Pulmonary Tuberculosis:

towards improved adjunctive therapies

Anna Ralph

July 2010

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy of The Australian National University

Front section: Author’s statement

Author’s statement

This thesis describes the development, implementation and preliminary results of the Arginine and Vitamin D Adjunctive Therapy in Pulmonary Tuberculosis (AVDAPT) randomised controlled trial.

I had a central role in developing and implementing the study protocol collaboratively with my PhD supervisors Associate Professor Paul Kelly (Australian National University, Canberra) and Professor Nicholas Anstey (Menzies School of Health Research, Darwin), and with additional input from the investigators listed in Appendix 1. I wrote the initial draft of the study protocol for submission to the relevant ethics committees and for trial registration purposes (http://clinicaltrials.gov/show/ NCT00677339). I supervised and participated in the collection of data, performed the data analyses, wrote the thesis, and wrote all published and submitted manuscripts arising from the thesis. The named co-authors made intellectual and writing contributions to the final manuscripts.

One section of data analysis was not performed by me: the interim safety analysis described in Chapter 10 was conducted by an independent biostatistician (Mr Joseph McDonnell, Menzies School of Health Research) in his role as a member of the AVDAPT study Data and Safety Monitoring Committee.

I am a named Chief Investigator on the successful National Health and Medical Research Council Project Grant Application 605806 entitled ―L-Arginine and Vitamin D Adjunctive Therapies in Pulmonary Tuberculosis‖.

______

Anna Ralph BMedSci, MBBS(Hons), MPH, DTM&H, FRACP July 2010

i Front section: Acknowledgements

Acknowledgements

I would like to acknowledge a number of people and organisations who have played significant roles in this research project. I am very grateful to the National Health and Medical Research Council for providing a Postgraduate scholarship and a 2009 research grant, and the Australian Respiratory Council and the Royal Australasian College of Physicians (Covance award) which generously provided funds for the project.

At the field research site in Timika, my very great thanks go to the research assistants Bapak Govert Waramori and Dr Gysje Pontororing, and director of the Timika Translational Research Facility Dr Enny Kenangalem, for their enthusiasm and commitment to the project, and for making the work so enjoyable. Profound thanks also to all Timika Translational Research Facility staff who make the project possible: Ferryanto Chalfein, Prayoga, Daud Rumere, Frans Wabiser, Yeni, Henwi Pieris and Baspak Gobay (laboratory staff); Natalia Dwi Haryanti and Sri Hasunik (data management), Sri Rahayu (administration), and Gertruida Bellatrix and Hendrix Antonius (research assistants). Thanks also to Dr Daniel Lampah for medical and logistical help, and Maikel Zonggonau for driving, errands, and making sense of my limited Bahasa Indonesia. At RSMM, I sincerely thank Dr Paulus Sugiarto for his support and for chairing the Data and Safety Monitoring Committee, and Dr Enny Malonda, for helpful discussions about TB management in Timika. Dr Rini Poespoprodjo and Drs Franciscus Thio have also offered very valuable assistance for which I thank them very much. Special thanks also to TB clinic staff Dr Andri Wiguna for medical support, Bapak Djonny Lempoy for frequent general assistance, and to Bapak Erstanto, Head, Timika TB laboratory, for diligently processing, recording and storing sputum slides for the study. I also greatly thank Drs Pasi Pennitien, Michael Bangs, and Michael Stone (Public Health / Malaria Control, Timika), for their vital support across many tasks, including facilitating access to consumables and transporting specimens to Jakarta. Finally in Timika, I am indebted to all the study participants, healthy volunteers and their families for their involvement in the study.

ii Front section: Acknowledgements

In Jakarta, I extend my thanks to colleagues at the Ministry of Health‘s National Institute of Health Research and Development who allowed this study to proceed. In particular, I thank Dr Sandjaja for his assistance in facilitating the project, and his intellectual and practical input to the project; Dr Dina Bisara Lolong for contributing to the development of the study protocol and visiting the field site, Ibu Merryani Girsang for contributing to laboratory quality control checks, and Dr Emiliana Tjitra for providing a co-ordinating role. Major thanks to Dr Retno Soemanto and Mbak Yuni Rukminiati at the University of Indonesia Faculty of Microbiology for taking on the large task of processing all specimens collected for this project (culture / DST), and for being readily available for frequent discussions about results.

In Darwin at Menzies School of Health Research (MSHR), I wish to convey deep thanks to my supervisor Professor Nicholas Anstey who has been a greatly valued mentor, and whose intellectual rigor is a constant inspiration. His attention to detail and boundless reserves of optimism, tenacity and diplomacy mitigated many potential problems, solved the seemingly insoluble, and kept the project afloat. He also provided greatly-appreciated contributions to the development of this thesis and the publications arising from it. I greatly thank Dr Ric Price (PhD co-supervisor) for trying to impart to me some of his knowledge regarding statistics, data management and data analysis; the databases created for this thesis relied heavily on his assistance, and his detailed statistical advice was greatly appreciated. Great thanks to Kim Piera for her meticulous approach to managing logistics and supplies, providing laboratory expertise, packaging medications, and helping with data entry, as well as providing good company in Timika. Other laboratory personnel including Drs Tonia Woodberry, Gabriella Minigo and Jutta Marfurt have been extremely helpful in educating both myself and the Timika staff in laboratory methods, and striving to maintain good laboratory standards. Administrative staff at MSHR essential to the operation of this project include Tania Paul, Ella Curry, Robi Cohalan, Asriana Kebon and Joanne Bex, to all of whom I am very grateful. Also at MSHR, thanks to Associate Professor Peter Morris (PhD Advisor) for his major contributions to development of the study protocol, advice regarding the analytical plan, and input to devising a composite clinical outcome score; to Dr Nick Douglas for greatly- appreciated help and company in Timika; to Dr Tsin Yeo for statistical advice and for making available his data and knowledge on exhaled nitric oxide measurement; to Dr

iii Front section: Acknowledgements

Joshua Davis for friendly and comprehensible statistical advice; to Dr Louise Maple- Brown and Joseph McDonnell for their roles in the Data and Safety Monitoring Committee.

At ANU, immense thanks to Associate Professor Paul Kelly who chaired my PhD supervisory panel. It was through enthusiastic discussions with him in 2006 that I was inspired to embark on this research. His earlier tuberculosis work in Timika and in other international settings provided a strong basis for the current project, and his contributions to discussions with collaborators in Timika and Jakarta have been vital for the project‘s operation. I also sincerely thank Paul for making available to me the TB data that he and others had previously obtained in Timika, for providing templates on which I could model the study protocol and data collection forms, and for providing essential feedback on manuscripts, including this thesis. My thanks also go to Professor Niels Becker (PhD co-supervisor) for providing patient and detailed statistical help, especially in relation to the x-ray analyses and the exhaled nitric oxide correction factors, and to Dr Mark Clemens for his major assistance in the complicated factorial study sample size calculation. To all other ANU staff who provided help (including Dr Robyn Lucas for vitamin D advice, and Sarah Geddes and Barbara Bowen for administrative help), to many fellow ANU PhD students who provided much-appreciated camaraderie and support, I convey my great thanks also.

I also wish to sincerely thank Associate Professor Graeme Maguire, James Cook University (PhD Advisor), for support with supplies, logistics, lung function testing, data analyses, and occasional accommodation (snakes notwithstanding). I am very grateful for the important mycobacteriological advice and expertise provided by Mr Richard Lumb at the Institute for Medical & Veterinary Science, including his visits to the Jakarta laboratory. Many thanks also to Dr Cheryl Salome, Woolcock Institute of Medical Research, for valuable exhaled nitric oxide advice and kind provision of materials. Thanks also to Dr Mairwen Jones for proof reading sections of this thesis.

Deepest thanks finally go to my partner Deborah for tolerating my absences and other difficulties this project has brought, and for supporting me unconditionally in every way.

iv Front section: Figure

FIGURE 1: NIHRD-MSHR Timika research staff outside the research buidling

L to R: Front row Maikel Zonggonau, Enny Kenangalem, Sri Rahayu, Basbak Gobay, Hendrix Antonius Back row: Yani Reyaan, Wendelina Fobia, Anna Ralph, Frans Wabiser, Gysje Pontororing, Adol Fobia, Sri Hasunik. Photo: Nick Anstey.

v Front section: Abstract

Abstract

The potential to improve pulmonary tuberculosis (PTB) treatment outcomes with adjunctive immunotherapies requires investigation. L-arginine and vitamin D have antimycobacterial properties which render them suitable candidates. Therefore the Arginine and Vitamin D Adjunctive therapy in Pulmonary TB (AVDAPT) trial evaluates these supplements in PTB. This large trial commenced in June 2008. The project is run in Timika, Papua Province, Indonesia by the International Health Division, Menzies School of Health Research (Darwin, Australia), the National Institute for Health Research and Development (Ministry of Health, Indonesia), and the Australian National University (Canberra).

Aims of this thesis were to design and commence the AVDAPT study and examine baseline data. Among the tested hypotheses were that exhaled nitric oxide (FENO), an L-arginine-derived antimycobacterial immunological mediator, would be elevated in PTB compared with healthy controls (HC), and inversely related to disease severity; secondly, that significant relationships would exist between different measures of TB severity.

Consenting, eligible adults with smear-positive PTB were enrolled at the Timika TB clinic according to the protocol. Assessments included sputum microscopy, culture and susceptibility, X-ray, weight, pulmonary function, FENO, 6-minute walk testing (6MWT) and quality of life (St George‘s Respiratory Questionnaire [SGRQ]). HC were enrolled for a single assessment.

Results from 162 TB patients and 40 HC included: (1) findings pertaining to the trial (development / validation of outcome measures, and establishment of locally-relevant reference ranges for 6MWT and SGRQ); (2) findings pertaining to improved understanding of TB (demonstration of relationships between clinical, physiological, immunological [FENO] and functional measures of disease severity), and (3) investigation of TB drug-resistance and HIV rates.

vi Front section: Abstract

A key finding was that FENO was not elevated in TB compared with HC and was lower still in worse disease. These findings suggest that an impaired ability to generate adequate NO (e.g. in L-arginine deficiency) might contribute to host inability to adequately contain TB or mitigate lung pathology. These findings support the rationale for conducting a trial of adjunctive L-arginine in TB.

New relationships were identified between sputum smear grade, X-ray, weight, pulmonary function, 6MWT and SGRQ. Patients with more-severe malnutrition had worse pulmonary function; 6MWT was independent of lung function; SGRQ results accurately captured people‘s perceived quality of life, correlating significantly with symptoms, 6MWT and pulmonary function; and sputum bacillary grade was significantly related to radiological extent and weight, but not to other results. These findings support the use of a range of outcome measures in TB trials, to provide a comprehensive assessment of TB severity, rather than focusing on bacteriology alone.

An x-ray severity score and a clinical outcome score were created, providing valuable tools for use in clinical trials. Interim analysis confirmed the safety of L-arginine and vitamin D adjunctive therapy. Multi-drug resistant TB rates remained low in new cases (2.0%), but HIV-TB co-infection rates rose significantly over 5 years, creating major challenges.

This thesis provides the basis for continuation of the AVDAPT study, produces original findings relating to clinico-immunological aspects of PTB, and provides information of major local importance to help guide TB service provision in Timika.

vii Front section: Glossary

Glossary

6MWT 6 minute walk test 6MWWD 6 minute weight.walk distance AE Adverse event AVDAPT Arginine and Vitamin D Adjunctive therapy in pulmonary tuberculosis AFB Acid-fast bacilli AIDS Acquired immunodeficiency syndrome ANU Australian National University BTA Basil tahan asam (acid-fast bacilli) CI Confidence interval Dinas Dinas Kesehatan (District Health Authority) DOT Directly Observed Treatment DOTS Directly Observed Treatment, Short-course DSMC Data and Safety Monitoring Committee E Ethambutol FDC Fixed-dose combination antituberculous therapy FENO Fractional exhaled nitric oxide FEV1 Forced expiratory volume in 1 minute FKUI Faculty of Microbiology, University of Indonesia GMP Good Manufacturing Practice H Isoniazid HIV Human Immunodeficiency Virus IFN-γ Interferon gamma IMVS Institute for Medical & Veterinary Science MDR-TB Multi-drug resistant TB MGIT Mycobacterium Growth Indicator Tube MIRU Micro-satellite Interstitial Repetitive Unit MSHR Menzies School of Health Research (Darwin, Australia) MTB Mycobacterium tuberculosis NCEPH National Centre for Epidemiology & Population Health (Australia) NHMRC National Health & Medical Research Council (Australia) NIHRD National Institute of Health Research & Development (Indonesia) NiOX FLEX Device for measurement of exhaled nitric oxide (non-portable) NiOX MINO Device for measurement of exhaled nitric oxide (portable) NO Nitric oxide NOS Nitric Oxide synthase NTP National TB Control Program PTB Pulmonary TB PT Perseroan Terbatus (Proprietary Limited) Puskesmas Pusat Kesehatan Masyarakat (Community Health Centre) R Rifampicin RA Research assistant RCT Randomised, controlled trial RSMM Rumah Sakit Mitra Mayarakat (Community hospital, Timika) RSUD Rumah Sakit Umum Daerah (Regional General Hospital, Timika) S Streptomycin SAE Serious adverse events SGRQ St George‟s Respiratory Questionnaire TB Tuberculosis Th1 T helper cell Type 1 TLR Toll-like receptor TNF Tumour necrosis factor UV Ultraviolet VCT Voluntary counselling and testing for HIV WHO World Health Organization XDR-TB Extensively drug-resistant TB Z Pyrazinamide

viii Tables of contents

Table of Contents

AUTHOR‟S STATEMENT ...... I ACKNOWLEDGEMENTS ...... II ABSTRACT ...... VI GLOSSARY ...... VIII 1 AIMS ...... 1 1.1 BACKGROUND ...... 1 1.2 AIMS ...... 2 2 INTRODUCTION I: TUBERCULOSIS ...... 1 3 INTRODUCTION II: ADJUNCTIVE TREATMENT IN TB ...... 5 3.1 WHY ARE NEW TREATMENT APPROACHES NEEDED? ...... 5 3.2 ADJUNCTIVE THERAPIES ...... 8 3.3 ARGININE AND VITAMIN D AS NOVEL POTENTIAL ADJUNCTIVE TREATMENTS IN TB ...... 10 4 INTRODUCTION III: NITRIC OXIDE AND ITS MEASUREMENT IN VIVO ...... 19 4.1 NITRIC OXIDE PATHWAYS ...... 19 4.2 EXHALED NITRIC OXIDE MEASUREMENT ...... 20 5 STUDY SETTING ...... 26 5.1 TIMIKA FIELD RESEARCH FACILITY ...... 26 5.2 TIMIKA TB CLINIC...... 28 5.3 OVERVIEW OF TIMIKA ...... 29 5.4 CONDUCT OF MEDICAL RESEARCH IN PAPUA ...... 34 5.5 CONCLUSION ...... 34 6 AVDAPT STUDY DESIGN AND METHODOLOGY ...... 36 6.1 BACKGROUND ...... 36 6.2 HYPOTHESES ...... 37 6.3 AIMS ...... 37 6.4 METHODS ...... 38 6.5 HEALTHY VOLUNTEER SUB-STUDY ...... 65 7 RESULTS I: BASELINE DATA IN STUDY PARTICIPANTS AND HEALTHY VOLUNTEERS ...... 67 7.1 INTRODUCTION ...... 67 7.2 METHODS ...... 68 7.3 RESULTS ...... 70 7.4 DISCUSSION ...... 85 7.5 CONCLUSION ...... 93 8 RESULTS II: RELATIONSHIPS BETWEEN MICROBIOLOGICAL, CLINICAL AND FUNCTIONAL MEASURES OF TB SEVERITY ...... 94 8.1 INTRODUCTION ...... 94 8.2 METHODS ...... 95 8.3 RESULTS ...... 95 8.4 DISCUSSION ...... 100 8.5 CONCLUSION ...... 103 9 RESULTS III: MEASURING TB RADIOLOGICAL SEVERITY ...... 104

ix Tables of contents

10 RESULTS IV: LONGITUDINAL FOLLOW UP ...... 105 10.1 INTRODUCTION ...... 105 10.2 METHODS ...... 107 10.3 RESULTS ...... 110 10.4 DISCUSSION ...... 123 10.5 CONCLUSION ...... 129 11 RESULTS V: EXHALED NITRIC OXIDE IN PULMONARY TB ...... 131 11.1 INTRODUCTION ...... 131 11.2 METHODS ...... 132 11.3 RESULTS ...... 134 11.4 DISCUSSION ...... 143 11.5 CONCLUSIONS ...... 150 12 RESULTS VI: HIV-TB CO-INFECTION ...... 152 12.1 INTRODUCTION ...... 152 12.2 METHODS ...... 154 12.3 RESULTS ...... 155 12.4 DISCUSSION ...... 160 12.5 CONCLUSION ...... 163 13 CONCLUSIONS AND FUTURE DIRECTIONS ...... 165 13.1 CLINICAL FINDINGS ...... 166 13.2 HIV-TB AND MDR-TB TRENDS...... 168 13.3 CONCLUSIONS FROM THE AVDAPT STUDY TO DATE ...... 168 14 REFERENCES ...... 171 15 APPENDICES ...... 187 15.1 STUDY INVESTIGATORS ...... 187 15.2 GOOD CLINICAL PRACTICE CERTIFICATION ...... 189 15.3 TRIAL REGISTRATION ...... 190 15.4 AVDAPT INFORMATION AND CONSENT FORMS ...... 191 15.5 AVDAPT DATA COLLECTION FORMS ...... 195 15.6 ST GEORGE‘S RESPIRATORY QUESTIONNAIRE ...... 205 15.7 ARGIMAX CERTIFICATE OF ANALYSIS ...... 207 15.8 CALCIFEROL STRONG CERTIFICATE OF ANALYSIS ...... 208 15.9 SAFTEY REPORTING PROCESS ...... 209 15.10 SERIOUS ADVERSE EVENT REPORT FORM ...... 211 15.11 HYPERCALCAEMIA MANAGEMENT GUIDELINE ...... 214 15.12 EXAMPLE OF EPIDATA DATABASE ...... 216 15.13 HEALTHY VOLUNTEER INFORMATION & CONSENT FORM ...... 217 15.14 HEALTHY VOLUNTEER DATA COLLECTION FORM ...... 219

TABLES Table 2.1: Chronology of TB milestones ...... 1 Table 4.1: Factors associated with abnormal exhaled nitric oxide ...... 23 Table 6.1: Follow-up schedule for AVDAPT study participants ...... 51 Table 6.2: Blood collection schedule for AVDAPT study participants ...... 52 Table 7.1: Baseline characteristics of AVDAPT study participants (TB patients) and healthy volunteers 72 Table 7.2: Symptoms ...... 74 Table 7.3: Baseline clinical findings and haemoglobin ...... 75 Table 7.4: Baseline laboratory and radiological findings ...... 79 Table 7.5: Agreement in sputum AFB grade ...... 81 Table 8.1:Association between sputum smear grade at diagnosis and other measures of disease severity ...... 96 Table 8.2: Regression coefficients from univariate linear regression models examining associations between clinical and laboratory measures* ...... 97 Table 8.3: Correlation matrices showing correlation coefficients (top number) and p values (bottom number) for baseline clinical and laboratory measures* ...... 98

x Tables of contents

Table 10.1: Composite clinical outcome score ...... 108 Table 10.2: Sputum MTB microscopy and culture results at baseline and 4 and 8 weeks ...... 112 Table 10.3: Predictors of sputum culture conversion, univariate analyses ...... 116 Table 10.4: Six-month treatment outcome...... 116 Table 10.5: Composite clinical outcome score at 8 weeks (end of intensive treatment phase) and 24 weeks (end of treatment) ...... 117 Table 10.6: Correlations between outcome scores and percentage change in other measures ...... 118 Table 10.7: Serious adverse events (SAE) ...... 121 Table 10.8: Number of study participants with hypercalcaemia ...... 122 Table 10.9: Summary of findings of the interim analysis ...... 123 Table 11.1: Correlation between FENO measures ...... 134 Table 11.2: Corrections applied to exhaled nitric oxide readings obtained from the NiOX MINO portable analyser ...... 137 Table 11.3: Characteristics and FENO in healthy volunteers and TB patients at enrolment ...... 138 Table 11.4: Summary of salient findings from the present study and previous investigations of exhaled nitric oxide in tuberculosis ...... 149 Table 12.1: Characteristics of 138 study participants with known HIV status ...... 156 Table ‎12.2: Treatment outcome in HIV positive and HIVnegative study participants ...... 159 Table 12.3: TB-HIV coinfection management ...... 160

FIGURES FIGURE 1: NIHRD-MSHR Timika research staff outside the research buidling ...... v Figure 4.1: NiOX FLEX ...... 22 Figure 4.2: NiOX MINO ...... 23 Figure 5.1: Map of Indonesia and Northern Australia showing Timika (Papua Province) ...... 26 Figure 5.2: Timika community hospital (Rumah Sakit Mitra Masyarakat) ...... 27 Figure 5.3: NIHRD-MSHR Timika research staff and visitors in the research buidling ...... 28 Figure 5.4: Timika TB clinic and staff ...... 29 Figure 5.5: Mimika district map ...... 30 Figure 5.6: HIV education banner near RSMM hospital ...... 34 Figure 5.7: Images of Timika ...... 35 Figure 6.1: Diagrammatic representation of AVDAPT trial and related studies ...... 39 Figure 6.2: Consent booklet ...... 41 Figure 6.3: Enrolment procedure ...... 44 Figure 6.4: Sample stickers applied to study medications ...... 46 Figure 6.5: Spirometer calibration using 3 litre syringe ...... 48 Figure 6.6: Mycobacterial growth indicator tubes (MGIT) ...... 51 Figure 6.7: Sample processing at the NIHRD-MSHR research facility ...... 53 Figure 7.1: Eligibility screening and enrolment of AVDAPT study participants ...... 71 Figure 7.2: Smoking rates in male and female AVDAPT study participants and healthy volunteers* ...... 73 Figure 7.3: Educational attainment in Papuan and Non-Papuan study participants* ...... 73 Figure 7.4: Employment status in Papuan and Non-Papuan study participants ...... 74 Figure 7.5: Symptoms reported among AVDAPT study participants at TB diagnosis ...... 76 Figure 7.6: Number of symptoms reported at the time of TB diagnosis in men and women ...... 76 Figure 7.7: Nutritional status in AVDAPT study participants ...... 77 Figure 7.8: Haemoglobin according to sex, ethnicity, HIV status and in healthy volunteers versus TB patients overall ...... 80 Figure 7.9: Educational attainment in healthy volunteers and TB patients* ...... 82 Figure 7.10: Telephone ownership in TB patients and healthy volunteers ...... 82 Figure 7.11: Employment status in TB patients and healthy volunteers ...... 83 Figure 7.12: BMI in healthy volunteers and AVDAPT study participants ...... 83 Figure 7.13: Six minute walk distances in healthy volunteers and AVDAPT study participants* ...... 84 Figure‎7.14:‎St‎George’s‎respiratory‎questionnaire‎total‎score‎in‎healthy‎volunteers‎and‎AVDAPT‎study‎ participants* ...... 85 Figure 8.1: Relationship between smear grade at TB diagnosis and CXR score and weight ...... 96 Figure 8.2: Relationships between lung function impairment and weight, haemoglobin, illness duration and quality of life total score (SGRQ) ...... 99 Figure 8.3: Relationship between number of reported symptoms and illness duration and quality of life (SGRQ) score ...... 100 Figure 8.4: Illness duration in relation to educational attainment ...... 100 Figure 10.1: Participant follow up profile ...... 110

xi Tables of contents

Figure 10.2: Microscopy and culture results at enrolment, and 4 and 8 weeks in participants followed for 8 weeks ...... 112 Figure 10.3: Kaplan-Meier survival curve showing probability of sputum smear conversion by ethnicity ...... 114 Figure 10.4: Kaplan-Meier survival curve showing probability of sputum smear conversion by HIV status ...... 114 Figure 10.5: Kaplan-Meier survival curve showing probability of sputum smear conversion by cavitary disease status ...... 115 Figure 10.6: Kaplan-Meier survival curve showing probability of sputum smear conversion by baseline sputum AFB grade ...... 115 Figure 10.7: Histograms demonstrating distributions of composite clinical outcome scores at 8 and 24 weeks ...... 118 Figure 10.8: Non-serious adverse events experienced by AVDAPT study participants ...... 120 Figure 10.9: Serial calcium readings in a representative individual receiving TB treatment and study medications ...... 122 Figure 10.10: Ionised calcium (mean indicated by bar and range) by week in AVDAPT study participants ...... 122 Figure 11.1: Bland-Altman plot: repeated exhaled nitric oxide measures on single analyser ...... 135 Figure 11.2: Bland-Altman plot: paired exhaled nitric oxide measures on 2 analysers ...... 135 Figure 11.3: Bland-Altman plot: paired exhaled nitric oxide measures on the NiOX MINO (portable analyser)‎and‎NiOX‎FLEX‎(‘gold‎standard’) ...... 136 Figure 11.4: Relationship between exhaled nitric oxide measures on the Niox Mino and Niox Flex ...... 136 Figure 11.5: Exhaled nitric oxide in healthy volunteers and TB patients at week 0 and during TB treatment ...... 140 Figure 11.6: Exhaled nitric oxide according to sex in TB patients at week 0 ...... 141 Figure 11.7: Exhaled nitric oxide according to weight in TB patients at week 0 ...... 141 Figure 11.8: Correlations between change in FENO and clinical outcome score at weeks 8 and 24 ...... 142 Figure 12.1: Rates of HIV-TB co-infection among study participants ...... 157 Figure 12.2: HIV-TB co-infection rates in Timika in 2003-4 compared with 2008-9 ...... 158 Figure 12.3: Meeting between AVDAPT research personell and local HIV care providers ...... 164

BOXES Box 3.1: Actions of Vitamin D ...... 13 Box 4.1: Principles of exhaled nitric oxide measurement ...... 21 Box 5.1: Approvals required for conducting medical research in Papua, Indonesia ...... 35 Box 6.1: Explanation regarding planned off-shore laboratory analyses ...... 55 Box 7.1: Definitions ...... 69 Box 7.2: Locally-relevant reference ranges for 6-minute walk testing ...... 85 Box 10.1: AVDAPT study primary outcome measures ...... 106

PUBLISHED AND SUBMITTED MANUSCRIPTS Manuscript 1: Ralph AP, Anstey NM, Kelly PM. Tuberculosis into the 2010s: is the glass half full? Clin Infect Dis 2009;49(4):574-83...... 3 Manuscript 2: Ralph A, Kelly P, Krause V. What's new in TB? Australian Family Physician 2009; 38(8):578-585...... 4 Manuscript 3: Ralph AP, Kelly PM, Anstey NM. L-arginine and vitamin D: novel adjunctive immunotherapies in tuberculosis. Trends Microbiol 2008;16(7):336-44...... 15 Manuscript 4: Ralph AP, Ardian M, Wiguna A, Maguire GP, Becker NG, Drogumuller D, Wilks MJ, Waramori G, Tjitra E, Sandjaja, Kenangalem E, Pontororing GJ, Anstey NA, Kelly PM. A simple, valid, numerical score for grading chest X-ray severity in adult smear positive pulmonary tuberculosis. Accepted, Thorax, July 2010...... 104

xii Chapter 1: Aims

1 Aims

1.1 BACKGROUND

Mycobacterium tuberculosis (MTB) is one of the most successful human pathogens. It infects an estimated third of the human population, and currently accounts for the greatest number of deaths from a curable infectious disease.1, 2 Tuberculosis (TB) is an historical disease whose existence may pre-date Homo sapiens,3 but it maintains major contemporary relevance. It re-emerged as a ‗global emergency‘ by the turn of the 21st century, and now is defying reduction targets in parts of the globe worst-affected by the overlapping pandemics of TB and HIV.2 New case numbers are estimated at around 9.3 million annually.2 Antibiotic treatment regimens for TB, largely unchanged in the last four decades, are cumbersome and protracted, contributing to cure rates frequently falling short of the 85% target set by the World Health Organisation (WHO).4 Innovative strategies therefore require investigation for their potential to accelerate responses to antibiotics, with the ultimate goals of reducing required TB treatment duration, reducing the period of infectivity, permitting earlier return to employment or school, and reducing post-TB residual lung pathology.

Recognising this priority research field, the AVDAPT (Arginine and Vitamin D Adjunctive therapy in Pulmonary TB) clinical trial investigates the use of L-arginine and vitamin D as immunotherapies supplementary to conventional TB treatment in pulmonary TB. This is a large randomised, double-blind, placebo-controlled trial which commenced in June 2008 and is projected to complete enrolments in early 2012, at the Timika Translational Research Facility in Papua, Indonesia, through Menzies School of Health Research‘s (MSHR) International Health Division in partnership with the National Institute for Health Research and Development (NIHRD), Indonesian Ministry of Health, and the Australian National University (ANU).

1 Chapter 1: Aims 1.2 AIMS

The aim of this thesis is to design and implement a clinical trial of the safety and efficacy of L-arginine and vitamin D as adjunctive therapies in pulmonary TB (the AVDAPT study). This includes methodological objectives (Aim 1) and analytical objectives (Aims 2 to 8).

In detail, this comprises development of methodologies relevant to operating the trial, testing of hypotheses regarding the validity of the measures used in the study, examination of data collected during the initial phase of study participant recruitment and follow up (June 2008 - October 2009), and determination of longitudinal epidemiological trends by comparing current data with an historical cohort at the same site.

Final results of the AVDAPT study will be presented at completion of the trial, anticipated to be in 2012.

AIM 1: DESIGN AND COMMENCE THE AVDAPT RCT

The first objective is to design and commence the AVDAPT RCT. This study aims to determine whether supplementation with L-arginine and / or vitamin D is safe and effective in TB, where efficacy includes more rapid improvement in clinical, mycobacterial, immunological, radiological, physiological and / or functional measures of treatment outcome. In order to gain understanding of the underlying immunology of relevance to this trial, the AVDAPT study further aims to determine whether exhaled nitric oxide (FENO) is inversely related to disease severity. The detailed aims of the AVDAPT trial are set forth in Chapter 6 (Methods). The hypotheses investigated in this study include:

Hypothesis 1(a): That L-arginine supplementation in pulmonary TB will be safe, will increase plasma arginine concentrations, will enhance pulmonary production of nitric oxide (NO) (a key arginine-dependent immunomodulator and downstream immune mediator of mycobacterial killing) and will improve the rapidity and magnitude of the microbiological and clinical response. Baseline pulmonary NO production will be elevated in pulmonary TB but inversely associated with disease severity. Both

2 Chapter 1: Aims

baseline and post-treatment increments in exhaled NO will be associated with rapidity and magnitude of the treatment response.

Hypothesis 1(b): That supplementation with vitamin D, the metabolite of which (1,25-

dihydroxyvitamin D3) has anti-mycobacterial activity, will be safe, will increase plasma vitamin D concentrations, and will improve the rapidity and magnitude of the treatment response in human PTB.

AIM 2: INVESTIGATE BASELINE CHARACTERISTICS OF TB STUDY PARTICIPANTS AND HEALTHY VOLUNTEERS

Aim 2(a): to explore the baseline demographic characteristics of AVDAPT study participants, and present clinical and laboratory findings at the time of their enrolment into the study, including results of Mycobacterium tuberculosis susceptibility testing.

Aim 2(b): to establish reference ranges for exercise tolerance (six-minute walk test) and a quality of life score (modified SGRQ), by performing these tests in locally recruited healthy volunteers, for comparison with values obtained in TB patients (AVDAPT study participants).

AIM 3: INVESTIGATE RELATIONSHIPS BETWEEN MICROBIOLOGICAL, CLINICAL AND FUNCTIONAL MEASURES AT BASELINE

Aim: to describe the relationships between clinical (symptoms and weight), mycobacterial (sputum smear grade), physiological (spirometry) and functional (six- minute walk test, SGRQ) measures, and to investigate any associations between socio- economic indicators and diagnostic delay or disease severity.

Hypotheses: That clinical and functional measures of TB disease severity at enrolment will significantly relate to bacteriological (sputum smear grade) measures. Specifically, that baseline weight, FEV1 and six-minute walk test will be inversely related, and modified SGRQ score, cough severity and number of symptoms will be directly related, to sputum smear grade. Also, that clinical, functional and physiological measures of TB disease severity at enrolment will significantly correlate with each other.

3 Chapter 1: Aims

AIM 4: MEASURE RADIOLOGICAL SEVERITY OF TB

Aim: To develop a valid method by which to grade chest X-ray severity in study participants with pulmonary TB, using a previously-collected dataset from a similar sample of adults with pulmonary TB in Timika, and to further examine the ability of this score to predict baseline clinical and microbiological severity and 2 month outcomes in AVDAPT study set.

Hypothesis: that a numerical score applied to an X-ray can provide a means of evaluating baseline severity and response to treatment.

AIM 5: DEVELOP A COMPOSITE CLINICAL OUTCOME MEASURE

Aim: to develop a composite clinical outcome score calculable at 2 and 6 months, and determine the relationship between this and microbiological, radiological and functional measures of TB severity

Hypothesis: That a composite clinical outcome score at 2 and 6 months will be significantly related to microbiological, radiological and functional measures.

AIM 6: EVALUATE INTERIM OUTCOMES

Aim: to document treatment outcomes among study participants, including sputum smear and culture conversion, and provide interim adverse event and safety data relating to the AVDAPT study.

AIM 7: MEASURE EXHALED NITRIC OXIDE IN PULMONARY TB

Aims: to determine the relationship between FENO and TB disease severity

(bacteriological, radiological and clinical) at TB diagnosis; to compare FENO measures from pulmonary TB patients with measures from contemporaneously evaluated local healthy controls and an historical healthy control group from the same research site; and to determine longitudinal changes in FENO in response to TB treatment.

4 Chapter 1: Aims

Hypothesis: That FENO will be increased in participants with pulmonary TB compared with healthy controls, will be inversely related to disease severity at baseline, and will return towards normal by the end of therapy.

AIM 8: EPIDEMIOLOGY OF HIV – TB COINFECTION

Aim: to describe the current epidemiology of HIV-TB co-infection in Timika, and compare the HIV-TB co-infection rates in 2008-2009 with 2003-2004 (a time period for which previous HIV-TB co-infection rates are published).

5

Chapter 2: Introduction 1: Tuberculosis

2 Introduction I: Tuberculosis

TB has been the subject of recent comprehensive reviews.5-7 My approach to reviewing the literature in this introductory chapter is therefore to summarise milestones in TB understanding and management (Table 2.1), and to focus on two specific questions: 1) What recent progress has been made globally in TB control? 2) What should Australian practitioners know about contemporary TB management? These questions are addressed, respectively, in publications reproduced hereafter: ―TB into the 2010s: is the glass half full?‖8 and ―What‘s new in TB?‖.9

Table 2.1: Chronology of TB milestones

Year Event 1882 Robert Koch (German physician), announced his identification of Mycobacterium tuberculosis as the causative agent of TB on March 24th, now commemorated as World TB Day. 1882 German pathologists Ziehl and Neelsen introduced a staining method using carbolfuchsin, an acid wash, and methylene blue to demonstrate the presence of acid- fast bacilli. 1890 Description of tuberculin by Koch, trialed (unsuccessfully) to treat TB. 1895 Discovery of x-ray techniques for diagnostic purposes by Conrad Roentgen, providing an important additional diagnostic tool for pulmonary TB. 1905 Koch received Nobel Prize in Physiology or Medicine. 1921 Development of vaccine, now known as BCG, from attenuated Mycobacterium bovis by Calmette and Guerin. 1940s Roll-out of mass TB vaccination using BCG. 1944 Streptomycin (SM) and Para-amino salicylic acid (PAS) discovered as effective antimicrobial therapies for the treatment of TB. 1947- First randomised curative trial to be conducted in the UK was performed, evaluating 1948 SM as an anti-tuberculosis agent. 1952 Addition of isoniazid (INH) to SM and PAS shown to increase cure rates from 70 to 95%, but required treatment for 18-24 months. 1959 Madras study showed domiciliary treatment to be as good as in sanitaria, and not resulting in more TB cases in family contacts, allowing TB treatment to be shifted to the community. 1965 Rifampicin discovered, leading to the advent of short course combination therapy.

1 Chapter 2: Introduction 1: Tuberculosis

1969 Given successes in the management of TB and other infectious diseases, U.S Surgeon General William Stewart famously stated in his address to Congress it was time to “close the book on infectious diseases”. This prevailing attitude led to TB- specific control being dismantled in many countries during the 1970-80s. 1981 HIV pandemic first recognized. 1985 TB incidence in the USA began to rise for first time in 30 years, described as an unprecedented resurgence during 1985-1992. 1991 World Health Organisation (WHO) specifies targets for case detection and cure: detect at least 70% and cure at least 85% of smear-positive TB cases. 1991 WHO Directly Observed Therapy, Short Course (DOTS) strategy rolled out (officially named „DOTS‟ in 1994) comprising: (1)Political commitment with increased, sustained financing; (2) Case detection through quality-assured bacteriology; (3) Standardized treatment, with supervision and patient support; (4) An effective drug supply and management system; (5) Monitoring and evaluation system, and impact measurement. 1992 Multi-drug resistant (MDR)-TB identified as a major new threat in New York City. 1993 World Health Assembly declared TB a “Global Emergency”. 2003 Early guidelines released on the use of interferon gamma release assays for latent TB diagnosis; understanding of test interpretation evolved during the reminder of the decade. 2006 Extensively drug-resistant TB labeled „XDR-TB‟. XDR-TB / HIV co-infection in Kwa- Zulu Natal province, South Africa, reported to have near 100% fatality. 2006 Stop TB partnership launched. 2008 WHO endorsed the use of rapid molecular resistance detection tests, able to provide MDR-TB diagnosis in 1-2 days. 2008 MDR and XDR-TB cure rates of >60% reported. 2008 Mortality significantly reduced in HIV-TB co-infection by commencing antiretroviral therapy during TB treatment instead of deferring until after TB treatment. 2009 Positive results from drug trials (e.g. moxifloxacin, TMC207) provide optimism for improvements of standard TB regimens and MDR-TB treatment.

Table 2.1 references5,10-20

2 Chapter 2: Introduction 1: Tuberculosis

Manuscript 1: Ralph AP, Anstey NM, Kelly PM. Tuberculosis into the 2010s: is the glass half full? Clin Infect Dis 2009;49(4):574-83.

3 REVIEW ARTICLE

Tuberculosis into the 2010s: Is the Glass Half Full?

Anna P. Ralph,1,4 Nicholas M. Anstey,1,2,3 and Paul M. Kelly1,4 1International Health Division, Menzies School of Health Research, 2Charles Darwin University, and 3Division of Medicine, Royal Darwin Hospital, Darwin, and 4National Centre for Epidemiology and Population Health Research, College of Medicine, Biology and Environment, Australian National University, Canberra, Australia

During the 16 years since the World Health Organization declared tuberculosis (TB) a global emergency, major new challenges have emerged—in particular the spread of extensively drug-resistant (XDR)-TB and its overlap with human immunodeficiency virus infection. However, during this period, we have also witnessed the creation of—and major commitments from—agencies dedicated to TB control, research, and funding, and tangible positive achievements have occurred; these include improvements in both new and existing TB diagnostics, a developmental pipeline of new candidate TB drugs, better treatment outcomes for multidrug-resistant TB and XDR-TB, heightened recognition of the importance of nosocomial transmission, and improved strategies to reduce mortality associated with concurrent human immunodeficiency virus infection and TB. We suggest updates to the 2006 International Standards of Tuberculosis Care to embrace these developments. The in- corporation of these recent advances into optimized directly observed treatment, short course (DOTS), pro- grams, in conjunction with more widespread deployment and enhanced political will, all provide grounds for improved control.

Mycobacterium tuberculosis is the consummate human velopment Goal 6.C of reduction in TB prevalence and pathogen. Millennia of evolution alongside human mortality by 50% by 2015 will be achievable in this hosts have led to elaborate immune evasion and trans- region [7, 8]. mission strategies [1, 2]; as such, M. tuberculosis is Amid these grim realities, however, exciting recent thought to infect one-third of humans, and in 2007, it developments with the potential to bring about im- accounted for an estimated 9.27 million new cases of portant reductions in TB-related burden of disease have tuberculosis (TB) and ∼1.7 million deaths [3]. Com- been achieved, including in achievements in under- pounding this already crippling burden are the ex- resourced settings. However, their implementation panding threats of TB drug resistance and of concurrent poses new challenges due to resource constraints, pol- human immunodeficiency virus (HIV) infection and icy-change inertia, and the need to prioritize basic TB TB. Primary transmission is the most common mode care, as articulated by the World Health Organization’s of acquisition seen in some settings for both extensively (WHO’s) directly observed therapy, short-course drug-resistant (XDR) and multidrug-resistant (MDR) (DOTS), and Stop TB partnership strategies [9, 10]. As TB [4–6]. The overlapping of HIV and TB epidemics has been clearly articulated, it is incumbent upon those in sub-Saharan Africa in particular creates a health care with expertise and resources to take a serious role in crisis and renders it unlikely that the Millennium De- bringing these developments into action in under- resourced TB-burdened settings [11]. Indeed, investing in TB control in resource-poor settings might be more cost-effective for developed nations in improving their Received 27 January 2009; accepted 24 April 2009; electronically published 7 July 2009. own TB control than alternative approaches [12]. Reprints or correspondence: Dr Anna Ralph, International Health Division, School The TB literature is characterized by bleak statistics of Health Research, PO Box 41096, Casuarina NT 0811, Australia ([email protected]). that provide substance for, in health-promotion terms, Clinical Infectious Diseases 2009;49:000–000 “fear-based” appeals; there is merit in directing atten- 2009 by the Infectious Diseases Society of America. All rights reserved. tion in this manner to the dire global state of TB. How- 1058-4838/2009/4904-00XX$15.00 DOI: 10.1086/600889 ever, there is an equal place for the alternative approach

Tuberculosis into the 2010s • CID 2009:49 (15 August) • 000 of positive appeals as effective strategies to promote a shift in midable barriers to dissemination of these messages to national mindset and uptake of new practices [13]. Here, we review TB control programs and the practitioners who implement important recent gains made in TB management and knowl- these programs. edge, discuss how these might be incorporated into existing DOTS programs, suggest a revision of several standards con- NEW DRUG REGIMENS tained within the comprehensive 17-point 2006 International New anti-TB drugs are required to permit shorter treatment Standards of TB Care (ISTC), and recommend an 18th Stan- durations for drug-susceptible TB [27], which mathematical dard [14]. Contrasting with negative appeals, we show that modeling indicates could effect major reductions in TB inci- there is scope for optimism. dence and mortality [28], and to provide less toxic, more ef- fective, and shorter regimens for MDR-TB. The TB literature TB DIAGNOSTICS has long lamented the absence of new drug developments since Rapid recent developments have occurred in the field of TB rifampicin in the 1960s. Barriers to TB drug development in- diagnostics, as evidenced by the need to establish a subgroup clude the need to evaluate drugs in combination over long within the Stop TB Partnership’s New Diagnostics Working follow-up periods in resource-limited settings; a lack of good Group to provide ongoing systematic reviews of diagnostic animal models for preclinical drug evaluation [29]; metabolic methods (Table 1) [15]. adaptability of M. tuberculosis, whereby genetic targets that ap- Of greatest priority are affordable ways to improve case de- pear promising have not proven to be so [30]; and the per- tection through smear microscopy at field laboratories. Simple ception by pharmaceutical companies that antibiotic devel- procedures recently shown to be of benefit include more clear opment is unrewarding [31]. Modeling performed in 2006 that instruction of people on how to produce an adequate sputum incorporated the high attrition in drug development, found specimen [16], a reduction in the required number of speci- only a 5% chance of a new TB drug being ready for clinical mens from 3 to 2 [17], more rapid specimen collection [18], use in humans by 2010 [32]. Despite these impediments, there and the processing of sputum specimens prior to examination are now ∼30 new drugs for TB under development (Table 2) (Table 1) [15, 19]. Fluorescence microscopy is more sensitive [29, 33–35]. PA-824 (a nitroimidazole) was shown to be suc- and rapid than conventional microscopy [19], and a light-emit- cessful against M. tuberculosis in vitro and in mouse models ting diode light source has recently been shown to be a reliable [36] and is now in phase II human trial stage (http://www alternative to the expensive, short-lived mercury vapor lamps .clinicaltrials.gov/show/NCT00567840). The diarylquinoline [20]. Such approaches provide solutions to the valid assertion TMC207 (also called R207910) has appeared to be particularly that substandard TB diagnostics are unacceptable in resource- promising in animal studies [37, 38], and a phase IIa random- poor countries [11]. ized trial involving people with smear-positive pulmonary TB Transportation of specimens to a reference laboratory can has been recently completed (http://www.clinicaltrials.gov/ be achieved despite challenging barriers: fresh sputum samples show/NCT00523926). Benzothiazinones (eg, BTZ043) are a can be stored at 4C for up to 6 weeks before unrefrigerated propitious new antimycobacterial class which kill M. tubercu- transportation, achieving excellent M. tuberculosis recovery losis in vitro and in mice by targeting M. tuberculosis cell wall without excessive contamination [21]. At laboratories with ad- synthesis [39], and have important potential in drug-susceptible equate capacity, early resistance detection—a critical tool in and drug-resistant TB. prevention of resistance amplification that is associated with The requirement for prolonged treatment has been hypoth- better treatment outcomes for management of MDR-TB [22]— esized to arise from the development of a nonreplicating, phe- is now achievable with rapid molecular and culture-based notypically drug-resistant phase of M. tuberculosis driven by methods. Line-probe assays (eg, Genotype MTBDRplus assay hypoxia and low-level nitric oxide; these factors, which char- [Hain Lifescience]), which detect M. tuberculosis gene muta- acterize the internal environment of granulomata, up-regulate tions that confer resistance to rifampicin (rpoB) and isoniazid dormancy genes to yield the metabolically inert state [30, 40– (katG and inhA), provide sensitive and specific results in 6 h 42]. To shorten treatment regimens, new drugs inhibiting to 2 days [23–25]. Culture-based rapid methods for resistance mechanisms underlying this nonreplicating state may have detection are outlined in Table 1. promise [30]. These improvements over traditional direct Ziehl-Neelsen A second strategy that may permit shorter TB treatment staining and use of solid-culture media require rapid dissem- regimens is the use of drugs that are bactericidal against rapidly ination and uptake. Promotion of new technologies, via WHO metabolizing M. tuberculosis. Replacement of ethambutol with endorsement [25], inclusion in guidelines [26], or internation- moxifloxacin has been shown to significantly increase the 2- ally accepted standards [14], is the first step in their deploy- month sputum culture conversion rate, from 63% to 80% [43]. ment, but innovative means are required to traverse the for- Both moxifloxacin and gatifloxacin improved the sterilizing

000 • CID 2009:49 (15 August) • Ralph et al Table 1. Methods to Improve Diagnosis and Accelerate Drug Susceptibility Results

Method, test Comments Sputum collection Improved sputum-submission guidance If smear positive pulmonary TB case detection is impaired by poor-quality specimen submission, case detection can be im- proved by provision of adequate instructions Reduce number of collections from 3 to 2 Because incremental yields from subsequent sputum specimens are small, WHO recommends examining 2 smears; this can al- leviate laboratory workloads, decrease time for diagnosis, and decrease the number of patients who “drop out” of the diag- nostic pathway Sputum smear microscopy Processing of sputum sample prior to smear examination (eg, This is 18%–23% more sensitive than direct microscopy use of bleach then centrifugation or use of bleach or sodium hydroxide then overnight sedimentation) Fluorescence microscopy This is 10% more sensitive than conventional microscopy; use to determine viability of Mycobacterium tuberculosis in follow-up sputum specimens to detect treatment failure Fluorescence microscopy using light-emitting diode light source These light sources are cheaper, last longer, and have less poten- tial for environmental contamination than do traditional lamps used in this method Culture-based methods Liquid culture (eg, automated mycobacteria growth indicator Faster and more sensitive than solid media; recommended stan- tube) dard practice Microscopic observation drug susceptibility assay Yields faster culture and DST results than do liquid or solid media and is inexpensive, but requires inverted microscope and skilled technician to interpret culture appearance of M. tuberculosis Thin-layer agar methodology Yields faster culture and DST results than do liquid or solid media and is inexpensive, but requires skilled technician to recognize M. tuberculosis colony formation. Colorimetric DST methods using redox indicators, tetrazolium These are low-cost, low-tech, and able to yield DST results within salts, or a nitrate reductase assay 2 weeks; potential for biosafety hazard Molecular methods Line probe assays (eg, Genotype MTBDRplus assay [Hain] and High sensitivity and specificity for detection of rifampicin (with or INNO-LiPA Rif.TB assay [Immunogenetics]) without isoniazid) resistance, with a 1–2 day turnaround time di- rectly for smear-positive sputum; requires DNA extraction and amplification facilities Nucleic acid amplification tests High specificity; important role in confirming mycobacterial iden- tity; poor negative predictive value for pulmonary and extrapul- monary TB; updated US CDC guidelines recommend sputum M. tuberculosis nucleic acid amplification tests for cases of sus- pected, unconfirmed TB if results would alter management [111] Cytokine assays: T cell interferon-g release assays Useful in targeted strategies for LTBI detection in low TB-inci- dence settings; more specific than tuberculin skin test; cannot distinguish active from treated TB or LTBI

NOTE. From [15–17, 19, 20, 112, 113]. CDC, Centers for Disease Control and Prevention; DST, drug susceptibility testing; LTBI, latent tuberculosis infection; TB, tuberculosis; WHO, World Health Organization.

activity of regimens for drug-susceptible TB [27] (gatifloxacin for drug-susceptible TB is lent credibility by these recent has since been withdrawn from some markets on the basis of findings. adverse glycemic effects). Although, in a multicenter study, moxifloxacin failed to accelerate sputum culture negativity at VACCINES AND ADJUNCTIVE 2 months, 1-month culture conversions were more frequently IMMUNOTHERAPIES achieved [44]. The later-generation fluoroquinolones have a well-established place in the treatment of MDR-TB; the pos- The need for an improvement on bacille Calmette-Gue´rin has sibility that they might also permit shorter treatment durations been long recognized as a major priority [45]. The status of

Tuberculosis into the 2010s • CID 2009:49 (15 August) • 000 Table 2. Antituberculosis Agents in Current Use and in Development

Antituberculosis agents Agent Agents Current antituberculosis agents, WHO classification [26] Group 1 First-line oral antituberculosis agents Isoniazid, rifampicin, ethambutol, pyrazinamide, rifabutin Group 2 Injectible antituberculosis agents Streptomycin, kanamycin, amikacin, capreomycin Group 3 Fluoroquinolones Moxifloxacin, levofloxacin, ofloxacin Group 4 Second-line oral bacteriostatic anti- Ethionamide or protionamide, cycloserine or terizidone, tuberculosis agents para-aminosalicylic acid Group 5 Antituberculosis agents with unclear Clofazimine, amoxicillin-clavulanate, linezolid, thioaceta- efficacy zone (contraindicated for HIV-infected patients), imi- penem-cilastin, high-dose isoniazid, clarithromycin New drugs Group 6 New drugs undergoing clinical Diarylquinolone (TMC-207, also known as R207910), ni- evaluation troimidazopyrans (PA-824 and OPC-67683), diamine (SQ-109) Group 7 New drugs at discovery stage Benzothiazinones (eg, BTZ043), LL-3858, cell-wall inhib- itors, multifunctional molecules, diaryl oxides, dihy- drolipoamide acryltransferase inhibitors, dipiperidine SQ-609, econazole, fatty acid synthase inhibitors, hy- drazones, InhA inhibitors, isocitrate lyase inhibitors, malate synthase inhibitors, mefloquine analogues, oxazolidinones, peptide diformylase inhibitors, pluero- mutilins, riminophenazines, thiolactomycin inhibitors, topoisomerase inhibitors, translocase inhibitors

NOTE. Multidrug-resistant tuberculosis treatment regimens should use all first-line drugs to which the organism is susceptible, a fluoroquinolone, a daily injectible (for at least 4 months after culture conversion and for at least 6 months in total), and other second- line drugs to provide at least 4 agents (for minimum of 18 months), with directly observed therapy, short course (DOTS), used throughout [26, 76]. In drug susceptibility testing–tailored regimens, mindfulness of the lack of universal clinical applicability of all drug susceptibility test results and cross-resistance is required [26]. From [26, 29, 33, 35, 38, 39]. HIV, human immunodeficiency virus. promising candidate recombinant TB vaccines, including the bacteriological end points [60–62]. Whole-food nutritional commencement of phase II trials of MVA85A, has been re- support poses logistical challenges and has yet to be shown to viewed in detail elsewhere [45–47]. be cost-effective or to improve bacteriological outcomes, but Investigation of adjunctive immunotherapies is identified as it can improve weight and possibly other parameters [63, 64]. one of several priority research areas [48–51]. They offer an The WHO recommends nutritional support in the manage- attractively novel strategy to shorten TB treatment and to con- ment of MDR TB [26], and many programs already incorporate serve antimicrobial efficacy in the face of growing resistance. this. Despite negative results from many studies using inactivated An alternative adjunctive therapeutic strategy, immunosup- Mycobacterium vaccae as an adjunct to TB chemotherapy [52, pression, has long been employed in TB-related meningitis and 53], hope persists that this immunotherapy given in multiple has proven to be beneficial, with corticosteroids shown to de- doses might yet have potential as an adjunctive treatment in crease risk of death and drug-induced hepatitis [65, 66]. On MDR-TB or previously treated TB [54] or as a preventative the basis of the hypothesis that amelioration of cytokine-me- vaccine in previously bacille Calmette-Gue´rin–vaccinated HIV- diated pathology might be advantageous in some forms of TB, infected persons [55, 56]. other combined immunosuppressive-therapeutic strategies The concept of micronutrients as potential adjunctive im- have also been advocated [67, 68]. munotherapy candidates has gained currency since specific an- timycobacterial mechanisms of action of vitamin D3 in mac- IMPROVED MANAGEMENT AND PREVENTION rophages (chiefly, up-regulation of LL-37/cathelicin) were OF MDR-TB AND XDR-TB demonstrated [57, 58]. The amino acid L-arginine has been found to influence antimycobacterial T cell responses via ex- Assigning a name to XDR-TB in 2005 brought it to public pression of the zeta chain of the CD3/T-cell receptor complex attention, accompanied by fears of a return to the preantibiotic [59], and the metabolic product of L-arginine, nitric oxide, era and the specter of untreatable TB [69]. Rapid fatality in mediates important macrophage antimycobacterial responses people with XDR-TB in Tugela Ferry, South Africa, illustrated [58]. Some multiple-micronutrient interventions in TB have the tragic consequences of this infection in the setting of high been associated with benefits in patient subgroups or in non- rates of concurrent HIV infection and nosocomial spread [5].

000 • CID 2009:49 (15 August) • Ralph et al Figure 1. Proposed updates and additions to the International Standards in TB Care [14]. ART, antiretroviral therapy; HIV, human immunodeficiency virus; MDR, multidrug-resistant; TB, tuberculosis; WHO, World Health Organization.

Although, in sub-Saharan Africa, this burden remains fearsome factors (higher use of alcohol, tobacco, and other drugs; mal- [6, 7, 70], and although a failure to detect MDR-TB obscures nutrition; and host immunity) and/or M. tuberculosis strain the true scale of this crisis [3], a new picture emerged during factors (differing virulence) [70], although meta-analysis did 2008 in which XDR-TB cure is a realistic aim and decreases in not find these factors to have a significant impact on MDR- MDR-TB rates can be achieved, including in resource-limited TB outcome [76]. settings [71–73]. Retrospective cohort studies from diverse lo- Even the best outcomes for MDR-TB treatment remain sub- cations reported their experiences with MDR-TB and XDR-TB stantially worse than the WHO’s cure target of 85% for drug- treatment in late 2008 [22, 71, 72, 74], offering an optimistic susceptible TB, and the pool of undiagnosed MDR-TB and “potentially favorable perspective for patients” with XDR-TB XDR-TB seriously threatens control efforts. Nevertheless, good [22] (eg, cure rates of 66.3% and 60.4% for MDR-TB and XDR- cure rates provide a notably more positive outlook than the TB, respectively [72]). Level 1 (randomized, controlled trial– dire reports from only 2–3 years ago. Despite the expenses of derived) evidence is required, as articulated by the Cambridge MDR-TB treatment, modeling including drug, laboratory, and Declaration on clinical trials for drug-resistant TB [75], but personnel costs indicates that MDR-TB treatment can be highly meta-analysis of retrospective observational cohort studies was cost-effective [77]. There is a clear imperative for managing recently performed. This has identified the WHO recommen- drug-resistant TB in accordance with current guidelines (eg, in dations of a treatment duration of у18 months after culture DOTSPlus projects, accessing second-line anti-TB drugs conversion and administration of DOT throughout as the fac- through the Green Light Committee) [26] while not diverting tors associated with the greatest chance (almost 70%) of treat- funds away from the core business of basic TB service provision. ment success [76]. Drug susceptibility testing–tailored regimens An additional cause for optimism is the recent decrease in were associated with nonstatistically significantly better out- MDR-TB rates documented in Estonia, Latvia, Hong Kong, comes than were standardized regimens, and inconsistent re- and the United States [73]; the next important step is to identify porting of HIV status rendered examination of this variable the factors responsible for these successes. Infection control is difficult [76]. MDR-TB and XDR-TB treatment programs pro- emerging as a critical focus for MDR-TB and XDR-TB pre- viding additional measures, such as nutritional and economic vention, because they are frequently nosocomially transmitted assistance for patients, have reported high rates of cure and [4–6, 78], requires renewed enthusiasm for more stringent re- treatment completion [71, 72], but the relative efficacy of such spiratory infection control. This need not be complicated: mod- supports is unknown. High rates of successful treatment out- eling of XDR-TB transmission in Tugela Ferry indicated that come may not be widely generalizable; major differences in appropriate use of existing resources (a combination of use of outcomes are seen in South Africa, even accounting for HIV face masks, reduced duration of hospitalization, and a shift to infection, possibly attributable to low use of quinolones; patient outpatient therapy) could prevent nearly one-third of XDR-TB

Tuberculosis into the 2010s • CID 2009:49 (15 August) • 000 Table 3. Summary of Recommendations for Antiretroviral Therapy (ART) in Patients with Concurrent Human Immunodeficiency Virus (HIV) Infection and Tuberculosis (TB)

Timing of ART initiationa ART CD4 T cell count recommendation Drug-susceptible TB Multidrug-resistant TB !200 cells/mm3 Yes 2–8 weeks after starting TB treatment 2 weeks or when anti-TB regimen is tolerated 200–350 cells/mm3 Yes After 8 weeks After 8 weeks 1350 cells/mm3 No Reevaluate at 8 weeks and every 6 Reevaluate at 8 weeks and every 6 months months thereafter thereafter Not available Yes 2–8 weeks after starting TB treatment 2–8 weeks or when anti-TB regimen is tolerated

NOTE. The recommended first-line ART regimen is efavirenz (600 mg daily) plus 2 nucleoside reverse-transcriptase inhibitors, with a rifampicin-based TB regimen given 5–7 times weekly [91, 92]. The rationale for using ART to prevent TB and to improve TB treatment outcome in persons with HIV infection is based on evidence that ART reduces the risk of developing active TB [85, 86] and improves survival when it is started during TB treatment, compared with if it is deferred [93–96], with the benefits of early ART initiation in people with TB outweighing the disadvantage of potential increased occurrence of immune reconstitution disease [7, 106]. Furthermore, ART achieves as-good virological and immunological responses as in people without TB [106]. a From [7, 89, 91]. cases [79]. Elsewhere, natural ventilation achieved by opening cell count), are areas of ongoing research (http://www.clinical windows and doors was estimated to achieve lower TB trans- trials.gov/NCT00463086). mission than costly, maintenance-requiring mechanical venti- First-line antiretroviral therapy recommended for persons lation systems [80]. Recent revisitation of 1950s experimental with concurrent HIV infection and TB by Centers for Disease designs, using exhaust air from a TB ward passed into guinea Control and Prevention and WHO comprises standard-dose pig enclosures, demonstrated that low-cost ultraviolet lights efavirenz plus 2 nucleoside reverse-transcriptase inhibitors [91, prevented 70% of TB infections, and negative air ionization 92], on the basis of the understanding that the 20% lower serum prevented 60% of TB infections [81]. Nosocomial XDR-TB efavirenz concentration caused by rifampicin remains effective transmission can occur even with good ventilation, limited pa- in suppressing HIV replication [98, 99]. Efavirenz may be safer tients per room, and ultraviolet lamps [4]. Nevertheless, evi- in pregnancy than hitherto realized [92, 100, 101], although it dence indicates that the ISTC [14] should require an additional remains a Category D listing. Nevirapine concentrations are 18th standard: that respiratory infection control measures more likely to be subtherapeutic in combination with rifam- should be implemented in facilities treating patients with newly picin [102], and virological failure risk may be higher [103]. diagnosed smear-positive TB, especially in areas with a high Despite this, nevirapine-based antiretroviral therapy is consid- prevalence of MDR-TB or XDR-TB. ered a suitable choice for second- or third-line treatment if efavirenz is contraindicated or unavailable and if rifamycins IMPROVED HIV-TB MANAGEMENT with less potency in inducing cytochrome P450 enzymes (ri- HIV infection increases the risk of latent TB reactivation 20- fapentine or rifabutin) are unavailable (as is generally the case fold [82, 83], and in southern Africa, TB is the leading cause where TB is endemic) [91, 92]. of death among people with HIV infection [84]. Identifying Determining optimal timing for antiretroviral therapy com- HIV infection presents an important TB control opportunity mencement after initiation of TB treatment is challenging be- through the use of antiretroviral therapy and isoniazid preven- cause of drug toxicities, interactions, the heavy pill burden and tive therapy, both of which reduce the risk of developing active attendant adherence risk, and immune reconstitution inflam- TB and require continued up-scaling [85–88]. Advances in the matory syndrome [104, 105]. Although immune reconstitution understanding of management of concurrent HIV infection and inflammatory syndrome may increase with wider and earlier TB include improved guidelines on HIV testing in persons with antiretroviral therapy use [94], it has not been associated with TB (ie, provider-initiated opt-out testing of all people with increased mortality, and to date, the risks appear to be out- newly diagnosed TB [7, 89–92]), growing knowledge of optimal weighed by the benefits of early antiretroviral therapy [7, 106]. antiretroviral therapy regimens to use in combination with an- Recent cohort studies and preliminary data from one trial in- timycobacterial agents [7, 89–92], and timing of antiretroviral dicate that the optimal strategy is to begin antiretroviral therapy therapy commencement in relation to TB [7, 89, 91, 93–96]. during TB treatment and that integration of HIV and TB care Benefits of isoniazid preventive therapy are also evident [97], offers valuable benefits [93–96]. WHO guidelines recommend although details, such as how to confidently exclude active TB, commencement of antiretroviral therapy 2–8 weeks after ini- safety in combination with antiretroviral therapy, and the rec- tiation of TB treatment if the CD4 cell count is !350 cells/mm3 ommended duration (eg, 12 months, lifelong, or base on CD4 or unknown [7, 89, 91]. Additional data, including data from

000 • CID 2009:49 (15 August) • Ralph et al the SAPIT (Starting Antiretrovirals at Three Points in Tuber- zation of DOTS programs provide grounds for improved TB culosis Therapy) trial [96], should provide further clarity. On control. the basis of the evidence summarized above, ISTC Standard 13, which recommends antiretroviral therapy in selected pa- Acknowledgments tients with concurrent HIV infection and TB, can be updated We thank the anonymous reviewers for their very helpful contributions to incorporate advice regarding antiretroviral therapy regimen to the final manuscript. selection and timing of commencement (Figure 1 and Table Financial support. The Australian National Health and Medical Re- 3). search Council. Potential conflicts of interest. All authors: no conflicts.

TOWARD THE STABILIZATION OF THE References “GLOBAL EMERGENCY” 1. Flynn JL, Chan J. What’s good for the host is good for the bug. Trends The reported number of TB cases per capita has been decreasing Microbiol 2005; 13:98–102. globally since 2003, and funding for TB control has improved, 2. Flynn JL, Chan J. Immune evasion by Mycobacterium tuberculosis: peaking in 2008 at US $3.3 billion [8]. Although the challenges living with the enemy. Curr Opin Immunol 2003; 15:450–5. 3. World Health Organization. Global tuberculosis control: epidemiol- posed by HIV infection and MDR-TB remain formidable ogy, strategy, financing. WHO report 2009. WHO/HTM/TB/2009.411. worldwide, 3 of the 6 WHO regions (the Americas, the Eastern Geneva: World Health Organization, 2009. Available at: http://www Mediterranean, and South-East Asia) will meet 2015 targets for .who.int/tb/publications/global_report/2009/pdf/full_report.pdf. Ac- cessed January 2009. reductions in TB case numbers and fatalities [3]. Progress in 4. Cox HS, Sibilia K, Feuerriegel S, et al. Emergence of extensive drug industrialized countries includes downward trends in TB rates resistance during treatment for multidrug-resistant tuberculosis. N in Indigenous populations. Over the past 20 years, the TB Engl J Med 2008; 359:2398–400. incidence has decreased among Australian Aborigines [107], 5. Gandhi NR, Moll A, Sturm AW, et al. Extensively drug-resistant tu- berculosis as a cause of death in patients co-infected with tuberculosis Canadian First Nations populations [108, 109], and Native and HIV in a rural area of South Africa. Lancet 2006; 368:1575–80. Americans [110], attributed to factors that include improve- 6. Andrews JR, Gandhi NR, Moodley P, et al. Exogenous reinfection as ments in case finding and treatment in the early 1990s [108, a cause of multidrug-resistant and extensively drug-resistant tuber- culosis in rural South Africa. J Infect Dis 2008; 198:1582–9. 109]. These figures do not permit relaxation in TB control but 7. Harries AD, Zachariah R, Lawn SD. Providing HIV care for co-in- illustrate that, where adequate resources can be directed toward fected tuberculosis patients: a perspective from sub-Saharan Africa. at-risk populations, major benefits can result, and downward Int J Tuberc Lung Dis 2009; 13:6–16. trends ought to be maintainable. 8. World Health Organization. Global tuberculosis control: surveillance, planning, financing: WHO report 2008. WHO/HTM/TB/2008.393 New challenges continually arise in TB, but these become ed.Geneva: World Health Organization, 2008. opportunities for the recognition and development of inno- 9. World Health Organization. An expanded DOTS framework for ef- vative TB control strategies. For these advances to translate into fective tuberculosis control. WHO/CDS/TB/2002.297. Geneva: World Health Organization, 2002. mainstream practice in a timely manner, national TB programs 10. Raviglione MC, Uplekar MW. WHO’s new Stop TB Strategy. Lancet need to embrace new evidence, and clinicians in areas where 2006; 367:952–5. TB is endemic require workable mechanisms to achieve con- 11. Dukes Hamilton C, Sterling TR, Blumberg HM, et al. Extensively tinuing education alongside their heavy workloads. Such mech- drug-resistant tuberculosis: are we learning from history or repeating it? Clin Infect Dis 2007; 45:338–42. anisms include the more vigorous promotion of international 12. Schwartzman K, Oxalade O, Barr R. Domestic returns from invest- standards at a national level and timely implementation by ment in the control of tuberculosis in other countries. N Engl J Med practitioners. The 2006 ISTC [14] has proven to be useful in 2005; 353:1008–20. convincing national professional societies and academic insti- 13. Corcoran N. Mass media. In: Communicating health: strategies for health promotion. London: Sage Publications, 2007:94. tutions to support implementation of internationally recog- 14. Hopewell PC, Pai M, Maher D, Uplekar M, Raviglione MC. Inter- nized standards of TB care [8]. Such has been the rapidity of national standards for tuberculosis care. Lancet Infect Dis 2006;6: progress in the past 3 years that we suggest that the ISTC be 710–25. 15. Pai M, Ramsay A, O’Brien R. Evidence-based tuberculosis diagnosis. updated to incorporate the elements summarized in Figure 1. PLoS Med 2008; 5:e156. In many parts of the world, such strategies may appear un- 16. Khan MS, Dar O, Sismanidis C, Shah K, Godfrey-Faussett P. Im- realistic. However, even in circumstances of poverty or insta- provement of tuberculosis case detection and reduction of discrep- ancies between men and women by simple sputum-submission in- bility, there can be scope for optimization of resource allocation structions: a pragmatic randomised controlled trial. Lancet 2007; 369: and mobilization of political will to allow modern TB man- 1955–60. agement to be adopted where it is needed most. Enthusiasm 17. World Health Organization. New WHO policies. 2007. Available at: for adopting such changes can be generated by disseminating http://www.who.int/tb/dots/laboratory/policy/en/index2.html. Ac- cessed March 2009. cost-effective TB success stories. Despite major new challenges, 18. Ramsay A, Yassin MA, Cambanis A, et al. Front-loaded sputum mi- wider incorporation of recent advances, and further optimi- croscopy services: an opportunity to optimise smear-based case de-

Tuberculosis into the 2010s • CID 2009:49 (15 August) • 000 tection of tuberculosis in high-prevalence countries. J Trop Med 2009 taining regimen exceeds activity of the standard daily regimen in [Epub ahead of print]. murine tuberculosis. Am J Respir Crit Care Med 2009; 179:75–9. 19. Steingart KR, Ramsay A, Pai M. Optimizing sputum smear micros- 39. Makarov V, Manina G, Mikusova K, et al. Benzothiazinones kill My- copy for the diagnosis of pulmonary tuberculosis. Expert Rev Anti cobacterium tuberculosis by blocking arabinan synthesis. Science Infect Ther 2007; 5:327–31. 2009; 324:801–4. 20. Marais BJ, Brittle W, Painczyk K, et al. Use of light-emitting diode 40. Connolly L, Edelstein P, Ramakrishnan L. Why is long-term therapy fluorescence microscopy to detect acid-fast bacilli in sputum. Clin required to cure tuberculosis? PLoS Medicine 2007; 4:e120. Infect Dis 2008; 47:203–7. 41. Wayne LG, Hayes LG. An in vitro model for sequential study of 21. Lumb R, Ardian M, Waramori G, et al. An alternative method for shiftdown of Mycobacterium tuberculosis through two stages of non- sputum storage and transport for mycobacterium tuberculosis drug replicating persistence. Infect Immun 1996; 64:2062–9. resistance surveys. Int J Tuberc Lung Dis 2006; 10:172–7. 42. Voskuil MI, Schnappinger D, Visconti KC, et al. Inhibition of res- 22. Bonilla CA, Crossa A, Jave HO, et al. Management of extensively piration by nitric oxide induces a Mycobacterium tuberculosis dor- drug-resistant tuberculosis in Peru: cure is possible. PLoS ONE mancy program. J Exp Med 2003; 198:705–13. 2008; 3:e2957. 43. Conde MB, Efron A, Loredo C, et al. Moxifloxacin versus ethambutol 23. Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid in the initial treatment of tuberculosis: a double-blind, randomised, molecular screening for multidrug-resistant tuberculosis in a high- controlled phase II trial. Lancet 2009; 373:1183–89. volume public health laboratory in South Africa. Am J Respir Crit 44. Burman WJ, Goldberg S, Johnson JL, et al. . Moxifloxacin versus Care Med 2008; 177:787–92. ethambutol in the first 2 months of treatment for pulmonary tuber- 24. Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the di- culosis. Am J Respir Crit Care Med 2006; 174:331–8. agnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur Res- 45. Senior K. Moving closer to a new tuberculosis vaccine. Lancet Infect pir J 2008; 32:1165–74. Dis 2009; 9:146. 25. World Health Organization. Molecular line probe assays for rapid 46. McShane H. Vaccine strategies against tuberculosis. Swiss Med Wkly screening of patients at risk of multidrug-resistant tuberculosis (MDR- 2009; 139:156–60. TB): policy statement. Availalable at: http://www.who.int/tb/ 47. Hoft DF. Tuberculosis vaccine development: goals, immunological features_archive/policy_statement.pdf. Geneva: World Health Orga- design and evaluation. Lancet 2008; 372:164–75. nization, 2008. 48. Onyebujoh P, Rodriguez W, Mwaba P. Priorities in tuberculosis re- 26. World Health Organization. Guidelines for the programmatic man- search. Lancet 2006; 367:940–2. agement of drug-resistant tuberculosis: emergency update. WHO/ 49. Roy E, Lowrie DB, Jolles SR. Current strategies in TB immunotherapy. HTM/TB/2008.402. Geneva: World Health Organization, 2008. Avail- Curr Mol Med 2007; 7:373–86. able at: http://whqlibdoc.who.int/publications/2008/9789241547581 50. Reljic R. IFN-gamma therapy of tuberculosis and related infections. _eng.pdf. Accessed March 2009. J Interferon Cytokine Res 2007; 27:353–64. 27. Rustomjee R, Lienhardt C, Kanyok T, et al. A Phase II study of the 51. Rook GA, Lowrie DB, Hernandez-Pando R. Immunotherapeutics for sterilising activities of ofloxacin, gatifloxacin and moxifloxacin in pul- tuberculosis in experimental animals: is there a common pathway monary tuberculosis. Int J Tuberc Lung Dis 2008; 12:128–38. activated by effective protocols? J Infect Dis 2007; 196:191–8. 28. Salomon JA, Lloyd-Smith JO, Getz WM, et al. Prospects for advancing 52. de Bruyn G, Garner P. Mycobacterium vaccae immunotherapy for tuberculosis control efforts through novel therapies. PLoS Med treating tuberculosis. Cochrane Database Syst Rev 2003:CD001166. 2006; 3:e273. 53. Mwinga A, Nunn A, Ngwira B, et al. Mycobacterium vaccae (SRL172) 29. Lenaerts AJ, Degroote MA, Orme IM. Preclinical testing of new drugs immunotherapy as an adjunct to standard antituberculosis treatment for tuberculosis: current challenges. Trends Microbiol 2008; 16:48–54. in HIV-infected adults with pulmonary tuberculosis: a randomised 30. Murphy DJ, Brown JR. Novel drug target strategies against Myco- placebo-controlled trial. Lancet 2002; 360:1050–5. bacterium tuberculosis. Curr Opin Microbiol 2008; 11:422–7. 54. Fan MY, Chen XR, Wang Ka, et al. Adjuvant effect of Mycobacterium 31. Charles PG, Grayson ML. The dearth of new antibiotic development: vaccae on treatment of recurrent treated pulmonary tuberculosis: A why we should be worried and what we can do about it. Med J Aust meta-analysis. Chinese Journal of Evidence-Based Medicine 2007;7: 2004; 181:549–53. 449–55. 32. Glickman SW, Rasiel EB, Hamilton CD, Kubataev A, Schulman KA. 55. von Reyn C, Arbeit R, Mtei L, et al. The DarDar prime-boost TB Medicine: a portfolio model of drug development for tuberculosis. vaccine trial in HIV infection: final results. In: Program and abstracts Science 2006; 311:1246–7. of the 39th World Conference on Lung Health of the International 33. Check E. After decades of drought, new drug possibilities flood TB Union Against Tuberculosis and Lung Disease (Paris). Int J Tuberc pipeline. Nat Med 2007; 13:266. Lung Dis 2008; 12:S318. 34. Kaiser Family Foundation. Open forum II: key issues in TB drug 56. Wise J. Tuberculosis vaccine for HIV-infected patients shows promise. development -day one: welcome and introduction; the TB drug pipe- Lancet Infect Dis 2008; 8:745. line—present and future. TB Alliance. London, United Kingdom. 57. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin 2006. Available at: http://www.kaisernetwork.org/health_cast/ D-mediated human antimicrobial response. Science 2006; 311:1770–3. uploaded_files/121206_tballiance_welcomeandpipeline.pdf. Accessed 58. Ralph AP, Kelly PM, Anstey NM. L-arginine and vitamin D: novel March 2009. adjunctive immunotherapies in tuberculosis. Trends Microbiol 35. van den Boogaard J, Kibiki GS, Kisanga ER, Boeree MJ, Aarnoutse 2008; 16:336–44. RE. New drugs against tuberculosis: problems, progress, and evalu- 59. Zea AH, Culotta KS, Ali J, et al. Decreased expression of CD3z and ation of agents in clinical development. Antimicrob Agents Che- nuclear transcription factork B in patients with pulmonary tuber- mother 2009; 53:849–62. culosis: potential mechanisms and reversibility with treatment. J Infect 36. Lenaerts AJ, Gruppo V, Marietta KS, et al. Preclinical testing of the Dis 2006; 194:1385–93. nitroimidazopyran PA-824 for activity against Mycobacterium tuber- 60. Abba K, Sudarsanam TD, Grobler L, Volmink J. Nutritional supple- culosis in a series of in vitro and in vivo models. Antimicrob Agents ments for people being treated for active tuberculosis. Cochrane Da- Chemother 2005; 49:2294–301. tabase Syst Rev 2008:CD006086. 37. Nuermberger E, Mitchison DA. Once-weekly treatment of tubercu- 61. Range N, Andersen AB, Magnussen P, Mugomela A, Friis H. The losis with the diarylquinoline R207910: a real possibility. Am J Respir effect of micronutrient supplementation on treatment outcome in Crit Care Med 2009; 179:2–3. patients with pulmonary tuberculosis: a randomized controlled trial 38. Veziris N, Ibrahim M, Lounis N, et al. A once-weekly R207910-con- in Mwanza, Tanzania. Trop Med Int Health 2005; 10:826–32.

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Ac- antiretroviral therapy on the risk of tuberculosis among persons with cessed January 2009. HIV infection. AIDS 2000; 14:1985–91. 64. Martins N, Morris P, Kelly P. Nutritional enablers to enhance TB 87. Golub JE, Saraceni V, Cavalcante SC, et al. The impact of antiretroviral treatment adherence. In: Program and abstracts of the 39th World therapy and isoniazid preventive therapy on tuberculosis incidence Conference on Lung Health of the International Union Against Tu- in HIV-infected patients in Rio de Janeiro, Brazil. AIDS 2007;21: berculosis and Lung Disease (Paris). Int J Tuberc Lung Dis 2008; 1441–8. 12(Suppl 2):S15. 88. Havlir DV, Getahun H, Sanne I, Nunn P.Opportunities and challenges 65. Prasad K, Singh MB. Corticosteroids for managing tuberculous men- for HIV care in overlapping HIV and TB epidemics. JAMA 2008; ingitis. Cochrane Database Syst Rev 2008:CD002244. 300:423–30. 66. Thwaites GE, Nguyen DB, Nguyen HD, et al. Dexamethasone for the 89. Scano F, Vitoria M, Burman W, Harries AD, Gilks CF, Havlir D. treatment of tuberculous meningitis in adolescents and adults. N Engl Management of HIV-infected patients with MDR- and XDR-TB in JMed2004; 351:1741–51. resource-limited settings. Int J Tuberc Lung Dis 2008; 12:1370–5. 67. Wallis RS. Reconsidering adjuvant immunotherapy for tuberculosis. 90. World Health Organization/Joint United Nations Programme for Clin Infect Dis 2005; 41:201–8. HIV/AIDS. Strengthening health services to fight HIV/AIDS: guidance 68. Wallis RS, van Vuuren C, Potgieter S. Adalimumab treatment of life- on provider-initiated HIV testing and counselling in health facilities. threatening tuberculosis. Clin Infect Dis 2009; 48:1429–32. Geneva: World Health Organization, 2007. 69. Centers for Disease Control and Prevention. Revised definition of 91. World Health Organization. Antiretroviral therapy for HIV infection extensively drug-resistant tuberculosis. 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J Acquir Immune Defic Syndr 2009; 50:148–52. 1–2. 96. Baleta A. Trial finds simultaneous HIV/tuberculosis treatment ben- 76. Orenstein EW, Basu S, Shah NS, et al. Treatment outcomes among eficial. Lancet Infect Dis 2008; 8:669. patients with multidrug-resistant tuberculosis: systematic review and 97. Woldehanna S, Volmink J. Treatment of latent tuberculosis infection meta-analysis. Lancet Infect Dis 2009; 9:153–61. in HIV infected persons. Cochrane Database Syst Rev 2004: 77. Resch SC, Salomon JA, Murray M, Weinstein MC. Cost-effectiveness CD000171. of treating multidrug-resistant tuberculosis. PLoS Med 2006; 3:e241. 98. Lopez-Cortes LF, Ruiz-Valderas R, Viciana P, et al. Pharmacokinetic 78. Horsburgh CR Jr. Primary transmission of multidrug-resistant and interactions between efavirenz and rifampicin in HIV-infected pa- extensively drug-resistant tuberculosis among HIV-infected persons: tients with tuberculosis. Clin Pharmacokinet 2002; 41:681–90. what does the future hold in store? 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Tuberculosis into the 2010s • CID 2009:49 (15 August) • 000 105. French MA. HIV/AIDS: immune reconstitution inflammatory syn- 110. Centers for Disease Control and Prevention. Trends in tuberculosis— drome: a reappraisal. Clin Infect Dis 2009; 48:101–7. United States, 2008. MMWR Morb Mortal Wkly Rep 2009; 58:249–53. 106. Hung CC, Chen MY, Hsiao CF, Hsieh SM, Sheng WH, Chang SC. 111. Centers for Disease Control and Prevention. Updated guidelines for Improved outcomes of HIV-1–infected adults with tuberculosis in the the use of nucleic acid amplification tests in the diagnosis of tuber- era of highly active antiretroviral therapy. AIDS 2003; 17:2615–22. culosis. MMWR Morb Mortal Wkly Rep 2009; 58:7–10. 107. Roche PW, Krause V, Konstantinos A, et al. Tuberculosis notifications 112. Grandjean L, Moore DA. Tuberculosis in the developing world: recent in Australia, 2006. Commun Dis Intell 2008; 32:1–11. advances in diagnosis with special consideration of extensively drug- 108. Clark M, Riben P. Tuberculosis in First Nations communities. Ca- resistant tuberculosis. Curr Opin Infect Dis 2008; 21:454–61. nadian Ministry of Health. 1999. Available at: http://www.hc-sc.gc 113. Dinnes J, Deeks J, Kunst H, et al. A systematic review of rapid di- .ca/fniah-spnia/alt_formats/fnihb-dgspni/pdf/pubs/tuberculos/1999 agnostic tests for the detection of tuberculosis infection. Health Tech- _commun-eng.pdf. Accessed March 2009. nol Assess 2007; 11:1–196. 109. Smeja C, Brassard P. Tuberculosis infection in an Aboriginal (First Nations) population of Canada. Int J Tuberc Lung Dis 2000; 4:925–30.

000 • CID 2009:49 (15 August) • Ralph et al Chapter 2: Introduction 1: Tuberculosis

Manuscript 2: Ralph A, Kelly P, Krause V. What's new in TB? Australian Family Physician 2009; 38(8):578-585.

4 THEME infectioNS THAT LAST

Anna Ralph Paul Kelly BMedSci, MBBS, MPH, DTM&H, FRACP, is a PhD Candidate, National MBBS, DTM&H, PhD, FAFPHM, is Director, Masters of Applied Centre for Epidemiology and Population Health, Australian National Epidemiology Program, National Centre for Epidemiology and University and Menzies School of Health Research, and a locum Population Health, Australian National University, Canberra, infectious diseases physician, Alice Springs, Northern Territory. Australian Capital Territory. [email protected] What’s new in TB?

Appreciation of what’s new in tuberculosis (TB) requires a Background perspective of what’s old: Mycobacterium tuberculosis (MTB) Australia has among the world’s lowest rates of tuberculosis and other members of the MTB complex (Table 1) have been (TB). However, it remains a leading global cause of morbidity infecting humans since antiquity.1,2 Being therefore and mortality. In Australia, TB remains more common in consummately adapted to life within the human host,3 MTB Indigenous than non-Indigenous Australians, and rates are rising among migrants, reflecting changing immigration patterns and infects up to one-third of the global population, is rising rates in their homelands. characterised by a dormant phase which confounds diagnosis and control, and in 2006 accounted for an estimated 9.2 million Objective new cases and 1.7 million deaths worldwide.4 This article reviews recent developments in TB of relevance to

Australian general practice and provides an update of advances th in the diagnosis and management of TB, and the role of the Australia largely avoided the late 20 century global TB resurgence, general practitioners in co-managing people with TB. which prompted the declaration of TB as a ‘global emergency’ by the World Health Organization (WHO) in 1993. The millennium Discussion development goals set a target of reducing prevalence and death First hand experience with imported multidrug resistant TB rates of TB by 50% by 2015. Although many countries are on track to (MDR-TB) is increasing and is anticipated to rise in Australia. attain these rates, highest burden countries, notably in sub-Saharan The reach of extensively drug resistant TB is also expanding. Although standard guidelines for management of drug Africa where multidrug resistant TB (MDR-TB) and the overlap of TB 4 susceptible TB remain unchanged, recent progress in the and HIV epidemics pose crippling challenges, are not. understanding, diagnosis and management of TB has occurred, Against this background, Australia in 2006 reported 1201 TB driven by the need to respond to the challenges of MDR-TB and cases, an incidence of 5.8 per 100 000 population,5 with preliminary HIV-TB co-infection. 2008 figures being similar.6 The national strategic plan for TB control recognises the importance of favourable socioeconomic circumstances; successful post-World War II national TB campaigns; specialised, multidisciplinary, free TB services; and effective premigration screening as contributors to Australia’s low TB rates.7 New challenges faced by Australian practitioners include addressing high rates of TB (~1360/100 000) in Indonesian fishermen entering Northern Territory waters,8 and MDR-TB (25% of cases) in Papua New Guinean nationals seeking treatment in the Torres Strait protected zone.9 This provides a preview of cases Australian practitioners are likely to be more frequently presented with in coming years as the distribution of MDR-TB continues to expand. Tuberculosis treatment in Australia is conducted by specialists within centralised, coordinated TB services. Failure to manage TB in this fashion is associated with higher risks of treatment failure and emergence of drug resistance.10 However, general practitioners have the key responsibilities of recognising at risk people and coordinating care for affected families. In particular, the most vital contribution GPs can make is to avoid TB diagnostic delay by sending sputum specimens

2 Australian Family Physician Vol. 38, No. 8, August 2009 Vicki Krause MD, DTM&H, FAFPHM, is Director, Centre Table 1. Mycobacterium TB and other members of the MTB complex for Disease Control and Head, Tuberculosis/ Leprosy Services, Department Community Mycobacterium genus and Families, Darwin, Northern Territory. • Gram positive, acid fast bacilli. Those pathogenic to humans include mycobacteria of the MTB complex, pathogenic non- tuberculous mycobacteria (NTM) and M. leprae • Tuberculosis causing group – MTB complex: – M. tuberculosis for specific examination for acid fast bacilli (AFB) and reviewing chest – M. bovis radiograph findings in at risk people with suspicious cough (>2 weeks – M. africanum or ‘chronic pneumonia’). This review presents recent developments – M. bovis Bacillus Calmette Guerin (BCG) (attenuated in TB relevant to Australian general practice. Comprehensive TB vaccine strain, able to cause disseminated tuberculosis in management guidelines are provided elsewhere (see Resources). immunocompromised hosts) Who is at risk in Australia? – Uncommon: M. microti, M. bovis subspecies capra, M. tuberculosis subspecies canettii, M. pinnipedii Tuberculosis requires particular consideration in migrants and refugees from high incidence countries. Approximately 85% of new treatment (Table 3). Detailed screening and LTBI treatment guidelines Australian TB cases occur in people born overseas.5 Highest global are provided elsewhere.11,21–25 per capita TB rates occur in sub-Saharan Africa,4 reflected in high TB notification rates in Australians of sub-Saharan origin.5 Commonest What’s new in TB diagnostics? countries of origin of people with TB in Australia (consistent with Globally, the commonest method for diagnosing TB remains sputum relative sizes of migrant populations) are India, Vietnam, the microscopy only; therefore improved access to inexpensive diagnostics is Philippines, China and Indonesia.5 Tuberculosis cases in migrants are a priority in under resourced settings. There has been promising progress usually attributable to reactivation of latent TB infection (LTBI) (often in the development of such tests, including improved microscopy and within the first 2 years after migration),11 with smaller contributions culture techniques, and rapid molecular methods for detection of from local transmission within migrant communities, and newly rifampicin and isoniazid resistance gene mutations.26 These can provide acquired infections after visits home.12 Negative premigration rifampicin/isoniazid susceptibility results within 1–2 days, compared or arrival TB screening does not therefore negate the possibility with approximately 42 days using liquid culture media.27 A reliable point of subsequent TB. of care test such as antigen detection on blood or sputum would be the Indigenous Australians (in some communities more than others) ‘holy grail’ of TB diagnostics; such tests are under investigation28 but not are at higher risk than non-Indigenous Australian born people (6.6 yet commercially available. Nucleic acid amplification tests, including vs. 0.9 per 100 000).5 Other commonly identified TB risk factors in polymerase chain reaction (PCR), have high specificity. Although negative Australia are household contact with TB, or residence in a TB high predictive value is low, the possibility of a positive result in smear prevalence country for more than 3 months.5 Returned travellers negative disease can allow more rapid diagnosis while awaiting culture; uncommonly present with TB.13 recently updated Centres for Disease Control (USA) guidelines therefore Human immunodeficiency virus confers the greatest single recommend sputum MTB nucleic acid amplification testing where risk for TB, increasing the chance of latent TB reactivation up to available and affordable, in cases of suspected, unconfirmed TB, if 20-fold.14,15 In Australia, HIV-TB co-infection is uncommon, but the results would alter management.29 reporting of HIV status in people with TB, at 37%, is much lower Interferon gamma release assays than recommended.5 A new diagnosis of TB must prompt an offer of HIV testing as per local and international recommendations.16–18 Of more relevance in Australia are interferon gamma release assays Other forms of immunosuppression also significantly increase the (IGRA), such as QuantiFERON-TB Gold, as potential supplements likelihood of LTBI reactivation. In people with LTBI, such as migrants or alternatives to tuberculin skin testing (TST) for detecting TB and indigenous people from high TB burden communities, chronic exposure. Currently, Australia’s National Tuberculosis Advisory renal failure and high dose corticosteroid use approximately doubles Committee discourages use of these tests in clinical settings pending the risk of developing active TB, while TNF-α blockers (eg. infliximab) further research into sensitivity, specificity and cost effectiveness increase the risk five-fold compared with the comparative population (Table 2), favouring use of clinical history and TST.21,30 Like the risk of LTBI reactivation.19,20 TST, IGRA detect T-cell responsiveness to MTB antigens, but this is These at risk groups require screening for LTBI if asymptomatic measured on a blood sample as interferon (IFN)-γ production rather (see below and Table 2), or investigation for active pulmonary or extra than an in vivo skin reaction, and the antigens used to elicit the pulmonary TB if symptomatic with a clinically relevant syndrome. response are more specific to MTB than those contained in purified People with LTBI at risk of progression to active TB, require LTBI protein derivative. Some diagnostic algorithms incorporate these tests

Australian Family Physician Vol. 38, No. 8, August 2009 3 theme What’s new in TB?

as adjuncts to,31 or replacements of,32,33 TST, hence some familiarity HIV-TB co-infection management with this test is required. Traditionally, delayed commencement of antiretroviral treatment A ‘take home message’ is that a positive result in either IGRA or TST (ART) was recommended in HIV-TB co-infection due to overlapping does not confirm, and a negative test does not exclude, TB exposure. drug toxicities, drug interactions, and the potential for paradoxical worsening of TB pathology due to immune restoration inflammatory What’s new in TB treatment? syndrome.40 However, it is becoming clearer that benefits of early ART Standard treatment regimens for active and latent disease caused outweigh these risks, and that despite rifampicin induced increased by drug susceptible MTB have not changed in Australia in recent clearance of non-nucleoside reverse transcriptase drugs (efavirenz/ decades (Table 3), however, new developments are discussed below. nevirapine), serum levels of these drugs can remain adequately efficacious to permit their use.41 Current guidelines recommend New drugs for TB starting ART within 2–8 weeks of TB treatment initiation if CD4 <200, The drought in new TB drug discovery appears to be breaking, with and after 8 weeks if CD4 200–350, preferably using an efavirenz about 30 new agents in the developmental pipeline.34,35 A metronidazole based regimen.17,40,41 related antibiotic, PA-824, has been found to be successful against MTB The importance of TB screening in people with HIV (eg. using in vitro and in mouse models36 and is now in phase 2 human trials. a combination of symptom check, sputum microscopy, TST), and Novel agents targeting specific MTB genes are under investigation.37 isoniazid preventive therapy if LTBI is identified, are increasingly Quinolones, with a well established place in the treatment of MDR-TB, recognised as critical and underutilised public health measures.42 are under investigation for their potential to shorten treatment durations MDR-TB management in drug sensitive TB.38 Of critical importance is the need to limit misuse of new drugs, eg. use of quinolones as monotherapy in TB misdiagnosed Multidrug resistant TB was initially recognised to result from poor as pneumonia is associated with development of resistance and treatment programs with low adherence, leading to selection of delayed TB diagnosis.39 drug resistance mutations in infecting MTB strains.43 However, it

Table 2. Advantages and disadvantages of interferon gamma release assays29,57–62 IGRA disadvantages compared with tuberculin skin testing Currently less collective experience with IGRA exists, and there are uncertainties in how to interpret results, eg. when there is discordance between IGRA and TST, discordance between different IGRA types, and when serial IGRA results change (reversion/conversion) Requires blood taking, which in infants (a common target group for screening) is more difficult, and often not done by rural/remote health workers and registered nurses Loss of patient educational opportunities which are provided when TB nurse performs and reads TST IGRA possibly less sensitive IGRA test kit more expensive than PPD Reversions from positive to negative can occur over time and are difficult to interpret (but usually only occur when the initial reading was low-positive) Requires laboratory reagents and staff not generally available in resource limited settings, thereby restricting accessibility in remote settings Indeterminate results can occur, eg. due to failures of controls, inadequate cell separation, cross contamination Greater incidence of discordance between IGRA and TST in children (usually TST positive, IGRA negative) IGRA advantages compared with TST IGRA only target antigens present in MTB ‘region of difference 1’ (RDI1) such as CFP10 and ESAT6, which are not shared by other mycobacteria except M. kansasii, M. marinum and M. szulgai, thereby increasing the specificity of the test Unaffected by prior BCG vaccination, for same reason as above (RDI antigens not present in M. bovis BCG) Only requires one clinic attendance if negative Disadvantages of both IGRA and TST Unable to distinguish active from latent disease False negatives occur especially in immunosuppression No gold standard for LTBI diagnosis, therefore the true sensitivity and specificity of both tests cannot be accurately evaluated A niche for IGRA use? QTF-G may be strategically used in settings such as previously BCG vaccinated TB contacts with a positive skin test, and BCG vaccinated health care workers in low TB incidence countries who require repeat testing TST= tuberculin skin test using purified protein derivative (PPD) (also known as Mantoux test)

4 Australian Family Physician Vol. 38, No. 8, August 2009 What’s new in TB? THEME

Table 3. Anti-TB agents and treatment strategies Anti-TB agents, WHO classification63 Drug Group 1 First line oral antituberculosis agents Isoniazid, rifampicin, ethambutol, pyrazinamide, rifabutin Group 2 Injectable antituberculosisagents Streptomycin, kanamycin, amikacin, capreomycin Group 3 Fluroquinolones Moxifloxacin, levofloxacin, ofloxacin Group 4 Second line oral bacteriostatic antituberculosis agents Ethionamide, protionamide, cycloserine, terizidone, para- aminosalicylic acid Group 5 Antituberculosis agents with unclear efficacy Clofazimine, amoxicillin/clavulanate, linezolid, thioacetazone, imipenem/cilastin, high dose isoniazid, clarithromycin Standard first line TB treatment Rifampicin, isoniazid*, pyrazinamide, ethambutol daily or thrice weekly for 2 month ‘intensive phase’ then rifampicin and isoniazid daily or thrice weekly for 4 month ‘continuation phase’. Dosing (see reference 23) Standard LTBI treatment** Isoniazid* daily 9 months23–25 MDR-TB treatment strategies Two phase regimen using at least five drugs to which the infecting isolate is sensitive. Drugs include remaining first line drugs to which the organism is sensitive (ethambutol and/or pyrazinamide) plus a quinolone (descending order of potency against MTB: moxifloxacin = gatifloxacin > levofloxacin > ofloxacin = ciprofloxacin) plus an injectible agent from Group 2 above, plus others as required. High dose isoniazid may retain efficacy against MDR isolates with low level isoniazid resistance64,65 Phase 1: includes injectible agent, minimum 6 month duration and at least 4 months past culture conversion Phase 2: after cessation of the injectible agent, continued for at least 18 months after culture conversion63,66 LTBI treatment where infecting strain is thought to be MDR-TB No Level 1 evidence. Two drug regimen tailored to the sensitivity profile of the MTB isolated from the contact may be efficacious, eg. pyrazinamide + ethambutol or a quinolone 6–12 months;65 alternatively, a monitoring approach may be appropriate, especially in children * Vitamin B6 (pyridoxine) 25 mg/day routinely accompanies isoniazid to reduce the incidence of peripheral neuropathy ** Combination therapy is needed in active but not latent TB, as active TB is characterised by high bacillary numbers, hence the number of bacilli with spontaneously arising resistance mutations at baseline, which can be selected out by inadequate regimens, is much greater than in LTBI67 is increasingly found to be a primary transmitted organism in high Avoid diagnostic delay MDR burden areas, emphasising the vital importance of infection By considering pulmonary TB in people with symptoms (eg. cough control.9,44,45 Multidrug resistant TB treatment regimens (Table 3) >2–3 weeks, fever ≥3 weeks, loss of >10% body weight) and are prolonged, costly, and associated with significant side effects epidemiological risk factors, GPs have the valuable opportunity and adherence difficulties. However, well functioning programs, to avoid diagnostic delay. Delayed diagnosis aggravates the especially those incorporating the WHO recommendations of ≥18 individual’s morbidity, increases public health risk, and complicates months treatment duration after culture conversion and directly the subsequent contact tracing required. observed therapy throughout, report cure rates of >60%.46 Similar Key actions to making a timely diagnosis are: success rates have recently been reported for extensively drug • request AFB examination on sputum in addition to standard resistant TB (XDR-TB) in some programs.47 (Note: XDR-TB = resistant microscopy/culture to rifampicin, isoniazid, any fluoroquinolone and at least one second • improve yield by requesting morning sputum specimens, and line injectible agent such as amikacin, capreomycin or kanamycin). arranging 2–3 collections, and This has overturned the spectre of ‘untreatable TB’ which has • assiduous follow up of chest radiography. surrounded XDR-TB,48 and comprehensive new recommendations for As a negative smear does not exclude pulmonary TB, clinical suspicion combating XDR-TB are available.49 in a person from a TB endemic country warrants referral to TB services (see Resources), regardless of AFB sputum smear result. How can GPs care for patients with TB? Infection control Although speciality TB treatment units have the responsibility for reporting and treating TB, managing medication adverse events General practices require a policy for ensuring adequate respiratory and ensuring adequate contact tracing (see Resources), GPs precautions for people with suspected or confirmed infectious are at the forefront of having to suspect TB and LTBI, and integrate pulmonary TB attending their practice, or whom they care for in TB care with other medical needs of the affected individual and nursing homes, prisons or other institutions, including avoidance of their family. communal areas and use of face masks.22,24

Australian Family Physician Vol. 38, No. 8, August 2009 5 theme What’s new in TB?

Avoid drug interactions Resources Rifampicin is a potent inhibitor of cytochrome P450 isoenzymes, Australian state and territory TB contacts especially CYP3A4 and CYP2C8/9, and therefore great attention • Australian Capital Territory Tuberculosis Service, c/o Thoracic Unit, The Canberra Hospital. Phone 02 6244 2066 to other medications metabolised by these enzymes, especially • New South Wales Tuberculosis Program, NSW Department of Health. anticonvulsants, oral contraceptives and warfarin, is required. Phone 02 9391 9277 Nutrition • Northern Territory Tuberculosis Unit, Centre for Disease Control. Phone 08 8922 8804 Malnutrition contributes to TB risk and complicates active TB.50 • Queensland Tuberculosis Control Centre. Phone 07 3896 3939 Specific micronutrients with immunological functions such as vitamin • South Australian Tuberculosis Services, Royal Adelaide Hospital Chest Clinic. Phone 08 8222 4867 D may be of particular importance,51,52 but trial outcomes examining • Tasmanian Tuberculosis Services, Southern Region – Respiratory Unit, use of these agents as supplements in TB treatment are pending. Royal Hobart Hospital, phone 03 6222 7353; Northern Region – Respiratory Food scarcity is obviously less problematic in Australia than in high Unit, Launceston General Hospital, phone 03 6348 7708; North-Western TB burden countries, but nutritional optimisation and replacement of Region – Chest Clinic, Community Nursing, phone 03 6421 7700 deficient nutrients in individuals with TB is in keeping with current • Victorian Tuberculosis Control Section, Department of Human Services. Phone 03 9096 5110 or 1300 651 160 WHO recommendations.53 • Western Australian Tuberculosis Control Program, Perth Chest Clinic. Smoking cessation Phone 08 9219 3222. Selected guidelines and resources Active and passive smoking are associated with elevated TB risk and • Australian National Tuberculosis Advisory Committee (NTAC). Available worse outcomes from TB infection.54 Smoking cessation is therefore an at www.health.gov.au/internet/main/publishing.nsf/Content/cdna-ntac- important potential intervention area, although the impact of quitting about.htm after diagnosis on individual patient outcomes remains to be determined. • Management, control and prevention of tuberculosis. Guidelines for health care providers (2002–2005). Rural and Regional Health and Aged Psychological support and adherence help Care Services Division, Victorian Government Department of Human Services, 2002. Available at www.health.vic.gov.au/__data/assets/ Psychological and practical supports are key requirements of TB pdf_file/0006/19986/tb_mgmt_guide.pdf care. Tuberculosis remains deeply stigmatised in many societies, and • Centre for Disease Control Northern Territory. Guidelines for the control of confinement in respiratory isolation can be traumatising.55 A diagnosis tuberculosis in the Northern Territory. 4th edn, 2008. Department of Health and Community Services, Northern Territory Government. Available at of TB can therefore require management of negative psychological www.health.nt.gov.au/library/scripts/objectifyMedia.aspx?file=pdf/25/05. effects. The prolonged duration of therapy requires assiduous pdf&siteID=1&str_title=Tuberculosis Control Guidelines.pdf measures to promote adherence. The most widely used of these • Australasian Society for Infectious Diseases Writing Group. Diagnosis, is the WHO sanctioned ‘DOTS’ (directly observed therapy – shourt management and prevention of infections in recently arrived refugees. corse). Other adherence promoting measures include addressing 2002. Available at www.asid.net.au/downloads/RefugeeGuidelines.pdf financial costs of leave from work by assisting in accessing short • World Health Organization TB publications. Available at www.who.int/tb/ publications/en/index.html term disability pensions, addressing any problematic beliefs about • World Health Organization. Toman’s tuberculosis case detection, treat- TB including those to do with law and migration status, and providing ment and monitoring: Questions & answers. 2nd edn, 2004. Available at ongoing education about the need to continue treatment even after whqlibdoc.who.int/publications/2004/9241546034.pdf symptom resolution.56 • Stop TB Partnership. Available at www.stoptb.org/. Fact sheets for patients Conclusion • www.health.vic.gov.au/ideas/diseases/tb_facts • www.health.nt.gov.au/Centre_for_Disease_Control/Publications/CDC_ Important new developments in TB diagnosis and treatment Factsheets/index.aspx. have occurred in recent years. These include the availability of new diagnostic tests, updated guidelines on managing HIV-TB Conflict of interest: none declared. co-infection, new data showing better prognoses for people with Acknowledgments MDR-TB/XDR-TB, and renewed emphasis on the importance of Anna Ralph is supported by the National Health and Medical Research Council infection control. Australia remains in the fortunate position of (NHMRC) and a Royal Australasian College of Physicians – Covance Award. having very low TB rates by global standards, low drug resistance Paul Kelly is supported by NHMRC. We thank the members of the National TB (although increasing cases are projected), infrequent cases of HIV Advisory Committee for providing the contacts listed in the resources table. co-infection, and access to MTB culture and susceptibility testing. References Maintaining this position despite the high global burden of TB 1. Taylor GM, Murphy E, Hopkins R, Rutland P, Chistov Y. First report of Mycobacterium bovis DNA in human remains from the Iron Age. Microbiology requires ongoing timely TB screening, a low threshold for considering 2007;153:1243–9. the diagnosis of active TB in people at risk, and psychosocial support 2. Kappelman J, Alcicek MC, Kazanci N, Schultz M, Ozkul M, Sen S. First homo to assist adherence to treatment. erectus from Turkey and implications for migrations into temperate Eurasia. Am J

6 Australian Family Physician Vol. 38, No. 8, August 2009 What’s new in TB? THEME

Phys Anthropol 2008;135:110–6. multidrug-resistant tuberculosis: A meta-analysis. Eur Respir J 2008;32:1165-74. 3. Flynn JL, Chan J. Immune evasion by Mycobacterium tuberculosis: Living with the 28. Perkins MD, Cunningham J. Facing the crisis:improving the diagnosis of tuberculo- enemy. Curr Opin Immunol 2003;15:450–5. sis in the HIV era. J Infect Dis 2007;196:S15–27. 4. World Health Organization. Global tuberculosis control: surveillance, planning, 29. Updated guidelines for the use of nucleic acid amplification tests in the diagnosis financing: WHO report 2008 WHO/HTM/TB/2008.393 ed. Geneva: WHO, 2008. of tuberculosis. MMWR Morb Mortal Wkly Rep 2009;58:7–10. 5. Roche PW, Krause V, Konstantinos A, et al. Tuberculosis notifications in Australia, 30. National Tuberculosis Advisory Committee. Position Statement on Interferon- 2006. Commun Dis Intell 2008;32:1–11. Release Immunoassays in the Detection of Latent Tuberculosis Infection. 2007. 6. Australian Government Department of Health and Ageing. National Notifiable Available at www.health.gov.au/internet/main/publishing.nsf/Content/cdna– Diseases Surveillance System. Available at www9.health.gov.au/cda/Source/ ntac-interferon.htm [Accessed January 2009]. Rpt_4.cfm [Accessed March 2009]. 31. Guo N, Marra F, Marra CA. Measuring health-related quality of life in tuberculosis: 7. Communicable Diseases Network of Australia. National Strategic Plan for TB a systematic review. Health Qual Life Outcomes 2009;7:14. Control in Australia Beyond 2000. Australian Government Department of Health 32. Gupta A, Street AC, Macrae FA. Tumour necrosis factor alpha inhibitors: screen- and Ageing, Canberra, 2002. Available at www.health.gov.au/internet/wcms/ ing for tuberculosis infection in inflammatory bowel disease. Med J Aust publishing.nsf/Content/cda-pubs-other-tb_plan.htm. [Accessed January 2009]. 2008;188:168–70. 8. Gray N, Hansen-Knarhoi M, Krause V. Tuberculosis in illegal foreign fishermen; 33. Mazurek GH, Jereb J, Lobue P, Iademarco MF, Metchock B, Vernon A. Guidelines whose public health are we protecting? Med J Aust 2008;188:144–7. for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis 9. Gilpin C, Simpson G, Vincent S, et al. Evidence of primary transmission of multi- infection, United States. MMWR Recomm Rep 2005;54(RR-15):49–55. drug resistant tuberculosis in the Western Province of Papua New Guinea. Med J 34. Lenaerts AJ, Degroote MA, Orme IM. Preclinical testing of new drugs for tubercu- Aust 2008;188:148–52. losis: Current challenges. Trends Microbiol 2008;16:48–54. 10. Rao SN, Mookerjee AL, Obasanjo OO, Chaisson RE. Errors in the treatment of 35. Check E. After decades of drought, new drug possibilities flood TB pipeline. Nat tuberculosis in Baltimore. Chest 2000;117:734–7. Med 2007;13:266. 11. Australasian Society for Infectious Diseases Writing Group. Diagnosis, manage- 36. Lenaerts AJ, Gruppo V, Marietta KS, Johnson CM, Driscoll DK, Tompkins NM, ment and prevention of infections in recently arrived refugees, 2002. Available at et al. Preclinical testing of the nitroimidazopyran PA-824 for activity against www.asid.net.au/downloads/RefugeeGuidelines.pdf [Assessed January 2009]. Mycobacterium tuberculosis in a series of in vitro and in vivo models. Antimicrob 12. Hargreaves S, Carballo M, Friedland JS. Screening migrants for tuberculosis: Agents Chemother 2005;49:2294–301. Where next? Lancet Infect Dis 2009;9:139–40. 37. Murphy DJ, Brown JR. Novel drug target strategies against Mycobacterium tuber- 13. O’Brien DP, Leder K, Matchett E, Brown GV, Torresi J. Illness in returned travelers culosis. Curr Opin Microbiol 2008;11:422–7. and immigrants/refugees: The 6-year experience of two Australian infectious 38. Rustomjee R, Lienhardt C, Kanyok T, et al. A Phase II study of the sterilising activi- diseases units. J Travel Med 2006;13:145–52. ties of ofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis. Int J 14. Allen S, Batungwanayo J, Kerlikowske K, et al. Two-year incidence of tuberculosis Tuberc Lung Dis 2008;12:128–38. in cohorts of HIV–infected and uninfected urban Rwandan women. Am Rev Respir 39. Singh A. Fluoroquinolones should not be the first-line antibiotics to treat com- Dis 1992;146:1439–44. munity-acquired pneumonia in areas of tuberculosis endemicity. Clin Infect Dis 15. Selwyn PA, Hartel D, Lewis VA, et al. A prospective study of the risk of tuberculo- 2007;45:133; author reply 4–5. sis among intravenous drug users with human immunodeficiency virus infection. 40. Harries AD, Zachariah R, Lawn SD. Providing HIV care for co-infected tuber- N Engl J Med 1989;320:545–50. culosis patients: a perspective from sub-Saharan Africa. Int J Tuberc Lung Dis 16. Bastian I, Krause VL. Tuberculosis: the dis-ease that didn’t dis-appear. Med J Aust 2009;13:6–16. 2008;188:131–2. 41. Centres for Diseases Control and Prevention. Managing drug interactions in the 17. World Health Organization. Antiretroviral therapy for HIV infection in adults and treatment of HIV-related tuberculosis. 2007. Available at www.cdc.gov/tb/publi- adolescents: Recommendations for a public health approach. Geneva: WHO, 2006. cations/guidelines/TB_HIV_Drugs/default.htm. Available at www.who.int/hiv/pub/guidelines/artadultguidelines.pdf [Accessed 42. Reid MJ, Shah NS. Approaches to tuberculosis screening and diagnosis in people January 2009]. with HIV in resource-limited settings. Lancet Infect Dis 2009;9:173–84. 18. Post JJ, Emerson CR. To routinely offer testing for HIV infection in all cases of 43. Cobelens FG, Heldal E, Kimerling ME, et al. Scaling up programmatic manage- tuberculosis: a rational clinical approach? Med J Aust 2008;188:162–3. ment of drug-resistant tuberculosis: A prioritized research agenda. PLoS Med 19. BTS recommendations for assessing risk and for managing Mycobacterium tuber- 2008;5:e150. culosis infection and disease in patients due to start anti-TNF-alpha treatment. 44. Andrews JR, Gandhi NR, Moodley P, et al. Exogenous reinfection as a cause of Thorax 2005;60:800–5. multidrug-resistant and extensively drug-resistant tuberculosis in rural South 20. Horsburgh CR, Jr. Priorities for the treatment of latent tuberculosis infection in the Africa. J Infect Dis 2008;198:1582–9. United States. N Engl J Med 2004;350:2060–7. 45. Cox HS, Sibilia K, Feuerriegel S, et al. Emergence of extensive drug resist- 21. Davis JS, Currie BJ, Fisher DA, et al. Prevention of opportunistic infections in ance during treatment for multidrug-resistant tuberculosis. N Engl J Med immunosuppressed patients in the tropical top end of the Northern Territory. 2008;359:2398–400. Commun Dis Intell 2003;27:526–32. 46. Orenstein EW, Basu S, Shah NS, et al. Treatment outcomes among patients with 22. Inge LD, Wilson JW. Update on the treatment of tuberculosis. Am Fam Physician multidrug-resistant tuberculosis: Systematic review and meta-analysis. Lancet 2008;78:457–65. Infect Dis 2009;9:153–61. 23. Therapeutic Guidelines: Antibiotic. Therapeutic Guidelines Limited. Available at 47. Mitnick CD, Shin SS, Seung KJ, et al. Comprehensive treatment of extensively http://proxy9.use.hcn.com.au/. drug-resistant tuberculosis. N Engl J Med 2008;359:563–74. 24. Management, Control and Prevention of Tuberculosis. Guidelines for Health 48. Emergence of Mycobacterium tuberculosis with extensive resistance to second- Care Providers (2002–2005). Rural and Regional Health and Aged Care Services line drugs worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 2006;55:301–5. Division, Victorian Government Department of Human Services, Melbourne 49. Plan to combat extensively drug-resistant tuberculosis: Recommendations of the Victoria. Available at www.health.vic.gov.au/_data/assets/pdf_file/0006/19986/ Federal Tuberculosis Task Force. MMWR Recomm Rep 2009;58(RR-3):1–43. tb_mgmt_guide.pdf. 50. Africa’s Health in 2010 Project, United States Agency for International 25. Centre for Disease Control Darwin. Guidelines for the Control of Tuberculosis Development. Nutrition and Tuberculosis: A review of the literature and consid- in the Northern Territory, 2002. Available at http://nt.gov.au/health/cdc/treat- erations for TB control programs, 2008. Available at www.aidsportal.org/repos/ ment_protocol/TB.pdf. Nutrition%20and%20TB_Final.pdf [Accessed January 2009]. 26. Pai M, Ramsay A, O’Brien R. Evidence-based tuberculosis diagnosis. PLoS Med 51. Ralph AP, Kelly PM, Anstey NM. L-arginine and vitamin D: Novel adjunctive immu- 2008;5:e156. notherapies in tuberculosis. Trends Microbiol 2008;16:336–44. 27. Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of 52. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated

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human antimicrobial response. Science 2006;311:1770–3. Cost-effectiveness of a new interferon-based blood assay, QuantiFERON(R)-TB 53. WHO. Guidelines for the programmatic management of drug-resistant tuberculo- Gold, in screening tuberculosis contacts. Int J Tuberc Lung Dis 2008;12:1414-24. sis. Geneva: World Health Organization, 2006. Available at http:whqlibdocwhoint/ 62. de Perio MA, Tsevat J, Roselle GA, Kralovic SM, Eckman MH. Cost-effectiveness pblications/2006/9241546956_engpdf [Accessed June 2007]. of interferon gamma release assays vs tuberculin skin tests in health care 54. World Health Organization, International Union against TB and Lung disease. A workers. Arch Intern Med 2009;169:179-87. WHO/The Union monograph on TB and tobacco control. Joining efforts to control 63. World Health Organization. Guidelines for the programmatic management of drug- two related global epidemics. Document no. WHO/HTM/TB/2007390, 2007. resistant tuberculosis: Emergency update. WHO/HTM/TB/2008.402, 2008. 2008. 55. Baral SC, Karki DK, Newell JN. Causes of stigma and discrimination associated Available at http://whqlibdoc.who.int/publications/2008/9789241547581_eng.pdf with tuberculosis in Nepal: A qualitative study. BMC Public Health 2007;7:211. [Accessed March 2009]. 56. Munro SA, Lewin SA, Smith HJ, Engel ME, Fretheim A, Volmink J. Patient adher- 64. Katiyar SK, Bihari S, Prakash S, Mamtani M, Kulkarni H. A randomised controlled ence to tuberculosis treatment: A systematic review of qualitative research. PLoS trial of high-dose isoniazid adjuvant therapy for multidrug-resistant tuberculosis. Med 2007;4:e238. Int J Tuberc Lung Dis 2008;12:139-45. 57. Pai M, Riley LW, Colford JM, Jr. Interferon-gamma assays in the immunodiagnosis 65. Schaaf HS, Gie RP, Kennedy M, Beyers N, Hesseling PB, Donald PR. Evaluation of of tuberculosis: A systematic review. Lancet Infect Dis 2004;4:761-76. young children in contact with adult multidrug-resistant pulmonary tuberculosis: A 58. Connell TG, Ritz N, Paxton GA, Buttery JP, Curtis N, Ranganathan SC. A three-way 30-month follow-up. Pediatrics 2002;109:765-71. comparison of tuberculin skin testing, QuantiFERON-TB gold and T-SPOT.TB in 66. National Tuberculosis Advisory Committee multi-drug resistant tuberculosis: children. PLoS ONE 2008;3:e2624. Information paper (October 2007). Commun Dis Intell 2007;31:406-9. 59. Pai M, Joshi R, Dogra S, Zwerling AA, Gajalakshmi D, Goswami K, et al. T-cell 67. World Health Organization. Toman’s tuberculosis case detection, treatment and assay conversions and reversions among household contacts of tuberculosis monitoring: Questions and answers. 2nd edn. 2004. Available at http://whqlibdoc. patients in rural India. Int J Tuberc Lung Dis 2009;13:84-92. who.int/publications/2004/9241546034.pdf [Accessed January 2009]. 60. Pai M, Zwerling A, Menzies D. Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann Intern Med 20085;149:177-84. 61. Marra F, Marra CA, Sadatsafavi M, Moran-Mendoza O, Cook V, Elwood RK, et al. correspondence [email protected]

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Chapter 3: Introduction II - Adjunctive treatment in TB

3 Introduction II: Adjunctive Treatment in TB

Problems with current standard TB treatment regimens, rising drug resistance and the challenges posed by HIV-TB co-infection are driving a need for novel therapeutic approaches in TB. Acknowledging this, the investigation of adjunctive immunotherapies (agents given in addition to antibiotics to beneficially modulate immune responses and thereby improve outcomes), was nominated by the UNICEF/UNDP/World Bank/WHO Special Programme on Research and Training in Tropical Diseases in 2006 as one of six priority TB research areas.10 In this chapter, I present the rationale for investigating candidate adjunctive immunotherapies in TB, specifically, L-arginine and vitamin D.

3.1 WHY ARE NEW TREATMENT APPROACHES NEEDED?

PROBLEMS WITH THE STANDARD TB TREATMENT REGIMEN

Progressive refinement of TB treatment regimens between the 1940s and 1970s culminated in the finding that the addition of rifampicin, by accelerating culture conversion rates, permitted treatment to be reduced from 18 to 6-9 months.11 The resulting current 6-month ‗short-course‘ regimen for drug-susceptible MTB infection comprises rifampicin, isoniazid, pyrazinamide and ethambutol for 2 months (intensive phase) then rifampicin, and isoniazid for 4 months (continuation phase). This complex and protracted regimen has high risks for adherence difficulty. Additional problems with the regimen are drug interactions (due to rifampicin‘s strong induction of cytochrome P450), antibiotic resistance, and toxicity. Thus a key goal in contemporary TB research is to work towards shorter treatment courses, permitting more rapid disease resolution and earlier cure. Achieving these goals could have important clinical, public health and economic benefits including a faster return to employment or school, and diminution of residual respiratory impairment. Exciting recent developments in this field include the findings that moxifloxacin as an ethambutol substitute accelerates culture conversion in infection with drug-susceptible MTB,12 and the diarylquinolone

5 Chapter 3: Introduction II - Adjunctive treatment in TB TMC207 (targeting ATP synthase) significantly reduces time to culture conversion when added to MDR-TB treatment regimens.13 Ongoing investigation of rifamycins, including higher doses of rifampicin with the potential to accelerate treatment response,14 are also under investigation.15

Adherence

Adherence to TB treatment is compromised by many factors, which have been summarised by Munro et al16 as structural (e.g. financial burden including work absenteeism during treatment, cost of regular transport to the clinic, distance between patient‘s home and clinic), personal (e.g. attitudes towards illness and treatment, lack of belief in tablet efficacy, poor patient-staff relations), social (family / community responses including stigmatisation of the TB sufferer), and health service factors (the lengthy treatment duration, pill burden and adverse effects).16-18

The most widely used method of promoting adherence is the use of directly observed therapy (DOTS). Introduction of DOTS has been associated with documented 19, 20 reductions in TB prevalence (although meta-analysis did not find clinic-based DOT to be superior to routine self-administration, or supervision at home by a family or community member, in achieving TB cure21).

Patient incentives such as a cash deposit paid at treatment commencement are used widely, including at the field research site at which the AVDAPT study is based. Pill burdens are reduced by use of fixed-dose combination (FDC) therapy, but people >55kg still require four FDC tablets per dose, FDC may only be intermittently unavailable (as at our field research site), and FDC is not suitable when adverse reactions require tailored regimens.

Rates of successful treatment completion vary according to the resources available for supporting DOTS or other adherence-promoting measures.16 Examples of reported treatment default rates are 20-50% in the US in the 1980s,22 and 28% in the Philippines in the 1990s.23 Treatment completion rates were as low as 12% at the RSMM community hospital near the Timika TB clinic in 2007 (personal communication, Drs

6 Chapter 3: Introduction II - Adjunctive treatment in TB Frans Thio, Director, Rumah Sakit Umum Daerah [Regional General Hospital], Timika, Indonesia).

Drug interactions

Rifamycins vary in the potency with which they induce the cytochrome P450 enzyme system; rifampin is the most potent, strongly inducing CYP3A4 and CYP2C8/9 isozymes and, to a lesser extent, CYP2C19 and CYPD6 isozymes. However, alternative rifamycins are not widely available at low cost in high TB burden countries. Therefore drug interactions are an important consideration; serum levels and therefore efficacy is significantly diminished for many potentially co-prescribed medications (e.g. oral contraceptives, anticonvulsants). Most problematic are potential interactions with antiretroviral therapy (ART). However recent data in fact indicate that while protease inhibitors are definitely contraindicated, adequate serum levels of efavirenz are maintained in combination with rifampicin, and the reduction in serum nevirapine is not as profound as previously supposed; thus there can be greater confidence in following guidelines recommencing early commencement of ART in people being treated for TB.8, 24

Toxicity

Although adverse events to antituberculosis drugs requiring treatment modification are thought to occur in less than 10% of people (e.g. ranging from 2.3% in those ≤19 years to 8.4% for those ≥60 years in one study25), milder adverse events such as pyrazinamide-induced arthralgia affect up to 40% of people,26 and can be an important cause of morbidity and non-adherence.27

Drug resistance

The above-mentioned difficulties in achieving TB course-completion, particularly where structural and health service factors impede careful adherence, promote the development of acquired and amplified resistance in Mycobacterim tuberculosis (MTB).28 Transmission of MDR-TB, rather than resistance-development during

7 Chapter 3: Introduction II - Adjunctive treatment in TB treatment, is now increasingly common.29 Unfortunately resistance mutations do not generally appear to be associated with any significant MTB fitness cost.30 WHO‘s ‗Anti-tuberculosis drug resistance in the world‘ reports document the growing problem of global MDR-TB, with the tally of countries reporting cases of XDR-TB inexorably rising.31

Treatment regimens for MDR or XDR-TB are more complex, toxic, expensive and prolonged than the standard TB regimen, creating major challenges for health systems and added difficulties for patient adherence.32, 33 However, comprehensive treatment of MDR and XDR-TB can achieve good outcomes,34 as reviewed above.8

As rates of MDR and XDR-TB rise and their geographical distribution expands, the need for new anti-tuberculous antibiotics and / or novel adjunctive therapies with different mechanism of resistance development becomes increasingly evident.

HIV-TB co-infection

As reviewed above,8 the HIV pandemic is a major factor responsible for failures to control TB into the 21st century. Pulmonary TB is a ubiquitous opportunistic infection in HIV infection, even in early stages before immune deficiency as defined by CD4 count becomes evident. Combating high TB rates among HIV positive people, even before they are eligible for antiretroviral therapy (i.e. CD4 count <200 in resource- limited settings),35 requires broad approaches including research into new anti- tuberculous chemotherapeutic agents.

3.2 ADJUNCTIVE THERAPIES

ADJUNCTIVE IMMUNOTHERAPEUTIC STRATEGIES

These points provide a clear rationale for the need for new approaches to TB treatment. While new antibiotics (reviewed elsewhere8) are arguably of greatest importance, the investigation of potential adjunctive immunotherapies is recognised as an additional important priority.10 Immunotherapy strategies in TB have been reviewed by other authors36, 37 and key points are summarised in the paper reproduced below.38 The two

8 Chapter 3: Introduction II - Adjunctive treatment in TB contrasting strategies of immune modulation to improve TB treatment outcomes can be broadly categorised as:

 Promotion of the host‘s innate or Th1 cell-mediated anti-mycobacterial immune responses;

 Mitigation of immunopathology-mediated tissue damage due to excessive inflammatory responses (immunosuppression).

L-arginine and vitamin D have anti-mycobacterial properties which render them suitable candidate adjunctive immunotherapies in TB,38 thus bringing together the fields of adjunctive immunotherapy and nutrition in TB.

NUTRITIONAL SUPPLEMENTATION IN TB

There is a clear bi-directional relationship between malnutrition and TB, with underweight individuals having higher risk of developing active TB, and TB causing cachexia.39-42 However the corollary that improved nutrition decreases TB risk, or improves outcomes in active TB, is difficult to demonstrate. Nevertheless, nutritional optimisation is recommended by the WHO, especially in the management of drug resistant TB,32 and is a feature of successful comprehensive treatment programs.34

There has long been interest in the use of nutritional agents to improve immune responses to a variety of infections. Some investigations have shown disappointing lack of efficacy (such as vitamin C in common cold43), while others have shown important benefits (such as vitamin A in measles44 and zinc in diarrhoeal disease45). Supplementation of specific micronutrients in TB may be required not only to replenish the deficiency states which accompany active TB, but because of the roles certain micronutrients have in mediating anti-mycobacterial immune responses. As well as considering which micronutrients would be most efficacious on the basis of their immunological mechanisms of action, other important questions include whether people with active or latent TB would be better targets for nutritional supplementation, whether micronutrients at achievable doses can offer sufficient potency to significantly influence TB treatment outcome, whether supplementation is of any benefit if given to non- deficient people, and whether a ‗magic bullet‘ micronutrient tablet approach is preferable to whole food supplementation. To date, a large number of randomised trials

9 Chapter 3: Introduction II - Adjunctive treatment in TB of nutrition in TB have sought to answer these questions, including investigation of individual supplements, multi-micronutrients of differing combinations and doses, and whole food.

Meta-analysis of nutritional supplements for people being treated for active TB recently identified some evidence for improved weight gain with energy supplements and some combinations of zinc with other micronutrients.46 For example, a trial investigating the effect of a high-energy food supplement on anthropometric and quality of life outcomes found improved weight and grip strength in those receiving the supplement.47 A Tanzanian study in almost 500 people found a small but significant increase in weight in people assigned to supplementation with vitamins A, B1, B2, B12, C, E, D3, folic acid, niacin, selenium, copper and zinc, but no effect on culture conversion.48 In a larger trial evaluating mortality as the outcome measure, no impact was found from supplementation of vitamins A, B1, B2, B6, B12, C, D, E, niacin, folate, zinc, iodine and selenium in HIV positive or negative TB patients.49 A more recent trial of micronutrient combination therapy (vitamins A, B1, B2, B6, B12, C, E, folic acid, niacin and selenium) found decreased risk of ‗TB recurrence‘ in the supplemented group (but ‗recurrence‘ was somewhat unusually defined as reversion to smear positivity during treatment) with a greater effect in HIV-infected patients.50 Supplementation in this study also increased CD3 and CD4 T cell counts. A wholefood trial comprising a nutritious daily meal was associated with significant improvements in weight gain in the intervention arm in pulmonary TB patients in Timor Leste.51 In summary, certain nutritional supplements may improve weight gain in people with TB, but no convincing impacts on bacteriological outcomes have been identified in trials to date.

3.3 ARGININE AND VITAMIN D AS NOVEL POTENTIAL ADJUNCTIVE TREATMENTS IN TB

L-ARGININE HYDROCHLORIDE

L-arginine is a conditionally essential amino acid found in a variety of food sources including nuts, meat and dairy products. Normal dietary intake in well-nourished people provides around 4 to 5g L-arginine per day.52 L-arginine is the precursor of nitric oxide (NO), a molecule with key vascular and immunological functions, and L-arginine also has T cell immunomodulatory effects.38

10 Chapter 3: Introduction II - Adjunctive treatment in TB Use of L-arginine in human trials

Clinical trials of L-arginine in oral, intravenous or inhaled form have been performed examining endovascular and immunological functions, including in cardiovascular conditions, pulmonary hypertension in sickle cell disease, cystic fibrosis, and as an adjunct to vaccination.27-37 Cardiovascular studies have found that at doses of 9 to 30 g daily orally or intravenously, L-arginine can significantly improve coronary blood flow and angina symptoms in patients with non-obstructive coronary artery disease,53 acutely reduce blood pressure,54 and improve claudication distance in patients with peripheral vascular disease.55 In people with severe falciparum malaria, who have lower serum L- arginine than healthy controls, administration of intravenous L-arginine 3 to 12 g has been shown to improve endovascular function, as measured using reactive hyperemia – peripheral arterial tonometry, and to increase exhaled NO by 55%.56 Use of 30g L- arginine daily in people with breast cancer was found to be associated with enhanced host defences, including lymphocyte mitogenic responses and NK cell activity 57.

L-arginine Pharmacokinetics

Healthy adult plasma arginine levels range from 20 to 180 mol/L.53 Orally administered L-arginine-hydrochloride bioavailability ranges from 21% after a 10g oral dose in one study58 to 68% after a 6g dose in another.54 Peak plasma concentrations

(Cmax) of 310 ± 152 mol/L occur at approximately 90 minutes after a 6g oral dose 54 (tmax), and half life is reported to be 77.5 ± 9.3 minutes.

VITAMIN D3 (CHOLECALCIFEROL)

Vitamin D3 is generated in the skin from 7-dehydrocholesterol on exposure to

Ultraviolet (UV) B light and is metabolised in the liver to 25-hydroxyvitamin D3 (cholecalciferol). Dietary sources (oily fish, fortified foods such as margarine) also provide a small contribution to total body vitamin D. 25-hydroxyvitamin D3 is converted by the enzyme 1-hydroxylase to the biologically active steroid hormone,

1,25-dihydroxyvitaminD3 (cholecalciferol or calcitriol, abbreviated 1,25(OH)2D3). It is now recognised that the gene encoding 1-hydroxylase, CPY27B, is expressed widely

11 Chapter 3: Introduction II - Adjunctive treatment in TB by human cells, and that 1,25(OH)2D3 has diverse roles in most organs including anti- proliferative and immunomodulatory effects.59

Vitamin D deficiency and its measurement

Traditional methods of measuring serum vitamin D (e.g. radioimmunoassay, chemiluminescent assay) tend to lack reliability.60, 61 A more robust and reproducible method using isotope-dilution liquid chromatography-tandem mass spectrometry has been described,62 and is now available in Australia.63

There have been calls to revise ‗normal‘ reference ranges for serum vitamin D, using the vitamin D level needed for maximal suppression of parathyroid hormone to signify sufficiency, rather than using population measures to develop normal reference ranges. Thus, some enthusiasts suggest that levels as high as 80 nmol/L should be considered the lower level of normal for serum vitamin D,64 while others accept 50 nmol/L.65 Australian guidelines define mild deficiency as being 25 - 50 nmol/L, moderate as 12.5 – 25 nmol/L, and severe as <12.5 nmol/L.66

Even using standard vitamin D reference ranges, high rates of vitamin D insufficiency and deficiency are being recognised globally, likely a consequence of modern lifestyles (indoor living and clothing). This ‗pandemic‘59 of vitamin D deficiency is not confined to high latitudes but affects all surveyed populations, including in tropical / equatorial and sunny regions in peoples with varying degrees of skin melanisation.67-69

Associations of adverse health outcomes with vitamin D deficiency

Coupled with the appreciation of the pandemic nature of vitamin D deficiency has been the discovery of the pleiotropic actions of 1,25(OH)2D3, thus generating many recent observational and some interventional studies demonstrating an association between vitamin D deficiency and a broad range of adverse health outcomes, beyond rickets and osteomalacia. These include neoplastic disease, auto-immune diseases, cardiovascular disease, diabetes, mental health problems, and infections.70-76 Meta-analysis has also found an association between vitamin D deficiency and all-cause mortality.77 The wide- ranging actions of vitamin D, reviewed by Holick59 and summarised in Box 3.1, partly explain these associations. The need for either adequate UV exposure, or oral vitamin D intake or supplementation, to avoid vitamin D deficiency-related diseases has led to

12 Chapter 3: Introduction II - Adjunctive treatment in TB calls for reconsideration of public health messages advising sun avoidance to reduce skin cancer risk.78

Vitamin D Pharmacokinetics

Vitamin D replacement can be given as large intermittent bolus doses, a concept embraced in the AVDAPT study design (see Chapter 5). The volume of distribution of vitamin D is large since it is fat soluble, necessitating large doses before any change in plasma vitamin D level is detectable. Intermittent depot vitamin D dosing known as ‗stoss‘ therapy is among the recommendations for restoration of vitamin D deficiency.79 A recent study of cholecalciferol pharmacokinetics in 30 healthy adults with normal baseline vitamin D concentration found that such dosing was safe, resulting in no cases of vitamin D intoxication and no hypercalcaemia.80

Box 3.1: Actions of Vitamin D

 Calcium and bone

o Maintenance of normal serum calcium and phosphorus levels. o Control of bone mineralisation and turnover.  Anti-proliferative effects

o 1,25(OH) D controls genes in healthy and malignant cells involved in 2 3 cellular proliferation, differentiation and apoptosis. This is thought to explain the beneficial effect of vitamin D in the hyper-proliferative skin disease psoriasis, and to explain the association between vitamin D

deficiency and increased cancer risk.

 Immunomodulation o Suppression of Th1 activity: anti-autoimmune effect (type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Crohn‟s disease).

o Promotion of macrophage activity: upregulation of genes leading to

cathelicidn gene upregulation and the generation of antimicrobial peptide LL-37.  Cardiovascular effects

o 1,25(OH) D reduces renin production, decreases proliferation of 2 3 myocardial and vascular smooth muscle cells, decreases LDL, increases HDL, and enhances insulin production.

13 Chapter 3: Introduction II - Adjunctive treatment in TB

USE OF L-ARGININE AND VITAMIN D IN TB

The antimycobacterial immunomodulatory properties of L-arginine and vitamin D, and the rationale for investigating their use as adjunctive immunotherapies in tuberculosis, is presented in the paper reproduced below.38

Further relevant studies pertaining to L-arginine or vitamin D in TB which have appeared since publication of this paper are discussed in the section ‗Literature update‘ below.

14 Chapter 3: Introduction II - Adjunctive treatment in TB

Manuscript 3: Ralph AP, Kelly PM, Anstey NM. L-arginine and vitamin D: novel adjunctive immunotherapies in tuberculosis. Trends Microbiol 2008;16(7):336-44.

NOTICE: this is the author’s version of a work that was accepted for publication in Trends Microbiology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Trends Microbiology, [16, 6, (2008)] doi:10.1016/j.tim.2008.04.003

L-arginine and vitamin D: novel adjunctive immunotherapies in tuberculosis Anna P. Ralph1,2, Paul M. Kelly1,2 and Nicholas M. Anstey1,3 1 International Health Division, Menzies School of Health Research, Building 58, Royal Darwin Hospital Campus, Casuarina, Northern Territory, 0810, Australia 2 National Centre for Epidemiology and Population Health Research, Building 62, Australian National University, Canberra, Australian Capital Territory, 0200, Australia 3 Charles Darwin University, Ellengowan Drive, Casuarina, Northern Territory, 0810, Australia Corresponding author: Ralph, A.P. ([email protected]). Worsening drug resistance and the need for prolonged macrophages [7]. Although early reports cast doubt on the treatment in tuberculosis (TB) require innovative solutions importance of the L-arginine–NO pathway in human including investigation of inexpensive, safe adjunctive macrophage killing of MTB, we review here the evidence immunotherapies. L-arginine, the precursor of nitric oxide, for an important functional role in humans. Within the and vitamin D recently have elucidated mycobactericidal past 2 years, evidence has emerged in humans for an and immunomodulatory actions against TB and are deficient immunomodulatory role of L-arginine via an NO- in people with TB. We review the potential of these agents independent pathway, namely via modulation of T-cell as adjunctive TB immunotherapies and explore how function through L-arginine’s effect on the expression of comparative clinical trials might help clarify their relative CD3ζ, a component of the T-cell receptor (TCR) [8]. The importance in the human TB immune response. By role of the vitamin D3 pathway in mycobacterial killing enhancing mycobacterial killing in macrophages, L-arginine within human macrophages has been carefully elucidated and vitamin D might have the potential to enable shorter in recent studies [6,9]. The importance of vitamin D3 in duration of treatment, reduced infectivity and improved human host resistance TB has been suspected since the response in drug-resistant TB. late nineteenth century [10,11]. Now, expanding recognition of the association between vitamin D Nitric oxide and vitamin D as adjunctive tuberculosis deficiency and TB risk and of the pleiotropic immunotherapies immunomodulatory functions of vitamin D3 provides Mycobacterium tuberculosis (MTB) is one of the most biological mechanisms for this supposition and has lead to successful human pathogens, infecting an estimated one- calls for the clinical evaluation of vitamin D as an third of the human population for a lifelong duration and adjunctive therapy in the treatment of active tuberculosis accounting for the greatest number of deaths from a [6,12]. In this review we explore the potential clinical curable infectious disease [1,2]. Shortening the duration of application of both L-arginine and vitamin D as adjunctive tuberculosis (TB) treatment by using strategies such as treatments in TB. revised regimens or modes of delivery of existing drugs, the development of new antimicrobial agents and Adjunctive therapy in tuberculosis successful adjunctive immunotherapies could profoundly Immunosuppressants and proimmune mediators (i.e. impact TB cure rates [3–5]. Here, we examine two agents enhancers of immunological function) both have roles as whose therapeutic potential as adjunctive TB adjuncts to TB treatment. Corticosteroids, possibly by immunotherapies derives from their predominant suppressing tumour necrosis factor-α (TNF-α) and other (although not exclusive) enhancement of macrophage proinflammatory cytokines, can ameliorate excessive host antituberculous responses. inflammatory T-cell responses and subsequent organ The L-arginine–nitric oxide (NO) and vitamin D damage and have the best recognized role as TB pathways comprise two parallel systems utilized by adjunctive treatments [13]. Positive results have been macrophages to kill MTB, an example of redundancy as an obtained in some trials with thalidomide, also acting via immune safeguard. They have been proposed to be of TNF-α [14]. Deliberate therapeutic disruption of varying importance among different mammalian species, granulomata using high-dose corticosteroids or etanercept, with nocturnal rodents using NO to combat TB and combined with standard antituberculous drugs, has been diurnal humans, who have the capacity to synthesize suggested as a novel means of accelerating TB cure [15]. vitamin D3 upon UV exposure, potentially being more By contrast interest in proimmune mediators as reliant upon vitamin D pathways [6]. This concept has adjunctive immunotherapies is growing and could become been debated, given the evidence for a significant role for increasingly important as drug-resistant TB becomes more + NO in human protective immune responses [7]. prevalent [3,4,16]. Enhancement of CD8 cytotoxic T- NO generated from the amino acid L-arginine has a lymphocyte responses and suppression of T helper 2 (Th2) clearly established role in MTB killing by murine processes appear promising in animal immunotherapy studies [3]. Human data from mostly small trials of Th1

cytokines, cytokine inhibitors or therapeutic vaccination associated with the plasma membrane [29]. Vitamin D is are reviewed in Ref. [4]. Interferon gamma (IFN-γ) has transported by the polymorphic, multifunctional DBP. As shown efficacy in small numbers of cases of refractory well as carrying vitamin D (which occupies only ~5% of human TB [17]. These proimmune mediators are mostly T- binding sites), DBP transports other immunoactive cell active agents, whereas immunotherapeutic agents molecules and when deglycosylated becomes DBP- targeting mycobacterial killing within macrophages have macrophage-activating factor (DBP-MAF), a potent been explored less. stimulator of macrophages and osteoclasts [30]. Whether Adjunctive therapies in TB-endemic settings must be altered DBP kinetics might modulate vitamin D handling affordable and easy to administer. Cheap and safe in the setting of macrophage activation in TB is worthy of approaches include food and nutritional components. The speculation. relationship between nutritional deficiency and TB is Epidemiology bidirectional: underweight individuals carry a higher TB Accumulating evidence of an association between low risk (and inadequate weight gain during treatment is serum vitamin D levels in individuals, certain ethnic associated with higher TB-relapse risk) [18], and TB groups, migrants and others, and a higher risk of active infection causes cachexia [19]. Specific micronutrient TB [6,31–35] now has been confirmed in a recent meta- ‘adjunctive therapy’ with vitamin A, zinc or multivitamins analysis [36]. Hypovitaminosis D in people with active TB or high-energy and protein food supplements have shown might be attributable to factors including genetic disappointingly modest benefits [20–22]. determinants; inadequate cutaneous synthesis due to L-arginine and vitamin D physiology environmental conditions or medically or socially imposed isolation; or reduced dietary intake due to preference, L-arginine availability or TB-induced anorexia. L-arginine is a conditionally essential (semiessential) amino acid in humans. In vivo, arginine derives from Genetics exogenous sources (diet) and endogenous sources (whole- VDR polymorphisms have been investigated as possible body protein degradation plus synthesis from citrulline) genetic determinants of TB risk. Meta-analysis has been [23]. In adults de novo arginine synthesis accounts for only inconclusive [37], but findings to date suggest that the 5%–15% of endogenous arginine flux [23]. Whole-body TaqI tt polymorphism might be associated with a shift protein turnover contributes the majority of endogenous towards beneficial Th1 responses and at least one t allele arginine turnover. Citrulline is synthesized in the gut and might be associated with better MTB-directed cell- then converted to arginine in the kidneys via actions of the mediated immune (CMI) responses [38]. NOS2A cytosolic enzymes argininosuccinate synthetase (ASS) and polymorphisms of the gene encoding NOS2 (nitric oxide argininosuccinate lyase (ASL) [23]. Arginine also can be synthase 2) (Figure 1) also have been investigated. In one produced in cells actively producing NO via the citrulline– study long repeats of a microsatellite in the promoter NO cycle (Figure 1). During hypercatabolic states such as region of the NOS2A gene were over-represented in Paisa infection or trauma, arginine synthesis can no longer Columbians with TB compared with controls [39]. match catabolism and an exogenous source of arginine Interpretation of findings can be difficult because many becomes essential to maintain the extracellular polymorphisms investigated have unknown functions. concentrations required for optimum NO synthesis. Furthermore, increasing recognition of non-VDR-mediated Hypoargininemia has been demonstrated in people with vitamin-D3-signaling pathways (via membrane-associated, pulmonary TB [8], malaria [24], sepsis [25] and pregnancy rapid-response receptors), especially within the immune [26]. Hypoargininemia in people with active TB, due to system, indicate that VDR polymorphisms cannot be excessive arginase activity, might be difficult to overcome expected to completely explain genetic differences in from dietary means alone due to disease-related anorexia. vitamin D metabolism. Vitamin D Immunomodulatory role of L-arginine in TB Cholecalciferol (vitamin D3 or 25(OH)D3) is converted into Nitric oxide as a mycobactericidal mediator the biologically active hormone 1,25-dihydroxyvitamin D3 L-arginine-derived NO plays a key role in the innate (1,25(OH)2D3) by 1α-hydroxylase, encoded by the gene immune system and, specifically, in the defense against CYP27b1. Exogenous determinants of vitamin D3 levels mycobacteria [7,40] (Table 1). NO kills MTB by direct include ultraviolet light B exposure, dietary intake and TB bacterial cell damage through deamination of bacterial drugs themselves, with isoniazid inhibiting and rifampicin DNA, proteins and lipids and by induction of apoptosis of inducing cytochrome 3A4, a vitamin D hydroxylase [27]. TB-harbouring macrophages [41]. NO is noted to be Endogenous determinants include genetic factors [e.g. skin capable of killing TB bacilli in vitro with a molar potency pigmentation and vitamin D receptor (VDR) comparable to that of antibiotics [42]. Early in vitro polymorphisms], vitamin-D-binding protein (DBP) studies of human monocyte and macrophage responses to concentration and macrophage activation. Activated MTB failed to demonstrate a role for NO due to macrophages generate active vitamin D3 due to methodological problems [41]. Despite initial doubts, there upregulation of CYP27b1 [28] (Figure 2). Vitamin D3 is now compelling evidence that human macrophages and actions are mediated either by ligation with the nuclear monocytes are capable of high level NOS2 expression and VDR, resulting in genomic responses, or via more recently identified rapid-response receptors, which might be

NO production in vivo [43], including in TB [44–45], and These have been the subject of recent reviews both in TB subsequent increased capacity for MTB killing (Table 1). [12,54] and the broader immunological context [55]. Induction of MTB dormancy gene expression by NO Role of activated macrophages NO at a low (nontoxic) concentration appears capable of Macrophages are important sites of vitamin D metabolism driving MTB to enter a dormant phase of inhibited in patients with TB. Hypercalcemia occurs in a minority of metabolism and consequent chemo-insensitivity, which people with TB and other granulomatous diseases due to might equate with latency (Ref. [46] and reviewed in Refs CYP27b1 upregulation in activated macrophages [28]. [47,48]). Thus, although high NO concentrations are Despite epidemiological data showing low vitamin D levels mycobactericidal, based on this literature low NO states in TB patients on the whole [36], in selected individuals resulting from L-arginine deficiency or inadequate NOS2 vitamin D3 levels can reach high concentrations in the expression could be doubly disadvantageous to the host by vicinity of the granuloma, with spillover into the systemic contributing to the development of latent TB infection. circulation resulting in hypercalcemia in up to 25% of TB patients, although this uncommonly reaches clinically Mycobacterial resistance to NO significant levels [56]. Emphasizing the fundamental role of NO in MTB immune Before the specific role of 1,25(OH)2D3 in the immune responses, MTB has developed specific strategies to response to TB was recognized, speculation regarding the mitigate NO-mediated microorganism toxicity [47]. MTB functional rationale for its production by activated strains also demonstrate variable degrees of susceptibility macrophages included (i) macrophage-derived vitamin D3 to NO. One TB strain accounting for a large proportion of acting as a local downregulator of T-cell activity [57], (ii) a new drug-susceptible TB cases in New York in the early modulator of monocyte-macrophage differentiation [58] or 1990s was relatively resistant to reactive nitrogen (iii) vitamin D3 attracting DBP to the vicinity of the intermediates (RNI) compared with other circulating MTB granuloma, with DBP-MAF further escalating macrophage strains [49]. Although this strain appeared to have an activation [30]. All of these possibilities might still have advantage allowing it to account for more cases than relevance, but the mechanism and rationale for CYP27b1 expected, the disease severity in affected patients was not upregulation is now clearer [6], as detailed below. described, whereas in guinea pigs, at least, RNI-resistance among MTB isolates does correlate with virulence [50]. Mechanism of vitamin D activity against MTB in cultured human cells L-arginine and TCR function The inhibitory effect of vitamin D3 on MTB in cultured L-arginine has a further important immunomodulatory human monocytes or macrophage cell lines has been role independent of NO through its effects on the CD3ζ recognized since 1986 and replicated in many subsequent component of the TCR. CD3ζ, expressed on the surface of studies [6,9,59–62]. Macrophage activation via the TLR natural killer (NK), CD4 and CD8 T cells, plays a central (toll-like receptor) 1/2 pathway initiates a cascade of role in initiation of signal transduction leading to T-cell events culminating in increased intracellular 1,25(OH)2D3 activation, proliferation and cytokine secretion [51]. and increased production of antimicrobial peptide LL-37, The causal pathway between increased arginase which kills intracellular MTB [6]. Specifically, TLR1/2 activity leading to L-arginine depletion and decreased activation leads to upregulation of the genes encoding CD3ζ expression leading to relative T-cell VDR and 1α-hydroxylase (CYP27b1); this allows hyporesponsiveness is increasingly understood. Peripheral utilization of 25(OH)D3 and conversion to 1,25(OH)2D3; blood mononuclear cells from people with pulmonary TB 1,25(OH)2D3 promotes the formation of the LL-37 have elevated levels of arginase, leading to L-arginine antimicrobial peptide from the cathelicidin gene hCAP18, catabolism and hypoargininemia; their consequently and LL-37 then mediates MTB cell death [6,9,54] decreased CD3ζ expression normalises with successful TB (Figure 2). There is now convincing evidence that the treatment [8]. Separate studies have shown that antimicrobial peptide LL-37 is an important mediator of restoration of extracellular L-arginine ex vivo (in L- this effect [6,9,63], but other downstream mediators also arginine-deficient human placental cell suspensions and might be recruited, including induction of NOS2A leukaemic cells) leads to CD3ζ re-expression and recovery [9,62,64] and phosphatidylinositol-3-kinase (PI3-K)- of T-cell proliferation [26,52]. These data collectively mediated superoxide production [60]. provide important evidence that reduced L-arginine is Successful induction of antimycobacterial activity associated with impaired CD3ζ expression and relative T- through cathelicidin and LL-37 production is dependent cell hyporesponsiveness. upon replete serum vitamin D levels [6]. Ethnic groups To date there have been no clinical studies in humans (including African Americans) at higher risk of TB have testing whether L-arginine supplementation can reverse lower serum levels of 25(OH)D3 and their serum is less CD3ζ-associated in vivo T-cell dysfunction, although L- efficient in supporting cathelicidin mRNA induction; arginine supplementation in normal subjects and patients supplementation of this serum with 25(OH)D3 restores with breast cancer enhanced NK and lymphocyte- TLR-mediated induction of cathelicidin mRNA [6]. The activated NK cell activity [53]. findings provide an immunological basis for differential Immunomodulatory role of vitamin D in TB susceptibility among human populations to mycobacterial Pre-antibiotic era efforts at TB treatment (Box 1) might infection; namely, that vitamin D deficiency portends a have exploited immunomodulatory effects of vitamin D. higher risk of TB disease.

Reversal of MTB-induced phagosome maturation arrest by depression, such as is achieved with the use of adjunctive vitamin D corticosteroids [13], are beneficial. Inhibition of phagosome maturation is an important NO inhibits activities including lymphocyte activation example of MTB immune evasion. Killing of phagocytosed and adherence and transmigration of monocytes that organisms is usually achieved through a calcium- contribute to granuloma formation, caseation and lung dependent fusion process between phagosomes and toxic- destruction in TB [69,71]. In TB-infected NOS2−/− acid-containing lysosomes [65]. By inhibiting the fusion knockout mice and mice treated with NOS2 inhibitors, step, through lipoarabinomannan-mediated inhibition of larger and more destructive lung lesions are seen in early sphingosine kinase [65] MTB is able to achieve latency in infection even before significant increases in mycobacterial a protected niche. Recent data demonstrate that MTB- numbers are seen [41,69,72,73]. NO also inhibits induced phagosome-maturation arrest in a human production of TNF-α(69), a mediator of caseating necrosis macrophage model is able to be reversed by vitamin D3 via of lung tissue and weight loss in TB. Alveolar macrophage a PI3-K pathway [66]. This PI3-K mediation of vitamin D3 NO production is significantly higher in patients with antimycobacterial activity demonstrates nongenomic milder disease than those with more extensive TB, (VDR-independent) signaling of vitamin D3 [60,66]. Thus, consistent with a disease-protective role for NO not only in both nuclear VDRs, via TLR activation, and non-nuclear mice but also in humans [74]. receptors, via PI3-K, mediate the effects of vitamin D3 in Although direct evidence for vitamin D ameliorating the macrophage. Although TLR-mediated MTB killing is TB-induced pathology is lacking, indirect evidence would an important first line defense against initial TB infection, support this hypothesis. The CMI-depressive effect of phagolysosome fusion could play a greater role later in the vitamin D has generated interest in vitamin D3 as an course of infection, including in the prevention of latent immunosuppressant treatment after organ TB reactivation. transplantation [55]. It also provides the theoretical basis for apparent associations between vitamin D deficiency Interactions between L-arginine–NO and vitamin D– and autoimmune disease [75]. The possibility of a cathelicidin pathways deleterious effect of replete or supplemented vitamin D Macrophages can be activated either by TLR triggering, stores on immune responses to TB does not appear to be leading to increased intracellular vitamin D3 and borne out by the literature to date, suggesting that cathelicidin induction, or by IFN-γ, acting via Janus possible harm from Th1 suppression is outweighed by the tyrosine kinase (JAK) and signal transducer and activator beneficial roles of vitamin D3 on macrophage function and of the transcription (STAT-1)-signaling pathway to in the possible mitigation of tissue damage by suppression upregulate NOS2 and thereby increase NO release of excess Th1 responses. (Figure 2). Interactions between the L-arginine–NO and vitamin D–TLR–cathelicidin pathways are probable. First, Clinical experience with L-arginine and vitamin D CYP27b1 requires an extracellular source of L-arginine for Trials of L-arginine and NO in human TB full expression and is upregulated by NO in an avian Extensive experience with therapeutic L-arginine in macrophage cell line [28]. Second, the antimycobacterial humans has been gained from clinical trials of L-arginine effect of vitamin D might be mediated in part by NO in the oral, intravenous or inhaled form in studies of both [9,62]. Third, nuclear factor kappa Β (NF-κB), a endovascular and immunological functions [53,76–78]. downstream mediator of TLR signaling, also might impact Pharmacokinetic and modest side-effect properties are on NOS2 regulation and NO production in murine well described elsewhere [79]. There has been one macrophages [67]. randomized controlled trial (RCT) of L-arginine in TB to These in vitro studies cannot determine how these date. Low dose L-arginine supplementation (1 g orally immunological strategies manifest in the infinitely daily) in Ethiopian TB patients was safe and well complex in vivo setting of human TB infection in which tolerated [80]. In subgroup analysis of those without HIV there are individual variations in vitamin D and L- infection, there was a modest but significant rise in arginine concentrations both systemically and locally in plasma levels of L-arginine, a significant improvement in the vicinity of the granuloma. Comparative clinical trials the resolution of cough but no significant effect on weight of both arginine and/or vitamin D as adjunctive therapies gain and sputum smear conversion in the arginine group. in TB could assess the relative importance of these two Although arginine was safe and promising, limitations of immune pathways in the human host. this study include the small sample size, low dose and Potential for attenuation of lung pathology apparent posthoc analysis. TB results in significant tissue destruction and long-term Inhaled NO has been trialled as a therapeutic adjuvant impairment of lung function [68]. Attenuation of lung in HIV-negative, smear-positive pulmonary TB patients. pathology has the potential to minimize long-term Administered at a dose of 80 ppm for 72 hr, NO was found pulmonary disability. Both NO and vitamin D share a to be safe and well tolerated, but no beneficial effects were propensity to bolster innate immune responses but demonstrated in eight cases compared with ten controls attenuate overexuberant CMI responses [54,55,69,70]. [81]. Definitive conclusions from this study are limited by Profound T-cell depression, exemplified by HIV, is the small size and short treatment duration. significantly detrimental to host containment of Measurement of serum NO metabolites can be used to mycobacterial growth, but lesser degrees of T-cell measure systemic, but not organ-specific, NO production,

but adherence to fasting protocols is required [82]. hypercalcemia and increased bone turnover [55]. However, Measurement of exhaled NO (eNO) has been validated as expensive or complicated regimens, if shown to be of a reliable measure of pulmonary NO production and can benefit, are unlikely to be widely adopted, especially in now be performed by using portable and simple resource-poor, high-prevalence TB settings. apparatuses [83,84]. eNO is elevated in human pulmonary Concluding remarks and further directions TB, decreases with treatment and appears inversely TB is a disease of the highest global public health associated with disease severity [70]. Furthermore, NOS2 significance, requiring the pursuit of innovative research expression in alveolar macrophages derived from directions (Box 2). Improvements in TB therapeutic bronchoalveolar lavage correlates significantly with eNO, regimens through the use of adjunctive immunotherapies supporting the validity of eNO in this setting [70]. Finally, could have profound impacts, and these are recognized as systemic administration of L-arginine orally or priority TB research fields [16]. Evidence for the intravenously increases eNO [76], making eNO a useful suitability of L-arginine and vitamin D as candidate means of monitoring response to adjunctive L-arginine. adjunctive TB or latent TB treatments include the Vitamin D trials in TB following: (i) human macrophages use activated vitamin D Martineau and colleagues [12] reviewed three RCT and and L-arginine-derived NO to kill TB bacilli, (ii) human T ten prospective case series in which vitamin D was cells require replete L-arginine levels to function optimally administered to patients with TB, but, overall, the impact because expression of the CD3ζ chain of the TCR requires of vitamin D supplementation on TB outcome could not be L-arginine, (iii) vitamin D and NO might both have the assessed; most studies were conducted in the 1950s, were potential to attenuate excessive organ pathology resulting identified as being of poor quality and used ergocalciferol from exaggerated CMI and (iv) active TB infection is (vitamin D2), which is less efficacious than cholecalciferol associated with deficiencies in L-arginine and vitamin D. (vitamin D3) [85]. A more recent trial of vitamin D The L-arginine and vitamin D immunological pathways supplementation in Indonesian pulmonary TB patients might work in parallel in TB-infected individuals, could suggested more rapid sputum clearance and radiological have differential importance during stages of TB infection improvement, but the trial was small, the process of or among individuals depending on prevailing genetic, randomisation was not described and the safety profile cytokine and substrate influences and are likely to be of including calcium levels was not assessed [86]. varying importance among different host species affected Some [36], but not all [35], epidemiological studies by mycobacteria. Comparative clinical trials of L-arginine suggest that a fall in serum vitamin D leads to activation and/or vitamin D could help to determine their relative of latent TB. If this is the case then an important potential importance in human TB. If L-arginine and/or vitamin D application for vitamin D is as an adjunctive therapy in can shorten time to sputum clearance and culture latently infected individuals to prevent TB reactivation. negativity, administration of these agents would have Clearly, any such clinical trials would need to be very potential for both population and individual benefits, large [87]. However, a recent trial of vitamin D including reduced transmission and possible shorter and, supplementation in latently infected TB patients therefore, more successful courses of therapy. demonstrated immunological improvement, as indicated Furthermore, with the increasing prevalence of multidrug- by growth restriction of luminescent recombinant Bacillus resistant TB, agents that improve innate mycobacterial Calmette-Guerin (BCG)-lux in whole blood from vitamin- killing mechanisms might improve cure rates in drug- D-supplemented people compared with controls [88]. resistant disease. There is, thus, a compelling rationale for Would vitamin D supplementation be of value only in clinical trials investigating the safety and efficacy of both those who are deficient or could supraphysiological L-arginine and vitamin D, singly or together, as adjunctive vitamin D levels also be beneficial in those replete at immunotherapies in TB. baseline? Ongoing debate regarding ideal vitamin D levels Acknowledgements complicates this question [89]; given current recommended We acknowledge the many authors who have contributed to the fields reviewed here reference ranges, vitamin D deficiency is increasingly whose work we have been unable to cite due to space constraints. In keeping with recognized, even in geographical regions considered to the journal’s requirements, we frequently have been obliged to cite reviews instead. have high UV ratings [90]. It could be hypothesized that The National Health and Medical Research Council (Australia) supports A.R. (Postgraduate Medical Research scholarship), P.K. (Career Development Award) because unregulated production of CYP27b1 by activated and N.A. (Practitioner Fellowship, ICRG and Program Grant). macrophages (achieving supraphysiological vitamin D References levels in some patients) has been naturally selected as an 1 Dye, C. (2006) Global epidemiology of tuberculosis. Lancet antimycobacterial innate immune response, then there 367, 938–940 might indeed be an immunological benefit of higher than 2 Maartens, G. and Wilkinson, R.J. (2007) Tuberculosis. Lancet normal vitamin D concentration in the direct vicinity of 370, 2030–2043 3 Rook, G.A. et al. (2007) Immunotherapeutics for tuberculosis the macrophage. The possibility of hypercalcemia would in experimental animals: is there a common pathway activated by necessitate careful vitamin D dose selection and effective protocols? J. Infect. Dis. 196, 191–198 monitoring and indeed could limit the potential of vitamin 4 Roy, E. et al. (2007) Current strategies in TB immunotherapy. D as an adjunctive therapy. The use of vitamin D3 Curr. Mol. Med. 7, 373–386 analogues or combination therapy with bisphosphonates 5 Spigelman, M.K. (2007) New tuberculosis therapeutics: a growing pipeline. J. Infect. Dis. 196 (Suppl 1), S28–S34 has been proposed to exploit the immunomodulatory properties of vitamin D while avoiding possible

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Arginase1, arginase2 NOS2

Plasma L-ornithine + urea L-citrulline + NO L-arginine

Arginosuccinate synthase

Diet Muscle turnover

TRENDS in Microbiology Figure 1. Metabolic fate of L-arginine and macrophage nitric oxide production. Sources of L-arginine (the active biological enantiomer of this conditionally essential amino acid) in the fed state include exogenous (food) and endogenous sources (whole-body protein degradation and, to a small extent, de novo synthesis from citrulline by renal arginosuccinate synthase) [22]. L-arginine is converted to NO by NOS2, the inducible product of the NOS2 gene. Th1 cytokines (principally IFN-γ) stimulate expression of macrophage NOS2 leading to NO production, whereas under the influence of Th2 cytokines (IL-4, IL-10 and IL-13), arginine is depleted by arginases. NO is one of several RNIs with antimicrobial activity [91].

Macrophage

MTB dormancy

? Nucleus Low-level NO NOS2 Th1 L-arginine and NK IFNγ JAK STAT-1 NOS2 cells MTB NOS2 VDR gene and κβ TLR1/2 MyD88 NF CYP27b1 High-level NO

Cathelicidin (hCAP18) MTB growth suppression

1α hydroxylase LL-37 1,25(OH)2D3

25(OH)D3 TRENDS in Microbiology Figure 2. Simplified schemata of macrophage antimycobacterial pathways. Parallel pathways [IFN-γ–JAK and STAT–L-arginine-derived NO] and [TLR2–MyD88 and NFκΒ– vitamin D–cathelicidin and LL37] lead to macrophage activation and mycobacterial control. IFN-γ release by Th1 and NK cells (stimulated by MTB-infected antigen-presenting cells) engages the JAK and STAT-1 pathways within the macrophage, leading to upregulation of genes including NOS2. Interaction of MTB with TLR in monocytes and macrophages (mainly via TLR1/2) leads to engagement with MyD88 and NF-κΒ. The subsequent cascade of antimycobacterial responses includes TNFα production, IL-12 release (recruiting Th1-mediated immunity, providing another means of IFN-γ production and further macrophage activation) and upregulation of the vitamin D3–cathelicidin pathway. High NO concentrations (e.g. resulting from high L-arginine concentration and NOS2 expression) are associated with MTB growth inhibition, whereas low-level NO (e.g. resulting from L-arginine or NOS2 insufficiency) appears to be associated with MTB dormancy. Abbreviations: IFNγ, Interferon gamma; JAK, Janus tyrosine kinase; MTB, Mycobacterium tuberculosis; MyD88, adaptor protein; NF-κΒ, nuclear factor-kappa B (cytoplasmic transcription factor); NK cell, natural killer cell; STAT, signal transducer and activator of transcription; TLR2, Toll-like receptor 2; Th1, Type 1 T helper cell; VDR, vitamin D receptor. Dotted arrow signifies non-dominant pathway.

Box 1. Chronology of vitamin D-based treatments in the pre-antibiotic era Although not meeting the level of evidence of modern RCTs, these early reports might have been exploiting immunomodulatory effects of vitamin D [10,11]. Heliotherapy (sunlight exposure) 1855 Rikli, a Swiss doctor, opened a thermal treatment station in Slovenia. 1901 Finsen was awarded the Nobel Prize for Medicine for successful treatment of cutaneous tuberculosis (lupus vulgaris) with UV radiation. 1903 Rollier from Switzerland opened a hospital to treat TB by using graded sun exposure. In 1914 he published a book based on results. 1922 Increasing popularity of sunlight treatment in the UK. Establishment in the UK of ‘Committee on Sunlight’ and Light Department at London Hospital; widespread adoption of sun-exposure practices. Cod liver oil 1771 Cod liver oil was introduced into the British pharmacopoeia. 1833 Henkel, a German doctor, reported on the successful use of cod liver oil in the treatment of TB. 1841 Bennett, a Scottish physician, published medical uses of cod liver oil, including in TB. Five case reports of improvement in TB symptoms, but two relapsed after stopping cod liver oil. 1849 Hospital for Consumption and Diseases of the Chest in London published outcomes of cod liver oil in TB: 18% disease arrested, 63% improved and 19% unchanged. 1855 Woods from Philadelphia, USA, attributed the 19% fall in deaths due to TB between 1847 and 1852 to the use of cod liver oil.

Box 2. Future research directions

• Adjunctive immunotherapies, new TB drugs, new regimens of existing drugs and novel means of drug delivery (e.g. liposomal rifabutin and oral streptomycin) are among the strategies being investigated to improve treatment regimens for both sensitive and multidrug-resistant TB. • Phase III clinical trials of adjunctive vitamin D monotherapy in active TB (http://clinicaltrials.gov/show/NCT00419068, http://clinicaltrials.gov/show/NCT00507000 and http://clinicaltrials.gov/show/NCT00366470, and http://www.controlled- trials.com/ISRCTN35212132), and a comparative trial of L-arginine and/or vitamin D (http://clinicaltrials.gov/show/NCT00677339) are in progress. • Pending results of such trials, nutrition needs to be optimized in people with TB and vitamin D deficiency should be identified and corrected if present. Table 1. Immunological evidence for a role of arginine and NO in TB control

Study Type Results Refs

L-arginine-derived nitric oxide and other RNIs In vitro MTB MTB isolates are sensitive to NO and other RNI and differ in their RNI-susceptibility. [50] culture Mouse Arginine-derived RNI in mouse macrophages effectively kill MTB. [41] macrophage MTB lacking genetic resistance to RNI cannot grow in mouse macrophages, in contrast with wild-type MTB. [42] studies. Human Alveolar macrophages from healthy humans infected ex vivo with MTB produce NO, and NO production correlates with [45] macrophage intracellular growth inhibition of MTB. studies Blood mononuclear cells from healthy donors infected ex vivo with MTB and from people with pre-existing TB infection produce [92] NO. Pulmonary macrophages kill mycobacteria only if they express NOS2; killing is prevented with a NOS inhibitor. [93] In vivo NOS2 is expressed at sites of disease in immunocompetent mice. Fulminant MTB disease develops in NOS2 knockout mice Reviewed mouse (NOS2−/−) and in mice with a variety of immune defects, in contrast with controls. in [40] studies MTB infection is poorly contained in mice treated with NOS inhibitors. [41] [40] In vivo In lung resection studies NOS2 and nitrotyrosine (a tissue marker of NO metabolism) are expressed in macrophages within [94] human granulomata and areas of TB pneumonitis. studies NOS2 expression is increased in peripheral blood monocytes from people with TB compared with healthy controls. [95] NOS2 is expressed in macrophages from lungs of patients with tuberculosis. [70] Pulmonary NO production measured by eNO is significantly higher in TB patients than in controls; eNO is inversely associated [70] with disease severity and decreases with anti-TB treatment. Relationship between CD3ζ expression and L-arginine in TB and other conditions Human T Depletion of L-arginine in vitro causes reduced CD3ζ expression, impaired T-cell signaling and diminished proliferation. [91] cells in vitro Arginase-mediated L-arginine depletion induces downregulation of CD3ζ; addition of L-arginine ex vivo leads to CD3ζ re- [26] expression and recovery of T-cell proliferation. In vivo Peripheral blood T cells from patients with pulmonary TB have decreased CD3ζ expression, correlating with arginase-mediated L- [23] human TB arginine deficiency, which normalises with successful TB treatment. study

Chapter 3: Introduction II - Adjunctive treatment in TB

LITERATURE UPDATE

Vitamin D in TB

Since publication of the above paper, results of a randomised trial of suopplemental cholecalciferol (100 000 IU) in patients with TB in Guinea-Bissau have been reported, demonstrating no mortality difference between vitamin D supplement and placebo arms.81 Due to funding constraints, the study was terminated after only 63% of the planned sample size was enrolled; furthermore, only 64% of those enrolled were able to contribute primary outcome data (a TB score generated by the authors). The study did include an assessment of smear positivity at 1 month (in 58% of participants) and there was a non-significant reduction in smear positivity in the vitamin D group (risk difference -6%, 95% CI -18 to 5). Respiratory function was not assessed. Interestingly, the study did not demonstrate any difference in vitamin D levels between the 2 groups, suggesting that the vitamin D formulation used (an injectable preparation given orally) may have been poorly absorbed. Finally, while the study was randomised, the use of treatment group medication labels (‗lot 204‘ or ‗lot 205‘) meant that allocation concealment and blinding could not be guaranteed.81 Therefore this study is inconclusive and indicates the need for further adequately powered trials of vitamin D in TB.

This publication and its accompanying editorial82 fuelled ongoing debate regarding the appropriate timing of administration of vitamin D in relation to onset of active TB. While reliable, reproducible clinical trial results in both latent or active TB are still lacking, some hypothesise that vitamin D supplementation would only be efficacious as a prophylactic agent for reducing the risk of activation of latent TB (for instance, as life- long therapy in vitamin D-deficient people with latent TB).83 Others argue that vitamin D has roles in immunological pathways other than just latent TB reactivation, thereby warranting trials of vitamin D as an adjunctive agent in active TB.84

In preliminary safety data from another study, 11 TB patients were administered a single dose of 100 000 IU ergocalciferol (vitamin D2, considered less efficacious than D385), resulting in a significant rise in serum vitamin D2 and no episodes of hypercalcaemia at 8 weeks; no rise in serum D2 occurred in 14 TB patients randomised to placebo.86 Although the most common time for hypercalcaemia occurrence has been

16 Chapter 3: Introduction II - Adjunctive treatment in TB reported to be around 2 to 4 weeks,87, 88 these authors only examined hypercalcaemia at 8 weeks, therefore potentially underestimating true hypercalcaemia occurrence.

Contrasting with the majority of reports showing that low plasma vitamin D correlates with active TB, Selvaraj et al recently reported that plasma 1,25(OH)2D3 levels were significantly increased among pulmonary TB patients compared to healthy controls.89

Only 1,25(OH)2D3 (not 25(OH)D3) was measured in this study. The authors hypothesised that the widely reported finding of low 25(OH)D3 in people with active 90 TB may be explained by excessive conversion of 25(OH)D3 to 1,25(OH)2D3, providing a mechanism for simultaneous findings of low 25(OH)D3, and 1- hydroxylase-driven hypercalcaemia which can characterise active TB.89

A further update since publication of our paper38 pertains to VDR polymorphisms: subsequent to Lewis et al‘s meta-analysis of VDR polymorphisms91 which we cited, Gao and colleagues have now reviewed the literature, and report that among Asians, the FokI ff VDR genotype is associated with increased TB risk, the BsmI bb genotype was protective, and TaqI and ApaI polymorphisms were only marginally significant.92

Nitric oxide in TB

A subsequent review of NO in mycobacterial infection, referencing our article, offered similar conclusions regarding the role of L-arginine-derived NO in TB.93 Also furthering the understanding of mechanisms of immune evasion and intracellular survival, El Kasmi and colleagues reported that arginase can be induced by MTB in macrophages, thus diverting L-arginine substrate away from NO production and instead promoting the breakdown of L-arginine to ornithine.94 This might serve as a micro- organism-mediated mechanism to aid the establishment of successful latent infection.

Of particular relevance, a recent study found that 1,25(OH)2D3, in combination with IFN, led to the up-regulation of NOS2 mRNA and NO production by human peripheral blood monocytes / macrophages after stimulation with MTB or PPD, and that this response was attenuated by selective TKR2 antibodies.95 These data indicate for the first time that NO production may comprise part of the anti-mycobacterial mechanism of action of vitamin D3, via a TLR2 mechanism.

17 Chapter 3: Introduction II - Adjunctive treatment in TB In a mouse study, Beisiegel et al96 have provided new evidence of a lung protective role of NO in the murine immune response to infection with virulent MTB, whereby NO is required for regulation of pulmonary neutrophil influx. When wild type and NOS2 (iNOS) knockout mice were infected with virulent (H37Rv) and attenuated (H37Ra) MTB strains, iNOS knockout mice showed grossly impaired responses, including abnormal neutrophil influx, when infected with the virulent strain.

These updates reinforce the arguments put forward in the review article38 of the importance of undertaking investigation of adjunctive therapies such as L-arginine / NO and vitamin D in TB.

18 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB

4 Introduction III: Nitric oxide and its measurement in vivo

―A startlingly simple molecule unites neuroscience, physiology, and immunology, and revises scientists' understanding of how cells communicate and defend themselves.‖97

The discovery of NO as not just a noxious environmental pollutant, but an important biological mediator, was made in the late 1980s, revealing this as the agent previously known as endothelium-derived relaxing factor.98, 99 Analogous with the current cross- disciplinary interest in vitamin D and its new-found pleiotrophic actions, NO was found to have potent roles in cell signalling in many organs, earning it the title of ‗molecule of the year‘ in 1992, and opening the door to enthusiastic interest in the therapeutic potential of NO and its precursor, L-arginine, and to ways of measuring NO in vivo.97

An understanding of the antimycobacterial function of NO, summarised above,38 underpins the use of L-arginine in the AVDAPT clinical trial. A key measure of interest in AVDAPT study participants is fractional exhaled nitric oxide (FENO). This chapter therefore reviews the literature pertaining to FENO measurement.

4.1 NITRIC OXIDE PATHWAYS

NO is derived from L-arginine by nitric oxide synthase (NOS) enzymes, including the 2 constitutive NOS enzymes (neuronal NOS [nNOS or NOS1] and endothelial NOS [eNOS or NOS3], and inducible NOS (iNOS or NOS2). NOS2 is the NOS enzyme subtype of chief interest in the lung and in immunity.99 Induction of NOS2 gene expression by pro-inflammatory cytokines has been summarised above.38 This gene is expressed widely in different cell lines in the respiratory tract (alveolar macrophages, airway and vascular smooth muscle cells, epithelial and endothelial cells, lung fibroblasts, mast cells and neutrophils), consistent with the wide actions of NO.99 Biologically beneficial or protective effects of NO in the lung (e.g. smooth muscle relaxation, downregulation of Th1 pro-inflammatory activities, and the killing of

19 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB invading microorganisms) are balanced by the potentially deleterious effects of NO and other reactive nitrogen species (e.g. pro-inflammatory activities, necrosis, apoptosis).99

NO is a free radical (it has one unpaired electron) making it highly reactive with a short half life in vivo of 1 to 5 seconds. This short half life precludes direct NO measurement in blood. Surrogate measures such as NO metabolic products (e.g. nitrite, NO2−), or urinary excretion rates of nitrate (NO3−), have been used in the past. Measurement of

FENO provides a means of directly determining NO production in a human organ system.100 Therefore this method has potential utility in examining NO dynamics in lower respiratory tract infections such as TB.

4.2 EXHALED NITRIC OXIDE MEASUREMENT

FENO can be used as a marker of airway inflammation. A joint position statement of the

European Respiratory and American Thoracic Societies provides guidelines for FENO measurement.100

METHODS

The current standard method for FENO measurement is a single-breath technique comprising inhalation to total lung capacity followed by exhalation at a constant flow rate of 50 ± 5 mL/s (achieved by real-time audio and visual feedback from the measuring device) for a 10s duration. Modern portable devices permit online measurement (i.e. providing a real-time display of FENO). Alternatively, offline testing techniques can be employed where exhalate is collected into an impermeable bag for delayed analysis. The principles of FENO measurement are listed in Box 4.1.

Devices available for FENO measurement include large chemiluminescence analyzers such as the NiOX FLEX® (Aerocine, Sweden), and hand-held portable machines such as the NiOX MINO® (Aerocine, Sweden) (See Figures 4.1 and 4.2). Constraints of using traditional non-portable FENO analysers in a tropical clinic setting include the need for a large research space and air conditioning. The advent of portable eNO analysers in part overcomes these barriers. The hand-held portable NiOX MINO® has been reported 101-103 to provide valid, reproducible measures of FENO.

20 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB

Box 4.1: Principles of exhaled nitric oxide measurement

 Inhalation of NO-free air

o Can be achieved by use of an NO scrubber in the mouthpiece which absorbs NO from inspired air.  Expiration at a fixed flow rate: o FE varies markedly with flow rate (rises non-linearly with increasing flow NO rate). o The standard current flow rate required for most analysers, considered to be reproducibly achievable by adults and children, is 50 mL/s.

 Avoidance of nasal NO

o Nasal NO is high relative to NO in lower airways. o High pressures created in the oral cavity during exhalation against resistance ensures velum closure; this is generally considered a satisfactory technique

to exclude nasal air, avoiding the need for a nose peg.

 Avoidance of breath-holding o Breath-holding results in accumulation of NO, therefore the inspiration should be immediately followed by expiration.

 Avoidance of NO or arginine-containing foods

o Foods rich in nitrates or nitrites (e.g. lettuce) or arginine (e.g. peanuts) should be avoided prior to testing; thus fasting for 2 hours prior to measurement is recommended.

 Appreciation of the impact of smoking o Smoking acutely decreases eNO,  Avoidance of exercising prior to eNO measurement

o Exercise has been reported to have variable effects on eNO, therefore rest for

1 hour is recommended prior to eNO testing.

Box 4.1 references:100, 104

NORMAL FENO RANGE

The understanding of ‗normal‘ FENO and its determinants is evolving. Upper limits of normal FENO at a flow rate of 50mL/s (FENO0.05) for healthy adults and children of 33 105 ppb and 25 ppb, respectively, have been suggested. FENO0.05 distribution shows a right skew; median FENO in healthy Swedish non-smoking subjects was found to be 10 ppb 106 (range ~5 to 55), and very similar findings (median FENO 16.5 ppb, range 4.9 – 67.1) were reported from healthy Papuan and Non-Papuan adults by Yeo and colleagues at the MSHR-NIHRD Timika field research site.56

21 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB

Positive associations were reported between FENO and height and age, but not sex, in a carefully conducted survey of FENO values in 1131 healthy, never-smoking Swedish 107 adults. The upper limit of normal FENO for 25-34 year olds <160cm was reported as 24.0 ppb, and for 65-75 year olds >190 cm, as 57.4 ppb.107 However even excluding asthma, other lung diseases, atopy and smoking, the authors of this study found FENO to be poorly predictable based on the explanatory variables height and age, suggesting that other factors including nutritional status, genetics and / or undiagnosed or low grade lung inflammation might contribute to inter-subject FENO variability.

Figure 4.1: NiOX FLEX Study investigator (Anna Ralph) demonstrating use of the NiOX FLEX. Photo: Daniel Lampah.

CLINICAL UTILITY

Use of FENO as a monitoring tool is best described in asthma. Increases herald an asthma exacerbation, and FENO evaluation has been suggested as a means of diagnosing asthma, monitoring response to treatment, verifying adherence, and predicting upcoming exacerbations.100, 108

22 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB

Figure 4.2: NiOX MINO Research assistant (Govert Waramori) instructing a healthy volunteer in the use of the portable NiOX MINO. Photo: Anna Ralph.

Although an understanding of what constitutes normal FENO remains incompletely defined, Table 4.1 illustrates selected states found to be associated with abnormal FENO.

Some factors have been associated with both increases and decreases in FENO (exercise, caffeine, sputum induction).109

Table 4.1: Factors associated with abnormal exhaled nitric oxide

Factors associated with increased exhaled Factors associated with decreased exhaled nitric oxide nitric oxide Asthma exacerbation Cystic fibrosis (chronically) Airway viral infection Active and passive smoking Alveolitis Alcohol Asbestosis Altitude Bronchiectasis Severe malaria Chronic bronchitis Lung transplant rejection Cystic fibrosis (during acute exacerbation) Increasing age and height Height Nitrate-rich diet Table 4.1 references56, 109

23 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB

EXHALED NITRIC OXIDE IN TB

Two research groups have investigated FENO in pulmonary TB and reported inconsistent findings. Wang and colleagues in Taiwan110 conducted a detailed study (findings from which are included in our paper above38) in a small number of people (19 adults with pulmonary TB and 14 control subjects), in which FENO was correlated with disease stage and severity, and with other markers of NO production (expression of NOS2 gene in alveolar macrophages obtained via bronchoalveolar lavage, and in lung tissue [TB patients only], obtained via transbronchial biopsy). FENO was measured using a now- obsolete method, whereby patients exhaled slowly (at an uncontrolled flow rate of around 200 mL/s) through an open tube, with a probe sampling from the stream of expired breath. Therefore results in parts per billion are not comparable to results obtained at flow rates of 50 mL/s. They found FENO to be significantly higher in people with newly diagnosed TB compared with controls, but with normalisation of FENO by 3 months of treatment. An inverse association between FENO and disease severity was found, which the authors hypothesised might indicate that people with a poorer capacity to generate NO from alveolar macrophages developed more extensive disease. NOS2 gene expression in alveolar macrophages derived from bronchoalveolar lavage correlated significantly with exhaled NO, supporting the validity of FENO as a measure of lung NO production in this setting.110

In contrast, Idh and colleagues a decade later, in a paper which appeared after 38 publication of our review, reported inconsistent FENO results in Ethiopian adults with smear positive pulmonary TB.111 Several methodological flaws were evident in their report. FENO was measured using a non-portable NiOX analyser but omitted the standard initial step of inhalation of NO-free air prior to exhalation. Analyses did not control for age or anthropometric measures. Performing separate pair-wise comparisons between 4 patient subgroups: people with TB and HIV (n=36); people with TB without HIV (n=59); household contacts (n=17); blood donors (n=46), the authors reported that mean

FENO was lower in HIV-TB co-infected patients (approximately 14 ppb) than in controls

(approximately 18 ppb), and TB patients without HIV were more likely to have FENO > 25 ppb (an arbitrary post-hoc cut-off) than TB patients with HIV. No correlation was found between FENO and radiological disease severity. While only limited conclusions can be drawn from this study, results do appear to support the hypothesis that a poorer capacity to generate NO, as seen in the HIV positive group of TB patients, might be

24 Chapter 4: Introduction III: Exhaled Nitric Oxide in TB consistent with these people having a defective immune response, while the immunocompetent group with TB were more likely to have elevated eNO readings, consistent with NOS2 upregulation as part of the antimycobacterial immune response.

The conflicting results from these studies may partly reflect change in FENO measurement methodology over time, particularly flow rate. They indicate the requirement for further investigation of FENO in people with pulmonary TB, to clarify questions of pathophysiology, addressed further in Chapter 11.

25 Chapter 5: Study setting

5 Study setting

5.1 TIMIKA FIELD RESEARCH FACILITY

The Timika Translational Research Facility in Timika, Mimika district, Papua Province, Republic of Indonesia, is operated by Menzies School of Health Research‘s (MSHR) International Health Division in partnership with the National Institute for Health Research and Development (NIHRD), Indonesian Ministry of Health. Since establishment of this facility in 1999, major research into malarial drug resistance and comparative drug studies in Plasmodium falciparum and P. vivax infections, have led to important policy changes in local malaria management. Previous MSHR-NIHRD tuberculosis research at the site in 2003-4 has documented MTB drug resistance rates,112 sputum transportation methods,113 TB-induced lung impairment,114 and described the model of TB care delivery in Timika115.

Figure 5.1: Map of Indonesia and Northern Australia showing Timika (Papua Province)

Map courtesy of ANU Cartography Unit

Additional to the TB research currently underway, malaria research at the field site includes ongoing surveillance of the impact of community-based artemisinin combination drug deployment on uncomplicated and severe falciparum and vivax malaria, impact on pregnancy outcomes, in vitro drug sensitivity testing for P. falciparum and P. vivax, and a phase 2 study of intravenous arginine as adjunctive

26 Chapter 5: Study setting therapy in patients with severe malaria. Studies are also being conducted in sepsis and Maternal and Child health (see http://www.menzies.edu.au/research/international- health). The Research Facility currently receives core infrastructure financial support from AusAID, and a National Health and Medical Research Council (Australia) grant was provided in 2009 for ongoing TB research.

Figure 5.2: Timika community hospital (Rumah Sakit Mitra Masyarakat) Photos: Anna Ralph (a) Hospital entrance

(b) Hospital sign and Papuan carvings

(c) Nicholas Anstey, Enny Kenangalem & Paul Kelly at RSMM

27 Chapter 5: Study setting

Strong collaborations are in place with important local health organisations, including the District Health Authority, the two local hospitals, the Amungme and Kamoro Community Development Institution, and the Public Health Malaria Control unit of Freeport Indonesia. The field research facility, comprising a laboratory, offices and accommodation for visiting research staff is located on the campus of the community hospital, Rumah Sakit Mitra Masyarakat (RSMM) (Figure 5.2), 15 km from the Timika town centre and TB clinic.

Figure 5.3: NIHRD-MSHR Timika research staff and visitors in the research buidling L to R: Back row: Gysje Pontororing, Meryani Girsang (NIHRD), Wendelina Fobia, Yani Reyaan, Natalia Dwi Haryanti Front row: Anna Ralph, Trivonita Ria Bless, Getruida Bellatrix, Denius (visitor). Photo: Daniel Lampah.

5.2 TIMIKA TB CLINIC

The Timika TB clinic (Figure 5.4) is situated at the Timika Community Health Centre (Pusat Kesehatan Masyarakat, abbreviated Puskesmas), run by the District Government Health Authority (Dinas Kesehatan). As well as the TB clinic, the Puskesmas campus includes a general clinic, two sexually transmitted infection (STI) clinics and a laboratory. The general clinic and one STI clinic are operated by Dinas Kesehatan, while the TB clinic and one STI clinic are funded by the Public Health Malaria Control unit of PT Freeport Indonesia, and the laboratory by both sources. The TB clinic is the main referral centre for outpatient management of TB in Timika. Patients requiring

28 Chapter 5: Study setting hospitalization are referred to either of the two Timika hospitals. Although some smaller Puskesmas clinics also provide TB management, they are regularly encouraged by the research staff to refer TB patients for management at the dedicated Timika TB clinic, in keeping with District Health Authority policy, since treatment success can be optimized there by TB specialist staff, including public health employees who trace non-attendees in the community. Space at the clinic was very limited at the time of study commencement, so an extension to the existing facility was built in 2008 (using Covance Award funding granted to me by the Royal Australasian College of Physicians) (Figure 5.4).

Figure 5.4: Timika TB clinic and staff Photos: Anna Ralph and Govert Waramori (a) Front entrance (b) Research staff L to R: Anna Ralph, Daud Rumere (NIHRD-MSHR L to R: Gysje Pontororing, Govert Waramori, lab technician), Kristina Palupi (NIHRD). Getruida Bellatrix.

(c) Before construction of extension (d) After construction of extension

5.3 OVERVIEW OF TIMIKA

Indonesia is the world‘s fourth most populous nation, at 237 million people. Defining characteristics of the Indonesian archipelago are cultural, linguistic and religious diversity. These are especially evident in its easternmost province of Papua, as

29 Chapter 5: Study setting photographs of staff and study participants in this thesis attest. Known outside of Indonesia as West Papua, this western half of the island of New Guinea was called Irian Jaya during Sukarno‘s presidency, but was re-named Papua Province in 1999.116 The provincial capital is Jayapura on the north coast.

Figure 5.5: Mimika district map

(a) Districts of Papua Province (Mimika region = 12) Map from ref 123 (IBBS 2006 survey)

(b) Mimika region showing Timika location Map courtesy of Public Health / Malaria Control, Timika.

GEOGRAPHY

30 Chapter 5: Study setting

The Mimika district on the southern coast of Papua covers an area of 21 522 square- kilometres with 12 sub-districts and 85 villages. Timika, the largest town in Mimika district, is in equatorial lowlands rainforest, 3 degrees south of the equator (Figures 5.1 and 5.5). Climatic conditions are dramatic, with rainfall estimates around Timika and mountain foothills areas between 4 and 11m annually. This quantity of rain and the accompanying storms, floods and power blackouts, impact significantly on day-to-day life. Timika lacks a distinct monsoon season, although rainfall peaks in July to September and again in December.117 The lack of major seasonal variation in UV exposure removes what might pose a confounder in this vitamin D-related research, making Timika a uniquely ideal environment for the AVDAPT study.

When the rain clouds infrequently clear, the mountain ranges of the Lorenz National Park, and its visible mountaintop glaciation, are revealed to be unexpectedly close to Timika. The dominant peak Puncak Jaya (Cartenz Pyramid) reaches 4883 m, making it the highest mountain in Australasia / Oceania / South-East Asia region (see http://www.papuaweb.org/). The area is thought to have one of the highest rates of biodiversity in South-east Asia.118

HISTORICAL AND SOCIAL BACKGROUND

―Papua‘s rapid transformation from being one of the world‘s most isolated societies to becoming part, initially, of a large multi-ethnic colonial empire (Dutch East Indies), then an independent state (Indonesia), together with the disjunction between the colonial era international boundaries and the complex messy intermingling of Malay and Melanesian cultural worlds, have framed the conflicts of the past century.‖119

Indonesian independence from the Netherlands was declared by the first Indonesian president, Sukarno, in August 1945. Papua was however the subject of an ongoing dispute between Indonesia and the Netherlands, until the Dutch administration ultimately departed in 1962 and the territory was transferred to Indonesia via the interim authority of the United Nations. An ―Act of Free Choice‖ led to Papua‘s incorporation into Indonesia and the subsequent development of the Free Papua movement, setting the scene for Papua‘s contemporary socio-political climate, which continues to attract adjectives such as ‗restive‘ or ‗politically sensitive‘.116, 120-122

The last 30 years in Timika have been characterised by immense cultural shifts due to the discovery of mineral wealth in the Grasberg Mountain range. Mining of Ertzberg commenced in the 1960s, and expanded to the nearby highly lucrative Grasberg deposit

31 Chapter 5: Study setting in 1988.118 The mine is now one of the largest global producers of copper and gold, operated by PT Freeport Indonesia and 86% owned by American Freeport McMoRan Copper and Gold. Mining operations have most particularly impacted the Papuan tribes- people of the seven Mimika district tribes (Amunge, Kamoro, Dani, Damal, Nduga, Moni and Mee). Since 1995, 1% of funds from gross annual revenue from the Freeport- McMoRan mine have been allocated to ―community development‖ for these seven tribes. Use of these funds, managed by the organisation Lembaga Pengembangan Masyarakat Amungme dan Kamoro (LPMAK) (Amungme and Kamoro Community Development Institution; see http://www.lpmak.org), has included construction and operation of the hospital (RSMM), and construction of the MSHR-NIHRD research facility building.

Shifting population dynamics resulting from mining activities and government- sponsored transmigrants have resulted in growth in Timika‘s population from ~3000 in 1967 to 130 000 in 2004117 and an estimated 200 000 in 2009, representing the highest rate of growth of any urban community in Indonesia.118, 123 The number of Non-Papuans in Timika is now approximately equal to numbers of indigenous Papuans, although a 2006 survey found the estimated population breakdown throughout the whole province to be 69% Papuan / 31% Non-Papuan.124 This survey also illustrated a bottom-heavy population pyramid (80% aged less then 40; average age 30 years), distributed throughout lowland (70%) and highland (30%) districts, many of which are difficult to access. Farming and fishing were common occupations (43% of survey respondents), and unemployment was high (31%). Close to half of females surveyed (42%) and 29% of males had never attended school.124

Many hundreds of Papuan languages are spoken.125 The official language throughout the country since 1962 is Bahasa Indonesia, a standardised dialect of Malay. Knowledge of Bahasa Indonesia is high among Timika residents, but more limited among Papuans from isolated areas or who have received limited or no schooling. Of the Papuan participants in the AVDAPT study, approximately 10% require a Papuan-Indonesian translator. Identification with a religious belief is considered an integral component of Indonesian identify. Further illustrating aspects of population diversity in Papua Province, Papuans mostly follow various Christian denominations introduced by earlier missionaries, and Non-Papuans identify predominantly as Muslim followed by Protestant, Catholic, Buddhist or Hindu.

32 Chapter 5: Study setting

HEALTH STATUS AND HEALTH CARE

In addition to the community hospital described above (RSMM), which provides free health care to the Papuans of the seven tribes, a second Regional General hospital, Rumah Sakit Umum Daerah, opened in September 2008, where low fees are charged for health care provision.

Health status and health determinants (education, socio-economic status) are poor in Papua Province compared with Indonesia as a whole.126 The Mimika district is characterised by malaria endemicity, with multidrug-resistant strains of P. falciparum and P. vivax being highly prevalent. The first reports of chloroquine-resistant P.vivax were from Papua (and Papua New Guinea) in 1989, and these regions now have the highest rates of drug-resistant P. vivax in the world.117 The calculated incidence of malaria is 512 and 322 per 1000 population per year for P. falciparum and P. vivax respectively.117

Maternal mortality and infant mortality rates in Mimika district are exceptionally high, exceeding those reported elsewhere in Indonesia, at 1145 maternal deaths per 100 000 live births, and 68 infant deaths per 1000 live births.127 Less than 40% of pregnant women access antenatal care visits or deliver in hospital.127

The reported HIV prevalence in Papua Province in 2007 was 60.9 per 100 000 population, 15 times the Indonesian national average, and cumulative AIDS cases in Papua reported to March 2007 were second highest in the country after Jakarta.124 In a 2006 population survey,124 52% of people had heard of HIV / AIDS, but the percentage was considerable lower (26%) in those with no or limited education. Only 35% of people surveyed knew that condom use was protective. Public awareness campaigns to redress this are becoming evident (Figure 5.6). HIV / AIDS is deeply stigmatised in Papua. The Jakarta Post reported on November 22nd 2008:

―Amid protests from Papuans and NGOs, the Papua provincial legislative council is set to pass a bylaw on HIV/AIDS that includes a controversial article requiring certain people living with the disease to be implanted with a microchip…the microchips would only be implanted in people living with HIV/AIDS who were deemed to be…‗actively seeking sexual intercourse‘.‖128

33 Chapter 5: Study setting

Although the microchip plan was subsequently overturned, such approaches to public health illustrate the formidable barriers that exist to the provision of optimal HIV care in Papua.

Figure 5.6: HIV education banner near RSMM hospital Photo: Anna Ralph

5.4 CONDUCT OF MEDICAL RESEARCH IN PAPUA

Authorisation to conduct medical research in the province is complex and regulated, currently requiring approval from 2 Indonesian government ministries (Ministry of Health and Ministry of Research and Technology). Other required approvals are listed in Box 5.1.

5.5 CONCLUSION

Working in Papua Province, alongside the many local and national research partners to undertake this doctoral research, has offered a unique and highly valued opportunity to stay in an infrequently-visited tropical rainforest region of great geographical and historical interest, and to gain personal insights into a richly complex society.

34 Chapter 5: Study setting

Box 5.1: Approvals required for conducting medical research in Papua, Indonesia

Ethics committee approvals gained from:  National Institute of Health Research Development, Indonesia  Menzies School of Health Research, Darwin  Australian National University, Canberra

Training in Good Clinical Practice (GCP)  Required by study investigators (see Appendix 15.2)

Authorisation to conduct research and receive multi-entry permit  The Study protocol, curricula vitae and health certificates of study investigators, forms of identification and letters of support from the Indonesian Ministry of Health, were submitted to the Ministry of Research and Technology, Indonesia. Permission to conduct research and a letter of support to Police headquarters were subsequently secured 3 months later from the Ministry of Research and Technology.  After registration with Police Headquarters in Jakarta and Papua, a letter of introduction facilitating research in Papua province was issued.

Permission from Badam POM (National Food and Drug Administration, Indonesia) to import medications  Prior to study commencement, Badam POM required details of study medications (ingredients, manufacturer, packaging, batch numbers, expiry dates, numbers of tablets to be imported, whether manufactured according to Good Manufacturing Practice standards and Halal requirements) and rationale for medication importation.  Permission to import medications granted June 2008.

Permission to perform laboratory tests at designated reference laboratories in Australia  Materials Transfer Agreements were signed as described in Chapter 6 (Box 6.1)

Figure 5.7: Images of Timika Photos: Anna Ralph

(a) Timika markets (b) View from aeroplane flying south from Timika

35 Chapter 6: Methods

6 AVDAPT Study design and methodology

In this methods chapter, I address Aim 1 (Chapter 1) of designing and commencing the AVDAPT study. The study protocol is registered online at http://clinicaltrials.gov/show/NCT00677339 (see Appendix 15.3). Information and consent sheets (English versions) and data collection forms are reproduced in Appendices 15.4 and 15.5.

6.1 BACKGROUND

The theoretical rationale for the AVDAPT study is provided in the introductory chapters of this thesis. Preliminary research at the Timika field site upon which this trial builds includes a study in 2003-4 of 115 people with pulmonary TB, examining risk factors for impaired lung function.112, 114 Participants in this study had severe disease at diagnosis (as measured by sputum positivity, X-ray changes, lung function tests and functional measurements), low rates of HIV co-infection (4%) and MDR-TB (2%), high rates of malnutrition (41%) according to WHO criteria (BMI<18.5kg/m2), and a default rate of less than 10%. Thirty one percent of patients remained in the malnourished range at 2 months. Adjunctive therapy which could accelerate mycobacterial killing, modify immunological responses to TB and/or facilitate a more rapid return to normal weight in TB patients would be of great benefit in this population.

Preliminary results from trials of arginine supplementation in patients with malaria in Timika demonstrated substantial increases in exhaled NO: 6g supplementation led to a mean increase of 55%.56 The immunohistopathological changes associated with TB suggest that NOS2 expression in TB is at least as high as in malaria. Therefore, if the malaria result is replicated in the AVDAPT study, 6g of L-arginine would be expected to increase exhaled NO by at least 55%.

In unpublished data from Timika, median serum vitamin D concentration in 26 Non- Papuans was in the deficient range (<50nmol/L), at 45.5 nmol/L (range: 36-111).

36 Chapter 6: Methods

Although Papuans (n=52) had significantly higher serum vitamin D (median 64.5, range 40-95, p=0.0003, Mann Whitney U test), 8% were still deficient, and vitamin D levels are likely to be lower still in those with active TB.

6.2 HYPOTHESES

The study hypotheses and aims set forth in Chapter 1 are reiterated as follows:

i. L-arginine supplementation in pulmonary TB will be safe, will increase plasma arginine concentrations, will enhance pulmonary production of nitric oxide (NO) (a key arginine-dependent immunomodulator and downstream immune mediator of mycobacterial killing) and will improve the rapidity and magnitude of the microbiological and clinical response. Baseline pulmonary NO production will be elevated in pulmonary TB but inversely associated with disease severity. Both baseline and post-treatment increments in exhaled NO will be associated with rapidity and magnitude of the treatment response.

ii. Supplementation with vitamin D, the metabolite of which (1,25-dihydroxyvitamin

D3) has anti-mycobacterial activity, will be safe, will increase plasma vitamin D concentrations, and will improve the rapidity and magnitude of the treatment response in human PTB.

6.3 AIMS

i. To determine whether supplementation with L-arginine and / or vitamin D is safe and results in more rapid improvement in clinical, mycobacterial, immunological, radiological, physiological and functional measures of treatment outcome. Patients with pulmonary TB will be randomised to receive, in addition to standard TB therapy, adjunctive L-arginine and vitamin D, singly or in combination, or matching placebos. Serial measurements of plasma concentrations of L-arginine and vitamin D will be examined in relation to immunological responses (pulmonary NO production, T cell function and phenotype) and measures of treatment outcome [mycobacterial (sputum smear clearance and culture conversion), physiological (spirometry), clinical (symptoms and weight),

37 Chapter 6: Methods

radiological (chest Xray) and functional (six-minute walk test, modified St George Respiratory Questionnaire, SGRQ)].

ii. To determine whether exhaled NO (FENO) is inversely related to disease severity at presentation. Baseline and serial measures of NO production will be correlated with disease severity and the magnitude and rapidity of clinical response.

6.4 METHODS

SETTING

The study is being conducted at the Timika TB Clinic, Timika, Papua Province, Indonesia by the NIHRD-MSHR health research collaboration. Study personnel (Investigators, Consultants and members of the Data and Safety Monitoring Committee) are listed in Appendix 15.1.

STUDY POPULATION

Patients diagnosed with pulmonary TB in Timika who are referred for outpatient treatment to the Timika TB clinic are eligible for enrolment in the study. All patients with suspected TB are diagnosed according to the Indonesian National TB Protocol (NTP) guidelines.129 This comprises three sputum smears examined for acid fast bacilli (Ziehl Neelsen [ZN] stain on direct sputum specimens) in people with symptoms suggestive of pulmonary TB. Posteroanterior chest radiograph is also routine at TB diagnosis.

STUDY DESIGN OVERVIEW

The study has a factorial 2x2 study design (4 arms containing 2 interventions or their matching placebos). It is a randomised double-blind, placebo controlled trial of adjunctive oral L-arginine and / or vitamin D for 8 weeks, in addition to standard anti- tuberculous treatment, for people commencing treatment for newly diagnosed with pulmonary TB (Figure 6.1). Additional to the randomised controlled trial (RCT), other related studies being undertaken in Timika are also shown in the Figure and described below.

38 Chapter 6: Methods

Figure 6.1: Diagrammatic representation of AVDAPT trial and related studies Recruitment of healthy volunteers is described in Section 6.5. PK / PD measures are described in the section titled Evaluation of L-Arginine Pharmacokinetics and Pharmacodynamics. Abbreviations: RCT: randomised controlled trial; 6MWT: 6 minute walk test, SGRQ: St George‘s Respiratory Questionnaire, FENO: fractional exhaled nitric oxide

AVDAPT RCT Healthy volunteers

Eligible adults with smear positive pulmonary TB Healthy subjects commencing TB treatment enrolled to establish n = 444 normal reference ranges for 6MWT, SGRQ, FENO, blood assays n = 100

ACTIVE ARG ACTIVE ARG PLACEBO ARG PLACEBO ARG + + + + Pharmacokinetic and ACTIVE VITD PLACEBO VITD ACTIVE VITD PLACEBO VITD for 2 months for 2 months for 2 months for 2 months Pharmacodynamic n = 111 n = 111 n = 111 n = 111 measures

AVDAPT participants have blood and exhaled nitric Primary outcomes: oxide measured at repeat 1) Proportion culture negative at 1 month time points after study 2) Composite clinical outcome score at 2 months medication Total follow up: ingestion 6 months n = 60

39 Chapter 6: Methods

INCLUSION CRITERIA

People aged 15 years or older are eligible if they have sputum smear positive pulmonary TB; have never received more that one month of anti-TB treatment in the past (that is, only new cases of TB are included); agree to continue treatment in Timika for the full six month course of treatment; are not pregnant, and consent to enrol in the study.

EXCLUSION CRITERIA

Eligible participants who consent but are subsequently found to have hypercalcaemia (ionized calcium >1.32 mmol/L prior to randomisation) are excluded from the study.

ENROLMENT PROCEDURE AND INFORMED CONSENT

All patients diagnosed with smear positive pulmonary TB who are commencing TB treatment are seen by the Timika TB clinic doctor (usually Dr Andri Wiguna). A log of all such patients commencing treatment, their eligibility for the study, and reasons if any for non-enrolment, is maintained by the TB clinic doctor and research assistant (RA) staff. All apparently eligible patients are referred to one of the three AVDAPT RAs, whose workspace is in the adjoining room (see diagrammatic summary of enrolment procedure, Figure 6.3). The RAs (2 nurses, Govert Waramori and Gertruida Bellatrix and 2 doctors, Gysje Pontororing and Enny Kenangalem) are employed by or seconded to the NIHRD-MSHR Health Research Collaboration, and have received specific training and supervision in all aspects of the study protocol. After being referred a potential study participant, they conduct a thorough eligibility assessment, and seek informed consent from eligible patients.

To obtain informed consent, patients receive a detailed verbal and written explanation regarding the reason for the study and what it involves, and regarding the voluntary nature of involvement in the study. The written information sheet is provided in Bahasa Indonesia (English version: Appendix 15.4). Verbal explanations are provided using a Papuan interpreter where required. A pictorial aid (Figure 6.2) is used to assist the consent process. Written consent is obtained using the signature or thumbprint of the participant, and of the parent or guardian for participants aged between 15 and 18.

40 Chapter 6: Methods

Figure 6.2: Consent booklet

Diskusi dan kuisioner Penelitian TBC “ADAPT” Informasi

Litbangkes-MSHR Health Research Collaboration ADAPT study, no 07/40

Discussions and completing Information about TB ‘AVDAPT ’ study questionnaires

Ambil darah Pemeriksaan darah

Blood tests include calcium Take blood at week 0, 1, 2, 4, 8, 24 measurement, and HIV testing if specific additional consent is given

Tes fungsi paru „nitric oxide‟ Tes fungsi paru „volume‟

napas napas tiup tiup Exhaled nitric oxide is measured at Pulmonary function tests are week 0, 1, 2, 4, 8, 24 performed at week 0, 4, 8 and 24

41 Chapter 6: Methods

Tes Jalan 6 menit Foto dada

Chest x-ray is performed at week 0 Six minute walk test is performed at (prior to enrolment in study) and week 0, 4 8 and 24 again at week 8 and 24

Minum obat TBC program Minum obat penelitian

Minggu ke 1 dan minggu ke 5

6 tiap hari selama 2 bulan

Standard TB medications are taken as Study medications (L-arginine &/or fixed-dose combination (number of vitamin D &/or matching placebos) tablets determined by body weight) are taken for the first 8 weeks Follow-up setiap minggu selama 8 minggu, kemudian minggu ke 12, 16, 20, 24

Study participants must attend at least weekly for the first 8 weeks, then monthly during the continuation phase of treatment

Only patients who understand the study and sign the consent form are enrolled. It is explicitly stated that involvement is voluntary, participants are able to say no, and if they do initially consent, they can leave the study at any time without prejudicing their treatment in any way. On withdrawing from the study, the researchers provide ongoing

42 Chapter 6: Methods positive encouragement and support for the participants to continue her/his TB treatment as usual, according to the NTP.

Consenting participants have blood drawn for point-of-care calcium measurement (ionised calcium using iSTAT® device). Hypercalcaemic patients (confirmed on 2 tests) are excluded from the study as per the exclusion criteria, and referred to the TB clinic doctor for ongoing management. Normocalcaemic patients are retained in the study, and are assigned to an unidentified (blinded) study arm, by receiving a sequential, unique study identification number stratified by ethnicity, printed on an opaque envelope containing the study medications (see also section below, ‗Randomisation and Blinding‘). Baseline evaluations outlined below are performed, and the first supervised dose of TB medications and study drugs is administered. All study participants are requested to attend the clinic daily or weekly during the intensive phase, and monthly during the continuation phase to receive their anti-TB medications, study medications, routine examinations and the study examinations as outlined below. If patients do not attend daily, a family member is requested to act as the medication supervisor on non-clinic days.

TB TREATMENT REGIMEN

The standard TB treatment regimen used in Timika is rifampicin (10mg/kg, max 600mg), isoniazid (10mg/kg, max 300mg), ethambutol (15mg/kg) and pyrazinamide (25mg/kg, max 2g) 7 times weekly during the intensive phase (2 months, or 3 months if smear status still positive at 2 months), then rifampicin (15mg/kg, max 600mg) and isoniazid (15mg/kg, max 600mg) 3 times weekly (continuation phase, 4 months). These antibiotics are compounded in fixed dose combinations (e.g. one intensive phase tablet contains rifampicin 150mg, isoniazid 75mg, ethambutol 275mg and pyrazinamide 400mg), allowing close approximations of the recommended doses per kg to be achieved by following standard guidelines on number of tablets to be taken per day according to weight.

STUDY MEDICATION REGIMENS

Study medications, administered separately from the pre-packaged TB medications, are allocated to the 4 study arms in the following way:

43 Chapter 6: Methods

 Group 1: supplementary L-arginine 6g daily for eight weeks plus vitamin D3 (cholecalciferol) 50 000 IU (1250mcg) once at baseline and once on day 28 (0 and 4 weeks);  Group 2: supplementary L-arginine 6g daily for eight weeks plus placebo vitamin

D3 once at baseline and once on day 28 (0 and 4 weeks);

 Group 3: placebo L-arginine daily for eight weeks plus vitamin D3 (cholecalciferol) 50 000 IU (1250mcg) once at baseline and once on day 28 (0 and 4 weeks);

 Group 4: placebo L-arginine daily for eight weeks plus placebo vitamin D3 once at baseline and once on day 28 (0 and 4 weeks).

Figure 6.3: Enrolment procedure

1. Diagnosis Patient diagnosed with smear positive pulmonary TB (clinical suspicion of TB and at least 2 AFB positive sputum specimens). TB doctor refers to AVDAPT research assistant (RA).

2. Eligibility screening: RA assesses whether referred patients fulfill inclusion criteria.

3. Consent process: Written, pictorial and verbal information provided about the study, with Papuan interpreter if necessary. Patient indicates consent by signing or providing thumbprint.

4. Blinded assignment to study arm / Allocation of study code: A unique, sequential study code, stratified by ethnicity, is allocated. The code comprises the letters TAD (TB, Arginine, Vitamin D) and a 4 digit number, starting with 1 for Papuans or 2 for Non-Papuans.

5. Allocation of medication pack: The study medications for the full 8 week course are allocated. They are contained in zip-lock bags inside a large, sealed, opaque envelope, identified with the study code.

44 Chapter 6: Methods

L-arginine hydrochloride formulation and dose

L-arginine hydrochloride tablets (Argimax®) used in the AVDAPT study are manufactured by Hankintatukku Oy, Finland according to Good Manufacturing Practice (GMP) standards, in 1g tablets packaged in blister packs (Certificate of Analysis, Appendix 15.7). Tablets are large, oblong, uncoated pills. Matching placebo tablets containing microcrystalline cellulose, maize starch and magnesium stearate, have been purposely manufactured for the AVDAPT study by the same manufacturer with identical appearance, colour, size, taste and packaging as active Argimax® tablets.

The choice of 6g daily is based on clinical experience with this dose (see Chapter 2), safety profile at this and higher doses, and the preference for a once daily regimen for TB patients to avoid dosing errors (as other TB medications are dosed once daily), and to optimise adherence.

Vitamin D3 (Cholecalciferol) formulation and dose

Cholecalciferol (Calciferol Strong®) is manufactured by API Consumer Brands, New Zealand, in 50 000 IU (= 1250 mcg) doses, packages in sterile bottles. They are manufactured according to GMP (Certificate of Analysis, Appendix 15.8), and glycerine used in the tablets is manufactured according to Halal requirements, as required for use in Indonesia. Tablets are small, round, coated white tablets, easily distinguishable from the L-arginine / L-arginine placebo and TB pills. A batch of placebo tablets identical in every way to active cholecalciferol tablets has also been specifically manufactured for this study by API Consumer Brands.

The dose of 50 000 IU once monthly for 2 months has been selected to provide adequate replacement for those who are vitamin D deficient, and avoid toxicity in those replete at baseline (see Chapter 2).

PREPARATION OF STUDY MEDICATION PACKS

Study medications are pre-labelled and packaged by an unblinded MSHR researcher (Ms Kim Piera, who is not involved in any clinical aspects of the study), according to the random allocation sequence.

45 Chapter 6: Methods

 Argimax® / placebo blister packs Each blister pack is labelled with a sticker containing all information required according to Good Clinical Practice (GCP) regulations (study participant‘s code, space for investigator to write name and date, drug information, study information. See sample sticker, Figure 6.4). Enough tablets for one week (42 tablets, 6 per day) are packaged into each of 8 zip-lock bags, labelled week 1-8.  Calciferol Strong® / placebo tablets One tablet is placed in a small zip-lock bag labelled with a sticker, similar to Figure 6.4 but stating that the medication is vitamin D or vitamin D placebo. One labelled, individually packaged tablet is placed in the week 1 study medication bag, and one in the week 5 study medication bag.

The clearly marked Study Drug Packs are stored in an air-conditioned room at the Timika TB Clinic until being assigned to a study participant, at which stage they are transferred to a refrigerator.

Figure 6.4: Sample stickers applied to study medications

(a) Indonesian (b) English LITBANGKES-MSHR PENELITIAN AVDAPT LITBANGKES-MSHR AVDAPT STUDY

KODE STUDI: TAD-1001 TANGGAL: ___|___| 20____ STUDY CODE: TAD-1001 DATE: ___|___| 20____

Nama______Name______

DOSE minum obat 6x tablet bersamaan dengan makanan setiap DOSE take 6x tablets daily with food. These tablets contain haro. Tablet ini bersini arginine 1g atau placebo. Pelajaran no. arginine 1g or placebo. Study no. 0740. 0740. Emergensi kontak: Dr Daniel 0811 491 699 MASA Emergency contact: Dr Daniel 0811 491 699 BERLAKU: Maret 2011 MEDICATION EXPIRY DATE: March 2011

PARTICIPANT EVALUATIONS

Height, weight and BMI Standing height is measured without shoes to the nearest centimetre using a stadiometer. Weight in kilograms is measured without shoes to the nearest 0.1kg using an adult weight(kg) balance. BMI is calculated in the standard way, i.e: BMI = height(m)2

Questionnaires An enrolment questionnaire is completed once per patient, and a regular questionnaire (Appendix 15.5) is completed weekly during weeks 0 to 8, then monthly until treatment completion. All data collection forms and other documents are labelled with the participant‘s study code. The regular questionnaire assesses medication adherence,

46 Chapter 6: Methods results of point-of-care blood tests (haemoglobin and calcium), clinical measures (lung function, exhaled nitric oxide, 6 minute walk test, lung function tests), symptoms including cough (mild, moderate, severe or very severe, as subjectively judged by the patient), and any adverse events. Any medications additional to TB and study medications are documented, and whether HIV counselling and testing has been performed is recorded.

Chest radiograph

A posteroanterior chest x-ray is performed as per standard clinical protocols at the TB clinic prior to patient enrolment in the study, and at week 8 and 24. Chest x-rays are reported by myself, using the method described in Chapter 9. Study arm allocation concealment is maintained at all times.

Pulmonary Function

Pulmonary function is measured using a handheld spirometer (ML3535C MicroLoopTM, Micro Medical, UK), which is calibrated daily using a standard 3L syringe (Figure 6.5). Individual-use filtered one-way mouthpieces suitable for use in smear-positive pulmonary TB patients are used (Sure-Gard®). Each patient performs at least three maximum effort expirations until volumes vary less than 200mls, with the highest values for FVC and FEV1 used as measures of lung function. RAs receive training and supervision in use of the device initially, and on a regular periodic basis thereafter. Measurements are performed outside (in the area adjacent to the TB clinic).

Exhaled nitric oxide

Fractional exhaled nitric oxide (FENO), described in Chapter 3, is used to evaluate pulmonary NO production, using a portable online device, the NiOX MINO® (Aerocrine, Sweden). This device is well validated,102, 103 easy to use, does not require calibration, and employs single-use disposable, filtered mouthpieces. The process does not pose any infection control risks. FENO is measured prior to spirometry and the 6 minute walk test, and subjects are requested to avoid eating or vigorously exercising for at least one hour before testing. Measurements comply with ATS 2005 Guidelines.100 To assess for sensor drift or inter-sensor variation, quality control (QC) measures

47 Chapter 6: Methods comparing results from biological controls (RA staff) are compared on a weekly basis between the NiOX MINO® and a ‗gold standard‘ non-portable NiOX FLEX®.

Figure 6.5: Spirometer calibration using 3 litre syringe Research assistant Bapak Govert Waramori at the Timika TB clinic

Modified St George Respiratory Questionnaire (SGRQ)

The SGRQ is a health-related quality of life instrument formulated for use in chronic obstructive pulmonary disease.130 The English version is supplied in Appendix 15.6. An overall score (range 0-100) and individual domain scores for symptoms, activity and impacts on daily life are obtained. Low scores indicate better lung health.

An Indonesian translation of the SGRQ was provided by the creators of the questionnaire (Professor P.W. Jones, St George‘s Hospital Medical School, London, United Kingdom), and slightly modified to reflect local conditions in Timika. Results of this modified SGRQ have previously been reported in TB patients in Timika.114 Standard recommendations are that patients complete the SGRQ without assistance; however given literacy rates at the field research site, the trained RAs administer the questionnaire in an impartial manner, and document participant responses.

48 Chapter 6: Methods

6 minute walk test (6MWT)

The 6 minute walk test is a practical, simple test measuring the distance a patient can quickly walk on a flat, hard surface (ideally 100 ft i.e. 30.5m in length) in a period of 6 minutes. It is widely used as a measure of functional exercise capacity.131 Multiplying the 6 minute walk distance by body weight to generate weight ∙ walk distance has been shown in some studies to correlate more closely with energy expenditure and lung function,132, 133 and hence 6 minute weight ∙ walk distance is also calculated.

6MWT is assessed in study participants on a 20m open air path beside the TB clinic. Conduct of the 6 minute walk test adheres to ATS guidelines, with the exception that a minimum 30m track length is preferred, although use of 20m tracks in other studies has not resulted in any significant difference in distance walked.131 The 20m track length is the maximum available at the study site, is consistent throughout the study, and healthy volunteers are tested on the same track to establish a comparative normal range (see below).

Sputum collection and processing

Prior to enrolment in the study, smear positive TB is diagnosed at the TB clinic (or at another community clinic or hospital outpatient centre, with subsequent referral to the

TB clinic), on the basis of ≥2 sputum specimens positive acid fast bacilli (AFB), in accordance with NTP standard protocols.

Additionally on the day of entry into the study, a further sputum specimen is obtained, results of which are recorded as the study participant‘s baseline AFB grade and culture result. Follow up samples are collected weekly up to week 8 for smear microscopy at the on-site laboratory. Mycobacterial culture facilities are unavailable throughout Papua Province. At weeks 0, 4 and 8, samples are therefore additionally dispatched for culture and drug susceptibility testing (DST) to the University of Indonesia Faculty of Microbiology (FKUI), Jakarta (see Table 6.1)

At the on-site laboratory at the Timika health clinic (Puskesmas) complex, a direct slide is prepared under an approved biological safety hood, and examined under a light microscope using standard ZN stain. WHO guidelines for grading smear results are 49 Chapter 6: Methods adhered to.134 Microscope slides labelled with the study code and date are stored at the NIHRD-MSHR Research Building in Timika for later quality control testing.

Samples collected at weeks 0, 4 and 8 are divided for use (in some instances 2 simultaneously-collected samples have been used); after slide preparation at the local laboratory, the sample is stored (batched for up to 2 weeks) in a 4°C refrigerator, then packaged by the RA (Govert Waramori) who is trained in International Air Transport Association regulations, and dispatched unrefrigerated by courier to FKUI. Unrefrigerated transportation of fresh sputum samples has previously been shown by MSHR researchers to be associated with good MTB recovery rates.113 At FKUI, slides prepared directly and from concentrated sputum are examined using the ZN method as above. Concentration is achieved by adding 4% sodium hydroxide, repeatedly homogenising the mixture using a vortex, adding phosphate-buffered saline, then obtaining the concentrated sample by centrifugation.

Culture is performed at FKUI for each specimen using both liquid and solid media. Sputum specimens are decontaminated using 2% sodium hydroxide and 0.5% N-acetyl- cysteine for 25 minutes, then neutralised to pH 7, concentrated by centrifugation (3,000xg for 15 min) and inoculated into a single Mycobacterium Growth Indicator Tube 960 (MGIT; Becton Dickinson Microbiology Systems, Sparks, Md) and two tubes of Lowenstein-Jensen media. DST is performed on week 0 specimens, using the BACTEC MGIT 960 TB system, using the recommended critical concentrations for the following antibiotics: isoniazid (0.1), rifampicin (1.0 mg/ml), ethambutol (5.0 mg/ml), streptomycin (1.0 mg/ml). In instances of multi-drug resistance, second line testing to ofloxacin, amikacin and kanamycin is performed.

Confirmation of MDR-TB or rifampicin monoresistance is achieved using the Hain GenoType® MTBDRplus assay. This assay permits the molecular genetic identification of MTB complex, and detects the most common mutations occurring within the rpoB gene that confer rifampicin resistance, and the mutations conferring low-level isoniazid resistance (inhA gene) and high-level isoniazid resistance (katG gene). DNA is extracted from cultured MTB isolates, followed by multiplex amplification and reverse hybridization steps as per the manufacturers instructions.135 Results are provided on paper strips with visible lines indicating the presence of wild type or mutation genes.

50 Chapter 6: Methods

Figure 6.6: Mycobacterial growth indicator tubes (MGIT) MGIT inoculated with MTB containing (L-R): ethambutol, rifampicin, isoniazid, streptomycin, no antibiotic (control tube), for determination of drug susceptibility (Photo: Anna Ralph, taken at FKUI, Jakarta).

FOLLOW UP Study participants are re-evaluated during the 6 month follow up period according to the schedule in Table 6.1 (tests are performed when indicated by a tick). Default tracing, including telephoning or text messaging for participants with telephones, visiting patient‘s homes and contacting neighbours or relatives, is conducted rigorously to attempt to trace participants who miss scheduled clinic appointments.

Table 6.1: Follow-up schedule for AVDAPT study participants

Week of TB treatment Test 0 1 2 3 4 5 6 7 8 12 16 20 24† Weight              Symptoms              Sputum microscopy*           Sputum culture    Chest x-ray    Spirometry     Exhaled nitric oxide       6 minute walk test     SGRQ    

*Sputum microscopy is repeated weekly if still positive at week 8, until negative. †If the patient has not completed treatment by week 24, a further follow up as for week 24 is repeated at treatment completion.

51 Chapter 6: Methods

BLOOD COLLECTION Blood is collected from participants at weeks 0, 2, 4, 8 and 24. Assays, tube types and volumes are indicated in Table 6.2.

Table 6.2: Blood collection schedule for AVDAPT study participants Week Blood test Tube 0 2 4 8 24 3ml HIV*  EDTA† 3ml Parathyroid hormone   EDTA 25(OH)D , 1,25(OH) D and amino 6mL 3 3 3      acids LiHep‡ 6mL Calcium and haemoglobin     LiHep Peripheral blood mononuclear cells 10mL     and Interferon gamma release assay LiHep 2mL Back-up serum for storage   LiHep Total blood volume 19ml 18ml 19ml 6ml 18ml

*HIV is ideally performed at week 0 but may be deferred depending on pre-test counselling availability. †EDTA = ethylene diamine tetra-acetic acid blood tube; ‡LiHep = Lithium heparin blood tube

Methods for on-site blood processing (i.e. in Timika):  HIV: Standard Diagnostic (SD)® point-of-care HIV test on plasma or whole blood. If positive, confirmation is performed using Abbott Determine® and Oncoprobe® point-of-care tests. In instances where SD kits have been unavailable, FOKUS® test kits have been used. All point-of-care tests have high sensitivity and specificity (e.g. Standard Diagnostic (SD)®: sensitivity 100%, specificity 99.8%).136 Written, informed consent is obtained as part of routine TB using the Indonesian national standard ‗Voluntary Counselling and Testing for HIV‘ form, by either the TB clinic doctor, AVDAPT RA (doctor), or on-site sexual health clinic doctor or trained counsellor.  Ionised calcium and haemoglobin: point-of-care iSTAT cartridges and iSTAT analyser. New batches of iSTAT cartridges are tested using standard QC vials.  Blood film, cell count and differential: A blood film is prepared for manual white cell count and differential  Interferon gamma release assay: Interferon- production in response to stimulus with mitogens, bacterial LPS and PPD will be determined using ex vivo Quantiferon® assays. These will performed when blood is available from all required time points (weeks 0, 2, 4, 24), for testing in parallel.

52 Chapter 6: Methods

Figure 6.7: Sample processing at the NIHRD-MSHR research facility Bapak Prayoga (Laboratory technician) performing blood count and differential

Methods for storage of blood prior to processing in Australia:  Peripheral blood mononuclear cell (PBMC) separation according to MSHR protocols. Samples are stored at -70 (plasma) or in liquid nitrogen (PBMCs in foetal calf serum/DMSO) in the Timika research laboratory, for transportation by dry shipper at -70°C to MSHR.

Methods for blood processing in Australia: For reasons described in Box 6.1, samples are currently in storage and are yet to be transported to Australia for analysis. Planned analysis methods are as follows:

 25(OH)D3: isotope-dilution liquid chromatography-tandem mass spectrometry (LC- MS) method, RMIT Drug Discovery Technologies Pty Ltd, Victoria

 1,25(OH) 2D3: IDS Radioimmunoassay, IMVS  Parathyroid hormone (PTH): IMMULITE 1000 Intact PTH®, IMVS  Quantitative L-arginine, citrulline and ornithine: High performance liquid chromatography, MSHR  White cell studies (PBMC phenotype, CD3 zeta expression in activated and naïve CD4 and CD8 T cells): will be determined using fluorescence activated cell sorting (FACS). Phenotypic analyses will be run in conjunction with functional assays to measure cell proliferation and cytokine secretion. Cell proliferation will be detected by using carboxyfluorescein diacetate succinimidyl ester (CFSE).

53 Chapter 6: Methods

EVALUATION OF L-ARGININE PHARMACOKINETICS AND PHARMACODYNAMICS

In a sub-set of 60 sequential patients who give additional verbal consent to wait at the clinic for up to 4 hours and have repeated venesection, the 18mL blood collected at week 2 is obtained in three divided aliquots before and up to 4 hours after dosing with study medications, accompanied by repeated FENO measures over the same time period, in order to:  Determine the pharamacokinetic profile of orally-administered L-arginine 6g by evaluating time to maximum plasma concentration (Tmax) and plasma half life, and  Determine the pharmacodynamic profile of orally-administered L-arginine 6g by

evaluating time to peak and normalization of FENO.

The times of blood draws, FENO measurements and tablet dosing are accurately recorded on a data collection sheet (Appendix 15.5).

54 Chapter 6: Methods

Box 6.1: Explanation regarding planned off-shore laboratory analyses

Prior to commencement of the AVDAPT study, Materials Transfer Agreements were signed by the Indonesian and Australian directors of the NIHRD-MSHR collaboration, and directors of the respective laboratories where assays were to be performed. These agreements included that the originating country (Indonesia) would be acknowledged in any publications arising from sample usage, and that there would be no further distribution of specimens outside the laboratories specified in the agreement.

However due to a subsequent directive from Indonesia‟s 2004-09 Minister of Health, Dr Siti Fadilah Supari, biological samples collected from AVDAPT study participants were prevented from being exported for analysis outside Indonesia, coinciding with the date of study commencement (June 12th 2008). Arising from grievances over usage of Indonesian H5N1 virus samples which had been shared with WHO in 2005, Indonesia claimed that biological samples are its “sovereign property and do not constitute resources that other countries or the international community can access and use”.137 Ramifications have included threats to close U.S. Naval Medical Research Unit 2 (NAMRU-2), a public health laboratory in Jakarta, and describing NAMRU-2 scientists as “profiteering off its „sovereign‟ viruses to manufacture the H5N1 bird flu in an alleged biological warfare scheme.”138 Dr Supari‟s concern that the US might be involved in a conspiracy to use the bird flu virus to develop biological weapons is also expressed in her book (endorsed by Indonesian President Yudhoyono) entitled It's Time for the World to Change: Divine Hands behind Bird Flu (Saatnya Dunia Berubah: Tangan Tuhan dibalik Flu Burung).139 Dr Supari and colleagues at NIHRD articulated the perceived unethical outcomes of sharing of biological samples in an article published in the Annals of the Academy of Medicine Singapore in 2008 as follows:140

“Disease affected countries, which are usually developing countries, provide information and share biological specimens/virus with the WHO system; then pharmaceutical industries of developed countries obtain free access to this information and specimens, produce and patent the products (diagnostics, vaccines, therapeutics or other technologies), and sell them back to the developing countries at unaffordable prices.”140

Indonesia‟s stance on withholding H5N1 virus samples from WHO and potential vaccine developers eventually led to a World Health Assembly resolution being adopted on May 23, 2007, stipulating that “transparent, fair and equitable sharing of the benefits arising from the generation of information, diagnostics, medicines, vaccines and other technologies”141 would be promoted. Indonesia thereafter resumed sharing influenza virus samples, but has remained protective of biological samples in general.

Ramifications for the AVDAPT study are that to date, those assays that were approved by the Indonesian Ministry of Health Ethics Committee (and the NIHRD Ministry of Health Materials Transfer processes) for processing in Australia are yet to be performed. The study protocol has been adapted where possible to accommodate the regulation. Rather than undertaking sputum culture and DST at the WHO supranational reference laboratory (IMVS) in Australia as planned, sputum is now processed at FKUI, Indonesia (this laboratory was fortuitously accredited for mycobacteriological culture by WHO in May 2008). The capability for performing the blood assays designated for processing in Australia, to Good Laboratory Practice recommended standards, is not currently possible in Indonesia. Therefore samples are stored at -70 degrees as described, for transfer for processing when this regulation has been amended.

55 Chapter 6: Methods

TRIAL OUTCOME MEASURES

A number of outcome measures will be evaluated. The primary outcome measures are:

 Proportion of pulmonary TB patients who are culture negative at 1 month.  Difference in improvement in composite clinical endpoint comprising weight, cough

clearance and FEV1 at 2 months.

The secondary outcome measures are:

 Safety.

 Difference in improvement in percent predicted FEV1 at 2 and 6 months.

 Change in plasma L-arginine and 25(OH)D3 concentrations.  Weight gain.  Cough clearance.  Sputum smear conversion time.  Functional improvement including Six minute walk test and quality of life assessment using modified St George‘s Respiratory Questionnaire.  Immunological improvement (exhaled NO, T cell CD3ζ expression and T cell function).  Radiological improvement (percentage lung involvement on CXR at 2 months).  Percentage obstructive and/or restrictive lung disease at 6 months.  Death, clinical failure and default independently, and ‗death or clinical failure or default‘.  Primary end points stratified by HIV status.  Primary end points stratified by baseline vitamin D and L-arginine status.  Primary end points stratified by ethnicity (Papuan and non-Papuan patients).

Use of mycobacterial culture as a primary outcome measure overcomes the potential problem of continued smear positivity of non-viable organisms. Although various outcomes have been selected by TB researchers as surrogates for 6-month treatment outcome and 2-year relapse, including 2-month sputum smear (a predictor of treatment failure142-144) or 2-month sputum culture conversion,145, 146 culture at one month has been selected in this study in order to capture any early microbiological effect of adjunctive therapy.

56 Chapter 6: Methods

The composite clinical outcome score, assigning weights to percentage improvement in weight gain and FEV1 improvement, and to respiratory symptoms (cough, sputum production and haemoptysis), is described in detail in Chapter 10.

STATISTICAL ANALYSES

Sample size

Because of the potential for interaction when 2 interventions are trialled in a single study, it is recommended that sample size in a 2x2 factorial study be calculated by estimating the size of any potential interaction, then using the Fleiss equation.147 Making a conservative assumption of sub-additive interaction between L-arginine and vitamin D (interaction coefficient of 0.5), then at a level of significance of 5%, a sample size of 444 (111 participants in each arm) was found using this method to be appropriate, providing 82% power to demonstrate that each treatment results in a 20% reduction in the proportion culture positive at one month (from 60% to 40%), assuming loss to follow up of 10%. As there have been no trials of co-administration of vitamin D and L-arginine, the characteristics of potential immunological interactions can be extrapolated only from in vitro macrophage studies, in which findings conflict: vitamin D was found to inhibit NOS2 expression in one study,148 yet upregulate it in another.149 The latter study also found the suppressive effect of vitamin D on MTB replication to be partially impaired if NO formation was inhibited. Including a sub-additive interaction term in the sample size calculation provides a margin a caution; if there is minimal or no interaction between the two interventions, power will be greater. A factorial design rather than a three-arm study allows investigation of whether it is safe and effective to co-administer the two interventions. Acknowledging the possibility of statistical interaction between the terms, the a priori plan is to analyse primary and secondary outcomes according to subgroups receiving single interventions, as well as according to the overall factorial model.147

Mean time to culture negativity in mostly drug sensitive TB has been reported to be 32 days (New York)150 to 57 days (Turkey).151 Based on prior observations of high bacillary burden and extent of cavitary disease in Timika TB patients, a minimum of 60% of Timika patients are estimated to be culture positive at 1 month. Power will be increased if this proportion is higher. This sample size will also be powered to detect a

57 Chapter 6: Methods

9% absolute difference in the mean improvement in percent predicted FEV1, a component of the composite endpoint, at 6 months. A previous study in Timika found mean baseline percent predicted FEV1 to be 63.6% predicted (SD 22) rising to 77.9% predicted (SD 23) at 6 months.114

Outcome Analysis

Data is double-entered and validated using EpiData 3.02 software (EpiData Association, Odense, Denmark) and analysis will be performed using Stata software (version 10.1; Statacorp) and GraphPad (version 5.0; Prism). An a priori analytical plan will be followed for evaluation of trial outcomes. The two primary outcomes (sputum culture positivity at one month and composite clinical outcome score at two months) will be determined by an independent observer, blinded to the intervention arm, and compared on an intention to treat basis. Descriptive statistics will be presented for all outcomes for each of the 4 factorial cells in the trial (L-arginine, vitamin D, L-arginine plus vitamin D, and placebo). We will assess the main effects of the 2 interventions (L-arginine versus no L-arginine and vitamin D versus no vitamin D) by estimating the average effect on the primary endpoints. In the primary analysis, results will be presented as adjusted odds ratio (and 95% confidence intervals [CI]) and mean difference in change in clinical score (and 95% CI). Regression models (logistic regression or multiple regression/analysis of variance) will be used to adjust for co-interventions, ethnicity and HIV status. Any interaction between the interventions will also be described (with its 95% CI). Secondary endpoints listed above will be assessed on a per protocol population. Time to smear negativity will be assessed by survival analysis using the

Kaplan Meier method. Changes in plasma L-arginine, 25(OH)D3 and exhaled NO will be compared between treatment arms, and primary and secondary outcomes will be related to baseline pulmonary NO production/L-arginine/25(OH)D3 and clinical, radiological, microbiological and functional measures of disease severity at enrolment.

ASSESSMENT OF SAFETY

Adverse events

As per GCP guidelines, adverse events (AE) are defined as any new signs or symptoms reported or observed during the course of the study which were not evident at baseline. Serious adverse events (SAE) are defined as death, life-threatening illness or 58 Chapter 6: Methods hospitalisation. Adverse events may be unrelated to the study, may be a consequence of TB per se or TB treatment, or may be a consequence of the study interventions. Adverse event reporting guidelines are reproduced in Appendix 15.9.

SAE are notified immediately to study investigators (usually myself) and a case summary is prepared the form supplied in Appendix 15.10. I then submit this report to the clinician members of the Data and Safety Monitoring Committee, who determine the likelihood of relatedness of the SAE to the study medications (unrelated, unlikely, possible, probable or definite, as per GCP definitions shown in Appendix 15.9). Non- serious Adverse Events (AE) are managed by the study investigators, RAs and the local TB clinic doctor as required, and are reported to the Data and Safety Monitoring Committee at the time of interim analyses.

AE from oral L-arginine are uncommon.52 Any potential adverse effects of L-arginine are carefully monitored. Use of up to 21g daily was not associated with adverse events in two studies.152, 153 Oral L-arginine 7g three times daily for 4 weeks was associated with diarrhoea in 3 of 27 patients, but this resolved on dose reduction to 7g twice daily.154 Minor gastrointestinal discomfort has been reported in up to 3% of patients.52, 155 Adverse events potentially attributable to L-arginine in this study include diarrhoea, bloating, nausea, vomiting, mild to moderate abdominal pain.

Although much less common than in other granulomatous diseases, vitamin D supplementation in TB has the potential for hypercalcaemia (iCa2+ > 1.32 mmol/L) from 156 extra-renal 1,25(OH)2D3 production. Based on clinical experience with vitamin D supplementation (Professor John Eisman, listed study consultant as per Appendix 15.1), and review of the literature,80, 87, 157-160 the risk of symptomatic hypercalcaemia as a complication of vitamin D supplementation at the dose used in this study in patients with pulmonary TB is anticipated to be very low. Ionised calcium is measured, using point-of-care iSTAT testing providing immediate results, at 0, 1, 2, 4 and 8 weeks. In the event of hypercalcaemia developing, the literature indicates that the most likely time of onset would be 2-4 weeks post commencement of anti-TB therapy. Potential symptoms of hypercalcaemia, including nausea, vomiting, abdominal pain, constipation, anorexia, fatigue, irritability, confusion, somnolence, coma and dehydration, are checked for on a weekly basis. Study participants with hypercalcaemia are managed as

59 Chapter 6: Methods per the flow chart (Appendix 15.11), including having vitamin D / vitamin D placebo withheld.

Data and Safety Monitoring Committee

The Data and Safety Monitoring Committee has Indonesian and Australian membership comprising experienced researchers and health practitioners independent of the research team: Dr Paulus Sugiarto, Clinical Director, RSMM Hospital, Timika (chair of the committee), Dr Louise Maple-Brown, Specialist Physician and Endocrinologist, MSHR, Dr Joseph McDonnell, Statistician, MSHR and Ibu Hapsari, Statistician, NIHRD. The clinicians (Dr Sugiarto and Dr Maple-Brown) provide reports on any SAEs and advise whether any action to address the SAE is required by the study investigators, and whether they deem the study to be safe to continue. Interim safety analyses are conducted by the statistician members of the committee (Dr McDonnell and Ibu Hapsari) at time points as indicated below. The Data and Safety Monitoring Committee has the power to immediately discontinue the study if severe adverse events attributable to the study medications occur.

Stopping rules

Interim safety analyses will be performed after 25% and 50% of participants have been followed to 2 months (first analysis complete, see Chapter 10), or as requested by the Data and Safety Monitoring Committee in the event of SAE related to study drugs. The committee will be able to recommend that enrolment into the study ceases if they believe there is evidence that individuals are being harmed through their participation in the study. Early termination of the study (or of one of the study arms) may also be recommended if there is strong evidence of benefit or harm associated with either of the interventions at the interim analysis (p<0.01) or if there is strong evidence that further enrolment will not provide any useful information.

RANDOMISATION AND BLINDING

A random allocation sequence assigning participants to an intervention group was computer-generated (Stata 9.2) by an independent statistician and will remain concealed from all investigators throughout the study. Block randomisation was used to maintain

60 Chapter 6: Methods similar numbers of participants in the intervention and control groups and to minimise the potential influence of time of enrolment. Allocation is stratified by place of TB enrolment (hospital or clinic, but enrolments have only taken place at one study site [clinic] to date) and ethnicity (Papuan/Non-Papuan), the latter because of the different Vitamin D status of these two groups (see ‗Background‘ at the start of this chapter). Allocation concealment conforms to the revised CONSORT guidelines which take account of the logistically challenging environment of the study site.161 This method has been used successfully in over 1,200 malaria patients (three RCTs) in Timika.162-164

The randomisation number corresponds to a study medication pack in a sealed opaque envelope, comprising the 2-month supply of study medications (as described above). Medications inside the envelope are indistinguishable as active or placebo medications in all respects. The unique code is used to identify all documents and samples for that study participant. The code comprises the letters TAD (TB: Arginine and Vitamin D) to distinguish the study from other studies being conducted at the field research site, and a unique 4-digit code (commencing TAD-1001 for Papuans and TAD-2001 for Non- Papuans). Local RAs ensure that patients have been randomised in the correct manner set out in this protocol. Any enrolment violations are clearly documented and notified to myself or other study investigators.

Only the study statistician and the Data and Safety Monitoring Committee see any unblinded data, and these individuals do not have contact with study participants. The randomisation code will be kept at MSHR by an independent person unrelated to the study. The randomisation sequence will not be broken until primary outcomes have been measured and agreed by the study investigators.

ETHICAL CONSIDERATIONS

Ethics committee approvals

This study has been approved by the Human Research Ethics Committees of Menzies School of Health Research, the National Institute for Health Research and Development, and the Australian National University.

61 Chapter 6: Methods

Ethical considerations of randomised design

The withholding of a potentially beneficial medication to half the enrolled study participants might be perceived as an ethical consideration. To alleviate these concerns, AVDAPT investigators explain clearly to staff, community leaders and patients: (a) the necessity for the blinded, randomised, placebo-controlled approach, and (b) the fact that there is as yet no evidence of benefit of L-arginine and/or vitamin D supplementation in TB, and hence those receiving placebo will not necessarily be disadvantaged. The verbal and written information provided by local research assistants and interpreters explain that half the patients enrolled will not get the active study drug. Patients are reminded that they can withdraw from the study at any time without compromising their care or relationship with clinic staff. MSHR researchers have successfully employed blinded RCT design in the local population in large malaria trials to date, with patients willing to participate in such a study design.

All patients, including those who decline to participate in the study, continue to receive all aspects of standard TB care. They may in fact experience improved care due to greater attention being given to the importance of TB medication adherence, quality control aspects of the study which will also have benefits for patients not enrolled in the study, and the presence of extra staff (i.e. research assistants and study investigators) being able to provide operational and medical assistance at the clinic and laboratory.

Potential for harm arising from study drugs or clinical testing

Although anticipated to be unlikely, potential medication side effects are monitored as detailed above. Filtered mouthpieces used with both the spirometer and FENO analyser are stated by the manufacturers to provide protection against transmission of TB. Occupational health and safety standards for staff are upheld, including safe practices in respiratory precautions and biohazard disposal.

Disincentive due to requirements of study participants

Additional time required to perform the extra investigations (e.g. questionnaires, exercise tolerance, lung function) might be a disincentive for some patients. Eligible patients may decline to consent if they deem that the amount of time required to fulfil

62 Chapter 6: Methods the study requirements is excessive. Research staff members ensure that the clinic environment is welcoming. A television has been installed to alleviate boredom in study participants, of particular use for those having PK / PD evaluations. RAs offer flexibility where possible (e.g. scheduling visits to suit the patient). RAs ensure that those who miss or decline some measures, or withdraw altogether, continue to receive their TB therapy.

Feasibility of continuation after study completion

Vitamin D and L-arginine are relatively cheap and readily available and therefore, if shown to be beneficial, would be able to be utilised as adjunctive treatments after completion of the trial at the study site, more widely in Indonesia and potentially in similar settings in other countries.

CONFIDENTIALITY AND DATA STORAGE

All data collected from individuals is stored confidentially on password-protected computers accessible only to the named investigators, data entry clerks and named research students. Paper records are stored in duplicate in separate filing cabinets at the NIHRD-MSHR research building in Timika. Appreciating the risk of loss of data due to computer failure, power blackout, flood or other events, data is backed up at least weekly, sent to and stored at the collaborating institutions.

QUALITY CONTROL AND QUALITY ASSURANCE

Study investigators evaluate and assess all aspects of the work to ensure that the study operates according to GCP guidelines, and that data collected is of as high quality as possible given the constraints of working in the resource-limited setting of the field research site.

ELIGIBILITY CRITERIA AND INFORMED CONSENT PROCESS

RAs receive thorough training in the processes of ensuring eligibility and obtaining informed consent. I check each enrolment form to ensure that the correct diagnostic criteria have been applied and that only eligible patients have been included. I check that all consent forms have been signed by the RA and study participant or guardian if

63 Chapter 6: Methods aged <18 years. During frequent visits to the field research site, I participate at the TB clinic in enrolling and following up patients to ensure that processes are in keeping with the study protocol and with GCP requirements, including that informed consent is being obtained in the appropriate manner as set out in this protocol.

ACCURACY OF DATA COLLECTION AND DATA ENTRY

RAs‘ techniques in all aspects of data collection and recording (performing accurate measurements, ensuring consistency between RAs, maintaining the equipment, transcribing results accurately to the data collection forms and writing legibly), are reviewed by me on an ongoing basis. Electronic entry of data is only performed after data collection forms have been checked for accuracy and completeness. Any apparent queries or errors are clarified with RAs. Clinical and laboratory data are double-entered by two data entry staff into an EpiData® 3.1 database, created by me for the purpose of this study (Appendix 15.12). Any discrepancies between the two records are checked against primary sources and corrected. SGRQ results are entered into a locked excel calculator provided by the questionnaire‘s creators (Professor P.W. Jones, St George‘s Hospital Medical School, London, UK).

DATA HANDLING AND RECORD KEEPING

The NIHRD and MSHR have co-ownership of the data, consistent with the Memorandum of Understanding (MOU) between the two institutions, under which the AVDAPT study was approved. Data is held in Timika for the duration of the trial. Back-up copies of data are regularly sent as required by the relevant ethical committees and for safe-keeping in electronic format to investigators directly involved in this research at NIHRD (Jakarta), MSHR (Darwin) and ANU (Canberra). The field data collected will be held by the principal investigators for 10 years after publication, in accordance with international best practice requirements, and then destroyed.

64 Chapter 6: Methods

6.5 HEALTHY VOLUNTEER SUB-STUDY

As shown in Figure 6.1, a sub-study also approved by the NIHRD, MSHR and ANU ethics committees is being conducted alongside the AVDAPT trial, in which healthy volunteers are recruited in order to establish normal Papuan and Non-Papuan population values for serum vitamin D, amino acids and T cell CD3 zeta expression, exhaled nitric oxide levels, the 6 minute walk test and the modified St George‘s Respiratory Questionnaire. Normal local reference ranges for pulmonary function have already been established at the field research site.165 The selected target sample size is 100 participants, comprising 50 each of healthy adult Papuans and Non-Papuans (25 males and females in each group).

SITE

Healthy volunteers are enrolled at the Timika TB clinic.

INCLUSION CRITERIA

To be eligible, healthy adults must be >18 years of age and must provide written informed consent to being enrolled. Chronic stable conditions such as hypertension which will not impact on ability to perform 6 minute walk or other tests are permitted. Smokers are included.

EXCLUSION CRITERIA

Exclusion criteria include pregnancy, hospitalisation currently or within the last month, current illness including asthma, diagnosis by a doctor of any acute illness or active infection, fever or history of fever in the last week, uncontrolled baseline hypertension: systolic blood pressure > 180mmHg, diastolic blood pressure > 100mmHg, uncontrolled baseline tachycardia, pulse > 120 bpm, or unstable angina pectoris.

ENROLMENT PROCEDURE

Healthy volunteers are sought from among friends and relatives of TB patients, after they have received TB contact tracing and are found to have no evidence of active TB,

65 Chapter 6: Methods and from local staff, friends and family members. The Research assistants seek written informed consent from potential control subjects after providing verbal and written information (see Appendix 15.13) regarding the purpose of their participation and what is required of them.

TESTS PERFORMED

Data collection forms are reproduced in Appendix 15.14. Data collected for each healthy volunteer includes: age, sex, ethnicity, educational and employment status, smoking status, pulse and blood pressure. Consistent with the methods described earlier in this chapter, exhaled nitric oxide is measured using the portable NiOX MINO® analyser, 6 minute walk testing is performed on the standard walking track, and the modified St George‘s Respiratory Questionnaire is completed.

Blood is collected for plasma vitamin D, plasma amino acids (and metabolites), and white cell function and phenotype (including T cell CD3 zeta expression), and processed in the same manner as blood collected from AVDAPT study participants. Haemoglobin is tested using a point-of-care HemoCue® analyser.

Many volunteers are also interested in having their pulmonary function tested, hence this is offered but not essential, as normal reference ranges for spirometric values have already been established in Timika.165

Healthy volunteers are issued with a certificate showing their results. If any results (e.g. haemoglobin, blood pressure) are found to be abnormal, standard local management is implemented (e.g. for anaemia: diagnostic workup [blood film for malaria, investigation of potential bleeding sources] and treatment [anti-malarials if indicated according to local guidelines, iron supplementation if indicated, further referral e.g. to gynaecology clinic if required]).

Data entry and record keeping are consistent with practices described above for the AVDAPT study.

66 Chapter 7: Results I - Baseline

7 Results I: Baseline data in study participants and healthy volunteers

Between June 12th 2008 and October 3rd 2009, 162 participants were enrolled in the AVDAPT study. The aims of this chapter are to describe baseline characteristics of these study participants, and to compare their baseline findings with healthy volunteers.

7.1 INTRODUCTION

A key marker of TB disease severity is sputum smear grade. However, additional indicators of disease severity measured in AVDAPT study participants at baseline and serially during treatment have been selected in order to capture a broader assessment of disease severity, encompassing clinical (symptoms and weight), physiological (spirometry), functional (6 minute walk test) and quality of life (modified St George‘s Respiratory Questionnaire, SGRQ) evaluations. Patient-reported quality of life outcomes are increasingly valued in the assessment of TB management and outcome, in addition to traditional clinical and biological indicators.166 Six minute walk testing (6MWT) and calculation of 6 minute weight ∙ walk distance are acknowledged as useful measures of functional exercise capacity.131-133 This low-technology, essentially cost- free test is ideally suited for measuring TB-related functional impairment in under- resourced TB-endemic settings. The SGRQ is a health-related quality of life instrument formulated for use in chronic obstructive pulmonary disease,130 but validated in a number of respiratory diseases, including TB.114, 167 Early reversible lung impairment in pulmonary TB114, 168 and long-term residual impairment after TB169 are evaluable using spirometric measures such as forced expiratory volume in 1 second (FEV1). The initial aims of this chapter are to explore the baseline demographic characteristics of AVDAPT study participants, and to present their clinical and laboratory findings at their time of enrolment into the study.

Reference ranges in the Timika population are currently lacking for the modified St George‘s Respiratory questionnaire (hereafter abbreviated SGRQ) and 6MWT. The second aim of this chapter is thus to report reference ranges for these measures among

67 Chapter 7: Results I - Baseline locally recruited healthy volunteers, and to compare these with values obtained in the TB patients.

7.2 METHODS

Recruitment of consenting, eligible subjects into the AVDAPT study and associated healthy volunteer study commenced on June 12th 2008 according to the methods detailed in Chapter 6. These studies remain underway, with completion projected by early 2012. Data analysed in this thesis comprise all which had been entered into the electronic database and checked for accuracy using the duplicate data entry verification system with cross referencing to original case record forms, up to and including October 3rd 2009; this includes all study participants enrolled up to that date.

Methods of participant testing are described in Chapter 6. Participant information, consent and data collection forms are reproduced in Appendix 15.5. Additional clarification of methods is provided in the following points:

 Quality control (QC) measures in place include: o Continual review and cross-checking of RA‘s techniques in conducting questionnaires, clinical measures and documenting results, and of laboratory technician‘s blood-processing methods and results documentation. o Spirometer: daily check of volume reading using a standard 3L syringe. o Grading of acid fast bacillus (AFB) density in sputum specimens: (1) specimens collected at weeks 0, 4 and 8 are examined (either the same or simultaneously collected specimens) at both the local Timika laboratory and the University of Indonesia Faculty of Microbiology laboratory, allowing comparison of smear grading; (2) blinded QC testing is performed on a select number of stored slides by visiting trained laboratory staff.

 To be eligible for enrolment, all AVDAPT study participants require demonstration of AFB in sputum (i.e. smear positive pulmonary TB) on at least 2 sputum specimens prior to referral of the patient to the study research staff. Additional to these diagnostic specimens, a further sputum specimen is obtained

68 Chapter 7: Results I - Baseline

at enrolment for culture and drug susceptibility testing at the reference laboratory, and for smear grade assessment at the Timika laboratory. The result of this specimen is the one reported here; thus a small proportion of study participants have a negative enrolment sputum smear.

 Standard definitions are used, shown in Box 7.1:

Box 7.1: Definitions

Nutritional status Defined according to body mass index (BMI) in kg/m2 (see reference170):  Normal BMI: ≥18.5, Mild malnutrition: BMI 17.0–18.49, Moderate malnutrition: BMI 16.0–16.99, Severe malnutrition: BMI<16.0

Pulmonary function 171 Defined according to FEV1 (see reference ):

 Normal lung function: ≥80% predicted FEV1; Mild lung function impairment: 70-79% predicted; Moderate: 60–69% predicted; Mod-severe: 50–59% predicted; Severe: 35– 49% predicted; Very severe: <35% predicted.

Outcome TB treatment outcome at 6 months (see reference172)  Cured: A patient who was initially culture or sputum smear microscopy at the beginning of the treatment but who was smear-negative in the last month of treatment and on at least one previous occasion.  Treatment completed: A patient who completed treatment but who did not meet the criteria to be classified as a cure or a treatment failure. This definition applies to pulmonary smear-positive and smear-negative patients and to patients with extrapulmonary disease.  Treatment failure: (i) A new patient who is culture or sputum smear microscopy positive at five months or later during treatment, or who is switched to Category IV treatment because sputum culture revealed MDR -TB; (ii) A previously-treated patient who is culture or sputum smear microscopy positive at the end of the re-treatment regimen or who is switched to Category IV treatment because sputum culture revealed MDR -TB.  Died: A patient who died from any cause during the course of treatment.

 Defaulted: A patient whose treatment was interrupted for ≥2 consecutive months.  Transferred out: A patient who was transferred to a health facility elsewhere and for whom the treatment outcome is not known.  Successfully treated: A patient who was cured or who completed treatment.

69 Chapter 7: Results I - Baseline

 Predicted FEV1 was calculated according to coefficients and constants previously established in the local Timika population165 as follows (where male=0, female=1):

o Papuans: Predicted FEV1 = (3.9 x height in metres) – (age x 0.020) – (gender x 0.551)- 2.558

o Non-Papuans: Predicted FEV1 = (4.13 x height in metres) – (age x 0.010) – (gender x 0.575)- 3.114

STATISTICAL ANALYSES

Statistical analyses were performed with Stata software (version 10.1; Statacorp), and graphs were prepared in GraphPad (version 5.0; Prism). Two-sided values of p<0.05 were considered significant. Proportions were compared between groups using Pearson‘s  test, or Fisher‘s exact test where appropriate. Differences in continuous data between groups were tested using 2-sample t-tests or analysis of variance (ANOVA) for normally distributed data. For non-normally distributed data, transformations were applied to achieve a normal distribution (e.g. natural logarithm), or non-parametric tests were used (Wilcoxon Rank-Sum or Kruskal-Wallis tests). Where the effect of more than one variable on continuous normally distributed data needed to be tested, multiple linear regression models were used, and residuals were examined to ensure that required assumptions were met. Weighted kappa scores (linear weighting) were used to measure inter-rater agreement for categorical variables. Kappa results were interpreted as follows: kappa<0.20: poor agreement; 0.21 - 0.40: fair; 0.41 - 0.60: moderate; 0.61 - 0.80: good; 0.81 - 1.00: very good.173

7.3 RESULTS

RECRUITMENT OF STUDY PARTICIPANTS AND HEALTHY VOLUNTEERS

Four hundred and ninety two patients were commenced on TB treatment at the Timika TB clinic in the 16 months between 12/06/08 and 03/10/09 (Figure 7.1). One hundred and seventy two eligible people (smear positive, non-pregnant adults with new TB,

70 Chapter 7: Results I - Baseline planning to live in Timika for the 6-month treatment duration) were referred to the AVDAPT research assistants; 8 did not consent, 2 were found to have hypercalcaemia, and 162 were enrolled. The data presented in this chapter represent 36.5% of the planned final sample size of 444 participants. Reasons for ineligibility are shown in Figure 7.1. Reasons cited for non-consent included being too busy with other duties (7), or family did not agree to the individual‘s involvement (1).

Figure 7.1: Eligibility screening and enrolment of AVDAPT study participants

Subjects commenced on TB treatment at Papuan: 293 (59.6%)

the Timika TB clinic Female: 181 (36.8%) 12/06/08 – 03/10/09:

492 Smear negative: 180

<15 years: 52 Ineligible for enrolment: Previously treated for TB: 43 320 Unable to stay in Timika: 26

Eligible: Pregnant: 1

172 Other or unknown: 18

Not enrolled: Hypercalcaemic: 2 10 Did not consent: 8

Enrolled: Papuan: 78 (48.2%) 162 Female: 54 (33.3%)

The proportion of females enrolled did not differ from the total proportion of females commenced on TB treatment during the recruitment period (33.3 versus 36.8%, p=0.4). However, the proportion of Papuans enrolled (48.2%) was significantly lower than the proportion starting TB treatment (59.6%) (p<0.0001, ), due to more Papuans being ineligible (more likely to be leaving Timika within 6 months or to be smear negative).

71 Chapter 7: Results I - Baseline

Forty adults comprising 20 Papuans and 20 Non-Papuans volunteered and were enrolled in the Healthy Control study. These included staff members and their colleagues, or relatives and friends of AVDAPT participants, once they had been shown on contact tracing to have no evidence of active pulmonary TB.

BASELINE DATA

Demographic and socio-economic characteristics

Table 7.1: Baseline characteristics of AVDAPT study participants (TB patients) and healthy volunteers

TB patients Healthy volunteers Number of study participants 162 40 Age in years: median (range) 27 (15 – 65) 26.5 (18 – 65) Papuan: no. (%) 78 (48.2) 20 (50.0) Female: no. (%) 54 (33.3) 9 (22.5) Smoking status Current or ex-smokers: no.(%)* 93 (57.4) 19 (47.5) Never smoked: no. (%) 69 (42.6) 21 (52.5) Highest educational attainment: no. (%) No schooling 14 (8.7) 1 (4.6) Primary school 32 (19.9) 3 (13.6) High school 112 (69.6) 17 (77.3) Academy or university 3 (1.8) 1 (4.6) Occupation: no. (%)† Employed 75 (49.0) 25 (78.1) Unemployed 66 (43.1) 3 (9.4) Attending school 12 (7.8) 4 (12.5) Owns telephone: no. (%) 90 (55.6) 24 (60.0)

*p= 0.07 calculated using  test. †p<0.0001 calculated using  test. All other differences between groups are non-significant.

Baseline characteristics of the 162 AVDAPT study participants (TB patients) and the 40 healthy controls are shown in Table 7.1. Median age was slightly lower in Papuan than Non-Papuan TB patients (25.5 vs 30.0 years respectively, p=0.01, Wilcoxon Rank Sum test). The gender ratio did not differ between ethnic groups (p=0.1; 2 test). Males were significantly more likely to be current or ex-smokers than women (Figure 7.2). Slightly more Papuans than Non-Papuans were either current or ex-smokers (50 vs 43%) but the difference was non-significant (, p=0.1).

72 Chapter 7: Results I - Baseline

Figure 7.2: Smoking rates in male and female AVDAPT study participants and healthy volunteers*

p=0.007

1.0 p<0.0005 p=0.001

0.8

0.6

0.4

(proportion, 95% CI) 0.2 Current & ex-smokers

0.0

Male Male Female Female

TB patients Healthy volunteers

*p values were calculated from 2 or Fisher‘s exact tests

Educational attainment did not significantly differ between men and women (test, p=0.9), but Papuans achieved significantly lower educational levels than Non-Papuans (Figure 7.3). Papuan people were also less likely than Non-Papuan people to be employed (Figure 7.4) or to own a telephone.

Figure 7.3: Educational attainment in Papuan and Non-Papuan study participants*

100

80

60 Non-Papuan Papuan

40

Percentage (mean, 95%CI) (mean, Percentage 20

0

High school No schooling Academy/Uni Primary school

*Papuans significantly more likely to have no or primary schooling only, p<0.0001 ( test)

73 Chapter 7: Results I - Baseline

Figure 7.4: Employment status in Papuan and Non-Papuan study participants

p<0.0005

100 76.9% Non-Papuan 80 Papuan

60 36.8%

40

20 Percentage (mean, 95%CI) (mean, Percentage 0 Employed or at school

*p value calculated using  test

Clinical findings

Table 7.2: Symptoms

TB patients Healthy volunteers N 162 40 Cough: no. (%) 162 (100) - Mild 14 (8.9) Moderate 69 (43.7) Severe 67 (42.4) Very severe 8 (5.1) Sputum: no. (%) 162 (100) - Haemoptysis: no. (%) 49 (30.4) - Diagnostic delay: median (range) months 2 (0.25-24) - Symptom tally: mean no. symptoms (95% CI) 7.7 (7.3 - 8.2) - Weight (kg): mean (95% CI) † 48.4 (47.3-49.6) 62.5 (59.2-65.8)

All patients reported cough and sputum production at baseline and 30.4% had haemoptysis (Table 7.2). Median reported diagnostic delay (duration of illness from onset until enrolment) was 2 months, with a positively skewed distribution; 17 patients (10.5%) reported illness duration of 1 year or more prior to being diagnosed with TB. Reported illness duration did not differ between men and women (p=0.2, Wilcoxon rank-sum test), but was longer in Papuans than Non-Papuans in univariate analysis; this

74 Chapter 7: Results I - Baseline difference did not remain significant after controlling for educational level in a linear regression model of illness duration (log-transformed to achieve a normal distribution).

Table 7.3: Baseline clinical findings and haemoglobin

TB patients Healthy volunteers Number 162 38 Height (m): mean (95% CI) 1.58 (1.57-1.60) 1.61 (1.58-1.63) BMI (kg/m2): mean (95% CI) † 19.3 (18.9-19.7) 24.3 (23.0-25.5) Normal BMI: no. (%) 101 (62.4) 36 (94.7) Mild malnutrition: no. (%) 34 (21.0) 2 (5.3) Moderate malnutrition: no. (%) 13 (8.0) 0 (0) Severe malnutrition: no. (%) 14 (8.6) 0 (0) Number 162 22* FEV1(L): mean (95% CI) † 1.84 (1.73–1.94) 2.86 (2.43-3.29)

% Predicted FEV1(L): mean % (95% CI) † 63.7 (60.6–66.7) 92.0 (83.2-100.8) Lung function class: no. (%)† Normal lung function 40 (24.7) 16 (72.7) Mild lung function impairment 21 (13.0) 3 (13.6) Moderate 25 (15.4) 2 (9.1) Mod-severe 27 (16.7) 1 (4.6) Severe 38 (23.5) 0 (0) Very severe 11 (6.8) 0 (0) Number 141 35 Modified St George‟s Respiratory Questionnaire: median (range) ‡ Symptoms 45.8 (0-90.8) 0 (0-21.5) Activity 40.8 (0-100) 0 (0-12.8) Impact 36.6 (0-89.0) 0 (0-15.1) Total 40.7 (5.2-91.9) 0 (0-9.2) Number 160 40 6 minute walk test (m): median (range) ‡ 410 (0 – 612) 485 (360-640) Number 162 40 Haemoglobin (g/dL): mean (95% CI)§ 12.3 (12.0 – 12.5) 13.7 (12.6-14.7)

*FEV1 not routinely included since normal reference ranges for FEV1 already established in Timika. †p values <0.0001 calculated from 2-sample t-tests. ‡ p values<0.0001 calculated using Wilcoxon rank sum tests. §p=0.0002 calculated form 2-sample t-test.

At diagnosis, study participants reported a broad range of symptoms; the average number of reported symptoms was 7.7. Most common symptoms are shown in Figure

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7.5. Females reported slightly more symptoms than men (women: 8.6 symptoms, 95% CI 7.8-9.5; men: 7.3 symptoms, 95% CI 6.8-7.8; p=0.004), but did not report more severe cough than men (p=0.5,  test).

Figure 7.5: Symptoms reported among AVDAPT study participants at TB diagnosis

100 90 80 70 60 50 40

(mean, 95%CI) (mean, 30 20 Percentage with symptom with Percentage 10 0

Itch Rash Cough Fever Nausea MyalgiaVomiting Bloating Sputum HeadacheDizzinessArthralgia Diarrhoea Back pain Weight loss Chest pain Haemoptysis Paraesthesia Constipation

Figure 7.6: Number of symptoms reported at the time of TB diagnosis in men and women

p=0.004 20

15

10

5

Number of symptoms of Number reported at TB diagnosis 0 Male Female

*p value calculated from 2-sample t-test. Bars represent median values and points represent individual‘s results.

Papuan males with TB had significantly higher mean BMI at baseline (20.3 kg/m2, 95% CI 18.1-19.3) than Non-Papuan males (18.7 kg/m2, 95% CI 19.8-20.9), since their weight was greater (52.3 vs 49.3 kg, p=0.0001) and height marginally lower (1.61 vs 1.62 m, p=0.3). Only 7.6% of Papuan males were classed as having malnutrition

76 Chapter 7: Results I - Baseline

(BMI<18.5), compared with 52.7% of Non-Papuan males; this trend was noted among females, but did not reach statistical significance (Figure 7.7). On sub-grouping Papuans into Highland and Lowland tribes, no differences in BMI were observed between these groups.

Lung function was impaired in three quarters of study participants (Table 7.3), with mean percentage of predicted FEV1 falling in the category of moderate impairment 171 (FEV1 60-69% predicted ). No differences in % predicted FEV1 were seen between gender or ethnic groups (p>0.5, 2-sample t-tests) or according to age (p=0.2 calculated from regression of % predicted FEV1 against age).

SGRQ total scores were available in 141 study participants at baseline; results were not recorded in 7 instances, and a total score could not be calculated in a further 14 people due to missing answers to parts of the questionnaire. Responses were normally distributed (but not among healthy volunteers, hence results are presented in Table 7.3 as medians), and did not differ among patient gender or ethnic groups. Results were high in each domain score and the total score. These respectively indicate presence of respiratory symptoms, restricted activities due to respiratory symptoms, perceived impact on daily living, and overall impairment in quality of life.

Figure 7.7: Nutritional status in AVDAPT study participants

(a) Males (b) Females

100 100 Papuan 90 90

80 80

70 70

60 60 Non-Papuan Papuan 50 50

40 40

30 30

Percentage (mean, 95%CI) (mean, Percentage 95%CI) (mean, Percentage

20 20

10 10

0 0

Normal BMI Normal BMI

Mild malnutritionMod malnutrition Mild malnutritionMod malnutrition Severe malnutrition Severe malnutrition p<0.0001 calculated using  test comparing p=0.6 calculated using  test comparing nutrition nutrition group in Papuan and Non-Papuan males group in Papuan and Non-Papuan females

77 Chapter 7: Results I - Baseline

Six minute walk distances were very limited in those patients who had significant malaise; 1 patient with copious vomiting scored 0m walk distance, and 19 of 160 participants who attempted the walk test (11.9%) walked less than 300m (Figure 7.13 illustrates 6MWT distributions). Males walked significantly further than females (median walk distances 428.0m and 373.5m respectively, p<0.0001; see Figure 7.13).

Laboratory and radiological findings

Consistent with expected haemoglobin (Hb) ranges, women had significantly lower mean Hb (11.5g/dL) than men (12.7 g/dL). These readings are below or at the lower limit of the standard international reference ranges for Hb of 13.5-17.5 g/dL for men and 11.5-15.5 g/dL for women (Figure 7.8). Papuan ethnicity and HIV positivity (but not age or smoking status) also significantly predicted lower Hb levels (Figure 7.8). Female sex, Papuan ethnicity and HIV positive status each remained independently predictive of lower Hb in a multivariate linear regression model (data not shown).

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Table 7.4: Baseline laboratory and radiological findings

TB patients* Ionised calcium (mmol/L): mean (95% CI) 1.21 (1.20 – 1.22) HIV positive: no./no.tested (%) 18 / 138 (13.0) Papuan† 14 / 67 (20.9) Non-Papuan 4 / 71 (5.6) Density of acid fast bacilli in sputum: no.(%) (Sputum smear grade) 0 10 (6.3) Scanty 26 (16.4) 1+ 49 (30.8) 2+ 41 (25.8) 3+ 33 (20.8) Chest radiograph Cavitation: no.(%) 88 (56.1) Percentage of lung affected: median (range) 40 (0-100) Score‡: median (range) 68 (0-140) Sputum culture at diagnosis N 157 No growth 14 (8.9) Mycobacterium tuberculosis identified 123 (78.3) Contaminated & negative after decontamination 2 (1.3) Report pending 18 (11.5) Mycobacterium tuberculosis susceptibility N 102 Isoniazid monoresistance 5 (4.9) Streptomycin monoresistance 4 (3.9) Ethambutol monoresistance 1 (1.0) Isoniazid & rifampicin resistance (MDR-TB) 2 (2.0) Rifampicin monoresistance or other patterns of resistance combination 0 (0.0) *N=162 unless otherwise stated. † p=0.01, Fisher‘s exact test. ‡Chest radiograph score developed as described in Results Chapter 9.

Ionised calcium levels at baseline were independent of age, sex, ethnicity, smoking status or HIV status. HIV results are discussed in detail in Chapter 12. Chest x-ray findings are presented in Chapter 9; preliminary examination revealed no significant variation in chest x-ray scores or presence of cavitary disease according to sex, ethnicity or age (p>0.05 in each instance).

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Figure 7.8: Haemoglobin according to sex, ethnicity, HIV status and in healthy volunteers versus TB patients overall

20 p<0.00005 p<0.00005 p=0.002 p=0.002

m 18

f 16

14

12

Hb Hb (g/dL) 10

8

6

4

HIV-

Male

HIV+

Female

Papuan

TBpatients Non-Papuan TB patients

Healthyvolunteers *Bars represent mean Hb. P values were calculated using 2-sample t-tests. Normal male and female reference ranges are marked adjacent to the y axis: Males: 13.5-17.5 g/dL; females: 11.5-15.5 g/dL

For reasons explained in the methods section of this chapter, the sputum specimen provided by a small proportion of study participants (6.3%) was AFB-negative at enrolment despite previous specimens having been positive. Sputum smear grade at diagnosis did not differ according to sex, ethnicity or smoking status (p>0.05 in each instance calculated using  tests) or age (p=0.6 calculated from regression of log- transformed age against sputum smear grade). HIV positive people had lower smear grades (13/18 = 72.2% had smear grade ≤1) than HIV negative people (61 of 117 =. 52.1% had smear grade ≤1) but the difference was not significant (p=0.1, Fisher‘s exact test).

Sputum culture was reported as negative in 14 study participants at diagnosis. The 14 specimens which were culture negative were smear positive in 8 instances and smear negative in 6. One patient with no growth from sputum at week 0 had MTB isolated from his week 4 sample. Appreciating the opportunities for cultures to be falsely negative, patients with negative baseline cultures are not currently excluded from

80 Chapter 7: Results I - Baseline analyses. Diagnostic culture results were unavailable in 5 study participants due to the specimens being lost in transit.

Two patients were found to have MDR-TB; both results were confirmed with Hain GenoType® MTBDRplus assay. Additionally, one case each of isoniazid and rifampicin resistance, rifampicin monoresistance, and combined isoniazid, rifampicin, ethambutol and streptomycin resistance were reported, but in each case, further evaluation including Hain testing revealed, in each case respectively: isoniazid resistance only; no resistance; mixed MTB / non-tuberculous mycobacterial growth with fully-susceptible MTB.

Sputum microscopy quality control results

Inter-laboratory agreement on sputum AFB grades was able to be determined where simultaneously-collected sputum samples obtained from study participants, or the same sample divided for each laboratory, were submitted to the local Timika laboratory and the Faculty of Microbiology, University of Indonesia laboratory. This routinely occurs at week 0, 4 and 8. Inter-rater agreement was moderate (weighted kappa=0.56, standard error 0.04). The paired results in 219 of 237 samples (92.5%) were within 1 grade of each other, which is generally considered to be acceptable.

Inter-rater agreement was additionally assessed by comparing results obtained from expert mycobacteriology laboratory technicians visiting the field site from the National Institute of Health Research and Development, Jakarta, and the Faculty of Microbiology, University of Indonesia. They provided an assessment of stored slides which had been made and reported at the Timika laboratory (Table 7.5). Slides assessed by the second assessor had been in storage for over 12 months in some cases, hence some deterioration of slide quality may have accounted for the lower agreement in that instance.

Table 7.5: Agreement in sputum AFB grade Compred between visiting technicians and Timika laboratory staff

Complete agreement with Agreement within 1 Weighted kappa original result: no. (%) grade: no. (%) (standard error) Assessor 1 13/18 (72) 18/18 (100) 0.83 (0.16) Assessor 2 11/20 (55) 16/20 (80) 0.48 (0.13)

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Comparisons between AVDAPT participants and healthy volunteers

Forty percent of the planned healthy volunteer sample size has been recruited to date. Despite relatively small numbers, preliminary comparisons are made here with the AVDAPT study participants and will be re-examined when full enrolment is completed. The group of 40 healthy volunteers had a similar ethnic and age profile as the TB patients, but fewer women (9/40 vs 54/162), although this difference was not statistically significant. Despite having similar educational backgrounds (p=0.7,  test) and similar telephone ownership rates (as a marker of socio-economic status), healthy volunteers were significantly more likely to be currently employed than people with TB (Figures 7.9-7.11).

Figure 7.9: Educational attainment in healthy volunteers and TB patients*

100 Healthy volunteers 80 TB patients

60

40

20 Percentage (mean, 95%CI) (mean, Percentage 0

High school No schooling Academy/Uni Primary school

*no significant difference in educational attainment, p=0.7 ()

Figure 7.10: Telephone ownership in TB patients and healthy volunteers

82 Chapter 7: Results I - Baseline

p=0.6

p=0.2 p<0.0005

100 70.0% 77.4% Non-Papuan 80 50.0% Papuan

60 32.1% 40

Percentage owning Percentage 20 a phone (mean, 95%CI) (mean, phone a

0 Healthy volunteers TB patients

*p values calculated using  test

Figure 7.11: Employment status in TB patients and healthy volunteers

p=0.001

100 87.5% Healthy Volunteers 80 TB patients 57.1% 60

40

20 Percentage (mean, 95%CI) (mean, Percentage 0 Employed or at school

*p value calculated using  test

Male TB patients were more likely to smoke than male healthy volunteers (83 of 108 i.e. 76.9% versus 19 of 40 i.e. 61.3%), but this difference did not achieve significance at the 0.05 level (p=0.07,  test) (Figure 7.2). None of the female healthy volunteers admitted to current or past smoking.

Consistent with their healthy status and in contrast with TB patients, volunteers‘mean BMI was in the normal range (Figure 7.12). No BMI difference was observed between the 2 ethnic groups, unlike that noted among the TB patients.

Figure 7.12: BMI in healthy volunteers and AVDAPT study participants

83 Chapter 7: Results I - Baseline

p<0.00005 35

30

25

20 BMI (kg/m2) BMI

15

10 Healthy volunteers TB patients

*bars represent mean BMI; each point represents an individual‘s result. P values calculated using 2- sample t-tests.

Figure 7.13: Six minute walk distances in healthy volunteers and AVDAPT study participants*

p<0.00005

p=0.003 p<0.00005

600

400

200 6min walk distance (m) distance walk 6min

0 Male Female Male Female Healthy AVDAPT study volunteers participants

*bars represent median 6-minute walk distance; each point represents an individual‘s result. P values calculated using Wilcoxon rank sum tests.

Healthy volunteers had largely normal lung function according to previously- established local reference ranges (Table 7.3), and mostly scored zero (i.e. normal lung- related quality of life) for the SGRQ total score and individual domain scores (Table 7.3 and Figure 7.14), with no difference between gender or ethnic groups (data not shown). Median distance walked in 6 minutes was significantly further for healthy volunteers (485m) than for TB study participants (410m), and further for men than women (Figure 7.13). Normal reference values in Timika for 6-minute walking testing based on the current group of 40 controls are summarised in Box 7.2.

84 Chapter 7: Results I - Baseline

Figure 7.14: St George‟s respiratory questionnaire total score in healthy volunteers and AVDAPT study participants*

p<0.00005

p=0.4 p=0.3 100

50 SGRQ SGRQ total score

0

Male Female Male Female Healthy AVDAPT study volunteers participants

*bars represent median SGRQ results; each point represents an individual‘s result. P values calculated using Wilcoxon rank sum tests.

Box 7.2: Locally-relevant reference ranges for 6-minute walk testing

Exercise tolerance in healthy volunteers

MALES: Mean distance in metres walked in 6 minutes: mean (± 1.96×SD):

511m (393-629)

FEMALES: Mean distance in metres walked in 6 minutes: mean ( ± 1.96×SD):

447m (356-538)

Hb was significantly higher in healthy volunteers than in people with TB (Table 7.3 and Figure 7.8). The healthy volunteers‘ mean Hb levels were within standard international reference ranges for men (13.9 g/dL, 95% CI 12.7-15.2) and women (12.8, 95% CI 11.0 - 14.5), but some were frankly anaemic (men: Hb range 4.9-18.6; females: Hb range 8.8-15.1). People found to have anaemia were offered malaria testing and treatment if required, further investigation as indicated, and iron supplementation, according to local treatment protocols. No healthy controls were found to have current malaria parasitaemia.

7.4 DISCUSSION

85 Chapter 7: Results I - Baseline

Key findings from this analysis of preliminary data from AVDAPT study participants and healthy volunteers include: over-representation of males compared with females among TB patients in Timika; socio-economic differences between Papuan and Non- Papuan TB patients; requirement for differing interpretation of BMI in Papuan and Non-Papuan people; substantial diagnostic delay, burden of disease and loss of employment in people with TB compared with healthy volunteers, and low rates of MDR-TB among cases of newly-diagnosed TB in Timika. Additionally, locally appropriate reference ranges for SGRQ and 6MWT have been established.

SEX DIFFERENCE IN TB RATES IN TIMIKA

Female to male ratios among all new clinic patients diagnosed with TB (181:311) and among enrolled AVDAPT study participants (54:108) were consistent with published female to male ratios of between 0.3 (South-East Asia) and 0.5 (Western Pacific region).174 The relative contributions to this imbalance of case-detection or notification discrepancies versus true rate differentials due to biological or exposure differences in Timika are unclear. Elsewhere, tuberculin skin test surveys showing that TB exposure rates are equal among the sexes until adolescence suggest that the greater number of contacts males have outside the household after adolescence may be relevant in increasing their TB risk.175 Evidence for under-reporting among females exists in some areas where the sex ratio is higher in active than passive case detection programs.176 Females may be more likely to have smear negative disease, with improvements in smear-positive detection rates achievable through provision of education in how to produce a good sputum sample.177 Higher rates of smoking (as observed in this study) and alcoholism in males may contribute to their increased pulmonary TB risk.175 Female gender is also identified as a risk factor for extra-pulmonary TB.178, 179 Together these data suggest that females may be less likely to be exposed to TB due to social networks, more likely to develop forms of TB other than smear-positive pulmonary TB, and less likely to have TB detected and treated. These and other biological / hormonal factor have been recently reviewed elsewhere.180

Data presented here contribute to the understanding of the uneven gender ratio in several ways: firstly, the recruitment data indirectly demonstrate that females were not more likely to be ineligible for recruitment into the study on the basis of smear negative disease or other factors, since the proportion of females entering the TB clinic was the

86 Chapter 7: Results I - Baseline same as the proportion being enrolled in the trial. Thus although other investigators have identified females as having poorer ability to expectorate good-quality sputum production (due for instance to cultural inhibitions) and hence lower likelihood of being diagnosed with smear positive TB,177 these data indirectly do not support this. Secondly, females were not less likely to consent to enrolment in the study than males, suggesting that women‘s engagement with health care providers at this particular clinic does not differ from men‘s. Thirdly, these data do not indicate that diagnosis was delayed among females compared with males, since they did not report longer illness duration, and were not found to have more extensive disease according to baseline sputum smear grade, X-ray score, % predicted FEV1 or SGRQ scores. Females did have significantly lower haemoglobin and 6 minute walk distance than males, but these associations are physiologically expected and unlikely to be representative of more severe disease in females. Females also reported slightly more symptoms, but this is a relatively subjective measure which may be subject to gender bias, and is not necessarily a definite representation of more severe organic disease. Overall, these findings provide indirect evidence that in the Timika population, delayed diagnosis due to different health care seeking behaviour (leading to more severe baseline disease) is unlikely to be a major factor in the gender imbalance among patients with pulmonary TB in Timika. Factors including the significantly higher male smoking and employment rates reported here, with attendant risks of TB disease and exposure respectively, might contribute to the gender imbalance.

SOCIO-ECONOMIC DATA

Timika has a rapidly-changing socio-cultural landscape characterized by an ethnically mixed population of native (Melanesian) Papuans and immigrant (Malay) Non-Papuan Indonesians. Using education, employment and telephone ownership as markers of socio-economic status, Papuan participants in the AVDAPT study were found to be more socio-economically disadvantaged according to each indicator than Non-Papuan people. Of particular relevance in this study is that the ability to contact people by telephone provides an important mechanism by which research assistants and other clinic staff can remind study participants to attend the clinic, re-schedule appointments, trace people who have defaulted, or contact people who have been transferred to other parts of Indonesia to ensure they are continuing TB treatment. Although all measures are taken to retrieve defaulting patients who are uncontactable by phone by visiting their

87 Chapter 7: Results I - Baseline house and enquiring of their whereabouts from neighbours or family, lack of a telephone remains disadvantageous in health care delivery in this setting.

Documentation of employment status in this study does not distinguish inability to continue in employment due to illness from pre-existing unemployment, which are both categorised as ‗unemployed‘. Educational attainment and telephone ownership were not significantly higher among the healthy volunteers, suggesting comparable socio- economic status, yet healthy people were more likely to be employed. Thus although it is possible that TB patients were unemployed prior to becoming sick, illness-related loss of employment (an indirect cost of TB) is an alternative explanation for this finding. The particularly low employment rates among Papuans compared with Non-Papuans may additionally represent pre-existing unemployment, in keeping with their lower educational attainment.

SMOKING

A high proportion (76.9%) of male AVADPT study participants were current or ex- smokers, and smokers appear to be over-represented in TB patients compared with male healthy volunteers (61.3% of whom were current or ex-smokers), consistent with the known association between smoking and increased TB risk,181 and suggesting smoking may be a risk factor for development of active TB in this population. Whether this difference in smoking rates between TB patients and the general population will be observed and reach statistical significance in larger sample sizes will be re-examined when recruitment has been completed. These high reported smoking rates are comparable to WHO estimates of 50-59% of adult males being current smokers in Indonesia.182 Females remain relatively protected due to the perceived unacceptability of female smoking among both main ethnic groups in Timika.

CLINICAL AND LABORATORY MEASURES

Body mass index

A striking difference in BMI was observed between Papuan and Non-Papuan males. Almost all Papuan males were in the normal nutritional range according to standard BMI criteria (BMI≥18.5 kg/m2),170 compared with only 47.3% of Non-Papuan males.

88 Chapter 7: Results I - Baseline

Contrastingly, lower haemoglobin levels were seen in Papuans than Non-Papuans, Papuan males did not have lesser disease severity on the various measures described, and weight loss was commonly reported by them to have preceded the diagnosis of TB. These observations indicate that standard BMI criteria may under-estimate malnutrition in Papuan males, who have classically muscular body habitus and short stature. There is an absence of literature replicating this observation in other Melanesian populations, possibly because the opportunity for direct comparisons of this kind between Asian and Melanesian populations are limited outside of Indonesia‘s Papua Province. On the other hand, lower BMI cut-offs are known to be necessary in Asian people (i.e. Non-Papuans in this dataset); while the same BMI definitions of malnutrition are used, Asian-specific lower cut-offs are recommended to define overweight and obesity.183 Thus a two-way bias probably exaggerates the BMI difference between Papuans and Non-Papuans. Hence subsequent analyses in this thesis include weight as well as BMI, and malnutrition is not considered to be absent merely on account of BMI being >18 kg/m2 in Papuan males. Any formal recommendation to revise definitions of nutritional status according to BMI in Papuan males (±females) would require a large population sample with evaluation of additional comparative markers of nutritional status.

Lung function

The significant burden of illness due to TB is illustrated by the high proportion of AVDAPT study participants with impaired pulmonary function, high SGRQ scores, and limited 6-minute walk distances. Three quarters of people with TB had impaired lung function according to FEV1, and almost half (47%) had <60% of predicted FEV1, indicating moderate-severe impairment. Given that locally-established FEV1 reference ranges have been used to calculate % predicted FEV1, and that lung function impairment was uncommon among the healthy volunteers despite their high smoking rates, these data indicate that TB is highly likely to be the cause of the poor lung function among these study participants.

These findings are comparable to (slightly exceeding) the extent of lung function impairment recently published from an observational study of TB patients at the same Timika research site. In this study conducted in 2003-4, 39% of people with newly- diagnosed smear positive pulmonary TB had moderately severe to severe impairment of lung function.114 Although improvement by 6 months was significant, 25% still had an

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FEV1 in the moderately-severe to severely impaired range at treatment completion. Residual pulmonary impairment after TB has also been identified in a cohort of South African TB patients, in whom 28% had residual airflow limitation and 24% had a restrictive pattern on spirometric testing at treatment completion,168 and in patients with a recent past history of TB in the USA, in whom >50% had significantly impaired pulmonary function (including restrictive pattern, obstructive pattern &/or low vital capacity).169 TB is acknowledged as an important cause of non-smoking related chronic obstructive pulmonary disease.184

St George’s Respiratory Questionnaire

The ability of the modified SGRQ to identify impaired lung-related quality of life in people with newly-diagnosed TB is evident from the high scores in AVADPT study participants compared with healthy volunteers. Testing the modified, translated questionnaire in healthy people has not previously been performed in Timika; the distribution of responses in healthy people (Figure 7.14: median score 0, range 0-9.8), confirms the appropriateness of this tool for measuring lung-related quality of life impairment in this population. Studies of the SGRQ in healthy populations are uncommon: one general population study recruited 862 Spanish adults but permitted inclusion of people with chronic airways disease (10.2% of participants); a mean score of 5.8 (range: 0 – 92.8) was reported in the youngest (39 - 49 year) age group.185 The low SGRQ scores obtained in the healthy Timika population are expected and appropriate, given their young age and specific exclusion of those with underlying disease.

On the scale of 0 (no lung-related quality of life impairment) to 100 (severe lung-related quality of life impairment), the mean SGRQ score of 41.8 in study participants with TB represents impaired quality of life approximately equivalent to that experienced by people with moderate chronic obstructive pulmonary disease (COPD); a study in the

Netherlands found that COPD patients with predicted FEV1 of ~43-67% had SGRQ total scores of 35.7 – 43.4.186 Similar to the data presented here, Maguire and colleagues in the previous study in Timika also assessed responses among TB patients to the modified Indonesian SGRQ, reporting a mean baseline score of 45.4.114 The SGRQ has also been shown to be valid and reliable in assessing post-TB lung-related quality of life in people with TB in the USA, in whom mean scores of 23.5 were reported.167

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6 minute walk test

Exercise capacity was very limited in the most unwell patients, and the median distance walked (410m) was low compared with healthy volunteers (485m). Six-minute walk testing, chiefly used in chronic respiratory or cardiac disease, is a simple and inexpensive test of functional exercise capacity which has rarely been reported in TB. Erhabor et al (in a conference abstract) reported mean 6-minute walk distances of 502.0m and 481.7m among male and female Nigerian adults with pulmonary TB respectively.187 Maguire et al established the feasibility and utility of this test in Timika, reporting low six minute weight ∙ walk distance among TB study participants at diagnosis, with significant increases during 6 months of follow up.114 I have chosen to report 6-minute walk distance rather than six minute weight ∙ walk distance (‗six minute work‘132). Multiplying the distance walked by weight favours heavier people by taking account of the extra work and oxygenation required to move a heavier body. However, weight in this under-nourished patient group has different physiological ramifications for oxygen consumption compared with weight in the heavy populations in whom weight ∙ walk distance has been studied.132, 133 Furthermore, work as it relates primarily to oxygenation capacity is less relevant in this patient group, in whom reasons observed for limited exercise tolerance at the time of TB diagnosis included factors such as fatigue and vomiting, not just impaired lung function.

The healthy Timika volunteers walked a substantially shorter distance than healthy people in other studies, in whom distances of 630m,188 and 580m in men and 500m in women189 have been reported in the USA and Japan respectively. Likely reasons for these differences include short stature (average height 1.61m), hot and humid ambient conditions not conducive to vigorous exercise, the walking track length, and cultural norms regarding usual walking pace. Thus the establishment of locally-relevant reference ranges for this test in men and women of both ethnic groups is particularly important, and will be published when recruitment of healthy volunteers has been completed.

LABORATORY RESULTS

Haemoglobin was, as expected, found to be lower in women and HIV positive people, but was additionally found to be lower in Papuans than Non-Papuans. Whether this

91 Chapter 7: Results I - Baseline reflects dietary factors, frequency of malaria, TB disease severity or other factors cannot be determined from this data.

Sputum culture results are currently incomplete. False negative culture results may arise for reasons such as loss of viability during the process of transporting specimens to the laboratory (Faculty of Microbiology, University of Indonesia, Jakarta), during specimen decontamination, or loss of power/heat in the incubator. Problems in transporting specimens from Timika to Jakarta (a distance of 3500km) were identified early in the study, including unacceptably long delays and loss of some consignments; hence an alternative transport method using a direct courier was instigated. Additionally, susceptibility results are missing for a proportion of patients due to laboratory difficulties in re-culturing the organism in the antibiotic-containing tubes for susceptibility testing. These issues have been directly addressed in quality improvement exercises undertaken regularly at this WHO-accredited laboratory by senior laboratory staff from the supranational Mycobacterium reference laboratory in South Australia (Mr Richard Lumb, IMVS, South Australia).

MDR-TB IN TIMIKA

MDR-TB is relatively uncommon among new cases of pulmonary TB in Timika, identified in 2 of 102 patients (1.96 %). This rate has not changed in the last 5 years.112 Although definitions of ‗low‘ and ‗high‘ MDR-TB rates are not established, the 2008 report on global MDR-TB found only 14 settings where MDR prevalence among new cases was higher than 5% (up to as high as 40.8% in Azerbaijan), all in eastern Europe / Eurasia and China.31 Global estimates are limited by extensive under-reporting of MDR-TB due to lack of susceptibility-testing facilities. Indonesian MDR-TB rates of 2% as quoted in the 2008 WHO report are derived from the 2003-4 Timika data.31, 112 Hence how the current prevalence of MDR-TB in new cases in Timika compares with other Indonesian provinces and internationally cannot be firmly established. Regardless of the uncommon nature of MDR-TB, each case creates enormous challenges in Timika, where access to second line drugs is limited to kanamycin and levofloxacin. The Indonesian National TB program‘s pilot project to commence MDR-TB treatment in 100 patients only at 2 designated hospitals in East and West Java provinces, have only recently commenced.

92 Chapter 7: Results I - Baseline 7.5 CONCLUSION

AVDAPT study participants are largely representative of the overall population of people seeking treatment at the Timika TB clinic. They have significant disease burden at baseline according to the objective measures of body weight, lung function, exercise tolerance and low haemoglobin, and according to their low perceived quality of life. They are significantly impaired in each of these measures in comparison with local healthy volunteers. Stratification by ethnicity will be important in subsequent analyses, given the differences identified between the 2 ethnic groups in socio-economic status and BMI. Further research to clarify ethnic differences in nutritional status definitions according to BMI is warranted. The establishment of reference ranges for SGRQ and the 6-minute walk test among healthy volunteers at the study site provides essential data by which disability among AVDAPT study participants can be compared, not only at baseline, but also at treatment completion.

93 Chapter 8: Results II- Relationships

8 Results II: Relationships between microbiological, clinical and functional measures of TB severity

This chapter explores the relationships between the TB disease severity measures in AVDAPT study participants described in the preceding chapter.

8.1 INTRODUCTION

Sputum AFB testing provides the basis for many aspects of TB diagnosis and management. Firstly, a finding of AFB in the sputum of a person with appropriate symptoms signifies a high likelihood of pulmonary TB in those with high pre-test probability, such as in high TB-burden countries. Secondly, the public health importance of smear positive status is well established, since smear positivity confers higher infectivity.190 Thirdly, smear positive status provides a measure of pulmonary pathology, since bacillary load correlates with the presence of cavitary disease, as discussed in the next chapter.191, 192 However, less is understood of the relationship between sputum smear grade and other measures of disease severity. The first aim of this chapter is therefore to determine whether baseline weight, FEV1 and six-minute walk test (6MWT) are inversely related, and modified St George‘s Respiratory Questionnaire (SGRQ) score, cough severity, symptom tally (number of reported symptoms) and illness duration (in months), are directly related, to sputum smear grade.

Although an association between diagnostic delay and increased TB-related morbidity is intuitive, such a relationship is not universally apparent. For instance, a recent study by Vree and colleagues failed to find an association between length of diagnostic delay and either sputum smear grade at diagnosis or mortality.193 Therefore the second aim of this chapter is to determine whether illness duration prior to TB diagnosis is associated with disease severity measures at baseline, and to further explore the correlations among the severity measures, which form secondary end-points of the AVDAPT study.

There is a clear association between socio-economic status and TB,194 although some studies suggest that associations between lower socio-economic status and pulmonary TB risk are not always apparent.195, 196 Having established in the previous chapter that Papuans in this dataset are relatively socio-economically disadvantaged, the third aim of 94 Chapter 8: Results II- Relationships this chapter is to investigate the relationship between socio-economic indicators and diagnostic delay or disease severity.

8.2 METHODS

Methods relating to recruitment and testing of AVDAPT study participants between June 12th 2008 and October 3rd 2009 are described in Chapters 6 and 7. Statistical analyses were performed using Intercooled Stata 10.1 and graphs were created in GraphPad Prism 5. Statistical tests were two-sided, with significance for p-value <0.05. Intergroup differences in means or medians were compared using 2-sample t-tests, Wilcoxon rank sum tests, analysis of variance or Kruskal-Wallis tests as appropriate. Intergroup differences in proportions were compared using Pearson‘s  or Fisher‘s exact tests. Regression models were used to examine relationships between variables and control for potential confounders. The goodness of fit of logistic regression models was assessed using the Hosmer-Lemeshow test. For linear regression models, residuals were examined to ensure that relevant assumptions were met. For multiple comparisons, a correlation matrix was constructed with and without application of Bonferroni corrections.

8.3 RESULTS

Results were obtained for 162 enrolled study participants, whose baseline characteristics are described in Chapter 7.

SPUTUM AFB GRADE VERSUS CLINICAL AND LABORATORY MEASURES

In Chapter 7, sputum AFB grade at baseline (graded as 0, scanty, 1, 2 or 3+) was shown to have no significant relationship to sex, ethnicity, age, or smoking status. Sputum AFB grade at diagnosis was highly predictive of sputum smear status after 8 weeks‘ treatment (p<0.0001, test): 58 of 66 (87.9%) people whose initial sample was graded <2+ achieved culture conversion by 8 weeks, compared with 37 of 59 (62.7%) whose initial sample was graded as 2 or 3+.

The sputum AFB grade was significantly related to radiological severity, including both presence of cavitation on X-ray (p=0.002, test) and overall X-ray score (p=0.007, Krsuskal Wallis test, see Table 8.1). It was also significantly related to nutritional status

95 Chapter 8: Results II- Relationships (weight, p=0.006 or BMI, p=0.03 see Table 8.1) in univariate analysis, and in multivariate models controlling for ethnicity, sex, age, smoking, HIV and MDR-TB. The magnitude of these associations is illustrated in Figure 8.1. Although there was an inverse relationship between AFB grade and exercise tolerance (6MWT), this did not reach statistical significance. No significant associations were identified in univariate or multivariate analyses between AFB grade and illness duration, symptom tally, FEV1, Hb or SGRQ score (Table 8.1).

Figure 8.1: Relationship between smear grade at TB diagnosis and CXR score and weight

(a) CXR score (p=0.007) (b) Weight (p=0.006)

120 60

100

80 50 60

40 40 Weight in Weightkg in

20 95%(mean, CI) CXR score (median, IQR) CXR (median, score 0 30 0 Scanty 1 2 3 0 Scanty 1 2 3 Sputum AFB grade at diagnosis Sputum AFB grade at diagnosis

Table 8.1:Association between sputum smear grade at diagnosis and other measures of disease severity

Variable 1 Variable 2 Test Significance

Sputum smear grade 2-month sputum smear  P<0.0001

Cough severity  p=0.7 Chest x-ray Score Kruskal-Wallis p=0.007 Presence of cavitation  p=0.002 Univariate regression models Independent variable Dependent variable Coefficient Significance Sputum smear grade Illness duration* 0.11 p=0.2 Symptom tally 0.33 p=0.1 Weight -1.53 p=0.006 Bmi -0.42 p=0.03 SGRQ† 0.16 p=0.2

6MWT‡ -9563.5 p=0.09 %predicted FEV1 -2.05 p=0.2 Hb -0.21 p=0.2 Transformations required to achieve normal distribution: *logarithmic transformation; †square root transformation; ‡square transformation.

96 Chapter 8: Results II- Relationships

RELATIONSHIPS AMONG OTHER VARIABLES AT BASELINE Relationships between the baseline clinical and laboratory measures were examined using pair-wise correlations, applying Bonferroni correction for multiple comparisons (correlation matrices, Table 8.3a and b). Any associations between these measures and demographic variables are presented in the previous chapter.

Correlations found to be significant before Bonferroni correction were investigated further in univariate linear regression models, and multivariate models controlling for age, sex, ethnicity, smoking status and HIV status. The associations listed below were found to be significant, including in the multivariate analyses. The regression coefficient and p value for each relationship is shown in Table 8.2. The magnitudes of the relationships are illustrated in Figures 8.2-8.3. (1) Longer duration of illness prior to diagnosis and treatment was associated with more symptoms, lower body weight, higher (worse) SGRQ total scores, shorter 6-minute

walk distances and lower %predicted FEV1;

(2) The %predicted FEV1 was associated with lower body weight and haemoglobin; (3) Higher reported number of symptoms at baseline was associated with higher (worse) SGRQ total scores and shorter 6-minute walk distances. (4) Shorter 6-minute walk distances were associated with higher (worse) SGRQ total scores. Table 8.2: Regression coefficients from univariate linear regression models examining associations between clinical and laboratory measures*

Dependent variable Independent variable Regression coefficient P value Symptom tally Illness duration 0.58 p=0.02 Weight Illness duration -1.47 p=0.01 6MWT Illness duration -14957.7 p=0.007 SGRQ Illness duration 0.54 p<0.0005 % Pred FEV1 Illness duration -6.73 p<0.0005 % Pred FEV1 Weight 0.65 p=0.001 % Pred FEV1 Hb 2.86 p=0.001 SGRQ % Pred FEV1 -0.28 p<0.0005 SGRQ 6MWT -0.01 p<0.0005 SGRQ Symptom tally 0.29 p<0.0005 6 minute walk Symptom tally -5347.3 p=0.02 *Each association shown remained significant in multivariate models controlling for age, sex, ethnicity, smoking status and HIV status. Transformations required to achieve normal distribution include logarithmic transformation (illness duration), square root transformation (SGRQ) and square transformation (6MWT), accounting for the high regression coefficients with the latter.

97 Chapter 8: Results II- Relationships

Table 8.3: Correlation matrices showing correlation coefficients (top number) and p values (bottom number) for baseline clinical and laboratory measures*

(a) Without Bonferroni correction

Illness Symptom Weight SGRQ 6 min %Pred Hb duration tally score walk FEV1 test Illness 1.00 duration Symptom 0.16 1.00 tally 0.04 Weight -0.20 -0.20 1.00 0.01 0.01 SGRQ 0.32 0.5037 -0.0881 1.00 score 0.0001 >0.0001 0.2989 6 min walk -0.21 -0.22 0.24 -0.48 1.00 test 0.007 0.006 0.003 >0.0001 %Pred -0.34 -0.08 0.25 -0.34 0.16 1.00 FEV1 >0.0001 0.3 0.001 >0.0001 0.04 Hb -0.14 -0.03 0.20 -0.16 0.23 0.25 1.0 0.07 0.75 0.01 0.06 0.004 0.001

(a) With Bonferroni correction

Illness Symptom Weight SGRQ 6 min %Pred Hb duration tally score walk FEV1 test Illness 1.00 duration Symptom 0.16 1.00 tally 0.8 Weight -0.20 -0.20 1.00 0.3 0.2 SGRQ 0.32 0.50 -0.09 1.00 score 0.003 >0.0001 1.0000 6 min walk -0.21 -0.22 0.2384 -0.48 1.00 test 0.2 0.1 0.05 >0.0001 %Pred -0.34 -0.08 0.25 -0.34 0.16 1.00 FEV1 0.0002 1.0000 0.03 0.001 0.9 Hb -0.14 -0.03 0.20 -0.16 0.23 0.25 1.00 1.00 1.00 0.3 1.00 0.08 0.02 *Coefficients and p values calculated using pair-wise correlation with Bonferroni correction for multiple comparisons in (b). Significant p values marked in bold.

98 Chapter 8: Results II- Relationships

Figure 8.2: Relationships between lung function impairment and weight, haemoglobin, illness duration and quality of life total score (SGRQ)

60

50

40

Weight in Weightkg in (mean, 95%(mean, CI)

30

Mild Mod 16 V.sev Normal Severe Mod-Sev Lung function according to % predicted FEV1 14

12 Hb in g/dL Hb in

10 (mean, 95%(mean, CI)

Mild Mod 100 V.sev Normal Severe Mod-Sev Lung80 function according to % predicted FEV1

60

40

(mean, 95%(mean, CI) 20 SGRQ SGRQ total score

0

Mild Mod 10 V.sev Normal Severe Mod-Sev 8Lung function according to % predicted FEV1

6

4

(median, IQR) (median, 2

0 Illness duration in months in duration Illness

Mild Mod V.sev Normal Severe Mod-Sev Lung function according to % predicted FEV1

Lung function impairment is defined according to established criteria: Normal >80% predicted FEV1; mild impairment 70-79; moderate impairment 60-69; moderate-severe impairment 50-59; severe impairment 35-49; very severe impairment <35.171

99 Chapter 8: Results II- Relationships

Figure 8.3: Relationship between number of reported symptoms and illness duration and quality of life (SGRQ) score

(a) Illness duration (b) SGRQ total score

6 80

70 5 60 4 50

3 40

30 2

SGRQ SGRQ total score 20 1 Illness duration (months) duration Illness 10

0 0 <5 5-10 >10 <5 5-10 >10 Number of symptoms reported Number of symptoms reported

Dots indicate median, whiskers show interquartile ranges.

A modest association between longer diagnostic delay and lower educational level was also identified (p=0.01, Figure 8.4). No other significant associations were found between the 3 socio-economic indicators (educational attainment, employment and telephone ownership) and either the length of diagnostic delay or the severity of disease.

Figure 8.4: Illness duration in relation to educational attainment

14 p=0.01*

12

10

8

6

(median, IQR) (median, 4

2 Illness duration in months in duration Illness

0

High school No schooling Academy/Uni Primary school

*p value calculated using a rank-sum test comparing no/primary school with high school/academy

8.4 DISCUSSION

Salient findings from this chapter include that, among participants in the AVDAPT study, sputum AFB grade was associated with baseline weight and radiological disease extent, but not other measures of disease severity; lesser educational attainment was associated with diagnostic delay, and diagnostic delay was in turn associated with higher morbidity. Disease severity measures reported here, encompassing objective 100 Chapter 8: Results II- Relationships physiological and functional measures, and patient-reported subjective assessments of quality of life and symptomatology, provide a broad assessment of illness impacts, and show varying degrees of correlation with each other.

RELATIONSHIPS BETWEEN CLINICAL AND LABORATORY MEASURES

Although sputum AFB grade was significantly related to two widely used measures of TB severity (radiological disease extent and weight), it was not significantly associated with a range of other clinical and functional measures. While bacteriological clearance is undoubtedly of prime individual and public health importance, focussing on bacteriology alone might therefore overlook other clinically important aspects of disease severity. Bacillary grade is determined in large part by cavitation,191 but extensive pulmonary TB can exist in the absence of cavitation (e.g. miliary disease or widespread consolidation), and thereby have significant impacts on lung function and quality of life, yet not be reflected in higher sputum smear grades. These novel findings support the choice of a range of secondary outcome measures in the AVDAPT study. Results of a single sputum specimen have been used in this analysis, which may not be representative of the individual‘s maximum baseline smear grade, but overall accuracy of results is supported by the clear association identified between week 0 and week 8 sputum smear results.

Exploration of the relationships between other baseline measures further highlights the concept that traditional methods of evaluating the TB patient via sputum smear grade and weight provide an incomplete assessment of disease severity. For example, people who were more symptomatic, including greater number of reported symptoms, poorer exercise tolerance and worse lung function, perceived their quality of life to be lower, but quality of life assessments did not correlate with bacillary burden or weight loss.

Limited 6-minute walk distances have previously been reported among people with TB.114, 187 However, the current data provide new understanding of the determinants and consequences of impaired exercise capacity specifically among pulmonary TB patients. Longer illness duration prior to TB diagnosis, a greater number of symptoms, and poorer quality of life scores, were associated with decreased exercise capacity. The absence of an association between FEV1 and exercise capacity might appear unexpected. However, a similarly absent relationship between these variables has also been reported in chronic obstructive airways disease.186 Most importantly, this finding

101 Chapter 8: Results II- Relationships confirms the observation (noted in Chapter 7) that determinants of 6-minute walk distance among people with newly-diagnosed TB include factors other than pulmonary function alone, which direct observation of study participants would suggest includes such factors as fatigue, malaise and dehydration. Other important determinants, such as a pre-existing musculoskeletal mobility disorder, are likely to impact similarly on serial walk test results, suggesting that percentage change rather than absolute values should be evaluated in longitudinal analyses. The 6-minute walk test is therefore a valuable assessment tool, being an objective functional measure which is able to identify different aspects of TB-related disease from other measures described here, and identify those who are most ill at diagnosis.

A 2004 study from Malawi demonstrated that the extent of pulmonary disease, as assessed by chest radiography, was associated with severity of malnutrition (measured using BMI and analyses of body composition) in people with pulmonary TB.197 However, the finding that TB patients with more evidence of malnutrition (lower body weight and haemoglobin) have worse lung function (% predicted FEV1, i.e. adjusted for sex, weight and ethnicity, using locally-derived FEV1 reference ranges) as described here, has not previously been reported. The direction of this relationship cannot be established from this cross-sectional data. While progressive weight loss and worsening lung disease are both an obvious consequence of progressive TB infection, additional interactions might exist. For example, low FEV1 in chronic airways disease is associated with increased metabolic rate, greater caloric requirements, and weight loss,198 and decreased nutritional intake is often clinically apparent as a consequence of dyspnoea; such relationships between impaired pulmonary function and nutrition may also exist in TB. Conversely, malnutrition may impair lung tissue response to pathological insults. This finding lends support to the hypothesis that nutritional interventions in TB (either specific micronutrients such as L-arginine and vitamin D, or wholefood nutritional supplementation), might have wider benefits than direct improvement of nutritional parameters alone.

DIAGNOSTIC DELAY

Diagnostic delay is multifactorial and has been extensively investigated elsewhere.199 Lower educational attainment was associated with delayed diagnosis in these study participants, thereby placing Papuans at greater risk of prolonged illness duration prior

102 Chapter 8: Results II- Relationships to diagnosis. This information can assist in targeting future public health strategies in Timika to this at-risk group. A range of additional explanations operating at patient and health service-provider levels are likely to play roles in diagnostic delay, although investigation of these is beyond the scope of this study.

Diagnostic delay is clearly associated with greater morbidity among the study participants. Although reported illness duration is subject to substantial recall bias, associations (which remained significant in multivariate analyses) were nevertheless identified between illness duration and body weight, exercise tolerance, lung function, symptom tally and quality of life score. Although the public health importance of early diagnosis to avoid community transmission is widely appreciated, the findings reported here additionally emphasise the major importance at the level of the individual of making a timely diagnosis of TB, in order to preserve functional capacity and quality of life. While impairments in functional capacity and quality of life respond to TB treatment, post TB residual deficits in these measures are more likely in those with more advanced disease at baseline.114, 169 Patients who have more advanced disease by virtue of delayed diagnosis could potentially be even more likely to benefit from adjunctive therapies which might accelerate recovery. This will be able to be investigated in future analyses of the AVDAPT trial.

8.5 CONCLUSION

Among the secondary outcomes which will be examined in the AVDAPT study are

FEV1, weight gain, cough clearance, sputum smear conversion time, exercise tolerance (6MWT) and quality of life assessment (SGRQ) (see Chapter 6). Other outcomes (composite clinical score, exhaled nitric oxide and radiological improvement) are addressed in subsequent chapters. Results presented here indicate that significant correlations exist between some but not all of these measures, indicating that they successfully capture a comprehensive range of physiological and functional aspects of pulmonary TB, rather than merely providing alternative measures of the same processes. Original findings identified in these interim analyses will be re-assessed and submitted for publication at the time of full sample size recruitment.

103 Chapter 9: Results III – X-ray

9 Results III: Measuring TB radiological severity

TB severity and response to treatment can be assessed on clinical, bacteriological and radiological grounds. Radiological improvement is a secondary outcome measure of the AVDAPT trial. On reviewing the literature, it became apparent that a variety of X-ray scores are in current use for grading radiological severity in adult pulmonary TB, raising the question of which method ought to be selected for use in the trial. Currently- available systems have not been validated against outcomes, and this area is lacking in standardisation. The opportunity to develop a simple x-ray scoring method arose since radiological data had been previously collected at the field research site, and a score derived from this older dataset could be validated in the current AVDAPT data.

This chapter therefore addresses Aim 5, described in Chapter 1, of developing and validating a numerical score for grading chest X-ray severity in adults with smear positive pulmonary TB, and monitoring response to therapy. This analysis is currently under review for publication. The submitted manuscript is inserted below.

Manuscript 4: Ralph AP, Ardian M, Wiguna A, Maguire GP, Becker NG, Drogumuller D, Wilks MJ, Waramori G, Tjitra E, Sandjaja, Kenangalem E, Pontororing GJ, Anstey NA, Kelly PM. A simple, valid, numerical score for grading chest X-ray severity in adult smear positive pulmonary tuberculosis. Accepted, Thorax, July 2010

104 Topics: xxx

Tuberculosis

1 65 2 A simple, valid, numerical score for grading chest 66 3 67 4 x-ray severity in adult smear-positive 68 5 69 6 pulmonary tuberculosis 70 7 71 8 Anna P Ralph,1,2 Muhamed Ardian,3,4,5 Andri Wiguna,4,5 Graeme P Maguire,6 72 9 2 7 8 4 73 10 Niels G Becker, Glen Drogumuller, Michael J Wilks, Govert Waramori, 74 9 9 3,10 10 11 Emiliana Tjitra, Sandjaja, Enny Kenagalem, Gysje J Pontororing, 75 12 Nicholas M Anstey,1,11 Paul M Kelly1,2 76 13 77 14 78 15 1Global Health Division, Menzies ABSTRACT in 1960,1 Simon in 19662 and the National TB and 79 16 School of Health Research and Background The grading of radiological severity in Respiratory Disease Association of the USA in 1969.3 80 Charles Darwin University, clinical trials in tuberculosis (TB) remains unstandardised. Despite this, no system has been validated in 17 Australia 81 18 2National Centre for The aim of this was to generate and validate a numerical predicting outcome in more than one patient popu- 82 19 Epidemiology and Population score for grading chest x-ray (CXR) severity and lation. Recent randomised controlled trials 83 e 20 Health Research, College of predicting response to treatment in adults with (RCTs)4 7 and observational studies in adults with 84 e 21 Medicine, Biology and smear-positive pulmonary TB. TB8 13 illustrate this lack of standardisation in the 85 Environment, Australian 22 National University, Australia Methods At a TB clinic in Papua, Indonesia, serial CXRs grading of radiological severity, with each of these 86 23 3District Health Authority, were performed at diagnosis, 2 and 6 months in 115 studies utilising different non-validated investigator- 87 24 Timika, Papua Province, adults with smear-positive pulmonary TB. Radiographic generated systems to grade CXR severity. 88 25 Indonesia findings predictive of 2-month sputum microscopy status The same problem of non-standardised radio- 89 4International SOS, Timika, 26 were used to generate a score. The validity of the score logical reporting has been recently articulated by 90 Papua Province, Indonesia et al 27 5Public Health & Malaria Control was then assessed in a second data set of 139 Dawson relating to TB screening, who evalu- 91 28 Department, PT Freeport comparable adults with TB, recruited 4 years later at the ated and recommended the Chest Radiographic 92 29 Indonesia, Papua Province, same site. Relationships between the CXR score and Reading and Reporting System14 for TB screening 93 Indonesia 15 30 6 other measures of TB severity were examined. in HIV-positive people. However, this and other 94 School of Medicine and 16e18 31 Dentistry, James Cook Results The estimated proportion of lung affected and screening tools seek to identify the presence of 95 32 University, Queensland, presence of cavitation, but not cavity size or other latent TB infection or active disease, and are not 96 33 Australia radiological findings, significantly predicted outcome and useful for researchers wishing to accurately document 97 7 34 Department of Radiology, Royal were combined to derive a score given by percentage of severity or response to treatment in established TB. 98 Darwin Hospital, Northern lung affected plus 40 if cavitation was present. As well Problems in CXR reporting arise from the hetero- 35 Territory, Australia 99 36 8Radiology Department, Royal as predicting 2-month outcome, scores were geneous CXR manifestations of pulmonary TB (eg, 100 37 Adelaide Hospital, North significantly associated with sputum smear grade at in primary vs postprimary disease, adults vs children, 101 e 38 Terrace, Adelaide, South diagnosis (p<0.0001), body mass index, lung function, immunocompetent vs immunocompromised)19 21 102 Australia 39 9 haemoglobin, exercise tolerance and quality of life and to inaccuracies inherent in CXR performance 103 National Institute of Health < 2 40 Research and Development, (p 0.02 for each). In the validation data set, baseline and interpretation, including limited interobserver 104 22 23 41 Jakarta, Indonesia CXR score predicted 2-month smear status significantly agreement on CXR findings. Despite these 105 42 10Menzies School of Health more accurately than did the proportion of lung affected shortcomings, the utility of CXR is well established 106 43 Research-National Institute of alone. In both data sets, CXR scores decreased over time in TB diagnosis and clinical monitoring. 107 Health Research and (p<0.0001). Associations between radiological extent and 44 Development Research 108 45 Program, and District Ministry of Conclusion This simple, validated method for grading other measures such as forced expiratory volume in 109 46 Health, Timika, Papua Province, CXR severity in adults with smear-positive pulmonary TB 1 s (FEV1), age or multidrug-resistant (MDR)-TB 110 Indonesia correlates with baseline clinical and microbiological have previously been identified,8 24 but a standard, 47 11 111 48 Division of Medicine, Royal severity and response to treatment, and is suitable for simple, numerical score, validated against TB 112 Darwin Hospital, Darwin, 49 Australia use in clinical trials. outcome, in repeated data sets, is lacking. We 113 50 therefore aimed to devise a simple CXR score for 114 51 Correspondence to use in adults with smear-positive pulmonary TB, 115 52 Dr Anna Ralph, International which predicts outcome and correlates with 116 53 Health Division, Menzies School bacteriological and clinical severity markers, for the 117 of Health Research, PO Box INTRODUCTION 54 41096, Casuarina, Northern Sputum smear microscopy, and culture where purpose of grading severity and monitoring treat- 118 55 Territory, 0811, Australia; available, are standardised modalities for diagnosing ment response in the context of TB clinical trials. 119 56 [email protected] and monitoring treatment response in pulmonary We then determined the utility of the score in 120 57 a separate, comparable patient population. 121 Received 8 February 2010 tuberculosis (TB). Chest radiography (CXR) 58 Accepted 30 June 2010 provides useful information regarding disease extent 122 59 and progress, but there is no agreed-upon, validated METHODS 123 60 system for grading the severity of CXR abnormali- Study setting 124 61 ties in bacteriologically proven pulmonary TB. The study was conducted at a community-based 125 62 Several methods were devised for this purpose at the TB clinic in Timika, Papua Province, Indonesia. 126 63 time of early TB treatment trials, such as those Timika has population of w200 000 and an 127 64 described by the Madras TB Chemotherapy Centre estimated TB incidence of 311/100 000.25 128

Ralph AP, Ardian M, Wiguna A, et al. Thorax (2010). doi:10.1136/thx.2010.136242 1 of 7 Tuberculosis

129 Participants of outcome,35 in the absence of suitable alternatives, it remains 193 130 Adults (>15 years) diagnosed with sputum smear-positive a commonly used surrogate end point. 194 131 pulmonary TB who gave written informed consent were eligible 195 132 for enrolment in the study. Study participants were recruited Data analysis 196 133 during two time periods: 2003e2004 (the training data set) and Statistical calculations were performed using Intercooled Stata 197 134 2008e2009 (the validation data set). The demographic, clinical 10.1 (StataCorp, College Station, Texas, USA); graphs were 198 135 and microbiological findings and outcomes in the first data set created in GraphPad Prism 5. Statistical tests were two sided, 2 199 136 have been reported previously.26 27 with a p value of <0.05 indicating statistical significance. 200 137 Intergroup differences in means or medians were compared 201 138 Chest radiography using two-sample t tests, Wilcoxon rank sum tests, analysis of 202 139 Standard full-size posteroanterior CXR were performed at the variance or KruskaleWallis tests as appropriate. 203 140 time of TB diagnosis and 2 and 6 months thereafter, with Agreement between reporters in the derivation data set was 204 fi 141 reports provided by a clinician at the field site (first data set, tested using the concordance coef cients, rhoc for continuous 205 142 PMK; second data set, APR) and, additionally for the first data variables or the kappa statistic for categorical variables. Preva- 206 143 set, by one of two radiologists (MJW or GD). During the first lence-adjusted, bias-adjusted kappa values were calculated 207 144 data collection period, the presence of small (1e2 mm) or large according to the method described by Byrt et al.36 Kappa values 208 145 (>2 mm) nodules, patchy or confluent consolidation, cavitation, were interpreted according to guidelines given by Landis and 209 146 bronchial lesions or fibrosis was reported for each of three zones Koch37 (kappa #0.00, poor; 0.00e0.20, slight; 0.21e0.40, fair; 210 147 (upper, mid or lower zones) in each lung. The presence of effu- 0.41e0.60, moderate; 0.61e0.80, substantial; 0.81e1.00, almost 211 148 sion or lymphadenopathy was reported, the total percentage of perfect). 212 149 each lung affected by any pathology was estimated, total cavity The relationships between radiographic findings and clinical 213 150 size in millimetres was recorded and the effusion volume outcome were examined by multivariable regression analysis, 214 151 (percentage of lung field) was estimated. To grade the percentage using a forward stepwise approach in which any radiological 215 152 of affected lung, visual estimation of the extent of opacification, variable found to be significant (p<0.05) in univariate analysis 216 153 cavitation or other pathology as a percentage of visible lung was was included in the initial model. Goodness of fitoffinal models 217 154 made; dense opacification of a zone was graded as 100% of that was assessed using the HosmereLemeshow test and compared 218 155 zone, while patchy opacification within a zone attracted scores using the likelihood ratio test. The weighting for a numerical 219 156 <100% depending on the extent of opacification. Other remarks radiological score was derived from the regression coefficients. 220 157 including presence of miliary disease were recorded. During the Its ability to predict outcome in the validation data set was 221 158 second data collection period, a simplified CXR report method determined using receiver operator characteristics (ROC; area 222 159 was used (percentage lung affected, cavitation (0, <4 cm, under the curve (AUC)). The relationships between this score 223 160 $4 cm), effusion (0, <25%, $25% of hemithorax), presence of and demographic, biological and clinical variables were deter- 224 161 consolidation, fibrosis, nodules, miliary disease). Reporters were mined in data sets 1 and 2 using regression models using the 225 162 blinded to HIV status, bacteriological and clinical parameters same principles. 226 163 and treatment outcome. 227 164 Ethics 228 165 Sputum microscopy and clinical evaluations Approval was granted by the ethics committees of the National 229 166 Baseline sputum microscopy was performed at the onsite labo- Institute of Health Research and Development (Jakarta, 230 167 ratory and repeated at the reference laboratory on samples Indonesia), Menzies School of Health Research (Darwin, 231 168 collected at 0, 2 and 6 months, and the density of acid-fast bacilli Australia) and the Australian National University (Canberra, 232 169 (AFB) was graded as 1, 2 or 3+ according to standard proto- Australia). Written informed consent was obtained from 233 170 cols.26 27 Baseline and follow-up evaluations included: body mass participants in Indonesian or an appropriate Papuan language. 234 171 235 index (BMI), FEV1 (spirometry performed using ML3535C, 172 1 MicroLoop, MicroMedical), haemoglobin (Hb), measured using RESULTS 236 173 point-of-care HemaCue or iSTAT tests, 6 min walk test Characteristics of study participants in the two data collection 237 174 (distance walked in 6 min on a straight walking track), measured phases are shown in table 1. All participants had smear-positive 238 175 according to American Thoracic Society guidelines, and pulmonary TB ($2 AFB smear-positive sputum samples); the 239 176 St George’s Respiratory Questionnaire (SGRQ) modified to result of an additional sample provided for microscopy and 240 177 reflect local conditions and translated into Indonesian.27 28 culture on the day of treatment commencement is reported here. 241 178 Standard definitions were used for nutritional category (normal, This was negative in 5.7% and 7.2% of participants in the two 242 179 mild malnutrition, moderate malnutrition or severe malnutri- data sets, respectively despite their previous samples being 243 180 tion) according to BMI,29 and for TB treatment outcome at positive. Initial smear grade predicted the likelihood of smear 244 181 6 months (cured, completed, transferred, defaulted, failed or conversion by 2 months. In the derivation data set, failure to 245 182 30 246 died). Impairment in FEV1 as a percentage of predicted values convert to smear negative by 2 months was observed in 60.9% of 183 was calculated using previously established local reference patients with a baseline smear grade of 3 and in 38.7% of 247 184 ranges.31 patients with a baseline smear grade of <3+ (p¼0.051). In the 248 185 validation data set, failure to convert to smear negative by 249 186 Outcome measure 2 months was observed in 48.4% of patients with a baseline 250 187 The outcome measure used in this study is 2-month sputum smear grade of 3 and in 11.8% of patients with a baseline smear 251 188 AFB microscopy status. Two-month smear positivity has been grade of <3+ (p<0.001). 252 189 previously shown to predict unfavourable outcomes including CXR reports were available at baseline, 2 and 6 months for 253 e 190 treatment failure and death,32 34 and determines the need for 112, 76 and 76 study participants in the first data set, and 136, 254 191 continued intensive-phase treatment versus switching to 93 and 76 study participants in the second data set (incomplete 255 192 continuation-phase therapy.30 Although an imperfect predictor in the second data set as 30 of 139 had not yet completed 256

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257 Table 1 Study participant characteristics Table 2 Chest radiograph (CXR) results 321 258 Derivation data set Validation data set Derivation Validation 322 259 data set data set 323 260 Demographic details 324 Number of CXR available for report 261 Number 115 139 325 Age in years: median (range) 30 (17e69) 27 (15e65) Baseline 112 136 262 326 Female gender, n (%) 33 (28.7) 48 (34.5) 2 months 76 93 263 327 Papuan ethnicity, n (%) 57 (49.6) 66 (47.5) 6 months 76 76* 264 328 Smokers, n (%) 38 (33.0) 41 (29.5) Baseline radiological findings 265 HIV positive: no./no. tested (%) 5/112 (4.5) 16/121 (13.2) Consolidation, n (%) 106 (94.6) 104 (76.5) 329 266 MDR-TB, n (%) 2 (1.7) 2 (1.4) Cavitation, n (%) 71 (63.4) 77 (56.6) 330 267 Baseline clinical findings Effusion, n (%) 37 (33.0) 24 (17.3) 331 268 BMI, median (range), kg/m2 18.6 (14.2e25.2) 19.0 (12.9e32.5) Miliary, n (%) 2 (1.7) 5 (3.7) 332 269 Haemoglobin, median (range), g/dl 11.2 (6.8e18.0) 12.2 (7.1e16.0) Nodules, n (%) 83 (72.2) 29 (21.3) 333 Fibrosis, n (%) 62 (55.3) 10 (7.4) 270 FEV1: median (range), litres 1.76 (0.49e4.12) 1.70 (0.59e3.56) 334 271 SGRQ total score, median (range) 45.3 (2.5e83.5) 37.8 (5.2e91.9) Proportion of lung affected, median % (range) 35 (0e100) 41.5 (0e100) 335 272 6 min walk distance, median (range) m 405 (185e625) 410 (20e612) CXR weighted score characteristics 336 273 Sputum AFB smear grade at diagnosis n (%) Baseline score, median (range) 65 (5e130) 69 (0e140) 337 274 0* 6 (5.7) 10 (7.2) 2 month score, median (range) 35 (0e130) 29 (0e140) 338 275 Scanty or 1+ 25 (23.6) 65 (45.7) 6 month score, median (range) 10 (0e105) 10 (0e115) 339 276 2+ 28 (26.4) 35 (25.2) Median score decrease in 2 months, % 27.3 38.8 340 277 3+ 47 (44.3) 29 (20.9) Median score decrease in 6 months, % 73.1 80.2 341 278 2-month smear status n (%) Baseline score by smear grade at diagnosis median (range) 342 0 15 (0.5e70) 10 (0e133) 279 Positive 25 (21.8) 31 (22.3) 343 Scanty or 1+ 50 (0e110) 66 (0e140) 280 Negative 81 (70.4) 95 (68.4) 344 2+ 70 (6.5e135) 76 (11e140) 281 No result available 9 (7.8) 13 (9.3) 345 3+ 80 (10e140) 85 (4e140) 282 6-month outcome n (%) 346 Cured/completed 88 (76.5) 92 (66.2) 283 *30 patients still under follow up. 347 Died 3 (2.6) 2 (1.4) 284 348 Failed 2 (1.7) 1 (0.7) 285 had worse lung function, with a mean percentage predicted 349 Default 13 (11.3) 8 (5.8) FEV of 59.0 (95% CI 54.4 to 63.6) in cavitary disease versus 68.7 286 Transferred 9 (7.8) 6 (4.3) 1 350 (95% CI 60.6 to 76.7) in non-cavitary disease (p¼0.03, 287 6 months not yet completed 0 30 (21.6) 351 288 two-sample t test). People with cavitary disease had slightly 352 *All study participants had at least two prior smear-positive sputum samples; some are 289 lower BMI (18.5, 95% CI 18.0 to 19.1) compared with those 353 reported as negative since this result pertains to the additional spot specimen provided at 2 290 enrolment into the study (see the methods section). with non-cavitary disease (19.2 kg/m , 95% CI 18.5 to 20.0), but 354 291 AFB, acid-fast bacilli; BMI, body mass index; FEV1, forced expiratory volume in 1 s; this difference was not statistically significantly (mean between- 355 MDR-TB, multidrug-resistant tuberculosis; SGRQ, St George’s Respiratory Questionnaire. 292 group difference 0.70 kg/m2, 95% CI 0.18 to 1.59). No signif- 356 293 icant associations were identified between cavitary disease and 357 6 months) (table 2). Reasons for missing CXR included patient 294 exercise tolerance (6 min walk distance), quality of life (SGRQ 358 failure to attend (died, defaulted or transferred prior to 295 total or individual domain scores) or Hb. 359 appointment), inability to obtain CXR (eg, electricity failure), 296 The amount (%) of lung affected significantly predicted all 360 CXR date >3 weeks before or after the due follow-up date, or 297 clinical and laboratory variables. Specifically, greater proportions 361 CXR unavailable for reporting. 298 of affected lung were significantly associated with decreasing BMI 362 e 299 Agreement on radiological abnormalities category (p¼0.002, Kruskal Wallis test), lung function category 363 300 Agreement between reporters on radiological abnormalities in 364 301 the derivation data set is shown in table 3. Agreement was Table 3 Agreement on radiological findings, derivation data set 3 365 302 relatively low overall. More substantial agreement was achieved Interpretation 366 303 for some variables after adjusting kappa values for variable Prevalence- of prevalence- 367 Inter-rater agreement adjusted, adjusted, 304 prevalence and reporter bias. 368 305 among dichotomous bias-adjusted bias-adjusted 369 variables Kappa kappa kappa 306 Development of score using the training data set 370 Presence of patchy consolidation 0.20 0.70 Substantial 307 Two-month sputum smear status in the initial data set (n¼115) 371 Presence of confluent consolidation 0.33 0.31 Fair 308 was significantly predicted in univariate logistic regression 372 309 Presence of any consolidation 0.24 0.81 Almost perfect 373 models by the presence of baseline cavitation (OR 3.26, 95% CI Presence of small nodules 0.12 0.06 Slight 310 1.11 to 9.56) and the total percentage of lung affected (OR 1.9, 374 311 Presence of large nodules 0.09 0.09 Slight 375 95% CI 1.3 to 2.7, calculated per each 20% increment of affected Presence of any nodules 0.19 0.19 Slight 312 376 lung), but not by cavitation size, presence or number of nodules, Presence of fibrosis 0.08 0.06 Slight 313 fi 377 brosis, effusion or lymph nodes (table 4). Presence of cavitation 0.33 0.37 Fair 314 378 The relationships between those radiological findings which Presence of effusion -0.35 -0.61 Poor 315 were independently predictive of 2-month outcome (cavitation Presence of lymphadenopathy 0.01 -0.09 Poor 379 316 and percentage of lung affected) and baseline clinical and 380 317 Concordance among 95% limits of agreement 381 bacteriological measures were then examined. Cavitary disease continuous variables rhoc (Bland and Altman) 318 on CXR at TB diagnosis was significantly associated with higher 382 Total amount (%) of lung affected 0.85 28.20 to 22.46 % 319 baseline AFB density in sputum (ie, smear microscopy grade) 383 Extent of cavity size (mm) 0.69 56.62 to 50.66 mm 320 (p¼0.007, c2 test for trend), and people with cavitary disease 384

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385 Table 4 Relationship between radiological and biological parameters 449 386 and 2-month sputum acid-fast bacilli density in the derivation data set, 450 387 showing results of univariate logistic regression analyses*. 451 388 Odds ratio 95% CI p Value 452 389 453 Radiological parameters 390 454 Presence of any consolidation 391 Per 1% increments 1.30 0.14 to 12.18 0.001 455 392 Per 20% increments 1.90 1.30 to 2.68 0.001 456 393 Cavitary disease 3.26 1.11 to 9.56 0.032 457 394 Effusion 1.50 0.59 to 3.82 0.590 458 395 Nodules 1.17 0.41 to 3.32 0.773 459 396 Fibrosis 2.23 0.86 to 5.79 0.097 460 397 Lymphadenopathy 1.43 0.57 to 3.56 0.448 461 398 *OR was unable to be calculated for the two instances of military disease (both had 462 399 negative sputum at 2 months). 463 400 464 401 (p<0.001, KruskaleWallis test), 6 min walk distance (0¼0.001, 465 402 linear regression) and Hb in males (p¼0.003, linear regression), 466 Figure 2 Chest x-ray (CXR) score according to body mass index (BMI), 403 though not in females (p¼0.4). A greater proportion of lung 467 404 fi percentage of predicted forced expiratory volume in1 s (FEV1) and 468 affected on the baseline CXR was also signi cantly associated haemoglobin (Hb) at diagnosis (derivation data set). 405 with SGRQ total scores (p<0.0001, linear regression) and with 469 406 sputum smear grade at diagnosis (p<0.001, KruskaleWallis test). 470 significantly associated with age in univariate or multivariate 407 To create a CXR score, cavitation and percentage of lung 471 analyses. Mean baseline CXR score in people with unfavourable 408 affected were included as independent variables in a logistic 472 (positive) 2-month outcomes was significantly higher (88.2; 95% 409 regression model for 2-month sputum smear status. The model 473 CI 76.5 to 99.9) than in those with a favourable outcome (56.8; 410 containing both variables was significantly better than the model 474 95% CI 49.7 to 64.0), but the range of scores in each smear grade 411 containing cavitaion alone (likelihood ratio test p<0.001) or 475 was wide (figure 1). Scores were also significantly associated 412 proportion of lung affected alone (likelihood ratio test p¼0.016) 476 with baseline microscopy grade (figure 1). CXR scores were 413 at predicting 2-month sputum smear status. Regression coeffi- 477 inversely related to BMI, FEV , Hb and 6 min walk distance, 414 cients were 0.03167 for proportion of lung affected and 1.26151 1 478 were directly related to SGRQ total score (higher SGRQ scores 415 for presence/absence of cavitation, indicating a relative weighting 479 indicate worse quality of life) and significantly decreased over 416 of 40.27 for cavitation (1.26151O0.03167), thereby generating an 480 time (figures 2e4). 417 equation for the weighted score as follows: 481 418 482 CXR score ¼ proportion of total lung affectedð%Þ 419 Performance of score using the validation data set 483 420 þ 40 if cavitation present The weighted score calculated for the new data set showed 484 421 similar characteristics (table 2), including a median baseline 485 fi 422 CXR score results score of 69, no signi cant relationship with demographic factors, 486 significant positive association with baseline smear grade 423 CXR score characteristics are shown in table 2 and figures 1e4. 487 (p¼0.009, KruskaleWallis test) and the same relationships as 424 Scores did not significantly differ according to sex, ethnicity or 488 425 smoking status (p>0.05, two-sample t tests), and were not 489 426 490 427 491 428 492 429 493 430 494 431 495 432 496 433 497 434 498 435 499 436 500 437 501 438 502 439 503 440 504 441 505 442 506 443 507 444 508 445 509 446 Figure 3 Chest x-ray (CXR) score according to St George’s Respiratory 510 447 Figure 1 Chest x-ray (CXR) score at enrolment according to sputum Questionnaire (SGRQ) (total score and 6 minute walk distance at 511 448 acid-fast bacilli (AFB) grade at diagnosis and 2 months (derivation data set). tuberculosis diagnosis (derivation data set). 512

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513 Figure 4 Chest x-ray (CXR) score at 577 514 diagnosis, 8 weeks (end of intensive 578 515 treatment phase) and 24 weeks (end of 579 516 treatment). 580 517 581 518 582 519 583 520 584 521 585 522 586 523 587 524 588 525 589 526 590 527 591 528 592 529 593 530 594 531 595 532 were found in the initial data set between CXR score and each of smear positivity at 2 months and provides a single numerical 596 533 the clinical/laboratory measures (BMI, Hb, FEV1, SGRQ total score for each CXR. The score shows good correlation with 597 < 534 score and 6 min walk distance; p 0.05 in each case). baseline bacteriological and clinical severity markers, and is 598 535 Comparing ROC scores to predict outcome, the weighted sensitive to changes over time. The score performs better than 599 fi fi 536 CXR score (AUC 0.75) was signi cantly better at predicting 2- its individual components: it was signi cantly better at 600 537 month smear status than the percentage lung affected alone predicting outcome than was the percentage of lung affected 601 fi fi 538 (ROC 0.69; p¼0.013, c2 test; gure 5). The optimal cut-off point alone, and was signi cantly associated with a broader range of 602 539 for weighted CXR score (value furthest from the diagonal) was baseline severity measures (BMI, |Hb, exercise tolerance and 603 540 71, at which value the sensitivity for predicting a positive quality of life) than presence of cavitation alone. Advantages of 604 541 sputum smear status at 2 months was 80% (95% CI 61.4 to this method are that CXR assessment does not require aids, grids 605 fi fi 542 92.3) and speci city 67.7% (95% CI 57.3 to 77.1). Comparative or rulers, and it is derived by tting a statistical model to 606 fi 543 sensitivity and speci city values are shown in table 5. outcome data rather than by assigning points based on assumed 607 544 relative importance of radiographic pathologies. It has been 608 545 DISCUSSION 609 546 The current need for a universal and standard system for Table 5 Sensitivity and specificity of baseline radiological and 610 547 reporting CXR in pulmonary TB is acknowledged.38 In order to bacteriological findings in predicting outcome (2-month sputum acid-fast 611 548 grade CXR severity and assess radiological treatment response, bacilli (AFB) status) 612 549 we have derived a simple equation from radiographic parameters Sensitivity for predicting Specificity for predicting 613 550 from adults with smear-positive pulmonary TB that predicts positive 2 month smear positive 2 month smear 614 551 Radiological parameters at diagnosis 615 552 Derivation data set 616 553 Total amount of lung affected 617 1.00 554 20% 96 35.9 618 555 37.5% (optimal cut-off point) 84 68.0 619 556 80% 20 89.7 620

557 0.75 Cavitation 80 44.9 621 558 Score 622 559 20 100 18.0 623 72.5 (optimal cut-off point) 80 71.2 560 0.50 624 130 8 98.7 561 Sensitivity 625 562 Validation data set 626 Total amount of lung affected

563 0.25 627 20% 96.7 22.6 564 628 42.0% (optimal cut-off point) 73.3 62.4 565 629 80% 30 87.1 566 630

0.00 Cavitation 83.3 52.7 631 567 0.00 0.25 0.50 0.75 1.00 Score 568 1-Specificity 20 96.7 22.6 632 569 71 (optimal cut-off point) 80 67.7 633 %Lung affected ROC area: 0.6867 Reference 570 130 10 91.4 634 CXR Score ROC area: 0.7471 Optimal cut-point 571 Bacteriological parameters: baseline smear grade 635 572 Derivation data set 4 636 Figure 5 Receiver operator characteristics (ROC) for weighted chest 573 637 x-ray (CXR) score (% lung affected + 40 if cavitation present) (solid line) AFB density* 60.9 61.3 574 compared with percentage of lung affected (dashed line) as predictors of Derivation data set 638 575 2-month smear status (positive or negative). Optimal cut-off point for AFB density * 48.4 88.2 639 576 score (circle) ¼71. *Baseline AFB density (smear grade) dichotomised as #2or3. 640

Ralph AP, Ardian M, Wiguna A, et al. Thorax (2010). doi:10.1136/thx.2010.136242 5 of 7 Tuberculosis

641 validated in an independent data set, and offers a single, stand- data did not indicate this. This method can be used where 705 642 ardised solution where there are currently multiple unvalidated a numerical score is required for the purpose of comparing 706 643 methods in use. radiographic severity between adults with smear-positive 707 644 The proportion of lung affected and/or cavitation feature as pulmonary TB, and to monitor an individual’s improvement 708 645 the most important measures in many TB CXR grading meth- over time, such as in clinical trials of drug efficacy in TB. 709 e 646 ods.1 5 7 Cavitation is well recognised to correlate with bacillary 710 647 load.739We confirmed the association between cavitation and Acknowledgements We thank the following for their support and assistance: 711 648 bacteriological measures (baseline and 2-month sputum smear Dr M Okoseray, Pak Penias and Pak E Meokbun and the Timika District Health 712 649 Authority; Dr Dina Bisara Lolong and Ibu Meryani Girsang and the National Institute 713 status), and additionally showed cavitary disease to be predictive of Health Research and Development, Jakarta; Dr P Penttinen, Dr M Bangs and 650 of worse lung function. The proportion of lung affected was Dr M Stone, Public Health & Malaria Control (PHMC) and International SOS; Pak 714 651 associated with both bacteriological and a range of clinical Istanto and PHMC laboratory staff; Pak J Lempoy and Timika TB clinic staff; 715 652 measures. Dr P. Sugiarto and Mimika Community Hospital (RSMM); Natalia Dwi Haryanti, 716 653 This score was derived in adult patients with TB with smear- Sri Hasmunik, Sri Rahayu, G Bellatrix and clinical and laboratory staff, NIHRD-MSHR 717 654 Timika research programme; and Mr R Lumb and Dr I Bastian at the Institute of 718 positive pulmonary disease, in a setting with relatively low rates Medical and Vetinerary Science. 655 of HIVeTB co-infection and MDR-TB. The score requires 719 656 Funding Australian Respiratory Council, the Royal Australasian College of Physicians 720 further evaluation in populations with high HIV prevalence, in (Covance award), Australian National Health and Medical Research Council. 657 whom CXR findings characteristic of HIVeTB co-infection 721 658 (subtle or absent pathology, non-cavitary disease, lower lobe Competing interests None. 722 659 infiltrates, hilar lymphadenopathy and pleural effusion)20 40 may Ethics approval This study was conducted with the approval of the Human Research 723 660 mean that a differently weighted score is needed. Nevertheless, Ethics Committees of the NT Department of Health & Families and Menzies School of 724 661 Health Research, Australia, the Australian National University, and the National 725 the score remained valid and applicable in the newer data set in Institute for Health Research and Development, Indonesia. 662 which HIVeTB co-infection rates were higher (13%); the rise in 726 663 HIV prevalence may account for some of the differences Provenance and peer review Not commissioned; externally peer reviewed. 727 664 observed between the two data sets. The presence of MDR-TB 728 665 729 would not be expected to alter radiographic patterns, other than REFERENCES 666 being associated potentially with higher scores and smaller 1. Tuberculosis Chemotherapy Centre Madras. A concurrent comparison of 730 667 incremental improvements over time. isoniazid plus PAS with three regimens of isoniazid alone in the domiciliary treatment 731 e 668 Potential limitations of the study include the use of 2 month of pulmonary tuberculosis in South India. Bull World Health Org 1960;23:535 85. 732 669 2. Simon G. Radiology in epidemiological studies and some therapeutic trials. BMJ 733 smear status as an outcome measure (rather than a longer term 1966;2:491e4. 670 measure such as 6-month outcome or recurrence). 3. Falk A, O’Connor J, Pratt P. Classification of pulmonary tuberculosis. In: Falk A, 734 671 The absence of suitable biomarkers or other surrogate end O’Connor JB, Pratt PC, et al, eds. Diagnostic standards and classification of 735 672 tuberculosis. Vol 12. New York, NY: National Tuberculosis and Respiratory Disease 736 points in TB research is readily acknowledged, and recent Association, 1969. 5 673 estimates derived from meta-analysis found a sensitivity of only 4. Diacon AH, Pym A, Grobusch M, et al. The diarylquinoline TMC207 for 737 674 57% and specificity of 81% for 2-month smear status in multidrug-resistant tuberculosis. N Engl J Med 2009;360:2397e405. 738 675 predicting treatment failure.35 Nevertheless, until more suitable 5. Burman WJ, Goldberg S, Johnson JL, et al. Moxifloxacin versus ethambutol in the 739 676 first 2 months of treatment for pulmonary tuberculosis. Am J Respir Crit Care Med 740 measures become available, 2-month smear status remains 2006;174:331e8. e 677 a suitable outcome measure.30 32 34 6. Katiyar SK, Bihari S, Prakash S, et al. A randomised controlled trial of high-dose 741 678 Another limitation was the inherent problem of limited isoniazid adjuvant therapy for multidrug-resistant tuberculosis. Int J Tuberc Lung Dis 742 679 2008;12:139e45. 743 inter-rater agreement in CXR assessment. The low rates of 7. Benator D, Bhattacharya M, Bozeman L, et al. Rifapentine and isoniazid once 680 clinicianeradiologist agreement between reporters on CXR a week versus rifampicin and isoniazid twice a week for treatment of drug- 744 681 findings identified in the derivation data set are not unusual, with susceptible pulmonary tuberculosis in HIV-negative patients: a randomised clinical 745 682 trial. Lancet 2002;360:528e34. 746 only fair or poor agreement between radiologists and clinicians 8. Heo EY, Chun EJ, Lee CH, et al. Radiographic improvement and its predictors in 22 23 683 also being reported elsewhere. This emphasises the impor- patients with pulmonary tuberculosis. Int J Infect Dis 2009;13:e371e6. 747 684 tance of using simple rather than complex scores and ensuring 9. Mankatittham W, Likanonsakul S, Thawornwan U, et al. Characteristics of 748 685 HIV-infected tuberculosis patients in Thailand. Southeast Asian J Trop Med Public 749 individuals allocating CXR scores participate in continuing e 686 Health 2009;40:93 103. 750 education to maximise agreement. The score derived from radi- 10. Lawson L, Yassin MA, Thacher TD, et al. Clinical presentation of adults with 687 ologist CXR evaluation in the first data set is simple. Moreover, it pulmonary tuberculosis with and without HIV infection in Nigeria. Scand J Infect Dis 751 688 was shown to be valid in the second data set when used by an 2008;40:30e5. 752 689 fi 11. Idh J, Westman A, Elias D, et al. Nitric oxide production in the exhaled air of patients 753 independent TB clinician, rather than a radiologist, con rming its with pulmonary tuberculosis in relation to HIV co-infection. BMC Infect Dis 690 practical utility in a clinical and trial setting. Some systematic 2008;8:146. 754 691 differences in CXR results were noted between the two data sets; 12. Thorson A, Long NH, Larsson LO. Chest X-ray findings in relation to gender and 755 692 symptoms: a study of patients with smear positive tuberculosis in Vietnam. 756 while this may represent systematic difference in reporting e 693 Scand J Infect Dis 2007;39:33 7. 757 styles, the findings are in keeping with the possibility of less 13. Liam CK, Lim KH, Srinivas P, et al. Hypercalcaemia in patients with newly diagnosed 694 severe disease in the validation data set, as indicated by their tuberculosis in Malaysia. Int J Tuberc Lung Dis 1998;2:818e23. 758 695 lower bacillary burden (with slides read by the same senior 14. Den Boon S, Bateman ED, Enarson DA, et al. Development and evaluation of a new 759 696 chest radiograph reading and recording system for epidemiological surveys of 760 laboratory technician during both data collection periods). tuberculosis and lung disease. Int J Tuberc Lung Dis 2005;9:1088e96. 697 15. Dawson R, Masuka P, Edwards DJ, et al. Chest radiograph reading and recording 761 698 CONCLUSION system: evaluation for tuberculosis screening in patients with advanced HIV. 762 699 Int J Tuberc Lung Dis 2010;14:52e8. 763 In summary, we have derived and validated a simple method for 16. Gatner EM, Burkhardt KR. Correlation of the results of X-ray and sputum culture in 700 grading CXR severity in adults with smear-positive pulmonary tuberculosis prevalence surveys. Tubercle. 1980;61:27e31. 764 701 TB that predicts baseline clinical and microbiological severity 17. Linh NN, Marks GB, Crawford AB. Radiographic predictors of subsequent 765 e 702 and response to treatment in two separate patient populations. reactivation of tuberculosis. Int J Tuberc Lung Dis 2007;11:1136 42. 766 703 fi 18. World Health Organization. Assessing tuberculosis prevalence through 767 Although ner discriminatory accuracy might be achieved by population-based surveys. WHO, Western Pacific Region 2007 [October 2009]. 704 collecting more detailed CXR findings (such as cavity size), our http://www.wpro.who.int/publications/PUB_978+92+9061+314+5.htm. 768

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769 19. Andreu J, Caceres J, Pallisa E, et al. Radiological manifestations of pulmonary 30. World Health Organization. Treatment of tuberculosis: guidelines for national 793 770 tuberculosis. Eur J Radiol 2004;51:139e49. programmes. WHO/CDS/TB/2003.313. Geneva, Switzerland: WHO, 2003. 794 771 20. Raviglione MC, Narain JP, Kochi A. HIV-associated tuberculosis in developing 31. Handojo T, Anstey N, Kelly P, et al. Normal spirometry, gas transfer and lung volume 795 countries: clinical features, diagnosis, and treatment. Bull World Health Organ values in Papua, Indonesia. Southeast Asian J Trop Med Public Health 772 1992;70:515e26. 2006;37:571e7. 796 773 21. Marais BJ, Gie RP, Schaaf HS, et al. A proposed radiological classification of 32. Wang JY, Lee LN, Yu CJ, et al. Factors influencing time to smear conversion in 797 774 childhood intra-thoracic tuberculosis. Pediatr Radiol 2004;34:886e94. patients with smear-positive pulmonary tuberculosis. Respirology 2009. 6 798 22. Balabanova Y, Coker R, Fedorin I, et al. Variability in interpretation of chest 33. Rieder HL. Sputum smear conversion during directly observed treatment for 775 radiographs among Russian clinicians and implications for screening programmes: tuberculosis. Tuber Lung Dis. 1996;77:124e9. 7 799 776 observational study. BMJ 2005;331:379e82. 34. Ramarokoto H, Randriamiharisoa H, Rakotoarisaonina A, et al. Bacteriological 800 777 23. Zellweger JP, Heinzer R, Touray M, et al. Intra-observer and overall agreement in follow-up of tuberculosis treatment: a comparative study of smear microscopy and 801 the radiological assessment of tuberculosis. Int J Tuberc Lung Dis 2006;10:1123e6. culture results at the second month of treatment. Int J Tuberc Lung Dis 778 24. Plit ML, Anderson R, Van Rensburg CE, et al. Influence of antimicrobial 2002;6:909e12. 802 779 chemotherapy on spirometric parameters and pro-inflammatory indices in severe 35. Horne DJ, Royce SE, Gooze L, et al. Sputum monitoring during tuberculosis 803 780 pulmonary tuberculosis. Eur Respir J 1998;12:351e6. treatment for predicting outcome: systematic review and meta-analysis. 804 e 781 25. Ardian M, Meokbun E, Siburian L, et al. A public-private partnership for TB control in Lancet Infect Dis 2010;10:387 94. 805 Timika, Papua Province, Indonesia. Int J Tuberc Lung Dis 2007;11:1101e7. 36. Byrt T, Bishop J, Carlin JB. Bias, prevalence and kappa. J Clin Epidemiol 782 26. Kelly PM, Ardian M, Waramori G, et al. A community-based TB drug susceptibility 1993;46:423e9. 806 783 study in Mimika District, Papua Province, Indonesia. Int J Tuberc Lung Dis 37. Landis JR, Koch GG. The measurement of observer agreement for categorical data. 807 784 2006;10:167e71. Biometrics 1977;33:159e74. 808 27. Maguire GP, Anstey NM, Ardian M, et al. Pulmonary tuberculosis, impaired lung 38. Daley C, ed. Chest radiography in the diagnosis of tuberculosis: specificity and 785 function, disability and quality of life in a high-burden setting. Int J Tuberc Lung Dis accuracy. 39th World Conference on Lung Health of the International Union Against 809 786 2009;13:1500e6. TB and Lung Disease. Paris, France: 2008:16e20. 810 787 28. Jones PW, Quirk FH, Baveystock CM, et al. A self-complete measure of health 39. Palaci M, Dietze R, Hadad DJ, et al. Cavitary disease and quantitative sputum 811 e 788 status for chronic airflow limitation. The St. George’s Respiratory Questionnaire. bacillary load in cases of pulmonary tuberculosis. J Clin Microbiol 2007;45:4064 6. 812 Am Rev Respir Dis 1992;145:1321e7. 40. Aderaye G, Bruchfeld J, Assefa G, et al. The relationship between disease 789 29. World Health Organization. Management of severe malnutrition: a manual for pattern and disease burden by chest radiography, M. tuberculosis load, and HIV 813 790 physicians and other senior health workers. Geneva: 1999[December 2009]. status in patients with pulmonary tuberculosis in Addis Ababa. Infection 814 791 http://whqlibdoc.who.int/hq/1999/a57361.pdf. 2004;32:333e8. 815 792 816

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Chapter 10: Results IV – Longitudinal

10 Results IV: Longitudinal follow up

This chapter reports on the progress over time of enrolled AVDAPT study participants, including treatment outcome in those who have completed 6 months, adverse events, interim safety analysis and the development and evaluation of a composite clinical TB outcome score (a primary outcome measure of the AVDAPT study).

10.1 INTRODUCTION

Progress of individuals enrolled in the AVDAPT study can be assessed according to standard DOTS program objectives, and also according to the additional parameters of interest for the clinical trial. Adverse events and serious adverse events among study participants have been documented and reported in keeping with Good Clinical Practice (GCP).200 Particular attention is required regarding the possibility of increased risk of hypercalcaemia among participants randomised to vitamin D-containing arms of the AVDAPT study, or gastrointestinal disturbance in those randomised to arginine- containing study arms. An interim safety analysis was performed by an independent statistician when a quarter of study participants had been enrolled and followed up for 2 months. The two stated primary endpoints of the AVDAPT study are listed in Box 10.1.

The aims of this chapter are two-fold: 1. To document treatment outcomes among study participants, including sputum smear and culture conversion, and provide interim adverse event and safety data. 2. Describe the development of the composite clinical outcome score (the second primary endpoint), and its performance at 2 months in relation to other markers of improvement. As study arm allocation remains concealed until study completion, the current analysis does not include comparison of this endpoint among study arms.

105 Chapter 10: Results IV – Longitudinal

Box 10.1: AVDAPT study primary outcome measures

(1) Proportion of pulmonary TB patients who are culture negative at 1 month (2) Difference in improvement in composite clinical endpoint comprising weight, cough clearance and FEV1 at 2 months.

The composite clinical endpoint comprises a nutrition component (% weight change) and a respiratory function component (% change in FEV1, and progress in cough, sputum and haemoptysis). Percentage weight gain is widely recognised as a valuable measure of response to TB treatment.201, 202 The long-established two-way relationship between malnutrition and increased susceptibility to infection203 is of particular importance in TB. Underweight individuals carry a higher TB risk202, 204 and TB infection causes cachexia.205, 206 Inadequate weight gain during treatment is associated with higher TB relapse risk,201 and low body mass index is associated with an increased risk of early death.207 A recent study reports that a nutritional risk score comprising BMI<18.5 kg/m2, hypoalbuminaemia, hypocholesterolaemia and severe lymphopaenia, was predictive of mortality in hospitalised TB patients.208 TB-HIV co-infected people are even more likely to be underweight209 or have nutritional deficiencies210 than HIV negative TB patients.

Impaired lung function due to TB can be evaluated using a variety of means, with FEV1 a frequently-used measure. Both early reversible lung impairment in pulmonary TB114, 168 169 and long-term residual impairment post-TB have been characterised using FEV1, described in Chapter 7.

Weight and FEV1 change are therefore appropriate measures to include in an outcome score, and can be objectively assessed. Addition of clinical symptomatology (cough, sputum production, haemoptysis), routinely used in clinical practice to gauge response to therapy, to an outcome score, could improve the sensitivity of the score to clinically- relevant improvements over time. Eight and 24 weeks of follow up are standard assessment points at which to calculate the score, the former being the usual transition timepoint between intensive-phase and continuation phase therapy, and the latter representing end of standard therapy.

Other investigators have generated scores for the purpose of evaluating TB response to therapy: Wejse and colleagues constructed a score using respiratory symptoms, night

106 Chapter 10: Results IV – Longitudinal sweats, pale conjunctivae, tachycardia, abnormal chest auscultation, temperature >37, body mass index and middle upper arm circumference.211 They showed that higher absolute scores at baseline and 8 months were predictive of mortality. We however aim to use a score incorporating relative change over time, which is likely to offer greater discriminatory ability in assessing degrees of treatment response, and to use fewer, physiologically well-justified score components. In this chapter, the composite clinical outcome score we have devised for use in the AVDAPT study is calculated in two ways, generating either an ordinal score of 0 to 12, or a dichotomous score. The performance of these scores and the relationships between them and percentage change in other parameters measured during follow up is examined.

10.2 METHODS

Sputum processing methods are described in Chapter 6 (Methods). In brief, sputum AFB smears were examined using ZN staining (Timika laboratory) and culture was performed using MGIT and Lowenstein-Jensen medium (at the FKUI laboratory, Jakarta). Sputum AFB results reported here are those obtained at the Timika laboratory unless otherwise stated.

Interim analysis was performed as scheduled when 111 study participants (25% of the sample size) had 8-week data available. This was undertaken by the statistician member of the DSMC (Mr Joseph McDonnell). Study arms to which the participants had been randomised were identified to the statistician as Groups A, B, C and D, but concealment was maintained regarding whether active or placebo medications had been administered, and study investigators including myself were not given access to the group allocations. Safety (any differences in adverse event rates in the study arms) and balance of baseline characteristics among participants in the different study arms were assessed. Safety measures included potential adverse effects of vitamin D or L-arginine i.e. hypercalcaemia, neurological symptoms including headache, dizziness and confusion which could be attributable to hypercalcaemia, gastrointestinal symptoms, and any new respiratory symptoms suggestive of illness exacerbation. The findings of the analysis were disseminated to other members of the DSMC to assess the safety of continuation of the trial.

107 Chapter 10: Results IV – Longitudinal

The composite clinical outcome score (Table 10.1) was devised pre hoc by consensus opinion of a panel of clinicians with collective experience in managing TB and in devising clinical trial outcome measures (Associate Professors Graeme Maguire, Paul Kelly, Peter Morris, Professor Nicholas Anstey, Dr Anna Ralph). We weighted the score components consistent with their recognised clinical importance in TB. Thus highest scores were assigned to weight gain, and the selected cut-off of weight gain <5% was guided by previously-published results.201 The lung function cut-offs were guided by knowledge of test repeatability (accuracy) and plausible improvements in 114 FEV1 during the given time frame. In developing the score, absolute values versus percentage changes were considered. The latter was chosen on the basis of percentage change being more able to accurately reflect an individual‘s response to treatment. The resulting score is ordinal, with possible results of 0 to 12, or can be summarised as a dichotomous score.

Table 10.1: Composite clinical outcome score

(a) Ordinal score Clinical parameter Outcome Points Weight % Weight change Decrease 0 parameter <5% weight gain 2 5.0-9.9% weight gain 4 ≥10% weight gain 6 Respiratory % FEV1 change ≥10% fall in FEV1 0 parameters <10% fall or <10% improvement in FEV1 1 ≥10% FEV1 improvement 2 Cough Worse or same 0 Improved 1 Ceased 2 Sputum Present 0 Absent 1 Haemoptysis Present 0 Absent 1

Total maximum score 12

108 Chapter 10: Results IV – Longitudinal a) Dichotomous score: Score 1 if the weight target plus at least one of the respiratory targets are met (i.e. score 0 if weight target not met).

Weight parameter ≥10% weight gain achieved No / Yes

Respiratory ≥10% FEV1 improvement achieved No / Yes parameters OR Improvement in cough and resolution of sputum / No / Yes haemoptysis achieved Possible scores 0 or 1

Adverse events in the AVDAPT study are defined and reported in keeping with GCP guidelines (non-serious adverse events [AE]: any new symptom or complaint arising during the course of the study which was not present at baseline; serious adverse events [SAE]: death, life-threatening illness or hospitalisation). All SAE are reported to the Data and Safety Monitoring committee members, who assess the case and determine the relatedness of the SAE to the study medications (although because allocation concealment is maintained, the study participant may not have received active study medications).

Statistical analyses were performed in Stata 10.1 as described in previous chapters. Additionally, Kaplan-Meier survival analysis was used to examine time-to-event data (time to sputum smear conversion using weekly sputum smear readings). Patient subgroup analysis was performed by Cox regression (proportional-hazards) models and hazard ratios and 95% confidence intervals. Log-log plots were used to establish whether proportional-hazards assumptions were met.

To account for repeated conversions / reversions in smear status during the course of treatment, smear negativity was defined as the time point (week after treatment) at which the first of 3 consecutive negative results was recorded, or the first negative result if fewer than 2 subsequent results were recorded, as described elsewhere.142, 150 McNemar‘s test was used to compare proportions for paired data. Wilcoxon‘s signed rank test for matched pairs was used to compare values for paired non-parametric data. For analysing associations of the composite clinical score, assumptions required for linear regression analyses could not be met, hence these were investigated using

Spearman‘s rank correlations.

109 Chapter 10: Results IV – Longitudinal 10.3 RESULTS

Of 162 participants enrolled since June 12th 2008, 155 were enrolled at least 4 weeks, and 139 at least 8 weeks, prior to database closure on October 3rd 2009. Findings reported below are confined to those with 8-week follow up data unless otherwise indicated.

Figure 10.1: Participant follow up profile Default= defaulted from TB treatment and study altogether. Withdrew from study = withdrew from study but continued TB treatment

Number Week 0 Default: 3

enrolled: Lost to 162 follow up Withdrew from study: 1 before week 4: 9 Transferred: 5

155 Week 4 Died: 0

Lost to Default: 0

follow up between Withdrew from study: 1 week 4 and

time each

t Transferred: 0

a 8: 2

d e

t Died: 1

c

e 139 Week 8

p

x e

s up follow to losses no if

point

Number

110 Week 24

STUDY PARTICIPANTS LOST TO FOLLOW UP

Losses to follow up comprised 9 study participants who defaulted from TB treatment altogether, withdrew from the study but continued TB treatment, or were transferred prior to their week 4 appointment (9/155 = 5.8% loss to follow up by 1 month), and a further 2 who withdrew or died prior to their week 8 appointment (11/139 = 7.9% loss to follow up by 2 months). See Figure 10.1. These 11 study participants who were lost to follow up did not differ in age, sex, ethnicity, HIV, education or employment status 110 Chapter 10: Results IV – Longitudinal from the rest of the study population (p>0.05 in each instance). Some differences were noted in baseline disease severity indicating the possibility of more severe disease according to some parameters, but none reached statistical significance. Specifically, the 11 people lost to follow up had higher (worse) baseline chest x-ray scores (median score

100 vs 68) and lower percentage of predicted FEV1 (median % predicted FEV1 49.4% vs 61.5%) than other study participants (p=0.1 and 0.2 respectively). There were no statistically significant differences in baseline sputum smear grade, 6-minute walk distance, quality of life score, illness duration or symptom tally (using Fisher‘s exact test or Wilcoxon rank sum tests).

BACTERIOLOGICAL RESULTS

SPUTUM SMEAR AND CULTURE CONVERSION

Of 139 study participants enrolled at least 8 weeks prior to database closure, sputum smear conversion (according to the Timika laboratory sputum microscopy result) was achieved in 60.9% and 75.6% by 4 and 8 weeks respectively, and culture conversion (according to the FKUI laboratory culture results) in 57.9% and 80.4% by 4 and 8 weeks respectively (Table 10.3 and Figure 10.2).

Reasons for culture results being unavailable included: contamination of culture and inability to successfully de-contaminate; samples not collected or lost in transit; inadequate sample present in the specimen jar; electricity failure at the laboratory resulting in incubator failure, or departure of the patient from the study (default, withdrawal from study, transfer or death).

111 Chapter 10: Results IV – Longitudinal

Table 10.2: Sputum MTB microscopy and culture results at baseline and 4 and 8 weeks

Baseline Week 4 Week 8 SPUTUM AFB MICROSCOPY Microscopy result available 139/139 (100) 128/139 (92.1) 127/139 (91.4) Smear positive: no. (%) 119/139 (85.6) 50/128 (39.1) 31/127 (24.4) Reasons for microscopy unavailability Patient left study - 9 11 Patient did not attend - 2 1 Patient unable to expectorate - 0 0 SPUTUM CULTURE Culture result available 123/139 (88.5) 107/139 (77.0) 92/139 (66.2) Culture Positive: no. (%) 111/123 (90.2) 45/107 (42.1) 18/92 (19.6) Reasons for culture unavailability Patient left study - 9 11 Culture contamination 4 8 7 Electricity failure 1 1 6 Sample inadequate or missing from consignment 2 0 1 Sample lost in transit 9 14 22 Patient unable to expectorate 0 0 0

Figure 10.2: Microscopy and culture results at enrolment, and 4 and 8 weeks in participants followed for 8 weeks

n=123 n=107 n=92 100

80

Smear pos Culture pos 60 Smear pos Culture neg Smear neg Culture pos 40 Smear neg Culture neg

20 Percentage of sputum samples sputum of Percentage

0 0 4 8 Week

112 Chapter 10: Results IV – Longitudinal

Twenty six of 92 study participants (28%) were still smear positive at 8 weeks, and of these, 12/26 (46%) were also culture positive, the remaining 14 (54%) were culture negative. Of the 66/92 (72%) participants who were smear negative at 8 weeks, 6/66 (9%) were culture positive (Figure 10.2).

Predictors of sputum AFB smear conversion

The median time to smear conversion was 4.5 weeks (range 1 to 28). Smear conversion times did not differ according to sex, age, ethnicity or smoking status (p>0.5 in each instance, Cox proportional hazards methods). Sputum smear conversions / reversions were common during individual‘s courses of treatment, providing justification for defining smear negativity as the first of 3 consecutive negative samples (as per Methods). Time to smear conversion over the 24 weeks of standard treatment is shown in Figure 10.3. Subsequent figures truncate follow up time at 8-10 weeks. HIV status did not significantly influence time to conversion; in fact conversion appeared initially more rapid in HIV positive people (Figure 10.4), but not significantly so (hazards ratio [HR] 0.7, 95% CI 0.41 - 1.3, p=0.2).

The presence of cavities on the baseline chest radiograph, and higher baseline AFB grade, were both associated with significantly slower time to sputum smear conversion (HR 0.6, 95% CI 0.4-0.96, p=0.03 and HR 0.6, 95% CI 0.5- 0.8, p<0.0001 respectively; Figures 10.5 and 10.6). The presence of isoniazid mono-resistance (identified in 5/139 study participants) did not significantly predict to time to smear conversion (HR 1.6, 95% CI 0.6-4.5, p=0.3). The impact of MDR-TB could not be modelled due to low numbers in this subgroup (1 MDR-TB patient experienced sputum smear conversion at week 11, the other remains smear positive to date).

In the following figures, the number at risk is indicated below each graph. Analyses are restricted to participants followed up for the indicated number of weeks. Number tallies are not the same in each graph since <100% of study participants were smear positive at baseline, or had known HIV status or documented CXR results.

113 Chapter 10: Results IV – Longitudinal

Figure 10.3: Kaplan-Meier survival curve showing probability of sputum smear conversion by

ethnicity

1.00

0.75

0.50

0.25

Probabilityremaining of smear positive 0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 week Number at risk ethnicity = Non-Papuan 55 29 9 4 4 3 ethnicity = Papuan 48 21 7 3 3 1 ethnicity = Non-Papuan ethnicity = Papuan

Figure 10.4: Kaplan-Meier survival curve showing probability of sputum smear conversion by HIV status

1.00 Hazards ratio 0.7, p=0.2

0.75

0.50

0.25

Probabilityremaining of smear positive 0.00 0 1 2 3 4 5 6 7 8 9 10 Week Number at risk hiv = 0 97 80 64 45 30 17 hiv = 1 16 13 6 3 2 2 HIV neg HIV pos

114 Chapter 10: Results IV – Longitudinal

Figure 10.5: Kaplan-Meier survival curve showing probability of sputum smear conversion by cavitary disease status

1.00

Hazards ratio 1.6, p=0.03

0.75

0.50

0.25

Probabilityremaining of smear positive 0.00 0 1 2 3 4 5 6 7 8 Week Number at risk cavity = 0 53 38 25 13 8 cavity = 1 68 62 49 36 26 Non-cavitary disease Cavitary disease

Figure 10.6: Kaplan-Meier survival curve showing probability of sputum smear conversion by baseline sputum AFB grade

Hazards ratio 1.6, p<0.0001 1.00

3+ 0.75

2+ 0.50

1+ 0.25

scanty 0.00

Probabilityremaining of smear positive 0 1 2 3 4 5 6 7 8 Week Number at risk AFBgrade = Scanty 16 11 9 4 2 AFBgrade = 1+ 32 27 16 10 6 AFBgrade = 2+ 32 31 25 17 12 AFBgrade = 3+ 24 24 20 17 13 Scanty 1+ 2+ 3+

PREDICTORS OF SPUTUM CULTURE CONVERSION

In univariate logistic regression analyses, higher sputum smear grade at diagnosis predicted a failure to achieve sputum culture conversion at 4 and 8 weeks, while presence of cavitary disease and lower BMI (but not weight) at baseline predicted a

115 Chapter 10: Results IV – Longitudinal failure to achieve sputum culture conversion at 4 weeks. In multivariate logistic regression analyses, the association between BMI and 4-week culture conversion became statistically non-significant (p=0.1), leaving baseline smear grade the only predictor of culture conversion. No associations were identified between culture results and sex, age, ethnicity, smoking status, HIV status, baseline weight or baseline pulmonary function. Significant results in univariate analyses are shown in Table 10.4.

Table 10.3: Predictors of sputum culture conversion, univariate analyses

Dependent Independent Odds ratio* 95% Confidence P value variable variable interval Culture week 4 BMI at baseline 0.8 0.7 – 0.99 p=0.04 Culture week 4 Cavitary disease 4.4 1.9 – 10.4 p=0.001 Culture week 4 Baseline AFB grade 2.2 1.5 – 3.4 p<0.0001 Culture week 8 Baseline AFB grade 1.9 1.1 – 3.2 p=0.02 *Odds ratios calculated from univariate logistic regression models

TREATMENT OUTCOME

Six-month treatment outcomes were assessed according to the standard WHO categories (Table 10.5) and grouped as Treatment Success (cured or completed) or Unfavourable outcome (failed, died or defaulted). Treatment success was achieved in 84.5% overall, almost reaching the 85% WHO target for completion or cure.

Table 10.4: Six-month treatment outcome

6-month treatment outcome: no. (%) Study participants: no. (%) N 110 Cured 86 (78.2) Completed 7 (6.4) Failed 1 (0.9) Default 8 (7.3) Died 2 (1.8) Transferred 6 (5.5)

Two deaths and one treatment failure occurred. Longer diagnostic delay appeared to be associated with death or failure: the illness duration prior to TB diagnosis in the 2 patients who died and 1 who failed treatment were 3, 12 and 24 months respectively,

116 Chapter 10: Results IV – Longitudinal significantly higher than the median illness duration (2 months) in those who achieved treatment success (p=0.02, Wilcoxon rank sum test). Overall, differences between outcome groups were difficult to ascertain with confidence due to the small numbers in categories other than the successful treatment group. Outcome was not found to differ according to age, gender, ethnic group, smoking status or HIV status (p>0.05 in each instance calculated from Fisher‘s exact tests). Furthermore, outcome was not predicted by baseline sputum smear grade, baseline weight or BMI, or baseline X-ray score (p>0.05 in each instance calculated from Kruskal-Wallis tests or one-way ANOVA).

Unfavourable outcomes according to WHO criteria are not identical to losses to follow up for the purposes of the AVDAPT study, since firstly only 110 participants who have completed the study were examined regarding final outcome, but losses to follow up reported above includes all who had been enrolled in the study for at least 8 weeks. Secondly, the final outcome was able to be determined (cured in both instances) in 2 of the patients lost to follow up who dropped out of the study but remained under DOTS.

COMPOSITE CLINICAL OUTCOME SCORES

TWELVE-POINT SCORE

Composite clinical outcome scores using the 12-point scale were able to be calculated in patients who had data recorded for each score component at baseline and the comparative time point at 8 or 24 weeks. Thus scores were available in 125 of 139 study participants who had completed 8 weeks, and 81 of 110 who had completed 24 weeks (78 matched pairs). Scores increased significantly between week 8 and 24 (Table 10.6 and Figure 10.7). Higher scores indicate greater improvement.

Table 10.5: Composite clinical outcome score at 8 weeks (end of intensive treatment phase) and 24 weeks (end of treatment)

Composite clinical outcome 8 weeks 24 weeks p value score Ordinal score: median (range) 6 (1-11) 9 (3-11) p<0.0001* Dichotomous score positive 18/127 (14.2) 38/88 (43.2) p<0.0001† (successful): no. (%) *p value calculated using Wilcoxon signed rank test for matched pairs. †p value calculated using McNemar  test

117 Chapter 10: Results IV – Longitudinal

At both 8 and 24 weeks, composite clinical scores correlated weakly but significantly with percentage changes in SGRQ score, 6 minute walk distance and haemoglobin, using Spearman‘s rank correlation (Table 10.7). (Note: a positive value for % change in SGRQ score signifies deterioration, accounting for the negative Rho values in the table below.) Median composite clinical scores at 8 weeks did not differ according to sputum microscopy and culture status at week 8 (p=0.8 and 0.9 respectively; Kruskal-Wallis tests).

Figure 10.7: Histograms demonstrating distributions of composite clinical outcome scores at 8 and 24 weeks

0 2 4 6 8 10 2 4 6 8 10 Composite clinical outcome score at week 8 Composite clinical outcome score at week 24

Table 10.6: Correlations between outcome scores and percentage change in other measures

Variable 1 Variable 2 Spearman’s Rho p value 8 week composite SGRQ -0.21 p=0.04 clinical outcome score 6MWT 0.26 p=0.005 Hb 0.25 p=0.006 24 week composite SGRQ -0.26 p=0.03 clinical outcome score 6MWT 0.27 p=0.02 Hb 0.46 p=0.04

DICHOTOMOUS SCORE

Dichotomous scores were available in 127 study participants at 8 weeks and 88 study participants at 24 weeks (87 matched pairs); a positive score, indicating success in achieving at least 10% increase in body weight plus at least 10% increase in lung

118 Chapter 10: Results IV – Longitudinal function or improvement in respiratory symptoms, was achieved by only 14.2% of participants by 8 weeks, and 43.2% by 24 weeks (Table 10.6).

Positive scores at 8 and 24 weeks were significantly associated with greater improvements in SGRQ score; also at 8 weeks, in greater improvement in 6-minute walk distance, and at 24 weeks, in rise in haemoglobin (all p values<0.05, Rank-Sum tests). Other associations were not significant. The dichotomous score at 8 weeks was not associated with 8 week sputum AFB status. The dichotomous score at 24 weeks could not be compared with outcome since those with unfavourable outcomes (i.e. died, defaulted or were transferred), did not reach week 24. The individual who failed treatment did not achieve a positive score by week 24.

ADVERSE EVENT REPORTING

Five serious adverse events (SAE) occurred: 2 deaths and 3 hospitalisations (Table 10.8). In each case a detailed clinical summary was provided to the Data and Safety Monitoring committee. All SAE were judged to be unlikely to be related, or unrelated, to the study medications (L-arginine / vitamin D / placebo L-arginine / placebo vitamin D) according to GCP definitions (provided on the SAE reporting guide, see Appendix 15.9).

Non-serious adverse events (any new symptom not reported at baseline) were very common, being reported in 128 of the 139 study participants (92.1%) who had been enrolled for at least 8 weeks. The most common AEs (Figure 10.8) were itch (reported at least once in 47.5% of people) followed by gastrointestinal disturbance (nausea, vomiting, abdominal pain, bloating, diarrhoea, epigastric pain and/or constipation), reported by 46.8% of all study participants, peaking at week 1 and 2 after commencing treatment. Of the gastrointestinal symptoms, nausea was most common, followed by bloating then vomiting.

While common, adverse events were mostly mild, often requiring reassurance only or symptomatic management (analgesics, antacid medication etc). However among the 139 participants who had 8-week follow up data, one case of severe desquamating rash occurred, with recurrence on re-challenge with both ethambutol then pyrazindamide,

119 Chapter 10: Results IV – Longitudinal necessitating a change of regimen. Hence the rate of major anti-tuberculosis drug reactions requiring regimen switch was 1/139 (0.7%).

Notably, 12 study participants developed symptomatic malaria (7 Plasmodium falciparum, 5 Plasmodium vivax) whilst undergoing TB treatment. Malaria diagnosis

(blood film) and treatment (dihydroartemesinin piperaquine ± primaquine) was provided in keeping with Timika protocols.

HIV positive status was associated with a significantly higher likelihood of vomiting (HIV+: 27.8%, HIV-: 9.2%, p=0.04) and bloating (HIV+: 33.3%, HIV-: 14.2%, p=0.05), and somewhat higher likelihood of malaise (HIV+: 27.8%, HIV-: 11.7%, p=0.08) during the first 8 weeks of treatment. Other adverse events, and overall likelihood of AE, were not significantly higher in HIV-TB co-infected particiapnts.

Figure 10.8: Non-serious adverse events experienced by AVDAPT study participants

60 55 50 45 40 35 30 25

(mean, 95%(mean, CI) 20 15

Percentage with symptom Percentage 10 5 0

Itch Rash Fever Malaria GI upsetMyalgia ArthralgiaChestBack painHeadache painDizziness Heamoptysis Paraesthesia

120 Chapter 10: Results IV – Longitudinal

Table 10.7: Serious adverse events (SAE)

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age (yrs) 21 60 38 48 21 Gender Male Male Female Male Female Ethnicity Papuan Papuan Non-Papuan Non-Papuan Non-Papuan

HIV status Positive Positive Positive Negative Negative

SAE type Death at Death in Hospitalisation Hospitalisatio Hospitalisation home hospital n Diagnosis Progressive Stroke Pneumothorax Vomiting and MDR-TB with respiratory complicated dehydration progressive illness due by pulmonary to TB or aspiration disease and secondary pneumonia large pleural pneumonia effusion Time of SAE 7 9 4 3 16 (weeks after TB treatment start) Relatedness of Unlikely Unlikely Unlikely Unlikely Unrelated SAE to study drugs SAE resulted No Already No Yes Already in study ceased ceased medication cessation? SAE Outcome Died Died Recovery; Recovery; Recovery; 2nd- cured cured line treatment ongoing

Hypercalcaemia, defined as a non-serious adverse event (unless resulting in hospitalisation, life-threatening illness or death), occurred in 23 / 139 (16.5%) people (Table 10.9), most commonly in week 2 (42.9% in week 2, 35.7 in week 4, 21.4% in week 8). None were symptomatic or required specific treatment or hospitalisation.

While 1 person had mild to moderate asymptomatic hypercalcaemia sustained over 5 weeks (Figure 10.9), and 2 people had elevated calcium readings on 2 occasions each,

121 Chapter 10: Results IV – Longitudinal the remaining 20 people with hypercalcaemia had only a single reading greater than the normal range.

Table 10.8: Number of study participants with hypercalcaemia Hypercalcaemia: no. (%) Study participants N 139 Hypercalcaemia None (ionised Ca <1.33 mmol/L) 116 (83.5) Mild (1.33-1.39 mmol/L) 18 (13.0) Moderate (1.40-1.49 mmol/L) 5 (3.6) Severe (>1.50 mmol/L) 0 (0.0)

Figure 10.9: Serial calcium readings in a representative individual receiving TB treatment and study medications

1.50 1.45 1.40 1.35 iCa 1.33 = hypercalcaemia 1.30 1.25 1.20

1.15 Ionised calicum (mmol/L) calicum Ionised 1.10 0 1 2 3 4 5 6 7 8 week

Overall, calcium increased by a small but statistically significant amount (but within the normal range i.e. <1.33 mmol/L) from 1.21 to 1.24 mmol/L between weeks 0 to 2, then remained elevated during the remainder of the intensive phase (Figure 10.10).

Figure 10.10: Ionised calcium (mean indicated by bar and range) by week in AVDAPT study participants

p<0.0001 1.50 Mean iCa Mean iCa Mean iCa 1.45 1.21 1.24 1.25 1.40 1.35 1.30 1.25 1.20 1.15

1.10 Ionised calcium (mmol/L) calcium Ionised 1.05 1.00 0 2 4 6 8 Week

*p value calculated using paired t-test. The differences between weeks 2 and 4 or 8 were not significant. 122 Chapter 10: Results IV – Longitudinal

INTERIM ANALYSIS AE potentially related to study drugs were assessed in the interim safety analysis performed by the statistician of the Data and Safety Monitoring Committee. Data from 111 study participants (25% of planned sample) were evaluated. As shown in Table 10.9, study groups were balanced in number and in most characteristics except for gender. No differences in rates of adverse events (hypercalcaemia, or new symptoms of gastrointestinal, neurological or respiratory origin) were observed between the 4 study arms.

Table 10.9: Summary of findings of the interim analysis Group A Group B Group C Group D P value* Baseline characteristics n 28 29 27 27 p>0.05 Age in yrs: mean 31.8 31.4 30.4 29.0 p>0.05 Female: no. (%) 7 (25.0) 14 (48.3) 13 (48.2) 5 (18.5) p=0.03 Papuan: % 50.0 44.8 48.2 48.2 p=0.98 Weight in kg: mean 47.8 48.3 47.2 48.4 HIV+: no. 3 8 2 1 p=0.08 MDR-TB 0 1 1 0 p=0.9 Haemoglobin in g/dL: mean 12.0 11.6 12.3 12.7 p=0.4 Adverse events Serious adverse event: no. 1 1 2 0 p=0.3 Hypercalcaemia: % 12.0 11.6 12.3 12.7 p=0.4 Any new gastrointestinal 39.3 44.8 37.0 48.2 p=0.8 symptoms: % Any new neurological 32.1 17.2 14.8 7.4 p=0.1 symptoms: % Any new respiratory 28.6 31.0 29.6 37.0 P=0.9 symptoms: % *p values were calculated using Fisher‘s exact or Pearson‘s Chi2 tests.

Members of the Data and Safety Monitoring committee concluded that results of the interim safety analysis revealed no concerns, and deemed the study safe to proceed.

10.4 DISCUSSION

The chief findings from analyses of longitudinal data in this chapter are that losses to follow up were modest, treatment outcomes were close to WHO targets, predictors of

123 Chapter 10: Results IV – Longitudinal bacteriological outcomes were consistent with previous reports in the literature, and no safety concerns for participants randomised to adjunctive therapy were identified. Furthermore, the 12-point clinical score developed as a primary outcome measure of the study is shown to be an appropriate measure of clinical improvement.

SAMPLE SIZE AND LOSSES TO FOLLOW UP

Assumptions which guided the AVADPT sample size calculation (see Methods, Chapter 6) included a 20% reduction in the proportion culture positive at one month (from 60% to 40%) with losses to follow up of 10%. In the current data, the 1-month culture positive rate of 42.1% includes participants in all 4 study arms (3 intervention arms, one placebo arm), and is therefore within the anticipated range on which the sample size calculation was based. Losses to follow up by 1 month were less than anticipated (5.8%). People who were lost to follow up were representative of the group as a whole, but with a non-significant trend towards more extensive radiological disease extent and worse lung function at baseline.

BACTERIOLOGICAL RESULTS

The estimation that 60% of Timika patients might remain culture positive at 1 month was made on the basis of previous data from Timika indicating high bacillary burden and cavitary disease,112, 114 and from other studies showing mean time to culture negativity (in mostly drug sensitive TB) of 32 to 57 days.150, 151 Other published comparative figures include 11.1% of smear-positive pulmonary TB patients remaining culture positive at 2 months in one study,142 and 38.6% at 1 month and 15.7% at 2 months in another study.212 Our reported rates of culture positivity at 1 month (42.1%) and 2 months (19.6%) are somewhat higher, possibly related to relative disease severity at baseline among the patients in these different settings.

However in examining predictors of sputum smear and culture conversion in these study participants, it is apparent that while expected variables such as baseline AFB grade and presence of cavities were predictive of smear conversion and culture at 4 weeks, factors predicting 8-week culture conversion were less readily evident. AFB smear grade at baseline did remain predictive of 8-week culture conversion, but the effect was relatively small (odds ratio 1.9). This result could suggest that other

124 Chapter 10: Results IV – Longitudinal determinants, such as the robustness of an individual‘s anti-mycobacterial immune response, potentially influenced by factors such as serum vitamin D or L-arginine levels, might have relevance in determining culture conversion. This will be evaluable later in the course of the AVADPT study.

Presence of cavitary disease and higher AFB density at TB diagnosis are well recognised to predict slower sputum smear conversion times.142, 150, 151, 213 Additional predictors of sputum smear conversion rates in one study included older age, male sex and smoking,151 but these associations have not been confirmed elsewhere,142, 150, 213, 214 nor in the data presented in this chapter. Also consistent with the findings presented here, isoniazid resistance has not been shown to influence sputum smear conversion rates142 or other outcome measures215 among people receiving standard isoniazid- containing regimens. HIV has been found to be associated with either no difference150 or with shorter times to smear conversion,213 again supporting the current findings in which a small but non-significant faster smear conversion during the early treatment phase was noted among HIV positive people, likely to be attributable to their lower baseline smear grade and lower likelihood of cavitary disease.

Time to smear conversion is an important public health measure due to the reliance on this measure, in many protocols, to guide duration of respiratory isolation, and the duration of intensive phase treatment. In Timika, according to standard practice, intensive phase treatment (the daily 4-drug regimen) is continued for an extra month if the sputum smear is still positive at week 8. However in this study as elsewhere, persistent smear positivity can over-estimate infectivity risk, due to persistence of non- viable or minimally viable bacilli in sputum.212 Here, around half (54%) of smear positive 8-week sputum specimens were culture negative. The most likely explanation is that the visualised bacilli were non-viable, but other explanations include loss of viability prior to the specimen reaching the laboratory, excessive de-contamination procedures or other potential laboratory errors (e.g. labelling or data entry error). These results confirm that the use of smear status to guide the duration of respiratory precautions may be an excessively cautious approach; however, in the Timika setting, delays in culture and sensitivity turn-around time, and the potential for false-negative culture results, mean that this approach remains sensible.

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TREATMENT OUTCOMES

Key targets set by the WHO for DOTS programs are to detect at least 70% of new sputum smear-positive TB cases, and cure at least 85% of these, where cure is defined as negative smears in the last month of treatment plus on at least one previous occasion.4 People who complete treatment but cannot be proven to be cured, because of absence of a second confirmatory negative sputum sample, are nevertheless classified as achieving a successful outcome. WHO TB annual reports consider the 85% target to be fulfilled if 85% attain treatment success (cure or completion, rather than cure alone).2 Cure was achieved in 78.2% of AVDAPT study participants, and overall treatment success in 84.5%. These data, only just reaching WHO targets, indicate the difficulties of confirming cure and retaining patients in care, even in a well-functioning DOTS setting. (The Timika TB clinic is considered to achieve better outcomes than other Puskesmas clinics and private practitioners in Timika.) Furthermore, study participants comprise only those well enough to be managed as outpatients at diagnosis, with the most unwell TB patients requiring initial hospital care excluded from this RCT. However, as the large majority of people with TB in Indonesian and globally are managed on an outpatient basis (an important component of nosocomial transmission reduction strategies), the disease severity of these study participants is therefore representative of the majority of Indonesian pulmonary TB cases.

Comparing with the most recently available national Indonesian reported outcomes (cure 83%, completed 8.5%, died 2.1%, failed 0.6%, defaulted 4.6%, transferred 1.7%),2 transfer and default rates were higher in this study, likely to be attributable to the high mobility of the Timika population. Death rates were low compared with nations with higher HIV-TB co-infection or MDR-TB rates (e.g. South Africa: 7.3%, Russian federation 12%).2 Direct comparison with national statistics may be somewhat misleading, since the careful scrutiny of each case in this study to determine that outcome is correctly classified may result in different reported outcomes compared with national reporting seeking to satisfy WHO targets. Indeed, in Indonesia overall in 2006, treatment success was reported to have been achieved in a surprisingly high 91% of cases.2

The WHO treatment outcome categories serve as an important guide in low-income countries where routine culture facilities are unavailable, providing an indication of DOTS program performance. They are less useful as a clinical measure at the level of 126 Chapter 10: Results IV – Longitudinal the individual, as has been previously acknowledged;216 for instance, cause of death, which may be unrelated to TB, is not taken into account. Therefore it is not surprising that in the data examined here, no firm associations were identified between favourable / unfavourable outcome and other markers of disease severity, apart from illness duration prior to diagnosis being longer in the 2 people who died and the one who failed treatment.

An additional important outcome measure for trials of TB treatment is TB relapse in the 24 months following the completion of treatment.217 Relapse is not assessed in the AVDAPT study because the outcomes of interest are impacts of adjunctive therapies on early treatment responses, because 2-month sputum culture results correlate strongly with later relapse risk,145, 217 and because the additional follow up time and MTB isolate typing which would be required to distinguish relapse from reinfection, pose feasibility problems at the research site.

DEVELOPMENT OF A COMPOSITE CLINICAL OUTCOME SCORE

Use of a composite clinical outcome score as a primary outcome measure of the AVDAPT study was considered to be prudent in order to avoid reliance on a single primary outcome measure requiring off-site processing in a reference laboratory, and to capture important clinical outcomes independent of bacteriological results. The resulting 12-point score, comprising components which were selected pre hoc on the basis of perceived clinical relevance and established importance in TB, was found to be significantly related to changes in other treatment response measures, and to decrease significantly over time. Furthermore, scores were broadly distributed at the two time points (8 and 24 weeks) indicating that this score is likely to provide adequate discriminatory capacity.

The dichotomous score shows evidence of being too stringent and therefore is less likely to be of good discriminatory value in assessing AVDAPT study outcomes. Positive (good) scores on the dichotomous measure were only achieved in 14% of patients by 8 weeks, and less than half by treatment completion, including in the majority of people who had a successful treatment outcome. Therefore this analysis establishes that the ordinal score is likely to be of more value than the dichotomous

127 Chapter 10: Results IV – Longitudinal score, and hence the former will be employed as the AVDAPT study‘s second primary endpoint.

ADVERSE EVENTS

Transient, asymptomatic hypercalcaemia was documented in 16.5% of study participants between 2 and 8 weeks, not more than has been reported elsewhere during the course of TB treatment with or without vitamin D supplementation.87, 158 In other recent published reports of vitamin D supplementation in TB, 11 TB patients were administered a single dose of 100 000 IU ergocalciferol, resulting in no episodes of hypercalcaemia at 8 weeks,86 and in a randomised trial of 100 000 IU cholecalciferol in patients with TB in Guinea-Bissau, there was no difference in hypercalcaemia symptoms or detection of biochemical hypercalcaemia in those randomised to the treatment arm.81 Other studies have also reported safe administration of vitamin D in TB.157, 159

Although numbers in each study arm were small at the time of the interim analysis, our interim safety analysis also indicated that hypercalcaemia and other adverse event rates did not differ between the study arms. Therefore this new data adds to the recently- growing body of literature indicating the safety of vitamin D use in active TB, in contrast to the view still mentioned in some contemporary guidelines that active TB contraindicates vitamin D administration.218 High rates of hypercalcaemia reported in 1984 by Narang et al219 in vitamin D-supplemented people with or without TB formed the basis of these concerns, but these findings have not been reproduced, and are speculated to have arisen due to a major underestimation of the actual amount of vitamin D administered in this study.220 A second reason to cite TB as a contraindication to vitamin D treatment might be the hypothetical concern of vitamin D- induced up-regulation of host immune responses leading to an immune reconstitution- type effect with transient worsening of TB symptoms. Such concerns have not been borne out by studies to date,74, 81, 86, 157, 159 nor in the data presented herein, (specifically, worsening of respiratory symptoms was not significantly higher in any of the treatment arms). The cell-mediated immune depressive effect of vitamin D3 (in contrast to its stimulatory effect on innate immune responses) might in fact be hypothesised to act to prevent excessive cell-mediated immune responses and thereby mitigate inflammatory immunopathology.38

128 Chapter 10: Results IV – Longitudinal

Adverse effects of oral L-arginine at the dose used in this study (6g daily) include diarrhoea, bloating or nausea, but these are uncommon, affecting only about 3 - 11% of people.52, 154, 155 Gastrointestinal symptoms affected almost half (46.8%) of the study participants overall, but were usually mild. Rates were approximately even in each study arm (between 37.0 and 48.2% at the time of the interim analysis), indicating no current evidence for additional gastrointestinal upset associated with L-arginine. True differences might have been undetectable due to the small numbers in each arm (27 to 29 people) and will be re-evaluated when 50% and 100% of participant data is available. Similarly, the uneven gender distribution due to the small numbers of females in the study (only 5 to 14 in each arm) may balance as the sample size increases. Randomisation is stratified on the basis of ethnicity but not gender, and the randomisation process has been successful in balancing all other baseline characteristics and maintaining even numbers among study arms.

Gastrointestinal disturbance, as well as being a potential study medication adverse effect, is also widely reported with anti-tuberculosis antibiotics, either as a direct medication side effect, or secondary to drug-induced hepatitis. However gastrointestinal disturbance rates are usually quoted as occurring in only around 1-10% of people taking anti-tuberculosis medications.221 The higher rates reported here may be attributable to the active surveillance undertaken, with weekly questioning about symptoms, which may encourage people to report mild symptoms they might usually not disclose. The rate of drug regimen switching due to side effects was not higher than expected (0.7%, compared with 5.1% reported elsewhere25), confirming the mild nature of most of the AE reported here.

10.5 CONCLUSION

Confirmation of previously-defined predictors of sputum smear and culture conversion provides confidence in the reliability of the methods used in the AVADPT study to grade sputum microscopy, perform MTB culture and classify the presence of cavitary disease on x-ray, as well as in the record-keeping and data entry processes employed. The observation that predictors of culture conversion become less important over the course of 8 weeks suggests the potential importance of other as-yet undefined factors in determining an individual‘s response to therapy. After evaluating two alternative

129 Chapter 10: Results IV – Longitudinal methods for coding the second primary endpoint, a composite clinical endpoint which allocates a total score between 0 and 12 to study participants, depending on their percentage improvement in weight and respiratory parameters, has been selected for use in the trial. The interim analysis and the Data and Safety Monitoring committee‘s recommendation adds weight to the argument that vitamin D at the dose used is safe in TB. Additionally, the administration of oral L-arginine in active TB appears to be associated with no apparent safety concerns.

130 Chapter 11: Results V – Exhaled Nitric oxide

11 Results V: Exhaled nitric oxide in pulmonary TB

In this chapter I describe the performance of the portable NiOX MINO® analyser for measurement of fractional exhaled nitric oxide (FENO), and examine FENO characteristics in AVDAPT study participants and healthy volunteers in Timika.

11.1 INTRODUCTION

FENO IN PULMONARY TB

As detailed in Chapter 4, nitric oxide (NO) is a key component of the macrophage anti- mycobacterial immune response,38 and is measurable in expired air using chemiluminescence analysers. Immunological, biochemical and haemodynamic determinants of NO in expired air are multiple, complex and incompletely defined,107 but primarily include macrophage activation, Nitric oxide synthase 2 (NOS2) production, and availability of NOS2 substrate (L-arginine).

Core hypotheses of the AVDAPT study include that baseline pulmonary NO production will be elevated in pulmonary TB but inversely associated with disease severity, that baseline and post-treatment increments in exhaled NO will be associated with rapidity and magnitude of the treatment response, and that L-arginine supplementation in pulmonary TB will enhance pulmonary production of NO. In this chapter I therefore compare FENO in TB patients versus healthy controls, investigate the relationships between baseline FENO and TB disease severity measures, and describe longitudinal trends in FENO. The impact of L-arginine supplementation on FENO will be evaluated at trial completion when the randomisation code has been revealed.

PERFORMANCE CHARACTERISTICS OF NIOX MINO FENO ANALYSER

A NiOX FLEX® non-portable exhaled NO analyser, considered the reference analyser, is installed at the Timika Research facility, housed in a custom-built, temperature- regulated room at 23°C. A handheld NiOX MINO® exhaled NO analyser is available

131 Chapter 11: Results V – Exhaled Nitric oxide for use by participants in the AVDAPT study, who are evaluated at the Timika TB clinic, 15 km from the Timika Research facility. NiOX MINO and FLEX analysers have been shown to provide comparable readings under controlled research settings (see Chapter 3).101-103 The portable analyser has a replaceable sensor providing a set number of tests (100 or 300 tests depending on the sensor type). It does not permit calibration, and therefore requires regular comparison against the reference analyser.

The manufacturer‘s guidebook states that NiOX MINO precision (repeatability) is ±5 ppb of measured values <50 ppb, or ±10% of measured values ≥50 ppb, and accuracy (compared with gold standard) should be <3 ppb of measured values <30 ppb or <10% of measured values ≥30 ppb.222 The optimal environmental conditions for NiOX MINO® sensor operation include an ambient temperature range of 16 to 30°C, and relative humidity of 20 to 60%.222 Performance characteristics of the portable analyser sensors have not previously been described in tropical settings.

Potential sources of inaccuracy in FENO measurement in AVDAPT study participants require consideration and, if necessary, correction. These include any systematic difference in results between the portable and reference analysers, or drift within NiOX MINO sensors over time, which has been noted by other investigators.223 Therefore prior to presenting FENO data, the results of quality control measurements to determine the need for correction factors are presented.

11.2 METHODS

Methods of study participant recruitment are as described in Chapter 6. The NiOX FLEX and handheld NiOX MINO exhaled NO analysers are operated according to the guidelines provided by the manufacturer (Aerocine) and the American Thoracic Society.100 The NiOX FLEX machine is calibrated fortnightly using nitric oxide (200 ppB), and is serviced regularly by the manufacturer. All FENO measurements from study participants or healthy controls are made using the NiOX MINO, since the TB clinic where they are seen is in a different location from the research building housing the NiOX FLEX.

FENO is measured once on the NiOX MINO analyser in healthy controls, and in TB patients, at weeks 0, 1, 2, 4, 8 and 24. Subjects are asked to refrain for eating and

132 Chapter 11: Results V – Exhaled Nitric oxide smoking for several hours prior to FENO measurement, and timing of the last meal is recorded. To measure FENO, subjects inhale NO-free air to total lung capacity by inhaling through a mouthpiece containing an NO scrubber, then exhale for a 10-second duration at a constant flow rate of 50 ± 5 mL/s, achieved by real-time audio and visual feedback from the measuring device indicating whether the flow rate is correct.

An additional historical dataset of FENO values in healthy controls was also made available for this analysis. This dataset comprises 44 Papuan and 4 Non-Papuan volunteers recruited in 2005 at the Timika research facility as the control group for a study of FENO in subjects with malaria, by NIHRD-MSHR research staff led by Dr Tsin 56 Yeo. FENO was measured on a single occasion using the NiOX FLEX (reference) analyser.

QUALITY CONTROL (QC) TESTING AND ANALYSIS

FENO values obtained using the portable analyser (MINO) used in the TB clinic are compared with the reference analyser (FLEX) at the research building once weekly. QC measures comprise the following 3 methods:

1. FENO readings are obtained sequentially from healthy human biological controls (AVDAPT study staff members) on both machines at the research building, with measures made not more than 15 minutes apart. Eating and exercising are

avoided for 2 hours prior to FENO measurement.

2. FENO readings are obtained using an impermeable foil bag filled with a mixture of exhaled breath and NO from a standard 200ppb NO cylinder, passed into the reference analyser then the portable analyser. This method permits higher readings than can be obtained from the available biological controls (but within the range obtained among study participants). Foil bags and accompanying tubing and 3-way taps were provided by Dr Cheryl Salome, Woolcock Institute of Medical Research, NSW. 3. In the instance of the reference analyser being unavailable (faulty or under service), ongoing weekly measures from the healthy human biological controls,

whose usual FENO readings are known, are continued on the NiOX MINO.

Statistical analyses were performed in Stata 10.1, with graphs produced either in Stata or Graph Pad Prism 5.0. Regression analyses were used to calculate correction factors 133 Chapter 11: Results V – Exhaled Nitric oxide and examine associations between FENO and predictor variables. Bland-Altman plots were constructed to evaluate differences in FENO results within and between analysers. Proportions were compared between groups using Pearson‘s  tests. Differences in continuous data between groups were tested using 2-sample t-tests for normally distributed data, and the Wilcoxon Rank-Sum test for non-normally distributed data.

Paired Students T-tests were used to compare FENO results obtained at 2 time points from the same individual (log-transformed since FENO data was non-parametrically distributed). Statistical tests were two-sided, with significance for p-value <0.05. Correlations between non-parametric data were examined using Spearman‘s method.

11.3 RESULTS

QUALITY CONTROL DATA

NiOX MINO precision (repeatability)

Precision of FENO measures was ascertained from repeated measures made using a single analyser and paired measures using 2 different NiOX MINO analysers. The average difference between repeated measures of exhaled nitric oxide made within 15 minutes by an individual on a single NiOX MINO analyzer was -0.4 ppb (95% limits of agreement -6.2 to 5.5) calculated using the Bland-Altman method (Table 11.1 and Figure 11.1). The average difference between paired measures made within 15 minutes by an individual on two different NiOX MINO analyzers (with different sensors) was 0.8 ppb (95% limits of agreement -4.3 to 5.8) (Table 11.1 and Figure 11.2).

Table 11.1: Correlation between FENO measures Table shows correlations between sequential FENO measures from biological controls (research staff members) on one NiOX MINO analyser, and paired measures on two analysers

n Bland-Altman method Correlation Difference between readings Pearson’s R P value in ppb: mean (95% limits of agreement) Repeated measures on 36 -0.4 (-6.2 to 5.5) 0.81 p<0.0001 one analyser Paired measures on two 16 0.8 (-4.3 to 5.8) 0.82 p<0.0001 analysers

134 Chapter 11: Results V – Exhaled Nitric oxide

Figure 11.1: Bland-Altman plot: repeated exhaled nitric oxide measures on single analyser

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observed average agreement 95% limits of agreement y=0 is line of perfect average agreement

Figure 11.2: Bland-Altman plot: paired exhaled nitric oxide measures on 2 analysers

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NiOX MINO accuracy (comparison with ‘gold standard’)

FENO values obtained from the Niox Mino portable analysers correlated highly with values obtained from the Niox Flex ‗gold standard‘ analyser (Pearson‘s R 0.95,

p<0.0001), but were consistently lower, with the difference being proportional to FENO values (see Bland Altman plot, Figure 11.3), indicating the need for a correction factor.

For example, as demonstrated in the scatter plot (Figure 11.4), for true FENO values of 20 and 80 ppb, the NiOX MINO gave readings of approximately 15 and 50 ppb respectively.

135 Chapter 11: Results V – Exhaled Nitric oxide

Figure 11.3: Bland-Altman plot: paired exhaled nitric oxide measures on the NiOX MINO

(portable analyser) and NiOX FLEX („gold standard‟)

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observed average agreement 95% limits of agreement y=0 is line of perfect average agreement

Figure 11.4: Relationship between exhaled nitric oxide measures on the Niox Mino and Niox Flex

(Scatter plot showing individual data points and line of best fit).

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FeNO (ppb), portableFeNO analyser(ppb), (NiOXMINO) 0

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Examination of the difference in FENOMINO and FENOFLEX values demonstrated that readings were almost identical between the two analysers for the first month of the study (June 12 2008 – July 7 2008), but lower readings were consistently obtained from all MINO sensors thereafter (July 8 2008 – October 30 2009), postulated to be due to local environmental conditions (high temperature and humidity). All efforts to maintain optimal operating environments were instigated at this point, including storing the portable analyser and its sensors in a dry, air-conditioned environment at all times that the analyser was not in use, and placing it in a 19°C refrigerator between tests at the TB clinic.

Additionally in the case of 2 NiOX MINO sensors, a significant downward drift was noted over time, generating spuriously low readings, including frequent readings less

136 Chapter 11: Results V – Exhaled Nitric oxide than the lower limit of detection (5 ppb), and culminating in sensor failure in both instances before all tests on the sensor had been used. Therefore FENO data for TB patients and healthy controls collected during these two time periods (14/7/08 - 7/9/08 and 17/03/09 - 24/03/09) prior to sensor failure were culled from further analyses

Correction factor applied to NiOX MINO results

Correction factors to apply to FENOMINO values obtained at the TB clinic, to allow an approximation of ‗true‘ (FENOFLEX) values, were calculated using the equation for a straight line: y = a + bx where y = FENOMINO (test value)

x = FENOFLEX (true value) a = constant derived from regression of MINO sensor on FLEX b = coefficient derived from regression of MINO sensor on FLEX Solving for x:

True FENO = (FENOMINO – a) / b

To avoid extrapolating far beyond the bounds of the quality control range of FENO data

(Figure 11.4) or beyond the bounds of physiological likelihood, the maximum FENOMINO value was considered to be 100 ppb, to which corrections were subsequently applied. This affected the results of 2 healthy controls but no TB patients. Corrections are summarised in Table 11.2.

Table 11.2: Corrections applied to exhaled nitric oxide readings obtained from the NiOX MINO portable analyser

Time period Correction applied to NiOX MINO FENO result

Correction factor 12/06/08 – 07/07/08 True FENO = (FENOMINO - 0.2795245) / 0.9560819 required 08/07/08 – 30/10/09 True FENO = (FENOMINO - 2.922176 ) / 0.5824131 Data not 14/07/08 - 07/09/08 Data coded as missing analysed due to 17/03/09 - 24/03/09 Data coded as missing faulty sensor

FENOMINO >100 12/06/08 - 24/03/09 FENOMINO =100

137 Chapter 11: Results V – Exhaled Nitric oxide

FENO IN ADULTS WITH PULMONARY TB VERSUS HEALTHY SUBJECTS

Table 11.3: Characteristics and FENO in healthy volunteers and TB patients at enrolment

Healthy volunteers TB patients P value* N 83 162 - Dataset 1 48 Dataset 2 35 Age in yrs: median (range) 27 (18-65) 27 (15 – 65) p=0.4 Dataset 1 27.5 (18 – 42) Dataset 2 27 (18-65) Female: no. (%) 26 (31.3) 54 (33.3) p=0.5 Dataset 1 17 (35.4) Dataset 2 9 (25.7) Papuan: no. (%) 62 (74.4) 78 (48.2) p<0.0001 Dataset 1 44 (91.7) Dataset 2 18 (51.4)† Current or ex-smoker: no. (%) 42 (51.9) 93 (57.4) p=0.3 Dataset 1 26 (56.5) Dataset 2 16 (45.7) Weight in kg: mean (SD) 60.8 (9.5) 48.4 (7.5) p<0.0001 Dataset 1 59.8 (9.0) Dataset 2 62.2 (10.2) Height in m: mean (SD) 1.61 (0.07) 1.58 (0.08) p=0.1 Dataset 1 - Dataset 2 1.61 (0.07)

Number with corrected FENO 83 133 available

FENO in ppb‡: median (range) 16.9 (2.5-166.7) 15.6 (2.5 – 106.6) p=0.4 Dataset 1 16.6 (4.9 – 64.6) Dataset 2 19.0 (2.5-166.7)§ *p values calculated from Pearson‘s chi2 tests, 2-sample t-tests or rank-sum tests. †proportion of Papuans in healthy volunteer Dataset1 vs Dataset2: p<0.0001. ‡ Corrected FENO used the calculated correction factors applied to Dataset 2 healthy volunteers and TB patients. §FENO in healthy volunteer Dataset1 vs Dataset2: p=0.6

Healthy subjects

FENO results were available for 48 healthy volunteers recruited in 2005 (Dataset 1) who had FENO measured using the NiOX FLEX. FENO results were also available for 35 healthy volunteers recruited in 2008-9 (Dataset 2) who had FENO measured using the NiOX MINO. The proportion of Papuans to Non-Papuans was significantly higher in

138 Chapter 11: Results V – Exhaled Nitric oxide

Dataset 1 than Dataset 2, and median FENO trended higher in Dataset 2 (19.0 vs 16.6), but the difference was not significant (Table 11.3).Height was not recorded in Dataset 1.

Predictors of FeNO in healthy volunteers

No variables were found to significantly predict FENO among the pooled or individual healthy volunteer groups. Statistically non-significant differences included that smokers had lower median FENO (15.5 ppb) than non-smokers (17.3 ppb, p=0.08), and people of

Papuan ethnicity had higher median FENO (17.3 ppb) than Non-Papuans (12.2 ppb, p=0.08); these associations diminished further in significance when controlling for each other and age, sex, weight and height. Notably, weight and height failed to significantly predict FENO (regression coefficient for weight: -0.004, p=0.7; for height: -0.18, p=1.0).

FENO was also found to be independent of age, sex and haemoglobin.

FeNO in TB patients versus healthy volunteers

At the time of TB diagnosis, no statistically significant difference was identifiable between people with TB and healthy volunteers. People with TB had somewhat lower median FENO than healthy volunteers (see Table 11.3), particularly when comparing TB patients with the new healthy volunteer sub-set (Dataset 2, 2008-9) whose FENO measurements were made using the same NiOX MINO method and application of correction factors as the AVDAPT study participants. Although these FENO difference was not statistically significant at week 0 (p=0.2, 2-sample t-test on log-transformed

FENO), FENO decreased further in TB patients immediately after starting treatment, such that by weeks 1 and 2 of TB treatment, patients had significantly lower FENO than did the healthy volunteers (p=0.004 at week 1, p=0.04 at week 2, Figure 11.5). Longitudinal

FENO trends are examined in more detail below.

DETERMINANTS OF FENO IN ADULT WITH PULMONARY TB

Predictors of FENO among the 162 AVDAPT study participants at enrolment were evaluated initially in univariate analyses (regression of log-transformed FENO on predictor variables). Significant predictors of FENO were found to be sex, weight and radiological disease severity. Females had lower median FENO than males (13.0 vs 17.3 ppb, p=0.05, Figure 11.6). Lower bodyweight (or BMI) predicted lower FENO (weight: p=0.002, Figure 11.7; BMI: p=0.02). More extensive radiological disease also predicted

139 Chapter 11: Results V – Exhaled Nitric oxide lower FENO (p=0.03). However in multivariate models controlling for sex, age, ethnicity, height and smoking status, only the association with weight remained significant.

Figure 11.5: Exhaled nitric oxide in healthy volunteers and TB patients at week 0 and during TB treatment

p=0.9 p=0.04 p=0.004 p=0.2* 35 16.9 15.6 14.7 13.9 13.9 15.6 20.7 n:83 n:156 n:88 n:138 n:125 n:118 n:75 30

25

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FeNO in ppb (median, IQR) (median, ppb in FeNO 10

5 0 1 2 4 8 24 Week Healthy TB patients volunteers

*All p values calculated from 2-sample t tests (log-transformed FENO). Numbers above data indicate median FENO and number of observations at each time point.

Other small differences were detected which failed to achieve statistical significance:

Non-Papuans had marginally lower median FENO than Papuans (15.5 vs 16.9 ppb, p=0.1) as was observed among healthy volunteers, and FENO was also lower in HIV+ compared with HIV- people (15.6 vs 17.3 ppb, p=0.4), and in current smokers compared with non-smokers (15.8 vs 16.4 ppb, p=0.8). No associations were evident between height, age or serum ionised calcium level and FENO.

In examination of other markers of disease severity, trends towards an association between greater TB severity and lower FENO were noted, but none were statistically significant. Illness duration, symptom tally and SGRQ scores were inversely related to

FENO, while percentage of predicted FEV1, 6-minute walk distance and haemoglobin 140 Chapter 11: Results V – Exhaled Nitric oxide were directly related to FENO (p>0.05 level in univariate analyses and when controlling for weight or other variables).

Figure 11.6: Exhaled nitric oxide according to sex in TB patients at week 0

p=0.05*

Median FeNO Median FeNO 120 17.3 13.0

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*p value calculated from regression of log-transformed FENO (dependent variable) on sex (independent variable). Bars indicate median FENO.

Figure 11.7: Exhaled nitric oxide according to weight in TB patients at week 0

p=0.002* 120

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0 <40kg 40-49.9kg 50-59.9kg >=60kg Weight

*p value calculated from regression of log-transformed FENO (dependent variable) on weight (independent variable). Bars indicate median FENO.

FENO TRENDS DURING TREATMENT FOR PULMONARY TB

As noted above, median FENO readings initially decreased, reaching a nadir between weeks 2 to 4, then gradually rose throughout the remainder of the 24-week treatment

141 Chapter 11: Results V – Exhaled Nitric oxide

duration. Using paired t-tests, the FENO fall between week 0 and week 1 was significant

(p=0.005, 87 data pairs), as were the differences in FENO between weeks 2 and 24 (p=0.04, 70 pairs) and weeks 4 and 24 (p=0.001, 67 pairs), although not between weeks 0 and 24 (p=0.3, 71 pairs). However, not all individuals followed this pattern, with

longitudinal FENO trends among individuals showing some variability (data not shown). Modest, statistically non-significant direct correlations were found between percentage

increases in FENO over time and higher composite clinical outcome scores at weeks 8 and 24 (Figure 11.9).

Figure 11.8: Correlations between change in FENO and clinical outcome score at weeks 8 and 24

Composite clinical outcome scors aree calculated as described in Chapter 10. Plots show individual data points and line of best fit.

(a) Week 8 (b) Week 24

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Fitted values Fitted values

Spearman‘s Rho: 0.1, p=0.2. Spearman‘s Rho: 0.2 , p=0.096

Associations between serum ionised calcium (potentially indicative of macrophage

activation and 1,25(OH)2D3 synthesis) and FENO were examined at baseline and during follow up. At baseline, no significant association was identified between calcium level

and FENO in a linear regression model (p=0.1). During follow up, 23 people developed

hypercalcaemia. Median FENO among those individuals at the time of hypercalcemia occurrence was 12.2 ppb, compared with 16.9 ppb in those same individuals at times of normocalcaemia (p=0.4), consistent with the observation that the timing of hypercalcaemia occurrence coincided with the period between weeks 2 and 4 when

minimum FENO also occurred. Examining individual trends, synchronous with the

development of hypercalcaemia, FENO rose in 6 people, fell or stayed the same in 15, and was unavailable in 2.

142 Chapter 11: Results V – Exhaled Nitric oxide 11.4 DISCUSSION

This is the largest study to date of FENO in patients with pulmonary TB. The findings provide important and unexpected new contributions to the literature regarding the nature of FENO responses in people with pulmonary TB, from which a further understanding of local antimycobacterial immune responses can be extrapolated.

Interpretation of these findings is contingent on an understanding of FENO measurement characteristics in the field, and FENO values in locally-recruited healthy volunteers.

These interim results have several limitations. Firstly, although longitudinal findings have been presented here as well as baseline results, the potential impact of the study interventions (especially L-arginine) on FENO is readily acknowledged; hence in the absence of randomisation arm information, these results are not intended to provide definitive answers on FENO trends over time. Secondly, the methodological issues with regards to use of the NiOX MINO portable analyser FENO indicate a need for caution in interpreting the results. Finally, while the sample size is the largest to date investigating

FENO in TB, full recruitment of TB pateitns and healthy volunteers will allow greater confidence in interpreting the findings.

NIOX MINO PERFORMANCE

NiOX MINO analysers were found to have high repeatability, supporting the manufacturer‘s instruction that a single measure is adequate for any given time point.

However, FENO measured in human biological controls at the field research site was lower when using the portable NiOX MINO analyser than when using the non-portable NiOX FLEX reference analyser. This is in contrast with the findings of Alving et al, who found NiOX MINO readings to be generally slightly higher than those obtained from the reference machine.103 Additionally, 2 sensors were unstable over time, suffering from significant downward drift. Although other authors have described the need to apply correction factors to account for drift within NiOX MINO sensors over time,223 overall the extent of the difference between portable and reference analyser results and the amount of drift seen in some sensors in this study were greater than anticipated.

143 Chapter 11: Results V – Exhaled Nitric oxide

Environmental conditions (high humidity and ambient temperature) at the field research site are the most likely explanations for these findings. Aerocrine® scientific staff contacted about this issue and an expert adviser in FENO measurement (Dr Cheryl Salome) also agree with this interpretation of the results. These findings clearly establish the need for regulation of ambient conditions when operating NiOX MINO analysers, and are instructive for other researchers seeking to measure FENO in tropical settings. Since differences between the analysers were first recognised, the MINO analysers and their sensors have been stored whenever possible in temperature and humidity-regulated environments. However, during use, the analyser is unavoidably exposed to standard ambient conditions at the non-airconditioned TB clinic (temperature 27 – 34°C, relative humidity 80-99%) (see Figure 4.2, Chapter 4).

Given these findings, appropriate correction factors have been calculated and applied to all FENO values obtained from AVDAPT study participants and healthy controls. Although a systematic difference between the portable and reference analyser might not impact on analysis of trends, application of a correction factor permits the FENO data obtained from TB patients to be compared with the historical controls, and to be compared with the published literature on FENO values obtained at the same flow rate of 50 mL/s.

FENO IN HEALTHY VOLUNTEERS

Among volunteers recruited contemporaneously with the AVDAPT study participants, exceptionally high FENO values were measured in some apparently healthy individuals.

As discussed in Chapter 3, delineations of a normal reference range for FENO are evolving, but those constructed from studies of Swedish adults suggest that, for people of an age and height comparable to the healthy controls described here, FENO > 25 ppb is considered abnormal.105, 223 A possible explanation is that participants labelled as healthy did have underlying unrecognised pulmonary or systemic disease causing elevated FENO. While research assistants are assiduous in ensuring that participants are free of any intercurrent or recent illness including cough in order to satisfy inclusion criteria for eligibility as a healthy control, it is possible that undiagnosed or subclinical asthma or other inflammatory conditions might exist which are unknown to the patient and missed in the screening process. Another possible explanation for the finding of higher-than-expected FENO readings among a supposedly healthy population might 144 Chapter 11: Results V – Exhaled Nitric oxide include ethnic or dietary determinants; other authors have proposed that as-yet unidentified factors including nutritional status, genetics and / or undiagnosed or low 107 grade lung inflammation might contribute to inter-subject FENO variability. Nevertheless, such underlying factors are likely to apply across both control and TB groups in this setting, and highlight why control data derived from the local population are important. Experience with evaluating FENO in this environment in the past is limited to the study by Yeo and colleagues,56 the healthy control data from which has been used in the analysis here. This previous study did not examine differences in FENO between healthy Papuans and Non-Papuans. The new data presented here demonstrate that Papuans may have higher FENO than Non-Papuans, both among healthy volunteers and TB patients, although these findings did not reach statistical significance.

Few predictors of FENO in healthy people have been identified in the past: initially, 100 factors such as age and height were not thought to significantly influence FENO, but a more recently-published study of over 1000 Swedish adults who had never smoked was able to show a significant relationship, whereby increasing age and height predicted 107 higher FENO values. That age and height were not found to significantly predict FENO among the healthy volunteers reported here may therefore reflect the relatively small sample size and the heterogeneous nature of the volunteer population, comprising a mix of ethnicities and smoking status. A potential limitation in interpreting the pooled results of historical and contemporary controls is the different methods used to measure

FENO (the reference analyser in the former, and the portable analyser with application of a correction factor in latter). Smoking is one of the few consistently-identified 100, 109 predictors of FENO, being associated with depressed FENO. Although smokers

(both healthy volunteers and AVDAPT patients) did have slightly lower FENO values than non-smokers, the differences were non-significant. Rather than bringing into question the reliability of the FENO assay, this may again be a product of the heterogeneity of the groups studied, and in the case of the volunteers, the relatively small sample size, such that other unidentified influences might have impacted on NO dynamics in the lung, obscuring the significance of the smoking effect.

FENO IN PEOPLE WITH PULMONARY TB

A key hypothesis articulated in the Aims of this thesis is that FENO will be increased in participants with pulmonary TB compared with healthy controls, will be inversely related to disease severity at baseline, and will return towards normal by the end of 145 Chapter 11: Results V – Exhaled Nitric oxide therapy. The theoretical basis for the generation of this hypothesis included the following principles, unpinned by the clinical findings reported by Wang et al:110 (1) in 100 general, FENO is increased in pulmonary inflammatory conditions; (2) specifically, NO as a mediator of anti-mycobacterial immune responses would be expected to be increased in response to infection with TB;38, 110, 224-229 (3) an inverse relationship with disease severity would arise due to people with insufficient NO responses being less able to contain MTB proliferation and TB-related pathology.38, 230, 231

In dramatic contrast to the stated hypothesis, the data presented here indicate that FENO appears to be no different, or in fact potentially lower (but not statistically significantly), in TB than in health. Why might people with pulmonary TB have non-elevated, or lower, FENO than healthy volunteers? As reviewed in the paper reproduced in Chapter 3, NO production from L-arginine by the enzyme NOS2 is a key component of the anti- mycobacterial response. An absence of elevated NO in active pulmonary TB infection could therefore indicate one or more of the following possible hypotheses.

Firstly, an impaired ability to generate adequate NO might be a precursor for the development of active TB, rather than low-normal FENO necessarily being a consequence of active TB. Thus, low NO-generating capacity in an individual‘s pulmonary tract might be a risk factor for active TB. This concept is lent plausibility by the additional inverse relationship identified between disease severity (chiefly lower weight) and FENO, a finding which is consistent with our original hypothesis. A defective capacity to generate NO from L-arginine might arise due to L-arginine deficiency. L-arginine deficiency can be due to malnutrition or disease-related L- arginine metabolism, since arginase can be induced by MTB in macrophages, thus diverting L-arginine substrate away from NO production and instead promoting the breakdown of L-arginine to ornithine.94 Alternatively, NOS2 production or activity might be impaired because of a cytokine profile favouring diminished activity in the IFN-JAK/STAT-1 pathway, resulting in down-regulation of the NOS2 gene and decreased NOS2 generation.38 With less NO available in alveolar macrophages and surrounding tissues, MTB proliferation might be less well controlled,224, 225, 228 and, extrapolating from animal models, more severe pulmonary pathology might develop.38, 230, 231 Alternatively NO quenching from disease-induced superoxide or other radicals may reduce local NO bioavailability in TB. These possibilities strengthen the rationale for trialling adjunctive L-arginine in people with active TB, and further suggest that

146 Chapter 11: Results V – Exhaled Nitric oxide maintaining adequate L-arginine levels through optimised nutrition may be an important preventive strategy in people with latent TB infection.

Secondly, the capacity to generate NO might be high, but actual NO generation at the level of the alveolar macrophage could be poorly reflected by the measurement of NO in expired breath after transiting the pulmonary tract. NO is a highly volatile gas with a short half life. It is rapidly removed by pulmonary and upper respiratory tract scavenger molecules for conversion in vivo to nitrite or nitrate. However, given the recognised validity of FENO measurement, including the known association between NOS2 110 concentration and FENO, this appears to be a less likely explanation.

Thirdly, it is possible that, in contrast to murine models of TB, NO production may not be as important in the human TB immune response as has been suggested by the in vivo and in vitro studies to date. However the evidence for NO importance is substantial: additional to the large body of literature on the role of NO in animal immune responses to MTB, summarised elsewhere,38 human pulmonary macrophages have been shown to kill mycobacteria only if they express NOS2, with killing prevented with a NOS inhibitor.224 Rich and colleagues showed that NO production by alveolar macrophages from healthy humans infected ex vivo with MTB correlates with intracellular growth inhibition of MTB.225 A number of other researchers have identified NO production by macrophages to be a standard human immunological response to MTB infection.110, 226- 229 While the evidence is strong that NO production is a usual human protective immune response to TB infection, it is possible that this response is of chief importance at the time of initial exposure to MTB, rather than at the later stage of disease (active TB) assessed in this study. NO production may be higher at earlier stages of active disease, but fall as disease progression leads to reduced L-arginine bioavailability and/or NOS2 activity or increased local quenching of NO. Each of these factors may then exacerbate disease progression.

Aside from obvious risk factors such as HIV infection, why it is that some individuals adequately contain latent TB infection, while others eventually progress to active TB, remains a major research question. The data presented here raise the hypothesis that a failure to generate adequate NO in response to MTB infection may be an example of a characteristic identifying those at increased risk of developing active TB. Furthermore, the more defective the NO response, the worse the TB disease severity may be. This

147 Chapter 11: Results V – Exhaled Nitric oxide possibility is supported by the finding that weight / BMI significantly inversely predicted FENO in TB patients but not in healthy controls, suggesting that the association is due to TB disease severity rather than body weight or BMI per se influencing FENO.

The additional inverse association between radiological disease extent and FENO supports this, as does the fact that FENO was marginally lower (but not significantly so) in those with longer illness duration, more symptoms, worse quality of life scores, and HIV-TB co-infection.

The next question raised by this data is: why might FENO decrease after TB treatment is started, as suggested by the AVDAPT data? As mentioned above, longitudinal trends cannot be interpreted with confidence without the availability of randomisation arm information. However, this finding deserves discussion since it is a reverse of the anticipated trend at least among the 50% of study participants who will have been randomised to receive L-arginine.

An initial worsening of disease severity prior to improvement, due to increased inflammatory activity in response to antibiotic treatment, is a well-recognised phenomenon in TB. These paradoxical reactions are noted to occur in approximately 10% of TB patients without HIV,232 and up to 43% of HIV-TB co-infected patients commencing antiretroviral therapy (in which case, if meeting the case definition, it is termed immune reconstitution inflammatory syndrome).233 This phenomenon is also thought to account for the development of hypercalcaemia after TB treatment commencement, as the host‘s granulomatous response is restored and macrophage activation via toll-like receptor (TLR) pathways leads to increased 1,25(OH)2D3 synthesis.234 Since the trend indicated in data from the AVDAPT study participants is for an initial decrease in FENO prior to a later rise, and a fall in FENO (statistically non- significant) in concert with a rise in serum calcium, macrophage activity during this period might become predominantly skewed towards TLR1/2-MyD88-NFkB pathways and away from the IFN-JAK/STAT-1 pathway (see Figure 2, ―L-arginine and Vitamin D: novel adjunctive immunotherapies in tuberculosis‖,38 Chapter 3), thereby resulting in increased 1,25(OH)2D3 synthesis rather than NOS2 up-regulation. Whether or not this is an immunologically beneficial outcome is unclear.

Since many of the FENO associations identified here were modest, definitive conclusions will be deferred until study completion. In particular, assessment of the longitudinal

148 Chapter 11: Results V – Exhaled Nitric oxide data will be more meaningful once study participants can be stratified according to the study medication arm to which they were randomised. The early reduction in FENO may be more apparent in those not receiving L-arginine supplementation.

COMPARISON WITH PREVIOUSLY-PUBLISHED FINDINGS

As described in Chapter 3, the two previously published reports investigating FENO in TB provided conflicting results. The data presented here agree with elements of both papers (Table 11.4), including the inverse association between FENO and disease severity identified by Wang et al,110 and the lower (but statistically non-significant) 111 FENO in TB infection identified by Idh et al.

Table 11.4: Summary of salient findings from the present study and previous investigations of exhaled nitric oxide in tuberculosis

AVDAPT study Wang et al, 1998110 Idh et al, 2008111 Number TB patients 162 19 95 Healthy controls 83 14 63 Method

FENO assay NiOX MINO (flow rate Slow exhalation (flow NiOX FLEX (flow rate methodology 50mL/s, inhalation of rate ~200 mL/s) 50mL/s, omission of NO-free air prior to through open tube, inhalation of NO-free exhalation) with with a probe sampling air prior to exhalation) application of NO from the stream correction factor of expired breath. Results

FENO in TB versus Same (non-significant Higher Same (non-significant healthy volunteers trend towards lower trend towards lower

FENO in TB) FENO in TB)

FENO in HIV+TB+ Same (non-significant Not evaluated Same (non-significant versus HIV-TB+ trend towards lower trend towards lower

FENO in HIV+TB+) FENO in HIV+TB+)

Association between FENO lower in more FENO lower in more FENO independent of

FENO and TB severity severe disease severe disease disease severity

Longitudinal trend FENO more closely FENO more closely Not evaluated approximates that of approximates that of healthy controls by 6 healthy controls by 3 months of treatment months of treatment

(net rise in FENO) (net fall in FENO)

149 Chapter 11: Results V – Exhaled Nitric oxide

110 Wang et al investigated FENO in 19 people with smear positive pulmonary TB (notably excluding people with poor nutritional status) and 14 non-smoking controls (6 with haemoptysis of unknown cause and 8 healthy volunteers) using a now-obsolete method, whereby participants exhaled slowly at a flow rate of around 200 mL/s through an open tube, with a probe sampling NO from the stream of expired breath. Therefore,

FENO values are not directly comparable to results obtained at flow rates of 50 mL/s, but nevertheless among TB patients were in a similar range to results reported here. They found higher FENO in people with newly diagnosed TB (16.2±1.2 ppb) compared with controls (6.5±0.9 ppb), net normalisation of FENO by 3 months of treatment (comprising a decrease in 18 patients, but an increase in one), and an inverse association between 110 FENO and disease severity. Their finding of higher baseline FENO in contrast to the data presented here might be attributable to the different FENO measurement method employed, the small sample size might have been poorly representative of usual populations of pulmonary TB patients, the ‗healthy‘ controls might not have been an ideal comparative group since 6 of them had haemoptysis, or since malnourished TB patients were specifically excluded, this may have ensured the inclusion only of people with replete NO substrate (L-arginine) levels, who were therefore capable of adequate exhaled NO production.

The study by Idh et al,111 also in smear positive pulmonary TB, suffered from methodological flaws including that inhalation of NO-free air was omitted prior to FENO measurement, and analyses did not control for anthropometric measures or age.

Although the difference in FENO was miniscule between the 36 HIV+/TB+ patients

(median FENO 14.2 ppb) and 59 HIV-/TB+ patients (median FENO 14.3 ppb), the authors nevertheless found FENO to be significantly lower in HIV+/TB+ than in healthy controls, but that the difference between HIV-/TB+ (and presumably in the 95 TB patients overall) was not significantly lower than in healthy controls. These trends are essentially similar to those found in the AVDAPT study participants.

11.5 CONCLUSIONS

Despite the recognised anti-mycobacterial role of NO in human macrophages, active pulmonary TB does not appear to be associated with elevated FENO compared with controls in the current study (the largest to date), nor in the only other study111 which

150 Chapter 11: Results V – Exhaled Nitric oxide employed a comparable method of evaluating FENO. An impaired ability to generate adequate NO might be a risk factor for the development of active TB, a consequence of active TB and/or contribute to more severe disease in those who do develop active TB. Thus, individuals at risk for impaired NO production, such as those who are relatively L-arginine deficient, may be individuals at risk for development of active TB. Further clarification of the relationship between FENO and TB at diagnosis and during treatment will be obtained through recruitment of the full AVDAPT and healthy volunteer samples, when serum L-arginine levels are available to include in analyses, and when the randomisation code is revealed.

Results reported here also indicate that researchers seeking to measure FENO in tropical environments using NiOX MINO® analysers need to be mindful of keeping the device in a temperature and humidity-regulated environment, to avoid FENO under-estimation.

151 Chapter 12: Results VI – HIV-TB

12 Results VI: HIV-TB co-infection

This chapter describes HIV-TB co-infection among AVDAPT study participants.

12.1 INTRODUCTION

EVOLVING RESPONSE TO HIV IN TIMIKA

The period of June 2008 - October 2009, during which the data analysed in this thesis was collected, witnessed changes in HIV management in Timika including: (1) evolving regulations regarding who is authorised to offer voluntary counselling and testing (VCT); (2) increased integration of HIV testing into routine TB services; (3) variable CD4 count availability; (4) the launch of a Timika District Health Authority TB-HIV collaborative team of medical and administrative staff, and (5) the opening of a second community hospital in September 2008 adding an extra antiretroviral therapy (ART)- prescribing site.

Timika had a fledgling VCT system in place at the commencement of the study in June 2008, and limited access to ART. Traditional VCT comprised an emphasis on pre-test counselling, on the basis that HIV had limited treatment options and a very poor prognosis. The ‗provider-initiated, opt-out‘ HIV testing approach aims to increase the focus on treatment, and hence make testing more routine, with greater emphasis on ‗opting out‘ (i.e. HIV testing undertaken as part of the diagnostic work-up unless specifically declined), and a focus on post-test rather than pre-test counselling, as for most other serious medical conditions.235 Provider-initiated opt-out HIV testing is increasingly being deployed internationally,236 along with wide advocacy for this strategy to improve HIV detection rates.24, 35, 235, 237, 238 In contrast, rising HIV awareness in Timika has led to the introduction of a stringent VCT process in early to mid 2008. This involves counselling only from a trained counsellor (either a doctor or other health professional with specific VCT certification) and written consent.

152 Chapter 12: Results VI – HIV-TB

In order to centralise HIV care, the District Health Authority, under instruction from national health authorities, has determined that ART be prescribed by designated prescribers at nominated sites only. Thus ART cannot be prescribed at the Timika TB clinic or the adjoining Timika sexual health clinic (although VCT can be provided at both sites). Patients must be referred for ART to one of the 2 district hospitals, where a doctor at each site has authority to prescribe ART. Additionally, a non-government organisation (International SOS) provides ART to employees of Freeport Mining Corporation and their families. Use of isoniazid preventive therapy, acknowledged as an important and underutilised strategy for preventing TB in people living with HIV/AIDS,239-241 is not currently routine in Indonesia, including Timika.

TB-HIV COINFECTION OVERVIEW

Tuberculosis (TB) and HIV infections are major global health threats. An estimated 1.4 million new TB cases in HIV-positive individuals were reported in 2007,2 posing wide- ranging diagnostic, management and economic challenges. HIV confers the greatest risk for TB, increasing the risk of latent TB reactivation 20-fold 242, 243. TB is a leading cause of death among people with HIV,244 and TB patients co-infected with HIV (HIV+/TB+) have significantly worse outcomes than those without HIV (HIV-/TB+).245

Priorities in addressing TB-HIV co-infection include instituting the World Health Organisation (WHO)‘s ‗3Is‘ (Intensified TB case-finding, Isoniazid preventive therapy and Infection control),246 ensuring routine ascertainment of HIV status in people with TB,35 close integration of TB and HIV services,235 and universal access to antiretroviral therapy (ART) in HIV-positive people,247 including early ART initiation in TB-HIV co- infection.8, 235, 248, 249. Acknowledging the improved outcomes achieved with ART,249 WHO guidelines have moved beyond 2009 recommendations guided by CD4 count235 to now recommend universal ART in people with TB-HIV co-infection.248

Despite these rapid advances in knowledge, substantial barriers persist to the achievement of optimised TB-HIV management goals, especially in lower-income countries. In tackling the challenge of HIV in people with TB, multifactorial barriers include failures to implement HIV testing, failure to prescribe ART or other elements of HIV care, pre-treatment loss to care and post-ART-initiation loss to follow up.250

153 Chapter 12: Results VI – HIV-TB

In Indonesia, HIV infection rates in TB are not routinely reported.251 A 2006 study utilising unlinked, anonymous testing in people with TB in Yogjakarta found an HIV seroprevalence rate among TB patients of 1.9%.252 The overall national estimate is 3%.2 Papua Province has long been recognised as having one of the highest HIV burdens in Indonesia.253 An ‗Integrated Bio-Behavioral Surveillance‘ study in 2006, of 6305 Papua Province residents aged 15-49 years, reported population HIV seroprevalence as 2.4%.124 This survey also revealed low levels of health knowledge: 51.8% overall had heard of HIV/AIDS (26.3% among those with no or limited education). Only 35.4% knew that condom use was protective.124 Especially high HIV rates of 26% have been documented in Papuan female commercial sex workers.253

Despite Indonesia having a policy to provide free ART since 2003,254 national capacity to widely roll out HIV care is limited.253-255 HIV VCT was taken up by just 4% of TB patients in one 2006 Indonesian study.252 Recent estimates of ART accessibility indicate that only about 24% of Indonesian people with advanced HIV infection receive ART; this figure is as low as 3% in Papua Province.254, 255

The objective of this chapter is to describe current TB-HIV epidemiology and management in Timika in order to tailor future interventions. Specifically, I aim to investigate the burden of HIV infection among adults with smear-positive pulmonary TB, to examine changes over time, to describe current HIV management and its relationship to WHO guidelines, and investigate TB treatment outcomes among TB- HIV co-infected people.

12.2 METHODS

Recruitment of study participants and statistical analyses are as described in preceding chapters. VCT is conducted by the TB clinic doctor, one of the medically-trained study Research Assistants, or a trained counsellor at the sexual health clinic which is adjacent to the TB clinic. It is done privately and confidentially, or with a spouse or family member if requested by the patient, or with a parent/guardian if the patient is aged <18 years. An Indonesian national VCT form is used. Detailed education about HIV/AIDS is provided; the patient may decline testing, or demonstrate their agreement by signing their name or applying their thumbprint to the form. The process takes around 30 to 60 minutes.

154 Chapter 12: Results VI – HIV-TB

HIV antibody testing is performed using SD BioLine HIV-1/2 3.0TM antibody test (Standard Diagnostics, Inc). If positive, confirmation is performed using Abbott DetermineTM HIV-1/2 (Inverness Medical), and OncoprobeTM (PT Oncoprobe Utama) point-of-care tests. Similar strategies for HIV testing (i.e. reliance on follow-up point- of-care tests to confirm positive results) have been shown to be valid elsewhere.256 SD BioLine HIV sensitivity is reported to be 100%, and specificity to be 99.8%, and for Determine, to be 100% and 99.6% respectively.136, 256 In instances where SD BioLine assays have been unavailable, FOKUSTM point-of-care HIV antibody test kits have been used. If there is discordance between tests, the result is reported as ‗intermediate‘ and is repeated on a second blood sample within 4 weeks. A fluorescence-activated cell sorter for performing CD4 T cell assays was intermittently available for use two mornings per week at the District Health Clinic laboratory. Its maintenance and operation is independent of the AVDAPT study, and measurement of CD4 count is not included in the study protocol.

WHO 2009 guidelines were used as the best-practice reference, recommending ART initiation within 2-8 weeks after TB treatment commencement for CD4 <200 or unknown, after 8 weeks if CD4 200-350, and deferral of ART if CD4>350 235.

12.3 RESULTS

As described in previous chapters, 162 smear-positive pulmonary TB patients were recruited (Table 12.1). One hundred and forty one (87.0%) were offered VCT. Uptake of HIV testing was high (138/141 = 97.9%); only 3 participants (all females, 1 Papuan and 2 Non-Papuan) declined an HIV test. Eighteen of 138 people (13.0%) who had an HIV antibody test were HIV positive, confirmed with 3 rapid assays.

Papuans were significantly more likely to be HIV positive than Non-Papuans (14/67=20.9% versus 4/71=5.6%, p=0.01). Papuan women comprised the highest-risk subgroup, although confidence intervals were wide (Figure 12.1). HIV+/TB+ participants were less likely than HIV-/TB+ to be employed (6/18 = 33.3% vs 67/114 =

58.8%, p=0.04,  test), and less likely to have achieved an educational level above primary school (10/18=55.6% vs 89/119=74.8%, p=0.05). However, these associations became non-statistically significant when controlling for ethnicity in multivariate logistic regression analyses, since Papuan ethnicity was associated with lower employment and education. HIV status was unrelated to age. 155 Chapter 12: Results VI – HIV-TB

Table 12.1: Characteristics of 138 study participants with known HIV status

All HIV+ HIV- p value (HIV+ vs HIV-) Number (%) 138 18 (13.0) 120 (87.0) Age in years: median (range) 27 (15-65) 31 (16-60) 27 (15-56) 1.0 Papuan: no. (%) 67 (48.6) 14 (77.8) 53 (44.2) 0.01 Female: no. (%) 43 (31.2) 7 (38.9) 36 (30.0) 0.4 Current smoker: no.(%) 42 (30.4) 5 (27.8) 37 (30.8) 0.8 Highest educational attainment: no. (%) 10 (7.2) 4 (22.2) 6 (5.0) No schooling 28 (20.3) 4 (22.2) 24 (20) 0.05* Primary school 96 (69.6) 10 (55.6) 86 (71.7) High school 4 (2.9) 0 (0) 4 (3.3) Academy or university Unemployed: no. (%) 59 (44.7) 12 (66.7) 47 (41.2) 0.04* Owns telephone: no. (%) 77 (55.8) 8 (44.4) 69 (57.7) 0.3 Clinical and laboratory investigations BMI in kg/m2: mean (range) 19.3 (12.9- 19.2 (12.9- 19.4 (13.3-32.5) 0.8 32.5) 26.7)

Percentage of predicted FEV1: 63.9 (16.6- 59.5 (16.5- 64.6 (23.9 - 0.3 mean (range) 108.5) 92.0) 108.5) 6 minute walk test in m: median 415 (0-612) 390 (0-485) 415 (75-612) 0.04 (range) St George‟s Respiratory Questionnaire† total score: 38.3 (5.2 – 41.7 (13.6- 38.1 (5.2-91.9) median (range) 91.9) 67.0) 0.4 Haemoglobin in g/dL: mean 12.4 (7.1- 11.2 (8.5-12.9) 12.6 (7.1-16) 0.002 (range) 16.0) White cell count x 109/L: mean 8.0 (1.6-22.7) 6.8 (2.4-13.2) 9.0 (1.6-22.7) 0.01 (range)

Sputum smear ≥2+: no. (%) 61 (45.2) 5 (27.8) 56 (47.9) 0.1 Chest X-ray Total score: median (IQR) 67 (4-140) 57 (19-121) 68 (6-140) 0.5 Cavitary disease on CXR: 74 (55.2) 6 (33.3) 68 (58.6) 0.04 no. (%) Pleural effusion: no. (%) 23 (17.2) 7 (38.9) 16 (13.8) 0.009 *Differences in education and employment were not significant in multivariate models controlling for ethnicity. †St George‘s Respiratory Questionnaire results were only available in 119 participants (15 HIV+ and 104 HIV-)

156 Chapter 12: Results VI – HIV-TB

Figure 12.1: Rates of HIV-TB co-infection among study participants p=0.01 60 p=0.5 p=0.6

50

Male 40 Female

30

20

10 HIV-TB co-infection rates (%, 95%CI) (%, rates co-infection HIV-TB

0 Papuan Non-Papuan

*p value calculated using Fisher‘s exact tests

LONGITUDINAL TREND IN TB-HIV CO-INFECTION RATE

In a previous study of smear-positive pulmonary TB patients at the same Timika TB clinic (2003-2004), HIV status was ascertained in all study participants; 5/112 (4.5%) were HIV positive, all Papuan. Compared with these earlier rates, data presented here demonstrate a significant rise in TB-HIV co-infection in Timika in the last 4 years (p=0.03, Figure 12.2).

CLINICAL CHARACTERISTICS

Neither weight nor BMI differed among HIV+/TB+ and HIV-/TB+ study participants, including when controlling for ethnicity and sex (Table 12.1). Exercise tolerance (6MWT) was significantly lower in HIV+/TB+ compared with HIV-/TB+, as was haemoglobin (p=0.002) and total white cell count (p=0.01). These associations remained significant when controlling for weight, ethnicity and sex. Mean pulmonary function results were in the moderately impaired category overall and in the HIV-/TB+ group (i.e. FEV1 60-69% of predicted), but in the moderate-severely impaired category

(i.e. FEV1 50-59% of predicted) in the HIV+/TB+ group, but this difference was not

157 Chapter 12: Results VI – HIV-TB statistically significant (p=0.3, Table 12.1). Quality of life scores (SGRQ) did not significantly differ according to HIV status (Table 12.1). No significant differences were found in the rates of reported symptoms at TB diagnosis (including fever, malaise, nausea, vomiting, diarrhoea, dyspnoea, chest pain, haemoptysis, rash etc).

Chest x-ray findings (Table 12.1) differed significantly in TB-HIV co-infected patients, with cavitary disease being less common (p=0.05), and pleural effusion (i.e. extra- pulmonary TB) being more common (p=0.009), although overall extent of radiological disease (chest x-ray score) did not differ (p=0.6). TB-HIV co-infected patients also had lower bacillary load in sputum, but this difference was not statistically significant (Table 12.1).

Figure 12.2: HIV-TB co-infection rates in Timika in 2003-4 compared with 2008-9 p=0.03 35

30

25 Papuan Non-Papuan 20

15

10

HIV-TB co-infection rate (%, 95%CI) (%, rate co-infection HIV-TB 5

0 2003-4 2008-9

*p value calculated using Fisher‘s exact test

OUTCOME

HIV status did not significantly influence time to sputum smear conversion (hazards ratio [HR] 0.7, 95% CI 0.41 - 1.3, p=0.2; see also Chpater 10). The HIV+/TB+ group had a similar overall likelihood of treatment success (HIV+/TB+ cure or completion: 85.7%; HIV-/TB+ cure or completion: 92.6%, p=0.2; Table 12.2). However, they were more likely to suffer an serious adverse event, although numbers were very small (Table

158 Chapter 12: Results VI – HIV-TB

12.2). These comprised in the HIV-/TB+ group: 2 deaths (1 progressive respiratory illness due to TB or secondary pneumonia, and 1 stroke complicated by aspiration pneumonia) and 1 hospitalisation (due to pneumothorax and severe malnutrition, BMI 12.9 kg/m2), and in the HIV-/TB+ group, no deaths and 2 hospitalisations (1 vomiting / dehydration and 1 multi-drug resistant-TB with progressive pulmonary disease and large pleural effusion). The relationship between HIV status and default rates could not be determined, since people who defaulted often did so prior to HIV testing being offered (of the original 162 study participants, 8 defaulted: of these 3 were HIV negative, none were known to be positive, and 5 had unknown status).

Table 12.2: Treatment outcome in HIV positive and HIVnegative study participants N=96 (known HIV status and 6 months follow up competed)

HIV positive HIV negative p value Outcome N 14 82 Cure 11 (78.6) 65 (79.3) Complete 1 (7.1) 10 (12.2) Failed 0 (0) 1 (1.2) 0.2 Default 0 (0) 3 (3.7) Died 2 (14.3) 0 (0) Transferred 0 (0) 3 (3.7) Serious adverse event: no. (%) 3/18 (16.7) 2/120 (1.7%) 0.02

HIV MANAGEMENT

CD4 counts were obtained in 6 patients (Table 12.2). According to 2009 WHO guidelines,235 14 people were eligible for early ART initiation (CD4<200 cells/L or unknown), 1 for ART initiation after week 8 (CD4 200-350), and 3 for deferred ART initiation (CD4>350). However, during their 6-month TB treatment period, only one person with TB-HIV co-infection was successfully commenced and maintained on ART, 1 patient with known HIV at the time of TB diagnosis was already taking and remained on ART throughout her involvement in the study, and 2 additional patients who had been referred for ART were commenced by the authorised ART prescriber after their TB treatment was completed.

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Table 12.3: TB-HIV coinfection management

TB-HIV co-infected study participants CD4+ T-cell count: no. (%) 6 (33) CD4+ T-cells/L: median (range)* 318 (18-739) CD4+ T-cells <200 2 CD4+ T-cells 200-350 1 CD4+ T-cells >350 3 CD4+ T-cells unknown 12 Anti-retroviral therapy: no. (%) 4/18 (22) Commenced prior to TB diagnosis 1 Commenced during TB treatment 1 Commenced after TB treatment completed 2 ART type AZT / 3TC / nevirapine 3 AZT / 3TC / efavirenz 1 Co-trimoxazole: no. (%) 11/18 (61) *CD4 results: 21yr Papuan female: 78; 19yr old Papuan female: 739; 16 yr Papuan female 275; 29yr Non-Papuan male: 361; 38yr Non-Papuan female: 18; 28yr Non-Papuan female: 640.

12.4 DISCUSSION

TB-HIV CO-INFECTION RATES

This study has identified that TB-HIV co-infection rates have significantly risen during 5 years in Timika, Indonesia. Among the Indigenous Papuan subgroup, HIV seroprevalence in TB patients rose from 4.5 to 20.9%. This remains lower than in some global regions; for example, the estimate for the WHO Africa region is 51%.2 However, it is among the highest reported from Asia, and the rate of change has been rapid, outstripping local capacity to adequately respond to the crisis. HIV co-infection rates differ markedly across Asia, ranging from 0.5% in a large study in Guangxi, China,257 to an estimated 3% overall for Indonesia and 17% in Thailand.2 Few published data are available for comparison from neighbouring Papua New Guinea, but the 2009 WHO report indicates 14.5%.2

DIFFERENCES BETWEEN HIV POSITIVE AND NEGATIVE TB PATIENTS

Reasons for ethnic differences in HIV rates were not investigated in this study but may comprise a combination of factors including sexual behaviours, knowledge of sexual

160 Chapter 12: Results VI – HIV-TB health and transmission prevention, and possibly, the difference in male circumcision rates (about 5% of Papuans and 70% of Non-Papuans are circumcised).124 Injecting drug use is not thought to contribute to HIV transimission in Papua Province.124 The trend towards women being at greater risk in Timika is in keeping with findings elsewhere.258 HIV+ people were less likely to be educated or employed, indicating that HIV education is need at the community level, not just targeting schools or workplaces.

Clinical differences according to HIV status in this study were not large. A limitation of the study is that the relatively small numbers mean it may be under-powered to adequately detect differences between HIV+ and HIV- groups. No significant differences in symptoms or perceived health-related quality of life were identified. Weight (or BMI) was also not different at baseline. This may indicate that HIV infections overall were not advanced, also supported by the relatively well-preserved CD4+ T-cell counts in some individuals (Table 2). These findings emphasise the importance of routine HIV testing in TB rather than restricting testing to those with additional features suspicious for HIV (such as oesophageal candidiasis), as has been practiced elsewhere.259 The HIV+/TB+ group did have significantly reduced exercise tolerance. Haemoglobin was also significantly lower in those with HIV; contributors to this are likely to be multifactorial in Timika, which is characterised by high rates of Plasmodium falciparum and vivax malaria.117 Insufficient dietary iron intake is also likely to be common. This illustrates the compounding effect of overlapping endemic illnesses, potentially contributing to other poor health outcomes such as the very high maternal and infant mortality in Timika.127 People with HIV+/TB+ status are known to have higher mortality rates than those who are HIV-/TB+,245 reinforced by present findings.

Lower rates of cavitary disease and higher likelihood of pleural effusion were identified among HIV positive people at TB diagnosis in this study. These findings are consistent with previous studies examining chest x-rays in TB-HIV co-infection260, 261 Since cavitation largely determines sputum bacillary grade,191, 217 TB-HIV co-infection is also associated with higher likelihood of smear-negative disease, or low bacillary density in smear-positive disease, in proportion to CD4 count.262 Our data support these findings, although not reaching statistical significance. Such findings emphasise the difficulty of establishing TB diagnoses (based on sputum smear and radiological appearance) in HIV infection. Problems in confidently excluding active TB in HIV contribute to some

161 Chapter 12: Results VI – HIV-TB persisting reluctance to widely roll-out isoniazid preventive treatment, an important and underutilised strategy for preventing TB in people with HIV/AIDS.241, 246 Indeed, this strategy is not currently routine in Indonesia. Improving diagnostic sensitivity through inexpensive measures in laboratories (e.g. sputum concentration prior to ZN staining, fluorescence microscopy using an inexpensive light-emitting diode light source, or simple culture-based techniques)8 and clinics (e.g. educating medical staff to appreciate radiological pathology characteristic of TB-HIV co-infection), are not necessarily beyond the means of low-income settings.

BARRIERS TO HIV TESTING AND TREATMENT

In this study, HIV status was established in 138/162 (85.2%) participants. The refusal rate was low. Missed opportunities for HIV diagnosis are widely recognised internationally as well as in Timika. For example, the HIV status of only 35% of Australian TB cases was ascertained in 2006,263 and nearly half of all TB patients in London in 2003-4 were not offered HIV testing.264 Barriers to HIV testing in the current study relate primarily to availability of a certified counsellor, availability of a private room, and the time required to provide pre-test counselling. Conducting VCT in a confidential manner is difficult in a crowded TB clinic, reinforcing previous findings of the requirement for the right structural conditions to be present at a clinic to support effective VCT.252 Given the lack of general knowledge about HIV, reasonably high pre- test probability of a positive result (especially among Papuans), and the degree of stigmatisation suffered by HIV positive individuals,255 the pre-test counselling process in Timika is justifiably lengthy. On identifying HIV positivity, access to appropriate care including ART and condoms is limited; thus the benefit of HIV status knowledge is reduced. Therefore prevailing attitudes among local medical staff has been to take a cautious approach, even if this leads to incomplete ascertainment of HIV status.

Deployment of provider-initiated opt-out HIV testing has been widely advocated236 as a strategy to improve HIV detection rates.24, 35, 235, 237, 238 However, a stringent VCT process was introduced in Timika in 2008, stipulating the requirement for written consent, and for only a doctor or professional with specific certification, to conduct VCT. A study investigating barriers to HIV testing among TB patients in another Indonesian province identified both patient and health provider factors as reasons for

162 Chapter 12: Results VI – HIV-TB low VCT uptake.265 Among patients, these included low HIV knowledge, the disincentive of having to access and endure VCT, and fear of knowing the test results; and among health care providers: low HIV knowledge, communication issues, concern about patients feeling offended, stigmatization and additional work load.265

Access to CD4+ T-cell testing was limited due to difficulties in maintaining the analyser and lack of staff familiarity with its operation. Only four (5.6%) of the HIV positive study participants successfully commenced ART, 2 after TB treatment completion, even though 15 were eligible for ART during TB treatment according to 2009 guidelines. WHO guidelines on the timing of ART initiation in TB have evolved rapidly on the basis of new trials,249 such that universal ART initiation regardless of CD4 count is now advocated in TB-HIV co-infection.248 Integration of TB-HIV is strongly advocated.235, 248 However, a chief barrier to ART initiation in Timika is the absence of such integration, with no authorised ART-prescriber available in the vicinity of the TB clinic. In Indonesia, ART can be prescribed only by a designated person at nominated sites (usually hospitals). Even if a patient is successfully referred to, and attends, a designated ART-prescription site, reasons for low ART prescription rates cited by medical staff include concern about potential poor adherence, and drug toxicities or paradoxical reactions. Education of healthcare providers about ways to support adherence and manage TB-HIV co-infection is therefore greatly needed.

12.5 CONCLUSION

Rising HIV rates in Timika are a serious concern. Timika has experienced large recent population fluxes related to the local mining industry attracting migrants from rural areas and other parts of Indonesia, and thus presents a classic demographic scenario for burgeoning HIV rates. The association between HIV and migrant labourers, especially mine workers and a thriving commercial sex industry, has been well established elsewhere.266, 267

These data provide an important South Asian perspective on the overlapping TB and HIV epidemics, and illustrate the added morbidity suffered by HIV+ compared with HIV- individuals with TB. This study demonstrates that, despite knowledge of TB-HIV management guidelines and even within the setting of a well-regarded TB Directly

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Observed Treatment facility, management of TB-HIV co-infection poses an enormous challenge in resource-limited environments. Multiple interventions are now required at community, patient and healthcare provider levels, to address rising HIV rates and the barriers to testing and treatment.

Some improvements have been effected by AVDAPT study investigators through the provision of up-to-date evidence, especially WHO guidelines, to local practitioners and authorities; in particular, we have enabled VCT to be conducted at the TB clinic and by AVDAPT research assistants, and promoted early referral for ART. Data arising from this study will continue to be regularly presented to the District Health Authority to assist in guiding the local response to HIV-TB management. The recent publication of revised WHO guidelines on TB-HIV management provides a timely opportunity to incorporate these into national guidelines, and support their implementation in community settings.

Figure 12.3: Meeting between AVDAPT research personell and local HIV care providers for discussion of HIV-TB management in Timika

Foreground (L to R): Dr Enny Malonda (RSMM), Dr Andri Wiguna (Timika TB clinic), Dr Dina Bisara Lolong (NIHRD), Dr Enny Kenangalem (NIHRD-MSHR Research Program and Dinas Kesehatan). Photo: Anna Ralph

164 Chapter 13: Conclusion

13 Conclusions and Future Directions

―Tuberculosis is arguably among the most written about diseases in the history of medicine. It reaches far into the past, as there was clear evidence of its existence 15000 years ago, and has received the attention of the Old Testament, Hipocritas, and Gallen. One would not have foreseen, however, that as we approach 2012—the 130th anniversary of Robert Koch‘s conclusive demonstration of the bacterial cause of tuberculosis—the disease would remain prominent in medical literature.‖268

TB is a disease of exceptional significance to individuals and to global public health. Major recent progress has been achieved in the collective effort to decrease TB rates. A key challenge is to implement these developments in parts of the globe that most need them. The mechanism by which AVDAPT researchers have sought to contribute to this process is through bringing research of an internationally-recognised standard, with associated potential to build capacity and assist in elevating local standards of care, to a high TB-burden area.

This thesis demonstrates both barriers and solutions to research implementation in such a setting. A notable barrier is evident in the omission from current analyses of blood results requiring off-shore processing. On the other hand, effective solutions to other barriers are evident, such as the use of inexpensive, locally-available tests (e.g. 6-minute walk test) where possible, and the use of an in-country laboratory as soon as it became accredited, to achieve the primary trial endpoint of sputum culture. Other creative approaches have been required to address problems familiar in high TB-burden settings, such as intermittent electricity supplies, the impact of environmental conditions on equipment, transportation problems (for people, consumables and sputum samples) and staff availability and training. The successful recruitment, randomisation, evaluation and follow up to date of 162 study participants, with less than anticipated losses to follow up, testifies to the ability to conduct complex trials in Timika despite these challenges.

Conclusions of the thesis can be drawn firstly from findings pertaining to epidemiological, clinical and immunological characteristics of people with pulmonary

165 Chapter 13: Conclusion

TB in Timika, and secondly, from findings relevant to the methodological development and operation of the AVDAPT clinical trial, as discussed below.

13.1 CLINICAL FINDINGS

A major contribution of this thesis relates to the investigation of exhaled nitric oxide in

TB. FENO was not elevated in TB patients compared with healthy volunteers, and was lower still in worse disease. These findings suggest that an impaired ability to generate adequate NO (e.g. due to absolute or relative L-arginine deficiency) might therefore contribute to the host‘s inability to adequately contain infection with MTB. Also, as hypothesised, demonstration of an inverse relationship between baseline pulmonary NO production and disease severity provides supportive data for a disease protective effect of NO (and hence L-arginine) in TB. These findings therefore support the rationale for conducting a trial of adjunctive L-arginine in TB.

A number of further original findings were identified in cross-sectional analyses. The standard malnutrition definition (BMI<18.5kg/m2) appeared to underestimate malnutrition in Papuans, especially Papuan males. While this finding is consistent with recognised typical Melanesian body habitus, no existing literature has been identified which remarks upon this. This indicates a need to further investigate and revise, if necessary, this definition in Melanesian people, to avoid under-diagnosis of malnutrition. In the AVDAPT study, BMI will not be relied on as a nutritional marker: the key nutrients of interest are L-arginine and vitamin D, and an individual‘s nutritional status with regards to these molecules will be defined according to serum levels, when available.

The reasons for male over-representation among TB cases need to be identified in different geographical settings, in order to ensure that appropriate TB prevention and care is reaching all parts of the population. In Timika, higher TB rates in men appear to be due to higher actual rates of TB, rather than higher rates of smear positivity or health care attendance, among males.

Another previously unreported finding arising from this data analysis is that TB patients with more evidence of malnutrition, according to lower body weight and haemoglobin, had worse pulmonary function (adjusting for height, sex and ethnicity). This raises

166 Chapter 13: Conclusion mechanistic questions of whether the increased caloric demands associated with tachypnoea and dyspnoea in TB might contribute to weight loss, or whether immunological consequences of malnutrition could impair lung reparative responses to tissue damage. Studies of nutritional supplementation in TB to date, summarised in Chapter 3, have mainly examined anthropometric and bacteriological outcomes. This new finding suggests that respiratory outcome measures ought to be included in such nutrition intervention studies, as will be the case in the AVDAPT study.

Exercise tolerance was found to be related to symptoms and illness duration rather than pulmonary function in TB. This measure therefore provides a useful additional means of assessing the systemic impacts of TB, beyond respiratory disability alone. It provides insights into TB-related burden of disease, specifically, the difficulty of achieving exercise-dependent activities including walking to the clinic for treatment, and engaging in employment. St George‘s Respiratory Questionnaire results were found to accurately capture people‘s perceived quality of life in active TB, correlating significantly with symptoms, exercise tolerance and lung function. These findings indicate that this essentially cost-free test therefore provides a further valuable tool with which disease burden and response to treatment can be measured. Sputum AFB grade, although significantly related to radiological disease extent and weight, was not significantly associated with other test results. This finding supports the use in the AVDAPT study of a range of measures to provide a comprehensive assessment of TB severity.

In the current era in which increasing numbers of TB treatment trials are needed in drug-susceptible TB, MDR-TB and HIV-TB co-infection, increasingly sophisticated outcome assessments are being sought, ranging from surrogate biomarkers (which might predict cure or relapse), to quality of life measures (which can provide insight into clinical and socioeconomic disease impacts). The findings reported here contribute important data on outcome assessments which grade different aspects of TB severity and are suitable for use in high TB-burden countries. Further, these results contribute to the understanding of TB pathology and its clinical consequences, and indicate specific areas needing further investigation.

167 Chapter 13: Conclusion 13.2 HIV-TB AND MDR-TB TRENDS

Compared with many other high TB-burden settings, Timika has relatively low MDR- TB and HIV-TB co-infection rates. Nevertheless, major challenges in health care delivery have arisen in Timika, as the rapid rise in HIV-TB co-infection has outstripped local resources and expertise. Implementation of WHO guidelines on integration of HIV and TB services, and improvements in comprehensive HIV care including access to antiretroviral therapy, will be a major focus of future work in Timika. In keeping with international trends, data from AVDAPT study participants indicate that the Indigenous population in Papua Province suffers poorer socio-economic and health status than the non-Indigenous population; this disparity is most particularly evident in their respective HIV-TB co-infection rates.

MDR-TB rates among new TB cases have remained unchanged over 5 years in Timika, indicating that transmitted MDR-TB remains fortunately uncommon relative to the dominant circulating drug-susceptible strains. Reasons for this are unclear, given the presence of a suitable recipe for rising MDR-TB rates (lack of available MDR-TB treatment and rising HIV rates). A potential rise in MDR-TB may therefore be lagging behind that of HIV, and hence continued monitoring and vigilance, and introduction of appropriate treatment regimens, are major local public health priorities. Data cannot be provided here on acquired resistance rates, since the study protocol excludes relapsed TB cases from enrolment. During the course of the study, the establishment of links with a reference laboratory capable of culture and drug susceptibility testing has increased the options for local practitioners to seek results for their patients with suspected MDR-TB, despite them not being enrolled in the study. This has identified at least one further case of MDR-TB in a re-treatment patient at the study site.

13.3 CONCLUSIONS FROM THE AVDAPT STUDY TO DATE

In this thesis I have confirmed the feasibility of performing the measures described in the study protocol, identified the relationships between these, and refined study endpoints, especially the composite clinical outcome score and the radiological score. A suitable composite clinical outcome score, which appears to be clinically valuable and adequately discriminatory in the current dataset, was devised. Radiological improvement was selected as a secondary endpoint, but literature review demonstrated a

168 Chapter 13: Conclusion surprising lack of standardisation in this field, prompting the investigation of ways to apply a single numerical score to an x-ray. The two scoring methods thus developed (and, in the case of the x-ray score, validated in a second dataset) for use in the AVDAPT study are likely to also have wider applicability, with potential utility for other TB clinical trialists.

Examination of adverse events in study participants indicated no safety concerns for participants randomised to adjunctive therapy were identified; specifically, the hypercalcaemia data corroborate mounting evidence for vitamin D safety in active TB, despite some persisting misconceptions on this issue lingering in contemporary literature. An additional insight provided by the analysis of adverse events was the high morbidity among TB patients (with half in this study reporting new onset of gastrointestinal disturbance during the first 8 weeks of treatment), which may be overlooked in standard practice when patients are not assiduously questioned about such symptoms on a weekly basis.

The importance of using a locally-derived reference range for exercise tolerance, for comparison with results in AVDAPT study participants, was illustrated by the finding of short 6-minute walk distances among healthy Timika volunteers relative to volunteers in other international settings (not unexpected, given short stature, cultural norms and climate). Establishment of a locally-appropriate normal reference range allows for an accurate estimation of the impact of active TB on exercise tolerance.

St George‘s Respiratory Questionnaire (SGRQ) has rarely been tested in ‗normal‘ healthy populations, and some points in the questionnaire (such as one relating to impacts on quality of life of taking medications, see Appendix 15.6) are irrelevant in healthy volunteers. Nevertheless, evaluating the SGRQ in the local Timika population was necessary in order to test the performance of the modified, translated SGRQ, and to control for any potential locally-relevant factors which could influence results, such as cigarette smoking rates, lung damage due to pollution from indoor cooking fires, or culturally-determined perceptions of disability and quality of life. Most healthy volunteers scored zero, thus clearly indicating the high specificity of this questionnaire for quality of life impairment due to respiratory-induced disability, as well as allowing for an accurate baseline against which to estimate the impact of TB on quality of life.

169 Chapter 13: Conclusion

When completed, data from the AVDAPT trial will be able to demonstrate whether the low-cost interventions of adjunctive L-arginine and vitamin D singly or together can shorten time to sputum clearance and culture negativity, which would have potential for public health benefit with reduced infectivity. Further, any acceleration in improvement in weight, symptoms and disability could enable an earlier return to work or school, thus translating to economic benefits to individuals and society. Even if only small benefits are observed, combining this with other recent TB developments (e.g. evolving TB drug regimens incorporating moxifloxacin, improved rifamycin formulations or doses, or TMC207 [discussed in Chapter 3]), could achieve more significant incremental improvements and contribute to much-needed improvements in standard TB treatment regimens. If adjunctive L-arginine and vitamin D are associated with no improvements, the trial will nevertheless provide answers to important research questions. Comparing mycobacterial clearance in each study arm will contribute to the current debate on the relative importance of the L-arginine-NO and vitamin D- cathelicidin pathways in mycobacterial killing in humans in vivo. In the current blinded analyses, the impact of L-arginine administration on FENO is unable to be assessed: if L- arginine is subsequently found to increase FENO and is associated with improved outcomes, this will provide mechanistic evidence for the effect. It would also identify

FENO as a readily measurable lung tissue immunological correlate of treatment outcome, useful in future clinical and immunological studies in TB. If L-arginine therapy improves T cell CD3ζ expression and T cell function (described in Chapter 3), this would support a dual effect of L-arginine therapy and provide the first evidence that this mechanism of impaired T cell function can be overcome with simple interventions potentially applicable to many other disease states.

Due to close links between MSHR-NIHRD‘s Timika Translational Research Facility and national policy makers in Indonesia, the study findings will be readily able to be translated into policy and practice. The AVDAPT study will be eligible for inclusion by the Cochrane Infectious Diseases Collaborative Review Group.

This thesis has described the development and implementation of the AVDAPT study, and provided analyses of baseline data from enrolled participants. It has illustrated the persisting high impact of TB in the 21st century, and provided directions for future progress in the improvement of TB management.

170 References

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186 Appendices

15 Appendices

15.1 STUDY INVESTIGATORS

Arginine and Vitamin D Adjunctive therapies in Pulmonary TB (AVDPAT) Investigators:

 Dr Sandjaja, PhD, Senior Researcher, Food and Nutrition Research and Development Center, National Institute for Health Research & Development (NIHRD), Bogor, Indonesia: Indonesian PI  Prof Nicholas Anstey MBBS FRACP PhD, Infectious Diseases Physician and Head, International Health Division, Menzies School of Health Research (MSHR) Darwin: Australian Co-PI  A/Prof Paul Kelly, MBBS, DTM&H, PhD, FAFPHM, Honorary Principal Research Fellow, MSHR & Director, Masters of Applied Epidemiology Program, National Centre for Epidemiology & Population Health, The Australian National University (ANU), Canberra, ACT: Australian Co-PI  Drg. Franciscus Thio, MPPM, Health Services Manager, District Ministry of Health, Timika, Papua, Indonesia and PhD Scholar, ANU  Dr Anna Ralph, MBBS, MPH, DTM&H, FRACP, Infectious Diseases Physician and PhD Scholar, ANU and Honorary Research Fellow, MSHR, Australia  Dr Dina Bisara Lolong, MD, MA, Clinical Researcher, Biomedical and Pharmaceutical Research and Development Centre, NIHRD, Jakarta  Ibu Merriani Girsang, Laboratory Researcher, Biomedical and Pharmaceutical Research and Development Centre, NIHRD, Jakarta  Dr Enny Kenangalem MD, Research Clinician, NIHRD-MSHR Research Program, Timika and Dinas Kesehatan, Timika, Papua, Indonesia  Ms. Kristina Retnoningtyas Palupi, SSi, Laboratory Scientist, NIHRD  Dr Emiliana Tjitra, MD, PhD. Senior researcher, NIHRD  Dr Firdy Permana, MD, B.Med.Sc, M.Sc.PH, Public Health Coordinator, Public Health Malaria Control, Timika, Papua, Indonesia  Dr Andri Wiguna, MD, Clinician, Tuberculosis Clinic, Timika, Papua, Indonesia  Dr Enny Malonda, MD, Tuberculosis clinician, Rumah Sakit Mitra Masyarakat (RSMM), Timika, Papua, Indonesia  Bapak Govert Waramori, Research Nurse, Malaria Control and Public Health Department, PTFI, Timika  A/Prof Graham Maguire, MBBS, PhD, FRACP, Respiratory Physician and Honorary Research Fellow, MSHR  A/Prof Peter Morris, MBBS, PhD, FRACP, Clinical Trials Specialist and Research Fellow, MSHR  Dr Ivan Bastian, MBBS, PhD, FRCPA, Microbiologist, WHO Supranational Reference laboratory, Institute for Medical & Veterinary Science (IMVS), Adelaide, Australia.  Dr Michael Stone, MD, Medical Advisor, Public Health Malaria Control, Timika, Papua, Indonesia.  Dr Ric Price, MBChB, FRCP, PhD, Infectious Diseases Specialist, MSHR

Consultants:

 Dr Jane Soepardi, MD, Director, National Tuberculosis Control Program, Ministry of Health, Jakarta (all aspects of TB Program delivery)  Mr Richard Lumb, BSc, TB Laboratory Scientist, WHO Supranational Reference laboratory, IMVS,IMVS, Adelaide (TB culture and sensitivity)  Prof Niels Becker, BSc, MSc, PhD. Professor of Biostatisics, National Centre for Epidemiology & Population Health, ANU, Canberra, ACT, Australia. (statistical methods).

187 Appendices

 Dr Cheryl Salome, PhD, Leader, Airway Physiology Group, Woolcock Institute of Medical Research, University of Sydney (exhaled NO measurements and quality control)  Prof John Eisman, BSc (Med), MBBS, FRACP, PhD. Director, Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney Australia (monitoring effects of Vitamin D)  Dr Tonia Woodberry, BSci, PhD, Laboratory scientist, MSHR (PBMC)  Dr Indri Rooslamiati, BPharm, Laboratory scientist, NIHRD (PK/PD)  Prof Stephen Duffull, MPharm, PhD, Professor of Pharmacy, University of Otago, Dunedin, New Zealand (PK/PD)  Dr Tsin Yeo, MD, PhD Candidate, NIHRD-MSHR Research Collaboration (PK/PD)

Data and Safety Monitoring Committee:

 Dr Paulus Sugiarto MD, Chair of Safety monitoring committee. Clinical Director, RSMM, Timika  Dwi Hapsari Tjandrarini, Biostatistician, NIHRD, Jakarta.  Dr Louise Maple-Brown, MBBS FRACP PhD:. Senior Lecturer and Specialist, Internal Medicine and Endocrinology, MSHR  Dr Joseph McDonnell, Statistician, Menzies School of Health Research.

188 Anna Ralph

Course AA. Site Personnel – Core GCP Training

14/04/2008 

    

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- Page 5 of 5 - NIHRD_MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40

15.4 AVDAPT INFORMATION AND CONSENT FORMS AVDAPT Study Participant / Guardian information sheet

Arginine and Vitamin D Adjuvant Therapies in Pulmonary Tuberculosis (AVDAPT) Research study at Timika Puskesmas and RS Mitra Masyarakat

Study Participant / Guardian information sheet Can extra medicine help make TB better more quickly?

This is for you to keep

Tuberculosis (TB) is a major cause of sickness in Indonesia. It can cause cough, weight loss and other problems. People with TB need to take antibiotics for a long time, usually 6 months, to be cured. We want to find out if new extra treatments taken with the antibiotics might help patients with TB to get better faster. We are asking people with lung TB to take part in this study. If you agree, we will ask you to take extra tablets for 2 months in addition to the usual TB antibiotics (which you take for 6 months). We will also ask you to do some extra lung and blood tests today, and again in 2, 4, 8 and 24 weeks.

The body needs nutrition, such as protein and vitamins, to stay healthy and help fight off infection. There is a substance all people need called arginine which is found in foods like nuts, but also comes in a tablet. Also, an important vitamin is Vitamin D which comes from sunshine through the skin, and also from some foods, and can be taken as a tablet too. Some doctors think that extra arginine and vitamin D may be good for people with TB, but there is no clear answer to this yet.

You can help us try to answer this important question. If you agree to be part of this study, you will be given some extra tablets. Some people will receive tablets that have the real arginine or vitamin D, and some people will be given tablets that contain nothing except powder (‗placebo‘ tablet). You, your doctor and the study investigators are not allowed to know until the end which you are taking, to make sure the results are not biased. If you are taking the real arginine and / or vitamin D, they should not cause you any harm. It is very uncommon, but some people get stomach upset from arginine, so we will be checking for this and advise to take tablets after food. Vitamin D can rarely make the calcium in blood high, so we will be checking for this in your blood tests. If you have high calcium already, or get high calcium later, we will stop the extra medications.

Whether or not you agree to participate in this study, you will receive the standard TB antibiotics that the health centre usually uses to treat people with tuberculosis. As part of normal care, the TB clinic will ask you to provide a sputum sample, have your height and weight measured, get a chest x ray and have blood taken (including an HIV test) and you will be asked questions about your health.

If you say yes to the study, then when the blood is taken, we will collect an additional 20 ml of blood (4 teaspoons). This will include testing for HIV if you agree, which is standard care for all people with TB. Then we will also ask you to do breathing tests, a walking test for 6 minutes, and answer some more questions about your breathing. When your blood test results are ready, you will get the extra medications. For the lung tests, we will ask you to breathe into the mouthpiece of a machine called a spirometer and a NIOX machine. This does not hurt or cause any harm, but tells us how your lungs are working. As part of routine care by the TB clinic, 191 NIHRD_MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 every week you will have a check up to see how you are feeling and check your weight. If you are in the study, you will also be asked to do the following tests: sputum test every week; NIOX lung test and blood test in 2, 4, 8 and 24 weeks; chest x ray, spirometer lung test, walk test and questionnaire in 4, 8 and 24 weeks.

Taking part in this study is voluntary; it does not cost you any money. All information collected is confidential. Results will be given to your doctor if they can help the doctor to treat your illness. No personal identification will be revealed to persons outside the study. You do not have to participate if you don‘t want to, this will not affect your medical treatment, If you decide to participate, you can withdraw from this study at any time for any reason and still receive standard treatment for tuberculosis by the staff at the health centre.

If you have any questions about the study you can telephone Dr Paulus Sugiarto at RS Mitra Masyarakat Hospital on 901-301 881. If you have any concerns or complaints about the conduct of the study, please contact Dr Sandjaja, Chair of the NIHRD Ethics Committee in Jakarta on 081319304040 or 6202104261088, or The Secretary, Human Research Ethics Committee of the NT Department of Health & Community services & Menzies School of Health Research in Australia on 61-8-8922 7922.

192 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 ADAPT Code TAD- |__|__|__|__|

AVDAPT Study Participant / Guardian Consent form

Can extra medicine help make TB better more quickly?

This form means you can say No.

I have read the Patient Information Sheet and have had the details explained to me by the witness below. I understand that I will receive the usual treatment and tests for tuberculosis. I understand that I will also have extra tablets which may be active arginine and / or vitamin D, or placebo. I will also have extra lung tests, blood tests, sputum tests, repeat chest X-rays and questions that are not needed by my doctors to treat my tuberculosis. The extra tablets are being tested to find out if they will help people with TB to get better faster. They are very unlikely to be harmful, but could potentially cause stomach upset or high calcium level. The extra tests will provide information about how my lungs and blood are working. I may not receive any direct benefit from this study, but the results will help answer questions about TB treatment, and therefore may be of help to other people in the future.

I understand that I have tuberculosis and I will be asked to come to Timika TB Clinic / RSMM (delete one) for repeat breathing tests, blood tests, chest X-ray, walk test and questionnaire in 2, 4, 8 and 24 weeks. I understand that this will take longer than a usual clinic appointment. I also understand that my medical records at the health centre will be read by research staff and some information about my health will be collected.

I understand that all information collected is confidential and no information will be available to anyone outside the study. Results will be given to my doctor if they can help the doctor to treat my illness. I understand that I do not have to participate in this study. I can withdraw from this study at any time for any reason and still receive standard treatment for tuberculosis by the staff at Rumah Sakit Mitra Masyarakat or Timika TB Clinic.

Page 1 of 2 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 ADAPT Code TAD- |__|__|__|__|

PARTICIPANT I (print name) …………………………………………………………………… agree to take part in this study. Signed Date …………………………………………………………………… ______/ ______/ 20_____

PARTICIPANTS AGED <18 YEARS: PARENT / GUARDIAN TO SIGN I (print name) agree for my child / person in …………………………………………………………………… my care to take part in this study. Participant name …………………………………………………………………… Signed Date …………………………………………………………………… ______/ ______/ 20_____ WITNESS I (print name) have explained the study & …………………………………………………………………… information sheet Signed Date …………………………………………………………………… ______/ ______/ 20_____

Page 2 of 2 AVDAPT Code TAD- |__|__|__|__| 15.5 AVDAPT DATA COLLECTION FORMS

FORMULIR PENGUMPULAN DATA DASAR

Baseline data collection form

Bagian A: Kriteria Inklusi dan Eksklusi Section A: Inclusion and Exclusion Criteria

Nama peserta [pertama]:______[keluarga]: ______Name First Surname

Tanggal hari ini: |__|__|/|__|__|/20__|__| Date today

Nama pewawancara : ______Data collector

1. BTA positif TB paru: Ya 1 Tidak 0 Smear positive pulmonary TB

2. TB Baru: Ya 1 Tidak 0 Never treated for TB before

3. Bersedia untuk menjalani pengobatan Ya 1 Tidak 0 di Timika selama 6 bulan: Agree to continue treatment in Timika for 6 months

4. Umur >= 15 th: Ya 1 Tidak 0 Age > OR = 15yrs

5. Persetujuan Consent untuk Ya 1 Tidak 0 berpartisipasi dalam penelitian ini: Consent given Bila menjawab “tidak” untuk pertanyaan no. 1-5, pasien tidak bisa dimasukkan dalam penelitian ini. If „No‟ to any questions 1 to 5, PATIENT IS NOT ELIGIBLE

6. Apakah dalam keadaan hamil ? Ya 1 Tidak 0 Pregnant?

7. Ionized calcium > 1.32 mmol/L? Ya 1 Tidak 0

Bila pasien menjawab “Ya” untuk pertanyaan no. 6-7, pasien tidak bisa diikutkan dalam penelitian ini. If „Yes‟ to question 6-7, PATIENT IS NOT ELIGIBLE

Investigator name and signature: ______195 AVDAPT Code TAD- |__|__|__|__| Bagian B: Informasi dasar peserta Section B: Respondent Baseline Information

Tempat: TB Clinic 1 Tanggal lahir: |__|__| / |__|__| / 19|__|__| Enrolment Site Date of birth: dd/mm/yyyy RSMM 2

Puskesmas 3 Umur: ______thn Age Other 4

Alamat:______Telepon:|__|__|__|__|__|__|__|__|__|__|__|__| Address Phone

Jenis Kelamin: Laki 1 Perempuan 0 Sex: M1,F0 Suku: Papuan 1 Non-Papuan 0 Ethnicity Agats Ekari 9 Merauke 17 Lain Amungme 1 Fak-Fak 10 Moni 18 25 Asmat Genyem 11 Nabire 19 Ayamaru 2 Jayapura 12 Nduga 20 Biak Kamoro 13 NonPapuan 21 Bintuni 3 Damal 14 Paniai 22 Damal Dani 15 Sarui 23 Dani 4 Mee 16 Sorong 24

5

6

7

8

Pendidikan: Sekolah Dasar 1 Education: (Primary1, SMP/SMA 2 High2, Further3, nil4) Akademi/Universitas 3 Tidak sekolah 4

Pekerjaan: Bekerja 1 sebutkan______Employment (work, Pergi ke sekolah 2 school, nil) Tidak ada 3

Riwayat merokok? Merokok 1 Smoker? Pernah merokok tetapi sudah berhenti 2 Tidak pernah merokok 3 Investigator name and signature: ______196 AVDAPT Code TAD- |__|__|__|__|

Jika merokok, Berapa banyak rokok yang diisap per hari ? cigs/day? ______

Sudah berapa lama sakit seperti ini? How long have you have this sickness? ______bulan Tinggi badan Height ______m

FORMULIR PENGUMPULAN DATA MINGGUAN Weekly data collection form

Bagian A: Data Peserta

Nama peserta [pertama]:______[keluarga]: ______Name First Surname

Tanggal: ____ | ____ | 20_____ Minggu pengobatan (0-24) |___|___| Date today Week of treatment

Nama pengumpul data: ______Data collector

Berapa bungkus obat yang dihabiskan selama Obat TB program |___| minggu ini? How many tablet packs taken? Arginine / placebo |___| Second Vitamin D / placebo dose given today? Ya 1 Tidak 0 If given on other day during this week, date given: ____ | ____ | 20_____

Comments regarding mediations:______

Seberapa sering pergi ke clinic? Setiap hari 1 every day Frequency of clinic visit Setiap mingguu 2 every week Setiap bulan 3 every month Lain-lain 4 other:

Bagian B : Petunjuk Langkah Demi Langkah Pengkajian Mingguan

Jam berapa makan terakhir? |___|___:___|___| Catat hasil pemeriksaan darah iSTAT What time did patient eat? Waktu pengambilan darah? Ionised calcium |___.______| mmol/L (dicatat juga di tabung) |___|___:___|___| What time was blood taken

Waktu pemeriksaan eNO Haemoglobin |______.___| g/dL What time eNO measured? |___|___:___|___| Investigator name and signature: ______197 AVDAPT Code TAD- |__|__|__|__|

Tes fungsi paru

Hasil eNO |______| ppB FEV1 |___| . |___|___| L/s

Tes jalan 6 menit |___|___|___| m FVC |___| . |___|___| L

Bagian C: Gejala Section C: Symptoms

Berat badan ______Kg Weight Batuk Ya 1 Tidak 0 Cough - Jika ada Ringan 1 Mild - Apakah Memburu 1 batuk: Sedang 2 Mod ada k Worse If cough present: perubahan? Parah 3 Severe Sama 2 Has it changed? Parah sekali 4 Very Lebih Same severe baik 3 Hilang Better 4 Gone - Apakah ada lendir? Ya 1 Tidak 0 Sputum?

- Apakah batuk darah? Ya 1 Tidak 0 Haemoptysis?

Demam Ya 1 Tidak 0 Fever Lemas Ya 1 Tidak 0 Malaise Bingung Ya 1 Tidak 0 Confusion Kembung Ya 1 Tidak 0 Bloating Konstipasi Ya 1 Tidak 0 Constipation Mual Ya 1 Tidak 0 Nausea Muntah Ya 1 Tidak 0 Vomiting Diare Ya 1 Tidak 0 Diarrhoea Sakit kepala Ya 1 Tidak 0 Headache Pusing Ya 1 Tidak 0 Dizziness Jaundice Ya 1 Tidak 0 Kuning

Investigator name and signature: ______198 AVDAPT Code TAD- |__|__|__|__|

Kemerahan Ya 1 Tidak 0 Rash Gatal-gatal Ya 1 Tidak 0 Itch Rasa terbakar, mati Ya 1 Tidak 0 rasa di tangan & kaki Burning, numbness in feet or hands Gangguan penglihatan Ya 1 Tidak 0 Poor vision, colour blind Gangguan persendian Ya 1 Tidak 0 Joint pain/ swelling Berat badan turun Ya 1 Tidak 0 Weight loss Mialgia Ya 1 Tidak 0 Myalgia Lain-lain, sebutkan Ya 1 Tidak 0 ______Other, specify Lain-lain, sebutkan Ya 1 Tidak 0 ______Other, specify Lain-lain, sebutkan Ya 1 Tidak 0 ______Other, specify

Bagian D: Sakit Lain-lain

HIV status known Ya 1 Tidak 0 If known: Result date ____ | ____ | 20_____ If known: CD4 count |______|

Apakah anda minum obat untuk lain- Ya 1 Tidak 0 lain? Are you taking any other medications? Jika ja, obat D4T Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____ apa? What medicines? 3TC Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____

AZT Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____

Nevirapine Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____

Efavirenz Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____

Cotrimoxazol Ya 1 Tidak 0 Tanggal: ____ | ____ | 20_____ Investigator name and signature: ______199 AVDAPT Code TAD- |__|__|__|__| e Obat lain

Investigator name and signature: ______200 AVDAPT Code TAD- |__|__|__|__|

Pharmacokinetic data

Nama peserta [pertama]:______[keluarga]:______

Tanggal: |__|__|/|__|__|/20__|__| Minggu |__|__|

Nama pewawancara : ______

Meal  Time of last meal |__|__| : |__|__| Time 0: Baseline  eNO time |__|__| : |__|__| eNO result |__|__|__| ppB  blood time |__|__| : |__|__|

Time 1: Medications given  tablet time |__|__| : |__|__|

Time 2: Follow-up reading  eNO time |__|__| : |__|__| eNO result |__|__|__| ppB  blood time |__|__| : |__|__|

Time 3: Follow-up reading  eNO time |__|__| : |__|__| eNO result |__|__|__| ppB  blood time |__|__| : |__|__|

Investigator name and signature: ______201 AVDAPT Code TAD- |__|__|__|__|

X-RAY REPORT FORM

Study participant: First name: ______Last name: ______

X-ray date ____ | ____ | 20______| ____ | 20______| ____ | 20_____

Total % affected lung |______| % |______| % |______| %

Cavitation Absent Absent Absent Mod (≤4cm) Mod (≤4cm) Mod (≤4cm) Severe (>4cm) Severe (>4cm) Severe (>4cm)

Effusion Absent Absent Absent Small (<25% of lung field) Small (<25% of lung field) Small (<25% of lung field) Large (≥25% of lung field) Large (≥25% of lung field) Large (≥25% of lung field)

Any consolidation Yes 1 No 0 Yes 1 No 0 Yes 1 No 0

Any nodules Yes 1 No 0 Yes 1 No 0 Yes 1 No 0

Any fibrosis Yes 1 No 0 Yes 1 No 0 Yes 1 No 0

Miliary disease Yes 1 No 0 Yes 1 No 0 Yes 1 No 0

Score |______| |______| |______|

Worse Worse Change Same Same Better Better

Comment ______

Reporter name and signature ______

Date of report ______|______| 20______|______| 20______|______| 20______

Investigator name and signature: ______202 NIHRD-MSHR Health Research Collaboration ADAPT study AVDAPT Code TAD- |__|__|__|__|

PENGKAJIAN TERAKHIR hanya MINGGU 24 OUTCOME, WEEK 24

Nama peserta [pertama]:______[keluarga]: ______Name First Surname

Tanggal: |__|__|/|__|__|/20__|__| Date

Outcome: Pengobatan selesai 1 (tick one) Completed

Sembuh 2 Cured

Gagal pengobatan 3 Failed (smear positive at 5 months)

Default 4 Defaulted

Meninggal 5 Died

Transfer 6 Tranferred

Investigator name and signature: ______203

PENGKAJIAN TERAKHIR OUTCOME

ENROLMENT DATE: |__|__| . |__|__| . 20 |__|__|

DATE LAST SEEN: |__|__| . |__|__| . 20 |__|__| BACTERIOLOGY UI SMEAR WEEK 0 0 1 WEEK 4 0 1 WEEK 8 0 1 CULTURE WEEK 0 0 1 2 3 WEEK 4 0 1 2 3 WEEK 8 0 1 2 3 OUTCOME CURED (2 CONSEC SMEAR -) 1 COMPLETED 2 FAILED (SMEAR + 5 MO) 3 DEFAULTED 4 DIED 5 TRANSFERRED 6 TB TREATMENT NON-STANDARD: NON FDC Yes 1 No 0 REGIMEN NON-STANDARD: >24 WEEKS Yes 1 No 0 NON-STANDARD: OTHER Yes 1 No 0 PROTOCOL ENROLMENT VIOLATION Yes 1 No 0

VIOLATIONS RANDOMISATION VIOLATION Yes 1 No 0 STUDY DRUG ERROR (MISLABBELED, Yes 1 No 0 MISSING) FOLLOW UP MISSED: 1-2 APPOINTMENTS Yes 1 No 0 FOLLOW UP MISSED: >=3 APPOINTMENTS Yes 1 No 0 COMPLIANCE <80% Yes 1 No 0 STUDY DRUGS STOPPED BY INVESTIGATOR Yes 1 No 0 CONSENT WITHDRAWN BEFORE WEEK 8 Yes 1 No 0 CONSENT WITHDRAWN AFTER WEEK 8 Yes 1 No 0 SECOND VITD/PLACEBO At week 4 1 DOSE ADMINISTRATION After 2 week4 Not given 3 Not 4 recorded OTHER details:______Yes 1 No 0 ADVERSE HYPERCALCAEMIA NO 0 MILD 1.33- EVENTS 1 1.39 MODERATE 1.40- 2 1.49 SEVERE >1.50 3 OTHER ADVERSE EVENT Yes 1 No 0 SERIOUS ADVERSE EVENT Yes 1 No 0 HIV NEGATIVE 0 POSITIVE 1 INDETERMINATE 2 NOT DONE 3

Investigator name and signature: ______204

15.6 ST GEORGE’S RESPIRATORY QUESTIONNAIRE English translation

SGRQ PART 1

1) Over the last year, I have coughed: 5) During the last year, how many Most days severe or very bad unpleasant attacks of Several chest trouble have you had? A few More than three Only ______days 3 attacks Not at all 2 attacks 1 attack 2) Over the last year, I have brought None up phlegm (sputum): Most days 6) How long did the worst attack of Several chest trouble last? A few a week or more Only ______days 3 or more days Not at all 1 or 2 days less than a day 3) Over the last year, I have had shortness of breath: 7) Over the last year, in an average Most days week, how many good days (with little chest Several trouble) have you had? A few None Only ______days 1 or 2 Not at all 3 or 4 nearly every day 4) Over the last year, I have had every day attacks of wheezing: Most days 8) If you have a wheeze, is it worse in Several the morning? A few No Only ______days Yes Not at all

SGRQ PART 2

9) How would you describe your chest condition? The most important problem I have Causes me quite a lot of problems Causes me a few problems Causes no problem

10) If you have ever had paid employment? My chest trouble made me stop work My chest trouble interferes with my work or made me change my work My chest trouble does not affect my work

11) Questions about what activities usually make you feel breathless. Sitting or lying still Getting washed or dressed Walking around the home Walking outside on the level Walking up a flight of stairs Walking up hills Playing sports or games

205

12) More questions about your cough and breathlessness. My cough hurts My cough makes me tired I get breathless when I talk I get breathless when I bend over My cough or breathing disturbs my sleep I get exhausted easily

13) Questions about other effects your chest trouble may have on you. My cough or breathing is embarrassing in public My chest trouble is a nuisance to my family, friends or neighbours I get afraid or panic when I cannot get my breath I feel that I am not in control of my chest problem I do not expect my chest to get any better I have become frail or an invalid because of my chest Exercise is not safe for me Everything seems too much of an effort

14) Questions about your medication. My medication does not help me very much I get embarrassed using my medication in public I have unpleasant side effects from my medication My medication interferes with my life a lot

15) Questions about how activities may be affected by your breathing. I take a long time to get washed or dressed I cannot take a bath or shower, or I take a long time I walk more slowly than other people, or I stop for rests Jobs such as housework take a long time, or I have to stop for rests If I walk up one flight of stairs, I have to go slowly or stop If I hurry or walk fast, I have to stop or slow down My breathing makes it difficult to do things such as walk up hills, carry things up stairs, light gardening such as weeding, dance, play bowls or play golf My breathing makes it difficult to do things such as carry heavy loads, dig the garden or shovel snow, jog or walk at 5 miles per hour, play tennis or swim My breathing makes it difficult to do things such as very heavy manual work, run, cycle, swim fast or play competitive sports

16) We would like to know how your chest trouble usually affects your daily life. I cannot play sports or games I cannot go out for entertainment or recreation I cannot go out of the house to do the shopping I cannot do housework I cannot move far from my bed or chair

17) Tick the statement which you think best describes how your chest affects you. It does not stop me doing anything I would like to do 0 It stops me doing one or two things I would like to do It stops me doing most of the things I would like to do It stops me doing everything I would like to do

206

31.03.2005/PI Revised 8.11.2005/PI PRODUCT DATA SHEET 207(241) ARGIMAX 15.7 ARGIMAX CERTIFICATE OF ANALYSIS

General information

Product name Argimax

Dosage form tablet

Product number 5146

Manufacturer Hankintatukku Oy, Finland

Package 60 tablets / 3 x 20 blister packed tablets in a carton

Ingredients

Raw material mg/tablet % L-argininehydrochloride (food quality) 1000,00 86,21 Microcrystalline cellulose (Ph. Eur.) 100,00 8,62 Hydroxypropyl cellulose (food quality) 48,00 4,14 Magnesium stearate (E 470b, Ph. Eur.) 12,00 1,03 ______1 160 mg 100 % label claim / 1 tablet contains 830 mg L-arginine

Specification of the finished product

Test Requirements

Appearance White, 20 mm long, 8 mm broad, capsule formed tablet

Weight 1 160 mg (Ph. Eur.)

Checking of the carton / label The right batch number and best before -marking

Content of the package 60 capsules in a carton

207

15.9 SAFTEY REPORTING PROCESS

AVDAPT Safety Reporting Process

Clinical Investigator: Determine if an Event is Serious (definitions on page 2)

Yes No

Serious Event Reporting NonSerious Event Reporting

Research Assistant in Timika: Research Assistant in Timika: • Determine causality • Record on Weekly Data • Notify Dr Anna Ralph and Dr collection form Dina Bisara Lolong WITHIN 24 • Discuss with TB clinic doctor HOURS. If Anna not available, and Study Investigators if medical notify Dr Paul Kelly or Nick advice needed Anstey. • Record on SAE form Dr Anna Ralph and Dr Dina Lolong: •Notify BPOM in routine report •Record event in clinical database

Dr Anna Ralph and Dr Dina Lolong: Ensure that event is recorded in Contacts safety database - Dr Anna Ralph, fax + 61 2 6125 0740, email [email protected]  Determine reportability. Is the - Dr Dina Bisara Lolong, fax 62 21 424 5386, email event: [email protected] o Serious AND - Dr Paul Kelly, fax + 61 2 6125 0740, email [email protected] o Unexpected AND - Dr Nick Anstey. [email protected]

o Related to study drug? Data and Safety Monitoring Committee: - Dr Paulus Sugiarto MD, Chair. 0811490658, [email protected] - Dr Louise Maple-Brown, [email protected]

Yes No

Is the event: Fatal or life-threatening? No

Yes

Dr Anna Ralph and Dr Dina Lolong should Dr Anna Ralph and Dr Dina Lolong should notify and supply the SAE form WITHIN notify and supply the SAE form WITHIN 24 HOURS to theData Safety Monitoring 24 HOURS to theData Safety Monitoring Committee by fax or email, who will Committee, who will prepare a report for prepare a report for HREC and decide HREC and decide whether BPOM needs to whether BPOM needs to be notified be notified WITHIN 15 DAYS WITHIN 7 DAYS. Other Investigators should also be notified Other Investigators should be notified as within 15 calendar days soon as possible

209

Adverse Events Definitions

Adverse Event  Any adverse medical occurrence in an AVDAPT participant. The adverse event may or may not be due to the study medications. It includes symptoms reported by study subjects, signs noted by RA, or out-of-range laboratory values, which were not noted at baseline.

Serious Adverse Event  Death  Life-threatening condition  Hospitalisation

Expected The following are expected (recognized potential adverse effects of L-arginine and / or vitamin D): L-arginine:  Diarrhoea  Bloating  Nausea, Vomiting  Mild to moderate abdominal pain Vitamin D:  Asymptomatic hypercalcaemia defined as iCa2+ > 1.32 mmol/L  Symptomatic hypercalcaemia i.e. iCa2+ > 1.32 mmol/L with one or more of the following symptoms or signs: o Gastrointestinal: Nausea, Vomiting, Abdominal pain, Constipation, Anorexia o Neurological / Psychiatric: Fatigue, Irritability, Confusion, somnolence, Coma o Renal: Polyuria, dehydration o Others: Bone pain, itch.

Unexpected Adverse events other than those listed above are considered to be unexpected.

Relationship to study drug from http://www.niaid.nih.gov/ncn/sop/adverseevents.htm Unrelated Adverse event is clearly due to extraneous causes (e.g., underlying disease, Tidak berhubungan environment) Unlikely (must have 2) 1. Timing of adverse event did not fit with start / finish of study drug Tidak 2. Adverse event could have been due to TB 3. Adverse event could be due to something else such as TB medications or external (environmental) factor. 4. Adverse event is not a recognized possible response to the medication. 5. Does not reappear or worsen with reintroduction of intervention

Possible (must have 2) 1. Timing of adverse event does fit with start / finish of study drug Mungkin 2. Adverse event not due to TB 3. Adverse event not due to something else such as TB medications or external (environmental) factor. 4. Adverse event is a recognized possible response to the medication.

Probable (must have 3) 1. Timing of adverse event does fit with start / finish of study drug Dicurigai 2. Adverse event not due to TB, TB medications or external (environmental) factor. 3. Adverse event is a recognized possible response to the medication. 4. Adverse event disappears or decreases when study drug reduced or stopped.

Definite (must have all 4) 1. Adverse event started after drug introduced Ya 2. Adverse event not due to TB, TB medications or external (environmental) factor. 3. Adverse event is a recognized possible response to the medication. 4. Adverse event disappears or decreases when study drug reduced or stopped, and recurs with re-exposure.

210 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 ADAPT SAE Report Form Code: TAD- 15.10 SERIOUS ADVERSE EVENT REPORT FORM

FORMULIR AKIBAT SAMPINGAN SERIUS (ASS) Serious Adverse Events Reporting Form

Nama peserta [pertama]:______[keluarga]: ______Tanggal SAE: |___|___| 20_____| Name First Surname Date of SAE

Tempat: TB Clinic 1 Nama pewawancara: ______Enrolment Site Data collector RSMM 2

Puskesmas 3 Lain 4 Jenis akibat Fatal 1 Fatal sampingan Mengancam jiwa 2 Life-threat serius (ASS) Rumah sakit 3 Hosp SAE type Kesudahan Sembuh 1 Resolved Outcome Terus berlangsung 2 Continued Meninggal 3 Death Tidak tahu 4 Don‟t know

Tanggal Kesudahan Outcome date |___|___| 20_____|

Hubungan dengan obat Tidak berhubungan 1 Unrelated Causal relationship to study drug Tidak 2 Unlikely Mungkin 3 Possible Dicurigai 4 Probably Ya 5 Definite

211 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 ADAPT SAE Report Form Code: TAD- Informasi obat penelitian: 1. Arginine / Arginine placebo

Tanggal mulai |__|__|/|__|__|/20__|__| Tanggal berakhir |__|__|/|__|__|/20__|__| Start date Stop date Berapa bungkus yang tidak diminum? __at least 1____ How many doses were missed? Perubahan dosis obat Tidak 1 No penelitian? Dikurangi 2 Reduced Study drug dose changed? Stop 3 Stopped Diberikan kembali Ya 1 rechallenged? Tidak 0 Tanggal diberikan kembali |__|__|/|__|__|/20__|__| Date rechallenged Apakah ASS terjandi kembali? Ya 1 Did SAE reappear? Tidak 0

Informasi obat penelitian: 2. Vitamin D / Vitamin D placebo

Waktu pertama minum Vitamin D / Vitamin D placebo? Ya 1 Tanggal: |__|__|/|__|__|/20__|__| Was the first vitD/plac dose taken ? Tidak 0 Waktu kedua minum Vitamin D / Vitamin D placebo? Ya 1 Tanggal: |__|__|/|__|__|/20__|__| Was the second vitD/plac dose taken ? Tidak 0 Hypercalcaemia? Ya 1 Hasil maximum |__|.|__|__| Tanggal |__|__|/|__|__|/20__|__| Tidak 0 Perubahan dosis obat Tidak 1 No penelitian? Withheld 2 Withheld Study drug dose changed? Stop 3 Stopped Diberikan kembali Ya 1 rechallenged? Tidak 0 Tanggal diberikan kembali |__|__|/|__|__|/20__|__| Date rechallenged Apakah ASS terjandi kembali? Ya 1 Did SAE reappear? Tidak 0

212 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 AVDAPT AE Report Form Code: TAD-|_|_|_|_| Apakah Investigator melihat kode studi? Ya 1 Peserta grup Active Arg / Active VitD 1 Has investigator unblinded treatment? Tidak 0 pengpbatan? Placebo Arg / Active VitD 2 Subject treatment arm Active Arg / Placebo VitD 3 Placebo Arg / Placebo VitD 4

Obat lain Ya 1 Obat Tanggal mulai Tanggal berakhir Other meds Tidak 0 |__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

|__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

|__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

|__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

|__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

|__|__|/|__|__|/20__|__| |__|__|/|__|__|/20__|__|

Catatan; Riwayat penyakit yang berhubungan Notes, relevant medical history

After completing adverse event form, notify Dr Anna Ralph [email protected] and Dr Dina Lolong [email protected]

Investigator name, signature, date: ______|___|___| 20____| 213 Investigator brochure NIHRD_MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40

15.11 HYPERCALCAEMIA MANAGEMENT GUIDELINE

Hypercalcaemia in AVDAPT patients

 Any patient found to have ionized calcium > 1.32 for the first time should have the test repeated.  If iCa > 1.32 mmol/L at baseline, patient not eligible for enrolment in the study.  If iCa > 1.32 mmol/L at any reading during treatment, follow the flow chart below.  Hospitalization should be avoided where possible (IV saline can be given in the TB clinic or outpatient clinic); requirement for hospitalisiation in severe, symptomatic people will be discussed on a case-by-case basis.

Hypercalcaemia Grade Ionised calcium Equivalent total calcium level (=iCax2) Mild 1.33 – 1.39 mmol/L 2.60 – 2.79 mmol/L Moderate 1.40 – 1.49 mmol/L 2.80 – 2.99 mmol/L Severe, potentially symptomatic 1.50 – 2.25 mmol/L 3.00 – 4.49 mmol/L Very severe > 2.25 mmol/L > 4.50 mmol/L

Symptomatic hypercalacaemia will be defined as one or more of the following symptoms or signs occurring in a person with ionised calcium > 1.5 mmol/L 1. Gastrointestinal: Nausea, Vomiting, Abdominal pain, Constipation, Anorexia 2. Neurological / Psychiatric: Fatigue, Irritability, Confusion, somnolence, Coma 3. Renal: Polyuria, dehydration 4. Others: Bone pain, itch.

214 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 AVDAPT AE Report Form Code: TAD-|_|_|_|_| iCa > 1.32 mmol/L

Mild Moderate Severe 1.32 – 1.39 1.40 – 1.49 >1.50

Observe and Increase oral fluid Asymptomatic Symptomatic repeat blood test intake. within next week Observe and repeat test within 3 days

Treat with IV saline Treat with IV saline +/- frusemide and oral prednisolone 15- 60mg/day

215

15.12 EXAMPLE OF EPIDATA 2. „CHECK file‟ then applied to DATABASE provide labels, calculations, internal consistency checks etc 1. „QES File‟ written to generate data entry program for Enrolment 3. „REC file‟: Data entry screen questionnaire

DATA DASAR

STUDY CODE #### ID DATE0 Enrolment Date @

Bagain A: Kriteria inklusi dan eksklusi ******************************* NAMAP ______NAMAF ______RA # Inclusion: SMPOS # TBNEW # STAYTIM # ADULT # CONSENT # Exclusion: PREG # CALHI # VIOLATION #

Bagain B: Informasi dasar peserta ****************************** SITE # DOB

age ## ADDRESS ______PHONE # SEX # 3. View data screen: creates PAPUAN # spreadsheet SUKU EDUC # EMPLOY1 # EMPLOY2 ______SMOK # SMOKNO ## ILLTIME ##.## HEIGHT #.## ****************************** RELATE TO OTHER DATABASES To jump to other database, place cursor in box and press return. To get back to the BASELINE file press F10 RELATE #

216 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 15.13 HEALTHY VOLUNTEER INFORMATION & CONSENT FORM Participant Information Sheet: Normal Healthy People in Timika, Indonesia

Understanding how TB causes lung disease

This is for you to keep

We would like to ask you to take part in a study testing how far you can walk in 6 minutes and how TB causes lung damage. You are suitable for this study because you are healthy. We want to compare your results with people with tuberculosis (TB), a lung illness which affects breathing. To find out how their lungs compare with normal, we need to test healthy people to see how far they can walk, what their breathing tests show, and how well their blood may fight infection.

If you want to take part, we will ask you a few questions about your health, check how much you weigh, test your breathing (exhaled nitric oxide) then ask you to walk along the marked track for 6 minutes. We will also ask you for a small amount of blood: 20 ml (about 4 teaspoons): this will be used to examine the ability of healthy people to fight infection, so we can compare this to people with TB. These tests will not cost you anything.

All information collected is confidential. No personal identification will be revealed to persons outside the study.

You do not have to participate if you don‘t want to, this is entirely voluntary.

These tests will not benefit you directly. But by helping us better understand how TB causes disease, we may be better able to prevent and treat TB in the future.

If you have any questions about the study you can telephone Dr Paulus Sugiarto at RS Mitra Masyarakat Hospital on 901-301 881. If you have any concerns or complaints about the conduct of the study, please contact Dr Sandjaja, Chair of the NIHRD Ethics Committee in Jakarta on 081319304040 or 6202104261088, or The Secretary, Human Research Ethics Committee of the NT Department of Health & Community services & Menzies School of Health Research in Australia on 61-8-8922 7922

217 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40

ADAPT Healthy Controls Consent Form

Understanding how TB causes lung disease

This form means you can say No.

I have read the information sheet and have had the details explained to me by the witness below. I understand that I am not sick and will only need to answer questions, have my weight measured, a blood test (20 mls = 4 teaspoons), and walk for 6 minutes. I understand that all information collected is confidential and no information will be available to anyone outside the study. I understand that I do not have to participate in this study. I can withdraw from this study at any time for any reason.

PARTICIPANT I (print name) ………………………………………………………… agree to take part in this study. Signed Date ………………………………………………………… ______/ ______/ 20_____

PARTICIPANTS AGED <18 YEARS: PARENT / GUARDIAN TO SIGN I (print name) agree for my child / person in ………………………………………………………… my care to take part in this study. Participant name ………………………………………………………… Signed Date ………………………………………………………… ______/ ______/ 20_____ WITNESS I (print name) have explained the study & ………………………………………………………… information sheet Signed Date ………………………………………………………… ______/ ______/ 20_____

218 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 AVDAPT Controls Code THC- |__|__|__|

15.14 HEALTHY VOLUNTEER DATA COLLECTION FORM

FORMULIR PENGUMPULAN DATA ORANG SEHAT Healthy people data collection form

Bagian A : Nama Peserta dan Pewawancara Section A: Study Participant and data collector name

1. Tanggal hari: |__|__|/|__|__|/20__|__| Date today

2. Nama [pertama]: ______First Name

3. Nama [keluarga]: ______Surname

4. Nama pengumpul data : ______Data collector

Bagian B: Kriteria Inklusi dan Eksklusi Section B: Inclusion and Exclusion Criteria

8. Umur > 15 th: Ya 1 Tidak 2 Age >15yrs

9. Pulse <120/min? Ya 1 Tidak 2

10. BP<180/100? Ya 1 Tidak 2

11. Persetujuan Consent untuk Ya 1 Tidak 2 berpartisipasi dalam penelitian ini: Consent given Bila menjawab “tidak” untuk pertanyaan no. 5-8, pasien tidak bisa dimasukkan dalam penelitian ini. If „No‟ to any questions 5-8, PATIENT IS NOT ELIGIBLE 12. Currently sick? (febrile illness ot Ya 1 Tidak 2 diagnosed by doctor with illness during the last week)? 10.Unstable angina present? Ya 1 Tidak 2

11. Cough present? (only tick ‘ja’ if severe or new. Mild Ya 1 Tidak 2 stable cough permitted) Bila pasien menjawab “Ya” untuk pertanyaan no. 9-11, pasien tidak bisa diikutkan dalam penelitian ini. If „Yes‟ to question 9-11, PATIENT IS NOT ELIGIBLE

219 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 AVDAPT Controls Code THC- |__|__|__| Bagian C : Data Peserta Section C: Study Participant data

Alamat: ______Address

Telepon: |__|__|__|__|__|__|__|__|__|__| Phone

Tanggal kelahiran: |__|__| / |__|__| / 19|__|__| Date of birth: dd/mm/yyyy

Umur: ______tahun Age yr

Jenis Kelamin: Laki 1 Perempuan 2 Sex: M1,F2

Suku: Papuan 1 Bukan Papuan 2 Ethnicity Agats 1 Ekari 9 Merauke 17 Lain 25 Amungme 2 Fak-Fak 10 Moni 18 Asmat 3 Genyem 11 Nabire 19 Ayamaru 4 Jayapura 12 Nduga 20 Biak 5 Kamoro 13 NonPapuan 21 Bintuni 6 Damal 14 Paniai 22 Damal 7 Dani 15 Sarui 23 Dani 8 Mee 16 Sorong 24

Pendidikan: Sekolah Dasar 1 Education: (Primary1, SMP/SMA 2 High2, Further3, nil4) Akademi/Universitas 3 Tidak sekolah 4

Pekerjaan: Bekerja 1 sebutkan______Employment (work, school, Pergi ke sekolah 2 nil) Tidak ada 3

Riwayat merokok? Merokok 1 Smoker? Pernah merokok tetapi sudah berhenti 2 Tidak pernah merokok 3

Jika merokok, Berapa banyak rokok yang diisap per cigs/day? hari ? _____ / hari

220 NIHRD-MSHR Health Research Collaboration ADAPT study Study HREC Number 07/40 AVDAPT Controls Code THC- |__|__|__| Bagian D : Section D: Clinical and laboratory measurements Jam berapa makan terakhir ? Jam yang lalu How long ago did patient last eat? (hrs) Pulse bpm

Systolic BP mmHg

Diastolic BP mmHg

Hb g/dL

Blood collected for lab Ya 1 Tidak 2 eNO ppb

Lung function: FEV1 L/s

Lung function: FVC L Berat badan kg weight

Height m height

Tes jalan 6 menit m 6-min walk test

Kuisioner Respiratori St Ya 1 Tidak 2 George

SGRQ done yes / no

Investigator name and signature:______221