A blood RNA signature for tuberculosis disease risk: a prospective cohort study.

BACKGROUND: Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease might lead to interventions that combat the tuberculosis epidemic. We aimed to assess whether global gene expression measured in whole blood of healthy people allowed identification of prospective signatures of risk of active tuberculosis disease. METHODS: In this prospective cohort study, we followed up healthy, South African adolescents aged 12-18 years from the adolescent cohort study (ACS) who were infected with M tuberculosis for 2 years. We collected blood samples from study participants every 6 months and monitored the adolescents for progression to tuberculosis disease. A prospective signature of risk was derived from whole blood RNA sequencing data by comparing participants who developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex quantitative real-time PCR (qRT-PCR), the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. Participants of the independent cohorts were household contacts of adults with active pulmonary tuberculosis disease. FINDINGS: Between July 6, 2005, and April 23, 2007, we enrolled 6363 participants from the ACS study and 4466 from independent South African and Gambian cohorts. 46 progressors and 107 matched controls were identified in the ACS cohort. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% CI 63·2-68·9) and a specificity of 80·6% (79·2-82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA sequencing and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6-64·3) and a specificity of 82·8% (76·7-86) in the 12 months preceding tuberculosis. INTERPRETATION: The whole blood tuberculosis risk signature prospectively identified people at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. FUNDING: Bill & Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union, and the South African Medical Research Council.

Serum indoleamine 2,3-dioxygenase activity is associated with reduced immunogenicity following vaccination with MVA85A.

BACKGROUND: There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function. METHODS: Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry. RESULTS: We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response. CONCLUSIONS: Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies. TRIAL REGISTRATION: Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558.

Prospects for a new, safer and more effective TB vaccine.

Tuberculosis in infants and young children remains an all too common cause of morbidity and mortality in high burden countries, despite the fact that the majority of these children receive vaccination with BCG in infancy. BCG confers incomplete and variable protection against pulmonary tuberculosis [PTB] and is unsafe in HIV positive persons. Newer TB vaccines, which, it is hoped, will either replace or complement BCG are being developed and a number of these have reached the stage of clinical trials, with two booster vaccines going into Phase IIB trials in 2009. Prospects for at least one new licensed TB vaccine within the next 5-10 years appear reasonable. This article explores some of the issues around the development of new vaccines against TB and details the leading candidates.

Structured approaches for the screening and diagnosis of childhood tuberculosis in a high prevalence region of South Africa.

OBJECTIVE: To measure agreement between nine structured approaches for diagnosing childhood tuberculosis; to quantify differences in the number of tuberculosis cases diagnosed with the different approaches, and to determine the distribution of cases in different categories of diagnostic certainty. METHODS: We investigated 1445 children aged < 2 years during a vaccine trial (2001-2006) in a rural South African community. Clinical, radiological and microbiological data were collected prospectively. Tuberculosis case status was determined using each of the nine diagnostic approaches. We calculated differences in case frequency and categorical agreement for binary (tuberculosis/not tuberculosis) outcomes using McNemar's test (with 95% confidence intervals, CIs) and Cohen's kappa coefficient (Kappa). FINDINGS: Tuberculosis case frequency ranged from 6.9% to 89.2% (median: 41.7). Significant differences in case frequency (P < 0.05) occurred in 34 of the 36 pair-wise comparisons between structured diagnostic approaches (range of absolute differences: 1.5-82.3%). Kappa ranged from 0.02 to 0.71 (median: 0.18). The two systems that yielded the highest case frequencies (89.2% and 70.0%) showed fair agreement (Kappa: 0.33); the two that yielded the lowest case frequencies (6.9% and 10.0%) showed slight agreement (Kappa: 0.18). CONCLUSION: There is only slight agreement between structured approaches for the screening and diagnosis of childhood tuberculosis and high variability between them in terms of case yield. Diagnostic systems that yield similarly low case frequencies may be identifying different subpopulations of children. The study findings do not support the routine clinical use of structured approaches for the definitive diagnosis of childhood tuberculosis, although high-yielding systems may be useful screening tools.

Evaluation of the quality of informed consent in a vaccine field trial in a developing country setting.

BACKGROUND: Informed consent is an ethical and legal requirement for research involving human participants. However, few studies have evaluated the process, particularly in Africa. Participants in a case control study designed to identify correlates of immune protection against tuberculosis (TB) in South Africa. This study was in turn nested in a large TB vaccine efficacy trial. The aim of the study was to evaluate the quality of consent in the case control study, and to identify factors that may influence the quality of consent. Cross-sectional study conducted over a 4 month period. METHODS: Consent was obtained from parents of trial participants. These parents were asked to complete a questionnaire that contained questions about the key elements of informed consent (voluntary participation, confidentiality, the main risks and benefits, etc.). The recall (success in selecting the correct answers) and understanding (correctness of interpretation of statements presented) were measured. RESULTS: The majority of the 192 subjects interviewed obtained scores greater than 75% for both the recall and understanding sections. The median score for recall was 66%; interquartile range (IQR) = 55%-77% and for understanding 75% (IQR = 50%-87%). Most (79%) were aware of the risks and 64% knew that they participated voluntarily. Participants who had completed Grade 7 at school and higher were more likely (OR = 4.94; 95% CI = 1.57 - 15.55) to obtain scores greater than 75% for recall than those who did not. Participants who were consented by professional nurses who had worked for more than two years in research were also more likely (OR = 2.62; 95% CI = 1.35-5.07) to obtain such scores for recall than those who were not. CONCLUSION: Notwithstanding the constraints in a developing country, in a population with low levels of literacy and education, the quality of informed consent found in this study could be considered as building blocks for establishing acceptable standards for public health research. Education level of respondents and experience of research staff taking the consent were associated with good quality informed consent.

The impact of a change in bacille Calmette-Guérin vaccine policy on tuberculosis incidence in children in Cape Town, South Africa.

BACKGROUND: A decision by the South African National Department of Health to change the route of administration and strain of bacille Calmette-Guérin (BCG) vaccine was implemented in Cape Town, South Africa, between July and December 2000. This provided an opportunity to compare the incidence of tuberculosis and proportion with disseminated disease in children less than 2 years old before and after the changeover from percutaneous (PC) Tokyo 172 BCG to intradermal (ID) 1331 Danish BCG immunization. METHODS: Clinical records of all tuberculosis patients aged less than 2 years at diagnosis and born between January 1, 1999, and June 30, 2000 (PC cohort) and between January 1, 2001, and June 30, 2002 (ID cohort) were collected. All cases were reviewed for likelihood of TB, its severity and disease dissemination. RESULTS: The number of reported patients with tuberculosis in the PC cohort was 1369 and in the ID cohort 1397, giving incidence rates of 866 (95% confidence interval [CI], 821-913) and 858 (95% CI, 814-904) per 100,000 person-years, respectively. The proportion who had disseminated disease (meningitis and/or miliary spread) was significantly lower in the ID cohort (4.7%) than in the PC cohort (8.6%) (relative risk, 0.54; 95% CI, 0.40-0.72). Those not vaccinated had a significantly higher proportion of disseminated disease cases (29.2%) than the PC and ID groups combined (6.6%) (relative risk, 4.4; 95% CI, 2.7-6.7). CONCLUSION: A program using Danish 1331 BCG given intradermally did not prevent more tuberculosis cases in children overall as compared with a program using Tokyo 172 BCG given percutaneously but reduced the proportion with disseminated disease.

Screening for TB in high school adolescents in a high burden setting in South Africa.

Screening for tuberculosis (TB) disease is important for TB control and TB vaccine efficacy trials but this has not been evaluated in adolescents. We conducted a study to determine the prevalence of active TB and performance of specific screening tests for TB in adolescents in a high burden setting. Adolescents aged 12-18 years were recruited from high schools in a rural town in South Africa. Participants were screened for active TB using symptoms, household TB contact, positive interferon gamma release assay (IGRA) and positive tuberculin skin test (TST). Of 6363 adolescents recruited, 21 were newly diagnosed with TB of whom 19 were culture positive. After exclusions, the derived prevalence of smear positive TB was 16/5682 = 3/1000 (95% confidence interval (CI) 1-4/1000). The sensitivity of TST and IGRA for active TB were 85% (95% CI 62-100%) and 94% (95% CI 79-100%) respectively. None of the methods alone or in combination had positive predictive values greater than 2%. The screening tools evaluated in this study may not be practical for routine use owing to low positive predictive values but may be useful in TB vaccine clinical trials.

TB incidence in an adolescent cohort in South Africa.

BACKGROUND: Tuberculosis (TB) is a major public health problem globally. Little is known about TB incidence in adolescents who are a proposed target group for new TB vaccines. We conducted a study to determine the TB incidence rates and risk factors for TB disease in a cohort of school-going adolescents in a high TB burden area in South Africa. METHODS: We recruited adolescents aged 12 to 18 years from high schools in Worcester, South Africa. Demographic and clinical information was collected, a tuberculin skin test (TST) performed and blood drawn for a QuantiFERON TB Gold assay at baseline. Screening for TB cases occurred at follow up visits and by surveillance of registers at public sector TB clinics over a period of up to 3.8 years after enrolment. RESULTS: A total of 6,363 adolescents were enrolled (58% of the school population targeted). During follow up, 67 cases of bacteriologically confirmed TB were detected giving an overall incidence rate of 0.45 per 100 person years (95% confidence interval 0.29-0.72). Black or mixed race, maternal education of primary school or less or unknown, a positive baseline QuantiFERON assay and a positive baseline TST were significant predictors of TB disease on adjusted analysis. CONCLUSION: The adolescent TB incidence found in a high burden setting will help TB vaccine developers plan clinical trials in this population. Latent TB infection and low socio-economic status were predictors of TB disease.

The tuberculin skin test versus QuantiFERON TB Gold® in predicting tuberculosis disease in an adolescent cohort study in South Africa.

SETTING: This study was conducted in a high tuberculosis (TB) burden area in Worcester, South Africa, with a notified all TB incidence rate of 1,400/100,000. MAIN OBJECTIVE: To compare the predictive value of a baseline tuberculin skin test (TST) with that of the QuantiFERON TB Gold (In-tube) assay (QFT) for subsequent microbiologically confirmed TB disease among adolescents. METHODS: Adolescents aged 12-18 years were recruited from high schools in the study area. At baseline, blood was drawn for QFT and a TST administered. Participants were followed up for up to 3.8 years for incident TB disease (median 2.4 years). RESULTS: After exclusions, 5244 (82.4%) of 6,363 adolescents enrolled, were analysed. The TB incidence rate was 0.60 cases per 100 person years (pyrs) (95% CI 0.43-0.82) for baseline TST positive (≥ 5 mm) participants and 0.64 cases per 100 pyrs (95% CI 0.45-0.87) for baseline QFT positive participants. TB incidence rates were 0.22 per 100 pyrs (0.11-0.39) and 0.22 per 100 pyrs (0.12-0.38) among those with a negative baseline TST and QFT respectively. Sensitivity for incident TB disease was 76.9% for TST and 75.0% for QFT (p = 0.81). Positive predictive value was 1.4% for TST and 1.5% for QFT. CONCLUSION: Positive TST and QFT tests were moderately sensitive predictors of progression to microbiologically confirmed TB disease. There was no significant difference in the predictive ability of these tests for TB disease amongst adolescents in this high burden setting. Therefore, these findings do not support use of QFT in preference to TST to predict the risk of TB disease in this study population.

HIV-specific gag responses in early infancy correlate with clinical outcome and inversely with viral load.

Many HIV-infected infants progress to AIDS during the first year of life when antiretroviral therapy (ART) is not given. The immune determinants of progression to AIDS are not known. We hypothesized that distinct HIV-specific T cell responses correlate with viral load and survival over the first year of life. Whole blood of infants at 3, 6, 9, and 12 months of age was incubated with HIV antigens Gag and Env. The frequency of specific T cells producing interferon (IFN)-γ was then measured by flow cytometry. Viral load and CD4% in HIV(+) infants were determined at each time point. ART was not available for this population at the time of sample collection. Those infants who survived to 12 months of age (n=12) had lower viral loads and higher Gag-specific CD8(+) T cell responses at 3 months, compared with infants who died (n=8). Furthermore, the frequency of Gag-specific CD4(+) T cells correlated inversely with viral load at 3 and 6 months of age. Together these data indicate that the early presence of quantitatively higher Gag-specific T cell responses in HIV-infected infants is associated with lower viral loads and decreased mortality in the first year of life. Our data support the design of a vaccine that preferentially elicits Gag responses, which may result in lower levels of viremia and possibly improve outcome.

Phenotypic variability in childhood TB: implications for diagnostic endpoints in tuberculosis vaccine trials.

The endpoint definition for infant tuberculosis (TB) vaccine trials should match the TB disease phenotype expected in the control arm of the study population. Our aim was to analyse selected combinations of the clinical, radiological, and microbiological features of pulmonary TB among children investigated under vaccine trial conditions, in order to estimate case frequency for a range of expected TB phenotypes. Two thousand one hundred and eighty five South African children were investigated over a nine-year period (2001-2009). Evidence of TB exposure and classical symptoms were several times more common than chest radiography (CXR) compatible with TB, or positive Mycobacterium tuberculosis culture. Discordance between clinical, radiological, and microbiological features was common in individual children. Up to one third of children with compatible CXR, and up to half the children who were M. tuberculosis culture positive, were asymptomatic. The culture positive rate fell over time, although rates of TB exposure and compatible chest radiography increased. Consequently, the annual incidence of diagnostic combinations that included M. tuberculosis culture fell to <0.2%. However, in this study population (children <2 years of age), annual incidence of the TB disease phenotype that included the triad of TB exposure, symptoms, and compatible CXR, approached 1% (n=848 per 100,000). These findings allow modelling of expected TB case frequency in multi-centre infant TB vaccine trials, based upon benchmarking of diagnostic data against the key indicator variables that constitute the building blocks of a trial endpoint.

New vaccines against tuberculosis.

The only currently licensed tuberculosis (TB) vaccine, bacille Calmette Guérin, confers incomplete protection against tuberculosis, and is not safe in infants infected with the human immunodeficiency virus. A new, safe vaccine regimen, which better protects against lung disease, is urgently needed to control TB in high-burden countries. Multiple candidate vaccines have shown promise in preclinical studies, and are now entering phase 1 to 2B clinical trials. This article discusses progress in the field and issues surrounding safety, reactogenicity, immunogenicity, and efficacy testing of new TB vaccines.

A New Vaccine for Tuberculosis: The Challenges of Development and Deployment.

Tuberculosis (TB) is one of the world's leading causes of death due to infection and efforts to control TB would be substantially aided by the availability of an improved TB vaccine. There are currently nine new TB vaccines in clinical development, and the first efficacy trials are due to commence in 2009. There are many complex ethical issues which arise at all stages of TB vaccine development, from the need to conduct trials in developing countries to informed consent and the process of ethical review. While it is important that these issues are discussed, it may also be timely to consider the challenges which may arise if a vaccine in clinical development proves to be highly effective. We examine a number of scenarios where decisions on the deployment of a new TB vaccine may impact on the rights and liberty of the individual.

Comparison of mantoux and tine tuberculin skin tests in BCG-vaccinated children investigated for tuberculosis.

BACKGROUND: Tuberculin skin tests (TSTs) are long-established screening methods for tuberculosis (TB). We aimed to compare agreement between the intradermal Mantoux and multipuncture percutaneous Tine methods and to quantify risk factors for a positive test result. METHODOLOGY/PRINCIPAL FINDINGS: 1512 South African children younger than 5 years of age who were investigated for tuberculosis (TB) during a Bacille Calmette Guerin (BCG) trial were included in this analysis. Children underwent both Mantoux and Tine tests. A positive test was defined as Mantoux >or=15 mm or Tine >or= Grade 3 for the binary comparison. Agreement was evaluated using kappa (binary) and weighted kappa (hierarchical). Multivariate regression models identified independent risk factors for TST positivity. The Mantoux test was positive in 430 children (28.4%) and the Tine test in 496 children (32.8%, p<0.0001), with observed binary agreement 87.3% (kappa 0.70) and hierarchical agreement 85.0% (weighted kappa 0.66). Among 173 children culture-positive for Mycobacterium tuberculosis, Mantoux was positive in 49.1% and Tine in 54.9%, p<0.0001 (kappa 0.70). Evidence of digit preference was noted for Mantoux readings at 5 mm threshold intervals. After adjustment for confounders, a positive culture, suggestive chest radiograph, and proximity of TB contact were risk factors for a positive test using both TST methods. There were no independent associations between ethnicity, gender, age, or over-crowding, and TST result. CONCLUSIONS/SIGNIFICANCE: The Tine test demonstrated a higher positive test rate than the Mantoux, with substantial agreement between TST methods among young BCG-vaccinated children. TB disease and exposure factors, but not demographic variables, were independent risk factors for a positive result using either test method. These findings suggest that the Tine might be a useful screening tool for childhood TB in resource-limited countries.

Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in healthy adults in South Africa.

BACKGROUND: The efficacy of bacille Calmette-Guérin (BCG) may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A (where "MVA" denotes "modified vaccinia virus Ankara"), has shown promising safety and immunogenicity in human trials performed in the United Kingdom. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed--but Mycobacterium tuberculosis-uninfected--healthy adults from a region of South Africa where TB is endemic. METHODS: Twenty-four adults were vaccinated with MVA85A. All subjects were monitored for 1 year for adverse events and for immunological assessment. RESULTS: MVA85A vaccination was well tolerated and induced potent T cell responses, as measured by interferon (IFN)-gamma enzyme-linked immunospot assay, which exceeded prevaccination responses up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A were comprised of multiple populations expressing combinations of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and IL-17, as measured by polychromatic flow cytometry. IFN-gamma-expressing and polyfunctional IFN-gamma+TNF-gamma+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response, which occurred 7 days after vaccination. CONCLUSION: The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials assessing the efficacy of MVA85A as a boosting vaccine in countries where TB is endemic. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00460590.

Childhood tuberculosis: old and new vaccines.

The world is witnessing an escalation of the tuberculosis (TB) epidemic, particularly in sub-Saharan Africa and South-East Asia. The problem has been compounded by the evolution of the human immunodeficiency virus pandemic, the increase in multidrug-resistant TB and the emergence of extensively drug-resistant TB. This has led to renewed interest in vaccines aimed at preventing tuberculosis. The current Bacille Calmette-Guérin (BCG) vaccine prevents the invasive complications of childhood tuberculosis, such as meningitis and miliary disease, but provides variable protection against adult pulmonary disease. This review discusses the history of the BCG vaccine, the reasons for its variable efficacy, protective immunity and TB, and the evolution of and obstacles to development of new candidate vaccines. Several new TB vaccines have demonstrated promising results in animal models; a number have gone into phase I clinical trials in humans, and it is anticipated that phase III trials will commence by 2009. Licensing of an effective new TB vaccine by 2015 is thus a possibility.

Determining causes of mortality in children enrolled in a vaccine field trial in a rural area in the Western Cape Province of South Africa.

AIM: A mortality surveillance system was developed to identify and document causes of death among children enrolled in a tuberculosis vaccine field trial in South Africa. The aims of this study were to describe causes of mortality in children enrolled in a phase IV trial comparing intradermal with percutaneous administration of Bacille Calmette Guerin, and to compare causes of mortality recorded on death certificates with those obtained by clinical record review combined with verbal autopsies (CR/VA). METHODS: For children who died, certified causes of death were compared with those determined by CR/VA. RESULTS: Among 11677 children enrolled, 177 deaths were notified over 4 years. The incidence rate of death was 6.8/1000 person-years. Follow-up time ranged from 0.03 to 35.3 months (median 4 months; interquartile range 1.4-8.5). The infant mortality rate was 12.5/1000 live births and the neonatal mortality was 3/1000 live births. Pneumonia, gastroenteritis and septicaemia were among top causes of mortality by both methods. 'Sudden unexplained' and 'ill-defined' causes were among top causes of mortality based on CR/VA, while tuberculosis and 'natural causes' were among top causes based on death certificates. Important underlying causes of mortality by CR/VA include HIV/AIDS, prematurity/low birth weight and malnutrition. In 47% of deaths there was agreement on immediate causes of death. This increased to 54% when 'natural causes' and 'sudden unexplained deaths' were included. CONCLUSION: In this cohort mortality was largely due to infectious diseases. While CR/VA provided additional information on most deaths, this was not always sufficient to assign specific causes of death.

Isolation of non-tuberculous mycobacteria in children investigated for pulmonary tuberculosis.

OBJECTIVE: To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community. METHODS: Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001-2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis. RESULTS: Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5-7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31-50), compared to 67% (95% CI 58-76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9-12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0-0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04). DISCUSSION: NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation.