L-arginine and Vitamin D Adjunctive Therapies in Pulmonary Tuberculosis: A Randomised, Double-Blind, Placebo-Controlled Trial

Background Vitamin D (vitD) and L-arginine have important antimycobacterial effects in humans. Adjunctive therapy with these agents has the potential to improve outcomes in active tuberculosis (TB). Methods In a 4-arm randomised, double-blind, placebo-controlled factorial trial in adults with smear-positive pulmonary tuberculosis (PTB) in Timika, Indonesia, we tested the effect of oral adjunctive vitD 50,000 IU 4-weekly or matching placebo, and L-arginine 6.0 g daily or matching placebo, for 8 weeks, on proportions of participants with negative 4-week sputum culture, and on an 8-week clinical score (weight, FEV1, cough, sputum, haemoptysis). All participants with available endpoints were included in analyses according to the study arm to which they were originally assigned. Adults with new smear-positive PTB were eligible. The trial was registered at ClinicalTrials.gov NCT00677339. Results 200 participants were enrolled, less than the intended sample size: 50 received L-arginine + active vitD, 49 received L-arginine + placebo vit D, 51 received placebo L-arginine + active vitD and 50 received placebo L-arginine + placebo vitD. According to the factorial model, 99 people received arginine, 101 placebo arginine, 101 vitamin D, 99 placebo vitamin D. Results for the primary endpoints were available in 155 (4-week culture) and 167 (clinical score) participants. Sputum culture conversion was achieved by week 4 in 48/76 (63%) participants in the active L-arginine versus 48/79 (61%) in placebo L-arginine arms (risk difference −3%, 95% CI −19 to 13%), and in 44/75 (59%) in the active vitD versus 52/80 (65%) in the placebo vitD arms (risk difference 7%, 95% CI −9 to 22%). The mean clinical outcome score also did not differ between study arms. There were no effects of the interventions on adverse event rates including hypercalcaemia, or other secondary outcomes. Conclusion Neither vitD nor L-arginine supplementation, at the doses administered and with the power attained, affected TB outcomes. Registry ClinicalTrials.gov. Registry number: NCT00677339


EXECUTIVE SUMMARY
Described herein is our proposed program of related work in the field of tuberculosis research in Timika, Papua Province, Indonesia.
TB, caused by Mycobacterium tuberculosis (MTB) is a disease of major public health significance to Indonesia. Our goal is to achieve better treatment outcomes in pulmonary tuberculosis (PTB) through simple and cost-effective interventions. Improvement in TB treatment resulting from successful adjuvant therapy has the potential to decrease duration of infectivity, disability, and duration of antimicrobial treatment. Each would have major clinical and public health benefits. Two major pathways by which human macrophages kill MTB are the arginine-nitric oxide (NO) and Vitamin D pathways. Both NO and activated vitamin D are potent mycobactericidal agents in vitro. We therefore propose a clinical RCT to investigate the in vivo potency of L-arginine and vitamin D, the substrates for these two major candidate human mycobactericidal pathways, in clearing mycobacteria from sputum and improving clinical outcomes.
A summary of the proposed studies is shown in diagrammatic form in Figure 1. The main study is a randomised controlled trial: 'Arginine or Vitamin D Adjuvant Therapy in Pulmonary Tuberculosis'the ADAPT Study. In this study we will enrol 444 consecutive newly diagnosed sputum smear positive pulmonary TB patients from two sites in Timika, Papua Province, Indonesia. Patients will be commenced on standard TB treatment and in addition, those who agree will be randomised into four groups (arginine/placebo, vitamin D/placebo, arginine/vitamin D or placebo) and followed for six months. A series of clinical, haematological, microbiological and pulmonary function measurements will be made to assess treatment outcome. The primary outcome measures of sputum culture at 1 month and pulmonary function 2 months after commencing treatment. Related studies include:

Evaluation of the burden of TB-HIV co-infection;
4. Evaluation of a rapid diagnostic TB antigen test, and 5. Evaluation of healthy non-TB infected control subjects in order to gain normal reference range values for the RCT outcome measures.
The proposed research program will include significant capacity building opportunities, including 2 PhDs, 1 Masters and a range of professional training opportunities in Indonesia and Australia.  The study will be based at Rumah Sakit Mitra Masyarakat (RSMM) and at the TB control program (NTP) at Timika Puskesmas, Papua province. It builds on existing research collaborations and Memoranda of Understanding (MOU) between NIHRD, RSMM, Timika District Health Department and MSHR. When the Government to Government agreement for health research cooperation was signed in 1996, and the NIHRD-MSHR MOU in 1998, the agreements specified two priority diseases of public health importance to Indonesia which should form the basis of the collaboration: malaria and tuberculosis (TB). While malaria has been the major focus of our collaborative efforts to date, we have undertaken a number of small studies in TB (see 1.1.2). We are now proposing to extend our initial TB studies in keeping with the MSHR-NIHRD agreement calling for an additional focus on TB-related studies.

Tuberculosis Research outputs from the Timika NIHRD-MSHR Study Site to date
We have undertaken a small series of TB studies at the NIHRD-MSHR study site in Timika as part of a collaboration among personnel from Timika Pusksesmas, Dinas Kesehatan, RSMM, NIHRD, MSHR and Public Health and Malaria Control, PTFI. Whilst these have been small projects, with limited funds, they have achieved good outputs including: Funding for Bapak Harun from NIHRD to travel to Adelaide (via MSHR) to undertake TB diagnostic training at the WHO Supranational Reference Laboratory, and supply of reagents and protocols to continue this work at NIHRD; Funding for a Papuan co-investigator, Bapak Govert Waramori, to enroll in the Masters of Public Health at MSHR; Capacity building in Timika including multiple seminars and on-the-job training in clinical, laboratory and public health interventions to improve TB control in Timika District; Two quality assurance visits from the WHO Supranational TB Reference Laboratory with subsequent improvements in TB laboratory performance in Timika Puskesmas and RSMM; Information on multi-drug resistant TB (MDR-TB) for the Indonesian National TB Control Program to fulfill their reporting requirements to WHO, and to inform the national drug resistance survey currently underway in several other sites in Indonesia; and Five peer reviewed papers (4 published, 1 under review), including 2 papers first authored by Indonesian co-investigators (Dr Muhamed Ardian, Timika Dinas Kesehatan doctor and Dr Tjandra Handojo, staff of the NIHRD-MSHR research collaboration in Timika), and all coauthored with Indonesian investigators.

Laboratory and Pulmonary Equipment on site
A major strength of this study is our existing research base at the proposed field site, established with support from the Wellcome Trust-NHMRC to NIHRD-MSHR. A well-equipped research laboratory, clinical research ward and accommodation block for visiting researchers has been established on the Timika Hospital campus. The NIHRD-MSHR Timika Laboratory has on-site equipment (including the NIOX NO analyser, pulmonary function testing equipment, refrigerated centrifuges, bio-safety cabinet, -70°C freezers, microscopes and a Coulter counter). The Timika Puskesmas has a well stocked and well maintained laboratory (safety cabinet, microscopy facilities, reagents for acid fast staining of sputum samples) and a radiological facility on-site. Well trained technicians are also employed at the Puskesmas. NIHRD-MSHR has on-site staff trained in all the proposed procedures. The research team in Timika is very familiar with RCTs, with randomisation of over 1200 patients in local malaria trials to date (1-4).

Data ownership, management and analysis
The NIHRD and Menzies School of Health Research will have co-ownership of the data, consistent with the Memorandum of Understanding and with previous practice of other collaborative studies between our two institutions. There will continue to be training opportunities in Timika and Darwin for members of the research team, subject to funding. These include clinical, radiological, pulmonary function testing (PFT) and public health training. For example, portable PFT equipment has been brought to RSMM, and two RSMM staff members have been trained in its use by MSHR staff.

Anticipated Benefits to local medical staff and the wider community
Preliminary discussions regarding TB treatment in Timika, undertaken in planning this study, have revealed that medical staff at RSMM and the TB Clinic have particular concerns about poor adherence to treatment by patients, loss to follow up, HIV-TB co-infection, and the emergence of drug-resistant TB. The structures and resources associated with this proposed study will help to address these concerns: in particular, two dedicated TB Research Assistant positions will be created. Anticipated benefits of the study, building on those listed in 1.1.2, are summarised as follows: Support for medical staff in implementing the National TB Protocol (NTP): o Two dedicated nurses to work as TB Research Assistants o Access to research vehicle to assist in follow up of treatment defaulters o In-services by NTP and Research staff to provide continuing professional development for local medical and nursing staff on the topic of TB, including highlighting HIV testing and treatment in TB Quality assurance practices including examination of all sputum smears at the WHO Supranational TB Reference Laboratory TB culture and sensitivity testing to be performed at Supranational TB Reference Laboratory as previously (during 2003-4 studies) to: o Provide confirmation of TB diagnosis or otherwise (e.g. rates of non-tuberculous mycobacterial pulmonary infections) o Complement the national drug resistance survey currently underway in several other sites in Indonesia by providing ongoing information to NTP and WHO on multi-drug resistant TB (MDR-TB) rates o Allow individual patient medication regimens to be tailored if resistant isolate identified Measures of Vitamin D status of people with TB and healthy controls will provide important information about vitamin D deficiency rates, recognised globally as an emerging health problem Potential benefits related to study intervention: if L-arginine and / or vitamin D are found to be beneficial as we and other researchers propose, this will have major positive impacts on TB outcomes for patients enrolled in the study who receive active intervention. If shown to be beneficial, wider roll-out of these treatments can then be undertaken.

Ongoing development of the study protocol
This study protocol is a dynamic collaboration between NIHRD and MSHR. Significant changes to an earlier draft were implemented in response to recommendations made by Drs Sadjaja and Dina Bisara Lolong, including: Change from a 3-arm design to a 4-arm factorial design Improved attention to HIV-TB co-infection Further recommendations will be incorporated where possible.

Scientific Background
Tuberculosis is the leading cause of death from a curable infectious disease (5). There are an estimated 1.7 billion people (a third of the world's population) infected with TB, 9 million new cases of TB disease each year and an estimated 2 million deaths annually(6). In the Asia-Pacific region, TB represents a major cause of mortality and morbidity and TB treatment consumes a high proportion of health budgets. In 2004, 59% of all TB cases notified to WHO occurred in the Asia-Pacific Region(6). In Indonesia, the TB incidence rate is 245 per 100,000 population per year with a death rate amongst TB patients of 19%(6). This represents 539,000 cases of TB and 100,000 deaths from the disease each year. As most of these cases occur in people in the productive years of life, TB has a major economic impact (7,8). In response to a resurgence in the disease, WHO declared TB a "global emergency" in 1993(9) and subsequently launched a comprehensive Stop TB Strategy, which includes the promotion of high-quality DOTS (directly observed therapy short-course) (10). Although very effective in achieving microbiological cure if compliance with 6-8 months' therapy is achieved, a major limitation of DOTS is this need for supervised therapy for such a prolonged period. Default from therapy is common (11). Moreover, antimicrobial therapy alone does not prevent the significant residual impairment of lung function at the completion of therapy shown in our longitudinal studies in Timika (see 'preliminary data').

TB Research Priorities
To improve TB treatment outcomes, the UNICEF/UNDP/World Bank/WHO Special Programme on Research and Training in Tropical Diseases has identified six research areas of priority importance (12). Our proposed study specifically targets two of these priorities: i) Development of new drug and treatment regimens and ii) Immunopathogenesis studies including the assessment of adjuvant immunotherapy. The search for new TB drugs has prioritized the discovery of agents with activity against latent, non-dividing bacilli. Latent bacilli become indifferent to antibiotics, i.e. phenotypically antibiotic resistant (13). Adjuvant immunotherapies which might prevent the switch from actively replicating to dormant forms, which might reverse the dormant state, or which in their own right might provide mycobacteriacidal activity against dormant forms, would be eagerly welcomed. Improvements in TB treatment resulting from successful adjuvant therapy has the potential to improve TB treatment by decreasing the duration of infectivity, duration of illness and disability, residual impairment in lung function and the duration of antimicrobial treatment. Each would have major clinical and public health benefits.

Mechanisms of immune protection against TB in humans
The nature of the immune response to Mycobacterium tuberculosis in humans has been extensively reviewed (14,15). Although complex, the adaptive immune response is characterised by T-cell populations capable of recognising mycobacterial antigens. The clearest demonstration of the importance of T cell function in the protective immune response to TB is the markedly increased risk of reactivation and progressive TB in patients with HIV infection and those on immunosuppressant therapy. Key Th1 cytokines such as IFN-γ activate macrophages to kill most, but not always all, tubercule bacilli, which in murine models is through a predominantly NO-mediated mechanism (16). In human macrophages there is evidence for both NO-mediated killing but also killing via Vitamin-D mediated pathways (see below). Both L-arginine and vitamin D have been proposed as adjuvant TB treatments in humans. L-arginine is the critical substrate for NO synthesis by inducible nitric oxide synthase (NOS2) in macrophages. Arginine and 25(OH)D 3 are taken up by macrophages and converted to the potent mycobacteriocidal molecules, NO and 1,25(OH) 2 D 3 respectively.

Role of NO in mycobacterial killing in TB
As reviewed by Nathan (16), "NO is the only molecule known to be produced by mammalian cells that can kill tubercle bacilli in vitro with a molar potency comparable to chemotherapy". Other in vitro/rodent evidence for the importance of NO in protection from disease include the following: i) Activated mouse macrophages expressing NOS2 kill MTB in vitro, an effect that is ablated by NOS2 inhibitors; ii) MTB-infected NOS2-/-knockout mice develop fulminant disease; iii) Infected mice treated with NOS2 inhibitors also develop fulminant TB (17,18). and iv) NOS2 is expressed in infected mouse lung where bacterial growth is minimal. It is clearly not ethical to administer NOS2 inhibitors to infected humans. Evidence for a functionally important role for NOS2/NO in human TB is less welldefined, but is accumulating. Bronchoalveolar lavage (BAL) macrophages obtained from TB patients (but not healthy controls) express significant NOS2 (19). This 1996 finding, and our own findings that year of NOS2 expression by peripheral blood monocytes in malaria-exposed children (20), were among the first studies to clearly demonstrate that human macrophages/monocytes are capable of high level NOS2 expression in vivo, notwithstanding the difficulties demonstrating such expression in vitro. In lung resection studies, NOS2 and nitrotyrosine are expressed in macrophages within granulomas and areas of pneumonitis, suggesting that they may be functionally important in TB (21). Wang et al. showed that pulmonary NO production measured by exhaled NO was significantly higher in TB patients than in controls (22). Importantly, NO production from alveolar macrophages derived from BAL was significantly correlated with exhaled NO in these patients, supporting the approach we will use in this study to use exhaled NO as a non-invasive measure of pulmonary NO production in TB (22).

Role of NO in attenuating lung destruction in TB
In TB disease there is evidence that NO and IFN-γ may mediate disease-protection not only from mycobacterial killing but also by anti-inflammatory effects and reduced immunopathology/lung destruction (17,18,23). In infected NOS2-/-knockout mice and infected mice treated with NOS2 inhibitors, larger and more destructive lung lesions are seen in early infection, before a significant increase in mycobacterial numbers are seen. (24) NO down-regulates lymphocyte activation, induces apoptosis of activated lymphocytes, and inhibits adherence and transmigration of monocytes, all of which are thought to contribute to granulomatous inflammation and to caseating necrosis, lung destruction and fibrosis. NO also down-regulates TNF production(25), a mediator of caseating necrosis of lung tissue and weight loss in TB. Alveolar macrophage NO production is significantly higher in patients with milder disease than those with more extensive TB, consistent with a disease-protective role for NO (22).

Adjuvant L-Arginine treatment in human TB
In the only human RCT to date, Schon et al. showed that low dose L-arginine supplementation (1g daily) in Ethiopian TB patients was safe and well-tolerated (26). There was no significant response in the group with HIV co-infection. In a subgroup analysis of those without HIV infection, there was a modest but significant rise in plasma levels of arginine, a significant improvement in resolution of cough and a non-significant trend to more rapid weight gain and sputum smear conversion in the arginine group. Although arginine was safe and promising, limitations of this study include the small 8 sample size and post-hoc analysis. Our trial in Timika will occur in a population with a high incidence of TB and a low-prevalence of HIV. It will overcome the deficiencies of the earlier trial by using larger patient numbers, an a priori analytical plan and a higher dose of arginine (6g/day), which has been used safely and effectively in clinical trials in other disease states (27,28). The Ethiopian study (26) did not measure exhaled NO, and could not correlate improvement with pulmonary NO production. In our trial we will determine the relationship between the treatment-induced increases in plasma Larginine/pulmonary NO and each of the clinical, radiological and physiological measures of TB outcome. We have demonstrated that exhaled NO can be reliably measured in illness at our field site in Timika (see below).

NO-independent importance of L-arginine in T cell function.
Over the last 5 years, a crucial NO-independent link between L-arginine and T cell function has emerged. CD3ζ is an essential component of the T cell receptor (TCR), specialized antigen recognition molecules expressed on the surface of all CD4 and CD8 T lymphocytes that initiate signal cascades leading to T cell activation, proliferation and cytokine secretion, including IFN-γ required for macrophage activation and mycobacterial killing. Recent studies have shown that peripheral blood T cells from patients with pulmonary TB have decreased CD3ζ expression which normalises with successful treatment at 4 and 9 months (29). T cells infiltrating MTB-infected tissue also express less CD3ζ (30). Depletion of L-arginine in vitro (but not other amino acids) causes reduced CD3ζ expression, impaired T cell signalling and diminished proliferation (31)(32)(33). Addition of excess Larginine ex vivo leads to CD3ζ re-expression and recovery of T cell proliferation. (34) These data and the elevated levels of arginase (which metabolises L-arginine) found in peripheral blood monuclear cell lysates from TB patients (29), suggest that reduced L-arginine causes impaired CD3ζ expression and cellular unresponsiveness. Our preliminary data from hypoargininemic patients with falciparum malaria shows significant impairment in CD3ζ expression by activated CD8 T cells compared to arginine-replete controls. To date, there have been no clinical studies in any disease state testing whether L-arginine supplementation can reverse CD3ζ-associated in-vivo T cell dysfunction. A novel component of our proposal will determine whether L-arginine therapy can accelerate the restoration of T cell-CD3ζ expression and mycobacterial cellular immune responses and determine the association with clinical recovery.

Vitamin D as an anti-mycobacterial immune mediator
The importance of vitamin D 3 in human host resistance to TB has been suspected since the latenineteenth century (35)(36)(37)(38). Now, expanding recognition of the association between vitamin D deficiency and TB risk, and the pleiotropic immunomodulatory functions of vitamin D 3 (summarised below) provide theoretical bases for this supposition. Vitamin D 3 is generated in the skin on exposure to UVB light and is metabolised in the liver to 25-hydroxyvitamin D 3 . This circulating metabolite is converted by the enzyme 1 -hydroxylase to the biologically active steroid hormone, 1,25-dihydroxyvitaminD 3 (1,25(OH) 2 D 3 , synonymous hereafter with Vitamin D 3 ). Its actions are mediated by ligation with the nuclear vitamin D receptor (VDR), resulting in genomic responses, or alternatively, via more recently identified rapid-response receptors which may be associated with the plasma membrane(39).

Vitamin D suppresses MTB growth in cultured human cells
Vitamin D 3 has been shown to exhibit inhibitory effects on MTB in cultured human monocytes (40,41) and human macrophages cell lines (42,43). While this action appeared in one study to be mediated by NO upregulation (43), subsequent studies have not replicated this vitamin D 3 -NO interplay. Peripheral blood mononuclear cells from TB-infected people were found by Chang et al. to produce vitamin D 3 more readily, but release less NO than control cells (44). Sly et al. found that Vitamin D 3 -induced antimycbacterial activity in human macrophages was not due to NO production but rather, was due to phosphatidylinositol-3-kinase (PI3-K)-mediated superoxide production (41).

Vitamin D reverses MTB-induced phagocyte maturation arrest
Antimycobacterial activity of vitamin D 3 is further demonstrated by studies of phagolysosome function. An important mechanism of MTB immune evasion is inhibition of phagocyte maturation. Killing of phagocytosed organisms is usually achieved through a calcium-dependent fusion process between phagosomes and toxic acid-containing lysosomes (45). By inhibiting the fusion step, through inhibition of sphingosine kinase (45), MTB is able to achieve latency in a protected niche. Hmama and colleagues have demonstrated that MTB-induced phagocyte maturation arrest in a human macrophage model is able to be reversed by vitamin D 3 , via a PI3-K pathway (46). This finding, and those of Sly et al., implicate PI3-K as an important mediator of the antimycobacterial action of vitamin D 3 , and provide an example of non-genomic (VDR independent) signaling of vitamin D 3 (41).

TLR activation triggers vitamin D-mediated MTB immune response; adequate responses require serum vitamin D sufficiency
In their seminal 2006 Science paper, Liu et al. demonstrated that TLR1/2 activation of human macrophages (an MTB-induced process in vivo) upregulated the expression of both the vitamin D receptor and 1 -hydroxylase genes, resulting in increased production of the antimicrobial peptide cathelicidin, with subsequent intracellular killing of MTB (47). Thus a second mechanism of vitamin D 3 -mediated mycobacterial killing, in this instance utilizing genomic VDR-mediated responses, has been identified.
The researchers went on to demonstrate that successful induction of antimycobacterial activity through cathelicidin production was reliant upon replete serum vitamin D levels. Serum from ethnic groups (African Americans) at higher risk for TB had lower levels of 25(OH)D 3 , and their serum was less efficient in supporting cathelicidin mRNA induction; supplementation of this serum with 25(OH)D 3 restored TLR-induction of cathelicidin mRNA. The findings provide an immunological basis for differential susceptibility among human populations to mycobacterial infection; namely, that vitamin D deficiency appears to portend higher risk of TB disease.

Epidemiological and clinical evidence for the importance of vitamin D in TB outcomes
Prior to the antibiotic era, nutritional supplementation (including vitamin D-rich cod liver oil) and sunlight exposure were centrepieces of therapy for TB (48)(49)(50). Finsen was awarded the Nobel Prize for Medicine in 1903 for demonstrating the use of light exposure (which acts by upregulating vitamin D production) to treat lupus vulgaris (cutaneous tuberculosis) (51). Deficiency of vitamin D has been demonstrated in people with TB (52), and in ethnic groups recognised to be at higher risk for TB (47), including darkly-pigmented migrants to cooler climates (53). who have elevated risk of development of active TB post-migration. Vitamin D receptor polymorphisms have been found to be under-represented in pulmonary TB patients (54) (and in lepromatous leprosy) (55), suggesting the importance of vitamin D-mediated pathways in determining TB susceptibility, although meta-analysis of VDR polymorphisms has been inconclusive(56).
Martineau and colleagues (57) identified and reviewed three RCTs and ten prospective case series in which vitamin D was administered to patients with TB; most were conducted in the 1950s, were identified as being of poor quality, and used vitamin D 2 . were identified as being of poor quality, and used Ergocalciferol (vitamin D 2 ), which is known to be less efficacious than cholecalciferol (D 3 )(58). Only one study investigated the impact of vitamin D supplementation on TB outcome, and although concluding that vitamin D was beneficial, there were no statistically significant differences in clinical response (59). A more recent trial of vitamin D supplementation in Indonesian pulmonary TB patients appeared to be safe, and suggested more rapid sputum clearance and radiological improvement(60), but the trial was small and the process of randomisation was not described. A larger study with rigorous randomisation is required, such as we propose. Liu et al. and Martineau et al. advocate trials of vitamin D supplementation in patients with TB(47), particularly those with vitamin D insufficiency. A study conducted in Surabaya found that three quarters of patients with TB, and the same proportion of control subjects without TB, had serum vitamin D levels of less than 21 ng/mL (=80 nmol/L)(61), consistent with vitamin D deficiency according to latest recommended reference ranges (62). A recent position statement on vitamin D supplementation emphasised that even in countries with apparent high ambient UV light, vitamin D remains a common and under-diagnosed problem(63).

Do macrophages kill MTB by predominantly NO-or Vit D-mediated mechanisms? Do the L-arginine-NO and Vitamin D-cathelicidin pathways interact?
A major debate is ongoing about the fundamental biology of the mechanisms by which human macrophages kill intracellular pathogens such as MTB (47). While rodent models have identified NO as the central mechanism for mycobacterial killing by macrophages and protection from tissue destruction, Liu et al. have postulated that these NO-mediated mechanisms are more important in nocturnal animals such as mice than in diurnal animals such as humans who are able to synthesise vitamin D 3 in the skin on exposure to UV light. Nathan has argued that the limitations of current in vitro and ex vivo models for inducing human iNOS and testing its role in mycobacterial killing mean we cannot exclude a major role for NO in humans (64). We have shown that notwithstanding the difficulties in inducing NOS2 in vitro, human monocytes/macrophages are capable of high level NOS2 expression in vivo, and that NO production/PBMC NOS2 expression is associated with protection from infectious disease in humans (20).
There are evident intersections between these two immunological pathways. Firstly, CYP27B1, the gene which encodes 1 -hydroxylase (the enzyme which converts 25-hydroxyvitamin D 3 to the biologically active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 ) gene, requires an extracellular source of L-arginine for full expression, and is upregulated by NO in an avian macrophage cell line (65)(66)(67). Secondly, the antimycobacterial effect of vitamin D may be mediated in part by NO. (43,68) The inhibitory effect of Vitamin D 3 on MTB in cultured human macrophage cell lines was initially thought to be mediated by NO upregulation, as demonstrated by Rockett and colleagues (43), but subsequent studies did not replicate this finding, as described in Section 1.2.4.1. However more recent work by Martineau and colleagues gives support to Rockett's finding that vitamin D 3 , when added to MTBinfected cultured human peripheral blood mononuclear cells, did upregulate NOS2a expression, but the effect was small in comparison with the strong upregulating impact of vitamin D 3 on the antimicrobial peptide cathelicidin (68), which is emerging as an important mediator of anti-TB immunity in humans (47).
Those on both sides of this deabte acknowledge the difficulty of answering this question with current in vitro tools (47,64). Our clinical trial is designed to determine the potential utility of L-arginine and Vitamin D as adjuvant therapies. An additional benefit of our study will be the comparison of the in vivo potency of these two major candidate immune pathways of mycobacterial killing in humans in clearing mycobacteria from sputum and ameliorating pathology. As well as the potential for clinical and public health benefit, this study will contribute to the understanding of the biological mechanisms of disease protection in human TB relative to mouse models.

Preliminary Studies Related to this Proposal
The NIHRD-MSHR research facility in Timika is well placed to undertake the 'Arginine and Vitamin D Adjuvant Therapy in Pulmonary Tuberculosis' (ADAPT) Study. ADAPT is a logical extension of our current field studies in pulmonary TB and the role of adjuvant L-arginine therapy in infectious diseases. We have completed a study of 115 patients with pulmonary TB in Timika and examined the risk factors for impaired lung function. We have been able to follow a high proportion of patients to six months, demonstrating the feasibility of longitudinal studies of TB patients in Timika (69). Preliminary data from the initial Timika TB studies show that TB patients in Timika have severe disease at diagnosis (as measured by sputum positivity, Xray changes, lung function tests and functional measurements), low rates of HIV co-infection (4%) and MDR-TB (2%) high rates (41%) of malnutrition according to WHO criteria (BMI<18.5kg/m 2 ) and a default rate of less than 10% (69).
Whilst TB patients show signs of clinical, radiological and anthropometric improvement after two months of treatment, respiratory function remains impaired in a significant number of patients, with over 45% having significant pulmonary restriction at 6 months despite high rates of microbiological cure ( Figure 2). Additionally, 31% of patients remain in the malnourished range at 2 months. Adjunctive therapy which accelerates mycobacterial killing, modifies the immunological response to TB and/or facilitates a more rapid return to normal weight in TB patients would be of enormous benefit in this population.
Preliminary results from our trials of arginine supplementation in patients with malaria in Timika have demonstrated substantial increases in exhaled NO: 6g supplementation led to a mean increase of 55%. 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 our TB study, 6g of Larginine would be expected to increase exhaled NO by at least 55%. 12

Hypotheses
1) 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.
2) Supplementation with vitamin D, the metabolite of which (1,25-dihydroxyvitamin D 3 ) has antimycobacterial activity, will be safe, will increase plasma Vitamin D concentrations, and will improve the rapidity and magnitude of the treatment response in human PTB.

Aims
1) 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. We will randomise patients with pulmonary TB to receive, in addition to standard TB therapy, adjuvant arginine, vitamin D, arginine and vitamin D, or placebo. We will relate serial measurements of plasma concentrations of L-arginine and vitamin D, and immunological responses (pulmonary NO production, T cell function and phenotype) to measures of treatment outcome [mycobacterial (sputum smear clearance and culture conversion), physiological (spirometry), clinical (symptoms and weight), radiological (chest Xray) and functional (six-minute walk test, modified St George Respiratory Questionnaire)].
2) Determine whether expired NO 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.

Setting
The study will be based at the Timika TB Clinic and community health centre (Puskesmas) and the community hospital, Rumah Sakit Mitra Masyarakat (RSMM), Timika, Papua Province, Indonesia.

Study Population
Patients diagnosed with pulmonary TB at RSMM, the Timika TB clinic or the Timika Puskesmas will be eligible to enrol in the study. All patients with suspected TB will be diagnosed in the usual way according to the NTP guidelines. This will include three sputum smears examined for acid fast bacilli and a chest Xray. During the study period, surveillance and evaluation of extrapulmonary TB cases will also take place (Appendix 10).

Inclusion criteria
Adults 15 years of age or older will be recruited 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 will be 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
Children less than 15 years of age, TB patients who have previously received treatment for TB for more than one month, patients unwilling to continue treatment in Timika for the full course, pregnant patients and those who are not willing to enrol in the study, will be excluded. Patients will be excluded from the study if hypercalcaemia (ionized calcium >1.32 mmol/L) is identified at baseline.

Informed consent
All patients will receive a detailed explanation regarding the reason for the study and what it involves, and regarding the voluntary nature of involvement in the study. Each eligible patient will be provided with verbal and written information in Bahasa Indonesia, or verbally using a Papuan translator if required.
Only patients who understand the study and provide informed consent will be enrolled. It will be 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 they wish without prejudicing their treatment in any way. On withdrawing from the study, the researchers will provide ongoing positive encouragement and support for the participants to continue her/his TB treatment as usual, according to the National TB Protocol.

Enrolment Procedure
All patients with confirmed smear positive pulmonary TB will be provided by the Research Assistant (RA) with verbal and written information in Bahasa Indonesia (see Patient Information Sheet, Appendix 3) regarding ADAPT, and informed consent will be obtained prior to patient enrolment. For participants who cannot read the information sheet and consent form, the study will be carefully and fully explained using a Papuan translator if required. Consenting participants will then receive their randomisation assignation. To achieve this, an envelope will be opened containing the code (Study Identification (ID) Number) assigning the patient to an unidentified (blinded) study arm. The Study ID Number will match a numbered Study Drug Packet containing the study drugs (see also Section 7).
Before any medications are administered, the baseline blood sample will be collected (see below) for immediate calcium results, along with the evaluations listed below (section 3.3.3.1). The first supervised dose of TB medication plus study drugs will then be administered. The patient name, time of randomisation, the randomisation number, and whether or not they were excluded from the vitamin D/placebo arm will all be noted.
All patients will be required to attend the clinic daily during the intensive phase and twice a month for the continuation phase to receive their anti-TB medications, study medications, routine examinations and the study examinations as outlined below (Follow up, section 3.3.5).

Medication administration
At enrolment, patients will be randomly assigned to one of three groups: 1) Group 1: supplementary L-arginine 6g daily for eight weeks plus placebo vitamin D 3 once every four weeks for two doses (0 and 4 weeks); 2) Group 2: supplementary L-arginine 6g daily for eight weeks plus vitamin D 3 (cholecalciferol) 50 000 IU (1250mcg) once every four weeks for two doses (0 and 4 weeks); 3) Group 3: placebo L-arginine daily for eight weeks plus vitamin D 3 (cholecalciferol) 50 000 IU (1250mcg) once every four weeks for two doses (0 and 4 weeks); 4) Group 4: placebo L-arginine daily for eight weeks plus placebo vitamin D 3 every four weeks for two doses (0 and 4 weeks); Study drugs will be administered separately from the pre-packaged TB medications, provided in standard Indonesian NTP 'Combipacks' (fixed dose combination therapy for TB).

L-Arginine
L-arginine hydrochloride powder is commercially available as a GMP standard nutritional supplement (Nutricia Hong Kong). It will be compounded into a 6g dose at RSMM pharmacy. A matching placebo from the same manufacturer with identical appearance, colour, size, and taste will also be prepared.
The study medication will then be packaged and numbered according to the randomisation sequence. Clearly marked Study Drug Packs will be stored in a cool, dry place in the medication cupboards of the Timika TB Clinic and RSMM.
The choice of 6g daily is based on clinical experience with this dose, safety profile at this and higher doses, and the need to adhere to a once daily regimen for TB patients to avoid dosing errors (all other medications are dosed once daily), and to optimise adherence.

Vitamin D 3 (Cholecalciferol)
We will use high dose vitamin D 3 (Calciferol Strong, 50 000 IU cholecalciferol; PSM Healthcare, Auckland, NZ), a formulation that allows monthly oral administration. This will be given orally at baseline and again at 4 and 8 weeks. Matching placebo will similarly be obtained. This is similar to but lower dose than standard stoss therapy (intermittent high dose depot vitamin D dosing) used for restoration of vitamin D deficiency (70). The large volume of distribution of vitamin D (since it is fat soluble) necessitates such large doses before any change in plasma vitamin D level is detectable.

Cessation of Study Medications
If medications are thought to be causing unacceptable symptoms, the treating doctor will determine the likely pharmacological cause (either of the two study drugs, or any of the four TB drugs) according to the strategy outlined in Appendix 5. If symptoms are determined to be due to arginine, this will be immediately ceased, and reintroduced at a lower dose as outlined in Appendix 5. If symptoms are due to vitamin D/hypercalcaemia, appropriate hypercalcaemia management will be instituted and any further scheduled dose of vitamin D will be cancelled. Any such events will be recorded on the 'Adverse drug events' sheet (Appendix 6) and notified immediately to the study investigators.

Clinical, Radiological, Physiological and Functional Evaluations 1) Height
Standing height in centimetres will be measured without shoes to the nearest centimetre using a stadiometer, ensuring that the tragus is level with the eyes.

2) Weight
Weight in kilograms will be measured without shoes to the nearest 0.1kg using an adult balance.

3) Body Mass Index (BMI)
BMI will be calculated in the standard way:

4) Questionnaire
A questionnaire to obtain demographic and clinical information will be completed (see TB Baseline Data Collection form, Appendix 1)

5) Chest radiograph
A postero-anterior chest x ray will be performed as per standard clinical protocols at the TB clinic and the RSMM, whether or not the patient is enrolled in the study. Chest Xrays will be evaluated by two study investigators using a previously validated scoring system. The percentage of abnormal lung will be assessed and averaged. The presence or absence of cavitation will also be recorded.

6) Lung Function
Respiratory function will be measured as outlined in Appendix 12 using the hand held spirometer (MicroLoop TM , Micro Medical, UK) used in our previous studies, which will be calibrated daily. Each patient will perform at least three maximum effort expirations until volumes vary less than 200mls, with the highest values for FVC and FEV 1 used as measures of lung function. Dr Graeme Maguire will supervise the QA/QC of this test.

7) Exhaled nitric oxide
Exhaled NO will be used to measure pulmonary NO production at the TB Puskesmas and RSMM, using the online NIOX Mino™ (Aerocrine, Sweden), as detailed in Appendix 13. .

8) Modified St George Respiratory Questionnaire (MSGRQ)
The SGRQ (see Appendix 17) is a valid self-complete measure of health status for patients with lung disease. An overall score and individual domain scores for symptoms, activity and impacts on daily life, are obtained. Patients will be requested to complete the questionnaire without assistance where possible in order to obtain objective responses, but with assistance from clinic staff if required.

9) 6 minute walk test (6MWT)
The 6 minute walk test will be performed in keeping with standard ATS guidelines as described in Appendix 13. The six minute weight.walk distance (6MWWD), calculated by multiplying this distance in kilometres by the subject's weight in kilograms, will be calculated.

10) Sputum collection
Sputum collections will be obtained at baseline from each TB patient in accordance with NTP standard protocols (minimum of 2 positive smears) prior to enrolment in the study. An additional sputum specimen will be obtained on the day of entry into the study. The study sputum specimen containers will be distinct from standard sterile specimen jars used at the clinic or hospital, and will be labelled with the patient's details (name, study ID number, age, sex), date of collection and laboratory number. A microscopy slide will be prepared from this specimen and examined using standard Zeihl Neelsen method, labelled using a diamond-tipped pen as above and stored at the NIHRD-MSHR Research Building for later QA. Sputum specimens will be divided to provide material for smear examination, antigen testing, culture, susceptibility testing and MIRU typing as detailed in Appendix 15.

11) Blood collection
Twenty mL venous blood (a safe amount) will be collected from participants at enrolment and at follow up weeks 2, 4, 8 and 24 as described in Appendix 15.

12) Urine Collection
A midstream urine specimen will be collected into a sterile specimen container for TB antigen testing as detailed in Appendix 15.

Follow up
Usual follow-up according to the NTP includes monthly weight, repeat chest x ray and sputum at 2 and 6 months, and recording of symptoms, adherence and side-effects. Study participants in addition will receive evaluations as indicated in the table below. Default tracing will comprise three attempts to trace participants who miss two consecutive scheduled clinic appointments, which is standard practice in Timika.

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 FEV 1 at 2 months.

2.
Difference in improvement in percent predicted FEV 1 at 2 and 6 months.
Functional improvement including Six minute walk test and quality of life assessment using modified St George Respiratory Questionnaire. 8.
Immunological improvement (exhaled NO, T cell CD3ζ expression and T cell function). 9.
Radiological improvement (percentage lung involvement on CXR at 2 months). 10. Percentage obstructive and/or restrictive lung disease at 6 months. 11. Death, clinical failure and default independently, and 'death or clinical failure or default'. 12. Primary end points stratified by HIV status. 13. Primary end points stratified by baseline vitamin D and L-arginine status. 14. 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. Culture at one month will capture any early microbiological effect of adjuvant therapy. As we have shown in previous work in Timika, FEV 1 at 2 months is predictive of longer term deficits in lung function that may be modified by early adjuvant therapy. We will also determine the relationship between baseline and serial measurements of pulmonary NO production, serum L-arginine and 25(OH)D 3 concentrations, and clinical, radiological, microbiological and functional measures of disease severity.

Sample size
Sample size has been calculated using the graphical method for 2x2 factorial designs informed by the Fleiss equation, described by Byth and Gebski (71). Making a conservative assumption of sub-additive interaction between the 2 interventions (interaction coefficient of 0.5), then at a level of significance of 5%, a sample size of 444 (111 participants in each arm) will provide 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 iNOS expression in one study (44); yet upregulate NOS2A in another (68); 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, our power will be greater. A factorial design rather than a three-arm study allows us to investigate whether if is safe and effective to co-administer the two interventions. Acknowledging the possibility of statistical interaction between the terms, we have an a priori plan to analyse primary and secondary outcomes according to subgroups receiving single interventions as well as according to the overall factorial model (71).
Mean time to culture negativity in mostly drug sensitive TB is 32 days (New York) (72) -57 days (Turkey) (73). Based on bacillary burden and extent of cavitatory disease, we estimate a minimum of 60% of Timika patients will be culture positive at 1 month. Our power will be increased if this proportion is higher. This sample size will also be powered to detect a 9% absolute difference in the mean improvement in percent predicted FEV1, a component of the composite endpoint, at 6 months. Mean baseline percent predicted FEV1 in Timika is 63.6% predicted (SD 22) rising to 77.9% predicted (SD 23) at 6 months. Each month, at least 25 eligible smear positive pulmonary TB patients are diagnosed and treated at the Timika study site. If 90% agree to be enrolled, recruitment will take <2yrs to complete. 20

Outcome Analysis
The primary analysis of all outcomes will be by intention to treat. All participants in the study will contribute outcomes for analysis whenever possible. Each participant's success or failure for the primary outcome will be determined by an independent observer who will be blinded to the intervention. Analyses will use Intercooled Stata 9.2 (Stata Corp, College Station, Texas, USA). 95% confidence intervals and/or p values <0.05 will be employed to signify statistical significance.
As per recent clinical journal requirements, ADAPT will be registered with the protocol registration system at Clinicaltrials.gov.

Intervention Procedure
Potential adverse effects of L-arginine and vitamin D will be carefully monitored. Adverse effects from oral L-arginine are uncommon. (27) Use of up to 21g daily was not associated with adverse events in two studies (28,74). 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 (75). Minor gastrointestinal discomfort has been reported in up to 3% of patients (27,76). We do not expect that this will be a problem but will monitor our study patients carefully for this adverse effect and treat accordingly.
Although much less common than in other granulomatous diseases, vitamin D supplementation in TB has the potential for hypercalcaemia from extra-renal 1,25(OH) 2 D 3 production(77). Based on our previous experience with vitamin D supplementation (Eisman, personal communication), and extensive review of the literature (60,(78)(79)(80)(81), the risk of symptomatic hypercalcaemia as a complication of vitamin D supplementation at the dose proposed in this study (63) in patients with pulmonary TB is anticipated to be very low. Ionised calcium will be measured, using portable i-STAT cartridges in Timika, at 0, 2, 4 and 8 weeks. In the unlikely 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. Patients found to have elevated iCa 2+ on follow up will have their Vit D/placebo dose withheld.

TB Medication side effects
All patients with pulmonary TB in the study will be treated with anti-tuberculous agents according to the standard NTP protocol. This comprises daily weight-adjusted doses of Rifampicin (R), Isoniazid (H), Pyrazinamide (Z) and Ethambutol (E). Medications are taken under direct observation by health care workers during the intensive phase. Treating doctors will comply with NTP guidelines with regards to standard monitoring for TB drug side effects.

Stopping rules
Adverse events defined as serious will be i) death and ii) ionised hypercalemia >1.6 mmol/L or hypercalcaemia requiring hospitalisation. All serious adverse events (SAE) will be reported to the chair of the DSMC. It is expected that death may occur during the 6 month treatment and follow up period in up to 5% of patients enrolled in the study related to TB itself (eg advanced/disseminated disease) or comorbidities (eg AIDS). An interim analysis will be performed after 25 and 50% of participants have been followed to 2 months, or as requested by the DSMC in the event of SAE related to study drugs. The DSMC 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 will be computer-generated (Intercooled Stata 9.2) and concealed from all investigators throughout the study. The random assignment to intervention group will be provided by the MSHR statistician. Block randomisation will be used to maintain similar numbers of participants in the intervention and control groups and to minimise the potential influence of time of enrolment. Allocation will be stratified by place of TB diagnosis (hospital or clinic) and ethnicity (Papuan/non-Papuan), the latter because of the different Vitamin D status of these two groups (as per preliminary data). Allocation concealment will conform to the revised CONSORT guidelines which take account of the logistically challenging environment at the study site (82). We have successfully used this method in over 1,200 malaria patients (three RCTs) in Timika (1, 2, 4). The random allocation will be in sealed, opaque envelopes in two boxes (Papuan and non-Papuan) in each study location. Prior to opening the envelope, the investigator will complete the randomisation form to check if the patient is eligible for enrolment, obtain informed consent and complete the enrolment questionnaire.
Placebo will be compounded to be indistinguishable by appearance, smell and taste from the matching active medication. All study personnel and participants will be blinded to treatment assignment for the duration of the study. Only the study statistician and the Data Safety Monitoring Committee will see any unblinded data and these individuals will 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.

Randomised controlled trial design
The perceived withholding of a potentially beneficial medication to half the enrolled study participants may arise as an ethical consideration. To alleviate these concerns, we will explain in advance very clearly to staff, community leaders and patients: (a) the necessity for the blinded, randomised, placebocontrolled 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 will explain the fact that half the patients enrolled will not get the active study drug. Patients will be reminded that they can withdraw from the study at any time without compromising their care or relationship with clinic staff. We 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, will 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 spill over into general practice, and the presence of extra staff (i.e. research assistants and study investigators) being able to provide operational and medical assistance at the clinic.

Potential for harm arising from study medications
Although anticipated to be unlikely, potential medication side effects will be monitored as detailed in 6.1, 6.2 and Appendices 5 and 6.

Disincentive due to requirements of study participants
Impacts on participants include the extra medications (active study drug or placebo) and the extra investigations required. The baseline evaluations (standard TB clinic questionnaire, collection of sputum sample, provision of information for informed consent, anthropometric measures, chest radiograph, 6 minute walk, eNO, lung function, modified St George Respiratory Questionnaire) are estimated to take approximately 1 hour per patient. Further appointments of similar duration will be required at 4, 8 and 24 weeks when some of these measures are repeated, but all other follow up visits will be much shorter. It is already routine according to NTP protocols for TB patients undergoing treatment to have a baseline questionnaire, clinical assessment, 3 sputum specimens and chest x ray, then monthly weight, repeat chest x ray and sputum collections at 2 and 6 months, and regular recording of symptoms, adherence and side-effects. The additional time required at the clinic could potentially serve as a disincentive for patients to attend for TB treatment. These extra requirements will be explained at the time of enrolment. In addition, we will be careful to question patients about whether this is the case, offer flexibility regarding time and day of the week on which they can attend, ensure efficiency in carrying out the investigations, and ensure that those who skip some measures, or withdraw altogether, continue to receive their TB therapy.

Potential for harm arising from baseline and follow up measurements
Measurements will be performed in the purpose-built open-air clinic annex. The filters used with both the spirometry and eNO will prevent transmission of TB. Standard occupational health and safety standards for staff will be upheld, including safe practices in biohazard disposal.

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 adjuvant 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 will be stored confidentially on password-protected computers accessible only to the named investigators, data entry clerks and named research students. Paper records will be stored in locked filing cabinets at the NIHRD-MSHR research building in Timika. We appreciate the risk of loss of data due to computer failure or power blackout and as per our current studies, data will be backed up at least weekly, sent to and stored at the collaborating institutions.

QUALITY CONTROL AND QUALITY ASSURANCE
The NIHRD and Menzies School of Health Research will have co-ownership of the data, consistent with the Memorandum of Understanding and with previous practice of other collaborative studies between our two institutions. The research team will be working on the basis of this protocol and the NTP manual. The team from NIHRD and MSHR/ANU will evaluate and assess the work to ensure a high standard procedure applies to this study and ensure the collection of high quality data.

Eligibility criteria
Dr Anna Ralph will perform weekly checks on all enrolment forms to ensure that the correct diagnostic criteria have been applied and that only patients eligible for inclusion have been included. These will be cross-checked by Dr Dina (NHIRD).

Informed consent process, including refusal rate and withdrawal rate
The local investigators Dr Firdy and Bapak Govert Waramori will perform weekly checks on all consent forms to ensure that the correct procedures have been followed and that informed consent has been obtained in the manner set out in this protocol.

Randomisation process
Local investigators will perform weekly checks to ensure that patients have been randomised in the correct manner set out in this protocol. The principal investigators will carry out spot checks on eligibility and the informed consent and randomisation processes during regular visits to Timika.

Accuracy of data collection and data entry
All data will be double entered and discrepant data will be checked with primary sources. Dr Firdy and Bapak Govert Waramori will carry out spot checks of data collection forms and check the accuracy of information directly from the patient.

Adverse events related to the study or to TB treatment
Research team members at the TB clinic and at RSMM will report all potential adverse events to the treating doctor and the local investigators (Dr Firdy and Bapak Govert Waramori) immediately. Presumed TB medication side effects will be reported to the treating physician as soon as possible, the strategy outlined in Appendix 5 should be followed, and if necessary, an Adverse Drug Reaction form completed by the treating doctor (Appendix 6). These will be reviewed by Dr Ralph and Dr Dina. Any serious adverse effects will be reported immediately to the principal investigators and the chair of the Data and Safety Monitoring Committee.

Data and Safety Monitoring Committee
A Data and Safety Monitoring Committee has Indonesian and Australian membership consisting of experienced researchers and health practitioners independent of the research team (NIHRD nominee, Dr Louise Maple-Brown, Specialist Physician and Endocrinologist, MSHR, Dr Peter Caley, Statistician, ANU and Dr Paulus Sugiarto, Clinical Director, RSMM Hospital, Timika). They will review the progress of the study and give advice to the research team on two occasions during the study. They will have the power to immediately discontinue the study if severe adverse events attributable to the study medications occur.

DATA HANDLING AND RECORD KEEPING
Data will be held in Timika for the duration of the trial, paper in a locked filing cabinet and electronic data in a password-protected regularly backed-up file. Back-up copies of data will be 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.

FINANCING
Financing has been secured (subject to ethical approval) from the following sources indicated in Table  4. Funding bodies have indicated that funds need to be spent by the dates indicated. In particular, major in-kind contributions have been committed by the out-going MALKON director, with the expectation of use of funds commencing prior to his departure in June 2008. Pending applications are listed, and further funding applications will be made as appropriate during subsequent years of the TB project.

PUBLICATION POLICY
The study results will be published by the investigators with acknowledgement of the funding bodies. All co-investigators will be offered authorship according to their involvement in the study and conforming with standard international definitions for authorship (83). An accurate list of publications to arise from the research cannot be clearly anticipated until research is underway. Possible outputs with identification of assigned researcher and key authors are listed below (Table 5). During the course of the project, there may be changes, which will be agreed upon by mutual agreement of NIHRD & MSHR, to both the list of investigators involved (e.g. in the event of transfer of investigator elsewhere, or requirement to seek further expertise from statisticians or other consultants), and to publications arising.
Research assistants and clinicians at the study site whose work is integral to the research will have the opportunity to be included as authors on publications as appropriate based on international standards of contribution (as in previous publications to date arising from the Timika NIHRD-MSRH research collaboration). NIHRD and MSHR endorse the internationally recognised requirement that first authorship requires leadership of the work involved in each paper (ie considerable ownership and leadership with regard to study design, analysis, writing the paper and major intellectual input to the paper). Co-authorship will apply for all who significantly contributed to the work, in keeping with international standards of authorship (i.e. input to study design and/or data collection and/or analysis as well as a substantial intellectual input to the paper).
How long ago did patient last eat? (hrs)

Selama minggu yang lalu, berapa kali minum obat TB?
During the last week, how many doses of TB tablets taken?

Jika ada hasil pemeriksaan darah yang relevan:
If any other relevant / abnormal blood tests are available, write results here:

Selama minggu lalu, berapa kali minum obat sirup
During the last week, how many doses of study medication were taken? Don't know

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, 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
Anda bisa membantu kita menjawab pertanyaan yang penting itu. Jika Anda setuju dengan menjadi terlibat dalam studi ini, akan Anda diberi tablet tambahan. Sebagian orang akan diberi tablet yang kosong, hanya berisi bubuk (tablet placebo 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.

Hypercalcaemia in ADAPT 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.

Appendix 7: Healthy (Control) Subjects
Control subjects will be required in order to obtain normal Papuan and Non-Papuan population values for serum vitamin D, amino acids and T cell CD3 zeta expression, the 6 minute walk test and the Modified St George Respiratory Questionnaire.

Methods
As per our previously approved malaria studies at the same study site, we will recruit 50 healthy adult Papuan and 50 healthy Non-Papuan people from among clinic and hospital staff or visitors. In each ethnic group, we will seek to enrol 25 men and 25 non-pregnant women who consent to participate.

Inclusion criteria
Healthy adults >15 years of age Consent to having one-off blood test and 6 minute walk test Chronic stable conditions (such as hypertension) not impacting on ability to perform 6 minute walk Exclusion criteria Pregnancy Hospitalisation currently or within the last month The Research assistant will seek informed consent from potential control subjects after providing verbal and written information (see Appendix) regarding the purpose of their participation and what is required of them. For participants who cannot read the information sheet and consent form in Bahasa Indonesia, the research assistants will carefully and fully explain the information using a translator if required. 20mL venous blood, a safe amount, will be collected in the manner described in Appendix 16 and transferred to lithium heparin tubes for separation and cryopreservation for plasma vitamin D, plasma amino acids (and metabolites), and white cell function and phenotype (including T cell CD3 zeta expression). Participants will be instructed on how to perform a 6 Minute Walk test (6MWT), and this will be undertaken as described in Appendix 13.

Appendix 10: Extrapulmonary TB Survey and Data Collection Sheet
Only patients who have smear positive pulmonary TB (with or without additional extrapulmonary sites of infection) will be eligible for ADAPT. In order to gain an understanding of the context of these cases, intercurrent instances of extrapulmonary TB (EPTB) in children and adults in RSMM and Timika Puskesmus will be monitored. Patients with both smear positive pulmonary and extrapulmonary disease could be enrolled in both studies.

Study design:
o Descriptive study comprising a prospective audit Background: EPTB is recognised internationally to be a difficult diagnostic entity. Preliminary work demonstrates differences in rates of extra-pulmonary TB in Papuans and Non-Papuans in Timika. However the details of EPTB type, diagnostic method, treatment and outcome have not been available for Timika to date. This proposed preliminary descriptive study of EPTB aims to answer these questions, and furthermore, to promote the use of existing protocols for diagnosing EPTB (e.g. Pedoman Nasional Penanggulangan TB Sistem Skoring for Children). Education in EPTB diagnosis and management will be provided to medical staff, and heightened efforts to retain patients in treatment for the full 6 months will be made, with treating doctors being requested to follow up patients and complete data collection forms at 2 and 6 months.
The potential change in HIV prevalence which might occur during the study period is also likely to have an impact on presentation of TB. Specific aspects of the presentation and management of TB-HIV co-infected patients will be evaluated, and education and support in HIV management will be provided as part of standard clinical care at RSMM and the TB clinic. This topic is well placed to be developed further by a future student into research which could form the basis of post-graduate studies.

Aims:
o To evaluate the burden and nature of extra-pulmonary TB in Papuan and Non-Papuan Timika residents

Methods:
o All adults and children presenting to RSMM or Timika TB Clinic with suspected EPTB will be eligible for enrolment in the study. o The treating doctor will be asked to follow standard NTP practice in diagnosing and treating EPTB, which includes offering HIV testing. The treating doctor will be asked to complete a form at baseline and again at 2 and 6 months to indicate demographic, clinical, laboratory and outcome characteristics (PLEASE SEE THE FOLLOWING 3 DATA COLLECTION FORMS: Baseline, 2-months, 6-months) o Given the planned study design (chart review), it will not be necessary for direct interaction between the study team and the patient except where this is in the course of standard clinical interaction. o If suitable biological specimens are collected during routine clinical care, portions of these will be saved for subsequent Rapid Diagnostic Protoeme TM antigen testing.

Timeline:
o No biological specimens additional to standard care are required for this study, therefore in anticipation of lack of signed consent requirement and rapid ethical clearance, this study could commence in early 2008.

Appendix 11: HIV-TB Co-infection
HIV rates are anticipated to be rising in Timika. To evaluate the burden of HIV-TB co-infection and uptake of antiretroviral treatment, the NTP policy of offering HIV testing to all people with TB will be supported. This is routinely offered as part of standard clinical practice in Timika. Dr Dina Bisara Lolong will be involved in the evaluation of HIV-TB co-infection.

Methods:
o Patients enrolled in either ADAPT or the EPTB chart review will be eligible for inclusion o HIV testing is routine for all TB patients, and will performed on site in Timika using two tests (ELISA using Determine TM , Abbott Diagnostics, Jakarta, Indonesia and a confirmatory ELISA or Western Blot) o Outcomes of TB treatment and contributing factors will be compared in patients with and without HIV o Antiretroviral treatment commencement, adherence and side effects will be recorded, with advice to treating doctors to adhere to WHO protocols relating to TB-HIV coinfection o Results will be compared with data previously collected by NIHRD

Sputum Collection and Handling
After collection in sterile specimen containers, sputum specimens will be kept in a domestic refrigerator at +4 C in the TB clinic or RSMM until ready for batched transport by air with a commercial courier via Darwin to the WHO Supranational Reference laboratory at the Institute of Medical and Veterinary Sciences (IMVS) in Adelaide, Australia. Preparation and packaging of the specimens will comply with International Air Transport Association (IATA) regulations. Specimens will not be decontaminated prior to storage, and will be sent at three to six week intervals during the study period. After leaving the TB clinic or RSMM Laboratory, specimens will be kept at room temperature until arrival at the reference laboratory. We have previously demonstrated success in culturing MTB in over 94% of smear positive sputum samples, despite considerable delays in transport to the Adelaide laboratory (mean 18.7, range 4-42 days).(89)

Location of testing
Wherever possible, specimens will be processed locally. Thus sputum microscopy and the novel antigen testing will be performed in Timika. There is no capacity for TB culture in Timika. It is our understanding that the nationally accredited TB culture laboratories in Indonesia (in Surabaya and Jakarta) are operating at maximum capacity and are fully committed to work related to the ongoing National TB drug resistance survey. Furthermore, it is mandatory for TB research to demonstrate participation in a quality assurance program. We therefore propose that sputum culture, sensitivity and typing should be performed at the WHO Supranational Reference Centre (IMVS, Adelaide) with whom the National TB Control Program is currently collaborating with a range of training, guideline development and quality assurance activities. Our previous experience of transferring samples from Timika to the WHO reference Laboratory has been successful. Additionally, processing at the WHO reference Laboratory will provide excellent training opportunities for NIHRD laboratory scientist (Ibu Palupi) to work on Indonesian samples in a supportive environment with world class training.

Ziehl Nielsen (ZN) staining and light microscopy
ZN staining is performed routinely at Timika Puskasmas and RSMM laboratories in accordance with NTP guidelines.

Proteome Systems' rapid diagnostic (RD) test
3 mL sputum will be reserved for antigen testing. Protease will be added and the sample will be frozen for subsequent batched antigen testing using Proteome Systems' rapid diagnostic (RD) test TM at RSMM laboratory.

Culture
Upon arrival at IMVS, Sputum specimens will be decontaminated using 2% sodium hydroxide and 0.5% N-acetyl-cysteine for 25 min, 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). Microscopy of sputum concentrates will be performed using fluorochrome and ZN stains and results recorded using standard IUATLD grading scales. Contaminated specimens will be re-decontaminated with 4% sulphuric acid.
Isolates of M. tuberculosis will be identified by ZN staining, hybridisation with commercial nucleic acid probes (Accuprobe, Gen-Probe, Inc., San Diego, Calif.), and biochemical investigations (eg. nitrate reduction) (90,91). The IMVS successfully participates in external quality assessment programmes through the WHO Global Supranational Reference Laboratory Quality Control Network and the Centers for Disease Control and Prevention, Atlanta, USA.

Micro-satellite Interstitial Repetitive Unit typing (MIRU)
MIRU is a DNA typing method using polymerase chain reaction (PCR) to detect differences in the number of repetitive units in specific regions of the MTB genome (known as MIRU loci), using genomic DNA from cultured MTB isolates. We will target 12 MIRU's in order to genotype MTB isolates is this study. PCR amplification of MIRU loci will be performed in a volume of 25 mL containing: 2.5 mL GeneAmp 10x PCR Buffer II (Perkin-Elmer Cetus), 2 mM MgCl2, 100nM each primter, 200 mM each of 4 dNTPs and 0.625 U AmpliTaq Gold DNA Polymerase (Perkin-Elmer Cetus). An initial denaturation will be followed by 35 cycles of detauration / annealing / extension. Negative and positive controls will be included with each PCR round. The presence and size of each PCR product will be determined by electrophoresis on an agarose gel followed by staining with ethidium bromide.

Instructions for specimen collection, storage & transport (SPUTUM)
A. On day specimen is collected:

Location of testing
In all instances, tests which are readily able to be performed locally will be processed in Timika. Where assays are unable to be performed in the local laboratory, samples will require storage for subsequent batched transport to MSHR and thence in some instances, to commercial laboratories for standard testing. Tests to be performed in Timika include: Calcium, Haemoglobin, HIV serology, and Proteome Systems' rapid diagnostic (RD) test on blood, urine and sputum. Tests which will be performed in Australia include vitamin D and parathyroid hormone, amino acids (arginine, citrulline, ornithine), and white cell studies. Standard operating protocols are as detailed below.

HIV antibody (as part of routine clinical protocols)
Following appropriate counselling as per normal clinical procedures in Timika, same-day testing for HIV using a rapid test (Determine TM , Abbott, Jakarta) and a confirmatory ELISA at the RSMM Laboratory, and any other tests deemed to be necessary by the treating doctor (not for the study), e.g. liver function, full blood count.

Calcium, Haemoglobin
Portable i-STAT cartridges already in routine use at the hospital or clinic will be used for immediate processing for ionised calcium (iCa 2+ ) and haemoglobin.

Proteome Systems' rapid diagnostic (RD) test (see also Point 3.3)
Plsama will be reserved for antigen testing using Proteome Systems' rapid diagnostic (RD) test TM at RSMM laboratory. The urine specimen will also be stored for this purpose. Protease will be added to the specimens prior to storage.

Blood tests in Australia (SEE ALSO APPENDIX B)
Heparinised blood samples will be separated within 3 hours of collection into plasma and peripheral blood mononuclear cells (PBMCs) [using Ficoll Hypaque as is routine in the Timika lab]), stored at -70 (plasma) or in liquid nitrogen (PBMCs in foetal calf serum/DMSO) in our Timika research laboratory, then shipped by dry shipper at -70°C to MSHR within one month of collection.

Vitamin D assay
25(OH)D 3 and 1,25(OH) 2 D 3 levels will be measured using DiaSorin LIASION® 25OH Vitamin D assay at a NATA-accredited commercial laboratory. Plasma 1,25(OH) 2 D 3 and parathormone will also be measured in order to evaluate the possibility of increases in systemic vitamin D metabolites that may relate to efficacy and any adverse effects.

Arginine assay
Plasma amino acids (including metabolites) will be measured by HPLC.

White cell studies
PBMC phenotype and CD3 zeta expression will be determined by FACS, measuring the amount of bound intracellular anti-CD3ζ PE fluorescent antibody (Immunotech, France), as is routine in our lab. The shift in expression of CD3ζ between activated and naïve CD4 and CD8 T cells will be compared among patients in different study arms. 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). Interferon-production in response to stimulus with mitogens, bacterial LPS and PPD will be determined using ex-vivo ELISpot assays. All assays are routinely run in the MSHR lab.

Proteome Systems' rapid diagnostic (RD) test
The Proteome Systems' rapid diagnostic (RD) test TM is in late stages of development and should be available for field testing by 2008. As noted above, this test will be performed on sputum and blood. Further samples which have been routinely collected as part of standard clinical care (e.g. ascitic fluid in extrapulmonary TB) which may be available for evaluation with the Proteome Systems' rapid diagnostic (RD) test TM , can also be tested. In the event of field evaluation of this diagnostic test commencing, it will be performed by laboratory scientist Ms. Kristina Retnoningtyas Palupi at the RSMM laboratory.

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How would you describe your chest condition?
The most important problem I have 10% of the overhead costs.

Effectiveness :
The effectiveness will be measured through the following indicators : Cure rate, Time of sputum conversion rate Clinical improvement

Cost-Effectiveness Analysis ( CEA)
Cost-effectiveness will calculated by dividing the total cost per activity by each different effectiveness measure using the formula: The cost per patient cure between intervention and control group will be compared and will make an inference about the cost-effectiveness of L-arginine or vitamin D intervention. Drg. Franciscus Thio is a Papuan dental graduate with many years experience in health administration at the district level and, more recently, as a research administrator and leader at our field site in Timika. He will be studying for his PhD as part of the capacity building aspects of this project. Dr Dina Bisara Lolong is a medical graduate with a masters in demography who has previously worked in Papua at Puskesmas, District and provincial administrative levels. In recent years she has been a principle investigator on a range of TB and HIV related research projects.
Dr Enny Kenangalem, Papuan medical doctor and experienced clinician, or her replacement, will provide research leadership at our field site in Timika. Ms. Kristina Retnoningtyas Palupi is a Laboratory Scientist who will continue to develop her research skills as part of the capacity building aspects of this project. Dr Lamria and Ibu Luxi are NIHRD researchers who will participate in the research. Dr Emiliana Tjitra Senior Research Clinician will provide a supervisory and advisory role in the study. Dr Andri Wiguna and Dr Enny Malonda (or her replacement) are the clinicians working at the hospital and the clinic where the patients will be enrolled and followed up. Bapak Govert Waramori is a Papuan nurse who has worked with the research team for the past 4 years and has developed many technical skills. He has a deep understanding of the equipment and procedures in the field site and will be working towards his MPH as part of the capacity-building aspects of this project. Dr Anna Ralph is an experienced infectious diseases physician and NHMRC postgraduate scholar who will be based in Timika and undertake these studies for her PhD.
Dr Ric Price has extensive research experience with the NIHRD-MSHR collaboration and will provide database and analytical support. Dr Graeme Maguire has completed studies of lung function in Timika (TB) and other remote settings, including RCTs to improve lung function. He will ensure training and QA in lung function testing. Dr Peter Morris has coordinated many successful RCTs, in northern Australia, Indonesia and East Timor in conjunction with Nick Anstey and Paul Kelly. Dr Ivan Bastian and Mr Richard Lumb have worked with microbiology partners in Indonesia and his laboratory is the WHO Supranational TB reference laboratory. Dr Tonia Woodberry is a MSHR laboratory scientist who provides expertise in PBMC work in partnership with NIHRD laboratory partners. Dr Indri Rooslamiati, Prof Stephen Duffull and Dr Tsin Yeo will provide expertise and advice for pharmacokinetic / pharmacodynamic studies as required. Dr Pasi Penttinen and Dr Firdy Permana are important local public health collaborators. Prof John Eisman is a world authority on Vitamin D replacement and monitoring; Dr Cheryl Salome on QA/QC of exhaled NO measurements; Prof Niels Becker, is an expert statistician in infectious diseases modelling; Dr Carmelia Basri is the head of the Indonesian National TB Control Program and will ensure that study results are transformed to policy where appropriate.

Rationale for location of specimen processing
The new focus by NIHRD on TB culture and molecular typing in 2008 will require external quality control and possible requirements for training in-country or abroad. Specimens collected for the ADAPT study will be ideally suited for use in a QA program for the developing NIHRD-University of Indonesia collaborative TB Laboratory. Options for the provision of training by ADAPT study investigators in sputum culture and typing in Jakarta or Australia are under investigation.
In all instances, tests which are readily able to be performed locally will be processed in Timika.
Where assays are unable to be performed in the local laboratory, samples will require storage for subsequent batched transport to MSHR and thence in some instances, to commercial laboratories for standard testing. Tests to be performed in Timika include: sputum smear microscopy, Calcium, Haemoglobin, HIV serology, and Proteome TM Systems' rapid diagnostic (RD) test on blood, urine and sputum (when available). Tests which will be performed in Australia include sputum culture, sensitivity testing and genotyping, vitamin D and parathyroid hormone, amino acids (arginine, citrulline, ornithine), and white cell studies. Standard operating protocols are as detailed below.

Sputum Culture
There is no capacity for TB culture in Papua. We therefore propose doing sputum culture at the WHO reference laboratory, Adelaide, Australia, for the following reasons: 1.
The laboratory in Adelaide is recognised by WHO as one of 7 global Supranational reference laboratories for TB testing. It provides this service for Australia, New Zealand, the Pacific and South-East Asia. It is within the mandate and capacity of this laboratory to provide a service to Indonesia. The WHO Supranational Reference Centre (IMVS, Adelaide) continues to collaborate with the Indonesian National TB Control Program with a range of training, guideline development and quality assurance activities.

2.
It is our understanding that the nationally accredited TB culture laboratories in Indonesia (in Surabaya and Jakarta) are operating at maximum capacity and are fully committed to work related to the ongoing National TB drug resistance survey.

3.
While these Indonesian laboratories therefore cannot take on the added burden of processing all sputum specimens from the ADAPT study, a regular random sample of divided specimens can be dispatched to the developing NIHRD-University of Indonesia collaborative TB Laboratory, when it becomes operational, for QA purposes.

4.
To maintain the strong reputation of NIHRD-MSHR Research collaboration in international research and permit ongoing publication of outcomes in leading international journals, ethical and scientific integrity principles mandating participation in a quality assurance program need to be upheld. This is standard international practice in the conduct of highquality TB research.

5.
Our previous experience of transferring samples from Timika to the WHO reference Laboratory has been successful.

6.
Processing of samples at the WHO reference Laboratory will provide excellent training opportunities for NIHRD laboratory scientist (Ibu Palupi) to work on Indonesian samples in a supportive environment with world class training.
Vitamin D 1. A particular problem recognised among vitamin D assays is that of variance in results, even at the most reputable laboratories, due to unavoidable fluctuations in reagents between batches and over time. A variety of vitamin D testing methods exist, and this further increases the problem of inter-laboratory variance. (92,93) It is recognised amongst vitamin D researchers that the variability accepted in clinical practice is too excessive according the stringent requirements of research where accurate determination within 10 nmol/L is critical. Use of standard (non-reference) laboratories for vitamin D research requires QA testing on a high proportion if not all samples, significantly elevating costs; even then, results can be difficult to interpret due to inter-test variability.
2. The newer liquid chromatography tandem mass spectrometry (LC-MS) method for vitamin D determination is increasingly recognised as the gold standard for vitamin D measurement. Laboratories using this method have demonstrated superior consistency of results compared with RIA or chemiluminescent assays. A laboratory offering this test has now become available in Australia (Division of Laboratory Medicine, Royal Melbourne Institute of Technology University, Victoria), and is in the process of publishing results showing superior results (lower coefficient of variance) compared with other standard vitamin D assay methodologies. This information will be forwarded to the NIHRD Ethics Committee as soon as it is available. Thus it would be in the interests of best quality research to utilise this laboratory for the measurement of vitamin D levels. The volume of blood required for LC-MS vitamin D testing is lower than that required by standard methods (100-300 microL).
3. Development of the LC-MS method is described by Maunsell Z et al. (94) In their discussion, they state: "Recent publications have highlighted the interlaboratory variability of 25-OH D analysis on patient samples measured by RIA and chemiluminescence assays (24 ) and quality assurance material (22 ). In view of this, some authors have suggested that there should be international standardization of assays and have suggested that until that is achieved, RIA techniques should be used for clinical analyses (24 ). However, the use of a routine LC-MS/MS method offers a real alternative and negates the use of radioactive tracers." 4. The LC-MS method has been chosen as the vitamin D assay method for the UK trial of vitamin D in tuberculosis which is currently recruiting patients. The same researchers have already published results of studies of vitamin D in latent TB using the same method (see Martineau AR et al, 2007). For our study to be optimally comparable to this research, the same vitamin D assay methodology should be used.

DRAFT MATERIALS TRANSFER AGREEMENT: MSHR (Final versions in PDF format included in Regulatory Binder)
The National Institute of Health Research and Development (NIHRD), hereby referred to as the First Party, agrees to provide (name, institution), hereby referred to as the Second Party, with the biological specimens and / or data (Material) described in Appendix A for use in the study and for specific assays (Research Plan) described in Appendix B, under the direct oversight and responsibility of (Scientist). Note that both Appendix A and Appendix B are hereby considered as integral parts of this document and that the terms described herein apply to both the Institution and Scientist irrespective of their association with each other. The First Party and The Second Party agree to the following conditions: 1. The Material shall be used exclusively by the Second Party and be limited to the Scientist, and others as expressly stated in Appendix B, for executing the Research Plan, and for no other purpose, unless this agreement is amended in writing by expressed written consent of both parties.
2. The Second Party will assure that the Material is not distributed, released, or disclosed by any means, either intentional or accidental, to any person or entity other than those listed in the Research Plan. It is understood that all persons listed in the Research Plan are under the direct responsibility of the Second Party as well as the Scientist and the actions of such persons, and consequences thereof, with respect to the Material and Research Plan are thereby considered the full responsibility of the Second Party and the Scientist.

In accordance with the signed Agreement on Research Collaboration Between The Menzies
School of Health Research and NIHRD (dated 12 June 2003), intellectual property rights arising from this Agreement shall be jointly owned by the First and Second Party. Both parties shall be allowed to use such property for non-commercial purposes free of royalty. Both parties are entitled to the royalties on the principle of equitable contribution if they are used for commercial purposes.

In accordance with the signed Agreement on Research Collaboration Between The Menzies
School of Health Research and NIHRD (dated 12 June 2003), both parties have the same rights and access to specimens and data collected from approved studies. Upon a reasonable request made by the First Party, taking into consideration the logistics of transporting samples and whether time has elapsed since the completion of the Research Plan, the Second Party will return any and all unused Material to the first Party within thirty (30) days of written request. Costs of specimen transport will be negotiated between the First and Second Parties.
5. The second Party will transfer, in confidence, to the First Party any and all raw data, records, and results derived from the Material and Research Plan, including detailed records of direct use of Material. In addition, such information will be provided, where possible, within thirty (30) days of written request by the First Party.
6. In accordance with both parties' institutional agreements with the Wellcome Trust (signed by both NIHRD and MSHR in 2003), both parties will promote the publication of data arising from research funded by The Wellcome Trust, where possible in open-access journals. The use of any data, results, or concepts, hereby termed outputs, derived from use of the Material in presentations, abstracts, publications (both peer-reviewed and not peerreviewed), grants, or other means of dissemination by the Second Party or Scientists will be determined by mutual agreement between the Parties, in accordance with the signed 7. It is fully acknowledged by the Second Party that the Material is experimental in nature and it is provided without warranty of fitness for the purposes described in the Research Plan. Moreover, the First Party makes no claims or warranty that the use of the material will not infringe on any unforeseen patents or proprietary rights and accepts no responsibility for such infringements. In no event shall the First Party be held liable in any way for any use, expenses, loss, claim, damage or liability of any kind which may arise from or in connection with this Agreement or the use, handling or storage of the Material.