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Background: Tuberculosis, a major cause of death in people living with HIV, remains challenging to diagnose. Diagnostic accuracy data are scarce for promising triage and confirmatory tests such as C-reactive protein (CRP), sputum and urine Xpert MTB/RIF Ultra (Xpert Ultra), and urine Determine TB LAM Ag (a lateral flow lipoarabinomannan [LF-LAM] test), without symptom selection. We evaluated novel triage and confirmatory tests in ambulatory people with HIV initiating antiretroviral therapy (ART)., Methods: 897 ART-initiators were recruited irrespective of symptoms and sputum induction offered. For triage (n=800), we evaluated point-of-care blood-based CRP testing, compared with the WHO-recommended four-symptom screen (W4SS). For sputum-based confirmatory testing (n=787), we evaluated Xpert Ultra versus Xpert MTB/RIF (Xpert). For urine-based confirmatory testing (n=732), we evaluated Xpert Ultra and LF-LAM. We used a sputum culture reference standard., Findings: 463 (52%) of 897 participants were female. The areas under the receiver operator characteristic curves for CRP was 0·78 (95% CI 0·73-0·83) and for number of W4SS symptoms was 0·70 (0·64-0·75). CRP (≥10 mg/L) had similar sensitivity to W4SS (77% [95% CI 68-85; 80/104] vs 77% [68-85; 80/104]; p>0·99] but higher specificity (64% [61-68; 445/696] vs 48% [45-52; 334/696]; p<0·0001]; reducing unnecessary confirmatory testing by 138 (95% CI 117-160) per 1000 people and number-needed-to-test from 6·91 (95% CI 6·25-7·81) to 4·87 (4·41-5·51). Sputum samples with Xpert Ultra, which required induction in 49 (31%) of 158 of people (95% CI 24-39), had higher sensitivity than Xpert (71% [95% CI 61-80; 74/104] vs 56% [46-66; 58/104]; p<0·0001). Of the people with one or more confirmatory sputum or urine test results that were positive, the proportion detected by Xpert Ultra increased from 45% (26-64) to 66% (46-82) with induction. Programmatically done haemoglobin, triage test combinations, and urine tests showed comparatively worse results., Interpretation: CRP is a more specific triage test than W4SS in those initiating ART. Sputum induction improves diagnostic yield. Sputum samples with Xpert Ultra is a more accurate confirmatory test than with Xpert., Funding: South African Medical Research Council, EDCTP2, US National Institutes of Health-National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of interests GT received in-kind donations from Cepheid and Boditech. BWPR received travel support from Cepheid to attend a conference and present unrelated data. RMW declares a salary paid by the South African Medical Research Council. MN declares a research grant funding by the Wellcome Trust and research funding by the UK National Institute for Health and Care Research Biomedical Research Centre at the University College London Hospitals NHS Foundation Trust. CCN declares grants or contracts from the US National Institutes of Health (K43TW012303) and the European and Developing Countries Clinical Trials Partnership (TMA2017CDF-1914) for career development fellowship. The authors have no financial involvement with any organisation or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary tuberculosis (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on noninvasive specimens, are limited. Fine-needle aspiration biopsy specimens (FNABs) from outpatients (≥18 years) with presumptive TBL ( n  = 135) underwent (i) routine Xpert MTB/RIF testing (later with Ultra once programmatically available), (ii) MGIT 960 culture (if Xpert or Ultra negative or rifampicin resistant), and (iii) study Ultra testing. Concentrated paired urine specimens underwent Ultra testing. Primary analyses used a microbiological reference standard (MRS). In a head-to-head comparison ( n  = 92) of an FNAB study Ultra and Xpert, Ultra had increased sensitivity (91% [95% confidence interval: 79, 98] versus 72% [57, 84]; P  = 0.016) and decreased specificity (76% [61, 87] versus 93% [82, 99]; P  = 0.020) and diagnosed patients not on treatment. Neither HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultra tests, the latter detected more cases (+20% [0, 42]; P  = 0.034), and false-negative study Ultra results were more inhibited than true-positive results. Study Ultra false positives had less mycobacterial DNA than true positives (trace-positive proportions, 59% [13/22] versus 12% [5/51]; P  < 0.001). "Trace" exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra tests had low sensitivity (18% [7, 35]). Ultra testing on FNABs is highly sensitive and detects more TBL than Xpert (Ultra still missed some cases due in part to inhibition). Patients with FNAB Ultra-positive "trace" results, most of whom will be culture negative, may require additional clinical investigation. Urine Ultra testing could reduce the number of patients needing invasive sampling.

Background: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood., Methods: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB., Findings: Cases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways., Interpretation: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB., Funding: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of Competing Interest GT received in-kind donations (GeneXpert cartridges and machines) from Cepheid and FIND. Cepheid had no role in study design or interpretation of results. BWPR received travel support from Cepheid to attend a conference and present unrelated data. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guérin (BCG) -the most widely used vaccine in history-has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts., (Copyright © 2020 Zatarain-Barrón, Ramos-Espinosa, Marquina-Castillo, Barrios-Payán, Cornejo-Granados, Maya-Lucas, López-Leal, Molina-Romero, Anthony, Ochoa-Leyva, De La Rosa-Velázquez, Rebollar-Vega, Warren, Mata-Espinosa, Hernández-Pando and van Soolingen.)

Background: Xpert MTB/RIF Ultra (Ultra) is a new test for tuberculosis undergoing global roll-out. We assessed the performance of Ultra compared with Xpert MTB/RIF (Xpert) in an HIV-endemic setting where previous tuberculosis is frequent and current test performance is suboptimal., Methods: In this two-cohort diagnostic accuracy study, we used sputum samples from patients in South Africa to evaluate the accuracy of Ultra and Xpert against a single culture reference standard. For the first cohort (cohort A), we recruited adults (aged ≥18 years) with symptoms of presumptive tuberculosis at Scottsdene clinic in Cape Town, South Africa. We collected three sputum samples from each patient in cohort A, two at the first visit of which one was tested using Xpert and the other was tested using culture, and one sample the next morning which was tested using Ultra. In a separate cohort of patients with presumptive tuberculosis and recent previous tuberculosis (≤2 years) who had submitted sputum samples to the National Health Laboratory Services (cohort B), decontaminated sediments were, after processing, randomly allocated (1:1) for testing with Ultra or Xpert. For both cohorts we calculated the sensitivity and specificity of Ultra and Xpert and evaluated the effects of different methods of interpreting Ultra trace results., Findings: Between Feb 6, 2016, and Feb 2, 2018, we recruited 302 people into cohort A, all of whom provided sputum samples and 239 were included in the head-to-head analyses of Ultra and Xpert. For cohort B, we collected sputum samples from eligible patients who had submitted samples between Dec 6, 2016, and Dec 21, 2017, to give a cohort of 831 samples, of which 352 were eligible for inclusion in analyses and randomly assigned to Ultra (n=173) or Xpert (n=179). In cohort A, Ultra gave more non-actionable results (not positive or negative) than did Xpert (28 [10%] 275 vs 14 [5%] 301; p=0·011). In the head-to-head analysis, in smear-negative patients, sensitivity of Ultra was 80% (95% CI 64-90) and of Xpert was 73% (57-85; p=0·45). Overall, specificity of Ultra was lower than that of Xpert (90% [84-94] vs 99% [95-100]; p=0·001). In cohort B, overall sensitivity was 92% (81-98) for Xpert versus 86% (73-95; p=0·36) for Ultra and overall specificity was 69% (60-77) for Ultra versus 84% (78-91; p=0·005) for Xpert. Ultra specificity estimates improved after reclassification of results with the lowest Ultra-positive semiquantitation category (trace) to negative (15% [8-22]). In cohort A, the positive predictive value (PPV) for Ultra was 78% (67-87) and for Xpert was 96% (87-99; p=0·004); in cohort B, the PPV for Ultra was 50% (43-57) and for Xpert was 70% (61-78; p=0·014). Ultra PPV estimates in previously treated patients were low: at 15% tuberculosis prevalence, half of Ultra-positive patients with presumptive tuberculosis would be culture negative, increasing to approximately 70% in patients with recent previous tuberculosis. In cohort B, 21 (28%) of 76 samples that were Ultra positive were rifampicin indeterminate (all trace) and, like cohort A, most were culture negative (19 [90%] of 21)., Interpretation: In a setting with a high burden of previous tuberculosis, Ultra generated more non-actionable results and had diminished specificity compared with Xpert. In patients with recent previous tuberculosis, a quarter of Ultra-positive samples were indeterminate for rifampicin resistance and culture negative, suggesting that additional drug-resistance testing will probably be unsuccessful. Our data have implications for the handling of Ultra-positive results in patients with previous tuberculosis in high burden settings., Funding: South African Medical Research Council, the EDCTP2 program, and the Faculty of Medicine and Health Sciences, Stellenbosch University., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Lesotho guidelines for the management of drug-resistant tuberculosis (TB) recommend initiation of patients diagnosed with rifampicin resistant (RR)-TB on a standardized drug resistant regimen while awaiting confirmation of rifampicin resistant TB (RR-TB) and complete drug susceptibility test results. Review of diagnostic records between 2014 and 2016 identified 518 patients with RR-TB. Only 314 (60.6%) patients could be linked to treatment records at the Lesotho MDR hospital. The median delay in treatment initiation from the availability of Xpert MTB/RIF assay result was 12 days (IQR 7-19). Only 32% (101) of patients had a documented first-line drug resistant test. MDR-TB was detected in 56.4% of patients while 33.7% of patients had rifampicin mono-resistance. Only 7.4% of patients assessed for second-line resistance had a positive result (resistance to fluoroquinolone). Treatment success was 69.8%, death rate was 28.8%, loss to follow up was 1.0%, and 0.4% failed treatment. Death was associated with positive or unavailable sputum smear at the end of first month of treatment (Fisher exact p < 0.001) and older age (p = 0.007). Urgent attention needs to be given to link patients with RR-TB to care worldwide. The association of death rate with positive sputum smear at the end of the first month of treatment should trigger early individualization of treatment.

The diverse microbial communities within our bodies produce metabolites that modulate host immune responses. Even the microbiome at distal sites has an important function in respiratory health. However, the clinical importance of the microbiome in tuberculosis, the biggest infectious cause of death worldwide, is only starting to be understood. Here, we critically review research on the microbiome's association with pulmonary tuberculosis. The research indicates five main points: (1) susceptibility to infection and progression to active tuberculosis is altered by gut Helicobacter co-infection, (2) aerosol Mycobacterium tuberculosis infection changes the gut microbiota, (3) oral anaerobes in the lung make metabolites that decrease pulmonary immunity and predict progression, (4) the increased susceptibility to reinfection of patients who have previously been treated for tuberculosis is likely due to the depletion of T-cell epitopes on commensal gut non-tuberculosis mycobacteria, and (5) the prolonged antibiotic treatment required for cure of tuberculosis has long-term detrimental effects on the microbiome. We highlight knowledge gaps, considerations for addressing these knowledge gaps, and describe potential targets for modifying the microbiome to control tuberculosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Multidrug-resistant tuberculosis (MDR-TB) jeopardizes global TB control. The prevalence and predictors of Rifampicin-resistant (RR) TB, a proxy for MDR-TB, and the treatment outcomes with standard and shortened regimens have not been assessed in post-conflict regions, such as the South Kivu province in the eastern Democratic Republic of the Congo (DRC). We aimed to fill this knowledge gap and to inform the DRC National TB Program., Methods: of adults and children evaluated for pulmonary TB by sputum smear microscopy and Xpert MTB/RIF (Xpert) from February 2012 to June 2017. Multivariable logistic regression, Kaplan-Meier estimates, and multivariable Cox regression were used to assess independent predictors of RR-TB and treatment failure/death., Results: Of 1535 patients Xpert-positive for TB, 11% had RR-TB. Independent predictors of RR-TB were a positive sputum smear (adjusted odds ratio [aOR] 2.42, 95% confidence interval [CI] 1.63-3.59), retreatment of TB (aOR 4.92, 95% CI 2.31-10.45), and one or more prior TB episodes (aOR 1.77 per episode, 95% CI 1.01-3.10). Over 45% of RR-TB patients had no prior TB history or treatment. The median time from Xpert diagnosis to RR-TB treatment initiation was 12 days (interquartile range 3-60.2). Cures were achieved in 30/36 (83%) and 84/114 (74%) of patients on 9- vs 20/24-month MDR-TB regimens, respectively (P = .06). Predictors of treatment failure/death were the absence of directly observed therapy (DOT; adjusted hazard ratio [aHR] 2.77, 95% CI 1.2-6.66) and any serious adverse drug event (aHR 4.28, 95% CI 1.88-9.71)., Conclusions: Favorable RR-TB cure rates are achievable in this post-conflict setting with a high RR-TB prevalence. An expanded Xpert scale-up; the prompt initiation of shorter, safer, highly effective MDR-TB regimens; and treatment adherence support are critically needed to optimize outcomes., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Biomarkers for TB treatment response and outcome are needed. This study characterize changes in immune profiles during TB treatment, define biosignatures associated with treatment outcomes, and explore the feasibility of predictive models for relapse. Seventy-two markers were measured by multiplex cytokine array in serum samples from 78 cured, 12 relapsed and 15 failed treatment patients from South Africa before and during therapy for pulmonary TB. Promising biosignatures were evaluated in a second cohort from Uganda/Brazil consisting of 17 relapse and 23 cured patients. Thirty markers changed significantly with different response patterns during TB treatment in cured patients. The serum biosignature distinguished cured from relapse patients and a combination of two clinical (time to positivity in liquid culture and BMI) and four immunological parameters (TNF-β, sIL-6R, IL-12p40 and IP-10) at diagnosis predicted relapse with a 75% sensitivity (95%CI 0.38-1) and 85% specificity (95%CI 0.75-0.93). This biosignature was validated in an independent Uganda/Brazil cohort correctly classifying relapse patients with 83% (95%CI 0.58-1) sensitivity and 61% (95%CI 0.39-0.83) specificity. A characteristic biosignature with value as predictor of TB relapse was identified. The repeatability and robustness of these biomarkers require further validation in well-characterized cohorts., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rationale: There is poor understanding about protective immunity and the pathogenesis of cavitation in patients with tuberculosis. Objectives: To map pathophysiological pathways at anatomically distinct positions within the human tuberculosis cavity. Methods: Biopsies were obtained from eight predetermined locations within lung cavities of patients with multidrug-resistant tuberculosis undergoing therapeutic surgical resection ( n  = 14) and healthy lung tissue from control subjects without tuberculosis ( n  = 10). RNA sequencing, immunohistochemistry, and bacterial load determination were performed at each cavity position. Differentially expressed genes were normalized to control subjects without tuberculosis, and ontologically mapped to identify a spatially compartmentalized pathophysiological map of the cavity. In silico perturbation using a novel distance-dependent dynamical sink model was used to investigate interactions between immune networks and bacterial burden, and to integrate these identified pathways. Measurements and Main Results: The median (range) lung cavity volume on positron emission tomography/computed tomography scans was 50 cm 3 (15-389 cm 3 ). RNA sequence reads (31% splice variants) mapped to 19,049 annotated human genes. Multiple proinflammatory pathways were upregulated in the cavity wall, whereas a downregulation "sink" in the central caseum-fluid interface characterized 53% of pathways including neuroendocrine signaling, calcium signaling, triggering receptor expressed on myeloid cells-1, reactive oxygen and nitrogen species production, retinoic acid-mediated apoptosis, and RIG-I-like receptor signaling. The mathematical model demonstrated that neuroendocrine, protein kinase C-θ, and triggering receptor expressed on myeloid cells-1 pathways, and macrophage and neutrophil numbers, had the highest correlation with bacterial burden ( r  > 0.6), whereas T-helper effector systems did not. Conclusions: These data provide novel insights into host immunity to Mycobacterium tuberculosis -related cavitation. The pathways defined may serve as useful targets for the design of host-directed therapies, and transmission prevention interventions.

The World Health Organization (WHO) recently recommended that linezolid be prioritized in treatment regimens for drug-resistant tuberculosis (TB), but there are limited data on its pharmacokinetics (PK) in patients with this disease. We conducted an observational study to explore covariate effects on linezolid PK and to estimate the probability of PK/pharmacodynamic target attainment in South African patients with drug-resistant TB. Consecutive adults on linezolid-based regimens were recruited in Cape Town and underwent intensive PK sampling at steady state. Noncompartmental analysis was performed. Thirty participants were included: 15 HIV positive, 26 on the initial dose of 600 mg daily, and 4 participants on 300 mg daily after dose reduction for linezolid-related toxicity. There was a negative correlation between body weight and exposure, with 17.4% (95% confidence interval [CI], 0.1 to 31.7) decrease in area under the concentration-time curve from 0 to 24 h (AUC 0-24 ) per 10-kg weight increment after adjustment for other covariates. Age was an independent predictor of trough concentration, with an estimated 43.4% (95% CI, 5.9 to 94.2) increase per 10-year increment in age. The standard 600-mg dose achieved the efficacy target of free AUC/MIC of >119 at wild-type MIC values (≤0.5 mg/liter), but the probability of target attainment dropped to 61.5% (95% CI, 40.6 to 79.8) at the critical concentration of 1 mg/liter. When dosed at 600 mg daily, trough concentrations were above the toxicity threshold of 2 mg/liter in 57.7% (95% CI, 36.9 to 76.6). This confirms the narrow therapeutic index of linezolid, and alternative dosing strategies should be explored., (Copyright © 2019 American Society for Microbiology.)

Background: Heteroresistant Mycobacterium tuberculosis infections (defined as concomitant infection with drug-resistant and drug-susceptible strains) may explain the higher risk of poor tuberculosis treatment outcomes observed among patients with mixed-strain M. tuberculosis infections. We investigated the clinical effect of mixed-strain infections while controlling for pretreatment heteroresistance in a population-based sample of patients with tuberculosis starting first-line tuberculosis therapy in Botswana., Methods: We performed 24-locus mycobacterial interspersed repetitive unit-variable number tandem-repeat analysis and targeted deep sequencing on baseline primary cultured isolates to detect mixed infections and heteroresistance, respectively. Drug-sensitive, micro-heteroresistant, macro-heteroresistant, and fixed-resistant infections were defined as infections in which the frequency of resistance was <0.1%, 0.1%-4%, 5%-94%, and ≥95%, respectively, in resistance-conferring domains of the inhA promoter, the katG gene, and the rpoB gene., Results: Of the 260 patients with tuberculosis included in the study, 25 (9.6%) had mixed infections and 30 (11.5%) had poor treatment outcomes. Micro-heteroresistance, macro-heteroresistance, and fixed resistance were found among 11 (4.2%), 2 (0.8%), and 11 (4.2%), respectively, for isoniazid and 21 (8.1%), 0 (0%), and 10 (3.8%), respectively, for rifampicin. In multivariable analysis, mixed infections but not heteroresistant infections independently predicted poor treatment outcomes., Conclusions: Among patients starting first-line tuberculosis therapy in Botswana, mixed infections were associated with poor tuberculosis treatment outcomes, independent of heteroresistance.

Background: The first-line TB antibiotic isoniazid (INH) serves as a central component of combined first-line anti-tuberculosis drug therapy. However, resistance to INH has hindered the functioning of this drug. Resistance is caused by several known and unknown mutations in genes/regions in Mycobacterium tuberculosis (M. tuberculosis), followed by selection of these mutants in the presence of the drug. INH resistance can be categorised as either "high-level" (minimum inhibitory concentration (MIC) of > 1µg/mL to INH) or "low-level" (MIC between 0.1-1.0 µg/L) resistance and is dependent on the specific mutation acquired. The level of resistance is relevant, as INH resistance is often considered to be the first step in development of Multi-Drug Resistant (MDR) and extremely resistant (XDR) TB. Isoniazid is a pro-drug in which first pass metabolism happens via N-acetyltransferase and is fast, intermediate or slow, depending on the genetics of the host. Thus, low-level INH resistance, particularly in the presence of fast metabolism, could allow additional mutations, development of high-level resistance and progression to multi-drug resistance., Methods: A structured search of bibliographic databases for peer-reviewed research literature was performed. Set parameters and specific inclusion criteria were used to filter the literature, based on our specified review questions. The quality and relevance of included papers was deduced using standard tools. The relevant content of cited papers was described, and an inferential qualitative content analysis methodology was utilised to analyse the inferences and findings of included studies using a conceptual framework., Results: Seventy-eight papers were included in the review, of which a sub-set (36) of the papers describe how different genetic mutations result in low or high-level resistance to INH. These papers were also used to set up a diagram detailing how each mutation affects INH functionality in order to visualise the interactome of INH and M.tuberculosis A further twenty-eight out of the seventy-eight papers detail the methods for testing for INH resistance, current treatment regimens and factors that influence treatment outcome in order to better understand the role of INH within the current anti-tuberculosis treatment therapy and how its use can be optimised., Conclusion: The findings of this review suggest that low-level INH resistance, in the presence of fast-acetylation, is an underrated component of the global TB epidemic worldwide, and may be a significant problem in terms of treatment outcome and progression to antibiotic resistance. Thus, more research must be done to test whether personalised diagnostics and targeted high dose treatment with INH will reduce the incidence of isoniazid mono-resistant and multi-drug resistant (MDR) tuberculosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Toll-like receptors (TLRs) are involved in the recognition of conserved microbial structures, leading to activation of an inflammatory response and formation of an adaptive immune response., Methods: Twenty-three polymorphisms in five TLR genes were genotyped in 729 tuberculosis cases and 487 healthy controls in a population-based case-control association study in a South African population., Results: We detected sex-specific associations for TLR8 polymorphisms, with rs3761624 (OR=1.54, p<0.001), rs3764879 (OR=1.41, p=0.011) and rs3764880 (OR=1.42, p=0.011) associated in females and rs3764879 (OR=0.72, p=0.013) and rs3764880 (OR=0.75, p=0.036) associated in males. Epistatic interactions between the TLR genes were investigated and the TLR1&#95;rs4833095 polymorphism was shown to interact with TLR2&#95;rs3804100 and (GT)n microsatellite (p=0.002) and alter susceptibility to TB. We also studied the role of TLRs in disease caused by different Mycobacterium tuberculosis genotypes in 257 tuberculosis cases, and identified associations between specific TLR polymorphisms and disease caused by specific strains., Conclusion: This study provides further evidence that the TLRs play an important role in the outcome of tuberculosis disease, and suggests a partial explanation for the male bias in tuberculosis ratios., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: There is increasing evidence from tuberculosis high-burden settings that exogenous reinfection contributes considerably to recurrent disease. However, large longitudinal studies of endogenous reactivation (relapse) and reinfection tuberculosis are lacking. We hypothesize a relationship between relapse vs reinfection and the time between treatment completion and recurrent disease., Methods: Population-based retrospective cohort study on all smear-positive tuberculosis cases successfully treated between 1996 and 2008 in a suburban setting in Cape Town, South Africa. Inverse gaussian distributions were fitted to observed annual rates of relapse and reinfection, distinguished by DNA fingerprinting of Mycobacterium tuberculosis strains recultured from diagnostic samples., Results: Paired DNA fingerprint data were available for 130 (64%) of 203 recurrent smear-positive tuberculosis cases in the 13-year study period. Reinfection accounted for 66 (51%) of 130 recurrent cases overall, 9 (20%) of 44 recurrent cases within the first year, and 57 (66%) of 86 thereafter (P < .001). The relapse rate peaked at 3.93% (95% confidence interval [CI], 2.35%-5.96%) per annum 0.35 (95% CI, .15-.45) years after treatment completion. The reinfection tuberculosis rate peaked at 1.58% (95% CI, .94%-2.46%) per annum 1.20 (95% CI, .55-1.70) years after completion., Conclusions: To our knowledge, this is the first study of sufficient size and duration using DNA fingerprinting to investigate tuberculosis relapse and reinfection over a lengthy period. Relapse occurred early after treatment completion, whereas reinfection dominated after 1 year and accounted for at least half of recurrent disease. This temporal relationship may explain the high variability in reinfection observed across smaller studies. We speculate that follow-up time in antituberculosis drug trials should take reinfection into account., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection., Aim: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes., Method: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes., Results: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype., Conclusion: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.

The Beijing genotype family is an epidemiologically important sub-group of Mycobacterium tuberculosis. It has been suggested that the high frequency of the Beijing isolates in some areas could be explained by selective advantages. Some evidence suggests that the emerging and most frequently isolated "Typical Beijing" lineage has the ability to circumvent BCG-induced immunity. To investigate the phylogeny of the Beijing genotype of M. tuberculosis, the genome of six Beijing strains from three different countries was sequenced with next-generation sequencing. The phylogeny of these strains was established using single nucleotide polymorphisms (SNPs). The three Typical Beijing strains clustered very tightly in the Beijing phylogeny suggesting that Typical Beijing strains represent a monophyletic lineage and resulted from recent diversification. Typing of 150 M. tuberculosis strains with a subset of the SNPs and comparison of the IS6110 restriction-fragment length polymorphism (RFLP) patterns of these strains to a database of 1522 Beijing RFLP patterns revealed that about 80% of all Beijing strains belong to the Typical Beijing subclone, which indicates clonal expansion. To identify the genomic changes that are characteristic for all Typical Beijing strains and to reconstruct their most recent common ancestor, the presence of SNPs was assayed in other Beijing strains. We identified 51 SNPs that define the minimal set of polymorphisms for all Typical Beijing strains. Nonsynonymous polymorphisms in genes coding for the regulatory network were over-represented in this set of mutations. We suggest that alterations in the response to environmental signals may have enabled Typical Beijing strains to develop the emerging phenotype., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report a case of extensive necrogranulomatous pneumonia due to infection with the dassie bacillus (Mycobacterium tuberculosis complex sp.) in a free-living pregnant adult female dassie (rock hyrax-Procavia capensis). A juvenile female dassie from the same colony also showed a focal lesion in the lungs suggestive of mycobacterial pneumonia. Our findings indicate the widespread occurrence of the dassie bacillus in free-living dassies and suggest very high infection rates in some populations. The introduction of South African dassies into novel environments should be considered in this light.

Background: In many communities where TB occurs at high incidence, the major force driving the epidemic is transmission. It is plausible that the typical long delay from the onset of infectious disease to diagnosis and commencement of treatment is almost certainly the major factor contributing to the high rate of transmission., Methodology/principal Findings: This study is confined to communities which are epidemiologically relatively isolated and which have low HIV incidence. The consequences of delays to diagnosis are analyzed and the existence of a threshold delay value is demonstrated. It is shown that unless a sufficient number of cases are detected before this threshold, the epidemic will escalate. The method used for the analysis avoids the standard computer integration of systems of differential equations since the intention is to present a line of reasoning that reveals the essential dynamics of an epidemic in an intuitively clear way that is nevertheless quantitatively realistic., Conclusions/significance: The analysis presented here shows that typical delays to diagnosis present a major obstacle to the control of a TB epidemic. Control can be achieved by optimizing the rapid identification of TB cases together with measures to increase the threshold value. A calculated and aggressive program is therefore necessary in order to bring about a reduction in the prevalence of TB in a community by decreasing the time to diagnosis in all its ramifications. Intervention strategies to increase the threshold value relative to the time to diagnosis and which thereby decrease disease incidence are discussed.

Rationale: Multiple infections with different strains of Mycobacterium tuberculosis may occur in settings where the infection pressure is high. The relevance of mixed infections for the patient, clinician, and control program remains unclear., Objectives: This study aimed to describe reinfection and mixed infection as underlying mechanisms of changing drug-susceptibility patterns in serial sputum cultures., Methods: Serial M. tuberculosis sputum cultures from patients diagnosed with multi-drug-resistant (MDR) tuberculosis were evaluated by phenotypic drug-susceptibility testing and mutation detection methods. Genotypic analysis was done by IS6110 DNA fingerprinting and a novel strain-specific polymerase chain reaction amplification method., Measurements and Main Results: DNA fingerprinting analysis of serial sputum cultures from 48 patients with MDR tuberculosis attributed 10 cases to reinfection and 1 case to mixed infection. In contrast, strain-specific polymerase chain reaction amplification analysis in 9 of the 11 cases demonstrated mixed infection in 5 cases, reinfection in 3 cases, and laboratory contamination in 1 case. Analysis of clinical data suggests that first-line therapy can select for a resistant subpopulation, whereas poor adherence or second-line therapy resulted in the reemergence of the drug-susceptible subpopulations., Conclusions: We have shown that, in some patients with MDR tuberculosis, mixed infection may be responsible for observations attributed to reinfection by DNA fingerprinting. We conclude that treatment and adherence determines which strain is dominant. We hypothesize that treatment with second-line drugs may lead to reemergence of the drug-susceptible strain in patients with mixed infection.

Rationale: In a high-tuberculosis (TB) incidence area of Cape Town, South Africa, there is a very high rate of unexplained recurrent TB. The incidence of new bacteriologically confirmed disease in the area is 313 per 100,000 individuals., Objective: To estimate the rate of recurrent TB attributable to reinfection after successful treatment., Methods: All patients with reported TB in the area between 1993 and 1998 were followed up to 2001 for disease needing retreatment (recurrences). Patients who were multi-drug-resistant or who had treatment failure, were transferred, or died during treatment were excluded. Analysis was restricted to patients for whom DNA fingerprinting of their Mycobacterium tuberculosis isolates was obtained. Reinfection TB was defined as a recurrent TB episode in which the strains of the separate episodes differed by more than four bands., Measurements and Main Results: 612 of 897 (68%) patients had a DNA fingerprint available at enrollment. Median duration of follow-up was 5.2 years. Recurrent TB occurred in 108 of 612 (18%) patients, of whom 61 of 447 (14%) experienced recurrence after successful treatment, and 47 of 165 (28%) experience recurrence after default. Of the 108 patients with recurrent TB, 68 (63%) had a DNA fingerprint in the second episode. Among these patients, 24 of 31 (77%) recurrences after successful treatment and 4 of 37 (11%) recurrences after default were attributable to reinfection. The reinfection disease rate after successful treatment was estimated at 2.2 per 100 person-years., Conclusions: The age-adjusted incidence rate of TB attributable to reinfection after successful treatment was four times that of new TB. People who had TB once are at a strongly increased risk of developing TB when reinfected.

Guidelines for the diagnosis and management of Bacillus Calmette-Guérin (BCG) disease in children are lacking, and there are limited data on drug resistance of Mycobacterium bovis BCG. A 6-month-old HIV-infected infant presented with right axillary adenitis ipsilateral to the site of BCG immunization. M. tuberculosis complex was cultured from axillary lymph nodes and gastric aspirates, and M. bovis BCG was isolated. Susceptibility testing before initiation of therapy demonstrated inherent resistance to isoniazid. The organism acquired rifampin resistance during therapy. This was confirmed by the presence of a mutation in codon 531 (Ser531Tyr) of the rpoB gene. Treatment guidelines for BCG disease with consideration of inherent and possible acquired drug resistance should be established in settings with high rates of vertical HIV transmission and routine BCG vaccination.

It is generally accepted that tuberculosis results from a single infection with a single Mycobacterium tuberculosis strain. Such infections are thought to confer protective immunity against exogenous reinfection. In this study, a novel polymerase chain reaction method was developed to specifically identify M. tuberculosis strains belonging to the Beijing and non-Beijing evolutionary lineages in sputum specimens collected from tuberculosis patients resident in an epidemiologic field site in Cape Town, South Africa. The sensitivity and specificity of the polymerase chain reaction-based strain classification method were 100% (95% confidence interval, 85-100%) when compared with DNA fingerprinting and spacer oligotyping (spoligotyping). Application of this method showed that 19% of all patients were simultaneously infected with Beijing and non-Beijing strains, and 57% of patients infected with a Beijing strain were also infected with a non-Beijing strain. Multiple infections were more frequent in retreatment cases (23%) as compared with new cases (17%), but were not associated with sex, age, or smear grading. These results suggest that multiple infections are frequent, implying high reinfection rates and the absence of efficient protective immunity conferred by the initial infection. This finding could influence our understanding of the epidemiology of disease in high-incidence regions and our understanding for vaccine development.

In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.