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 i 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 Front section: Acknowledgements ii 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. Front section: Acknowledgements iii 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 Front section: Acknowledgements iv 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. Front section: Figure v 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. Front section: Abstract vi 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 (FE NO ), 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, FE NO , 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 [FE NO ] and functional measures of disease severity), and (3) investigation of TB drug-resistance and HIV rates. Front section: Abstract vii A key finding was that FE NO 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. Front section: Glossary viii 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 FE NO Fractional exhaled nitric oxide FEV 1 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 [...]... II: ADJUNCTIVE TREATMENT IN TB 5 3.1 3.2 3.3 4 INTRODUCTION III: NITRIC OXIDE AND ITS MEASUREMENT IN VIVO 19 4.1 4.2 5 NITRIC OXIDE PATHWAYS 19 EXHALED NITRIC OXIDE MEASUREMENT 20 STUDY SETTING 26 5.1 5.2 5.3 5.4 5.5 6 WHY ARE NEW TREATMENT APPROACHES NEEDED? 5 ADJUNCTIVE THERAPIES 8 ARGININE AND VITAMIN D AS NOVEL POTENTIAL ADJUNCTIVE. .. 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... 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... Both moxifloxacin and gatifloxacin improved the sterilizing Table 1 Methods to Improve Diagnosis and Accelerate Drug Susceptibility Results Method, test Sputum collection Improved sputum-submission guidance Reduce number of collections from 3 to 2 Comments If smear positive pulmonary TB case detection is impaired by poor-quality specimen submission, case detection can be improved by provision of adequate... (SRL172) immunotherapy as an adjunct to standard antituberculosis treatment in HIV-infected adults with pulmonary tuberculosis: a randomised placebo-controlled trial Lancet 2002; 360:1050–5 Fan MY, Chen XR, Wang Ka, et al Adjuvant effect of Mycobacterium vaccae on treatment of recurrent treated pulmonary tuberculosis: A meta-analysis Chinese Journal of Evidence-Based Medicine 2007; 7: 449–55 von Reyn C,... antimicrobial response Science 2006; 311:1770–3 Ralph AP, Kelly PM, Anstey NM L-arginine and vitamin D: novel adjunctive immunotherapies in tuberculosis Trends Microbiol 2008; 16:336–44 Zea AH, Culotta KS, Ali J, et al Decreased expression of CD3z and nuclear transcription factork B in patients with pulmonary tuberculosis: potential mechanisms and reversibility with treatment J Infect Dis 2006; 194:1385–93 Abba... 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,... 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... micronutrient supplementation on treatment outcome in patients with pulmonary tuberculosis: a randomized controlled trial in Mwanza, Tanzania Trop Med Int Health 2005; 10:826–32 62 Villamor E, Mugusi F, Urassa W, et al A trial of the effect of micronutrient supplementation on treatment outcome, T cell counts, morbidity, and mortality in adults with pulmonary tuberculosis J Infect Dis 2008; 197:1499–505 63 Africa’s... 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 . Pulmonary Tuberculosis: towards improved adjunctive therapies Anna Ralph July 2010 . Project Grant Application 605806 entitled ―L-Arginine and Vitamin D Adjunctive Therapies in Pulmonary Tuberculosis‖. _________________________