuberculosis than other mycobacterial species. Thus it is presented as a straight line from the hypothesized progenitor in Fig. 1.1. The position in time of the common progenitor implies that whatever diversity had occurred during preceding millennia became severely constricted before giving rise to modern species. Present-day TB is caused by six or seven clades – strains with common ancestors – of M. tuberculosis, which have separate geographic origins.5–7 Dating methods applied to members of two of these strains suggest that they emerged in their present form between 250 and 1,000 years ago.6
The earliest archaeological evidence of human TB comes from Egyptian art and mummies; there is ample evidence of spinal TB (Pott’s disease) as early as 5,500 years ago.8–10 While early workers attributed these infections to M. bovis, there is now good evidence from studies of amplified DNA recovered from mummies that M. tuberculosis was the cause of disease in ancient Egyptians.11,12 There are unequivocal references to TB in the Old Testament books of Deuteronomy and Leviticus at the time when Jews were in exile in Egypt.13 In fact, although the archaeological record is sparse or non-existent, there is reason to believe that TB was widespread, if not uniformly distributed, in Africa long before Arabians and Europeans entered the continent.14 There is general agreement that TB first appeared as a human disease in East Central Africa and that it travelled with early peoples as they migrated into Asia Minor and across the globe. There are imprecise prehistoric references to TB from India and China, but no archaeological evidence.15 Migrating early peoples reached the Americas across the land bridge connecting Siberia and Alaska and along its coast, perhaps in several waves, reaching as far south as Chile by 15,000 years ago. Tuberculosis was common in a number of western hemisphere locations before the arrival of Columbus and the Spanish conquistadores.16 As in Egypt, Andean mummies have yielded mycobacterial DNA. In both Africa and the Americas the TB epidemic wave seems to have crested and receded at early times, leaving naive populations susceptible to the reintroduction of TB by European colonizers.
WORLD HISTORY During my elementary school education, which took place during the 1930s, several earnest teachers embarked upon lessons of world history for me and my classmates. Each time the courses started with classical Greece, perhaps after a passing mention of Egyptian pharaohs, progressed to Rome, skipped hurriedly over the Middle Ages, and arrived at the Industrial Revolution at about the time the school year ended. This time course might well serve as a model for the rising wave of TB that crested in Europe and North America in the nineteenth century. In classical Greece, Hippocrates, Plato, and Aretaeus described TB, and the word phthisis for pulmonary TB is Greek.15 ‘Phthisis makes its attacks chiefly between the age of eighteen and thirtyfive’, Hippocrates wrote.17 Tuberculosis was described by Galen, the Greek physician who settled in Rome and became its preeminent physician. He recommended fresh air, milk, and sea voyages
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Numerous polymorphisms in house-keeping genes
Common ancestor of the M. tuberculosis complex Loss of 26 spacers present in M. canettii
can
M. canetti
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ancestral
TbD 1
RD 9
M. tuberculosis
modern
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RD 7 RD 8
RD 10 M. africanum
RDmic Rights were not granted to include this content in electronic media. Please refer to the printed book. RD12 RDseal RD 13
RD 4
M. microti oryx seal
goat M. bovis
RD 1 RD 2
classical BCG Tokyo
RD 14 BCG Pasteur
Fig. 1.1 Proposed scheme for the evolution of modern strains of Mycobacterium tuberculosis complex organisms based on the presence or absence of conserved deleted regions and on sequence polymorphisms in five selected genes. All modern strains of M. tuberculosis are shown as direct descendants of a common ancestor hypothesized to have existed 15,000–20,000 years ago. Reproduced with permission from Brosch R, Gordon SV, Marmiesse M, et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. PNAS 2002;99:3684–3689. Copyright 2002 National Academy of Sciences, USA.
for his TB patients, treatment modalities that would persist well into the nineteenth and twentieth centuries. With the fall of the Roman Empire in the fifth century AD, Europe entered a millennium that has been called the Dark Ages. Written records of disease are virtually non-existent. However, there is evidence of bony TB from archaeological sites throughout Europe.18 There is also abundant evidence that scrofula – TB of the cervical lymph nodes – was common in mediaeval Europe. Beginning with the Frankish king Clovis in 496, European rulers practiced healing of this affliction by the royal touch, and later rulers supported some of the large expenses of the throne by charging the thousands of supplicants who sought their cures.16 Protocols became established that probably did much to verify the TB nature of the adenopathy. Much of the scrofula may have been bovine TB resulting from the ingestion of contaminated milk. The Renaissance began in southern Europe and moved northwards. With it, our record of TB re-emerges, perhaps simply because the record is clearer, but more likely because increasing urbanization led to rising TB incidences. St. Francis died of TB in 1226; Baruch
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Spinoza succumbed in 1677.16 In 1660 John Bunyon called TB ‘The Captain among these Men of Death’.19 At that time, case rates in London probably reached 1,000–1,250/100,000/year.20 As the epidemic wave of TB crested in Europe and North America in the eighteenth and nineteenth centuries, the disease became romanticized.16,21 Byron is said to have remarked, ‘I should like to die of consumption’. ‘Why?’ he was asked. ‘Because the ladies would all say, “Look at that poor Byron, how interesting he looks in dying.”’21 Fre´de´ric Chopin suffered from TB throughout his adult life, dying of cardiorespiratory failure secondary to that destructive disease in 1849. His lover, Georges Sand, called him her ‘poor melancholy angel’.15 Charles Dickens wrote, ‘There is a dread disease . . . in which the struggle between body and soul is so gradual, quiet, and solemn . . . [that as] the mortal part wastes and withers away, so the spirit grows light and sanguine.’22 Perhaps the contrast between the affluence of those who profited from the Industrial Revolution and the grinding poverty and tortured life circumstances of those newly crowded into factories and slums who were most likely to be afflicted mandated a rosier view of TB.
CHAPTER
The history of tuberculosis
CONQUESTS AND EMPIRES Even before Europe emerged from the Dark Ages, European seamen were exploring the world’s oceans and their rulers were claiming hegemony over the lands they discovered. By the sixteenth and seventeenth centuries, conquests, colonization, and empire building were underway on large scale. Tuberculosis went with the Europeans, and it found fertile ground in the populations of much of the world that they conquered. Although TB had reached most of the globe in prehistoric times, it had long since receded from the Americas and much of Africa, leaving the peoples of those regions immunologically naive with respect to M. tuberculosis. Following Charles Darwin’s exploration of Tierra del Fuego, the British established a Protestant Mission at Ushuaia. Local Fuegians were relocated from their villages to this mission. In the winter of 1884, half of them died; TB was the principal cause of death.23 This tragic story was repeated many times in Alaska, Amazonia, and throughout the western hemisphere.16
EBB TIDE All epidemics wane as susceptible individuals decrease in the population, perhaps because death claims those most susceptible, perhaps because survivors display immunity. The time scale of the European and North American TB epidemic was such that it is more likely that other factors were the determinants of the decrease in TB incidence. Tuberculosis crested in Europe and North America at the middle of the nineteenth century and has been falling since that time to present historic low case rates.15 Figure 1.2 presents the decline of TB deaths in comparison with deaths from all causes in England and Wales between 1850 and 1910. Tuberculosis deaths declined more rapidly than those from all causes. Thomas McKeown, a highly respected population scientist, examined many possible causes for this decline and concluded that better nutrition played the major role.24 Leonard Wilson argued that removal of infectious individuals to treatment facilities was a major factor.25
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DISCOVERY OF THE CAUSE Tuberculosis so commonly appeared in multiple members of a family that it was easily concluded that it was a familial, inherited disease. This view predominated well into the nineteenth century in northern Europe, although contagiousness of TB was generally accepted in Mediterranean countries. Chopin was ejected from the house he had rented in Mallorca because, as Georges Sand wrote, ‘Phthisis [is] extremely rare in those latitudes, and, moreover considered contagious!’15,27 Sand and Chopin, escaping from the winter of Paris in hopes of ameliorating Chopin’s TB, considered the Spanish view outlandish. In 1720 Benjamin Marten published a remarkably clairvoyant book entitled, A New Theory of Consumptions: More Especially of a Phthisis or Consumption of the Lungs. ‘The original and Essential Cause, then,’ he wrote, ‘may possibly be some certain species of Animalcula or wonderful minute living creatures.’28 His terminology suggests that he was referring to the animalcula that had been described by Anton van Leeuwenhoek in the scurf of his teeth in 1626. It was Jean-Antoine Villemin, a French military surgeon, who convincingly demonstrated the infectious nature of TB. He injected a rabbit with material from the pulmonary cavity of an individual who had died of TB and observed that the animal had extensive TB when it was sacrificed and autopsied 14 days later.29 The great pioneer of bacteriology, Robert Koch (Fig. 1.3), demonstrated conclusively that a bacterium, which he called Bacillus tuberculosis and is now known as Mycobacterium tuberculosis, is the
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E. R. N. Grigg concluded that genetic herd immunity which developed as a result of selection influenced by the high death rates in young TB adults resulted in the falling incidence.26 Whatever the cause, the decline was dramatic, but it was limited to the populations of western Europe and a few of those regions which they colonized, such as Australia.
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Fig. 1.2 Declining death rates for TB, left-hand scale (dashed line), and all causes, right-hand scale (solid line), as deaths/million/year for England and Wales from 1850 to 1910. Reproduced with permission from Davies RPO, Tocque K, Bellis MA, et al. Historical declines in tuberculosis in England and Wales: improving social conditions or natural selection. Int J Tuberc Lung Dis 1999;3:1051–1054.
Fig. 1.3 Robert Koch. Pencil drawing by Theresa S. K. Chung.
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HISTORY AND EPIDEMIOLOGY OF TUBERCULOSIS
aetiological agent of TB.30 His dramatic presentation of his findings on 24 March 1882 was immediately hailed, and he was acclaimed throughout the world.
DIAGNOSIS Since the time of Koch, the diagnosis of TB has rested primarily on the microbiology laboratory. Staining and culturing techniques introduced by Koch were soon improved upon, although not fundamentally changed. They remain the benchmarks of diagnosis to the present. Koch’s studies of TB led to his report of tuberculin, as noted below. He recognized its diagnostic potential, and Danish veterinarians soon were using it to diagnosis tuberculosis in cattle.31 In 1907 Viennese paediatrician Clemens Freiherr von Pirquet (Fig. 1.4), one of the founders of the science of allergy, developed the intracutaneous tuberculin test much as we know it today. Using it he recognized that many of the children in his clinic were latently infected.15 The further work of Florence Seibert and others in the 1930s led to the production of tuberculin-purified protein derivative, the antigen used today for tuberculin skin testing. In November 1895 Conrad Wilhelm Ro¨ntgen, a distinguished physicist interested in radiation, discovered X-rays.32 Within a month he had imaged his wife’s hand. The American inventor Thomas Alva Edison developed the first practical fluoroscope, making clinical radiology a practical reality.33 In May 1897, scarcely 18 months after Ro¨ntgen’s discovery, Francis Williams reported on the fluoroscopic examination of the lungs in more than 100 patients at the Boston City Hospital.34 During the Second World War, France, the United States, and Germany introduced radiographic screening of military recruits for TB. After the war,
radiographic surveys for TB case finding were commonplace for about 25 years before declining incidences made them unproductive. Chest radiology remained important for the evaluation of tuberculous disease under treatment, however.
THE SEARCH FOR A CURE Koch, now famous and revered, followed his announcement of the cause of TB with news of his discovery of a substance that would ‘render harmless the pathogenic bacteria . . . without disadvantage to the body’.35 His product was tuberculin, a concentrated, bacteria-free, filtrate of liquid cultures of M. tuberculosis. While tuberculin was ineffective in treating disease, it became of great importance in the diagnosis of tuberculous infection. If no specific remedy was at hand, then treatment must rely on such long-favored measures as rest, fresh air, sea voyages, and a healthy diet. None of these measures benefitted the dying poet John Keats, however.15 Herman Brehmer opened the first sanatorium for TB patients in Goerbersdorf in the Silesian Mountains of Prussia in 1859. Others followed; Davos in the Alps of Switzerland and Saranac Lake in the Adirondacks of New York became especially well known. There is no convincing evidence that sanatorium care benefitted tuberculous patients. It may have reduced transmission of M. tuberculosis in some communities, however. If rest for the body was good, perhaps rest for diseased lungs would ameliorate TB. As early as 1696, Giorgio Baglivi observed that a patient with pulmonary TB improved after a sword wound resulted in a pneumothorax. Pneumothorax was first introduced for therapy by Carlo Forlanini in 1894 with beneficial results.36 The procedure was rapidly accepted and widely used to collapse pulmonary cavities during the next 50 years. Pneumoperitoneum was used for lower lobe cavities. About the same time, thoracoplasty came into use in those cases in which satisfactory collapse could not be achieved by pneumothorax.
CHEMOTHERAPY
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Fig. 1.4 Clemens von Pirquet. Pencil drawing by Theresa S. K. Chung.
4
The idea that it might be possible to find an agent that was specifically curative was espoused by Paul Ehrlich, who heard Koch’s presentation. He improved on Koch’s staining techniques and reasoned that if it was possible to stain organisms differently than tissues, it ought to be possible to find a drug that would attack the microbes but not the host. His work led to treatments for trypanosomiasis and syphilis. In 1932 Gerhardt Domagk, a pathologist working at the laboratories of Bayer, the German chemical company, discovered Prontosil, the first sulphonamide. Others followed, and a new era of anti-infective drugs was born. During the Second World War Domagk continued to work, and his efforts led to thioacetazone, the first mycobacteriostatic drug.37 Shortly thereafter Jorgen Lehman in Sweden discovered para-amino salicylic acid (PAS), also mycobacteriostatic. Selman Waksman (Fig. 1.5) with his junior colleagues Albert Schatz and Elizabeth Bugie discovered streptomycin in 1943, the first mycobactericidal drug and an early member of a class of drugs that Waksman called antibiotics.15 The drug was first used the following year by Corwin Hinshaw and William Feldman to treat a patient who had failed collapse therapy. Her response was dramatic. It was soon discovered that drug resistance could be prevented by using two drugs;
CHAPTER
The history of tuberculosis
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1
Report, in which it recommended that BCG should be used as widely as possible. Many trials of BCG efficacy have been conducted, some more rigorous than others. There is a striking disparity of results.39 This disparity is illustrated in Fig. 1.6, which is taken from a review by Paul Fine. Ultimately, an expert consensus emerged that BCG is useful in the prevention of miliary and meningeal TB in young children but has no epidemiological impact and no utility in prevention-oriented TB control programmes. Epidemiologists of the US Public Health Service never embraced BCG. Rather, they focused on prophylactic treatment of latent TB with isoniazid, an approach first suggested by Edith Lincoln.40 Large randomized control studies demonstrated the efficacy of this preventative measure in a variety of populations.41 Other countries have been slow to follow the American lead, however, and current control strategies in most nations emphasize treatment under direct observation using optimal drug regimens to reduce the further spread of tubercle bacilli.
CHALLENGES
Fig. 1.5 Selman Waksman. Pencil drawing by Theresa S. K. Chung.
streptomycin was coupled with PAS. Isoniazid was discovered almost simultaneously by three pharmaceutical companies in 1952 and rifampicin in 1963. With these bactericidal agents the modern treatment era was born. Other drugs followed and their therapeutic roles and optimum drug regimens were developed in clinical trials conducted in many countries.
PREVENTION AND CONTROL Without dismissing the importance of their efforts at case finding and treatment, public health workers interested in TB sought measures that might prevent this disease. With knowledge of Edward Jenner’s vaccinia prevention of smallpox and Louis Pasteur’s immunization treatment of rabies, Albert Calmette decided to turn his efforts at the Pasteur Institute in Lille, France, to developing a vaccine against TB. Together with his colleague, Camille Gue´rin, he began efforts to attenuate M. bovis by serial passage in 1902.15,38 During the devastating German siege of Lille in 1914 and the subsequent German occupation, they managed to maintain their cultures. In 1921 Calmette, now in Paris, was ready to try the vaccine known as Bacillus Calmette-Gue´rin (BCG) in a human subject. He approached Drs. Benjamin Weill-Halle´ and Raymond Turpin at the Hoˆpital Charite´, and, on 18 July 1921, the new vaccine was administered to a 3-day-old infant whose mother had just died of TB and who would be raised by its tuberculous grandmother. The infant lived and thrived. During the next 4 years more than 100,000 doses of BCG were administered, and the TB death rate in vaccinated children was thought to be reduced by 10-fold. BCG came into widespread use in Europe following the Second World War, and in December 1973 the World Health Organization Expert Committee on Tuberculosis issued its Ninth
History tells the story of things past. The future of TB must concern all of us now. Whatever the past may have taught us, we face a future challenged by rising TB incidence in much of the world. That HIV infection fosters the spread of TB and that multidrugresistant disease is an increasing problem add to the challenges and call for new initiatives. New drugs and a better vaccine are needed. The spread of TB is now being approached with new tools made possible by genetic typing of mycobacteria. What we learned by contact tracing has been augmented with new knowledge, for now it is possible to connect sources and targets of air-borne tubercle bacilli with great certainty. The contacts are not always obvious, and often are buried in the forgotten past. New approaches to the diagnosis of TB are being made possible by new knowledge of the antigens of mycobacteria. There is a vast panoply of these proteins; some are limited to specific species or strains, while others are widespread among members of the genus. ESAT-6, for example, is an antigen that has been lost from BCG, so it is possible to use the presence or absence of immune recognition of it to distinguish BCG infections from true tuberculous infection. Immunization has served since the time of Edward Jenner as a major weapon in the battle against infectious diseases. However, BCG has fallen short of initial hopes for controlling TB. As more is learned about the immunopathogenesis of TB, it is becoming more possible to target candidate vaccines against specific components of the tubercle bacillus, perhaps increasing protective efficacy. New vaccines are currently in development; field trials to assess their efficacy will pose large challenges. Drug discovery efforts have long neglected TB, but this has changed in recent years. New agents targeting new microbial receptors are being produced by pharmacologists now alerted to the challenges of multidrug-resistant TB. Research workers – immunologists, epidemiologists, microbiologists, pharmacologists, molecular biologists, experimental pathologists – are attacking these challenges. One can hope that expanding knowledge emanating from their laboratories will produce new and unanticipated tools for control of the ‘Captain among these Men of Death’.
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500 300 200 100
Vaccine efficacy (%) 0 20 40 50 60
70
80
90
Population Haiti British school children N. American Indians USA (Chicago infants) Puerto Rico gen. pop. S. India (Madanapalle) USA (Georgia + Alabama) S. India (Chingleput) USA (Illinois children) USA (Georgia children)
Controlled trials
900
Case control studies
Brazil (Sao Paulo) Brazil (Belo Horizonte) Argentina (Buenos Aires) Cameroun (Yaounde) Canada (Manitoba Indians) Indonesia (Jakarta) Burma (Rangoon) Sri Lanka (Colombo) Colombia (Cali) Argentina (Santa Fe)
Contact studies
´ Togo (Lome) Thailand (Bangkok)
Fig. 1.6 Summary of the estimates of the efficacy of BCG vaccine against TB in randomized controlled trails, case–control studies, and householdcontact studies with 95% confidence intervals. Reproduced with permission from Fine PEM. The BCG story: Lessons from the past and implications for the future. Rev Infect Dis 1989;11(suppl):S353–S359.
REFERENCES 1. Hayman J. Mycobacterium ulcerans: An infection from Jurassic time? Lancet 1984;2:1015–1016. 2. Gutierrez MC, Brisse S, Brosch R, et al. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PloS Pathog 2005;1:e5. 3. Kapur V, Whittam TS, Musser JM. Is Mycobacterium tuberculosis 15,000 years old? J Infect Dis 1994;170: 1348–1349. 4. Brosch R, Gordon SV, Marmiesse M, et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 2002;99:3684–3689. 5. Sola C, Filliol I, Gutierrez MC, et al. Spoligtype database of Mycobacterium tuberculosis: Biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerg Infect Dis 2001;7:390–396. 6. Hirsh AE, Tsolaki AG, DeReimer K, et al. Stable association between strains of Mycobacterium tuberculosis and their human populations. Proc Natl Acad Sci USA 2004;101:4871–4876. 7. Gagneux S, DeReimer K, Tran V, et al. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006;103: 2869–2873. 8. Cave AJE. The evidence for the incidence of tuberculosis in ancient Egypt. Br J Tuberc 1939; 33:142–152.
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9. Morse D, Brothwell DR, Ucko PJ. Tuberculosis in ancient Egypt. Am Rev Respir Dis 1964;90:5224–5241. 10. Morse D. Tuberculosis. In: Brothwell D, Sandison AT (eds). Diseases in Antiquity. A Survey of the Diseases, Injuries and Surgery of Early Populations. Springfield, IL: CC Thomas, 1967. 11. Nerlich AG, Haas CJ, Zink A, et al. Molecular evidence for tuberculosis in an ancient Egyptian mummy. Lancet 1997;350:1404. 12. Crube´zy E´, Ludes B, Poveda J-D, et al. Identification of Mycobacterium DNA in an Egyptian Pott’s disease of 5400 years old. C R Acad Sci III 1998;321:941–951. 13. Daniel VS, Daniel TM. Old Testament biblical references to tuberculosis. Clin Infect Dis 1999; 29:1557–1558. 14. Daniel TM. The early history of tuberculosis in central East Africa: Insights from the clinical records of the first twenty years of Mengo Hospital and review of the relevant literature. Int J Tuberc Lung Dis 1998;2:1–7. 15. Daniel TM. Captain of Death: The Story of Tuberculosis. Rochester, NY: University of Rochester Press, 1997. 16. Daniel TM. The origins and precolonial epidemiology of tuberculosis in the Americas: can we figure them out? Int J Tuberc Lung Dis 2000;4:395–400. 17. Coar T. The Aphorisms of Hippocrates with a Translation into Latin, and English. Birmingham, AL: Gryphon Editions, 1982. Sectio VIII. 18. Roberts CA, Buikstra JE. The Bioarchaeology of Tuberculosis. A Global View on Reemerging Disease. Gainesville, FL: University of Florida Press, 2003.
19. Bunyon J. The Life and Death of Mr. Badman. New York: R.H. Russell, 1900. 20. Krause AK. Tuberculosis and public health. Am Rev Tuberc 1928;18:271–322. 21. Priestly JB. Tom Moore’s Diary [a selection edited, with an introduction]. Cambridge: Cambridge University Press, 1925. 22. Dickens C. Nicholas Nickleby. London: Penguin Books, 1986. 23. Martinic M. The meeting of two cultures. In: McEwan C, Borrero LA, Prieto A (eds). Patagonia: Natural History, Prehistory and Ethnography at the Uttermost End of the Earth. Princeton, NJ: Princeton University Press, 1997: 110–126. 24. McKeown T. A historical appraisal of the medical task. In: McLachlan G, McKeown T (eds). Medical History and Medical Care: A Symposium of Perspectives. London: Oxford University Press, 1971: 29–55. 25. Wilson LG. The historical decline of tuberculosis in Europe and America: Its causes and significance. J Hist Med Allied Sci 1990;45:366–396. 26. Grigg ERN. The arcana of tuberculosis with a brief epidemiologic history of the disease in the U.S.A. Part III. Am Rev Tuberc Pulm Dis 1958;78: 426–453. 27. Sand G. George Sand and Chopin: A Glimpse of Bohemia [a letter written to M. Francois Rollinat dated March 8, 1838; trans. Lewis Buddy]. Canton, PA: The Kirgate Press, 1902.
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The history of tuberculosis 28. Doetsch RN. Benjamin Marten and his ‘new theory of consumptions’. Microbiol Rev 1978;42:521–528. 29. Major RH. Classic Descriptions of Disease, 3rd edn. Springfield, IL: CC Thomas, 1945. 30. Koch R. Die Aetiologie der Tuberculose [a translation by Berna Pinner and Max Pinner with an introduction by Allen K. Krause]. Am Rev Tuberc 1932;25: 285–323. 31. Edwards PQ, Edwards LB. Story of the tuberculin test from an epidemiologic viewpoint. Am Rev Respir Dis 1960;81(suppl):1–47. 32. Glasser O. Wilhelm Conrad Ro¨ntgen and the discovery of the roentgen rays. In: Glasser O (ed.). The Science of Radiology. Springfield, IL: CC Thomas, 1933:1–14.
33. Eisenberg RL. Radiology: An Illustrated History. St. Louis, MO: Mosby Year Book, 1992. 34. Williams FH. The Ro¨ntgen rays in thoracic disease. Am J Med Sci 1897;114:661–687. 35. Koch R. A further communication on a remedy for tuberculosis. Deutsche Medicinische Wochenschrift, November 15, 1890. English translation published in BMJ 1890;2:1193. 36. Brown L. The Story of Clinical Pulmonary Tuberculosis. Baltimore, MD: Williams and Wilkins, 1941. 37. Ryan F. The Forgotten Plague: How the Battle against Tuberculosis Was Won – and Lost. Boston, MA: Little, Brown, 1992. 38. Daniel TM. Leon Charles Albert Calmette and BCG vaccine. Int J Tuberc Lung Dis 2005;9:205–206.
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39. Comstock GW. Field trials of tuberculosis vaccines: how could we have done them better? Controlled Clin Trials 1994;15:247–276. 40. Lincoln EM. The effect of antimicrobial therapy on the prognosis of primary tuberculosis in children. Am Rev Tuberc 1954;69:682–689. 41. Ferebee SH. Controlled chemoprophylaxis trials in tuberculosis. A general review. Adv Tuberc Res 1970;17:28–106.
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2
Transmission of Mycobacterium tuberculosis Ashwin S Dharmadhikari and Edward A Nardell
INTRODUCTION AND HISTORICAL PERSPECTIVE Considering the centuries during which TB has been afflicting humanity, awareness that the disease is the result of a transmissible airborne infection is surprisingly recent. Although scientists such as Aristotle and his contemporaries believed that TB was contagious, they had no understanding of what caused it or exactly how it was spread. During the Middle Ages through to the seventeenth century, clinical practice and societal laws in Europe attested to the fact that the contagiousness of TB was suspected, if imperfectly understood. In Italy and Spain, cases of TB had to be reported to city authorities, and, when a patient with TB died, his personal belongings were burned in order to eradicate any traces of the disease that might spread to others. Moreover, physicians of the time, such as Valsalva and Morgagni, refused to perform autopsies on victims of TB, concerned that the procedure would spread the disease, as indeed it does.1 In the seventeenth century, however, a contrary view began to emerge. Eminent scientists and clinicians in Europe started to question whether TB was really transmissible. As proof, they offered observations that many healthcare workers who came into contact with TB patients did not themselves acquire the disease. Under situations ideal for transmission, they reasoned, how could TB possibly be considered a transmissible disease? Instead, noting the clustering of cases within families, they concluded that infection with TB was probably a hereditary phenomenon.1 Given the long latency periods that routinely occur between exposure and disease development, firm conclusions about transmissibility were understandably elusive. By 1910, decades after Koch discovered the tubercle bacillus, Chapin, in his influential book The Sources and Modes of Infection argued against the airborne mode of transmission of any organism: Bacteriology teaches that former ideas in regard to the manner in which diseases may be airborne are entirely erroneous; that most diseases are not likely to be dust-borne, and they are spray-borne for only 2 or 3 feet, a phenomenon which after all resembles contact infection more than it does aerial infection as ordinarily understood.2
Chapin did concede that, if any infection was airborne, it would likely be TB. But, in Thomas Mann’s 1927 novel The Magic Mountain, visitors to the fictitious alpine sanatorium lived with patients for weeks at a time with no apparent fear of contagion.3 As recently as 1932, Fishberg wrote that it was not risky for ‘healthy adults to be coughed at by patients suffering from pulmonary or laryngeal
8
tuberculosis’.4 The partial immunity acquired by infection early in life undoubtedly contributed to the misleading observation that many of those exposed did not develop disease. By 1947, the American Public Health Association (APHA) stated that ‘conclusive evidence is not available at present that the airborne mode of transmission is predominant for any particular disease’.5 Instead, it was postulated that TB might be spread by direct contact with infected sputum. Public health campaigns to discourage spitting were a logical consequence. The prevailing perception through the early 1900s, therefore, was that the airborne spread of TB beyond the immediate proximity of the source was unlikely. Yet even as many in the medical, public health, and scientific communities accepted Chapin’s view on airborne transmission, other researchers were working to better understand the propagation of infections, including TB. It was not until the late 1950s, however, a full decade after the APHA’s conclusion, cited above, that the tide of scientific opinion once again turned on this matter. Among the landmark investigations in this area were those of William Firth Wells, who while working as a sanitary engineer at Harvard University first conceptualized how certain infectious agents such as measles and TB might become airborne and travel from person to person. In his seminal 1934 paper entitled ‘On air-borne infections. II. Droplets and droplet nuclei’, Wells introduced the notion that transmissible infections fell into two major categories: those spread by local, direct, person-to-person contact and those spread more widely without direct contact by the airborne route.6 In 1931 Wells had developed an air centrifuge, a device that allowed him to concentrate airborne microorganisms from air samples. He had been commissioned by the Massachusetts Department of Health to investigate the aetiology of respiratory infections among textile mill workers. He speculated that the source of these infections was bacteria from contaminated standing water that had been aerosolized to keep dust down in the mill. Using his air sampler, he identified the same organisms in the air and in the standing water.7 The next intellectual leap was to theorize that aerosols produced by coughing and sneezing could also be responsible for person-to-person airborne transmission. He was assisted in this investigation by then Harvard medical student Richard Riley, with whom he shared credit for making the distinction between ordinary large respiratory droplets and so-called droplet nuclei – the dried residua of larger respiratory droplets. He ultimately published a comprehensive exposition of his theories and observations in his now classic 1955 text ‘Airborne contagion and air hygiene’, much of which remains valid to this day.8 Riley later updated and summarized this work in his 1961 monograph Airborne Infection and Control.9
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Transmission of Mycobacterium tuberculosis
The distinction between airborne and droplet-borne (contact) spread of infection blurs for some organisms, and a predominant mode is sometimes difficult to establish. The gold standard definition of airborne infection is transmission between individuals who have not had direct or indirect contact through contaminated fomites or surfaces. Smallpox, for example, is generally believed to be spread by direct contact with virus shed from cutaneous sores, but this cannot explain spread between patients on different floors of a German hospital where there was no direct or indirect person-to-person contact. A patient with respiratory mucosal involvement was believed to be responsible.10 Smallpox is now thought to be spread predominantly through large droplets, but with a poorly defined but definite potential for airborne spread. Smallpox is also considered a potential bioterrorism agent, presumably through aerosol attack. Several factors determine whether a given infectious agent travels from person to person through large or small respiratory droplets, and often both mechanisms appear to play a role. Besides smallpox, other infections with ambiguous or unclear modes of transmission include the common cold, Legionella, and influenza. In contrast, Mycobacterium tuberculosis (MTB) is almost exclusively spread by the airborne route, for several reasons peculiar to its pathogenesis. Most importantly, TB infection usually begins in the alveolar macrophage, the very cells intended to destroy invading pathogens. MTB has evolved mechanisms to evade killing by immunologically naı¨ve macrophages, which, ironically, provide the essential intracellular environment for its initial growth. The respiratory mucosa provides no such haven for MTB and is quite resistant to infection. The requirement for MTB to reach the alveoli of the peripheral lung essentially defines particles in the size range 1–3 mm. Such particles have so little inertia that they more likely evade impaction on the upper respiratory mucosa, which also means that they are light enough to have a negligible settling tendency in ordinary room air. Submicron particles are also generated by coughs and even quiet breathing, but are too small to contain tubercle bacilli or to settle on the alveolar surface. Larger respiratory droplets could contain several tubercle bacilli, but, unless they desiccate into droplet nuclei, they are less likely to reach the vulnerable outer reaches of the lung (Table 2.1). Wells detailed the increasingly rapid evaporation of larger into smaller particles as the ratio of surface area to mass increases, culminating in the dried residue of droplets, which he called droplet nuclei, as already noted. Another determinant of the transmission mode of a microorganism is the dose required to cause infection. For MTB, the dose is believed to be as little as a single organism, favouring its spread by minute airborne particles. For other infections such as some common bacterial pneumonias, host defences are more effective and doses larger than
2
can be carried by airborne particles dispersed in air may be required. People frequently experience microaspirations of mouth bacterial flora without ill effects, but, with gross aspiration containing larger numbers of organisms, pneumonia can result. It is believed that some respiratory pathogens, such as Pneumococcus and Staphylococcus, first colonize the upper respiratory tract where concentrations of organisms reach sufficiently high numbers to cause infection by aspiration. Most bacterial respiratory pathogens and many viruses appear to be spread by large respiratory droplets, justifying the contact precautions employed in hospitals and clinics. Perhaps the best way to prove that an infection is predominantly airborne is to successfully interrupt transmission by air disinfection alone. Wells showed unequivocally in the early 1940s that the transmission of measles was airborne and could be substantially interrupted in schools by the use of upper room ultraviolet germicidal irradiation (UVGI).11 Yet the experimental evidence that TB was airborne and could be interrupted did not emerge until studies conducted by Riley between 1958 and 1962 at the Baltimore Veterans Administration Hospital were published. Bringing to fruition a study design that had been conceived by Wells, Riley carried out a series of experiments using patients with active pulmonary TB (including untreated, treated, and drug-resistant cases) and highly susceptible guinea pigs to show definitively that TB was airborne and that its transmission could be interrupted by germicidal irradiation within the ventilation duct system. Riley used a six-bed hospital TB ward that was renovated in such a way that exhaust air from the ward passed through one or two penthouse exposure chambers containing hundreds of guinea pigs (Fig. 2.1). There was no other contact between the patients and the guinea pigs. Riley used guinea pigs because these animals were so exquisitely vulnerable to MTB that exposure to dilute artificial aerosols (which permitted inhalation of single infectious droplet nuclei) was sufficient to establish pulmonary infection. Over the course of the first 2 years of Riley’s study, an average of 150 guinea pigs per month breathed TB-infected air from the patient ward and subsequently became infected, as determined by conversion of their tuberculin skin test, with subsequent bacteriological or histological confirmation. The timing of guinea pig infections relative to patient occupancy on the ward combined with matching of drug susceptibility patterns of human and animal bacterial isolates allowed the infectious source of most of the guinea pig infections to be determined.12 Since the guinea pigs’ only exposure to TB was breathing the air ventilated to the animal chamber from the patient ward, this experiment proved that TB was an airborne infection. A second 2-year experiment was designed to show that other factors, such as contact with the animal handlers, were not responsible for the guinea pig infections.13 The initial
Table 2.1 Key distinguishing features of droplet nuclei and respiratory droplets Droplet nuclei transmission (airborne infection)
Respiratory droplet transmission (an extension of direct contact)
1- to 5-mm-diameter particles (dried residua of larger particles) Remain suspended indefinitely Alveolar deposition Contain few microbes UVGI susceptible in air Examples: TB, measles
>100-mm-diameter particles Settle out within 1 m of the source Upper airway deposition Contain many microbes UVGI resistant on surfaces Examples: Staphylococcus respiratory syncytial virus
UVGI, ultraviolet germicidal irradiation. Rom & Garay, Tuberculosis, 2nd edition. Lippincott Williams and Wilkins, 2003.
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Rights were not granted to include this content in electronic media. Please refer to the printed book.
Fig. 2.1 A schematic diagram of the hospital unit, ventilation ducts, and guinea pig exposure chambers used in the Riley experiments. Riley RL, Wills FW, Mills CC, et al. Air hygiene in tuberculosis: quantitative studies of infectivity and control in a pilot ward. Am Rev Tuberc Pulmonary Dis 1957;75:420–431. # American Thoracic Society.
2-year experiment was repeated with half the ward air being heavily irradiated with UVGI before reaching the guinea pigs. Despite the same handlers and other conditions, no infections occurred in the guinea pigs protected by germicidal UV. Additionally, Riley and colleagues were able to show that patients on the ward varied greatly in infectiousness, that drug-resistant strains were less infectious, and that effective TB treatment rapidly reduced infectiousness.
EPIDEMIOLOGICAL DATA ON THE DETERMINANTS OF TUBERCULOSIS TRANSMISSION Successful passage of the TB bacillus from one person to the next requires an infectious source case, a virulent microorganism, a vulnerable host, and favourable environmental conditions. Upon closer inspection, there are complex factors that contribute to this process, including factors that determine the strength and infectivity of the source, the integrity of the host defences of the exposed individual, intrinsic properties of the bacillus itself, including viability, vulnerability or resistance to environmental stressors, virulence factors, genetic mutations, drug resistance, and virulence for a particular host. Figure 2.2 depicts this model of TB propagation. 1. Source factors: Smear + Cough strength and frequency Lung cavitation Effective treatment
Source
2. Environmental factors: Ventilation, room volume, humidity, UV 3. Microbial factors Genetic virulence Airborne M.tb
Fig. 2.2 TB transmission factors.
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Host
An important caveat to this biomedical model, however, is that it only indirectly alludes to some of the biosocial influences that have been associated with TB incidence and prevalence in many parts of the world, such as socioeconomic status, access to healthcare, or crowded living conditions. Nonetheless, the simplified model in Fig. 2.2 helps to frame a discussion of the ways in which the public health, scientific, engineering, and medical communities may understand and attempt to interrupt MTB propagation. Within both high and low TB prevalence areas, high rates of transmission have occurred in congregate settings such as hospitals, clinics, prisons, refugee camps, schools, and shelters. Although the average concentration of airborne TB infectious droplet nuclei in such settings has been estimated to be quite low, there appear to be situations in which airborne concentrations are transiently quite high or contact time of vulnerable individuals is sufficiently long to account for high rates of transmission. In hospitals, for example, the risk of TB transmission to healthcare workers (HCWs) remains an issue in both high- and low-prevalence areas. Ironically, concern has generally been much higher in low-prevalence, resource-rich settings where the risk is generally low and quite unpredictable, often from unsuspected source cases. However, implementation of existing international infection control guidelines is especially urgent in resource-limited settings where an already stretched pool of HCWs, many human immunodeficiency virus (HIV)-infected, is suffering from attrition due to TB or fear of acquiring drug-resistant disease.14,15 The risk of transmission and progression to active TB disease is also extremely high for patients, prisoners, and refugees in regions where both HIV and MTB infections are common. Of all congregate settings, the risk of TB transmission for HCWs has been especially well documented in the medical literature. In the 1930s and 1940s, Israel, a lung specialist, carried out an epidemiological study in which he tracked nurses and physicians working in an urban Philadelphia hospital over several years for TB exposure and infection, using a tuberculin skin test (TST) reaction of 5 mm induration as the criterion for infection.16 At the time of joining the hospital staff, approximately one out of three nurses and physicians had a positive TST. Since these individuals had not yet had occupational TB exposure, Israel used this rate of TST positivity to represent the latent infection rate from household exposure for the socioeconomic group from which these nurses and doctors hailed. Israel then tracked annual rates of TST positivity and conversion over 3 years for the cohort and found that, by the end of 3 years, nearly all the nurses and doctors had become TST positive and that about 10% had developed clinical TB disease. He surmised that this rate of TST conversion represented an estimate of occupational exposure intensity to unsuspected TB cases in an urban hospital. He emphasized exposure to unsuspected cases of TB because the standard of care at that time was to rapidly transfer known or suspected TB cases to specialized sanatoria for long-term care. More contemporary studies of the epidemiology of latent TB infection show that while 5% of the US population is TST positive, HCWs in urban American hospitals have a 20–30% TST positivity rate, whereas their counterparts in US community hospitals have just slightly lower TST positive rates.17 A study by the US Centers for Disease Control (CDC) has shown that TST conversion rates among US HCWs is 1% annually – deemed unacceptably high in a low-prevalence country. However, a carefully done, multicenter, prospective study of skin test conversions among HCWs conducted by the CDC found weak correlation with patient exposure, but a strong correlation with foreign birth
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Transmission of Mycobacterium tuberculosis
and BCG vaccination, suggesting lingering skin test boosting as a cause of skin test conversion despite base-line two-step testing intended to minimize the effects of boosting.18 However, other studies have documented higher than average risks among certain categories of HCWs, including respiratory therapists, pathologists, TB laboratory workers, nurses, and physicians.19 Among doctors, one study found that fellows in training who did bronchoscopy, a cough-generating procedure, were at much higher risk than colleagues who did not, such as infectious disease fellows, even though both specialists saw patients with TB.20–34 Between 1985 and 1992, there was a resurgence in the rates of TB in North America and western Europe, largely attributable to outbreaks in congregate settings such as homeless shelters, residential facilities for patients with acquired immunodeficiency syndrome (AIDS), prisons, and healthcare facilities and to a breakdown in public health TB control programmes.35–42 Using restriction fragment length polymorphism fingerprinting (RFLP) data and drug resistance patterns to determine which strains were represented in the source cases and the secondary infections in these outbreaks, it was shown that up to 30–40% of the observed cases of TB in New York City, for example, were due to recently transmitted infection rather than the result of reactivation of old foci of infection.43 Prior to these data, it was commonly believed that the large majority of new cases of TB in low-prevalence settings like the United States were due to reactivation disease rather than recent transmission. However, with advances in epidemiological and molecular investigations it became clear that well-localized and recent transmission in congregate settings contributed significantly to the rise in TB rates. After the epidemic came under control, Freiden et al.44 considered improved infection control as well as improved treatment to have been essential factors. In 1994, in response to the rise in TB rates and the emergence of multidrug-resistant TB (MDR-TB), the CDC published comprehensive guidelines for improved control of institutional TB transmission.45 The guidelines promoted a three-tiered hierarchical control strategy adapted from industrial hygiene: administrative, environmental/engineering, and personal protective equipment. These strategies are discussed in detail later in this book in chapters on TB infection control.
HUMAN SOURCE FACTORS Some patients with TB appear to be much more infectious than others based on the investigation of their contacts. Substantial epidemiological, clinical, and experimental data support these observations. Beyond a few conventional criteria, however, there are still generally no reliable ways to predict which patients will be more or less infectious to others. Those conventional criteria include the presence of lung cavities on chest radiographs; active, forceful coughing; sputum smears that are positive for acid-fast bacilli; and absence of effective drug treatment. These same criteria are generally used by hospitals to determine who gets isolated and how long such isolation lasts. While a review of the immunopathology of TB infection is beyond the scope of this chapter and is covered elsewhere in this textbook, a few concepts deserve mention here. Dannenberg46 has argued that lung cavitation, the end result of liquefaction necrosis due to delayed type hypersensitivity (DTH), is the event responsible for ongoing propagation of MTB in human populations. It has been estimated that lung cavities may harbour 108–109 organisms in the extracellular debris
2
associated with liquefaction necrosis. When the caseous foci of pulmonary TB rupture into a bronchi, countless MTB gain access to the large airways, and thus to the outside environment. In the prechemotherapy era, TB physicians focused on cavity formation as a sentinel event portending a downhill clinical course, presumably due to a larger bacterial burden, chronic cough, reduced lung function, and also a propensity to haemorrhage – the most feared and often fatal event. Rest as well as surgery and a variety of mechanical collapse therapies were all intended to effect cavity closure, with mixed success. In theory, if it were possible to prevent lung cavitation while retaining the beneficial aspects of DTH and acquired cell-mediated immunity (CMI), transmission would be lessened. The predisposition to drug resistance associated with spontaneous mutations occurring among such large numbers of organisms would also be lessened (see Microbial Factors, below). While HIV coinfection is associated with less lung cavitation (i.e. less DTH), it is also associated with less CMI, and large numbers of organisms still occur in lung tissue, associated with rates of transmission not much different than among patients without HIV and with lung cavities.47 The site of TB disease also determines whether and how infectious a source case may be. In his classic air-sampling study, Riley13 observed that one patient with laryngeal TB infected 15 guinea pigs during the 3 days he resided on the experimental ward, far more than other patients with pulmonary TB even though they resided on the ward for longer periods of time. Tuberculosis disease in other parts of the body, such as the bone and kidneys, for example, is normally not infectious because these sites do not communicate with the environment. However, in certain circumstances even extrapulmonary TB can be infectious, as illustrated by the case of a thigh abscess that was drained and then irrigated with a high-pressure water jet, or several reports of autopsies associated with extensive spread of infection, presumably the result of aerosol-generating procedures.21,30 Fennelly and colleagues have focused on the human infectious source determinants of transmission (Fig. 2.2). Building upon earlier reports by Loudon48 that overnight cough frequency of TB patients correlated with the rates of transmission to household contacts, Fennelly49 examined differences in the rates of culturing airborne MTB using a novel cough aerosol-sampling system. Effective treatment, he reported, was associated with a decline in the number of cough-generated aerosol cultures from samples obtained from MDR-TB patients within the first 3 weeks of treatment. However, among 12 sputum smear-positive patients who were evaluated, only four cough samples yielded positive cultures, reinforcing the notion that factors beyond sputum smear status alone are likely playing a role in transmission. He also measured the size distribution of cough-generated, culturable particles, confirming in his experimental system earlier animal experiments that showed that 1- to 2-mm particles were those most likely to serve as vehicles for MTB.49,50 Source factor determinants require further research. For example, it is generally believed that patients who have negative sputum smears are likely to be less infectious than those with a sputum that is smear positive. In clinical practice, two or three negative sputum smears are used as the main criteria for ending respiratory isolation. Yet, as shown by Fennelly, only a fraction of sputum smear-positive patients generate culturable aerosol. Other, poorly understood transmission factors are likely at work. Differences in sputum quality, for example, might influence transmission. Less viscous sputum might be more easily aerosolized into droplet nuclei than thick sputum. The physical or chemical properties of respiratory secretions could also influence transmission by shielding MTB organisms from injury during the violent process of aerosolization
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and airborne transport through a generally hostile environment. A better understanding of such factors might permit a more accurate assessment of patient infectiousness, or lead to novel infection control interventions, such as agents to modify sputum physical characteristics in order to reduce respiratory particle generation rate, or change particle size.51 Little is known of the effects on MTB transmission of ambient temperature, humidity, oxygen tensions, irradiation, or air pollution52,53 A better understanding of these factors might lead to interventions to render the environment less favourable for MTB survival during transport.54 A final source factor that requires mention is treatment status. With effective treatment, most patients become non-infectious within about 2–3 weeks.55–58 On Riley’s experimental ward, patients on at least 2 weeks of effective treatment were less likely to infect guinea pigs. In Madras, India, additional household converters ceased after about 2 weeks of effective treatment.59 These findings support current respiratory isolation guidelines. However, when therapy has been ineffective, as occurred during the 1985–1992 TB resurgence when patients with unsuspected and inadequately treated MDR-TB were prematurely released from isolation, transmission continued. Effective disease treatment remains the single most important intervention to prevent MTB transmission.
MYCOBACTERIAL FACTORS, STRAIN DIFFERENCES, AND INTERACTIONS WITH SOURCE, ENVIRONMENT, AND HOST As mycobacteria multiply during the course of infection, they acquire point mutations, frame shift mutations, deletions, and genetic sequence insertions as a result of chance DNA transcriptional errors and recombinations. As discussed earlier, there may be as many as 108 or 109 organisms within a lung cavity, and, within this population, genetic mutations may occur at a rate of 1 in 107 to 1 in 1010 per bacterium per generation.60 These genetic mutations can be associated with a variety of phenotypic outcomes, including alterations in drug resistance, nutrient uptake, metabolism, or interaction with host immune cells, that is, altered virulence. How often do these mutations result in reduced mycobacterial fitness, how often do compensatory mutations occur that restore fitness to the wild-type state, and how important are these microbial determinants of infection or disease amid the many other factors depicted in Fig. 2.2? These are among the most important questions being addressed by molecular epidemiological and laboratory researchers. Anecdotal observations, combined with molecular typing data, suggest that some strains propagate more readily or are more ‘fit’ than others. In the natural history of TB infection, there are several stages that might be altered by genetic mutations that could influence the propagation of TB, including the ability of the bacillus to:
12
initiate infection as measured by an immune response, i.e. infectivity and DTH stimulation; evade the host immune system and replicate within a host and cause disease, i.e. virulence; disseminate haematogenously and to cause lung cavitation, i.e. virulence and DTH-induced tissue destruction; and travel between hosts, i.e. transmissibility, including resistance to the stresses of aerosolization and airborne transport.
In 1998, Valway et al.61 reported on the results of a contact investigation in the setting of an unusual outbreak of TB. Interestingly, while the rate of skin test conversion was unusually high and the reactions unusually large among exposed individuals, the rate of disease development was lower than expected, suggesting that this strain of TB was highly infectious, a strong stimulator of DTH, but not particularly virulent. Conversely, the strong induced host response itself might explain the lower rate of disease progression. Several decades earlier, Middlebrook and Cohn62 described transmission patterns for isoniazid (INH) susceptible and resistant strains of MTB among guinea pigs exposed via the aerosol route. INH resistant strains were less virulent for the guinea pigs than the susceptible strains, as evidenced by smaller and less numerous foci of infection on autopsy, fewer organs involved, a milder clinical course, and prolonged survival. Early on, researchers attributed the loss of virulence of INH resistant strains to a loss of catalase activity, the enzyme responsible for metabolizing INH from a prodrug to its metabolically active form.63–65 Subsequent work has found associations between the type of KatG mutation responsible for catalase activity and the apparent virulence of the organism. As an example, it was shown that, although both point mutations and deletions in the gene sequence disrupt the production of catalase, the deletions confer a more profound reduction in catalase levels, a higher degree of resistance to INH, and a more marked attenuation in virulence than point mutations.66 In contrast to these observations for INH mutations, mutations which lead to pyrazinamide resistance alone tend not to alter the fitness of MTB.67 Whether mutations which contribute to drug resistance necessarily reduce the reproductive fitness of the MTB has recently been the subject of considerable debate. Ordway et al.68 examined 15 different clinical isolates of MTB, some of which were fully drug sensitive, INH resistant, or multidrug resistant. They found that, although growth rates in experimentally infected mice varied for each of the 15 isolates, these growth rates did not correlate with the extent of drug sensitivity or resistance.68 Likewise, Cohen and colleagues69 recently evaluated epidemiological data available on transmission of MDR-TB using mathematical models to determine whether drug resistance mutations also result in a loss of reproductive fitness. In their analysis they conclude that the fitness of drug-resistant strains is quite heterogeneous and that attempting to discover an average degree of reproductive fitness for MDR-TB upon which to base programmatic and policy decisions may be misleading.69 Instead, they argue, it is important to determine the distribution of highly resistant and less resistant strains, so that outbreaks can be averted and handled appropriately. As a real-life example of this heterogeneity, we might consider another contact investigation conducted by Texeira et al.70 in Brazil, who evaluated rates of TST conversion among household contacts of index cases of MDR-TB. They found no association between the degree of drug resistance and rates of TST conversion or disease development among the household contacts, even though the drug-resistant index cases were infectious for a longer period of time than the drug-sensitive cases in their study. Using IS6100 pattern molecular typing techniques, they also confirmed that the secondary cases of TB among household contacts were from the same strain as the index case for any given household, thereby eliminating any doubt that secondary cases in a household were from a non-household exposure.70 As discussed elsewhere in this volume, developments in the molecular biology and genetics of MTB have permitted closer
CHAPTER
Transmission of Mycobacterium tuberculosis
analysis of the interplay of genes, proteins, and bacterial enzymes in the life cycle of this organism. We mention this here only in relation to transmission. Mycobacterial growth in the face of exogenous stressors has been shown to be dependent upon the elaboration of gene products called sigma factors, which regulate defence regulons within the mycobacterial genome. Laboratory studies in which several sigma factors were mutated demonstrated that mutation of the sigma factors had no impact on the ability of the H37Rv strain of mycobacterium to grow in culture dishes or within in vitro macrophage systems, but had a substantial impact on the in vivo growth and replication of H37Rv within a guinea pig infection model. Among the factors studied, mutations in SigC affected the adaptive survival of H37Rv in guinea pigs more than mutations in SigF.71
HUMAN HOST FACTORS WHICH AFFECT VULNERABILITY TO TUBERCULOSIS INFECTION Once the TB bacillus finds an entry into the human lung, its interaction with the host immune system and the impact of the host’s other comorbid medical conditions ultimately determines whether infection is established and whether it progresses to clinical disease. Substantial immunological research has begun to elucidate key steps in the human immune system’s attempt to contain and eliminate TB. Simply stated, once MTB breaches the structural defences of the upper airways and reaches the distal lung, innate mechanisms involving macrophages are the first defence against infection, followed by the acquired CMI and DTH, which together usually contain further spread of infection within the host. Defects in any portion along this elaborate and still incompletely understood defence pathway could render a host more vulnerable to MTB infection, thereby enhancing transmission. Compared with what is known about the immune defences operative during active TB, relatively little is known about the immune response that contains latent MTB infection. Similarly, the events that trigger reactivation of latent MTB infection are poorly understood. CD4 T cells certainly have an important role in this process, since numerous studies document the higher rates of reactivation in HIV-infected individuals.72–74 There is debate, however, about whether HIV infection increases the risk of acquiring TB infection in the first place since this appears to be predominantly determined by macrophage function.75 Exposure to both silica dust and silicosis increases the rate of reactivation TB.76–78 Inhaled silica particles damage alveolar macrophage cell membranes, the same cells which MTB typically encounters upon inhalation and deposition in the distal lung. Allison and Hart79 showed that sublethal doses of silica enhanced the growth of MTB in macrophage cultures, and guinea pig exposure studies revealed that inhalation of quartz (a chief component of silica dust) reactivated TB lesions that were healing.80 Still other studies suggest that humoral and cell-mediated immunity may also be altered in silica exposure.81 One would expect silica exposure to enhance MTB transmission as well as disease progression. It is known from human postmortem studies in which single isolated tubercles are found in the lungs that even a single inhaled droplet nucleus is sufficient in some individuals to initiate MTB infection. Yet as seen by TST or interferon gamma release assay (IGRA) results, not all exposed individuals acquire TB infection, even after comparable types of exposures. Observations like these
2
underscore the variability in human susceptibility to TB, which is generally attributed to differences in immune function, genetics, and concomitant health conditions that predispose to infection and disease. Epidemiological studies by Stead and colleagues82,83 specifically suggest an enhanced risk of MTB infection, but not disease reactivation, associated with ethnic or racial background. The authors maintained that these factors were surrogates for the historical selection of populations with innate MTB resistance resulting from the evolutionary pressure of the several hundred-year-old TB epidemic in Europe and North America. Persons in central Africa, Aboriginals, and others isolated from this epidemic pressure remained vulnerable because there was no such selection.82,83 It has long been an accepted tenet of medicine that, once infected with MTB, the latent infection is lifelong, with a small but persistent risk of reactivation to active disease. Occasional observations of skin test reversions in the absence of anergy, with or without treatment, had been dismissed as the exceptions to the rule. However, these observations are now supported by reversions in IGRA results. The possibility that transient MTB infection may be part of the pathogenesis of the disease has several implications for transmission. First, skin test surveys may greatly underestimate transmission in populations since infected persons may have already reverted their skin test or IGRA back to negative by the time they were tested. Second, if infection can be transient, progression to disease may depend on reinfection to a degree not previously suspected. Indeed there is evidence that what appears to be high rates of reactivation TB disease among recent arrivals to the United States from high-prevalence areas may in fact represent recent infection or reinfection.84 Finally, if natural MTB infection is often transient, and reinfection is important to pathogenesis, then perhaps vaccines may not be as beneficial as hoped.
MATHEMATICAL MODELLING OF M. TUBERCULOSIS TRANSMISSION With few exceptions, every case of TB results from the airborne transport of MTB from an infectious source to a vulnerable host. Primarily an intracellular pathogen, MTB is adapted to replicate extracellularly in necrotic lung cavities, to endure the rigors of aerosolization in mucus as tiny respiratory droplets, to survive the process of rapid drying into droplet nuclei, and to remain infectious after airborne transport through a variety of harsh environments. Potentially lethal environmental exposures include the extremes of air temperature and humidity, ambient levels of air pollution, and natural ozone and irradiation (Fig. 2.2). Little is known about these adaptive processes, but the selective pressure is strong since only adapted strains can propagate. Finally, organisms surviving the gauntlet of airborne transmission face innate and adaptive host defences honed, in some human populations, by generations of natural selection. Virulence, the ability of the pathogen to overcome host defences and cause infection, is also selected for by the necessity of transmission. Microbes, of course, adapt much faster than can humans, even to the antimicrobials invented to tilt the contest in our favour. The mathematical modelling that follows attempts to quantify the relationship between the infectious doses generated by the source case(s) and the number of infected humans, but ignores the fact that most tubercle bacilli released from the source case are unlikely to cause infection due to factors cited earlier. In addition, probability plays a role in whether a vulnerable host inhales an infectious dose of MTB.
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In an attempt to characterize the transmission process as a probability that a vulnerable host will acquire TB infection, Wells and Riley (Richard’s brother, Edward, an engineer) developed a mathematical model that expands upon and modifies the Soper mass balance equation for epidemiological investigations, incorporating the following operational assumptions: 1. steady-state conditions in which the infectious source is constant; 2. complete air mixing within a defined volume or space being studied; 3. equal susceptibility among exposed individuals to infection; 4. Poisson’s law of small chances which employs the natural logarithm e; 5. uniform virulence of organisms released into the air space; and 6. random distribution of infectious particles within a defined space. This model states that for a single generation of infection: C ¼ Sð1 eIqpt=Q Þ; where: ¼ number of new cases, ¼ number of susceptibles exposed, ¼ natural logarithm, ¼ number of infectious sources, ¼ number of quanta (infectious doses) generated per unit time in minutes, p ¼ human ventilation rate in L/minute, t ¼ exposure duration, and Q ¼ infection-free ventilation in the room in L/second. C S e I q
Although this model applies to situations that meet the assumptions above (i.e. a single room or enclosed space with a defined ventilation), it can be applied, although with less validity, to spaces served by a single central ventilation system (i.e. HVAC system) and whose air spaces are connected – an essential component for infections which are airborne. The larger the space being considered, the less evenly distributed (mixed) airborne particles may be, thus also potentially reducing the validity of this model for that air space. Despite these limitations, the model has been useful in examining the relative importance of transmission factors in real-life exposure situations. In epidemiological investigations of epidemics in which C, S, I, p, t, and Q were known or estimated, values for q could be calculated as a representation of the infectiousness of the index source case. This was done for a few scenarios of TB transmission: the patients
on Riley’s TB ward in the 1950 experiment with guinea pigs, an office outbreak, and a bronchoscopy case on a patient with TB in an intensive care unit (Table 2.2). What follows is a discussion of how the Wells–Riley equation has been used to compare the intensity of exposure to TB and how it can inform approaches to reducing transmission. In the first situation, Riley’s TB patients were receiving TB drug treatment and the infectiousness values represent that of the entire six-bed ward. In the second scenario, a 30-year-old woman returned to work in a welfare office for an additional month before she was diagnosed with cavitary, sputum smear-positive TB, at which time contact with fellow employees ended.85 Of 67 co-workers who were initially tuberculin skin test negative, 27 (40%) had conversions to positive upon repeat testing 3 months later. One non-infectious secondary case resulted in a worker who had declined treatment of latent infection. The office building had been the subject of repeated air quality complaints, and several air quality assessments had been done before and after the TB exposure. A mathematical analysis of the exposure was prompted by the suspicion of several workers that inadequate ventilation was responsible for the large number of infected workers. All values of the Wells–Riley equation were known or estimable except q, the number of infectious quanta generated by the source case. By calculation, the source generated 13 infectious quanta per hour. Further calculations showed that outdoor air ventilation at the low end for acceptable air quality (15 cubic feet per minute (cfm) per occupant, based on average room CO2 values of 1000 ppm) contributed to transmission. However, the model was useful also for indicating that doubling the ventilation would reduce the risk of infection by approximately half (Fig. 2.3). Thirteen workers would still have been infected, according to the model. Moreover, an additional doubling of ventilation, to 60 cfm per occupant, would again reduce the risk by half, leaving approximately six workers unprotected. Both the potential of a moderately infectious patient to infect many contacts over a prolonged period of time and the limitations of building ventilation to prevent transmission were demonstrated, within the assumptions and limitations of the model. For the third scenario, Catanzaro86 applied the Wells–Riley equation to an episode of transmission in an intensive care unit where an unsuspected patient, initially smear negative for TB, underwent intubation and bronchoscopy. During the 2½ hours of the procedures, 10 of 13 susceptible room occupants became infected. By calculation, the source produced a remarkable 250 infectious quanta per hour. However, the ventilation rate in the intensive care unit was extremely low, and further calculations predicted substantial improvements by increases in ventilation that were
Table 2.2 Transmission factors and exposure conditions for three different scenarios using the modified Wells–Riley equation Factors
VA hospitals
Office buildings
Bronchoscopy
Source (I) Exposure time (t) Susceptibles (S) Air sampled Infected (C) Ventilation (Q) Infectivity (q) Concentration
6 patients 730 days 120 guinea pigs 693,000 cf 63/120 (52%) 38 cfm/person 1.25 qph 1/11,000 cf
1 patient 6.7 days 67 people 230,000 cf 27/67 (40%) 15 cfm/person 13 qph 1/8500 cf
1 patient 150 min 13 people 688 cf 10/13 (77%) 11.5 cfm/person 249 qph 1/70 cf
Source: Friedman: Tuberculosis: Current Concepts and Treatment, 2nd edition. Informa Healthcare, 2000.
14
CHAPTER
Transmission of Mycobacterium tuberculosis The effect of ventilation on the probabilty of infection
PProbabilty of infection (%)
100 80
P=1q
60 40 Nardell
0
Catanzaro
0
2000 4000 6000 Q Germ-free ventilation (cfm)
8000
Fig. 2.3 Two different examples of transmission and the effect of
ventilation. # Joint Commission Resources: Joint Commission Journal on Quality and Patient Safety. 20(1):25–31, 1993. Reprinted with permission.
achievable (Fig. 2.3). The episode illustrates the ever-present risk of the unsuspected case, the wide range of infectivity, and the potential for environmental controls to greatly reduce transmission when existing ventilation is low.
FUTURE PROSPECTS AND OTHER IMPORTANT CONSIDERATIONS IN INFECTION CONTROL (BOX 2.1) Although the basic mechanism of MTB transmission has been understood for more than 50 years, there have been no real advances in infection control over at least that long. Such advances will likely require renewed interest and new research on the basic aerobiology of MTB. Each component shown in Fig. 2.1 is a potential target for novel infection control innovations. For example, new interventions may reduce the production of airborne particles or reduce their viability. New environmental interventions may inactivate MTB in its vulnerable airborne state. New
REFERENCES 1. Sepkowitz KA. Tuberculosis and the health care worker: a historical perspective. Ann Intern Med 1994;120(1):71–79. 2. Chapin C. Infections by air. In: The Sources and Modes of Infection. New York: Wiley; 1910. 3. Mann T. The Magic Mountain. New York: Knopf; 1924. 4. Fishberg M. Pulmonary Tuberculosis, 4th edn. Philadelphia: Lea and Febiger, 1932. 5. Subcommittee for the Evaluation of Methods of Control of Air-Borne Infections. Present status of the control of airborne infections. Am J Public Health 1947;37:13–22. 6. Wells WF. On air-borne infection. II. Droplets and droplet nuclei. Am J Hyg 1934;20:611–618. 7. Wells WF, Riley EC. An investigation of the bacterial contamination of the air of textile mills with special reference to the influence of artificial humidification. J Indust Hyg Toxicol 1937;19(10):513–561. 8. Wells WF. Airborne Contagion and Air Hygiene. Cambridge, MA: Harvard University Press, 1955.
Box 2.1 Infection control strategies in relation to source, environmental, and host transmission factors Infection control is covered in a separate chapter. Here, for completeness, we list how the traditional control and prevention strategies fit into the source, organism, environment, and host model that we have used.
·lqpl/Q
20
2
Control measures focused on the infectious source Administrative measures: case surveillance, triage, isolation, and diagnosis. Case treatment, both pharmacological and surgery in selected cases. Contact investigation and treatment of LTBI. Masks on patients. Cough suppression. Control measures focused on the organism Case treatment. Drug susceptibility testing. Control measures focused on the environment Air disinfection by ventilation, filtration, or germicidal irradiation. Dilution. Isolation. Control measures focused on the host Immunization—preventive. Immunomodulation—boosting already infected persons to better control the pathogen. Nutrition. Antiretroviral therapy for HIV coinfection. Respiratory protection.
vaccines and administration routes may reduce the chance of infection or reinfection. A major challenge, however, is that TB is predominantly a disease of resource-limited countries. Innovations must be effective and they must also be affordable where they are most needed. Finally, greater efforts are needed to increase awareness of the importance of transmission, and to implement existing guidelines as well as future novel interventions to prevent transmission. The convergence of the HIV and TB epidemics, including drug-resistant strains, in many parts of the world is providing a strong incentive to end the neglect of this vital aspect of TB control, but much work remains to be done.
9. Riley RL, O’Grady F. Airborne Infection. New York: Macmillan, 1961. 10. Wehrle PF, Posch J, Richter KH, et al. An airborne outbreak of smallpox in a German hospital and its significance with respect to other recent outbreaks in Europe. Bull World Health Organ 1970;43(5): 669–679. 11. Wells WF, Wells MF, Wilder TS. The environmental control of epidemic contagion. I. An epidemiologic study of radiant disinfection of air in day schools. Am J Hyg 1942;35:97–121. 12. Riley RL, Mills CC, Nyka W, et al. Aerial dissemination of pulmonary tuberculosis. A two-year study of contagion in a tuberculosis ward. Am J Hyg 1959;70:2. 13. Riley RL, Mills CC, O’Grady F, et al. Infectiousness of air from a tuberculosis ward. Ultraviolet irradiation of infected air: comparative infectiousness of different patients. Am Rev Respir Dis 1962;85:511–525. 14. WHO. Guidelines for the Prevention of Tuberculosis in Health Care Facilities in Resource-Limited Settings. Geneva: World Health Organization, 1999.
15. WHO. International Migration of Health Personnel: A Challenge for Health: Eight Plenary Meeting Committee A. Geneva: World Health Organization, 2004. 16. Israel H, Hetherington H, Ord J. A study of tuberculosis among student nurses. JAMA 1941;117:839. 17. Nardell E, Sepkowitz K. Chapter 56: Tuberculosis transmission and control in hospitals and other institutions. In: Rom WN, Garay SM (eds). Tuberculosis, 2nd edn. Philadelphia: Lippincott Williams & Wilkins, 2004. 18. Panlilio AL, Burwen DR, Curtis AB, et al. Tuberculin skin testing surveillance of health care personnel. Clin Infect Dis 2002;35(3):219–227. 19. Daniels M, Ridehalgh F, Springett VH. Tuberculosis in Young Adults: Report on the Prophit Tuberculosis Survey. 1936–1944. London: HK Lewis, 1948. 20. Lundgren R, Norrman E, Asberg I. Tuberculosis infection transmitted at autopsy. Tubercle 1987;68(2):147–150. 21. Templeton GL, Illing LA, Young L, et al. The risk for transmission of Mycobacterium tuberculosis at the bedside and during autopsy. Ann Intern Med 1995;122(12):922–925.
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SECTION
1
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22. Meade G. The prevention of primary tuberculous infections in medical students: The autopsy as a source of primary infection. Am Rev Tuberc 1948;58:675–683. 23. Kantor HS, Poblete R, Pusateri SL. Nosocomial transmission of tuberculosis from unsuspected disease. Am J Med 1988;84(5):833–838. 24. Collins CH, Grange JM. Tuberculosis acquired in laboratories and necropsy rooms. Commun Dis Public Health 1999;2(3):161–167. 25. Malasky C, Jordan T, Potulski F, et al. Occupational tuberculous infections among pulmonary physicians in training. Am Rev Respir Dis 1990;142(3):505–507. 26. Blumberg HM, Sotir M, Erwin M, et al. Risk of house staff tuberculin skin test conversion in an area with a high incidence of tuberculosis. Clin Infect Dis 1998;27(4):826–833. 27. Do AN, Limpakarnjarat K, Uthaivoravit W, et al. Increased risk of Mycobacterium tuberculosis infection related to the occupational exposures of health care workers in Chiang Rai, Thailand. Int J Tuberc Lung Dis 1999;3(5):377–381. 28. Eyob G, Gebeyhu M, Goshu S, et al. Increase in tuberculosis incidence among the staff working at the Tuberculosis Demonstration and Training Centre in Addis Ababa, Ethiopia: A retrospective cohort study (1989–1998). Int J Tuberc Lung Dis 2002;6(1):85–88. 29. Fagan MJ, Poland GA. Tuberculin skin testing in medical students: a survey of US medical schools. Ann Intern Med 1994;120(11):930–931. 30. Hutton MD, Stead WW, Cauthen GM, et al. Nosocomial transmission of tuberculosis associated with a draining abscess. J Infect Dis 1990;161(2):286–295. 31. Kassim S, Zuber P, Wiktor SZ, et al. Tuberculin skin testing to assess the occupational risk of Mycobacterium tuberculosis infection among health care workers in Abidjan, Cote d’Ivoire. Int J Tuberc Lung Dis 2000;4(4):321–326. 32. Kruuner A, Danilovitsh M, Pehme L, et al. Tuberculosis as an occupational hazard for health care workers in Estonia. Int J Tuberc Lung Dis 2001;5(2):170–176. 33. Silva VM, Cunha AJ, Oliveira JR, et al. Medical students at risk of nosocomial transmission of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2000;4(5):420–426. 34. Wilkinson D, Gilks CF. Increasing frequency of tuberculosis among staff in a South African district hospital: impact of the HIV epidemic on the supply side of health care. Trans R Soc Trop Med Hyg 1998;92(5):500–502. 35. Centers for Disease Control and Prevention. Nosocomial transmission of multi-drug resistant tuberculosis to health-care workers and HIV-infected patients in an urban hospital—Florida. Morb Mortal Wkly Rep 1990;39(40):718–722. 36. Centers for Disease Control and Prevention. Nosocomial transmission of multidrug-resistant tuberculosis among HIV-infected persons—Florida and New York, 1988-91. Morb Mortal Wkly Rep 1991;40(34):585–591. 37. Centers for Disease Control and Prevention. Transmission of multi-drug resistant tuberculosis among immunocompromised persons in a correctional system—New York, 1991. Morb Mortal Wkly Rep 1992;41(28):507–509. 38. Centers for Disease Control and Prevention. Tuberculosis transmission in a state correctional institution--California, 1990-1991. Morb Mortal Wkly Rep 1992;41(49):927–929. 39. Coronado VG, Beck-Sague CM, Hutton MD, et al. Transmission of multidrug-resistant Mycobacterium tuberculosis among persons with human immunodeficiency virus infection in an urban hospital: epidemiologic and restriction fragment length polymorphism analysis. J Infect Dis 1993;168(4):1052–1055. 40. Edlin BR, Tokars JI, Grieco MH, et al. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome. N Engl J Med 1992;326(23):1514–1521. 41. Guerrero A, Cobo J, Fortun J, et al. Nosocomial transmission of Mycobacterium bovis resistant to 11 drugs in people with advanced HIV-1 infection. Lancet 1997;350(9093):1738–1742.
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42. Sepkowitz KA. Occupationally acquired infections in health care workers. Part I. Ann Intern Med 1996;125(10):826–834. 43. Alland D, Kalkut GE, Moss AR, et al. Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. N Engl J Med 1994;330(24):1710–1716. 44. Frieden TR, Fujiwara PI, Washko RM, et al. Tuberculosis in New York City—turning the tide. N Engl J Med 1995;333(4):229–233. 45. Centers for Disease Control. Guidelines for preventing the transmission of Mycobacterium tuberculosis in health-care facilities, 1994. Morb Mortal Wkly Rep 43(RR 13):1–132. 46. Dannenberg AM Jr. Delayed-type hypersensitivity and cell-mediated immunity in the pathogenesis of tuberculosis. Immunol Today 1991;12(7):228–233. 47. Barnes PF, Bloch AB, Davidson PT, et al. Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med 1991;324(23):1644–1650. 48. Loudon RG, Spohn SK. Cough frequency and infectivity in patients with pulmonary tuberculosis. Am Rev Respir Dis 1969;99(1):109–111. 49. Fennelly KP, Martyny JW, Fulton KE, et al. Coughgenerated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness. Am J Respir Crit Care Med 2004;169(5):604–609. 50. Wells WF, Ratcliff H, Crumb C. On the mechanism of droplet nuclei infection, II: quantitative experimental airborne infection in rabbits. Am J Hyg 1948;47(11). 51. Edwards DA, Man JC, Brand P, et al. Inhaling to mitigate exhaled bioaerosols. Proc Natl Acad Sci USA 2004;101(50):17383–17388. 52. Cox CS. Airborne bacteria and viruses. Sci Prog 1989;73(292 Pt 4):469–499. 53. Cox CS. Roles of water molecules in bacteria and viruses. Orig Life Evol Biosph 1993;23(1):29–36. 54. Puck T. The mechanism of aerial disinfection by glycols and other chemical agents: II. An analysis of the factors governing the efficiency of chemical disinfection of the air. J Exp Med 1947;85:741–757. 55. Brindle R, Odhiambo J, Mitchison D. Serial counts of Mycobacterium tuberculosis in sputum as surrogate markers of the sterilising activity of rifampicin and pyrazinamide in treating pulmonary tuberculosis. BMC Pulm Med 2001;1:2. 56. Gunnels JJ, Bates JH, Swindoll H. Infectivity of sputumpositive tuberculous patients on chemotherapy. Am Rev Respir Dis 1974;109(3):323–330. 57. Jindani A, Aber VR, Edwards EA, et al. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 1980;121(6):939–949. 58. Jindani A, Dore CJ, Mitchison DA. Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days. Am J Respir Crit Care Med 2003;167(10):1348–1354. 59. Tuberculosis Chemotherapy Centre, Madras. A concurrent comparison of isoniazid plus PAS with three regimens of isoniazid alone in the domiciliary treatment of pulmonary tuberculosis in South India. Bull World Health Organ 1960;23:535–585. 60. David HL. Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis. Appl Microbiol 1970;20(5):810–814. 61. 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. 62. Middlebrook G, Cohn ML. Some observations on the pathogenicity of isoniazid-resistant variants of tubercle bacilli. Science 1953; 118(3063):297–299. 63. Cohn ML, Kovitz C, Oda U, et al. Studies on isoniazid and tubercle bacilli. II. The growth requirements, catalase activities, and pathogenic properties of isoniazid-resistant mutants. Am Rev Tuberc 1954;70(4):641–664. 64. Middlebrook G, Cohn ML, Schaefer WB. Studies on isoniazid and tubercle bacilli. III. The isolation, drugsusceptibility, and catalase-testing of tubercle bacilli
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81. 82.
83.
84.
85.
86.
from isoniazid-treated patients. Am Rev Tuberc 1954;70(5):852–872. Peizer LR, Widelock D, Klein S. Effect of isoniazid on the viability of isoniazid-susceptible and isoniazidresistant cultures of Mycobacterium tuberculosis. Am Rev Tuberc 1954;89(6):1022–1028. Li Z, Kelley C, Collins F, et al. Expression of katG in Mycobacterium tuberculosis is associated with its growth and persistence in mice and guinea pigs. J Infect Dis 1998;177(4):1030–1035. Behr M. Genetic diversity of the Mycobacterium tuberculosis complex. In: Rom WN, Garay SM (eds). Tuberculosis, 2nd edn. Philadelphia: Lippincott Williams & Wilkins, 2004. Ordway DJ, Sonnenberg MG, Donahue SA, et al. Drug-resistant strains of Mycobacterium tuberculosis exhibit a range of virulence for mice. Infect Immun 1995;63(2):741–743. Cohen T, Sommers B, Murray M. The effect of drug resistance on the fitness of Mycobacterium tuberculosis. Lancet Infect Dis 2003;3(1):13–21. Teixeira L, Perkins MD, Johnson JL, et al. Infection and disease among household contacts of patients with multidrug-resistant tuberculosis. Int J Tuberc Lung Dis 2001;5(4):321–328. Karls RK, Guarner J, McMurray DN, et al. Examination of Mycobacterium tuberculosis sigma factor mutants using low-dose aerosol infection of guinea pigs suggests a role for SigC in pathogenesis. Microbiology 2006;152(Pt 6):1591–1600. Markowitz N, Hansen NI, Hopewell PC, et al. Incidence of tuberculosis in the United States among HIV-infected persons. The Pulmonary Complications of HIV Infection Study Group. Ann Intern Med 1997;126(2):123–132. Selwyn PA, Hartel D, Lewis VA, et al. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 1989;320(9):545–550. Selwyn PA, Sckell BM, Alcabes P, et al. High risk of active tuberculosis in HIV-infected drug users with cutaneous anergy. JAMA 1992;268(4):504–509. Hopewell PC, Chaisson RE. Chapter 20: Tuberculosis and human immunodeficiency virus infection. In: Reichman LB, Hershfield ES (eds). Tuberculosis: A Comprehensive International Approach, vol. 144, 2nd edn. New York: Marcel Dekker, 2000. Cowie RL, Langton ME, Becklake MR. Pulmonary tuberculosis in South African gold miners. Am Rev Respir Dis 1989;139(5):1086–1089. Hnizdo E, Murray J. Risk of pulmonary tuberculosis relative to silicosis and exposure to silica dust in South African gold miners. Occup Environ Med 1998;55(7): 496–502. Sherson D, Lander F. Morbidity of pulmonary tuberculosis among silicotic and nonsilicotic foundry workers in Denmark. J Occup Med 1990;32(2):110–113. Allison AC, Hart PD. Potentiation by silica of the growth of Mycobacterium tuberculosis in macrophage cultures. Br J Exp Pathol 1968;49(5):465–476. Gardner L. Studies on experimental pneumoconiosisthe reactivation of healing primary tubercles in the lung by inhalation of quartz, granite and carborundum dusts. Am Rev Tuberc 1929;20:833–875. Uber CL, McReynolds RA. Immunotoxicology of silica. Crit Rev Toxicol 1982;10(4):303–319. Stead WW. Genetics and resistance to tuberculosis. Could resistance be enhanced by genetic engineering? Ann Intern Med 1992;116(11):937–941. Stead WW, Senner JW, Reddick WT, et al. Racial differences in susceptibility to infection by Mycobacterium tuberculosis. N Engl J Med 1990;322(7):422–427. Cohen T, Murray M. Incident tuberculosis among recent US immigrants and exogenous reinfection. Emerg Infect Dis 2005;11(5):725–728. Nardell EA, Keegan J, Cheney SA, et al. Airborne infection. Theoretical limits of protection achievable by building ventilation. Am Rev Respir Dis 1991;144(2):302–306. Catanzaro A. Nosocomial tuberculosis. Am Rev Respir Dis 1982;125(5):559–562.
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3
The global epidemiology of tuberculosis Catherine J Watt, S Mehran Hosseini, Knut Lo¨nnroth, Brian G Williams, and Christopher Dye
NATURAL HISTORY OF TUBERCULOSIS TRANSMISSION, INFECTION, DISEASE Tuberculosis is a rare disease, whose prevalence is measured or estimated in cases per 100,000 population. TB is also a slow-moving disease – the time scale of epidemics is decades rather than weeks or years. The natural history of TB helps us understand the driving forces behind these ‘slow epidemics of a rare disease’,1 and the temporal and geographical patterns in its distribution. In Fig. 3.1, arrows represent the processes by which individuals enter and leave each of the states represented by the boxes. An individual can be uninfected, latently infected, or can have primary or post-primary disease. Infection with Mycobacterium tuberculosis, the causative agent of TB, results from inhaling droplets containing the bacilli, which are produced when a person with infectious TB coughs, talks, or sneezes (see Chapter 14 for a more detailed discussion). A widely used rule of thumb in TB epidemiology is that each untreated, infectious TB case infects, on average, about another 10 individuals each year.2,3 The estimated prevalence of smearpositive disease was just under 0.1% (90 per 100,000) in 2005,4 which corresponds to an annual risk of infection of just under 1%. A recent assessment of new infections caused by all infectious TB cases (treated and untreated) suggests an average of six new infections per case, which would imply an even lower annual risk of infection.5 Of infected individuals, only about 5% (in the absence of other predisposing conditions) develop ‘progressive primary’ disease following infection.6 Progression is typically slow, with time to development of primary disease averaging 3–4 years.7 For the remainder, who enter the pool of ‘latently infected’ individuals, there is a low annual risk of developing TB by ‘reactivation’ of infection. Whether latent bacteria remain viable for the full lifespan of all infected people is unknown, but the risk of reactivation certainly persists into old age for many. Infection is associated with only partial immune protection from reinfection.7–9 Thus, particularly in areas where infection transmission is high, infected persons remain at risk of disease resulting from reinfection. At the population level, the relative importance of primary disease, of post-primary disease resulting from reactivation and of post-primary disease following reinfection varies according to past and current patterns of transmission and breakdown to
disease. The majority of individuals infected with M. tuberculosis (but not with HIV) do not develop TB disease; the lifetime risk of pulmonary disease among infected individuals has been estimated at 12% for England and Wales in the second half of the twentieth century.8 It is this large pool of infected, healthy individuals and the typically long interval from acquiring infection to developing disease which give the slow-moving epidemics of TB their momentum, and mean that they generally respond slowly to control efforts. While the low rate of infection and breakdown to disease make TB a relatively rare disease, it is principally the high case-fatality rate which makes it one of major public health significance. Left untreated, and in the absence of HIV, about two-thirds of smearpositive cases will die, mostly within 2 years.3 For untreated smear-negative cases, case fatality rates are lower: 10–15%.10,11 Even on treatment, over 10% of smear-positive patients die in settings where adherence to treatment is low, or rates of HIV infection or drug resistance are high, although in other settings as few as 2% of smear-positive patients die while on treatment.4
RISK FACTORS The rates at which individuals move along the various arrows from one box to another in Fig. 3.1 determine the burden and distribution of disease. Factors which affect those rates, or which affect the proportion of individuals who take each path when an arrow branches, will in turn affect the size and dynamics of the epidemic. These ‘risk factors’ result from inherent characteristics of the biology of the human host and of the mycobacterial pathogen and from characteristics of the environment. Some key risk factors and their effects are shown in Table 3.1. While the association between certain risk factors and TB disease is clear, it is in practice difficult to determine which part of the life cycle of TB is affected by a particular risk factor (e.g. whether it is the risk of infection or the risk of breakdown to disease which is affected). The problem of confounding further complicates the study of risk factors; factors such as crowded living conditions, malnutrition, and exposure to indoor air pollution from cooking fires are clearly all linked to poverty, and difficult to study independently. The likelihood of transmission depends largely on the proximity and duration of contact with an infectious TB case, and is therefore increased by poorly ventilated, overcrowded housing. People in close contact with infectious cases (family members, health workers, prisoners) are at elevated risk of infection.
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Birth
Pre-exposure vaccination
Uninfected (susceptible)
Protected
Exposure and infection Preventive treatment
Postexposure vaccination
Latently infected Cure (spontaneous or following treatment)
Reactication or re-infection
Primary or post-primary disease Death
Fig. 3.1 Processes of transmission, infection and disease for TB.
Table 3.1 Risk factors for tuberculosis Risk factor
Affects
Sources
Crowded living conditions Smoking Malnutrition
Exposure to infectious cases
12, 13
Susceptibility to infection Susceptibility to infection, likelihood of developing primary disease, likelihood of breakdown from latent infection, likelihood of recovery from disease Susceptibility to infection Exposure to infectious cases, likelihood of developing primary disease, likelihood of breakdown from latent infection, likelihood of recovery from disease Susceptibility to infection, likelihood of breakdown, likelihood of recurrence Severity and infectiousness of disease, likelihood of treatment failure or relapse Exposure, susceptibility to infection, likelihood of developing primary disease, or of breakdown to disease from latent infection, type of disease Exposure, susceptibility to infection, likelihood of breakdown to disease, type of disease, health-seeking behaviours, tendency to default from treatment Susceptibility to infection, or to certain types of disease
14–17 18–20
Indoor air pollution Alcohol HIV Diabetes Age Sex/gender Genetic factors
Susceptibility to tuberculosis may be affected by factors such as tobacco smoking, silicosis, exposure to smoke from cooking fires and excessive alcohol use as well as by HIV infection, but it is difficult in practice to distinguish between the effect of such factors on susceptibility to infection and the likelihood of progression to disease. Malnutrition influences breakdown to disease,18 although to what extent requires careful investigation in order to distinguish pre-existing malnutrition from wasting resulting from TB. Nutrition is likely also to influence the likelihood of recovery from disease. HIV infection has a dramatic effect on the likelihood of breakdown to disease,24–27 increasing the likelihood of breakdown from a lifetime risk of between 10% and 20% to an annual risk of over 10%.7,8,37,38 Both age and sex have biological and social effects which are difficult to distinguish. The risk of developing primary disease is
18
21, 22 23 24–27 28 29 11, 30–33 34–36
lower in children than in adults,6,39 and children are more likely to develop severe forms of disease in organs other than the lungs (e.g. tuberculous meningitis). Young women (15–44 years) may be more likely than men to develop active TB following infection, but the socially driven effects of gender are generally more marked than the biological effects of sex.11,31–33 Men and women experience different environmental risk factors, and demonstrate different health-seeking behaviours and tendencies to adhere to treatment.40
Population attributable fractions The overall contribution of an individual risk factor to determining the path of the global TB epidemic depends both on the strength of the effect of that risk factor and on how widespread that risk factor is. By quantifying the relative risks associated with various risk factors and the prevalence of those risk factors one can calculate the
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The global epidemiology of tuberculosis
population attributable fraction (PAF) for those risk factors. The study of risk factors with high prevalence, and which can be altered (not, for example, age and sex), is one possible approach to identifying activities which have the potential to help prevent TB. In taking this approach, however, it is important to acknowledge that the sum of the PAFs for a set of risk factors can be greater than 1. Furthermore, the size of the PAF gives no indication of how quickly one would see the effect of any intervention aimed at the risk factor in question. Bearing these limitations in mind, a comparison, for example of smoking and HIV, illustrates the value of considering the prevalence of a risk factor as well as the relative risk. The relative risk of TB associated with HIV is almost certainly considerably higher than that associated with tobacco smoking. However, the prevalence of smoking in the 22 countries which account for 80% of the annual incidence of TB is estimated at 18%,41 compared with an estimated prevalence of HIV of 1.1%.42 Thus preliminary estimates suggest that the PAF for smoking is several times that for HIV globally. Only in the World Health Organization (WHO) African region is the PAF for HIV higher than that for smoking, and even there the PAF for smoking is substantial. These preliminary results suggest that much could be gained in terms of preventing TB by addressing tobacco smoking, and by exploring other prevalent but preventable risk factors.
TUBERCULOSIS CONTROL The path of TB epidemics is clearly determined not only by the biological and social phenomena discussed above under Risk Factors, but also by explicit efforts to control the disease. Prior to the development of antibiotic therapy for TB, the main methods of control available were reducing transmission by isolating infectious patients, and increasing the likelihood of spontaneous recovery by providing patients with rest and improved nutrition. Both these aims were achieved by treating TB patients in sanatoria, where case-fatality rates were about 50%.3 Modern TB control is based on early detection and treatment, particularly of infectious cases. Treatment is generally effective; the average global cure rate was 84.7% for smear-positive cases treated in 2005 by programmes following international recommendations.4 Treatment has, therefore, a direct impact on the prevalence of disease and on mortality. Furthermore, transmission is reduced, as infectious cases quickly become non-infectious once treatment is started. In addition to the primary goal of TB control (early diagnosis and treatment), national TB programmes or health authorities can (and, to varying degrees, do) influence the flow of individuals along the paths shown in Fig. 3.1 in the following ways: by implementing appropriate ‘infection control’ strategies (ensuring adequate ventilation in healthcare centres, minimizing contact between infectious and susceptible individuals in order to reduce transmission43,44); by providing preventive treatment (isoniazid preventive therapy, IPT) to infected individuals who do not have TB disease (thus returning them from the latently infected pool to the pool of susceptibles); by collaborating with national acquired immunodeficiency syndrome (AIDS) programmes to identify and provide appropriate care for HIV-infected TB patients; and by providing nutritional support to TB patients and their families, thus improving their nutritional status and perhaps increasing the likelihood of recovery for patients, decreasing the likelihood of infection for family members and encouraging patients to complete treatment. Finally, roughly 100 million infants (>80% of the
3
annual cohort) are vaccinated each year with Bacillus CalmetteGue´rin (BCG), the effect of which is mainly to prevent serious forms of disease in children: meningitis and miliary TB. The potential of existing and possibly future tools for reducing the burden of TB, particularly with reference to international targets, is discussed further below.
GLOBAL AND REGIONAL BURDEN AND TRENDS Based on surveys of the prevalence of infection and of disease, on assessments of the performance of surveillance systems and on death registrations, there were an estimated 9.2 million new cases of TB in 2006, of which 4.1 million were smear-positive.4,45,46 The WHO African region had the highest estimated incidence rate (363 per 100,000 population), but the majority of TB patients live in the most populous countries of Asia. Five countries – Bangladesh, China, India, Indonesia, and Pakistan – have almost half the world’s population (46%) and produced about half (48%) of all new TB cases arising worldwide in 2006. Figure 3.2 illustrates the global distribution of new TB cases in 2006, in terms of numbers and rates per 100,000 population. Much of the work of the WHO and partners focuses on the 22 countries known as the high-burden countries (HBCs). These are the countries which, in 2002, were estimated to have had the highest numbers of incident TB cases in the year 2000: Afghanistan, Bangladesh, Brazil, Cambodia, China, the Democratic Republic of the Congo, Ethiopia, India, Indonesia, Kenya, Mozambique, Myanmar, Nigeria, Pakistan, Philippines, the Russian Federation, South Africa, Thailand, Uganda, the United Republic of Tanzania, Viet Nam and Zimbabwe.45 Changes in estimates due to new data or techniques, and likely changes in incidence rates and population sizes, mean that this list no longer exactly matches the list of the 22 countries with the largest number of new cases each year, but it is still true that, between them, the HBCs account for 80% of new TB cases arising annually. Table 3.2 shows the estimated incidence and prevalence of TB and mortality from TB for 2006. Globally an estimated 1.7 million people died from TB in 2006, 231,000 of them infected with HIV. Estimates of TB incidence, prevalence and mortality are uncertain, and rely to varying degrees on assumptions about the quality of surveillance data, about the quality and impact of treatment and about the duration of disease and case-fatality rates.45,46 New methods for evaluating the burden of TB and impact of control are needed. In our view these should be based principally on assessments of the quality of surveillance systems, backed by data obtained from surveys of the prevalence of disease. These methods and the resulting data would help to meet increasing demands from donor agencies (such as the Global Fund to Fight AIDS, Tuberculosis, and Malaria) to demonstrate the impact of the activities which they fund, and of international experts to increase the transparency surrounding statistics provided through databases such as those of the WHO (e.g. http://www.who.int/tb/country/global_tb_database, and http://www.who.int/whosis) and of the United Nation Statistical Division (http://mdgs.un.org).47 A recently established task force on measurement of TB will guide work on improving estimates of the burden of TB and the impact of control.48 Estimates of the incidence of multidrug-resistant TB (MDR-TB; caused by strains of M. tuberculosis resistant to at least isoniazid and rifampicin) are even more uncertain than those of overall TB incidence. Drug susceptibility testing is not widely available, although
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Estimated numbers of new cases, 2006
Estimated number of TB cases per year (all forms) No estimate 0–999 1000–9999 10,000–99,999 100,000–999,999 1,000,000 or more
Estimated TB incidence rate, 2006
Estimated TB cases per year (all forms) per 10, 000 population No estimate 0–24 25–49 50–99 100–299 300 or more
Fig. 3.2 Estimated incidence (numbers and rates) of TB cases, 2006.
WHO guidelines for incorporating the diagnosis and treatment of drug-resistant TB into the routine activities of national TB control programmes are likely to lead to improved information about the proportion of TB cases which are MDR.49 Current estimates (based on multivariate analysis) are that 489,000 cases of MDR-TB arose in 2006 among new and previously treated TB cases.50 Extensively drug-resistant TB (XDR-TB) is defined as TB due to bacilli resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin), in addition to isoniazid and rifampicin.51 The magnitude of the XDR-TB problem globally is not yet known. Where the transmission of M. tuberculosis has been stable or increasing for many years, the incidence rate is relatively high among
20
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 comes 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, while patients who are immigrants from highincidence countries tend to be young adults. Allowing for the difficulties of diagnosing childhood TB, estimation exercises indicate that there are relatively few cases among 0–14 year olds; while this age group accounts for nearly 30% of the world’s population, it accounts for only 12% of estimated cases. In 2006, countries reported 1.6 million smear-positive TB cases among men, but only 884,000 among women. In some instances
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The global epidemiology of tuberculosis
3
Table 3.2 Estimated TB incidence, prevalence and mortality, 2006 WHO region
Incidence (per year) All forms
Africa The Americas Eastern Mediterranean Europe South-East Asia Western Pacific Global
Prevalence (all forms) Smear-positive
Number (thousands) (% of global total)
Per 100,000 population
Number (thousands)
Per 100,000 population
2,808 (31) 331 (4) 570 (6)
363 37 105
1,203 165 256
155 18 47
433 (5) 3,100 (34) 1,915 (22) 9,157 (100)
49 180 109 139
194 1,391 860 4,068
22 81 49 62
women have poorer access to diagnostic facilities,30 but the broader pattern also reflects real epidemiological differences between the sexes: while 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.11,31–33
Tuberculosis prevalence and death rates have probably been falling globally for several years, and it is likely that the TB incidence rate had reached a peak worldwide in about 2003 (Fig. 3.3). However, because the populations of the countries heavily affected by TB are still growing, the total number of new TB cases arising each year was also still slowly increasing in 2006.4 This rather static picture of the global epidemic close to its peak conceals much variation in the dynamics of TB among regions. The countries of sub-Saharan Africa and the former Soviet Union showed striking increases in case loads during the 1990s. These rises offset the fall in case numbers in other parts of the world, principally West and Central Europe, the Americas and the Eastern Mediterranean regions. Industrialized countries are typically seeing fewer cases among nationals each year, but steady or rising numbers of cases among immigrants. In 12 out of 28 Western European countries providing data in 2005, the majority of TB patients were foreign-born or foreign citizens.52 Prevalence
Per Number 100,000 (thousands) population
Per 100,000 population
4,234 398 826
546 44 152
639 41 108
83 4.5 20
478 4,975 3,513 14,424
54 289 199 219
62 515 291 1,656
7.0 30 17 25
Mortality
300
Incidence 144
280 260 240 220
1995
2000
2005
cases per 100,000 pop/year
33 deaths per 100,000 pop/year
cases per 100,000 pop
Number (thousands)
An estimated 1.7 million people died of TB in 2006. Tuberculosis is the world’s second largest killer among infectious agents, behind HIV/AIDS.53 In terms of years of healthy life lost, TB remains among the top 10 causes of illness, death and disability. The 231,000 TB deaths in people infected with HIV were 14% of all TB deaths and 10% of AIDS deaths in 2006, and the vast majority (205,000) were in Africa.4 Death registrations have been increasing in former Soviet countries since the 1980s, but falling in Central Europe, Latin America and the industrialized world. Estimates suggest that, while TB deaths were probably increasing during the 1990s, driven mainly by the steep rise in HIV-related mortality in Africa, the global TB death rate peaked before year 2000. This was after prevalence began to fall, but before the peak in incidence (Fig. 3.3). In Eastern Europe (principally countries of the former Soviet Union), case reports increased dramatically in the early 1990s. This resurgence can be explained by economic decline and the failure of TB control and other health services since 1991,54 along with other factors such as alcoholism, cardiovascular disease and the mixing of prison and civilian populations.55–57 Over the past 3–6 years, the increase in notification rates has stopped or reversed in Estonia, Romania, Kazakhstan, Kyrgyzstan, Latvia, Lithuania and the Russian Federation, suggesting a levelling off in the incidence of TB in the region.4 It is estimated that 13% of all new TB cases arising each year in Eastern Europe, and 46% of re-treatment cases, are MDR.50 Drug
TRENDS
200 1990
TB mortality (per year)
31 29 27 25 23 1990
1995
2000
2005
140 136 132 128 124 120 1990
1995
2000
2005
Fig. 3.3 Estimated global prevalence, mortality and incidence rates, 1990–2006. Note different y axes.
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IMPLEMENTATION OF THE STOP TUBERCULOSIS STRATEGY Progress in TB control has been measured principally in terms of the implementation of DOTS (see Chapters 106 and 107).63,64 Data collected by the end of 2007 allowed the WHO to assess in which countries and regions the targets for 70% case detection and 85% treatment success, originally set for 2005, were met by the end of 2006.4,65
Case detection Over the 12 years 1994–2006, a total of 30 million TB patients were diagnosed and reported under DOTS. In 2006, DOTS programmes worldwide reported 5.3 million new and relapse cases,
22
WHO target 70 60 50 40 30
Average rate of progress 1995–2000
DOTS begins 20 10 0 1990
1995
2000
2005
2015
2010
Year
Fig. 3.4 Case detection of new smear-positive cases and of all new cases under DOTS, 1995–2006. Open circles mark the number of new smearpositive cases notified under DOTS 1995–2006, expressed as a percentage of estimated new cases in each year. The solid line through these points indicates the average annual increment from 1995 to 2000 of about 134,000 new cases, compared with the average increment from 2000 to 2006 of about 243,000 cases. Closed circles show the total number of smear-positive cases notified (DOTS and non-DOTS) as a percentage of estimated cases.
among which 2.5 million were smear-positive. This gives a smear-positive case detection rate of 61% (95% confidence interval (CI) 55–75%), short of the 70% target. The estimated case detection rate by DOTS programmes increased almost linearly from 12% globally in 1995 to 23% in 2000. Case detection then accelerated, though it has slowed somewhat since 2004 (Fig. 3.4). The global acceleration in case detection has been driven principally by South-East Asia (mostly India) since 2000, supported by the Western Pacific Region (mostly China) since 2002. However, only eight of the 22 high-burden countries (including China), 77 countries and territories in total and one WHO region (Western Pacific) reached the 70% target by 2006.4,65
Treatment success Of 2.4 million smear-positive patients registered for treatment under DOTS in 2005, 2.0 million (84.7%) were successfully treated (i.e. cured, as judged by sputum smear conversion, or completed treatment without final smear examination), just short of the 85% target. The global treatment success rate under DOTS has been high since the first observed cohort in 1994 (77% of 245,000 patients), and has remained above 80% since 1998 (Fig. 3.5).4,65 Treatment success target: 85% 85
2500
80 2000 75 70
1500
65
1000
60 500 55 50 1993
Thousands of patients treated
TUBERCULOSIS CONTROL: IMPLEMENTATION, TARGETS AND PROJECTIONS
80 Case detection rate, smear-positive cases (%)
resistance is likely to be a byproduct 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, but 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 increase in caseload to excess transmission from treatment failures. Tuberculosis notification rates, and probably incidence, have been rising dramatically in Africa since 1990. It is likely that this increase is largely due to HIV infection;45 in 2006 an estimated 22% of new TB cases arose in people infected with HIV, and the HIV prevalence among new TB cases was over 40% in eight countries.4 The HIV epidemic is probably now in decline in most countries of sub-Saharan Africa,42 and it appears that the incidence of TB may also have reached a peak.4 Based on case reports, TB has been in decline in Western Europe for 150 years.1,29 There are numerous plausible explanations for this decline, and it is not possible to determine the exact contribution of the various factors. The first main explanation is that transmission fell when housing conditions improved and people began to live at lower density in better ventilated housing, and when patients were isolated in sanatoria.12 Contact rates may also have fallen as the average age of cases increased.13 Secondly, it is likely that improved nutrition led to reduced susceptibility to disease.18–20,58 The failure of the case-fatality rate to decline before the widespread introduction of anti-TB drugs in the late 1940s suggests that improved nutrition had little effect on the outcome of disease. Thirdly, it has been proposed, but not demonstrated, that M. tuberculosis has become less pathogenic.59–61 The decline in case notifications slowed in many developed countries around 1990 and has stopped or almost stopped falling in some countries, notably Hong Kong, Japan, Singapore, the United Kingdom and the United States. This is likely to be the result of at least two phenomena – first, more cases are arising by reactivation from an aging TB epidemic in an aging human population; second, an increasing proportion of notified cases is in immigrants.62
Treatment success rate
1
0 1995
1997
1999
2001
2003
2005
Fig. 3.5 Treatment success rate (closed circles) and numbers of patients treated (open circles), new smear-positive cases treated under DOTS, 1994–2005.
CHAPTER
The global epidemiology of tuberculosis target: 15% or less (success 85% or more) Africa The Americas Eastern Mediterranean Europe South-East Asia Western Pacific 0 Died
Failed
Defaulted
15 % of cohort Transferred
30 Not evaluated
Fig. 3.6 Unsuccessful treatment outcomes for new smear-positive cases treated under DOTS, 2005, by WHO region.
For patients diagnosed in 2005, the 85% target was reached by eight of the 22 high-burden countries, and by two WHO regions (South-East Asia, Western Pacific). In the four regions that did not meet the target, the reasons for poor results were, as shown in Fig. 3.6, high rates of death (Africa, Europe), treatment failure (Europe) and unknown outcomes through default, transfer without follow-up or no evaluation (Africa, Americas, Eastern Mediterranean, Europe).
IMPACT OF DOTS ON INCIDENCE, PREVALENCE AND MORTALITY Although the decline in TB has almost certainly been accelerated by good chemotherapy programmes, which have been implemented for decades in countries such as Chile, Cuba and Uruguay, there have been few recent, unequivocal demonstrations of impact in high-burden countries. This is because large-scale public health programmes are not carried out as controlled experiments, and because there are other factors that influence transmission or susceptibility to disease (housing, nutrition, coinfection and others; see discussion under Risk Factors, above). However, Morocco and Peru provide two persuasive examples. Between 1994 and 2000, the incidence of pulmonary TB among Moroccan children 0–4 years of age fell at more than 10% per year, suggesting that the risk of infection was falling at least as quickly (Ministry of Health Morocco, unpublished data). The 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. In Peru, DOTS was launched in 1991, and high rates of case detection and cure pushed down the incidence rate of pulmonary TB by 6% per year.66 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. There have been few direct measures of the reduction in TB prevalence over time. The Republic of Korea carried out seven surveys at 5-year intervals between 1965 and 1995, during which time the prevalence of bacteriologically positive cases (smearand/or culture-positive) disease fell from 940 per 100,000 to 219 per 100,000.67 Prevalence surveys done in China in 1990 and in 2000 showed the reduction in the prevalence per capita of all forms of TB in DOTS areas was 32 percentage points (95% CI 9–51%) greater than the reduction in other parts of the country.68 A national survey in Indonesia in 2004 found that the prevalence
3
of smear-positive TB had fallen by a factor of 3 since a set of regional surveys were carried out between 1979 and 1982.69,70 But not all of this reduction can be attributed to the DOTS programme, or even to the direct effects of chemotherapy. Some investigations of the impact of DOTS programmes have shown that, after several years of implementation, incidence is not falling as expected. Viet Nam has apparently exceeded the targets for case detection and treatment success since 1997, and yet the case notification rate remained approximately stable over that period.71 Closer inspection of surveillance data shows that, while case notification rates are falling among adults aged 35–64 years (especially women), they are increasing among 15–24 year olds (especially men).4 These increases and decreases in different age groups, and for men and women, are almost equal in magnitude. This pattern of change in case notifications by age and sex is also visible in routine surveillance data from Sri Lanka. In Indonesia and Myanmar, TB patients in the age group 15–54 years are becoming younger on average, but the average age should be increasing if transmission and incidence are falling.4 Some states of India have been implementing DOTS since 1998, and yet there was no detectable decline in case notification rates at state level by 2006 (Revised National TB Control Programme, personal communication). The case notification rates in different states and in different years are closely correlated with the number of TB suspects examined. This suggests that the number of cases diagnosed and reported is determined by the performance of the public health service, as well as by the true incidence of TB (and contrasts with the situation in Peru, described above, where the number of TB cases found was decreasing despite an increasing number of suspects examined). The southern states of Kerala and Tamil Nadu show an increase in the average age of TB cases, which may reflect falling transmission. This has yet to be translated into a clear demonstration of falling incidence across a whole state, though transmission and prevalence have been reduced on the smaller scale of Tiruvallur District in Tamil Nadu.72,73 Calculations carried out for the WHO’s 2008 report on Global TB Control suggest that, although prevalence and death rates are falling globally, the rate of decline is not fast enough to meet the Millennium Development Goals (MDGs) by 2015.4 While the surveillance data from certain Asian countries show some worrying signs, the biggest challenges in cutting TB burden are in subSaharan Africa and Eastern Europe.
ACHIEVING THE MILLENNIUM DEVELOPMENT GOALS BY 2015 Since the launch of the DOTS strategy during the 1990s, a series of specific, emerging problems in TB epidemiology and control have demanded specific solutions. These include M. tuberculosis and HIV coinfection, drug resistance, the quality of treatment in the private sector and the need to evaluate the epidemiological impact of TB control (not simply the implementation of DOTS). For this reason, DOTS has been extended as the Stop TB Strategy, with the additional elements listed in Table 105.2 of Chapter 105.4,63 The blueprint for implementing the Stop TB Strategy over the next decade is The Global Plan to Stop TB, 2006–2015, discussed in detail in Chapter 106.74 The plan imagines and compares three scenarios. Scenario 1: No DOTS. This assumes that the strategy was never introduced in any region, so chemotherapy would continue as it was pre-DOTS, with variable rates of case detection
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and typically lower rates of cure. This gives a baseline against which to compare gains that have already been made, and which might be made in future. Scenario 2: Sustained DOTS. Case detection and treatment success increase until 2005, and then remain steady until 2015. Approximately 50 million patients would be treated under DOTS between 2006 and 2015, as compared with over 20 million in the previous decade, 1996–2005. Scenario 3: Enhanced DOTS. Case detection and treatment success continue to increase beyond 2005, up to 2015. 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). To reach high rates of case detection and cure requires various additions to the basic DOTS strategy, including communitybased 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. 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. Approximately 200 million people infected with HIV will be screened for TB, and 24 million will be offered IPT.
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. 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 in any other region. The proportional reduction in deaths would be greatest in Africa (high HIV countries) and Eastern Europe. In regions of the world other than Africa and Eastern Europe, 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. And TB epidemiology in these other regions, notably Asia, governs the global trend. Thus, enhanced DOTS (scenario 3), as compared with sustained DOTS (scenario 2), would save only an additional 2.7 million deaths globally over the next decade. But if scenario 3 is considered to be the logical extension of the programme of global DOTS expansion that began in the early 1990s, then enhanced DOTS will save an additional 13.7 million deaths between 2006 and 2015 (compared with scenario 1). In continuing this programme of DOTS expansion, most cases and deaths saved will be in the South-East Asia and Western Pacific Regions.
The potential impact of scenario 3, as compared with scenarios 1 and 2, has been evaluated with a mathematical transmission model describing how the planned interventions determine incidence, prevalence and death rates through time.9,75–77 Model calculations show that scenarios 2 and 3 should both satisfy MDG target 8, to ensure that the incidence rate of TB is falling globally. In fact, the annual incidence of new cases appears to have peaked and may already have been in decline in 2006.4 Ambitious plans for the South-East Asia and Western Pacific regions are expected to generate relatively rapid declines in incidence rate of 7–9% per year by 2015. To halve prevalence and death rates between 1990 and 2015 will be more challenging. Projections suggest that these targets can be met globally with full implementation of the enhanced DOTS strategy (scenario 3), but not in Africa or Eastern Europe. Based on the calculated rate of decline in mortality from 2006 to 2015 in the African countries most affected by HIV, the target death rate 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 even later than 2025. In Eastern Europe, prevalence rates are also expected to remain high compared with 1990 levels because a relatively high proportion of patients have chronic TB, which is commonly multidrug resistant. In Africa, in the absence of ART, patients infected with HIV do not suffer from TB for long; their illness typically progresses quickly, and they are either cured or die.78 The widespread use of ART will alter the dynamics of the coepidemic in ways which are not yet fully understood. The 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. While not ignoring the epidemiology of the recent past, more relevant here is the impact of interventions in the near future. The same
ELIMINATING TUBERCULOSIS IN THE TWENTY-FIRST CENTURY
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This discussion of control via the Stop TB Strategy has focused on the chemotherapy of active disease, because drug treatment is likely to remain the principal option for TB over the next decade. In the longer term, TB elimination might be achieved through a combination of measures to prevent infection, to stop progression from infection to active disease and to treat active disease. An examination of the principles of TB elimination would show how we may exploit, or even drive, the development of new drugs, diagnostics and vaccines.79–81 Mathematical modelling shows that, with the diagnosis and treatment of 70% of new infectious cases arising each year and a cure rate of 85%, the incidence rate is expected to fall from a steady state at about 10% annually for the first 10 years, and more slowly thereafter.82 This is the pattern of decline observed in Western European countries after effective TB drugs became available during the 1950s. The net effect is to reduce the incidence rate by a factor of about 10 over a period of 45 years. Applying this result to TB in regions of the world other than sub-Saharan Africa, the incidence rate by 2050 would still be of the order of 100 per million population. A reduction of this magnitude would be an important achievement for public health, but it leaves an incidence rate mid-century that is still about 100 times greater than the elimination target. This is similar to the result obtained under scenario 3 of the Global Plan to Stop TB. To eliminate TB by 2050 (outside sub-Saharan Africa), the incidence rate must fall at an average of 15% annually. This rate of decline might be achieved for a few years, but it is unlikely to be sustainable. The reason, as observed earlier, is that when transmission and incidence fall, a growing proportion of cases arise from the slow reactivation of long-standing latent infections, rather than from the rapid progression of recent infections. Thus, the initial rate of decline in incidence is controlled by primary
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The global epidemiology of tuberculosis
progression, but the long-term rate of decline is governed by reactivation. Combinations of interventions can, in principle, push TB closer to elimination, but some combinations are more effective than others. An exploration of the likely impact of pre-exposure vaccination (to prevent infection) combined with the treatment of TB patients (which also reduces infection) reveals that the effects of the two approaches are similar when used alone, and intensifying either control method yields sharply diminishing returns. The two methods are somewhat more effective in combination, but the additional impact is small. This is because drug treatment to stop transmission at source has effects similar to those of vaccination, which protects those exposed to infection. One intervention partly substitutes for the other; they do not act independently or synergistically. Therefore, a pre-exposure vaccine will be most useful in addition to treatment when the detection rate of active TB cases is low, and vice versa. The campaign to eliminate TB will be more effective if two interventions attack different aetiological pathways. The treatment of latent TB, by either preventive drug therapy or post-exposure vaccination, is relatively ineffective when used alone, but powerful in combination with the treatment of active TB or with preexposure vaccination. When these combined approaches are implemented intensively, TB incidence can be forced close to or below the elimination threshold by 2050.
CONCLUSIONS The countries of sub-Saharan Africa and Eastern Europe (mainly the former Soviet Union) suffered large increases in TB during the 1990s, but there are signs that the incidence rates in these two regions, and, as a result, globally, are now stable or falling. This would mean that MDG target 8 has already been satisfied, 10 years in advance of the 2015 deadline. But there are two important caveats. First, the decline in incidence per capita was slower than the increase in human population in 2006; consequently, the total number of new cases was still rising each year. And second, prevalence and mortality rates are not being reduced quickly enough to meet the MDGs by 2015. These are sharp reminders that, despite having treated more than 30 million patients between 1994 and 2006, DOTS has so far had limited epidemiological impact. It is clear, too, that the fall in case load in some countries is only partially due to the impact of DOTS. In sub-Saharan Africa, the incidence of HIV infection was probably at its highest during the 1990s, and the fall in HIV incidence has weakened the main driving force behind the TB epidemic. In Eastern European countries, many components of the TB control system (diagnostic services, drug supplies, etc.) have been restored after the collapse of the Soviet Union. But this is unlikely to be the full explanation for the stabilization in case notifications in Eastern Europe, because the initial resurgence could also have been due to changes in susceptibility among people who were malnourished, stressed and unemployed. In many countries of Latin America, the Eastern Mediterranean region and Asia, TB case notification rates were falling well before the implementation of DOTS programmes.
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The epidemiological impact of DOTS has been discernible in a few countries (e.g. Peru, China), but remains a matter of conjecture in most. There will always be analytical difficulties in distinguishing the impact of public health intervention outside a randomized controlled trial. Nonetheless, surveillance in India, Indonesia, Myanmar, Sri Lanka and Viet Nam raises doubts about whether the fall in incidence in Asia will be as great as expected from the post-war experience in Europe,3 or as anticipated by mathematical models.9,83 The causes of, for example, rising incidence rates among young adults are not yet clear. HIV infection plays a part, but is unlikely to be the full explanation. There is a strong case for re-examining some basic assumptions about TB epidemiology, such as the magnitude of reactivation and reinfection rates in relation to risk factors including indoor air pollution, drug resistance, malnutrition, diabetes and tobacco smoking.84–89 None of these observations are reasons for abandoning DOTS or the Stop TB Strategy. Rather, the strategy must be reinforced as the only feasible mechanism for achieving the MDGs by 2015. In evaluating progress towards those goals, it will be vital to measure epidemiological impact through a combination of survey methods and surveillance. Periodic population-based surveys of the prevalence of active disease and infection can reveal trends, which could be attributable to specific interventions. Tuberculosis deaths need to be counted in more countries, and, more accurately, either as a component of general cause-of-death surveys or through systems of routine death registration.90 The evidence from surveys of prevalence and deaths should be supplemented by fuller analyses of the huge body of surveillance data that is routinely collected by national control programmes (following the example of Peru). Beyond the Stop TB Strategy, beyond the Global Plan and beyond the MDGs, TB elimination in the twenty-first century will require a new armoury of diagnostics, drugs and vaccines. Tuberculosis cannot be eliminated by 2050 solely by cutting transmission, no matter how well current programmes of drug treatment are implemented. To eliminate TB on this time scale it will be essential to stop the progression from latent infection to active disease, in addition to preventing new infections. This is unlikely to be possible on a large scale unless existing procedures to carry out preventive therapy can be greatly simplified, or replaced. That will require, first, a diagnostic test for latent infection that is more sensitive and specific than the current tuberculin skin test but easy to administer and read. That need might be satisfied, in part, by interferon-g release assays.91 Preventive therapy also requires a course of treatment shorter than 9 months. In this context, a 3-month treatment regimen now appears feasible with combinations of drugs such as isoniazid with rifapentine.92 A major step forward in preventive therapy would be a test to identify which infected people are at relatively high risk of progressing to active disease. The alternative to preventive therapy is immunization, but TB elimination will be possible only with a vaccine that is far superior to BCG. The ideal, dual-action vaccine would prevent both new infection (pre-exposure) and reactivation of latent infection (postexposure). But whatever technological solutions are devised to neutralize latent infection they must be combined with methods to stop transmission. Only by attacking both aetiological pathways can we hope to eliminate TB within the next half century.
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REFERENCES 1. Dye C. Epidemiology. In: Davies PDO, Barnes P, Gordon SB, eds. Clinical Tuberculosis, 4th edn. London: Arnold, 2008. 2. Styblo K. The relationship between the risk of tuberculous infection and the risk of developing infectious tuberculosis. Bull Int Union Tuberc Lung Dis 1985;60:117–119. 3. Styblo K. Epidemiology of Tuberculosis, 2nd edn. The Hague: KNCV Tuberculosis Foundation, 1991. 4. World Health Organization. Global Tuberculosis Control: Surveillance, Planning, Financing. Geneva: World Health Organization, 2008. 5. Bourdin Trunz B, 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. 6. Sutherland I, Svandova E, Radhakrishna S. The development of clinical tuberculosis following infection with tubercle bacilli. 1. A theoretical model for the development of clinical tuberculosis following infection, linking from data on the risk of tuberculous infection and the incidence of clinical tuberculosis in the Netherlands. Tubercle 1982;63:255–268. 7. Sutherland I. The Ten-year Incidence of Clinical Tuberculosis Following ‘Conversion’ in 2,550 Individuals Aged 14 to 19 Years. The Hague: Tuberculosis Surveillance and Research Unit Progress Report, KNCV, 1968. 8. Vynnycky E, Fine PEM. Life time risks, incubation period, and serial interval of tuberculosis. Am J Epidemiol 2000;152:247–263. 9. Dye C, Garnett GP, Sleeman K, et al. Prospects for worldwide tuberculosis control under the WHO DOTS strategy. Directly observed short-course therapy. Lancet 1998;352:1886–1891. 10. Krebs W. Die Fa¨lle von Lungentuberkulose in der aargauischen Heilsta¨tte Barmelweid aus den Jahren 1912–1927. Beitr Klin Tuberk 1930; 74:345–379. 11. Rieder HL. Epidemiologic Basis of Tuberculosis Control. Paris: International Union against Tuberculosis and Lung Disease, 1999. 12. McFarlane N. Hospitals, housing and tuberculosis in Glasgow. Soc Hist Med 1989;2:59–85. 13. Vynnycky E, Fine PEM. Interpreting the decline in tuberculosis: the role of secular trends in effective contact. Int J Epidemiol 1999;28:327–334. 14. Bates MN, Khalakdina A, Pai M, et al. Risk of tuberculosis from exposure to tobacco smoke: a systematic review and meta-analysis. Arch Intern Med 2007;167:335–342. 15. Hassmiller K. The impact of smoking on populationlevel tuberculosis outcomes. PhD dissertation, University of Michigan, 2006. 16. International Union against Tuberculosis and Lung Disease. Association Between Exposure to Tobacco Smoking and Tuberculosis: a Qualitative Systematic Review. Paris: International Union against Tuberculosis and Lung Disease, 2007. 17. Vazquez-Gallardo R, Anibarro L, Fernandez-Villar A, et al. Multidrug-resistant tuberculosis in a lowincidence region shows a high rate of transmission. Int J Tuberc Lung Dis 2007;11:429–435. 18. Edwards LB, Livesay VT, Acquaviva FA, et al. Height, weight, tuberculous infection, and tuberculous disease. Arch Environ Health 1971;22:106–112. 19. 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(3):286–298. 20. Cegielski JP, Kohlmeier L, Cornoni-Huntley J. Relative and Attributable Risks of Tuberculosis due to Undernutrition in a Population-based Sample of Adults. TSRU. The Hague: KNCV, 2007.
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21. Lin HH, Ezzati M, Murray M. Tobacco smoke, indoor air pollution and tuberculosis: a systematic review and meta-analysis. PLoS Med 2007;4(1):e20. 22. World Health Organization. Fuel for Life: Household Energy and Health. Geneva: World Health Organization, 2006. 23. Lo¨nnroth K, Williams BG, et al. Alcohol use as a risk factor for tuberculosis – a systematic review. BMC Public Health 2008;8:289. 24. Corbett EL, Marston B, Churchyard GJ, et al. Tuberculosis in sub-Saharan Africa: opportunities, challenges, and change in the era of antiretroviral treatment. Lancet 2006;367(9514):926–937. 25. Nunn P, Williams BG, Floyd K, et al. Tuberculosis control in the era of HIV. Nature Rev Immunol 2005;5:819–826. 26. Williams BG, Dye C. Antiretroviral drugs for tuberculosis control in the era of HIV/AIDS. Science 2003;301:1535–1537. 27. Reid A, Scano F, Getahun H, et al. Towards universal access to HIV prevention, treatment, care, and support: the role of tuberculosis/HIV collaboration. Lancet Infect Dis 2006;6:483–495. 28. Stevenson CR, Forouhi NG, Roglic G, et al. Diabetes and the risk of tuberculosis: a neglected threat to public health? Chronic Illn 2007;3(3):228–245. 29. Vynnycky E. An Investigation of the Transmission Dynamics of M. tuberculosis. London: London University, 1996. 30. Hudelson P. Gender differentials in tuberculosis: the role of socio-economic and cultural factors. Tuberc Lung Dis 1996;77:391–400. 31. Borgdorff MW, Nagelkerke NJ, Dye C, et al. 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. 32. Hamid Salim A, Declercq E, Van Deun A, et al. Gender differences in tuberculosis: a prevalence survey done in Bangladesh. Int J Tuberc Lung Dis 2004;8:952–957. 33. 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. 34. Goldfeld AE, Delgado JC, Thim S, et al. Association of an HLA-DQ allele with clinical tuberculosis. JAMA 1998;279:226–228. 35. Hoal EG. Human genetic susceptibility to tuberculosis and other mycobacterial diseases. IUBMB Life 2002;53:225–229. 36. Alm JS, Sanjeevi CB, Miller EN, et al. Atopy in children in relation to BCG vaccination and genetic polymorphisms at SLC11A1 (formerly NRAMP1) and D2S1471. Genes Immun 2002;3:71–77. 37. Bucher HC, Griffith LE, Guyatt GH, et al. Isoniazid prophylaxis for tuberculosis in HIV infection: a metaanalysis of randomized controlled trials. AIDS 1999;13:501–507. 38. Selwyn PA, Hartel D, Lewis VA, et al. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 1989;320:545–550. 39. Vynnycky E, Fine PEM. The natural history of tuberculosis: the implications of age-dependent risks of disease and the role of reinfection. Epidemiol Infect 1997;119:183–201. 40. Uplekar MW, Rangan S, Weiss MG, et al. Attention to gender issues in tuberculosis control. Int J Tuberc Lung Dis 2001;5:220–224. 41. Mackay J, Eriksen M. Tobacco Atlas. Geneva: World Health Organization, 2006. 42. UNAIDS, World Health Organization. AIDS Epidemic Update: Special Report on HIV/AIDS: December 2006. Geneva: World Health Organization, 2006. 43. World Health Organization. Guidelines for the Prevention of Tuberculosis in Health Care Facilities in Resource-Limited Settings. Geneva: World Health Organization, 1999.
44. Centers for Disease Control and Prevention, World Health Organization, International Union Against Tuberculosis and Lung Disease. Tuberculosis infection control in the era of expanding HIV care and treatment: addendum to WHO Guidelines for the prevention of tuberculosis in health care facilities in resourcelimited settings; 2007. 45. 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. 46. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. JAMA 1999;282:677–686. 47. Murray CJ. Towards good practice for health statistics: lessons from the Millennium Development Goal health indicators. Lancet 2007;369(9564):862–873. 48. World Health Organization. Measuring progress in TB control - recommendations of a WHO task force, 15–16 June 2006. [Online]. Accessed 2007 January 22. Available from URL:http://www.who.int/tb/ events/archive/measuring_progress_june06/en/ index.html 49. World Health Organization. Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis. Geneva: World Health Organization, 2006. 50. Zignol M, Hosseini MS, Wright A, et al. Global incidence of multidrug-resistant tuberculosis. J Infect Dis 2006;194:479–485. 51. WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance. Anti-tuberculosis Drug Resistance in the World. Fourth Global Report. Geneva: World Health Organization, 2008. 52. Euro TB and the National Coordinators for Tuberculosis Surveillance in the WHO European Region. Surveillance of Tuberculosis in Europe. Report on Tuberculosis Cases Notified in 2005. Paris: Institut de Veille Sanitaire, 2007. 53. Lopez AD, Mathers CD, Ezzati M, eds. Global Burden of Disease and Risk Factors. New York: Oxford University Press and The World Bank, 2006. 54. Shilova MV, Dye C. The resurgence of tuberculosis in Russia. Philos Trans R Soc Lond B Biol Sci 2001;356:1069–1075. 55. Leon DA, Chenet L, Shkolnikov VM, et al. Huge variation in Russian mortality rates 1984-94: artefact, alcohol, or what? Lancet 1997;350:383–388. 56. Shkolnikov V, McKee M, Leon DA. Changes in life expectancy in Russia in the mid-1990s. Lancet 2001;357(9260):917–921. 57. Walberg P, McKee M, Shkolnikov V, et al. Economic change, crime, and mortality crisis in Russia: regional analysis. BMJ 1998;317(7154):312–318. 58. McKeown T, Record RG. Reasons for the decline in mortality in England and Wales in the nineteenth century. Popul Stud 1962;16:94–122. 59. Kato-Maeda M, Rhee JT, Gingeras TR, et al. Comparing genomes within the species Mycobacterium tuberculosis. Genome Res 2001;11:547–554. 60. Mostowy S, Cousins D, Brinkman J, et al. Genomic deletions suggest a phylogeny for the Mycobacterium tuberculosis complex. J Infect Dis 2002;186:74–80. 61. Valway SE, Sanchez MPC, Shinnick TF, et al. An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N Engl J Med 1998;338:633–639. 62. Cain KP, Haley CA, Armstrong LR, et al. Tuberculosis among foreign-born persons in the United States: achieving tuberculosis elimination. Am J Respir Crit Care Med 2007;175(1):75–79. 63. Raviglione MC, Uplekar MW. WHO’s new Stop TB Strategy. Lancet 2006;367:952–955. 64. Uplekar M. The Stop TB Strategy: Building on and Enhancing DOTS to Meet the TB-Related Millennium Development Goals. Geneva: World Health Organization, 2006. 65. Dye C, Hosseini M, Watt C. Did we meet the 2005 targets for tuberculosis control? Bull World Health Organ 2007;85:364–369.
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The global epidemiology of tuberculosis 66. 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. 67. Hong YP, Kim SJ, Lew WJ, et al. The seventh nationwide tuberculosis prevalence survey in Korea, 1995. Int J Tuberc Lung Dis 1998;2:27–36. 68. China Tuberculosis Control Collaboration. The effect of tuberculosis control in China. Lancet 2004;364:417–422. 69. Aditama TY. Prevalence of tuberculosis in Indonesia, Singapore, Brunei Darussalam and the Philippines. Tubercle 1991;72:255–260. 70. Soemantri S, Senewe FP, Tjandrarini DH, et al. Threefold reduction in the prevalence of pulmonary tuberculosis over 25 years in Indonesia. Int J Tuberc Lung Dis 2007;11(4):398–404. 71. Huong NT, Duong BD, Co NV, et al. Tuberculosis epidemiology in six provinces of Vietnam after the introduction of the DOTS strategy. Int J Tuberc Lung Dis 2006;10:963–969. 72. Gopi PG, Subramani R, Narayanan PR. Trend in the prevalence of TB infection and ARTI after implementation of a DOTS programme in south India. Int J Tuberc Lung Dis 2006;10:346–348. 73. Subramani R, Santha T, Frieden TR, et al. Active community surveillance of the impact of different tuberculosis control measures, Tiruvallur, South India, 1968–2001. Int J Epidemiol 2007; 36(2):387–393. 74. Stop TB Partnership and World Health Organization. The Global Plan to Stop TB, 2006–2015. Geneva: Stop TB Partnership, 2006.
75. Blower SM, McLean AR, Porco TC, et al. The intrinsic transmission dynamics of tuberculosis epidemics. Nature Med 1995;1:815–821. 76. Dye C, Williams BG. Criteria for the control of drugresistant tuberculosis. Proc Natl Acad Sci USA 2000;97:8180–8185. 77. Dye C, Espinal MA. Will tuberculosis become resistant to all antibiotics? Proc Biol Sci 2001;268:45–52. 78. 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. 79. Salomon JA, Lloyd-Smith JO, Getz WM, et al. Prospects for advancing tuberculosis control efforts through novel therapies. PLoS Med 2006;3:e273. 80. Keeler E, Perkins MD, Small P, et al. Reducing the global burden of tuberculosis: the contribution of improved diagnosis. Nature 2006;444(Suppl 1):49–57. 81. Young DB, Dye C. The development and impact of tuberculosis vaccines. Cell 2006;124:683–687. 82. Dye C, Williams BG. Eliminating human tuberculosis in the twenty-first century. J R Soc Interface 2008; 5(23):653–662. 83. Borgdorff MW, Floyd K, Broekmans JF. Interventions to reduce tuberculosis mortality and transmission in low- and middle-income countries. Bull World Health Organ 2002;80(3):217–227. 84. Gajalakshmi V, Peto R, Kanaka TS, et al. 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.
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85. Ponce-De-Leon A, Garcia-Garcia Md Mde L, GarciaSancho MC, et al. Tuberculosis and diabetes in southern Mexico. Diabetes Care 2004;27:1584–1590. 86. Coker R, McKee M, Atun R, et al. Risk factors for pulmonary tuberculosis in Russia: case-control study. BMJ 2006;332:85–87. 87. Kim SJ, Hong YP, Lew WJ, et al. Incidence of pulmonary tuberculosis among diabetics. Tuberc Lung Dis 1995;76:529–533. 88. Jick SS, Lieberman ES, Rahman MU, et al. Glucocorticoid use, other associated factors, and the risk of tuberculosis. Arthritis Rheum 2006;55:19–26. 89. Verver S, Warren RM, Beyers N, et al. Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis. Am J Respir Crit Care Med 2005;171:1430–1435. 90. Whiting DR, Setel PW, Chandramohan D, et al. Estimating cause-specific mortality from communityand facility-based sources in the United Republic of Tanzania: options and implications for mortality burden estimates. Bull World Health Organ 2006; 84:940–948. 91. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: Part I. Latent tuberculosis. Expert Rev Mol Diagn 2006; 6:413–422. 92. Schechter M, Zajdenverg R, Falco G, et al. Weekly rifapentine/isoniazid or daily rifampin/ pyrazinamide for latent tuberculosis in household contacts. Am J Respir Crit Care Med 2006; 173:922–926.
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Molecular methods and their application in tuberculosis epidemiology Christopher RE McEvoy, Robin M Warren, and Paul D van Helden
INTRODUCTION It is now over 50 years since the introduction of the first effective antituberculosis chemotherapy. Despite this TB remains a major global health concern which is exacerbated by the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) pandemic, the emergence of drug-resistant TB strains, and inefficient health services combined with poverty and malnutrition in developing nations. Worldwide, it is estimated that TB is responsible for over 1.6 million deaths per year.1 Molecular epidemiology aims to complement classical epidemiology by determining the natural history and disease dynamics of Mycobacterium tuberculosis strains. Its purpose is to determine the genetic relatedness between isolates and the introduction of molecular tools has greatly increased accuracy to the point where the movements of specific strains and substrains can now be followed through both time and space. These studies can help determine genetic and environmental risk factors that may then aid health authorities in implementing the most effective control and treatment approaches as well as highlighting weaknesses in current control methods. Molecular epidemiology may be used to answer a variety of questions relating to TB infection, transmission, and control. Some of these include:
Where does disease transmission occur? What is the average latency period between infection and disease? What is the proportion of cases caused by endogenous reactivation versus exogenous reinfection? Do different strains show different pathologies? Do different strains show differences in transmission dynamics? Are drug-resistant strains able to transmit as efficiently as drug-susceptible strains? Are some strains more likely to become drug resistant than others? Are some strains more susceptible to anti-TB agents? How often does mixed infection (more than one strain) occur? What are the population risk factors and do these change between populations? How frequently does laboratory-based error occur?
Prior to the use of molecular methods, researchers in TB epidemiology were limited to bacterial factors such as colony morphology, growth rates in vitro, drug resistance profiles, and phage typing for differentiating between populations of M. tuberculosis. These aspects,
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while providing some valuable information at the time, lacked the specificity needed to define the vast majority of TB strains and thus the application of epidemiologically based TB research was greatly limited. It was not until the mid-1980s, over a century after Robert Koch’s seminal discovery that TB is caused by an infectious agent, that molecular biological methods and knowledge of the M. tuberculosis genome were sufficiently advanced to usher in a new range of epidemiological tools. Since that time a variety of molecular-based methods have been described, each with its own particular advantages and disadvantages. Each one, however, is far superior in its strain discrimination ability than previous methods. Thus, since the early 1990s our knowledge of the epidemiological characteristics of the current TB epidemic has grown rapidly and scientists have been provided with unprecedented insights into the factors that drive it. This has resulted in many previously held dogmas being challenged and has, in turn, assisted greatly in public health interventions. For additional information on the molecular epidemiology of TB readers are directed to a recent review by Mathema and co-authors.2
THE GENOME OF M. TUBERCULOSIS AND REGIONS OF EPIDEMIOLOGICAL SIGNIFICANCE The genome of M. tuberculosis holds all the information required for it to grow and reproduce or, in the view of the epidemiologist or clinician, to transmit and cause disease. This information is unique to each individual organism and can thus, in theory, be used to follow the natural history, including the growth, spread, and transmission, of any single lineage. The ultimate epidemiological tool would consist of the total genetic sequence of each TB isolate. While DNA sequencing technology is advancing at a spectacular rate and this may be a viable approach in the future it is not currently feasible and researchers are therefore compelled to concentrate on the small regions of the genome that are most informative. A disadvantage for the TB epidemiologist is that the genomes of all M. tuberculosis strains are remarkably homogeneous.3 Mycobacterium tuberculosis belongs to a closely related group of species known as the M. tuberculosis complex (MTBC) (Fig. 4.1). Recent genetic evidence has suggested that all members of this group are derived from the clonal expansion of a single progenitor that underwent a severe evolutionary bottleneck 20,000–35,000 years ago.4,5 The extreme genetic similarity exhibited by all members of the
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M. bovis BCG
M. bovis
M. caprae
M. pinnipedii
M. microti
M. africanum
M. tuberculosis modern
M. tuberculosis ancestral
M. canetti
Molecular methods and their application in tuberculosis epidemiology
4
methods available it is important that the most appropriate method or combination of methods be selected for the specific study in question. This will depend on factors including the size and geography of the study group, the genetic and cultural makeup of the population, the time period analysed, and whether the study is tracking endemic disease or a disease outbreak. The section below lists the most common genotyping methods used in the molecular epidemiology of TB. In each case background information on the genetic basis for the method, a brief methodology, and advantages and disadvantages of the method are discussed.
IS6110 RFLP ANALYSIS
Fig. 4.1 Phylogenetic tree of the M. tuberculosis complex (MTBC) determined from the analysis of the presence/absence of conserved deleted regions and of DNA polymorphisms in selected genes. Mycobacterium canetti split from the MTBC prior to an evolutionary bottleneck, which has resulted in high genomic homology between MTBC members. Adapted from 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. Copyright 2002 National Academy of Sciences, USA.
MTBC is demonstrated by the fact that the average sequence divergence between the two MTBC species M. tuberculosis and Mycobacterium bovis is less than 0.05% while the divergence between two strains of Escherichia coli is 1.6%.6,7 This fact has led to the recent proposal that members of the MTBC be reclassified as host-adapted ecotypes of the same species.8 Genomic regions that differ between TB strains and which can be exploited by molecular epidemiologists do occur however. These polymorphic regions, termed markers, are often located in non-coding regions and many involve repetitive sequences that vary in copy number. Apart from ease of use, cost, and standardization issues, an ideal genetic marker evolves at a constant rate in all strains, is selectively neutral, and exhibits extensive polymorphism so that convergent evolution (the same marker profile being displayed in unrelated strains) is avoided. Crucially, the ideal marker will evolve at a rate fast enough to distinguish between epidemiologically unrelated strains but slow enough to show no or limited differences between epidemiologically related strains. While no single marker satisfies all these criteria, many markers have been shown to display characteristics sufficient to be exploited for epidemiological purposes.
CURRENT GENOTYPING METHODS USED IN EPIDEMIOLOGICAL STUDIES OF TUBERCULOSIS There are many molecular methods available for TB epidemiological research, each of which presents its own distinct characteristics of discrimination ability, labour intensiveness, cost, speed, and ease of intra- and interlaboratory standardization. Given the variety of
The genome of M. tuberculosis contains numerous distinct small insertion sequences (ISs).9,10 These genetic elements encode a transposase that enables the element to ‘jump’ from one region to another. Thus these elements are mobile and in addition to displaying positional polymorphism they may also show polymorphism in the number of elements per genome. IS6110 is a MTBC-specific 1355-bp IS originally described by Thierry and colleagues10 that presents at 0–25 copies per genome. IS6110 restriction fragment length polymorphism (RFLP) methodology involves the following steps. Briefly, DNA is extracted and purified from the M. tuberculosis isolate, and it is then restricted with PvuII. PvuII cuts the genomic DNA into convenient sized fragments and also recognizes a single site within the IS6110 sequence. Following agarose gel electrophoresis and Southern blotting of the restricted DNA onto a nylon membrane, a labelled hybridization probe that recognizes an IS6110-specific site at the 30 end of the sequence is applied. Visualization of probebinding sites is then achieved using autoradiography. The results take the form of a biological barcode or fingerprint with the number of bands representing the number of IS6110 elements in the isolates’ genome and the position (size) of each band relating to its position of integration relative to the downstream PvuII site (Fig. 4.2). Because of its high interstrain polymorphism (in both a positional and numerical sense), and convenient mutation rate, IS6110 RFLP has become the most widely used genetic marker in TB epidemiology and is recognized as the international standard for M. tuberculosis genotyping. A standardized methodology has been implemented and dedicated computer software programmes have enabled results to be internationally standardized, thus allowing for interlaboratory comparisons.11 These results can then be used to construct dendrograms that illustrate the genetic relatedness of isolates obtained from a patient population of epidemiological interest (Fig. 4.3). However, several limitations to the technique apply. The standardized RFLP methodology requires at least 2 mg of DNA and subculturing of sputum samples is therefore required. Because of the slow-growing nature of M. tuberculosis this is extremely time consuming. The fingerprinting method itself, although technically quite simple, is also a laborious procedure and this, in conjunction with the long bacterial growth phase, results in a turnaround time of at least 5 weeks. The length of time that this method requires means that it cannot track epidemiological events in real time and is unsuitable for tasks such as rapid outbreak management. Another disadvantage of this technique is its inability to provide reliable typing for low IS6110 copy number (<6) strains since the resolution of the method is inversely proportional to the number of elements present.
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DNA. The method has only recently been described and it remains to be seen whether it becomes used generally in laboratories worldwide. Possible impediments to its use include the current lack of a standardized protocol for both the methodology and results analysis and the inherent disinclination of laboratories to move from older well-established and reliable techniques. As with many PCR-based genotyping methods DNA purity may also be an issue since poor quality DNA is difficult to amplify. The limitation of the IS6110 RFLP method in genotyping low copy number strains also applies equally to this method.
SPOLIGOTYPING
Fig. 4.2 Autoradiograph of IS6110 RFLP fingerprinting patterns for
13 clinical isolates of M. tuberculosis along with the standard laboratory strain H37Rv577 (far right). Each band represents an individual IS6110 element with the different band positions representing different locations within the genome. Strain relatedness can generally be inferred from the similarity of RFLP patterns.
PCR-BASED METHODS OF IS6110 GENOTYPING In an attempt to overcome the time and labour shortcomings that hinder IS6110 RFLP genotyping, several polymerase chain reaction (PCR)-based IS6110 genotyping techniques have been developed. An overview of the reproducibility and discriminatory power of these methods has been published by Kremer and colleagues.12 They found that in blinded tests conducted by laboratories with experience in the chosen technique, most of these methods suffered to varying degrees from poor reproducibility and/or discriminatory power. One method, fast ligation-mediated PCR (FLiP)13 (a further development of the previously described mixed-linker PCR),14 shows considerable promise however. This method is reported to allow IS6110 strain typing in under 7 hours from less than 1 ng of starting DNA and has only slightly less discriminatory power than conventional IS6110 RFLP analysis. The comparative methodology study by Kremer and colleagues also found it to be extremely reproducible.12 In addition, it has the potential to be adapted for automated analysis using fluorescent primers and, although no standardized protocol has been suggested, there are no reasons why this could not be done. The method involves the restriction of genomic M. tuberculosis DNA with HhaI followed by ligation of a DNA linker molecule to the fragment ends in a single reaction. In one strand the linker contains uracil in place of thymidine and specific amplification is obtained by digestion of this strand with uracil N-glycosylase followed by PCR using one primer specific for IS6110 and a second primer complementary to the linker ligated to the restricted genomic
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The direct repeat (DR) chromosomal region is found in the genomes of all MTBC members and comprises multiple identical 36-bp directly repeated sequences (DRs) interspersed by unique spacer regions ranging from 35 to 41 bp in length.15 One DR and its adjacent spacer is termed a direct variable repeat (DVR). Extensive polymorphism is observed between M. tuberculosis isolates within this region. Polymorphism may result from homologous recombination between DR sequences (either adjacent or distal), IS6110 insertions (which are extremely common within the DR region), homologous recombination between DR-associated IS6110 elements, and single nucleotide polymorphisms (SNPs) within the DR spacer regions. Apart from spacer SNPs all of the above mutations result in the deletion of DRs from the genome. Spacer oligonucleotide typing, or spoligotyping, is a simple technique used to determine the presence or absence of 43 spacer regions in M. tuberculosis isolates.16 Membranes are first spotted with synthetic oligonucleotides specific for each of the spacer regions. These are then hybridized with labelled spacer regions PCR-amplified from the isolate of interest resulting in a binary pattern that can be visualized using chemoluminescence and autoradiography. This genotyping method has two major attributes that have resulted in it becoming the most commonly used PCR-based strain discrimination method. The first relates to its simplicity, speed, reproducibility, and ease of use. Since it is based on the PCR reaction only minute quantities of starting DNA are required; thus the lengthy culturing step required for IS6110 RFLP is avoided and the typical time taken for a result to be obtained is less than 2 days. In addition, the robustness of the PCR reactions means that DNA purity is less of an issue and impure DNA, paraffin-embedded material, and material from Ziehl-Neelsen stainings, as well as DNA from other non-viable specimens, can be used. Spoligotyping’s second major attribute concerns the simplicity of its binary (present/absent) output. This means that data can be easily digitized, interpreted, and compared in an interlaboratory fashion. A global spoligotyping database (SpolDB4) has been launched, which by late 2006 contained over 39,295 different spoligotype entries from 122 countries and a Web-based programme, SPOTCLUST, that places spoligotypes into families, has recently been developed.17,18 The major drawback of spoligotyping is that it generally lacks the extensive discriminatory power of many other alternative genotyping methods. For example, the W-Beijing lineage, an extremely large and important group of strains that have attracted special attention due to their ability to cause frequent epidemic outbreaks, may be divided into many families of strains and substrains based on IS6110 RFLP and MIRU/VNTR (see below) marker variation. IS6110 RFLP in particular is capable of detecting hundreds of similar yet unique genotype patterns, enabling the
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Molecular methods and their application in tuberculosis epidemiology
4
Fig. 4.3 Dendrogram of IS6110 fingerprint patterns. IS6110 RFLP analysis was performed on 63 M. tuberculosis isolates taken from various regions of South Africa. Fingerprint patterns are ordered by similarity in a dendrogram that was constructed using GelCompar software (Applied Math, Kortrijk, Belgium) analysis. The scale at the top left-hand side represents a similarity index. Isolates showing similar fingerprints are grouped into family clusters.
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detection of clustering and family groupings. In contrast, spoligotyping reveals W-Beijing isolates to possess extremely similar or identical genotypes that all lack spacers 1–34.19 Spoligotyping has, however, been shown to be able to discriminate between many lS6110 low copy number strains that IS6110 RFLP analysis is unable to do.20 Thus, a combination approach using both IS6110 RFLP and spoligotyping has been shown to provide an extremely accurate and discriminatory genotyping method for M. tuberculosis isolates.21
Unfortunately the PGRS is found in the genome at a far higher frequency than IS6110 (61 copies in H37Rv) and this results in extremely complex fingerprints that may show variation in band intensity. For this reason reproducible scoring and computerization of results is difficult. This, coupled with the time-consuming and labour-intensive methodology (as for IS6110 RFLP genotyping), has greatly restricted its use.
MIRU/VNTR ANALYSIS
Both large and small deletion events are common in the M. tuberculosis genome and result in approximately 5% of genes and 4% of the entire genome being variably absent in clinical isolates.27 Genomic deletions may occur through recombination between IS elements as well as other unknown mechanisms. Large sequence deletions are considered to be unique events that occur once in the phylogeny of a lineage. Since these events are irreversible they arise sequentially and, if an assumption of selective neutrality is made, they allow the epidemiologist to genotype strains and construct strain phylogenies (Fig. 4.1).4,27–29 For example, Tsolaki et al.29 have defined a genetic deletion unique to the W-Beijing strain family that can therefore serve as a useful marker. Furthermore, additional deletions subdivide this family into four subgroups. On a more distant evolutionary scale, the MTBC species may be defined by specific deletion events present in the lineage of each species.4 In particular, ‘modern’ M. tuberculosis has been found to have undergone deletion of the ‘M. tuberculosis specific deletion 1’ (TbD1) genomic region while all ancestral strains and species possess this locus. While the power of large deletion analysis has been demonstrated in evolutionary and phylogenetic studies, its use in epidemiology remains in its infancy. In some cases a unique deletion associated with a single strain is known in advance and a simple deletion-specific PCR assay can enable the tracking of the strain through a population.29,30 However, in most epidemiology studies where numerous strains are encountered the analysis of multiple deletions is required. The use of multiple deletion-specific PCRs in multiple isolates is laborious and costly and microarray-based methods also suffer from technical complexity and high cost.27,28,31 A method designed for high-throughput analysis of genomic deletions has recently been reported.32 In this method, termed deligotyping, 43 variably deleted regions were chosen and amplicons of these regions generated from the isolate of interest were hybridized to a membrane spotted with these target sequences. This method is extremely similar to spoligotyping and shares its advantages including speed, simplicity, and an easily interpretable binary result. Care must be taken in the choice of deletion regions selected for analysis, however, since many deletions occur in ‘hot-spots’ and can occur independently in epidemiologically unrelated strains resulting in convergent evolution. By mapping deletion events onto a synonymous SNP-based phylogenetic tree Alland and colleagues33 have made progress in determining which events can be traced to a unique ancestral event (and therefore are probably selectively neutral and suitable for phylogenetic/genotype studies) and those that occur independently multiple times, thus precluding them as genotypic markers. Small deletions may also represent useful epidemiological markers but these are difficult to detect using current methods. A new microarray-based method, termed high-density DNA tiling arrays, promises to aid in their detection. This method is also amenable for SNP detection (see below). Its usefulness has recently been reported in investigating diversity between E. coli strains.34
Analysis of the complete M. tuberculosis genome has revealed the presence of 41 variable nucleotide tandem repeats (VNTRs).22 These represent DNA sequences of 40–100 bp that are directly repeated in a manner reminiscent of eukaryotic minisatellites. In the original study, Supply and colleagues22 found that 12 of these VNTRs, termed mycobacterial interspersed repetitive units (MIRUs), exhibited polymorphisms between isolates based on differences in repeat unit number. PCR analysis using primers flanking each MIRU is a simple method of genotyping and the 12-MIRU system of genotyping has become a widely used epidemiological tool. Apart from its simplicity other advantages are that results can be digitized, allowing for easy interlaboratory comparisons and that mixed infections are easily identified due to the sensitivity of the PCR reaction. However, in most cases the discrimination power of this method, even when used in conjunction with spoligotyping, is less than for IS6110 RFLP typing. Also, the number and combination of MIRUs used has varied among researchers and this, along with differences in nomenclature used to describe the MIRUs, has led to some confusion in the interpretation and comparison of results. Supply and co-workers have recently proposed a 15-loci set for routine epidemiological analysis that is claimed to have the same discriminatory power as IS6110 genotyping when used alongside spoligotyping.23 A further nine MIRU loci are proposed for isolates requiring higher resolution discrimination.23 It is still necessary to thoroughly evaluate these loci and research into the most informative and practical MIRU sets for use in specific settings, including low- and highincidence communities, is ongoing. The discriminatory power of MIRU genotyping is directly proportional to the number of loci analysed but additional loci also increase the time, cost, and labour required. In order to streamline the approach a multiplex PCR system using fluorescently labelled primers in combination with capillary fractionation and computerized automation of genotyping has been described, although this may not be widely adopted because of its cost and complexity.24
PGRS RFLP The polymorphic GC-rich repetitive sequence (PGRS) consists of a 96-bp consensus sequence that is found to be both numerically and positionally polymorphic within the M. tuberculosis genome. Like the IS6110 RFLP technique, PGRS RFLP also utilizes DNA restriction followed by gel electrophoresis, Southern blot hybridization, and sequence-specific probe recognition. It has been demonstrated to show identical banding patterns between closely related strains while being able to distinguish between epidemiologically unrelated strains.25 In some cases the method has been shown to be more discriminatory than IS6110 RFLP, especially when low copy number IS6110 strains are considered.26
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GENOMIC DELETION ANALYSIS
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Molecular methods and their application in tuberculosis epidemiology
SNPs Sequencing analysis of M. tuberculosis genomic regions has revealed very little genetic diversity compared with most other bacterial organisms.3,35,36 Despite this, more than sufficient SNP variation exists to exploit for genotyping purposes. SNPs may be divided into two categories. Non-synonymous SNPs (nsSNPs) occur in intragenic regions and are associated with amino acid changes. Hence they may alter the phenotype of the organism and be subject to selection pressure. Synonymous SNPs (sSNPs) either occur in intergenic regions or occur in intragenic regions without altering the amino acid sequence. They are generally considered to be selectively neutral, especially in comparison with genotypic markers that generate large chromosomal alterations such as IS6110 integrations and large deletion events. sSNPs are also unlikely to undergo convergent evolution and their mutation rate is hypothesized to be relatively constant, enabling them to be used to measure divergence times. These attributes make them attractive targets for epidemiological and phylogenetic studies. The methodologies generally used to detect SNPs, such as the SNaPshot primer extension method (used by Gutacker et al.37) and hairpin primer assays are PCR-based.37,38 They therefore benefit from the advantages of all PCR-based methodologies. Several studies have demonstrated the usefulness of SNP genotyping in the construction of phylogenetic trees of M. tuberculosis strains (Fig. 4.1).3,37–41 In the most extensive of these, Filliol and colleagues41 employed 212 SNPs to determine the phylogenetic relationships between 294 M. tuberculosis and 29 M. bovis isolates collected globally. All isolates were found to divide into one of six phylogenetically distinct groups and a minimal subset of just six highly informative SNPs was found to be able to discriminate all isolates into their specific groups. This finding may have important consequences since analysis of the large number of SNPs (often in the hundreds) used in most studies is expensive and time consuming despite it being PCR-based and a single standardized SNP set is required for interlaboratory comparisons.
THE INTERPRETATION OF MOLECULAR EPIDEMIOLOGY DATA The genomes of all species are continually evolving and this must be considered when interpreting molecular epidemiology data. It is generally considered that patient-derived isolates that display identical, or near-identical (fewer than two band shifts in an IS6110 fingerprint), genotypes (termed clusters) represent examples of recent transmission where infection in the recent past (i.e. within 2 years) has resulted in relatively rapid progression to disease. Genotypes that do not match those of other cases are considered to reflect reactivation of an infection that occurred many years prior to disease onset. By determining the relative proportion of clustered TB cases in a community an estimate of the proportion of either recent (using the ‘n1’ calculation) or ongoing (using the ‘n’ calculation) transmission can be made.42,43 These calculations are widely used as public health tools to estimate the efficacy of public health programmes. Several potential sources of error should be considered when interpreting epidemiological results. First, it is important to note the mutation rate of the marker being analysed before a decision on the clustering status of M. tuberculosis isolates is made. For example, slowly evolving markers, such as spoligotypes, may
4
show identical genotypes in epidemiologically unrelated strains. A marker that displays a high mutation rate (such as IS6110) will be more discriminatory although it then follows that genotype similarity measures in the definition of a cluster need to be relaxed. Second, undersampling of the population may result in an apparent reduction of strain clustering. This may subsequently lead to an underestimation of the frequency of recent transmission.44 Third, study duration is an important variable in epidemiology calculations. The difficulty of predicting events that occurred before or after the study duration (‘edge effects’) is particularly relevant to TB, which may remain latent for several years between initial infection and disease. Because of this, a 2-year lead-in and lag phase is recommended during which strains already present in the community (during the lead-in phase) and newly detected strains (during the lag phase) are disregarded. Finally, studies must ideally account for population migrations into or out of the study community. This is particularly relevant in mobile, high-incidence communities since immigrant patients will often be infected with strains not present in the community. This can then result in a false diagnosis of reactivation and an underestimation of the frequency of recent transmission.
THE APPLICATION OF MOLECULAR EPIDEMIOLOGY TO TUBERCULOSIS Molecular epidemiology has now clarified many aspects of TB dynamics that were impossible to determine using previous methods. Below we list several of these and show how molecular epidemiology has expanded our knowledge of each.
THE TRANSMISSION AND REACTIVATION DYNAMICS OF TUBERCULOSIS By comparing clinical isolates using clustering analysis, epidemiologists can estimate the proportion of disease caused by recent transmission (clustered) versus reactivation of latent infection (non-clustered). Several previously held assumptions regarding the dynamics of transmission/reactivation have been challenged since the introduction of molecular epidemiology techniques. It has now been shown, for example, that recent transmission is far more common in low-incidence communities than was previously thought.42,43 Furthermore, the proportion of cases due to recent transmission may be in excess of 70% in higher incidence communities.45 Another surprising finding is that transmission, in both high- and low-incidence settings, is less reliant on repeated close contact than was expected, with a high proportion of cases resulting from casual contact.42,46 Molecular epidemiology techniques combined with patient demographic information have also proved invaluable in establishing risk factors associated with M. tuberculosis transmission between patients. Age, ethnicity, socioeconomic status, and HIV status have all been associated with transmission but not all factors are common to all communities or geographic regions.42 This information suggests that control strategies should target prevention of transmission rather than prophylaxis for the prevention of reactivation.
RECURRENCE OF TUBERCULOSIS Recurrent TB is defined as a secondary disease episode following an initial disease event that has been considered cured. Of primary
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interest to epidemiologists is whether the recurrent disease represents the reactivation of the original strain (demonstrating that treatment was not fully effective) or whether it represents a new infection. When bacterial isolates from both disease episodes are available for analysis, molecular epidemiology techniques are ideal for distinguishing between the two alternative scenarios. Compliance to treatment regimens does not always result in complete sterilization and for many years it was assumed that recurrent TB was the result of the endogenous reactivation of surviving organisms from the initial infection.47,48 This dogma was first challenged using phage typing methods but was not confirmed unequivocally until molecular epidemiology methods were used.49,50 Reports from many geographical regions have demonstrated that the level of exogenous reinfection increases proportionally according to the local disease incidence. In The Netherlands, for example, where TB incidence is low, exogenous reinfection has been reported at 16%, whereas in high-incidence settings such as described by van Rie and colleagues the rate may be up to 75%.50,51 HIV-infected individuals have also been found to be more at risk of exogenous reinfection.52 These findings raise the possibility that certain individuals are predisposed to TB and that an initial disease episode may itself be a predisposing factor for later disease.53 In light of the above information it is suggested that the term ‘relapse’ be avoided unless it has been proven as such by genotyping.
MIXED INFECTIONS Multiple M. tuberculosis strains may be simultaneously present during disease in a patient, through either multiple recent infections or a recent infection combined with reactivation of a primary infection. The strains present in mixed infections rarely contribute equally to the bacterial load with one generally dominating the population. Because of their sensitivity, PCR-based genotyping methods are best employed for detection of the underlying strain. Molecular epidemiology has revealed that in high-incidence communities the frequency of mixed infection can be far higher than previously supposed. Using a PCR-based method to detect the presence of an IS6110 insertion within gene Rv2820 that is unique to the Beijing family lineage, Warren and colleagues54 reported that 19% of patients from a high-incidence community in Cape Town, South Africa, were coinfected with Beijing and non-Beijing strains and 57% of patients infected with a Beijing strain were also infected with a non-Beijing strain. The high frequency of observed mixed infection suggests that a prior M. tuberculosis infection provides little immune protection against subsequent infection. This has implications for disease control measures and vaccine development. Mixed infection has also been shown to be an important mechanism for the development of drug resistance.54
MOLECULAR EPIDEMIOLOGY IN OUTBREAK STUDIES Tuberculosis outbreaks are defined as an unusual increase in disease incidence within a specific community over a defined time period. The determination of the factors that can result in sudden changes in TB incidence are one of the major successes of molecular epidemiology techniques. The ability to differentiate between M. tuberculosis strains has allowed the tracking of outbreak strain transmission events in hospitals, prisons, mines, and communities and has highlighted deficiencies in disease control measures.55 The identification of outbreak strains has also allowed host- or
34
strain-specific risk factors to be determined. The tracking and characterization of drug-resistant outbreak strains has been of particular importance and one example is given in the next section.
DRUG RESISTANCE Drug-resistant isolates of M. tuberculosis were first identified less than 1 year after the first introduction of anti-TB chemotherapy.56 The frequency with which spontaneous drug resistance can develop has forced clinicians to treat TB with an antibiotic cocktail designed to ensure that drug resistance does not develop during treatment. A major contributing factor to the emergence of drug resistance is the inherent toxicity of the drugs combined with the prolonged treatment time (6 months) required by the current treatment regimen. This results in a high proportion of patients who fail to complete their treatment or who complete it at suboptimal levels. This produces the ideal environment for mutant bacterial clones that exhibit decreased sensitivity to the treatment to spread and eventually dominate the population. Outbreaks of multidrugresistant (MDR) (and even so-called extensively drug resistant; XDR) strains are now increasing in frequency in many parts of the world and this has sparked intense public health concerns since they are far more difficult (and costly) to treat, show a higher mortality rate, and may be transmitted to other individuals during the prolonged duration of treatment. Most drug resistance is caused by micromutations (point mutations or small insertions/deletions) in key genes involved in drug metabolism or transport. Fortunately, these genes often display ‘hot-spots’ where the majority of resistance-conferring mutations occur. This allows researchers to focus their analysis on a relatively short DNA sequence. For example, over 95% of rifampin-resistant M. tuberculosis isolates possess mutations within an 81-bp region of the rpoB gene and the majority of these mutations occur at specific defined sites within this region.57 This allows for simple sequencing or PCR-based mutation detection techniques to be implemented in the majority of cases. Isolates that fail to reveal mutations in initial studies may then undergo further analysis either using PCRbased methods directed at less common mutation sites or complete gene sequencing. Many epidemiological studies directed at drug resistance aim to follow a specific drug-resistant outbreak strain. In these cases once the specific resistance-conferring mutation is located a single mutation detection method can be conveniently implemented for the entire study. Molecular epidemiological tools have been used to study many aspects of drug-resistant TB disease dynamics. These include studies that have sought to determine the frequency of drug resistance in specific strains, the frequency of drug resistance in certain populations, patient risk factors for acquiring drug resistance, the proportion of primary (infection by a previously resistant organism) versus acquired (the emergence of drug resistance from a previously drug-susceptible organism de novo) resistance, and the prevalence of specific resistance-associated mutations within a population. The results of such studies have provided valuable insights. For example, studies have shown that primary resistance is most commonly observed, apart from cases involving drug-resistant outbreak strains. High-incidence communities may also display increased rates of acquired drug resistance.59 Molecular epidemiology has also been used to study the hypothesis that MDR invariably produces a fitness cost that results in reduced transmissibility. This generally appears to be true although the study of Bifani and colleagues,58 who followed a large MDR outbreak in New York, has
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Molecular methods and their application in tuberculosis epidemiology
shown that it is not always the case. Studies have also found that the acquisition of drug-resistant TB may be due to reinfection, and that drug resistance may develop as a result of selective pressure after mixed infection.59,60 Studies aimed at determining the relationship between the frequency of drug resistance acquisition and genotype have also been performed with some results suggesting that the Beijing family is more prone to develop drug resistance.61 The tracking of drug-resistant outbreaks has also been aided by molecular epidemiology. An example of the role it can play in understanding an MDR outbreak is provided by Dahle and colleagues.62 In this case 20 individuals, of whom 19 were immigrants from East Africa, were diagnosed with drug-resistant or MDR TB in Oslo, Norway. The initial patient presented with TB that was found to be resistant to the frontline anti-TB drug isoniazid (INH) and 1 year later was hospitalized with disease that was found to have acquired additional resistance to rifampicin. In the following 6 years a further 19 patients presented with TB that was found to be either drug resistant or MDR. IS6110 RFLP analysis revealed all isolates to be clustered, demonstrating recent transmission, and comparisons of the outbreak RFLP fingerprint with those from other countries strongly supported a Somalian origin for the strain. Spoligotyping was used to further confirm the genetic similarity of the 20 isolates. All strains were INH resistant and katG gene (which confers INH resistance when mutated) analysis demonstrated identical sequences, all with three alterations from wild type. All MDR strains (11/20) showed a single point mutation in the rpoB gene conferring rifampicin resistance while all non-MDR strains showed wild-type rpoB sequence. Thus the mutations in drug resistance genes provide explanations for the resistant phenotype as well as further confirming the close relatedness of the strains. Social links were found between 14 of the patients. Thus the study demonstrates that the outbreak strain was imported from Somalia (where it was probably already INH resistant) but acquired multidrug resistance and was transmitted in Norway. The authors use these findings to highlight the high communicability of some drug-resistant and MDR strains, especially among immigrant communities. They also make several suggestions hoped to make health and immigration policies more efficient in preventing future outbreaks.
EVOLUTIONARY AND PHYLOGENETIC STUDIES An important benefit of the introduction of molecular epidemiology has been its ability to provide high-resolution analysis of specific M. tuberculosis strains. It is now known that TB is caused by organisms that represent many different families and that each of these families can be divided into strains and substrains based on genotypic similarities. Several recent reports have shed light on the phylogeny of M. tuberculosis strains.4,37,40,41,63 Some inconsistencies exist, seemingly based on the number of polymorphisms analysed and the extent of sampling, but the studies are generally consistent and, overall, support the division of M. tuberculosis into three major groups that can be subdivided into nine major strains. Of note is the extent of geographic specificity found with certain strains. For example, strains I and II (which include the W-Beijing strains) are found to a greater extent among patients of East Asian origin, supporting this location as the origin of the clade. Molecular epidemiology methods, in particular deletion, SNP analysis, and extensive sequencing, are also useful in the construction of phylogenetic trees representing more distant evolutionary events. These may then be used to determine the time and location of the
4
origin of M. tuberculosis as well as its subsequent spread throughout the world. These studies have found that M. tuberculosis is not derived from M. bovis, as was previously thought, but that the M. tuberculosis lineage predates the emergence of M. bovis (Fig. 4.1).4 Sequence-based studies examining all MTBC members confirm a severe evolutionary bottleneck that occurred in the MTBC 20,000–35,000 years ago and suggest that Mycobacterium canetti and other smooth bacilli group lineages predate this bottleneck while all other MTBC members represent the clonal expansion of one of these progenitor species.4 Moreover, the extant representatives of this ancient group (including M. canetti ) are localized to East Africa and are also human pathogens, suggesting that M. tuberculosis has coevolved with the human lineage since at least the time of early hominids between 2.6 and 2.8 million years ago.5 While the above information is not directly relevant to the study of TB epidemiology it is important because it allows us to establish the rate of change (mutation) of genotypic markers, a factor that influences the interpretation of epidemiological data.
PHENOTYPIC VARIATION BETWEEN M. TUBERCULOSIS STRAINS AND HOST–PATHOGEN COMPATIBILITY Of epidemiological interest is the fact that strains may exhibit phenotypic variation in many aspects of virulence. This phenomenon has been especially highlighted in animal models and can include Box 4.1 Definitions This section provides definitions of terms, particularly molecular, that may not be familiar to the casual reader. Agarose gel electrophoresis: The separation of DNA according to size by the migration through an agarose gel in an electrical field. Cluster: A grouping of two or more TB cases that are proximally and temporarly related. If a cluster is of sufficient size and importance it may be re-evaluated as an outbreak. Genotype: The genetic constitution of an organism, as distinct from its expressed features or phenotype. Insertion sequence: A small, mobile DNA sequence that can replicate and insert copies of itself at random sites within chromosomes. They code for the enzyme transposase, which catalyses their replication. Microarray: A collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to chemically suitable matrices. Non-coding DNA: DNA regions that do not represent a polypeptide code. These regions may regulate the expression of coding regions, have other more enigmatic functions, or simply be ‘junk’ DNA without any apparent function. Outbreak: See Cluster. PCR (polymerase chain reaction): A technique for massive in vitro amplification of specific DNA fragments. Polymorphism: The occurrence in the same population of multiple discrete allelic states. Repetitive DNA: Nucleotide sequences in DNA that are present in the genome as numerous copies. Much repetitive DNA is tandemly repeated. Selectively neutral: When neither of two (or more) alternative polymorphic variants possesses an evolutionary selective advantage. Southern blot: A technique used for searching for a specific DNA fragment that has been electrophoresed and bound to a nitrocellulose membrane.
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differences in transmission ability, disease manifestation, immunological responses, replication rate, and possibly the frequency of drug resistance and ability to evade vaccination.61,64–71 This implies that a population experiencing a high incidence of TB that presents multiple strains should be viewed as presenting a combination of distinct subepidemics rather than a single uniform epidemic. A recent analysis of the global population structure of M. tuberculosis has shown that different lineages are associated with specific sympatric human populations.28 Furthermore, in mixed, urban settings these lineages were found to be more likely to spread in sympatric than allopatric populations, suggesting host–pathogen compatibility. International databases that collate genotype data gathered throughout the world (such as the global spoligotype database) should be valuable in the further investigation of this interesting phenomenon.17
TRANSMISSION FROM SMEAR-NEGATIVE CASES The Ziehl-Neelsen sputum smear is a rapid, inexpensive diagnostic test for the presence of active TB and it has long been assumed that smear-negative patients are far less infectious.71 The potential for smear-negative patients to transmit disease at a high frequency was confirmed using molecular genotyping methods in 1999.72 In this study at least 17% of TB transmission was found to be from patients who had produced a smear-negative sputum sample.
DETECTION OF LABORATORY ERROR Incorrect sample labelling, contamination of clinical devices, or inefficient measures of cross-contamination control can result in a false diagnosis or the misidentification of drug resistance status and may seriously compromise patient welfare. Burman and
REFERENCES 1. World Health Organization. Global Tuberculosis Control: Surveillance, Planning, Financing. Geneva: (WHO/HTM/TB/2007.376). 2. Mathema B, Kurepina NE, Bifani PJ, et al. Molecular epidemiology of tuberculosis: current insights. Clin Microbiol Rev 2006;19(4):658–685. 3. 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. 4. 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. 5. Gutierrez MC, Brisse S, Brosch R, et al. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog 2005; 1(1):e5. 6. Garnier T, Eiglmeier K, Camus JC, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci USA 2003;100(13): 7877–7882. 7. Perna NT, Plunkett G III, Burland V, et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 2001;409(6819):529–533. 8. Smith NH, Kremer K, Inwald J, et al. Ecotypes of the Mycobacterium tuberculosis complex. J Theor Biol 2006;239(2):220–225. 9. Gordon SV, Heym B, Parkhill J, et al. New insertion sequences and a novel repeated sequence in the
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10.
11.
12.
13.
14.
15.
16.
17.
Reves73 have shown that M. tuberculosis-associated laboratory errors can occur more frequently than suspected and are often not recognized by laboratory personnel. Genotyping of samples by IS6110 RFLP has frequently enabled errors to be identified. Identification of laboratory error usually occurs when identical RFLP fingerprints are observed from different patients whose samples were processed at a similar time or when serial isolates from the same patient show different fingerprints. The presence of mixed infections, which may be common in high-incidence regions, must also be considered.54 In these cases both isolates may be represented and this can result in the erroneous conclusion of cross contamination.
SUMMARY In recent years our understanding of the structure of the M. tuberculosis genome has grown immensely. The discovery of polymorphic genomic regions combined with molecular methods for their analysis has allowed unprecedented insights into the structure of M. tuberculosis populations and has revealed that it consists of a huge assortment of genetically distinct families, strains, and substrains. This insight has allowed epidemiologists an unprecedented understanding of TB biology and has resulted in many previous assumptions of the disease to be revised. However, health control and treatment measures have benefited in only very few cases and the TB pandemic has continued to worsen. In order to reverse this situation new diagnostic tests, vaccines, and treatment options are desperately needed. The rapid increase in knowledge of many aspects of TB biology provides hope, however, and molecular epidemiology will continue to play a crucial role in our ongoing attempt to fully understand and control the disease. The challenge before us, given this new understanding, is to revise control programmes to reflect this knowledge.
genome of Mycobacterium tuberculosis H37Rv. Microbiology 1999;145(Pt 4):881–892. Thierry D, Cave MD, Eisenach KD, et al. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 1990;18(1):188. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993; 31(2):406–409. Kremer K, Arnold C, Cataldi A, et al. Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains. J Clin Microbiol 2005;43(11):5628–5638. Reisig F, Kremer K, Amthor B, et al. Fast ligationmediated PCR, a fast and reliable method for IS6110based typing of Mycobacterium tuberculosis complex. J Clin Microbiol 2005;43(11):5622–5627. Haas WH, Butler WR, Woodley CL, et al. Mixedlinker polymerase chain reaction: a new method for rapid fingerprinting of isolates of the Mycobacterium tuberculosis complex. J Clin Microbiol 1993;31(5): 1293–1298. Hermans PW, van Soolingen D, Bik EM, et al. Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun 1991;59(8):2695–2705. Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35(4):907–914. Brudey K, Driscoll JR, Rigouts L, et al. Mycobacterium tuberculosis complex genetic diversity: mining the
18.
19.
20.
21.
22.
23.
24.
fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 2006;6(1):23. Vitol I, Driscoll J, Kreiswirth B, et al. Identifying Mycobacterium tuberculosis complex strain families using spoligotypes. Infect Genet Evol 2006;6(6):491–504. van Soolingen D, Qian L, de Haas PE, et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J Clin Microbiol 1995;33(12):3234–3238. Bauer J, Andersen AB, Kremer K, et al. Usefulness of spoligotyping to discriminate IS6110 low-copynumber Mycobacterium tuberculosis complex strains cultured in Denmark. J Clin Microbiol 1999;37(8): 2602–2606. 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. Supply P, Mazars E, Lesjean S, et al. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 2000;36(3):762–771. Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006;44(12):4498–4510. Supply P, Lesjean S, Savine E, et al. Automated highthroughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 2001;39(10):3563–3571.
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Molecular methods and their application in tuberculosis epidemiology 25. Ross BC, Raios K, Jackson K, et al. Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool. J Clin Microbiol 1992;30(4):942–946. 26. Chaves F, Yang Z, el Hajj H, et al. Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis. J Clin Microbiol 1996; 34(5):1118–1123. 27. 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. 28. Gagneux S, DeRiemer K, Van T, et al. Variable hostpathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006;103(8):2869–2873. 29. Tsolaki AG, Gagneux S, Pym AS, et al. Genomic deletions classify the Beijing/W strains as a distinct genetic lineage of Mycobacterium tuberculosis. J Clin Microbiol 2005;43(7):3185–3191. 30. Freeman R, Kato-Maeda M, Hauge KA, et al. Use of rapid genomic deletion typing to monitor a tuberculosis outbreak within an urban homeless population. J Clin Microbiol 2005;43(11):5550–5554. 31. Kato-Maeda M, Rhee JT, Gingeras TR, et al. Comparing genomes within the species Mycobacterium tuberculosis. Genome Res 2001;11(4):547–554. 32. Goguet de la Salmoniere YO, Kim CC, Tsolaki AG, et al. High-throughput method for detecting genomic-deletion polymorphisms. J Clin Microbiol 2004;42(7):2913–2918. 33. Alland D, Lacher DW, Hazbon MH, et al. The role of large sequence polymorphisms (LSPs) in generating genomic diversity among clinical isolates of Mycobacterium tuberculosis and the utility of LSPs in phylogenetic analysis. J Clin Microbiol 2007;45(1):39–46. 34. Jackson SA, Mammel MK, Patel IR, et al. Interrogating genomic diversity of E. coli O157:H7 using DNA tiling arrays. Forensic Sci Int 2007; 168(2–3):183–199. 35. Fleischmann RD, Alland D, Eisen JA, et al. Wholegenome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 2002; 184(19):5479–5490. 36. Hughes AL, Friedman R, Murray M. Genomewide pattern of synonymous nucleotide substitution in two complete genomes of Mycobacterium tuberculosis. Emerg Infect Dis 2002;8(11):1342–1346. 37. 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. 38. Hazbon MH. Recent advances in molecular methods for early diagnosis of tuberculosis and drug-resistant tuberculosis. Biomedica 2004;24(Suppl 1):149–162. 39. 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. 40. Gutacker MM, Mathema B, Soini H, et al. Singlenucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 2006;193(1): 121–128. 41. Filliol I, Motiwala AS, Cavatore M, et al. Global phylogeny of Mycobacterium tuberculosis based on single
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
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. 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(24): 1703–1709. Alland D, Kalkut GE, Moss AR, et al. Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. N Engl J Med 1994;330(24):1710–1716. Glynn JR, Crampin AC, Yates MD, et al. The importance of recent infection with Mycobacterium tuberculosis in an area with high HIV prevalence: a long-term molecular epidemiological study in northern Malawi. J Infect Dis 2005;192(3):480–487. van der Spuy GD, Warren RM, Richardson M, et al. Use of genetic distance as a measure of ongoing transmission of Mycobacterium tuberculosis. J Clin Microbiol 2003;41(12):5640–5644. Verver S, Warren RM, Munch Z, et al. Proportion of tuberculosis transmission that takes place in households in a high-incidence area. Lancet 2004; 363(9404):212–214. Manabe YC, Bishai WR. Latent Mycobacterium tuberculosis—persistence, patience, and winning by waiting. Nat Med 2000;6(12):1327–1329. Stead WW. Pathogenesis of a first episode of chronic pulmonary tuberculosis in man: recrudescence of residuals of the primary infection or exogenous reinfection? Am Rev Respir Dis 1967;95(5):729–745. Raleigh JW, Wichelhausen R. Exogenous reinfection with Mycobacterium tuberculosis confirmed by phage typing. Am Rev Respir Dis 1973; 108(3):639–642. van Rie A, Warren R, Richardson M, et al. Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N Engl J Med 1999;341(16):1174–1179. de Boer AS, Borgdorff MW, Vynnycky E, et al. Exogenous re-infection as a cause of recurrent tuberculosis in a low-incidence area. Int J Tuberc Lung Dis 2003;7(2):145–152. Sonnenberg P, Murray J, Glynn JR, et al. HIV-1 and recurrence, relapse, and reinfection of tuberculosis after cure: a cohort study in South African mineworkers. Lancet 2001;358(9294):1687–1693. Verver S, Warren RM, Beyers N, et al. Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis. Am J Respir Crit Care Med 2005;171(12):1430–1435. Warren RM, Victor TC, Streicher EM, et al. Patients with active tuberculosis often have different strains in the same sputum specimen. Am J Respir Crit Care Med 2004;169(5):610–614. 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(3):305–314. Youmans GP, Raleigh GW, Youmans AS. The tuberculostatic action of para-aminosalicylic acid. J Bacteriol 1947;54(4):409–416. Williams DL, Waguespack C, Eisenach K, et al. Characterization of rifampin-resistance in pathogenic mycobacteria. Antimicrob Agents Chemother 1994; 38(10):2380–2386.
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58. Bifani PJ, Plikaytis BB, Kapur V, et al. Origin and interstate spread of a New York City multidrugresistant Mycobacterium tuberculosis clone family. JAMA 1996;275(6):452–457. 59. van Rie A, Warren R, Richardson M, et al. Classification of drug-resistant tuberculosis in an epidemic area. Lancet 2000;356(9223):22–25. 60. van Rie A, Victor TC, Richardson M, et al. Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns. Am J Respir Crit Care Med 2005;172(5):636–642. 61. Glynn JR, Whiteley J, Bifani PJ, et al. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 2002;8(8):843–849. 62. Dahle UR, Sandven P, Heldal E, et al. Deciphering an outbreak of drug-resistant Mycobacterium tuberculosis. J Clin Microbiol 2003;41(1):67–72. 63. Baker L, Brown T, Maiden MC, et al. Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis. Emerg Infect Dis 2004; 10(9):1568–1577. 64. 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. [Published erratum appears in N Engl J Med 1998;338(24):1783.] 65. Dormans J, Burger M, Aguilar D, et al. Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model. Clin Exp Immunol 2004; 137(3):460–468. 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. Manca C, Reed MB, Freeman S, et al. Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis. Infect Immun 2004;72(9):5511–5514. 68. Chacon-Salinas R, Serafin-Lopez J, Ramos-Payan R, et al. Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 2005;140(3):443–449. 69. Zhang M, Gong J, Yang Z, et al. Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis 1999; 179(5):1213–1217. 70. Abebe F, Bjune G. The emergence of Beijing family genotypes of Mycobacterium tuberculosis and low-level protection by bacille Calmette-Guerin (BCG) vaccines: Is there a link? Clin Exp Immunol 2006; 145(3):389–397. 71. Shaw JB, Wynn-Williams N. Infectivity of pulmonary tuberculosis in relation to sputum status. Am Rev Tuberc 1954; 69(5):724–732. 72. Behr MA, Warren SA, Salamon H, et al. Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 1999;353(9151):444–449. [Published erratum appears in Lancet 1999;353(9165):1714.] 73. Burman WJ, Reves RR. Review of false-positive cultures for Mycobacterium tuberculosis and recommendations for avoiding unnecessary treatment. Clin Infect Dis 2000;31(6):1390–1395.
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Epidemiology of childhood tuberculosis Robert P Gie, Nulda Beyers, and Donald A Enarson
INTRODUCTION Relatively few accurate data are available on the epidemiology of childhood TB partly because TB is sometimes difficult to diagnose in children and also partly because children are not seen as being important in the epidemiology of TB. However, TB infection and TB disease in children are sentinel events and can give a fairly accurate indication of how well the TB control programme is functioning and of the level of transmission of TB taking place in a community. Children may thus be seen as ‘sentinel’ indicators of the trend for TB in a specific country or region.
DEFINITIONS
TUBERCULOSIS INFECTION Tuberculosis infection has been traditionally identified using the tuberculin skin test, by the Mantoux method. Induration in reaction to the tuberculin skin test, measured as the largest transverse diameter in response to intradermal injection of tuberculin, has been the traditional approach, with reaction sizes of 5 (or 10) mm or greater taken as significant, indicating the presence of infection. This size of reaction was defined as significant in that it has been shown to accurately predict risk of subsequent development of TB following infection.3 In recent years, newer tests such as the interferon-g release assays (IGRAs) have been introduced that promise to have more efficient characteristics than the tuberculin skin test but they have not yet been sufficiently evaluated to know whether these claims will be upheld.
EPIDEMIOLOGY
DEATH FROM TUBERCULOSIS (FATALITY)
Epidemiology is defined as the study of distribution and determinants of health-related states or events in specified populations, and the application of this study to control of health problems.1
While notification of death from any cause while on TB treatment is the event reported routinely in cohort reports on the outcome of TB treatment, they are not sufficiently precise for use in epidemiology. The term ‘fatality’ refers, rather, to death due to the condition. To determine this correctly requires a review and classification of deaths in patients with TB to determine whether the death was due to TB, whether TB was a contributing factor or whether the death was unrelated to TB.4
CHILDHOOD The definition of who is considered a ‘child’ varies from one location to another and is frequently determined in health services for administrative reasons (where and by whom health services are provided). For the purposes of this chapter, all persons aged less than 15 years is considered a child. For reasons that will be illustrated by the discussion of the distribution and determinants of disease, we further classify children by the following age groups: under 2 years of age; 2–4 years of age; and 5–14 years of age.
CASE OF TUBERCULOSIS For purposes of routine notification, it is recommended that every person given treatment for active TB be recorded as a case of TB,2 but that those who are sputum smear-positive and those who have received previous treatment for TB for 1 month or more be reported separately. In the setting where children under 5 years of age are living in the same household with a patient who is sputum smear-positive, the following recommendation is given: ‘Any child in the household under 5 years of age who has symptoms that suggest TB should be given treatment as a case of TB.’
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CURE The definition of cure from TB requires bacteriological proof that the disease is no longer active. In current practice, this implies confirmation of bacteriological conversion, in a patient initially bacteriologically positive, sustained from the fifth month of treatment onwards.2
TRANSITION FRAMEWORK In studying the epidemiology of TB, it is vital to set a framework from which to work. This framework should take into account a model of the transitions that take place in the pathogenesis of TB.5 This is important because each transition has its own predictors and transition probabilities that vary from one setting to another. Key transitions that must be carefully distinguished in understanding the epidemiology of childhood TB are those from
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exposure to infection, from infection to disease, from disease to death and from disease to cure. Conflating several transitions (e.g. studies that entail an analysis of determinants of the transition from exposure to disease) may identify determinants of the two key transitions that are either powerful determinants of a single transition or may fail to identify ‘conflicting’ determinants associated with the two transitions. Mortality studies are particularly fraught as they include all the key transitions.
SOURCES OF INFORMATION Determining the total burden, distribution and determinants of TB in children requires valid and reliable information on the disease. This information may be obtained from a variety of sources. The two main sources of information on the disease are routine notifications of the disease (mandatory in most countries) and information derived from specific scientific investigations and published in the peer-reviewed scientific literature.
PRE-CHEMOTHERAPY LITERATURE Since the introduction of chemotherapy for treatment of TB, the ‘natural history’ of TB in human populations has changed dramatically. Treatment of disease has radically changed the fate of TB by preventing death and permanently curing most patients. Moreover, treatment of infection has drastically reduced the progress from infection to disease particularly in children and most efficiently within households of TB patients where contact investigation is actively pursued. Consequently, because treatment with anti-TB medications purposely aims to change much in the epidemiology of TB, information derived from studies prior to the chemotherapy era (before the Second World War) is particularly relevant in providing a picture of the disease at that time. The transitions most immediately affected by chemotherapy are the transitions from disease to cure or death; transitions from infection to disease and from exposure to infection are affected at later stages of widespread implementation of TB services. Several comprehensive reviews of scientific publications on the TB situation in children prior to the chemotherapy era have been published.6,7 This exercise identified all published articles in the English language literature reporting studies undertaken between 1920 and 1950 that included at least 1,000 children followed for a minimum of 10 years.8–15
ROUTINE NOTIFICATIONS Annual reports on TB notifications are prepared by the World Health Organization (WHO), summarizing information obtained from all countries of the world (the most recent being from 200716). These reports are compiled from information collected routinely at the clinic level, and at district level, using standardized data collection forms, reporting on all individuals put on treatment for active TB in the country from which the report is submitted. These reports provide information on the total number of cases according to whether they are new or re-treatment cases and, for new cases, the number of cases that are sputum smear-positive, sputum smear-negative pulmonary or extrapulmonary. Age-specific reports are provided routinely only for new smear-positive cases and, among these, the information on children is reported for the
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age group under 15 years, combined in the vast majority of settings. As the greatest proportion of cases in children (and particularly in small children) is not sputum smear-positive, these routine reports have limited usefulness in determining the epidemiology of TB in childhood. Other factors further limit the usefulness of these reports. The comprehensiveness of reporting may vary widely and is influenced by the priority given to ensure completeness of the information collected.17 It varies as well by the criteria used to define a case of TB in children,18 and on the extent of active case finding (in particular, household contact investigation) carried out in a particular location. Particular caution must be used in interpreting routinely reported cases as they may specifically under-report more serious forms of disease in children.19
DISEASE PREVALENCE SURVEYS Surveys of disease prevalence should be able to provide useful information on the distribution and determinants of TB in childhood. Despite this, they have provided very limited data upon which study of the epidemiology of TB in childhood can be based. Disease prevalence surveys for the epidemiology of TB in childhood have several inherent limitations. First, TB is a relatively rare disease in most locations. Because of this, the statistical power of such surveys is limited by the necessity of examining a large sample of the population to obtain reliable estimates of disease prevalence. In most instances, such surveys are powered to provide such estimates for only the entire population of a country and do not have sufficient power to study subsets of the population (including subsets of age that permit generalizations concerning children). A second limitation is related to the tools used in such surveys to detect cases. In most surveys, the measurements are based on questionnaires, chest radiographs and bacteriological examination of sputum. These methods are distinctly limited in their usefulness in identifying children with TB. As noted earlier, the majority of children with TB are not sputum smear-positive. Moreover, the symptoms of TB in children are not as straightforward as in adults (in whom they already have significant limitations). Chest radiography is the most useful of the three tools for measurement but even chest radiography has limitations in that standardization of reading of chest radiographs has not been undertaken for children.
TUBERCULIN SKIN TEST SURVEYS The specific public health objective of modern TB services is the reduction in prevalence of infectious TB in order to reduce the incidence of infection in children. The ultimate aim is to create a generation of children that is ‘infection-free’ and, thus, to eliminate TB from the community. Infection is measured classically by the tuberculin skin test survey. Studying the distribution (especially the trend over time) of infection in children is the only way to directly measure progress towards this objective. Tuberculin skin test surveys are usually undertaken in school children and therefore provide important information on TB in childhood.20 The size of induration resulting from the tuberculin skin test has been demonstrated to accurately predict who is most likely to develop TB in future.3 This test is therefore used to identify TB infection. Such surveys have the possibility of determining the burden and distribution of TB infection in children. The target population is school children aged 6–8 years and, as the test identifies the
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prevalence of infection in this age group, the results are a summary of the incidence of infection multiplied by the person-years at risk. Thus, the survey gives an average experience of the children over the entire period of the child’s life (centred on the date defined as the current date minus the mean age of the group of children, divided by 2). In most instances, therefore, the measurement derived from a tuberculin skin test survey measures the average experience over the previous 7 years, centred on 3.5 years prior to the date of the study. This ‘averaging’ thus adds imprecision to the measurement as it obscures (or is adversely affected by) changes that might have taken place over the period studied (whether prevalence of TB is falling – as is the case in the indigenous populations of most industrialized countries – or rising – as is the case in much of sub-Saharan Africa over the past decade). Tuberculin skin test surveys are also limited by poor technique (indicated by ‘terminal digit preference’) and by cross-reactivity of the antigens with those of Bacillus Calmette–Gue´rin (BCG) vaccination and with other environmental mycobacteria. Moreover, as the prevalence of significant tuberculin skin reactions declines, this imprecision becomes greater,21 drastically limiting the usefulness of the test where TB is infrequent. Extensive surveys were undertaken by the WHO in many countries in the 1950s and 1960s, often in preparation for mass campaigns for BCG vaccination. The studies undertaken in Africa during this period have been reported.22 A number of more recent tuberculin skin test surveys have been reported from Africa and India.23–25
HOUSEHOLD CONTACT STUDIES Because exposure is most prolonged and intense among persons living within the household of a TB patient, active investigation to detect those in the household (especially children under 5 years of age) with infection and with TB is recommended.2 Treatment is recommended for all those found to have infection or disease. This policy on the one hand substantially changes the epidemiology of TB by significantly reducing the risk of progression from infection to disease and by permanently curing a high proportion of those with disease. On the other hand, it provides systematic investigation within households (and sometimes more broadly) that provides information on the transition from infection and, to a more limited extent, from infection to disease. Three large studies of household contacts established the basis for this literature.26–28
INTERVENTION TRIALS At the point that interventions were first introduced to address the problem of TB, several very large trials were conducted in which placebo controls were compared with those assigned to an intervention arm of investigation. The trial of preventive chemotherapy for treatment of TB infection in contacts of active cases of TB was undertaken in children.3 The study participants were followed for an extensive period of time after the trial to determine the development of disease. The placebo control group thus provides information on the transition from infection to disease in the absence of intervention.
HEALTH SERVICES RESEARCH The final source of information on the epidemiology of childhood TB comes from a series of health service-based studies of TB in children.
40
GLOBAL ESTIMATES OF BURDEN OF DISEASE IN CHILDREN Several publications have explored the global epidemiology of childhood TB.29,30 Estimates of the global burden of disease vary widely. Nelson and Wells noted that, in 1989, the Expanded Programme on Immunization of WHO reported a total of 1.3 million cases of TB in children under 15 years of age leading to the death of 450,000 children.31 A second report from the WHO estimated that this number was 650,000 cases in children out of 7.5 million total cases in 1990,32 9% of the total estimated cases. A further report providing estimates of the trend expected over the decade 1990–2000 reported that the expected number of cases in the year 2000 would be 10.2 million.33 There are not good estimates of the percentage of cases occurring in children, but it is estimated that the proportion of children with TB is much higher in countries with a high incidence of TB than in low-incidence countries.34 Clearly, the variation in these estimates is much greater than could be explained by biological factors, indicating the extent of uncertainty regarding the size of the problem. Some authors have concluded that reliable estimates of the burden of disease can only be obtained from industrialized countries.35 Even this optimism may be misplaced for the reasons given earlier in discussion of routine notifications as sources of information. Because of the unavailability of actual reports on TB in children, the problem has been addressed using modelling exercises. One such exercise used the reported positive cases in 2000 in the 22 ‘high-burden’ countries (those nations estimated to contain more than 80% of all cases of TB) to estimate the number of all cases expected in children under 15 years of age.36 The total number of expected cases in children under 15 years of age was 884,019, 10.7% of all estimated cases, of which 659,397 (74.6%) were in the 22 high-burden countries. The proportion of children under 15 years of age among all cases varied from 2.7% (Indonesia and Thailand) to 25.3% (Afghanistan and Pakistan), with the largest numbers in India (185,233) and China (86,978). Reports of TB in children in Europe have been published in the UK,37–40 in Scandinavia41,42 and in the USA.43 These reports indicate the role of children as ‘sentinel’ indicators of the trend for TB in general. Other publications have reported on TB in children from Africa.34,44–47
EPIDEMIOLOGY OF TRANSITIONS POPULATION AT RISK The population at risk is influenced by the composition of the population. In developed countries children make up 5% or less of the population while in developing countries children less than 15 years make up between 40% and 50% of the total population.46 In developing countries young adults, who have the highest prevalence of disease, expose a large pool of children to the risk of infection so that as the incidence of TB rises in adults the rise in childhood cases increases exponentially.46 In low-incidence countries TB occurs mainly in older men and with fewer children the case load of children with TB is less than 5%, while in highincidence countries it has been calculated that children are responsible for about 25% of cases.29
CHAPTER
Epidemiology of childhood tuberculosis
FROM EXPOSURE TO INFECTION The majority of but by no means all, children exposed in the same household for prolonged periods to a sputum smear-positive case of TB become infected. The proportion becoming infected has been variously estimated at between 60% and 80%.10,13–15
Demographic factors: age, sex, ethnic group The probability of developing infection is not constant across age in childhood. The probability has been observed to be higher in ages 5–7 years.12 This has been interpreted as indicating a higher risk of becoming infected when children leave their homes and when the social network of the child increases with school entry. From tuberculin surveys in Africa,22 the prevalence of TB infection by age and sex has been reported and the prevalence rises steadily with age. The prevalence of infection is higher in young girls than in boys, but this ratio reverses in later childhood and throughout adult life. Host factors: immunological status As the human immunodeficiency virus (HIV) epidemic progresses in a community the number of children infected with TB increases. In a Kenyan study in districts where the notification rates increased, the HIV prevalence rates in TB patients were greater than 50%, while in districts where the notification rates had not increased the prevalence of HIV infection in TB patients was 28%. In the districts where the notification rates had increased, the TB infection prevalence rates increased strongly (odds ratio (OR) ¼ 3.1, 95% confidence interval (CI) 2.3–4.1).24 Environmental factors: exposure (intensity, proximity, duration), socioeconomic status The probability of being infected among those in contact with a bacteriologically positive case of pulmonary TB is highest if that case is sputum smear-positive (as compared with the sputum smear-negative pulmonary source case),11,14,15,27 and greater if the contact is within the same household.27 Additional factors associated with a higher risk of becoming infected given contact with an active case of TB include the grade of sputum smear-positivity in the source case and the duration of symptoms.10,48,49 While it seems logical that the physical characteristics of the household (size, space, ventilation) should be associated with the probability of becoming infected, this has not been confirmed in studies where it has been investigated, although the peak of infection in winter months might suggest that reduced ventilation in the household during this period might be the cause.15 Moreover, the inverse association of risk of infection following household contact with social class would also suggest that the household characteristics might be a determinant.12,14 Drug resistance Multidrug-resistant (MDR) cases of TB often have a longer duration of illness related to the poor response to treatment than drugsusceptible cases and therefore it is plausible that there may be a longer time for transmission to occur; however, studies of household contacts of such cases have failed to demonstrate a higher probability of infection among children in the household than among those exposed to cases harbouring drug-susceptible organisms.50–54 FROM INFECTION TO DISEASE The risk of developing disease following infection is greatest in the immediate period following infection and declines exponentially with time since infection.3
5
Demographic factors: age, sex, ethnic group The progression from infection to disease (and particularly to severe disease such as disseminated TB and tuberculous meningitis) is substantially more likely in young children (and particularly in infants) than in older children or adults.11,12,15 This has been suggested to be due to immaturity of the cellular immunity in young children.55 Host factors: immunological status Children who are living with HIV infection have been reported to have a higher probability of progressing from infection to disease.56 In an isoniazid (INH) prophylaxis trial in HIV-infected children the prevalence of TB in the placebo arm was 23,700 per 100,000 children.57 This study gives an indication of the tremendous rate of progression to disease in severe immune-suppressed HIVinfected children. The effect of HIV infection in peri-urban areas of Africa is illustrated in an article where over the course of 9 years TB notifications increased by 2.5-fold as the prevalence of HIV infection increased from 6% to 22%. The increase in adolescent TB notifications was even more dramatic. In the initial year of the study no adolescents were notified, whereas notification increased in the last year to 436 cases per 100,000 persons.58 Environmental factors: exposure (intensity, proximity, duration), socioeconomic status Investigation of household contacts of pulmonary TB patients reports a higher rate of progression from infection to disease among contacts infected by sputum smear-positive patients than among those only positive on sputum cultures.27 This has been interpreted as a result of the size of the ‘infecting dose’. In a study from a large urban African city childhood TB notification was inversely correlated with parental education (r ¼ 0.64) and annual income (r ¼ 0.6) and positively with household crowding (r ¼ 0.32).34 Several studies have shown a correlation between childhood TB notification rate and crowding, income, rate of unemployment, infant mortality and social deprivation.59–61 FROM DISEASE TO DEATH Demographic factors: age, sex, ethnic group Owing to the fact that infants are more likely to develop more severe forms of TB following infection than older children, the fatality rate for TB is higher in very young children – in one report 13% of children under the age of 3 months with TB died.62 Such children may die rapidly from their disease, highlighting the importance of rapidly investigating such children who are contacts of active cases of TB in their households in order that their disease may be detected at an early stage or, even better, its appearance prevented by providing preventive chemotherapy. Host factors: immunological status African children with TB who are coinfected with HIV are much more likely to die than those with TB who are HIV-uninfected.63,64 Although it is not clear that death in these cases was due to TB, a substantial proportion of African children dying of respiratory illnesses who are infected with HIV have active TB evident (and often first diagnosed) at postmortem examination.65 This would suggest that TB was a cause of death in these children, likely as a result of the fact that many of them received no treatment. Drug resistance While there are many studies reporting on the outcome of adult patients with MDR-TB there is a paucity of papers addressing this aspect of childhood TB. In recent trials the mortality rate varied
41
SECTION
1
HISTORY AND EPIDEMIOLOGY OF TUBERCULOSIS
from 2.5% to 10%.66,67 The death rate of MDR-TB in HIVinfected children has not been widely reported but Schaaf et al.67 reported that two out of six HIV-infected children treated for MDR-TB died, giving an indication that the mortality in HIVinfected children could be considerably higher.
MDR-TB in HIV-infected children has not been reported but might be worse than that in HIV-uninfected children.67
THE WAY FORWARD QUALITY OF INFORMATION CURRENTLY AVAILABLE
FROM DISEASE TO CURE Demographic factors: age, sex, ethnic group Children with TB due to susceptible organisms would be expected to respond to appropriate treatment regardless of age, sex or ethnic group. However, the results of treatment of children in Malawi are reported to be substantially less satisfactory than those among adults with only 45% with successful treatment, 17% who died while on treatment, and 34% who defaulted or in whom the outcome was unknown.47 In clinical trials the outcome of children treated for TB are excellent with 97% being regarded as successfully treated and only one recurrence.68 Trials like these must be interpreted with care as they include only children treated in peripheral clinics and exclude children admitted to hospital with complicated disease.
Until recently the only childhood TB statistics reported by countries were those of the number of sputum smear-positive cases in children aged 0–14 years. As sputum collection is limited especially in small children and with the proportion of smear-positive cases being small the information on the effect of TB on children is very limited. Little attention has been given to this information, leading to poor quality data. An audit of a number of clinics revealed that 15.7% of children treated for TB were not recorded in the TB register and of those treated 19.4% did not have TB when the case was reviewed.19 This limited study reflects the care that needs to be taken to ensure accurate information.
Host factors: immunological status In culture-proven TB in a hospital-based study the hospital-related mortality was higher among HIV-infected children (17.5%) than among HIV-uninfected children (11.4%).69 Other studies have reported a sixfold higher mortality in HIV-infected children than in HIV-uninfected children receiving treatment for TB with the HIV-infected children having a 38% mortality rate.64 In a study from Malawian children receiving highly active antiretroviral therapy (HAART) the 12-month outcome of children in whom HAART was started because of TB (0.86) was no worse than that among those who had previous TB (0.86) or those who did not have active or previous TB (0.88).70 This indicates that the outcome on HAART is not worsened by active TB or a history of previous TB.
With 95% of children developing disease within 12 months of being infected, the information on childhood TB gives an indication of the recent transmission within communities – and reconfirms that TB infection and TB disease in children are sentinel events indicating the burden of TB and the effects of control strategies in a community. A decrease in the number of childhood cases, especially in those less than 5 years of age, who are mostly infected in their households, would be the first indication that transmission is decreasing in a community. This is one of the reasons the recording of children in the age band 0–4 years is important.
Drug resistance Very little information is known on the outcome of children treated for drug-resistant TB. Recently it was reported that 36 out of 38 (95%) children treated for MDR were cured or presumed cured while only one child died.66 These results are similar to another study from Africa in which 35 of 39 children with culture-confirmed MDR-TB were successfully treated.67 The outcome of
REFERENCES 1. Last JM. A Dictionary of Epidemiology, 4th edn. International Epidemiological Association. London: Oxford University Press, 2001: 62. 2. Enarson DA, Rieder HL, Arnadottir T, et al. Management of Tuberculosis. A Guide for Low Income Countries, 5th edn. St-Just-La-Pendue: Compogravure Impression, Broachage Imprimeriie, 2000: 10, 25. 3. Ferebee SH. Controlled chemoprophylaxis trials in tuberculosis. A general review. Adv Tuberc Res 1969;17:29–106. 4. Enarson DA, Grzybowski S, Dorken E. Failure of diagnosis as a factor in tuberculosis mortality. Can Med Assoc J 1978;118:1520–1522.
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CHILDHOOD TUBERCULOSIS AS A SENTINEL EVENT
RECENT DEVELOPMENTS The importance of childhood TB as a worldwide problem has been recognized and the WHO has developed new international guidelines for the management of childhood TB.71 The STOP TB Strategy has included a Childhood TB subgroup to ensure that children are included in treatment, recording and reporting strategies. In future children suffering from TB are more likely to be diagnosed, receive treatment and be recorded and reported. With these new data the extent of the childhood TB epidemic will become clearer.
5. Enarson DA, Ait-Khaled N. Tuberculosis. In: Annesi-Maesano I, Gulsvik A, Viegi G (eds). Respiratory Epidemiology in Europe. Huddersfield: Charlesworth Group, 2000: 67–91. 6. Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402. 7. Marais BJ, Gie RP, Schaaf HS, et al. The clinical epidemiology of childhood pulmonary tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004; 8:278–285. 8. Opie E, McPhedran FM, Putnam P. The fate of children in contact with tuberculosis: the exogenous infection of children and adults. Am J Hygiene 1935;22:644–682.
9. Popoe AS, Sartwell MD, Zacks D. Development of tuberculosis in infected children. Am J Public Health 1939;29:1318–1325. 10. Brailey MA. A study of tuberculous infection and mortality in the children of tuberculous households. Am J Hyg 1940;31:Sec. A1–A43 11. Gedde-Dahl T. Tuberculous infection in the light of tuberculin matriculation. Am J Hyg 1952;56:139–214. 12. Bentley FJ, Grzybowski S, Benjamin B. Tuberculosis in Childhood and Adolescence. The National Association for the Prevention of Tuberculosis. London: Waterlow, 1954: 217–238. 13. Davies PDB. The natural history of tuberculosis in children. Tubercle 1961;42(suppl):1–40. 14. Zeidberg LD, Gass RS, Dillon A, et al. The Williamson County tuberculosis study: a twentyfour-year epidemiologic study. Am Rev Respir Pulm Dis 1962;87:1–41.
CHAPTER
Epidemiology of childhood tuberculosis 15. Miller FJW, Seal RME, Talor MD. Tuberculosis in Children. London: Churchill, 1963: 79–163. 16. World Health Organization. Global Tuberculosis Control. WHO/CDS/TB/2007. Geneva: World Health Organization, 2007. 17. Chiang C-Y, Enarson DA, Yang S-L, et al. The impact of national health insurance on the notification of tuberculosis in Taiwan. Int J Tuberc Lung Dis 2002;6:974–979. 18. Marais BJ, Hesseling AC, Gie RP, et al. The burden of childhood tuberculosis and the accuracy of community based surveillance data. Int J Tuberc Lung Dis 2006;10:259–263. 19. Berman S, Kibel M, Fourie P, et al. Childhood tuberculous meningitis: high incidence in the Western Cape of South Africa. Tuber Lung Dis 1992;73:349–355. 20. Arnadottir T, Rieder HL, Trebucq A, et al. Guidelines for conducting tuberculin skin test surveys in high prevalence countries. Tuber Lung Dis 1996; 77(Suppl):1–20. 21. Neuenschwander BE, Zwahlen M, Kim SJ, et al. Determination of the prevalence of infection with Mycobacterium tuberculosis among persons vaccinated against Bacillus Calmette-Guerin in South Korea. Am J Epidemiol 2002;155(7):654–663. 22. Roelsgaard E, Iversen E, Blocher C. Tuberculosis in tropical Africa: an epidemiological study. Bull World Health Organ 1964;30:459–518. 23. Tanzanian Tuberculin Study Collaboration. Tuberculosis control in the era of the HIV epidemic: risk of tuberculosis infection in Tanzania, 1983–1998. Int J Tuberc Lung Dis 2001;5:103–112. 24. Odhiambo J, Borgdorff M, Kambih F. Tuberculosis and the HIV epidemic: Increasing annual risk of tuberculosis infection in Kenya, 1986-1996. Am J Public Health 1999;89:1078–1082. 25. Tuberculosis Research Center. Trends in the prevalence and incidence of tuberculosis in South India. Int J Tuberc Lung Dis 2001;5:142–157. 26. Shaw JB, Wynn-Williams NW. Infectivity of pulmonary tuberculosis in relation to sputum status. Am Rev Tuberc 1954;69:724–732. 27. Grzybowski S, Barnett GD, Styblo K. Contacts of cases of active pulmonary tuberculosis. Bull Int Union Tuberc 1975;50:90–106. 28. Van Geuns HA, Meijer J, Styblo K. Results of contact examinations in Rotterdam 1967–1969. Bull Int Union Tuberc 1975;50:107–121. 29. Nelson LJ, Wells CD. Global epidemiology of childhood tuberculosis. Int J Tuberc Lung Dis 2004;8:636–647. 30. Walls T, Shingadia D. Global epidemiology of paediatric tuberculosis. J Infect 2004;48:13–22. 31. World Health Organization. Expanded Program on Immunization. Update August, 1989. Geneva: World Health Organization, 1989. 32. Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organisation. Tubercle 1991;72:1–6. 33. Dolin PJ, Raviglione MC, Kochi A. Global tuberculosis incidence and mortality during 1990–2000. Bull World Health Organ 1994;72: 213–220. 34. van Rie A, Beyers N, Gie R, et al. Childhood tuberculosis in an urban population in South Africa: burden and risk factors. Arch Dis Child 1999;80: 433–437.
35. Hershfield E. Tuberculosis in children: guidelines for diagnosis, prevention and management. A statement of the scientific committees of the IUATLD. Bull Int Union Tuberc Lung Dis 1991;66:61–67. 36. Corbett EL, Watt CJ, Wlaker N, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 2003;163:1009–1021. 37. Public Health Laboratory Service. Available at URL: www.phls.org.uk/topics_az/tb/data_menu.htm 38. Surinder S, Hawker J, Ali S. The epidemiology of tuberculosis by ethnic group in Birmingham and its implications for future trends in tuberculosis in the UK. Ethn Health 1997;2:147–153. 39. Parslow R, El-Shimy N, Cundall D, et al. Tuberculosis, deprivation and ethnicity in Leeds, UK, 1982–1997. Arch Dis Child 2001;84:109–113. 40. Atkinson P, Taylor H, Sharland M, et al. Resurgence of paediatric tuberculosis in London. Arch Dis Child 2002;86:264–265. 41. Eriksson M, Bennet R, Danielsson N. Clinical manifestations and epidemiology of childhood tuberculosis in Stockholm, 1976-95. Scand J Infect Dis 1997;29:569–572. 42. Rosenfeldt V, Paerregaard A, Fuursted K, et al. Childhood tuberculosis in a Scandinavian metropolitan area 1984-93. Scand J Infect Dis 1998;30:53–57. 43. Ussery XT, Valway SE, McKenna M, et al. Epidemiology of tuberculosis among children in the United States: 1985–1994. Pediatr Infect Dis J 1996;15:697–704. 44. Harries A, Parry C, Nyong’onya M, et al. The pattern of tuberculosis in Queen Elizabeth Central Hospital Blantyre, Malawi: 1986–1995. Int J Tuberc Lung Dis 1997;1:346–351. 45. Kiwanka J, Graham SM, Coulter JBS, et al. Diagnosis of pulmonary tuberculosis in children in an HIV-endemic area, Malawi. Ann Trop Paediatr 2001;21:5–14. 46. Donald P. Childhood tuberculosis: out of control? Curr Opin Paediatr 2002;8:178–182. 47. Harries AD, Hargreaves NJ, Graham SM, et al. Childhood tuberculosis in Malawi: nationwide casefinding and treatment outcomes. Int J Tuberc Lung Dis 2002;6:424–431. 48. Liippo KK, Kulmala K, Tala EO. Focusing tuberculosis contact tracing by smear grading of index cases. Am Rev Resp Dis 1993;148(1):235–236. 49. Golub JE, Bur S, Cronin WA, et al. Delayed tuberculosis diagnosis and tuberculosis transmission. Int J Tuberc Lung Dis 2006;10(1):24–30. 50. Snider DE Jr, Kelley GD, Cauthen GM, et al. Infection and disease among contacts of tuberculosis cases with drug-resistant and drug-susceptible bacilli. Am Rev Respir Dis 1985;132:125–132. 51. Steiner P, Rao M, Mitchell M, et al. Primary drugresistant tuberculosis in children. Correlation of drugsusceptibility patterns of matched patient and source case strains of Mycobacterium tuberculosis. Am J Dis Child 1985;139:780–782. 52. Schaaf HS, Vermeulen HA, Gie RP, et al. Evaluation of young children in household contact with adult multidrug-resistant pulmonary tuberculosis cases. Pediatr Infect Dis J 1999;18:494–500. 53. Schaaf HS, et al. Transmission of multidrug-resistant tuberculosis. Pediatr Infect Dis 2000;19:695–699. 54. Texeira L, Perkins MH, Johnson J. Infection and disease among household contacts of patients with
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
5
multidrug-resistant tuberculosis. Int J Tuberc Lung Dis 2001;5:321–328. Smith S, Jacobs RF, Wilson CB. Immunobiology of childhood tuberculosis: a window on the ontogeny of cellular immunity. J Pediatr 1997;131:16–26. Thomas P, Bornschlegel K, Singh T, et al. Tuberculosis in human immunodeficiency-exposed children in New York City. Pediatr Infect Dis J 2000;19:700–706. Zar HJ, Cotton MF, Strauss S, et al. Effect of isoniazid prophylaxis on mortality and incidence of tuberculosis in children with HIV: randomized controlled trial. BMJ 2007;334:136–148. Lawn SD, Bekker L-G, Middelkoop K, et al. Impact of HIV infection on the epidemiology of tuberculosis in a peri-urban community in South Africa: the need for age-specific interventions. Clin Infect Dis 2006;42: 1040–1047. Drucker E, Alcabes P, Bosworth W, et al. Childhood tuberculosis in the Bronx, New York. Lancet 1994;343:1482–1485. Reinhard C, Paul WS, McAuley JB. Epidemiology of pediatric tuberculosis in Chicago, 1974–1994: a continuing public health problem. Am J Med Sci 1997;313:336–340. Chaulk CP, Khoo L, Matuszak DL, et al. Case characteristics and trends in pediatric tuberculosis: Maryland 1986–1993. Public Health Reports 1997;112:146–152. Schaaf HS, Gie RP, Beyers N, et al. Tuberculosis in infants less than 3 months of age. Arch Dis Child 1993;69:371–374. Mukadi Y, Wiktor S, Coulibaly I, et al. Impact of HIV infection on development, clinical presentation, and outcome of tuberculosis among children in Abidjan, Cote d’Ivoire. AIDS 1997;11:1151–1158. Palme I, Gudetta B, Bruchfeld J, et al. Impact of immunodeficiency virus 1 infection on clinical presentation, treatment and survival in a cohort of Ethiopian children with tuberculosis. Pediatr Infect Dis J 2002;21:1053–1061. Chintu C, Mudenda V, Lucas S. Lung diseases at necropsy in African children dying from respiratory illnesses: a descriptive necropsy study. Lancet 2002;360:985–990. Drobac PC, Mukhergee JS, Joseph KJ, et al. Community-based therapy for children with multidrugresistant tuberculosis. Pediatrics 2006;117:2002–2009. Schaaf HS, Shean K, Donald PR. Culture-confirmed multidrug-resistant tuberculosis in children: diagnostic delay, clinical features, response to treatment and outcome. Arch Dis Child 2003;88:1106–1111. Te Water Naude JM, Donald PR, Hussey GD, et al. Twice weekly vs. daily chemotherapy for childhood tuberculosis. Pediatr Infect Dis J 2000;19:405–410. Jeena PM, Pillay P, Pillay T, Coovadia HM. Impact of HIV-1 co-infection on presentation and hospitalrelated mortality in children with culture proven pulmonary tuberculosis in Durban, South Africa. Int J Tuberc Lung Dis 2002;6:672–678. Bong C-N, Chen SCC, Jong Y-J, et al. Outcomes of HIV infected children with tuberculosis who are started on antiretroviral therapy in Malawi. Int J Tuberc Lung Dis 2007;11:534–538. World Health Organization. Guidance for National Tuberculosis Programs on the Management of Childhood Tuberculosis. WHO/HTM/TB/2006.371. Geneva: World Health Organization, 2006.
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SECTION 2 CHAPTER
6
BASIC SCIENCE
The genus Mycobacterium and the Mycobacterium tuberculosis complex John M Grange
THE MYCOBACTERIA AND THE TUBERCLE BACILLUS – AN HISTORICAL INTRODUCTION On the evening of 24 March 1882, Robert Koch astounded the audience at a meeting of the Berlin Physiological Society with a presentation of his meticulous work that had led to the discovery of the cause of TB. So thorough was Koch’s technique that his discovery was soon ratified by other bacteriologists and his opponents who postulated a constitutional or hereditary cause of TB were silenced.1 Koch (Fig. 6.1) referred to the causative agent of TB simply as the Tuberkelbazillus; the binomial term Mycobacterium tuberculosis was introduced by Lehmann and Neumann in the first edition of their Atlas of Bacteriology published in 1891. The name Mycobacterium – fungus bacterium – alludes to the mold-like pellicle produced by members of this genus on the surfaces of liquid media. At that time, the only other known member of this genus was the leprosy bacillus – Mycobacterium leprae – which was included as it shared the characteristic acid-fast staining property with the tubercle bacillus. The leprosy bacillus, which has never been cultivated in vitro, was first observed microscopically in the tissues of leprosy patients by the Norwegian physician Gerhard Armauer Hansen in 1874, preceding Koch’s discovery of the tubercle bacillus by 8 years.2 In 1898, small differences between tubercle bacilli isolated from humans and cattle were observed.3 Strains isolated from cattle were termed bovine tubercle bacilli but the specific name Mycobacterium bovis was not formally introduced until 1970. Other acid-fast bacilli were isolated in the late nineteenth and early twentieth centuries, some from granulomatous lesions in mammals, birds, and coldblooded animals and some from environmental sources such as grass and compost. In the early nineteenth century, four types of ‘tubercle bacilli’ were recognized – human, bovine, avian, and ‘cold-blooded’. These correspond, respectively, to M. tuberculosis, M. bovis, Mycobacterium avium, and a group of rapidly growing mycobacteria including Mycobacterium fortuitum (previously Mycobacterium ranae) and Mycobacterium chelonae isolated, respectively, from frogs and turtles. The exact relationship between mycobacteria other than typical tubercle (MOTT) bacilli as they were once referred to and the typical tubercle bacilli was a source of considerable confusion. As a result of some flawed experiments it was claimed that the various types of tubercle bacilli were the result of adaptation in different hosts. Thus it was erroneously claimed that M. tuberculosis changed into M. avium if injected into birds. In view of its clinical
44
importance, M. tuberculosis was selected to be the ‘type species’ of the genus Mycobacterium and all other species were referred to as ‘atypical mycobacteria’, a term in common use until recently. In 1939 the Dutch microbiologist den Dooren de Jong had the inspiration to realize that the environmental mycobacteria were the typical ones and that by evolving, or more likely devolving, to obligate pathogenicity, M. tuberculosis was an atypical exception.4 Indeed, she termed the tubercle bacillus ‘the wayward son of honourable parents’. Notwithstanding, the ‘tuberculocentric’ attitude is still propagated by the use of the term ‘non-tuberculous mycobacteria’ (NTM) to describe the environmental mycobacteria. During the first half of the twentieth century, many species names were given to mycobacteria isolated from human and animal lesions and from the environment and 128 names were listed in the 1966 edition of Index Bergeyana. In practice, it was so difficult to assign any isolate apart from the major pathogens to one of the many names that they became generally known as the ‘anonymous mycobacteria’. Order replaced chaos in the 1950s when Ernest Runyon observed that mycobacteria isolated from human clinical material could be divided into four groups according to their pigmentation and rate of growth on culture media (Table 6.1).5 The slow growers were divisible into those that formed a pigment, usually bright yellow, only on or after exposure to light (photochromogens), those forming pigments in the dark (scotochromogens), and those with no pigment (non-chromogens). The rapid growers that Runyon studied were isolated from clinical specimens and were all non-chromogens but environmental isolates are often photochromogens or scotochromogens. Runyon’s pioneering work led to a resurgence of interest in the classification of mycobacteria and eventually to the establishment of the International Working Group on Mycobacterial Taxonomy (IWGMT), which undertook detailed studies of collections of organisms in Runyon’s four groups, principally by the use of Adansonian taxonomy.6,7 In this system, a large number of characteristics, notably metabolism of a range of substrates including carbohydrates, amides, and organic acids, as well as physiological characteristics such as growth at various temperatures, were used to develop trees of relatedness according to similarity, with selected degrees of relatedness delineating genera, species, and subspecific variants. The IWGMT studies also delineated reliable tests for the routine laboratory identification of mycobacteria. Although the work of the IWGMT revolutionized the classification of the mycobacteria and their laboratory identification, one
CHAPTER
The genus Mycobacterium and the Mycobacterium tuberculosis complex
6
epidemiological purposes and their susceptibility to some of the anti-TB drugs to be determined with a minimum of delay.9 Furthermore, complete sequencing of the major pathogens in the genus is proving a great help in the determination of the mechanisms of virulence and pathogenicity which could lead to the rational development of novel vaccines and therapeutic agents. Of particular interest, and not without relevance to disease control, are modern insights into the evolutionary diversification of the species within the group causing human TB, the M. tuberculosis complex, as described below.
CLASSIFICATION OF THE GENUS MYCOBACTERIUM
Fig. 6.1 Robert Koch (1845–1910), the discoverer of the tubercle bacillus.
Table 6.1 Runyon’s four groups of mycobacteria Group
Pigmentation
Growth rate
Examples
I II
Photochromogens Scotochromogens
Slow Slow
III
Non-chromogens
Slow
IV
Rapid growers
Rapida
M. M. M. M. M. M. M.
kansasii, M. marinum scrofulaceum, gordonae avium, malmoense fortuitum, chelonae
a
Defined as visible growth on solid culture media within 1 week on subculture.
problem remained. Until 1980, by international convention, the oldest known name for a given species had priority over later names and there were several examples of well-recognized species being shown to be identical to older and more obscure ones, the name of which then became the official one. Understandably, this led to confusion, particularly among clinicians, and it was therefore decided to produce Approved Lists of Bacterial Names, which were published in 1980 and which are now the sole historical source of reference.8 The approved list of mycobacteria contained 41 species but it was not long before some workers, upset that their species had been omitted, reintroduced them and an increasing number of new species were described. By 2006, the number of new species had grown to over 100, principally as a result of the introduction of molecular methods for identification. In recent years, the application of molecular technology has revolutionized bacteriological studies on TB and other mycobacterial diseases. Not only do new techniques hold out great hope for rapid, sensitive, and specific diagnostic tests but they enable clinical isolates of the M. tuberculosis complex to be typed for
The genus Mycobacterium is divisible into two main groups – slow growers and rapid growers, which on genomic and antigenic analysis are distinct subgenera,10 with the rapid growers being closely related to the genus Nocardia. The species within these two broad divisions were originally determined by Adansonian taxonomy as described above, but determination of differences in highly conserved regions of the genome is now accepted as the ‘gold standard’ for speciation. In particular, analysis of the DNA coding for 16S and 23S ribosomal RNA (‘ribotyping’) has been used for this purpose.10,11 In most cases, results obtained by classical methods and by ribotyping coincide closely but an over-reliance on the latter has led to splitting, confusing from the clinical standpoint, of some rapidly growing pathogenic strains into separate species. Thus M. fortuitum and Mycobacterium peregrinum are now officially separate species rather than variants of the former. Likewise, M. chelonae is now divided into M. chelonae and Mycobacterium abscessus. It should be noted that there are no absolute criteria for speciation of mycobacteria and the current nomenclature is not entirely logical. Thus, the members of the M. tuberculosis complex are divided into seven named species as described below because the very small genomic difference between them (less than 0.1% difference) is not reflected in the very great differences in their significance for human and animal health. By contrast, members of the M. avium complex which were once accorded separate species status are now subspecies, even though they also differ considerably in respect to their host ranges and nature of the disease they cause. Doubtless, despite advances in techniques for characterizing bacteria at the genomic level, taxonomists will always remain either ‘lumpers’ or ‘splitters’ and it is worth bearing in mind the words of the English empiricist philosopher John Locke (1632–1704), ‘The boundaries of the species, whereby men sort them, are made by men.’ One of the clearest systems for division of the genus Mycobacterium into subgenera and species is based on the distribution of soluble cytoplasmic antigens determined by immunodiffusion in gel, as described below.
THE ECOLOGY OF MYCOBACTERIA The genus is divided into obligate pathogens (the M. tuberculosis complex and M. leprae) and the many species that live freely in the environment and are therefore often termed environmental mycobacteria. Some of the latter, as described in Chapter 7, are able to cause disease in susceptible humans and animals. The environmental mycobacteria are found principally in wet environments including marshes, lakes, and rivers. A particularly
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high concentration has been found in sphagnum marshes, including some strains that could not be cultivated in vitro but could be propagated in mouse footpads.12 Some species such as Mycobacterium marinum and Mycobacterium gordonae (previously known as Mycobacterium aquae or the tap-water scotochromogen) appear to prefer water while others, including Mycobacterium terrae, prefer soil. Mycobacteria have been found at a high density in the biofilm on the inner surfaces of domestic and industrial water pipes.13 The chlorination of water may aid the proliferation of mycobacteria in these biofilms by inhibiting the growth of other bacteria that compete for this ecological niche.14 As a result of their lipid-rich, water-repellent cell walls, mycobacteria are hydrophobic and thus easily enter aerosols generated by showers, spas, and whirlpool baths and may thus be inhaled. Indeed, use of aerosol-generating bathing facilities is a risk factor for the development of pulmonary disease due to M. avium, a condition sometimes referred to as ‘hot tub lung’.15 Being distributed widely in nature, including piped water supplies, contamination of the human body, especially the mouth, pharynx, and external genitalia, by environmental mycobacteria readily occurs. Accordingly, they are frequently cultivated from sputum and urine samples and, as described in Chapter 18, care must be taken in determining the clinical significance of such isolated mycobacteria. ‘Pseudo-epidemics’ of disease due to environmental mycobacteria in care facilities have been traced to the collection of specimens into unsterile containers, and in faults in equipment for cleaning endoscopes.16,17 Disease may also follow traumatic inoculation of environmental mycobacteria into the skin.18 Two named mycobacterial skin diseases, Buruli ulcer due to M. ulcerans and swimming pool granuloma due to M. marinum, are associated with traumatic inoculation, and post-injection abscesses due to rapidly growing mycobacteria including the aptly named M. abscessus and the closely related M. chelonae have been reported. Indeed, injected suspensions of viable M. chelonae, originally isolated from a turtle, were used for the immunotherapy of TB in the early twentieth century and the patients accepted the abscesses as an indication that the therapeutic agent was working! Exposure of the human population to environmental mycobacteria may be sufficient to have measurable effects on the immune system. Thus, in some regions, such ‘immunologically effective contact’ has been sufficient to induce low levels of tuberculin reactivity due to antigens shared with M. tuberculosis.19 In some regions, exposure appears to induce some degree of protective immunity against TB but in others it may interfere with the protection conferred by Bacillus Calmette–Gue´rin (BCG) vaccination by the mechanisms described in Chapter 74. Such environmental sensitization may, at least in part, explain the considerable regional variation in the protective efficacy of BCG vaccination.20,21 Some environmental mycobacteria, including members of the M. avium complex, are able to replicate within various protozoans and also to survive within amoebic cysts under conditions of environmental stress. It has been postulated that such intracellular replication may, by analogy with Legionella pneumophila, have played a central role in the evolution of mycobacteria as intracellular pathogens.22 Furthermore, a relationship between mycobacteria and protozoa may explain some enigmatic features of M. leprae, an obligate pathogen with a degenerate genome which does not grow on any known culture medium in vitro. With very rare exceptions M. leprae only causes disease in humans and it appears, from skeletal evidence, only to have done so for the past 3,000 years, a very
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short time in evolutionary history. Although assumed to be transmitted directly between humans, 50–70% of sporadic cases of leprosy in well-studied populations occur in those who have had no known contact with human source cases, suggesting an environmental source, possibly soil.23 Accordingly, it has been suggested that M. leprae could be an obligate endoparasite of a protozoan and that the human macrophage is an alternative unicellular host.24
GROWTH CHARACTERISTICS AND METABOLISM OF MYCOBACTERIA The mycobacteria are non-sporing and non-flagellate. Their cells are mostly straight or slightly curved rods although members of some species are coccoid. Some, notably Mycobacterium xenopi, M. marinum, and Mycobacterium kansasii, may be long and filamentous under some growth conditions and the latter two may contain lipid storage granules that give the cells a distinct beaded appearance (Fig. 6.2). Branching of cells may be seen in older cultures and colonies of some species, including M. xenopi, may bear small arial hyphae. The morphology of mycobacteria in clinical specimens is very variable and is affected by prior antimicrobial therapy. Thus, reports of microscopical examinations should merely state that acid-fast bacilli are present. Identification at the species level requires the application of cultural or molecular methods. Mycobacteria are Gram positive, although they are poorly stained by this method. They are acid-fast, retaining the color of arylmethane dyes when treated with a dilute mineral acid. Thus the term acid-fast bacilli (AFB) is often used as a synonym for mycobacteria. They also retain the colour on treatment with acid and alcohol and are thus sometimes termed acid-alcohol-fast bacilli (AAFB). Contrary to a widespread view, the addition of alcohol does not enable members of the M. tuberculosis complex to be differentiated from environmental mycobacteria; it merely reduces the risk of staining artefacts. Acid-fastness is not unique to the mycobacteria – bacterial spores and members of the genus Nocardia are acid-fast to varying degrees. The acid-fast staining technique in regular use today bears the names of Ziehl and Neelsen although these workers merely modified a technique first described by Paul Ehrlich, who used it to detect tubercle bacilli in human sputum, including his own! In the classical Ziehl–Neelsen (ZN) technique
Fig. 6.2 Mycobacterium kansasii in sputum stained by the Ziehl–Neelsen method, showing elongated cells with a distinct banded or beaded appearance.
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Fig. 6.3 Acid-fast bacilli in sputum demonstrated by (A) Ziehl–Neelsen staining and light microscopy and (B) auramine staining and fluorescence microscopy. Courtesy of Dr. P. D. O. Davies.
specimens are stained with hot carbol fuchsin, decolourized with a dilute mineral acid in water or alcohol, and counterstained with a green or blue dye so that mycobacteria appear as red organisms against the counterstained background (Fig. 6.3). Fluorescent methods, based on the same principle of acid-fastness, are also available and enable more specimens to be examined in a given time as they can be scanned at a low magnification. Full technical details for the microscopical detection of mycobacteria and the organization of laboratory services are given elsewhere.25 As mentioned earlier, mycobacteria are, with the exception of a few non-cultivable species, divisible into slow and rapid growers. The latter are defined as those giving visible growth on solid media within 1 week on subculture. Most mycobacteria, both slow and rapid growers, have simple nutritional requirements and are able to utilize a range of sugars and organic acids as carbon sources and various amides and amino acids as nitrogen sources. Most species are therefore able to grow on simple synthetic media. The most common medium for the cultivation of mycobacteria in clinical laboratories is Lo¨wensteinJensen (LJ) medium, containing glycerol, mineral salts, and whole eggs which, in addition to being a source of nutrients, enable the medium to be solidified by heating at 80–85 C, a process termed inspissation. This medium is suitable for the cultivation of most mycobacterial species encountered in clinical practice with the notable exception of M. bovis. There are defects in several genes of M. bovis involved in carbohydrate metabolism, including those responsible for the phosphorylation of glucose and the formation of pyruvate.26 Growth of this species is therefore feeble on media containing glycerol, resulting in small, flat ‘dysgonic’ colonies, unlike the heaped-up ‘eugonic’ colonies of M. tuberculosis, likened to small cauliflowers or breadcrumbs, on such media. Growth of M. bovis is enhanced by replacing glycerol with sodium pyruvate. For research purposes mycobacteria may be grown on simple egg-free media solidified with agar.25 Various simple liquid media are also used for the cultivation of mycobacteria, especially in the automated culture systems described in Chapter 23. Mycobacteria are aerobic, although a few species, notably M. bovis, grow better under conditions of low oxygen concentration. Simple media contain sufficient iron and trace elements to sustain growth
although, as suggested by the name, Mycobacterium haemophilum requires media supplemented with blood or other substrates rich in iron for growth. Some members of the M. avium complex are deficient in mycobactin, a lipid required for acquisition of iron, and this must be added to the medium for the cultivation of these organisms (p. 49).
ANTIGENIC STRUCTURE OF MYCOBACTERIA Mycobacterial antigens are divisible into three main groups – actively secreted, cell wall-bound, and cytoplasmic. The first two are those initially encountered by the immune system and are thus likely to play a role in the induction of protective defence mechanisms and in pathogenicity. The nomenclature and classification of mycobacterial antigens is confusing. Some, especially the various polysaccharide antigens, are known by their chemical names, such as lipoarabinomannan, phenolic glycolipid, and trehalose dimycolate. Protein antigens are defined by their size in kilodaltons, their N-terminal amino acid sequencing, and their mass spectroscopic patterns.27 Numerous soluble and cell-bound mycobacterial antigens have been described but only a few have been well characterized functionally. Some have been utilized for the development of various diagnostic tests based on antibody or cell-mediated host immune responses. Around 200 antigenic culture filtrate proteins (CFPs) are demonstrable in two-dimensional immunoelectrophoresis in gel.27 Double immunodiffusion in gel reveals around 15 soluble antigens in each species which form the basis of a taxonomic system particularly developed by Stanford and Grange in the early 1970s.28 In this system, soluble antigens are divisible into those shared by all mycobacteria and variably by related genera (group I), those restricted to slowly growing species (group II), those found in rapidly growing species and shared with nocardiae (group III), and those unique to each species (group IV). A very few species, notably M. leprae, do not have antigens that distinguish slowly growing from rapidly growing species, as shown in Table 6.2. In some species, variation among the group IV antigens corresponds with recognized variants within them. Thus, the avium, intracellulare, brunense, paratuberculosis, and lepraemurium
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Mycosides
Table 6.2 Distribution of soluble antigens in the genus Mycobacterium Antigen group
Distribution
I
All mycobacteria and sharing of some with related genera Slowly growing mycobacteria Rapidly growing mycobacteria and the genus Nocardia Restricted to individual mycobacterial species and showing some intraspecific variation
II III IV
Trehalose dimycolates and sulpholipids Lipoarabinomannan Cell wall Mycolic acid Arabinogalactan Peptidoglycan Cell membrane
M. avium avium Bacterial cell, with nuclear body and lipid inclusions
M. avium brunense M. avium intracellulare M. avium lepraemurium M. avian paratuberculosis
v
v
Fig. 6.4 Variation in species-specific antigens in the M. avium complex demonstrable by immunodiffusion-in-gel.
subspecies of M. avium differ in their group IV antigenic structure (Fig. 6.4). Speciation by analysis of soluble cytoplasmic antigens should not be confused with the typing of smooth and emulsifiable mycobacteria by agglutination serology. This is based on differences in cell surface mycoside antigens and divides those species to which it can be applied into many variants. Before the advent of nucleic acid-based technology, agglutination typing was widely used to subdivide the M. avium complex for epidemiological purposes.29 A panel of antisera divides smooth isolates of M. avium subsp. avium into three types and M. avium subsp. intracellulare into 24 types, although some strains are untypable. Many of the CFPs are liberated into the medium by cell leakage or autolysis but a few have been shown to be actively secreted and may be involved in virulence and induction of immune responses. These include the 6-kDa early secreted antigenic target (ESAT-6) and the antigen 85 (Ag85) complex, which are described elsewhere in this chapter. In addition, by destroying toxic reactive oxygen intermediates produced within phagocytic cells, catalase-peroxidase and superoxide dismutase contribute to intracellular survival but it is not clear whether these enzymes are actively secreted by M. tuberculosis.30
MACROMOLECULAR STRUCTURE OF MYCOBACTERIA In common with other bacteria, the mycobacteria have a cell membrane consisting of two phospholipid layers. The bacterial cell membrane is, like the mammalian mitochondria, associated with various enzymes involved in energy production. The pigments responsible for the yellow color of some strains of mycobacteria are found within the bilayer. The outstanding characteristic feature of the mycobacteria, one that is responsible for acid-fastness, their slow rate of growth, and some aspects of their pathogenicity, is their very complex and
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Fig. 6.5 A diagrammatic representation of the mycobacterial cell wall.
lipid-rich cell wall external to the cell membrane (Fig. 6.5).31 The innermost layer is, in common with most other bacteria, a net-like macromolecule, peptidoglycan or murein, consisting of long polysaccharide chains cross-linked by short peptides consisting of four amino acids.32 This layer gives the bacterial cells their shape and rigidity. External to the murein layer is another macromolecule – arabinogalactan – a branched polysaccharide consisting of arabinose and galactose.32 Attached to this layer are the mycolic acids – long chain fatty acids that give the cell wall its thickness and acid-fast staining properties. These molecules have a long chain, containing 50–56 carbon atoms, and a shorter side chain with 22–26 carbon atoms. Similar but smaller mycolic acids are found in related genera such as Nocardia and Corynebacterium. In their basic structure the mycolic acids are aliphatic chains terminating in the acidic -COOH group but they may contain unsaturated bonds, oxygen-containing groups, methyl side chains, and cyclopropane rings. The latter contribute to the structural integrity of the cell wall and, by protecting against toxic oxygen derivatives such as hydrogen peroxide, they play a role in pathogenicity although the details are far from clear and observations are contradictory.33 The synthesis of mycolic acids is a complex one requiring multidomain fatty acid synthase (FAS) enzymes and additional enzymes involved in the synthesis of the cyclopropane rings and other components. The synthetic processes and the genes and enzymes involved have been well characterized and reviewed in detail.34 The processes by which the newly synthesized mycolic acids are transported to, and positioned in, the cell wall and attached to the arabinogalactan in the cell wall is less well understood. The final steps in the cell wall require the activity of a group of proteins termed the antigen 85 (Ag85) complex composed, in M. tuberculosis, of three structurally related proteins Ag85A, B, and C, which are among the predominant secreted proteins of this species.35 Mycolic acid synthesis is the target of the anti-TB agents isoniazid and ethionamide and a full elucidation of such synthesis may pave the way to the development of new therapeutic agents. The mycolic acids form the inner of two principal layers of lipids in the cell wall and the outer layer is formed by a related class of cell wall lipids, the acylated trehaloses, which contains two groups,
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The genus Mycobacterium and the Mycobacterium tuberculosis complex
Fig. 6.6 Fluorescent stained microcolonies of M. tuberculosis showing ‘serpentine cording’.
trehalose dimycolates and sulpholipids. The trehalose dimycolates were formerly termed ‘cord factors’ as they were thought to be responsible for the characteristic ‘serpentine cord’ arrangement of cells of M. tuberculosis in micro-colonies (Fig. 6.6). They were also thought to be major determinants of virulence of M. tuberculosis and although this is controversial there is now evidence that they are powerful inducers of several cytokines including tumor necrosis factor and that they have a number of effects, some beneficial but mostly harmful, on the host–pathogen relationship.36 In particular, trehalose dimycolates containing cyclopropane rings play a role in pathogenicity by adversely influencing early interactions of the infecting organism with the innate immune system.37 The cell wall content of the strongly acidic sulpholipids varies among the pathogenic members of the M. tuberculosis complex and their relevance to pathogenicity is uncertain. One of the most biologically important molecules in the mycobacterial cell wall is lipoarabinomannan (LAM), which is a branched molecule composed of the carbohydrates arabinose and mannan and anchored by a phospholipid to the cell membrane.38 LAM stretches from the cell membrane up to the surface of the cell wall and may be an important structural component, possibly acting as an anchor for several cell wall components. LAM is a dominant mycobacterial antigen and, as described in Chapters 8 and 23, plays an important role in determining the nature of the host immune response.39 In particular it is an adjuvant that plays a part in determining the pattern and regulation of the immune response by interacting with Toll-like receptors on antigen-presenting cells.40 Although it is widely accepted that the immune responses in TB are principally cell-mediated, there is evidence that antibodies, especially those against LAM, induced by BCG vaccination enhances innate and acquired immune responses to mycobacteria possibly by enhancing phagosome–lysosome fusion and intracellular killing of the pathogen.41 Two types have been described – AraLAM and ManLAM – in which the branches are capped with, respectively, arabinose and mannan. The ManLAM variant, found in the M. tuberculosis complex, has been shown to enhance entry of the bacilli into macrophages and survival within these cells.42 The outermost layer of the mycobacterial cell wall contains complex molecules termed mycosides.43 Most mycosides contain lipids (mycoserosic acids), carbohydrates, and peptides and are
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termed peptidoglycolipids and members of a related class, the phenolic glycolipids or phenol-phthiocerol dimycoserosates, lack the peptide component. The mycosides are responsible for a number of biological activities including determination of colony morphology and, in the case of strains forming smooth colonies, agglutination serotypes and they are also receptor sites for the attachment of some bacteriophages. They appear as ribbon-like structures under the electron microscope and they may be abundant enough in the cell wall to form pseudo-capsules. Smooth-to-rough mutational variation of the colony morphology, observed in many mycobacterial species, is associated with a loss of some mycosides from the cell surface. Mycosides contain a wide range of antigenic sugars, some of them being unusual ones, which form the basis of the agglutination serotypes, principally of M. avium. Phenolic glycolipids have been detected in many mycobacteria and large amounts of an apparently unique one, phenolic glycolipid-I (PGL-I), is found in the cell walls of M. leprae and has been used as an antigen in serological studies of leprosy.44,45 Intriguingly, non-cultivable mycobacteria isolated from sphagnum marshes by passage in mouse footpads contain PGL-1, suggesting that these might be an ancestral form of M. leprae.12 Another phenolic glycolipid has been found in strains of M. tuberculosis principally isolated in South India and of a distinct phage type (type I, p. 53) and, as these strains are attenuated in guinea pigs, it has been named the ‘attenuation indicator lipid’ but its link to attenuation is almost certainly coincidental.46 By contrast, certain phenolic glycolipids may contribute to virulence as one from strains of M. tuberculosis of particularly high virulence has been shown to inhibit innate immune responses.47 The complex mycobacterial cell wall forms a permeability barrier and is, for example, 100–1000 times less permeable to hydrophilic molecules than Escherichia coli. Permeability is facilitated by channel-forming protein structures termed porins, of which there are at least two types in M. tuberculosis.48 Some forms of pyrazinamide resistance are due to mutational changes in porin structure.49 A further functionally important class of cell wall lipids are the mycobactins, which are iron-chelating agents involved in the transport of iron across the cell wall and its subsequent storage.50 These lipids, which show considerable structural variation between species, are synthesized by almost all mycobacteria. Exceptions are M. avium paratuberculosis, M. avium sylvaticum, and some other members of the M. avium complex which produce little or no mycobactin. The in vitro cultivation of these organisms therefore requires the addition of mycobactin extracted from other mycobacteria to the culture medium. A group of 10 genes involved in mycobactin production and iron transport have been identified in M. tuberculosis and one of these is truncated in M. avium paratuberculosis, suggesting but not proving that this is responsible for the defect in mycobactin production in this species.51
DEFINITION OF THE M. TUBERCULOSIS COMPLEX The various so-called species that cause TB in humans and other mammals are grouped within the M. tuberculosis complex. Some, but not all, have been given separate species status, despite the fact that they are genetically very closely related. The use of separate species names has been justified on historical grounds and because,
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Table 6.3 Members of the M. tuberculosis complex with specific names Species
Principal host
M. M. M. M. M. M. M.
Human Cattle, deer, elk, bison, badger, opossum Goats Human Vole Human Seal
tuberculosis bovis caprae africanum microti canetti pinnipedii
although genetically very closely related, they differ considerably in their natural host ranges and, accordingly, their importance in respect to public and veterinary health. The named species and their principal host ranges are listed in Table 6.3. The human tubercle bacillus, M. tuberculosis, is not homogeneous. One of the first major variants in this species to be described was one common in South India and among those migrating from that region to other countries. This variant, termed the Asian or South Indian type, differs from other strains in being susceptible in vitro to the isoniazid analogue thiophen-2-carboxylic acid hydrazide (TCH), being attenuated in the guinea pig, having the so-called attenuation indicator lipid (see above) in its cell wall and being of bacteriophage type I.46 In the United Kingdom this type is much more common among immigrants of Indian subcontinent ethnic origin than among the indigenous population but, although the former have a relatively higher incidence of non-pulmonary TB, there is no relationship between the type of disease and the causative strain.52 More recently, a number of different genotypes, lineages or clades within the M. tuberculosis complex have been determined by DNA analysis as described below (p. 55). Another, though rare, variant of M. tuberculosis produces smooth colonies on egg-based solid media rather than the usual rough bread crumbs-like colonies. Originally termed the Canetti type, it is now often referred to as M. canetti.53 There is evidence, discussed later, that it is closely related to the supposed ancestral or progenitor type from which other members of the M. tuberculosis complex have devolved. Intraspecific variation has also been demonstrated in M. bovis, of which one of the generally accepted defining characteristics is resistance to the anti-TB agent pyrazinamide. In recent years, a few isolates of M. bovis from animals, principally goats, and also from humans in Spain and Germany have been found to be sensitive to this agent. Most of these strains differ in genomic structure from M. bovis and have been given the separate species name M. caprae.54 A very few strains of M. bovis that do not fit into the species M. caprae are also sensitive to pyrazinamide. Somewhat surprisingly, a study of strains of M. bovis isolated between 1999 to 2001 from 166 patients living in Germany showed that one-third were M. caprae, with the highest concentration (80%) being isolated in South Germany.55 This high incidence appears unique to Germany and it is possible that a new genotype is emerging in that country but, as the patients were mostly elderly and in the same age range as those from whom typical M. bovis was isolated, reactivation of old infections, acquired before the effective control of bovine TB, is a more likely explanation. Bacillus Calmette-Gue´rin (BCG) was derived from a tubercle bacillus, isolated by Nocard in the late nineteenth century from a
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case of bovine mastitis and termed Lait Nocard, by a total of 230 subcultivations on potato slices dipped in bile over a 13-year period, between 1908 and 1921. Initially, three lines of BCG maintained on different media were distributed by the Institute Pasteur to laboratories world-wide but after 1932 only one line, maintained on an antigen-free medium, was continued. The earliest daughter strains, all obtained before 1932, include the Brazilian, Japanese, Romanian, Russian, and Swedish. These differ from the other daughter strains in having two copies of the insertion sequence IS6110 (previously termed IS986), rather than only one, in the presence of methoxy groups in the cell wall mycolic acids and in actively secreting large amounts of the antigenic protein MPB70.56 In these respects, the earlier daughter strains more closely resemble M. bovis but, as the original strain from which BCG was derived was lost, it is not possible to determine the exact pathways by which the variants arose. As there have been no field studies in which BCG daughter strains of the two types have been directly compared, and as the protective efficacy of BCG varies greatly from region to region, the relative ability of the two genotypes to induce protective immunity is unknown. Strains of BCG also differ from M. bovis in being aerobic rather than microaerophilic and in utilizing glycerol as a carbon source. As in the case of the above variations, it is not clear how or when these metabolic differences occurred, or whether the original Lait Nocard strain had these properties. It seems, however, highly likely that the original strain was a typical M. bovis as genomic analysis of BCG shows it to be very closely related to a group of strains of M. bovis recently isolated in France.57 Mycobacterium africanum is the name given to a rather heterogeneous group of strains isolated from humans in equatorial Africa and in migrants from that region to other countries. Superficially, strains of this species appear to bridge the phenetic gap between M. tuberculosis and M. bovis and they are divided into type I which is found principally in West Africa and resembles M. bovis and type II which is of East African origin and has features in common with M. tuberculosis.58 When first encountered in the United Kingdom, type I strains were termed the ‘Afro-Asian bovine type’. Mycobacterium microti is a rarely encountered species originally isolated from the vole, Microtus agrestis. In humans it has the same level of virulence as BCG vaccine and it was used as a vaccine together with the latter in extensive clinical trials organized by the British Medical Research Council in which the two were found to afford the same level of protection against TB.59 It is highly likely that, if BCG had not been developed and was already in world-wide use, M. microti would have become the standard anti-TB vaccine. Mycobacterium pinnepedii is the name given to a group of tubercle bacilli isolated from tuberculous lesions in various species of seals and sea lions, captive and free, in Australia, New Zealand, and South America, and in an Australian seal trainer.60 A further case occurred in a captive seal, and in a tapir in an adjacent enclosure, in a zoo in Great Britain. Experimentally, M. pinnipedii causes disease in rabbits and guinea pigs. Genomic differences, revealed particularly by spoligotyping (p. 52), show that strains in this species form a cluster clearly different from those of other members of the M. tuberculosis complex. Doubtless, these named species do not cover the entire range of variation within the M. tuberculosis complex. Strains with characteristic differentiating features have been isolated from the rock hyrax or dassie, oryx, water buffaloes, and cats.
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The genus Mycobacterium and the Mycobacterium tuberculosis complex
THE GENOME OF M. TUBERCULOSIS AND OTHER MYCOBACTERIA The entire genome of M. tuberculosis H37Rv, a widely used reference strain, was completely sequenced in 1998.61 This analysis showed that the genome of this strain contained 4,411,529 base pairs (bp) and around 4,000 genes. This genome is larger than that of many bacteria, though not as large as that of E. coli. It has a high guanine + cytosine content (65.6%) and this is uniform throughout the genome, suggesting that it has evolved with little or no incorporation of DNA from other bacterial genera (Fig. 6.7). In contrast to other bacteria, many of the genes of M. tuberculosis are involved in synthesis and metabolism of lipids. Indeed every known lipid biosynthetic system, including those in plants and animals, as well as some unique ones, are found in mycobacteria and M. tuberculosis contains around 250 enzymes involved in lipid metabolism, compared to a mere 50 in E. coli. Mycobacteria are unique among the bacteria in possessing the enzyme fatty acid synthase I (FASI), which is also found in many eukaryotic cells, as well as fatty acid synthase II (FASII), which is found in most other prokaryotes and plants.62 Much of this metabolic capability enables the mycobacteria to synthesize their very complex lipid-rich cell walls. Another unusual feature of the mycobacterial genome is the large number of genes, over 4% of the total, containing polymorphic GCrepetitive sequence (PGRS) and coding for two unrelated families, PE and PPE, of acidic, glycine-rich proteins.61 The function of these proteins is unknown but, as many of them are found in the cell membrane and cell wall, they may mediate antigenic variation and thereby affect virulence and pathogenicity.62,63 At least one PE protein of M. tuberculosis binds to fibronectin and may therefore be of relevance to pathogenicity by facilitating adhesion to tissues and entry into host cells.64
Fig. 6.7 A diagrammatic representation of the genome of M. tuberculosis H37Rv. From Cole et al.61
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The genome of the strain of M. bovis selected for sequencing, a strain isolated in 1997 from a cow with pulmonary TB lesions, shows over 99.95% similarity with that of M. tuberculosis although, with 4,345,492 bp, the former is slightly smaller.26 The genome of M. bovis differs from that of M. tuberculosis by genetic deletion – it has no unique genes although there are several differences in gene expression and regulation between the two species. Most of the differences between the two species are in genes encoding cell wall lipids and secreted antigenic proteins. There are several differences in the cell wall PE proteins described earlier and it is possible that these may determine the differences in mammalian host ranges of M. bovis and M. tuberculosis.64 In addition, as discussed earlier, there are defects in several genes of M. bovis involved in carbohydrate metabolism. Being genetically highly conserved, many of the differences between strains within the M. tuberculosis complex involve just single nucleotides, the so-called single nucleotide polymorphisms (SNPs).66 A total of 1,075 SNP differences have been found between M. tuberculosis H37Rv and a clinical isolate CDC1551 and 2,437 between H37Rv and the fully sequenced strain of M. bovis described earlier. These differences are small considering that there are over 4 million bp in the genomes of members of the M. tuberculosis complex. In addition to single nucleotide polymorphisms, there are some interstrain differences involving several sequential nucleotides.67 These, the large-sequence polymorphisms (LSPs), are much less common than the SNPs and include around 20 so-called regions of difference (RDs), which, as described later, have been used to establish the evolutionary relatedness of strains within the M. tuberculosis complex. In common with all forms of life, the mycobacterial genome contains small nucleotides, up to 6 bp in length, termed microsatellites. By insertion or deletion, these cause reversible frame shift mutations at a relatively high frequency and it has been postulated that this imparts a ‘plasticity’ to the genome facilitating, by phenotypic variation, adaptation of pathogens to different hosts.68 In addition to the above genetic differences, the mycobacterial genome contains a number of repetitive genetic elements that have been utilized in the development of typing schemes for epidemiological purposes. These include various classes of mobile genetic elements (‘jumping genes’) which contribute to genetic diversity and evolution. One of these classes contains the so-called insertion sequences (IS) and 56 different ones, belonging to various families, have been detected in the genome of M. tuberculosis strain H37Rv. Several copies of one of these, IS6110, are found in the genomes of almost all strains of M. tuberculosis and, as their numbers and positions show considerable interstrain variation, they have been utilized in a typing system known as restriction fragment length polymorphism (RFLP) typing. Strains usually contain from 1 to 25 copies although a few strains contain none. In general, strains of M. bovis contain fewer copies than M. tuberculosis – BCG daughter strains, for example, contain either 1 or 2 copies. It has been postulated that strains with high and low copy numbers of IS6110 reflect different evolutionary lineages. Repetitive elements useful for typing purposes also include a region of repeat sequences found in all bacteria and termed ‘clustered regularly interspaced short palindromic repeats’ (CRISPA). The function of the CRISPAs is unknown but they have structural similarities with the centromeres of eukaryotic chromosomes and may therefore be involved in the initiation and regulation of replication of the bacterial genome. The CRISPA
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in the M. tuberculosis complex is structurally unique and is termed the direct repeat (DR) locus and consists of repetitive 36-bp units of DNA separated by non-repetitive 34- to 41-bp spacer oligonucleotides which can be amplified by PCR employing just one pair of primers. Variation in the patterns of the latter, of which there are thousands of possible combinations, in the M. tuberculosis complex, forms the basis of spacer oligonucleotide typing, or ‘spoligotyping’ for short.69 Spoligotyping has the great advantage over RFLP typing that it can be performed on PCR products, therefore avoiding the need for cultivation of the strains. Spoligotypes are more stable than RFLP types based on IS6110 variation and, as discussed later, the spoligotype of BCG has remained stable for around a century, despite multiple subcultivations and division into many daughter strains with differences detectable by RFLP typing. Other typing systems are available such as the VNTR (variable nucleotide tandem repeats), which, together with spoligotyping, provides a very discriminating system that may well replace the slower and technically more difficult RFLP typing system.70,71 Standardized methods for the typing systems currently in use and their various epidemiological uses are described in Chapter 4. The leprosy bacillus, M. leprae, has never been cultivated in vitro and was included in the genus Mycobacterium on account of its acidfast staining characteristics. Sequencing of the entire genome of M. leprae has confirmed that it is a mycobacterium.72 Its genome contains 3.27 million bp and is therefore only around threequarters the size of that of M. tuberculosis and less than half of its genome is composed of functional genes. Many of its genes are pseudogenes – non-functional counterparts of genes present in other mycobacteria – and this has deprived it of many crucial metabolic activities although it has retained the functional genes required for synthesis of its elaborate lipid-rich cell wall, and those enabling it to be a major human pathogen.73,74 The complete genome sequence of M. avium paratuberculosis has also been determined.51 This very slowly growing organism causes hypertrophic enteritis or Johne’s disease in cattle and other ruminants and there is also evidence, though controversial, that it is a cause of Crohn’s disease in humans. The complete sequencing of its genome should lead to the development of more efficient diagnostic tests, more rapid in vitro cultivation, and resolution over the controversy concerning its role as a human pathogen.
GENOMIC ASPECTS OF RESISTANCE TO ANTI-MYCOBACTERIAL AGENTS Mycobacteria, especially the slowly growing species including the M. tuberculosis complex, are naturally resistant to many of the classes of anti-bacterial agents used for treatment of other bacterial infections. On the other hand, certain distinctive metabolic processes, including those involved in the synthesis of the unique lipid-rich cell wall, are the targets of some agents used specifically for the treatment of TB, including three of the first-line drugs – isoniazid, ethambutol, and pyrazinamide. Advances in techniques for analyzing the genomic structure of mycobacteria has enabled the mode of action and the target molecule(s) of most of the anti-mycobacterial agents, and the genes involved, to be determined. These are summarized in Table 6.4. The same advances raise hopes for a rational development of additional agents active on other specific metabolic pathways and structures. Furthermore, determination of the mutation(s) in the
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Table 6.4 Targets of anti-mycobacterial agents and associated genetic loci Agent
Target molecule or function
Genes encoding targets or the site of resistancedetermining mutations
Isoniazid
Mycolic acid synthesis
Rifampicin
DNA-dependent RNA polymerase Cell membrane energy function Arabinogalactan synthesis Ribosomal protein S12 Mycolic acid synthesis
katG, inhA and its promoter region, oxyR-ahpC intergenic region rpoB
Pyrazinamide Ethambutol Streptomycin Ethionamide and prothionamide Capreomycin and viomycin Cycloserine Clofazimine p-aminosalicylic acid Dapsone
pncA embA, embB, embC
50S and 30S ribosomal subunit Peptidoglycan synthesis ? RNA polymerase Folic acid synthesis
rpsL ethA, inhA and its promoter region vicA (50S), vicB (30S) alrA Unknown Unknown
Folic acid synthesis
Unknown
relevant genes responsible for drug resistance is facilitating the development of rapid methods for detecting such resistance to several anti-TB agents.9,75 Various systems, including microarrays of oligonucleotides on chips for the detection of mutations determining resistance to rifampicin and isoniazid, are commercially available.76,77 The mode of action of rifampicin against mycobacteria is the same as in other bacterial genera and involves the prevention of synthesis of mRNA by inhibiting bacterial DNA-dependent RNA polymerase, thereby blocking the protein synthesis. Resistance is due to one of several tightly clustered single amino acid mutational changes in a short region of the rpoB gene, which encodes for the b subunit of the polymerase.78 Isoniazid is a pro-drug requiring oxidative activation by the mycobacterial catalase-peroxidase enzyme KatG, an enzyme involved in protection against oxidative stress. The most commonly reported mutations causing isoniazid resistance occur in the katG gene but mutations in the inhA locus or its promoter region and in the intergenic region of the oxyR-ahpC locus have also been associated with resistance. The inhA locus codes for a NADH-dependent enoyl-acyl carrier protein reductase which is involved in mycolic acid synthesis and is a target for isoniazid. The oxyR-ahpC locus is, like the katG locus, involved in protection against oxidative stress but the role of mutations in this locus in causing resistance to isoniazid is not clear. The relative frequency of these mutations in isoniazid-resistant strains varies from study to study and in different geographical regions and is, at least in part, related to the distribution of the genotypes of M. tuberculosis in given regions.79,80 Isoniazid (INH) is inactivated in vivo by acetylation and humans are divisible into rapid and slow inactivators.81 In addition, mycobacteria
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possess a polymorphic arylamine N-acetyltransferase (NAT) enzyme which, by local acetylation of INH, may contribute to resistance to this agent.82 NAT is essential for mycolic acid synthesis and mutants of BCG lacking this enzyme activity are more susceptible to killing by macrophages. Thus, although NAT is not a target for isoniazid, it could be a target for novel anti-TB agents. Pyrazinamide is an unusual drug as it has a powerful sterilizing activity against slowly replicating tubercle bacilli in acidic and anoxic or hypoxic inflammatory lesions.83 It is therefore most effective during the early stage of therapy before the inflammation has subsided. Pyrazinamide first requires conversion to the active metabolite pyrazinoic acid by mycobacterial pyrazinamidase enzymes encoded by the pncA gene and this enzymatic activity is not detectable in most pyrazinamide-resistant mutants of M. tuberculosis or in strains of M. bovis, which are naturally resistant to this agent. The mode of action of pyrazinoic acid is not fully understood but there is evidence that it disrupts bacterial membrane energetics and transport function. A wide range of mutations in the 600-bp pncA gene cause resistance to pyrazinamide and techniques for the detection of point mutations over a relatively large length of DNA are required for the molecular determination of resistance. One such technique is denaturing gradient gel electrophoresis, which has also been used to detect mutations in the rpoB gene causing rifampicin resistance, and a microarray for the detection of all known pncA mutations has been developed and evaluated.84,85 A few pyrazinamide-resistant strains, however, lack mutations in the pncA gene, indicating alternative mechanisms for resistance to this agent, including defects in transportation of the agent into the bacterial cell.49 Pyrazinamide also inhibits mycobacterial fatty acid synthase I and this activity may therefore contribute to its bactericidal property.62 Ethambutol inhibits the synthesis of the polysaccharide arabinogalactan, a macromolecule essential for the structural integrity of the mycobacterial cell wall, by inhibiting the enzyme arabinosyl transferase. Acquisition of resistance is a multistep process involving several genes in the embA, embB, and embC gene cluster (principally embB), which encode for this enzyme. One particular mutation in codon 306 of the embB gene is commonly associated with ethambutol resistance but it has been postulated that it does not cause classical resistance to this agent but predisposes the bacteria to develop resistance to a range of anti-TB agents, yielding strains which clustering studies indicate are readily transmissible.86 There is only a weak association between isoniazid and ethambutol although isoniazid-resistant strains with one particular mutation in the katG gene are more likely to acquire high-level resistance to ethambutol.87 Ethionamide and the closely related prothionamide are structurally related to isoniazid and have the same target molecule – the enoyl acyl carrier protein reductase involved in mycolic acid synthesis and encoded by the inhA locus.88 Also, in common with isoniazid, ethionamide, and prothionamide are pro-drugs requiring activation. An enzyme able to activate these drugs is encoded by the ethA locus but complete sequencing of this locus in ethionamide-resistant isolates revealed mutations in only about half of them, suggesting that other mechanisms of resistance occur.89 Mutations in the inhA locus or its promoter region are associated with resistance to ethionamide and prothionamide and explain the partial cross-resistance with isoniazid seen in some strains but, in contrast to isoniazid, mutants in the katG locus do not induce resistance to the former drugs.
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MYCOBACTERIOPHAGES, LYSOGENY, AND PHAGE TYPING Numerous bacteriophages able to infect, replicate in, and lyse a range of mycobacterial species, including members of the M. tuberculosis complex, have been isolated from environmental sources including soil and water. These have been detected by incubating environmental samples in liquid media inoculated with various strains of mycobacteria and then applying filtrates of the culture to lawns of the same strains and observing whether plaques due to cell lysis appear. In addition a few mycobacteria isolated from clinical and environmental material have been found to carry whole or defective phages.90 In some cases these phages integrate stably into the genome of the host mycobacterium and are transmitted to all progeny, a state termed lysogeny. In others, the relationship is a pseudolysogenic one, in which stable integration does not occur and the phages are therefore only transmitted to some of the progeny. Thus, if a phage enters the virulent cycle and lyses the host cell, surrounding phage-free cells in a bacterial lawn on a solid medium are infected and lysed, so that lytic plaques appear spontaneously. Lysis occurring in cultures of lysogenic and pseudolysogenic mycobacteria results in the liberation of cell contents including DNA and, when grown on solid media, there may be enough extracellular DNA to make the colonies very mucoid and sticky. Some lysogenic strains of environmental mycobacteria have anomalous properties, including changes in cell wall lipid structure, due to phage conversion but there is no evidence that lysogeny plays a role in mycobacterial virulence. Defective prophages have been indirectly detected in mycobacteria in recombination studies. Thus, infection of a strain by a single phage may result in the appearance of several morphologically different phage plaques on bacterial lawns, or phages with quite different electron microscopical appearances may be seen in culture filtrates. Infection of the H37Rv reference strain of M. tuberculosis with a phage (LEO) resulted in the appearance of three quite different types of lytic plaques on bacterial lawns, suggesting the presence in the strain of more than one defective phage, and genomic sequencing studies have confirmed that this strain contains two defective prophages.61,91 Although no whole phages have been isolated from M. tuberculosis, several phages from environmental sources have wide host ranges and are able to infect and lyse this species. These have been used to develop typing systems for M. tuberculosis although the number of clearly delineated types is small and for this reason, as well as technical ones, this typing method has been superseded by DNA fingerprinting. Three major phage types of M. tuberculosis have been delineated – A, B, and I – and these correlate with other major biological differences between the strains.46 Thus strains of type A have high contents of strongly acidic lipids and sulpholipids in their cell walls and those of type I are attenuated in the guinea pig and correspond to the variant of M. tuberculosis known as the Asian or South Indian type (p. 50). Phages containing the gene coding for firefly luciferase have been utilized in an ingenious technique for the rapid detection of viable M. tuberculosis. In the presence of the substrate luciferin, viable mycobacteria emit light which is detectable by a luminometer or a Polaroid photographic film as in a simple low-technology system termed the ‘Bronx box’.9,92 This system detects bacterial
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killing by anti-TB agents and provides a simple and inexpensive method for determining drug resistance.
PLASMIDS OR EPISOMES In addition to the circular genome, many bacteria contain smaller closed circles of DNA, termed plasmids or episomes. Plasmids bear a few genes which in most cases have no proven major function but in some bacterial genera plasmids accumulate genes determining resistance to antibacterial agents and can be transferred from one bacterial cell to another, a process termed infectious drug resistance. This phenomenon has not been demonstrated in the genus Mycobacterium and plasmids have not been detected in members of the M. tuberculosis complex or in M. leprae. They have been detected in some environmental mycobacteria including members of the M. avium complex in which they have been most intensively sought. As mycobacterial plasmids lack specific functional markers including determinants of drug resistance, most are very difficult to detect and isolate and, for this reason little is known of their effect, if any, on their host bacteria. A technique for the ‘rescue’ of mycobacterial plasmids has, however, been described and used to isolate four otherwise cryptic plasmids from M. avium.93 Further use of this technique may shed useful light on the properties of mycobacterial plasmids and their role, if any, in pathogenicity.
THE EVOLUTION AND DEVOLUTION OF THE M. TUBERCULOSIS COMPLEX It was, until recently, widely believed that TB first emerged in animals and that the original or ancestral member of the M. tuberculosis complex might well therefore have resembled M. bovis. It was also assumed that human TB, caused by M. tuberculosis, resulted from a species jump from animals, possibly when they were first domesticated, with a subsequent diminution of host range. Detailed genomic analysis strongly suggests a quite different scenario and it now seems likely that the ancestral member of the complex, ‘M. prototuberculosis’, arose around 3 million years ago and may have caused disease in the ancestors of modern humans.94 It has also been a general assumption that, in common with other forms of life, bacteria evolve and diversify by acquisition of new gene-coded functions and by transfer of genes from one species, or even one genus, to another. While undoubtedly horizontal gene transfer does occur in many species, it appears to be a rare occurrence in the mycobacteria. In 1973 it was postulated that variants within mycobacterial species are largely derived, directly or indirectly, from a common progenitor type with deletional mutation playing a key role.95 It required the development of techniques for DNA sequencing to confirm this postulate and to show that it applied to the M. tuberculosis complex. It has been shown that the present-day species within the M. tuberculosis complex differ from one another by their pattern of genomic deletions, particularly in the presence or absence of parts of the genome termed regions of difference.96 Fourteen such regions are present in M. tuberculosis H37Rv, the strain selected for sequencing of the entire genome, but absent from the Institute Pasteur strain of BCG. These, designated RD1 to RD14, vary in size from 2 to 12.7 kilobases (kb). Two RD regions, RD3 and RD11, contain, respectively, the defective prophages phiRv1
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and phiRv2. A further six regions are absent from strain H37Rv but present to a varying extent in other strains of M. tuberculosis. The loss of these from H37Rv may be due to the fact that this strain was isolated in 1905 and repeatedly subcultivated in vitro over a 35year period, and although it retained its virulence it could perhaps, by analogy with BCG, have undergone various genetic losses.97 The missing regions are designated RvD1 to RvD5 and TbD1 (M. tuberculosis specific deletion). Despite the name, the latter is deleted in many strains within the M. tuberculosis complex and is not deleted in all strains of M. tuberculosis. On the basis of the presence or absence of TbD1, strains of M. tuberculosis are divisible into ‘ancestral’ strains that posses this marker and ‘modern’ strains in which it is deleted.94,96 The latter group contains the strains, classified as described below, that appear to be associated with major contemporary epidemics while some strains in the Indo-Oceanic lineage described below belong to the ‘ancestral’ group. Almost all strains of M. microti and M. bovis possess the six regions deleted in H37Rv but they lack several of the other RDs, particularly RD7 to RD10. Mycobacterium microti also has a specific deletion termed RDmic. Most strains of M. africanum likewise possess the six regions deleted in H37Rv but lack some of the other RDs, notably RD9. Exceptions were two isolates from Uganda which possessed RD1 to 14 but lacked TbD1 and some of the other RvDs. As mentioned earlier, isolates of M. africanum from Uganda more closely resemble M. tuberculosis in their cultural characteristics than isolates from West Africa which have more in common with M. bovis. A PCR-based technique for the differentiation of the various species in the M. tuberculosis complex and for distinguishing them from the environmental mycobacteria on the basis of their RD profiles has been developed.98,99 The Canetti strains have almost all the RD and RvD markers as well as TbD1 and may therefore most closely resemble the postulated ancestral form of the M. tuberculosis complex. Thus a probable evolutionary scenario is that the Canetti strains separated from the common progenitor early on, followed later by the divergence of M. africanum, M. bovis, and M. microti. The common progenitor then underwent the loss of some of the RvD markers and, perhaps relatively recently, the loss of TbD1 to give the various ‘modern’ genotypes of M. tuberculosis. The devolution of M. prototuberculosis to the present-day species is shown diagrammatically in Fig. 6.8. Studies M. canetti
RDcan RD9
TBD1
RD7, 8, 10
M. tuberculosis ancestral M. tuberculosis modern M. africanum Type I M. africanum Type II
mic
RD
RD12, 13
M. microti seal
RD
RD4
M. pinnipedii M. caprae
RD1
M. bovis BCG
Fig. 6.8 Devolution of the M. tuberculosis complex from the putative common progenitor, showing the regions of difference (RDs) lost at the various devolutionary stages. The figure does not show the numerous single nucleotide changes. Adapted from Brosch et al., with simplification.96
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The genus Mycobacterium and the Mycobacterium tuberculosis complex
on ancient DNA of the M. tuberculosis complex are confirming and shedding further light on this devolutionary scenario.100 The presence or absence of certain RDs may affect virulence. Thus RD1, a 9.5-kb region, is present in M. bovis but absent from all daughter strains of BCG and also M. microti, which is of comparable low virulence for humans.101 As described below, RD1 contains genes relevant to virulence, including those coding for ESAT-6. In addition, the strains of M. tuberculosis that appear particularly virulent and are spreading world-wide are ‘modern’ strains that have lost TbD1. Spoligotyping has also shed light on the lines of diversification of the M. tuberculosis complex as there is evidence that the variations in the DR locus is the result of loss of various spacer oligonucleotides due to various mutational events, including disruption by the translocation of the insertion sequence IS6110 into the locus.102 Such variation occurs at a very slow rate and serves as an evolutionary ‘clock’. In this context, BCG daughter strains, although subcultivated repeatedly since the introduction of the vaccine in 1921, share the same spoligotype, which is identical to that of about a quarter of strains of M. bovis currently isolated between 1979 and 2000 from cattle in France, the country where the progenitor of BCG was isolated by Nocard in the late nineteenth century.57 Members of the M. tuberculosis complex have been divided into a number of groups, termed lineages, superfamilies, or clades, for epidemiological purposes. At the time of writing, the grouping and nomenclature of these lineages is confusing. Some workers identify the lineages on the basis of spoligotyping,103 some on single nucleotide polymorphisms,66 and some on large-sequence polymorphisms,104 and grouping by these methods does not completely coincide. While some workers are primarily interested in epidemiology others, as discussed earlier, are more concerned with the phylogenetic relationships between strains. There is no internationally recognized nomenclature of the lineages but published names include Beijing (or W/Beijing), Central Asian, East Asian (which includes the Beijing lineage), East African-Indian, West African 1 and 2 (including some strains of M. africanum), Indo-Oceanic, Haarlem, Euro-American, Latin American Mediterranean, Manu, S, T, and X. On the basis of spoligotyping, 62 lineages and sublineages in the M. tuberculosis complex have been defined, including 3 sublineages of M. bovis and 2 of M. pinnipedi.103 Doubtless, more lineages will be described and hopefully a clear and internationally agreed nomenclature will eventually be proposed. A minimal standard set of 16 single nucleotide polymorphisms that resolve M. tuberculosis into 6 lineages and distinguish these from M. bovis have been proposed, but these do not correlate exactly with the lineages defined by spoligotyping.66 There is evidence that lineages vary in virulence for humans and elicit different patterns of immune responses.105 Although other factors may be involved, the lineages vary in their ability to spread world-wide which may be expressed mathematically as their Spreading Index (SI).106 Lineages with a high SI are defined as ‘epidemic’ and include the Beijing lineage, which is now found in many parts of the world.107 The percentages of the major lineages in different geographical regions and maps of the distributions of six common lineages have been published.103 There is also evidence that lineages vary in the extent to which they lose virulence on mutating to drug or multidrug resistance.80 Members of the Beijing lineage appear to retain virulence on mutation to drug resistance and have been implicated in the
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epidemic spread of multidrug-resistant TB in some parts of the world.108
DETERMINANTS OF VIRULENCE AND PATHOGENICITY Numerous possible determinants of virulence of members of the M. tuberculosis complex, including secreted antigens and other molecules, cell surface components, enzymes involved in general cellular metabolism, and transcriptional regulators, have been described.30 Host–pathogen interactions appear to be multifactorial and involve the initial entry of the infecting organism into macrophages and other host cells and the subsequent determination of the host immune response, including the induction of responses that act in favour of the pathogen. There is evidence that most of the genes of M. tuberculosis are required for the maintenance of TB in the human population, although many may play only a minor role.109 Virulence of mycobacteria appears to be due much more to the immune interactions between the bacillus and the host rather than to any toxic properties of the bacilli. On the other hand, there is some evidence, discussed later, that a secreted antigen (ESAT-6) of M. tuberculosis may have a deleterious toxic effect on host cell membranes. An exceptional example of a toxin being involved in the pathogenesis of a mycobacterial disease is seen in the case of Buruli ulcer, caused by M. ulcerans. Gross tissue necrosis resulting in the characteristic large and deeply undermined skin ulcers in this disease is due largely if not entirely to a toxic substance termed mycolactone, which also has locally acting immunosuppressive properties.110 Three major problems have been encountered in studies on mycobacterial virulence. First, there are several stages in the course of TB – initial establishment of infection, a period of latency or dormancy, and post-primary disease due to either endogenous reactivation or exogenous reinfection. Secondly, virulence in experimental animal models does not correlate closely with that in humans and, thirdly, there are observations suggesting that mechanisms of virulence of M. tuberculosis may vary according to the genotypic lineages of the strains which may have diversified as adaptations to human populations of differing genetic constitution. Thus infection by a strain of M. tuberculosis may be more likely to proceed to overt TB in the human population to which it has adapted (sympatric hosts) than in a different population (allopatric hosts).104 This concept is supported by the demonstration that the lineages of M. tuberculosis differ considerably in the nature of the disease they cause, and the immune responses that they elicit, in the experimental mouse.111 Although the regional differences in the protective efficacy of BCG vaccination have generally been attributed to ecological factors, geographical differences in host–pathogen interactions may also be involved and require consideration in the development of novel vaccines.104 Although dissemination of disease from the site of infection, usually the lung, is affected by the host immune responses, bacterial factors are also involved. A study of patients infected with more than one strain of M. tuberculosis and in whom one strain was isolated from extrapulmonary sites and the other only from the lung showed that the former strains infected macrophages in vitro more efficiently than the latter and that they were more infectious in vivo in an experimental murine model.112
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These findings lead to the conclusion that TB is not a homogeneous disease. It is continuously changing and evolving as it spreads around the world and encounters different host populations including those containing many individuals with compromised immunity, as well as being subject to the various organizational and other factors that favour the selection of drug-resistant mutants. The Beijing lineage, which appears to be more virulent than other lineages even when multidrug resistant, is appearing in many countries and is a source of concern.107 In the words of Paracelsus, ‘There are never again the same causations; things are more virulent now as both the philosophies of heaven and the elements sufficiently prove.’ In primary human infection, the tubercle bacillus enters phagocytic cells, and possibly other cells, where it survives and replicates. Subsequently, if the primary infection is rendered quiescent by the host defence mechanisms, bacilli enter a state of poorly understood dormancy which, in humans, may continue for several years or decades until reactivation occurs. Such reactivation or exogenous reinfection results in post-primary tuberculosis which is characterized by gross tissue necrosis, which, by generating open pulmonary cavities, enables the organism to be transmitted from one host to another. Thus, the steps in pathogenicity are initial entry into cells, particularly macrophages, intracellular survival and replication, the establishment of the persister state, ‘awakening’ from the persister state, and subversion of the immune responses to induce the gross tissue necrosis seen in post-primary TB. These steps depend on quite different mechanisms of virulence which are therefore clearly multifactorial.
EARLY EVENTS The early events in the establishment of TB cannot be investigated in humans and are thus extrapolated from experimental animal studies and in vitro observations on the uptake of mycobacteria into macrophages. The first step in pathogenesis after initial infection is the entry of the mycobacterium into macrophages and, possibly, other cells. In common with other bacteria, phagocytosis of M. tuberculosis may involve complement activation and mannose binding. Within the macrophage, survival of the bacteria depends largely on their ability to inhibit phagosome maturation and to block phagosome– lysosome fusion.113 Mannose-capped lipoarabinomannan (ManLAM) of M. tuberculosis not only facilitates entry into macrophages by binding to mannose receptors but has these effects on phagosomal activity.42 Other poorly understood mechanisms also appear to be involved. Studies on M. marinum, the cause of swimming pool granuloma (Chapter 7), have led to the identification of two groups of genes termed mycobacterial-enhanced infection loci (mel ) 1 and 2.114 Genetic transfer of these loci to non-pathogenic mycobacteria showed that they enhanced both attachment to macrophages and intracellular survival. These loci contain 11 genes but the exact relevance of the various gene products to the observed effects is not clear. Although this study was conducted on M. marinum, the presence of both loci in M. tuberculosis but not in non-pathogens suggests a similar role in this species. Various glycine-rich fibronectin-binding mycobacterial proteins may also be involved in the establishment of infection by facilitating adherence to host tissues.63,65 Early secreted antigens have attracted attention as determinants of virulence and inducers of protective immunity. Particular attention has been focused on a group of secreted antigens coded for by
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the ESX-I locus in the RD1 region of the genome which is missing from BCG, M. microti, and other strains attenuated for humans. The ESX-1 locus codes for two antigens, ESAT-6 and CFP-10, and a protein, EspA, which is essential for the secretion of the two antigens.115 The function of ESAT-6 has not been fully determined but there is evidence that it may disrupt host cell membranes, resulting in lysis of infected cells, spread of infection from cell to cell, and possibly escape of intracellular pathogens from the phagosomes. Strains of M. tuberculosis vary in their ability to cause cytotoxicity and necrosis of murine macrophages in vitro and the extent of cytotoxicity correlates with other criteria of virulence.116 In addition, the early secreted antigens are potent inducers of protective immune responses by recruiting large numbers of the appropriate T cells. Thus, BCG complemented with RD1 (BCG::RD1) is more virulent than the parent BCG strain but also elicits a much more powerful immune response.117 A construct of BCG and RD1 possessing the immunogenicity but lacking virulence might prove to be a useful vaccine. Once engulfed by phagocytic cells, the next crucial step in pathogenesis is survival and replication of the mycobacterium within these cells. Various mechanisms enhancing survival of pathogenic mycobacteria within macrophages, including inhibition of the fusion of phagosomes and lysosomes, inhibition of the acidification of phagosomes, and resistance to killing by various reactive oxygen and nitrogen molecules, have been postulated.118 The exact mechanism of intracellular survival is poorly understood but one study based on gene transfer between M. tuberculosis and the nonpathogenic species M. smegmatis have led to the identification of a genetic locus termed the enhanced intracellular survival (eis) gene, which is present in members of the M. tuberculosis complex, including BCG, but not in M. smegmatis and 10 other non-pathogenic mycobacterial species.118 Preliminary evidence suggests that this gene codes for a 42-kDa protein situated on the surface of the mycobacterial cell and which might therefore modify the interactions between the bacterial and host cells. As mentioned earlier, the products of the mel1 and mel2 genetic loci also appear to affect intracellular survival.
PERSISTENCE, LATENCY, AND DORMANCY Very little is known about the morphological and physiological nature of tubercle bacilli in latent TB.119,120 Although animal models for studying latency have been developed, these appear rather artificial and therefore, perhaps, bear little relevance to human TB. In addition, it is possible that the bacilli persisting after the natural resolution of primary TB are different to those that persist after a course of treatment of post-primary disease and are responsible for early recurrence of the disease. One hypothesis is that persisting bacilli are truly dormant and are sequestered in dense, anoxic, scar tissue. In this context there is evidence that the establishment, and ‘awakening’ from the dormant state is controlled by genes transcribed in hypoxic environments.121 While some tubercle bacilli may adapt to a state of physiological dormancy in anoxic situations, this may not be the only mechanism of persistence. The problem with the dormancy hypothesis is that isoniazid is known to prevent reactivation of endogenous infection and that it only kills actively replicating tubercle bacilli. Thus an alternative explanation is that persisting bacilli exist in a more or less balanced cycle of replication and destruction by immune mechanisms. This
CHAPTER
The genus Mycobacterium and the Mycobacterium tuberculosis complex
would explain the association between a reactivation and immune compromise and the continuing production of gamma-interferon in those with latent TB.119 The morphological features of persisting mycobacteria remain a mystery. Although various forms have been described, including cell-wall-free forms and minute sporelike particles (Much’s granules), none have actually been clearly demonstrated. They may, however, be analogous to minute nonreplicating bacterial cells in the genera Rhodomicrobium and Rhodococcus that develop in response to adverse conditions.122 The situation of persisters within host cells and tissues is also uncertain. A common assumption is that they are within dense and anoxic fibrous or even calcified scar tissue, but the use of in situ PCR reveals that DNA supposedly specific to the M. tuberculosis complex is found throughout the lung in those with latent TB dying of other, mostly traumatic, causes.123 No acid-fast bacilli were seen at the sites of this DNA, suggesting that the persisters were cell-wall-free forms. In view of the rather puzzling observations to date, it has been postulated that the classical acid-fast rods seen in clinical specimens and in vitro cultures may just be one of a number of physiological and morphological variants of the mycobacteria.120
LATE EVENTS The principal difference between primary and post-primary TB is the extensive and excessive tissue necrosis in the latter, resulting in cavity formation in the lung and transmissibility of the infection. This process is principally determined by the nature of the host immune responses, particularly the action of interleukin-4 and other cytokines produced or induced by Th2 T cells as described in detail in Chapter 8 and 9. Pathogenic mycobacteria appear to drive detrimental Th2-mediated immune responses while some avirulent species including M. vaccae enhance protective Th1 responses and down-regulate Th2 responses and may therefore be useful immunotherapeutic agents for the treatment of TB and other conditions characterized by Th2-induced immunopathology.124
CONCLUSIONS Robert Koch discovered the cause of TB over 120 years ago but the application of this discovery to strategies for the diagnosis, prevention, and treatment of this and other mycobacterial diseases has been painfully slow. The fact that today the number of people contracting TB and dying of it each year are counted in millions is a sure indication that powerful new tools, or a much more
REFERENCES ¨ ber tuberculose.” A tribute 1. Grange JM, Bishop PJ. “U to Robert Koch’s discovery of the tubercle bacillus, 1882. Tubercle 1982;63:3–17. Also published, in part, in Bull Int Union Tuberc 1982;57:116–121. 2. Pallamary P. Translation of Gerhard Armauer Hansen: Spedalskheden Aarsager (Causes of leprosy). Int J Leprosy 1955;23:307–309. 3. Smith T. A comparative study of bovine tubercle bacilli and of human bacilli from sputum. J Exp Med 1898;3:451–511. 4. Den Dooren de Jong LE. De tuberculose van bacteriologisch stanpunt bezien. Nederl Tijdschr Geneeskinde 1939;83:5896–5906.
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determined deployment of existing tools, are required. Our principal diagnostic tool, tuberculin testing, was introduced in 1907, the only vaccine in present use was first used in 1921, and no drug superior to rifampicin has emerged since the late 1960s. There are signs, however, that we are on the verge of a scientific revolution. The past few years have seen enormous strides being taken in unravelling the hidden mysteries of the mycobacteria, the means by which they cause disease and the immune strategies which the infected host can utilize for protection. In addition, molecular techniques have led to the rapid detection and identification of mycobacteria in clinical specimens and, in the case of the M. tuberculosis complex, the rapid determination of patterns of drug susceptibility and resistance and the typing of strains for epidemiological purposes. Modern techniques have yielded specific antigens for use in diagnostic tests and for the unravelling of the complexities of the immune responses resulting in either protection or immunopathology. These developments have also raised the hopes of developing highly effective vaccines, including non-viable ones that could safely be given to those with HIV disease and other immunosuppressive conditions. The mammoth task of sequencing the entire genome, initially of M. tuberculosis and subsequently M. bovis, M. leprae, and M. avium paratuberculosis, has recently been completed successfully and is shedding enormous light on the structure and properties of these major pathogens, including the regulation of their metabolic activity which is likely to prove of enormous benefit in the rational development of novel anti-mycobacterial agents. The same work has also made it possible to determine the evolution of these pathogens which, in addition to being a most fascinating academic exercise, is revealing key aspects of host–pathogen interactions which may vary significantly across the globe. Never before in the very long history of TB and other mycobacterial disease, including leprosy, has the human race had such a wonderful opportunity to develop powerful tools for the final conquest of these afflictions. Whether the human race will grasp this opportunity to control one of the world’s principal preventable causes of suffering or will squander resources on weapons of mass destruction remains to be seen. It is perhaps worth reflecting on the words of the American public health worker John B. Huber, who a century ago, and well before the introduction of vaccines and anti-TB agents, wrote, ‘The tubercle bacillus is an index, by inversion, of the real progress of the human race. By it the claim of civilization to dominate human life may fairly be judged. Tuberculosis will decrease with the substantial advance of civilization, and the disease will as surely increase as civilization retrogrades.’125
5. Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am 1959;43:273–290. 6. Wayne LG. Numerical taxonomy and cooperative studies: roles and limitations. Rev Infect Dis 1981;3:822–828. 7. Wayne LG, Good RC, Krichevsky MI, et al. Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the international Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol 1991;41:463–472. 8. Skerman VDB, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol 1980;30:225–420. 9. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: Part II. Active tuberculosis and drug resistance. Expert Rev Mol Diagn 2006;6:423–432.
10. Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin Microbiol Rev 2003;16:319–354. 11. Rogall T, Flohr T, Bottger EC. Differentiation of mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol 1990;136:1915–1920. 12. Kazda J, Irgens LM, Kolk AH. Acid-fast bacilli found in sphagnum vegetation of coastal Norway containing Mycobacterium leprae-specific phenolic glycolipid-I. Int J Lepr Other Mycobact Dis 1990;58:353–357. 13. Schultze-Robbicke R, Janning B, Fischeder R. Occurrence of mycobacteria in biofilm samples. Tuberc Lung Dis 1992;73:141–144. 14. Falkinham JO, Norton CD, Lechavallier MW. Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other mycobacteria in
57
SECTION
2
15.
16. 17.
18.
19.
20.
21.
22.
23.
24. 25.
26.
27.
28.
29.
30.
31. 32.
33.
34.
35.
36.
37.
38.
58
BASIC SCIENCE
drinking water systems. Appl Environ Microbiol 2001;67:1225–1231. Khoor A, Leslie KO, Tazelaar HD, et al. Diffuse pulmonary disease caused by nontuberculous mycobacteria in immunocompetent people (hot tub lung). Am J Clin Pathol 2001;115:755–762. Collins CH, Grange JM, Yates MD. Mycobacteria in water. J Appl Bacteriol 1984;57:193–211. Gubler JGH, Salfinger M, von Graevenitz A. Pseudoepidemic of non-tuberculous mycobacteria due to a contaminated bronchoscope cleaning machine - report of an outbreak and a review of the literature. Chest 1992;101:1245–1249. Grange JM, Noble WC, Yates MD, et al. Inoculation mycobacterioses. Clin Exptl Dermatol 1988;13: 211–220. Palmer CE, Long MW. Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. Am Rev Respir Dis 1969;94:553–568. Stanford JL, Shield MJ, Rook GAW. How environmental mycobacteria may predetermine the protective efficacy of BCG. Tubercle 1981;62:5–62. Fine PEM. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 1995;346:1339–1345. Primm TP, Lucero CA, Falkinham JO. Health impacts of environmental mycobacteria. Clin Microbiol Rev 2004;17:98–106. Chakrabarty AN, Dastidar SG. Is soil an alternative source of leprosy infection? Acta Leprol 2001–2002; 12:79–84. Grange JM, Lethaby JI. Leprosy of the past and today. Semin Respir Crit Care Med 2004;25:271–282. Collins CH, Grange JM, Yates MD. Tuberculosis Bacteriology. Organization and Practice, 2nd edn. Oxford: Butterworth Heinemann, 1997. Garnier T, Eiglmeier K, Camus J-C, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci USA 2003;100:7877–7882. Sonnenberg MG, Belisle JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectroscopy. Infect Immun 1997;65:4515–4524. Stanford JL, Grange JM. The meaning and structure of species as applied to mycobacteria. Tubercle 1974;55:143–152. Schaefer WB. Serologic identification and classification of the atypical mycobacteria by their agglutination. Am Rev Respir Dis 1965;92(Suppl): 85–93. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 2003;16:463–496. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem 1995;64:29–63. Dean C, Crick DC, Mahapatra S, et al. Biosynthesis of the arabinogalactan–peptidoglycan complex of Mycobacterium tuberculosis. Glycobiology 2001; 11:107R–118R. Lee W, Riley LW. Of mice, men, and elephants: Mycobacterium tuberculosis cell envelope lipids and pathogenesis. J Clin Invest 2006;116:1475–1478. Takayama K, Wang C, Besra GS, et al. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 2005;18:81–101. Armitige LY, Jagannath C, Wanger AR, et al. Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages. Infect Immun 2000;68:767–778. Geisel RE, Sakamoto K, Russell DG, et al. In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Gue´rin is due principally to trehalose mycolates. J Immunol 2005;174:5007–5015. Rao V, Fujiwara N, Porcelli SA, et al. Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule. J Exptl Med 2005;201:535–543. Chatterjee D, Khoo KH. Mycobacterial lipoarabinomannan: an extraordinary
39.
40.
41.
42.
43.
44. 45.
46.
47.
48.
49.
50. 51.
52.
53.
54.
55.
56.
57.
58.
59.
lipoheteroglycan with profound physiological effects. Glycobiology 1998;8:113–120. Rook GAW, Zumla A. Advances in the immunopathogenesis of pulmonary tuberculosis. Curr Opin Pulmon Med 2001;7:116–123. Means TK, Jones BW, Schromm AB, et al. Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses. J Immunol 2001;166:4074–4082. De Valliere S, abate G, Blazevic A, et al. Enhancement of innate and cell-mediated immunity by antimycobacterial antibodies. Infect Immun 2005;73:6711–6720. Kang PB, Azad AK, Torrelles JB, et al. The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exptl Med 2005;202:987–999. Chatterjee D, Hunter SW, McNeil M, et al. Structure and function of mycobacterial glycolipids and glycopeptidolipids. Acta Leprol 1989;7(suppl 1):81–84. Smith PG. The serodiagnosis of leprosy. Lepr Rev 1992;63:97–100. Cho S-N, Cellona R, Villahermosa LG, et al. Detection of phenolic glycolipid I of Mycobacterium leprae in sera from leprosy patients before and after start of multidrug therapy. Clin Diagn Lab Immunol 2001;8:138–142. Goren MB, Grange JM, Aber VR, et al. Role of lipid content and hydrogen peroxide susceptibility in determining the guinea-pig virulence of Mycobacterium tuberculosis. Br J Exp Pathol 1982;63:693–700. Reed MB, Domenech P, Manca C, et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 2004;431:84–87. Kartmann B, Stengler S, Niederweis M. Porins in the cell wall of Mycobacterium tuberculosis. J Bacteriol 1999;181:6543–6546. Raynaud C, Laneelle MA, Senaratne RH, et al. Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity. Microbiology 1999;145:1359–1367. Ratledge C. Iron, mycobacteria and tuberculosis. Tuberculosis (Edinb) 2004;84:110–130. Li L, Bannantine JP, Zhang Q, et al. The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci USA 2005;102:12344–12349. Yates MD, Collins CH, Grange JM. ‘Classical’ and ‘Asian’ variants of Mycobacterium tuberculosis isolated in South-East England, 1977-1980. Tubercle 1982;63:55–61. Pfyffer GE, Auckenthaler R, van Embden JD, et al. Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg Infect Dis 1998;4:631–634. Aranaz A, Cousins D, Mateos A, et al. 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;53:1785–1789. Kubica T, Ru¨sch-Gerdes S, Niemann S. Mycobacterium bovis subsp. caprae caused one-third of human M. bovis-associated tuberculosis cases reported in Germany between 1999 and 2001. J Clin Microbiol 2003;41:3070–3077. Fomukong NG, Dale JW, Osborn TW, et al. Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains. J App Bacteriol 1992;72:126–133. Haddad N, Ostyn A, Karoni C, et al. Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979-2000. J Clin Microbiol 2001;39:3623–3632. Grange JM, Yates MD, de Kantor IN. Guidelines for Speciation within the Mycobacterium tuberculosis Complex. Second edition. Geneva: World Health Organization, 1996. WHO/EMC/ZOO/96.4. Hart PD, Sutherland I. BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life. Br Med J 1977; 2:293–295.
60. 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;53:1305–1314. 61. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537–544. 62. Zimhony O, Vilche`ze C, Jacobs WR. Characterization of Mycobacterium smegmatis expressing the Mycobacterium tuberculosis fatty acid synthase I (fasI) gene. J Bacteriol 2004;186:4051–4055. 63. Banu S, Honore N, Saint-Joanis D. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Mol Microbiol 2002;4:9–19. 64. Talarico S, Cave MD, Marrs CF, et al. Variation of the Mycobacterium tuberculosis PE_PGRS33 gene among clinical isolates. J Clin Microbiol 2005; 43:4954–4960. 65. Espitia C, Laclette JP, Mondrago´n-Palomino M, et al. The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins? Microbiology 1999;145:3487–3495. 66. 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;188:759–772. 67. Mostowy S, Cousins D, Brinkman J. Genomic deletions suggest a phylogeny for the Mycobacterium tuberculosis complex. J Infect Dis 2002;186:74–80. 68. Sreenu VB, Kumar P, Nagaraju J, et al. Microsattelite polymorphism across the M. tuberculosis and M. bovis genomes: implications on genome evolution and plasticity. BMC Genomics 2006;7:78 (Epub). 69. Savine E, de Haas P, van Groenen PM, et al. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis and its use as an epidemiological tool. Mol Microbiol 1993;10: 1057–1065. 70. Gopaul KK, Brown TJ, Gibson AL, et al. Progression toward an improved DNA amplification-based typing technique in the study of Mycobacterium tuberculosis epidemiology. .J Clin Microbiol 2006;44:2492–2498. 71. 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;37:2607–2618. 72. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the leprosy bacillus. Nature 2001; 409:1007–1011. 73. Brennan PJ, Vissa VD. Genomic evidence for the retention of the essential mycobacterial cell wall in the otherwise defective Mycobacterium leprae. Lepr Rev 2001;72:415–428. 74. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 2003;48:77–84. 75. Takakura S, Tsuchiya S, Fujihara N, et al. Isothermal RNA sequence amplification method for rapid antituberculosis drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2005;43:2489–2491. 76. Aragon LM, Navarro F, Heiser V, et al. Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent lowdensity DNA microarrays. J Antimicrob Chemother 2006;57:825–831. 77. Park H, Song EJ, Song ES, et al. Comparison of a conventional antimicrobial susceptibility assay to an oligonucleotide chip system for detection of drug resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol 2006;44:1619–1624. 78. Williams DL, Waguespack C, Eisenach K, et al. Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob Ag Chemother 1994;38: 2380–2386.
CHAPTER
The genus Mycobacterium and the Mycobacterium tuberculosis complex 79. Baker LV, Brown TJ, Maxwell O, et al. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC-46A polymorphism. Antimicrob Ag Chemother 2005;49:1455–1464. 80. Gagneux S, Burgos MV, DeRiemer K, et al. Impact of bacterial genetics on the transmission of isoniazidresistant Mycobacterium tuberculosis. PLoS Pathog 2006; 2(6):e61. 81. Price-Evans DA, Manley KA, McKusick VA. Genetic control of isoniazid metabolism in man. Br Med J 1960;2:485–491. 82. Bhakta S, Besra GS, Anna M, et al. Arylamine N-Acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target. J Exptl Med 2004;199:1191–1199. 83. Zhang Y, Mitchison D. The curious characteristics of pyrazinamide: a review. Int J Tuberc Lung Dis 2003;7:6–21. 84. McCammon MT, Gillette JS, Derek P, et al. Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States–Mexico border region. Antimicrob Ag Chemother 2005;49:2210–2217. 85. Denkin S, Volokhov D, Chizhikov V, et al. Microarray-based PncA genotyping of pyrazinamideresistant strains of Mycobacterium tuberculosis. J Med Microbiol 2005;54:1127–1131. 86. Hazbo´n MH, del Valle MB, Guerrero MI, et al. Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance. Antimicrob Ag Chemother 2005;49:3794–3802. 87. Parsons LM, Salfinger M, Clobridge A, et al. Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol. Antimicrob Ag Chemother 2005;49:2218–2225. 88. Larsen MH, Vilcheze C, Kremer L, et al. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol Microbiol 2002;46:453–466. 89. Morlock GP, Metchock B, Sikes D, et al. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob Ag Chemother 2003;47:3799–3805. 90. Grange JM, Redmond WB. Host-phage relationships in the genus Mycobacterium and their clinical significance. Tubercle 1978;59:203–225. 91. Mankiewicz E, Redmond WB. Lytic phenomena of phage LEO isolated from a sarcoid lesion. Am Rev Respir Dis 1968;98:41–46. 92. Hazbo´n MH, Guarı´n N, Ferro BE, et al. Photographic and luminometric detection of luciferase reporter phages for drug susceptibility testing of clinical Mycobacterium tuberculosis isolates. J Clin Microbiol 2003;41:4865–4869. 93. Kirby C, Waring A, Griffin TJ, et al. Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue. Mol Microbiol 2002;43:173–186.
94. Gutierrez MC, Brise S, Brosch R, et al. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathogens 2005; 1(1):e5. 95. Grange JM. Intra-specific variation in the mycobacteria—a taxonomic aid. Ann Soc Belge Med Trop 1973;53:339–346. 96. Brosch R, Gordon SV, Marmiesse M, et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 2002;99:3684–3689. 97. Steenken W, Gardner LU. History of H37 strain of tubercle bacillus. Am Rev Tuberc 1946;54:62–66. 98. Huard RC, Lazzarini C de O, Butler WR, et al. PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J Clin Microbiol 2003; 41:1637–1650. 99. Huard RC, Fabre M, de Haas P, et al. Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex. J Bacteriol 2006;188:4271–4287. 100. Donoghue H, Spigelman M, Greenblatt CL, et al. Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA. Lancet Infect Dis 2004; 4:584–592. 101. Mahairas GG, Sabo PJ, Hickey MJ, et al. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:1274–1282. 102. Legrand E, Filliol I, Sola C, et al. Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis. J Clin Microbiol 2001;39:1595–1599. 103. Brudey K, Driscoll JR, Rigouts L, et al. Mycobacterium tuberculosis complex genetic diversity: mining the forth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiology 2006;6:23. 104. Gagneux S, DeRiemer K, Tran Van, et al. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006; 103:2869–2873. 105. Sun Y-J, Lim TK, Ong AKY, et al. Tuberculosis associated with Mycobacterium tuberculosis Beijing and non-Beijing genotypes: a clinical and immunological comparison. BMC Infect Dis 2006;6:105. Published online 2006 July 5. doi: 10.1186/1471-2334-6-105. 106. Filliol I, Driscoll JR, van Soolingen D, et al. Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study. J Clin Microbiol 2003;41:1963–1970. 107. Drobniewski F, Balabanova Y, Nikolayevsky V, et al. Drug-resistant tuberculosis, clinical virulence, and the dominance of the Beijing strain family in Russia. JAMA 2005;293:2726–2731. 108. Toungoussova OS, CaugantDA, Sandven P, et al. Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype. FEMS Immunol Med Microbiol 2004;42:281–290. 109. Yesilkaya H, Dale JW, Strachan NJC, et al. Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need? J Bacteriol 2005;187:6726–6732.
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110. George KM, Chatterjee D, Gunawardana G, et al. Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 1999; 283:854–857. 111. 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;133:30–37. 112. Garcia de Viedma D, Lorenzo G, Cardona PJ, et al. Association between the infectivity of Mycobacterium tuberculosis strains and their efficiency for extrarespiratory infection. J Infect Dis 2005;192: 2059–2065. 113. Deretic V, Singh S, Master S, et al. Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism. Cell Microbiol 2006;8:719–727. 114. El-Etr SH, Subbian S, Cirillo SLG, et al. Identification of two Mycobacterium marinum loci that affect interactions with macrophages. Infect Immun 2004;72:6902–6913. 115. Fortune SM, Jaeger A, Sarracino DA, et al. Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci USA 2005;102:10676–10681. 116. Park JS, Tamayo MH, Gonzalez-Juarrero M, et al. Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages. J Leukocyte Biol 2006;79:80–86. 117. Majlessi L, Brodin P, Brosch R, et al. Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system. J Immunol 2005;174:3570–3579. 118. Wei J, Dahl JL, Moulder JW, et al. Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol 2000;182:377–384. 119. Orme IM. The latent tubercle bacillus (I’ll let you know if I ever meet one). Int J Tuberc Lung Dis 2001;5:589–593. 120. Grange JM. Would you know one if you met one? Int J Tuberc Lung Dis 2001;5:1162–1163. 121. He H, Zahrt TC. Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 2005;187:202–212. 122. Shleeva MO, Bagramyan K, Telkov MV, et al. Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase. Microbiology 2002;148:1581–1591. 123. Hernandez-Pando R, Jeyanathan M, Mengistu G, et al. Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection. Lancet 2000;356:2133–2138. 124. Dlugovitzky D, Fiorenza G, Farroni M, et al. Immunological consequences of three doses of heatkilled Mycobacterium vaccae in the immunotherapy of tuberculosis. Respir Med 2006;100:1079–1087. 125. Huber JB. Civilization and tuberculosis. Br J Tuberc 1907;1:158–168.Cited in Grange JM, Zumla A. The global emergency of tuberculosis: what is the cause? JR Soc Health 2002;122:78–81.
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Other mycobacteria causing human disease Charles L Daley and Leonid Heifets
INTRODUCTION The non-tuberculous mycobacteria (NTM) comprise over 125 different species widely distributed throughout the environment, most of which have not been implicated as a cause of disease in humans.1 The NTM have been referred to as mycobacteria other than TB (MOTT), atypical mycobacteria, and environmental mycobacteria. Despite their frequent isolation in the environment and human specimens, the NTM were not widely recognized as a cause of human disease until the late 1950s. Since that time, the number of new species of NTM have grown dramatically primarily related to the availability of DNA sequencing, which has allowed identification of new species simply by detecting relatively small differences in DNA sequences. In addition to the proliferation of new species, the rate of disease related to NTM has also increased, overtaking the rate of TB in some areas. NTM have several features that distinguish them from organisms of the Mycobacterium tuberculosis complex, such as a wide range of pathogenicity and, unlike M. tuberculosis, they are not transmissible from human to human. Unfortunately, the pathogenic NTM are relatively drug resistant compared with M. tuberculosis and, thus, difficult to treat with high rates of treatment failure and recurrence. Because of our poor understanding of the transmission and pathogenesis of most NTM infections, we have little insight into how to prevent these infections and thus our public health strategy to control disease caused by these ubiquitous organisms is often lacking.
EPIDEMIOLOGY AND RISK FACTORS EPIDEMIOLOGY NTM are ubiquitous bacteria that have been isolated from soil and water sources, including both natural and treated water sources, from throughout the world. The source of infection appears to be through environmental exposure and transmission from human to human has not been described. Studies that address the epidemiology of NTM infections can be broadly divided into three types.2 Cutaneous delayed-type hypersensitivity to NTM antigens has been used to assess the frequency of infection with these organisms. Skin test studies have indicated that a substantial proportion of adults have had prior exposure to NTM. Between 1943 and 1959, 22,000 nursing students were tested with 5 tuberculin units of PPD-S.3 Students who did not react to 5 tuberculin units were retested with 250 units to detect reactivity to cross-reacting antigens. Sixty-five per cent of
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students who came from the southeast of the USA reacted to the higher dose of tuberculin compared with only 29% of those from other areas of the country. Skin test surveys of US Navy recruits in the 1960s demonstrated higher rates of reactivity in those from the southeast of the USA (56%) than in those from the northern states (15–25%), suggesting higher rates of infection.4 These surveys demonstrated the striking geographic differences in NTM infection. Another type of study utilizes consecutive isolates from a welldefined catchment area to estimate the frequency of infection but, because this approach lacks clinical information, it is unable to estimate the prevalence of disease. Survey data from state laboratories in the early 1980s estimated a prevalence of NTM infection of 1–2 cases per 100,000 population.5 A similar survey from 1993 to 1996 reported an annual case rate of 7–8 per 100,000, documenting an increase in isolation of NTM when compared with the previous survey.6 The most useful studies combine clinical and microbiological data to provide estimates of disease rates. The incidence of pulmonary NTM was studied prospectively in France between 2001 and 2003 at 32 sentinel surveillance sites: 262 patients met the American Thoracic Society (ATS) criteria for disease (see below).7Mycobacterium avium complex was the cause of disease in almost half of the patients, followed by Mycobacterium xenopi in 25%, Mycobacterium kansasii in 13% and rapidly growing mycobacteria in 10%. The overall incidence was estimated to be stable and approximately 0.72–0.74 per 100,000; however, the method for calculating the incidence rate probably underestimated the true incidence. Recent data support the view that the incidence of NTM infections is increasing.8 In a retrospective cohort review from 1997 to 2003 in Ontario, Canada, 222,247 pulmonary isolates from 10,231 patients were identified. The prevalence was 9.1/100,000 in 1997 and increased to 14.1/100,000 by 2003 (p < 0.0001) with an average annual increase of 8.4%. Increases were noted among M. avium complex, M. xenopi, rapidly growing mycobacteria and M. kansasii. Of note, the rate of TB declined 4.0% over the study period. Two hundred patients were evaluated in more detail and 33% fulfilled the clinical, radiological and bacteriological ATS criteria. The most common NTM pathogens vary geographically. Mycobacterium avium complex has been reported as the most common cause of NTM-related pulmonary disease in almost all studies.1 However, the next most common NTM that causes disease varies by location. For example, in the USA, it is M. kansasii, followed by Mycobacterium abscessus. In Canada, and some parts of Europe, M. xenopi is the second most common, whereas in northern Europe and Scandinavia, Mycobacterium malmoense is second.
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Mycobacterium marinum resides in both fresh and marine water environments, and infections are acquired by inoculation in these environments. Initial infections were reported from swimming pool exposures,9 although home aquariums have become increasingly implicated as the source of infection.10 The reported incidence of M. marinum infection is variable, ranging from 0.05 to 27 per 100,000 in adults in North Carolina, USA.11 Buruli ulcer, a skin disease caused by Mycobacterium ulcerans, was first described in central Africa during the late 1800s and early 1990s.12 The disease occurs primarily in humid rural tropical areas and predominantly affects children between 5 and 15 years of age although disease can occur at any age. Buruli ulcer is most common in some parts of Africa but it has also been reported in Papua New Guinea, French Guiana, Mexico and southeast Australia. Although the epidemiology is not well understood, the disease seems to occur in areas where water is standing or slow moving. Use of polymerase chain reaction (PCR) in West Africa has identified positive signals in the salivary glands of water bugs.13 The organism has now been cultured from these salivary glands.14
RISK FACTORS Recent skin testing data from Palm Beach, Florida, reported that 32.9% of 447 participants in a population-based random household survey had a positive reaction to M. avium sensitin.15 Independent predictors of a positive reaction included black race, birth outside the USA, and more than 6 years’ cumulative exposure to soil. Exposure to water, food and pets was not associated with skin test reactivity. However, the assessment of water exposure was crude and reactivity to M. avium sensitin may be associated with infection but not necessarily disease; thus the risk factors identified in this study may not be relevant to persons with disease. Patients who have pulmonary disease caused by NTM often have structural lung disease such as chronic obstructive pulmonary disease,1,16 bronchiectasis, cystic fibrosis (CF), pneumoconiosis, prior TB, alveolar proteinosis and chronic aspiration.17–20 In the study from France noted previously, over 50% of the patients who met ATS disease criteria (except for those with M. kansasii) had underlying predisposing factors such as pre-existing pulmonary or immune deficiency. There are reports of an association between abnormal CF genotypes21 and a1-antitrypsin phenotypes22,23 and NTM disease. Among 50 patients with bronchiectasis and pulmonary NTM infection, 20% were diagnosed with CF and 50% of the patients had a cystic fibrosis transmembrane conductance regulator (CFTR) mutation identified.21 Among 98 patients with bronchiectasis in the UK, 10 had NTM isolated from sputum specimens and CF was more likely in those with NTM than without.24 The incidence of NTM disease in gold miners in South Africa was reported to be 47.6 per 100,000 and that of M. kansasii, the most commonly isolated NTM, was 37.0/100,000, 34% of whom were human immunodeficiency virus (HIV)-infected.25 Patients who have abnormalities in the interferon-gamma (IFN-g) or interleukin-12 (IL-12) pathways are predisposed to severe NTM infections.26,27 In addition, patients receiving tumour necrosis factor (TNF) antagonist are also at increased risk of developing mycobacterial infections, particularly TB. HIV-infected patients who have advanced disease develop disseminated disease. Of note, many women with NTM infection have nodular bronchiectasis and similar body types: scoliosis, pectus excavatum, mitral valve prolapse and joint hypermobility.28 The reason for this association has not been definitively determined.
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BACTERIOLOGY ORGANISMS Mycobacteria and actinomycetes belong to the order of Actinomycetales. The taxonomy and nomenclature of the genus Mycobacterium (Mycobactericeae family) within this order has been addressed in special reviews.29–31 In order to improve the standardization of the naming of new species, the International Committee on Systematic Bacteriology of the International Union of Microbiological Societies provided recommendations on minimal standards for publications of new species.32 Currently, the definition of a new species is 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. In 1998, there were already 71 validly described species of the Mycobacterium genus, selected from several hundred previously published and proposed names.29 Since 1998, more new species of mycobacteria have been reported.31 There are hundreds of mycobacterial species existing in the environment, but, so far, fewer than 80 of them have been found in diagnostic specimens from patients.33,34 Finding mycobacteria in a patient’s specimen leads to the question of whether the isolate is causing infection/disease in the patient. Isolation from blood or other sterile sites, along with a consistent clinical manifestation of the disease, is reasonable grounds for considering the NTM isolate as the causative agent. The decision on diagnosis is more complex in cases of suspected pulmonary disease. To exclude the transient occurrence of NTM in the patient’s respiratory specimen due to exposure to the bacteria, aerosolized from waters or soil, specific criteria have been recommended for the diagnosis of pulmonary disease caused by NTM (see below).1,35 Contrary to previous theories,19,36 it is now well established that so-called colonization with NTM without tissue invasion is likely a rare occurrence. NTM are typically divided into rapid- and slow-growing mycobacteria based on their ability to produce visible colonies on solid culture medium under optimal conditions in less or more than 7 days. Based on isolation from patients in whom the isolate was identified as a causative agent (sometimes only in a few cases), at least 39 organisms among slow-growing mycobacteria (listed in Table 7.1) are considered potential pathogens. In addition, other slow-growing mycobacteria not considered definitely ‘pathogenic’ for humans can be found in diagnostic specimens, some of them due to contamination from the environment or contact with animals or animal products: M. avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis), Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium hiberniae, Mycobacterium nonchromogenicum, Mycobacterium terrae, Mycobacterium triviale, Mycobacterium lepraemurium and Mycobacterium microti. Some species among the rapid-growing mycobacteria are considered definite opportunistic pathogens for humans29 (upper part of Table 7.2). More recently this list has been expanded by adding more species occasionally isolated from patients’ specimens;31,33 however, their role in human pathology is not clear yet (lower part of Table 7.2).
ISOLATION OF MYCOBACTERIA The bacteriological diagnosis of NTM is based on isolation of these organisms from diagnostic specimens using standard mycobacteriology laboratories techniques. Frequency of isolation and semiquantitative assessment of the bacteriological load in the specimen
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Table 7.1 Slow-growing NTM and reported sites of infection Species
Infection sites Bacteraemia
M. arupense M. aviuma M asiaticum M. bohemicum M. branderi M. chemaera M. selatum M. conspicuum M. doricum M. florentinum M. genavense M. haemophilum M. heckeshornense M. heidelbergense M. intermedium M. interjectum M. intracellulare M. kansasii M. kubicae M. lacus M. lentiflavum M. malmoense M. marinum M. nebraskense M. parmense M. parascrofulaceum M. palustre M. saskatchewanse M. scrofulaceum M. selatum M. sherrissii M. shottsii M. shimodei M. simiae M. szulgai M. tusciae M. triplex M. ulcerans M. xenopi
þ
þ þ þ þ
þ þ
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Pulmonary
Skin
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þ þ þ þ þ þ þ þ
þ þ þ þ
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þ
þ
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CSF
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Bonesa bursitisa synovitis
Other
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þ þ
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Lymph nodes
þ þ
þ þ þ
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þ þ þ
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a
Subspecies: avium, colombiense, silvaticum, hominissuis, paratuberculosis. CSF, cerebrospinal fluid. References: Good and Snider,19 Goodfellow and Magee,29 Tortoli,31,33 Wallace et al.,35 Kent and Kubica.39, Woods and Washington.109
is necessary in cases when differentiation between invasive disease and colonization is an issue, as addressed below in cases of pulmonary NTM disease. A special issue is the bacteriological diagnosis of disseminated NTM disease, which is based on isolation of NTM from blood. The most convenient approach is to submit a blood sample collected in a sodium polyanetholesulphonate (SPS)-containing yellow-top vacutainer to the laboratory. Such a sample can be used for either culture isolation only and/or quantitation of the bacterial load in blood (quantitative culture) as a tool for monitoring the patient’s response to therapy.37,38 It is important that a properly processed specimen is inoculated into both liquid medium and agar plates rather than only on the Lo¨wenstein-Jensen slant, which is the least favourable for cultivation of NTM.
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MYCOBACTERIAL IDENTIFICATION Both rapid- and slow-growing NTM vary by their phenotypic characteristics such as optimal conditions for cultivation, appearance of the colonies on solid medium (pigmented and nonpigmented), enzymatic activity and tolerance to different reagents and antimicrobial agents. These features, described in a number of reviews and textbooks, are often used for so-called conventional methods (or ‘biochemical tests’) for identification of NTM among clinical and environmental isolates.37,39,40 Along with traditional techniques, the modern clinical laboratory includes a broad variety of rapid methods for mycobacterial speciation.41–50 These methods include nucleic acid probes, PCR
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Table 7.2 Rapid-growing NTM and reported sites of infection Species
M. M. M. M. M. M. M.
abscessus chelonae fortuitum a mucogenicum peregrinum porcinum senegalense
M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M.
alvei boenickei bollettii brumae canariasense confluentis cosmeticum elephantis goodii hassiacum holsaticum immunogenum mageritence novocastrense phocaicum septicum smegmatis thermoresistible wolinskyi
Infection sites Bacteraemia
Pulmonary
þ þ þ þ
þ þ þ þ þ
þ
Skin
þ
þ þ
þ þ þ þ
þ þ þ
þ
Wounds
þ
þ
Other
þ þ
þ þ
þ
þ þ
þ
þ þ
þ
þ
Bonesa bursitisa synovitis
þ þ
þ
þ
Soft tissue þ
þ
þ þ
þ
Lymph nodes
þ
þ þ þ þ þ
þ þ
a
Subspecies fortuitum and acetamydoliticum. References: Good and Snider,19 Goodfellow and Magee,29 Tortoli,31,33 Wallace et al.,35 Kent and Kubica,39 Woods and Washington.109
and other amplification methods, high-performance liquid chromatography (HPLC) and nucleic acid sequencing. The commercially available AccuProbe technology (Gen-Probe, San Diego, CA) is usually the preferred technique among the nucleic acid probes hybridization methods. It is currently recommended for identification of M. tuberculosis complex, M. avium complex (as well as M. avium and M. intracellulare separately), M. kansasii and M. gordonae. The method requires a substantial amount of bacterial harvest, and it takes at least 3 weeks for cultivation on agar medium or 10–12 days in a liquid medium to achieve sufficient growth for this test. Amplification of discrete fragments of bacterial DNA or RNA, by PCR or other techniques, made the nucleic acid probes hybridization technique highly sensitive and, thus, provided an opportunity to expedite the laboratory report by testing the patients’ raw specimens directly.51–55 The commercially available amplification techniques (for example, the Mycobacterium tuberculosis Direct (MTD) test from the Gen-Probe Company) are useful tools for rapid identification of M. tuberculosis complex and its differentiation from NTM. The subsequent identification among the NTM has been done in most clinical laboratories on the basis of the conventional methods mentioned earlier. One alternative to this approach is HPLC, which analyses the chromatographic profile of the mycolic acids extracted from the bacterial cell wall (Fig. 7.1). This method became highly sensitive after introduction of the fluorescent detection system incorporated into an automated HPLC
system.45,46,48,56 High sensitivity of the new HPLC system provides reliable results with a relatively small number of bacteria present in the sample. Testing a culture with HPLC at the minimal growth in a liquid medium may allow identification in a shorter period of time than with AccuProbe technology. One attraction of the modern HPLC technology is the opportunity for rapid and reliable identification of M. tuberculosis complex from cultures at their initial growth in liquid media. At least 63 chromatographic patterns, which represent 73 mycobacterial species, were recently reviewed.46,48 HPLC alone (performed with cultures submitted on various media) can rapidly identify M. tuberculosis complex, M. avium complex, M. gordonae, M. marinum, Mycobacterium simiae, M. xenopi, Mycobacterium asiaticum, M. malmoense, Mycobacterium scrofulaceum (from solid medium), Mycobacterium chelonae-abscessus group, Mycobacterium fortuitum-smegmatis group, Nocardia/Rhodococcus species, and genus Tsukamurella. A much broader range of species can be reliably identified by combining information from the HPLC test results with other data, such as growth requirements, growth rate, colonial morphology and pigmentation. Identification of mycobacteria by 16S ribosomal DNA sequencing is a new tool implemented in only a few clinical laboratories to date.43,47,49,50 This technique provides more accurate determination of the species, and it is especially useful in cases where any combination of the above-mentioned methods does not provide reliable identification of the mycobacterial species. The procedure includes disruption of the bacterial cells, followed by PCR using
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Auto-scaled chromatogram
800 700
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E4 3.202
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R-65 1.741
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Fig. 7.1 Mycolic acid peaks are depicted with the use of high-pressure liquid chromatography HPLC. The pattern represents the mycolic acid profile seen with M. avium complex. HPLC is unable to reliably differentiate M. avium from M. intracullulare.
primers 264 and 285 (complementary to the A and B regions) to amplify a 1-kb fragment of 16S ribosomal DNA. The species identification is based on a comparison between the sequences obtained and those in the libraries of already known species. The supplies for the test and the libraries are commercially available, for example the MicroSec 500 16S rDNA system from Applied Biosystems (Foster City, CA). Though this methodology requires further development and standardization, it can now be considered a useful
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supplement to the tools available in large mycobacteriology laboratories dealing with identification of NTM species. The algorithm combining various methods for mycobacterial identification should take into account both the need for rapid identification of M. tuberculosis and the optimal combination and costeffectiveness of all available methods. At National Jewish, the first step, the amplification assay (MTD test), is performed only when requested. Along with or without this procedure, the processed
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raw specimens, or cultures received from other laboratories, are inoculated into four units of medium, including agar plates and 7H12 broth (e.g. Bactec-460 or 960). Employment of the agar plates is essential for detection of mixed mycobacterial cultures, as well as for isolation of some NTM that either do not grow or grow poorly on Lo¨wenstein-Jensen and other egg-based media. The parallel (or next step) is the HPLC procedure. Either the next day (with the culture submitted from another laboratory) or within 8–12 days (at very early indications of growth in the broth culture), the HPLC report on identification of M. tuberculosis complex, M. avium complex and a number of other mycobacteria is issued. Additional testing using the Gen-Probe technique is required if differentiation between M. avium and M. intracellulare is requested, as well as when confirmation is needed for some other species (e.g. M. kansasii or M. gordonae). Additional tests are also required for differentiation between M. tuberculosis and Mycobacterium bovis (niacin production, nitrate reduction, pyrazinamidase, susceptibility to thiopene-2-carboxylic acid hydrazide (TCH)). The final step is the nucleic acid sequencing test (using MicroSec 500 16S rDNA system), sometimes in combination with conventional tests, to evaluate the isolates not identified through the previous steps or to resolve contradictory data from other tests.
DRUG SUSCEPTIBILITY TESTING Determination of drug susceptibility of NTM as a guiding tool for selection of appropriate antimicrobial therapy has less in common with TB (with only few exceptions, such as M. kansasii) than it has with infections caused by other Gram-positive bacteria. The most common methods for drug susceptibility testing of M. tuberculosis clinical isolates are qualitative tests using ‘critical concentrations’ of the drugs in question, which are reported as ‘susceptible’ or ‘resistant’. This approach was based on the fact that initial pre-treatment M. tuberculosis isolates that have never been exposed to TB drugs (often called ‘wild strains’) are very uniform in the degree of susceptibility to these agents, and any detectable change (decrease) in susceptibility would reflect development of resistance. The situation with NTM is different. First of all, there is a range of susceptibility/resistance to drugs considered for therapy against most NTM. Because of this, unlike in the case of TB, selection of drugs is often based on the correlation between the attainable drug serum concentrations and the degree of susceptibility of the patient’s isolate expressed quantitatively as the minimal inhibitory concentration (MIC) of the drug, determined in an in vitro laboratory test. Such a correlation is called ‘dual individualization’ if it is based on the data of both pharmacokinetics and pharmacodynamics determined for an individual patient.57-60,67 The specific technique for MIC determination and choice of drugs included in the test depends on the species of NTM. There is no uniform consensus on the preferred method or selection of drugs included, and there is a variety of opinion on the clinical predictability of these tests. With M. avium complex infection, a multicentre study using standardized methodology made it possible to express the level of susceptibility/resistance of the isolate to any drug in a quantitative manner as a broth-determined MIC.60 Most of the M. avium clinical isolates (but not all) from newly diagnosed patients are susceptible to clarithromycin and azithromycin, and the MIC determination in this case has shown a very clear predictability value of drug susceptibility testing.59,61,62 The situation with other available drugs is different. The isolates have shown a very broad range of susceptibility/resistance patterns, and from 30% to 75% of the isolates, depending on the drug, were resistant even in the
7
most favourable in vitro conditions, with MICs exceeding any possibility of being achieved in blood and tissues. It seems illogical (and to some extent even damaging) to administer such a drug to the patient unless data from clinical trials support its use. On the other hand, only a fraction of isolates (from 20% to 40%) were susceptible to the drug concentrations attainable in vivo, and there is a likelihood that under these conditions a drug may work in the patient. At the same time, a substantial proportion of isolates were in the ‘grey zone’ with MICs at an intermediate level, when the drug susceptibility test results may not have any value in directing the selection of the drugs for therapy. This variability of the M. avium isolates, along with improper MIC determination methods, has led some authors to conclude that there is a lack of correlation between the test results and the outcome of therapy.1 Drug susceptibility testing is probably a more important element in the management of patients who have already received treatment. It is especially important in cases when there is a need to diminish the toxicity of the regimen by removing from it those drugs to which the patient’s isolate is highly resistant. Though of limited value, as compared with TB, drug susceptibility testing has been found to be useful in a number of studies.63–65 In one study, patients whose isolates were susceptible to rifampicin and ethambutol had better outcomes, although the authors did not fully recognize the value of drug susceptibility testing in their conclusions.66 Pulmonary disease caused by M. kansasii has many features in common with TB, including a drug susceptibility pattern of wild strains isolated from patients who have not been treated with antimicrobial agents. Accordingly, there are two options for drug susceptibility of M. kansasii isolates: one is similar to that for M. tuberculosis (qualitative test with critical concentrations, for example, using proportion method); the other is determination of MICs in liquid medium. Unlike M. tuberculosis, most of the M. kansasii isolates are resistant to para-aminosalicylic acid (PAS), capreomycin, low concentrations of isoniazid (0.1–0.2 mg) and, as with any other NTM, pyrazinamide. Some strains are also resistant to low concentrations of aminoglycosides.67 Some authors suggest that drug susceptibility of the M. kansasii isolates can be limited to a test with rifampicin as the most essential element in the treatment of this infection.68 An alternative and perhaps better approach would be to perform drug susceptibility testing for all drugs with potential use against this infection from the very beginning. Testing against rifampicin only or other selected drugs is not likely to be cost-effective compared with a battery of drugs when using the agar proportion method. Unlike the case with other NTM, there are no arguments against the usefulness of such testing for the rapid-growing mycobacteria, though the predictability value of laboratory data for infections caused by organisms is not any clearer than in cases of infections by slow-growing mycobacteria, due to the lack of data from controlled clinical trials. The susceptibility test is an MIC determination procedure with a series of concentrations of a number of drugs incorporated into a microtitre plate. Because the test is performed simultaneously with the species identification procedures, results are available within 4–5 days. For these two reasons, the test is performed with all drugs incorporated into the plate, regardless of the possible usefulness of some of the results for treatment regimen selection. Therefore, recommendations on selective drug testing depending on the species identification may not be practical.69 There are various microtitre plates commercially available and a specific set of drugs to be tested can be ordered to accommodate testing either mycobacteria only or also Nocardia and other aerobic Actinomycetes species. For isolates identified as rapid-growing mycobacteria,
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inclusion of the following 14 drugs is recommended: amikacin, kanamycin, tobramycin, imipenem, doxycycline, clarithromycin, azithromycin, trimethoprim/sulfamethoxazole, augmentin, cefoxitin, ciprofloxacin, gatifloxacin, moxifloxacin, linezolid.
TRANSMISSION AND PATHOGENESIS The source of NTM infection appears to be through environmental exposure and transmission from human to human has not been described. Genotyping methods have demonstrated similar patterns between isolates cultured from patients and their environments.70–72 In most cases, the source of infection has been water, although soil has also been implicated.71 Once the organism enters the body, there are three possible fates for the organism: 1. the host can clear the organism; 2. the organism can ‘colonize’ the airways; or 3. the organism can cause infection and disease. Which of these three pathways is followed is determined by an interplay between the host and organism. The importance of host immunity has been demonstrated in individuals and families with mutations in the IFN-g/IL-12 pathway genes.26,27,73 These defects have led to disseminated mycobacterial infections. In a study from Japan, 170 patients with M. avium complex (MAC) lung infection were studied and, out of 622 siblings of these patients, three had MAC lung disease also.74 The authors concluded that the rate of infection among the siblings was higher than previously estimated among the general population. In patients with HIV infection, disseminated NTM infections typically occur only after the CD4 lymphocyte count falls below 50 cells/mL, suggesting that specific T-cell products or activities are required for mycobacterial resistance.1 MAC presumably enters the body through ingestion of contaminated food or liquid. There is a striking association between bronchiectasis, nodular pulmonary NTM infections and a particular body habitus, predominantly in postmenopausal women.28 In the latter instance, it remains to be seen whether these women have some sort of subtle immune deficiency that predisposes them to NTM pulmonary infections or whether their predispostion is related to ineffective mucociliary clearance or poor tracheobronchial secretion drainage. Unlike with M. tuberculosis, the NTM do not appear to live in a state of dormancy. Moreover, simply isolating NTM from a respiratory specimen does not mean that the patient has NTM-related disease. For years the term ‘colonization’ has been used to differentiate those who have a single or small number of positive cultures over time from those in whom progressive disease cannot be demonstrated. For example, Ahn and colleagues75 described 497 patients with pulmonary MAC and 226 patients with M. kansasii. Twenty-four per cent of the patients converted cultures to negative within 4 months with bronchial hygiene alone, while 74% converted cultures to negative on chemotherapy, 27 within 1 week. The authors concluded that many of these patients were unlikely to have disease related to these infections. However, with longer follow-up many patients demonstrate clinical and/or radiographic progression of disease, so it may be more appropriate to think of these patients as having indolent infection. Two additional observations suggest that colonization is uncommon. First, Prince and colleagues76 described 21 patients with pulmonary MAC who had no underlying lung disease, most of whom had non-cavitary disease that was slowly progressive. Subsequently, Erasmus and colleagues77 reported the findings on computed
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tomography (CT), demonstrating multiple small nodules and associated cylindrical bronchiectasis in these patients. Taken together, these studies suggest that some patients thought to have been colonized did in fact have evidence of disease on CT scans and demonstrated slow clinical and radiographic progression. Puncture of the skin is the usual mechanism of entry for cutaneous infections due to NTM. For M. marinum, the source of infection is usually contaminated water in a swimming pool or home aquarium or through puncture of the skin from a fish. The mechanism of entry for M. ulcerans is not so clear. As noted previously, the organism has been isolated from the salivary glands of water insects but a history of insect bite at the site of infection is seldom revealing.12 Whether other environmental exposures are important is not known but environmental studies are underway to attempt to better describe the transmission of this important pathogen. Once the organism enters the skin, it produces a polyketide called mycolactone that caused tissue necrosis, a hallmark of Buruli ulcer.78
CLINICAL PRESENTATION AND DIAGNOSIS CLINICAL PRESENTATION The most common clinical manifestation of NTM infection is chronic lung disease.1 However, lymphatic, skin/soft tissue, bone/joint involvement as well as disseminated disease are also important manifestations of infection. The propensity of a specific manifestation varies with the specific species and certain host factors. For example, HIVinfected patients typically present with disseminated disease, whereas persons with cystic fibrosis and elderly white females present with pulmonary disease. The rapid growers, while a common cause of lung disease, have a propensity for producing skin and soft-tissue infections.
Pulmonary disease Chronic pulmonary disease is the most common clinical presentation of NTM disease and usually presents with chronic cough, fatigue, malaise, dyspnoea, fever, haemoptysis, chest pain and weight loss. Physical examination may identify in postmenopausal women, and occasionally men, certain morphological characteristics that include thin body habitus, scoliosis, pectus excavatum and mitral valve prolapse. The radiographic features in patients with MAC pulmonary disease are typically divided into primarily fibrocavitary disease and nodular bronchiectatic disease. In patients with fibrocavitary disease, the radiographic abnormalities consist of upper lobe opacities with evidence of cavitation and volume loss. Classically, this pattern of disease was recognized in older men with underlying lung disease although women can also present with upper lobe cavitary disease (Fig. 7.2). Patients with predominantly non-cavitary nodular bronchiectatic disease have opacities in the mid- and lower lung fields (Fig. 7.3A). High-resolution computed tomography (HRCT) scans demonstrate bronchiectasis often in the middle lobe and lingula with evidence of small nodules, centrilobular in location and often described as ‘tree-in-bud’ (Fig 7.3B). Patients with M. kansasii infection usually present with upper lobe cavitary opacities and the cavities are typically thin walled (Fig. 7.4). The chest radiograph in patients with rapid-growing mycobacterial infections usually shows multilobar, patchy, reticulonodular or mixed interstitial alveolar opacities with an upper lobe predominance (Fig. 7.5A). Cavitation is reported to occur in 15%. HRCT will show bronchiectasis and small nodules similar to that of MAC (Fig. 7.5B).
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Fig. 7.2 A 65-year-old man with M. avium complex pulmonary infection. (A) Chest radiograph demonstrates bilateral upper lobe opacities with areas of cavitation. In addition there are nodular opacities noted in the middle and lower lobes. (B) Chest computed tomography of the same patient demonstrating bilateral large upper lobe cavities with bronchiectasis.
Fig. 7.3 A 78-year-old woman with M. avium complex pulmonary infection. (A) Chest radiograph demonstrates kyphoscoliosis with nodular opacities primarily in the middle lobe, lingula and lower lobes. (B) Chest computed tomography of the same patient with evidence of pectus excavatum, right middle lobe and lingular bronchiectasis and multilobar centrilobular nodules. The arrows are pointing at tree-in-bud opacities.
Mycobacterium xenopi is second to MAC as a cause of pulmonary disease in Canada and parts of Europe. Typically, patients with disease due to M. xenopi present with underlying chronic obstructive pulmonary disease and the radiographic appearance is similar to that seen in upper lobe cavitary MAC disease.
Lymphadenitis The most common form of NTM disease in children is cervical lymphadenitis.1 Mycobacterium avium is the most common aetiology accounting for 80% of culture-proven cases. Mycobacterium scrofulaceum is the second most common cause in the USA and Australia,
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Fig. 7.4 A 45-year-old woman with M. kansasii pulmonary infection. (A) The chest radiograph demonstrates upper lobe thin-walled cavities, typical of M. kansasii infection. (B) Chest computed tomography of the same patient shows one of the thin-walled cavities with adjacent bronchiectasis.
Fig. 7.5 A 65-year-old woman with M. abscessus pulmonary infection. (A) Chest radiograph demonstrating bilateral lower lobe and right middle lobe nodular opacities and bronchiectasis. (B) Chest computed tomography of the same patient shows atelectasis and bronchiectasis of the right middle lobe with nodular opacities in the right and left lower lobes.
whereas M. malmoense and Mycobacterium haemophilum are more common in Scandinavia, the United Kingdom and other areas of northern Europe. Mycobacterium tuberculosis is isolated in only approximately 10% of culture-proven mycobacterial cervical lymphadenitis in the USA but in adults 90% are due to TB.1 Infection usually involves the submandibular, submaxillary, cervical and preauricular lymph nodes in children between 1 and 5 years of age. The disease occurs insidiously and is rarely associated with systemic symptoms. Involvement of the lymph nodes is
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usually unilateral and non-tender. The lymph nodes may enlarge and eventually rupture producing sinus tracts just like tuberculous lymphadenitis.
Soft-tissue, skin and bone infection Although virtually any NTM can cause skin, soft-tissue, and bony infection, the most common species to do so are the rapid growers, M. marinum and M. ulcerans.79,80 Rapid growers often produce infections at the site of punctures or surgery and present with tender
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subcutaneous nodules. Mycobacterium marinum causes ‘swimming pool granuloma’ and ‘fish tank granuloma’. The lesions usually appear as papules on an extremity and eventually progress to shallow ulceration and scar formation. In cases of swimming pool granuloma the site of infection is typically the elbow or knee,10 whereas in fish tank granuloma the site of infection is usually the hands. The median incubation time from skin puncture to clinically evident lesion is 21 days but ranges from 5 to 270 days.81 Although most lesions are solitary they may spread and produce arthritis and tenosynovitis, particularly of the hand and wrist. Mycobacterium ulcerans causes indolent progressive ulceration of the skin and underlying tissues, producing scalloped edges called Buruli ulcers.82 The infection often starts as a painless, mobile nodule and in 90% of cases the lesions are on the limbs, with nearly 60% of all lesions on the lower extremities.
Disseminated infections Disseminated infections are most commonly associated with HIV infection and other forms of severe immunosuppression. Over 90% of reported disseminated infection in HIV-infected patients are due to MAC and almost all of these are due to M. avium.1 The next most common cause of disseminated NTM disease in HIV patients is M. kansasii but a number of other species have also been implicated. Interestingly, M. intracellulare and the rapid growers are uncommon causes of disseminated disease in HIVinfected patients. Most patients present with advanced HIV disease and complain of fever, night sweats and weight loss. Abdominal pain and diarrhoea may also be reported. In HIV-uninfected patients, fever of unknown origin is a common presentation. INVESTIGATIONS Pulmonary disease Patients suspected of having a pulmonary NTM infection should be evaluated with a chest radiograph and/or HRCT, particularly when there is no evidence of cavitation on the radiograph. If there is evidence of radiographic abnormalities consistent with an NTM infection, at least three sputum specimens should be obtained for acid-fast bacilli (AFB) examination and mycobacterial culture. Tuberculosis as well as other disorders should be excluded. In order to diagnose pulmonary NTM infection the clinician must weigh clinical, bacteriological and radiographic information (Table 7.3).1 NTM pulmonary infections should be suspected when a patient presents with a compatible clinical picture and has nodular or cavitary opacities on the chest radiograph or an HRCT scan that shows multifocal bronchiectasis with multiple small nodules. In addition to these clinical criteria, the patient should have at least two positive cultures from separate sputum specimens or a positive culture from at least one bronchial wash or lavage. A previous study by Tsukumura83 demonstrated that 98% of patients with two or more positive sputum cultures progressed without treatment. Additional diagnostic criteria include transbronchial or other lung biopsy with mycobacterial histopathological features and positive culture for NTM or biopsy showing mycobacterial histopathological features and one or more sputum or bronchial washings that are culture positive. In patients who do not meet the above definition for disease, close follow-up should occur since many of the patients will demonstrate progression over time. Lymphadenitis Diagnosis is usually made by fine needle aspiration or surgical excision of the involved lymph nodes. Only 50–82% of excised nodes will be culture positive.1
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Table 7.3 Clinical and microbiological criteria for diagnosing non-tuberculous mycobacterial lung disease Clinical (both required) 1. Pulmonary symptoms, nodular or cavitary opacities on chest radiograph, or a HRCT scan that shows multifocal bronchiectasis with multiple small nodules and 2. Appropriate exclusion for other diagnoses Microbiological 1. Positive culture results from at least two separate expectorated sputum samples. If results are non-diagnostic, consider repeat sputum AFB smears and cultures or 2. Positive culture result from at least one bronchial wash or lavage or 3. Transbronchial or other lung biopsy with mycobacterial histopathological features (granulomatous inflammation or AFB) and positive culture for NTM or biopsy showing mycobacterial histopathological features (granulomatous inflammation or AFB) and one or more sputum or bronchial washings culture positive for NTM AFB, acid-fast bacilli. Adapter from Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4):367–416. Official Journal of the American Thoracic Society. (c) American Thoracic Society.
Soft tissue Tissue biopsies that are cultured and examined histopathologically are the most sensitive way to diagnose infections due to rapid growers and M. marinum. Unfortunately, visualization of mycobacteria in the specimen is seen in only about 10% of cases and the yield of culture is not known since most cases that have been reported were culture confirmed.11,84 Buruli ulcer is usually diagnosed based on clinical findings although laboratory tests can be used to confirm the diagnosis. Direct smear examination performed on swabs from ulcers or smears from tissue biopsies is of low sensitivity (40%) and the sensitivity of cultures ranges from 20% to 60% with an incubation time of 6–8 weeks.82 Identification of M. ulcerans can be performed directly from clinical specimens or from culture media and the sensitivity of a PCR test is around 98%.12 Disseminated disease Diagnosis is usually through detection of the causative organism in the blood. In HIV-infected patients M. avium has been isolated in over 90% of cases.1
TREATMENT GENERAL PRINCIPLES OF TREATMENT There are several notable differences regarding the treatment of TB and that of infections caused by NTM. First, when a patient grows M. tuberculosis, treatment is always indicated assuming that the isolate was not due to laboratory cross-contamination. However, with NTM, isolation should not always lead to treatment. The decision to treat is based on the potential risks and benefits for the individual patient. Second, unlike with M. tuberculosis, in vitro susceptibility results for many NTM do not correlate well with clinical response to antimicrobial therapy; thus clinicians should use such data with a
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Table 7.4 Doses of drugs used to treat non-tuberculous mycobacterial infections in adults
Box 7.1 Controversies and unresolved questions in the management of Mycobacterium avium complex lung disease
Drug
Daily dose (max)
Three times/ week dose (max)
Isoniazid
5 mg/kg/day (300 mg)
Rifampicin
10 mg/kg/day (600 mg)
Rifabutin
5 mg/kg/day (300 mg)
Ethambutol
15–20 mg/kg/day (1.6 g)
Azithromycin
250 mg/day
Clarithromycin Doxycycline Trimethoprimsulfmethoxazole Ciprofloxacin Moxifloxacin Tobramycin Streptomycin
500–1000 mg/day 100 mg/day (100 mg) 160 mg trimethoprim/800 mg sulfmethoxazole twice daily 500–750 mg twice daily 400 mg/day (400 mg) 4–7 mg/kg/day 10–15 mg/kg/day (1 g)
Amikacin/ kanamycin Cefoxitin
10–15 mg/kg/day (1 g)
Imipenem
100–200 mg/kg (12 g/day) in divided doses 500–1000 mg 2 or 3 times/day
15 mg/kg/day (900 g) 10 mg/kg/day (600 g) 5 mg/kg/day (300 mg) 20–35 mg/kg/ day (2.5 g) 500–600 mg/ day 1000 mg/day — — — — — 15–20 mg/kg (1.5 g) 15–20 mg/kg (1.5 g) — —
clear understanding of the limitations. Third, while most patients with TB can be treated with a single four-drug regimen, the many species of NTM dictate different drugs and regimens (Table 7.4). Fourth, NTM infections usually require a duration of therapy longer than that with TB, partly because of the lack of potent bactericidal drugs and high levels of in vitro resistance. And, finally, surgical resection/excision is used more frequently in the management of NTM infection than with TB. Nevertheless, NTM like TB should be treated with more than one antimycobacterial drug in order to prevent the emergence of resistance. The role of in vitro susceptibility testing for managing patients with NTM disease remains controversial. In vitro susceptibility results and clinical outcome do not always correlate and this is particularly true of infections due to MAC, M. xenopi and M. simiae.1 On the other hand, there appears to be better correlation with M. marinum, M. kansasii and the rapid growers. Clarithromycin testing is currently recommended for new, previously untreated MAC isolates, those who fail macrolide-based treatment regimens or prophylaxis regimens.1 Previously untreated M. kansasii isolates should be tested in vitro against rifampicin. Isolates resistant to rifampicin should also be tested against rifabutin, ethambutol, isoniazid, clarithromycin, fluoroquinolones, amikacin and sulphonamides.
TREATMENT OF PULMONARY INFECTIONS Mycobacterium avium complex (Box 7.1) Untreated M. avium pulmonary disease is quite variable in its progression. In some patients progression can be seen over a matter of months and in others it may take years to demonstrate progressive disease. Prior to the availability of the newer macrolides, the long-term
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Usefulness of in vitro drug susceptibility results in management of patients. Which is the most effective macrolide? Which is the most effective rifamycin? Does addition of an aminoglycoside improve patient outcomes? Would two-, three- or four-drug regimen be most effective? What is the appropriate duration of therapy? What is the role of fluoroquinolones and other drugs in the management of patients?
treatment outcomes for patients treated with anti-TB regimens was approximately 50%.1 Small, non-comparative studies of azithromycin- and clarithromycin-containing regimens have been associated with higher bacteriological response rates but long-term follow-up is often lacking. Treatment responses are also variable with cures rates ranging from 60% to 85%.85–91 Genotyping data suggest that reinfection is common in women with nodular bronchiectasis so a patient in whom cure is achieved may become infected again.92 The ATS currently recommends that the treatment regimen be based on the presence or absence of cavitary disease and whether the patient has been treated previously (Table 7.5).1 For patients with non-cavitary nodular bronchiectasis disease a three-times-a-week regimen should be considered. In a recently published study examining the efficacy of inhaled IFN-g therapy for severe or previously treated MAC disease, the bacteriological response to three-times-aweek therapy was poor overall.90 Factors associated with a good bacteriological response were having non-cavitary disease, being AFB smear negative, no previous treatment for MAC lung disease, older age and longer duration of ethambutol use. For patients with noncavitary disease, 71% demonstrated improvement in culture results compared with 20% in those with cavitary disease. Because this study was stopped owing to lack of efficacy of the interferon, no long-term follow-up was available. Three-times-a-week dosing may be associated with less optic neuritis related to ethambutol: 8 of 139 patients with daily ethambutol therapy developed optic neuritis compared with 0 of 90 on intermittent therapy.93 For patients with cavitary and/or advanced disease and in those who have been treated previously, daily therapy is recommended.1 An aminoglycoside should be considered in these patients, at least for the first 2–3 months of therapy, although a recent study suggested that there was no long-term benefit to this practice. In a small randomized study, patients with pulmonary MAC were randomized to receive a standard three-drug treatment regimen with or without streptomycin.91 Although patients who received the aminoglycoside were shown to have a higher rate of sputum conversion at the end of treatment than those who did not receive the injectable agent, the sputum relapse rate and clinical improvement were no different between groups. Surgery should be considered in patients who have an underlying macrolide-resistant strain of MAC, have failed treatment or have primarily focal cavitary disease. Surgery should be performed by a surgical team experienced in managing these difficult cases.94–98
Mycobacterium kansasii Untreated M. kansasii is usually progressive much like TB. However, with appropriate treatment almost all patients respond well and both treatment failure and relapse are uncommon. Among 180 patients in three studies, the 4-month culture conversion rates were 100% and there was only one long-term relapse.99–101
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Table 7.5 Treatment of pulmonary disease due to Mycobacterium avium complex infections Type of disease
Regimen
Comment
Nodular/ bronchiectatic disease
Clarithromycin 1000 mg tiw or azithromycin 500–600 mg tiw Ethambutol 25 mg/kg tiw
When choosing a macrolide, the clinician should weigh such issues as drug tolerance and drug interactions. Intermittent ethambutol may be associated with a lower incidence of optic neuritis than with daily therapy. Rifampicin is recommended over rifabutin in these patients because it is better tolerated than rifabutin. When choosing a macrolide, the clinician should weigh such issues as drug tolerance and drug interactions.
Rifampicin 600 mg tiw Cavitary disease
Advance or previously treated
Clarithromycin 500–1000 mg/day or azithromycin 250–300 mg/day Ethambutol 15 mg/kg daily Rifampicin 450–600 mg daily Streptomycin or amikacin 15 mg/kg tiw Clarithromycin 500–1000 mg/day or azithromycin 250–300 mg/day Ethambutol 15 mg/kg daily Rifabutin 150–300 mg daily or rifampicin 450–600 mg daily Streptomycin or amikacin 15 mg/kg tiw
Rifampicin is recommended over rifabutin in these patients because it is better tolerated than rifabutin. The clinician should consider using an aminoglycoside in these patients. When choosing a macrolide, the clinician should weigh such issues as drug tolerance and drug interactions. Given this more difficult to treat scenario some experts feel that rifabutin may be worth using in place of rifampicin. The clinician should strongly consider using an aminoglycoside in these patients.
tiw, three times a week. Adapted from Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4):367–416.
Patients with lung disease due to M. kansasii should be treated with isoniazid, rifampicin and ethambutol.1 However, based on in vitro activity, the newer macrolides could probably be substituted for isoniazid. In one study no relapses were seen after 46 months of follow-up in patients who received clarithromycin, rifampicin and ethambutol.102 As with other pulmonary NTM infections, the treatment duration should include 12 months of negative sputum cultures. For patients whose isolate is resistant to the rifamycins, a three-drug regimen is recommended based on in vitro susceptibility data to clarithromycin or azithromycin, moxifloxacin, ethambutol, sulfamethoxazole or streptomycin.1 Surgical resection is seldom necessary in patients infected with M. kansasii.
Mycobacterium xenopi The optimal treatment regimen and duration of therapy is not known. In vitro susceptibility results may be difficult to interpret because the response of the organism does not always correlate. Most patients are treated with a typical anti-MAC regimen that includes a newer macrolide, ethambutol and rifampicin.1 Responses may be enhanced by the addition of a fluoroquinolone. The mortality rate with M. xenopi infection has been reported to be as high as 57% but this probably reflects the severity of the underlying lung disease.66 Rapid-growing mycobacteria The clinical course of untreated M. abscessus is generally slow and progressive but more rapidly progressive presentations have been described, particularly in patients with CF or oesophageal disorders associated with aspiration. Treatment outcomes with M. abscessus are generally poor in part because the organism is resistant to almost all of the standard anti-TB drugs. In vitro susceptibility testing is recommended for selection of a treatment regimen.1 Unfortunately, M. abscessus is susceptible to only a few antimicrobials including the macrolides, imipenem, cefoxitin, amikacin, tigecycline, and occasionally linezolid. Moreover, no antibiotic regimen has demonstrated long-term sputum conversion in patients with pulmonary disease. Current recommendations are to provide periodic drug administration of multidrug therapy including a macrolide and one
or more parenteral agents such as amikacin, cefoxitin or imipenem for 4–6 months to help control symptoms and prevent progression.1 For patients who are good candidates, surgical resection should be considered but only if done by an experienced surgeon and after a period of intensive antimicrobial therapy. Mycobacterium chelonae is typically susceptible to tobramycin, macrolides, linezolid, imipenem and amikacin and may demonstrate susceptibility to fluoroquinolones and doxycycline.1 Isolates are usually resistant to cefoxitin. Treatment should consist of at least two drugs to which there has been demonstrated in vitro drug susceptibility. The duration should be for at least 12 months of culture negativity. Mycobacterium fortuitum isolates are typically susceptible to newer macrolides, fluoroquinolones, doxycycline, minocycline, sulphonamides, cefoxitin and imipenem.1 Therapy should be with at least two agents with in vitro activity for at least 12 months of culture negativity.
TREATMENT OF LYMPHADENITIS Cervical lymphadenitis is usually caused by MAC or M. scrofulaceum infection.1 Surgical excision of the involved lymph nodes without chemotherapy is usually curative with success rates of approximately 95%. In patients who fail surgical excision a macrolide-based treatment regimen with or without repeat excision is usually successful.
TREATMENT OF SKIN, SOFT-TISSUE AND BONE/JOINT INFECTIONS For most patients with MAC infection of the skin and soft tissues, a combination of surgical excision plus multidrug chemotherapy is successful. The optimal duration of therapy is not known but should probably be 6–12 months.1 For serious infections caused by rapidgrowing mycobacteria, a newer macrolide should be combined with a parenteral medication (amikacin, cefoxitin or imipenem) or perhaps another oral agent with M. fortuitum.1 Therapy should continue for a minimum of 4 months for skin infections and at least 6 months for bone infections. Surgery is generally indicated for extensive
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disease and removal of foreign objects such as breast implants, percutaneous catheters and joint prostheses is necessary. Mycobacterium marinum isolates are usually susceptible to rifampicin, rifabutin and ethambutol with intermediate susceptibility to streptomycin and resistant to isoniazid and pyrazinamide.1 Isolates are susceptible to clarithromycin, azithromycin, sulphonamides and susceptible or intermediately susceptible to doxycycline and minocycline. Although some experts believe that M. marinum infections can be treated with a single drug, two active drugs seems more reasonable. The current recommendation is to give two active drugs for 1–2 months beyond resolution of the lesion or typically 3–4 months.1 In a study from France, 42 (93%) of 63 patients with localized disease who were treated for 3.5 months with two drugs (usually rifampicin and clarithromycin) resolved.103 For those with more invasive disease 13 (72%) resolved. In general, outcomes of treatment with clarithromycin and ethambutol or clarithromycin and rifampicin have been excellent. Surgical debridement should be reserved for extensive disease or infection involving closed spaces of the hand. The World Health Oraganization recommends a combination of rifampicin and streptomycin/amikacin for 8 weeks as a first-line treatment for all forms of active disease due to M. ulcerans.104 Among 224 patients diagnosed with Buruli ulcer in Benin who were treated with this regimen, 96% were considered treatment successes: 102 (47%) were treated with antibiotics only and 113 (53%) with a combination of antibiotics and surgical debridement.105 Chemotherapy alone was particularly effective in those with ulcers of less than 5 cm in diameter.
TREATMENT OF DISSEMINATED INFECTIONS Treatment of disseminated MAC infection in HIV-infected patients should include clarithromycin 500 mg twice daily, ethambutol 15 mg/kg daily with or without rifabutin 300 mg daily.1 Azithromycin 500 mg daily could be used as an alternative to clarithromycin. Treatment should be considered lifelong unless immune restoration is achieved by antiretroviral therapy. MAC
REFERENCES 1. Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4): 367–416. 2. Marras TK, Daley CL. Epidemiology of human pulmonary infection with nontuberculous mycobacteria. Clin Chest Med 2002;23:553–567. 3. Palmer CE. Tuberculin sensitivity and contact with tuberculosis; further evidence of nonspecific sensitivity. Am Rev Tuberc 1953;68(5):678–694. 4. Edwards LB, Acquaviva FA, Livesay VT, et al. An atlas of sensitivity to tuberculin, PPD-B, and histoplasmin in the United States. Am Rev Respir Dis 1969;99:1–131. 5. O’Brien RJ, Geiter LJ, Snider DE. The epidemiology of nontuberculous mycobacterial diseases in the United States: results from a national survey. Am Rev Respir Dis 1987;135:1007–1014. 6. Centers for Disease Control and Prevention (CDC). Nontuberculous mycobacteria reported to the public health laboratory information system by state public health laboratories United States 1993–1996. Atlanta: US Department of Health and Human Services, CDC, 1999. Available at URL:http://www.cdc.gov/tb/ Laboratory_Services/NontuberculousMycobacteria.pdf 7. Dailloux M, Abalain ML, Laurain C, et al. Respiratory infections associated with nontuberculous mycobacteria in non-HIV patients. Eur Respir J 2006;28(6):1211–1215.
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treatment can be stopped for patients who are asymptomatic and have achieved a CD4 lymphocyte count of over 100 cells/mL for at least 12 months.106 Prophylaxis should be reintroduced if the count falls below 100 cells/mL. The treatment for disseminated disease due to M. kansasii is the same as that for pulmonary disease. Unlike with MAC, there is no known prophylactic regimen.
PREVENTION OF NTM INFECTIONS Preventive therapy for disseminated MAC is recommended for HIVinfected patients with fewer than 50 CD4 lymphocytes/mL.106 Azithromycin given as 1200 mg once weekly is the preferred agent.107 Alternative regimens include clarithromycin 500 mg twice daily or rifabutin 300 mg daily. Primary prophylaxis should be discontinued when patients have responded to antiretroviral therapy with an increase in CD4 cell count to more than 100 cells/mL for more than 3 months.106,108 Prophylaxis should be reintroduced if the cell count falls to less than 50–100 cells/mL. NTM infections in healthcare settings have been associated with contamination of water sources, biologicals and multidose vials used in post-surgical infections.1 In these settings, specific steps can be taken to prevent the use of tap water for washing wounds or equipment and use of multidose vials should be avoided for injections. Prevention of community-acquired pulmonary infections remains elusive. The organisms have been isolated in tap water and water distribution systems and some NTM, such as M. xenopi, M. simiae and M. avium, survive well at water temperatures over 40 C.1 Moreover, these organisms are resistant to our typical decontamination methods. Therefore it is not clear how to best decrease our environmental exposure. One practical approach for avoiding potentially contaminated aerosols would be to avoid indoor hot tubs or even showers. However, because these potential sources have not been implicated in most NTM infections, it is difficult to predict whether this approach would decrease the risk of NTM infections.
8. Marras TK, Chedore P, Ying AM, et al. Isolation prevalence of pulmonary non-tuberculous mycobacteria in Ontario 1997–2003. Thorax 2007;62 (8):661–666. 9. Linell F, Norden A. Mycobacterium balnei: a new acid fast bacillus occurring in swimming pools and capable of producing skin lesions in humans. Acta Tuberc Scand 1954;33 (Suppl):1–84. 10. Casal M, Casal MM. Multicenter study of incidence of Mycobacterium marinum in humans in Spain. Int J Tuberc Lung Dis 2001;5(2):197–199. 11. Edelstein H. Mycobacterium marinum skin infections. Report of 31 cases and review of the literature. Arch Intern Med 1994;154(12):1359–1364. 12. Wansbrough-Jones M, Phillips R. Buruli ulcer: emerging from obscurity. Lancet 2006;367(9525): 1849–1858. 13. Portaels F, Elsen P, Guimaraes-Peres A, et al. Insects in the transmission of Mycobacterium ulcerans infection. Lancet 1999;353(9157):986. 14. Marsollier L, Brodin P, Jackson M, et al. Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches and Buruli ulcer pathogenesis. PLoS Pathog 2007;3(5):e62. 15. Reed C, von Reyn CF, Chamblee S, et al. Environmental risk factors for infection with Mycobacterium avium complex. Am J Epidemiol 2006;164(1):32–40. 16. Hadjiliadis D, Adlakha A, Prakash UB. Rapidly growing mycobacterial lung infection in association with esophageal disorders. Mayo Clinic Proc 1999; 74(1):45–51.
17. Oliver A, Maiz L, Canton R, et al. Nontuberculous mycobacteria in patients with cystic fibrosis. Clin Infect Dis 2001;32:1298–1303. 18. Olivier KN, Weber DJ, Wallace RJ Jr, et al. Nontuberculous mycobacteria in cystic fibrosis study group. Nontuberculous mycobacteria. I. Multicenter prevalence study in cystic fibrosis. Am J Respir Crit Care Med 2003;167:828–834. 19. Good RC, Snider DE Jr. Isolation of nontuberculous mycobacteria in the United States, 1980. J Infect Dis 1982;146:829–833. 20. Witty LA, Tapson VF, Piantadosi CA. Isolation of mycobacteria in patients with pulmonary alveolar proteinosis. Medicine 1994;73(2):103–109. 21. Ziedalski TM, Kao PN, Henig NR, et al. Prospective analysis of cystic fibrosis transmembrane regulator mutations in adults with bronchiectasis or pulmonary nontuberculous mycobacterial infection. Chest 2006;130(4):995–1002. 22. Kaminska AM, Huitt GA, Fulton KE, et al. Prevalence of alpha-1-antitrypsin (AAT) mutations in 100 bronchiectatic patients with rapid-growing mycobacterial (RGM) infections. Am J Respir Crit Care Med 2003;167:A708. 23. Kim JS, Tanaka N, Newell JD, et al. Nontuberculous mycobacterial infection: CT scan findings, genotype, and treatment responsiveness. Chest 2005;128(6): 3863–3869. 24. Fowler SJ, French J, Screaton NJ, et al. Nontuberculous mycobacteria in bronchiectasis: Prevalence and patient characteristics. Eur Respir J 2006;28(6):1204–1210.
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Other mycobacteria causing human disease 25. Corbett EL, Blumberg L, Churchyard GJ, et al. Nontuberculous mycobacteria: defining dissease in a prospective cohort of South African miners. Am J Respir Crit Care Med 1999;160:15–21. 26. Casanova JL, Abel L. Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol 2002;20:581–620. 27. Dorman SE, Holland SM. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev 2000;11:321–333. 28. Iseman MD, Buschman DL, Ackerson LM. Pectus excavatum and scoliosis. Thoracic anomalies associated with pulmonary disease caused by Mycobacterium avium complex. Am Rev Respir Dis 1991;144:914–916. 29. Goodfellow M, Magee JG. Taxonomy of mycobacteria. In: Gangadharam PRJ, Jenkins PA (eds). Mycobacteria I—Basic Aspects. New York: Chapman and Hall, 1998: 1–71. 30. Goodfellow M, Wayne LG. Taxonomy and nomenclature. In: Ratledge C, Stanford J (eds). The Biology of the Mycobacteria. London: Academic Press, 1982: 471–521. 31. Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990’s. Clin Microbiol Rev 2003;16:319–354. 32. Sneath PHA. International Code of Nomenclature of Bacteria. Washington, DC: American Society for Microbiology, 1992. 33. Tortoli E. The new mycobacteria: an update. FEMS Immunol Med Microbiol 2006;48:159–178. 34. Heifets L. Mycobacterial infections caused by nontuberculous mycobacteria. Semin Respir Crit Care Med 2004;25:283–295. 35. Wallace RJ Jr, Cook JL, Glassroth J, et al. American Thoracic Society Statement: Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med 1997;156:S1–S25. 36. Wolinsky E. Nontuberculous mycobacteria and associated diseases. Am Rev Respir Dis 1979; 110:107–159. 37. Heifets LB. Quantitative cultures and drug susceiptibility testing of M. avium clinical isolates before and during the antimicrobial therapy (review). Res Microbiol 1994;145:188–196. 38. Sanchez T, Vanderkolk J, Seay S, et al. Quantitation of mycobacteria in blood specimens from patients with AIDS. Tuberc Lung Dis 1994;75:386–390. 39. Kent PT, Kubica GP. A Guide for the Level III Laboratory. In: Public Health Mycobacteriology. Atlanta: US Department of Health and Human Services, 1985. 40. Heifets LB, Jenkins PA. Speciation of mycobacteria in clinical laboratories. In: Gangadharam PRJ, Jenkins PA (eds). Mycobacteria 1—Basic Aspects. Philadelphia: WB Saunders, 1998: 308–350. 41. Rogall T, Flohr T, Bottger EC. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol 1990;136: 1915–1920. 42. Butler WR, Jost KC, Kilburn JO. Identification of mycobacteria by high-performance liquid chromatography. J Clin Microbiol 1991;29: 2468–2472. 43. Kirschner P, Springer B, Vogel U, et al. 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. 44. Thibert L, Lapierre S. Routine application of highperformance liquid chromatography for identification of mycobacteria. J Clin Microbiol 1993;31:1759–1763. 45. Glickman SE, Kilburn SO, Butler WR. Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library. J Clin Microbiol 1994;32:740–749. 46. Roberts GD, Bottger EC, Stockman L. Methods for the rapid identification of mycobacterial species. In: Heifets LB (ed.). Clinical Mycobacteriology. Philadelphia: WB Saunders; 1996:603–639. 47. El Amin NM, Hanson HS, Pettersson B, et al. 16S rRNA gene sequence analysis vs. conventional methods. Scand J Infect Dis 2000;32:47–50.
48. Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin Microbiol Rev 2001;14:704–726. 49. Claud JL, Neal J, Rosenberry R, et al. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol 2002;40:400–406. 50. Hall L, Doerr KA, Wohlfiel SL, et al. Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 2003;41:1447–1453. 51. Eisenach KD, Sifford MC, Cave MD, et al. Detection of Mycobacterium tuberculosis in sputum samples using polymerase chain reaction. Am Rev Respir Dis 1991;144:1160–1163. 52. 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. 53. Pfyffer GE, Kissling P, Wirth R, et al. Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by a target-amplified test system. J Clin Microbiol 1994;32:918–923. 54. 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. 55. 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; 39:1993–1995. 56. Jost KC Jr, Dunbar DF, Barth SS, et al. 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. 57. Heifets LB, Iseman MD. Choice of antimicrobial agents for M. avium disease based on quantitative tests of drug susceptibility. N Engl J Med 1990;323:419. 58. Heifets LB, Iseman MD. Individualized therapy vs. standard regimens in the therapy of M. avium infections (editorial). Am Rev Respir Dis 1991;144:1–2. 59. Heifets L. Susceptibility testing of Mycobacterium avium complex isolates. Antimicrob Agents Chemother 1996;40(8):1759–1767. 60. Siddiqi SH, Heifets LB, Cynamon MH. Rapid broth macrifilution method for determination of MICs for M. avium isolates. J Clin Microbiol 1993;31:2332–2338. 61. Heifets L, Mar N, Vanderkolk J. Mycobacterium avium strains resistant to clarithromycin and azithromycin. Antimicrob Agents Chemother 1993;37:2364–2370. 62. Craft JC, Notario GF, Grosset JH, et al. Clarithromycin resistance and susceptibility pattern in M. avium strains isolated during prophylaxis of disseminated infection in patients with AIDS. Clin Infect Dis 1998;2:430–434. 63. Davidson PT, Khanijo V, Goble M, et al. Treatment of disease due to Mycobacterium intracellulare. Rev Infect Dis 1981;3:1052–1059. 64. Horsburgh CR Jr, Mason UG, Heifets LB III, et al. Response to therapy of pulmonary Mycobacterium avium-intracellulare infection correlates with results of in vitro susceptibility testing. Am Rev Respir Dis 1987;135:418–421. 65. Tsukamura M. Evience that antituberculosis drugs are really effective in the treatment of pulmonary infection caused by M. avium complex. Am Rev Respir Dis 1988;137:144–148. 66. Research Committee of the British Thoracic Society. First randomized trial of treatments for pulmonary disease caused by Mycobacterium avium intracellulare, Mycobacterium malmoense, and Mycobacterium xenopi in HIV negative patients: rifampicin, ethambutol and isoniazid versus rifampicin and ethambutol. Thorax 2001;56:167–172. 67. Heifets LB. Drug Susceptibility in the Chemotherapy of Mycobacterial Infections. Boca Raton: CRC Press, 1991. 68. Griffith DE. Management of disease due to M. kansasii. In: Catanzaro A, Daley CL (eds). Lung Disease due to Nontuberculous Mycobacterial Infections. Philadelphia: WB Saunders, 2003: 613–625.
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69. Woods GL, Brown-Elliot BA, Desmond PE. Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic actinomycetes; approved standard. In: National Committee for Clinical Laboratory Standards; 2003. 70. Marras TK, Wallace RJ Jr, Koth LL, et al. Hypersensitivity pneumonitis reaction to Mycobacterium avium in household water. Chest 2005;127:664–671. 71. De Groote MA, Pace NR, Fulton K, et al. Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils. Appl Environ Microbiol 2006;72 (12):7602–7606. 72. Falkinham JO. Nontuberculous mycobacteria in the environment. Clin Chest Med 2002;23:529–551. 73. Dorman SE, Picard C, Lammas D, et al. Clinical features of dominant and recessive interferon gamma receptor 1 deficiencies. Lancet 2004;364(9451): 2113–2121. 74. Tanaka E, Kimoto T, Matsumoto H, et al. Familial pulmonary Mycobacterium avium complex disease. Am J Respir Crit Care Med 2000;161:1643–1647. 75. Ahn CH, McLarty JW, Ahn SS, et al. Diagnostic criteria for pulmonary disease caused by Mycobacterium kansasii and Mycobacterium intracellulare. Am Rev Respir Dis 1982;125:388–391. 76. Prince DS, Peterson DD, Steiner RM, et al. Infection with Mycobacterium avium complex in patients without predisposing conditions [see comments]. N Engl J Med 1989;321:863. 77. Erasmus JJ, McAdams HP, Farrell MA, et al. Pulmonary nontuberculous mycobacterial infection: radiologic manifestations. Radiographics 1999;19(6): 1487–1505. 78. van der Werf TS, Stinear T, Stienstra Y, et al. Mycolactones and Mycobacterium ulcerans disease. Lancet 2003;362(9389):1062–1064. 79. Liao CH, Lai CC, Ding LW, et al. Skin and soft tissue infection caused by non-tuberculous mycobacteria. Int J Tuberc Lung Dis 2007;11(1):96–102. 80. Bartralot R, Garcia-Patos V, Sitjas D, et al. Clinical patterns of cutaneous nontuberculous mycobacterial infections. Br J Dermatol 2005;152(4):727–734. 81. Jernigan JA, Farr BM. Incubation period and sources of exposure for cutaneous Mycobacterium marinum infection: case report and review of the literature. Clin Infect Dis 2000;31(2):439–443. 82. Portaels F, Johnson P, Meyers WM. Buruli Ulcer. Diagnosis of Mycobacterium ulcerans Disease. Geneva: World Health Organization, 2001. 83. Tsukamura M. Diagnosis of disease caused by Mycobacterium avium complex. Chest 1991;99(3): 667–669. 84. Blackwell V. Mycobacterium marinum infections. Curr Opin Infect Dis 1999;12(3):181–184. 85. Wallace RJ Jr, Brown BA, Griffith DE, et al. Clarithromycin regimens for pulmonary Mycobacterium avium complex. The first 50 patients. Am J Respir Crit Care Med 1996;153:1766–1772. 86. Griffith DE, Brown BA, Girard WM, et al. Azithromycin-containing regimens for treatment of Mycobacterium avium complex lung disease. Clin Infect Dis 2001;32:1547–1553. 87. Tanaka E, Kimoto T, Tsuyuguchi K, et al. Effect of clarithromycin regimen for Mycobacterium avium complex pulmonary disease. Am J Respir Crit Care Med 1999;160:866–872. 88. Kobashi Y, Matsushima T. The effect of combined therapy according to the guidelines for the treatment of Mycobacterium avium complex pulmonary disease. Intern Med 2003;42(8):670–675. 89. Griffith DE, Brown BA, Cegielski P, et al. Early results (at 6 months) with intermittent clarithromycin-including regimens for lung disease due to Mycobacterium avium complex. Clin Infect Dis 2000;302:288–292. 90. Lam PK, Griffith DE, Aksamit TR, et al. Factors related to response to intermittent treatment of Mycobacterium avium complex lung disease. Am J Respir Crit Care Med 2006;173(11):1283–1289. 91. Kobashi Y, Matsushima T, Oka M. A double-blind randomized study of aminoglycoside infusion with
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93.
94.
95.
96.
97.
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combined therapy for pulmonary Mycobacterium avium complex disease. Respir Med 2007;101(1):130–138. Wallace RJ Jr, Zhang Y, Brown-Elliot BA, et al. Repeat positive cultures in Mycobacterium intracellulare lung disease after macrolide therapy represent new infections in patients with nodular bronchiectasis. J Infect Dis 2002;186:266–273. Griffith DE, Brown-Elliott BA, Shepherd S, et al. Ethambutol ocular toxicity in treatment regimens for Mycobacterium avium complex lung disease. Am J Respir Crit Care Med 2005;172(2):250–253. Nelson KG, Griffith DE, Brown BA, et al. Results of operation in Mycobacterium avium-intracellulare lung disease. Ann Thorac Surg 1998;66:325–330. Pomerantz ML, Madsen L, Goble M, et al. Surgical management of resistant mycobacterial tuberculosis and other mycobacterial pulmonary infections. Ann Thorac Surg 1991;52:1108–1112. Shiraishi Y, Nakajima Y, Takasuna K, et al. Surgery for Mycobacterium avium complex lung disease in the clarithromycin era. Eur J Cardiothoracic Surg 2002; 21:314–318. Shiraishi Y, Fukushima K, Komatsu H, et al. Early pulmonary resection for localized Mycobacterium avium complex disease. Ann Thorac Surg 1998;66:183–186.
98. Pomerantz ML, Denton JR, Huitt GA, et al. Resection of the right middle lobe and lingula for mycobacterial infection. Ann Thorac Surg 1996; 62:990–993. 99. Pezzia W, Raleigh JW, Bailey MC, et al. Treatment of pulmonary disease due to Mycobacterium kansasii: recent experience with rifampin. Rev Infect Dis 1981; 3:1035–1039. 100. Ahn CH, Lowell JR, Ahn SA, et al. Chemotherapy for pulmonary disease due to Mycobacterium kansasii: efficacies of some individual drugs. Rev Infect Dis 1981;3:1028–1034. 101. Ahn CH, Lowell JR, Ahn SS, et al. Short-course chemotherapy for pulmonary disease caused by Mycobacterium kansasii. Am Rev Respir Dis 1983; 128:1048–1050. 102. Griffith DE, Brown-Elliot BA, Wallace RJ Jr. Thrice-weekly clarithromycin-containing regimen for treatment of Mycobacterium kansasii lung disease: results of a preliminary study. Clin Infect Dis 2003;37:1178–1182. 103. Aubry A, Chosidow O, Caumes E, et al. Sixty-three cases of Mycobacterium marinum infection: clinical features, treatment, and antibiotic susceptibility of causative isolates. Arch Intern Med 2002;162(15): 1746–1752.
104. World Health Organization. Provisional Guidance on the Role of Specific Antibiotics in the Management of Mycobacterium ulcerans Disease. Geneva: World Health Organization, 2004. 105. Chauty A, Ardant MF, Adeye A, et al. Promising clinical efficacy of the combination streptomycin— rifampin for the treatment of Buruli ulcer (Mycobacterium ulcerans disease). Antimicrob Agents Chemother 2007;51(11):4029–4035. 106. Kaplan JE, Masur H, Holmes KK. Guidelines for preventing opportunistic infections among HIVinfected persons. MMWR Morb Mortal Wkly Rep 2002;RR-8:1–52. 107. Oldfield EC 3rd, Fessel WJ, Dunne MW, et al. Once weekly azithromycin therapy for prevention of Mycobacterium avium complex infection in patients with AIDS: a randomized, double-blind, placebocontrolled multicenter trial. Clin Infect Dis 1998;26:611–619. 108. El-Sadr WM, Burman WJ, Grant LB, et al. Discontinuation of prophylaxis against Mycobacterium avium complex disease in HIV-infected patients who have a response to antiretroviral therapy. N Engl J Med 2000;342:1085–1092.
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The basic immunology of tuberculosis Brian S Eley and David W Beatty
INTRODUCTION The term Mycobacterium tuberculosis is used throughout this chapter to describe the M. tuberculosis complex group of organisms (M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti) as well as M. tuberculosis sensu stricto. Interaction between M. tuberculosis and host macrophages leads to the establishment of infection. This initial encounter typically occurs in the terminal respiratory structures such as the distal alveoli or alveolar sacs, following the inhalation of pathogenic organisms primarily contained in infected airborne droplets. After phagocytosis by macrophages and the initial innate immune responses, which include macrophage and dendritic cell activation, antigen processing and presentation, cell-surface expression of co-stimulatory molecules, cytokine and chemokine elaboration, and lymph node targeting, M. tuberculosisspecific adaptive cell-mediated immunity develops. The combined innate and adaptive immune responses enhance killing by macrophages and is usually successful in containing but not completely eliminating the pathogen. Containment or clinical latency and mycobacterial latency usually extend over the lifespan of the individual. The initial encounter with the innate immune system, directed primarily by macrophages and dendritic cells, may theoretically lead to immediate mycobacterial elimination, aborting the development of specific T-cell immunity. This is, however, not considered the usual path of infection. Instead the development of a primary TB complex at the initial site of infection and in the associated regional lymph nodes, during which specific immunity is optimized, precedes the containment of infection. Approximately 10% of infected individuals will progress to active TB over their lifespan but in those infected with human immunodeficiency virus (HIV) the risk for active TB disease, either localized or disseminated, rises to approximately 8–10% per annum. The risk of children progressing to active pulmonary and disseminated disease is much higher than that in adults. Children under the age of 1 year are at a much higher risk for disseminated disease. The elderly are also more susceptible to TB (Fig. 8.1). Following containment of infection, reactivation of disease may occur in settings in which M. tuberculosis-specific immunity is undermined. In high TB-endemic communities or geographical regions reinfection with a new M. tuberculosis strain may lead to the establishment or re-establishment of disease in individuals with compromised immunity.1 In this chapter we describe the key immunological responses to M. tuberculosis that help to clarify this pathogenesis framework.
PROTECTIVE IMMUNITY Development of protective immunity following infection by M. tuberculosis depends on the interaction between innate and adaptive immunity (Box 8.1). Innate immunity (or natural immunity), induced independently of prior contact with M. tuberculosis, is mediated mainly by macrophages and dendritic cells. Recent studies have shown that the innate immune system has a greater degree of specificity than previously appreciated. This system is governed by the interaction between pathogen-associated molecular patterns (PAMPs) and specific pattern recognition receptors (PRRs), which are expressed on macrophages and dendritic cells. Activation of innate immunity is a prerequisite for the induction of adaptive immunity.2 In contrast, adaptive immunity (known as specific immunity) develops after exposure to M. tuberculosis-specific antigens that have been processed by, and presented on, antigen-presenting cells, usually in association with major histocompatibility complex (MHC) class I and II or CD1 molecules and co-stimulatory molecules together with soluble cytokine signals. Adaptive immune responses are directed by antigen-specific T-cell subsets, notably in order of decreasing importance CD4þ ab T-lymphocytes, CD8þ ab T-lymphocytes, gd T-lymphocytes, and CD1-restricted ab T-lymphocytes.3 Granuloma formation and function, determined by the interplay between innate and cellular adaptive immunity, is central to the containment of infection and induction of mycobacterial latency. Granuloma formation is the most important immunopathological response following infection by M. tuberculosis. Natural killer (NK) cells and neutrophils are recruited and contribute to granuloma function.4 Although the role of humoral-mediated adaptive immunity in M. tuberculosis infection remains uncertain, there is sufficient experimental evidence to indicate that antibody-mediated immune reactions may modify the course of infection.5
PHAGOCYTOSIS BY MACROPHAGES Internalization of M. tuberculosis by macrophages is an important initial step in the establishment of infection. Phagocytosis is mediated via several receptors on the surface of the macrophages which bind either opsonized or non-opsonized organisms (Box 8.2).6 A classic example of uptake of opsonized bacteria involves complement receptors of which type 1 (CR1) binds to the complement components C3b and C4b but not C3bi, whereas types 3 and 4 (CR3 and CR4) bind C3bi. Mycobacterium tuberculosis
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Inhalation of M TB Primary complex (PPD+)
Immediate killing of M TB(PPD)
Localized disease (Primary TB)
Dissemination of M TB Acute disease (TBM, miliary TB) Containment (latency)
Reactivation or reinfection
Fig. 8.1 Key pathogenic steps following M. tuberculosis inhalation.
Box 8.1 Major components of protective immunity
Innate responses including antigen processing are directed by macrophages, dendritic cells, and NK cells. Adaptive immunity is directed by CD4þ, CD8þ, gd, and CD1-restricted T-lymphocytes. Microbial containment and elimination is determined by cooperation between innate and adaptive immunity. Granuloma function is pivotal for microbial containment.
Box 8.2 Macrophage receptors that promote the uptake of Mycobacterium tuberculosis
Complement receptors: CR3, CR4, CR1. Mannose receptors. Scavenger receptors. Surfactant protein A receptors. Fcg receptors.
is able to non-specifically and directly activate the alternative complement pathway leading to the generation of C3b and C3bi. Once bacteria are sufficiently coated with either or these molecules phagocytosis can proceed through CR1, CR3, and CR4 on macrophages. Mycobacteria may also recruit C2b directly to form a C3 convertase and generate C3b in the absence of early activation of the complement pathway, leading to predominately CR1-mediated uptake. Mycobacteria are able to bind directly or non-opsonically to CR3.7 This complement pathway-independent binding is achieved by direct interaction between the bacterial oligosaccharide lipoarabinomannan (LAM) or cell wall antigen 85C and specific CR3 domains. CR3 may also recognize LAM after forming a trimolecular complex with CD14 and Toll-like receptor 4. There is also evidence supporting non-opsonic uptake via CR4. CR3 is considered the major complement receptor involved in phagocytosis of mycobacteria. Using CR3-deficient macrophages derived from knockout
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mice, it has been established that CR3 mediates about 50% of non-opsonic binding and 60% of opsonized binding.8 A widely expressed receptor on the surface of the macrophage is the mannose receptor, which mediates non-opsonized phagocytosis. Entry of mycobacteria into the macrophage is mediated by direct interaction with mannosyl residues on LAM, other mannose-containing glycoconjugates, or adhesins including the 19-KkDa antigen of M. tuberculosis.9 Interferon-g (IFN-g) is able to down regulate the expression of macrophage-expressed mannose receptors. It is possible that these receptors play an important phagocytic role during early infection before granuloma formation has occurred. Several other surface-expressed receptors promote the phagocytosis of mycobacteria including scavenger receptors which bind polyanionic surface molecules, surfactant protein A receptors that facilitate phagocytosis of organisms opsonized with surfactant A or mannose-binding protein, and CD14 and Fcg receptors.7,9 Following receptor binding, cytoskeletal activation precipitates internalization and subsequent events including phagosome–lysosome fusion, antigen processing and presentation, cytokine upregulation, and intracellular killing via several mechanisms. Macrophages may also function as vectors for translocating mycobacteria across pulmonary epithelial barriers. There is some evidence that the nature of the receptor interaction influences subsequent immunological events.6
DENDRITIC CELLS Dendritic cells (DCs) are a system of cells that have the specialized function of presenting antigens to T-lymphocytes and are considered the most important antigen-presenting cell population. They are located throughout the body in mucosal tissues and are regarded as important immunological sensors. They detect pathogens, internalize and process pathogen antigens, and relay the information to lymphocytes, thereby inducing specific adaptive immune responses. The early interaction between DCs and M. tuberculosis occurs in the respiratory mucosa. Dendritic cells are able to internalize pathogens by several routes including endocytosis, phagocytosis, and macropinocytosis. Two specific surface-expressed receptors play a role in phagocytosis of M. tuberculosis – DC-specific intracellular adhesion molecule-3 grabbing non-integrin (DC-SIGN), a C-type lectin, and the mannose receptor. Experimental evidence has shown that DC-SIGN is the major DC receptor for M. tuberculosis. The mannose receptor plays a minor role. DC-SIGN binds strongly to the mannose-capped cell wall component ManLAM of M. tuberculosis.10 Once phagocytosed, antigen processing and presentation proceeds, and the DCs undergo maturation, resulting in the expression of high levels of MHC molecules and the upregulation of co-stimulatory molecules such as CD80 and CD86, which are subsequently expressed on the surface of DCs. Activated DCs acquire migratory capacities, allowing them to exit the respiratory mucosa and enter regional lymph nodes where they can present antigens to naı¨ve T-lymphocytes. Dendritic cells are thus able to modulate the immune response through antigen presentation with consequent T-lymphocyte activation, as well as through cytokine elaboration resulting in the polarization of T-lymphocytes towards either Th1 or Th2 phenotypes. During the course of TB T-lymphocytes are predominantly directed towards a Th1 response. Therefore DCs link the innate and adaptive immune responses to infection by M. tuberculosis.11
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The basic immunology of tuberculosis
THE ROLE OF TOLL-LIKE RECEPTORS Toll-like receptors (TLRs) are an important family of specific PRRs, which serve to link microbial recognition to the activation of antigen-presenting cells (macrophages and dendritic cells) involved in T-lymphocyte activation and induction of adaptive immunity. They are a structurally conserved family of transmembrane glycoproteins which are homologous with the Toll proteins of fruit flies (Drosophila) in which they are essential for establishing dorsoventral polarity during embryogenesis and for innate immune responses against fungal infection. To date at least 13 mammalian TLRs have been identified, and the first to be characterized, TLR4, was cloned in 1997.12 In humans, signalling through the TLRs is important in inducing dendritic cell and macrophage activation and function, and facilitating adaptive immunity. The innate immune system uses a set of PRRs to identify conserved PAMPs on molecules such as lipopolysaccharide (LPS), peptidoglycan, mannan, hypomethylated CpG DNA motifs, and double-strand RNA. These molecular signatures, although present in a wide range of pathogens, are rarely found in the host, permitting discrimination between foreign pathogens and ‘self’ molecular signatures. Ligation of PRRs by microbial products leads to the activation of signalling cascades critical for the induction of an immune response to a specific microbial challenge.2,13 Signalling through TLRs induces maturation of DCs. Mature DCs up regulate surface expression of MHC class II and co-stimulatory molecules, elaborate cytokines, and migrate to peripheral lymph nodes where they induce the maturation of antigen-specific naı¨ve T-lymphocytes. Although components of the various TLR signalling pathways are shared, there are considerable differences. The extracellular domains of TLRs are important for pathogen recognition: they contain between 24 and 29 leucine-rich repeat sequences and form horseshoe-shaped structures which are involved with pathogen recognition. Leucine-rich repeat domains of different TLRs recognize structurally unrelated ligands. The cytoplasmic domains of all TLRs are homologous with the signalling domain of the interleukin (IL)-1 receptor and is currently referred to as the Toll/IL-1 receptor (TIR) domain. After ligand binding, TLRs form dimers and undergo conformational change, which permits the interaction of the cytoplasmic TIR domain with downstream adaptor molecules, myeloid differential primary-response protein 88 (My D88), TIR-domain-containing adaptor protein (TIRAP), TIR-domaincontaining adaptor protein inducing interferon (IFN)-b (TRIF), and TRIF-related adaptor molecule (TRAM). These adaptor proteins initiate complex molecular cascades leading to the activation of NF-kB and/or IFN-regulatory factor-3 transcription factors. These transcription factors translocate from the cytoplasm to the nucleus where they induce gene upregulation leading to DC maturation and cytokine elaboration (Fig. 8.2).2,14 Several M. tuberculosis-specific ligand–receptor interactions have been identified. The 19-kDa lipoprotein induces activation of TLR2. A soluble fraction of M. tuberculosis which contains mannosylated phosphatidylinositol (PIM) activates macrophages and DCs via TLR2 and TLR6. Viable and killed mycobacteria may signal via TLR2 and TLR4. Proinflammatory signals are up regulated by TLR2 and TLR4 after being bound to heat shock protein (HSP) 65 and HSP70, respectively. Furthermore, other TLRs may be involved in the regulation of the immune response to infection by M. tuberculosis. There is, for example, evidence to suggest that mannose-capped lipoarabinomannan
Transmembrane toll-like receptor
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M. tuberculosis ligand Plasma membrane Cytosol
My D88-independent signalling
My D88-dependent signalling
IRF-3 activation
NF-KB activation
Upregulation of gene expression
Effector functions
Fig. 8.2 Schematic representation of Toll-like receptor signalling pathways.
signals through a TLR other than TLR2 or TLR4. Signalling utilizes both My D88-dependent and -independent pathways. Although details of the specific TLR pathways activated by M. tuberculosis and its components are still being elucidated and remain the subject of contention, many immune functions have been identified. These include maturation of DCs, proinflammatory cytokine elaboration, maturation of Th1-biased T-lymphocytes, upregulation of macrophage phagocytosis, macrophage apoptosis, and induction of inducible nitric oxide synthetase (iNOS), resulting in the production of NO, a molecule with direct antimicrobial activity (Fig. 8.2).15
PHAGOSOMAL MATURATION AND ANTIGEN PROCESSING Receptor engagement leads to the internalization of microbes or pathogen-derived soluble proteins by macrophages and DCs. Following the degradation of the internalized material, a wide range of processed M. tuberculosis-derived antigens are presented to T-lymphocytes through several antigen-presenting molecules, notably MHC class I, MHC class II, or CD1 molecules. Dendritic cells and macrophages are mainly responsible for proteolytic processing and antigen presentation (Table 8.1).16,17 A phagosome is a vesicle that forms by invagination of the plasma membrane with its associated lipids and proteins during phagocytosis of microbes or microbial proteins. Antigen processing occurs after internalized phagosomes mature within macrophages and DCs (Fig. 8.3), a process involving a complex series of sequential fusion interactions with components of the endocytic pathway. These interactions cause major changes to the composition and function of the original phagosome. Progressive transfer of endosomal or lysosomal membrane and luminal constituents to phagosomes occurs during these fusion events. The endocytic pathway is organized as a continuum of organelles, progressing from early endosomes through late endosomes to lysosomes. Phagosomal maturation is a rapid process, which requires Ca2þ ions to activate calmodulin and the calmodulin-dependent protein kinase, CaMKII. Calcium is
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Table 8.1 The antigen-presenting functions of MHC class and CD1 molecules MHC class I
MHC class II
CD1a, CD1b, CD1c (group 1 molecules)
CD1d (group 2 molecule)
Genomic locus
MHC locus (chromosome 6)
MHC locus (chromosome 6)
CD1 locus (chromosome 1)
CD1 locus (chromosome 1)
Antigens presented
Endogenous/ exogenous peptides
Exogenous peptides
Endogenous/exogenous glycolipids
Endogenous/exogenous glycolipids
T-lymphocyte subsets primed
CD8þ TCRab
CD4þ TCRab
CD4–CD8– TCRab, CD4þ TCRab, CD8þ TCRab, TCRgd
CD4–CD8– TCRab, diverse TCRab, TCRa NK cells
Major T-lymphocyte functions
Cytotoxicity
Th1/Th2 cytokine productions
Th1/cytotoxicity
Th0/cytotoxicity?
Adapted from Gumperz and Brenner.27
Plasma membrane
Rab 5
Microbial contents
Rab 5
Early endosome
Endoplasmic reticulum pH: ~6.0
Early phagosome Rab 7
Rab 5
Late endosome pH: 5.5 6.0
Late phagosome
Lysosome pH: 4.5 5.5
Phagolysosome
Fig. 8.3 Simplified representation of Export of peptideMHC I complexes to the plasma membrane after cross-presentation
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Export of peptideMHC II complexes to plasma membrane surface
phagolysosome formation (adapted from Koul A, Herget T, Klebl B, et al. Interplay between mycobacteria and host signaling pathways. Nat Rev Microbiol 2004;2:189–202.)
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derived from endoplasmic reticulum stores and via open plasmalemmal channels. Phagosomal maturation also involves the acquisition of vacuolar-proton ATPase (V-ATPase), which catalyses the progressive acidification of phagosomes during maturation.18–20 Rab proteins are small GTPases or GTP-binding proteins that control the integrity of intracellular organelles, direct membrane trafficking and protein sorting, and play an important role in the maturation of the phagosome. The newly formed phagosomes acquire Rab5 either from the surrounding plasma membrane or by fusion with early endosomes. Rab5 controls the fusion of phagosomes with the early or sorting endosomes, where the phagocytosed material and receptors are sorted for degradation and/or recycling. Rab5 together with activated CaMKII recruits phosphatidylinositol-3-kinase (PI3K) to the early endosome, where it generates phosphatylinositol3-phosphate (PI3P) which then initiates the recruitment of early endosomal autoantigen 1 (EEA1) from endosomes. EEA1 is regarded as a ‘Rab5 effector’ that triggers the fusion of phagosomes with late endosomes, thereby facilitating Rab conversion, i.e. rapid acquisition of Rab7 and Rab9 from late endosomes with the simultaneous loss of Rab5.18,19 Rab7 is important in the regulation of late endosomal trafficking. It regulates the transition from early to late endosomes and controls the fusion of late phagosomes with lysosomes to form phagolysosomes. Phagolysosomes possess several degradative properties due to a low pH, between 4.5 and 5.5, generated by the vacuolar ATPase, hydrolytic enzymes such as cathepsin D, defensins, other bactericidal peptides, and the ability to generate oxidative compounds. Once phagolysosomes form, degradation of the internalized material by the hydrolytic enzymes can proceed. The processed antigens can be cycled to the cell surface bound to MHC class II molecules for antigen presentation as described below.19 Although TLRs are not involved in the uptake and internalization of microbes, they play a role in regulating phagosomal maturation and antigen processing. During phagocytosis, surfaceexpressed TLRs including TLR2 and TLR4 are recruited to the phagosome and become activated by microbial constituents. Phagosome maturation may then be influenced through My D88 and mitogen-associated protein kinase (MAPK) p38 activation. Although the detailed molecular mechanisms of TLR regulation are currently unknown, possible functions of MAPK p38 include the assembly of V-ATPase and regulation of Rab5 function. Furthermore, TLRs may be responsible for increasing the rate of movement of phagosomes along the endocytic pathway. Other receptor–microbe interactions may be involved in phagosome maturation.20,21
ANTIGEN PRESENTATION MHC CLASS II PRESENTATION A wide repertoire of clones of M. tuberculosis-specific CD4þ Tlymphocytes that play a dominant role in the protective immune response against TB has been identified. The maturation of these clones is induced by a broad spectrum of processed mycobacterial antigens. Many immunodominant antigens including ESAT-6, CFP-10, the 19- and 38-kDa lipoproteins, and several mycolyl transferases have been identified. CD4þ T-lymphocytes recognize epitope configurations on processed mycobacterial peptide fragments when presented by MHC class II molecules in association
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with co-stimulatory molecules. In this way exogenously derived antigens internalized through the endocytic pathway are presented by MHC class II molecules. Signalling through TLRs plays a role in improving the efficiency with which phagolysosomes execute their antigen-presenting function. The environment of the phagolysosome is important for antigen processing as well as for the modification of the MHC class II-associated invariant chain.16,22 Newly synthesized a- and b-chains of MHC class II molecules form a complex with an invariant chain in the endoplasmic reticulum. The cytoplasmic domain of the invariant chain contains a targeting signal that directs the MHC class II complexes to the endocytic pathway. These complexes enter the endocytic pathway and, within the hydrolytic environment of the late phagosomes and phagolysosomes, the invariant chain is proteolytically degraded so that an N-terminal fragment, the MHC class II-associated invariant chain derived peptide (CLIP), remains. This fragment occupies the peptide groove of MHC class II molecules. Within phagolysosomes, CLIP is exchanged for processed antigenic peptides. This pivotal antigenprocessing step is catalysed by the MHC-encoded molecules, HLA-H2-DM and HLA-DO. Toll-like receptors trigger the processing of the invariant chain after interaction with microbial constituents within phagolysosomes. The exact mechanisms by which this occurs have, however, yet to be defined. Once peptide–MHC class II complexes have formed, they are transported to the plasma membrane and presented to CD4þ T-lymphocytes in association with TLR-induced co-stimulatory molecules.20,23 During antigen presentation, signalling through the CD4þ cell receptor is necessary for the consequent maturation and activation of naı¨ve CD4þ T-lymphocytes. Mice with genes deleted for either the CD4 receptor or MHC class II molecules are extremely susceptible to disease after infection by M. tuberculosis, affirming the importance of CD4þ T-lymphocytes in protection. Cytokines such as IFN-g regulate cell-surface expression of MHC and co-stimulatory molecules, thereby influencing antigen presentation (Table 8.1).
MHC CLASS I ANTIGEN PRESENTATION MHC class I processing and the consequent induction of CD8þ T-lymphocyte-specific responses was first recognized in viral infections. In this setting newly synthesized viral proteins undergo posttranslational modification within the endoplasmic reticulum before being exported to the cytosol where they may be processed by proteosomes, resulting in the generation of antigenic peptides. Antigenic peptides are then transported back to the endoplasmic reticulum and undergo further trimming before associating with MHC class I molecules to form complexes. MHC class I complexes, formed in the endoplasmic reticulum, consist of a heavy chain and a light chain (or b2-microglobulin) together with peptide bound to the antigen groove of the MHC class I molecule. MHC class I complexes are exported to the plasma membrane surface via the Golgi apparatus where they are able to prime CD8þ T-lymphocytes.24 Specific CD8þ T-lymphocytes recognize mycobacterial peptides in association with MHC class I molecules. Mycobacteria located within macrophages are confined to the phagosomal compartment and their antigens are separated from the classical MHC class I presentation pathway. However, Mycobacterium tuberculosis antigens do gain access to the MHC class I presentation pathway although until recently the routes of entry have been difficult to define.25
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Box 8.3 The pathways by which segregated antigens gain access to the MHC class I processing and presentation
Cross-presentation: There is evidence that the endoplasmic reticulum membrane and its constituents, including the machinery for MHC class I presentation, are recruited to the early phagosome. The endoplamic reticulum-derived protein sec 61 allows bacterial proteins to shuttle from the phagosome to the cytosol for processing by proteasomes. Thereafter processed antigens are transported back to the phagosome where they are assembled on MHC class I molecules before being transported to the plasma membrane via the Golgi apparatus (Fig. 8.3). Cross-priming: Although M. tuberculosis located within the phagosome is relatively segregated from the cytosol, it is, by inducing apoptosis of the macrophage, able to release antigen-containing blebs into the microevironment. These antigens are then endocytosed by non-infected DCs, enter the phagosomal compartment, and are subsequently directed to the MHC class I presentation pathway for processing and presentation via the lysosome-to-cytosol pathway, which is present in DCs but not macrophages. Antigens may escape from phagosomes within macrophages, enter the cytosol, and be processed in a proteosome-dependent manner, gaining access to the classical MHC class I pathway (Table 8.1).25,26
The pathways by which segregated antigens gain access to the MHC class I processing and presentation are shown in Box 8.3.
MACROPHAGE AND DENDRITIC CELL CYTOKINE PRODUCTION The cytokine network and its interactions are complex in the setting of infection by M. tuberculosis. Conventional understanding supports a pivotal role for cytokines in the overall immune response to TB. Although cytokines are produced by a wide variety of cells, primed lymphocytes are the major producers, particularly two sub populations of CD4þ T-lymphocytes – the T-helper type 1 (Th1) cells that produce IL-2, IFN-g, lymphotoxin-a, and other proinflammatory cytokines, and the T-helper type 2 (Th2) cells that produce IL-4, IL-5, IL-10, IL-13, and other anti-inflammatory cytokines. During TB CD4þ T-lymphocyte production is biased towards a Th1, or proinflammatory, response that supports the killing of intracellular pathogens.30 There is, however, evidence to suggest that strains or genotypes of M. tuberculosis vary in the profile of cytokines that they induce and that the ability to control infection may be due in part to the induced profile.31 Cytokines produced by the major antigen-presenting cells (APCs), the macrophages and dendritic cells, have important regulatory functions during infection by M. tuberculosis. They help regulate the overall immune response, initiate APC activation, direct the CD4þ T-lymphocytes towards a Th1 profile, activate NK cells, stimulate and augment IFN-g production, direct antigeninduced cytotoxicity, and facilitate granuloma formation and function (Fig. 8.4).6 A few important cytokines elaborated by APCs during infection by M. tuberculosis are briefly discussed below.
CD1 PRESENTATION The CD1 family consists of antigen-presenting molecules encoded by genes outside the MHC. They have specific roles in the presentation of lipids, glycolipids, and lipopeptides to T-lymphocytes, and therefore play a major role in presenting M. tuberculosis cell wall-associated lipid antigens to naı¨ve T-lymphocytes. CD1 molecules resemble MHC class I molecules structurally, consisting of a transmembrane heavy chain that associates with b2-microglobulin.27 Signalling through TLRs induces the upregulation of CD1 expression, thereby facilitating lipid antigen presentation. There are currently five known CD1 isoforms: CD1a, CD1b, CD1c, and CD1d are involved in antigen presentation on cell surfaces, and CD1e facilitates the processing of complex glycolipids within the endocytic pathway.28,29 CD1 molecules are assembled within the endoplasmic reticulum and present antigens to several different T-lymphocyte subsets, including CD4–CD8–TCRab T-lymphocytes (double-negative T-lymphocytes) and CD8þ T-lymphocytes (Table 8.1).27 CD1 molecules function at different stages within the endocytic pathway. CD1a is present in early recycling endosomes where they load antigens in the absence of chaperone molecules. CD1b and CD1d molecules target endosomes and lysosomes, whereas CD1c is found throughout the endocytic pathway and is able to present antigens independent of lysosomal acidification. CD1c controls the induction of gd cells. CD1d is the only antigen-presenting molecule known to activate NK cells, thus recruiting these cells to the specific immune response. Mycobacterium tuberculosis-specific CD1-restricted T-lymphocyte clones mediate cellular cytotoxicity and/or are biased towards secreting Th1 cytokines. Although lipid presentation is a relatively novel concept, evidence suggests that CD1 presentation is important for the overall cellular adaptive response to infection by M. tuberculosis.16,29
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IL-12P70 AND RELATED CYTOKINES IL-12p70 Interleukin-12p70 (IL-12), a heterodimer composed of p40 and p35 subunits, is produced mainly by macrophages and DCs. Its early production by these cells is important for the induction of NK cells, the elaboration of IFN-g, and, subsequently, the activation, differentiation, and proliferation of M. tuberculosis-specific Th1 cells. It also induces IFN-g production by Th1 cells. It forms a link between the innate and adaptive responses and binds to the IL-12 receptor, consisting of two subunits, IL-12Rb1 and IL-12Rb2, expressed on Th1 and NK cells. In addition, IL-12 augments cytotoxicity of CD8þ T-lymphocytes and NK cells by inducing the transcription of genes encoding perforin and granzymes and by up regulating the expression of adhesion molecules. It also enhances antigen presentation by macrophages and DCs.32,33 IL-18 Several cytokines with IL-12-like function have been identified, including IL-18, IL-23, and IL-27. They are also produced by APCs, and complement the function of IL-12. Interleukin-18 is synthesized as an inactive precursor which undergoes proteolytic cleavage by caspase-1 before being secreted. It enhances CD8þ T-lymphocyte and NK cell cytotoxicity, and induces IFN-g production by NK cells together with IL-12 during the early stages of Th1 development but, alone, does not appear to induce IFN-g secretion. Instead it acts synergistically with IL-12 to augment IFN-g production. Interleukin-12 up regulates the expression of the IL-18 receptor, which is necessary for the functioning of IL-18.34 A newly identified proinflammatory cytokine, IL-32,
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Th2 cytokine expression IL-4, IL-10, TGFb, IL-27
M. tuberculosis
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Stimulation of T cells and NK cells IL-12, IL-15, IL-18, IL-23, IL-27
Acute phase response: IL-6 Macrophage of dendritic cell
IFNg
Autoinduction: TNFa, IL-1b, IFNg
Leukocyte chemotaxis: IL-8, MCP-1, MIP-1a, RANTES
Fig. 8.4 Major functions of macrophages and dendritic cells in containing M. tuberculosis infections (adapted from Van Crevel R, Ottenhoff THM, van der Meer JWM. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002;15:294–309.)
is induced during infection by M. tuberculosis in an IL-18-dependent manner and it may also induce the production of tumour necrosis factor-a (TNF-a).35
IL-23 Interleukin-23 consists of a p19 subunit that associates with the IL-12p40 subunit. It interacts with a receptor complex, which is composed of the IL-12Rb1 subunit and an IL-23R subunit that shares homology with the IL-12b2 subunit. Along with IL-12, IL-23 plays an important bridging role between the innate and adaptive immune responses. It induces the production of IFN-g from activated or memory CD4þ T-lymphocytes but not from naı¨ve lymphocytes. In contrast IL-12 promotes IFN-g production from both naı¨ve and memory subsets. Interleukin-23 also stimulates the proliferation of memory T-lymphocytes but not naı¨ve T-lymphocytes, and it enhances the elaboration of IL-17 primarily from gd T-lymphocytes.33,36 This IL-17 cell lineage is believed to have developed separately but in parallel with specialized Th1 and Th2 effectors that address intracellular pathogens and parasitic infections, respectively. Interleukin-17 receptors are expressed on a broad range of cell types. Upon ligation, IL-17 induces production of several cytokines including granulocyte colony-stimulating factor, IL-1, IL-6, IL-8, and TNF-a and may therefore mediate anti-TB activity through increased phagocytosis and proinflammatory actions. Interleukin-17 does not appear to play a role in the formation or maintenance of the granuloma.37,38 IL-27 Interleukin-27 is also required for the development of Th1 responses during infection by M. tuberculosis. It induces proliferation of naı¨ve T-lymphocytes, secretion of IFN-g by naı¨ve T-lymphocytes, and the production of IFN-g by NK cells in association with IL-12 or IL-18. It may therefore act in the early stages of the immune response.39 Studies on mice in which the gene encoding WSX-1, a type 1 cytokine receptor, was deleted showed that IL-27 is able to exert a paradoxical anti-inflammatory function in the setting of
Granuloma development
Initiation of adaptive immunity MCH, CD1, co-stimulatory molecules, Th1 cytokines
Containment or elimination of M. tuberculosis
Antigen-specific T-cell response: IFNg, LTa3, TNFa, perforin, granulysin
M. tuberculosis infection. By signalling through WSX-1, to which it binds with high-affinity, IL-27 was shown to attenuate the magnitude of the Th1 response and hence may control potentially pathological sequelae arising from the chronic inflammatory state induced by infection by M. tuberculosis.39,40
TUMOUR NECROSIS FACTOR-a Tumour necrosis factor-a, a proinflammatory cytokine that belongs to the TNF cytokine family, is produced mainly by DCs and macrophages. It exists in soluble and transmembrane isoforms which, respectively, activate the TNF type 1 and type 2 receptors. During infection by M. tuberculosis, soluble TNF-a regulates the immune response and consequent immunopathology. The importance of TNF-a in human infection has been confirmed in patients with chronic inflammatory conditions such as rheumatoid arthritis, where the administration of anti-TNF-a monoclonal antibodies significantly increases the risk of reactivation TB.41,42 Specific actions of TNF-a include the promotion of apoptosis of macrophages infected with M. tuberculosis, thereby contributing to the clearance of the infection, induction of DC maturation, and migration to regional lymph nodes. This facilitates antigen presentation, T-lymphocyte priming within lymph nodes, upregulation of expression of endothelial adhesion molecules, and chemokine elaboration and chemokine receptor expression, thus promoting the recruitment of APCs and T-lymphocytes to the infection site. Gramuloma formation and function, which leads to the control and containment of the infection and mycobacterial dormancy, is dependent on TNF-a production. In mice, TNF-a in combination with IFN-g plays a key role in mycobacterial killing within macrophages by stimulating the production of reactive nitrogen intermediates. In human infection, this does not appear to be a function of TNF-a. Circulating TNF-a is considered responsible for the cachexia that accompanies TB.42,43
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In addition to TNF-a, there are several other cytokines elaborated primarily by APCs that drive the proinflammatory response to TB including IL-1b and IL-6.
INTERLEUKIN-4 AND OTHER ANTI-INFLAMMATORY CYTOKINES Several cytokines, including transforming growth factor (TGF)-b, IL-4, IL-10, and IL-13, elaborated during infection by M. tuberculosis, exert anti-inflammatory effects.6 Interleukin-4 is produced by various cells including CD4þ T-lymphocytes, CD8þ T-lymphocytes, NK cells and APCs. Interleukin-4 levels are higher in patients with TB who reside close to the equator than in those in Europe. The exact regulatory role of IL-4 has not been completely elucidated. During the early stages of M. tuberculosis infection, IL-4 may be involved in driving the Th1 response; during the later stages of infection it probably down regulates Th1 responses, and may be associated with progressive disease and TNF-a-associated immunopathology. Interleukin-4 knockout mice are able to control M. tuberculosis infection as well as wild-type mice but they experience less TNF-a-mediated toxicity and pulmonary fibrosis, implying that IL-4 is involved in these effects.44,45
THE ACTIVATION OF T-LYMPHOCYTES Activation of CD4þ and CD8þ T-lymphocytes are antigen-specific events resulting from the interaction between DCs and naı¨ve T-lymphocytes. Activation primarily occurs within regional lymph nodes following the migration of DCs from the site of infection. Each T-lymphocyte expresses a unique T-cell receptor (TCR) which recognizes antigenic peptides in association with MHC class molecules. Each TCR consist of two transmembrane subunits, a and b. The hypervariable region of each subunit is formed by random genetic rearrangement of the V, D, and J functional segments. Genetic rearrangement is the major mechanism responsible for generating receptor diversity and it confers on each TCR a unique antigen recognition site, which is formed by the hypervariable regions of the two subunits. Binding of the TCR to peptide-bound MHCs with high affinity induces signalling downstream of the receptor, resulting in a range of cellular responses including proliferation, differentiation, elaboration of cytokines, induction of a memory response, and, in CD8þ T-lymphocytes, cytotoxicity.46,47 Several signals are required for T-lymphocyte activation. Following antigen ligation the cytoplasmic domains of TCR subunits associate non-covalently with downstream molecules, activating the signalling cascade. Surface-expressed co-receptors are important for signalling. The CD4 co-receptor expressed on CD4þ T-lymphocytes and the CD8 co-receptor expressed on CD8þ T-lymphocytes bind to the non-variable portion of the MHC class II and class I molecules, respectively.46 Co-stimulation is provided by two T-lymphocyte expressed receptors, CD28 and CTLA-4 (CD152), which bind to either CD80 or CD86 expressed on APCs. CD28 is constituently expressed while CTLA-4 is rapidly up regulated on the surface of T-lymphocytes following T-cell activation. Additional co-stimulation pathways including the B7-H1/B7-DC/PD-1 and B7-H2/ICOS pathway have been identified. Stimulation of ICOS on T-lymphocytes augments proliferation and Th1 and Th2 cytokine production. Polarization of T-lymphocytes into various phenotypes such as Th1, Th2, and Th17, and regulatory T-cell (Tr) subsets, requires additional signals provided by the
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existing cytokine milieu and, through co-signalling via dendritic cell subsets, NK cells and other cellular elements of the innate immune system. During infection by M. tuberculosis IL-12 and related cytokines appear to play an important role in directing a predominantly Th1 response.48
T-LYMPHOCYTE AND NK CELL FUNCTIONS Following priming, specific T-lymphocyte subsets play important roles, determined by their modified gene expression, following infection by M. tuberculosis. Activated T-lymphocytes express chemokine receptors which allow them to migrate to the site of infection where they participate in the immune response.
CD4þ T-LYMPHOCYTES The pivotal role of CD4þ T-lymphocytes has been clearly demonstrated in the setting of HIV infection in which CD4þ T-lymphocyte dysfunction and attrition significantly predisposes both adults and children to an increased risk of TB. Th1 cells elaborate several cytokines including IFN-g, IL-2, and lymphotoxin-a, which is a member of the TNF family of cytokines. These cytokines are instrumental in optimizing macrophage killing, augmenting macrophage TNF-a production, suppressing Th2 cytokine elaboration, and influencing cytotoxic responses by CD8þ T-lymphocytes, NK cells, and other CD1-restricted T-lymphocytes. Th1 cells and their cytokines play an important role in granuloma formation and organization.49
CD8þ T-LYMPHOCYTES Activated CD8þ T-lymphocytes lyse M. tuberculosis-infected macrophages by two major mechanisms. First, cytolysis via the granule exocytosis pathway depends on perforin, a pore-forming protein, which is synthesized and stored in cytotoxic secretory granules within CD8þ T-lymphocytes. Antigen-specific CD8þ T-lymphocytes form an immunological synapse with M. tuberculosis-infected macrophages. The lymphocytes then discharge the contents of their secretory granules, including perforin and granzymes A and B, into the synapse. Perforin induces the formation of pores within the plasma membrane of the macrophages, permitting entry of granzymes A and B which initiate caspase-dependent and -independent apoptosis leading to rapid cell death of targeted macrophages. Secondly, caspasedependent apoptosis follows upregulation of Fas-ligand expression on CD8þ T-lymphocytes and consequent signalling via Fas (CD95) expressed on target macrophages. In addition, activated CD8þ T-lymphocytes produce granulysin which is capable of exerting direct microbicidal activity against M. tuberculosis and, in the presence of perforin, kills intracellular bacilli. CD8þ T-lymphoctyes elaborate IFN-g and TNF-a, which are important in influencing macrophage effector responses.50
CD1-RESTRICTED T-LYMPHOCYTES CD1-restricted T-lymphocytes fulfil helper and effector functions, and interact with other cell types including macrophages and dendritic cells. In TB most activated CD1-restricted clones respond to many lipid antigens (Table 8.2). The effector CD1-restricted cells are of the Th1 type and they produce IFN-g and TNF-a and lyse M. tuberculosis-infected macrophages in a similar manner to lysis by
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Table 8.2 The survival of M. tuberculosis within macrophages is dependent upon several strategies Inhibition of macrophage activation ManLAM prevents the activation of mitogen-activated protein kinase, thereby inhibiting synthesis of TNF-a, chemokines, and the respiratory burst. Prevention of phagosome maturation Recruitment of the host protein, coronin 1, or tryptophan–aspartatecontaining coat protein (TACO) blocks the fusion of phagosome and lysosome. Mycobacterial cell wall components, e.g. ManLAM, inhibit phagosome– lysosome fusion. Mycobacterial kinase G blocks lysosomal delivery. Mycobacterial inhibition of Ca2þ-mediated signalling, generation of phosphatidylinositol-3-kinase, and the retention of Rab5 prevents phagosome maturation. Inhibition of MHC class II processing and presentation 19-kDa lipoprotein inhibits class II processing and presentation. Resistance against mycobacterial destruction Rearrangement of the cytoskeleton prevents the concentration of nitric oxide within phagolysosomes, thereby reducing its destructive effects. Nitric oxide damage is countered by the mycobacterial proteosome. Formation of anti-oxidant complexes prevent oxidant damage.
CD8þ T-lymphocytes. CD1-restricted CD4þ and CD8þ lymphocytes both elaborate granulysin and therefore exhibit direct microbiocidal activity.29
gd T-LYMPHOCYTE SUBSET The gd T-lymphocyte subset exhibits features of both innate and adaptive immunity.51 The majority of circulating gd T-lymphocytes express TCRs comprising Vg2 and Vd2 segments and are referred to as Vg2Vd2þ T-lymphocytes.52 gd T-lymphocytes recognize classical type I and II MHC molecules and non-classical molecules such as CD1 and MHC class 1b. Chemokine-receptor-mediated trafficking allows activated cells to migrate to the site of infection. Their functions include elaboration of Th1 cytokines, chemokines, perforin, and granulysin. Activated gd T-lymphocytes are capable of functioning as APCs. They express TLRs, transiently express CCR7 (a lymph node-honing chemokine receptor), up regulate surface expression of MHC class II and co-stimulatory molecules, and facilitate the proliferation of CD4þ ab T-lymphocytes.52 They also induce CD8þ ab T-lymphocytes to proliferate and differentiate into cytotoxic T-lymphocytes and they regulate macrophage and DC function through cytokine-mediated cross-talk.51
NATURAL KILLER (NK) CELLS Natural killer cells are, in common with all T-lymphocytes, derived from precursor CD34þ CD7þ cells. In general, NK cells do not require antigen-specific recognition to kill target cells but they do, however, recognize MHC class I molecules through surface receptors that deliver inhibitory rather than activating signals. A subset of NK cells, the NK T-lymphocytes (NKT cells), express the common NK cell marker, CD161, but are activated by processed lipid antigens presented in association with CD1d molecules, and therefore respond in an antigen-specific manner.27 In response to infection NK cells elaborate cytokines, particularly IFN-g, that are critical for early macrophage activation, mediate
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cytotoxicity primarily through the granule exocytic mechanism, and appear to maintain M. tuberculosis-specific CD8þ T-lymphocyte cytotoxic function by producing IFN-g, which induces macrophages to produce IL-15 and IL-18.53
INTERFERON-g Interferon-g, a homodimer produced by CD4þ, CD8þ, and gd T-lymphocytes and NK cells, is an important mediator of macrophage activation and effector function. Its importance in the immune response against mycobacteria has been confirmed by the demonstration of increased susceptibility, disease severity, and poor outcome in individuals with genetic or acquired defects/deficiencies of IFN-g, IFN-g receptor subunits, and the STAT-1 protein, all components of the IFN-g cytokine pathway.54 The IFN-g receptor complex expressed on a variety of cell types consists of IFN-gR1 and IFN-gR2 subunits. Ligand binding to the functional receptor complex usually involves the interaction of one IFN-g dimer and two IFN-g receptors. Upon binding of the IFN-g dimer to the extracellular domains of adjacent receptors, association of downstream signalling molecules leads to the activation of the Janus kinase (JAK)/signal inducer and activation of transcription1 (STAT-1) signalling cascade and consequent upregulation of IFN-g-inducible gene expression. Interferon-g induces the production of multiple proteins involved in the MHC class I and II antigen-presenting pathways, apoptotic mechanisms, nitric oxide production, and leucocyte trafficking, thus having important regulatory functions.55
CHEMOKINES, ADHESION MOLECULES AND CELLULAR MIGRATION Chemokines and their complementary chemokine receptors, as well as adhesion molecules including vascular addresins and lymphocyte- and macrophage-associated integrins, play a major role in regulating the trafficking of the cellular elements of the immune response during infection by M. tuberculosis, as well as influencing granuloma formation and organization. The inflammatory milieu that develops at the site of infection includes microbial products and a variety of cytokines, and is responsible for inducing the expression of chemokines, their receptors, and adhesion molecules.56,57 The chemokines are small molecules that mediate both constitutive and induced cell recruitment. They are grouped into several structurally defined families, including CXC, CC, C, and CX3C. Specific chemokines are able to bind more than one chemokine receptor and most receptors bind several different chemokines. Extensive chemokine expression is induced in infection by M. tuberculosis. Knockout mice experiments have established the importance of chemokines in directing the immune response to TB.57,58 Chemokines contribute to trafficking of cells, involving rolling, adhesion, and migration across endothelial barriers, by binding to G-protein-coupled transmembrane receptors, which result in actin-dependent effects such as membrane ruffling, pseudopod formation, and adhesion complex assemblage. Constitutive recruitment is responsible for the continual accumulation of naı¨ve and memory T cells in secondary lymphoid tissue including lymph nodes. Induced recruitment directs activated regulatory lymphocytes
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and effector cells including macrophages, NK cells, and CD8þ T-lymphocytes to the site of infection. Different chemokines and chemokine receptors are involved in constitutive and induced recruitment.49,57,58
MICROBIAL ELIMINATION The mechanisms by which M. tuberculosis is killed during human infection are only partly understood (Box 8.4). Macrophage effector functions augmented by the adaptive immune responses are believed to play a central role in eliminating the microbe. A key factor in the success of M. tuberculosis is its ability, through several mechanisms (Table 8.2), to survive within macrophages by obstructing macrophage activation, phagolysosomal formation, and antigen processing.59 The immune system must overcome this parasitic tendency and cross-talk, largely directed by antigen-specific cellular elements of adaptive immunity, is able to circumvent phagosomal maturational arrest through an as-yet incompletely characterized mechanism, thereby facilitating microbial killing.60 Resistance to killing of M. tuberculosis is overcome by specific induction of the production of LRG-47, a 47 kDa guanosine triphosphatase (p47 GTPase) protein.60 This is one of at least six proteins belonging to the newly identified IFN-g-inducible p47 GTPase family of proteins widely expressed in nature and important for resisting microbial infection by both plants and animals. In the absence of infection, macrophage mRNA levels of p47 genes are extremely low, but upon encountering bacteria there is a rapid and dramatic increase in p47 GTPase expression.61 IFN-g induces p47 GTPase production through the adaptor protein STAT-1, which binds directly to the genes encoding p47 GTPase proteins, thereby increasing specific mRNA production. LRG-47 is the only member of the p47 GTPases that participates in infection by M. tuberculosis in humans.60 Furthermore, experiments in LRG-47 knockout mice have shown increased susceptibility to mycobacterial infection.62 Toll-like receptor stimulation may also increase LRG-47 production through increased IFN-b production. LRG-47 is expressed mainly on endoplasmic reticulum and the Golgi apparatus, but is recruited directly to phagosomes containing M. tuberculosis where it assists in overcoming resistance to killing by facilitating phagosomal maturation. The hostile environment of the mature phagolysome, characterized by acidification and the processing of immature hydrolases, eliminates the contained mycobacteria. The mechanism by which LRG-47 facilitates maturation of phagosomes containing M. tuberculosis has not yet been elucidated.60,61
Box 8.4 Intracellular and extracellular mechanisms by which human M. tuberculosis infection is contained
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Acidified phagolysosome environment. Lysosomal hydrolases. Reactive nitrogen intermediates. Downregulation of macrophage transferrin receptors. Defensins. Indoleamine 2,3-dioxygenase-mediated containment. Macrophage apoptosis. Granulysin. The role of specific antibodies remains unknown.
IFN-g is important for inducing several macrophage antimycobacterial mechanisms. The generation of nitric oxide and related metabolites is catalysed by the IFN-g-inducible form of nitric oxide synthetase 2. Maximal induction of iNOS2 requires additional stimuli including TNF-a. Although the role of reactive nitrogen intermediates is controversial, evidence suggests that they do contribute to the elimination of M. tuberculosis and control of the infection in humans.55 Downregulation of transferrin receptor expression limits the availability of intracellular iron and therefore restricts M. tuberculosis metabolism and growth.63 Defensins are leucocytic antimicrobial peptides capable of killing a wide range of microorganisms. In mice, IFN-g induces the production of b-defensin 3, a homolog of the defensin b-2 lysin, which has been shown to be tuberculocidal.60 Furthermore, the production of indoleamine 2,3-dioxygenase enhances the anti-mycobacterial ability of macrophages and may be important in infection by M. tuberculosis.64 Apoptosis or programmed cell death is characterized by digestion of the genomic DNA of target cells by endonuclease and is mediated through cell signalling involving several ‘death’ receptors expressed on the surface of cells targeted for apoptosis. In the context of mycobacterial infection, apoptosis is a major effector mechanism by which antigen-specific T-lymphocytes and NK cells eliminate infected macrophages. Mycobacterial growth is restricted because apoptosis destroys the protected environment of the phagosome as a result of the dissolution of macrophage organelles. The released mycobacteria are taken up by uninfected macrophages or eliminated by the direct action of granulysin, thereby reducing mycobacterial load. Mycobacterium tuberculosis is a potent inducer of apoptosis, mainly mediated through the expression of TNF-a. Furthermore, the 19 kDa lipoprotein of this pathogen activates TLR2, leading to the induction of apoptosis. Fas ligandinduced apoptosis, mediated by T-lymphocytes and NK cells, results in the destruction of intracellular organelles, the uptake of apoptotic bodies by adjacent phagocytes, and the inhibition of mycobacterial spread and growth. Apoptosis occurs in granulomas and may therefore modulate cell turnover and limit the spread of infection.50
GRANULOMA FORMATION AND FUNCTION Granuloma formation correlates with the development of delayed hypersensitivity revealed by a positive tuberculin test, emergence of activated T-cell subsets, improved macrophage responsiveness in areas of localized infection, and improved phagocytosis and mycobacterial killing. The granuloma plays a pivotal role in orchestrating the interaction between immune cells leading to an effective response, inhibiting and killing M. tuberculosis, containing infection, preventing the spread of the organism, and localizing the inflammatory response and tissue damage.57 Granuloma formation may be divided into four phases: initiation, accumulation, effector, and resolution. Very little is known about the initiation phase, but macrophages migrate into the site of infection and initiate granuloma formation. Both initiation and accumulation phases involve cellular recruitment.65 Knockout mice experiments have established the importance of both TNFa and lymphotoxin-a in granuloma formation. These cytokines achieve their effect by regulating the expression of adhesion molecules and chemokine receptors, and establishing chemokine gradients.49,57 T-lymphocytes are involved in all four phases.65
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accumulate within granulomas and research in mice suggests that these may assist in down regulating the inflammatory responses.65,68
IMMUNE RESPONSES IN CHILDREN
Fig. 8.5 A mature, caseating tuberculous granuloma with an area of central necrosis surrounded by a typical cellular infiltrate.
A fully formed tuberculous granuloma consists of a central zone of caseating necrosis, surrounded by cellular infiltrate comprising activated macrophages (epithelioid cells), DCs, T-lymphocytes, Blymphocytes, and fibroblasts. Macrophages are centrally located and have the ability to differentiate into multinucleated giant cells. The outer layer of the human granuloma structurally resembles secondary lymphoid follicles and contains APCs, CD4þ T-lymphocytes, CD8þ T-lymphoctes, and B-lymphocytes where the host and pathogen probably interact (Fig. 8.5).4 T-lymphocyte proliferation is limited in granulomata. T-cell receptor diversity is determined by cellular migration and possibly T-lymphocyte priming, which is thought to occur in the outer layers of the granuloma.65 In productive granuloma without recognizable caseation very few cells show apoptotic features but large numbers of macrophages and lymphocytes undergoing apoptosis are seen within caseating granulomas.66 Granuloma formation is accompanied by a change in the mycobacterial gene expression pattern as the organisms adapt to the environment of the granuloma. Loss of acid-fastness during latency suggests that the cell wall structure of dormant M. tuberculosis is altered.67 The location of the latent organisms remains uncertain but one study indicates that they are present in tissue outside the areas of caseating necrosis.14 During the effector phase macrophage–lymphocyte cooperation plays a determining role in reducing the pathogen burden within granulomas. Resolution of granulomas involves immunomodulatory cytokines including TGF-b and IL-10. Interleukin-13 and TGF-b induce fibrosis, the final step in resolution. gd T-lymphocytes
REFERENCES 1. Warren R, Victor TC, Streicher EM, et al. Patients with active tuberculosis often have different strains in the same specimen. Am J Respir Crit Care Med 2004;169:610–614. 2. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:498–511. 3. Kaufmann SHE. How can immunology contribute to the control of tuberculosis? Nat Rev Immunol 2001;1:20–30. 4. Ulrichs T, Kaufmann SHE. New insights into the function of granulomas in human tuberculosis. J Pathol 2006;208:261–269.
There are several immunocompromised states that predispose infected humans to active TB, including the relative immaturity and naivety of the immune system of very young children, waning immune competency in the elderly, primary immunodeficiency disorders, particularly genetic disorders of the IFN-g cytokine pathway, and secondary immunodeficiencies such as HIV infection. Young children, particularly those less than 1 year of age, are at a greater risk for developing TB than older children or adults and are at an increased risk for disseminated disease including tuberculous meningitis. These clinical vulnerabilities reflect the underlying immunological susceptibility of young children in whom both innate and adaptive responses are relatively weak. Evidence for impaired innate responses is mainly derived from animal studies and support suboptimal macrophage functionality. Dendritic cells play a critical role in regulating several immunological responses to infection by M. tuberculosis and deficiencies in their function have been demonstrated in both children and young animals. Studies in young children with congenital infections suggest that antigen-specific CD4þ T-lymphocyte responses are impaired, neonatal T-lymphocytes demonstrate reduced capacity to elaborate IFN-g and TNF-a, and following antigenic stimulation, cytokine production is biased towards a Th2 pattern. Furthermore, virus-specific CD8þ T-lymphocyte responses are suboptimal in young children.69,70
CONCLUSION The immune response following infection by M. tuberculosis is only partly understood. Recent progress has defined in great detail many intricate molecular events that underpin mycobacterial containment and clinical latency observed in the majority of individuals who encounter the organism. Key immunological processes include antigen recognition, adaptive immune responses, granuloma formation, microbial containment, and killing. Recent advances have, unfortunately, not been matched by improved therapeutic options. More attention should be given to translating fundamental immunological concepts into effective antimicrobial and immunomodulatory interventions so that therapy and preventive measures may be improved, particularly in resource-poor countries.
5. Glatman-Freedman A, Casadevall A. Serum therapy for tuberculosis revisited: Reappraisal of the role of antibody-mediated immunity against Mycobacterium tuberculosis. Clin Microbiol Rev 1998;11:514–532. 6. Van Crevel R, Ottenhoff THM, van der Meer JWM. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002;15:294–309. 7. Ernst JD. Macrophage receptors for Mycobacterium tuberculosis. Infect Immun 1998;66:1277–1281. 8. Velasco-Vela´zquez MA, Barrera D, Gonza´lez-Arenas A, et al. Macrophage-Mycobacterium tuberculosis interactions: role of complement receptor 3. Microbial Pathogenesis 2003;35:125–131. 9. Ehlers MRW, Daffe´ M. Interactions between Mycobacterium tuberculosis and host cells: are
10.
11.
12.
13.
14.
mycobacterial sugars the key? Trends Microbiol 1998;6:328–335. Van Kooyk Y, Geijtenbeek TBH. DC-SIGN: Escape mechanisms for pathogens. Nat Rev Immunol 2003;3:697–709. Hope JC, Thom ML, McCormick PA, et al. Interaction of antigen presenting cells with mycobacteria. Vet Immunol Immunopathol 2004;100:187–195. Doherty TM, Arditi M. TB or not TB: that is the question - does TLR signalling hold the answer? J Clin Invest 2004;114:1699–1703. Barton GM, Medzhitov R. Control of adaptive immune responses by Toll-like receptors. Curr Opin Immunol 2002;14:380–383. Barton GM, Medzhitov R. Toll-like receptor signalling pathways. Science 2003;300:1524–1525.
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15. Krutzik SR, Modlin RL. The role of toll-like receptors in combating mycobacteria. Semin Immunol 2004;16:35–41. 16. Kaufmann SHE, Schaible U. Antigen presentation and recognition in bacterial infections. Curr Opin Immunol 2005;17:79–87. 17. Koul A, Herget T, Klebl B, et al. Interplay between mycobacteria and host signaling pathways. Nat Rev Microbiol 2004;2:189–202. 18. Russell DG. Mycobacterium tuberculosis: Here today, and here tomorrow. Nat Rev 2001;2:1–9. 19. Viera OV, Botelho RJ, Grinstein S. Phagosome maturation: aging gracefully. Biochem J 2002;366: 689–704. 20. Blander JM, Medzhitov R. On regulation of phagosome maturation and antigen presentation. Nat Immunol 2006;7:1029–1035. 21. Blander JM, Medzhitov R. Regulation of phagosome maturation by signals from toll-like receptors. Science 2004;304:1014–1018. 22. Broom WH, Canaday DH, Fulton SA, et al. Human immunity to M. tuberculosis: T cell subsets and antigen processing. Tuberculosis 2003;83:98–106. 23. Ramachandra L, Noss E, Boom WH, et al. Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules. Cell Microbiol 1999;1:205–214. 24. Flutter B, Gao B. MHC class I antigen presentation – recently trimmed and well presented. Cell Mol Immunol 2004;1:22–30. 25. Winau F, Kaufmann SHE, Schaible UE. Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria. Cell Microbiol 2004;6:599–607. 26. Lewinsohn DM, Grotzke JE, Heinzel AS, et al. Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I-antigenprocessing pathway. J Immunol 2006,177: 437–442. 27. Gumperz JE, Brenner MB. CD1-specific T cells in microbial immunity. Curr Opin Immunol 2001; 13:471–478. 28. De La Salle H, Mariotti S, Angenieux C, et al. Assistance of microbial glycolipid antigen processing by CD1e. Science 2005;310:1321–1324. 29. De Libero G, Mori L. Mechanisms of lipid-antigen generation and presentation to T cells. Trends Immunol 2006;27:485–492. 30. Spellberg B, Edwards JE. Type 1/Type 2 immunity in infectious diseases. Clin Infect Dis 2001;32:76–102. 31. Chaco´n-Salinas R, Serafin-Lo´pez J, Ramos-Paya´n R, et al. Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 2005;140:443–449. 32. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–146. 33. Langrish CL, McKenzie BS, Wilson NJ, et al. IL-12 and IL-23: Master regulators of innate and adaptive immunity. Immunol Rev 2004;202:96–105. 34. Reddy P. Interleukin-18: recent advances. Curr Opin Hematol 2004;11:405–410.
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35. Netea MG, Azam T, Lewis EC, et al. Mycobacterium tuberculosis induces interleukin-32 production through a caspase-1/IL-18/interferon-g-dependent mechanism. Plos Med 2006;3:e277. URL: http:// www.plosmedicine.org 36. Happel KI, Lockhart EA, Mason CM, et al. Pulmonary interleukin-23 gene delivery increases local T-cell immunity and controls growth of Mycobacterium tuberculosis in the lungs. Infect Immun 2005;73:5782–5788. 37. Aggarwal S, Gurney AL. IL-17: prototype member of an emerging cytokine family. J Leukoc Biol 2002;71:1–8. 38. Lockhart E, Green AM, Flynn JL. IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006;177:4662–4669. 39. Villarino AV, Hunter CA. Biology of recently discovered cytokines: discerning the pro- and anti-inflammatory properties of interleukin-27. Arthritis Res Ther 2004;6:225–233. 40. Ho¨lscher C, Ho¨lscher A, Ru¨ckerl D, et al. The IL-27 receptor chain WSW-1 differentially regulates antibacterial immunity and survival during experimental tuberculosis. J Immunol 2005;174: 3534–3544. 41. Pfeffer K. Biological functions of tumour necrosis factor cytokines and their receptors. Cytokine Growth Factor Rev 2003;14:185–191. 42. Gardam MA, Keystone EC, Menzies R, et al. Antitumour necrosis factor agents and tuberculosis risk: mechanisms of action and clinical management. Lancet Infect Dis 2003;3:148–155. 43. Stenger S. Immunological control of tuberculosis: role of tumour necrosis factor and more. Ann Rheum Dis 2005;64:24–28. 44. Rook GAW, Hernandez-Pando R, Dheda K, et al. IL-4 in tuberculosis: implications for vaccine design. Trends Immunol 2004;25:483–488. 45. Jung Y, LaCourse R, Ryan L, et al. Evidence inconsistent with a negative influence of T helper 2 cells on protection afforded by a dominant T helper 1 response against Mycobacterium tuberculosis lung infection in mice. Infect Immun 2002;70:6436–6443. 46. Choudhuri K, Kearney A, Bakker TR, et al. Immunology: How do T cells recognize antigen? Curr Biol 2005;15:R382–R385. 47. Van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol 2003;21:659–684. 48. McGuirk P, Mills KHG. Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends Immunol 2002;23:450–455. 49. Peters W, Ernst JD. Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis. Microbes Infect 2003;5:151–158. 50. Grotzke JE, Lewinsohn DM. Role of CD8þ T lymphocytes in control of Mycobacterium tuberculosis infection. Microbes Infect 2005;7:776–788. 51. Brandes M, Willimann K, Moser B. Professional antigen presentation function by human gd T cells. Science 2005;309:264–268.
52. Chen ZW, Letvin NL. Vg2Vd2þ T cells and antimicrobial immune responses. Microbes Infect 2003; 5:491–498. 53. Vankayalapati R, Klucar P, Wizel B, et al. NK cells regulate CD8þ T cell effector function in response to an intracellular pathogen. J Immunol 2004;172;130–137. 54. Ottenhoff THM, Verreck FAW, Lichtenauer-Kaligis EGR, et al. Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae. Nat Genet 2002;32:97–105. 55. Schroder K, Hertzog PJ, Ravasi T, et al. Interferon-g: an overview of signals, mechanisms and functions. J Leukoc Biol 2004;75:163–189. 56. Kerr JR. Cell adhesion molecules in the pathogenesis of and host defense against microbial infection. J Clin Pathol Mol Pathol 1999;52:220–230. 57. Algood HMS, Chan J, Flynn JL. Chemokines and tuberculosis. Cytokine Growth Factor Rev 2003;14: 467–477. 58. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol 2000;18:217–242. 59. Vergne I, Chua J, Singh SB. Cell biology of Mycobacterium tuberculosis: phagosome. Annu Rev Cell Dev Biol 2004;20:367–394. 60. MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFN-g-inducible LRG-47. Science 2003;302:654–659. 61. MacMicking JD. Immune control of phagosomal bacteria by p47 GTPases. Curr Opin Microbiol 2005;8:74–82. 62. Feng CG, Collazo-Custodio CM, Eckhaus M, et al. Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia. J Immunol 2004;172:1163–1168. 63. Olakanmi O, Schlesinger LS, Britigan BE. Hereditary hemochromatosis results in decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages. J Leukoc Biol 2007;81:195–204. 64. Hayashi T, Rao SP, Takabayashi K, et al. Enhancement of innate immunity against Mycobacterium avium infection by immunostimulatory DNA is mediated by indoleamine 2,3-dioxygenase. Infect Immun 2001;69:6156–6164. 65. Co DO, Hogan LH, Il-Kim S, et al. T cell contributions to the different phases of granuloma formation. Immunol Lett 2004;92:135–142. 66. Fayyazi A, Eichmeyer B, Soruri A, et al. Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis. J Pathol 2000;191:417–425. 67. Seiler P, Ulrichs T, Bandermann S, et al. Cell-wall alterations as an attribute of Mycobacterium tuberculosis in latent infection. J Infect Dis 2003;188:1326–1331. 68. Ladel CH, Blum C, Dreher A, et al. Protective role of gamma/delta T cells and alpha/beta T cells in tuberculosis. Eur J Immunol 1995;25:2877–2881. 69. Lewinsohn DA, Gennaro ML, Scholvinck L, et al. Tuberculosis immunology in children: diagnostic and therapeutic challenges and opportunities. Int J Tuberc Lung Dis 2004;8:658–674. 70. Smith S, Jacobs RF, Wilson CB. Immunobiology of childhood tuberculosis: a window on the ontogeny of cellular immunity. J Pediatr 1997;131:16–26.
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9
Host susceptibility and resistance to Mycobacterium tuberculosis Genetic, neuroendocrine, and acquired factors Graham AW Rook and T Mark Doherty
INTRODUCTION Susceptibility to TB, like susceptibility to other infections, is controlled by genetic factors, as well as by environmental factors and interactions between the two. The role of host genetic variation in susceptibility to TB is evident from animal models of the disease, ethnic clustering of TB cases, and increased concordance rates for TB among monozygotic twins compared with dizygotic twins. However, for some of the genes discussed below there is striking disagreement between studies in different populations. It is important to bear in mind the fact that an association between protection/susceptibility and a particular allele can be misleading. The allele in question might not be the true disease susceptibility allele, and the pattern of linkage disequilibrium between the allele and the true disease-causing allele might be different in different populations. For instance, different studies give precisely reverse associations between single nucleotide polymorphisms (SNPs) of tumour necrosis factor-a (TNF-a) and susceptibility to TB, but there is strong linkage disequilibrium with HLA genes, so we do not discuss these TNF-a SNPs below. Finally, the relevance of a genetic variant often depends upon the environment in which the individual lives: gene–environment interactions. For instance one might guess that there will be polymorphisms relevant to people with simultaneous helminth infections, but irrelevant in people without such infections. Moreover the Mycobacterium tuberculosis strains are also variable. Humans and M. tuberculosis have co-evolved over thousands of years, so not all strains of M. tuberculosis are equivalent. Indeed, it has been suggested that different strains have tropisms for specific human populations so the relevance of a particular human polymorphism might be determined by interaction with polymorphisms in the bacteria that infect the population in question.1 In this chapter we deal first with genetic factors, and then with environmental ones, though, because of these interactions, there is inevitably some overlap. The basic immunology of TB and the impact of human immunodeficiency virus (HIV) are covered in their own chapters.
GENETIC FACTORS AND SUSCEPTIBILITY TO TUBERCULOSIS Both animal and human studies have identified cell-mediated immunity, particularly those pathways involving interferon (IFN)-g as
crucial to protection against M. tuberculosis. There are multiple factors that can influence this, but this topic is discussed in detail elsewhere in this volume. Here, the focus is not on immunology, but on those factors where genetic evidence for a role in immunity is known.
COMPONENTS OF THE Th1 PATHWAY IL-12 and IL-23 and their receptors Interleukin (IL)-12 consists of two subunits, p35 and p40, encoded by IL12A and IL12B respectively. IL-23 also uses the p40 subunit, but has a unique second subunit, p19. A child who was homozygous for a large loss-of-function deletion in IL12B suffered from infections caused by Bacillus Calmette–Gue´rin (BCG) and Salmonella enteritidis. Both IL-12 and IL-23 will have been non-functional in this child. Several other loss-of-function genotypes have been detected and patients with these variants also tend to suffer from mycobacterial infections.2,3 Polymorphisms that do not cause loss of function, but cause less extreme modulation of the expression of the p40, can affect susceptibility to TB, protection being associated with genotypes leading to high production and vice versa (Fig. 9.1).4 The IL-12 receptor has two subunits, IL-12Rb1 (shared with the IL-23 receptor) and IL-12Rb2. The second (non-shared) subunit of the IL-23 receptor is designated IL-23Rb3. Defects in the gene encoding IL-12Rb1 (IL12Rb1) are more common than defects in the cytokine itself. Since IL-12Rb1 also forms part of the IL-23 receptor, the defects again affect the function of both cytokines. Severe defects lead to Mendelian susceptibility to mycobacterial disease.5 More recently it has been noted that less severe polymorphisms that do not cause loss of function can also have significant effects on susceptibility, but this is not seen in all studies.6–8 Defects in IL12Rb2 have not been found, probably because this would leave signalling via IL-23 intact, and this activity may compensate adequately. IFN-g and IFN-gR Associations between TB and polymorphisms of IFN-g have been reported, but the most striking data come from the study of genetic variants of the IFN-g receptor (Fig. 9.1).9,10 This receptor has two chains, the ligand binding chain, IFN-gR1, and a second chain, IFN-gR2, encoded by IFNGR1 and IFNGR2, respectively. Complete absence of functional IFN-gR can result from recessive mutations (most often in IFNGR1), and leads to a severe defect.11
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M. tuberculosis CD8 MHC Class I MCH Class II
CD4+ Th1 IL-12R & IL-23R
Mac or DC IL-12 IL-23 IL-1 & IL-1RA
IFN-g IFN-g receptor STAT-1
Mac
Nucleus
Fig. 9.1 Pathways immediately relevant to the induction of interleukin (IL)-12/IL-23/interferon (IFN)-g-mediated immunity, in which it is claimed that there are polymorphisms relevant to susceptibility to TB.
There is striking susceptibility to progressive BCG infection and to non-tuberculous mycobacteria such as members of the Mycobacterium avium/intracellulare group, but not usually increased susceptibility to Salmonellae. Complete lack of IFN-gR1 has been seen in several TB patients.3 However there are other genetic defects leading to less severe syndromes, and there are conflicting data as to whether partial IFN-gR defects have any effect on susceptibility to, or severity of, TB.12,13 This raises fascinating issues. It might simply be that exposure to BCG or M. avium is much more frequent than exposure to M. tuberculosis. Alternatively, TB patients with such defects might die too quickly or they might not be investigated properly. However, the possibility remains that the IL-12/IL-23/IFN-g axis is less crucial for resistance to M. tuberculosis perhaps because it is able to block release of IL-12 and the response to IFN-g.14,15 In such a case, other pathways such as cytotoxicity or bactericidal peptides may play a larger role.16,17
STAT-1 STAT-1 encodes the signal-transducer and activator of transcription associated with signalling via the IFN-gR (Fig. 9.1). Partial deficiencies have been detected in kindreds with mycobacterial infections, and complete deficiency was found in two unrelated infants that had experienced disseminated BCG infection. While BCG could be successfully cleared by anti-mycobacterial therapy, these children eventually died from viral infections, perhaps because signalling via IFN-b and IFN-a was also impaired.18 Again it is not clear how crucial these pathways are for immunity to M. tuberculosis itself – the importance is inferred from susceptibility to more common mycobacteria. IL-1 Both IL-1b and its natural inhibitor, the IL-1 receptor antgonist (IL-1RA) are the products of polymorphic genes (Fig. 9.1). There are allelic variants that result in relatively higher or lower levels of production. The most proinflammatory haplotype (high IL-1b, low IL-1RA) was significantly associated with tuberculous pleurisy (92%) in comparison with healthy M. tuberculosis-sensitized control subjects or patients with other disease forms. In contrast, the high IL-1RA haplotype was associated with a reduced Mantoux response to purified protein derivative (PPD).19 The conclusion is that, while IL-1 polymorphisms may contribute to the pathogenesis of TB, they are not the major factor.19,20
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HLA MHC class II. The HLA class II molecules expressed by antigen-presenting cells present peptide antigens to CD4 T cells (Fig. 9.1). DQ and DR are two of five isotypes of HLA class II proteins; heterodimers composed of a and b chains, encoded by the highly polymorphic loci HLA-DQ and -DR, respectively. The HLA class II serotype, DR2, encoded by alleles DRB115 and DRB116, is associated with TB in several Indian populations, but no such association was found in studies in Poland or Cambodia.21 However, the DQB10503 HLA class II allele was associated with TB in Cambodia.22 Further work suggested that this association might be due to HLA alleles encoding an aspartic acid at codon 57 of the HLA-DQ b-chain. It was revealed that homozygosity for this SNP was strongly associated with progressive pulmonary TB.23 Moreover this variant was shown to bind a peptide from the central region of the M. tuberculosis-secreted protein ESAT-6 with low affinity and to stimulate less IFN-g production from CD4 T cells from TB patients than when this peptide was presented by an HLA-DQ-b allele encoding an alanine at codon 57.23 These observations appear important. In the Venda people in the Limpopo Province of South Africa, TB is significantly associated with HLA class II variants, common in Africa, and previously associated with resistance to malaria and hepatitis.21 Interestingly, most of the HLA-DQB1 alleles associated with TB in the Venda (alone or in haplotypes) matched alleles associated with progressive TB in Cambodia when present in homozygous form. All these alleles also encode Asp at position 57 of the HLA-DQ b-chain. Thus, although the associations of class II MHC alleles and SNPs with TB are different in different communities, there are some potentially important commonalities.21 MHC class I. There have been fewer studies of MHC class I, which presents antigens to CD8 T cells (Fig. 9.1), though recent work suggests that class 1 restricted CD8 T cells may be very important.16 A study in India suggested that HLA-B52 might be protective, while HLA-B51 was associated with susceptibility to pulmonary TB.24 CYTOKINES AND CHEMOKINES THAT MIGHT BE DETRIMENTAL TO IMMUNITY (FIG. 9.2) IFN-a/b In a mouse model, the virulence of a strain of M. tuberculosis was attributed to its tendency to drive production of IFN-a and -b rather than a Th1 response.25 It also emerged that IFN-a and -b inhibit the antimycobacterial effect of human macrophages and that hypervirulent Beijing strains of M. tuberculosis tend to up regulate IFN-a/b.26,27 However, a genetic variant that results in high levels of IFN-a/b was associated with susceptibility to sarcoidosis, but not to TB in a Japanese population.28 Th2-biasing chemokines (CCL2 and CCL18) and cytokines (IL-4) A polymorphism of the promoter region of the gene encoding CCL2 (also known as MCP-1) that results in excessive production of this chemokine is common in Mexico (50% of the population) and is strongly associated with TB both in Mexico and in Korea.29 This polymorphism might account for a significant fraction of susceptibility to TB in these communities, and has been described as the most substantial impact ever described of a human allele on adult TB at the population level.30 CCL2 drives recruitment of Th2 cells in the mouse and is associated with Th2 cytokines in man.31,32
CHAPTER
Host susceptibility and resistance to Mycobacterium tuberculosis Drive Th2 and / or Treg
M. tuberculosis
TGF-b IL-10 IL-4 Mac (or DC)
Tregs
different populations, but the associations with TB are inconsistent.44 Several studies have also looked at polymorphisms of TGF-b, but here too the results are variable, as discussed by Amirzargar et al.37
Th2
RECEPTORS FOR M. TUBERCULOSIS AND ITS COMPONENTS
Suppress anti-mycobacterial effects IFN-a/b CCL18 CCL2
9
Attract Th2 cells
Suppress IL-12
Fig. 9.2 Some relevant effects of cytokines and chemokines that might undermine the IL-12/IL-23/IFN-g pathway, and in which it is claimed that there are polymorphisms relevant to susceptibility to TB. All abbreviations are explained in the main text. Most of these mediators can be released by antigen-presenting cells and macrophages, in which case the response may be deviated towards Th2 or regulatory T cells. These T cells then become further sources of the potentially detrimental mediators. It is unlikely that any of these mediators is wholly detrimental, but they may become so when the balance of mediators is suboptimal. In particular, some regulatory cells probably play an essential role in controlling immunopathology.
The polymorphism described in Mexico and Korea is associated with asthma in Europe.33 Similarly, another Th2-associated chemokine, CCL18, might be associated with susceptibility to TB in Brazil.34 CCL18 also preferentially recruits Th2 cells and basophils, and is associated with asthma and atopic dermatitis.35,36 In conclusion part of the increased IL-4 that accompanies TB in developing countries might have a genetic basis. There is a single report that a C-T substitution in the promoter (590) of the gene encoding IL-4 is associated with protection from TB in an Iranian population.37 The significance of this is unclear. In an American population the 590 T allele was said to be associated with higher IL-4 and IgE, but in Taiwanese infants the 590 C allele was associated with higher IgE. At this stage all we can say is that it is interesting that a SNP likely to affect regulation of IL-4 might be relevant to TB. A splice variant of IL-4, designated IL-4d2, acts as an inhibitor of IL-4 and is increased in the unstimulated blood cells of individuals with stable latent TB.38 Interestingly there is, as one might expect, decreased allergic sensitization in these people with stable latent TB, possibly attributable to expression of this IL-4 antagonist.39 In sharp contrast, patients in whom TB progresses have increased expression of the agonist, IL-4, a corresponding increase in allergic sensitization, and reduced CMI responses.40–42
IL-10 and TGF-b These two cytokines are released by various regulatory cell types (described below), and can be present at high levels in progressive TB. Some authors have suggested that they are implicated in the failure of immunity to M. tuberculosis. In an African population, a variant of Slc11a1 (this gene is dicussed in detail later) associated with susceptibility to adult TB was found also to associate with higher release of IL-10 by macrophages in response to lipopolysaccharide (LPS).43 This suggested that the monocytes of people susceptible to TB might be inherently more prone to exaggerated IL-10 release, and raised the possibility that this is how SLC11A1 influences susceptibility to TB. Subsequently various polymorphisms of IL-10 have been studied in
M. tuberculosis interacts with numerous soluble molecules that modulate subsequent interactions with cells, and numerous receptors on human cells that influence immediate signalling to the cell, the initial cytokine response, and what happens to the organism after uptake (Fig. 9.3). In addition to antibody and complement, M. tuberculosis binds the surfactant proteins (SP-A and SP-D) and mannose-binding lectin (MBL) and dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), all of which are C-type lectins. M. tuberculosis also contains multiple ligands for Toll-like receptors. A number of these have genetic variants that influence susceptibility, confirming their importance.
Surfactant proteins A and D (SP-A, SP-D) Pulmonary surfactant and its components are essential for normal lung function but also play poorly defined roles in local host defence. Surfactant proteins are lectins, and bind to M. tuberculosis via carbohydrates in a saturatable and Ca2þ-dependent manner, and also bind to alveolar macrophages. SP-A enhances attachment of M. tuberculosis to alveolar macrophages, while SP-D agglutinates the organisms, though it is not clear whether (or if ) this helps the host or the organism.45 Two studies, one in a Mexican population and the other among Indians, suggested that some polymorphisms in these molecules are relevant to susceptibility to TB (Fig. 9.3).45,46 Mannose-binding lectin Mannose-binding lectin plays an important role in the early stages of primary infections and during the decay of maternal antibodies in infants. There are haplotypes resulting in low, intermediate, or high serum levels of MBL. It binds to mannose on the surface of M. tuberculosis and fixes complement, so high levels might cause
Mac (or DC)
M. tuberculosis
SP-A or SP-D
MBL NOD2
Modulate antimycobacterial function
SP110 Slc11a1
? DC-SIGN
TLR
Signals that regulate cellular functions
VDR
P2X7 purinergic receptor Vitamin D3
Nucleus
Fig. 9.3 Molecules that modulate the initial interaction between M. tuberculosis and the macrophage or dendritic cell (DC) are shown on the left. The diagram is not comprehensive, and shows only those factors in which it is reported that there are polymorphisms relevant to susceptibility to TB. By altering this initial interaction they also alter the signals that drive subsequent cellular functions. On the right are four other genetically variable molecules thought to modulate anti-mycobacterial functions. Vitamin D3, signalling via the vitamin D3 receptor (VDR), increases expression of anti-mycobacterial cathelicidins. TLR, Toll-like receptor.
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the organisms to be taken up via complement receptors (Fig. 9.3). On the other hand, when levels of MBL are low the mannose will bind to membrane mannose receptors, so uptake will be differently mediated.47 This might explain one study that has concluded that haplotypes resulting in low serum levels of MBL are protective though the reverse was found in a Chinese population.48,49
DC-SIGN DC-SIGN is another C-type lectin expressed mainly on the membranes of DC (Fig. 9.3). A recent study in a coloured population in South Africa suggested that two SNP variants (871G and 336A) confer protection against TB. Curiously, in the South African study, one of these (336A) was associated with increased expression of DC-SIGN, despite the fact that previous work had suggested that, by interacting with this receptor on DC, M. tuberculosis is able to pervert the immune response, and trigger release of IL-10.50 However, no significant association was found in a Colombian population.51 Toll-like receptors and NOD2 A polymorphism of TLR2 (arginine to glutamine substitution at residue 753 (Arg753Gln)) has been associated with decreased TLR2 function. If TLR2 is important for triggering protective mechanisms such as the cathelicidin pathway, one might expect this polymorphism to be associated with susceptibility to TB.17 This was found to be the case, particularly in homozygotes.52 Another polymorphism (TLR2 Arg677Trp polymorphism, due to a C2029T nucleotide substitution) results in non-functional TLR2 and is associated with lepromatous leprosy.53 This substitution appeared to be a risk factor for TB in Tunisia.54 These findings are supported by a study of Toll–interleukin 1 receptor domain containing adaptor protein (TIRAP), an adaptor protein that mediates signals from Toll-like receptors activated by mycobacteria. The SNP C558T was associated with susceptibility to TB, particularly TB meningitis, and appeared to result in decreased signalling.55 Several components of M. tuberculosis signal via TLR2, but signalling via TLR4 is much weaker. The Asp299Gly polymorphism in human TLR4 is associated with in vivo hyporesponsiveness to LPS in Caucasians. However, no association with susceptibility to TB was found in the Gambia.56 The nucleotide oligomerization binding domain 2 gene (NOD2) encodes an intracellular receptor for bacterial components, which is expressed in monocytes and a polymorphism in this gene is associated with Crohn’s disease, for which some authors suspect a mycobacterial cause. However, no association was found with TB in a Gambian population.57 MACROPHAGE FUNCTION Vitamin D3 (calcitriol) In the 1940s attempts were made to treat TB with vitamin D3 (VitD3). When patients with skin TB (Lupus vulgaris, often due to Mycobacterium bovis) were treated with VitD3, the chronic non-healing granulomatous lesions often underwent necrosis followed by resolution.58 However, necrosis and liquefaction also occurred in deep lesions in the spine and lungs if these were present, so this therapy was discontinued, and the role of VitD3 remained uncertain.59 Circulating levels of VitD3 can be high in TB patients, because IFN-g causes upregulation of a 1-a-hydroxylase, which generates VitD3 from a circulating precursor, 25-hydroxyvitamin D3.60 A clue that this might be a protective mechanism came from the observation that notifications of TB peaked in the summer season in England and Wales, particularly among immigrants from the
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Indian subcontinent. The suggestion was that individuals with darker skins living in the UK became VitD3 deficient during the winter, leading to increased reactivation of TB, manifested clinically a few months later.61 This hypothesis assumed that VitD3 was beneficial. VitD3 was shown in 1986 to enhance the ability of human macrophages to control the growth of M. tuberculosis.62 This observation has been consolidated by the discovery that stimulation of macrophages or monocytes via TLR-2/TLR-1 heterodimers leads to upregulation not only of 1-a-hydroxylase but also of VitD3 receptors (VDR). Then VitD3 generated from 25-hydroxyvitamin D in the serum signalled via the specific VDR to induce the antimicrobial peptide cathelicidin and killing of intracellular M. tuberculosis.17 It was also noted that serum from African American individuals was less able to support this process, and this was attributed to the presence of lower levels of circulating 25-hydroxyvitamin D. This provides a potentially satisfying explanation for the apparent greater susceptibility of African Americans to TB. Further support has come from the study of genetic polymorphisms of the VDR. Homozygotes for a polymorphism at codon 352 were significantly underrepresented among individuals with TB.63 Numerous studies have revealed similar findings, but complete haplotypes tend to correlate with susceptibility or resistance to TB rather than individual polymorphisms.21 Thus it appears that the influence of polymorphisms on VDR activity is collective rather than individual. Haplotypes of VDR polymorphisms located in the promoter region, coding regions for ligand- or DNA-binding domains, and the 30 UTR, respectively, influence VDR expression, VDR transactivation activity, and mRNA stability, respectively, and collectively influence function. Protective haplotypes appear to represent more active forms of the receptor.21
Slc11a1 (formerly Nramp1) Solute carrier 11a1 (Slc11a1; formerly Nramp1; natural-resistanceassociated macrophage protein) is a proton/bivalent cation transporter that in phagocytosing macrophages is rapidly recruited to the membrane of late endosomes/lysosomes.64 Its relevance to immunity to TB was first observed in genetic studies of susceptible and resistant strains of mice where it was found to affect immunity to multiple different intracellular pathogens. Recent studies in mouse macrophages and in Dictyostelium amoebae suggest that Slc11a1 is involved in both influx and efflux of iron and other cations from late endosomes/lysosomes, the direction of transport depending upon other factors such as the pH and availability of ATP and the V-Hþ ATPase.64,65 The evidence suggests that, under physiological conditions of low intravacuolar pH, Slc11a1 transports bivalent cations into, rather than out of, late endosomes. So if Slc11a1 is involved in removing from the cell any iron taken up during phagocytosis, it might do so via a lysosomal secretory pathway, rather than by pumping iron out of the phagosome and into the cytoplasm. This means that, if M. tuberculosis is able to disturb maturation of the phagosome, regulation of intraphagosomal pH, and expression or function of V-Hþ ATPases, then it is possible that it can exploit Slc11a1 in order to scavenge iron by profiting from the increased iron in late endosomes. On the other hand Peracino et al.,65 working admittedly with Dictyostelium, suggest that if the organisms were in non-acidic, Slc11a1-positive vesicles in the post-lysosomal pathway, in the presence of abundant V-Hþ ATPase, the role of Slc11a1 might be phagosomal depletion of iron and other metal ions, which are essential for bacterial growth. However, a different view is that iron, whether moving into or out of the endosomes, is not the whole story. Slc11a1 is involved in cation fluxes that abrogate pathogen-induced blockage of phagosome maturation, and also, as mentioned earlier, seems to modulate IL-10 release.43 The truth is
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position 762 of the gene encoding the P2X7 receptor (P2RX7) in a Gambian population.69
that the role of Slc11a1 in human macrophages is not known, but it is clear that we should not be surprised that the interactions of polymorphisms of Slc11a1 with susceptibility to TB are remarkably complex. Bellamy and colleagues66 typed polymorphisms of Slc11a1 in a case–control study of TB in the Gambia, West Africa. Four polymorphisms were significantly associated with TB. Some subsequent studies have yielded similar results, but many have not.67 A metaanalysis suggests no associations with TB in subjects of European descent.67 However, a recent paper looked at associations with paediatric TB, and might have cast light on the discrepancies.68 In this paediatric study the direction of Slc11a1 allele association with TB was inverted compared with previous studies in adult pulmonary TB. Thus common Slc11a1 alleles associated with protection from adult TB emerged as risk factors for paediatric TB. The authors suggested that the common alleles are risk factors for TB with an early onset following exposure. Thus the discrepancies can be rationalized by suggesting that the common Slc11a1 alleles predispose to rapid progression, but, if that does not occur, are then associated with stable latency. In view of the complexity of the role of Slc11a1 in control of iron and of phagosome maturation and cytokine profiles, complexity at this level is not surprising.
Ipr1 and the putative human homologue, SP110 Studies in mice identified Ipr1 (intracellular pathogen resistance 1) as a gene responsible for innate resistance of macrophages to intracellular proliferation of M. tuberculosis. The closest human homologue of Ipr1 is SP110, though it has only 41% identity at the amino acid level. SP110 is induced by IFN-g and is a component of the nuclear body, a multi-protein complex thought to participate in gene regulation. Three polymorphisms of this gene were associated with disease in a population in the Gambia.70 However, large studies in Ghana and Russia failed to find associations.71,72
ENVIRONMENTAL FACTORS AND SUSCEPTIBILITY TO TUBERCULOSIS Inevitably some environmental factors (such as lack of sunlight leading to low VitD3) have been discussed above in relation to relevant polymorphisms and the varying results in different populations imply such interactions are the rule, rather than the exception. Here we discuss the major additional factors.
P2X7 purinergic receptor When extracellular ATP binds to the P2X7 purinergic receptor on macrophages, large membrane pores permeable to molecules of <0.9 kDa develop, resulting in macrophage death by a combination of apoptosis and osmotic lysis (apolysis). There is rapid simultaneous induction of killing of intracellular mycobacteria. A significant protective association against TB was found for a SNP at nucleotide
ADRENAL STEROIDS IN TUBERCULOSIS The effects of stress Psychological stress causes activation of the hypothalamus–pituitary– adrenal (HPA) axis, resulting in release of cortisol and other glucocorticoid hormones from the adrenal cortex (Fig. 9.4). Glucocorticoids bias
Stress BRAIN
Pituitary
Sympathetic efferents
Parasympathetic efferents (vagus nerve)
Noradrenaline adenosine
Acetylcholine
ACTH Adrenal cortex
Cortisone (inactive)
Biased towards Th2 and Treg
11-bhydroxysteroid dehydrogenase
Cortisol (active)
T-cells
Nucleus TNF-a Ø IL-12Ø IL-10 ¹ Microbicidal effectØ
Mac/DC
a7 nicotinic receptor
Nicotine from smoking
Fig. 9.4 Pathways from the central nervous system to the immune system activated by psychological stress. The pathways are also activated by feedback from inflammation signalled via sensory afferents, and by cytokines passing from the blood into the brain via the circumventricular organs. Cortisol is the most important in chronic stress, and, in animal models, stress-induced activation of TB can be mimicked by slow release glucocorticoid pellets. These pathways tend to alter the cytokine profile of macrophages and dendritic cells, in such a way that anti-mycobacterial mechanisms are likely to be compromised (this has been shown for cortisol) and the T-cell response deviated towards Th2 and regulatory cells. Nicotine, taken in when smoking, will suppress tumour necrosis factor (TNF) release, while leaving interleukin (IL)-10 release unaltered, by triggering the a7 nicotinic acetylcholine receptor.
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the response to type 2 cytokine production via effects on dendritic cells, which secrete less IL-12 and more IL-10 in their presence, and also directly down regulate the anti-mycobacterial effects of macrophages.73–76 Therefore reactivation of latent TB can be caused by glucocorticoid therapy or by activation of the HPA axis by stress. For example living in war zones or poverty are both associated with TB.77 The TB-promoting effects of stress have been demonstrated under more controlled conditions in mice as well as in cattle and cervids.78,79
The HPA axis and dysregulation of the cortisol–cortisone shuttle in human tuberculosis In TB patients who are rested and acclimatized to hospital and so are no longer stressed by their environment, the cortisol diurnal rhythm is normal and so are the responses of the adrenals to corticotropin-releasing hormone (CRH) and to very low doses (i.e. physiological) of ACTH.80,81 The total 24-hour cortisol output may be normal or modestly raised. However, there is a change in the pattern of metabolism of cortisol, indicating a large alteration in the equilibrium point of the cortisol–cortisone shuttle.80 A major mechanism for the regulation of local tissue cortisol levels is the interconversion of active cortisol (11-hydroxy) and inactive cortisone (11-keto) by 11b-hydroxysteroid dehydrogenases (11bHSD). Thus cortisol concentrations in different organs can be very different from the values found in the serum. Gas chromatography and mass spectrometry revealed a striking excess of metabolites of cortisol relative to metabolites of cortisone in 24-hour urine collections from TB patients.80 Subsequent analysis of alveolar lavage samples revealed that the site of abnormal conversion of inactive cortisone to active cortisol is the infected lung itself, probably because both TNF-a and IL-1b increase the expression levels and reductase activity of 11b-HSD-1.81,82 The result is a local increase in cortisol levels not apparent from measurements of serum cortisol. This cortisol excess would presumably cause a shift towards type 2 cytokine expression, deactivation of the anti-mycobacterial effects of macrophages, increased IL-10, and increased TGF-b, so it may contribute to the changes seen in the human disease. Another pathway that might be activated by stress involves downregulation of TNF-a with concomitant maintenance of IL-10 levels, due to an effect on macrophages of acetylcholine released from vagal efferents.83 This mechanism is discussed later in the context of the effects of smoking, which also act via this pathway. Dehydroepiandrosterone and its metabolites in tuberculosis The most abundant steroid secreted by the adrenal cortex in man is dehydroepiandrosterone. Secretion is greatly decreased in TB patients.80 While this is not disease-specific because a similar fall occurs in most illnesses, it might be important. In a mouse model of pulmonary TB, dehydroepiandrosterone and a derivative, androstenediol, were protective and partly therapeutic, but not a practical therapy for humans because they are converted in the gonads into sex steroids.84 A synthetic analogue, 16a-bromoepiandrosterone (HE-2000), which has the advantage that it cannot enter sex steroid pathways, was also both protective and therapeutic in this model, and is currently in human clinical trials.85 SMOKING Epidemiological evidence from the UK, China, India, and the USA, implicating smoking as a risk factor for TB, has been reviewed elsewhere.86 Where an association has been found there seems to be an increase in TB case rates by between two- and fourfold for those
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smoking in excess of 20 cigarettes a day, but in some environments it is difficult to control for other factors associated with smoking such as poverty, poor educational standards, and excessive alcohol consumption. The effect of smoking is likely to be due to suppression of TNF-a levels in the lungs. Acute effects of nicotine on the immune system are mediated via the HPA axis, but adrenalectomy does not eliminate the chronic effects. It was then noted that nicotine caused increased proliferation of Legionella within a macrophage line in vitro, presumably inhibiting activation via nicotinic acetylcholine receptors,. Acetylcholine released from cholinergic vagal efferents strongly inhibits release of TNF-a from tissue macrophages.83 The effect is so potent that mice with severed vagus nerves are much more susceptible to fatal LPS-induced shock, and die with exaggerated circulating levels of TNF-a.83 The animals can be protected by electrical stimulation of the peripheral end of the cut vagus, which reduces the circulating TNF-a levels that result from the LPS challenge.83 Macrophages express high levels of the a-7 subunit of the nicotinic acetylcholine receptor, and downregulation of TNF-a via the vagus is eliminated in a-7 knockout animals.87 The anti-inflammatory cytokine IL-10 is not inhibited by nicotine acting on nicotinic receptors, so smokers will have a high IL-10/ TNF-a ratio in their lungs. Thus smoking, like neutralizing antibodies to TNF, might be expected to encourage activation of TB.
POVERTY AND MALNUTRITION Malnutrition tends to occur together with poverty, stress, and smoking, so it is difficult to isolate the nutritional component. Similarly, malnutrition could follow rather than precede development of TB. However, malnutrition is thought to impair Th1 responses. A recent study compared the incidence of TB in individuals with and without coeliac disease, which is a disorder of the gut often associated with malnutrition. There was a marked increase in TB in those with this disorder.88 In Indonesia TB was associated with malnutrition and with diabetes mellitus.89 Much more work is needed to define the roles of overall protein or carbohydrate malnutrition, or of specific deficiencies such as vitamin A and zinc. Multiple deficiencies tend to occur in the same individuals.
INTERFERENCE BY EXPOSURE TO ENVIRONMENTAL MYCOBACTERIA AND HELMINTHES The mycobacteria include hundreds of saprophytic environmental species common in soil and untreated water. Therefore in developing countries, even healthy individuals not exposed to M. tuberculosis have a background response to mycobacteria that is cross-reactive with M. tuberculosis as a result of contact with environmental species. Skin-test studies with reagents prepared from these species show that in developing countries almost everyone is skin-test positive to many mycobacterial antigens, whereas in developed countries at higher latitudes this is increasingly unusual, presumably because of changes in lifestyle, and chlorination of water supplies.90 There is strong evidence that contact with environmental mycobacteria can induce a Th1 response and provide some protection from TB. This effect can sometimes mask the effects of BCG.91 However, contact with environmental mycobacteria is very variable in different environments, and can also drive Th2 responses and regulatory T cells.
Th2 RESPONSES The evidence that a Th2 response is detrimental in TB has been reviewed in detail, and Fig. 9.5 illustrates some of the mechanisms
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Alternative activation of macrophages
REGULATORY T CELLS
Transferrin receptors ¹
Intracellular iron¹
TNF-aØ
sTNFR¹
Apoptosis Ø
iNOSØ
Arginase ¹
RNI Ø
Regulatory T cells, of which many different types are being identified, are involved in the control of the strength, nature, and duration of the immune response. Thus they fill a necessary role as modulators of excessive inflammatory reactions. Since TB is largely a disease of immunopathology it is possible that a defect in regulatory pathways, whether caused by genes or environment, could lie behind the pathogenesis of TB. On the other hand excessive regulation that suppresses a key protective pathway may be a factor leading to susceptibility in some people. In short, TB might be accompanied by too little regulation leading to immunopathology, too much regulation leading to suppressed effector mechanisms, or the wrong type of regulation leading to inappropriate effector functions such as Th2. It is inevitable that some environmental mycobacteria prime regulatory T cells. In the case of Mycobacterium vaccae these regulatory cells switch off Th2 responses, which might be beneficial.95,96 However, it is likely that different environmental species prime different types of regulation. This issue has not yet been explored. TB is accompanied by IL-10-secreting T cells that might be Tr1 regulatory cells, TGF-bsecreting cells, which include T cells possibly corresponding to Th3 regulatory cells, CD4+ CD25+ Foxp3+ regulatory cells, Th1-like cells that secrete both IFN-g and IL-10, and CD8+ LAG-3+ cells driven by living mycobacteria in vitro.97–102 In conclusion, while the role of environmental organisms in priming different subsets of effector and regulatory T cells is almost entirely unexplored, it is likely to be a major area of work for the future.
Reduced microbial activity
Immunoregulation FOXP-3+
Treg ¹
T-cell function Ø
Immunopathology VCAM-1¹
Cell infiltration¹
IL-4/TNF synergy ¹
Toxicity TNF¹
b transcript of GC receptor ¹
Inflammation¹
Arginase ¹
Collagen¹
ornithine ¹
TGF-b ¹ LTBP Ø MMP-9 ¹
Activated TGF-b
Necrosis
Fibrosis
Fig. 9.5 Some of the ways in which excessive Th2 activity, particularly IL-4, is likely to be detrimental to immunity to TB. STNFR, soluble TNF receptors; iNOS, inducible nitric oxide synthase; VCAM-1, vascular cell adhesion molecule-1; GC, glucocorticoid; LTBP1, latent transforming growth factor-b binding protein-1; MMP-9, matrix metalloproteinase 9.
involved.40 The size of the background Th2 response is strikingly higher in countries close to the equator where BCG vaccination fails to protect adults from TB, and the Th2 response that accompanies the disease is similarly greatest in these areas.92 Genetic factors that might influence this were outlined earlier. The response induced by environmental mycobacteria in developing countries is not a ‘pure’ Th1 response. This was shown in blood samples from Malawians, in which PPD (antigens precipitated from M. tuberculosis culture supernatant) induced secretion of the Th2 cytokine IL-5, whereas little IL-5 was seen in samples from the UK run in parallel. BCG vaccination failed to down regulate this IL-5 response to M. tuberculosis in Malawians (Dockrell HM, Black GF, Weir RE, personal communication). The Th2 component of the response might be enhanced by the exposure of mother and child to helminthes, which commonly induce Th2 immune responses. BCG vaccination induced a Th2-biased response in babies that had been sensitized in utero to antigens of the Th2-inducing helminthes Wuchereria bancrofti or Schistosoma haematobium as a result of maternal infection.93 Helminthes also induce regulatory T cells, and the presence of helminthes will modulate not only the response of these babies to BCG but also their response to cross-reactive environmental mycobacteria. It is therefore inevitable that populations in developing countries have mixed Th1, Th2, and Treg responses to mycobacteria.94
REFERENCES 1. Gagneux S, Deriemer K, Van T, et al. Variable host– pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006;103(8): 2869–2873. 2. Picard C, Fieschi C, Altare F, et al. Inherited interleukin-12 deficiency: IL12B genotype and clinical phenotype of 13 patients from six kindreds. Am J Hum Genet 2002;70:336–348.
CONCLUDING REMARKS In this chapter we have discussed a selection of genetic and environmental factors that modulate susceptibility to TB. We have concentrated on those that cast light on pathogenesis. Perhaps the most useful thing to emerge from the genetics is the fact that defects in the IL-12/IL-23/IFN-g pathways seem more likely to cause increased susceptibility to infection with mycobacteria of low virulence than susceptibility to M. tuberculosis itself. This observation, together with the association of TB with factors such as SNPs of vitamin D receptors, might imply that the major effector mechanisms needed for control of M. tuberculosis are only partly linked to the standard Th1 pathway. The second major conclusion is that there is so much variation in the genetics of the organisms, the genetics of the populations they infect and the nature of the environments with which these genes interact that the pathogenesis of TB might be different in different parts of the world. If this is so we might need different vaccines, diagnostic reagents, and immunotherapies in different places. We clearly have much to learn, and current ideas about vaccine design might need to be modified as our knowledge increases.
3. Ottenhoff TH, Verreck FA, Hoeve MA, et al. Control of human host immunity to mycobacteria. Tuberculosis (Edinb) 2005;85:53–64. 4. Tso HW, Lau YL, Tam CM, et al. Associations between IL12B polymorphisms and tuberculosis in the Hong Kong Chinese population. J Infect Dis 2004;190:913–919. 5. de Jong R, Altare F, Haagen IA, et al. Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 1998;280:1435–1438.
6. Remus N, El Baghdadi J, Fieschi C, et al. Association of IL12RB1 polymorphisms with pulmonary tuberculosis in adults in Morocco. J Infect Dis 2004;190:580–587. 7. Akahoshi M, Nakashima H, Miyake K, et al. Influence of interleukin-12 receptor beta1 polymorphisms on tuberculosis. Hum Genet 2003;112:237–243. 8. Lee HW, Lee HS, Kim DK, et al. Lack of an association between interleukin-12 receptor beta1 polymorphisms and tuberculosis in Koreans. Respiration 2005;72:365–368.
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9. Lio D, Marino V, Serauto A, et al. Genotype frequencies of the +874T–>A single nucleotide polymorphism in the first intron of the interferongamma gene in a sample of Sicilian patients affected by tuberculosis. Eur J Immunogenet 2002;29:371–374. 10. Tso HW, Ip WK, Chong WP, et al. Association of interferon gamma and interleukin 10 genes with tuberculosis in Hong Kong Chinese. Genes Immun 2005;6:358–363. 11. Jouanguy E, Altare F, Lamhamedi S, et al. Interferon-g receptor deficiency in an infant with fatal Bacille Calmette-Gue´rin infection. N Engl J Med 1996;335:1956–1961. 12. Awomoyi AA, Nejentsev S, Richardson A, et al. No association between interferon-gamma receptor-1 gene polymorphism and pulmonary tuberculosis in a Gambian population sample. Thorax 2004;59:291–294. 13. Cooke GS, Campbell SJ, Sillah J, et al. Polymorphism within the interferon-gamma/ receptor complex is associated with pulmonary tuberculosis. Am J Respir Crit Care Med 2006;174:339–343. 14. Nau GJ, Richmond JF, Schlesinger A, et al. Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci USA 2002; 99:1503–1508. 15. Ting LM, Kim AC, Cattamanchi A, et al. Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1. J Immunol 1999; 163:3898–3906. 16. Woodworth JS, Behar SM. Mycobacterium tuberculosis-specific CD8þ T cells and their role in immunity. Crit Rev Immunol 2006;26:317–352. 17. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:1770–1773. 18. Dupuis S, Jouanguy E, Al-Hajjar S, et al. Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency. Nat Genet 2003;33:388–391. 19. Wilkinson RJ, Patel P, Llewelyn M, et al. Influence of polymorphism in the genes for the interleukin (IL)-1 receptor antagonist and IL-1beta on tuberculosis. J Exp Med 1999;189:1863–1874. 20. Awomoyi AA, Charurat M, Marchant A, et al. Polymorphism in IL1B: IL1B-511 association with tuberculosis and decreased lipopolysaccharideinduced IL-1beta in IFN-gamma primed ex-vivo whole blood assay. J Endotoxin Res 2005;11:281–286. 21. Lombard Z, Dalton DL, Venter PA, et al. Association of HLA-DR, -DQ, and vitamin D receptor alleles and haplotypes with tuberculosis in the Venda of South Africa. Hum Immunol 2006;67:643–654. 22. Goldfeld AE, Delgado JC, Thim S, et al. Association of an HLA-DQ allele with clinical tuberculosis. JAMA 1998;279:226–228. 23. Delgado JC, Baena A, Thim S, et al. Aspartic acid homozygosity at codon 57 of HLA-DQ beta is associated with susceptibility to pulmonary tuberculosis in Cambodia. J Immunol 2006;176:1090–1097. 24. Vijaya Lakshmi V, Rakh SS, Anu Radha B, et al. Role of HLA-B51 and HLA-B52 in susceptibility to pulmonary tuberculosis. Infect Genet Evol 2006;6:436–439. 25. 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 IFN-alpha/beta. Proc Natl Acad Sci USA 2001;98:5752–5757. 26. Bouchonnet F, Boechat N, Bonay M, et al. Alpha/beta interferon impairs the ability of human macrophages to control growth of Mycobacterium bovis BCG. Infect Immun 2002;70:3020–3025. 27. Manca C, Tsenova L, Freeman S, et al. Hypervirulent M. tuberculosis W/Beijing strains upregulate type I IFNs and increase expression of negative regulators of the Jak-Stat pathway. J Interferon Cytokine Res 2005;25:694–701.
94
28. Akahoshi M, Ishihara M, Remus N, et al. Association between IFNA genotype and the risk of sarcoidosis. Hum Genet 2004;114:503–509. 29. Flores-Villanueva PO, Ruiz-Morales JA, Song CH, et al. A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis. J Exp Med 2005;202:1649–1658. 30. Alcais A, Fieschi C, Abel L, et al. Tuberculosis in children and adults: two distinct genetic diseases. J Exp Med 2005;202:1617–1621. 31. Gu L, Tseng S, Horner RM, et al. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 2000;404: 407–411. 32. Ip WK, Wong CK, Lam CW. Interleukin (IL)-4 and IL-13 up-regulate monocyte chemoattractant protein-1 expression in human bronchial epithelial cells: involvement of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 and Janus kinase-2 but not c-Jun NH2-terminal kinase 1/2 signalling pathways. Clin Exp Immunol 2006;145:162–172. 33. Szalai C, Kozma GT, Nagy A, et al. Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. J Allergy Clin Immunol 2001;108:375–381. 34. Jamieson SE, Miller EN, Black GF, et al. Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians. Genes Immun 2004;5:46–57. 35. de Nadai P, Charbonnier AS, Chenivesse C, et al. Involvement of CCL18 in allergic asthma. J Immunol 2006;176:6286–6293. 36. Pivarcsi A, Gombert M, Dieu-Nosjean MC, et al. CC chemokine ligand 18, an atopic dermatitisassociated and dendritic cell-derived chemokine, is regulated by staphylococcal products and allergen exposure. J Immunol 2004;173:5810–5817. 37. Amirzargar AA, Rezaei N, Jabbari H, et al. Cytokine single nucleotide polymorphisms in Iranian patients with pulmonary tuberculosis. Eur Cytokine Netw 2006;17:84–89. 38. Demissie A, Abebe M, Aseffa A, et al. Healthy individuals that control a latent infection with M. tuberculosis express high levels of Th-1 cytokines and the IL-4 antagonist IL-4d2. J Immunol 2004; 172:6938–6943. 39. Obihara CC, Kimpen JL, Gie RP, et al. Mycobacterium tuberculosis infection may protect against allergy in a tuberculosis endemic area. Clin Exp Allergy 2006;36:70–76. 40. Rook GA, Dheda K, Zumla A. Immune responses to tuberculosis in developing countries: implications for new vaccines. Nat Rev Immunol 2005;5:661–667. 41. Ellertsen LK, Wiker HG, Egeberg NT, et al. Allergic sensitisation in tuberculosis and leprosy patients. Int Arch Allergy Immunol 2005;138:217–224. 42. Baliko Z, Szereday L, Szekeres-Bartho J. Th2 biased immune response in cases with active Mycobacterium tuberculosis infection and tuberculin anergy. FEMS Immunol Med Microbiol 1998;22:199–204. 43. Awomoyi AA, Marchant A, Howson JM, et al. Interleukin-10, polymorphism in SLC11A1 (formerly NRAMP1), and susceptibility to tuberculosis. J Infect Dis 2002;186:1808–1814. 44. Henao MI, Montes C, Paris SC, et al. Cytokine gene polymorphisms in Colombian patients with different clinical presentations of tuberculosis. Tuberculosis (Edinb) 2006;86:11–19. 45. Floros J, Lin HM, Garcia A, et al. Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population. J Infect Dis 2000;182:1473–1478. 46. Madan T, Saxena S, Murthy KJ, et al. Association of polymorphisms in the collagen region of human SP-A1 and SP-A2 genes with pulmonary tuberculosis in Indian population. Clin Chem Lab Med 2002;40:1002–1008. 47. Selvaraj P, Jawahar MS, Rajeswari DN, et al. Role of mannose binding lectin gene variants on its protein levels and macrophage phagocytosis with
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67.
68.
live Mycobacterium tuberculosis in pulmonary tuberculosis. FEMS Immunol Med Microbiol 2006;46:433–437. Soborg C, Madsen HO, Andersen AB, et al. Mannose-binding lectin polymorphisms in clinical tuberculosis. J Infect Dis 2003;188:777–782. Liu W, Zhang F, Xin ZT, et al. Sequence variations in the MBL gene and their relationship to pulmonary tuberculosis in the Chinese Han population. Int J Tuberc Lung Dis 2006;10: 1098–1103. Barreiro LB, Neyrolles O, Babb CL, et al. Promoter variation in the DC-SIGN-encoding gene CD209 is associated with tuberculosis. PLoS Med 2006;3:e20. Gomez LM, Anaya JM, Sierra-Filardi E, et al. Analysis of DC-SIGN (CD209) functional variants in patients with tuberculosis. Hum Immunol 2006;67:808–811. Ogus AC, Yoldas B, Ozdemir T, et al. The Arg753GLn polymorphism of the human toll-like receptor 2 gene in tuberculosis disease. Eur Respir J 2004;23:219–223. Bochud PY, Hawn TR, Aderem A. Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling. J Immunol 2003;170:3451–3454. Ben-Ali M, Barbouche MR, Bousnina S, et al. Toll-like receptor 2 Arg677Trp polymorphism is associated with susceptibility to tuberculosis in Tunisian patients. Clin Diagn Lab Immunol 2004;11:625–626. Hawn TR, Dunstan SJ, Thwaites GE, et al. A polymorphism in Toll-interleukin 1 receptor domain containing adaptor protein is associated with susceptibility to meningeal tuberculosis. J Infect Dis 2006;194:1127–1134. Newport MJ, Allen A, Awomoyi AA, et al. The toll-like receptor 4 Asp299Gly variant: no influence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia. Tuberculosis (Edinb) 2004;84:347–352. Stockton JC, Howson JM, Awomoyi AA, et al. Polymorphism in NOD2, Crohn’s disease, and susceptibility to pulmonary tuberculosis. FEMS Immunol Med Microbiol 2004;41:157–160. Macrae DE. Calciferol treatment of Lupus vulgaris. BMJ 1947;59:333–338. Brincourt J Le calcife´rol a-t-il une action lique´fiante sur le caseum? Poumon Coeur 1967;23:841–851. Cadranel J, Hance AJ, Milleron B, et al. Vitamin D metabolisim in tuberculosis. Production of 1,25 (OH)2 D3 by cells recovered by bronchoalveolar lavage, and the role of this metabolite in calcium homeostasis. Am Rev Respir Dis 1988;138:984–989. Davies PD. The role of vitamin D in tuberculosis. Am Rev Respir Dis 1989;139:1571. Rook GAW, Steele J, Fraher L, et al. Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology 1986;57:159–163. Bellamy R, Ruwende C, Corrah T, et al. Tuberculosis and chronic hepatitis B virus infection in Africans and variation in the vitamin D receptor gene. J Infect Dis 1999;179:721–724. Mulero V, Searle S, Blackwell JM, et al. Solute carrier 11a1 (Slc11a1; formerly Nramp1) regulates metabolism and release of iron acquired by phagocytic, but not transferrin-receptor-mediated, iron uptake. Biochem J 2002;363:89–94. Peracino B, Wagner C, Balest A, et al. Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection. Traffic 2006;7:22–38. Bellamy R, Ruwende C, Corrah T, et al. Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 1998;338:640–644. Li HT, Zhang TT, Zhou YQ, et al. SLC11A1 (formerly NRAMP1) gene polymorphisms and tuberculosis susceptibility: a meta-analysis. Int J Tuberc Lung Dis 2006;10:3–12. Malik S, Abel L, Tooker H, et al. Alleles of the NRAMP1 gene are risk factors for pediatric
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69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
tuberculosis disease. Proc Natl Acad Sci USA 2005;102:12183–12188. Li CM, Campbell SJ, Kumararatne DS, et al. Association of a polymorphism in the P2X7 gene with tuberculosis in a Gambian population. J Infect Dis 2002;186:1458–1462. Tosh K, Campbell SJ, Fielding K, et al. Variants in the SP110 gene are associated with genetic susceptibility to tuberculosis in West Africa. Proc Natl Acad Sci USA 2006; 103:10364–10368. Thye T, Browne EN, Chinbuah MA, et al. No associations of human pulmonary tuberculosis with Sp110 variants. J Med Genet 2006;43:e32. Szeszko JS, Healy B, Stevens H, et al. Resequencing and association analysis of the SP110 gene in adult pulmonary tuberculosis. Hum Genet 2007;121: 155–160. Brinkmann V, Kristofic C. Regulation by corticosteroids of Th1 and Th2 cytokine production in human CD4+ effector T cells generated from CD45RO- and CD45RO+ subsets. J Immunol 1995;155:3322–3328. Ramirez F, Fowell DJ, Puklavec M, et al. Glucocorticoids promote a Th2 cytokine response by CD4+ T cells in vitro. J Immunol 1996; 156:2406–2412. Vieira PL, Kalinski P, Wierenga EA, et al. Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential. J Immunol 1998;161:5245–5251. Rook GA, Steele J, Ainsworth M, et al. A direct effect of glucocorticoid hormones on the ability of human and murine macrophages to control the growth of M. tuberculosis. Eur J Respir Dis 1987; 71:286–291. Spence DP, Hotchkiss J, Williams CS, et al. Tuberculosis and poverty. BMJ 1993; 307:759–761. Brown DH, Sheridan J, Pearl D, et al. Regulation of mycobacterial growth by the hypothalamuspituitary-adrenal axis: differential responses of Mycobacterium bovis BCG-resistant and -susceptible mice. Infect Immun 1993;61:4793–4800. Griffin JF, Buchan GS. Aetiology, pathogenesis and diagnosis of Mycobacterium bovis in deer. Vet Microbiol 1994;40:193–205.
80. Rook GAW, Honour J, Kon OM, et al. Urinary steroid metabolites in tuberculosis; a new clue to pathogenesis. Q J Med 1996;89:333–341. 81. Baker RW, Walker BR, Shaw RJ, et al. Increased cortisol-cortisone ratio in acute pulmonary tuberculosis. Am J Respir Crit Care Med 2000; 162:1641–1647. 82. Escher G, Galli I, Vishwanath BS, et al. Tumour necrosis factor and interleukin 1 enhance the cortisone/cortisol shuttle. J Exp Med 1997; 186:189–198. 83. Borovikova LV, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 2000;405:458–462. 84. Hernandez-Pando R, de la Luz Streber M, Orozco H, et al. The effects of androstenediol and dehydroepiandrosterone on the course and cytokine profile of tuberculosis in Balb/c mice. Immunology 1998;95:234–241. 85. Hernandez-Pando R, Aguilar-Leon D, Orozco H, et al. 16 alpha Bromoepiandrosterone restores T helper cell type 1 activity and accelerates chemotherapy-induced bacterial clearance in a model of progressive pulmonary tuberculosis. J Infect Dis 2005;191:299–306. 86. Davies PD, Yew WW, Ganguly D, et al. Smoking and tuberculosis: the epidemiological association and immunopathogenesis. Trans R Soc Trop Med Hyg 2006;100:291–298. 87. Wang H, Yu M, Ochani M, et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003;421:384. 88. Ludvigsson JF, Wahlstrom J, Grunewald J, et al. Coeliac disease and risk of tuberculosis: a population-based cohort study. Thorax 2007; 62:23–28. 89. Alisjahbana B, van Crevel R, Sahiratmadja E, et al. Diabetes mellitus is strongly associated with tuberculosis in Indonesia. Int J Tuberc Lung Dis 2006;10:696–700. 90. Fine PE, Floyd S, Stanford JL, et al. Environmental mycobacteria in northern Malawi: implications for the epidemiology of tuberculosis and leprosy. Epidemiol Infect 2001;126:379–387. 91. Tuberculosis Research Centre (ICMR) C. Influence of sex, age and nontuberculous infection at intake on the efficacy of BCG: re-analysis of 15-year data
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
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from a double-blind randomized control trial in south India. Indian J Med Res 2006;123:119–124. Rook G, Dheda K, Zumla A. Do successful tuberculosis vaccines need to be immunoregulatory rather than merely Th1-boosting? Vaccine 2005;23: 2115–2120. Malhotra I, Mungai P, Wamachi A, et al. Helminthand Bacillus Calmette-Guerin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. J Immunol 1999;162:6843–6848. Elias D, Akuffo H, Britton S. Helminthes could influence the outcome of vaccines against TB in the tropics. Parasite Immunol 2006;28:507–513. Zuany-Amorim C, Sawicka E, Manlius C, et al. Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regulatory T-cells. Nat Med 2002;8:625–629. Hernandez-Pando R, Pavon L, Orozco EH, et al. Interactions between hormone-mediated and vaccine-mediated immunotherapy for pulmonary tuberculosis in Balb/c mice. Immunology 2000;100:391–398. Delgado JC, Tsai EY, Thim S, et al. Antigenspecific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia. Proc Natl Acad Sci USA 2002;99: 7576–7581. Toossi Z, Ellner JJ. The role of TGF beta in the pathogenesis of human tuberculosis. Clin Immunol Immunopathol 1998;87:107–114. Guyot-Revol V, Innes JA, Hackforth S, et al. Regulatory T cells are expanded in blood and disease sites in tuberculosis patients. Am J Respir Crit Care Med 2005;173:803–810. Ribeiro-Rodrigues R, Resende Co T, Rojas R, et al. A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis. Clin Exp Immunol 2006;144:25–34. Gerosa F, Nisii C, Righetti S, et al. CD4(+) T cell clones producing both interferon-gamma and interleukin-10 predominate in bronchoalveolar lavages of active pulmonary tuberculosis patients. Clin Immunol 1999;92:224–234. Joosten SA, van Meijgaarden KE, Savage ND, et al. Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4. Proc Natl Acad Sci USA 2007;104(19):8029–8234.
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Co-pathogenesis of tuberculosis and HIV Stephen D Lawn and Linda-Gail Bekker
INTRODUCTION The human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) pandemic has had a devastating impact on TB control globally and TB has emerged as one of the principal causes of HIV-associated morbidity and mortality worldwide.1,2 The countries of sub-Saharan Africa have been hit hardest, with annual notification rates rising two- to threefold in many countries since 1990; more than half of patients with TB in these countries also have HIV-1 coinfection.1 Whereas TB notification rates continue to decrease in most countries of the world, the African epidemic fuelled by HIV was reported in 2005 to be driving up the global incidence of TB by 1% per year.3 While the effects of HIV on the epidemiology of TB became quickly apparent, it also emerged that the interaction between these diseases was bidirectional. In the 1990s, studies showed that immunological activation associated with TB could also enhance the pathogenesis of HIV infection, potentially causing accelerated decline in immune function in coinfected patients.4,5 The bidirectional synergy between these two infections continues to exact a very heavy toll particularly in communities in resourcelimited settings with a high prevalence of HIV. In this chapter, a brief overview of these interactions as revealed by epidemiological, clinical, and histopathological data is followed by an exploration of the immunological mechanisms underlying these observations. The specific defects in cellular antimycobacterial immunity caused by HIV-1 infection are reviewed in detail. Thereafter we examine the impact of TB-induced immune activation on the replication and immunopathogenesis of HIV at sites of TB disease and systemically.
IMPACT OF HIV ON TB EPIDEMIOLOGICAL DATA Infection with HIV-1 has emerged as the single strongest risk factor for development of active TB. This may be the result of a number of different processes and underlying immunological mechanisms. HIV-infected individuals may be more frequently exposed to Mycobacterium tuberculosis infection and outbreaks of TB have been described among patients attending healthcare facilities in both high-income and resource-limited settings.6–8 It is not known, however, whether HIV-infected individuals have a greater risk of
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acquiring M. tuberculosis infection than HIV-uninfected individuals following a similar exposure to the organism. The fact that certain aspects of innate immune function are impaired by HIV infection might potentially increase host susceptibility to M. tuberculosis infection following exposure. Data from outbreaks among drug abusers in New York, however, found no increased risk of M. tuberculosis infection among those individuals who were HIV-infected.9 This study was limited, though, as these data were derived using the tuberculin skin test (TST), which has lowered sensitivity to detect M. tuberculosis infection in the context of HIV coinfection. The high incidence of active TB reported in outbreaks among HIV-infected people may instead reflect more rapid progression of M. tuberculosis infection to active primary disease rather than an increased incidence of initial M. tuberculosis infection.10 The risk of progressive primary disease is indeed high following infection with M. tuberculosis. Attack rates of 30–40% have been reported among HIV-infected patients exposed to M. tuberculosis in healthcare settings, with many patients developing disease within 1–2 months of initial exposure.6,10 This suggests that the host granulomatous response to early M. tuberculosis infection is impaired by HIV infection. A great majority of HIV-uninfected individuals with latent M. tuberculosis infection do not develop TB disease as their cellular immune function is adequate to maintain the mycobacterial infection in a clinically latent state. The lifetime risk of active TB among HIV-uninfected individuals with latent M. tuberculosis infection is estimated to be 2–23%.11 In contrast, HIV-infected patients with latent M. tuberculosis infection have a high risk of reactivation, with approximately 10% of such individuals developing active TB disease each year.9 This observation strongly suggests that the ability to maintain M. tuberculosis latency within granulomas long term is abrogated by HIV-1 coinfection.12 An estimated one-third of the world’s population is infected with M. tuberculosis, with much higher rates in poor communities in resource-limited settings that have high HIV prevalence. The broad geographical overlap of these infections is a critical factor explaining the huge impact of HIV on global TB control.
CLINICO-RADIOLOGICAL DATA The HIV-1 epidemic has not only had a great impact on the epidemiology of TB, but the clinical, radiological, and histopathological features of the disease are often modified in coinfected patients. In contrast to many other opportunistic infections, TB occurs across a wide spectrum of immunodeficiency.13 This reflects the greater virulence
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of M. tuberculosis compared with other opportunistic pathogens such as Mycobacterium avium complex, Pneumocystic jiroveci, and Cryptococcus neoformans, which typically only cause disease among patients with advanced immunodeficiency. The features of TB in HIV-1-infected individuals with wellpreserved CD4 T cell counts are indistinguishable from those of individuals with TB but no HIV-1 coinfection. However, progressive immunodeficiency is associated with an increasing frequency of miliary and disseminated forms of disease, reflecting failure of containment of the mycobacterial infection.14,15 Impaired tissue inflammatory response to infection affects the radiographic appearances of pulmonary TB, with reduced consolidation, fibrosis, and cavitation, and also results in sputum smear microscopy for acidfast bacilli (AFB) being less frequently positive.15,16 Mortality rates among patients with TB and HIV-1 coinfection are high and may be due to either TB itself or another HIV-associated illness.17,18 HIV coinfection is also associated with high rates of cutaneous anergy to TST, indicating failure of delayed type hypersensitivity responses to mycobacterial antigens.15,19 Interestingly, though, TST responses do not gradually diminish with progressive immunodeficiency as one might expect but instead appear to be an ‘all-ornothing’ phenomenon.19 Although the proportion of HIV-infected
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patients who have cutaneous anergy to TST increases with progressive immunodeficiency, positive TST responses of HIV-infected patients are nevertheless similar in magnitude to the positive responses of HIV-uninfected patients.15,19
HISTOPATHOLOGICAL DATA Autopsy specimens from patients with AIDS who died of TB reveal a very high frequency of extrapulmonary and disseminated disease with multiorgan involvement.20 Occult-disseminated TB is a frequent finding in postmortem studies of people from subSaharan Africa who died with AIDS.21,22 Histological studies of tissue specimens from individuals with TB and HIV-1 coinfection reveal a spectrum of appearances that reflects the degree of immunosuppression. Lucas et al.23 described three histological stages of cellular immune response that correlate with depletion of the peripheral blood CD4 T-cell count (Fig. 10.1): 1. Immunocompetent individuals with HIV-1 infection develop TB granulomas characterized by abundant epithelioid macrophages, Langhans giant cells, peripherally located CD4 T cells, and a paucity of AFB.
B
A
C Fig. 10.1 (A) Low-power photomicrograph of haematoxylin and eosin-stained tissue containing a typical well-formed tuberculous granuloma in an immunocompetent individual showing central caseous necrosis, mononuclear cell infiltrate, and surrounding fibrosis. (B) Medium-power photomicrograph of tissue from an HIV-infected patient with TB and advanced immunodeficiency showing a mononuclear cell infiltrate but complete absence of granuloma formation. (C) High-power tissue section from the specimen in (B) with Ziehl–Neelsen staining showing numerous bacilli of M. tuberculosis that have multiplied in the absence of an effective host response. Photomicrographs kindly supplied by the Department of Anatomical Pathology, Faculty of Health Sciences, University of Cape Town, South Africa.
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2. In those with moderate HIV-associated immunodeficiency, Langhans giant cells are not seen, epithelioid differentiation and activation of macrophages are absent, there is CD4 lymphocytopenia, and AFB are more numerous. 3. In patients with advanced HIV-associated immunosuppression and AIDS, there is a striking paucity of granuloma formation with little cellular recruitment, very few CD4 T cells, and even larger numbers of AFB. These data indicate that progressive HIV-associated immunodeficiency is associated with progressive profound impairment of the granulomatous host response to M. tuberculosis. This spectrum of histological appearances bears a striking resemblance to that of leprosy, which is also the result of broadly divergent immune responses to the causative organism, Mycobacterium leprae. Patients with tuberculoid leprosy have strong cellular immune responses to M. leprae and so their disease is paucibacillary and is characterized by abundant granuloma formation. This resembles the histological appearances of TB in HIV-infected patients who have well-preserved immunity. In contrast, patients with lepromatous leprosy have very poor cellular responses to M. leprae; their disease is multibacillary with no granuloma formation, similar to the histological appearances of TB in HIV-infected patients who have advanced immunodeficiency.
IMPAIRMENT OF IMMUNE RESPONSES TO M. TUBERCULOSIS The immune response to M. tuberculosis is multifaceted, engaging several arms of the immune system. Macrophages are the key cell type effecting anti-mycobacterial immune responses. However, M. tuberculosis may evade intracellular killing mechanisms and persisting organisms then replicate within the intracellular environment of macrophages. Antigen presentation to CD4 T cells leads to the development of cell-mediated immune responses and delayed-type hypersensitivity (DTH) within 2–4 weeks of initial infection.24,25 Secretion of interleukin-12 (IL-12) by macrophages facilitates clonal expansion of CD4 T-helper type-1 lymphocytes, which secrete interferon-g (IFN-g). This cytokine is a potent activator of macrophages, promoting mycobacterial killing.26 Further release of chemokines, such as IL-8, and proinflammatory cytokines, such as tumour necrosis factor-a (TNF-a), facilitate mononuclear cell recruitment and activation, respectively. Subsequent orchestration of the granulomatous host response provides the critical environment within which the host limits M. tuberculosis infection.27 The TB granuloma is characterized by activation and epithelioid differentiation of macrophages and by development of multinucleate Langhans giant cells.28 CD4 T cells located within the periphery of the granuloma play a major role in orchestrating cell-mediated immune function. Central caseous necrosis inhibits extracellular bacillary growth. When granulomas fail to limit M. tuberculosis infection, however, the caseous centre liquefies, permitting rampant, uninhibited extracellular bacterial replication. Subsequent spreading tissue destruction and cavity formation result in TB disease.24 The pivotal role of CD4 T cells in orchestrating the acquired cellular response to M. tuberculosis has been emphatically demonstrated by the impact of HIV coinfection on these processes.12,29 Here we describe the impact of HIV on the critical cellular processes involved in these responses.
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IMPACT ON MONONUCLEAR PHAGOCYTES Macrophages are central to the pathogenesis of both TB and HIV-1 infection. They serve as the principal effector cells within both the innate and adaptive immune responses to M. tuberculosis. However, these cells also provide the preferred intracellular environment for mycobacterial growth and support HIV replication. Mononuclear phagocytes are not numerically depleted during HIV infection in vivo. However, monocyte/macrophage functions essential for adequate innate and adaptive responses to pathogens, such as chemotaxis, phagocytosis, intracellular killing, antigen presentation, and cytokine production, may all be affected by HIV-1 infection.12,30,31 Most data concerning this are derived from in vitro studies using non-mycobacterial organisms, although studies have reported that HIV impairs phagocytosis of M. avium complex and is associated with increased growth of M. tuberculosis within macrophages.32,33 Alveolar macrophages account for >85% of the immune cells of the lung and play a central role in the innate immune response to numerous pulmonary pathogens, including M. tuberculosis. Defects in mononuclear cell function may therefore render individuals more susceptible to M. tuberculosis infection following aerosol inhalation. However, the relevance of much of these data generated in vitro using non-mycobacterial organisms and using cell lines rather than primary alveolar macrophages must be limited. Furthermore, as discussed earlier, there is no epidemiological evidence that HIV-1-infected individuals have increased rates of M. tuberculosis infection following exposure. Thus, any impact of HIV on host innate immune function is likely to be less important than the effects on acquired immune responses that require the complex coordinated interplay between CD4 T cells and macrophages. Dendritic cells (DCs) are vital in the defence against many pathogens, serving as efficient antigen-presenting cells and playing an important role in determining the phenotype of subsequent acquired immune responses. Both plasmacytoid (pDC) and myeloid (mDC) subsets are reduced in numbers in HIV infection but the impact of this on immune responses to M. tuberculosis is unknown.34 However, in recent years it has emerged that both HIV and M. tuberculosis are independently able to subvert DC functions to escape immune surveillance.35,36 HIV-1 targets DC-SIGN (DC-specific-intercellular-adhesion-molecule-3-grabbing-nonintegrin) to protect it from antigen processing and to facilitate its transport to lymphoid tissue. Mycobacterium tuberculosis is also able to bind to DC-SIGN, leading to IL-10 production and inhibition of the immuno-stimulatory function of dendritic cells.
IMPACT ON CD4 T CELLS Although HIV infection is characterized by progressive CD4 T-cell loss, CD4 T cells in HIV-infected persons are functionally impaired prior to significant decreases in CD4 T-cell counts in peripheral blood.37 The various mechanisms potentially responsible for this are summarized in Fig. 10.2 and cause sequential loss of responses to recall antigens, antigens, alloantigens, and finally mitogens.37 These defects progress as the CD4 T-cell count decreases. Functional impairment of M. tuberculosis-specific CD4 T cells provides a likely explanation for the doubling of TB risk following acquisition of HIV infection among patients despite well-preserved CD4 T-cell counts.38 A number of different mechanisms may inhibit TB-specific CD4 T-cell function. Activation and proliferation of both HIV-infected
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Co-pathogenesis of tuberculosis and HIV ØIL-12, ¹IL-10 5.
ØIL-2
ØIFN-g 4.
1. 3. Macrophage
2.
CD4+ lymphocyte
¹¹TNF-a
Fig. 10.2 Mechanisms that may contribute to the impact of HIV-1 on
generation of cell-mediated immune responses to M. tuberculosis include: (1) binding and phagocytosis of M. tuberculosis by macrophages; (2) M. tuberculosis intracellular killing; (3) M. tuberculosis antigen processing and presentation; (4) CD4 T-cell activation and proliferation; and (5) effector cytokine production. Adapted from Lawn SD, Butera ST, Shinnick TM. Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect 2002 May;4:635–46.
and -uninfected CD4 T cells is impaired through inhibition of the IL-2 signalling pathway.39 The HIV-1 major surface envelope glycoprotein 120 (gp120) inhibits both antigen-driven IL-2 production and surface IL-2-receptor-a (IL-2R-a) expression.40 This is mediated, at least in part, by HIV gp120 binding to CD4 receptors. This hinders the physiological binding of the natural ligand, class II human lymphocyte antigen (HLA class II), which is essential to T-cell recognition of foreign antigen. HIV-1 also directly downregulates CD4 receptors and blocks expression of HLA class II, further impeding antigen presentation.41,42 Furthermore, HIV-1 tat transactivator also directly inhibits both IL-2 and IL-2Ra expression in HIV-infected CD4 T cells.43 Together these mechanisms result in partial inhibition or failure of expansion and activation of M. tuberculosis-specific CD4 T cells at sites of M. tuberculosis infection. Effective cell-mediated immunity to M. tuberculosis is characterized by strong T-helper type-1 IFN-g-secreting lymphocyte responses, which develop in the presence of macrophage-derived IL-12. The pivotal role of this cytokine pathway in mediating immune responses to mycobacteria is clearly illustrated by the greatly increased susceptibility of individuals with IL-12-receptor or IFN-g-receptor deficiencies to diseases caused by these organisms.44 During progression of HIV-1 infection, mononuclear cells lose their ability to secrete T-helper type-1 cytokines (IL-2, IFN-g, IL-12) and instead produce increased levels of the T-helper type-2 cytokines, IL-4 and IL-10.45 IL-10 antagonizes M. tuberculosis-induced Thelper type-1 responses and diminishes macrophage effector function in mycobacterial infection.46,47 TNF-a, which is secreted in high concentrations in individuals with TB and HIV-1 coinfection, also negatively regulates IL-12 production.48 Moreover, HIV-1 infection decreases expression of CD40 ligand (CD40L) on CD4 T cells, leading to diminished activation of macrophages via the CD40 receptor and thereby decreasing IL-12 secretion.49 Thus, Thelper type-2 skewing of lymphocyte responses in individuals with HIV-1 infection may further undermine their ability to generate effective cell-mediated responses to M. tuberculosis infection. Generalized loss of CD4 T cells in HIV-1-infected persons is the result of a complex pattern of gradual changes in the composition
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of T-cell subsets with depletion of both the CD4 and CD8 naı¨ve (CD45RA) subsets and the CD4 memory (CD45RO) subset.50 Several distinct processes contribute to loss of these cell subsets, including virus-induced cell death, the immune destruction of infected cells, apoptosis, and impaired lymphocyte regeneration. These losses may be accelerated in patients with TB as discussed later in this chapter. However, in addition to depletion of the systemic pool of CD4 T cells, cellular response at sites of TB disease are particularly impaired. Examination of bronchoalveolar lavage (BAL) fluid obtained from diseased lung segments of patients with pulmonary TB reveals a failure of recruitment and activation of CD4 T cells in patients who are HIV infected.51 Local depletion of CD4 T cells at sites of TB may be the result of impaired chemotaxis and inhibition of T-cell proliferation. Rates of cell loss may be high due to virus-induced cytolysis, with HIV-1 replication being greatly enhanced at sites of TB infection. Moreover, immune activation associated with TB and HIV-1 coinfection provides a potent stimulus for activation-induced cell apoptosis, depleting both HIV-1-infected CD4 T cells and uninfected bystander CD4 and CD8 T cells at the site of disease.52–56
IMPACT ON THE TUBERCULOSIS GRANULOMA The development of the TB granuloma can be divided into three steps: 1. development of a monocytic infiltrate; 2. aggregation, maturation, and organization of mononuclear cells into a granuloma; and 3. further evolution into an epithelioid granuloma.28 Although granulomatous lesions have been well characterized at the histological level, the cellular mechanisms and cytokine and chemokine pathways involved in the initiation, organization, and maintenance of granulomas are poorly understood.27 However, many of the HIV-induced defects in mononuclear cell function described above are likely to culminate in failure of the assembly and function of effective TB granulomas. These defects include impaired chemotaxis, cytokine dysregulation, bactericidal functioning of macrophages, and proliferative T-cell responses. Furthermore, progressive immunosuppression associated with the development of AIDS also results in failure of epithelioid differentiation of macrophages, formation of Langhans giant cells, and caseous necrosis.23 Thus, HIV impairs the formation of granulomas needed to contain new or spreading M. tuberculosis infection. Many patients living in high TB burden countries are likely to have acquired latent M. tuberculosis infection prior to acquisition of HIV infection. In such patients, increased risk of TB may largely be due to the impact of HIV on pre-existing granulomas, leading to reactivation TB. Very little is known about the mechanisms whereby HIV-1 coinfection actually disrupts established granulomas but the virus must presumably enter those granulomas. Various lines of evidence suggest that latent M. tuberculosis infection represents a dynamic host–pathogen relationship in which the granuloma serves to restrain bacilli, which are present as viable and metabolically active forms rather than inactive spore-like structures. Unlike the relatively inactive foreign-body granulomas formed in response to inert particulate matter, tuberculous hypersensitivitytype granulomas are immunologically active structures with a continual level of mononuclear cell death and cell replacement by active recruitment.28 It is likely that HIV-1 infection impairs granuloma functions via two basic means:
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HIV-infected mononuclear cell within the granuloma serves as a source of HIV production and transmission to other cells
Cell-free HIV-1 particles
HIV-infected mononuclear cells being recruited to the TB granuloma by chemotaxis
Fig. 10.3 A granuloma containing latent M. tuberculosis (red dots) infection in an HIV-infected person. Ongoing dynamic cell turnover within the granuloma requires continual mononuclear cell recruitment (arrows). Entry of HIV-infected cells is likely to lead to replication and intercellular transmission of HIV, leading to spreading infection, cell dysfunction and death (blue cells), and subsequent failure of the granuloma permitting uncontrolled M. tuberculosis replication.
1. systemic depletion of the mononuclear cells required for the ongoing maintenance and functioning of granulomas; and 2. the effects of HIV-1-infected cells (either lymphocytes or macrophages) trafficking into the granuloma itself 12 (Fig. 10.3). After gaining access to the granuloma, the immunologically active microenvironment may quickly promote HIV-1 replication and intercellular spread of the virus. Resulting virus-induced cellular dysfunction and cytopathic effect may permit active mycobacterial proliferation. By a process of cyclic augmentation, additional inflammatory activity may further accelerate HIV-1 replication, promote mononuclear cell apoptosis, and further increase recruitment of potentially HIV-infected mononuclear cells. In this way, the critical host–pathogen equilibrium may be skewed in favour of M. tuberculosis, leading to unchecked mycobacterial proliferation and active TB.12
IMPACT OF ANTIRETROVIRAL TREATMENT ON THE HOST RESPONSE TO M. TUBERCULOSIS The development of highly active antiretroviral treatment (ART) in 1996 revolutionized the care and prognosis of HIV-infected individuals. For the first time, robust suppression of viral replication permitted substantial immunological recovery, resulting in reduced morbidity and mortality risk. This has markedly altered both the risk and manifestations of TB among patients receiving ART. Risk of TB decreases by 80–90% in the first 1–2 years of ART with smaller gains thereafter.57–59 Cutaneous hypersensitivity responses to tuberculin are restored and pulmonary TB developing during ART is less likely to be radiographically atypical than that developing in untreated patients. Mortality in patients with HIVassociated TB is also greatly reduced.58,60,61
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The impact of ART on TB reflects the fact that ART reverses the effects of HIV on the host response to M. tuberculosis.58 Proliferative responses and IFN-g secretion to mycobacterial antigens by T cells in vitro are restored.62–64 The rapidity of this immune recovery is vividly illustrated by immune reconstitution disease (or immune reconstitution inflammatory syndrome (IRIS)).65 This typically occurs among patients with TB within the initial weeks of ART. Rapid recovery of immune responses to persisting mycobacteria and to shed antigen trigger clinical deterioration of the TB, which can occasionally be severe. These phenomena are associated with rapid expansion of mycobacteria-specific T cells in the peripheral circulation and rapid restoration of T-cell proliferation.66,67 Histopathological examination of affected tissues shows that these manifestations are associated with recovery of the ability of the host to form granulomas in response to M. tuberculosis, and this response may be unusually florid.65 Reports of the development of hypercalcaemia during TB-associated immune reconstitution disease also provide indirect evidence of the restoration of granuloma physiology.68 Despite the dramatic effects of ART in reversing the impact of HIV on TB, these effects are nevertheless only partial. Laboratory evidence indicates that restoration of functional immune responses to M. tuberculosis and other pathogens during long-term ART is incomplete.58 As a result, the risk of TB among patients receiving ART long term in a study in South Africa, although very substantially reduced, nevertheless remained 5- to 10-fold greater than the risk among HIV-uninfected patients living in the same community.69 Thus, strategies whereby restoration of M. tuberculosisspecific immunity is maximized during ART need to be identified. This may require initiation of ART prior to the development of severe immunodeficiency. In addition, future development of adjunctive immunotherapeutic measures or a novel vaccine may help to further improve M. tuberculosis-specific immune recovery.
IMPACT OF TUBERCULOISIS ON HIV EPIDEMIOLOGY Data from various studies support the hypothesis that active TB causes an accelerated rate of immunological decline among HIV-infected patients. The first such study conducted in the USA by Whalen et al.4 was a retrospective analysis. Survival among 106 HIV-infected patients with TB (cases) was compared with survival among HIVinfected control patients who remained TB free and who were matched for baseline degree of immunodeficiency and patient characteristics. The adjusted odds for mortality among cases were significantly increased (approximately twofold). Subsequent prospective studies in France, Uganda, and South Africa have produced findings consistent with this.70–72 In the two studies from sub-Saharan Africa, significantly increased mortality risk was observed only among TB patients with CD4 T-cell counts 200 cells/ml, suggesting that any long-term impact of TB on HIV pathogenesis occurs among those with greater immunological reserve. A substantial body of laboratory data is consistent with the hypothesis that TB has a co-factor effect on the pathogenesis of HIV-1 infection. The interpretation of the epidemiological data highlighting this potential co-factor has been questioned.73 A suggested alternative explanation for the data is that TB is a marker of advanced immunodeficiency independent of CD4 T-cell count and clinical stage of disease. Contradictory data have also been published, although
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0
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In vitro and in vivo data show that immune activation associated with M. tuberculosis increases HIV-1 replication.5 In vitro M. tuberculosis and certain mycobacterial cell wall components not only induce HIV-1 replication within cells of the monocytic lineage and CD4 T cells but also enhance viral infectivity for this cell type and increase transmission of HIV-1 from antigen-presenting cells to lymphocytes.75–82 Proinflammatory cytokines (TNF-a, IL-1, and IL-6) released by monocytes and macrophages up regulate HIV-1 expression by acting in an autocrine fashion and in a paracrine fashion on virus-infected lymphocytes.76,83–85 Intercellular adhesion between macrophages and activated lymphocytes that occurs within the inflammatory microenvironment at sites of TB disease is a further key mechanism causing marked cellular activation and increased HIV replication.86–88 Goletti et al.75 reported the first in vivo data showing that development of active TB was associated with marked (5- to 160-fold) increases in plasma HIV-1 load. In a cross-sectional study in Uganda, both cell-free and cell-associated plasma viral loads were also higher in HIV-infected patients with TB than in patients with similar CD4 T-cell counts but who were TB-free.89 Higher viral loads were found only among the subset of patients with wellpreserved CD4 T-cell counts (> 500 cells/ml), suggesting the impact of active TB on HIV pathogenesis may be most marked earlier in the course of HIV infection. This is broadly consistent with the two epidemiological studies from Uganda and South Africa.71,72 Prospective data from three studies in Uganda, Ethiopia, and South Africa each found that increases in plasma HIV-1 load occur with development of active TB.89–91 In contrast, latent M. tuberculosis infection has no impact on systemic viral load.92 In addition to increases in viral load in the systemic circulation, the impact of TB on viral replication may be greatest at anatomic sites of TB disease. Among patients with pulmonary TB, HIV-1 load is greater in BAL fluid obtained from diseased lung segments than in fluid from segments not involved.93 In patients with pleural TB, cell-free and cell-associated HIV-1 load are greater in pleural fluid than in plasma94,95 (Fig. 10.4). Similarly, among HIV-infected persons with TB meningitis, the viral load in cerebrospinal fluid is higher than that in blood.96 In the study by Goletti et al.75 plasma HIV-1 load in a small number of subjects studied in the USA and Italy decreased following successful treatment of TB. However, several studies from Africa have since found that increases in viral load associated with development of TB are generally sustained despite successful TB treatment.53,89– 91,97 One other study found that decreases occurred only in a subset of patients.98 The chronic nature of TB may be an important factor leading to these sustained increases in viral load, possibly causing development of a new higher viral load set-point. The effect of immune activation on viral load is not specific to TB. A wide variety of other immune-activating stimuli including viral, bacterial, parasitic, and fungal infections and immunizations
P = 0.003
400
TNF-a pg/ ml
comparison of survival among patients with TB and those with AIDS-defining illnesses such as Kaposi’s sarcoma or pneumocystis pneumonia is perhaps a less informative approach, especially when the co-factor effect may predominantly occur during the earlier stages of HIV infection.74 Nevertheless, the importance of any co-factor effect of TB on HIV pathogenesis at a population level in countries with a high TB burden does remain unclear.
10
5.0 4.0 3.0 2.0
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Pleural fluid
Fig. 10.4 Concentrations of (A) tumour necrosis factor (TNF)-a and (B) HIV-1 load (log RNA copies/mL) in paired plasma and pleural fluid samples from patients with pleural TB and HIV-1 coinfection (n ¼ 9). In (A) box and whisker plots indicate the median, 25th and 75th centiles, and the range of TNF-a concentrations. (B) Lines join the paired viral load values and the median is indicated by a square symbol. The compartmentalized proinflammatory cytokine response in the pleural fluid at the site of TB was associated with higher HIV-1 load at that site. Data from Lawn SD, Pisell TL, Hirsch CS, et al. Anatomically compartmentalized human immunodeficiency virus replication in HLA-DR+ cells and CD14+ macrophages at the site of pleural tuberculosis coinfection. J Infect Dis 2001 Nov 1;184:1127–33.
have also been found to be associated with increases in HIV-1 load.5,99 However, the effects on plasma viral load of many of these stimuli appear to be more transient than that caused by TB, suggesting that TB has the greater potential to affect HIV-1 pathogenesis.100,101 Furthermore, compared with many other opportunistic infections, TB develops across a wide spectrum of CD4 T-cell counts and the co-factor effect may be more marked among patients with higher CD4 T-cell counts.
MECHANISMS OF ENHANCEMENT OF HIV-1 PATHOGENESIS Stimulation of monocytes with M. tuberculosis in vitro results in production of high levels of proinflammatory cytokines, including TNF-a, IL-1, and IL-6.102,103 In vivo, immunological activation at the site of disease and in blood are greatly heightened among patients with TB and HIV coinfection.53–55,104,105 The importance of this to HIV-1 pathogenesis is that the HIV-1 life cycle is intimately related to the level of activation of the mononuclear cell reservoirs of HIV. Mononuclear cell activation during responses to TB, other coinfections, and immunizations enhances viral replication by promoting three key stages of the viral life cycle – viral cellular entry, reverse transcription, and proviral transcription5 (Fig. 10.5).
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BASIC SCIENCE 1. Immune cell activation causes upregulation of chemokine co-receptors CCR5 and CXCR4, facilitating entry of HIV to cells CD4 receptor
2. Immune cell activation facilitates completion of reverse transcription of HIV-1, converting the RNA genome into DNA
CCR5 receptor HIV RNA
CXCR4 receptor Human DNA
HIV DNA Human DNA
HIV DNA 3. Immune cell activation enhances pro-viral transcription to produce new virus particles
Fig. 10.5 Diagram of the HIV-1 life cycle showing the three key stages of the life cycle that may be facilitated by immune activation at sites of TB: (1) viral cellular entry, (2) reverse transcription of HIV-1 RNA, and (3) proviral transcription.
HIV-1 cellular entry HIV-1 particles typically enter host cells by binding to the cell surface CD4 receptor and a chemokine co-receptor – most commonly CCR5 or CXCR4. These chemokine receptors are inducibly expressed and immune activation associated with coinfections may cause upregulation of expression of these coreceptors, facilitating viral cellular entry. Mycobacterium avium, for example, causes upregulation of CCR5 on peripheral blood mononuclear cells in vitro, whereas blood CD4 T cells from patients with TB have increased membrane expression of both CXCR4 and CCR5.95,106,107 HIV-1 reverse transcription Compared with activated cells, completion of reverse transcription of HIV-1 RNA is less likely to be successful in immunologically quiescent cells.108 Cellular activation, however, increases cytoplasmic concentrations of certain mediators such as nuclear factor of activated T cells (NFATc) that facilitate reverse transcription.109
TB and other coinfections may thereby increase the pool of activated cells able to support productive viral infection.
HIV-1 proviral transcription The rate of transcription of proviral DNA is highly related to the activation state of the cell and is regulated by sequences within the long terminal repeat (LTR) at the 50 end of the viral genome110 (Fig. 10.6). These sequences include receptors for host-encoded transcription factors, including nuclear factor-kB (NF-kB). Proinflammatory cytokines released in response to TB act in an autocrine and paracrine fashion on mononuclear cell surface receptors, triggering release of high intracellular concentrations of physiologically active NF-kB and other transcription factors.111,112 As well as enhancing transcription of host cell genes, these transcription factors also activate the HIV-1 LTR, greatly increasing rates of HIV-1 proviral transcription.110,112 In this way, HIV-1 provirus harnesses the host cell transcriptional regulatory mechanisms to promote its own replication. Compared with the effect on HIV-1 infection, the
NF-kB from cytoplasm
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Fig. 10.6 Structure of the long terminal repeat (LTR) of the HIV-1 genome. The HIV-1 promoter is functionally divided into the modulatory enhancer region, core promoter region, and transactivation response region (TAR). Following cellular activation due, for example, to coinfections such as TB, increased concentrations of cellular transcription factors including nuclear factor-kB (NF-kB) activate receptors in the HIV-1 promoter, resulting in marked upregulation of HIV-1 transcription. Adapted from Lawn SD, Butera ST, Shinnick TM. Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect 2002 May;4:635–46.
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impact of TB and other coinfections on the pathogenesis of HIV2 infection is likely to be less since the HIV-2 LTR has only a single functionally active binding site for NF-kB, whereas the HIV-1 LTR has two.113 In addition to the role of proinflammatory cytokines, it has also been suggested that the beta-chemokine macrophage chemoattractant protein-1 (MCP-1) may play an important role in promoting HIV-1 transcription at sites of TB disease.95,114
Immune activation and CD4 lymphocyte depletion As well as increasing HIV-1 replication, coinfections may also accelerate CD4 T-cell loss by a number of mechanisms.5 Firstly, increased HIV-1 replication accelerates cell loss by virus-induced lysis or immune destruction of infected cells. Immune activation associated with coinfections may also have a generally suppressive effect on haematopoiesis. However, most importantly, generalized immune activation augments apoptotic cell loss, which is numerically the dominant mechanism of mononuclear cell loss in HIV infection.50 Apoptosis leads to loss of both HIV-infected and -uninfected bystander lymphocytes. Studies of patients with pleural TB and HIV-1 coinfection show very high rates of apoptosis of M. tuberculosis-specific T cells at sites of TB disease.56 These cell losses may undermine the host response to M. tuberculosis, favouring persistence of the organism and an ongoing inflammatory process. Immune activation and genotypic diversification Chronic HIV-1 infection is characterized by marked genotypic heterogeneity of the systemic virus pool. Increased rates of viral replication associated with immune-activating stimuli may promote further genotypic diversification by mutation or by leading to additional expression of previously latent proviral genotypes.100 It is hypothesized that, by these means, new viral quasi-species able to evade the established host anti-viral responses, increasing viral replication and disease progression, may be expressed. In patients with pulmonary TB, Nakata et al.93 found increased genotypic heterogeneity within BAL samples obtained from lung segments affected by TB compared with samples from uninvolved segments. HIV-1 heterogeneity in the systemic circulation is similarly increased in patients with TB compared with that in matched patients without TB.115 Comparison of the viral genotypes expressed in the plasma and pleural compartments of patients with pleural TB showed that this heterogeneity is likely to arise largely at sites of TB disease driven by increased HIV-1 replication in response to the proinflammatory drive.94,116 Further data suggest that increased heterogeneity can lead to the emergence of quasi-species that have increased replicative fitness and this may be an important factor in disease progression.117 Cellular sources of HIV-1 replication CD4 T cells and cells of the monocyte–macrophage lineage both support HIV replication. Mathematical modelling estimates, though, suggest that macrophages contribute very little (<2%) to the total plasma viral pool in patients with chronic HIV-1 infection.118 However, the number of HIV-infected macrophages is greatly expanded at sites of TB and these are highly activated. As a result, the proportion of HIV-1 particles derived from macrophages is therefore greatly increased both at sites of TB and within the systemic circulation.94,119–121 In contrast to lymphocytes which are estimated to have an average lifespan of approximately 2 days, macrophages do not succumb to virus-induced cytolysis and are resistant to Fas-mediated apoptosis.122 They therefore serve as much longer-lived reservoirs of HIV infection at sites of TB disease and are therefore likely to play a central role in the impact of TB on HIV-1 pathogenesis. These cells not only support high rates
MTB-specific CD4 T cells
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Fig. 10.7 Replication and intercellular transmission of HIV-1 is increased during antigen presentation at the site of M. tuberculosis infection. Following phagocytosis of M. tuberculosis, M. tuberculosis-specific CD4 T cells are attracted by chemotaxis (A). Subsequent intimate intercellular apposition and integrin–ligand interactions trigger marked cellular activation and secretion of proinflammatory cytokines, including TNF-a. Transmission of HIV to recruited CD4 T cells occurs and the inflammatory environment supports high rates of HIV-1 replication in both cell types. High rates of CD4 T-cell loss occur via virus-induced or immune-mediated lysis together with activation-induced apoptosis. Adapted from Lawn SD, Butera ST, Shinnick TM. Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect 2002 May;4:635–46.
of HIV-1 replication but also serve as a source of transmission of HIV infection to M. tuberculosis-specific CD4 T cells during the process of antigen presentation at the site of disease (Fig. 10.7). The cellular source of HIV replication may affect the surface phenotype of the virions produced. As virions bud from host mononuclear cells, host cell-surface molecules are incorporated into the virion envelope.121,123 Cellular expression of human lymphocyte antigen direct repeat (HLA-DR) reflects mononuclear cell activation and this molecule is incorporated at high levels into the envelope of budding virions. The proportion of the virus pool bearing HLA-DR in the envelope is greatly increased in patients with TB, especially at local sites of TB disease.94,124 This reflects high rates of viral replication within highly activated mononuclear cells. Interestingly, some data suggest that presence of HLA-DR in the viral envelope enhances viral infectivity for CD4 T cells since this molecule is the natural ligand for the CD4 T-cell receptor and is therefore likely to enhance virus–cell interactions.125 This is a further potential mechanism promoting HIV replication at sites of TB.
The inflammatory microenvironment at sites of tuberculosis While systemic immune activation associated with TB in HIVinfected patients is likely to have important effects on HIV pathogenesis, it is clear that these effects are maximal at the actual sites of TB disease. Table 10.1 summarizes the effects of the host inflammatory response on HIV-1 pathogenesis at sites of TB disease. The intense inflammatory milieu provides the ideal microenvironment for high rates of HIV-1 replication and genotypic diversification, further undermining host responses to the M. tuberculosis infection.93–95,116,126 However, genotypic studies of HIV replication at pleural sites of TB also show that the anatomic compartmentalization of these effects is only partial and that there is interplay between this HIV-1 pool and that in the systemic circulation. Thus, localized TB may also have important effects on HIV-1 pathogenesis systemically.
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Table 10.1 Summary points: effects of the host inflammatory response on the pathogenesis of HIV-1 at sites of tuberculosis disease
Increased HIV-1 replication within macrophages and CD4 T cells, leading to increased cell-associated and cell-free virus load. Enhanced susceptibility of mononuclear cells to infection with HIV. Transmission of HIV-1 from antigen-presenting cells to M. tuberculosis-specific CD4 T cells during antigen presentation. Increased loss of M. tuberculosis-specific CD4 T cells by activationinduced apoptosis or virus-induced/immune-mediated lysis. Increased HIV-1 genotypic heterogeneity, increasing potential for evolution of quasi-species with increased replicative capacity. Expansion of macrophages as long-lived reservoirs of HIV-1. Increased incorporation of HLA-DR into the virion envelope enhancing infectivity.
USE OF IMMUNOTHERAPY TO ADDRESS TUBERCULOSIS/HIV-1 CO-PATHOGENESIS The fact that immune activation and the effects of proinflammatory cytokines such as TNF-a are central to the impact of TB on the pathogenesis of HIV-1 infection has led to various trials of immunotherapeutic strategies to block these effects. Thalidomide and pentoxifylline are both drugs that provide inhibition of the TNF-a pathway and monoclonal antibodies directed against TNF-a have provided an even more targeted approach. Trials of these agents and of corticosteroids, which have more generalized immunosuppressive activity, have been conducted among patients with HIV-associated TB. Despite some indicators of beneficial immunomodulatory effect among such patients treated with thalidomide or pentoxifylline, trials of these agents found either no reductions in plasma TNF-a concentration and viral load or only small reductions unlikely to be of prognostic benefit.127–129 A trial of monoclonal TNF-a antibody showed a trend towards an increased CD4 T-cell count but no change in plasma viral load.130 Use of prednisolone therapy similarly showed an increase in CD4 T-cell count but paradoxically was associated with a transient increase in plasma viral load and a high rate of adverse events.131 The failure of these strategies probably reflects the complexity of the co-pathogenesis of TB and HIV-1. It currently seems unlikely that any such agents will prove to be useful, cost-effective
REFERENCES 1. World Health Organization. Global tuberculosis control: surveillance, planning, financing. WHO report 2005. WHO/HTM/TB/2005.349. Geneva: World Health Organization. 2. 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. 3. Nunn P, Williams B, Floyd K, et al. Tuberculosis control in the era of HIV. Nat Rev Immunol 2005; 5:819–826. 4. Whalen C, Horsburgh CR, Hom D, et al. Accelerated course of human immunodeficiency virus infection after tuberculosis. Am J Respir Crit Care Med 1995;151:129–135. 5. Lawn SD, Butera ST, Folks TM. Contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection. Clin Microbiol Rev 2001;14:753–777, table.
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adjunctive treatments in clinical practice. Many of the trials of immunomodulatory adjunctive treatments were conducted in resource-limited settings prior to the widespread availability of ART. Availability of this treatment has revolutionized the prognosis of patients with HIV-associated TB and is now expanding in resource-limited settings. Thus, the need of immunomodulatory treatment for such patients has largely been superseded.
CONCLUSIONS HIV-1 infection is one of the principal factors underlying the resurgence of TB over the past 20 years. An effective host cellular immune response with granuloma formation is required to contain M. tuberculosis infection and prevent the reactivation of latent infection, which is present in approximately one-third of the world’s population. The devastating impact of HIV-1 on the natural history of TB highlights the critical impact of the virus on the establishment and functioning of the granulomatous host response to M. tuberculosis. Via multiple mechanisms HIV-1 coinfection leads to functional and numeric depletion of M. tuberculosis-specific CD4 T cells and diminished type-1 cytokine responses. Dysfunction of the CD4 T cell–macrophage axis impairs the host’s ability to generate cell-mediated immune responses and form immunologically competent granulomas. Sites of TB disease and granulomas themselves provide the ideal microenvironment for the propagation of HIV-1, thereby maximizing the deleterious impact of HIV-1 at the critical interface between M. tuberculosis and the host. Moreover, immune activation generated during the host response to M. tuberculosis drives HIV-1 replication and genotypic diversification, potentially accelerating immunological decline. Substantial reversal of these effects is currently achievable only through use of ART, further emphasizing the need for rapid expansion of access to this treatment in resourcelimited settings where the burden of HIV-associated TB is greatest.
ACKNOWLEDGEMENTS Dr Stephen D. Lawn is funded by the Wellcome Trust, London, UK. Dr Linda-Gail Bekker is funded, in part, by the National Institutes of Health, USA, through CIPRA Grant 1U19AI53217-01.
6. Di Perri G, Cruciani M, Danzi MC, et al. Nosocomial epidemic of active tuberculosis among HIV-infected patients. Lancet 1989;2:1502–1504. 7. Dooley SW, Villarino ME, Lawrence M, et al. Nosocomial transmission of tuberculosis in a hospital unit for HIV-infected patients. JAMA 1992; 267:2632–2634. 8. Gandhi NR, Moll A, Sturm AW, et al. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 2006;368:1575–1580. 9. Selwyn PA, Hartel D, Lewis VA, et al. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 1989;320:545–550. 10. Daley CL, Small PM, Schecter GF, et al. An outbreak of tuberculosis with accelerated progression among persons infected with the human immunodeficiency virus. An analysis using restrictionfragment-length polymorphism. N Engl J Med 1992;326:231–235. 11. Gedde-Dahl T. Tuberculous infection in the light of tuberculin matriculation. Am J Hyg 1952;56:139–214.
12. Lawn SD, Butera ST, Shinnick TM. Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect 2002; 4:635–646. 13. Ackah AN, Coulibaly D, Digbeu H, et al. Response to treatment, mortality, and CD4 lymphocyte counts in HIV-infected persons with tuberculosis in Abidjan, Cote d’Ivoire. Lancet 1995;345:607–610. 14. Gilks CF, Brindle RJ, Otieno LS, et al. Extrapulmonary and disseminated tuberculosis in HIV-1-seropositive patients presenting to the acute medical services in Nairobi. AIDS 1990;4:981–985. 15. Elliott AM, Halwiindi B, Hayes RJ, et al. The impact of human immunodeficiency virus on presentation and diagnosis of tuberculosis in a cohort study in Zambia. J Trop Med Hyg 1993;96:1–11. 16. Lawn SD, Evans AJ, Sedgwick PM, et al. Pulmonary tuberculosis: radiological features in west Africans coinfected with HIV. Br J Radiol 1999;72:339–344. 17. Mukadi YD, Maher D, Harries A. Tuberculosis case fatality rates in high HIV prevalence populations in sub-Saharan Africa. AIDS 2001;15:143–152.
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Co-pathogenesis of tuberculosis and HIV 18. Greenberg AE, Lucas S, Tossou O, et al. Autopsyproven causes of death in HIV-infected patients treated for tuberculosis in Abidjan, Cote d’Ivoire. AIDS 1995;9:1251–1254. 19. Cobelens FG, Egwaga SM, van GT, et al. Tuberculin skin testing in patients with HIV infection: limited benefit of reduced cutoff values. Clin Infect Dis 2006;43:634–639. 20. Lucas SB, Hounnou A, Peacock C, et al. The mortality and pathology of HIV infection in a west African city. AIDS 1993;7:1569–1579. 21. Lucas SB, De Cock KM, Hounnou A, et al. Contribution of tuberculosis to slim disease in Africa. BMJ 1994;308:1531–1533. 22. Rana F, Hawken MP, Meme HK, et al. Autopsy findings in HIV-1-infected adults in Kenya. J Acquir Immune Defic Syndr Hum Retrovirol 1997;14:83–85. 23. Lucas SB, Nelson AM. Pathogenesis of tuberculosis in human immunodeficiency virus-infected people. In: Bloom BR (ed.). Tuberculosis: Pathogenesis, Protection and Control. Washington DC: ASM Press, 1994: 503–513. 24. Dannenberg AM Jr. Immune mechanisms in the pathogenesis of pulmonary tuberculosis. Rev Infect Dis 1989;11(Suppl 2):S369–S378. 25. Dannenberg AM Jr. Delayed-type hypersensitivity and cell-mediated immunity in the pathogenesis of tuberculosis. Immunol Today 1991;12:228–233. 26. Orme IM, Cooper AM. Cytokine/chemokine cascades in immunity to tuberculosis. Immunol Today 1999;20:307–312. 27. Saunders BM, Cooper AM. Restraining mycobacteria: role of granulomas in mycobacterial infections. Immunol Cell Biol 2000;78:334–341. 28. Adams DO. The granulomatous inflammatory response. A review. Am J Pathol 1976;84:164–191. 29. Boom WH. The role of T-cell subsets in Mycobacterium tuberculosis infection. Infect Agents Dis 1996;5:73–81. 30. Verani A, Gras G, Pancino G. Macrophages and HIV-1: dangerous liaisons. Mol Immunol 2005;42: 195–212. 31. Kedzierska K, Crowe SM. The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. Curr Med Chem 2002;9:1893–1903. 32. Kedzierska K, Mak J, Mijch A, et al. Granulocytemacrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type 1-infected monocytes/macrophages in vitro and in vivo. J Infect Dis 2000;181:390–394. 33. Imperiali FG, Zaninoni A, La Maestra L, et al. Increased Mycobacterium tuberculosis growth in HIV-1infected human macrophages: role of tumour necrosis factor-a. Clin Exp Immunol 2001;123:435–442. 34. Barron MA, Blyveis N, Palmer BE, et al. Influence of plasma viremia on defects in number and immunophenotype of blood dendritic cell subsets in human immunodeficiency virus 1-infected individuals. J Infect Dis 2003;187:26–37. 35. Kaufmann SH, Schaible UE. A dangerous liaison between two major killers: Mycobacterium tuberculosis and HIV target dendritic cells through DC-SIGN. J Exp Med 2003;197:1–5. 36. Van KY, Appelmelk B, Geijtenbeek TB. A fatal attraction: Mycobacterium tuberculosis and HIV-1 target DC-SIGN to escape immune surveillance. Trends Mol Med 2003;9:153–159. 37. Clerici M, Stocks NI, Zajac RA, et al. Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virusseropositive patients. Independence of CD4þ cell numbers and clinical staging. J Clin Invest 1989; 84:1892–1899. 38. Sonnenberg P, Glynn JR, Fielding K, et al. How soon after infection with HIV does the risk of tuberculosis start to increase? A retrospective cohort study in South African gold miners. J Infect Dis 2005;191:150–158. 39. Rosenberg ZF, Fauci AS. The immunopathogenesis of HIV infection. Adv Immunol 1989;47:377–431. 40. Oyaizu N, Chirmule N, Kalyanaraman VS, et al. Human immunodeficiency virus type 1 envelope
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59.
60.
glycoprotein gp120 produces immune defects in CD4þ T lymphocytes by inhibiting interleukin 2 mRNA. Proc Natl Acad Sci USA 1990;87:2379–2383. Hoxie JA, Alpers JD, Rackowski JL, et al. Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV. Science 1986;234:1123–1127. Kanazawa S, Okamoto T, Peterlin BM. Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection. Immunity 2000;12:61–70. Puri RK, Leland P, Aggarwal BB. Constitutive expression of human immunodeficiency virus type 1 tat gene inhibits interleukin 2 and interleukin 2 receptor expression in a human CD4þ T lymphoid (H9) cell line. AIDS Res Hum Retroviruses 1995; 11:31–40. Ottenhoff TH, Kumararatne D, Casanova JL. Novel human immunodeficiencies reveal the essential role of type-I cytokines in immunity to intracellular bacteria. Immunol Today 1998;19:491–494. Spellberg B, Edwards JE Jr. Type 1/type 2 immunity in infectious diseases. Clin Infect Dis 2001;32:76–102. Gong JH, Zhang M, Modlin RL, et al. Interleukin-10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA-4 expression. Infect Immun 1996;64:913–918. Murray PJ, Wang L, Onufryk C, et al. T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J Immunol 1997;158:315–321. Ma X, Sun J, Papasavvas E, et al. Inhibition of IL-12 production in human monocyte-derived macrophages by TNF. J Immunol 2000;164:1722–1729. Vanham G, Penne L, Devalck J, et al. Decreased CD40 ligand induction in CD4 T cells and dysregulated IL-12 production during HIV infection. Clin Exp Immunol 1999;117:335–342. Hazenberg MD, Hamann D, Schuitemaker H, et al. T cell depletion in HIV-1 infection: how CD4þ T cells go out of stock. Nat Immunol 2000;1:285–289. Law KF, Jagirdar J, Weiden MD, et al. Tuberculosis in HIV-positive patients: cellular response and immune activation in the lung. Am J Respir Crit Care Med 1996;153:1377–1384. Vanham G, Edmonds K, Qing L, et al. Generalized immune activation in pulmonary tuberculosis: co-activation with HIV infection. Clin Exp Immunol 1996;103:30–34. Lawn SD, Shattock RJ, Acheampong JW, et al. Sustained plasma TNF-a and HIV-1 load despite resolution of other parameters of immune activation during treatment of tuberculosis in Africans. AIDS 1999;13:2231–2237. Lawn SD, Rudolph D, Wiktor S, et al. Tuberculosis (TB) and HIV infection are independently associated with elevated serum concentrations of tumour necrosis factor receptor type 1 and beta2microglobulin, respectively. Clin Exp Immunol 2000;122:79–84. Hertoghe T, Wajja A, Ntambi L, et al. T cell activation, apoptosis and cytokine dysregulation in the (co)pathogenesis of HIV and pulmonary tuberculosis (TB). Clin Exp Immunol 2000;122:350–357. Hirsch CS, Toossi Z, Johnson JL, et al. Augmentation of apoptosis and interferon-gamma production at sites of active Mycobacterium tuberculosis infection in human tuberculosis. J Infect Dis 2001;183:779–788. Badri M, Wilson D, Wood R. Effect of highly active antiretroviral therapy on incidence of tuberculosis in South Africa: a cohort study. Lancet 2002;359: 2059–2064. Lawn SD, Bekker LG, Wood R. How effectively does HAART restore immune responses to Mycobacterium tuberculosis? Implications for tuberculosis control. AIDS 2005;19:1113–1124. Lawn SD, Badri M, Wood R. Tuberculosis among HIV-infected patients receiving HAART: long term incidence and risk factors in a South African cohort. AIDS 2005;19:2109–2116. Girardi E, Palmieri F, Cingolani A, et al. Changing clinical presentation and survival in HIV-associated tuberculosis after highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2001;26:326–331.
10
61. Dheda K, Lampe FC, Johnson MA, et al. Outcome of HIV-associated tuberculosis in the era of highly active antiretroviral therapy. J Infect Dis 2004;190: 1670–1676. 62. Autran B, Carcelain G, Li TS, et al. Positive effects of combined antiretroviral therapy on CD4þ T cell homeostasis and function in advanced HIV disease. Science 1997;277:112–116. 63. Li TS, Tubiana R, Katlama C, et al. Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral therapy in advanced HIV-1 disease. Lancet 1998;351: 1682–1686. 64. Schluger NW, Perez D, Liu YM. Reconstitution of immune responses to tuberculosis in patients with HIV infection who receive antiretroviral therapy. Chest 2002;122:597–602. 65. Lawn SD, Bekker LG, Miller RF. Immune reconstitution disease associated with mycobacterial infections in HIV-infected individuals receiving antiretrovirals. Lancet Infect Dis 2005;5:361–373. 66. Bourgarit A, Carcelain G, Martinez V, et al. Explosion of tuberculin-specific Th1-responses induces immune restoration syndrome in tuberculosis and HIV co-infected patients. AIDS 2006;20:F1–F7. 67. Foudraine NA, Hovenkamp E, Notermans DW, et al. Immunopathology as a result of highly active antiretroviral therapy in HIV-1-infected patients. AIDS 1999;13:177–184. 68. Lawn SD, Macallan DC. Hypercalcemia: a manifestation of immune reconstitution complicating tuberculosis in an HIV-infected person. Clin Infect Dis 2004;38:154–155. 69. Lawn SD, Myer L, Bekker LG, et al. Burden of tuberculosis in an antiretroviral treatment programme in sub-Saharan Africa: impact on treatment outcomes and implications for tuberculosis control. AIDS 2006;20:1605–1612. 70. Leroy V, Salmi LR, Dupon M, et al. Progression of human immunodeficiency virus infection in patients with tuberculosis disease. A cohort study in Bordeaux, France, 1988-1994. The Groupe d’Epidemiologie Clinique du Sida en Aquitaine (GECSA). Am J Epidemiol 1997;145:293–300. 71. Badri M, Ehrlich R, Wood R, et al. Association between tuberculosis and HIV disease progression in a high tuberculosis prevalence area. Int J Tuberc Lung Dis 2001;5:225–232. 72. Whalen CC, Nsubuga P, Okwera A, et al. Impact of pulmonary tuberculosis on survival of HIV-infected adults: a prospective epidemiologic study in Uganda. AIDS 2000;14:1219–1228. 73. Del AJ, Malin AS, Pozniak A, et al. Does tuberculosis accelerate the progression of HIV disease? Evidence from basic science and epidemiology. AIDS 1999;13:1151–1158. 74. Del Amo J, Perez-Hoyos S, Herna´ndez Aguado I, et al. Impact of tuberculosis on HIV disease progression in persons with well-documented time of HIV seroconversion. J Acquir Immune Defic Syndr 2003;33:184–190. 75. Goletti D, Weissman D, Jackson RW, et al. Effect of Mycobacterium tuberculosis on HIV replication. Role of immune activation. J Immunol 1996;157:1271–1278. 76. Lederman MM, Georges DL, Kusner DJ, et al. Mycobacterium tuberculosis and its purified protein derivative activate expression of the human immunodeficiency virus. J Acquir Immune Defic Syndr 1994;7:727–733. 77. Shattock RJ, Friedland JS, Griffin GE. Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells. J Gen Virol 1994;75(Pt 4):849–856. 78. Zhang Y, Nakata K, Weiden M, et al. Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat. J Clin Invest 1995;95:2324–2331. 79. Bernier R, Barbeau B, Olivier M, et al. Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells. J Gen Virol 1998;79(Pt 6):1353–1361.
105
SECTION
2
BASIC SCIENCE
80. Toossi Z, Sierra-Madero JG, Blinkhorn RA, et al. Enhanced susceptibility of blood monocytes from patients with pulmonary tuberculosis to productive infection with human immunodeficiency virus type 1. J Exp Med 1993;177:1511–1516. 81. Mancino G, Placido R, Bach S, et al. Infection of human monocytes with Mycobacterium tuberculosis enhances human immunodeficiency virus type 1 replication and transmission to T cells. J Infect Dis 1997;175:1531–1535. 82. Tsunetsugu-Yokota Y, Akagawa K, Kimoto H, et al. Monocyte-derived cultured dendritic cells are susceptible to human immunodeficiency virus infection and transmit virus to resting T cells in the process of nominal antigen presentation. J Virol 1995;69:4544–4547. 83. Clouse KA, Powell D, Washington I, et al. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 1989;142:431–438. 84. Michihiko S, Yamamoto N, Shinozaki F, et al. Augmentation of in-vitro HIV replication in peripheral blood mononuclear cells of AIDS and ARC patients by tumour necrosis factor. Lancet 1989;1:1206–1207. 85. Nabel G, Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 1987; 326:711–713. 86. Warwick-Davies J, Watson AJ, Griffin GE, et al. Enhancement of Mycobacterium tuberculosis-induced tumor necrosis factor alpha production from primary human monocytes by an activated T-cell membranemediated mechanism. Infect Immun 2001;69:6580–6587. 87. Shattock RJ, Burger D, Dayer JM, et al. Enhanced HIV replication in monocytic cells following engagement of adhesion molecules and contact with stimulated T cells. Res Virol 1996;147:171–179. 88. Hoshino Y, Nakata K, Hoshino S, et al. Maximal HIV-1 replication in alveolar macrophages during tuberculosis requires both lymphocyte contact and cytokines. J Exp Med 2002;195:495–505. 89. Toossi Z, Mayanja-Kizza H, Hirsch CS, et al. Impact of tuberculosis (TB) on HIV-1 activity in dually infected patients. Clin Exp Immunol 2001;123:233–238. 90. Wolday D, Hailu B, Girma M, et al. Low CD4þ T-cell count and high HIV viral load precede the development of tuberculosis disease in a cohort of HIV-positive Ethiopians. Int J Tuberc Lung Dis 2003;7:110–116. 91. Day JH, Grant AD, Fielding KL, et al. Does tuberculosis increase HIV load? J Infect Dis 2004;190:1677–1684. 92. Manoff SB, Farzadegan H, Munoz A, et al. The effect of latent Mycobacterium tuberculosis infection on human immunodeficiency virus (HIV) disease progression and HIV RNA load among injecting drug users. J Infect Dis 1996;174:299–308. 93. Nakata K, Rom WN, Honda Y, et al. Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication in the lung. Am J Respir Crit Care Med 1997;155:996–1003. 94. Lawn SD, Pisell TL, Hirsch CS, et al. Anatomically compartmentalized human immunodeficiency virus replication in HLA-DRþ cells and CD14þ macrophages at the site of pleural tuberculosis coinfection. J Infect Dis 2001;184:1127–1133. 95. Toossi Z, Johnson JL, Kanost RA, et al. Increased replication of HIV-1 at sites of Mycobacterium tuberculosis infection: potential mechanisms of viral activation. J Acquir Immune Defic Syndr 2001;28:1–8. 96. Brew BJ, Pemberton L, Cunningham P, et al. Levels of human immunodeficiency virus type 1 RNA in cerebrospinal fluid correlate with AIDS dementia stage. J Infect Dis 1997;175:963–966.
106
97. Morris L, Martin DJ, Bredell H, et al. Human immunodeficiency virus-1 RNA levels and CD4 lymphocyte counts, during treatment for active tuberculosis, in South African patients. J Infect Dis 2003;187:1967–1971. 98. Kizza HM, Rodriguez B, Quinones-Mateu M, et al. Persistent replication of human immunodeficiency virus type 1 despite treatment of pulmonary tuberculosis in dually infected subjects. Clin Diagn Lab Immunol 2005;12:1298–1304. 99. Bentwich Z, Maartens G, Torten D, et al. Concurrent infections and HIV pathogenesis. AIDS 2000;14:2071–2081. 100. Ostrowski MA, Krakauer DC, Li Y, et al. Effect of immune activation on the dynamics of human immunodeficiency virus replication and on the distribution of viral quasispecies. J Virol 1998; 72:7772–7784. 101. Stanley SK, Ostrowski MA, Justement JS, et al. Effect of immunization with a common recall antigen on viral expression in patients infected with human immunodeficiency virus type 1. N Engl J Med 1996;334:1222–1230. 102. Kaplan G, Freedman VH. The role of cytokines in the immune response to tuberculosis. Res Immunol 1996;147:565–572. 103. Toossi Z. Cytokine circuits in tuberculosis. Infect Agents Dis 1996;5:98–107. 104. Vanham G, Edmonds K, Qing L, et al. Generalized immune activation in pulmonary tuberculosis: co-activation with HIV infection. Clin Exp Immunol 1996;103:30–34. 105. Lawn SD, Labeta MO, Arias M, et al. Elevated serum concentrations of soluble CD14 in HIV- and HIVþ patients with tuberculosis in Africa: prolonged elevation during anti-tuberculosis treatment. Clin Exp Immunol 2000;120:483–487. 106. Wahl SM, Greenwell-Wild T, Peng G, et al. Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression. Proc Natl Acad Sci USA 1998;95:12574–12579. 107. Juffermans NP, Speelman P, Verbon A, et al. Patients with active tuberculosis have increased expression of HIV coreceptors CXCR4 and CCR5 on CD4(þ) T cells. Clin Infect Dis 2001;32: 650–652. 108. Zack JA, Arrigo SJ, Weitsman SR, et al. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990;61:213–222. 109. Kinoshita S, Chen BK, Kaneshima H, et al. Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells. Cell 1998;95:595–604. 110. Gaynor R. Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS 1992;6:347–363. 111. Baeuerle PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta 1991;1072:63–80. 112. Pereira LA, Bentley K, Peeters A, et al. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res 2000;28:663–668. 113. Tong-Starksen SE, Welsh TM, Peterlin BM. Differences in transcriptional enhancers of HIV-1 and HIV-2. Response to T cell activation signals. J Immunol 1990;145:4348–4354. 114. Vicenzi E, Alfano M, Ghezzi S, et al. Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1. J Leukoc Biol 2000;68: 405–412. 115. Collins KR, Mayanja-Kizza H, Sullivan BA, et al. Greater diversity of HIV-1 quasispecies in
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
HIV-infected individuals with active tuberculosis. J Acquir Immune Defic Syndr 2000;24:408–417. Collins KR, Quinones-Mateu ME, Wu M, et al. Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity. J Virol 2002;76:1697–1706. Troyer RM, Collins KR, Abraha A, et al. Changes in human immunodeficiency virus type 1 fitness and genetic diversity during disease progression. J Virol 2005;79:9006–9018. Perelson AS, Neumann AU, Markowitz M, et al. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 1996;271:1582–1586. Orenstein JM, Fox C, Wahl SM. Macrophages as a source of HIV during opportunistic infections. Science 1997;276:1857–1861. Wahl SM, Greenwell-Wild T, Peng G, et al. Co-infection with opportunistic pathogens promotes human immunodeficiency virus type 1 infection in macrophages. J Infect Dis 1999;179(Suppl 3): S457–S460. Lawn SD, Roberts BD, Griffin GE, et al. Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection. J Virol 2000;74:139–145. Perlman H, Pagliari LJ, Georganas C, et al. FLICEinhibitory protein expression during macrophage differentiation confers resistance to Fas-mediated apoptosis. J Exp Med 1999;190:1679–1688. Tremblay MJ, Fortin JF, Cantin R. The acquisition of host-encoded proteins by nascent HIV-1. Immunol Today 1998;19:346–351. Lawn SD, Butera ST. Incorporation of HLA-DR into the envelope of human immunodeficiency virus type 1 in vivo: correlation with stage of disease and presence of opportunistic infection. J Virol 2000; 74:10256–10259. Cantin R, Fortin JF, Lamontagne G, et al. The acquisition of host-derived major histocompatibility complex class II glycoproteins by human immunodeficiency virus type 1 accelerates the process of virus entry and infection in human T-lymphoid cells. Blood 1997;90:1091–1100. Garrait V, Cadranel J, Esvant H, et al. Tuberculosis generates a microenvironment enhancing the productive infection of local lymphocytes by HIV. J Immunol 1997;159:2824–2830. Bekker LG, Haslett P, Maartens G, et al. Thalidomide-induced antigen-specific immune stimulation in patients with human immunodeficiency virus type 1 and tuberculosis. J Infect Dis 2000;181:954–965. Wallis RS, Nsubuga P, Whalen C, et al. Pentoxifylline therapy in human immunodeficiency virus-seropositive persons with tuberculosis: a randomized, controlled trial. J Infect Dis 1996; 174:727–733. Klausner JD, Makonkawkeyoon S, Akarasewi P, et al. The effect of thalidomide on the pathogenesis of human immunodeficiency virus type 1 and M. tuberculosis infection. J Acquir Immune Defic Syndr Hum Retrovirol 1996;11:247–257. Wallis RS, Kyambadde P, Johnson JL, et al. A study of the safety, immunology, virology, and microbiology of adjunctive etanercept in HIV-1associated tuberculosis. AIDS 2004;18:257–264. Mayanja-Kizza H, Jones-Lopez E, Okwera A, et al. Immunoadjuvant prednisolone therapy for HIVassociated tuberculosis: a phase 2 clinical trial in Uganda. J Infect Dis 2005;191:856–865.
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11
Tuberculosis vaccines Gregory D Hussey, Tony Hawkridge, and Willem A Hanekom
INTRODUCTION The world is witnessing an escalation of the TB epidemic, particularly in sub-Saharan Africa and South-East Asia.1 The problem has been compounded by the evolution of the human immunodeficiency virus (HIV) epidemic, the increase in multidrug-resistant (MDR) TB and the emergence of extensively drug-resistant (XDR) TB.2 The escalation of the TB epidemic worldwide has occurred despite the widespread use of Bacillus Calmette–Gue´rin (BCG) vaccine, currently the only licensed vaccine.3 BCG is safe and may induce long-lasting protective immunity,4 but we do not fully understand the mechanisms underlying this immunity. BCG affords approximately 80% protection against TB meningitis and miliary TB in infancy and in young children, but protection against lung disease, at all ages, is highly variable.5 In some trials, efficacy in excess of 70% against lung TB has been reported; however, in the majority, efficacy has been closer to 0%.4 The failure of BCG vaccine and increasing global mortality due to TB has emphasized the urgent need to develop more effective vaccines against the disease. New genetic technology and the sequencing of the Mycobacterium tuberculosis genome in the late 1990s have made rational development of new TB vaccines a reality.6 To date, more than 200 new candidate TB vaccines have been tested in animal models, and five have undergone phase I trials in humans.7 The licensing of an effective new TB vaccine by 2015 is now becoming a possibility. The introduction of new TB vaccines is seen as an essential part of the global strategy to eliminate TB by 2050.8 This chapter reviews the development of new TB vaccines, strategies for implementation and the challenges to such strategies.
THE IDEAL TUBERCULOSIS VACCINE An ideal new TB vaccine should have all or most of the characteristics listed in Table 11.1. It is unlikely that a single candidate will meet all or even most of these requirements and it is likely that more than one candidate vaccine may be needed.
NEW TUBERCULOSIS VACCINE APPROACHES IN RELATION TO THE NATURAL HISTORY OF THE DISEASE The choice of a new vaccine candidate will depend, to some extent, upon which phase of the natural history of TB is targeted.9
Three general approaches to new vaccines are being pursued (Fig. 11.1). The first aims to deliver vaccines prior to exposure of individuals to mycobacterial infection, a pre-infection vaccination strategy. In high-burden countries this would be the most ideal option. As TB is common in early life in these settings, vaccination would ideally have to be as close to birth as possible. This vaccine would also have to be more effective than the current BCG vaccine. Alternatively, a new TB vaccine may be used to boost immunity induced by neonatal BCG vaccination. A boost vaccine should preferably be administered together with other Expanded Programme on Immunization (EPI) vaccines.10 The second approach is to enhance or boost immunity in individuals already primed through natural infection with M. tuberculosis, a post-infection vaccine strategy. This approach is attractive because more than two billion persons are already infected with the pathogen worldwide and thus are at risk of progression to disease. This is also an attractive option for countries with high rates of HIV infection, if such a vaccine will be safe in this population. A therapeutic vaccine as an adjunct to treatment to shorten therapy or reduce the risk of relapse is a third option, and may be particularly relevant in settings where MDR- or XDR-TB are common, and in areas where TB control programmes are not managing to hold cases adequately.9 It is unlikely that a single vaccine will be able to be used at all stages.
NEW CANDIDATE TUBERCULOSIS VACCINES TYPES OF VACCINES Traditionally, vaccines have been divided into inactivated, live attenuated, toxoid and subunit vaccines. Over the past two decades novel approaches to vaccine development have emerged, with resulting conjugate, recombinant vectored and DNA vaccines. The chief characteristics of each vaccine type are summarized in Table 11.2.
LEADING CANDIDATE TUBERCULOSIS VACCINES Several novel TB vaccine candidates appear to afford protection against TB that is either similar or superior to that afforded by BCG, in animal models. To date, six of these candidates have entered clinical trials; data on the status of five accessible at the time of writing this chapter will be addressed: MVA85A, rBCG30, Mtb72f/M72, Aeras-402 and Mycobacterium vaccae. Other candidates, some of which appear very promising (Table 11.3), have recently been described in excellent reviews.11,12
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Table 11.1 Characteristics of the ideal tuberculosis vaccine
Table 11.2 Types of vaccines
1. 2. 3. 4. 5.
Type
6. 7. 8. 9.
Easily administered. Low cost. Single dose administered at or soon after birth. Safe, immunogenic and effective in newborns, children and adults. Safe, immunogenic and effective in mycobacterial naı¨ve as well as exposed individuals, including persons infected with non-tuberculous mycobacteria and previously vaccinated with BCG. Does not induce tuberculin reactivity. Effective in preventing primary TB, reinfection and reactivation disease. Effective in preventing pulmonary, extrapulmonary and disseminated disease. Safe, immunogenic and effective in preventing TB in individuals with HIV infection or AIDS, other immune deficiencies and malnutrition.
Uninfected person
Cure
Therapeutic vaccine
Disease
Pre-infection vaccine
Post-infection vaccine
Infected person
Fig. 11.1 Tuberculosis vaccine strategies in relation to the natural history of TB.
Clinical trials generally allow for a step-wise increase in mycobacterial exposure among participants, as a precaution against a possible Koch-like reaction. The Koch phenomenon is development of immunopathology in a person with TB when an exaggerated mycobacterial immune response is stimulated.13 The risk of a Koch-like reaction is likely to increase in the presence of a high mycobacterial load, such as in the presence of TB disease,14 while the risk in the setting of asymptomatic latent infection is likely to be low.
MVA85A MVA85A was developed at Oxford University by Helen McShane and Adrian Hill. The vaccine consists of Modified Vaccinia Ankara (MVA) engineered to express the immunodominant M. tuberculosis antigen 85A. This secreted protein is a member of the antigen 85 complex,15 which is involved in the binding of mycobacteria to fibronectin. The mycolyltransferase activity of the proteins is critical for the biogenesis of cord factor. The antigen 85 complex is highly conserved across mycobacterial species, including all strains of the BCG vaccine and environmental mycobacteria sequenced to date. MVA is a replication-incompetent strain of vaccinia virus which can still synthesize proteins during its abortive infection. The viral genome remains stable during passage in cell culture. MVA has been administered to more than 120,000 human vaccinees in the smallpox eradication programme of the 1970s, with no serious adverse effects.16,17 MVA-based vaccines are safe and immunogenic when given to immunosuppressed recipients; recombinant MVA vaccines expressing HIV antigens have been administered to HIV-infected participants receiving or not receiving anti-retroviral therapy in phase I trials, with no serious adverse events reported.18,19
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Traditional types of vaccines Inactivated vaccines Organisms innately capable of causing disease that have undergone treatment with chemicals or heat, which has rendered them unable to cause the disease. This approach has been tested in TB, but has not been found useful, with the possible exception of the M. vaccae vaccine. Live, attenuated Live organisms which through culture under vaccines certain conditions have lost their virulent properties. BCG is an example. Toxoids Illness-causing components produced by pathogens that have been inactivated. This approach is not so far proved relevant to TB. Subunit vaccines A part of the organism, rather than the whole organism, is used to create an immune response. Examples include TB vaccines which consist of TB proteins (antigens) delivered with adjuvants or in specialized vehicles. Newer approaches to vaccine development Conjugate vaccines Linking the outer polysaccharide coats of certain organisms to proteins can lead to a better immune response. This approach is not relevant to TB vaccine development. Recombinant A gene thought to code for a protective protein vectored vaccines is isolated and then recombined with DNA of a vector. Examples include the vaccinia and adenovirus vectored vaccines currently in phase I and II trials. DNA vaccines The vaccine is created from the infectious agent’s DNA. Delivering this to human or animal cells leads to expression of the infectious agent’s proteins and triggers immune system recognition. Some TB DNA vaccine candidates have shown promise in animal models, but none have so far reached clinical testing.
Preclinical studies In mice, a BCG prime followed by a MVA85A boost induced higher levels of antigen-specific interferon-gamma (IFN-g)-secreting CD4þ and CD8þ T cells and greater levels of protection against aerosol TB challenge than a BCG prime with no boost.20 In guinea pigs, a BCG prime followed by boosting with MVA85A plus a second fowlpox vector expressing antigen 85A induced greater protection against high-dose TB challenge than BCG prime alone.21 This regimen was also immunogenic and protective in rhesus macaques.22 Clinical studies Phase I trials of MVA85A were first conducted in the UK, then in the Gambia and then in South Africa, as the expected degree of exposure of participants to M. tuberculosis is least in the UK, greater in the Gambia and greatest in South Africa. The primary immunological outcome was the ex vivo IFN-g Elispot assay, which assessed specific T-cell responses to M. tuberculosis purified protein derivative (PPD), antigen 85 and its constituent peptides. In all studies latent TB infection was defined by T-cell reactivity to ESAT-6 and CFP-10, antigens present in M. tuberculosis but absent from BCG and most environmental mycobacteria.
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Tuberculosis vaccines
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Table 11.3 Summary of new-generation tuberculosis vaccine work in progress Name
Description
Developmental stage
Researcher
Sponsor
rBCG30 BCG
BCG Tice strain engineered to overexpress Ag85B
Phase I trial in USA, completed in 2004; no serious adverse events, immunogenicity shown
M Horwitz, D Hoft, T Littlejohn
rBCG-Aeras 403
BCG Danish strain with an endosome escape mechanism (perfringolysin) and over expression of Ag85A, Ag85B and TB10.4 BCG Pasteur strain with an endosome escape mechanism (listeriolysin) BCG Pasteur strain with reintroduction of RD-1 locus which contains presumedly protective Ag’s BCG Tice strain with diminished superoxide dismutase activity
Ongoing pre clinical studies and GMP production
R Sun, D Hone, M Stone, J Sadoff
Sequella, Aeras Global TB Vaccine Foundation Aeras Global TB Vaccine Foundation
Ongoing pre clinical studies and GMP production Ongoing pre clinical studies
S Kaufmann, L Grode, S Cole
Ongoing pre clinical studies
D Kernodle
Deletion of the virulence-associated gene phoP from Mtb MT103 strain Mtb H37Rv with deletion of panCD and RD1 locus Mtb H37Rv with deletion of the lysA and the panCD locus Recombinant fusion protein (Mtb39 and Mtb32) in AS02A and AS01B adjuvants
Ongoing pre clinical studies and GLP production Ongoing pre clinical studies and GMP production Ongoing pre clinical studies and GMP production Phase I trials completed in the USA and Europe; no serious adverse events; additional trials ongoing in Europe Phase I clinical trials commenced
B Martin, B Gicquel W Jacobs
NIH
W Jacobs
NIH
Y Skeiky, S Reed
GlaxoSmithKline, Aeras Global TB Vaccine Foundation SSI, TBVAC
rBCGDUre:Hlyþ BCG::RDI
Pro-apoptotic BCG
M. tuberculosis PhoP M. tuberculosis mc26030 M. tuberculosis mc26020 Mtb72F
Hybrid-1 (Ag85B– ESAT6) HyVac-4 (Ag85B– TB10.4)
Recombinant fusion of Ag85B–ESAT-6 in IC31 adjuvant Recombinant fusion of Ag85B–TB10.4 in IC31 adjuvant
Heatshock protein
Hsp65 (GroEL) MVA85A
P Andersen
Ongoing pre clinical studies and GLP production
P Andersen
Nascent BCG proteins associated with purified heatshock proteins
Ongoing pre clinical studies
C Colaco
DNA: conserved antigen from Mycobacterium leprae for immunotherapy Attenuated strain of vaccinia expressing Ag85A
Ongoing pre clinical studies
D Lowrie
Completed and ongoing phase I and II clinical trials; immunogenic, no serious adverse events reported Clinical studies started 2006 and ongoing
A Hill, H McShane
Ongoing pre clinical studies
J Fulkerson, D Hone, M Stone, D Onyabe, J Sadoff
Aeras 402
Non-replicating Ad35-expressing multiple TB proteins including Ag85A, Ag85B and TB10.4
Double-stranded RNA capsids
Double-stranded RNA capsids encoding TB antigens for oral delivery
NIH, Aeras Global TB Vaccine Foundation
SSI, Aeras Global TB Vaccine Foundation ImmunoBiology, Aeras Global TB Vaccine Foundation Sequella Inc. Wellcome Trust
Aeras Global TB Vaccine Foundation, Crucell Aeras Global TB Vaccine Foundation
Adapted from Macmillan Publishers Ltd: Nature Reviews Microbiology, Skeiky Y, Sadoff J. Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 2006;4:469–476. Copyright 2006.
In the first UK trial, 14 mycobacteria-naı¨ve, i.e. ESAT-6 and CFP-10 Elispot negative and no history or physical evidence of prior BCG vaccination, asymptomatic adults were vaccinated with intradermal MVA85A, administered twice, 3 weeks apart.23 All participants experienced some local side effects, predominantly redness and itching, which lasted for 3–7 days following vaccination. One-third of the vaccinees experienced systemic symptoms, principally myalgia and headache, for the first 12–24 hours after vaccination. All adverse effects resolved spontaneously. There were no
severe adverse events. The vaccine induced high levels of specific effector T-cell responses 1 week after vaccination. The safety of MVA85A was then tested in 17 UK adults who had been vaccinated with BCG from 6 months to 37 years previously.23 A safety profile similar to that of vaccinated BCGnaı¨ve persons was observed. Higher peak levels of antigen-specific T cells 1 week post-vaccination were shown, compared with those who received MVA85A alone. Prior BCG vaccination was associated with maintenance of higher levels of antigen-specific
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T cells after MVA85A boost, for up to 24 weeks after vaccination, compared with prior BCG vaccination or MVA85A alone. The next UK trial investigated the effect of boosting MVA85A 1 month after BCG vaccination in 10 healthy, BCG-naı¨ve adults. Safety and boosting efficacy were comparable to that shown following prolonged intervals between BCG and MVA85A boosting.24 This was followed by a UK trial in asymptomatic adults who were latently infected with M. tuberculosis.25 Individuals with latent TB infection (LTBI) are thought to have a very low-level persistent bacterial infection, no clinical or radiological evidence of disease but a detectable immune response against M. tuberculosis. Twelve participants were vaccinated with a single dose of MVA85A and followed up with detailed chest radiological and clinical safety assessments. The safety and immunogenicity profile of the vaccine in this trial was identical to that seen in previous studies of MVA85A boosting BCG vaccination. There was no clinical, radiological or immunological evidence of a Koch reaction. Ongoing UK trials involve MVA85A vaccination of 20 HIVinfected individuals who are otherwise healthy. The first volunteer was vaccinated in November 2006. In collaboration with the British Medical Research Council (BMRC) unit in the Gambia, MVA85A was first evaluated in 11 Gambian BCG-naı¨ve participants, and subsequently in 10 BCGprimed participants.26 The safety and immunogenicity profiles obtained were comparable with those seen in the UK studies. A current ongoing study in the Gambia is evaluating the immune response of the vaccine given to BCG-vaccinated infants, and whether the vaccine would interfere with other routine childhood vaccines. In South Africa, a phase II study in 24 healthy, M. tuberculosisnaı¨ve and HIV-uninfected adults, half of whom were previously vaccinated with BCG, was performed at the South African Tuberculosis Vaccine Initiative (SATVI) site at Worcester, near Cape Town. This population was very different from those in the UK and the Gambia, in terms both of ethnic background and of mycobacterial exposure. The annual risk of M. tuberculosis infection is 3–4% in Worcester, but exposure to environmental mycobacteria is less than in the Gambia. The safety and immunogenicity results were comparable with the experience in the UK and the Gambia.27 Twenty-four healthy adolescents were then vaccinated; to date, safety and immunogenicity in the adolescents vaccinated appear similar to those seen in adults. The next South African studies are testing the vaccine’s safety and immunogenicity in HIV-infected, TB-infected and HIV/TB coinfected individuals in this high-prevalence setting, and in healthy children and infants, leading to a phase IIB proof of concept study in infants planned to start in 2009.
rBCG30 This live recombinant vaccine was constructed by Marcus Horwitz and his research team at the University of California in Los Angeles. The Tice strain of BCG was modified to over expresses Ag85B, another member of the antigen 85A family.28,29 In vitro, rBCG30 produces up to five times more extracellular Ag85B than Tice BCG. Preclinical studies rBCG30 has been shown to stimulate a cell-mediated immune response in guinea pigs, as indicated by a DTH reaction against Ag85B, with significantly greater induration than that induced by Tice BCG or sham vaccination.28 The vaccine provided greater
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protection against virulent M. tuberculosis in this animal model than did various strains of BCG alone, including Tice BCG. At week 10 post-challenge, animals immunized with rBCG30 had significantly fewer M. tuberculosis bacilli in the lungs and spleen than animals immunized with Tice BCG.28 In another 62-week experiment, animals immunized with rBCG30 survived significantly longer than animals immunized with Tice BCG.28
Clinical studies A phase I rBCG30 study was conducted in the USA among 30 healthy adults at the Center for Vaccine Development, St. Louis University, Missouri, and in Winston-Salem, North Carolina.30 There were no significant safety issues. Increased immunogenicity, compared with the parent BCG strain, was reported. After completion of the trial the sponsor decided, for various technical reasons, to delay further trials and re-engineer the vaccine. At the time of writing, no further trials of the vaccine have commenced. Mtb72F/M72 Mtb72F was developed by Yasir Skeiky and Steve Reed at Corixa Corporation under an agreement with GlaxoSmithKline Biologicals (GSK Bio). The vaccine antigen is a fusion protein of two immunogenic M. tuberculosis proteins, Mtb39a and Mtb32a, which are also expressed in BCG. Adjuvants in the vaccine have included an oil-in-water emulsion (AS02) or a liposomal formulation (AS01). The developers of the vaccine have made minor changes to the amino acid sequence of one of the component proteins in order to make it more stable; the vaccine is now referred to as M72. Preclinical studies The adjuvanted polyprotein vaccine has been shown to protect against virulent M. tuberculosis in several animal models of TB, and also to boost and prolong the protective effect of BCG.31,32 Some data from mouse studies suggest that M. tuberculosis-specific CD8þ T cells may be targeted by vaccination with Mtb72F.33 Recombinant Mtb72F protein in AS02A has been shown to enhance the Th1 response to BCG in mice.34 Immunization of mice with Mtb72F DNA resulted in IFN-g responses and a strong CD8þ T-cell response, whereas Mtb72F protein in AS02A stimulated a moderate IFN-g response and a weak CD8þ T-cell response.34 Immunization with Mtb72F protein in AS01B generated a comprehensive and robust immune response.34 All three forms of Mtb72F immunization (DNA, protein in AS02A and protein in AS01B) resulted in protection of mice against aerosol challenge with virulent M. tuberculosis.34 Mtb72F in selected adjuvants protects guinea pigs as well and as long as BCG following M. tuberculosis challenge.31–35 When coadministered with BCG, the mixture substantially increased the survival time of infected guinea pigs, compared with BCG alone, with some animals still alive and healthy in appearance more than 100 weeks post-aerosol challenge and the majority of animals living well over 100 weeks. The majority had clear lungs with minimal granulomatous lesions. Adding Mtb72F to BCG appears to prevent progressive consolidating lung pathology and promotes lesion healing and reabsorption. In the cynomolgus monkey model, Mtb72F was shown to provide long-term protection against a fatal M. tuberculosis challenge when administered as a boost following immunization with BCG, which alone does not confer such protection.32 Immunogenicity in mice and protection in guinea pigs of the adjuvanted new vaccine formulation (M72) are unchanged.
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Clinical studies Preclinical studies in mice, guinea pigs, rabbits and monkeys had supported the initiation of clinical development of Mtb72F in AS02A. In 2004, the vaccine formulation (three doses of 10 mg in adjuvant) entered a phase I clinical trial in 12 healthy M. tuberculosis-uninfected adult volunteers in the USA. The vaccine was found to be highly immunogenic, safe and well tolerated in this trial.36,37 Subsequently, the 40-mg dose of Mtb72F in adjuvant or adjuvant alone was evaluated in 38 adult volunteers in Switzerland, all of whom were PPD-reactive, 20 due to prior exposure to BCG and/or environmental (non-tuberculous) mycobacteria and 18 due to prior exposure to M. tuberculosis. The formulations were found to be highly immunogenic and well tolerated in the former group, and highly immunogenic but more reactogenic in the latter group. The adverse events reported were usually self-limiting and resolved without sequelae.38 GSKBio is proceeding with development of the vaccine to prevent primary or reactivation of TB in PPD-negative or -reactive individuals who are at low risk of active TB disease. The adjuvanted M72 vaccine is currently undergoing clinical trials in Europe, at the SATVI site in South Africa and further trials are planned at other sites in Asia and Africa. AERAS-402 This vaccine was developed by Crucell at Leiden in The Netherlands as a boosting vaccine in BCG-primed individuals. A serotype 35 adenovirus (Ad35), incapable of replicating, has been modified to express a fusion protein of three immunodominant M. tuberculosis antigens: 85A, 85B and TB10.4. Antigen TB10.4 is a member of the ESAT-6 group of secreted M. tuberculosis proteins. Ad35 is capable of infecting human and animal cells in culture, including epithelial, muscle and lymphoid cells.39 Five per cent of individuals in Europe, North America and Japan have been found to have pre-existing cross-reactive neutralizing antibodies to Ad35, compared with around 50% against Ad5, another prominent candidate vaccine vector.39,40 Preclinical studies The vaccine was shown to be safe, immunogenic and protective in animal models.41,42 Three doses of the vaccine were given to guinea pigs with and without BCG priming. No toxicity was detected either during life or in autopsies. The vaccine is immunogenic in mice, both when given alone and when given as a booster in mice which have been primed with BCG. The vaccine given alone gives some protection to mice subsequently challenged with M. tuberculosis. Clinical studies A trial of the vaccine was started in November 2006 and is in progress in Kansas in the USA, but no results have yet been published. The first trial of AERAS-402 in South Africa, a dose escalation study targeting four groups of 10 healthy, HIV-uninfected, TB-naive adults, started at the SATVI site in April 2007. Further trials of the vaccine in adults with evidence of TB disease or just TB infection are due to start in 2008. Mycobacterium vaccae Mycobacterium vaccae is an environmental saprophyte. The hypothesis underlying use of adjunctive M. vaccae in TB treatment is that entrenched Th2 (humoral) dominant responses in the lung lead to inflammation, necrosis and cavitation. Mycobacterium vaccae is believed to promote Th1 responses, important to host defences against intracellular pathogens.43 The hope is that immunotherapy with M. vaccae will shorten short-course anti-TB chemotherapy. Clinical studies Heat-killed M. vaccae vaccine was first tested in a three-dose (0, 2 and 10 months) intradermal schedule in healthy adults.44
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Immunization was safe and well tolerated. Seven participants completed the three-dose schedule; pre-existing immunologic responses to mycobacteria were boosted in three, and a new response was elicited in one. The safety and immunogenicity of M. vaccae in patients with HIV infection were then assessed.45 Fifteen (seven healthy and eight HIV-infected participants) received five doses of the vaccine. The vaccine was well tolerated in all 15 participants. The safety of the vaccine in 35 HIV-infected children, aged 1 to 8 years, with CD4þ T-lymphocyte counts 300/mm3 was then assessed.46 Immunization was found to be safe and well tolerated. No effects on viral load or CD4þ T-lymphocyte count were observed. In 1999, a South African group reported on a trial designed to test the hypothesis that the addition of M. vaccae to standard short-course anti-TB chemotherapy would decrease the time to achieve a negative sputum culture.47 Patients with newly diagnosed TB were randomly assigned an injection of saline (placebo, n ¼ 175) or M. vaccae (n ¼ 175) on day 8. All patients received fourdrug anti-TB chemotherapy. At 8 weeks, 70 patients in the M. vaccae group and 65 patients in the placebo group had a negative culture; there was no difference between groups in the time to a negative culture. The group concluded that M. vaccae immunotherapy has no benefit when added to standard anti-TB chemotherapy. A further trial was conducted in Zambia: five doses of inactivated M. vaccae were administered intradermally to 22 HIVinfected patients, whose CD4þ lymphocyte counts were 200 cells/mm3, 11 of whom were BCG-exposed.48 Immunization was found to be safe in these patients and to induce lymphocyte proliferation responses to the vaccine antigen. In 2002 the LUSKAR collaboration reported the results of a trial which aimed to assess the effect on mortality of immunotherapy with single-dose SRL172 (M. vaccae) added to standard anti-TB chemotherapy in HIV-infected patients treated for TB.49 No significant effect on survival or bacteriological outcome was shown, though it was safe and well tolerated. The attention to M. vaccae has begun to shift to an investigation of its role in MDR-TB. In a randomized trial from China, sputum conversion, cavity closure and relapse were significantly better in patients with MDR-TB treated with M. vaccae every 3–4 weeks for 6 months plus susceptibility-directed chemotherapy than in those on chemotherapy alone.43 De Bruyn and Garner have conducted a Cochrane review of studies in which the effects of M. vaccae as an adjunct to chemotherapy for treating TB were investigated. Up to the year 2000 they identified only six trials for inclusion in their review. The main findings were that M. vaccae had no effect on mortality and no consistent effect on sputum negativity or sputum culture. They concluded that immunotherapy with M. vaccae does not benefit patients with TB.40
CHALLENGES TO NEW TUBERCULOSIS VACCINE DEVELOPMENT LACK OF UNDERSTANDING OF THE PROTECTIVE IMMUNE RESPONSE AGAINST TUBERCULOSIS An optimal knowledge both of vaccination-induced immune correlates of protection against TB and of protective immunity against TB is critical for vaccine development. Our knowledge is so incomplete that many regard current novel TB vaccine development to be in a
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first phase: the TB epidemic is so devastating that vaccines of limited efficacy, or of which we understand immune protection inadequately, should reach clinical trials. The second phase of vaccine development is likely to be guided by improved understanding of protective immunity, and may therefore yield vaccines with greater efficacy. Here, we will focus primarily on immune correlates of protection, as description of these markers would have an immediate impact on vaccine development. Studies of novel TB vaccine candidates in animal models provide some insight, regardless of differences between host responses to mycobacteria in animals and humans.31,50,51 The message from recent reports is that components of immunity shown to be critical for protection may not necessarily correlate with vaccination-induced protection. For example, specific IFN-g production has been used widely as a marker of vaccination-induced responses, as this cytokine has a central role in host protection against mycobacteria.52,53 Multiple animal studies have shown that increased vaccine efficacy is associated with enhanced specific IFN-g production.54–56 This has also been the case when the frequency of specific IFN-gproducing T cells, as measured by intracellular cytokine or Elispot assays, has been measured.20,54,57,58 However, recent studies have questioned whether IFN-g production should be used as an immune correlate of protection against TB. For example, Hovav et al.59 reported that, although strong IFN-g responses were induced in mice by vaccination with recombinant 27-kDa M. tuberculosis lipoprotein, M. tuberculosis challenge was associated with a worse outcome than that in unvaccinated mice. In studies of BCG vaccination of newborn calves, Hope et al.60 found that there was no correlation between specific IFN-g induced by the vaccine and subsequent protection, as measured by tissue pathology and bacterial load following virulent M. bovis challenge; rather, high levels of IFN-g were associated with more extensive lesions. Also in neonatal calves, Buddle et al.61 showed that double vaccination with BCG was associated with poorer protection against TB, despite significantly stronger specific IFN-g and interleukin-2 (IL-2) induction, compared with animals who received single vaccination. These sobering findings may be confounded by the assay systems used, by the time point and age at which the immune response was measured62,63 and by measurement at an immunological site distant from the disease site, the lung.58,64 Regardless, a critical foundation for directing human investigation into vaccination-induced immune correlates of protection against TB will only be achieved once renewed effort is made to delineate immune correlates in animal studies. A report by Bennekov et al.65 clearly illustrates why much more detailed examination of the induced immune response may be warranted. Mice were vaccinated with a fusion protein of Ag85A and ESAT-6 administered either within an adenovirus vector or within a liposomal adjuvant. After vaccination, specific IFN-g production was quantitatively greater when the adenovirus construct was used than when the liposome-based vaccine was used. However, the latter vaccine induced far better protection against virulent M. tuberculosis challenge. The adenovirus-based vaccine induced a CD8þ T-cell response primarily directed at a single ESAT-6 epitope, whereas the liposome-based vaccine induced a CD4þ T-cell response primarily directed at a single Ag85B epitope. Administration of the adenovirus construct within liposomes resulted in protection, and induction of the CD4þ T-cell responses directed at the Ag85B epitope. Most animal studies of immune correlates of protection have focused on CD4þ T cells, known to be critical for protection against mycobacteria. However, CD8þ T cells may be important
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for controlling the chronic phase of M. tuberculosis disease.66 This raises the possibility that T-cell responses other than a classical CD4þ T-cell IFN-g response may be attractive correlates. Although various candidates, such as IL-2, IL-10, IL-12, IL-13, IL-18 and tumour necrosis factor-alpha (TNF-a) production, have been suggested, none of these have, to date, been demonstrated to be correlates of protection in animal studies.61 Regulation of the immune response, through for example regulatory T cells or innate immune cells, has received virtually no attention. Similarly, humoral measures, such as the antibody response, have found limited application in evaluation as immune correlates of vaccinationinduced protection.67,68 In humans the tuberculin skin test reaction induced by BCG vaccination was initially suggested to be a surrogate of protection, only to be discarded as a poor correlate.69 No correlates, using modern immunological tools, exist today. However, some advances have been made in description of the BCG-induced immune response, which may guide immune analysis in the large projects. BCG does induce a robust type 1 immune response, at all ages, including infancy.70–74 This is characterized by IFN-g production and T-cell proliferation in peripheral blood mononuclear cells (PBMCs) or whole blood incubated with mycobacterial antigens. It has been demonstrated that Malawian vaccine recipients make significantly less IFN-g than their British counterparts, strongly suggesting that genetic and environmental factors, such as environmental mycobacterial exposure, may modulate this response.70,75 BCG induces both CD4þ and CD8þ T cells. Specific CD8þ T-cell cytotoxic activity has been demonstrated.76,77 In infancy, BCG-induced CD8þ T cells appear to have the capacity either to produce IFN-g or to have cytotoxic activity, but not both.77 Whereas subsets of specific CD4þ T cells induced by newborn vaccination produce combinations of IFN-g, IL-2 and/or TNF-a, CD8þ T cells appear to produce mostly IFN-g and/or IL-2, and little TNF-a.72 BCG vaccination of adults results in inhibition of mycobacterial growth in PBMCs or whole blood incubated with M. tuberculosis or M. bovis BCG, suggesting that the induced T cells are indeed functional.73 Finally, recent data indicate that BCG vaccination of newborns induces functional regulatory CD4þ T cells, and a strong specific antibody response.72 To date, it is not clear which of these immune responses are associated with protection against TB disease. However, linking results from our recent immunological study of BCG vaccination with those of a much larger phase IV clinical trial of BCG vaccine efficacy should have a sobering effect. We have demonstrated that percutaneous vaccination of newborns with a Japanese BCG strain resulted in quantitatively greater type 1 immune responses, i.e. IFN-g production measured in plasma and T cells from whole blood incubated with BCG, compared with intradermal vaccination with the same strain.71 Given the dogma of the importance of type 1 immunity for protection against TB, one would presume that intradermal vaccination would result in superior protection. However, when 11,680 newborns were randomized to receive either percutaneous or intradermal vaccination with the same Japanese BCG strain, and followed up for at least 2 years to compare occurrence of TB disease, no difference in efficacy between the two routes of vaccination could be demonstrated.78 Once again, these findings question use of type 1 immunity markers traditionally proposed as immune correlates of protection against TB disease. Multiple large collaborative efforts are currently ongoing to look for immune correlates of protection against TB. Once discovered, the correlates may only be validated in a phase III trial of a very
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efficacious TB vaccine. Until then, immunogenicity testing in phase I and II trials of novel TB vaccines have necessarily involved measurement of type 1 immunity, particularly of a specific IFN-g response with assays such as the Elispot and detection of intracellular cytokine expression with flow cytometry.23
DIFFICULTY IN IDENTIFYING LATENT INFECTION AND DIFFERENTIATING INFECTION FROM DISEASE Any new TB vaccine will first have to be tested in mycobacterianaı¨ve individuals. The Mantoux tuberculin skin test (TST) is currently the ‘gold standard’. However, it lacks sensitivity in immunocompromised individuals and is not specific.79 False-positive results are relatively common following exposure to BCG and environmental (non-tuberculous) mycobacteria.80 New immunodiagnostic tests have been developed and are based on the measurement of IFN-g secretion after stimulation with antigens said to be specific to M. tuberculosis. Two such tests are currently available commercially, i.e. T-spot TB (Oxford Immuntec) and QuantiFERON (Cellestis). In 2005, the QuantiFERON-TB Gold received approval from the US Food and Drug Administration as an aid for diagnosing M. tuberculosis infection.81 The performance of QuantiFERON assays to identify LTBI was compared with TST in a high-TB-burden community.82 Among 358 participants 81% had a TST result of 10 mm induration or more, and 52% 15 mm or more. QuantiFERON-TB was positive in 60%, QuantiFERON-TB Gold in 38% and QuantiFERON-TB Gold (In-Tube method) in 56%. There was poor agreement between TST and QuantiFERON tests, and between the different generations of QuantiFERON tests (k ¼ 0.12–0.50). In individuals with TST indurations of 15 mm or more, 30–56% had negative QuantiFERON tests.
DIFFICULTY IN DEFINING CLINICAL ENDPOINTS OF TRIALS The current gold standard for diagnosing TB disease is M. tuberculosis culture. Most immunocompetent adults with TB, who classically present with cavitary lung disease, are culture positive. However, children classically have paucibacillary disease, which manifests as hilar or mediastinal adenopathy with or without complications, and are culture positive in only about 30% of cases.83 The diagnosis of TB in most situations is thus based on clinical grounds including the TST and chest radiograph. This problem has been highlighted by the recent randomized clinical trial comparing the efficacy of two routes of vaccination in the prevention of TB in the first 2 years of life, mentioned earlier.78 The cumulative incidence rates over 2 years of definite plus probable and of definite plus probable plus possible TB in the trial was a staggering 6.3%. Of the 236 participants who had positive cultures, 70 (30%) had a Mantoux test with no (0 mm) induration and 108 (46%) a Mantoux test result of less than 15 mm, 130 (55%) had relatively few significant clinical symptoms (scored below the cut-off on a standard clinical scoring system) and only 142 (60%) had a chest radiograph judged to be compatible with TB.
UNCERTAINTY ABOUT SAFETY OF NEW TUBERCULOSIS VACCINES The evaluation of safety in target populations, including infants, mycobacteria-exposed individuals and HIV-infected persons is critical in novel vaccine trials.
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Koch phenomenon Robert Koch, in search of a cure for TB, found that individuals with TB when injected subcutaneously with a culture filtrate of whole organisms developed exaggerated disease.13 Although unlikely, it has been postulated that the Koch phenomenon could have contributed to failure of BCG vaccine in a trial conducted in Chingelput, South India, as many had prior mycobacterial exposure.84 Animal studies suggest that such reactions occur in situations where a high bacillary load pre-exists; it is unlikely that the Koch phenomenon will occur in latently infected persons who receive immunogenic vaccines.85 Safety in HIV-infected individuals HIV infection is a major risk factor for developing TB. A latently infected HIV-infected person has a 10% annual risk of developing TB disease, while this risk is 10% in the lifetime of HIV-uninfected persons. An estimated 60% of persons newly diagnosed with TB in sub-Saharan Africa are coinfected with HIV. Preventing TB in HIV-infected individuals is thus a high priority.1 BCG poses a risk to HIV-infected children. The risk of disseminated BCG disease in HIV-infected infants is likely to be several hundred-fold greater than that in HIV-uninfected infants.86 The WHO Global Advisory Committee on Vaccine Safety has recently recommended that BCG vaccine should not be used in children who are known to be HIV infected.87 The committee recognizes the difficulty in identifying infants infected with HIV at birth in settings where diagnostic and treatment services for mothers and infants are limited. In such situations, BCG vaccination should continue to be given at birth to all infants regardless of HIV exposure, especially considering the high endemicity of TB in populations with high HIV prevalence. Close follow-up of infants known to be born to HIV-infected mothers and who received BCG at birth is recommended in order to provide early identification and treatment of any BCG-related complication. In settings with adequate HIV services that could allow for early identification and administration of antiretroviral therapy to HIVinfected children, consideration should be given to delaying BCG vaccination in infants born to mothers known to be infected with HIV until these infants are confirmed to be HIV-uninfected. Live attenuated mycobacterial vaccines are unlikely to be recommended for use in HIV-infected persons or other immunocompromised individuals. However, auxotrophic mutant vaccines, which maintain their infective ability and have a limited period of replication in the host may be ideal candidates to use in this situation. Such attenuated mutant vaccines have been shown to be safer than BCG in immunocompromised animals.88 THE NEED FOR PHASE III TRIAL SITES Phase III trials of novel TB vaccines will require multiple sites for testing, for sample size reasons. Developing a field site for the assessment of new TB vaccines is difficult. Such sites should ideally be located in developing countries where the rates of TB are high. However, in most areas the infrastructure necessary to conduct clinical trials is suboptimal, the health services are overwhelmed, there is a shortage of trained professional staff and laboratory and diagnostic facilities are suboptimal. In the absence of a well-defined surrogate marker of protection, studies using clinical endpoints are essential to determine efficacy and require long-term follow-up. Cohort retention is thus essential and good surveillance systems for morbidity and mortality are vital for monitoring outcomes.
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A single site, the SATVI site in South Africa, is currently ready for phase III trials. The main aim of this site was to develop the capacity to do phase I to phase IV TB vaccine trials, to characterize the TB epidemic epidemiologically and to identify clinical, microbiological and immunological endpoints of key importance to all stages of vaccine trials. In developing the site a number of challenges had to be overcome. These included working with a research naı¨ve population in a poor, socially disadvantaged rural area, renowned for its political conservatism. The distance from microbiological and immunological laboratories in Cape Town was also a challenge. Significant investment in infrastructural development, training of research staff and the development of a non-exploitative and supportive partnership model between the research team, the sponsors, the health services, the community and the research collaborators were essential for success. Other sites in Kenya and Uganda, and in India and Indonesia, are being developed with funding from the European and Developing Countries Trials Partnership (EDCTP) and the Aeras Global TB Vaccine Foundation.
SPECIAL STRUCTURES ARE NEEDED FOR MORBIDITY AND MORTALITY SURVEILLANCE Routine health information systems in most high-burden countries do not provide age-stratified admission and discharge data with recorded diagnoses. The quality of case notes commonly varies widely, and in most situations is not adequate for vaccine trial purposes. Statutory reporting of deaths is also generally incomplete, of poor quality and slow. Pathological postmortem examinations are very seldom done and may be culturally unacceptable.89 SATVI has attempted to improve the quality of morbidity and mortality surveillance and attempts to verify TB as a cause for hospital admission or death have been developed. Health workers in the region are encouraged to refer all possible cases of TB to a special research case verification ward which has been established at the regional TB hospital for a comprehensive diagnostic work-up. The case notes of any trial participant admitted to the district hospitals in the region are reviewed as soon as the research team is notified of such an event. We are also putting in place a mechanism whereby we can do postmortems as soon as we have been notified of a trial participant’s death and we have validated a verbal autopsy methodology for use in situations where postmortems have not been done.
ETHICAL CONSIDERATIONS Informed consent is an ethical and legal requirement for research involving human participants.90 Developing countries which lack
REFERENCES 6. 1. Dye C. The global epidemiology of tuberculosis. Lancet 2006;367:938–940. 2. Lawn SD, Wilkinson R. Extensively drug resistant tuberculosis. BMJ 2006;333:559–560. 3. Fine P, Carneiro I, Milstein J, et al. Issues Relating to the Use of BCG in Immunization Programmes: A Discussion Document. WHO/V&B/199923. Geneva: World Health Organization, 1999. 4. Aronson NE, Santosham M, Comstock GW, et al. Long-term efficacy of BCG vaccine in American Indians and Alaska Natives: a 60-year follow-up study. JAMA 2004;291:2086–2091. 5. Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and miliary
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9. 10.
well-organized Institutional Review Boards or Research Ethics Committees may have problems in protecting the rights of participants involved in clinical trials. In addition, concepts such as ‘voluntary participation’, ‘randomization’ and ‘benefits and risks’ may be difficult to explain to uneducated study participants. Poverty, disease, poor literacy and social insecurity make participants vulnerable to research exploitation. The informed consent process thus poses significant logistical and ethical challenges. SATVI has invested significantly in ensuring that our research staff are fully conversant with informed consent process and that the participants in our clinical trial understand what it means to be involved in clinical research projects.
ECONOMIC CONSIDERATIONS Given the magnitude of the problem worldwide it is imperative that there is a global strategy to coordinate collaborative efforts to develop vaccines that are effective in reducing TB. Strategic investments made by the European Union, the National Institutes of Health, the Bill and Melinda Gates Foundation and other agencies and governments have gone a long way towards meeting the intended goal. However, significant additional resources and commitments are needed to support additional research, preclinical development, vaccine production and clinical trials. In 2005, it was estimated that the investment in TB research and development in general amounted to approximately $400 million, with less than a quarter directed to vaccine research.91 The Stop TB Working Group on New TB Vaccines has estimated that over $3000 million will be needed to ensure that we get a new TB vaccine into the field by 2015.8
CONCLUDING REMARKS The challenges facing developers of new TB vaccines are considerable. Regardless, significant progress in the development of new vaccines has been made over the past two decades and continues to be made. Even when new TB vaccines are developed and shown to be superior to BCG in animal studies, the task of translating these laboratory findings into clinical practice and public health policy, and ultimately impacting on the burden of TB infection and disease, is daunting. Nevertheless we are optimistic that the goal of getting a more effective vaccine to those who most need one before 2015 is realizable.
tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 2006; 367:1173–1180. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537–544. Brennan MJ, Morris SL, Sizemore CF. Tuberculosis vaccine development: research, regulatory and clinical strategies. Expert Opin Biol Ther 2004;4:1493–1504. World Health Organization. Global Plan to Stop TB 2006–2015. WHO/HTM/STB/200635. Geneva: World Health Organization, 2006. Ginsberg AM. What’s new in tuberculosis vaccines? Bull World Health Organ 2002;80:483–488. McShane H, Hill A. Prime-boost immunisation strategies for tuberculosis. Microbes Infect 2005;7: 962–967.
11. Kaufmann SH, McMichael AJ. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat Med 2005;11:S33–44. 12. Skeiky YA, Sadoff JC. Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 2006;4:469–476. 13. Koch R. Fortstzung uber ein Heilmittel gegen Tuberculose. Deutsche Med Wochenschr 1891;17: 101–102. 14. Taylor JL, Turner OC, Basaraba RJ, et al. Pulmonary necrosis resulting from DNA vaccination against tuberculosis. Infect Immun 2003;71:2192–2198. 15. Wiker HG, Harboe M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev 1992;56:648–661. 16. Mahnel H, Mayr A. [Experiences with immunization against orthopox viruses of humans and animals using vaccine strain MVA]. Berl Munch Tierarztl Wochenschr 1994;107:253–256.
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Tuberculosis vaccines 17. Stickl H, Hochstein-Mintzel V, Mayr A, et al. [MVA vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (MVA)]. Dtsch Med Wochenschr 1974;99:2386–2392. 18. Cosma A, Nagaraj R, Buhler S, et al. Therapeutic vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine 2003;22:21–29. 19. Harrer E, Bauerle M, Ferstl B, et al. Therapeutic vaccination of HIV-1-infected patients on HAART with a recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antivir Ther 2005; 10:285–300. 20. Goonetilleke NP, McShane H, Hannan CM, et al. Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara. J Immunol 2003;171:1602–1609. 21. Williams A, Hatch GJ, Clark SO, et al. Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb) 2005;85:29–38. 22. Verreck F. Personal communication to H. McShane. 2007. 23. McShane H, Pathan AA, Sander CR, et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004;10:1240–1244. 24. Pathan AA, Sander CR, Fletcher HA, et al. Boosting BCG with Recombinant Modified Vaccinia Ankara expressing antigen 85A: Different boosting intervals and implications for efficacy trials. PLoS ONE 2007;2:e1052. 25. McShane H. Personal communication, 2007. 26. Ibanga HB, Brookes RH, Hill PC, et al. Early clinical trials with a new tuberculosis vaccine, MVA85A, in tuberculosis-endemic countries: issues in study design. Lancet Infect Dis 2006;6:522–528. 27. Hawkridge A. Phase one trial of Modified Vaccinia Ankara tuberculosis vaccine in adults in a high TB prevalence setting in South Africa. In: Proceedings of Tuberculosis Vaccines for the World Conference, Vienna, Austria, 19–21 April 2006. 28. Horwitz MA, Harth G. A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect Immun 2003;71:1672–1679. 29. Horwitz MA, Harth G, Dillon BJ, et al. Recombinant bacillus Calmette-Guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc Natl Acad Sci USA 2000;97:853–13,858. 30. Hoft DF. Results of the 1st phase I trial of a recombinant BCG TB vaccine (rBCG30). In: Proceedings of 40th Tuberculosis and Leprosy Research Conference; Seattle, WA, 28–30 July 2005. 31. Orme IM. Mouse and guinea pig models for testing new tuberculosis vaccines. Tuberculosis (Edinb) 2005;85:13–17. 32. Reed S, Lobet Y. Tuberculosis vaccine development; from mouse to man. Microbes Infect 2005;7:922–931. 33. Irwin SM, Izzo AA, Dow SW, et al. Tracking antigen-specific CD8 T lymphocytes in the lungs of mice vaccinated with the Mtb72F polyprotein. Infect Immun 2005;73:5809–5816. 34. Skeiky YA, Alderson MR, Ovendale PJ, et al. Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J Immunol 2004;172:7618–7628. 35. Brandt L, Skeiky YA, Alderson MR, et al. The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs. Infect Immun 2004;72:6622–6632. 36. Morrison R. Personal communication to O. OforiAnyinam, 2007.
37. Leroux-Roels I, Lerroux-Roels G, Clement F, et al. Safety and immunogenicity of the Mtb72f/ASO2A tuberculosis vaccine in PPD-negative Belgian adults. In: Proceedings of Tuberculosis Vaccines for the World Conference, Vienna, Austria, 19–21 April 2006. 38. Spertini F, Audran R, Lurati F, et al. Safety and immunogenicity of the Mtb72f/ASO2A tuberculosis vaccine in PPD-positive Swiss adults. In: Proceedings of Keystone Conference: Tuberculosis: from Lab Research to Field Sites; Vancouver, Canada; 20–25 March 2007. 39. Vogels R, Zuijdgeest D, van Rijnsoever R, et al. Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol 2003;77:8263–8271. 40. Kostense S, Koudstaal W, Sprangers M, et al. Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector. AIDS 2004;18:1213–1216. 41. Radosevic K, Wieland CW, Rodriguez A, et al. Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon. Infect Immun 2007;75: 4105–4115. 42. Skeiky Y. Personal communication, 2007. 43. Fourie PB, Ellner J, Johnson JL. Whither Mycobacterium vaccae—encore. Lancet 2002;360: 1032–1033. 44. Von Reyn CF, Arbeit RD, Yeaman G, et al. Immunization of healthy adult subjects in the United States with inactivated Mycobacterium vaccae administered in a three-dose series. Clin Infect Dis 1997;24:843–848. 45. Von Reyn CF, Marsh BJ, Waddell R, et al. Cellular immune responses to mycobacteria in healthy and human immunodeficiency virus-positive subjects in the United States after a five-dose schedule of Mycobacterium vaccae vaccine. Clin Infect Dis 1998;27:1517–1520. 46. Johnson D, Waddell RD, Pelton SI, et al. Randomised trial of intradermal Mycobacterium vaccae or intradermal hepatitis B immunisation in children with HIV infection. Vaccine 1999;17:2583–2587. 47. Durban Immunotherapy Trial Group. Immunotherapy with Mycobacterium vaccae in patients with newly diagnosed pulmonary tuberculosis: a randomised controlled trial. Lancet 1999;354:116–119. 48. Waddell RD, Chintu C, Lein AD, et al. Safety and immunogenicity of a five-dose series of inactivated Mycobacterium vaccae vaccination for the prevention of HIV-associated tuberculosis. Clin Infect Dis 2000;30: S309–315. 49. Mwinga A, Nunn A, Ngwira B, et al. Mycobacterium vaccae (SRL172) immunotherapy as an adjunct to standard antituberculosis treatment in HIV-infected adults with pulmonary tuberculosis: a randomised placebo-controlled trial. Lancet 2002;360:1050–1055. 50. Flynn JL. Lessons from experimental Mycobacterium tuberculosis infections. Microbes Infect 2006;8:1179–1188. 51. Orme IM. Preclinical testing of new vaccines for tuberculosis: a comprehensive review. Vaccine 2006;24:2–19. 52. Flynn JL. Immunology of tuberculosis and implications in vaccine development. Tuberculosis (Edinb) 2004;84(1–2):93–101. 53. Kaufmann SH. New issues in tuberculosis. Ann Rheum Dis 2004;63(Suppl 2):ii50–ii56. 54. Agger EM, Rosenkrands I, Olsen AW, et al. Protective immunity to tuberculosis with Ag85BESAT-6 in a synthetic cationic system IC31. Vaccine 2006;24:5452–5460. 55. Freidag BL, Melton GB, Collins F, et al. CpG oligodeoxynucleotides and interleukin-12 improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis. Infect Immun 2000;68:2948–2953. 56. Langermans JA, Doherty TM, Vervenne RA, et al. Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine 2005;23(21):2740–2750.
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57. Martin E, Kamath AT, Triccas JA, et al. Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(þ) T cells. Infect Immun 2000;68:3090–3096. 58. Santosuosso M, McCormick S, Zhang X, et al. Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect Immun 2006;74: 4634–4643. 59. Hovav AH, Mullerad J, Davidovitch L, et al. The Mycobacterium tuberculosis recombinant 27-kilodalton lipoprotein induces a strong Th1-type immune response deleterious to protection. Infect Immun 2003;71:3146–3154. 60. Hope JC, Thom ML, Villarreal-Ramos B, et al. Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis. Clin Exp Immunol 2005;139:48–56. 61. Buddle BM, Wedlock DN, Denis M, et al. Identification of immune response correlates for protection against bovine tuberculosis. Vet Immunol Immunopathol 2005;108:45–51. 62. Chen L, Wang J, Zganiacz A, et al. Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis. Infect Immun 2004;72:238–246. 63. Goter-Robinson C, Derrick SC, Yang AL, et al. Protection against an aerogenic Mycobacterium tuberculosis infection in BCG-immunized and DNAvaccinated mice is associated with early type I cytokine responses. Vaccine 2006;24:3522–3529. 64. Ha SJ, Park SH, Kim HJ, et al. Enhanced immunogenicity and protective efficacy with the use of interleukin-12-encapsulated microspheres plus AS01B in tuberculosis subunit vaccination. Infect Immun 2006;74:4954–4959. 65. Bennekov T, Dietrich J, Rosenkrands I, et al. Alteration of epitope recognition pattern in Ag85B and ESAT-6 has a profound influence on vaccineinduced protection against Mycobacterium tuberculosis. Eur J Immunol 2006;36:3346–3355. 66. van Pinxteren LA, Cassidy JP, Smedegaard BH, et al. Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells. Eur J Immunol 2000; 30:3689–3698. 67. Giri PK, Verma I, Khuller GK. Enhanced immunoprotective potential of Mycobacterium tuberculosis Ag85 complex protein based vaccine against airway Mycobacterium tuberculosis challenge following intranasal administration. FEMS Immunol Med Microbiol 2006;47:233–241. 68. Watanabe Y, Watari E, Matsunaga I, et al. BCG vaccine elicits both T-cell mediated and humoral immune responses directed against mycobacterial lipid components. Vaccine 2006;24:5700–5707. 69. Comstock GW. Identification of an effective vaccine against tuberculosis. Am Rev Respir Dis 1988; 138:479–480. 70. Black GF, Weir RE, Floyd S, et al. BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies. Lancet 2002;359:1393–1401. 71. Davids V, Hanekom WA, Mansoor N, et al. The effect of bacille Calmette-Guerin vaccine strain and route of administration on induced immune responses in vaccinated infants. J Infect Dis 2006;193:531–536. 72. Hanekom WA. The immune response to BCG vaccination of newborns. Ann NY Acad Sci 2005;1062:69–78. 73. Hoft DF, Worku S, Kampmann B, et al. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 2002;186:1448–1457. 74. Marchant A, Goetghebuer T, Ota MO, et al. Newborns develop a Th1-type immune response to
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77.
78.
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Mycobacterium bovis bacillus Calmette-Guerin vaccination. J Immunol 1999;163:2249–2255. Weir RE, Black GF, Nazareth B, et al. The influence of previous exposure to environmental mycobacteria on the interferon-gamma response to bacille Calmette-Guerin vaccination in southern England and northern Malawi. Clin Exp Immunol 2006;146:390–399. Smith SM, Brookes R, Klein MR, et al. Human CD8þ CTL specific for the mycobacterial major secreted antigen 85A. J Immunol 2000;165: 7088–7095. Murray RA, Mansoor N, Harbacheuski R, et al. Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8þ T cell response. J Immunol 2006;177:5647–5651. Hawkridge AJ, Hanekom W, Geiter L, et al. Results from a randomised controlled trial comparing intradermal and percutaneous administration. Int J Tuberc Lung Dis 2006;10:S13. Cobelens FG, Egwaga SM, van Ginkel T, et al. Tuberculin skin testing in patients with HIV infection: limited benefit of reduced cutoff values. Clin Infect Dis 2006;43(5):634–639.
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80. Farhat M, Greenaway C, Pai M, et al. False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? Int J Tuberc Lung Dis 2006;10:1192–1204. 81. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: part I. Latent tuberculosis. Expert Rev Mol Diagn 2006; 6:413–422. 82. Mahomed H, Hughes EJ, Hawkridge T, et al. Comparison of Mantoux skin test with three generations of a whole blood IFN-gamma assay for tuberculosis infection. Int J Tuberc Lung Dis 2006;10:310–316. 83. Enarson PM, Enarson DA, Gie R. Management of tuberculosis in children in low-income countries. Int J Tuberc Lung Dis 2005;9:1299–1304. 84. Fifteen year follow up of trial of BCG vaccines in south India for tuberculosis prevention. Tuberculosis Research Centre (ICMR), Chennai. Indian J Med Res 1999;110:56–69. 85. Rook GA, al Alliyah R. Cytokines and the Koch phenomenon. Tubercle 1991;72:13–20.
86. Hesseling AC, Marais BJ, Gie RP, et al. The risk of disseminated Bacillus Calmette-Guerin (BCG) disease in HIV-infected children. Vaccine 2007;25:14–18. 87. World Health Organization. Global advisory committee on vaccine safety, 29–30th November 2006. Weekly Epidemiol Rep 2006; 82:2–8. 88. Guleria I Teitelbaum R, McAdam RA, et al. Auxotrophic vaccinesfor tuberculosis. Nat Med 1996;2:334–337. 89. Moyo S, Hawkridge T, Mahomed H, et al. Determining causes of mortality in children enrolled in a vaccine field trail in a rural area in the western Cape Province of South Africa. J Paed Child Health 2007;43:178–183. 90. Joffe S, Cook EF, Cleary PD, et al. Quality of informed consent: a new measure of understanding among research subjects. J Natl Cancer Inst 2001;93:139–147. 91. Kaufmann SHE, Parida SK. Changing funding patterns in tuberculosis. Nat Med 2007;13:299–303.
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12
Pathology and pathogenesis of tuberculosis Juanita Bezuidenhout and Johann W Schneider
Tuberculosis is a mycobacterial infection that is usually pulmonary and may lead to severe destruction and lung dysfunction. It is a chronic disease, often associated with some form of underlying immune deficiency, such as malnutrition or human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). It may be seen in adults and children, depending on age at infection. In developing countries with a high annual risk of infection and a high proportion of the population less than 14 years of age children may constitute 20% of the TB case load. With the advent of HIV/AIDS the profile of TB has changed, especially in adults, and it is now frequently found in multiple extrapulmonary sites often with a more severe course than isolated pulmonary TB. The pathogenesis and macroscopic and microscopic morphology lie at the heart of the clinical manifestations and complications of TB. A clear understanding of the disease and its morphological manifestations in the various organs can be obtained only by understanding the pathogenesis and impact of the infection on the body.
BASIC CONCEPTS AND DEFINITIONS DEFINITION OF A GRANULOMA A granuloma comprises a microscopic aggregation of epithelioid (activated) macrophages, usually surrounded by a collar of lymphocytes (Fig. 12.1).1 Lymphocytes may be abundant or sparse, but they are always present. The epithelioid macrophages may occur in tight groups, or may be loosely associated. Although granulomas may display characteristic histological features in specific diseases, the various subtypes of granulomas are not pathognomonic of a particular disease and additional investigations are always required to determine the exact cause of the granulomatous disease. This will be discussed further in Chapter 21.
THE CELLS INVOLVED IN GRANULOMA FORMATION Antigen-presenting cells and T-lymphocytes are the main cell types involved in an immunogenic granuloma.
Antigen-presenting cells Antigen-presenting cells (macrophages and dendritic cells) are specialized to capture and process microbial and other antigens, present them to lymphocytes, and provide signals that stimulate proliferation and differentiation of lymphocytes.2
Macrophages The activated macrophage produces a wide range of chemical mediators that drive and orchestrate chronic inflammation.3 It forms part of the mononuclear phagocyte system that comprises blood monocytes and tissue macrophages arising from a common bone marrow precursor. Tissue macrophages are scattered in connective tissue or clustered in organs such as the liver (Kupffer cells), spleen, lymph nodes (sinus histiocytes), and lungs (alveolar macrophages). They originate from blood monocytes (with a half-life of about 1 day) that migrate into various tissues where they transform into macrophages with a half-life of months. This whole process is regulated by a variety of growth and differentiation factors, cytokines, adhesion molecules, and cellular interactions.3 Macrophages are primarily phagocytic cells, but once activated they become larger with an epithelioid appearance and display increased lysosomal enzyme levels and metabolism that enhance their efficiency to phagocytose and kill ingested organisms. The simultaneous increased expression of major histocompatibility complex (MHC) class II molecules on the cell surface facilitates antigen presentation, because CD4þ lymphocytes recognize antigens only in the presence of MHC class II expression. Several growth factors that stimulate fibroblast proliferation and collagen synthesis are also secreted. After activation the macrophage secretes a wide variety of biologically active products.4 In a haematoxylin–eosin-stained slide epithelioid cells have abundant pale-pink cytoplasm with indistinct cell borders, creating the impression of merging into one another. The cell has a vesicular nucleus, often with a distinct nucleolus. Epithelioid cells frequently fuse to form multinucleated giant cells of various configurations. These multinucleated giant cells are usually seen at the periphery of a granuloma, but can be present anywhere in the granuloma (Fig. 12.1). Dendritic cells Dendritic cells play an important role in antigen capture and the induction of T-cell responses, especially in naı¨ve T cells.5 They are present in lymphoid organs, the skin, the gastrointestinal and respiratory tracts, and most parenchymal organs. Dendritic cells have spine- or finger-like projections of their cell membranes, resulting in a vastly increased surface area of each cell. These cells capture protein antigens and transport them to the draining lymph nodes. During this journey the dendritic cells mature and become extremely efficient in presenting antigens to naı¨ve T cells.6
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BASIC SCIENCE Antigen presenting cell
Receptor Antigen Receptor
IL-12
CD4 + T-lymphocyte Activated macrophage (epithelioid macrophage)
IL-2 IFN-g
TNF Blood vessel Monocyte CD4 + T-lymphocytes
Granuloma Multinucleated giant cell
A
Epithelioid macrophage
Fig. 12.1 (A) A schematic illustration of granuloma formation during the delayed type hypersensitivity (DTH) reaction.39 After capture of the antigen, antigen-presenting cells present the antigen to the CD4þ lymphocytes, while producing interleukin (IL)-12, which stimulates Th1 differentiation. The proinflammatory cytokines tumour necrosis factor (TNF)-a and interferon (IFN)-g recruit and stimulate monocytes to transform into activated epithelioid macrophages, resulting in granuloma formation. (B) Microscopic appearance of a well-formed granuloma containing epithelioid macrophages, with a rim of lymphocytes and Langhans giant cells. (C) A granuloma with central caseous necrosis apparent as amorphous pink material.
Lymphocytes Lymphocytes are the only cells in the body capable of specifically recognizing and distinguishing different antigens.7 They are therefore responsible for both the specificity and memory that are the defining characteristics of the adaptive immune response. Lymphocytes that have not been stimulated by antigens are called naı¨ve
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lymphocytes and are small cells, measuring 8–10 mm in diameter. Once stimulated they become larger, with more cytoplasm, organelles, and cytoplasmic RNA. The lymphocytes involved in the cell-mediated adaptive immune response are CD3þ T cells, usually of the T-helper type (CD4þ). Once activated they differentiate into effector and memory cells.7,8
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PATHOGENESIS OF DELAYED-TYPE HYPERSENSITIVITY AND GRANULOMA FORMATION A detailed description of the immunopathogenesis of the granuloma can be found in Chapter 8. There are five distinct phases of adaptive immunity, namely:9,10 1. recognition of antigens; 2. activation of lymphocytes; 3. effector phase (elimination of antigens); 4. decline (return to resting state); and 5. memory. The current model for granuloma formation is based on the delayedtype hypersensitivity (DTH) reaction, as epitomized by the tuberculin reaction.11 With the first exposure of a person to tubercle bacilli, naı¨ve CD4þ T cells recognize captured peptides derived from these bacilli in association with class II molecules on antigenpresenting cells. This initiates the differentiation of these T cells into sensitized Th1 cells. Some of these Th1 cells enter the circulation and remain in the T-cell memory pool for years.8,12 On subsequent intracutaneous injection of tuberculin, the DTH process is initiated by these sensitized Th1 cells, following antigen presentation by macrophages.2,13 In a previously sensitized person, a response appears on site within 8–12 hours after injection (Mantoux test), in the form of reddening and induration. This reaches a peak within 24–72 hours and thereafter recedes. The redness and induration are due to vasodilation with perivascular cuffing of predominantly CD4 (helper) T-lymphocytes around small veins and venules.8,13 An increase in vascular permeability results in the escape of plasma proteins and fluid, causing dermal oedema and fibrin deposition, leading to induration. With persistence of the antigen, the lymphocytic infiltrate is largely replaced by macrophages over the next 2–3 weeks. These macrophages undergo morphological transformation to epithelioid cells and thus a granuloma is formed14 (Fig. 12.1B).
ANTIGEN CAPTURE, PROCESSING, AND PRESENTATION Microorganisms stimulate the innate immune response and induce the secretion of inflammatory cytokines in an effort to kill the organisms. Innate immunity against intracellular organisms may limit bacterial growth, but usually fails to eradicate the organisms; adaptive cell-mediated immunity is required for eradication. This process starts with the capturing of an antigen by an antigenpresenting cell.6,7,15 Once the protein antigen is captured and phagocytosed by the antigen-presenting cells, it is converted to a peptide and then displayed as a peptide–MHC II complex on the surface of the antigen-presenting cells, for recognition by T cells.7
ACTIVATION, PROLIFERATION, AND DIFFERENTIATION OF LYMPHOCYTES During this process the naı¨ve lymphocytes are activated. This results in a clonal expansion of antigen-specific lymphocytes and the differentiation of these cells into effector cells and memory cells. Some of these cells re-enter the systemic circulation and migrate to the site of infection. Here the cells again encounter the antigen and respond to eliminate the antigen.15
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Effector phase In cell-mediated immunity, the main effector function of the CD4þ cells is to stimulate the microbiocidal activities of macrophages and other leucocytes. Memory phase Memory T cells are an expanded population of T cells specific for an antigen that can respond rapidly to subsequent encounters with the same antigen, differentiate into effector cells, and eliminate the antigen. The mechanisms of memory cell generation are not known.
PATHOGENESIS OF TUBERCULOSIS Based on whether it is the first time a patient is infected, TB is divided into a primary and secondary type. Previously primary TB was called ‘childhood’ type, but over the years more adults presented for the first time with TB and the terminology was therefore changed to ‘primary TB’, indicating that a person is infected by TB for the first time. Secondary TB is usually seen in adults, subsequent to primary infection in childhood and a third type, ‘progressive-primary’, follows directly on primary TB. Figure 12.2 summarizes the typical spectrum of events that may follow primary and secondary TB, respectively.
PRIMARY TUBERCULOSIS This phase occurs in a previously unexposed host. The lungs are typically the initial site of contact between the host and Mycobacterium tuberculosis organisms, but less commonly other organs including the skin, oral cavity, and gastrointestinal tract may be involved. The initial phases of primary TB have seldom been seen in the human lung, and our understanding of the early pathogenesis is based solely on experimental animal models. In primary pulmonary TB the organism is inhaled and, usually in the periphery of the lung, is phagocytosed and innate immunity initiated. The phagocytosed organism is transported to the hilar lymph nodes and presented to naı¨ve T cells, stimulating the process of activation, proliferation, and differentiation of these T cells. If the organism is not killed, the process of adaptive immunity takes control and a DTH response commences at the primary site of infection. Initially very little reaction can be seen and small numbers of neutrophils and exudate surround the organisms. In the following days macrophages collect to engulf the organisms in an attempt to eradicate the infection. If this is unsuccessful, granuloma formation ensues. The macrophages first form microscopic aggregates that may distort the normal lung architecture. They become activated and develop into epithelioid cells, acquiring abundant pale-pink cytoplasm with indistinct cell borders, a vesicular nucleus, and often a distinct nucleolus. These changes represent the change from phagocytic to secretory function that enhances the microbiocidal properties of macrophages, but unfortunately can also induce necrosis of lung tissue. The macrophages may fuse to form multinucleated giant cells. Interspersed between and surrounding the macrophages are lymphocytes. This collection of macrophages, giant cells, and lymphocytes form the granuloma. After a few weeks, the granuloma can be macroscopically visible as a small nodule. At first it is grey in colour, but eventually turns yellow as it enlarges. On closer examination at this stage, necrosis is evident in the centre of the granuloma. Adjacent granulomas
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M. tuberculosis Ghon focus PRIMARY TB Poor immune status Ghon complex
PROGRESSIVE PRIMARY TB
Good immune status
FIBROSIS = latency
Reactivation or reinfection
Haemoptysis, haemorrhage
SECONDARY TB
Destroy bronchial walls Cavitation = ‘open’ TB
APICAL LUNG LESIONS Endotracheal Laryngeal
Break through pulmonary vein
P R - heart SWALLOW
Pulmonary arteries
L - heart
Both lungs
Intestinal TB Bronchogenic dissemination
TB bronchopneumonia
Systemic circulation
TB pleuritis + effusion
Fibrous pleuritis, empyema
Pulmonary military TB
Liver, bone marow, spleen, adrenal, CNS, uro-genital GIT, skin, lymph nodes
Systemic miliary TB
Single organ involvement
Break into pulmonary arteries
Segment of lung
Fig. 12.2 A summary of the typical spectrum of events that may follow primary and secondary TB, respectively. CNS, central nervous system; GIT, gastrointestinal tract.
often develop at this stage, and can also undergo necrosis and coalesce with later confluence of necrosis and more extensive tissue damage. The necrosis seen in TB is usually described as caseous, because of its crumbly, cheesy appearance. It is formed due to a combination of coagulative and liquefaction necrosis. The primary granulomatous reaction characteristically occurs in the peripheral part of the lung close to the pleura in virtually any part of the lung. Classically, this lesion is called the Ghon focus. In most patients infected in this manner the infection is contained, usually by fibrosis. Subsequent calcium salt deposition may occur. The calcium salts are usually deposited diffusely throughout the necrosis, but in certain cases may be deposited in a concentric fashion. The subsequent area of pulmonary calcification can be detected on chest radiograph about 9–16 months after recovery from primary TB. In isolated cases these calcifications may undergo ossification after several years. Dissemination of organisms from the primary infected lung tissue to the hilar lymph nodes may lead to granulomatous lymphadenitis. The subsequent hilar lymphadenopathy and Ghon focus are then referred to as a Ghon complex.5,16 Organisms may survive the initial immune reaction and persist for years as viable bacilli within the healed Ghon focus and hilar lymph nodes.
PROGRESSIVE PRIMARY TUBERCULOSIS AND CONSEQUENCES In a small number of patients, usually due to immune suppression and most often in young children under 2 years of age, active disease develops immediately following primary infection. This
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process is called progressive primary TB and often leads to a disseminated form of TB. Progressive granulomatous inflammation and necrosis of lung tissue result in erosion of infected debris into a bronchus or a blood vessel with consequent tuberculous bronchopneumonia or miliary TB (see below), respectively. Airway obstruction, segmental pulmonary TB, bacteraemia and miliary TB, and pulmonary haemorrhage may complicate primary TB.
Obstruction In cases where hilar lymph nodes are massively enlarged, they may compress and obstruct a large bronchus and lead to collapse of the distal pulmonary lobe or segment. Partial bronchial obstruction may result in a valve effect with air-trapping and overdistension of a lobe or segment. Respiratory distress, secondary bacterial infection and pneumonia, or bronchiectasis may be life threatening and require surgical intervention to alleviate the obstruction. Enlargement of paratracheal lymph nodes may compress the trachea and lead to stridor. On occasion a necrotic lymph node may rupture into a bronchus, causing acute obstruction and asphyxia. Involvement of the bronchus and oesophagus may lead to a broncho-oesophageal fistula. Segmental tuberculosis More commonly, progressive granulomatous inflammation and necrosis affect the relevant lung segment, leading to segmental TB. Rupture of a lymph node with extensive necrosis into a segmental bronchus results in infected necrotic debris being distributed throughout the airways, causing expansion of the inflammatory response. Macroscopically the affected area is pale
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Pathology and pathogenesis of tuberculosis
grey. On microscopy an exudate with macrophages and interstitial lymphocytes can be seen. Numerous small granulomas that may undergo necrosis are also present. In time organization by fibrosis may transpire, which may cause obstruction, or, in rare cases, a bronchial diverticulum.
Bacteraemia and miliary tuberculosis Early in primary TB, a mycobacterial bacteraemia frequently occurs. Usually the organisms are contained by macrophages, but may cause changes akin to the Ghon focus in various organs. In some cases granulomatous angiitis develops. Progressive caseous necrosis leads to ulceration of the blood vessel intima and further release of infected debris into the blood vessel. The organisms now have the opportunity to spread haematogenously, causing systemic miliary TB. This is often a complication of hilar lymph node involvement in primary TB, as the lesions in the hilar lymph nodes are often much bigger than the pulmonary component and the adjacent pulmonary veins are large, providing easy access. Macroscopically miliary TB consists of countless numbers of minute yellow–white nodules less than 2 mm in size that can be seen in all affected organs. It most commonly involves the liver, spleen, bone marrow, lungs, and meninges. The most likely reason for this distribution is that these organs have numerous phagocytic cells in their sinusoidal walls. Even though minute in size, these miliary tubercles are just as capable of undergoing necrosis and microscopically have the same appearance as any other granuloma. This is usually an aggressive form of the disease with a fulminant course, especially if the central nervous system is also involved, causing tuberculous meningitis.5 Haemorrhage Occasionally rupture into the aorta may occur, with massive haemorrhage and sudden death. In adults progressive primary TB may present as a bacterial pneumonia, usually in the lower and mid-lobes. Hilar lymphadenopathy and pleural effusions are often present, but cavitation is seldom seen. The organisms may also spread haematogenously or lymphatically, resulting in devastating disease when meningitis or miliary TB develops. Any of the changes that occur in secondary TB may also occur in progressive primary TB.
the bacteria and progressive and often extensive caseous necrosis and tissue destruction develop.17
Course of secondary tuberculosis The extent of granulomatous inflammation, necrosis, and tissue destruction depends on many factors and the subsequent course of the disease is thus variable:
Organization and healing of localized inflammation by fibrosis, scarring, and calcification results in a fibro-calcified nodule. Progression of the inflammatory process as caseating granulomas with local destruction of pulmonary tissue and cavitation facilitates growth of organisms due to the favourable oxygen tension and an alkaline pH (Fig. 12.3). Encapsulation and localization of pulmonary cavities by fibrosis and scarring is a frequent outcome and assists to limit progressive disease. Occasionally, infective material may disseminate to other anatomical regions or organs by means of one or more pathogenic mechanisms.17 M. tuberculosis bacilli favour extrapulmonary organs such as the kidneys, meninges, and skeletal metaphyses that are very well vascularized with relatively high oxygen tension.
Dissemination of M. tuberculosis organisms can happen in one or more of the following ways.
Rupture into a bronchus The caseating granulomatous inflammation may erode bronchi with subsequent release of infective caseous material into the airways (Fig. 12.4). The infective material may be coughed up and spread in the following way(s):
SECONDARY (ADULT-TYPE) TUBERCULOSIS Some patients become reinfected with M. tuberculosis or experience reactivation of dormant disease in the Ghon focus or complex. Latent M. tuberculosis infection is present in one-third of the world’s population. Less than 10% of infected individuals, however, develop active disease. The remainder generate an effective immune response, which allows containment of the bacilli within granulomas. The bacilli persist for long periods of time in this dormant state, and there is no transmission of infection. Reactivation of the dormant infection and the onset of active disease will follow immunosuppression for whatever reason and may lead to the transmission of disease to a new host. The clinical and morphological pictures of these events are similar. Granulomatous inflammation in secondary TB occurs most often in the lungs, but it may be widely disseminated, depending on a large variety of factors. M. tuberculosis is a strict aerobic organism, and due to higher oxygen tension and poor lymphatic drainage in the apices of the lungs, the organism favours these areas. In secondary TB the granulomatous inflammation fails to contain
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It can spread proximally along the airways and affect the trachea, larynx, and the rest of the upper respiratory and digestive tract. The infective material can be inhaled, and spread distally throughout other parts of the lungs, causing tuberculous bronchopneumonia in especially the lower lobes of the lungs. Sometimes a whole lower lobe may be involved by rapidly developing confluent tuberculous bronchopneumonia. On histology, numerous acid-fast bacilli can be identified. The infected debris can be swallowed with subsequent intestinal TB, usually in the region of the distal ileum or ileocaecal junction, as this is where the lymphoid tissue of the gastrointestinal tract is present in the form of Peyer’s patches.
Rupture into pulmonary artery The caseating granulomas may spread into a branch of the pulmonary artery, leading to the following:
The pulmonary artery haemorrhages into a pulmonary cavity or larger airway, resulting in haemoptysis. Usually, tissue destruction occurs slowly, causing obliterative endarteritis of the blood vessels with occlusion of blood vessel lumina before penetration of the blood vessel wall occurs. Rapid progression of granulomatous arteritis can lead to an aneurysm of the blood vessel wall (Rasmussen’s aneurysm) that may rupture and lead to sudden massive and often fatal haemorrhage. Distal spread of the infective material through a segment of the pulmonary artery will cause localized pulmonary miliary TB in the lung parenchyma supplied by that branch of the pulmonary artery.
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Fig. 12.3 Stages of cavity formation. (A) Extensive confluent areas of necrotizing granulomatous inflammation in the upper lobe, with lymph node involvement around the bronchus. Note the size of the lymph nodes and the prospect of bronchus compression. (B) Early cavity formation with adjacent tuberculous bronchopneumonic changes. (C) Extensive cavity formation is apparent.
Rupture into lymphatics The infective agent may spread through the lymphatics to lymph nodes, causing granulomatous lymphadenitis. Further lymphatic spread can lead to:
retrogressive dissemination through other lymphatics to other areas of the lung, or to other organs; and spread to both lungs via the right heart and back to the lungs through the pulmonary arteries, causing diffuse pulmonary miliary TB (Fig. 12.5).
Rupture into pulmonary veins The organisms may spread into the pulmonary veins, again resulting in:
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haemoptysis; and spread to the systemic circulation via the pulmonary veins and the left heart, leading to widely disseminated granulomas in
many organs, especially the kidneys, adrenal glands, bone marrow, meninges, liver, and spleen.
Morphology of typical pulmonary tuberculosis The lungs in established secondary pulmonary TB have a typical macroscopic appearance. The disease is usually bilateral, with one side often less affected than the other. The upper lobes show large cavities and smaller cavities are often present in the apices of the lower lobes. The cavities often contain caseous material, but may also be empty following the release of the necrotic content into the adjacent communicating bronchi. These cavities can be quite large and may measure up to several centimetres in diameter. The cavity walls contain fibrosis and often the remnants of a bronchial wall or branches of the pulmonary arteries. Histological examination of a cavity wall reveals different zones. The cavity itself is surrounded by a rim of caseous material flanked by organizing granulomatous inflammation and variable fibrosis and scarring
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Fig. 12.4 Bronchopneumonic TB. (A) A low magnification of a lung section with granuloma formation surrounding bronchioles. (B) Rupture into the bronchus is clearly illustrated by the erosion of the respiratory epithelium and the underlying necrotic granuloma. (C) The patchy, bronchopneumonic nature of the process in the lung.
towards the periphery of the wall. Ziehl–Neelsen stains usually reveal acid-fast bacilli in the necrotic material or on the edge of necrosis adjacent to the granulomatous inflammation. Satellite granulomas may occur in the adjacent lung tissue. When there is communication between a cavity and a bronchus infective material is coughed up and released into the environment as a droplet aerosol with the potential for transmission of the organisms. Cavitation is usually absent or much less pronounced in the lower lobes. Hilar lymph nodes are less frequently involved than in primary TB, but microscopic examination will often show residual granulomatous lymphadenitis.
Local complications of secondary pulmonary tuberculosis
Advanced secondary pulmonary TB leads to an extensive loss of lung parenchyma with subsequent diminished lung capacity and progressive respiratory failure. Large cavities with thick, fibrotic walls often persist and may develop an epithelial lining. This lining may be of respiratory type, but more often it comprises squamous metaplasia, simple or stratified, with or without keratinization. The cavity may contain keratin, mimicking an epidermoid cyst. In rare cases, squamous carcinoma may develop in the cyst wall.
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or opposite effusion should raise the possibility of miliary TB.19 Primary pleural TB usually resolves without treatment. However, especially in young adults, it correlates with an increased risk of developing into other forms of TB, usually within 5–10 years. Full therapy of primary pleural TB is therefore justified. Local complications that may ensue include focal or diffuse fibrosis, a large caseous lesion that may rupture into the lung, creating a pulmonary– pleural fistula or spontaneous pneumothorax, paravertebral abscess, and osteitis of the ribs.
Fig. 12.5 (A) Disseminated miliary tubercles can be seen in the lung, with (B) a close-up of tubercles in the inset, as well as in the liver and spleen.
The environment of the cavity, especially when not communicating with a bronchus, is conducive to secondary bacterial infection, with subsequent abscess formation. Occasionally fungal colonization, especially by Aspergillus flavus, results in a ball of fungi known as an aspergilloma. Bronchiectasis is a common complication of destructive pulmonary TB and can be complicated by the usual problems associated with bronchiectasis. Progression of pulmonary disease often involves the pleura by means of serous effusions, tuberculous empyema, or massive obliterative fibrous pleuritis.17
Spread via the systemic circulation can lead either to individual organs being affected or to miliary TB, depending on the immune status of the patient and the virulence of the organism. Organs often affected include the kidneys, brain, liver, spleen, adrenals, and testes. The respective chapters on organ TB provide more detail pertaining to the relevant organ-specific pathology and clinicopathological features.
PLEURAL TUBERCULOSIS Primary pleural effusion, as a presenting feature of TB, usually occurs in adolescents and adults. It occurs on the same side as the primary Ghon complex, suggesting that a subpleural tuberculous focus or lymph node has extended into the pleural space.18 Bilateral
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Pathogenesis of primary pleural tuberculosis The current hypothesis for the pathogenesis of primary tuberculous pleural effusion is that a subpleural caseous focus in the lung ruptures into the pleural space 6–12 weeks after a primary infection.19 Mycobacterial antigens enter the pleural space and elicit a delayed hypersensitivity reaction and an accumulation of fluid. It appears that the DTH reaction results in an increased permeability of pleural capillaries to serum proteins, leading to increased oncotic pressure in the pleural fluid and the development of a pleural effusion.20 In primary pleural effusions, typical TB granulomas occur on both the visceral and the parietal pleural surfaces. These granulomas tend to follow the lymphatics in the visceral pleural surface and focal or diffuse pleural fibrosis may follow. Impaired clearance of proteins from the pleural space has been reported in human tuberculous effusions.20 Clearance of proteins and fluid from the pleural space occurs via lymphatics in the parietal pleura. Access to the lymphatics is through openings in the parietal pleura called stomata.21 In tuberculous pleuritis, with diffuse involvement of the parietal pleura, damage to or obstruction of the stomata could be an important mechanism leading to accumulation of pleural fluid. It appears thus that tuberculous pleurisy is due to the release of isolated mycobacterial antigens into the pleural space, eliciting a delayed hypersensitivity reaction rather than to direct tuberculous infection of the pleura.22 On histological examination, acid-fast bacilli are demonstrated in only 5–18% of cases.23 However, the diagnosis can be confirmed in more than 95% of patients with the performance of a combination of culture of pleural fluid and tissue, and histology of pleural tissue.24 Typical granulomas with caseous necrosis are also present in pleural tissue.
THE EFFECT OF HIV ON TUBERCULOSIS A detailed discussion of the effect of HIV on TB will be found in Chapter 10. Increased nosocomial and community exposure to M. tuberculosis may play a role in the increased risk of contracting TB in HIV-infected patients, but HIV-associated impairment of one or more immunological mechanisms plays an important role.25–28 These mechanisms include impairment of pulmonary innate immune defences, impairment of cellular recruitment and establishment of the cell-mediated granulomatous response to recent M. tuberculosis infection, and functional impairment of established granulomas containing latent M. tuberculosis infection. HIV-1 infects macrophages and CD4þ T cells essential in granuloma formation in TB. Coinfection will therefore impair cell-mediated immune responses to M. tuberculosis infection. Several possible levels of impairment exist.29–34 Tuberculosis and HIV-1 coinfection is also likely to be associated with extensive virus-induced and activation-induced cell loss and with the suppression of lymphocyte regeneration and maturation.35,36 HIV may also block
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the ability of the host to mount an effective proliferative T-lymphocyte response to M. tuberculosis.37 The risk of developing TB disease following infection in HIV-infected individuals is no longer 10% lifelong but close to 10% per year.
In 1993 Lucas et al.39 described three histological stages of cellular immune response that correlate with depletion of the peripheral blood CD4þ lymphocyte count, namely:
THE EFFECT OF HIV ON GRANULOMATOUS INFLAMMATION The changes described above, as well as the progressive immunosuppression associated with the development of AIDS, result in failure of epithelioid differentiation of macrophages, and lack of Langhans giant cells and caseous necrosis. Tuberculous hypersensitivity-type granulomas are not considered immunologically quiescent structures, but rather have a continual level of mononuclear cell death and cell replacement by active recruitment.38 It is therefore possible that HIV-1 infection affects granuloma function via two processes:
systemic depletion of the mononuclear cells required for the ongoing maintenance and functioning of granulomas; and the effects of HIV-1-infected cells (either lymphocytes or macrophages) trafficking into the granuloma itself.
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In immunocompetent individuals with HIV-1 infection, tuberculous granulomas are characterized by abundant epithelioid macrophages, Langhans giant cells, peripherally located CD4þ lymphocytes, and a paucity of bacteria. In individuals with moderate HIV-associated immunodeficiency, Langhans giant cells are not seen, epithelioid differentiation and activation of macrophages are absent, there is CD4þ lymphocytopenia, and acid-fast bacilli are more numerous. In individuals with advanced HIV-associated immunosuppression and AIDS, there is a striking paucity of granuloma formation with little cellular recruitment, very few CD4þ lymphocytes, and even larger numbers of acid-fast bacilli.
It is therefore apparent that HIV-1 coinfection weakens the granulomatous host response to M. tuberculosis and affects the ability of established granulomas to contain M. tuberculosis, resulting in increased reactivation of latent mycobacteria (Fig. 12.6).
A
C
D
Fig. 12.6 The effect of HIV on granuloma formation. (A) A loose collection of inflammatory cells, vaguely resembling a granuloma, can be seen. (B) On high magnification of this area a few isolated epithelioid macrophages are visible. (C) A high magnification of a confluent area of inflammation with numerous neutrophils and some lymphocytes, but no recognizable epithelioid macrophages. (D) A Ziehl–Neelsen stain of the areas highlights the vast number of acid-fast bacilli present in this biopsy.
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THE EFFECT OF TUBERCULOSIS ON TISSUE The effect of TB on tissue varies enormously, depending on the extent of involvement and also the organs involved (Fig. 12.7). In some cases, especially when tissue destruction has not yet occurred, and treatment is early and effective, tissue may heal without any signs of residual damage. More often, one or more of the following will occur.
DESTRUCTION OF TISSUE
Caseating necrosis, associated with granulomatous inflammation, has a very destructive effect, often resulting in loss of function of the organ affected.
Tissue destruction occurs, especially in the lung, leading to fibrosis and distortion of lung tissue. Impairment of lung function will follow, depending on the extent of fibrosis. Destruction of tissue centred around the bronchi will result in bronchiectasis, with all its complications.
Destruction of blood vessel walls will cause haemorrhage, and, depending on the size of the vessel, the haemorrhage may be insignificant or devastating. In the adrenals TB is one of the causes of chronic primary insufficiency, due to destruction of the adrenal cortex and medulla. The breast is sometimes involved in TB and usually a fibrocaseous mass with numerous sinuses develops. In those cases where fibrosis develops, the mass may be clinically indistinguishable from carcinoma. In the kidney TB may form either a single mass, usually in the parenchyma, or multiple smaller nodules, resembling abscess formation. In the case of long-standing TB, pyelonephrosis develops due to rupture of the mass into the pelvis. Complete destruction of the renal pelvis may ensue. Involvement of the vertebral column is relatively frequent, also in children. Osteomyelitis causes extensive destruction of vertebral bodies, which is complicated by collapse. This condition is also known as Pott’s disease. In children, tuberculous arthritis occurs, with destruction of cartilage and subsequent fibrosis.
Fig. 12.7 Potential complications of TB. (A) A fibrinous pericarditis is demonstrated. On histology, numerous granulomas with caseous necrosis are evident. (B) Massively enlarged lymph nodes in the mesentery are demonstrated. This may lead to functional bowel obstruction. (C) Enlarged, necrotic lymph nodes can be seen around the main bronchi. It is apparent that obstruction is imminent and necrosis through the bronchus wall may follow.
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Granulomatous endometritis is an unusual, but not uncommon, cause of infertility in females. Numerous small granulomas form throughout the endometrium and ulceration with haemorrhage may develop. If this occurs during pregnancy, congenital TB may occur. Numerous other organs, including the epididymis, skin, gastrointestinal tract, and central nervous system, are also affected. For a discussion on the organ pathology of TB, please refer to the appropriate organ-specific chapters.
the extensive fibrosis may lead to further tissue destruction with dire consequences.
SPACE-OCCUPYING LESIONS Granulomas may enlarge and, depending on the organ, this may have a space-occupying effect.
In the brain, tuberculomas develop, mainly supratentorial, but may occasionally affect the cerebellum, basal ganglia, and brainstem. Although children frequently present with convulsions as the first sign, other symptoms and signs of a spaceoccupying lesion may develop. In the prostate, enlarging granulomas may clinically mimic benign prostate hyperplasia. Only on microscopic examination can the granulomas be seen and the diagnosis suggested.
FIBROSIS Extensive fibrosis is typical of organizing granulomatous inflammation associated with TB and tends to surround granulomas in an attempt to contain the organism within granulomas. Unfortunately
REFERENCES 1. Kumar V, Abbas A, Fausto N. Acute and chronic inflammation. In: Kumar V, Abbas AK, Fausto N (eds). Robbins and Cotran Pathologic Basis of Disease, 7th edn. Philadelphia: Elsevier Saunders, 2005: 47–86. 2. Beutler B. Innate immunity: an overview. Mol Immunol 2004;40:845–859. 3. Flynn JL. Immunology of tuberculosis and implications in vaccine development. Tuberculosis (Edinb) 2004;84:93–101. 4. Allen EA. Tuberculosis and other mycobacterial infections of the lung. In: Thurlbeck W, Churg A (eds), Pathology of the Lung, 2nd edn. New York: Thieme Medical, 1995: 229–265. 5. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997;90:3245–3287. 6. Sallusto F, Lanzavecchia A. The instructive role of dendritic cells on T-cell responses. Arthritis Res 2002; 4(Suppl 3):S127–S132. 7. von Andrian UH, Mackay CR. T-cell function and migration. Two sides of the same coin. N Engl J Med 2000;343:1020–1034. 8. Chaplin DD. Overview of the immune response. J Allergy Clin Immunol 2003;111(2 Suppl):S442–459. 9. Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 2000;343:338–344. 10. Abbas AK, Lichtman AH, Prober JS. Effector mechanisms of T cell-mediated immune reactions. In: Abbas AK, Lichtman AH, Prober JS (eds). Cellular and Molecular Immunology, 4th edn. Philadelphia: Saunders, 2000: 278–296. 11. Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 2001;19:47–64.
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In body cavities, organization through fibrosis results in adhesions. Depending on the site and extent of the adhesions, signs and symptoms may develop: ○ In tuberculous meningitis, obstructive hydrocephalus, with all its sequelae may develop. ○ In the pleura the fibrosis may be followed by loculated zones of empyema. ○ In the pericardium, a restrictive pericarditis develops. ○ In the peritoneum bowel obstruction with its complications may result. In hollow organs it may lead to obstruction: ○ In the fallopian tubes infertility may be ascribed to complete, or partial, obstruction of the lumina. ○ In the small bowel, where TB is present in the Peyer’s patches, ulceration and haemorrhage of the mucosal surface occurs early with circumferential granuloma formation and mural fibrosis later, culminating in intestinal obstruction.
One of the complications of TB that used to be very common, but currently is seldom seen, is amyloidosis. This is a condition associated with long-standing chronic diseases, but is at present uncommon. For the handling, processing, interpretation, fine needle aspiration technique and differential diagnosis of specimens, please refer to Chapter 21.
12. Alam R, Gorska M. Lymphocytes. J Allergy Clin Immunol 2003;111(2 Suppl):S476–485. 13. Sinigaglia F, D’Ambrosio D. Regulation of helper T cell differentiation and recruitment in airway inflammation. Am J Respir Crit Care Med 2000;162 (4 Pt 2):S157–160. 14. Algood HM, Chan J, Flynn JL. Chemokines and tuberculosis. Cytokine Growth Factor Rev 2003;14: 467–477. 15. Miceli MC, Parnes JR. Role of CD4 and CD8 in T cell activation and differentiation. Adv Immunol 1993;53:59–122. 16. Husain AN, Kumar V. In: Kumar V, Abbas AK, Fausto N (eds). Robbins and Cotran Pathologic Basis of Disease, 7th edn. Philadelphia: Elsevier, 2005: 711–772. 17. Hasleton PS. Pulmonary bacterial infection. In: Hasleton PS (ed.). Spencer’s Pathology of the Lung, 5th edn. New York: McGraw-Hill, 1996: 189–255. 18. Stead WW, Eichenholz A, Stauss HK. Operative and pathologic findings in twenty four patients with syndrome of idiopathic pleurisy with effusion, presumably tuberculous. Am Rev Respir Dis 1955;71:473–502. 19. Leckie WJH, Tothill P. Albumin turnover in pleural effusions. Clin Sci 1965;29:339–352. 20. Harley RA. Pathology of pleural infections. Semin Respir Infect 1988;3:291–297. 21. Wang NS. The preformed stomas connecting the pleural cavity and the lymphatics in the parietal pleura. Am Rev Respir Dis 1975;111:12–20. 22. Ellner JJ, Barnes PF, Wallis RS, et al. The immunology of tuberculous pleurisy. Semin Respir Infect 1988;3:335–342. 23. Reese O Jr, Mclean RL, Raaen TD. Acid-fast bacilli in pleural biopsy specimens. Arch Intern Med 1961;108:438–441.
24. Diacon AH, Van de Wal BW, Wyser C, et al. Diagnostic tools in tuberculous pleurisy: a direct comparative study. Eur Respir J 2003;22:589–591. 25. Di Perri G, Cruciani M, Danzi MC, et al. Nosocomial epidemic of active tuberculosis among HIV-infected patients. Lancet 1989;2:1502–1504. 26. Dooley SW, Villarino ME, Lawrence M. Nosocomial transmission of tuberculosis in a hospital unit for HIV-infected patients. JAMA 1992;267:2632–2635. 27. Centers for Disease Control and Prevention. Tuberculosis outbreak among persons in a residential facility for HIV-infected persons, Florida and New York. MMWR Morb Mortal Wkly Rep 1991;40:585–591. 28. Lawn SD, Butera ST, Shinnick TM. Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect 2002;4:635–646. 29. Polyak S, Chen H, Hirsch D, et al. Impaired class II expression and antigen uptake in monocytic cells after HIV-1 infection. J Immunol 1997;159:2177–2188. 30. Zembala M, Pryjma J, Plucienniczak A, et al. Modulation of antigen-presenting capacity of human monocytes by HIV-1 gp120 molecule fragments. Immunol Invest 1994;23:189–199. 31. Ieong MH, Reardon CC, Levitz SM, et al. Human immunodeficiency virus type 1 infection of alveolar macrophages impairs their innate fungicidal activity. Am J Respir Crit Care Med 2000;162:966–970. 32. Baldwin GC, Fleischmann J, Chung Y, et al. Human immunodeficiency virus causes mononuclear phagocyte dysfunction. Proc Natl Acad Sci USA 1990;87:3933–3937. 33. Imperiali FG, Zaninoni A, La Maestra L, et al. Increased Mycobacterium tuberculosis growth in HIV-1infected human macrophages: role of tumour necrosis factor-alpha. Clin Exp Immunol 2001;123:435–442.
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34. Kallenius G, Koivula T, Rydgard KJ, et al. Human immunodeficiency virus type 1 enhances intracellular growth of Mycobacterium avium in human macrophages. Infect Immun 1992;60:2453–2458. 35. Clark DR, de Boer RJ, Wolthers KC, et al. T cell dynamics in HIV-1 infection. Adv Immunol 1999;73:301–327. 36. Lawn SD, Rudolph D, Wiktor S, et al. Tuberculosis and HIV infection are independently associated with
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elevated serum concentrations of tumour necrosis factor receptor type 1 and b-microglobulin, respectively. Clin Exp Immunol 2000;122:79–84. 37. Lawn SD, Rudolph D, Coulibaly D, et al. Lack of induction of interleukin-2-receptor alpha expression in patients with tuberculosis and human immunodeficiency virus coinfection: implications for pathogenesis. Trans R Soc Trop Med Hyg 2001;95; 449–452.
38. Lawn SD, Butera ST, Folks TM. Contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection. Clin Microbiol Rev 2001;14: 753–777. 39. Lucas SB, Hounnou A, Peacock C, et al. The mortality and pathology of HIV infection in a west African city. AIDS 1993;7:1569–1579.
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The natural history of Mycobacterium tuberculosis infection in adults Dermot Maher
INTRODUCTION Our understanding of the natural history of TB goes back at least to the tenth century when Avicenna (also known as Ibn Sina) described the contagious nature of pulmonary TB in his book Qanun fi’l-Tibb (The Canon of Medicine).1 Nine hundred years later, Koch confirmed Avicenna’s description with his identification of the tubercle bacillus and its causative role in TB.2 Although the pace of scientific discoveries concerning the natural history of TB has sped up since Koch’s seminal discovery in 1882, much still remains to be discovered. As indicated by G. Bacelli in 1882, ‘Il bacillo non e´ ancora tutta la tuberculosi’ (‘The bacillus is not yet all there is to tuberculosis’).3 Tubercle bacilli are a necessary, but not a sufficient, cause of TB. Tuberculosis can only occur following infection with Mycobacterium tuberculosis, but occurs in a small minority of those infected. The risk of M. tuberculosis infection is largely exogenous in nature, determined by the characteristics of the source case, environment, and duration of exposure. In contrast, the risk of TB following M. tuberculosis infection is largely endogenous, determined by the individual’s immune status. This chapter describes the sequence of events that constitute the natural history of M. tuberculosis infection in adults: the transmission of infection, the process of becoming infected, and the pathogenesis of TB as one of the consequences of becoming infected. A good grasp of the natural history of M. tuberculosis infection underpins our understanding of TB epidemiology and control, as well described by Rieder.4
INFECTION WITH M. TUBERCULOSIS TRANSMISSION OF INFECTION The transmission of M. tuberculosis is almost exclusively by the airborne route. The most important source of infection is the patient with pulmonary TB who is coughing. Coughing produces droplets – as does talking, sneezing, spitting or singing – that may contain tubercle bacilli.5 As the droplets expelled into the air evaporate, some form droplet nuclei, which are infectious particles of respiratory secretions usually less than 5 mm in diameter containing one or a few tubercle bacilli. A single cough can produce 3,000 droplet nuclei and they can remain suspended in the air for several hours. Whereas larger particles either fall to the ground or, if inhaled, are trapped either
in the nose or in the mucociliary system of the tracheobronchial tree, droplet nuclei are so small that they avoid the defences of the bronchi and penetrate into the terminal alveoli of the lungs where infection begins.6 Since the particles containing tubercle bacilli on the clothing, bedcovers, or belongings of a TB patient cannot be dispersed in aerosols, they do not play a significant part in infection.7
CHARACTERISTICS OF AN INFECTIOUS PATIENT Patients with extrapulmonary TB do not generally constitute a source of infection, unless by direct inoculation. The production of airborne infectious droplets is by people with respiratory tract TB. Those who produce the most tubercle bacilli are the most infectious. The number of tubercle bacilli found in sputum specimens reflects infectiousness.8 Studies of the close contacts of patients with culture-proven pulmonary TB showed that a higher proportion of contacts of sputum smear-positive cases than of smearnegative source cases were infected.9–11 The most potent sources of infection are therefore patients with sputum smear-positive pulmonary TB. Classical contact tracing has also shown the importance of patients with sputum smear-positive pulmonary TB as sources of secondary cases of TB. For example, in a 2-year contact followup study in Finland, among the 609 contacts of 134 culture-proven source cases, four developed TB, all of them among contacts of the 69 source cases with heavily sputum smear-positive TB.12 The use of molecular fingerprinting techniques has also shed light on the relative importance of sputum smear-positive and sputum smear-negative source cases in generating clusters of secondary cases of cultureproven TB sharing identical DNA fingerprints and therefore assumed to have arisen from recent transmission. A study in San Francisco, USA, found that, of 183 secondary cases, the number attributed to infection by sputum smear-positive and sputum smear-negative source cases was 151 (83%) and 32 (17%), respectively, and the relative transmission rate from sputum smear-negative compared with sputum smear-positive source cases was 0.22.13 Patients with sputum smear-positive pulmonary TB are therefore also the most potent source cases of secondary TB.
RISK OF INFECTION Risk of infection depends on the extent of an individual’s exposure to droplet nuclei and on susceptibility to infection.
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Exposure to droplet nuclei Two factors determine an individual’s risk of exposure: the concentration of droplet nuclei in contaminated air and the length of time spent breathing that air. The extent of an individual’s exposure to droplet nuclei is determined by the proximity and duration of contact with an infectious source case, since the concentration of droplet nuclei to which the person is exposed depends on proximity, and the length of time spent breathing the contaminated air depends on duration of contact. The concentration of droplet nuclei depends on the number of infectious droplets expelled and the volume of air into which they are expelled. Risk of exposure is therefore much greater in an enclosed poorly ventilated space indoors than outdoors. Ventilation dramatically dilutes the concentration of droplet nuclei and is therefore the most important environmental measure to decrease risk of infection among exposed persons. The simplest and least expensive way of ventilating a room or hospital ward is to maximize natural ventilation through open windows.14 Since the concentration of droplet nuclei in contaminated air falls very quickly outdoors, and direct sunlight kills tubercle bacilli in 5 minutes (although they can survive in the dark for long periods of time), transmission generally occurs indoors. The risk of infection of a susceptible individual is therefore high with close, prolonged, indoor exposure to a person with sputum smear-positive pulmonary TB. These are the conditions generally thought of as constituting ‘close contact’. A contact tracing study in The Netherlands, with the contact tracing ‘net’ cast very widely, showed that the closer the contact of a susceptible individual to an infectious source case, the greater the chance of infection.15 The number of cases of infection in a particular exposure group (defined by closeness to the source case) is the product of the risk and the number of people in the group. Thus, exemplifying the Rose axiom, there is a larger number of cases of infection in the large group of distant, low-risk contacts than in the small group of close, high-risk contacts.16 Conventional contact tracing generally identifies the close, high-risk contacts and therefore identifies a minority of the contacts infected by a source case. The extent of an individual’s exposure to infection determines not only risk of infection but also affects the risk of disease, since those with greater intensity of exposure are at greater risk of developing disease. For example, in the micro-outbreak of TB described among the crew members of the US submarine Byrd, the presence of one man with TB resulted in 139 M. tuberculosis infections among the 308 crew, with six out of the seven cases of active TB occurring in men who were sleeping in the same cabin and working closely together.17 Susceptibility to infection The risk of M. tuberculosis infection is largely exogenous in nature, determined by the characteristics of the source case, environment, and duration of exposure. It has been difficult to separate the influence of the genetic and exogenous (socio economic and environmental) factors that determine susceptibility to infection. A study in the USA of nursing home residents with apparently the same risk of exposure found a higher risk of infection among black than white residents, suggesting the role of genetic factors in determining susceptibility.18 Susceptibility to infection is affected by the ability of macrophages to phagocytose and destroy the bacilli that reach the terminal alveoli and begin the process of implantation and infection.19 Since several genes are involved in this process, the study of human genomics has the potential to increase our
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understanding of differences between individuals and populations in their susceptibility to infection.20 Some exogenous factors that may cause increased susceptibility to infection by impairing the local immune response in the respiratory tract, e.g. silicosis and inhalation of smoke from cooking fires and industrial pollution, have been suggested. Long-term cigarette smoking causes disruption and injury of the respiratory tract endothelial cells (peribronchial inflammation, abnormal vascular and epithelial permeability) and decreased mucociliary clearance of inhaled particles that may favour infection by inhaled pathogens such as M. tuberculosis.21 In practice it has been difficult to separate the possible influence of these exogenous factors on susceptibility to infection versus progression of infection to disease. Human immunodeficiency virus (HIV) may increase susceptibility to infection with M. tuberculosis. The evidence for this mainly depends on comparisons in hospital outbreaks of the outcome of exposure to source cases of TB, with higher rates of development of TB in HIV-infected than in HIV-uninfected patients exposed. The assumption is that the secondary cases have primary TB, so the higher rates of TB in HIV-infected people reflect higher rates of M. tuberculosis infection and therefore increased susceptibility to infection with M. tuberculosis.
PATHOGENESIS OF TUBERCULOSIS PRIMARY INFECTION Primary infection occurs in persons without previous exposure to tubercle bacilli. Droplet nuclei inhaled into the lungs and small enough to avoid the mucociliary defences of the bronchi lodge in the terminal alveoli. Non-specifically activated alveolar macrophages ingest the inhaled bacilli. If the bacilli are virulent enough to withstand the proteolytic enzymes in the macrophage phagolysosomes, they multiply and initiate infection. About 2–4 weeks after infection, cell-mediated immunity results in the formation of granulomas which usually constrain the spread of the bacilli. A granuloma is an aggregation of macrophages activated by cytokines produced by CD4þ T-lymphocytes. The initial focus of infection in the lungs is the Ghon focus. Lymphatics drain the bacilli to the hilar lymph nodes. The Ghon focus and related hilar lymphadenopathy form the primary complex. The development of the primary complex is asymptomatic. The development of the immune response (delayed hypersensitivity and cellular immunity) about 4–6 weeks after the primary infection is indicated by a positive tuberculin skin test and occasionally by clinical hypersensitivity reactions (e.g. erythema nodosum, phlyctenular conjunctivitis, dactylitis).
OUTCOMES OF PRIMARY INFECTION The balance between host immunity and bacillary multiplication determines what happens next. In most cases, the immune response stops the multiplication of bacilli. A calcified nodule visible on chest radiography is a common outcome of successful immune containment of infection with killing of bacilli. The immune response in a few cases is not strong enough to prevent multiplication of bacilli, and progression from infection to disease occurs within a few months. Primary TB results from local bacillary multiplication and spread in the lung or spread in the blood from the primary complex throughout the body, with seeding of bacilli in
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The natural history of Mycobacterium tuberculosis infection in adults Outcome of primary infection No clinical disease positive tuberculin skin test (indicating latent infection) (usual outcome: 90% of cases)
Primary complex
Hypersenitivity reactions e.g. erythema nodosum phlyctenular conjunctivitis dactyllitis Pulmomary and pleural complications e.g. tuberculous pneumonia hyper inflation and collapse/consolidation pleural effusion Disseminated disease e.g. lymphadenopathy (usually cervical) meningitis percarditis miliary disease
Fig. 13.1 Outcomes of primary infection with M. tuberculosis.
various tissues and organs. In some cases dormant bacilli may persist and cause disease on later reactivation. Figure 13.1 summarizes the possible outcomes of primary infection with M. tuberculosis.
PROGRESSION OF M. TUBERCULOSIS INFECTION TO DISEASE Once infected with M. tuberculosis, a person stays infected for many years, probably for life. The vast majority (90%) of people without HIV infection who are infected with M. tuberculosis do not develop TB. In these healthy, asymptomatic, but infected individuals, the only evidence of infection may be a positive tuberculin skin test. In contrast to the risk of M. tuberculosis infection largely determined by exogenous factors, the risk of TB following M. tuberculosis infection is largely endogenous, determined by the individual’s immune status. A significant improvement in our understanding of M. tuberculosis latency is necessary to be able to answer the question why some people with latent M. tuberculosis infection develop TB and others do not. As Bishai22 has put it, ‘understanding how M. tuberculosis lies quiescent for years if not decades, how the immune system fails to detect and eradicate it, and the nature of the stimuli for its reactivation remain nearly as uncertain today as in Robert Koch’s time.’ About 5–10% of people infected with M. tuberculosis develop TB in their lifetime, mostly within 5 years of infection.23 Various physical or emotional stresses may trigger progression of infection to disease. The most important trigger is weakening of immune resistance, especially by HIV infection. The chance of developing disease is greatest shortly after initial infection and then steadily lessens as time goes by.
PRIMARY, POST-PRIMARY, AND RECURRENT TUBERCULOSIS A first episode of TB may be primary (i.e. occurring through progression of primary infection) or post-primary (i.e. occurring after a latent period of months or years after primary infection, either through reactivation of the dormant tubercle bacilli acquired from a primary infection or by reinfection). A recurrent episode of TB may occur through relapse (reactivation) or reinfection.
13
Primary tuberculosis Progression of primary infection may result in pulmonary and pleural complications, e.g. tuberculous pneumonia, hyperinflation and collapse/consolidation, pleural effusion, and disseminated disease, e.g. lymphadenopathy (usually cervical), meningitis, pericarditis, and miliary disease. Since most people infected with M. tuberculosis are infected in childhood, primary TB mainly occurs in childhood. Since HIV dramatically increases the risk of rapid progression of M. tuberculosis infection to disease, adults with HIV infection who become infected with M. tuberculosis often develop primary TB. The incidence of primary TB is therefore higher in countries with high HIV prevalence. Post-primary tuberculosis Post-primary TB occurs after a latent period of months or years after primary infection. It may occur either by reactivation of the dormant tubercle bacilli acquired from a primary infection or by reinfection. Reactivation means that dormant bacilli persisting in tissues for months or years after primary infection start to multiply. This may be in response to a trigger, such as weakening of the immune system by HIV infection. Reinfection means a repeat infection in a person who has already previously had a primary infection. The immune response of the patient results in a pathological lesion that is characteristically localized, often with extensive tissue destruction and cavitation. Post-primary TB usually affects the lungs but can involve any part of the body. The characteristic features of post-primary pulmonary TB are the following: extensive lung destruction with cavitation; positive sputum smear; upper lobe involvement; and usually no intrathoracic lymphadenopathy. Patients with these lesions are the main transmitters of infection in the community. Table 13.1 shows the main forms of postprimary TB. Recurrent tuberculosis A recurrence of TB is a second episode of TB occurring after a first episode had been considered cured. Recurrent TB may be due to relapse (reactivation) or reinfection.24
Table 13.1 The main forms of post-primary tuberculosis Pulmonary TB e.g. Cavities Upper lobe infiltrates Fibrosis Progressive pneumonia Endobronchial disease Extrapulmonary TB Common Pleural effusion Lymphadenopathy (usually cervical) Central nervous system (meningitis, cerebral tuberculoma) Pericarditis (effusion/constrictive) Gastrointestinal (ileocaecal, peritoneal) Spine, other bone and joint
Less common Empyema Male genital tract (epididymitis, orchitis) Female genital tract (tuboovarian, endometrium) Kidney Adrenal gland Skin (lupus vulgaris, tuberculids, miliary)
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COINFECTION WITH M. TUBERCULOSIS AND HIV HIV infection increases the risk of progression to active TB in both people with recently acquired and those with latent M. tuberculosis infection (for which HIV infection is the most powerful known risk factor for reactivation).25,26 This risk increases with increasing immunosuppression. As HIV infection progresses, CD4þ T-lymphocytes steadily decline in number and function and therefore the ability to restrict tubercle bacilli to a few infected macrophages decreases. The impact of HIV on the outcome of primary infection is an increased risk and speed of progression to primary TB through local and disseminated bacillary spread. The tuberculin skin reaction is also suppressed. HIV infection increases not only the risk but also the speed of progression of latent M. tuberculosis infection to disease. Overall, compared with an individual not infected with HIV, a HIV-infected individual has a 10 times increased risk of developing TB. HIV also increases the rate of recurrent TB, which may be due to either endogenous reactivation (true relapse) or exogenous reinfection.27–29
NATURAL HISTORY OF UNTREATED TUBERCULOSIS Nowadays it would be unethical to study the natural history of untreated TB. However, a study of TB in a rural population in South India, carried out during the 1960s, found that five out of
REFERENCES 1. Ahmed M. Ibn Sina (Avicenna)—Doctor of doctors. [online] Available at URL:http://www.ummah.net/ history/scholars/ibn_sina 2. Koch R. Die aetiologie der tuberculose. Berl Klin Wochenschr 1882;19:221–230. 3. Quoted in: Bloom BR (ed.). Tuberculosis: Pathogenesis, Protection and Control. Washington, DC: ASM Press, 1994. 4. Rieder HL. Epidemiologic Basis of Tuberculosis Control. Paris: International Union against Tuberculosis and Lung Disease, 1999. 5. Loudon RG, Roberts RM. Droplet expulsion from the respiratory tract. Am Rev Respir Dis 1966;95: 435–442. 6. Sonkin LS. The role of particle size in experimental air-borne infection. Am J Hyg 1951;53:337–354. 7. American Thoracic Society. Infectiousness of tuberculosis, a statement of the ad hoc committee on treatment of tuberculosis patients in general hospitals. Am Rev Respir Dis 1967;96:837. 8. Toman K. Tuberculosis Case-Finding and Chemotherapy: Questions and Answers. Geneva: World Health Organization, 1979. 9. Shaw JB, Wynn-Williams N. Infectivity of pulmonary tuberculosis in relation to sputum status. Am Rev Tuberc 1954;69:724–732. 10. Grzybowski S, Barnett GD, Styblo K. Contacts of cases of active pulmonary tuberculosis. Bull Int Union Tuberc 1975;50:90–106. 11. Van Geuns HA, Meijer J, Styblo K. Results of contact examination in Rotterdam, 1967–1969. Bull Int Union Tuberc 1975;50:107–121.
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10 untreated patients with sputum smear-positive pulmonary TB died within 5 years, three self-cured, and two remained ill with chronic, infectious TB.30 As a rule of thumb in TB epidemiology, this finding is embodied in practically every conceptual model – qualitative or quantitative – of the way TB affects populations.31,32
THE CYCLE OF TRANSMISSION In the absence of HIV infection, about 10% of people infected with M. tuberculosis will develop active TB (whether from progression of recent infection or from reactivation), of whom about onehalf will be infectious (usually with sputum smear-positive pulmonary disease).33 Thus, only one in 20 people infected with M. tuberculosis develops infectious TB, and each infectious case in its turn needs to infect about 20 people in order to generate one further infectious case. This is the situation of stable TB incidence (i.e. the case reproduction number is 1). Key determinants of the case reproduction number are the number of people infected by an infectious case, and the proportion of people infected with M. tuberculosis who develop active TB. Any factors which increase the number of people infected by an infectious case (e.g. lack of or inadequate antituberculosis treatment) or which increase the proportion of people infected with M. tuberculosis who develop active TB (e.g. HIV infection) will push the case reproduction number above 1, with consequent increasing TB incidence. The primary stratagem of TB control is to reduce the average number of people infected by each infectious case so that the case reproduction number is less than 1.
12. Liippo KK, Kulmala K, Tala EOJ. Results of tuberculosis contact tracing by smear grading of index cases. Am Rev Respir Dis 1993;148:235–236. 13. Behr MA, Warren SA, Salamon H, et al. Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 1999;353:444–449. 14. World Health Organization. Guidelines for the Prevention of Tuberculosis in Health Care Facilities in Resource-Limited Settings. Geneva: World Health Organization, 1999. 15. Veen J. Microepidemics of tuberculosis: the stone-in-the-pond principle. Tuberc Lung Dis 1992;73:73–76. 16. Rose G. Sick individuals and sick populations. Int J Epidemiol 1985;14:32–38. 17. Houk VN, Rent DC, Baker JH, et al. The Byrd study, an analysis of a micro-outbreak of tuberculosis in a closed environment. Arch Environmental Health 1968:16;26. 18. Stead WW, Senner JW, Reddick WT, et al. Racial differences in susceptibility to infection by M. tuberculosis. N Engl J Med 1990;322:422–427. 19. Schluger NW, Rom WN. The host immune response to tuberculosis. Am J Respir Crit Care Med 1998;157:679–691. 20. Davies P, Grange J. The genetics of host resistance and susceptibility to tuberculosis. Ann NY Acad Sci 2001;953:151–156. 21. Aubry MC, Wright JL, Myers JL. The pathology of smoking-related lung diseases. Clin Chest Med 2000;21:11–35. 22. Bishai WR. Rekindling old controversy on elusive lair of latent tuberculosis (Commentary). Lancet 2000;356:2113–2114. 23. Comstock GW, Cauthen GM. In: Reichman LB, Hershfield ES (eds). Tuberculosis. A Comprehensive
24.
25.
26.
27.
28. 29.
30.
31.
32.
33.
International Approach, Vol. 66. New York: Marcel Dekker, 1993: 23–48. Lambert M-L, Hasker E, Van Deun A, et al. Recurrence in tuberculosis: relapse or reinfection? Lancet Infect Dis 2003;3:282–287. DiPerri G, Cruciani M, Danzi MH, et al. Nosocomial epidemic of active tuberculosis in HIV infected patients. Lancet 1989;2;1502–1504. Rieder HL, Cauthen GM, Comstock GW, et al. Epidemiology of tuberculosis in the United States. Epidemiol Rev 1989;11:79–98. Korenromp EL, Scano F, Williams BG, et al. Effects of human immunodeficiency virus infection on recurrence of tuberculosis after rifampin-based treatment: an analytical review. Clin Infect Dis 2003;37:101–112. Daley CL. Tuberculosis recurrence in Africa: true relapse or re-infection? Lancet 1993;342:756–757. Sonnenberg P, Murray J, Glynn JR, et al. HIV-1 and recurrence, relapse, and reinfection of tuberculosis after cure: a cohort study in South African mineworkers. Lancet 2001;358:1687–1693. National Tuberculosis Institute, Bangalore. Tuberculosis in a rural population of South India: a five-year epidemiological study. Bull World Health Organ 1974;51:473–488. Styblo K. Epidemiology of Tuberculosis, 2nd edn. The Hague: Royal Netherlands Tuberculosis Association, 1991: 1–136. Dye C. India’s leading role in tuberculosis epidemiology and control (editorial). Indian J Med Res 2006;123:481–484. 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.
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14
The natural history of tuberculosis infection and disease in children Ben J Marais and Peter R Donald
BACKGROUND During the twentieth century major advances occurred in the diagnosis and treatment of TB. Although the most important advance in the diagnosis of adult TB occurred with the detection of Mycobacterium tuberculosis by direct sputum microscopy, this offered little diagnostic value to children with paucibacillary disease. However, the tuberculin skin test (TST) allowed diagnosis of M. tuberculosis infection and more accurate TB diagnosis became possible as chest radiography became more widely available after the First World War. Therefore, from 1920 the tools for diagnosing M. tuberculosis infection and disease were widely available in developed countries, enabling them to document the evolution of disease in children. The first anti-TB drugs were introduced after the Second World War, and the most effective drug, isoniazid (INH), only became widely available in the early 1950s. Studies of childhood TB conducted during the period from 1920 to 1950 offer a picture of the natural history of TB in children, which can no longer be obtained. During this time, TB was still a major public health problem in many Western countries, and diseased children were frequently admitted to child sanatoria, where they were observed for prolonged periods of time. Numerous prospective observation studies were conducted, with meticulous long-term follow-up of patients providing detailed descriptions of disease presentation and progression. Active screening and prospective disease surveillance of household contacts documented primary infection (TST conversion) and progression to active disease. Two recent reviews summarized the results of these seminal prechemotherapy studies, conducted between 1920 and 1950.1, 2 They included all major studies published in the English literature; major studies were defined as those reporting on more than 1,000 children and with a follow-up period of more than 10 years.3–16 Three exceptions were made: 1. Avrid Wallgren’s findings were included as, although he reported on only 100 exposed children who became infected under close observation, he described the classic time-table of primary TB and based his conclusions on a lifetime of personal experience and careful observation.11–13 2. Tobias Gedde-Dahl included 3,138 children, but the study was interrupted after 8 years of follow-up due to the Second World War.6 The study’s contribution is important, because it provides the only accurate description of active community surveillance.
3. Edith Lincoln reported on 964 children, who were followed for up to 25 years and the study provided unique descriptions of the clinical signs and symptoms that develop during disease progression.15 The studies reviewed included both community and hospital-based cohorts, which reduces selection bias and ensures representation of the whole disease spectrum. The combined studies represent an impressive body of evidence that clarifies some important epidemiological concepts, although it fails to describe the influence of human immunodeficiency virus (HIV) infection.
CLINICAL EPIDEMIOLOGY Table 14.1 provides a brief description of the individual studies and Table 14.2 summarizes the key findings and major limitations.
EXPOSURE TO INFECTION The Mantoux TST, using 5 tuberculin units (TU) and an induration cut-off of 10 mm, was identified as the optimal way to diagnose M. tuberculosis infection.8,9 The induration induced by Bacillus Calmette–Gue´rin (BCG) vaccination was usually less than 10 mm, compared with the response after natural infection with M. tuberculosis, which usually exceeded 10 mm.7 BCG-induced delayed hypersensitivity responses diminished with time. The waning of the TST response varied depending on the timing of the BCG vaccination and the specific strain used. TST reversion occurred in less than 0.5% of children after natural M. tuberculosis infection, although temporary inhibition of TST responses did occur; this was mainly associated with severe malnutrition, viral illnesses, or extensive TB.6,8–10 The probability of infection was influenced by the infectivity as well as the proximity and duration of contact with the source case. Household exposure to a sputum smear-positive source case posed the greatest infection risk. Following prolonged household contact with a sputum smear-positive source case, 60–80% of children became infected.5,8–10 Household exposure to a sputum smearnegative source case posed a reduced, but still appreciable, risk; when the source case was smear-negative, 30–40% of children became infected.6,9,10 The majority of children who became infected did so within 3 months of symptom onset in the adult source case, but infection was frequently delayed in household contacts under 2 years of age if the primary caregiver was not the
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Table 14.1 Description of the original studies and individual study designs documenting the clinical epidemiology of childhood pulmonary tuberculosis Individual study reference
Time frame
Study type
Study population
Opie E, McPhedran FM, Putnam P (1935), Henry Phipps Institute, Philadelphia, PA, USA3
1907–1934, follow-up period of 1–27 years
Retrospective descriptive, outpatient based
Pope AS, Sartwell MD, Zacks D (1939), State Department of Public Health (Chadwick Clinics), Boston MA, USA4 Brailey M (1940), Johns Hopkins (Harriet Lane Clinic), Baltimore, MD, USA5
1924–1939, follow-up period of 5–15 years
Prospective TST survey, school based
Gedde-Dahl T (1951), Kinn District, Bergen, Norway6
1937–1944, follow-up period of 1–8 years
Retrospective descriptive, outpatient based Prospective TST survey, community based
Bentley FJ, Grzybowski S, Benjamin B (1954), High Wood Hospital for Children, Brentwood, Essex, UK7
1901–1952, notification data used
Davies PDB (1961), Brompton Hospital, London, UK8
1930–1954, follow-up period of 1–25 years
Miller FJW, Seal RME, Taylor MD (1963), Royal Victoria Infirmary, Newcastle upon Tyne Children’s Sanatorium, Stannington, Northumberland, UK9
1. 1947–1954 2. 1951–1961 Follow-up period of 1–10 years
Retrospective descriptive, outpatient based Retrospective descriptive, outpatient based
Zeidberg LD, Gass RS, Dillon A, et al. (1963), Tennessee Department of Public Health, USA10
1931–1955, follow-up period of 1–24 years
1928–1937, follow-up period of 1–10 years
Audit of TB notifications Mathematical deductions
Prospective community cohort study
Children < 15 years from 1,000 families with an adult source case Mortality data for the city of Philadelphia 400,330 school children 6–16 years TB notification data for Boston 1383 children < 15 years from 285 families with an adult source case Mortality data for Baltimore 6,739 people of all ages 3,138 children < 15 years
UK national TB notification data British MRC national TST survey
2377 children < 15 years in household contact with an adult source case 1. Children < 7 years from 1,000 families with an adult source case 2. 1,500 children < 5 years in household contact with an adult source case
1746 children < 15 years from 828 families with an adult source case Tennessee mortality data
Data collection methods
Adult source cases were selfselected Annual clinical follow-up of all source case contacts Annual CXR (if available) TST survey (Von Pirquet) CXR done if TST positive Annual CXR if the initial CXR was abnormal All children from tuberculous households screened Old tuberculin TST (0.1 or 1 mg) Annual CXR if TST positive Annual community-based TST survey (Von Pirquet) Documented TST conversion/ matriculation Annual CXR once TST positive Reviewed TB notifications for the whole UK and for London (1901–1952) Reviewed TST surveys D’Arcy Hart (1929), Prophit (1944), British MRC (1950) Included all asymptomatic household contacts Different TSTs were compared Annual CXR 1. 1,000 family study Household contacts < 7 years Annual CXR+TST (until positive) 2. Household contact study Household contacts < 5 years Annual CXR+TST (until positive) Voluntary inclusion of all household contacts Detailed questionnaire and physical examination Old tuberculin TST (0.1 or 1 mg), 6–12 monthly CXR
TST, tuberculin skin test; CXR, Chest radiograph; MRC, Medical Research Council. Adapted from Marais BJ, Gie RP, Schaaf HS et al. The clinical epidemiology of childhood pulmonary tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:278–285.
source case.5 This observation suggests that the limited social contact of very young children reduces their likelihood of becoming infected, unless the caregiver is the source case.5 Infection rates peaked during winter months, but it was uncertain whether this was due to the increased crowding and reduced ventilation brought about by the cold winter months, or whether viral upper respiratory tract infections may have increased the vulnerability of children to become infected.9 Similar findings were documented in the 1990s in the Western Cape Province of South Africa; although the exact reasons underlying the seasonal variation observed remains uncertain.17 The vast majority of very young children were infected by a household source case, although additional caregivers, especially
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grandparents or extended family members who took care of the children during the day, were also important.5,7,9 The majority of older children with a positive TST reported no household contact with source case, and were therefore likely to have been infected in the community.4,5,9,11 As it is a public health priority to identify and treat all sputum smear-positive cases as quickly as possible to limit disease transmission, prudent public health policy should encourage active case finding amongst household contacts of very young children (< 3 years of age) with proven infection or disease. This type of ‘reverse’ contact tracing can be expanded to children of all ages in low-prevalence areas, where household exposure remains the most likely source. These studies provide the scientific basis for current contact investigation practices, which focus on children less than
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Table 14.2 Summary of key findings and major limitations of the original studies, documenting the clinical epidemiology of childhood pulmonary tuberculosis Citation
Features
Key findings
Major limitations
Opie et al.3
First to focus on families and children
Pope et al.4
TST positivity school survey
Brailey5
Focus on children < 2 years of age. Racial comparison
GeddeDahl6
TST conversion community survey
Bentley et al.7
TB disease and mortality specified per age group. Concept of relative contribution
Davies8
Long-term follow-up. Persistence of TST conversion
Miller et al.9
Comprehensive literature review
Zeidberg et al.10
Long-term follow-up in the community
Sputum-positive exposure increased the frequency and severity of disease in childhood contacts The lifetime risk for the development of cavitating disease depended on the age at primary infection, increasing significantly after 10 years of age Only 30% of infected school children had household contact with a known source case Cavitating pulmonary TB was seen only in children older than 10 years of age Infected children < 2 years of age indicated an active household source case The majority of household contacts infected within 3 months of symptom onset in the adult source case No racial difference in infection following exposure Definite racial difference in disease and mortality following infection The rate of TB infection and TB-related mortality was increased in urban areas Radiological abnormalities were visible in 75% of children following primary TB infection The ARI was not constant; varied between different age groups The majority (90%) of TB-related radiological abnormalities not detected in routine clinical practice The risk of disease and death following infection was the highest during infancy The relative contribution of TB to age-specific all-cause mortality was the lowest during infancy TB contributed significantly to all-cause mortality throughout childhood The Mantoux skin test outperformed other TSTs A positive TST response persisted for > 20 years Exposure to a sputum smear-positive vs a smear-negative source case doubled the risk of infection Infection after exposure to a sputum smear-positive source case doubled the risk for disease and death Clarified the confusion surrounding different TST techniques and doses Described the importance of cultural influences and the extended family Viral respiratory infections might have contributed to the seasonal variation in TB infection Identified critical periods of risk for disease development (infancy, puberty) Documented a drastic reduction in TB-related disease and mortality over the 24-year study period in black patients
Tuberculin skin test not recorded Age groups poorly defined Limited CXR availability Sputum positivity not specified (smear or culture)
Tuberculin skin test conversion not recorded Selective follow-up (only those with initial chest radiograph abnormalities were followed) Documented cavitating disease only Public health entry point selected the poor Response to high dose (1 mg) tuberculin, used in a small minority of patients, is not specific for M. tuberculosis infection Sputum positivity not specified (smear or culture) Socioeconomic differences were not evaluated
Pre-school children were selectively represented (only contacts and symptomatic cases included) Results from this isolated community may be difficult to generalize Response to high-dose (100 TU) tuberculin, used in 24% of subjects in the British MRC survey, is not specific for M. tuberculosis infection Relied exclusively on TB notification data for disease and mortality analysis
Majority of patients were already infected at study entry Selected only asymptomatic children at study entry, in order to ensure clinical uniformity
Few deductions were made from own studies Validity of quoted studies were not evaluated Relied extensively on notification data
Age at primary infection was not documented Entry criteria were adapted during the study A controversial finding, not supported by mortality data or results from other studies reviewed, was the delayed progression of disease reported in children infected between 1 and 15 years of age
ARI, annual rate of infection; TST, tuberculin skin test; CXR, chest radiograph; MRC, Medical Research Council; TU, tuberculin units. Adapted from Marais et al.1
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5 years of age in most developing countries (although reverse contact tracing is not advocated) and all household contacts in most industrialized countries. The risk of infection following household exposure was reduced under good socioeconomic conditions, and in American studies there were no racial differences in the rate of infection following household exposure.5,7,10 An interesting observation rarely taken into consideration was that the annual risk of infection (ARI) was not constant across age groups; there were clearly identifiable periods when infection rates increased.5,7,9 These age periods seemed to correlate with times of widening social contact; with increased mobility after 2 years of age, with school entry at 5–7 years of age, and with school exit at 15–20 years of age.7,9 The age-specific infection rate was found to be the single most important public health indicator of disease prevalence and active transmission within a given community.7,9,10
INFECTION TO DISEASE MORBIDITY With active contact tracing and regular radiographic screening, suggestive chest radiograph changes were noted in 50–60% of children following TST conversion.1,4,6–8 When comparing observations from prospective surveillance with routine notification data, it was calculated that only 5–10% of children in whom chest radiograph abnormalities were expected were ultimately notified. This implied that the majority of radiographic signs were transient and passed undetected in routine clinical practice.5,7 Nearly all disease manifestations developed within the first year following primary infection, identifying the first year following exposure as the time period of greatest risk.1,3–5,7 Specific time- and age-related disease patterns are discussed under ‘Clinical Disease Manifestations’.
MORTALITY The highest risk for TB-related mortality following primary infection (5–10%) occurred during infancy.3,5–7,9,10 This risk declined to 1% between 1 and 4 years of age, with the lowest levels (< 0.5%) maintained in the age group 5–14 years, before rising to more than 2% after 15 years of age.7,9 In children less than 10 years of age the majority of deaths occurred within the first year following primary infection, but, in older children who frequently developed adult-type cavitary disease, mortality lagged further behind.3,5–7,9,10 The relative TB-related mortality best describes the impact of TB on all-cause mortality within a specific age group.7 TB contributed significantly to all-cause mortality in all age groups, except in infancy.5,7 This contradiction is explained by the relatively small number of infants infected and the high rate of mortality from other causes.5,7,10 Although the relative contribution of TB to all-cause mortality is low in infancy, this does not detract from the important observation that, if infected, infants are at high risk of severe disease and death. Black children suffered double the TB-related mortality and four times the all-cause mortality compared with white children, but in the Tennessee study the TB-related mortality in black children declined by 80% within 20 years.5,9,10 In addition, a 10-fold decline in TB-related mortality occurred in Britain between 1900 and 1950, without a comparable decrease in TB infection.7,9 These dramatic declines in TB-related disease and mortality, documented within a single generation and without a comparable decrease in infection, emphasize the considerable influence of socioeconomic
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improvement.7,10 The nature versus nurture issue is complex, but the findings from these old studies suggest that improvement in the environment, rather than genetic selection, probably contributed most to the dramatic reduction in TB prevalence witnessed in the developed world during the twentieth century.18 Notification data often provide unreliable information due to under- or over-reporting of disease and inaccurate cause of death identification. In order to gain a better understanding of the crucial transition from infection to disease, it is important to look at studies that followed exposed children who became infected and/or diseased longitudinally and that described the clinical disease manifestations that occurred in sufficient detail.
CLINICAL DISEASE MANIFESTATIONS The optimal way to define risk and to describe exact disease entities is by prospectively following a cohort of children with recent primary infection for subsequent disease development and death. A number of studies from the pre-chemotherapy era achieved this. A brief description of each individual study is provided in Table 14.3. In order to facilitate the comparison and combination of results from different studies, a uniform template was developed to classify disease according to the disease descriptions and concepts of pathology derived from the pre-chemotherapy literature (Table 14.4). It is important to note that more than one disease entity may coexist at the same time or develop during the course of disease. Table 14.5 summarizes the key findings and major limitations.
‘TIME-TABLE OF CHILDHOOD TB’ Avrid Wallgren summarized the sequence of pathology following primary M. tuberculosis infection as the so-called ‘time-table of childhood TB’.11 Pulmonary infection occurs when a few TB bacilli, contained in a small infectious aerosol droplet, reaches a terminal airway and succeeds in establishing infection. A localized inflammatory process occurs within the lung and this is called the primary (Ghon) focus. From the Ghon focus, bacilli drain via lymphatics to the regional lymph nodes. The Ghon focus with associated tuberculous lymphangitis and involvement of the regional lymph nodes is called the primary (Ghon) complex. From the regional lymph nodes bacilli enter the systemic circulation directly or via the lymphatic duct. This occult haematogenous spread occurs during the incubation period, before adequate immune responses contain the disease. After dissemination, bacilli may survive in target organs for prolonged periods. The future course of the disease at each of these sites depends on the dynamic balance between host immunity and the pathogen.19
Phase 1 occurred 3–8 weeks after primary infection.11,13 The end of the initial asymptomatic incubation period was heralded clinically by hypersensitivity reactions such as initial fever, erythema nodosum, a positive TST response, and formation of the primary complex visible on chest radiograph.11,13 Phase 2 occurred 1–3 months after primary infection.13 This period followed the occult haematogenous spread that occurred during incubation, and represented the period of highest risk for the development of tuberculous meningitis (TBM) and disseminated (miliary) TB in young children.11,13 However, these disease entities may occur after any time interval; haematogenous dissemination frequently acted as
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14
Table 14.3 Description of original studies and individual study designs documenting the natural history of childhood intrathoracic tuberculosis Individual study reference
Time frame
Study type
Study population
Data collection methods
Wallgren A (1935, 1938, 1948), Children’s Hospital, Gothenburg and the Karolinska Medical Institute, Stockholm, Sweden11–13
1930-1950, followup period not specified
Prospective descriptive, hospital based Personal experience
100 newly infected children. All children with TB seen on referral
Brailey M (1940), Johns Hopkins (Harriet Lane Clinic), Baltimore, MD, USA14
1928-1937, followup period of 1–10 years
Retrospective descriptive, outpatient based
285 families with 1,383 children < 15 years 40% white 60% black
Gedde-Dahl T (1951), Kinn District, Bergen, Norway6
1937-1944, followup period of 1– 8 years
Prospective TST survey, community based
6,739 people 3,138 children < 15 years
Bentley FJ, Grzbowski S, Benjamin B (1954), High Wood Hospital for children, Brentwood, Essex, UK7
1942–1952, followup period of 5–10 years
Davies PDB (1961), Brompton Hospital, London, UK8
1930–1954, followup period of 2–25 years
Retrospective descriptive, outpatient based
Lincoln EM, Sewell EM (1963), Bellevue Hospital, New York City, NY, USA15
1930–1960, followup period of 10–25 years
Prospective descriptive, hospital based
Sanatorium patients 954 < 15 years 50% white 25% black 25% Puerto Rican
Miller FJW, Seal RME, Taylor MD (1963), Royal Victoria Infirmary, Newcastle upon Tyne and Children’s Sanatorium at Stannington, Northumberland, UK16
1. 1941–1951 2. 1951–1961 Follow-up period of 1–10 years
1. Children < 7 years from 1,000 families with an adult source case 2. 1,500 children < 5 years in household contact with an adult source case
1. Sanatorium patients 1,049 children < 16 years 2. Death investigation 100 consecutive TB deaths notified in children 2,377 children < 15 years
Retrospective descriptive, hospital based Literature review
Retrospective descriptive, outpatient based Literature review
Children observed after household exposure Meticulous documentation of signs/ symptoms following infection In addition, Wallgren drew from vast personal experience All children from tuberculous households TST (old tuberculin at 0.1 or 1 mg) Annual CXR if TST positive Annual community-based TST survey (Von Pirquet) Documented TST conversion Annual CXR if TST positive Successive referrals admitted over a 10-year period Observation and CXR in hospital Review CXR 5–10 years after hospital discharge
All asymptomatic household contacts Different TSTs compared Annual CXR 70% follow-up achieved Children referred with evidence of recent (uncalcified) TB Observation and CXR in hospital Hospitalized for extended periods with careful documentation of disease progression Annual CXR after discharge 90% follow-up achieved 1. 1,000 family study Household contacts < 7 years Annual CXR+TST (until positive) 99 TST converted 2. Household contact study Household contacts < 5 years Annual CXR+TST (until positive) 72 TST converted
TST, tuberculin skin test; CXR, chest radiograph; TB, tuberculosis. Adapted from Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis – a critical review of the literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402.
the final terminal pathway following uncontrolled disease progression in the absence of treatment.15,16 Phase 3 occurred 3–7 months after primary infection.13 This was the period of secondary airway involvement due to diseased lymph nodes in children less than 5 years of age, while large reactive pleural effusions also occurred during this time period, but mostly in children older than 5 years of age.13,15,16 Phase 4 lasted until the primary complex was calcified, 1– 3 years after primary infection, and represented the period of osteoarticular TB in children under 5 years of age, and adulttype disease in adolescents.12–19 As a general rule the risk of disease progression following primary infection had passed by the time calcification appeared.14–19 However, in adolescent
children adult-type disease sometimes had a delayed clinical onset, occurring after the appearance of calcification.8,16 Phase 5 occurred after calcification was complete, more than 3 years after primary infection. By this time the highest risk period has passed, although some of the very late manifestations of TB, including pulmonary reactivation, were rarely observed.7,16
The features of the classic time-table first described by Wallgren were later confirmed and expanded by observations from other studies. It is important to point out that the timeline reflected in Fig. 14.1 summarizes common clinical patterns of disease and does not represent dogmatic rules regarding the course of TB in children; also that the vast majority (> 90%) of disease manifestations occurred in the first 6–12 months following primary infection.7,8,16
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Table 14.4 Disease classification of childhood intrathoracic tuberculosis used as template Pulmonary infection Pulmonary disease
Tuberculosis infection uncomplicated by clinical symptoms (other than self-limiting, viral-like illness) or radiological abnormalities (other than the primary complex). The primary complex includes the Ghon focus with associated TB lymphangitis and affected regional lymph nodes. Tuberculosis infection complicated by marked clinical symptoms or additional radiological abnormalities apart from the primary complex. Pulmonary disease includes a diverse spectrum of pathology, described as separate disease entities.
Separate disease entities Ghon focus with/without cavitation Lymph node disease Complicated by airway involvement
—With obstruction —With collapse/ hyperinflation —With allergic consolidation —With bronchopneumonic consolidation —With caseating consolidation Pleural disease
—With effusion —With empyema Adult-type disease
Haematogenous spread
—With disseminated (miliary) disease
Progressive parenchymal caseation surrounding the Ghon focus represents poor organism containment. The caseated area may discharge into a bronchus, resulting in the formation of a cavity with possible endobronchial spread. Regional lymph node enlargement forms part of the primary complex, but the presence of marked clinical symptoms differentiates lymph node disease from pulmonary infection. With pulmonary infection, affected regional lymph nodes attach to the bronchus, but rarely progress to clinical or radiological disease. If disease progression follows this ‘lympho-bronchial involvement’, the affected bronchus may become partially or totally obstructed as a result of nodal compression, inflammatory oedema, polyps, granulomatous tissue, or caseous material extruded from ulcerated lymph nodes. Parenchymal disease may result from aspiration of caseous material. Variation in the degree of airway obstruction, dose and virulence of the bacilli aspirated and the immune status of the host determines the degree of pathology. Airway obstruction occurs because of enlarged matted nodes encircling and compressing an airway, together with associated inflammation or additional processes described above. Complete airway obstruction leads to resorption of distal air and collapse, while partial airway obstruction may cause a ball-valve effect with hyperinflation of the segment or lobe supplied. Nodal perforation into an airway with endobronchial aspiration of allergic products causes an acute hypersensitivity response (epituberculosis) with dense consolidation. Nodal perforation into an airway with endobronchial aspiration of live bacilli causes local areas of caseation surrounding the airways, resulting in patchy consolidation. Nodal airway obstruction with perforation and endobronchial aspiration of live bacilli causes extensive parenchymal caseation, resulting in dense expansile consolidation of the affected lobe. Pleural involvement occurs after direct spread of bacilli from a subpleural parenchymal or lymph node focus, or from haematogenous spread. Variation in the dose and virulence of bacilli that enter the pleural space and the immune status of the host determines the degree of pathology. The presence of caseous material in the pleural space triggers a hypersensitivity inflammatory response with the accumulation of serous straw-coloured fluid, containing few TB bacilli. Active caseation in the pleural space causes thick loculated pus, containing many TB bacilli. Excessive local containment may cause parenchymal destruction and resultant cavity formation. Tubercle bacilli flourish in these cavities from where they disseminate to other parts of the lung via endobronchial spread. Endobronchial spread occurs directly from these infected cavities and is not dependent on lympho-bronchial breakthrough, as with bronchial disease. TB bacilli may enter the blood stream via pulmonary lymphatic drainage, from affected regional lymph nodes or directly from the parenchymal focus. Haematogenous spread is a condition of infinite gradation, depending on the frequency, dose, and virulence of the bacilli released as well as host immunity. During occult spread bacilli are seeded into susceptible organs, while the child remains asymptomatic. Following invasion of the blood stream, TB bacilli lodge in small capillaries, where they may progress to form tubercles, visible on chest radiograph as typical even-sized, miliary lesions (< 2 mm) or atypical lesions of differing size.
Adapted from Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis – a critical review of the literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402.
INTRATHORACIC MANIFESTATIONS PULMONARY INFECTION Pulmonary infection was usually identified after active contact tracing or presentation of children by anxious parents.7,15,16 Pulmonary infection was associated with TST conversion and non-specific, self-limiting, viral-like, respiratory symptoms.11–13,16 Enlarged regional lymph nodes on chest radiograph were the hallmark of pulmonary infection with or without a visible Ghon focus.6,7,15,16 Following primary infection, 50–70% of children showed these radiological signs, irrespective of symptoms.6,8,16 Good quality anteroposterior and lateral radiographic views were required for optimal visualization of enlarged regional lymph nodes.15,16 The Ghon focus showed no predilection for any specific part of the lung.7,15,16 The regional lymph
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nodes affected depended on the anatomical location of the Ghon focus; a Ghon focus in the apex of the lung affected the ipsilateral paratracheal nodes,7,16 a Ghon focus in other parts of the right lung caused right-sided hilar adenopathy, and a Ghon focus in the left lung usually caused bilateral hilar adenopathy.7,16 Paratracheal nodes reflected a Ghon focus in the apex, spread from hilar glands, or haematogenous spread.7 It occurred more frequently in children under 2 years of age and showed a consistent trend to increased haematogenous spread, even with correction for age.7 During the first 3–4 months the lymph nodes were in a phase of ‘full activity’, vaguely defined, cloudy and homogeneous.7 Over the following months the radiological signs of ‘activity’ regressed, the shadow became denser and better defined.7 With serial radiographs, 40% of lesions cleared within 6 months, a further 30% within 1 year, and the remaining 30% persisted up to 4 years.7,16 Calcification
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The natural history of tuberculosis infection and disease in children
Table 14.5 Summary of key findings and major limitations of the original studies that documented the natural history of childhood intrathoracic tuberculosis Citation
Age groups
Unique feature
Key findings
Major limitations
Wallgren11–13
2 groups: < 3, 3–14 years
Meticulous observation Detailed description of symptoms and signs following primary infection Relevant age groups Racial differences
TST conversion in the community
Brailey14
5 groups: < 1, 1–2, 2–4, 5–9, 10–14 years
3 groups: < 5, 5–9, 10–14 years
4 groups: < 1, 1–4, 5–9, 10–15 years
4 groups: < 1, 1–4, 5–9, 10–14 years
Lincoln et al.15
4 groups: < 1, 1–4, 5–9, 10–14 years
Miller et al.16
5 groups: < 1, 1–2, 2–4, 5–9, 10–14 years
GeddeDahl6
Bentley et al.7
Davies8
First dedicated childhood TB study in UK
UK study with longest follow-up period
Detailed description of disease progression Relevant age groups Comprehensive literature review
Age at primary infection and time since infection were major determinants of risk for disease development. It also influenced the type of disease manifestation Host immunity was influenced by age and considered to be of crucial importance Documented the time-table of disease In all children < 2 years and in black children < 5 years segmental lung lesions predominated Black children suffered increased morbidity and mortality Enlarged nodes were visible on CXR in the vast majority of recently infected children All CXR changes apart from cavitation and calcification seen within 1 year after infection Described the slow rate of radiological regression with lymph adenopathy Suggested a focus on high-risk groups: < 2 years and > 10 years of age Progression of disease documented even after calcification became visible Risk of cavitating disease dependent on age at primary infection (> 10 years) Documented disease progression together with the signs, symptoms and outcome associated with specific disease entities
Informative illustrations of lymph drainage and TB lung pathology Re-emphasized high-risk groups following primary infection (< 2 years and adolescents) Cavitary disease may follow primary infection, reinfection or reactivation
Study methodology not specified Observations illustrated with case studies Guidelines provided very dogmatic
Public health entry point selected the poor Type of segmental lesion not specified Socioeconomic differences not evaluated Pre-school children were poorly and selectively represented Isolated community Excessive pre-selection occurred due to referral and long waiting periods Disease progression was not well documented Selected only asymptomatic children at study entry, to ensure clinical unity Majority of children were already infected at study entry Study inclusion was selective (symptomatic children with CXR evidence of recent infection) Limited racial subanalysis Cavitation with visible calcification was accepted as proof of reactivation. Validity of studies quoted were not evaluated
TST, tuberculin skin test; CXR, chest radiograph. Adapted from Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis – a critical review of the literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402.
0 0 1 Infection
I
II 2
III 3 4 Months
IV 6
8 Time
10
12
V 2
Years
3
4
13
Fig. 14.1 Phase of disease, adapted from the time-table of TB described by Wallgren.13
Phase of disease adapted from the time-table of tuberculosis described by Wallgren 0 Incubation phase I Hypersensitivity phase II Phase of miliary tuberculosis and tuberculous meningitis II Phase of segmental lesions in children < 5 years and pleural effusion in those > 5 years IV Phase of osteo-articular tuberculosis in children < 5 years and adult-type disease in those >10 years V Phase of late manifestations including pulmonary re-activation Not all these disease manifestations (phases) are equally common and while hypersensitivity is a nearly universal phenomenon following primary infection, the late manifestations are extremely rare. Table 6 provide an indication of how common the most important of these disease manifestations are in specific age groups. The vast majority of complications occur in the first 3 12 months following primary infection.
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Table 14.6 Age-specific risk for disease development following primary infection
50
Age at primary infection (years)
Risk of disease following primary infection in immune-competent children (dominant disease entity indicated in brackets)
40
<1
No disease 50% Pulmonary disease (Ghon focus and/or lymph node) 30–40% TBM or miliary disease 10–20% No disease 70–80% Pulmonary disease (Ghon focus and/or lymph node) 10–20% TBM or miliary disease 2–5% No disease 95% Pulmonary disease (lymph node) 5% TBM or miliary disease 0.5% No disease 98% Pulmonary disease (lymph node, pleural effusion or adult-type) 2% TBM or miliary disease < 0.5% No disease 80–90% Pulmonary disease (pleural effusion or adult-type) 10–20% TBM or miliary disease < 0.5%
5–10
> 10
TBM, tuberculous meningitis. Adapted from Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis – a critical review of the literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402.
developed in 20–50% of infected children with visible lymph node involvement.7,8,16 Calcification usually occurred between 12 and 24 months, but was sometimes delayed up to 4 years after primary infection.7,16 Calcification in young children tended to be more extensive and developed earlier (within 6–12 months) than in older children.7,16 In general, calcification was an indication of clinical quiescence, but not a guarantee thereof.15,16 The disappearance of calcification was rare and attributed to either resorption or bronchial escape of a pneumolith.16 The prognosis of pulmonary infection was generally favourable and the risk depended mainly on the age at the time of primary infection (Table 14.6).11–16
PULMONARY DISEASE Host immunity was considered to be the major determinant of risk for disease development following infection.11 Infants with immature immune systems were at highest risk, 11–16 with pulmonary disease developing in 30–40% and TBM and/or disseminated miliary disease in 10–20% (Fig. 14.2).14–16 The risk decreased considerably in the second year of life, but stayed significant with 10–20% of infected children developing pulmonary disease and a further 2–5% TBM or miliary disease.15,16 The risk decreased to less significant levels in the age group 2–5 years, before reaching its lowest level at 5–10 years of age.14–16 Lymph node disease with/without airway involvement predominated in children under 5 years.7,8,15,16 Disease occurred less frequently in children aged 5–10 years; pleural effusion became more common throughout this period,16–19 but lymph node disease was mainly restricted to the younger group and adulttype disease to the older group (> 7–8 years of age).15,16 Black children had an increased risk of developing complicated lymph node disease across all age groups.14 Primary infection during adolescence
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Disseminated
30 20 10 0
<1
1 to 2
2 to 5 Age in years
5 to 10
10 to 15
Fig. 14.2 Age-related risk of progression to active disease following primary M. tuberculosis infection.
Frequency of disease manifestation
2–5
PTB
%
1–2
Age-related risk
(1) 0
1
(2) 2
(3) 4
6
8 10 Age in years
(4) 12
14
16
(1) Complicated Ghon focus and/or disseminated disease (2) Uncomplicated Ghon focus and/or lymph node disease Complicated lymph node disease (3) Pleural effusion (4) Adult-type disease Adapted from Marais BJ et al.
Fig. 14.3 Age-related manifestations of intrathoracic TB in immunecompetent children.
(> 10 years of age) was associated with a high risk (10–20%) of developing adult-type disease.7–9,11–16 A summary of the age-related disease patterns described are demonstrated in Fig. 14.3.
GHON FOCUS WITH/WITHOUT CAVITATION A Ghon focus complicated by cavitation was rare. It occurred predominantly in infancy.14–16 Clinical symptoms of Ghon focus cavitation included weight loss, fatigue, fever, and chronic cough, and in these immune immature infants the underlying mechanism is probably poor disease containment.15,16 In those with cavitation of the Ghon focus, disease quickly progressed to death in the majority of cases and the few who survived the initial illness ultimately died from TB or TB-associated complications.15,16 Cavitation following primary infection was not uncommon during adolescence,7–9,12,15,16 but in this age group the parenchymal breakdown probably reflects excessive rather than poor disease containment;19 this is discussed under ‘Adult-Type Disease’.
CHAPTER
The natural history of tuberculosis infection and disease in children
LYMPH NODE DISEASE Enlarged regional lymph nodes, visible on chest radiograph, usually caused symptoms associated with airway inflammation and/or obstruction, but massive caseating nodes were often associated with persistent fever and excessive weight loss.16 The subcarinal nodes were most commonly involved, but any of the hilar or paratracheal group of nodes may be involved.16
Complicated by airway involvement Airway involvement was indicated by different degrees of obstruction and/or secondary parenchymal disease. Airway involvement occurred predominantly in children under 5 years of age and it occurred more frequently in younger children and boys, 7,8,15,16 as well as black children.14 The airways most frequently affected were those supplying the right upper lobe (anterior segment), the right middle lobe, and the left upper lobe.16–19 The airways most frequently affected in combination were those of the right middle and lower lobes, indicating involvement of the bronchus intermedius.16–19 Rebound enlargement of segmental lesions, attributed to immune reconstitution, were described after cessation of high-dose steroid treatment.19 The spectrum of airway pathology demonstrated on bronchoscopy included the following: no visible involvement, obstruction from external compression, endobronchial breakthrough with caseous drainage, granulation tissue with polyps, and fistula formation.7,15 A very rare sequel was the expectoration of a pneumolith after perforation of a calcified lymph node into an airway.15,16 Symptoms varied according to the degree of airway irritation and obstruction. Infants frequently developed a persistent cough, sometimes mimicking pertussis.15,16 With disease progression the cough became more prominent, often brassy or bi-tonal with associated large airway wheeze or stridor.11,15,16 With collapse/hyperinflation Collapse and hyperinflation were mostly a radiological diagnosis with minimal clinical symptoms unless large lung segments collapsed, or hyperinflation caused symptoms related to pressure on surrounding structures.7,16 With ‘allergic’ consolidation (epituberculosis) This represented a type of hypersensitivity response to dead TB organisms and TB antigens that were aspirated (or in experimental animal studies, deposited) into a particular part of the lung. The onset of symptoms could be dramatic, with a high fever, acute respiratory symptoms, and signs of consolidation.15 Chest radiography revealed a densely consolidated segment or lobe with minimal volume change.15,16 Consolidation resolved completely within months with no permanent sequellae.15 With bronchopneumonic consolidation Bronchopneumonic consolidation was rare. Symptoms were not well described, but depended on the extent of involvement. On chest radiograph patchy infiltration usually involved more than one lobe of a single lung, reflecting airway spread.7 Bilateral patchy infiltration was mostly due to protracted disease or due to haematogenous spread with an atypical slowly progressive course.7 With caseating consolidation Children with caseating consolidation were ill, with a high undulating fever, chronic cough, and sometimes even haemoptysis.15 On chest radiograph dense lobar consolidation was visible, usually with volume increase (expansile), with or without areas of breakdown. Mycobacterium tuberculosis cultures were positive in more than 80% of cases.7,15,16
14
Secondary bacterial infection often complicated the picture.7,16 Bronchoscopy showed total airway obstruction and surgical re-establishment of airway patency together with penicillin gave dramatic symptomatic relief.15,16 Following resolution of the consolidation, non-collapsing parenchymal bullae appeared with extensive fibrotic scarring in the surrounding lung tissue.7,15 Without intervention the prognosis was poor, with frequent haematogenous spread terminating in disseminated (miliary) TB and/or TBM.7,15 If resolution occurred, the end result of bronchopneumonic or caseating consolidation was a contracted, fibrotic area and contracted segments were often impossible to visualize on later chest radiographs.7,15 Bronchiectasis was a common sequel to peribronchial caseation.7,8,15,16 Airway damage ranged from saccular bronchiectasis to bronchial stenosis.15,16 Most children with bronchiectasis remained asymptomatic on long-term follow-up.7,8,15,16 Apical lesions hardly ever caused complications, but large basal bronchiectatic lesions did predispose to future suppurative disease.7,8,15,16 Surgery was indicated only when a bronchiectatic lobe caused recurrent symptomatic disease.15,16 Very rarely, massive fibrosis and contraction of a whole lung (chronic fibroid lung) caused mediastinal shift and also resulted in severe scoliosis.7,15
PLEURAL DISEASE With effusion Localized pleurisy overlying a peripheral Ghon focus was common.15 Limited adhesions developed between the visceral and parietal pleura, but this did not cause symptoms or lung function abnormality.15 Effusions were rare in children under 5 years of age and most common in adolescent boys.7,8,15,16 A seasonal variation was observed with the lowest incidence during late summer and autumn, accounted for by reduced primary infection in the preceding summer months.7 Pleural effusion had a characteristic clinical course, starting with an acute pleuritic pain in the chest, accompanied by a high fever in the absence of acute illness, an ill-defined loss of vigour, and a dry cough.7,8,15,16 The TST was usually highly reactive.7,15 On chest radiography the size varied from small effusions obliterating only the costophrenic angle to massive fluid collections that caused opacification of a whole lung with mediastinal shift to the opposite side.7,15 In most cases, a third to a half of the lung was obliterated with a clear meniscus sign.7 With fluid in the pleural space it is nearly impossible to exclude lesions in the underlying lung.7,15 Localized interlobular effusions required radiological differentiation from segmental lesions.16 A unilateral effusion, ipsilateral to the parenchymal focus, indicated direct spread to the pleural space from a subpleural focus. Bilateral effusions indicated haematogenous spread or bilateral primary foci.15,16 Pleural fluid was a straw-coloured exudate with high protein content and lymphocyte predominance, 15,16 although the amount of polymorphonuclear cells depended on the acuteness of onset.16 Direct microscopy was negative, but culture yields were as high as 70% with immediate inoculation.15 In children the prognosis of effusion was generally good. The high fever showed gradual defervescence over 3–4 weeks,7,15 while the fluid collection resolved more slowly over 3–6 months.7,16 Obliteration of the costophrenic angle and slight pleural thickening remained permanently.7,15,16 The main complication described was future adult-type disease, which was not a complication of the effusion per se, but reflected the risk associated with primary infection at an older age.7,8,15,16 Rarely, extensive pleural fibrosis caused contraction of the affected hemithorax with scoliosis.7,14 Bilateral
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effusions were associated with a higher risk for haematogenous spread and future adult-type disease.7,15,16
With empyema The presence of caseating empyema was indicated by a persistent high swinging fever and a loculated pleural collection on chest radiograph.16 Aspiration was difficult because of thick pus. Tubercle bacilli were usually visible on microscopy.16 Caseating empyema was rare and the prognosis was variable with slow disease progression and death or slow resolution with pleural calcification and fibrosis.16 ADULT-TYPE DISEASE Adult-type disease resulted after primary infection, endogenous reactivation, or exogenous reinfection.16 All these processes operated in the same community at the same time.15 Adult-type disease was most common after recent primary infection in children over 10 years of age.7,8,12,14–16 The interval from primary infection to adult-type disease was widely variable (3 months to 20 years) and depended mostly on the child’s age at the time of primary infection.7,8,15,16 The shortest time intervals and highest risk followed primary infection during adolescence, especially in girls of perimenarchal age.15,16 Disease started off with minimal symptoms such as cough, loss of appetite, and fatigue.15 With disease progression typical TB symptoms of chronic cough, chest pain, lethargy, anorexia, and weight loss became evident.7,8,15,16 Children with advanced disease became anaemic, and developed an oscillating fever and haemoptysis.15 A frequent complaint, even in the absence of fever, was night sweats.15 On chest radiograph an initial rounded homogeneous shadow, 2–3 cm in diameter situated in the vicinity of the clavicle was typical, followed by parenchymal breakdown and cavity formation.15,16 Cavities did not contain a fluid level and were characteristically surrounded by inflammation.16 Bilateral disease was common, mainly involving the apical segments of the upper lobes; lower lobe involvement was far less frequent.16 Previous radiographic appearances were non-predictive and highly variable, ranging from no visible abnormality detected to a densely calcified primary complex.7 A correlation existed between those lesions that represented primary infection in the older age group, such as pleural effusion, and adult-type disease.7,8,15,16 The prognosis of adult-type disease was poor, with 50–60% mortality within 5–10 years.7,8,15,16 These children were sputum smear-positive and could transmit infection.
HAEMATOGENOUS SPREAD During incubation and occult spread, bacilli seed to susceptible organs, especially the spleen, bone, kidney, and cerebral cortex and possibly to the apices of the lungs (Simon foci).11,15,16 The age at the time of infection and the time since infection were the major determinants of risk for the development of ‘metastatic’ disease. Infection under 2 years of age carried a significant risk of serious disease, even if the radiograph was considered normal.7,16 TBM was present in over 30% of children who presented with TB before 2 years of age.7,16 The risk of TBM after 3 years of age was extremely low and those who did develop TBM had significant preceding symptoms.15,16
With disseminated (miliary) disease Infants were most vulnerable to develop disseminated (miliary) disease.11,15,16 The symptoms included prolonged pyrexia, lassitude, anorexia, and weight loss.7,15,16 Children appeared acutely ill with minimal physical signs apart from tachypnoea and
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hepatosplenomegaly.7,15 Radiological mottling followed 7–21 days after febrile onset, starting as barely visible nodules that slowly progressed to large poorly defined patches.15 The initial miliary lesions were often difficult to visualize with 30–40% of autopsy-proven miliary lesions missed on chest radiograph before death.6 Bone marrow biopsy and ophthalmoscopy were useful diagnostic aids.7 The reported presence of choroidal tubercles varied widely, from 13% to more than 50%.7,8,15,16 The majority of children were TST-positive and, in those children with an initial negative TST, skin test conversion occurred within 1–4 months when effective treatment became available.15 The prognosis of disseminated (miliary) disease was poor. Clinical progression with persistent fever increased irritability and weight loss frequently terminated in TBM.15,16 The majority died within 6 months, but chronic forms were occasionally seen where children eventually died from ‘toxaemia’, malnutrition, or amyloidosis.15 Typical even-sized miliary mottling pointed to an acute invasion of the blood stream. Protracted release of bacilli from a chronic focus, e.g. a matted lymph node mass or rarely a skeletal lesion, also occured.15 The symptoms of protracted seeding were similar to acute invasion, but were initially more intermittent, presumably corresponding to periods of bacilli or toxic product release.15 This repetitive seeding was sometimes manifested by successive crops of papulonecrotic tuberculids (skin manifestation).15 Chest radiograph images revealed large hilar lymph node masses with mottling of variable size, and eventually progression did occur with either acute or chronic deterioration.15,16
PRINCIPLES OF DISEASE DYNAMIC BALANCE The clinical manifestation of an infectious disease depends on the balance that exists between the pathogenicity of the organism and the immune competence of the host. In tuberculosis, an important dynamic dimension is added to this balance, because initial organism containment rarely ensures organism eradication.19,20 Persistence of dormant bacilli inside sequestered foci (latent infection) provides an ‘ever-present’ risk of reactivation whenever the balance shifts in favour of the organism. In addition to the risk of reactivation following previous primary infection, high levels of transmission implies an additional risk of reinfection in highburden settings.21,22 The delicate and dynamic balance established between the pathogen and the host may be influenced by numerous variables (Fig. 14.4). Pathogen
Host immunity Dynamic balance
Pathogen Infecting dose (limited) Virulence Persistence (Preferential growth in lung apices)
Host immunity Innate immunity including local defences Acquired immunity (Pronounced lymphadenopathy <5 years) (Excessive tissue necrosis >10 years)
*Adapted from Marais BJ et al. Diversity of disease manifestations in childhood pulmonary tuberculosis19
Fig. 14.4 Factors underlying different manifestations of pulmonary TB. Adapted from Marais BJ, Donald PR, Gie RP, et al. Diversity of disease manifestations in childhood pulmonary tuberculosis. Ann Trop Paediatr 2005;25:79–86.
CHAPTER
The natural history of tuberculosis infection and disease in children
THE PATHOGEN Variables related to the pathogen include the number of organisms inhaled (size of the infecting dose), the virulence of these organisms, and their ability to resist eradication (persistence). With M. tuberculosis infection, variation in the infecting dose seems negligible. Lung deposition studies indicate that only the tiniest aerosol droplets, containing fewer than five bacilli, are likely to reach the terminal airways and establish a pulmonary focus of infection.23–25 Larger droplets are either not inhaled (since they are less likely to remain suspended in the air), or they are deposited in the proximal airways where infection is effectively resisted.24,25 Epidemiological evidence and experimental evidence from laboratory animals suggest that the intensity of exposure influences the risk of both infection and disease.26,27 It is well established that variables such as the duration of exposure and the number of infectious particles in the ambient air influence the risk of infection. However, natural infection in humans seems a fairly uniform event with little variation in the infecting dose, being ascribed to a single aerosol droplet containing fewer than five bacilli. Other variables, including multiple infections or increased virulence of the infecting organisms, may explain the increased tendency to progress to disease following high-intensity exposure, such as household exposure to a sputum smear-positive source case. Primary infection is usually visualized as a single parenchymal (Ghon) focus,2 but exposure to multiple infective doses may increase the likelihood that a single infective dose is ultimately successful in establishing infection and/or disease. Variation in the virulence of the infecting organisms may offer another explanation, as the phenotypic virulence may differ according to the sputum smear status of the source case, while the proximity of the source case will determine the influence of environmental factors such as exposure to ultraviolet irradiation or drying.24,25 Genetic variation in organism virulence is well documented, 28,29 but it is unlikely that strain differences can explain the consistent epidemiological finding that household contacts of sputum smear-positive source cases are more likely to progress to disease following infection. Persistence describes the ability of the pathogen to evade eradication and to remain dormant for extended periods of time. Several mechanisms which may enable M. tuberculosis to persist both intracellularly (inside macrophages) and extracellularly (inside the caseous centres of granulomas) have been described.30,31 The presence of dormant M. tuberculosis bacilli is not harmful to the host unless circumstances favour their reactivation, which explains why progressive immune compromise poses such a high risk of reactivation disease.
HOST IMMUNITY Total host immunity includes a combination of both innate and acquired immune responses, together with local pulmonary defences, which seem more important than was previously appreciated. Compromised organism clearance, decreased mucosal immunity, and a favourable microenvironment at the point of organism deposition may all contribute to an increased risk of infection and disease. The importance of these local pulmonary influences is demonstrated by the comorbidity that exists between pulmonary TB and silicosis,32 or tobacco smoking.33,34 The protection provided by the innate immune response seems limited, because bacilli grow unrestrained within naı¨ve macrophages and occult haematological dissemination occurs frequently during
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the first 4–6 weeks, following primary infection.11,19,35 Acquired cellular immunity is of crucial importance to contain the organism and prevent uncontrolled disease progression.11,19,35 In the absence of immune compromise, age is the dominant variable that determines the effectiveness of acquired cellular immunity to contain the organism. This is illustrated by the striking age-related risk to develop TB following primary infection and by the age-related differences seen between particular disease manifestations (Figs 14.2 and 14.3).19,36 Apart from the age and immune status of the child, the time since infection is another important variable that influences the particular disease manifestation, as illustrated by the time-table of TB in children (Fig. 14.1). To improve our understanding of the mechanisms that underlie the different manifestations of childhood TB, as described in the pre-chemotherapy literature and confirmed by more recent reports,37 requires a renewed focus on the ontogeny of the host immune response towards infection with M. tuberculosis.19 The knowledge gained may be essential for directing future research in the field of TB prevention, vaccine development, and treatment.
SUMMARY Clinicians and researchers have limited access to the important prechemotherapy literature that documented the natural history of TB in children. Since the discovery of safe and effective treatment, conducting studies on the natural history of disease became unethical and therefore these historic disease descriptions remain invaluable today. The pre-chemotherapy literature provides a strong body of evidence; multiple studies followed large cohorts of children for prolonged periods of time and carefully documented the development of disease following primary infection with M. tuberculosis. In summary, the natural history of disease demonstrates three central concepts that are important to consider when addressing current and/or future challenges in the field of childhood TB: 1. the need for accurate case definitions; 2. the importance of risk stratification; and 3. the diverse spectrum of disease pathology, which necessitates accurate disease classification.36,38
CASE DEFINITION Accurate case definition revolves mainly around the ability to differentiate primary infection from active disease. Primary infection is believed to occur when a previously uninfected child inhales an infectious aerosol droplet; a localized pneumonic process (the Ghon focus) forms at the site of organism deposition. Initially (for the first 4–6 weeks), unrestrained multiplication occurs within the Ghon focus and bacilli drain via local lymphatics to the regional lymph nodes and beyond. Occult dissemination occurs frequently during this early proliferative phase before cell-mediated immunity is fully activated. Bacteriologic cultures collected at this time may be positive; Arvid Wallgren demonstrated that M. tuberculosis can be recovered from recently infected children who are not diseased. Therefore, with active contact tracing and aggressive screening that includes the collection of mycobacterial cultures in asymptomatic children it is not unexpected that some positive cultures will be found in recently infected children who are not diseased. This illustrates the overlap that exists between recent primary infection and
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case definitions of disease that rely exclusively on bacteriology. It is important to consider this overlap when case definitions are formulated for research purposes, particularly within the contact setting, although it is less relevant in everyday practice where there is no reason to obtain cultures from completely asymptomatic children. Uncomplicated hilar adenopathy remains the most common disease manifestation in children and is usually regarded as the hallmark of primary TB. However, the pre-chemotherapy literature documented transient hilar adenopathy in the majority of children following recent primary pulmonary infection, of whom only a small minority progressed to disease. The natural history of disease illustrates that progression to disease is indicated by the onset of persistent, non-remitting symptoms, while the complete absence of symptoms usually indicates good organism containment. By convention, asymptomatic hilar adenopathy is currently treated as active disease, but, in terms of pathophysiology, microbiology, and natural history, asymptomatic hilar adenopathy is more indicative of recent primary infection than active disease. This indicates that radiologic signs should be interpreted with caution in the absence of clinical data. The entity of so-called ‘asymptomatic childhood TB’, where the case definition rests exclusively on radiographic criteria, is a case in point. In reality, differences in patient selection may result in the use of different functional case definitions even though the definitions appear similar on paper. In non-endemic areas where active contact tracing is diligently enforced, more children with transient radiological signs indicative of recent primary infection will be identified, and those with active disease will be diagnosed at an earlier, less advanced stage. Active contact tracing is rarely enforced in endemic areas and children usually present to healthcare facilities with suspicious symptoms and more advanced disease. Unlike asymptomatic contacts in whom visible radiologic signs probably indicate recent primary infection only, radiologic signs in symptomatic children indicate active disease. From a research perspective it is important to be aware of these differences, as inconsistent case definitions may confound the scientific interpretation of results. In everyday practice, distinguishing between the signs and symptoms of recent primary infection and active disease is less relevant in high-risk children (< 3 years of age and/or immune compromised) in whom infection more frequently (and sometimes more rapidly) progresses to disease.
RISK STRATIFICATION The natural history of disease demonstrates that age is the most important variable that determines the risk of progression to disease following primary M. tuberculosis infection in immune-competent children (Fig. 14.2). Infants are at highest risk; the risk drops but stays appreciable in the second year of life, to reach its lowest level in children infected between 5 and 10 years of age. Children with HIV infection and/or other forms of immune compromise, such as severe malnutrition, seem to experience a similar high risk as very young (< 2 years of age) immune immature children.39–42 The vast majority (> 95%) of children who progress to disease do so within 12 months of primary infection, and therefore it seems prudent to categorize all children < 3 years of age and/or immune-compromised children as high risk. Because of the frequency and rapidity with which disease progression may occur, exposure to and/or infection with M. tuberculosis warrants preventive chemotherapy in this high-risk group.
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Immune-competent children 3 years of age are at low risk of progression to disease following primary infection. However, as the vast majority of children in endemic areas become infected after 2– 3 years of age, these low-risk children still contribute a significant percentage of the total disease burden.43 In addition, although these children are at low risk of progression to disease, latent infection with M. tuberculosis poses a small risk of future reactivation disease. In non-endemic areas, where transmission rates are low and eradicating the pool of latent infection is an achievable aim, the provision of preventive therapy to these low-risk children is warranted. In endemic areas, where the majority of disease in immune-competent adults results from ongoing transmission and not from reactivation,44 the provision of preventive therapy following exposure and/or infection becomes less relevant. The major diagnostic challenge in this low-risk group is the differentiation between latent infection and active disease. Fortunately, the natural history of disease demonstrates that active disease is accompanied by persistent, non-remitting symptoms and disease progression is usually slow, which provides a window of opportunity for symptom-based diagnosis.
DISEASE DIVERSITY Childhood TB is often reported as a single disease entity, although it represents a diverse spectrum of pathology, and one of the obstacles has been the lack of standard descriptive terminology. Accurate disease classification is important, because of its prognostic significance and to facilitate scientific communication and optimal case management. Within the Ghon focus containment is usually successful, but disease progression may result from either poor or ‘excessive’ containment.19,36 Poor containment and unrestrained organism proliferation may cause progressive parenchymal damage, with ultimate breakdown of the Ghon focus. Infants and severely malnourished and/or HIV-infected children, who have poor cellmediated immune responses, are most vulnerable to this type of cavitation.2,19,39,45 These are also the groups at greatest risk of disseminated (miliary) disease. In contrast, immune-competent adolescents seem to mount an ‘excessive’ (damaging) immune response in an attempt to contain the organism. The exact immune mechanisms underlying adult-type disease remain uncertain, but it is a striking observation that it only emerges as children enter into puberty. It is important to remember that children with adult-type disease are frequently sputum smearpositive,46,47 and that they do contribute to disease transmission, particularly in congregate settings such as schools. Complications that arise from affected lymph nodes are most common in children < 5 years old, due to exuberant lymph node enlargement and small airway size. Radiologic signs vary from segmental or lobar hyperinflation with partial obstruction and a check-valve effect, to segmental or lobar collapse with total obstruction and resorption of distal air. The pathology that results when a diseased lymph node erupts into an airway and caseous material is aspirated depends on the dose and virulence of the bacilli aspirated. The pathology may range from transient parenchymal consolidation, resulting from a pure hypersensitivity response to dead bacilli and/or toxic products, to an expansile pneumonic process with progressive caseating pneumonia, which frequently leads to parenchymal destruction and cavity formation within the affected lobe. Thus cavitary disease in children may result from three distinct pathologic processes:
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The natural history of tuberculosis infection and disease in children
1. poor containment at the site of organism deposition (very young and/or immune-compromised children); 2. aspiration of live bacilli when a diseased lymph node erupts into an airway, with destructive caseating pneumonia in the distal segment or lobe (children < 5 years of age); and 3. from adult-type disease (mainly children > 10 years of age). In conclusion, the natural history of disease identifies two major challenges regarding the diagnosis of TB in childhood. The first challenge is to identify any untreated infection (primary or reinfection), with a
REFERENCES 1. Marais BJ, Gie RP, Schaaf HS, et al. The clinical epidemiology of childhood pulmonary tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:278–285. 2. Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis — a critical review of the literature from the prechemotherapy era. Int J Tuberc Lung Dis 2004;8:392–402. 3. Opie E, McPhedran FM, Putnam P. The fate of children in contact with tuberculosis: the exogenous infection of children and adults. Am J Hyg 1935;22:644–682. 4. Pope AS, Sartwell MD, Zacks D. Development of tuberculosis in infected children. Am J Public Health 1939;29:1318–1325. 5. Brailey M. A study of tuberculous infection and mortality in the children of tuberculous households. Am J Hyg 1940;31:Sec A1–43. 6. Gedde-Dahl T. Tuberculous infection in the light of tuberculin matriculation. Am J Hyg 1952;56:139–214. 7. Bentley FJ, Grzybowski S, Benjamin B. Tuberculosis in Childhood and Adolescence. The National Association for the Prevention of Tuberculosis. London: Waterlow, 1954:1–253. 8. Davies PDB. The natural history of tuberculosis in children. Tubercle 1961;42(suppl):1–40. 9. Miller FJW, Seal RME, Taylor MD. Tuberculosis in Children. London: Churchill, 1963: 79–163. 10. Zeidberg LD, Gass RS, Dillon A, et al. The Williamson County tuberculosis study: a twentyfour-year epidemiologic study. Am Rev Resp Pulm Dis 1962;87:1–41. 11. Wallgren A. Primary pulmonary tuberculosis in childhood. Am J Dis Child 1935;49:1105–1136. 12. Wallgren A. Pulmonary tuberculosis—relation of childhood infection to disease in adults. Lancet 1938;1:5973–5976. 13. Wallgren A. The time-table of tuberculosis. Tubercle 1948;29:245–251. 14. Brailey M. Prognosis in white and colored tuberculous children according to initial chest x-ray findings. Am J Public Health 1943;33:343–352. 15. Lincoln EM, Sewell EM. Tuberculosis in Children. New York: Mc Graw-Hill, 1963:1–315. 16. Miller FJW, Seal RME, Taylor MD. Tuberculosis in Children. London: Churchill, 1963: 163–275, 466-587. 17. Schaaf HS, Nel ED, Beyers N, et al. A decade of experience with Mycobacterium tuberculosis culture from children: a seasonal influence on incidence of childhood tuberculosis. Tuberc Lung Dis 1996;77:43–46.
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high degree of sensitivity and specificity in immune-compromised children, as this would identify children at high risk of disease progression who may benefit from preventive chemotherapy. The second challenge is to identify active disease as early as possible so that these children can be treated appropriately to reduce the high morbidity and mortality associated with childhood TB. It seems important to incorporate the lessons learnt from the natural history of disease in order to develop a rational and well-focused approach to childhood TB that will improve service delivery to children in high-burden settings with limited resources.
18. Grange JM, Gandy M, Farmer P, et al. Historical declines in tuberculosis, nature, nurture and the biosocial model. Int J Tuberc Lung Dis 2001; 5:208–212. 19. Marais BJ, Donald PR, Gie RP, et al. Diversity of disease manifestations in childhood pulmonary tuberculosis. Ann Trop Paediatr 2005;25:79–86. 20. Rich A. The Pathogenesis of Tuberculosis, 2nd edn. Springfield, IL: CC Thomas, 1951: 314–880. 21. Van der Spuy GD, Warren RM, Richardson M, et al. Use of genetic distance as a measure of ongoing transmission of Mycobaterium tuberculosis. J Clin Microbiol 2003;41:5640–5644. 22. Van Rie A, Warren R, Richardson M, et al. Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N Engl J Med 1999,341:1174–1179. 23. Riley RL. Airborne infection. Am J Med 1974;57:466–475. 24. Balasubramanian V, Wiegeshaus EH, Taylor BT, et al. Pathogenesis of tuberculosis: pathway to apical localization. Tuberc Lung Dis 1994;74: 168–178. 25. Dannenberg AM. Immune mechanisms in the pathogenesis of pulmonary tuberculosis. Rev Infect Dis 1989;11(Suppl 2):S369–S378. 26. Rieder HL. The Epidemiologic Basis of Tuberculosis Control. Paris: International Union against Tuberculosis and Lung Disease, 1999. 27. Converse PJ, Dannenberg AM, Estep JE, et al. Cavitary tuberculosis in rabbits by aerosolized virulent tubercle bacilli. Infect Immunol 1996;64: 4776–4787. 28. 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;133:30–37. 29. Manabe YC, Dannenberg AM, Tyagi SK, et al. Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis. Infect Immunol 2003;71:6004–6011. 30. Zahrt TC. Molecular mechanisms regulating persistent Mycobacterium tuberculosis infection. Microb Infect 2003;5:159–167. 31. Boom WH, Canaday DH, Fulton SA, et al. Human immunity to M. tuberculosis: T cell subsets and antigen processing. Tuberculosis 2003;83:98–106. 32. Cowie RL. The epidemiology of tuberculosis in gold miners with silicosis. Am J Resp Crit Care Med 1994;15:1460–1462. 33. Alcaide J, Altet MN, Plans P, et al. Cigarette smoking as a risk factor for tuberculosis in young adults: case control study. Tuberc Lung Dis 1996;77:112–116.
34. Gajalakshmi V, Peto R, Kanaka TS, et al. Smoking and mortality from tuberculosis and other diseases in India: a retrospective study of 43000 adult male deaths and 35000 controls. Lancet 2003;362: 1243–1244. 35. Dannenberg AM. Roles of cytotoxic delayed-type hypersensitivity and macrophage-activating cellmediated immunity in the pathogenesis of tuberculosis. Immunobiology 1994;191:461–473. 36. Marais BJ, Gie RP, Schaaf HS, et al. A proposed radiologic classification of childhood intra-thoracic tuberculosis. Pediatr Radiol 2004;33:886–894. 37. Marais BJ, Gie RP, Schaaf HS, et al. The spectrum of disease in children treated for tuberculosis in a highly endemic area. Int J Tuberc Lung Dis 2006;10:732–738. 38. Marais BJ, Gie RP, Schaaf HS, et al. Childhood pulmonary tuberculosis—old wisdom and new challenges. Am J Resp Crit Care Med 2006;173: 1078–1090. 39. Palme IB, Gudetta B, Giesecke J, et al. Impact of HIV 1 infection on clinical presentation, treatment, outcome and survival in a cohort of Ethiopian children with tuberculosis. Pediatr Infect Dis J 2002;21(11):1053–1061. 40. Madhi SA, Huebner RE, Doedens L, et al. HIV-1 coinfection in children hospitalised with tuberculosis in South Africa. Int J Tuberc Lung Dis 2000;4:448–454. 41. Hesseling AC, Westra AE, Werschkull H, et al. Outcome of HIV-infected children with cultureconfirmed tuberculosis. Arch Dis Child 2005;90: 1171–1174. 42. Marais BJ, Hesseling AC, Gie RP, et al. The burden of childhood tuberculosis and the accuracy of community-based surveillance data. Int J Tuberc Lung Dis 2006;10:259–263. 43. Shimeles D, Lulseged S. Clinical profile and pattern of infection in Ethiopian children with severe protein-energy malnutrition. East Afr Med J 1994;71:264–267. 44. Weber HC, Beyers N, Gie RP, et al. The clinical and radiological features of tuberculosis in adolescents. Ann Trop Paediatr 2000;20:5–10. 45. Marais BJ, Gie RP, Hesseling AC, et al. Adult-type pulmonary tuberculosis in children aged 10-14 years. Pediatr Infect Dis J 2005;24:743–744. 46. Curtis AB, Ridzon R, Vogel R, et al. Extensive transmission of Mycobacterium tuberculosis from a child. N Engl J Med 1999;341:1491–1495. 47. Goussard P, Gie RP, Kling S, et al. Expansile pneumonia in children caused by Mycobacterium tuberculosis: clinical, radiological, and bronchoscopic appearances. Pediatr Pulmonol 2004;38:451–455.
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Human tuberculosis due to Mycobacterium bovis and related animal pathogens John M Grange and Alimuddin I Zumla
INTRODUCTION AND HISTORY A zoonosis is a human infectious disease due to a microorganism for which the maintenance host is an animal. In the case of mycobacteria, the zoonosis of major importance is human TB of bovine origin, with the causative organism being Mycobacterium bovis. With the introduction of more discriminating methods for identifying members of the Mycobacterium tuberculosis complex, other species have been found to be of zoonotic importance, principally Mycobacterium caprae, which causes disease in goats. Although disease due to Mycobacterium avium occurs in humans, as described in Chapter 7, this cannot be regarded as a zoonosis as the principal reservoir from which humans are infected is the environment.
BOVINE TUBERCULOSIS A chronic wasting disease of cattle has long been recognized but its relationship to human disease, though suspected, was not at all clear until the late nineteenth century. In the eighteenth century a disease of cattle, now identified as bovine TB, was known in Germany as Perlsucht (pearl disease) because of the characteristic pearl-like granulomas found on the pleura of affected animals. The disease was considered to be a variant of syphilis and legislation to ensure safe disposal of affected carcasses was introduced. The relationship between bovine and human TB was confirmed in a meticulous series of studies, published in 1868, by Jean Antoine Villemin (Fig. 15.1) in which he demonstrated that rabbits inoculated with material from lesions in affected cattle and humans developed an identical TB-like disease.1 He observed, however, that bovine material appeared more virulent. The causative agent of TB was discovered by Robert Koch in 1882 but he did not differentiate between isolates of human and bovine origin. Indeed, Koch believed that all ‘tubercle bacilli’ were identical and urged the development of public health measures to protect the human population from infection from bovine sources. It was therefore in a climate of scepticism that, in 1898, Theobold Smith2 published his findings that tubercle bacilli from humans and cattle differed in small but constant ways. He also found, thereby confirming the observations of Villemin, that bovine isolates were more virulent for the rabbit than human isolates. Although referring to his isolates as the human and bovine tubercle bacilli, Smith warned against the assumption that disease due to these variants was limited to the species from which they were isolated.
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Unfortunately, Robert Koch made this assumption and radically altered his views on the importance of bovine TB control measures. At the British Congress on Tuberculosis held in London in 1901 he stated that ‘the human subject is immune against infection by bovine bacilli or is so slightly susceptible that I do not consider it necessary to take any measures to counteract the risk of infection’. Fortunately, the meeting was attended by several distinguished veterinary surgeons and medical scientists, including Lord Lister, who were certain that Koch was mistaken and were able to convince the British Government to establish a Royal Commission to investigate the matter. The Royal Commissioners undertook a 10-year programme of research which established beyond doubt that, although the human and bovine tubercle bacilli did differ in certain characteristics, humans must, in the words of the Royal Commission’s final report published in 1911, ‘be added to the list of animals notably susceptible to bovine tubercle bacilli’. The Royal Commission also laid the scientific foundations for the test-and-slaughter programmes that were eventually to have such a dramatic effects on the control of TB in cattle and, thereby, on human health.3 The bovine TB eradication schemes rank among the most successful campaigns ever waged against an infectious disease and the great debt owed to the scientists employed by the Royal Commission as well as to the veterinary profession in general should never be forgotten.
TUBERCULOSIS IN CATTLE AND OTHER MAMMALS As discussed in Chapter 6, M. bovis is a member of the M. tuberculosis complex and, although having very close, over 99%, genetic similarity with M. tuberculosis, it differs in several important features, notably the broad range of mammals susceptible to disease. Tuberculosis in cattle is almost always acquired by inhalation and the lung is the primary focus of disease. Tuberculin conversion occurs before the development of detectable lesions and such animals are said to be non-visibly lesioned (NVL) and are generally regarded as being non-infectious. Subsequently, granulomatous lesions containing large numbers of acid-fast bacilli develop in the lungs and the mediastinal and subpleural lymph nodes. The latter have a pearl-like appearance and, as mentioned earlier, this condition has been termed pearl disease. Dissemination to other organs may occur and, although TB mastitis occurs in only around 1% of overtly diseased cattle, this is important as a cause of transmission of infection to humans.
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Human tuberculosis due to Mycobacterium bovis and related animal pathogens
15
BOVINE TUBERCULOSIS IN THE DEVELOPING NATIONS A World Health Organization survey conducted in 1998 showed that information on the occurrence and prevalence of bovine TB in developing countries is rather fragmentary, as summarized in Table 15.1.5 A full account of the distribution of TB in cattle world-wide is beyond the scope of this chapter but detailed surveys covering all countries, and specifically Africa, have been published.6–8 Rights were not granted to include this content in electronic media. Please refer to the printed book.
TUBERCULOSIS IN OTHER ANIMALS The many hosts of M. bovis include feral and wild animals, including those in zoos and game parks, and farmed animals, especially cattle.9 Only a few of the many animals susceptible to TB are maintenance (reservoir) hosts – most are spillover (end) hosts. In addition to cattle, important maintenance hosts of disease are African buffalos, bison, deer, badgers (especially in the UK), the brush tailed possum (in New Zealand), and the elk in Canada.10 It is now appreciated that some closely related strains within the M. tuberculosis complex given separate species names (see Chapter 6) are also primarily pathogens of animals although humans may be infected. These include M. caprae (previously M. bovis subsp. caprae) originally isolated from goats but also from cattle, pigs, captive wild boar, and red deer, and Mycobacterium pinnipedii isolated from seals.11,12
Fig. 15.1 Jean-Antoine Villemin (1827–1892). The first to demonstrate experimentally the transmissibility of TB. Grange JM. Mycobacteria and Human Disease. 2nd edition. London: Arnold. 1996. Page 3.
BOVINE TUBERCULOSIS IN THE INDUSTRIALLY DEVELOPED NATIONS Tuberculosis was once widespread in cattle throughout Europe and the USA but the incidence has been very greatly reduced by the control measures described below. Complete elimination has not been achieved as a result of infection of cattle by wild animals and by humans with TB of bovine origin. In the UK, there is strong evidence that cattle in some regions acquire TB from the badger (Meles meles). Overall, in the UK, the number of tuberculin-positive cattle slaughtered rose from 638 in 1986 to 5,884 in 1998 and 22,571 in 2004, amounting to an increase of around 20% annually. This poses a serious veterinary and public health problem to which there is no easy solution. Although the exact impact of infected badgers on the incidence of bovine TB is not clear, it is noteworthy that the great majority of infected cattle have been detected in regions where diseased badgers have been found. Vaccination or selective culling of infected badgers is considered impractical and widespread culling has met with public opposition, especially as its efficacy has not been determined. Indeed there is some evidence that this strategy may even increase the reactor rate in cattle by extending the territorial range of surviving badgers and, thereby, opportunities for contact with cattle.4
THE NATURE OF ZOONOTIC TUBERCULOSIS Humans acquire TB from animals, notably cattle, by three routes – inhalation, ingestion of derivative products, and traumatic inoculation.
INHALATION As the lung is the main site of TB in cattle, farmers, veterinary surgeons, and other workers in close contact with diseased animals are principally infected by the aerogenous route. In pastoralist communities in developing countries, notably Africa, humans often live in close proximity to their cattle, increasing the risk of aerogenous transmission of disease. Workers in abattoirs are at risk of pulmonary infection by aerosols generated during the handling of diseased animal carcasses.13 Although cattle are the usual source of human infection, tuberculin conversion and, rarely, overt disease have followed exposure to other diseased animals including elk, seals, and zoo animals. In one illustrative case, a seal trainer became tuberculin positive while working with an infected animal, and in another case seven of 24 zoo attendants
Table 15.1 The occurrence of bovine tuberculosis in the developing nations Region
Africa Asia Latin America and Caribbean
Number of countries in region Total
High occurrence
Low and sporadic occurrence
Not reported
No data
Test and slaughter policy
55 36 34
8 1 8
25 16 12
4 10 12
18 9 2
7 7 12
Data taken from 5. Cosivi O, Grange JM, Daborn CJ, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4:59–70.
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converted to tuberculin reactivity following occupational exposure to a rhinoceros with tuberculous rhinitis over a 6-month period; all received isoniazid preventive therapy and none became ill.14,15 In an outbreak of TB in farmed elk in Alberta, Canada, 81 of 394 people in contact with diseased animals became tuberculin positive but only one developed TB.16 Strains of M. caprae have been isolated from humans, including one isolate from a veterinary surgeon with a recent history of working with tuberculous goats.17 More surprisingly, a third of cases of human TB in Germany, mostly in the south of the country, between 1999 and 2001 and originally thought to be due to M. bovis were found to be due to M. caprae.11 The patients were mostly elderly, and in the same age range as those with disease due to M. bovis and were therefore thought to have disease due to reactivation of infection acquired years or decades ago.
INGESTION OF DERIVATIVE PRODUCTS Town dwellers not exposed directly to animals are at risk of disease if they consume milk containing M. bovis. Although only a small proportion of tuberculous cattle, around 1%, have overt mastitis and excrete tubercle bacilli in their milk, the practice of pooling milk from several animals in churns, or even from several herds in tankers, renders many people prone to infection from a small number of animals. In 1945, M. bovis was isolated from 8% of churn milk samples and from the great majority of samples from 3,000-gal. tankers in the UK.18 In developing countries, milk drawn directly from cows forms an important component of the diet of adults and children. In some communities the milk is soured before consumption by adding fresh milk to some soured milk and storing it for a few days but it is doubtful if it kills mycobacteria to a significant degree.19 Although bovine TB control programmes and pasteurization of milk (see below) have almost eliminated the risk of milk-borne infection in countries where these are applied, importation of milk derivatives from other regions poses a health risk. Between 2001 and 2004, 35 cases of newly diagnosed TB, about 1% of the total, in New York City were due to M. bovis.20 Most patients were of Mexican or South American origin and many reported eating unpasteurized cheese imported from Mexico which could therefore have been a vector of the disease. In this context, M. bovis may survive for up to 100 days in butter and for almost 1 year in certain cheeses prepared from unpasteurized milk.
areas were less likely to develop overt TB after infection than those living in towns and cities.21 It was postulated that the differences in the proportion of those infected developing overt TB (the disease ratio) was due to a higher proportion of those in rural areas being infected with M. bovis. This postulate does not, however, take into account many confounding factors, such as the timing of the initial infection, the infective dose and route of infection, and various factors in the rural environment that might enhance resistance to disease. Human TB due to M. bovis can certainly be as severe as that due to M. tuberculosis, but so can disease due to environmental mycobacteria. Indeed, those infected by the aerogenous route are at risk of developing pulmonary TB which, in its clinical manifestations, is indistinguishable from that caused by M. tuberculosis.22 Another aspect of the pathogenesis of M. bovis is its postulated ability to generate protective immunity against disease due to M. tuberculosis. In the early twentieth century there was a belief, known as Marfan’s Law, that children who developed and recovered from tuberculous lymphadenitis due to consumption of milk were immune to more serious pulmonary TB later in life. Although regarded by some as a highly dubious proposition, it may explain the finding in Burkino Faso that the incidence of human pulmonary TB is five times greater in regions where bovine TB is rare than in regions where it is common, suggesting a protective effect of milk-borne infection.23 In this context, a temporary rise in the incidence of human TB occurred in Scandinavia at the time when bovine TB was controlled, a finding that even led to the suggestion that, by removing natural immunization, bovine TB control schemes had an adverse effect on the prevalence of human TB.24 Ingestion of bacilli, principally in raw milk, leads to implantation in the tonsil or the intestine, usually in the ileocaecal region. Lymphatic spread to the draining lymph nodes results, respectively, in cervical and mesenteric lymphadenopathy. Cervical lymphadenopathy, also known as scrofula, following ingestion of M. bovis usually involves the tonsillar nodes around the angle of the jaw and, less often, the preauricular nodes (Fig. 15.2). This is in contrast to superclavicular
TRAUMATIC INOCULATION Workers handling contaminated carcasses and meat in abattoirs and butchers’ shops are at risk of skin lesions due to traumatic inoculation of bacilli. The resulting lesions, usually on the hands, are termed butchers’ warts and are similar in pathogenesis and appearance to prosectors’ warts acquired by pathologists and anatomists.
PATHOGENESIS AND CLINICAL FEATURES OF ZOONOTIC TUBERCULOSIS It is widely assumed that M. bovis is much less virulent for humans than M. tuberculosis, an assumption never formally proven. One major difficulty in addressing the question of relative virulence is the inability of the tuberculin test to distinguish between latent TB due to the two species. Tuberculin testing surveys in Denmark before bovine TB was eradicated indicated that those living in rural
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Fig. 15.2 Tuberculous cervical lymphadenitis (scrofula) showing enlarged lymph nodes and sinuses around the angle of the jaw.
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lymphadenopathy in the lower part of the neck that sometimes presents as an upward extension of a primary pulmonary complex due, usually, to M. tuberculosis. In the early stages of the disease, the affected lymph nodes are painless and have a firm rubbery texture. Subsequently the nodes undergo central caseous necrosis and become fluctuant on palpation. The necrosis may spread out of the lymph node and through the fascia of the neck, resulting in a superficial abscess. Rupture of the disease process through the skin results in sinus formation and chronic spreading skin lesions with the characteristics of lupus vulgaris – a condition termed scrofuloderma. Involvement of the intestine and mesenteric lymph nodes leads to the various forms of abdominal TB as described in Chapters 39 and 40. Further lymphatic and haematogenous spread may lead to disease in other organs including bones, joints, the central nervous system, and the kidney. As discussed below, the kidney is a frequent site of reactivation disease. The reason for this is unknown, one possibility being that the relatively low oxygen tension may favour the growth of M. bovis which, in contrast to M. tuberculosis, is microaerophilic.25 The clinical and pathological features of renal TB caused by M. bovis are identical to those of disease caused by M. tuberculosis with, in some cases, extensive destructive lesions leading to renal failure.26 Many cases of lupus vulgaris, a very chronic form of skin TB which often involves the face and, if untreated, may lead to gross disfigurement, are caused by M. bovis. In the UK half the cases of lupus vulgaris reported by Griffith in 1932 were caused by M. bovis, and a study of six cases in the late 1960s showed that five were caused by M. bovis and one by an organism with features intermediate between M. bovis and M. tuberculosis.27 Subsequently this form of TB became extremely rare in the UK and a single case reported in 1986, in a 75-year-old woman, was caused by M. bovis.28 These findings suggest, but do not prove, that M. bovis is more likely to cause this form of TB than M. tuberculosis.
CHANGING TRENDS IN THE OCCURRENCE OF ZOONOTIC TUBERCULOSIS IN THE INDUSTRIALLY DEVELOPED NATIONS The bovine TB eradication programmes conducted in many industrially developed nations have made very significant impacts on the epidemiology of zoonotic TB.5,29–33 In the early part of the twentieth century, when bovine TB was common in Europe and North America, most cases of human TB due to M. bovis occurred in young people and were non-pulmonary. Table 15.2 shows that M. bovis was responsible for a high percentage of cases of non-pulmonary TB, particularly in children, with the notable exception of genitourinary disease. At that time, pulmonary disease due to M. bovis was considered a rarity – by 1922 only four cases had been reported in the UK – but more cases were found when actively sought and were relatively more common in rural areas.27 The few cases of TB due to M. bovis seen in the older population in countries that have had effective bovine TB in place for many years are assumed to be predominantly the result of late endogenous reactivation after a long period of latency – a concept supported by spoligotyping, which shows that bacilli isolated from elderly patients are of types not currently seen in cattle.34 The annual number of new cases of human TB due to M. bovis in the
15
Table 15.2 Cases of non-pulmonary tuberculosis reported in England and Wales, 1901–1932, and the percentage of cases caused by M. bovis Type of disease
Number of cases
Cervical node Lupus vulgaris Scrofuloderma Bone/joint Genitourinary Meningitis Other
Percentage of cases caused by M. bovis (%)
126 191 60 553 23 265 23
<5 years of age
5–15 years of age
All ages
91 58 53 30 0 28 33
53 44 43 19 0 25 9
50 49 37 20 17 25 9
UK in the period 1990–2003 varied from 17 to 50 – between 0.5% and 1.5% of cases of TB confirmed by culture.35 Irrespective of the site of primary infection, the lung is the most frequent site of post-primary TB, so that this organ is the commonest site of disease in older patients in countries where primary infection by M. bovis is now very rare.36 The distribution of types of disease in some countries and regions after the completion of their respective bovine control programmes are shown in Table 15.3. In addition, 13 patients (0.95% of all cases of TB) with disease due to M. bovis were seen in a hospital in Madrid, Spain, between 1994 and 1999; the mean age of the patients was 50 years (range 23–83 years) and 10 of the 13 patients, including four who were human immunodeficiency virus (HIV)-infected, had pulmonary disease.37 Genitourinary disease has also become relatively more common. In the years 1975–1980 in the German state of Hessen, where bovine TB had been virtually eradicated by 1961, M. bovis was isolated from 47 of 4498 patients (1%) with pulmonary TB and 23 of 3834 patients with non-pulmonary lesions (0.8%) and, of the latter, 15 had genitourinary TB.38 Since the completion of the bovine TB eradication programmes in the UK, cattle-to-human spread of infection resulting in overt disease has become extremely rare but one illustrative case, confirmed by spoligotyping, has been reported.34
Table 15.3 Type of tuberculosis caused by M. bovis in countries and regions with a low risk of infection from cattle Type of disease
England South-east Ontario, Southern and Wales, England, Canada, Sweden, 1962–1966 1977–1990 1964–1970 1936–1939
Pulmonary Non-pulmonary Genitourinary Lymphadenitis Bone/joint Meningeal Abdominal Other
65 37 25 6 4 2 0 0
94 138 53 39 26 8 8 4
13 18 12 4 1 0 1 0
54 40 12 12 9 4 3 0
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THE OCCURRENCE OF ZOONOTIC TUBERCULOSIS IN THE DEVELOPING WORLD Tuberculosis due to M. bovis is under-recognized and underreported in the developing nations owing to a lack of laboratory facilities for differentiating this species from M. tuberculosis.39 In Tanzania there is a definite relation between the incidence of non-pulmonary TB and the number of cattle owned by the human population in rural regions and in one high-incidence area M. bovis was grown from four of 11 biopsies from patients with cervical lymphadenitis.40 In a further study in the same region, 65 specimens from 457 patients with suspected mycobacterial lymphadenitis were culture positive: seven M. bovis, 27 M. tuberculosis, and 31 environmental mycobacteria.41 Risk factors for disease due to M. bovis were HIV infection and ingestion of raw milk. Studies in other parts of Africa have revealed cases of pulmonary TB due to M. bovis. In Egypt, three surveys on the percentage of such pulmonary cases were, respectively, 0.4%, 5.4%, and 6.4%, and in Nigeria four of 102 cases were caused by M. bovis.42,43 In a further study in Egypt, nine of 20 randomly selected cases of tuberculous peritonitis were caused by M. bovis.44 A report in the year 2000 showed that a third of positive cultures from children living on the USA–Mexico border were M. bovis.45 Most of these children were of Mexican origin and over half had pulmonary disease. Epidemiological studies in Zambia showed that pastoralist families with a case of TB during the previous year were seven times more likely to own herds of cattle containing tuberculin reactors than unaffected families but the cause of the disease in the patients was not confirmed bacteriologically.46 In South America, a conservative estimate is that 2% of cases of pulmonary TB and 8% of nonpulmonary TB cases are due to M. bovis, with higher incidences in areas where the dairy industry is concentrated.5 There is limited information on human TB of bovine origin in Asia. The use of discriminative polymerase chain reaction-based technology in one centre in India has shown that disease due to M. bovis occurs in humans and cattle and that 8.7% and 35.7% of non-pulmonary lesions from humans and cattle, respectively, contained both M. bovis and M. tuberculosis.47 The significance of such apparent mixed infection for transmission of disease between humans and cattle and vice versa requires careful investigation.
THE EPIDEMIOLOGY OF ZOONOTIC TUBERCULOSIS Humans are spillover hosts of M. bovis, which is principally transmitted between animals, either wild or farmed. There is, however, evidence that cycles of animal-to-human, human-to-human, and human-toanimal transmission may occasionally occur. The advent of the HIV/ acquired immunodeficiency syndrome (AIDS) pandemic, by reducing resistance of the human population, could well lead to an increased frequency of human-to-human and human-to-animal transmission, principally by the aerogenous route, with serious public and veterinary health consequences.40,48,49
HUMAN-TO-HUMAN TRANSMISSION: THE IMPACT OF IMMUNOSUPPRESSION Until recently there have been only a few well-documented cases of human-to-human transmission of M. bovis leading to overt disease.50,51 While the apparent rarity of disease due to such transmission has been
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attributed to the presumed lower virulence of M. bovis for humans, it could also be the result of the difficulties in establishing proof, especially as many years may separate the presentation of linked cases. As described in Chapter 10, HIV infection greatly increases the risk of developing active TB after infection by a tubercle bacillus. Thus the advent of the HIV/AIDS pandemic has led to an increase in reports of TB resulting from human-to-human transmission of M. bovis, including some mini-epidemics. In a nosocomial outbreak in Paris, human-to-human transmission of a multidrug-resistant strain of M. bovis resulted in five HIV-infected patients presenting with overt disease within 10 months of exposure to the source case.52 In Spain, 19 profoundly immunosuppressed patients with HIV disease developed TB caused by M. bovis resistant to 11 anti-TB drugs and all the patients died.53
HUMAN-TO-ANIMAL TRANSMISSION There have been several reports of farm workers with open TB due to M. bovis infecting cattle.22,54,55 In North Bavaria, Germany, 49 infected herds were detected between 1973 and 1984 and a human source was found to be the cause in 16 (33%) of these. Exposure to diseased cattle was considered responsible for eight of the other 33 outbreaks but no cause was found for the remainder.56 A case with clear evidence of transmission from cattle to a human and back to cattle has been documented in Switzerland.57 The usual route of transmission of infection from humans to cattle is by cough spray from farm workers with open pulmonary lesions but a substantial minority of workers have genitourinary TB and infect cattle by the apparently common practice of urinating on the hay in cowsheds. In an investigation of 50 cases of infection of cattle by farm workers in Europe, 24 of the source cases were found to have genitourinary TB.58 Between 1968 and 1972, 12 farmers in the German state of Hessen infected 114 cattle in 16 herds.59 Nine of the 12 farmers had genitourinary TB and one of them infected 48 cattle in four herds. Other more unusual activities, including certain traditional customs, could also result in infection of animals. These include the practice of spitting well-chewed tobacco into the mouths of cattle with the aim of restoring appetite and the bizarre but more physiologically sound practice of blowing forcibly into the vagina of a cow to inflate the uterus, thereby causing release of oxytocin which stimulates milk production.48 The effect of HIV disease on the risk of developing overt TB of bovine origin after infection has been mentioned earlier. As a result, it is possible that a high incidence of HIV disease in agricultural communities could result in more cases of human TB of bovine origin and a transmission of the disease back to cattle, with serious public and veterinary health consequences (Fig. 15.3). 40,48,49 Humans can infect cattle with M. tuberculosis by the aerogenous route, resulting in transient tuberculin conversion and, in some cases, small self-limiting lesions in the lungs and mediastinal lymph nodes but very rarely overt disease.55,60 In one case in Slovenia, 16 tuberculin reactors were detected in a previously non-reactive herd and the three strongest reactors were slaughtered.61 The mandibular, mediastinal, and portal lymph nodes were cultured and M. tuberculosis was isolated from one animal and was identical on DNA fingerprinting to a strain isolated from a farm worker with pulmonary TB. Human TB should therefore be excluded as a cause of tuberculin reactivity in previously non-reactive herds, especially in regions where this disease is common in the human population.
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A
15
B
Fig. 15.3 The cycle of infection between cattle and human beings. (A) In a non-immunocompromised community. (B) In a community containing immunocompromised individuals.
PREVENTION OF ZOONOTIC TUBERCULOSIS The risk of infection of the human population has been greatly reduced in many countries by bovine TB control programmes and pasteurization of milk. In early control programmes animals with overt disease were removed from herds and slaughtered, but with limited effect. Following studies by the British Medical Research Council, the importance of removing infected animals before the development of detectable lesions was established and this formed the basis of all subsequent effective eradication programmes.62 At the end of the Second World War, it was estimated that 30–35% of dairy cows in the UK were tuberculin reactors and a compulsory test-and-slaughter programme commenced in 1950. By 1961 the annual reactor rate had dropped to 0.16% and by 1965 it had dropped to 0.06%, with reactors in only 1% of herds. By 1979 only 0.18% of herds contained infected animals and this may be an irreducible minimum owing to infection from wild animals, such as the badger in the UK. The bovine TB control programmes caused a rapid and sharp decline in the incidence of tuberculous cervical lymphadenopathy in children.63 Other forms of the disease also appeared to decline sharply in incidence, but this may in part have been due to a failure or unwillingness of laboratories to discriminate between the types of tubercle bacilli. Physicians in the UK were unwilling to mention M. bovis on notification forms as this led to enquiries from veterinary agencies and breaches of patient confidentiality. Thus, although the Public Health Laboratory Service identified 556 strains of M. bovis isolated from humans in England and Wales between 1977 and 1981, only 125 had been notified by physicians to the health authorities.62 Pasteurization is a process of heat treatment of milk that kills tubercle bacilli and most other non-sporing bacteria. Various different time/temperature combinations, such as heating to 71.7 C for 15 seconds, are used. In regions where bovine TB occurs, and facilities for the formal pasteurization of milk are unavailable, simple boiling of milk before consumption is an effective disease prevention measure.
THERAPY OF ZOONOTIC TUBERCULOSIS With rare exceptions, strains of M. bovis are naturally resistant to pyrazinamide, but susceptibility to other anti-TB agents is similar to that of M. tuberculosis. No specific recommendations for treatment are made in the World Health Organization guidelines on the therapy of TB.64 In practice most patients are likely to be treated with the standard short course of anti-TB regimens as few laboratories in the developing nations differentiate between M. tuberculosis and M. bovis or conduct drug susceptibility tests. If pyrazinamide resistance is identified, this agent may be omitted from the regimen.65 On the other hand, the American Thoracic Society recommends an initial 2-month regimen of isoniazid, rifampicin, and ethambutol followed by a 7-month continuation phase of isoniazid and rifampicin given either daily or twice weekly.66 In a study of 167 patients with TB due to M. bovis in San Diego County, California, the median time to completion of treatment was 3 months longer than for patients with disease due to M. tuberculosis.67 For reasons not clear, although the numbers of patients completing therapy were very similar, mortality among those with disease due to M. bovis (15%) was higher than that in 928 patients with disease due to M. tuberculosis (7%). Drug and multidrug resistance may develop and two miniepidemics in care facilities for patients with HIV disease have been mentioned already.52,53 The management of such cases is the same as that of disease due to drug-resistant M. tuberculosis (Chapters 52 and 53).
CONCLUSIONS Tuberculosis in animals, especially cattle, and human disease resulting from contact with such animals or consumption of derivative products including milk and cheese is a veterinary and public health problem throughout the world. Although bovine TB eradication programmes have been highly successful in eliminating the
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threat to human health in the industrially developed nations, cattle still acquire infection from human attendants and wildlife. In the UK, infection of cattle from badgers is a serious, costly, and increasing problem to which there is no straightforward solution. The contribution of bovine TB to animal and human health in many developing nations is poorly documented owing to a lack of investigational facilities but sporadic investigations indicate that the detected cases in animals and humans are like the tip of an
REFERENCES 1. Villemin JA. E´tudes Experimentales et Cliniques sur Tuberculose. Paris: Bailliere et Fils, 1868. 2. Smith T. A comparative study of bovine tubercle bacilli and of human bacilli from sputum. J Exp Med 1898;3:451–511. 3. Francis J. The work of the British Royal Commission on Tuberculosis, 1901-1911. Tubercle 1959;40:124–132. 4. Donnelly CA, Woodroffe R, Cox DR, et al. Impact of localized badger culling on tuberculosis incidence in British cattle. Nature 2003;426:834–837. 5. Cosivi O, Grange JM, Daborn CJ, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4:59–70. 6. Thoen CO, Steele JH (eds). Mycobacterium bovis Infection in Animals and Humans. Ames, IA: Iowa State University Press, 1995: 169–345. 7. Report of the WHO working group on zoonotic tuberculosis (Mycobacterium bovis), with the participation of FAO. Geneva: WHO Veterinary Public Health Unit, 1994. Publication WHO/CDS/VPH/94.137. 8. Cosivi O, Meslin FX, Daborn CJ, et al. Epidemiology of Mycobacterium bovis infection in animals and humans, with particular reference to Africa. Rev Sci Tech 1995;14:733–746. 9. O’Reilly LM, Daborn CJ. The epidemiology of Mycobacterium bovis infection in animals and man: a review. Tubercle Lung Dis 1995;76(Suppl):1–46. 10. Biet F, Boschiroli ML, Thorel MF, et al. Zoonotic aspects of Mycobacterium bovis and Mycobacterium aviumintracellulare complex (MAC). Vet Res 2005;36:411–436. 11. Kubica T, Ru¨sch-Gerdes S, Niemann S. Mycobacterium bovis subsp. caprae caused one-third of human M. bovis-associated tuberculosis cases reported in Germany between 1999 and 2001. J Clin Microbiol 2003;41:3070–3077. 12. Cousins DV, Ricardo Bastida R, Angel 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: 1305–1314. 13. Robinson P, Morris D, Antic R. Mycobacterium bovis as an occupational hazard in abattoir workers. Aust NZ Med J 1988;18:701–703. 14. Thompson PJ, Cousins DV, Gow BL, et al. Seals, seal trainers and mycobacterial infection. Am Rev Respir Dis 1993;147:164–167. 15. Dalovisio JR, Setter M, Mikota-Wells S. Rhinoceros’ rhinorrhea: cause of an outbreak of infection due to airbourne Mycobacterium bovis in zookeepers. Clin Inf Dis 1992;15:598–600. 16. Fanning A, Edwards S. Mycobacterium bovis infection in human beings in contact with elk (Cervus elaphus) in Alberta, Canada. Lancet 1991;338:1253–1255. 17. Gutierrez M, Samper S, Jimenez MS, et al. Identification by spoligotyping of a caprine genotype in Mycobacterium bovis strains causing human tuberculosis. J Clin Microbiol 1997;35:3328–3330. 18. Francis J. Bovine Tuberculosis. London: Staples, 1947. 19. Idrisu A, Schnurrenberger P. Public health significance of bovine tuberculosis in four Northern states of Nigeria; a mycobacteriologic study. Nigerian Med J 1977;7:384–387. 20. Centers for Disease Control and Prevention. Human tuberculosis caused by Mycobacterium bovis—New York City, 2001–2004. MMWR Morb Mortal Wkly Rep 2005;54:605–608.
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iceberg. Although humans are usually spillover or end hosts, with human-to-human or human-to-animal transmission of infection being rare, the advent of the HIV/AIDS pandemic raises the distinct possibility of the establishment of cycles of transmission between humans and animals in pastoralist communities. Accordingly, cooperation between veterinary and medical workers on the problems of TB in animals and the human consequence of such disease needs to be intensified world-wide.
21. Magnus K. Epidemiological basis of tuberculosis eradication. 3. Risk of pulmonary tuberculosis after human and bovine infections. Bull World Health Organ 1966;35:483–508. 22. Magnusson H. The relationship between bovine and human tuberculosis from the veterinary point of view. Acta Med Scand 1941;135:201–239. 23. Rey JL, Villon A, Saliou P. La tuberculose bovine dans le Sahel Voltaique. Correlation avec la tuberculose humaine. Technical Document 6.678. Paris: Organisation de Cooperation et de Coordination pour la lute contre les Grandes Endemies, 1977. 24. Sjo¨gren I, Sutherland I. Studies of tuberculosis in man in relation to infection in cattle. Tubercle 1974;56: 113–127. 25. Grange JM, Yates MD, de Kantor IN. Guidelines for Speciation within the Mycobacterium tuberculosis Complex, 2nd edn. Geneva: World Health Organization, 1996. Publication WHO/EMC/ ZOO/96.4 26. Yaqoob M, Goldsmith HJ, Ahmad R. Bovine genitourinary tuberculosis revisited. Q J Med 1990;74:105–109. 27. Griffith AS. Bovine tuberculosis in man. Tubercle 1937;18:528–543. 28. Wilkins EG, Grifiths RJ, Roberts C. Bovine tuberculosis of the skin. J Infect 1986;12:280–281. 29. Grange JM, Collins CH. Tuberculosis and the cow. J R Soc Health 1997;117:119–122. 30. Grange JM, Yates MD. Zoonotic aspects of Mycobacterium bovis infection. Vet Microbiol 1994;40:137–151. 31. Grange JM. Human disease caused by Mycobacterium bovis and its attenuated derivative, bacillus CalmetteGue´rin (BCG). In: James DG, Zumla A (eds). Granulomatous Disorders. Cambridge: Cambridge University Press, 1999: 161–172. 32. Grange JM. Mycobacterium bovis infection in human beings. Tuberculosis (Edinb) 2001;81:71–77. 33. Moda G, Daborn CJ, Grange JM, et al. The zoonotic importance of Mycobacterium bovis. Tubercle Lung Dis 1996;77:103–108. 34. Gibson AL, Hewinson G, Goodchild T, et al. Molecular epidemiology of disease due to Mycobacterium bovis in humans in the United Kingdom. J Clin Microbiol 2004;42:431–434. 35. de la Rua-Domenech R. Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb) 2006; 86:77–109. 36. Balasubramanian V, Wiegeshaus EH, Taylor BT, et al. Pathogenesis of tuberculosis: pathway to apical localization. Tubercle Lung Dis 1994;75:168–178. 37. Esteban J, Robles P, Soledad Jimenez M, et al. Pleuropulmonary infections caused by Mycobacterium bovis: a re-emerging disease. Clin Microbiol Infect 2005;11:840–843. 38. Salfelder T, Schliesser T, Jungbluth H. Folgerungen aus dem derzeitigen Vorkommen von Mycobacterium bovis bei Mensch und Tier. Fortschr Vet Med 1983;37:141–145. (Summarized in English by Schliesser T. Prevalence of M. bovis in man 20 years after eradication of bovine tuberculosis in cattle. Bull Int Union Tuberc 1986;61(abstract book): 58–59. 39. Ayele WY, Neill SD, Zinsstag J, et al. Bovine tuberculosis: an old disease but a new threat to Africa. Int J Tuberc Lung Dis 2004;8:924–937.
40. Daborn CJ, Grange JM. HIV/AIDS and its implications for the control of animal tuberculosis. Br Vet J 1993;149:405–417. 41. Mfinanga SG, Morkve O, Kazwala RR, et al. Mycobacterial adenitis: role of Mycobacterium bovis, non-tuberculous mycobacteria, HIV infection, and risk factors in Arusha, Tanzania. East Afr Med J 2004;81:171–178. 42. Elsabban MS, Lofty O, Awad WM, et al. Bovine tuberculosis and its extent of spread as a source of infection to man and animals in the Arab Republic of Egypt. In: Proceedings of the International Union against Tuberculosis and Lung Disease Conference on Animal Tuberculosis in Africa and the Middle East, 1992 Apr 28–30, Cairo. Paris: IUATLD, 1992: 198–211. 43. Idigbe EO, Anyiwo CE, Onwujekwe DI. Human pulmonary infections with bovine and atypical mycobacteria in Lagos, Nigeria. J Trop Med Hyg 1986;89:143–148. 44. Nafeh MA, Medhat A, Abdul-Hameed A-G, et al. Tuberculous peritonitis in Egypt: the value of laparoscopy in diagnosis. Am J Trop Med Hyg 1992;47:470–477. 45. Dankner WM, Waecker NJ, Essey MA, et al. Mycobacterium bovis infections in San Diego: a clinicoepidemiological study of 73 patients and a historical review of a forgotten pathogen. Medicine 1993;72:11–37. 46. Cook AJC, Tuchili LM, Buve A, et al. Human and bovine tuberculosis in the Monze district of Zambia—a cross-sectional study. Br Vet J 1996;152:37–46. 47. Prasad HK, Singhal A, Mishra A, et al. Bovine tuberculosis in India: potential basis for zoonosis. Tuberculosis (Edinb) 2005;85:421–428. 48. Daborn CJ, Grange JM, Kazwala RR. The bovine tuberculosis cycle—an African perspective. J Appl Bacteriol 1996;81(Symposium Suppl):27S–32S. 49. Grange JM, Daborn C, Cosivi O. HIV-related tuberculosis due to Mycobacterium bovis. Eur Respir J 1994;7:1564–1566. 50. Sigurdsson J. Studies on the risk of infection with bovine tuberculosis in the rural population. With special reference to pulmonary tuberculosis. Acta Tuberc Scand 1955(Suppl 15):1–250. 51. Kubin M, Heralt Z, Morongova I. Two cases of probable man-to-man transmission of Mycobacterium tuberculosis. Z Erkrank Atmungsorgane 1984;163:285–291. 52. Bouvet E, Casalino E, Mendoza-Sassi G, et al. A nosocomial outbreak of multidrug-resistant Mycobacterium bovis among HIV-infected patients. A case-control study. AIDS 1993;7:1453–1460. 53. Cobo J, Asensio A, Moreno S, et al. Risk factors for nosocomial transmission of multidrug-resistant tuberculosis due to Mycobacterium bovis among HIVinfected patients. Int J Tuberc Lung Dis 2001;5:413–418. 54. Stenius R. Differential by tuberculin testing of infection in cattle due to human, bovine and avian types of tubercle bacilli. Vet Rec 1983;50:633–636. 55. Lesslie IW. Cross infections with mycobacteria between animals and man. Bull Int Union Tuberc 1968;41:285–288. 56. Lutz H. Untersuchungen u¨ber das Auftreten und die Erkennung von Reinfektion mit Mycobacterium bovis in Rinderbestanden in Nordbayern in den Jahren 1973 bis 1984. Inaugural dissertation, Justus Liebig Universita¨t, Giessen, Germany, 1987. 57. Fritsche A, Engel R, Buhl D, et al. Mycobacterium bovis tuberculosis: from animal to man and back. Int J Tuberc Lung Dis 2004;8:903–904.
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Human tuberculosis due to Mycobacterium bovis and related animal pathogens 58. Huitema H. The eradication of bovine tuberculosis in cattle in the Netherlands and the significance of man as a source of infection in cattle. Selected Papers of the Royal Netherlands Tuberculosis Association 1969;12:62–67. 59. Schliesser T. Die Beka¨mpfung der Rindertuberkulose—Tierversuch der Vergangenheit. Prax Pneumonol 1974;28(Suppl):870–874. 60. Krishnaswami KV, Mani KR. Mycobacterium tuberculosis humanis causing zoonotic tuberculosis among cattle. Indian J Public Health 1983;27:60–63. 61. Ocepek M, Pate M, Zolnir-Dovc M, et al. Transmission of Mycobacterium tuberculosis from human to cattle. J Clin Microbiol 2005;43:3555–3557.
62. Collins CH, Grange JM. The bovine tubercle bacillus. J Appl Bacteriol 1983;55:13–29. 63. Meissner G. Tuberkelbacterien von typus bovinus beim Menschen in den Jahren 1953-1957. Tuberk Arzt 1959;13:74–90. 64. World Health Organization. Treatment of Tuberculosis: Guidelines for National Programmes, 3rd edn. Geneva: WHO, 2003. Publication WHO/CDS/TB2003.313. 65. O’Donahue WJ, Bedi S, Bittner SJ, et al. Shortcourse chemotherapy for pulmonary infection due to Mycobacterium bovis. Arch Intern Med 1985;145: 703–705.
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66. American Thoracic Society, CDC, and Infectious Diseases Society of America. Treatment of tuberculosis. MMWR Morb Mortal Wkly Rep 2003;52 (RR11):1–77. 67. LoBue PA, Moser KS. Treatment of Mycobacterium bovis infected tuberculosis patients: San Diego County, California, United States, 1994-2003. Int J Tuberc Lung Dis 2005;9:333–338.
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GENERAL CLINICAL FEATURES AND DIAGNOSIS
Clinical features and index of suspicion of tuberculosis in children Stephen M Graham, Ben J Marais, and Robert P Gie
Tuberculosis can present with a wide variety of clinical manifestations in children. The commonest form is pulmonary TB and this can be the most challenging to diagnose. Extrapulmonary TB accounts for around 20–25% of all childhood TB and the commonest forms are TB lymphadenitis, spinal TB, pleural effusion, abdominal TB, miliary TB, and tuberculous meningitis.1–4 The greatest burden of TB in children occurs in highly TB-endemic communities. This is often a resource-limited setting where childhood protein-energy malnutrition and/or human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) may also be common. These problems lead to an increased risk of disease and challenges of management. In children at risk for TB infection, it is important to maintain a high index of suspicion for the possibility of TB as a diagnosis in many clinical contexts, especially when the child’s clinical condition fails to respond to recommended first-line care such as antibiotics for pneumonia. This chapter will outline clinical features of TB in children and discuss their diagnostic value in the context of where most cases of childhood TB present. Particular attention is given to the diagnostic approach to suspected pulmonary TB in children and to the diagnosis of sputum smear-negative pulmonary TB including in regions endemic for malnutrition and HIV. Later chapters give detail of the clinical abnormalities associated with pulmonary TB and extrapulmonary TB in children.
INTRODUCTION TO DIAGNOSIS The diagnosis of TB in children is a common clinical challenge that can be successfully overcome in the majority of cases by taking a systematic approach that makes full use of the epidemiological context, clinical information, and relevant investigations. Important factors to consider in all children with suspected TB is the endemic setting as well as the age and immune status of the child.
EPIDEMIOLOGICAL CONTEXT In countries or communities with a very low incidence of TB where childhood TB is normally rare, it is important for the clinician to be alert to the possibility of TB especially in certain groups, such as children of immigrants from TB-endemic countries.4 In this setting, diagnosis is more straightforward. A positive contact history with an infectious pulmonary TB case and suggestive symptoms such as chronic cough or failure to thrive are more specific
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for TB in non-endemic settings. In addition, this is usually in a resource-rich setting where protein-energy malnutrition and HIV infection are rare, and a wider range of investigations is readily available. A history of TB contact is also important in TB-endemic countries or communities but is less sensitive because, when the incidence of infectious TB within the community is high, transmission often occurs through unknown source cases, especially for the older child.5,6 On the other hand, because previous TB exposure and/or infection is not uncommon in older children, a history of recent contact may be less specific. A contact history has become more difficult to interpret in HIV-endemic regions where the majority of sputum smear-positive pulmonary TB cases are also HIV-infected. The children of adults with TB/HIV coinfection are at greater risk for acquiring either or both infections, yet there is considerable overlap in the clinical presentation of TB and HIV in young children. Adults with TB/HIV coinfection are also more likely to develop sputum smear-negative pulmonary TB, in which case the exposure of the children may not be appreciated.
AGE AND CLINICAL PRESENTATION The epidemiology of TB in children in high-TB-endemic countries and the importance of young age as a major risk factor for disease mean that the majority of cases of pulmonary TB present before 5 years of age.1,2,5,7 This adds to the challenge of diagnosis for a number of reasons: 1. symptoms and signs are often less specific than in older children or adults; 2. infants and young children are also at risk for many other infectious diseases of childhood including HIV infection; 3. in poor communities, this is the age group most at risk for protein-energy malnutrition; and 4. it is particularly difficult to obtain specimens for sputum smear and microscopy for acid-fast bacilli (AFB) and culture in infants and young children. Although the risk of developing active disease following TB exposure is much less in older children, they are more likely to be infected owing to their mobility within the community. Therefore, despite children older than 5 years of age being at relatively low risk of developing TB, a considerable number of child cases in endemic areas do occur in this age group. It is not only the difficulty of obtaining sputum samples from young children that makes it difficult to confirm pulmonary TB,
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but young children present with primary TB that is paucibacillary and so the yield from smears for AFB is low even when sputum is available for examination. Older children and adolescents are more able to provide sputum and are also more likely to have sputum smear-positive pulmonary TB.1–4 Age is also important when considering the diagnosis of extrapulmonary TB. Children of less than 2–3 years of age are at greatest risk of severe, disseminated disease following primary TB infection.1 In the pre-Bacillus Calmette–Gue´rin (BCG) era, the risk of miliary TB and TB meningitis were particularly high in this age group and arguably the greatest benefit of routine neonatal BCG immunization has been to reduce the incidence of severe disseminated (miliary) TB.7,8 Other forms of TB, such as pleural effusion, pericardial TB, peritoneal (abdominal) TB, or spinal TB, are more likely to present in children of 3 years and older.1–3 The different presentations with age are likely to be explained by different cellular immune response as the immune system matures with age.
16
Box 16.1 Recommended approach to diagnose tuberculosis in children 1. Careful history including history of TB contact and symptoms consistent with TB. 2. Clinical examination including growth assessment. 3. Tuberculin skin testing. 4. Bacteriological confirmation whenever possible. 5. Investigations relevant for suspected pulmonary TB and suspected extrapulmonary TB. 6. HIV testing in high HIV prevalence areas. From: Guidelines for national tuberculosis programmes on the management of tuberculosis in children. World Health Organization, Geneva, Switzerland. WHO/HTM/TB/2006.371.
with latent TB infection and non-TB-related coincident symptoms from the child with true TB disease.
IMMUNE STATUS AND CLINICAL PRESENTATION The cellular immune response to TB infection affects the likelihood and type of disease presentation in a number of ways. As already implied the effectiveness of the immune response to contain a recent TB infection improves with age as the immune system matures. Infants are at far greater risk for developing disease following infection than school-aged children and are particularly susceptible to disseminated disease. Further, the presentation of pulmonary TB in infants can be more acute and more generalized with a higher bacillary load than in older children.9,10 The presentation of pulmonary TB in children is usually characterized by chronic cough and a lack of respiratory distress but the possibility of pulmonary TB needs to be considered in children, especially infants with acute severe pneumonia not responding to first-line antibiotics.11,12 Children with immunosuppression for other reasons such as HIV infection or severe malnutrition are also at greater risk of disseminated disease if infected with TB.1,3 There are important clinical correlates. The immunodeficient response to TB infection is indicated by a non-reactive response to tuberculin skin test (TST), which is common in TB-infected children with HIV infection, severe malnutrition, or disseminated TB. On the other hand, the same child may become more symptomatic of TB when immune function is restored by antiretroviral therapy (ART) or nutritional rehabilitation. The immune response to TB infection in the immunocompetent child is also relevant to disease presentation. A strong cellular immune response can result in characteristic clinical disease such as in the pathogenesis of TB lymphadenitis or pleural effusion, usually associated with a reactive TST response and more common in older children.
DIAGNOSIS RECOMMENDED APPROACH TO TUBERCULOSIS DIAGNOSIS The approach to the diagnosis of TB in children as recommended in the World Health Organization (WHO) guidelines of 2006 is summarized in Box 16.1.13 The approach is based on clinical experience, expert opinion, and published evidence. Published evidence is generally consistent but interpretation is always limited by a lack of a gold standard for diagnosis to compare definite TB from definitely not TB and the challenge to differentiate the child
CLINICAL FEATURES THAT SUGGEST TUBERCULOSIS Positive tuberculosis contact A child is usually infected with Mycobacterium tuberculosis by a person with pulmonary TB. When seeking a history of TB contact, it is important to determine the closeness of contact and whether the contact is sputum smear-positive, as these are important risk factors for infection (Chapter 5). Close contact is defined as living in the same household as, or in frequent contact with, a source case with sputum smear-positive pulmonary TB. Source cases that are sputum smear negative but culture positive can also infect child contacts but the risk is much lower. The timing of contact is also important as natural history data suggest that the majority of children infected with TB who develop disease will do so within 1 year of infection.5 Therefore a contact history that goes beyond 1 year makes the history less relevant to the clinical presentation of most forms of TB such as pulmonary TB or disseminated disease, at least in HIV-uninfected children. The later presentation of reactivation disease should be considered if there is severe immunosuppression. If there is no known contact with a TB source case it is important to enquire further about anyone in regular contact with the child who has a chronic cough and/or other symptoms suspicious of TB. The presentation of suspected TB in a child can be an opportunity to identify and treat an infectious case in the community and an effort should be made to detect the source case for any child diagnosed with TB.14 Another potential source of infection in some communities is consumption of non-pasteurized milk from cattle infected with Mycobacterium bovis. The usual presentation of M. bovis disease is cervical adenitis or gastrointestinal TB. Persistent cough Pulmonary TB commonly presents with a persistent cough defined as an unremitting cough for more than 21 days that is not improving despite appropriate first-line management. A careful history of the character and duration of the cough is important and helpful.15–17 The characteristic cough of pulmonary TB is persistent and unremitting. Recurrent viral respiratory tract infections and mild asthma, for example, are common causes of cough in children but the cough is usually characterized by an acute onset with steady improvement or a recurrent/relapsing pattern. Table 16.1 lists causes of chronic (persistent or recurrent) cough in children with typical characteristics of their clinical presentation.
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Table 16.1 Characteristics of causes of persistent or recurrent cough in children Causes
Nature of cough
Presence of fever
Typical nutritional assessment
Relevant clinical information
Pulmonary TB
Persistent and unremitting
Variable
Not resolved with antibiotics
Recurrent viral respiratory tract infection Recurrent bacterial pneumonia Asthma Bronchiolitis
Acute onset with delayed recovery or relapse
Recurrent
FTT or malnourished Normal
Recurrent
Pertussis
Acute onset with improvement or relapse Variable. Worse at night Marked at onset with steady improvement Persistent and spasmodic
LIP
Persistent
Not typicala At onset then settles At onset then settles Variablea
Bronchiectasis
Persistent
Variablea
Cystic fibrosis
Persistent
Variablea
Foreign body aspiration Cardiac failure
Persistent Persistent. Worse at night
Not typicala Not typicala
Viral symptoms, e.g. coryza. Not resolved with antibiotics. Improves between episodes
FTT or malnourished Normal Normal
Resolves with antibiotics. Improves between episodes. HIV related Wheeze. Responds to bronchodilators Wheeze. Infant
FTT or malnourished Variable
Not immunized. Subconjunctival haemorrhages
FTT or malnourished FTT or malnourished Normal FTT
HIV-infected. Parotid enlargement, PGL, clubbing. Response to steroids Copious sputum. Clubbing Ethnic group. Copious sputum. Clubbing Choking episode at onset Other signs of cardiac disease
LIP, lymphoid interstitial pneumonitis; FTT, failure to thrive; PGL, persistent generalized lymphadenopathy. Occasionally associated with infection or an increased risk of secondary infection.
a
In TB/HIV-endemic countries, the commonest clinical challenge is to differentiate pulmonary TB from other HIV-related lung disease. This will be addressed in more detail later in the chapter.
Persistent fever Fever is very common in children and is also a variable feature of TB.17 The diagnosis of TB is not usually considered in a febrile child unless the fever is persistent (> 7 days) or other clinical features suspicious of TB are present. It is important that other common causes of fever such as viral respiratory tract infection, malaria, bacteraemia, or focal sepsis have been considered and treated or excluded. Tuberculosis should be considered as a cause of ‘pyrexia of unknown origin’ defined as a daily temperature > 38 C for more than 14 days. Weight loss or failure to thrive Tuberculosis is a debilitating disease that can also cause anorexia and therefore it is unusual for a child with TB disease to thrive. The impact on the nutritional state is affected by the age of the child and type of TB disease. Infants and young children are particularly vulnerable to failure to thrive and malnutrition due to the high caloric demands for rapid growth at that age. This also makes documentation of failure to thrive more sensitive in the younger age group as greater changes are expected over shorter time periods. The importance of conscientiously documenting data on immunization, acute illnesses, and weight gain in a small booklet or growth chart kept by mothers cannot be overemphasized. It is an extremely valuable tool for assessment of the child with suspected TB. Most forms of childhood TB (except TB lymphadenitis) are strongly associated with weight loss and/or failure to thrive.17 The greatest risk of infection or disease for infants and young children is when the mother or primary caregiver has sputum smear-positive pulmonary TB. A diagnosis of TB would be strongly considered if the infant is failing to thrive. It is important
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to keep in mind that in poor communities many infants are at risk of significant malnutrition and mortality if the mother is too sick to adequately care for the baby or the mother dies. This is a common and difficult scenario especially in HIV-endemic countries where TB/HIV coinfection is common in mothers; TB treatment is less effective, maternal comorbidities are common, the infants are at risk of infection with TB and HIV, and there is the likelihood of inadequate care and nutrition. It is difficult to formulate a strict definition of weight loss or failure to thrive for TB diagnosis for the aforementioned reasons. A flat or falling weight that crosses centile lines during the past 3–6 months is the most reliable sign,17 but other causes such as inadequate nutrition and/or episodes of diarrhoea should be considered as well. In poor communities it is common for the weight gain to start to falter around 6 months of age when breast milk alone is not enough for nutritional demands and usual added feeds such as dilute cereal porridge are not sufficient to meet increased demands. The diagnosis of TB and of HIV should be considered in any child who fails to thrive or loses weight despite adequate nutritional rehabilitation, including deworming and iron supplementation as indicated. The cachexia and increased energy demands resulting from TB is more likely to cause marasmus than kwashiorkor, but children with kwashiorkor are extremely vulnerable to develop TB due to immune dysfunction. These children are markedly immunosuppressed, which may suppress some of the clinical and radiological manifestations of TB. Immune reconstitution inflammatory syndrome may also occur on nutritional rehabilitation.
Night sweats Night sweats is a commonly asked-for symptom of TB, especially in adults but also in children. It is a very non-specific symptom and probably only of value if the mother or caregiver spontaneously complains that the child sweats a lot at night. Only if the night sweats are
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severe enough to require a change of clothes, because the clothes are drenched, does the symptom need to be taken seriously. However, night sweats could also be caused by other diseases such as malaria and is therefore of even less value in malaria-endemic regions.
Other symptoms Fatigue is a common symptom of TB though also non-specific.15,17 It is more readily noticeable in children of 3 years or older and may manifest as a decrease in playfulness, activity, or performance at school. Other symptoms such as chest pain or haemoptysis are uncommon but do occur and are more likely to present in older children. Dyspnoea and a raised respiratory rate are not common but may be present especially in young infants.9 Lethargy and/or sleepiness are symptoms of particular concern in very young children in whom the risk of developing TB meningitis is high. The presence of a combination of well-defined symptoms provides added diagnostic value. Cough is a common symptom in children but is more likely to be associated with pulmonary TB if there is also weight loss.16 A recent study of South African children living in a high-burden community found that the combined presence of three well-defined symptoms at presentation (persistent, unremitting cough of over 2 weeks’ duration; objective weight loss, i.e. documented failure to thrive during the preceding 3 months; and reported fatigue) provided good diagnostic accuracy in HIV-uninfected children of 3 years of age or older, with clinical follow-up providing additional value.17 CLINICAL EXAMINATION There are no specific examination findings that confirm pulmonary TB. There are clinical signs that strongly suggest extrapulmonary TB such as the typical non-painful asymmetrical often matted cervical lymphadenopathy, sometimes with fistula formation, that occurs with TB lymphadenitis; the non-painful gibbus of spinal TB; or the painless ascites of peritoneal TB. These are outlined in other chapters. Clinical abnormalities associated with pulmonary TB may be absent or are non-specific, but careful clinical examination is worthwhile (see Box 16.2). Note the nutritional state of the child and record weight, temperature and respiratory rate. Examination of the chest is normal in most cases of pulmonary TB but may reveal focal abnormalities such as inspiratory crackles, bronchial breathing, or the reduced air entry and stony dullness suggesting an effusion. In case of obvious abnormalities on auscultation, note whether the child has signs of respiratory distress. The distinctive clinical features of uncomplicated TB pleural effusion are a school-aged child, not acutely ill but complaining of unilateral chest pain with associated stony dullness. It is typical of pulmonary and miliary TB to be associated with considerable focal lung pathology without marked respiratory distress, which is in contrast to the typical presentation of pneumonia due to other pathogens such as Streptococcus pneumoniae in which the child looks more ill and is more breathless. Bacterial pneumonia can also be complicated by a pleural effusion or empyema. Empyema usually occurs at a younger age than TB pleural effusion and children are usually acutely ill. A diagnostic tap is helpful to distinguish empyema from TB pleural effusion although TB can also rarely cause empyema. The finding of wheeze on auscultation is typical of asthma in children or of viral bronchiolitis or pneumonia in infants. Wheeze does occasionally occur with pulmonary TB as a result of airway narrowing by enlarged peribronchial lymph nodes. Take note of the length of history, response to bronchodilators (good response in
16
Box 16.2 Important clinical points in assessment of a child with suspected pulmonary TB 1. Note and record nutritional status – children with pulmonary TB are often thin or undernourished. 2. Think of pulmonary TB if abnormalities on examination of the respiratory system are focal, marked and persistent in the ambulant child not in respiratory distress. 3. Think of pulmonary TB if radiological abnormalities are focal, marked and persistent in the ambulant child not in respiratory distress. 4. Negative tuberculin skin test does not exclude pulmonary TB. 5. Examine for clinical markers of HIV infection. 6. Examine for abnormalities of the cardiovascular system.
those with asthma), and nutritional state of the child. Asthma and bronchiolitis are typically conditions of immunocompetent children and infants of normal nutrition so that, if the child is malnourished, it makes these diagnoses less likely while increasing the possibility of pulmonary TB. Similarly, inspiratory stridor, barking cough, and hoarse voice (or cry) are typically associated with viral laryngotracheitis in children, commonly known as croup. TB involving the larynx or large airways can present with similar signs as croup. Useful features that might help distinguish TB from croup are length of history, persistence of symptoms, and regional lymphadenopathy. Examine for features that suggest HIV infection such as generalized symmetrical lymphadenopathy, digital clubbing, bilateral non-tender parotid swelling, extensive oropharyngeal candidiasis, or typical skin findings such as shingles scarring, extensive fungal infections, molluscum contagiosum, papulo-pruritic eruption, or Kaposi’s sarcoma lesions (Fig. 16.1). Any child in an HIV-endemic setting with suspected pulmonary TB should be offered an HIV test.13 Being HIVinfected increases the risk for TB as well as increasing the possibility of other diagnoses. Clubbing is also a feature of bronchiectasis which is further characterized by a cough productive of copious, purulent, sometimes blood-stained sputum and focal crackles on auscultation. Always examine the cardiac system to exclude pericardial TB but also because cardiac failure can present with persistent cough,
Fig. 16.1 Kaposi of the skin.
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failure to thrive, and fatigue. Congenital heart disease may be diagnosed in the infant and cardiomyopathy or rheumatic heart disease in the older child.
TUBERCULIN SKIN TEST The TST provides important additional information in the assessment of the child with suspected TB. The recommended method is the Mantoux test using either 5 tuberculin units of tuberculin purified protein derivative (PPD) or 2 tuberculin units of PPD RT23. A positive or reactive TST indicates TB infection and is defined as 10 mm diameter of induration when read 48–72 hours after administration in any child irrespective of BCG immunization.13 There is a large amount of data to support this cut-off. It is currently recommended that an induration of 5 mm is significant and be read as positive if the child is HIV-infected or severely malnourished. This recommendation is not based on robust evidence but in practice a reading of 5–10 mm is uncommon. The potential problem of poor specificity (often false-positive) due to BCG immunization or infection with environmental (non-tuberculous) mycobacteria is often mentioned. In practice, a TST of 10 mm should still be considered indicative of TB infection in developing countries where BCG is routinely given during the neonatal period.18 The main limitation of the TST is that it has a low sensitivity (often false-negative), especially in children in whom the diagnosis of TB is already the most difficult, i.e. the child with HIV infection or severe malnutrition. In children with confirmed culture-positive pulmonary TB, the TST is positive in 60–80%. Sensitivity is unknown in children with non-confirmed pulmonary TB but likely to be lower. Some of the other causes of false-negative TST are listed in Table 16.2. There are novel T-cell-based assays that utilize M. tuberculosis-specific antigens in the blood and so are more specific for M. tuberculosis infection than the TST.19 They also show promise as being more sensitive in immunosuppressed children. There are still unanswered questions as to their possible superiority to the TST and they are expensive. More data are needed from different settings of their value and applicability in the routine diagnostic approach to childhood TB.
Box 16.3 Key features suggestive of TB The presence of three or more of the following should strongly suggest a diagnosis of TB:
chronic symptoms suggestive of TB; physical signs highly suggestive of TB; positive tuberculin skin test; and chest radiograph suggestive of TB.
From: Guidelines for national tuberculosis programmes on the management of tuberculosis in children. World Health Organization, Geneva, Switzerland. WHO/HTM/TB/2006.371.
TST, and limitations of sputum examination outlined in the next section mean that CXR is relied upon to make the diagnosis in most parts of the world.4,20,21 The radiological picture depends on how the child presented to the healthcare facility. The majority of children who present through contact screening have hilar lymph node enlargement only. In contrast, children who present because they are symptomatic often (> 50%) have a radiological picture of complicated lymph node disease, pleural disease, or miliary TB.22 The diagnostic accuracy of the CXR depends on the experience of the interpreter and there is only fair to moderate intra- and interobserver agreement.23 More accurate diagnosis requires a good knowledge of the different radiological pictures caused by pulmonary TB in children,24 and these are presented in more detail in Chapter 32 . The use of CXR to diagnose pulmonary TB in HIV-infected children is complicated by other HIV-related lung disease discussed in a later section.25 Lymphoid interstitial pneumonitis (LIP) can cause an image difficult to distinguish on radiological grounds from miliary TB (Figs 16.2–16.4). The chronic radiological changes caused by repeated bacterial infections in an HIV-infected child are easily confused with pulmonary TB as the changes often do not completely resolve after a course of antibiotics.
RADIOLOGICAL FINDINGS The chest radiograph (CXR) is widely used to make the diagnosis of pulmonary TB in children (see Box 16.3). The lack of specificity of suggestive symptoms, the lack of sensitivity and specificity of the Table 16.2 Causes of false-negative tuberculin skin tests Incorrect administration Incorrect interpretation Improper storage of tuberculin Tested too early following infection HIV infection Protein-energy malnutrition Severe TB disease Viral infections (e.g. measles, mumps, varicella) Bacterial infections (e.g. typhoid fever, pertussis, brucellosis) Diseases of lymphoid tissue (e.g. lymphoma, leukaemia, sarcoidosis) Primary immunodeficiencies Neonatal age group Immunosuppressive drugs Chronic renal failure Low protein states
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Fig. 16.2 Chest radiograph of a 4-year-old HIV-infected child with a clinical diagnosis of lymphoid interstitial pneumonitis showing bilateral, diffuse reticulonodular infiltration. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
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Fig. 16.3 Chest radiograph of a 3-year-old HIV-infected child with a clinical diagnosis of lymphoid interstitial pneumonitis showing bilateral adenopathy and reticular infiltration. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
16
and before food (or drink) is taken. This method is not always practical as it requires hospital admission, it is not always well accepted by parents or nursing staff (or the child), and yield from smear is low. For detection using smears, 5,000 – 10,000 bacilli/mL of specimen must be present, whereas a positive culture may result from as few as 10 organisms/mL.4 However, culture of M. tuberculosis is often unavailable and, when it is, the result is not available for 6–8 weeks. It is usually neither practical nor appropriate to wait so long for confirmation before commencing anti-TB therapy if pulmonary TB is strongly suspected. Alternative methods have been employed to obtain sputum. These include nasopharyngeal aspiration, laryngeal swab, bronchoalveolar lavage, and induced sputum using hypertonic saline and chest physiotherapy.26–30 The induced sputum technique shows the most promise for improved yield in young children and can be employed in the outpatient ambulatory setting. Additional studies are needed to compare the feasibility and diagnostic value of these and other sampling methods in different settings.31 Because of the paucibacillary nature of pulmonary TB in young children, there is still a need for culture of sputum specimens to optimize diagnostic yield whatever method of obtaining sputum is employed. Newer diagnostic techniques that might improve accuracy and rapidity of diagnosis are being studied in children and are dealt with in detail in subsequent chapters.32,33
SCORING SYSTEMS
Fig. 16.4 Chest radiograph showing the typical micronodular pattern of miliary TB. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
BACTERIOLOGICAL CONFIRMATION Pulmonary TB is usually not confirmed by culture in children. Sputum smear microscopy for AFB is positive in less than 10–15% of children with probable TB and the yield from culture is around 30–40%.26,27 The yield is higher from older children and adolescents, and in children with advanced intrathoracic disease.4,28 Sputum samples should be obtained whenever possible and can be obtained by expectoration from children older than 6 years of age. The traditional method for obtaining sputum from children unable to provide an adequate sputum sample is by obtaining swallowed sputum from two or three consecutive early-morning gastric aspirate samples before the child is ambulant
As a result of the difficulty of confirming TB in children, there have been a variety of scoring systems proposed to assist the clinician. They usually rely on the key features that suggest TB diagnosis but are not able to be properly validated due to a lack of a gold standard especially for pulmonary TB in children.34 This limits their applicability to being perhaps more useful as a screening tool rather than as a standard diagnostic test but at what cutoff is uncertain. As the score increases, so does specificity but the trade-off is a reduced sensitivity and a good screening tool should have a high sensitivity. The value of these tools is also affected by the setting. They are likely to perform better in the low-TBendemic setting. In contrast, they are not so useful in the HIVendemic setting as the common clinical indicators for assessment of TB are affected by the possibility of coinfection with HIV (Table 16.3).35 Table 16.3 Impact of HIV infection on the value of features commonly used for diagnosis of pulmonary tuberculosis in children Diagnostic feature of pulmonary TB
Impact of HIV infection
Persistent symptoms Sputum smear-positive contact Malnutrition or failure to thrive Positive TST Characteristic CXR abnormalities Satisfactory response to TB treatment
Less Less Less Less Less Less
specific specific or sensitive specific sensitive specific sensitive
TST, tuberculin skin test; CXR, chest radiograph. Adapted from Graham SM, Coulter JBS, Gilks CF. Pulmonary disease in HIV-infected children. Int J Tuberc Lung Dis 2001:5:12–23.
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PULMONARY TUBERCULOSIS IN HIV-INFECTED CHILDREN This chapter has already referred to the important problem of assessing a child with suspected pulmonary TB in the HIV-endemic setting. There are epidemiological aspects to consider, added clinical and diagnostic challenges, and problems of poorer response to anti-TB treatment in the child with TB/HIV, especially in the absence of ART.
EPIDEMIOLOGICAL ASPECTS The incidence of TB is low in HIV-infected children living in areas where the incidence of pulmonary TB is low in adults, such as the United States or Western Europe. In the early stages of the HIV epidemic in Africa, it was uncertain as to how common TB was in HIVinfected African children.36 Autopsy studies indicated that TB was uncommon in HIV-infected children while clinical studies using mainly clinical and radiological criteria documented that HIV prevalence was high in children diagnosed with TB. Recent clinical studies from the region with large numbers of confirmed TB cases show that the incidence of TB is much higher in HIV-infected than in HIV-uninfected children and that HIV prevalence is high in children with pulmonary TB.3,37,38 This depends on the prevalence of pulmonary TB and HIV in the community. Most studies have reported on hospitalized children, which is likely to show a higher prevalence than community-based studies. The higher TB incidence among HIV-infected children is explained by an increased risk of TB infection and an increased risk of progression to active disease following infection. Notwithstanding, the risk of TB is high for all children living in communities where TB/HIV is endemic so that, in contrast to adults, most children with TB are not HIV-infected.1,3,38
DIAGNOSTIC ASPECTS Problems of TB diagnosis are more pronounced in HIV-infected children for many reasons:
clinical features that suggest pulmonary TB lack specificity or sensitivity in HIV-infected children (Table 16.3);17 most HIV-infected children are infected by mother-to-child transmission, which means that the peak age prevalence for HIV in children is the youngest children, also the most difficult age group in which to identify cause of respiratory disease; HIV-infected children have a very high incidence of acute and chronic respiratory disease due to a wide range of causes other than pulmonary TB (Table 16.4);25 TST has a significantly lower sensitivity in HIV-infected children with TB than in HIV-uninfected children;3,38 there is substantial overlap of clinical and radiological findings between TB and other HIV-related lung disease;3,25 confirmation of aetiological diagnosis is difficult in the lowresource setting for many potential causes of chronic lung disease including pulmonary TB;39 and HIV-infected children not uncommonly have more than one cause of lung disease which can mask response to empiric therapy.
There are two common clinical scenarios that challenge the clinician working in TB/HIV-endemic countries. The first is the child, often an infant, with acute severe pneumonia who is not responding to firstline antibiotics such as penicillin and gentamicin. HIV infection status for this age may be difficult to confirm in the resource-poor setting but
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Table 16.4 Possible causes of lung disease in HIV-infected children in TB endemic countries Age group
Common causes
Less common causes
Infants
Bacterial pneumonia PCP Viral pneumonia, e.g. RSV TB CMV pneumonitis Mixed infection Bacterial pneumonia TB LIP Bronchiectasis Mixed infection
Measlesa LIP
Children
Viral pneumonia Measlesa Malignancy Pulmonary KS Lymphoma Nocardiosis PCP Fungal pneumonia Candidiasis Cryptococcosis Aspergillosis Penicilliosisb Melioidosisb Paracoccidioidomycosisc Histoplasmosisc
PCP, Pneumocystis jiroveci pneumonia; CMV, cytomegalovirus; LIP, lymphoid interstitial pneumonitis; KS, Kaposi’s sarcoma. a In regions with poor measles immunization coverage. b In older children in endemic regions of South-East Asia. c In older children in endemic regions of Latin America. Adapted from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
an antibody test is still useful. If HIV-seropositive with severe pneumonia and not improving, it is better to treat presumptively as HIVinfected and this will often be the case.40 Bacterial pneumonia is the commonest cause of severe pneumonia in HIV-infected children due to a wider range of bacterial pathogens than for HIV-uninfected children. Therefore, if first-line antibiotic treatment is failing, the choice of second-line treatment should cover for Gram-negative bacteria such as Klebsiella pneumoniae and for Staphylococcus aureus.11,37Pneumocystis jiroveci is also a common cause of severe pneumonia and death in HIV-infected infants.11,25Pneumocystis jiroveci pneumonia (PCP) is characterized by absence of fever and marked hypoxia and the recommended treatment is high-dose cotrimoxazole although treatment response is often poor. Infants with PCP are commonly coinfected with cytomegalovirus, influencing treatment response and management. Despite appropriate broad-spectrum antibiotics and treatment for PCP, the infant remains febrile with respiratory distress. This is when the possibility of pulmonary TB is often considered. Pulmonary TB can present as acute pneumonia especially in immune immature infants especially if HIV-infected. TST and CXR should be done but are likely to lack sensitivity and specificity, respectively. The typical CXR features of PCP are diffuse whereas pulmonary TB presents with more focal abnormalities (Figs 16.5 and 16.6). Sputum (or gastric aspirate) should be investigated by smear and culture. An important investigation is to identify a contact with a TB source case; this is frequently the mother or primary caregiver. Pulmonary TB is a much less likely cause of pneumonia in an infant if there is no source case identifiable.
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Fig. 16.5 Chest radiograph of an HIV-infected infant with confirmed
Pneumocystis jiroveci pneumonia showing bilateral diffuse infiltration. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
Fig. 16.6 Chest radiograph of an HIV-infected infant with confirmed
Pneumocystis jiroveci pneumonia showing hyperinflation. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
The other more common scenario is the older child with persistent respiratory symptoms either as an inpatient or as an outpatient not responding to broad-spectrum antibiotics. Pulmonary TB will be suspected and any child with suspected TB should be tested for HIV following consent. A negative HIV test will shorten considerably the list of possible diagnoses, increasing the likelihood of pulmonary TB as a cause. If HIV-infected, examination may reveal clinical features more consistent with other HIV-related lung disease.25 Digital clubbing is rare with uncomplicated pulmonary TB but common with LIP or bronchiectasis. Other features of LIP are persistent generalized lymphadenopathy and bilateral non-tender parotid swelling. Examine carefully for Kaposi’s sarcoma lesions including on the palate. Investigations again should include TST, CXR, and sputum for smear and culture if available. There are typical radiological features for LIP (Figs 16.2 and 16.3), bronchiectasis (Fig. 16.7), and pulmonary Kaposi’s sarcoma (Fig. 16.8). LIP typically causes diffuse bilateral abnormalities while
16
Fig. 16.7 Chest radiograph of a 6-year-old child with chronic cough and finger clubbing showing bronchiectatic changes in the right lower lobe. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
Fig. 16.8 Chest radiograph of an 8-year-old HIV-infected child with typical Kaposi’s sarcoma lesion on the palate showing bilateral adenopathy and infiltration with right pleural effusion. Reproduced with permission from Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int J Tuberc Lung Dis 2005;9:592–602.
pulmonary TB and bacterial pneumonia tend to cause more focal abnormalities, but there is the problem of possible coinfection especially with bacterial pneumonia but also with pulmonary TB. A lack of positive contact history becomes less useful for excluding pulmonary TB as the child gets older, especially in TB endemic areas where unrecognized TB exposure is common.
MANAGEMENT APPROACH Treatment for TB in children should not be undertaken lightly as it takes at least 6 months to complete and, although relatively rare in children, may cause serious side effects. It is important to resist the temptation to commence anti-TB therapy in children with suspected pulmonary TB on the basis that the diagnosis is difficult to exclude and that treatment response is a way of confirming or
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excluding the diagnosis. HIV-uninfected children with drugsusceptible disease usually have an excellent response to standard anti-TB therapy, with symptom resolution and weight gain within 2 months of therapy. However, it is well established that children with definite pulmonary TB do not always respond well to antiTB therapy, especially if they are severely immunosuppressed.3,38 Therefore a poor treatment response does not necessarily exclude TB. On the other hand, it is often quoted that children without pulmonary TB, but with recurrent bacterial infection, may improve on anti-TB therapy because of the broad-spectrum antibiotic action of rifampicin. Most children with suspected pulmonary TB are not acutely ill and so can be safely observed and reassessed over time. Careful clinical follow-up can provide very useful additional information, as symptom resolution occurs within 2–4 weeks in the vast majority of children without TB.17 The importance of HIV testing has already been emphasized, as the result affects the list of diagnostic possibilities. It also may be the first opportunity for the HIVinfected child and family to access HIV-related care such as cotrimoxazole chemoprophylaxis and antiretroviral therapy.
Box 16.4 Standard case definitions of pulmonary tuberculosis in children Pulmonary TB, sputum smear-positive two or more initial sputum smear examinations positive for AFB; or one sputum smear examination positive for AFB plus CXR abnormalities consistent with active pulmonary TB, as determined by a clinician; or one sputum smear examination positive for AFB plus sputum culture-positive for M. tuberculosis. Pulmonary TB, sputum smear-negative at least three sputum specimens negative for AFB; and radiological abnormalities consistent with active pulmonary TB; and no response to a course of broad-spectrum antibiotics; and decision by a clinician to treat with a full course of anti-TB chemotherapy. AFB, acid-fast bacilli, CXR, chest radiograph.
STANDARD CASE DEFINITIONS OF PULMONARY TUBERCULOSIS IN CHILDREN Once a child has been diagnosed as having pulmonary TB, he/she should be registered with the National TB Control Programme for treatment according to standard regimens (Chapter 61). The registration for pulmonary TB will be either as ‘sputum smear-positive’ or as ‘sputum smear-negative’. The latter is the commonest registered
REFERENCES 12. 1. Marais BJ, Gie RP, Schaaf HS, et al. The spectrum of disease in children treated for tuberculosis in a highly endemic area. Int J Tuberc Lung Dis 2006;10:732–738. 2. Harries AD, Hargreaves NJ, Graham SM, et al. Childhood tuberculosis in Malawi: nationwide casefinding and treatment outcomes. Int J Tuberc Lung Dis 2002;6:24–431. 3. Palme IB, Gudetta B, Bruchfeld J, et al. Impact of human immunodeficiency virus 1 infection on clinical presentation, treatment outcome and survival in a cohort of Ethiopian children with tuberculosis. Pediatr Infect Dis J 2002;21:1053–1061. 4. Mandalakas AM, Starke JR. Current concepts of childhood tuberculosis. Semin Pediatr Infect Dis 2005;16:93–104. 5. Marais BJ, Gie RP, Schaaf HS, et al. The clinical epidemiology of childhood pulmonary tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 2004;8:278–285. 6. Verver S, Warren RM, Munch Z, et al. Proportion of tuberculosis transmission that takes place in households in a high-incidence area. Lancet 2004;363:212–214. 7. Marais BJ, Gie RP, Schaaf HS, et al. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the prechemotherapy era. Int J Tuberc Lung Dis 2004;8: 392–402. 8. Rieder HL. Interventions for Tuberculosis Control and Elimination. Paris: International Union against Tuberculosis and Lung Disease, 2002. 9. Schaaf HS, Gie RP, Beyers N, et al. Tuberculosis in infants less than 3 months of age. Arch Dis Child 1993;69:371–374. 10. Maltezou HC, Spyridis P, Kafetzis DA. Tuberculosis during infancy. Int J Tuberc Lung Dis 2000;4:414–419. 11. Zar HJ, Hanslo D, Tannenbaum E, et al. Aetiology and outcome of pneumonia in human
162
13.
14.
15.
16.
17.
18.
19.
20.
21.
form of TB in children but in most cases would be more accurately referred to as ‘sputum not examined’ rather than ‘sputum smearnegative’. There are recommended criteria for standard case definitions and those for pulmonary TB are listed in Box 16.4.13
immunodeficiency virus-infected children hospitalized in South Africa. Acta Paediatr 2001;90:119–125. Chintu C, Mudenda V, Lucas S, et al. Lung diseases at necropsy in African children dying from respiratory illnesses: a descriptive necropsy study. Lancet 2002;360:985–990. World Health Organization. Guidance for National Tuberculosis Programmes on the Management of Tuberculosis in Children. Geneva: World Health Organization, 2006. WHO/HTM/TB/2006.371. Schaaf HS, Donald PR, Scott F. Maternal chest radiography as supporting evidence for the diagnosis of tuberculosis in childhood. J Trop Pediatr 1991;37:223–225. Marais BJ, Gie RP, Obihara CC, et al. Well defined symptoms are of value in the diagnosis of childhood pulmonary tuberculosis. Arch Dis Child 2005;90: 1162–1165. Marais BJ, Obihara CC, Gie RP, et al. The prevalence of symptoms associated with pulmonary tuberculosis in randomly selected children from a high burden community. Arch Dis Child 2005;90:1166–1170. Marais BJ, Gie RP, Hesseling AC, et al. A refined symptom-based approach to diagnose pulmonary tuberculosis in children. Pediatrics 2006;118: e1350–e1359. Farhat M, Greenaway C, Pai M, et al. False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? Int J Tuberc Lung Dis 2006;10:1192–1204. Pai M, Riley LW, Colford JM Jr. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004;4:761–776. Theart AC, Marais BJ, Gie RP, et al. Criteria used for the diagnosis of childhood tuberculosis at primary health care level in a high-burden, urban setting. Int J Tuberc Lung Dis 2005;9:1210–1214. Weismuller MM, Graham SM, Claessens NJ, et al. Diagnosis of childhood tuberculosis in Malawi: an audit of hospital practice. Int J Tuberc Lung Dis 2002;6:432–438.
22. Marais BJ, Gie RP, Hesseling AC, et al. Radiographic signs and symptoms in children treated for tuberculosis: possible implications for symptom-based screening in resource-limited settings. Pediatr Infect Dis J 2006;25:237–240. 23. Swingler GH, du TG, Andronikou S, et al. Diagnostic accuracy of chest radiography in detecting mediastinal lymphadenopathy in suspected pulmonary tuberculosis. Arch Dis Child 2005;90:1153–1156. 24. Gie R. Diagnostic Atlas of Intrathoracic Tuberculosis in Children: A Guide for Low Income Countries. Paris: International Union against Tuberculosis and Lung Disease, 2003. 25. Graham SM. Non-tuberculosis opportunistic infections and other lung diseases in HIV-infected infants and children. Int.J Tuberc Lung Dis 2005;9:592–602. 26. Zar HJ, Tannenbaum E, Apolles P, et al. Sputum induction for the diagnosis of pulmonary tuberculosis in infants and young children in an urban setting in South Africa. Arch Dis Child 2000;82:305–308. 27. Abadco DL, Steiner P. Gastric lavage is better than bronchoalveolar lavage for isolation of Mycobacterium tuberculosis in childhood pulmonary tuberculosis. Pediatr Infect Dis J 1992;11:735–738. 28. Marais BJ, Hesseling AC, Gie RP, et al. The bacteriologic yield in children with intrathoracic tuberculosis. Clin Infect Dis 2006;42:e69–e71. 29. Thakur A, Coulter JB, Zutshi K, et al. Laryngeal swabs for diagnosing tuberculosis. Ann Trop Paediatr 1999;19:333–336. 30. Franchi LM, Cama RI, Gilman RH, et al. Detection of Mycobacterium tuberculosis in nasopharyngeal aspirate samples in children. Lancet 1998;352:1681–1682. 31. Vargas D, Garcia L, Gilman RH, et al. Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru. Lancet 2005;365:150–152. 32. Montenegro SH, Gilman RH, Sheen P, et al. Improved detection of Mycobacterium tuberculosis in Peruvian children by use of a heminested IS6110 polymerase chain reaction assay. Clin Infect Dis 2003;36:16–23.
CHAPTER
Clinical features and index of suspicion of tuberculosis in children 33. Oberhelman RA, Soto-Castellares G, Caviedes L, et al. Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method. Pediatrics 2006;118:e100–e106. 34. Hesseling AC, Schaaf HS, Gie RP, et al. A critical review of diagnostic approaches used in the diagnosis of childhood tuberculosis. Int J Tuberc Lung Dis 2002;6:1038–1045. 35. Van Rheenen P. The use of the paediatric tuberculosis score chart in an HIV-endemic area. Trop Med Int Health 2002;7:435–441. 36. Coovadia HM, Jeena P, Wilkinson D. Childhood human immunodeficiency virus and tuberculosis co-
infections: reconciling conflicting data. Int J Tuberc Lung Dis 1998;2:844–851. 37. Madhi SA, Petersen K, Madhi A, et al. Increased disease burden and antibiotic resistance of bacteria causing severe community-acquired lower respiratory tract infections in human immunodeficiency virus type 1-infected children. Clin Infect Dis 2000;31:170–176. 38. Madhi SA, Huebner RE, Doedens L, et al. HIV-1 co-infection in children hospitalised with tuberculosis in South Africa. Int J Tuberc Lung Dis 2000;4:448– 454. 39. Kiwanuka J, Graham SM, Coulter JB, et al. Diagnosis of pulmonary tuberculosis in children in an
16
HIV-endemic area, Malawi. Ann Trop Paediatr 2001;21:5–14. 40. Thirsk ER, Kapongo MC, Jeena PM, et al. HIVexposed infants with acute respiratory failure secondary to acute lower respiratory infections managed with and without mechanical ventilation. S Afr Med J 2003;93:617–620. 41. Graham SM, Coulter JBS, Gilks CF. Pulmonary disease in HIV-infected African children. Int J Tuberc Lung Dis 2001;5:12–23.
FURTHER READING Donald PR, Fourie PB, Grange JM. Tuberculosis in Childhood. Pretoria, IL: JL van Schaik, 1999. Miller FJW. Tuberculosis in Children. Edinburgh: Churchill Livingstone, 1982.
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Clinical features and index of suspicion in adults (HIV-negative and HIV-positive) Dermot Maher
INTRODUCTION The role that clinicians play in good clinical care of TB patients through prompt diagnosis and effective treatment is also key to the public health approach to TB control. This chapter reviews the importance of clinical features and index of suspicion for both individual patient care and public health, paving the way for a detailed description of clinical presentations in Section 5.
THE CLINICIAN’S ROLE IN GOOD PATIENT CARE AND GOOD PUBLIC HEALTH The focus of the primary stratagem of TB control is on people with TB and the aim is to reduce the average number of people infected by each infectious case so that the case reproduction number is less than 1.1 With proper treatment, a person with infectious TB very quickly becomes non-infectious and so can no longer spread disease to others. With the correct application of anti-TB drugs, it is possible to cure over 90% of new smear-positive TB patients who neither have bacillary resistance to first-line drugs nor are infected with human immunodeficiency virus (HIV). Before the spread of HIV, countries that met the two international targets of at least 70% case detection and at least 85% treatment success could expect to see a decline in TB incidence rates of 5–10% per year or more.1,2 This expected epidemiological impact has been demonstrated in, for example, Peru.3 It has been supported by observed reductions in prevalence, for example in the areas of China that have implemented the directly observed treatment, short course (DOTS), strategy.4 Prompt, accurate diagnosis and effective, standardized treatment of TB are thus not only essential for good patient care but they are the key elements in the public health response to TB and the cornerstone of TB control.5,6 The clinician diagnosing and treating TB patients works at the interface of clinical medicine and public health, since the identification and cure of infectious cases has been recognized as the most cost-effective measure currently available to control TB.7,8 Case-finding and treatment success lie at the heart of World Health Organization’s (WHO) Stop TB Strategy,9 which incorporates and goes beyond the DOTS strategy.10 Good clinical management of TB patients, with rapid diagnosis and successful treatment outcomes for individuals with TB, contributes to global TB control. Conversely, inadequate clinical management (delayed diagnosis and failure to ensure adherence to a recommended standardized treatment regimen) results in a poor
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outcome for the individual patient and exacerbation of the TB epidemic. Patients with infectious TB who receive inadequate treatment are at high risk of remaining infectious, often with drug-resistant TB. The pool of infectious cases therefore increases. The generation of drug resistance is converting a treatable epidemic into an untreatable epidemic. The clinician therefore has both an individual patient responsibility and a public health responsibility in ensuring proper case management.11,12
APPROACH TO DIAGNOSIS OF TUBERCULOSIS The first step in diagnosing TB is recognizing the clinical features of the disease. For the clinician, the index of suspicion consists of detecting clinical features consistent with TB in a patient following the question, ‘Could TB be the cause of this patient’s illness?’ Once this question is asked, the answer is pursued by diagnostic evaluation which in all cases involves seeking microbiological, or sometimes histopathological, confirmation of TB.11 However, in some cases TB is presumed as the most likely diagnosis without such gold standard confirmation, e.g. sputum smear-negative pulmonary TB.13
THE CLINICAL FEATURES OF TUBERCULOSIS Tuberculosis presents with clinical features due to systemic disturbance and related to pathology at the local site or sites of disease. Systemic symptoms include fever, night sweats, tiredness, loss of appetite, weight loss, and secondary amenorrhoea. Systemic signs include fever, wasting, and tachycardia. Since extrapulmonary and pulmonary TB can occur concurrently, the diagnostic evaluation of a patient with extrapulmonary TB should always include evaluation for pulmonary TB with a chest radiograph and sputum microbiology. The clinical features of TB are generally the same irrespective of whether the patient presents in a setting with low or high TB incidence. However, patients in settings with high TB incidence tend to present at a more advanced stage of illness and therefore with more pronounced clinical features, on account of barriers to access within often weak health systems in developing countries, where 95% of TB patients live. The following account of the clinical features of TB draws substantially on the WHO publication TB/HIV: A Clinical Manual.14
CHAPTER
Clinical features and index of suspicion in adults (HIV-negative and HIV-positive)
Pulmonary tuberculosis Since the predominant site of TB is the lungs, the commonest presenting symptoms are those of pulmonary TB – cough, sputum, chest pain, haemoptysis, and breathlessness. Haemoptysis is often the result of cavitating lung disease causing erosion of pulmonary blood vessels – one large cavity or several smaller cavities may be associated with haemoptysis. Chest wall pain may be due to extensive lung inflammation, particularly if the pleural surface is involved. Breathlessness is an uncommon symptom in pulmonary TB, and usually indicates extensive lung disease or disease complications/ forms, such as pneumothorax, pleural effusion, or endobronchitis. Weight loss and fever are more common in HIV-positive pulmonary TB patients than in those who are HIV-negative. Conversely, cough and haemoptysis are less common in HIV-positive pulmonary TB patients than in those who are HIV-negative. This is probably because there is less cavitation, inflammation, and endobronchial irritation in HIV-positive patients. Since the sensitivity of cough as a symptom of TB is very high, all persons with otherwise unexplained productive cough lasting 2–3 weeks or more should be evaluated for TB.15 The specificity of cough as a symptom of TB is low, since cough occurs in a wide range of respiratory conditions, including acute respiratory tract infections (upper and lower), asthma, chronic obstructive pulmonary disease, bronchiectasis, and lung cancer. In patients presenting with chronic cough, the proportion of cases attributable to TB will depend on the prevalence of TB in the community.15 An overall focus on adults and children presenting with chronic cough maximizes the chances of identifying patients with pulmonary TB. Inadequate evaluation of patients with respiratory symptoms for TB results in missed opportunities for earlier detection of TB and leads to increased disease severity at diagnosis for patients and a greater likelihood of transmission of Mycobacterium tuberculosis to family members and others in the community. Failure to perform a proper diagnostic evaluation before initiating treatment also potentially exposes the patient to the risks of unnecessary or wrong treatment with no benefit or even harm. Moreover, such an approach may delay accurate diagnosis and proper treatment of the non-TB condition. The physical signs in patients with pulmonary TB are non-specific. They do not help to distinguish pulmonary TB from other chest diseases. Finger clubbing may be present as a general sign and chest signs on auscultation may include crackles, wheezes, bronchial breathing, and amphoric breathing. There are often no abnormal signs in the chest. If there is concurrent extrapulmonary TB there may be consistent clinical findings, e.g. lymphadenopathy, especially in patients with HIV infection. Extrapulmonary tuberculosis In addition to systemic features, patients with extrapulmonary TB present with features related to the pathology at the local site of disease. Tuberculosis lymphadenopathy Cervical lymph node enlargement (regardless of HIV serostatus) is the commonest presentation. Lymphadenopathy can also be found in the axilla and the groin. Initially, lymph nodes are firm and discrete, but later they become matted together and fluctuant. The overlying skin may break down with the formation of abscesses and chronic discharging sinuses, which heal with scarring. In the patient with HIV infection, lymphadenitis can be acute and resemble acute pyogenic infection. A number of other conditions present with lymphadenopathy. In areas with high HIV prevalence the commonest differential diagnosis is persistent generalized lymphadenopathy (PGL), which
17
Box 17.1 Features of lymph nodes which indicate increased likelihood of tuberculosis
Size greater than 4 cm in diameter. Asymmetrical enlargement. Rapid increase in size. Pain or tenderness not associated with local inflammation. Matted or fluctuant. Marked systemic disturbance such as fever and weight loss. Associated hilar and mediastinal lymphadenopathy.
presents as symmetrical non-painful lymph node enlargement, often involving the posterior cervical chain or axilla. As it is impossible to investigate all patients with enlarged lymph nodes it is important to distinguish features which indicate the possibility of TB (see Box 17.1). The differential diagnosis includes lymphoma, carcinoma, Kaposi’s sarcoma in HIV-positive patients, and sarcoidosis.
Serous effusions Serous (i.e. inflammatory) effusions may occur in the pleural cavity, in the pericardial cavity, and in the peritoneal cavity. They are more common in HIV-positive patients than in HIV-negative patients, although the clinical presentation in both groups of patients is similar. Pleural effusions are often accompanied by chest pain and breathlessness. Physical signs reveal the presence of fluid within the pleural space, giving rise to reduction of air entry on auscultation and dullness on percussion. HIV-positive patients with a pleural effusion usually have a primary infection newly acquired. There is a large number of differential diagnoses including malignancy, pneumonia, and pulmonary embolism. Pericardial effusions are potentially serious because of fluid developing in a tightly enclosed sac causing external pressure on the heart with a fall in cardiac output and death unless the pressure is relieved. Presentation is usually with precordial chest pain, shortness of breath, orthopnoea, and oedema of the legs. Physical signs reveal a low cardiac output, elevated jugular venous pressure, peripheral oedema, an impalpable apex beat, and quiet heart sounds. Many conditions cause pericardial effusion, and a careful clinical assessment needs to be done to particularly rule out malignancies and chronic renal failure. Pericardial effusion may be the result of rupture of a mediastinal lymph node into the pericardial space or haematogenous dissemination, and in HIV-positive patients it is probably the result of a primary TB infection. Peritoneal effusions (or ascites) present with a distended abdomen and the physical signs of fluid within the abdominal cavity. Peritoneal TB often arises as a result of spread from mesenteric lymph nodes, and the clinical presence of palpable abdominal masses is a pointer to the diagnosis. There is a very wide range of differential diagnoses from transudates caused by liver cirrhosis, congestive cardiac failure, and nephrotic syndrome to exudates caused by malignancy and other infections within the abdominal cavity. Tuberculosis meningitis Tubercle bacilli usually gain access to the cerebrospinal fluid via small subependymal cerebral tuberculomas, but direct spread can occur as part of miliary TB. There is usually evidence of a chronic meningitis with gradual onset of fever, headache, and altered consciousness. Involvement of the base of the brain can cause cranial
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GENERAL CLINICAL FEATURES AND DIAGNOSIS
nerve palsies. Tuberculomas, vascular occlusions, and hydrocephalus can all give rise to focal neurological defects and seizures. There is again a wide differential diagnosis, especially in patients with HIV infection. The two main differential diagnoses are partially treated bacterial meningitis and in HIV-positive patients cryptococcal meningitis.
Miliary tuberculosis Miliary TB occurs when tubercle bacilli are spread through the blood stream. This disease occurs either from a recent primary infection or from reactivation of a tuberculous lesion with erosion of a blood vessel. The disease presents with gradual fever, malaise, and weight loss. Physical examination may reveal enlargement of the liver and spleen, and occasionally characteristic choroidal tubercles can be found on fundoscopy. A high index of suspicion is required. The main differential diagnoses are bacteraemia, especially typhoid fever, and the acquired immunodeficiency virus (AIDS) wasting syndrome. Tuberculosis of the spine Starting as an inflammation of the intervertebral disc (most commonly in the thoracic spine), TB then spreads along the anterior and longitudinal ligaments to involve adjacent vertebral bodies. The disease is important because the consequences of missing the diagnosis can lead to irreversible paraplegia. The disease presents as a painful spine, sometimes with a gibbus (a visible protrusion of the spine) and in the case of neurological involvement a spinal cord compression syndrome with paraplegia. Other forms of extrapulmonary tuberculosis Box 17.2 shows the key clinical features of other forms of extrapulmonary TB. IMPACT OF HIV ON CLINICAL PRESENTATION OF TUBERCULOSIS Tuberculosis can appear at any stage of HIV infection, and its presentation varies with the stage. When cell-mediated immunity is only partially compromised, pulmonary TB presents in a typical manner with upper lobe infiltrates and cavitation, without significant lymphadenopathy or pleural effusion. In late stages of HIV infection, a primary TB-like pattern, with diffuse interstitial or miliary infiltrates, little or no cavitation, and intrathoracic lymphadenopathy, is more common. Overall, sputum smears may be positive less frequently among TB patients with HIV infection than among those without; thus the diagnosis of TB may be unusually difficult, especially in view of the variety of HIV-related pulmonary conditions mimicking TB.
Box 17.2 Clinical features of other forms of extrapulmonary tuberculosis Site of disease
Clinical features
Bone Peripheral joints Ileocaecal disease Renal and urinary tract Adrenal gland Female genital tract Male genital tract
Chronic osteomyelitis Usually monoarthritis Diarrhoea and palpable mass right iliac fossa Micturition frequency, dysuria, haematuria, loin pain/swelling Features of hypoadrenalism Infertility, pelvic inflammatory disease
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Epididymitis
Extrapulmonary TB is common among HIV-positive patients. In various series, extrapulmonary TB – alone or in association with pulmonary disease – has been documented in 40–60% of all cases in HIV coinfected individuals. The most common forms are lymphatic, disseminated, pleural, and pericardial. Mycobacteraemia and meningitis are also frequent, particularly in advanced HIV disease. The diagnosis of TB in HIV-positive patients may be difficult not only because of the increased frequency of sputum-smear negativity (up to 40% in culture-proven pulmonary cases) but also because of atypical radiographic findings, a lack of classic granuloma formation in the late stages, and a negative tuberculin skin test. Delays in treatment may prove fatal.
INDEX OF SUSPICION An awareness of the main groups at risk of TB serves to alert clinical suspicion of TB. This section highlights some common TB risk factors – those interested in a full account of TB risk factors should see Rieder’s Epidemiologic Basis of Tuberculosis Control.16 Environmental risk factors include substance abuse (smoking, alcohol abuse, and injecting drug use) and nutrition, and medical conditions identified as risk factors include silicosis, diabetes, malignancy, renal failure, measles, gastrectomy, and corticosteroid treatment. In this section some common risk factors in suspecting TB are discussed separately for developing and developed countries since their usefulness generally depends on the setting. However, because HIV is such a strong risk factor for TB, anywhere in the world the presence of HIV infection in a patient with consistent clinical features should always alert the clinician to the possibility of TB.
DEVELOPING COUNTRIES Poverty Although poverty is a risk factor for TB it is not sufficiently specific, where poverty is common, to be of much use in alerting the clinician as to the likelihood of TB. Hence the recommendation is that all persons with otherwise unexplained productive cough lasting 2–3 weeks or more should be evaluated for TB.15 HIV infection With 11% of the world’s population, 70% of the world’s HIV-positive people, and 85% of the world’s HIV-positive new TB cases, sub-Saharan Africa bears a disproportionate burden of HIV infection and of HIV-related TB.17,18 In sub-Saharan Africa, TB is often the first manifestation of HIV infection, the leading cause of respiratory illness among HIV-positive patients in hospitals and primary health clinics, and the leading cause of death among HIV-positive patients.17,18 DEVELOPED COUNTRIES In developed countries, TB is uncommon among young adults of European descent, who have only rarely been exposed to M. tuberculosis infection during recent decades. In contrast, because of a high risk in the past, the prevalence of M. tuberculosis infection is relatively high among elderly Caucasians, who remain at increased
CHAPTER
Native-born
14000 Number of reported cases
60 50 40 30 20
Norway
Sweden
Denmark
Netherlands
UK
Belgium
Luxembourg
France
Italy
Germany
Austria
Iceland
0
Greece
10
Fig. 17.2 Contribution of the foreign-born to TB in countries in Europe in 2002.
HIV infection The impact of HIV on TB in Western Europe has been largely limited to certain countries (e.g. Portugal, Spain) and cities (e.g. Amsterdam, Paris).24 In most countries in Western Europe, the proportion of AIDS cases diagnosed with TB is low. The two notable exceptions are Portugal and Spain,25 where the overlap between the population infected with HIV and the population infected with M. tuberculosis is greater than in the other countries of Western Europe. Poverty Poverty, with its attendant social disadvantage, has been strongly associated with the incidence of TB.26,27 It can be difficult to disentangle the relative effect of low socioeconomic indicators on the risk of TB incidence, but overcrowded living conditions are conducive to increased transmission of TB bacilli, and poor nutrition tends to increase the risk of progression of M. tuberculosis infection to disease. Poverty may also result in reduced access to healthcare services, leading to delayed diagnosis and treatment, with prolongation of the period of infectiousness of TB patients and increased likelihood of an unsatisfactory clinical outcome.28
CONCLUSION
16000 Foreign-born
12000 10000 8000 6000 4000 2000 0
70
Portugal
Immigration Effectively applied chemotherapy in the latter half of the twentieth century further accelerated the already declining TB case notifications in industrialized countries. From the mid-1980s onwards, however, several countries saw a slowdown in the decline, while others saw the trend reversed, with case notifications increasing for the first time in many years. For example, in the USA, after 30 years of steady decline, TB incidence increased regularly between 1985 and 1992.19 Factors responsible for this reversal included increased poverty among marginalized groups in inner city areas, immigration from countries with high TB prevalence, the impact of HIV, and most importantly the failure to maintain the necessary public health infrastructure (as in the case of New York City), under the mistaken belief that TB was a problem of the past.20 Many countries in Europe, including Denmark, The Netherlands, Sweden, and the UK, reported this slowdown in the decline, or even a steady rise, in TB cases.21 The high proportion of cases in the foreign-born (e.g. 24% in France, 51% in The Netherlands, 54% in Sweden, 68% in Switzerland) suggested immigration as the main cause of this change in trend.22 Annual case rates among the foreign-born often exceed 50 per 100,000 and may even exceed 100 per 100,000 (e.g. in The Netherlands), in contrast to rates in native-born populations of usually less than 15 per 100,000. In many countries, TB has declined steadily among the native-born, while rising among the foreign-born (see Fig. 17.1). The foreign-born now account for a large proportion of TB cases in developed countries, as shown, for example, by many countries in Europe (see Fig. 17.2).23
17
80
Finland
risk of developing TB. In many developed countries TB has re-emerged as an important public health problem mainly as a result of cases among immigrants from countries with high TB incidence. However, risk of TB is also increased in people who are HIV-positive or disadvantaged, e.g. the homeless. The commitment to ensuring universal access to quality TB diagnosis and treatment implies particular efforts to reach those groups at increased risk of TB, including the poor, the homeless, and immigrants (whether legal or illegal).
TB cases among foreigners in 2002 (%)
Clinical features and index of suspicion in adults (HIV-negative and HIV-positive)
1998
1999
2000
2001
2002
Fig. 17.1 The number of TB cases in 16 European countries among native-born and foreign-born. The 16 countries include Austria, Belgium, Czech Republic, Denmark, Finland, Greece, Hungary, Ireland, Netherlands, Slovakia, Slovenia, UK, Iceland, Norway, and Switzerland. EuroTB. Surveillance of tuberculosis in Europe. Report on tuberculosis cases notified in 2002, Saint-Maurice, France. December 2004.
Although new and improved drugs, methods of diagnosis, and vaccines will be developed eventually, which could greatly decrease the global burden of TB, until then control of the disease is mainly based on interruption of its transmission through the rapid identification and cure of infectious cases. The rapid identification of TB that is essential to ensure good outcomes for TB patients and good public health control depends on clinicians’ knowledge of the clinical features of TB, linked to an index of suspicion. In developing countries it is difficult to ascertain the extent to which people die from unrecognized TB because most of these cases never become known. Even in developed countries a small but significant proportion of TB cases are diagnosed at death. For example, in a 4year period from 1985 to 1988, 5% of all TB cases notified in the USA never received anti-TB treatment.29 In each country closing the gap between diagnosed and true incident TB cases depends on improved clinical diagnostic performance, for which the starting point is knowledge of the clinical features of the disease, linked to a judicious index of suspicion.
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REFERENCES 1. Dye C, Garnett GP, Sleeman K, et al. Prospects for worldwide tuberculosis control under the WHO DOTS strategy. Directly observed short-course therapy. Lancet 1998;352:1886–1891. 2. Dye C, Maher D, Weil D, et al. Targets for global tuberculosis control. Int J Tuberc Lung Dis 2006; 10(4):460–462. 3. Sua´rez 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. 4. China Tuberculosis Control Collaboration. The effect of tuberculosis control in China. Lancet 2004;364:417–422. 5. Maher D. Smear-positive pulmonary tuberculosis: good clinical management is good public health. Africa Health 1999;21(4):6–9. 6. International Standards for Tuberculosis Care. The Hague: Tuberculosis Coalition for Technical Assistance, 2006. 7. Murray CJ, De Jonghe E, Chum HJ, et al. Cost effectiveness of chemotherapy for pulmonary tuberculosis in three sub-Saharan African countries. Lancet 1991;338:1305–1308. 8. World Bank. World Development Report 1993: Investing in Health. New York: Oxford University Press, 1993. 9. Raviglione M, Uplekar, M. The new Stop TB Strategy of WHO. Lancet 2006;367:952. 10. World Health Organization. What is DOTS? A Guide to Understanding the WHO-Recommended Tuberculosis Control Strategy Known as DOTS. Geneva: World
168
11.
12.
13.
14.
15.
16.
17.
18.
19.
Health Organization, 1999. WHO/CDS/CPC/TB/ 99.270. International Standards for Tuberculosis Care. The Hague: Tuberculosis Coalition for Technical Assistance, 2006. Hopewell P, Pai M, Maher D, et al. International standards for tuberculosis care. Lancet Infect Dis 2006;6:710–725. World Health Organization. Treatment of Tuberculosis: Guidelines for National Programmes, 3rd edn. Geneva: WHO, 2003. WHO/CDS/TB/2003.313. Harries AD, Maher D. TB/HIV: A Clinical Manual, 2nd edn. Geneva: World Health Organization, 2004. WHO/HTM/TB/2004.329. Luelmo F. What is the role of sputum microscopy in patients attending health facilities? In: Frieden TR (ed.). Toman’s Tuberculosis: Case Detection, Treatment and Monitoring, 2nd edn. Geneva: World Health Organization, 2004: 7–10. Rieder HL. Epidemiologic Basis of Tuberculosis Control. Paris: International Union against Tuberculosis and Lung Disease, 1999. World Health Organization. Global Tuberculosis Control: Surveillance, Planning and Financing. Geneva: World Health Organization, 2006. WHO/HTM/ TB/2006.362. 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. Cantwell MF, Snider DE Jr, Cauthen GM, et al. Epidemiology of tuberculosis in the United States, 1985 through 1992. JAMA 1994;272:535–539.
20. Frieden TR, Fujiwara PL, Washko RM, et al. Tuberculosis in New York City: turning the tide. N Engl J Med 1995;333:229–233. 21. Raviglione MC, Sudre P, Rieder HL, et al. Secular trends of tuberculosis in Western Europe. Bull World Health Organ 1993;71:297–306. 22. Rieder HL, Zellweger JP, Raviglione MC, et al. Tuberculosis control in Europe and international migration. Report of European Task Force. Eur Respir J 1994;7:1545–1553. 23. EuroTB. Surveillance of tuberculosis in Europe. Report on tuberculosis cases notified in 2002. SaintMaurice, France. December 2004. 24. Centers for Disease Control and Prevention. Tuberculosis—Western Europe, 1974-1991. MMWR Morbid Mortal Wkly Rep 1993;42:628–631. 25. European Centre for the Epidemiological Monitoring of AIDS. HIV/AIDS Surveillance in Europe: Quarterly Report, No. 46, 30 June 1995. 26. Enarson DA, Wang JS, Dirks JM. The incidence of active tuberculosis in a large urban area. Am J Epidemiol 1989;129:1268–1276. 27. Cantwell MF, Snider DE, Cauthen GM, et al. Epidemiology of tuberculosis in the United States, 1985 through 1992. JAMA 1994;272:535–539. 28. Bergner L, Yerby AS. Low income and barriers to use of health services. N Engl J Med 1968:278:541–546. 29. Rieder HL, Kelly GD, Bloch AB, et al. Tuberculosis diagnosed at death in the United States. Chest 1991;100;678–811.
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18
Microbiological testing for Mycobacterium tuberculosis Andrew C Whitelaw and Willem A Sturm
INTRODUCTION Approximately one-third of the world’s population is infected with Mycobacterium tuberculosis, making this microbe one of the most successful human pathogens. This is, amongst others, attributable to its ability to survive in the host for prolonged periods of time without inducing any symptoms and its capability to switch between this asymptomatic non-infectious phase and a clinically apparent infectious phase. Its close association with poverty and human immunodeficiency virus (HIV)-induced immunodeficiency makes it into one of the most important public health problems in underdeveloped parts of the world. Addressing these issues is crucial to control TB. However, interventions that convert an infectious individual into a non-infectious one as early as possible in the course of the disease are also important. It would be even better to prevent those who are developing clinical disease from becoming infectious. From a therapeutic point of view, the tools to achieve this are available. Despite that, the global TB burden remains extremely high, as well as the infectious disease with the highest mortality worldwide. This indicates that TB control programmes are still far from successful. The main reason for that is an inability to detect sufficient numbers of cases before transmission to uninfected individuals has occurred. The effectiveness of early case finding depends on a number of factors, of which access to healthcare facilities, contact tracing, and laboratory diagnosis are the most important. Efficient and rapid laboratory diagnostic tests are essential to make improvements in other interventions work. This is becoming even more important with the growing prevalence of effectively transmissible drug-resistant strains of M. tuberculosis. These strains, such as W-Beijing and F15/LAM4/KZN, pose a serious threat to the efficacy of TB control programmes.1,2 Their presence in significant numbers results in initiation of treatment of increasing numbers of patients on inadequate treatment regimens if rapid susceptibility tests are not available. In general, laboratory tests for the diagnosis of infections can be grouped in two main categories, as shown in Box 18.1. Whole microbes can be detected by means of microscopy or by culture. The sensitivity of these depends on the quality of the specimen, the concentration of microorganisms, and the volume of specimen received. Microbial macromolecules can be either secreted or released from dying organisms. As macromolecules are antigenic, antibodies can be produced against them, and then used to detect the macromolecules in test systems such as enzyme-linked immunosorbent assay (ELISA) or immuno-chromatography. Such tests are rapid and can be designed
with a high specificity for the microbe in question. The sensitivity is dependent on the concentration of the microbial components in the specimen. For generalized infections or infections with microbes that secrete vast amounts of such molecules, blood can be used. However, for many infections a specimen from the site of infection is needed. Detection of nucleic acids is becoming more popular. Nucleic acid detection tests can be performed without or with an amplification step. The commonest nucleic acid amplification test (NAAT) is the polymerase chain reaction (PCR), although other NAATs have also been described.3 The second category of tests measures the activity of the immune system against microbe-specific antigens in the possibly infected host. The problem with these tests is that immune activity is downregulated after the antigenic stimulus has disappeared. The time that it takes for the immune parameters measured in the test to decrease significantly varies per antigen and depends on whether there is repeat exposure. Such repeat exposure is present in endemic environments. Tests measuring immune activity may not reliably differentiate between current infection and past infection, and this problem is most prominent in populations in which the microbe to be detected is endemic. In this chapter, microscopy, culture, and antigen detection tests for TB will be discussed, while tests that measure immune activity and nucleic acid detection tests will be discussed in Chapters 19 and 20, respectively. This chapter is not a comprehensive laboratory manual, and serves mainly to highlight some of the important issues regarding current methods of laboratory-based detection, identification, and susceptibility testing of mycobacteria.
SPECIMEN COLLECTION, STORAGE, AND TRANSPORT One of the most important parameters affecting the performance of a microbiological diagnostic test is the quality of the specimen. Specimens need to be representative of the site of infection, preferably collected aseptically, and stored and transported to the laboratory to minimize multiplication of contaminating organisms. Tuberculosis is a disease that can affect all parts of the body and therefore all different specimens that can be collected for microbiological investigations need to be considered. Specimens for microbiological investigations are grouped according to the level of contamination, assuming applying optimal collection methods have been used.4,5 Examples of specimens with contamination levels are shown in Table 18.1.
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Box 18.1 Laboratory tests to diagnose infection 1. Detection of microbes or components of microbes: a. microscopy; b. culture; c. antigen detection; d. nucleic acid detection. 2. Detection of components of the immune response to the microbe: a. antibody detection; b. activated T cells.
Table 18.1 Common specimens for mycobacterial investigation showing likely degree of contamination Disease manifestation
Specimen
Contamination level
Generalized TB
Blood Percutaneous liver biopsy Expectorated sputum Induced sputum Bronchoalveolar lavage Bronchoscopic protected brush Gastric aspirate Percutaneous lung biopsy Open lung biopsy Pleural fluid aspirate Pleural fistula fluid Cerebrospinal fluid
None None
Pulmonary TB
Tuberculous pleuritis Tuberculous meningitis Ocular TB Bone and joint TB
Abdominal TB Cutaneous TB
Mucosal TB Tuberculous lymphadenitis ‘Cold’ abscesses TB in any other tissue
Corneal ulcer scrape Vitreous fluid aspirate Joint aspirate Bone marrow aspirate Bone chip Peritoneal fluid Stool Nodule biopsy Ulcer scrape Blood Ulcer scrape Fine needle biopsy Sinus aspirate Transcutaneous aspirate Percutaneous/surgical biopsy Transmucosal biopsy/ aspirate
Moderate Moderate Low Low Low to moderate Low Low None Moderate None Low None None None None None Heavy None Heavy None Heavy None Moderate to heavy None None Moderate
Specimens collected aseptically from a site without commensal organisms should not be contaminated. These specimens can be obtained by aspiration, biopsy, or surgical excision and include cerebrospinal fluid, lymph node aspirates, aspirates from deepseated abscesses, bone marrow and joint aspirates, and tissue biopsies such as liver biopsies. These specimens do not require a decontamination procedure. The second group of specimens are secretions from parts of the body with no or minimal commensal organisms, but that are
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connected to the body’s surface by means of an open connection which harbours commensal organisms. These commensal organisms will be mixed with the secretions from the infected site as they pass through the opening to the surface. Such specimens are therefore always contaminated. This group of specimens includes those from the respiratory tract, gastric aspirates, urine as well as menstrual fluid aspirates and uterine specimens. These specimens can usually be effectively decontaminated before culture is performed. The third group of specimens are specimens from parts of the body colonized with commensal and/or environmental organisms. This not only includes normally colonized areas such as the skin, oropharyngeal cavity, colon, and vagina but also secondarily colonized areas such as ulcers, open wounds, and fistulating lymph nodes. Specimens such as stool, scrapings from ulcers in mucosa and skin, and draining lymph nodes or abscesses belong to this group. These specimens are more heavily contaminated, which can affect the accuracy of microscopy and increases the chances of an unsuccessful mycobacterial culture. Many pathogenic organisms die during prolonged specimen storage or transport, due to:
exposure to lower temperatures; increased oxygen concentration; or decreased pH due to metabolic products of contaminants.
Mycobacteria are hardy organisms not easily killed in such adverse circumstances. However, the circumstances in which specimens are stored and/or transported may allow multiplication of contaminants. The generation time of M. tuberculosis is approximately 12 hours at 37 C versus 20–45 minutes for most contaminants. At moderate temperatures M. tuberculosis stops multiplying while many contaminating species only slow down their replication rate. As the level of contamination affects the sensitivity (not the specificity) of culture and to a lesser extent that of microscopy, the duration of storage should be minimized while transport lines should be as short as possible. Ideally, storage and transport should take place in temperature-controlled circumstances between 4 and 10 C,5,6 and specimens should arrive in the laboratory on the day of collection. In specimens from the respiratory tract a significant decrease in culture sensitivity due to contamination occurs if specimens arrive in the laboratory more than 48 hours after collection.6 Unfortunately TB is commonest in developing countries where patient care facilities are far away from laboratories with culture facilities. The difficulties in obtaining a reliable culture also render susceptibility testing impossible. If prolonged storage or transport is unavoidable, preservatives can be added to the samples to inhibit growth of contaminants and thus improve the yield from culture. Examples of these preservatives include sodium carbonate, cetylpyridinium chloride, and sodium borate. There are concerns that some of these compounds may not be compatible with some of the newer liquid-based culture systems such as the Bactec 460 or Mycobacterial Growth Indicator Tube (MGIT) system, and they may also reduce the sensitivity of microscopy.7–9
MICROSCOPY Microscopic detection of microbes in clinical specimens is the oldest approach to the laboratory diagnosis of infectious diseases, including TB. Based on its cell wall structure, M. tuberculosis is a
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Gram-positive bacterium. Indeed, on Gram-stained smears of clinical specimens of patients with TB, mycobacteria can be detected. However, it needs an alert microscopist to recognize the thin-beaded Gram-positive bacilli as mycobacteria. Therefore, microscopic detection of mycobacteria relies on the acid-fast nature of the organisms, resulting from the high lipid content of the mycobacterial cell wall. Two staining methods make use of this characteristic:
In the Ziehl-Neelsen (ZN) stain, carbol fuchsin enters the bacteria when heated but does not leave the cells when exposed to an alcohol/hydrochloric acid mixture. Mycobacteria are therefore seen as red-stained rods in a methylene blue-stained background. A modification of the ZN stain, the Kinyoun or cold stain, uses a higher concentration of phenol with the carbol fuchsin, and the slide does not need to be heated. In the Auramine-O stain, carbol fuchsin is replaced by auramine. Auramine fluoresces when bound to DNA and viewed under a fluorescence microscope. After decolourization of non-acid-fast bacteria and other material, only the acid-fast bacilli fluoresce.
While ZN-stained smears need to be viewed under high-power magnification (800–1000), auramine-stained smears can be viewed at a lower magnification (450–500) because of the easier visual detection of the fluorescence. The advantages of the auramine stain are an improved sensitivity (by about 10%) over the ZN stain, with less time required to read the smears since they are examined at lower magnification. There is also limited evidence that fluorescent stains are more sensitive than conventional microscopy in HIV-infected adults.10 There are concerns that the fluorescent stains may be less specific than the ZN stain, especially in low-prevalence areas. Therefore, it was recommended that auramine-stained smears with scanty acidfast bacilli (< 1þ) should be re-examined with the ZN stain. However, in areas with a high prevalence of TB the specificity of the auramine stain is much higher, and the need to restain low positives is probably unnecessary.10,11 The sensitivity of microscopic detection of TB depends on the duration of viewing, the viewing technique, the expertise of the microscopist, and the concentration of bacteria in the specimen. The advised maximum duration of viewing for ZN-stained smears is 20 minutes. About half this time is needed to view auraminestained smears, owing to the lower magnification used. The sensitivity of microscopy on sputum samples can vary considerably, but, in HIV-uninfected adults, it is probably of the order of 60–70%. Sensitivity can be increased by various techniques such as use of fluorescent stains as just discussed, specific processing methods, and examination of multiple samples.11–13 Liquefaction of sputum specimens is part of the preparation of specimens for culture and/or PCR. However, this is also recommended in settings where only microscopy is performed. If culture is to follow, the treatment should not kill the mycobacteria (see later). However, if only microscopy is performed, killing the mycobacteria in the process renders the specimen safe to handle. This can be achieved by the use of diluted household bleach. The use of bleach as a liquefying agent along with concentration of the specimen by centrifugation has been shown to improve the sensitivity of microscopy by about 18%.12 With respect to multiple samples, examination of a second sample significantly increases sensitivity of microscopy, but a third sample probably adds little to this. The World Health Organization (WHO) has recently recommended that only two sputum samples
18
Table 18.2 Grading of tuberculosis microscopy results for Ziehl–Neelsen- or auramine-stained smears Number of acid-fast bacilli per number of fields
Report
Ziehl–Neelsen (x1,000)
Auramine-O (x450)
0
0
1–9/100 fields 10–99/100 fields 1–10/field > 10/field
1–18/50 fields 4–36/10 fields
Negative (no acid-fast bacilli observed) Scanty positive (record exact count) Positive–1þ
4–36/field > 36/field
Positive–2þ Positive–3þ
Note: The grading of auramine-stained smears depends on the magnification used. This table shows the numbers of bacilli needed for grading if an auramine-stained smear is examined at a 450 magnification.
be used for microscopy-based diagnosis of TB.14 In extrapulmonary TB, in HIV-infected patients and in children, the sensitivity of microscopy is significantly reduced, and alternative approaches including culture-based methods are often required.15,16 Microscopy results are usually reported as shown in Table 18.2, corresponding semi-quantitatively with the concentration of acidfast bacilli per volume of specimen.17
NEW DEVELOPMENTS Apart from improvements in specimen preparation and staining, automated systems for reading ZN-stained smears have been described.18 This is still in the very early stages, but if available at an affordable cost may significantly reduce technologist time. Microscopes that use light-emitting diodes (LEDs) rather than traditional mercury vapour lamps for reading fluorescent stained smears have also been developed. They are cheaper and the LEDs last considerably longer than mercury vapour lamps. This may allow for the introduction of fluorescent microscopy in resource-limited settings. The performance of LED microscopes has been shown to be virtually identical to traditional fluorescent microscopes.18a
CULTURE SPECIMEN PREPARATION Specimens likely to be contaminated with commensal flora are first processed to remove viable organisms other than mycobacteria. This is essential as the contaminating bacteria or fungi invariably grow faster than M. tuberculosis. This not only depletes the culture media of nutrients but it makes it difficult to recover the mycobacteria in pure culture, which is essential to speciate the organism and to perform susceptibility tests. However, decontamination can also affect the mycobacterial cells and decrease their numbers. Therefore, uncontaminated specimens are inoculated directly onto the culture media (after centrifugation if necessary). Decontamination of specimens for mycobacterial culture involves striking a balance between effective killing of contaminants and
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survival of the mycobacteria. The more effective the decontamination, the less sensitive the culture, as more mycobacteria are lost. Whether this balance is reached is shown in the contamination rate which needs to be consistently monitored. A contamination rate of 0% indicates too harsh decontamination and loss of culture sensitivity. However, contamination also results in decreased sensitivity owing to the faster growth of the contaminants. A contamination rate of 3–5% is thought to be acceptable,5 although this varies with the culture system used (discussed later). Specimens from the respiratory tract are usually mucoid, and mycobacteria are embedded in this mucus. Recovery of these mycobacteria by culture improves if the mucus is digested to liquefy it. This also makes it possible to concentrate the bacteria present by means of centrifugation. Decontamination and digestion is performed in the same process. Of the various different digestion/decontamination methods, the most frequently used are the NaOH method and the NALC-NaOH method. In the NaOH method, the sodium hydroxide is both mucolytic and decontaminant. To achieve both effectively in a heavily contaminated mucoid specimen, a 2% NaOH concentration is needed in the specimen. This kills mycobacteria as well, but at a slower pace than the contaminants. Therefore, different concentrations of NaOH (from 2% to 4%) are used, depending on consistency of the specimen and the expected level of contamination. With this method, culture contamination rates need careful monitoring as these are influenced by the choice of the NaOH concentration, the shaking method, and the accuracy of the timing. The NALC-NaOH method uses N-acetyl-L-cysteine (NALC) to digest the mucus and other organic debris. Its lytic effect at a concentration of 1–2% is highly effective and provides full exposure of all microbes in the specimen to the NaOH. The NaOH concentration in the specimen need thus not exceed 1%. As a result fewer mycobacteria will be killed, providing increased sensitivity of the culture compared with the NaOH method. Although the NALC-NaOH method has more steps than the NaOH method, the process is better controlled and not significantly more time-consuming.
CULTURE METHODS After decontamination, specimens are inoculated into one or more culture media to allow for growth of mycobacteria. A variety of different culture media and culture systems (commercial and inhouse) have been used for this purpose. The most convenient way of dividing culture media is into solid and liquid, each with its own advantages and limitations.
Solid media These can be either egg- or agar-based. Egg-based media, of which Lo¨wenstein–Jensen (LJ) is the most well known, have a long shelf life and support growth of many mycobacterial species. However, quality may vary from batch to batch, depending on the quality of the eggs, and it can be difficult to detect the presence of mycobacterial colonies early on in the culture process. Agar-based media (such as Middlebrook 7H10 or 7H11) are transparent, and the presence of colonies can be detected earlier than with egg-based media. Standardization from batch to batch is more easily achieved, which makes agar-based media potentially more reliable for susceptibility testing. However, the shelf life of some of the agar-based media is shorter than that of egg-based, and they are more expensive.4 In some circumstances, antimicrobial agents (such as penicillin, nalidixic acid, lincomycin and cycloheximide) can be added to solid media
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to help prevent contamination by other flora. Growth supplements such as hemin or ferric ammonium citrate can also be added to support the growth of Mycobacterium haemophilum. Supplemented media should only be used when clinically indicated, and should always be used in conjunction with non-selective solid or liquid media.4
Liquid media Commonly used liquid media preparations are Middlebrook 7H9 and Dubos Tween albumin broths.4,19 Although solid media formed the mainstay of laboratory-based culture for many years, and are still used in many centres, the use of liquid media offers a number of advantages. Chief amongst these is that mycobacterial growth in liquid media is significantly more rapid than on solid media, reducing the delay associated with mycobacterial culture. Additionally, isolation rates using liquid media are generally higher than with solid media, especially with commercially available liquid-based culture systems. A number of commercial liquid-based mycobacterial culture systems, including the Bactec 460TB system, the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system, VersaTREK (ESP II) culture system and MB/BacT system, are available. While a full description of the merits of each commercial system is beyond the scope of this chapter, a few short and pertinent points follow. The Bactec 460 (Becton Dickinson) is a semi-automated system and was the first commercial liquid-based mycobacterial culture system. It is often still regarded as the gold standard with which other systems are compared. The culture medium contains radiolabelled palmitic acid as a carbon source in the medium. This is metabolized by growing organisms, and radiolabelled CO2 is given off. The amount and rate of the CO2 emitted is monitored by repeated aspiration of the atmosphere in each vial to detect growth. The need for needles to sample the atmosphere in each vial and the need for disposal of radioactive waste are disadvantages to this system. The Bactec MGIT system (Becton Dickinson) uses modified 7H11 broth, with an indicator at the base of the tube that fluoresces under UV light. Oxygen quenches the fluorescence, but, if the oxygen in the medium is consumed, the indicator will fluoresce. The fluorescence can be read manually (using a Woods lamp), or, if a Bactec MGIT 960 instrument is used, the fluorescence is read automatically and continuously. The MB/BacT system (bioMerieux) consists of a bottle with a colorimetric sensor. As increasing amounts of CO2 are produced by growing organisms, the sensor changes colour, which is detected by the instrument. In the VersaTREK system (TREK diagnostics), previously marketed as the ESP II culture system, growth is detected by monitoring gas-related pressure changes brought about by multiplication of organisms in the bottle. A limitation of the MGIT 960 system is that blood cannot be directly inoculated into the tubes. The Bactec 460 system supports the culture of blood or bone marrow samples directly inoculated into vials, as do other mycobacterial culture systems such as the BacT/Alert MB and VersaTREK systems. The use of Myco/F Lytic (Becton Dickinson) blood culture bottles for mycobacterial culture of blood or marrow has been shown to be at least as good as other systems.20 A summary of the performance of the above four systems compared with solid media is shown in Table 18.3. As can be seen, time to detection in all the non-radiometric automated systems is faster than on solid media, although slightly slower than the Bactec 460. Mycobacterial recovery rates for all systems are similar, although the MGIT 960 and Bacetec 460 may be slightly better.
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Table 18.3 Comparison of the performance of various mycobacterial culture methods System
Time to positivity (days)
Recovery a rate (%)
Contamination rate (%)
Bactec MGIT 960 Bactec 460 MB/BacT ESP II / VersaTREK Solid media (LJ slopes)
10–22
88–100
5.0–13
8–14 14–18 3–34
86–98 90–93 79–87
3.7–11.7 4.6–7.7 7.0–8.6
23–30
55–87
3.0–14
a
Recovery rate reflects recovery of all mycobacteria, not specifically M. tuberculosis. Data from Drobniewski et al.,19 Somoskovi et al.,22 Chew et al.,23 Pfyffer et al.,24 Piersimoni et al.,25 Woodes et al.,26 Tortolli et al.27
The lack of needles and radioactive waste, as well as full automation, provide some advantages over the Bactec 460. However, contamination rates in the MGIT system tend to be higher than for Bactec 460, and specimen-processing procedures may need to be modified to counteract this.21 A major disadvantage of many of the commercial liquid-based systems is cost – in terms of both instrumentation and reagents. In laboratories that process relatively small numbers of specimens this is always going to be one of the deciding factors in choosing which culture system to use.
IDENTIFICATION FROM CULTURE Once there is growth on either solid or liquid media, the organism must be identified. The first step is to perform a ZN stain (and possibly also a Gram stain) to confirm that the organisms growing are mycobacteria and not contaminants. If a culture is contaminated, it can either be re-decontaminated or discarded. There are a number of ways of identifying acid-fast bacilli growing in the culture. A full description of all available identification methods is beyond the scope of this chapter, and only some of the relevant issues will be mentioned.
Phenotypic methods Phenotypic tests, which determine growth rates, pigmentation and growth at different temperatures, as well as a range of biochemical tests allow a laboratory in most cases to differentiate M. tuberculosis complex organisms from other mycobacteria (the non-tuberculous mycobacteria, NTM). However, phenotypic tests can also be used to identify specific non-tuberculous mycobacteria. One of the commoner tests used to identify M. tuberculosis is the niacin test. Niacin is excreted by M. tuberculosis due to a block in the NADscavenging pathway, and the detection of niacin can be used to presumptively identify an isolate as M. tuberculosis.28 However, other NTM (such as M. simiae) can also occasionally produce niacin, and rare niacin-negative strains of M. tuberculosis may occur.29,30 An alternative is to determine whether p-nitro-benzoic acid inhibits the growth of the organism – M. tuberculosis will show inhibition of growth.31 Biochemical tests can be time-consuming and often require the organism to be re-grown on specific media. In the case of identification for NTM, a wide range of biochemical tests need to be
18
performed, which is labour-intensive and expensive, and results can be difficult to interpret.
Mycolic acid analysis Analysis of mycolic acids (a component of mycobacterial cell walls) can be used to identify mycobacteria, and is one of the recommended tests when a new mycobacterial species is described. The analysis is usually performed by high-performance liquid chromatography (HPLC), and is generally used only in a research setting.32 Genotypic analysis Given the complexities associated with phenotypic identification, as well as the delays associated with these methods, an increasing number of identification methods based on DNA analysis have been described – both commercial and in-house. The major advantages of the nucleic-acid-based assays are speed and (usually) ease of interpretation. The drawbacks are often expense (especially commercial assays) and a real (or imagined) lack of expertise among laboratories in performing ‘high tech’ tests. The MicroSeq 500 system (Applied Biosystems, California, USA) is based on amplification of a portion of the 16S rDNA gene, DNA sequencing and comparison of the resultant sequence with the library of sequence data in the MicroSeq 500 bacterial database. The system allows for identification of a large number of mycobacteria (as well as other bacteria – an added advantage). However, certain of the nontuberculous mycobacteria are difficult to identify using this system (for example, the system cannot distinguish between Mycobacterium avium and Mycobacterium paratuberculosis, Mycobacterium abscessus and Mycobacterium chelonae) and it similarly cannot differentiate between different members of the M. tuberculosis complex.33,34 The Accuprobe (Gen-Probe, San Diego, CA, USA) test targets ribosomal RNA, and can be used to identify M. tuberculosis complex, M. avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium kansasii. A separate probe must be used for each species, and a limited number of species can be identified. However, for identification of M. tuberculosis complex, this test is rapid and reliable, showing excellent agreement with traditional phenotypic methods as well as other genotypic methods.35–37 There have been reports of misidentification of Mycobacterium celatum as M. tuberculosis using the Accuprobe. The Inno-LiPA Mycobacteria assay (Innogenetics, Ghent, Belgium) involves hybridization of PCR amplification products of a portion of the 16–23S rDNA spacer region to oligonucleotide probes on a membrane strip, which allows for identification of M. tuberculosis as well as a range of the commoner NTM. This assay can be used on cultures from both the MGIT and Bactec 460 system, and has shown a high degree of correlation (> 95%) with reference methods.35,36,38 The GenoType assays (Hain Lifesciences, Nehren, Germany) are more recent identification systems, and different versions of the kit can be used either for detection of M. tuberculosis directly from clinical samples or for identification of mycobacteria from culture. The underlying principle involves multiplex amplification of extracted mycobacterial DNA by PCR, with subsequent hybridization of the biotin-labelled amplicons to oligonucleotide probes bound to a membrane strip. Identification is done by evaluation of the resultant banding pattern. Kits are available either to differentiate different members of the M. tuberculosis complex (GenoType MTBC)39–41 or to identify a variety of NTM (GenoType Mycobacterium CM/ AS).42,43 Again, the kits have been shown to have excellent correlation with reference methods, usually over 95%.
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Table 18.4 Examples of in-house assays that have been used to identify Mycobacterium tuberculosis (and other mycobacteria) from culture Methodology
Target
Species identified
Ref
PCR Multiplex PCR PCR with RE digestion of product PCR with DNA sequencing
IS6110, pncA, Ag85 (among others) RD1, RD9 65-kDa hsp gene 16S rDNA gene
M. tuberculosis M. bovis BCG (differentiated from other MTBC) Wide range of mycobacteria (> 30 species including MTBC) Wide range of mycobacteria (> 50 species including MTBC)
44–46 44,47 48 49
MTBC: M. tuberculosis complex; BCG, Bacillus Calmette–Gue´rin; PCR, polymerase chain reaction.
A large number of in-house assays have also been described, with the purpose of identifying both M. tuberculosis complex and a variety of NTM. Examples of some of these assays are summarized in Table 18.4. This is by no means a comprehensive listing, and serves only to illustrate some of the different in-house methods that have been described.
NEW DEVELOPMENTS Two exciting developments deserve mention. The microscopic observation of drug susceptibility (MODS) assay combines culture and susceptibility testing in one step, reducing turnaround times for susceptibility testing, and is also a relatively low-cost process. It will be discussed in more detail later in the chapter under Susceptibility Testing. A new solid medium has also been described. TK medium (Salubris Inc, Cambridge, MA, USA) is a solid medium that contains various dyes that enable early detection of mycobacterial growth as well as detection of contamination. It can be read by the naked eye, making it a relatively low-cost culture option. Preliminary studies have shown that the time to positivity is faster than on LJ slopes (15 vs 26 days), but still slower than Bactec 460.50 The medium is unfortunately not yet available commercially, and further studies are planned to evaluate its performance in a routine setting.
NON-CULTURE-BASED METHODS OF DETECTING M. TUBERCULOSIS ANTIGEN DETECTION METHODS Lipoarabinomannan (LAM) is a mycobacterial cell wall component excreted in the urine. Detection of LAM has been evaluated as a diagnostic tool,51 and recently a commercial kit to detect urinary LAM in an ELISA plate format has been marketed (Chemogen, Portland, ME, USA). Quoted sensitivity ranges from 57% to 81% in HIV-infected and uninfected patients, respectively. While the concept sounds attractive, more research is needed to evaluate the product in different settings.
PHAGE ASSAYS The use of mycobacteriophages to diagnose TB is also a relatively recent development. This is discussed more fully in Chapter 23. Phages can also be used to perform susceptibility testing as discussed later.
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SUSCEPTIBILITY TESTING Resistance to anti-mycobacterial drugs occurs spontaneously at a rate of about 107 to 1010/cell division,52,53 varying with the drug. In cavities where one may typically find 107 organisms, there will almost certainly be a small proportion naturally resistant to at least one of the antimycobacterial agents. Use of a single drug as therapy may select for this resistance, and this is one of the reasons why combination therapy is now the standard of care. If combination therapy is not well managed, as may happen with poor adherence to therapy, poor supply of drugs and poorly managed treatment programmes, resistance is likely to emerge. Drug resistance in M. tuberculosis is a growing concern. Multidrug-resistant TB (MDR-TB), defined as resistance to at least isoniazid and rifampicin, occurs world-wide and is associated with longer, more expensive and more toxic treatment courses as well as with worse outcomes. In addition, emergence of extensively drug-resistant TB (XDR-TB) has focused attention on the need for more regular susceptibility testing as well as more rapid availability of susceptibility test results.1,54 There are a number of old and new methods available to the clinical laboratory to test susceptibility to antimycobacterial drugs, as shown in Boxes 18.2 and 18.3. Many current phenotypic methods require weeks for a result, and new methods are being developed to reduce this delay. This section will provide an overview of the commonly used methods, and describe some of the newer research in the field of drug susceptibility testing.
CONVENTIONAL (PHENOTYPIC) SUSCEPTIBILITY TESTING Conventional susceptibility testing essentially determines whether an isolate is resistant to an agent by evaluating growth (or metabolic Box 18.2 Commercial/established tests Phenotypic Absolute concentration method Resistant ratio method Proportion method Agar or broth. Direct or indirect. Genotypic Commercial line probe assays Phage-based assays Phage amplification assays
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Microbiological testing for Mycobacterium tuberculosis
Box 18.3 Non-commercial/in-house tests Phenotypic Microscopic observation of drug susceptibility (MODS) assay Colorimetric Genotypic Various in-house assays Phage based Luciferase reporter phages
activity) in the presence of the drug. The three standard methods using solid media are:
absolute concentration method; resistant ratio method; and proportion method.
The details of these methods have been described in numerous books and reviews,52,53,55,56 and only a brief summary of the principles of the first two methods is laid out here.
Absolute concentration method This is essentially a method of determining a minimal inhibitory concentration (MIC). The drug is incorporated into medium (usually solid medium) in twofold dilutions, and the organism inoculated onto each concentration. After incubation, the lowest concentration of drug that inhibits growth is read and used to determine whether the organism is resistant or susceptible. It is important to ensure that drug concentrations and inoculum size are carefully standardized; otherwise, erroneous results will be obtained. Resistant ratio method This is similar to the absolute concentration method; however, the MIC of the test isolate is compared with the MIC for a reference strain tested simultaneously. If the ratio of the MICs for the test strain to the reference strain is 2, the isolate is susceptible. If the ratio is 8, the isolate is resistant. Since this method compares to a reference strain, it is less prone to errors related to preparation of media. Inoculum preparation is, however, still important. Proportion method This test relies on the premise that, if more than 1% of a given population of organisms are resistant to a drug, the strain is regarded as resistant to that drug. When the proportion method is performed on solid media, agar plates containing the drug in question at a set concentration are prepared. This concentration is known as the critical concentration of the drug. The organism is inoculated onto both drug-free and drug-containing medium, and incubated for up to 3 weeks. The number of colonies on the drug-free medium is compared with the number on the drug-containing medium, and the proportion of resistant colonies calculated. The proportion method has been modified to be performed in broth culture, and the proportion method performed on the Bactec 460 radiometric culture system is now one of the most commonly used susceptibility testing methods. In essence, a vial containing a critical concentration of the drug and a drug-free vial are both inoculated with the test organism; however, the inoculum in the drug-free vial is a 1:100 dilution of the inoculum in the drugcontaining vial. If the drug-free vial registers growth before the drug-containing vial, the isolate is regarded as susceptible. If the drug-containing vial registers growth at the same time as, or before, the drug-free vial, the isolate is resistant. The Bactec 460
18
proportion method is quicker than the agar proportion method, but has the drawback of using radioisotopes. It is important to remember that the critical concentration of the drug varies, depending on the type of medium used, and use of the incorrect critical concentration can lead to incorrect results. When testing isoniazid, ethambutol, and streptomycin, two concentrations may be tested – low and high critical concentrations. There is some debate about the value of doing this, particularly for ethambutol, given the fact that presence of resistance mutations is more associated with resistance at the high concentration. For the other drugs, if only the low concentration is tested and the isolate is resistant, the high concentration should also be tested. Resistance at the high concentration indicates resistance, while an isolate resistant to the low concentration but susceptible to the high concentration suggests low-level resistance. Dosage adjustment may be necessary and careful follow-up to monitor for the emergence of resistance may be warranted.56,57
Direct and indirect testing Direct testing refers to testing directly from a clinical sample. It is possible to inoculate a smear-positive specimen directly onto the drug-containing and drug-free agar after decontamination, and compare the amount of growth. Direct testing in the liquid-based Bactec 460 system is thought to be less reliable, and there is little evidence to support its use. Indirect testing refers to testing performed on a culture of M. tuberculosis that has been grown from a clinical sample. Pyrazinamide testing Susceptibility testing of pyrazinamide (PZA) is difficult, since the drug is active at a low pH, which can inhibit mycobacterial growth.52 However, both agar and broth proportion methods for performing PZA testing have been described. Of concern are reports showing that some phenotypic PZA resistance tests do not always correlate with the presence of resistance mutations in the pncA gene.58 The pyrazinamidase assay (for detecting PZA-resistant strains) has been advocated as an alternative; however, later studies have shown that there is little correlation between presence of the enzyme and susceptibility or resistance.52 Non-radiometric broth-based methods Given the problems associated with disposal of radioactive materials in the Bactec 460 system, it is not surprising that non-radioactive liquid-based culture systems have also been adapted to perform susceptibility testing. The principle is the same as for the Bactec 460 system, although the details of inoculation, growth monitoring, and critical concentration obviously differ from system to system. The Bactec MGIT 960 system, the MB/BacT system and the VersaTREK have all been used as non-radiometric susceptibility testing systems. While the MGIT 960 system has been most extensively described, all the systems appear to perform very well in comparison with the Bactec 460 or agar proportion methods, although the ideal critical concentrations for some of the systems still need to be clarified.59 It is also worth considering that a thorough correlation of the results from these methods and clinical outcomes has not yet been performed (and is probably unlikely to happen).55 MOLECULAR METHODS As with the molecular identification systems, a number of in-house and commercial assays for detecting resistance mutations in M. tuberculosis have been described. Most work has been done on detecting resistance to isoniazid and rifampicin – partly because the resistance mutations (especially for rifampicin) are well described, and partly because these two drugs form the cornerstone of therapy for TB.
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Rifampicin in particular lends itself to molecular testing as the vast majority of resistance (> 95%) has been associated with mutations in a fairly limited area of the rpoB gene. Resistance to isoniazid (INH) is slightly more difficult, as at least four different genes may be involved, and in about 10–15% of cases the molecular basis of resistance is still unclear.
Commercial assays Two commercial reverse-hybridization assays have been released for the detection of rifampicin and INH resistance – the Inno-LiPA Rif TB assay (Innogenetics, Ghent, Belgium) and the GenoType MTBDR assay (Hain Lifesciences, Nehren, Germany). The principles underlying the tests have been described earlier in this chapter, although the probes bound to the membrane strip now correspond to sequences in the rpoB and katG/inhA genes. The Inno-LiPA Rif assay only detects resistance to rifampicin, while the MTBDR detects resistance to both rifampicin and INH. The performance of both systems in detecting rifampicin resistance appears to be excellent, ranging from 95% to 100% sensitivity, compared with phenotypic tests. The ability of the MTBDR system to detect INH resistance is slightly poorer (unsurprisingly given the wider range of resistance mutations), but still of the order of 73–90% sensitivity. The specificity of both assays approaches 100%.55,60,61 Both systems have also been used to detect resistance directly from smear-positive clinical samples (usually sputum), and the performance in this setting, while not quite as good as when performed on cultured isolates, is still excellent.62 The main drawback to the routine implementation of these methods for susceptibility testing (whether it be from culture or specimen) is expense. In-house assays A wide range of in-house assays designed to detect resistance mutations have been described. It is again impractical to discuss them all in detail, but Table 18.5 serves to illustrate some examples of the different approaches that have been taken. PHAGE-BASED ASSAYS Mycobacteriophages have been used both to detect viable organisms in specimens (as a rapid diagnostic method) and to perform rapid susceptibility testing to rifampicin, using either clinical samples or cultured organisms. Phage-based assays have a turnaround time of 48–72 hours, compared with 3–6 weeks for current phenotypic tests. When performing susceptibility testing, the sample is first incubated in the presence of rifampicin. If the isolate is rifampicin resistant, viable bacilli will still be present after this step. The sample or Table 18.5 Examples of in-house assays for detecting resistance in Mycobacterium tuberculosis Methodology
Target
Antibiotic
Ref
Line probe assay
pncA gyrA rpoB katG,inhA-mabA rpoB katG embB
PZA Quinolones Rifampicin INH Rifampicin INH Ethambutol
63,64
Macroarrays Pyrosequencing
PZA, pyrazinamide; INH, isoniazid.
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65 66
culture is then inoculated with a mycobacteriophage, which will infect any viable bacilli in the sample, and this can be detected in two ways: 1. A virucide is added to kill off any phages outside the mycobacteria. The phages inside infected bacilli replicate, lyse the bacilli and, release more phage. This is inoculated onto a lawn of a rapidly growing mycobacterium (Mycobacterium smegmatis), and, if phage is present, it will infect the M. smegmatis and cause the formation of plaques. The absence of plaques indicates no phage; thus there were no viable bacilli after the initial exposure to rifampicin. 2. Luciferase reporter phages (LRPs) are phages containing a luciferase gene, which can be detected by light emission without the need to infect M. smegmatis. Although no commercial assays have yet been developed using LRPs, commercial assays using phage amplification are available (FASTPlaque-TB Rif, Biotec Laboratories, London, UK). The performance of these assays was reviewed in a meta-analysis in 2005,67 and both LRPs and phage amplification assays were shown to have very good sensitivity and specificity for detection of rifampicin resistance – although the majority (19/21) of studies reviewed had performed the test on cultured isolates rather than clinical samples. Sensitivity ranged from 81% to 100%, and specificity from 73% to 100%. The obvious concern is the lower than expected performance in some studies, and more research identifying the underlying reasons for the poor performance need to be identified. More recently, LRPs have also been used to test INH resistance, with promising results (96.9% agreement with Bactec 460).68
COLORIMETRIC METHODS A relatively new method of rapid susceptibility testing uses colorimetric methods to indicate growth and these were recently reviewed.69 A coloured indicator is added to the medium after exposure of M. tuberculosis to the anti-mycobacterial drug (thus far, usually only INH and rifampicin). The presence of viable mycobacteria is detected by a change in colour of the indicator. The assays are usually performed in microtitre plates with a range of concentrations of the drugs, and in this way an MIC can be derived. The indicators that have been used to date include tetrazolium salts (XTT and MTT), Alamar blue and resazurin. The turnaround time of these tests is usually 7–14 days, which again compares very favourably with current methods. The pooled sensitivities for detection of rifampicin and INH resistance were both 98%, while the pooled specificities were 99% and 98%, respectively. There does not appear to be a difference in performance amongst the different dyes.
MICROSCOPIC OBSERVATION DRUG SUSCEPTIBILITY (MODS) The assay was first described in Peru in 2000 and entails inoculating a sputum sample into the wells of a tissue culture plate containing Middlebrook 7H9 broth.70 Some wells contain antibiotics at set concentrations. The wells are examined with an inverted microscope on a daily basis, and assessed for the presence of the typical corded appearance of M. tuberculosis. The presence of growth in the control well but not in the drug-containing wells indicates susceptibility to that drug. Although initially only INH and rifampicin were tested, more recent studies have included ethambutol and streptomycin.71
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The assay has proven extremely reliable in terms of diagnosing TB and compares favourably with both the MB/BacT and MGIT 960 systems. Time to detection of growth is at least as good as automated systems, with some reports of better time to detection.71,72 The accuracy of the susceptibility tests has been equally impressive, with most studies showing excellent correlation between the MODS result and that of a reference method (agar proportion or Bactec 460). Two reservations about the assay are that, while it is relatively cost-effective in terms of reagents and equipment, it is labour-intensive. There have also been concerns about the potential for crosscontamination. This is alleviated in part by ensuring that each tray is in its own plastic bag, and cross-contamination can be avoided.73
SUMMARY The mainstay of TB diagnosis, especially in resource-poor settings, is still microscopy. Although this has limited sensitivity there are a
REFERENCES 1. Pillay A, Sturm AW. Evolution of the extensively drug resistant F15/LAM4/KZN strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa. Clin Infect Dis 2007;45:1409–1414. 2. Hanekom M, van der Spuy GD, Streicher E, et al. A recently evolved sublineage of the Mycobacterium tuberculosis Beijing Strain Family is associated with an increased ability to spread and cause disease. J Clin Microbiol 2007;45:1483–1490. 3. Tenover FC. Rapid detection and identification of bacterial pathogens using novel molecular technologies: infection control and beyond. Clin Infect Dis 2007; 44:418–423. 4. Pfyffer GE, Brown-Elliott BA, Jost KC, et al. Mycobacterium: general characteristics, isolation and staining procedures. In: Murray PR, Baron EJ, Jorgensen JH (eds), Manual of Clinical Microbiology, 8th edn. Washington, DC: ASM Press, 2003. 5. Kent PT, Kubica GP. Public Health Mycobacteriology: A Guide for the Level III Laboratory. Atlanta, GA: Centers for Disease Control, 1985. 6. Paramasivan CN, Narayana A, Prabhakar R, et al. Effect of storage of sputum specimens at room temperature on smear and culture results. Tubercle 1983;64:119–124. 7. Aparna S, Krishna MKV, Gokhale S. From microscopy centre to culture laboratory: a viable ride for mycobacteria. Int J Tuberc Lung Dis 2006;10:447–449. 8. Bobadilla-del-Valle M, Ponce-de-Leo´n A, Kato-Maeda M, et al. Comparison of sodium carbonate, cetyl-pyridinium chloride and sodium borate for preservation of sputa for culture of Mycobacterium tuberculosis. J Clin Microbiol 2003;41:4487–4488. 9. Selvakumar N, Sydhamathi S, Duraipandian M, et al. Reduced detection of by Ziehl-Neelsen method of acid-fast bacilli in sputum samples preserved in cetylpyridinium chloride solution. Int J Tuberc Lung Dis 2004;8:248–252. 10. Gilpin C, Kim SJ, Lumb R, et al. Critical appraisal of current recommendations and practices for tuberculosis sputum smear microscopy. Int J Tuberc Lung Dis 2007;11:946–952. 11. Steingart KR, Henry M, Ng V, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006;6:570–581. 12. Steingart KR, Ng V, Henry M, et al. Sputum processing methods to improve the sensitivity of
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number of ways of optimizing the sensitivity of microscopy, including proper specimen preparation and use of fluorescent stains if possible. Although there are some promising new tests for rapid diagnosis of TB available or on the horizon (see also Chapters 19 and 20), the implementation of these in resource-limited settings where TB is endemic is going to be challenging. If culture is to be carried out, commercial broth-based systems are probably still regarded as the first prize, although affordability is the main obstacle. Newer developments such as the MODS assay should be considered as alternatives. Although it may not be practical to perform susceptibility tests on every isolate, especially in areas with a high TB prevalence, every effort should be made to provide susceptibility test results as soon as possible if they are indicated. One of the new challenges facing TB control programmes may be deciding in what circumstances susceptibility testing should be performed, and which method to use, to facilitate early detection of patients with drugresistant strains, while not over-burdening already stretched healthcare budgets.
smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006;6:664–674. 13. Al Zahrani K, Al Jahdali H, Poirer L, et al. Yield of smear, culture and amplification tests from repeated sputum induction for the diagnosis of pulmonary tuberculosis. Int J Tuberc Lung Dis 2001;5:855–860. 14. World Health Organization. Tuberculosis (TB): New WHO policies: Background documentation. [online]. Accessed 26 Jan 2008. Available at URL:http://www. who.int/tb/dots/laboratory/policy/en/index.html 15. Marais BJ. Childhood tuberculosis—risk assessment and diagnosis. South Afr Med J 2007;10(Pt 2):978–982. 16. Getahun H, Harrington M, O’Brien R, et al. Diagnosis of smear negative pulmonary tuberculosis in people with HIV infection or AIDS in resourceconstrained settings: informing urgent policy changes. Lancet 2007;368:2042–2049. 17. World Health Organization. Chapter 17— Tuberculosis. Blood Safety and Clinical Technology: Guidelines on Standard Operating Procedures for Microbiology. [online]. Accessed 25 Jan 2008. Available at URL:http://www.searo.who.int/EN/Section10/ Section17/Section53/Section482_1799.htm 18. Veropoulos K, Learmonth G, Campbell C, et al. Automated identification of tubercle bacilli in sputum. A preliminary investigation. Anal Quant Cytol Histol 1999;21:277–282. 18a. Marais BJ, Brittle W, Painczyk K, et al. Use of lightemitting diode fluorescence microscopy to detect acid-fast bacilli in sputum. Clin Infect Dis 2008;47:203–207. 19. Drobniewski F, Caws M, Gibson A, et al. Modern laboratory diagnosis of tuberculosis. Lancet Infect Dis 2003;3:141–147. 20. Crump J, Tanner D, Mirrett S, et al. Controlled comparison of Bactec 13A, Myco/F Lytic, BacT/ Alert and Isolator 10 systems for detection of mycobacteraemia. J Clin Microbiol 2003;41: 1987–1990. 21. Cornfield D, Beavis K, Greene J, et al. Mycobacterial growth and bacterial contamination in the Mycobacteria Growth Indicator Tube and Bactec 460 culture systems. J Clin Microbiol 1997;35:2068–2071. 22. Somoskovi A, Clobridhge A, Larsen S, et al. Does the MGIT 960 system improve the turnaround times for growth detection and susceptibility testing of the Mycobacterium tuberculosis complex. J Clin Microbiol 2006;44:2314–2315. 23. Chew W, Lasaitas R, Schio F, et al. Clinical evaluation of the Mycobacteria Growth Indicator Tube (MGIT) with radiometric (Bactec) and solid media for isolation of Mycobacterium species. J Med 1998;47:821–827. 24. Pfyffer G, Welscher H, Kissling P, et al. Comparison of the Mycobacteria Growth
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
Indicator Tube (MGIT) with radiometric and solid culture for recovery of mycobacteria. J Clin Microbiol 1997;35:364–368. Piersimoni C, Scarparo C, Callegaro A, et al. Comparison of MB/BacT Alert 3D system with radiometric Bactec system and Lowenstein-Jensen Medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study. J Clin Microbiol 2001;39:651–657. Woodes GL, Fish G, Plaunt M, et al. Clinical evaluation of Difco ESP Culture System II for growth and detection of mycobacteria. J Clin Microbiol 1997;35:121–124. Tortolli E, Cichero P, Chirillo MG, et al. Multicenter comparison of ESP Culture System II with Bactec 460TB and with Lowenstein-Jensen medium for recovery of mycobacteria from different clinical specimens including blood. J Clin Microbiol 1998;36:1378–1381. Gruft H. Three simple tests as an adjunct for the small mycobacteriology laboratory. Health Lab Sci 1976;13:179–183. Weiszfeiler J, Karasseva V, Karczag E. Mycobacterium simiae and related mycobacteria. Rev Infect Dis 1981;3:1040–1045. Gruft H, Osterhout M. Niacin negative strain of Mycobacterium tuberculosis. Am Rev Respir Dis 1978;118:157. Stager C, Libonati J, Siddiqi S, et al. Role of solid media when used in conjunction with the Bactec system for mycobacterial isolation and identification. J Clin Microbiol 1991;29:154–157. Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin Microbiol Rev 2001;14:704–726. Hall L, Doerr K, Wohlfiel S, et al. Evaluation of the MicroSeq System for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 2003;41:1447–1453. Cloud JL, Neal H, Rosenberry R, et al. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol 2002; 40:400–406. Somoskovi A, Song Q, Mester J, et al. Use of molecular methods to identify the Mycobacterium tuberculosis complex (MTBC) and other mycobacterial species and to detect rifampicin resistance in MTBC isolates following growth detection with the Bactec MGIT 960 system. J Clin Microbiol 2003;41:2822–2826. Scarparo C, Piccoli PP, Rigon A, et al. Direct identification of mycobacteria from MB/BacT alert
177
SECTION
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37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
GENERAL CLINICAL FEATURES AND DIAGNOSIS
3D bottles: comparative evaluation of two commercial probe assays. J Clin Microbiol 2001;39:3222–3227. Alcaide F, Benitez MA, Escriba` J, et al. Evaluation of the Bactec MGIT 60 and the MB/BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA Accuprobe. J Clin Microbiol 2000;38:398–401. Lebrun L, Go¨nu¨llu¨ N, Boutros N, et al. Use of INNOLiPA assay for rapid identification of mycobacteria. Diagn Microbiol Infect Dis 2003;46:151–153. Richter E, Weizenegger M, Fahr AM, et al. Usefulness of the GenoType MTBC assay for differentiating species of the Mycobacterium tuberculosis complex in cultures obtained from clinical samples. J Clin Microbiol 2004;42:4303–4306. Sarkola A, Ma¨kinen J, Marjama¨ki M, et al. Prospective evaluation of the GenoType assay for routine identification of mycobacteria. Eur J Clin Microbiol Infect Dis 2004;23:642–645. Romero Go´mez M, Herrera-Leo´n L, Jime´nez M, et al. Comparison of GenoType MTBC with RFLPPCR and multiplex PCR to identify Mycobacterium tuberculosis complex species. Eur J Clin Microbiol Infect Dis 2007;26:63–66. Gitti Z, Neonakis I, Fanti G, et al. Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece. J Clin Microbiol 2006;44:2244–2246. Ma¨kinen J, Marjama¨ki M, Marttila HJ, et al. Evaluation of a novel test strip, GenoType Mycobacterium CM/AS, for species identification of mycobacterial cultures. Clin Microbiol Infect 2006; 12:481–483. Sion C, Degraux J, Delme´e M. Early identification of Mycobacterium tuberculosis and Mycobacterium avium using the polymerase chain reaction on samples positive by a rapid commercial culture system. Eur J Clin Microbiol Infect Dis 1999;18:346–351. Brown TJ, Tansel O, French GL. Simultaneous identification and typing of multi-drug-resistant Mycobacterium tuberculosis isolates by analysis of pncA and rpoB. J Med Microbiol 2000;49:651–656. Kidane D, Olobo JO, Habte A, et al. Identification of the causative organism of tuberculous lymphadenitis in Ethiopia by PCR. J Clin Microbiol 2002; 40:4230–4234. Talbot E, Williams D, Frothingham R. PCR identification of Mycobacterium bovis BCG. J Clin Microbiol 1997;35:566–569. Brunello F, Ligozzi M, Cristella E, et al. Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 2001;39:2799–2806. Harmsen D, Dostal S, Roth A, et al. RIDOM: comprehensive and public sequence database for identification of Mycobacterium species. BMC Infect Dis 2003;3:26.
178
50. Baylan O, Kisa O, Albay A, et al. Evaluation of a new automated, rapid, colorimetric culture system using solid medium for laboratory diagnosis of tuberculosis and determination of anti-tuberculosis drug susceptibility. Int J Tuberc Lung Dis 2004; 8:772–777. 51. Tessma TA, Hamasur B, Bjune G, et al. Diagnostic evaluation of urinary lipoarabinomannan at an Ethiopian tuberculosis centre. Scand J Infect Dis 2003;33:279–284. 52. Inderlied CB, Nash KA. Antimycobacterial agents: in vitro susceptibility testing and mechanisms of action and resistance. In: Lorian V (ed.). Antibiotics in Laboratory Medicine, 5th edn. Philadelphia: Lippincott Williams and Wilkins, 2005. 53. Drobniewski F, Rusch-Gerdes S, Hoffner S. Antimicrobial susceptibility testing of Mycobacterium tuberculosis (EUCAST document E.DEF 8.1)—report of the Subcommittee on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Clin Microbiol Infect 2007;13:1144–1156. 54. Centers for Disease Control and Prevention. Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs worldwide, 2000–2004. MMWR Nord Mortal Wkly Rep 2006;55:301–305. 55. Kim SJ. Drug susceptibility testing in tuberculosis: methods and reliability of results. Eur Respir J 2005;25:564–569. 56. Inderlied CB, Pfyffer GE. Susceptibility test methods: mycobacteria. In: Murray PR, Baron EJ, Jorgensen JH (eds). Manual of Clinical Microbiology, 8th edn. Washington, DC: ASM Press, 2003. 57. Schaaf HS, Victor TC, Engelke E, et al. Minimal inhibitory concentration of isoniazid in isoniazid resistant Mycobacterium tuberculosis isolates from children. Eur J Clin Microbiol Infect Dis 2007; 26:203–205. 58. Dormandy J, Somoskovi A, Kreswirth BN, et al. Discrepant results between pyrazinamide susceptibility testing by the reference Bactec 460TB method and pncA DNA sequencing in patients infected with multidrug resistant W-Beijing Mycobacterium tuberculosis strains. Chest 2007; 131:497–501. 59. Piersimoni C, Olivieri A, Benacchio L, et al. Current perspectives on drug susceptibility testing of Mycobacterium tuberculosis complex: the automated nonradiometric systems. J Clin Microbiol 2006: 44;20–28. 60. Makinen J, Marttila HJ, Marjamaki M, et al. Comparison of two commercially available DNA line probe assays for detection of multidrug-resistant Mycobacterium tuberculosis. J Clin Microbiol 2006; 44:350–352.
61. Barnard M, Albert H, Cotezee G, et al. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa. Am J Respir Crit Care Med 2008; 177:787–792. 62. Hillemann D, Rusch-Gerdes S, Richter E. Application of Genotype MTBDR assay directly on clinical specimens. Int J Tuberc Lung Dis 2006; 10:1057–1059. 63. Sekiguchi J, Nakamura T, Miyoshi-Akiyama T, et al. Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis strains. J Clin Microbiol 2007;45:2802–2807. 64. Giannoni F, Iona E, Sementilli F, et al. Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2005;49:2928–2933. 65. Brown TJ, Hererra-Leon L, Anthony RM, et al. The use of macroarrays for the identification of MDR Mycobacterium tuberculosis. J Microbiol Methods 2006; 65:294–300. 66. Zhao JR, Bai YJ, Wang Y, et al. Development of a pyrosequencing approach for rapid screening of rifampicin, isoniazid and ethambutol resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2005; 9:328–332. 67. Pai M, Kalantria S, Pascopella L, et al. Bacteriophagebased assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis. J Infect 2005;51:175–187. 68. Banaiee N, January V, Barthus C, et al. Evaluation of a semi-automated reporter phage assay for susceptibility testing of Mycobacterium tuberculosis isolates in South Africa. Tuberculosis (Edin) 2008;88:64–68. 69. Martin A, Portaels F, Palomino JC. Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 2007;59:175–183. 70. Caviedes L, Lee TS, Gilman RH, et al. Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopy observation of broth cultures. The Tuberculosis Working Group in Peru. J Clin Microbiol 2000;38:1203–1208. 71. Moore DA, Evans CA, Gilman RH, et al. Microscopic-observation drug-susceptibility for the diagnosis of TB. N Engl J Med 2006;355:1539–1550. 72. Arias M, Mello FC, Pavo´n A, et al. Clinical evaluation of the microscopic-observation drugsusceptibility assay for detection of tuberculosis. Clin Infect Dis 2007;44:674–680. 73. Moore DA, Caviedes L, Gilman RH, et al. Infrequent MODS culture cross-contamination in a high-burden resource-poor setting. Diagn Microbiol Infect Dis 2006;56:35–43.
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Immune-based tests for tuberculosis Dick Menzies, Kevin Schwartzman, and Madhukar Pai
INTRODUCTION Immune-based tests have great appeal for the diagnosis of TB, because the disease is notoriously difficult to diagnose. Active disease is a diagnostic challenge; because of the protean clinical manifestations, radiographic tests are insensitive and/or non-specific, while microbiological tests tend to be slow and labour intensive, and have suboptimal sensitivity, especially for extrapulmonary disease. The diagnosis of latent TB infection (LTBI) has assumed ever greater importance in many high-income countries. As rates of disease have fallen, emphasis has shifted to identification of latent infection and treatment to prevent later reactivation. Immune-based tests include some of the oldest and some of the newest tests for TB. In this chapter we will review two tests for detecting latent TB infection – the tuberculin skin test (TST) and interferong release assays (IGRA) – and one designed to diagnose active TB disease – serological assays. Both tests of latent infection measure cellmediated response to TB antigens, but otherwise differ markedly. The TST measures a complex in vivo response to intradermal injection of a fairly crude mixture of protein antigens derived from cultures of Mycobacterium tuberculosis – reflecting its origins of almost 100 years ago. IGRA have been introduced as commercial tests in many countries within the past 5 years. They are elegant in vitro tests that measure interferon-g (IFN-g) production by sensitized lymphocytes after in vitro exposure to antigens found in M. tuberculosis but not in several other organisms including Mycobacterium avium and Mycobacterium bovis Bacillus Calmette–Gue´rin (BCG). These new tests are promising because of their operational advantages and better specificity compared with the TST. Although many questions remain, this is a very active area of research in many settings, and the role of these tests is being constantly redefined. On the other hand serological tests measure humoral, or antibody, response to antigens from M. tuberculosis. Amazingly, the first serological assay was described in 1896! Despite having the longest history of use, and despite the efforts of many investigators in that time, serological assays remain the least utilized because of suboptimal test performance for diagnosis of latent or active TB in many populations and settings.
TUBERCULIN SKIN TESTING INTRODUCTION The tuberculin skin test was first introduced almost 100 years ago, giving it the distinction of being one of the oldest tests in current
clinical use. It is still the primary method of diagnosis of latent TB infection; an estimated 40 million TSTs are applied worldwide. Despite this long history, and common use, certain aspects of its use and interpretation remain controversial. And, with the advent of new in vitro immune-based tests for diagnosis of LTBI, the utility and limitations of the TST are important to review. The first tuberculin test material was prepared by Robert Koch, who filtered heat-sterilized cultures of M. tuberculosis grown on veal broth and then evaporated the filtrate to 10% of the original volume.1 This became known as old tuberculin (OT). In 1907 von Pirquet recognized its potential value for detection of persons infected with TB.2 The next year, Mantoux introduced the intradermal technique, which still bears his name.3 After considerable standardization work to create a purified protein derivative (PPD), a large quantity was carefully prepared by Dr Seibert in 1939 – termed PPD-standard, or PPD-S.4 With this, and other technical improvements, highly reproducible tuberculin test results could be obtained.5,6 In Europe, the Staten Serum Institute of Copenhagen, Denmark, developed a tuberculin test material termed RT-23. After considerable standardization work, this product was accepted as a standard tuberculin by the World Health Organization (WHO).7,8 Results from studies indicate that 2 TU of RT-23 provides equivalent results to testing with 5 TU of PPD-S.9 The available tuberculin tests and materials are summarized in the glossary of terms (Box 19.1). Injection of tuberculin material intradermally into a person previously infected with M. tuberculosis will result in infiltration of previously sensitized lymphocytes circulating in peripheral blood. At the site of injection, CD4 and CD8 T-lymphocytes, as well as monocytes and macrophages, will accumulate. These release inflammatory mediators, which produce oedema and erythema. Although there is increased blood flow, the locally increased metabolic activity of these inflammatory cells results in relative hypoxia and acidosis, which may be severe enough to result in ulceration and necrosis.10 Tuberculin reactions have often been equated with immune status. Even though both result from exposure and acquisition of infection, TST reactions and protective immunity are independent phenomena.
TECHNICAL ASPECTS OF TUBERCULIN SKIN TESTING Administration of the test Tuberculin test materials are commercially available in strengths ranging from 1 to 250 TU per test dose. Administration of 1 TU is not recommended because this preparation has reduced
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SECTION
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GENERAL CLINICAL FEATURES AND DIAGNOSIS
Box 19.1 Glossary of Terms Heaf
Multipuncture method of tuberculin testing introduced by Heaf in 1951. The prongs are dipped in tuberculin solution and then pressed into the skin. No longer in use.
IGRA (interferon-g release assays)
New in vitro tests for diagnosis of LTBI. Employ M. tuberculosis-specific antigens to stimulate lymphocytes from whole blood overnight, then measure interferon-g produced by sensitized circulating lymphocytes.
Mantoux
Intradermal method of tuberculin skin testing, first described by Mantoux in 1912. Tuberculin material is injected intradermally, and the resultant induration is measured 48–72 hours later.
NTM (nontuberculous mycobacteria)
Also know as MOTT (mycobacteria other than TB) and environmental mycobacteria. Includes diverse organisms such as M. avium, M. intracellulare, M. scrofulaceum, and M. kansasii. Not contagious although may be pathogenic. Important as may cause positive TST through cross-reactivity to very similar mycobacterial antigens.
PPD (purified protein derivative)
Material for tuberculin testing, prepared from culture of mycobacterial species, filtered, and purified by precipitation with ammonium sulphate or trichloroacetic acid. This is standardized against one large batch of standard PPD (PPD-S) produced from M. tuberculosis by Dr Florence Seibert in 1941. Stored by the Food and Drug Administration (FDA) (in the USA) and used worldwide as standard lot. One tuberculin unit (1 TU) is defined as 0.02 mg of PPD-S. Standard dose is 5 TU ¼ 0.1 mg. All commercial lots of PPD must be tested for bio-equivalence against PPD-S. This PPD is used mainly in North America.
QuantiFERONTB Gold
Commercial IGRA test, in which whole blood is incubated overnight with TB-specific antigens, then interferon-g produced by sensitized lymphocytes is measured using an enzyme-linked immunosorbent assay (ELISA) technique.
RT-23
Commercial PPD produced from M. tuberculosis, validated against PPD-S, and standardized by the WHO. This tuberculin test material is the most commonly used outside North America. Manufactured by Statens Serum Institute (Copenhagen). Standard dose is 2 TU, which is equivalent to 5 TU of PPD.
TINE
Multipuncture method of tuberculin testing. Four prongs (tines) coated with dried tuberculin material are pressed into the skin for about 2 seconds.
T-SPOT-TB
Commercial IGRA test, in which lymphocytes are separated, incubated with TB-specific antigens overnight, then lymphocytes expressing interferon-g are counted (Elispot technique).
sensitivity in children and adults, and it is not as safe.11–13 Higher strength formulations such as 100 or 250 TU are not recommended because positive reactions are very non-specific, have no correlation with risk of infection, and are more likely to revert later.14–16 Despite clear recommendations, published studies continue to use substandard tuberculin doses, reducing sensitivity, while others have used
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excessive doses such as 10 TU PPD-S, worsening specificity.17–23 The tuberculin skin test can be administered using the Mantoux method of intradermal injection, or using multipuncture techniques such as the Tine test. The multipuncture tests have lower sensitivity, and are less reproducible.24–28 It is recommended that for all tuberculin testing a dose bioequivalent to 5 TU of PPD-S, or 2 TU of RT23, be administered using the Mantoux technique of intradermal injection with a 26-G needle.8 The size of the wheal produced following intradermal injection is affected by age and gender – so is not reliable for verifying the amount injected.8 If injections are given subcutaneously, larger, more diffuse reactions will result, which are more difficult to read.15,29 The site of injection is not important, although the inner or volar aspect of the forearm is generally used for convenience.15
Reading the test Reactions that appear after 6 hours are non-specific, and readings at 24 hours are less sensitive and specific than readings at 48 hours.30,31 Readings made at 7 days following tuberculin administration will be 19–21% less sensitive.12,32 When two-step testing is performed, reading the first tuberculin test after 7 days has been suggested because the second test can be administered immediately for those with negative reactions. This approach is more practical, but is not recommended, because of the 20% lower sensitivity, plus 5% lower specificity.32 As well, all information regarding risk of TB (reviewed below) is based on TST measured 48–72 hours after testing. Hence, readings made later will be harder to interpret. Induration can be defined by palpation, or the ballpoint method, introduced by Sokal – the methods are equivalent.33–35 Selfreading by patients is not recommended as it is inaccurate. In two studies, 63–94% of patients with positive reactions believed they were negative.31,36 The variability of results with the Mantoux technique from two simultaneous tuberculin tests is remarkably small given the inherent variability resulting from administration and reading. Differences in readings, leading to misclassification errors, are less frequent when the same readers repeat their measurements (i.e. intrareader variability) than when different persons are asked to measure the same reaction (inter-reader variability).8 In two studies systematic differences between one reader and the others contributed the majority of variance and misclassification errors – a potentially correctable problem.37,38 Other potential causes of reader error are terminal digit preference or rounding, and conscious or unconscious reader bias. These problems can be minimized by using simple measuring calipers, such as those used by mechanics or tailors. Adverse reactions Adverse reactions to TSTs are rare. Vasovagal reactions can occur as with any injection. Immediate wheal and flare with a local rash was seen in 2% of allergy clinic patients.39 These reactions were associated with atopic history but not with positive tuberculin reactions at 48–72 hours. Well-documented anaphylaxis has been reported once following Mantoux testing.13 There have been two well-documented cases of anaphylaxis, one of them fatal, associated with Tine testing.40,41 In patients with severe blistering and ulceration, hydrocortisone cream is often given but was of no benefit in the only randomized controlled trial to assess its use.42 There is no evidence whatsoever that tuberculin testing poses any risk in pregnancy nor that tuberculin reactions are influenced by pregnancy, although in past years the manufacturers’ product monograph mentioned this as a potential precaution.43–45
CHAPTER
Immune-based tests for tuberculosis
INTERPRETING THE TST: FALSE-NEGATIVE AND FALSE-POSITIVE REACTIONS False-negative tests The TST may be false negative because of technical problems in the preparation or storage of material, or in the administration or reading of the test. Most of these problems can be avoided by meticulous technique in test administration and reading. Proper storage is important because test material will deteriorate if exposed to light or heat, or if frozen. Biological causes of false-negative results are more difficult to avoid. False-negative tests may occur in patients with active TB disease: estimates range from 5–8% in cross-sectional studies of patients already on treatment to 17% at the time of diagnosis, 30% among elderly patients, and 50% in patients with advanced disease in Nigeria.46–49 False-negative tests in TB patients are associated with more advanced forms of TB, malnutrition, and elevated serum creatinine levels.50–52 False-negative tests increase with older age.53 In most highincome countries, the proportion with a positive tuberculin reaction increases up to the age of 65, after which it declines.32,54,55 The proportion of false-negative reactions in dually infected (human immunodeficiency virus (HIV) and TB) patients ranges from 15–28% in those with CD4 counts greater than 400–500 up to 100% in patients with CD4 counts less than 200.56–58 Anergy testing, reviewed extensively elsewhere, has been suggested for the assessment of individuals with possibly false-negative TSTs.59,60 Among HIV-infected patients with negative tuberculin tests, the incidence of TB was significantly higher in those who were anergic than in those who were not.61–63 However, in individual patients results of anergy testing can be very misleading.64,65 Perhaps the most important finding is that in five placebo-controlled randomized trials, isoniazid (INH) was of no benefit in TST-negative, HIV-infected individuals.66 Accordingly anergy testing is not recommended for patient management.67
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Table 19.1 Summary of effect of BCG vaccination on TST Parameter
Value
10 years > 10 years 10–14 mm 15þ mm 10–14 mm 15þ mm
Interval TST size TST size if interval 10 years
Age when BCG vaccination given Infancy (% 10þ mm)
Older (% 10þ mm)
8.7 1.0 4.6 1.9 1.4 0
43 21 20 19 11 8
From meta-analysis of 24 studies with 240,203 subjects BCGvaccinated in infancy, and 12 studies of 12,728 subjects BCG-vaccinated after the age of 1 year. Adapted from Farhat M, Greenaway C, Pai M, et al. False positive tuberculin skin tests - What is the absolute effect of BCG and nontuberculous mycobacteria? Int J Tuberc Lung Dis 2006;10(11):1–13.
NTM may result in disease in humans.82,83 Testing with NTM antigens is not recommended for clinical use to diagnose nontuberculous mycobacterial disease.84,85 Antigens from NTM and M. tuberculosis are similar, which results in cross-reactivity when tuberculin testing. In experimental studies, animals infected with different mycobacteria developed the largest reactions to antigens prepared from that specific mycobacteria, and smaller reactions to antigens from other mycobacteria.86,87 In experimental animals infected with NTM, the proportion
25%
Fig. 19.1 Effect on TST reactions of BCG vaccination in infancy. Dashed
False-positive tests: non-tuberculous mycobacteria Non-tuberculous mycobacteria (NTM) exist in soil and water in the environment, particularly where the climate is warm and moist.80,81 In many parts of the world, a high proportion of individuals will have sensitivity to at least one NTM antigen by the age of 20. Although much less pathogenic than M. tuberculosis, these
line: weighted average effect in studies where the interval from BCG until TST was < 10 years. Dotted line: weighted average effect in stud
