Search

Background: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly., Objective: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates., Method: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2., Result: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages., Conclusion: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jobarteh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1-9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2-4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2-4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT., Importance: This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny., Competing Interests: C.U.K. is a consultant for Becton Dickinson, the Foundation for Innovative New Diagnostics, the TB Alliance, and the WHO Global TB Programme. C.U.K.'s consulting for Becton Dickinson involves collaboration with Janssen and Thermo Fisher Scientific. C.U.K. is collaborating with PZA Innovation and is an unpaid advisor to Cepheid and GenoScreen (GenoScreen covered related travel and accommodation expenses only). C.U.K. worked as a consultant for the Stop TB Partnership and the WHO Regional Office for Europe. C.U.K. gave a paid educational talk for Oxford Immunotec. C.U.K. was an unpaid advisor to BioVersys.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zwyer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains., Method: In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media., Results: For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9-∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3-∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6-710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0-26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98)., Conclusion: Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates., Competing Interests: None

Background: Outcomes of treatment of tuberculosis patients with regimens including pretomanid have not yet been systematically reviewed., Objectives: To appraise existing evidence on efficacy and safety of pretomanid in tuberculosis., Data Sources: Pubmed, clinicaltrials.gov. and Cochrane library., Study Eligibility Criteria: Quantitative studies presenting original data on clinical efficacy or safety of pretomanid., Participants: Patients with tuberculosis., Interventions: Treatment with pretomanid or pretomanid-containing regimens in minimum one study group., Methods: Two authors independently extracted data and assessed risk of bias. Data on efficacy (early bactericidal activity, bactericidal activity, end-of-treatment outcomes and acquired resistance) and safety were summarized in tables. Mean differences in efficacy outcomes between regimens across studies were calculated., Results: Eight studies were included; four randomized controlled trials on 2-week early bactericidal activity in rifampicin-susceptible tuberculosis, three trials with randomized rifampicin-susceptible tuberculosis arms and a single rifampicin-resistant tuberculosis arm (two on 8-week bactericidal activity, one on end-of-treatment outcomes), one single-arm trial with end-of-treatment outcomes in highly resistant tuberculosis. Activity of pretomanid-moxifloxacin-pyrazinamide was superior to standard treatment on daily change in colony-forming units at days 0-2, 0-56 and 7-56 and time to culture conversion in rifampicin-susceptible tuberculosis (hazard ratio: 1.7; 95% CI 1.1-2.7), but not at end of treatment in one study. This study was stopped due to serious hepatotoxic adverse events, including three deaths, in 4% (95% CI 2-8) patients on pretomanid-moxifloxacin-pyrazinamide and none in controls. In patients with uncomplicated rifampicin-resistant tuberculosis on pretomanid-moxifloxacin-pyrazinamide treatment, 91% (95% CI 59-100) had favourable end-of-treatment outcomes. In patients with highly resistant tuberculosis, 90% (95% CI 83-95) on pretomanid-bedaquiline-linezolid had favourable outcomes six months after treatment, but linezolid-related toxicity was frequent. No acquired resistance to pretomanid was reported., Conclusions: Evidence suggests an important role for pretomanid in rifampicin-resistant and highly resistant tuberculosis. Trials comparing pretomanid to existing core and companion drugs are needed to further define that role., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100-1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1-99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare " Mycobacterium canettii " strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 out of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment., Competing Interests: Conflict of interest: A. Jouet is an employee of GenoScreen. Conflict of interest: C. Gaudin is an employee of GenoScreen. Conflict of interest: N. Badalato is an employee of GenoScreen. Conflict of interest: C. Allix-Béguec is an employee of GenoScreen. Conflict of interest: S. Duthoy is an employee of GenoScreen. Conflict of interest: A. Ferré is an employee of GenoScreen. Conflict of interest: M. Diels has nothing to disclose. Conflict of interest: Y. Laurent is an employee of GenoScreen. Conflict of interest: S. Contreras is an employee of GenoScreen. Conflict of interest: S. Feuerriegel has nothing to disclose. Conflict of interest: S. Niemann reports grants from the German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation EXC 2167, Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG) and ECDC public tender: OJ/2017/OCS/7766 “Pilot study on the use of Whole Genome Sequencing for molecular typing and characterisation of M. tuberculosis in the EU/EEA”, during the conduct of the study. Conflict of interest: E. André has nothing to disclose. Conflict of interest: M.K. Kaswa has nothing to disclose. Conflict of interest: E. Tagliani has nothing to disclose. Conflict of interest: A. Cabibbe has nothing to disclose. Conflict of interest: V. Mathys has nothing to disclose. Conflict of interest: D. Cirillo has nothing to disclose. Conflict of interest: B.C. de Jong has nothing to disclose. Conflict of interest: L. Rigouts has nothing to disclose. Conflict of interest: P. Supply reports personal fees for consultancy from GenoScreen, grants from the European Union PathoNGen-Trace project (FP7-278864), during the conduct of the study., (Copyright ©ERS 2021.)

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto , as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

Background: Since a meta-analysis showed little or no effect of second-line injectables on treatment success, and using injectables may induce ototoxicity, injectable-free rifampicin-resistant tuberculosis (RR-TB) treatment regimens are recommended. However, acquired resistance preventing activity was overlooked. No previous study assessed the effect of shortening the duration of kanamycin administration to 2 months during the intensive phase of the RR-TB shorter treatment regimen (STR)., Methods: Retrospective cohort study of the effect of using 2 months of kanamycin instead of the standard 4(+) months (extension if lack of smear conversion at 4 months) on recurrence (either treatment failure or relapse) and fluoroquinolone acquired drug resistance, in patients treated with a gatifloxacin-based STR in Damien Foundation supported clinics in Bangladesh. Logistic regression was used to estimate associations., Results: Five of 52 (9.6%) treated with a STR containing two months of kanamycin had recurrence, compared to 21 of 738 (2.8%) patients treated with the standard STR containing 4(+) months of kanamycin (OR 3.7; 95%CI:1.5-10.3). In those with initially fluoroquinolone-susceptible TB, acquired resistance to fluoroquinolone was detected in none of 639 patients treated with 4(+) months of kanamycin and two (4.5%) of 44 treated with two months of kanamycin (OR 75.2; 95%CI:3.6-1592.1)., Conclusion: Two months of kanamycin was insufficient to prevent recurrence with acquired resistance to gatifloxacin, the core drug of the most effective RR-TB STR. Injectable mediated resistance prevention is important to reach high effectiveness, to safeguard all treatment options after recurrence, and to prevent the spread of resistant TB. Studies on all-oral regimens should also assess the effect of regimen composition on resistance acquisition. Until evidence shows that other drugs can assure at least the same strong resistance preventing activity of the injectables, it seems wise to continue using this group of drugs, and adapt the regimen if any ototoxicity is detected., Competing Interests: The authors have declared that no competing interests exist.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.

We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI ( n = 125, 94%) than with ETOH ( n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage ( n = 10, 41.7%) than with CPC ( n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI ( n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle ( C T ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC ( P = 0.915) or OMNI ( P = 0.33). For one patient's ETOH- and CPC-stored specimens with a C T of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.)., (Copyright © 2019 Sanoussi et al.)

Purpose: Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5)., Methodology: Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination., Results: In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5., Conclusion: The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.

After an alert regarding ≈31 tuberculosis (TB) cases, 3 of which were rifampin-resistant TB cases, in Mbuji-Mayi Central Prison, Democratic Republic of the Congo, we conducted an outbreak investigation in January 2015. We analyzed sputum of presumptive TB patients by using the Xpert MTB/RIF assay. We also assessed the Mycobacterium tuberculosis isolates' drug-susceptibility patterns and risk factors for TB infection. Among a prison population of 918 inmates, 29 TB case-patients were already undergoing treatment. We found an additional 475 presumptive TB case-patients and confirmed TB in 170 of them. In March 2015, the prevalence rate of confirmed TB was 21.7% (199/918 inmates). We detected an additional 14 cases of rifampin-resistant TB and initiated treatment in all 14 of these case-patients. Overcrowded living conditions and poor nutrition appeared to be the driving factors behind the high TB incidence in this prison.

Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult., Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005-2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated., Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off., Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches.

Objective: Paragonimiasis is a foodborne trematode infection of the lungs caused by Paragonimus spp., presenting clinically with similar symptoms to active tuberculosis (TB). Worldwide, an estimated 20.7 million people are infected with paragonimiasis, but relatively little epidemiological data exists for Africa. Given a recently reported case, we sought to establish whether paragonimiasis should be considered as an important differential diagnosis for human TB in The Gambia, West Africa., Results: We developed a novel PCR-based diagnostic test for Paragonimus species known to be found in West Africa, which we used to examine archived TB negative sputum samples from a cross-sectional study of volunteers with tuberculosis-like symptoms from communities in the Western coastal region of The Gambia. Based on a "zero patient" design for detection of rare diseases, 300 anonymised AFB smear negative sputum samples, randomly selected from 25 villages, were screened for active paragonimiasis by molecular detection of Paragonimus spp. DNA. No parasite DNA was found in any of the sputa of our patient group. Despite the recent case report, we found no evidence of active paragonimiasis infection masking as TB in the Western region of The Gambia.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration., Competing Interests: Statement The authors have no competing interests as defined by Springer Nature, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Introduction: Early diagnosis and initiation to appropriate treatment is vital for tuberculosis (TB) control. The XpertMTB/RIF (Xpert) assay offers rapid TB diagnosis and quantitative estimation of bacterial burden through Cycle threshold (Ct) values. We assessed whether the Xpert Ct value is associated with delayed TB diagnosis as a potential monitoring tool for TB control programme performance., Materials and Methods: This analysis was nested in a prospective study under the routine TB surveillance procedures of the National TB Control Program in Manhiça district, Maputo province, Mozambique. Presumptive TB patients were tested using smear microscopy and Xpert. We explored the association between Xpert Ct values and self-reported delay of Xpert-positive TB patients as recorded at the time of diagnosis enrolment. Patients with >60 days of TB symptoms were considered to have long delays., Results: Of 1,483 presumptive TB cases, 580 were diagnosed as TB of whom 505 (87.0%) were due to pulmonary TB and 302 (94.1%) were Xpert positive. Ct values (range, 9.7-46.4) showed a multimodal distribution. The median (IQR) delay was 30 (30-45) days. Ct values showed no correlation with delay (R2 = 0.001, p = 0.621), nor any association with long delays: adjusted odds ratios (AOR) (95% confidence interval [CI]) comparing to >28 cycles 0.99 (0.50-1.96; p = 0.987) for 23-28 cycles, 0.93 (0.50-1.74; p = 0.828) for 16-22 cycles; and 1.05 (0.47-2.36; p = 0.897) for <16 cycles. Being HIV-negative (AOR [95% CI]), 2.05 (1.19-3.51, p = 0.009) and rural residence 1.74 (1.08-2.81, p = 0.023), were independent predictors of long delays., Conclusion: Xpert Ct values were not associated with patient delay for TB diagnosis and cannot be used as an indicator of TB control program performance., Competing Interests: The authors have declared that no competing interests exist.

Background: In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments., Methods: From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis., Results: Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16-101)/mm(3) and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical., Conclusions: Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly.

Background: Phylogenetically distinct Mycobacterium tuberculosis lineages differ in their phenotypes and pathogenicity. Consequently, understanding mycobacterial population structures phylogeographically is essential for design, interpretation and generalizability of clinical trials. Comprehensive efforts are lacking to date to establish the West African mycobacterial population structure on a sub-continental scale, which has diagnostic implications and can inform the design of clinical TB trials., Methodology/principal Findings: We collated novel and published genotyping (spoligotyping) data and classified spoligotypes into mycobacterial lineages/families using TBLineage and Spotclust, followed by phylogeographic analyses using statistics (logistic regression) and lineage axis plot analysis in GenGIS, in which a phylogenetic tree constructed in MIRU-VNTRplus was analysed. Combining spoligotyping data from 16 previously published studies with novel data from The Gambia, we obtained a total of 3580 isolates from 12 countries and identified 6 lineages comprising 32 families. By using stringent analytical tools we demonstrate for the first time a significant phylogeographic separation between western and eastern West Africa not only of the two M. africanum (West Africa 1 and 2) but also of several major M. tuberculosis sensu stricto families, such as LAM10 and Haarlem 3. Moreover, in a longitudinal logistic regression analysis for grouped data we showed that M. africanum West Africa 2 remains a persistent health concern., Conclusions/significance: Because of the geographical divide of the mycobacterial populations in West Africa, individual research findings from one country cannot be generalized across the whole region. The unequal geographical family distribution should be considered in placement and design of future clinical trials in West Africa.

In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 "orphan" and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10&#95;CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs., (Copyright © 2015. Published by Elsevier B.V.)

Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (n = 463) of the isolates, most of which belonged to the Haarlem family (n = 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P = 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P < 0.0001), while Beijing (OR, 0.6; P = 0.006) and LAM (OR, 0.7; P = 0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8; P = 0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8; P = 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T-cell responses from 19 Maf- and 29 Mtb-infected HIV-negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB-specific antigens. Before treatment, percentages of early secreted antigenic target-6(ESAT-6)/culture filtrate protein-10(CFP-10) and purified protein derivative-specific single-TNF-α-producing CD4(+) and CD8(+) T cells were significantly higher while single-IL-2-producing T cells were significantly lower in Maf- compared with Mtb-infected patients. Purified protein derivative-specific polyfunctional CD4(+) T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3(+) CD11b(+) T cells was similar in both groups pretreatment, but was significantly lower with higher TNF-α, IL-2, and IFN-γ production in Mtb- compared with that of Maf-infected patients posttreatment. Our data provide evidence of differences in T-cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: A common complication of starting antiretroviral therapy (ART) for human immunodeficiency virus (HIV) is the development of immune reconstitution inflammatory syndrome (IRIS) in approximately 25% of patients. Despite similarities with paradoxical reactions to tuberculosis and reversal reactions in leprosy, the exact mechanisms, and therefore potential determinants, of IRIS are still unknown., Methods: In this longitudinal cohort study, we analyzed 20 patients who developed IRIS following initiation of ART and 16 patients who did not, matched for ART time point. Peripheral blood mononuclear cells were stimulated overnight with a positive control antigen and 2 tuberculosis-specific antigens (purified protein derivative [PPD] and ESAT-6/CFP10), followed by polychromatic flow cytometry for analysis of cytokine production from CD4(+) and CD8(+) T cells., Results: Responses to PPD were significantly higher in IRIS patients compared to controls during the IRIS time point, but CD4(+) and CD8(+) T-cell responses to the positive control stimulation were significantly lower in IRIS patients at all time points. Furthermore, whereas control patients had rejuvenated polyfunctional T-cell responses by 3 months after ART, IRIS patients were strikingly monofunctional (generally interferon γ alone), even up to 6 months of ART in response to all stimulations., Conclusions: Our findings suggest that the peripheral T-cell responses to the underlying pathogen are exaggerated in IRIS patients but that the overall quality of the peripheral T-cell pool is significantly reduced compared to non-IRIS patients. Furthermore, these effects are apparent at least up to 3 months after cessation of IRIS.

The rapid diagnosis of rifampin resistance is hampered by a reported insufficient specificity of molecular techniques for detection of rpoB mutations. Our objective for this study was to document the prevalence and prognostic value of rpoB mutations with unclear phenotypic resistance. The study design entailed sequencing directly from sputum of first failure or relapse patients without phenotypic selection and comparison of the standard retreatment regimen outcome, according to the mutation present. We found that among all rpoB mutations, the best-documented "disputed" rifampin resistance mutations (511Pro, 516Tyr, 526Asn, 526Leu, 533Pro, and 572Phe) made up 13.1% and 10.6% of all mutations in strains from Bangladesh and Kinshasa, respectively. Except for the 511Pro and 526Asn mutations, most of these strains with disputed mutations tested rifampin resistant in routine Löwenstein-Jensen medium proportion method drug susceptibility testing (DST; 78.7%), but significantly less than those with common, undisputed mutations (96.3%). With 63% of patients experiencing failure or relapse in both groups, there was no difference in outcome of first-line retreatment between patients carrying a strain with disputed versus common mutations. We conclude that rifampin resistance that is difficult to detect by the gold standard, phenotypic DST, is clinically and epidemiologically highly relevant. Sensitivity rather than specificity is imperfect with any rifampin DST method. Even at a low prevalence of rifampin resistance, a rifampin-resistant result issued by a competent laboratory may not warrant confirmation, although the absence of a necessity for confirmation needs to be confirmed for molecular results among new cases. However, a result of rifampin susceptibility should be questioned when suspicion is very high, and further DST using a different system (i.e., genotypic after phenotypic testing) would be fully justified.

Background: Human tuberculosis (TB) in West Africa is not only caused by M. tuberculosis but also by bacteria of the two lineages of M. africanum. For instance, in The Gambia, 40% of TB is due to infections with M. africanum West African 2. This bacterial lineage is associated with HIV infection, reduced ESAT-6 immunogenicity and slower progression to active disease. Although these characteristics suggest an attenuated phenotype of M. africanum, no underlying mechanism has been described. From the first descriptions of M. africanum in the literature in 1969, the time to a positive culture of M. africanum on solid medium was known to be longer than the time to a positive culture of M. tuberculosis. However, the delayed growth of M. africanum, which may correlate with the less virulent phenotype in the human host, has not previously been studied in detail., Methodology/principal Findings: We compared the growth rates of M. tuberculosis and M. africanum isolates from The Gambia in two liquid culture systems. M. africanum grows significantly slower than M. tuberculosis, not only when grown directly from sputa, but also in growth experiments under defined laboratory conditions. We also sequenced four M. africanum isolates and compared their whole genomes with the published M. tuberculosis H37Rv genome. M. africanum strains have several non-synonymous SNPs or frameshift mutations in genes that were previously associated with growth-attenuation. M. africanum strains also have a higher mutation frequency in genes crucial for transport of sulphur, ions and lipids/fatty acids across the cell membrane into the bacterial cell. Surprisingly, 5 of 7 operons, recently described as essential for intracellular survival of H37Rv in the host macrophage, showed at least one non-synonymously mutated gene in M. africanum., Conclusions/significance: The altered growth behaviour of M. africanum might indicate a different survival strategy within host cells.

Determining what constitutes protective immunity to TB is critical for the development of improved diagnostics and vaccines. The comparison of the immune system between contacts of TB patients, who later develop TB disease (progressors), versus contacts who remain healthy (non-progressors), allows for identification of predictive markers of TB disease. This study provides the first comprehensive analysis of the immune system of progressors and non-progressors using a well-characterised TB case-contact (TBCC) platform in The Gambia, West Africa. 22 progressors and 31 non-progressors were analysed at recruitment, 3 months and 18 months (time to progression: median[IQR] of 507[187-714] days). Immunophenotyping of PBMC, plasma cytokine levels and RT-MLPA analysis of whole blood-derived RNA was performed to capture key immune system parameters. At recruitment, progressors had lower PBMC proportions of CD4+ T cells, NKT cells and B cells relative to non-progressors. Analysis of the plasma showed higher levels of IL-18 in progressors compared to non-progressors and analysis of the RNA showed significantly lower gene expression of Bcl2 but higher CCR7 in progressors compared to non-progressors. This study shows several markers that may predict the onset of active TB at a very early stage after infection. Once these markers have been validated in larger studies, they provide avenues to prospectively identify people at risk of developing TB, a key issue in the testing of new TB vaccines.

Background: Helicobacter pylori, a lifelong and typically asymptomatic infection of the stomach, profoundly alters gastric immune responses, and may benefit the host in protection against other pathogens. We explored the hypothesis that H. pylori contributes to the control of infection with Mycobacterium tuberculosis., Methodology/principal Findings: We first examined M. tuberculosis-specific IFN-gamma and H. pylori antibody responses in 339 healthy Northern Californians undergoing routine tuberculin skin testing. Of 97 subjects (29%) meeting criteria for latent tuberculosis (TB) infection (LTBI), 45 (46%) were H. pylori seropositive. Subjects with LTBI who were H. pylori-seropositive had 1.5-fold higher TB antigen-induced IFN-gamma responses (p = 0.04, ANOVA), and a more Th-1 like cytokine profile in peripheral blood mononuclear cells, compared to those who were H. pylori seronegative. To explore an association between H. pylori infection and clinical outcome of TB exposure, we evaluated H. pylori seroprevalence in baseline samples from two high risk TB case-contact cohorts, and from cynomolgus macaques experimentally challenged with M. tuberculosis. Compared to 513 household contacts who did not progress to active disease during a median 24 months follow-up, 120 prevalent TB cases were significantly less likely to be H. pylori infected (AOR: 0.55, 95% CI 0.0.36-0.83, p = 0.005), though seroprevalence was not significantly different from non-progressors in 37 incident TB cases (AOR: 1.35 [95% CI 0.63-2.9] p = 0.44). Cynomolgus macaques with natural H. pylori infection were significantly less likely to progress to TB 6 to 8 months after M. tuberculosis challenge (RR: 0.31 [95% CI 0.12-0.80], p = 0.04)., Conclusions/significance: H. pylori infection may induce bystander effects that modify the risk of active TB in humans and non-human primates. That immunity to TB may be enhanced by exposure to other microbial agents may have important implications for vaccine development and disease control.

Mycobacterium africanum, first described in Senegal in 1968, causes up to half of the smear-positive pulmonary tuberculosis cases in West Africa, but it has not been found in other geographical areas except among recent West African migrants. The reasons for the geographic restriction of M. africanum are unknown. We used molecular tools to determine the population structure of the Mycobacterium tuberculosis complex in a cohort study of consecutive smear-positive tuberculosis cases in The Gambia. We collected and genotyped 386 clinical isolates using spoligotype analysis and PCRs for large sequence polymorphisms (LSPs) and compared the genotype patterns to the patterns in an international database. The results of spoligotyping and LSP analysis for the study population were also compared to determine the correlation between them. The main lineages within the Mycobacterium tuberculosis complex identified in The Gambia included M. africanum type I (38.4%), characterized by an LSP in region of difference 702 (RD702; West African type 2). Among the M. tuberculosis sensu stricto isolates, lineages characterized by RD182 and by RD174 were the most common. We also detected a gradient in the prevalence of M. africanum that extended from neighboring Guinea-Bissau. The genotypic diversity of the spoligotype patterns was greater among the isolates of M. africanum than among the isolates of M. tuberculosis. We postulate that M. africanum became endemic in West Africa first, before the introduction of different lineages within M. tuberculosis sensu stricto.

Background: There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease., Methods: In a cohort study of patients with tuberculosis and their household contacts in The Gambia, we categorized 1808 HIV-negative tuberculosis contacts according to exposure to M. tuberculosis or M. africanum. Positive skin test results indicated transmission, and development of tuberculosis during 2 years of follow-up indicated progression to disease., Results: Transmission rates were similar, but rates of progression to disease were significantly lower in contacts exposed to M. africanum than in those exposed to M. tuberculosis (1.0% vs. 2.9%; hazard ratio [HR], 3.1 [95% confidence interval {CI}, 1.1-8.7]). Within M. tuberculosis sensu stricto, contacts exposed to a Beijing family strain were most likely to progress to disease (5.6%; HR relative to M. africanum, 6.7 [95% CI, 2.0-22])., Conclusions: M. africanum and M. tuberculosis transmit equally well to household contacts, but contacts exposed to M. africanum are less likely to progress to tuberculosis disease than those exposed to M. tuberculosis. The variable rate of progression by lineage suggests that tuberculosis variability matters in clinical settings and should be accounted for in studies evaluating tuberculosis vaccines and treatment regimens for latent tuberculosis infection.

Background: Studies of Tuberculosis (TB) case contacts are increasingly being utilised for understanding the relationship between M. tuberculosis and the human host and for assessing new interventions and diagnostic tests. We aimed to identify the incidence rate of new TB cases among TB contacts and to relate this to their initial Mantoux and ELISPOT test results., Methods and Findings: After initial Mantoux and ELISPOT tests and exclusion of co-prevalent TB cases, we followed 2348 household contacts of sputum smear positive TB cases. We visited them at 3 months, 6 months, 12 months, 18 months and 24 months, and investigated those with symptoms consistent with TB. Those who were diagnosed separately at a government clinic had a chest x-ray. Twenty six contacts were diagnosed with definite TB over 4312 person years of follow-up (Incidence rate 603/100,000 person years; 95% Confidence Interval, 370-830). Nine index and secondary case pairs had cultured isolates available for genotyping. Of these, 6 pairs were concordant and 3 were discordant. 2.5% of non-progressors were HIV positive compared to 12% of progressors (HR 6.2; 95% CI 1.7-22.5; p = 0.010). 25 secondary cases had initial Mantoux results, 14 (56%) were positive ; 21 had initial ELISPOT results, 11 (52%) were positive; 15 (71%) of 21 tested were positive by one or the other test. Of the 6 contacts who had concordant isolates with their respective index case, 4 (67%) were Mantoux positive at recruitment, 3 (50%) were ELISPOT positive; 5 (83%) were positive by one or other of the two tests. ELISPOT positive contacts, and those with discordant results, had a similar rate of progression to those who were Mantoux positive. Those negative on either or both tests had the lowest rate of progression., Conclusions: The incidence rate of TB disease in Gambian TB case contacts, after screening for co-prevalent cases, was 603/100,000 person years. Since initial ELISPOT test and Mantoux tests were each positive in only just over half of cases, but 71% were positive by one or other test, positivity by either might be the best indication for preventive treatment. These data do not support the replacement of the Mantoux test by an ELISPOT test in The Gambia or similar settings.

Rationale: The optimal length of tuberculosis treatment in patients coinfected with HIV is unknown., Objectives: To evaluate treatment outcomes for HIV-infected patients stratified by duration of rifamycin-based tuberculosis therapy., Methods: We retrospectively reviewed data on all patients with tuberculosis reported to the San Francisco Tuberculosis Control Program from 1990 through 2001. Patients were followed for up to 12 months after treatment completion., Measurements and Main Results: Of 700 patients, 264 (38%) were HIV infected, 315 (45%) were not infected, and 121 (17%) were not tested. Mean duration of treatment was extended to 10.2 months for HIV-infected patients versus 8.4 months for uninfected/unknown patients (p < 0.001). Seventeen percent of the HIV-infected and 37% of the HIV uninfected/unknown patients received 6 months of rifamycin-based therapy. The relapse rate among HIV-infected was 9.3 per 100 person-years versus 1.0 in HIV-uninfected/unknown patients (p < 0.001). HIV-infected individuals who received a standard 6-month rifamycin-based regimen were more likely to relapse than those treated longer (adjusted hazard ratio, 4.33; p = 0.02). HIV-infected individuals who received intermittent therapy were also more likely to relapse than those treated on daily basis (adjusted hazard ratio, 4.12; p = 0.04). The use of highly active antiretroviral therapy was associated with more rapid conversion of smears and cultures and with improved survival., Conclusions: HIV-infected patients who received a 6-month rifamycin-based course of tuberculosis treatment or who received intermittent therapy had a higher relapse rate than HIV-infected subjects who received longer therapy or daily therapy, respectively. Standard 6-month therapy may be insufficient to prevent relapse in patients with HIV.

Background: Very little longitudinal information is available regarding the performance of T cell-based tests for Mycobacterium tuberculosis infection. To address this deficiency, we conducted a longitudinal assessment of the enzyme-linked immunosorbent spot test (ELISPOT) test in comparison to the standard tuberculin skin test (TST)., Methods and Findings: In tuberculosis (TB) contacts we repeated ELISPOT tests 3 mo (n = 341) and 18 mo (n = 210) after recruitment and TSTs at 18 mo (n = 130). We evaluated factors for association with conversion and reversion and investigated suspected cases of TB. Of 207 ELISPOT-negative contacts, 51 (24.6%) had 3-mo ELISPOT conversion, which was associated with a positive recruitment TST (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.0-5.0, p = 0.048) and negatively associated with bacillus Calmette-Guérin (BCG) vaccination (OR 0.5, 95% CI 0.2-1.0, p = 0.06). Of 134 contacts, 54 (40.2%) underwent 3-mo ELISPOT reversion, which was less likely in those with a positive recruitment TST (OR 0.3, 95% CI 0.1-0.8, p = 0.014). Between 3 and 18 mo, 35/132 (26.5%) contacts underwent ELISPOT conversion and 28/78 (35.9%) underwent ELISPOT reversion. Of the 210 contacts with complete results, 73 (34.8%) were ELISPOT negative at all three time points; 36 (17.1%) were positive at all three time points. Between recruitment and 18 mo, 20 (27%) contacts had ELISPOT conversion; 37 (50%) had TST conversion, which was associated with a positive recruitment ELISPOT (OR 7.2, 95% CI 1.4-37.1, p = 0.019); 18 (32.7%) underwent ELISPOT reversion; and five (8.9%) underwent TST reversion. Results in 13 contacts diagnosed as having TB were mixed, but suggested higher TST sensitivity., Conclusions: Both ELISPOT conversion and reversion occur after M. tuberculosis exposure. Rapid ELISPOT reversion may reflect M. tuberculosis clearance or transition into dormancy and may contribute to the relatively low reported ELISPOT conversion rate. Therefore, a negative ELISPOT test for M. tuberculosis infection should be interpreted with caution.

Background: Repeat tuberculin skin tests may be false positive due to boosting of waned immunity to past mycobacterial exposure. We evaluated whether an ELISPOT test could identify tuberculosis (TB) contacts with boosting of immunity to non-tuberculous mycobacterial exposure., Methodology/principal Findings: We conducted tuberculin and ELISPOT tests in 1665 TB contacts: 799 were tuberculin test negative and were offered a repeat test after three months. Those with tuberculin test conversion had an ELISPOT, chest X-ray and sputum analysis if appropriate. We compared converters with non-converters, assessed the probability of each of four combinations of ELISPOT results over the two time points and estimated boosting with adjustment for ELISPOT sensitivity and specificity. 704 (72%) contacts had a repeat tuberculin test; 176 (25%) had test conversion, which increased with exposure to a case (p = 0.002), increasing age (p = 0.0006) and BCG scar (p = 0.06). 114 tuberculin test converters had ELISPOT results: 16(14%) were recruitment positive/follow-up positive, 9 (8%) positive/negative, 34 (30%) negative/positive, and 55 (48%) were negative/negative. There was a significant non-linear effect of age for ELISPOT results in skin test converters (p = 0.038). Estimates of boosting ranged from 32%-41% of skin test converters with increasing age. Three converters were diagnosed with TB, two had ELISPOT results: both were positive, including one at recruitment., Conclusions/significance: We estimate that approximately one third of tuberculin skin test conversion in Gambian TB case contacts is due to boosting of immunity to non-tuberculous mycobacterial exposure. Further longitudinal studies are required to confirm whether ELISPOT can reliably identify case contacts with tuberculin test conversion that would benefit most from prophylactic treatment.

Background: Mycobacterium africanum, a member of the M. tuberculosis complex that is infrequently found outside of western Africa, is the cause of up to half of the tuberculosis cases there., Methods: We genotyped mycobacterial isolates obtained from a study of patients with tuberculosis and their household contacts and compared T cell responses and tuberculin skin test results by infecting genotype., Results: The T cell response to early secreted antigenic target, 6 kDa (ESAT-6), was attenuated in patients with tuberculosis (odds ratio [OR], 0.41 [95% confidence interval {CI}, 0.19-0.89]; P = .024) and household contacts (OR, 0.56 [95% CI, 0.38-0.83]; P = .004) infected with M. africanum, compared with the response in those infected with M. tuberculosis. In these same groups, responses to culture filtrate protein, 10 kDa (CFP-10), were nonsignificantly attenuated (P = .22 and P = .16, respectively), as were tuberculin skin test results (P = .30 and P = .46, respectively). Sequencing of region of difference 1 of M. africanum revealed that Rv3879c is a pseudogene in M. africanum; however, this finding does not provide an obvious mechanism for the attenuated ESAT-6 response., Conclusions: This is the first evidence, to our knowledge, that strain differences affect interferon- gamma -based T cell responses. Our findings highlight the need to test new diagnostic candidates against different strains of mycobacteria. Integrating additional immunologic and genomic comparisons of M. tuberculosis and M. africanum into further studies may provide fundamental insights into the interactions between humans and mycobacteria.

Mycobacterium tuberculosis remains a major cause of morbidity and mortality worldwide. Studies have reported human pathogens to have geographically structured population genetics, some of which have been linked to ancient human migrations. However, no study has addressed the potential evolutionary consequences of such longstanding human-pathogen associations. Here, we demonstrate that the global population structure of M. tuberculosis is defined by six phylogeographical lineages, each associated with specific, sympatric human populations. In an urban cosmopolitan environment, mycobacterial lineages were much more likely to spread in sympatric than in allopatric patient populations. Tuberculosis cases that did occur in allopatric hosts disproportionately involved high-risk individuals with impaired host resistance. These observations suggest that mycobacterial lineages are adapted to particular human populations. If confirmed, our findings have important implications for tuberculosis control and vaccine development.

Globally, the HIV and tuberculosis epidemics are stoking each other, creating a public health crisis of enormous proportions. At the level of individuals, contemporaneous infection with M. tuberculosis and HIV poses great challenges to clinical management. This chapter provides an overview of active and latent tuberculosis treatment in HIV-infected and -uninfected individuals. The discussion focuses on medication issues, including interactions between antitubercular drugs, antiretroviral drugs, and medicines used for opportunistic infections and treatment in the face of comorbidities. Clinical questions specific to coinfection are discussed, including duration and timing initiation of therapy and immune reconstitution. Most of the data presented were generated in industrialized settings and are presented to assist patient management in such settings. However, given the disproportionate amount of TB/HIV in less-developed nations and the increasing availability of antiretroviral therapy in resource-limited settings, the issues presented will become increasingly relevant globally.

BackgroundIt is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis.MethodsWe collected sputum specimens before treatment from Gambians and Ugandans with pulmonary tuberculosis, revealed by positive results of acid-fast bacillus smears. We quantified expression of 2179 M. tuberculosis genes and 234 human immune genes via quantitative reverse transcription-polymerase chain reaction. We summarized genes from key functional categories with significantly increased or decreased expression.ResultsA total of 24 of 65 patients with tuberculosis were HIV infected. M. tuberculosis DosR regulon genes were less highly expressed among HIV-infected patients with tuberculosis than among HIV-uninfected patients with tuberculosis (Gambia, P < .0001; Uganda, P = .037). In profiling of human genes from the same sputa, HIV-infected patients had 3.4-fold lower expression of IFNG (P = .005), 4.9-fold higher expression of ARG1 (P = .0006), and 3.4-fold higher expression of IL10 (P = .0002) than in HIV-uninfected patients with tuberculosis.ConclusionsM. tuberculosis in HIV-infected patients had lower expression of the DosR regulon, a critical metabolic and immunomodulatory switch induced by NO, carbon monoxide, and hypoxia. Our human data suggest that decreased DosR expression may result from alternative pathway activation of macrophages, with consequent decreased NO expression and/or by poor granuloma formation with consequent decreased hypoxic stress.

BackgroundTreatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli.MethodsM. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis.ResultsWithin 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days.ConclusionsTranscriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens.

BackgroundTuberculosis is a leading cause of death worldwide, yet the determinants of death are not well understood. We sought to determine risk factors for mortality during treatment of drug-susceptible pulmonary tuberculosis under program settings.MethodsRetrospective chart review of patients with drug-susceptible tuberculosis reported to the San Francisco Tuberculosis Control Program from 1990-2001.ResultsOf 565 patients meeting eligibility criteria, 37 (6.6%) died during the study period. Of 37 deaths, 12 (32.4%) had tuberculosis listed as a contributing factor. In multivariate analysis controlling for follow-up time, four characteristics were independently associated with mortality: HIV co-infection (HR = 2.57, p = 0.02), older age at tuberculosis diagnosis (HR = 1.52 per 10 years, p = 0.001); initial sputum smear positive for acid fast bacilli (HR = 3.07, p = 0.004); and experiencing an interruption in tuberculosis therapy (HR = 3.15, p = 0.002). The association between treatment interruption and risk of death was due to non-adherence during the intensive phase of treatment (HR = 3.20, p = 0.001). The median duration of treatment interruption did not differ significantly in either intensive or continuation phases between those who died and survived (23 versus 18 days, and 37 versus 29 days, respectively). No deaths were directly attributed to adverse drug reactions.ConclusionsIn addition to advanced age, HIV and characteristics of advanced tuberculosis, experiencing an interruption in anti-tuberculosis therapy, primarily due to non-adherence, was also independently associated with increased risk of death. Improving adherence early during treatment for tuberculosis may both improve tuberculosis outcomes as well as decrease mortality.

In its 2020 guidelines for the treatment of rifampicin-resistant TB (RR-TB), the WHO recommends all-oral fluoroquinolone-based regimens, with bedaquiline replacing the second-line injectable drugs (SLIDs). SLIDs were used for their strong acquired resistance-preventing activity. Data from three cohorts showed acquired bedaquiline resistance ranging between 2.5% and 30.8%, with no protection from a SLID in most cases. If bedaquiline resistance is that easily acquired, it will fail to protect fluoroquinolones and other drugs from acquiring resistance. Until evidence on resistance-preventing activity shows that SLIDs can safely be replaced, we call for more prudent use of the few potent second-line TB drugs available. Studies on new treatment regimens need to prioritize the prevention of acquired resistance along with treatment success. Meanwhile, reducing the dosing of SLIDs to thrice weekly from Day 1, and their replacement for any degree of audiometry abnormalities before or during treatment will largely avoid serious ototoxicity. [ABSTRACT FROM AUTHOR]

Abstract Background The prevalence of drug resistant tuberculosis (TB) in Ethiopia in general, and Jimma area in particular, is not well documented. We conducted a study at Jimma University specialized hospital in southwest Ethiopia among new cases of smear positive TB patients to determine the pattern of resistance to first-line drugs. Methods A health institution based cross sectional study was conducted from November 2010 to September 2011. Any newly diagnosed smear positive TB patient 18 years and above was included in the study. Demographic and related data were collected by trained personnel using a pretested structured questionnaire. Mycobacterial drug susceptibility testing (DST) to the first line drugs isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STM) was performed on cultures using the indirect proportion method. M. tuberculosis complex (MTBC) was identified with the Capilia TB-Neo test. Results 136 patients were enrolled in the study. Resistance to at least one drug was identified in 18.4%. The highest prevalence of resistance to any drug was identified against INH (13.2%) followed by STM (8.1%). There was no statistically significant difference in the proportion of any resistance by sex, age, HIV status and history of being imprisoned. The highest mono resistance was observed against INH (7.4%). Mono resistance to streptomycin was associated with HIV infection (crude OR 15.63, 95%CI: 1.31, 187). Multidrug-resistance TB (MDR-TB) was observed in two patients (1.5%). Conclusion Resistance to at least one drug was 18.4% (INH-13.2% and STM-8.1%). STM resistance was associated with HIV positivity. There was relatively low prevalence of MDR-TB yet INH resistance was common around Jimma. The capacity of laboratories for TB culture and DST should be strengthened, in order to correctly manage TB patients and avoid amplification of drug resistance.

The surveillance of drug resistance among tuberculosis (TB) patients is central to preventing the spread of antimicrobial resistance. The Democratic Republic of the Congo (DR Congo) is classified by the World Health Organization (WHO) as a country with a high burden of TB and multidrug-resistant TB (MDR-TB), but there are no nationally representative data on drug resistance. In 2016–2017, a national survey of TB patients was conducted in 108 microscopy centres across all 11 provinces of the country using innovative molecular approaches. Sputum samples were collected from 1,545 new and 163 previously treated patients. These were tested by the Xpert MTB/RIF assay, followed by targeted next-generation sequencing performed directly on sputum. The prevalence of rifampicin resistance was low, at 1.8% (95% CI: 1.0–3.2) among new and 17.3% (95% CI: 11.9–24.4) among previously treated patients. Resistance to pyrazinamide, fluoroquinolones and second-line injectables was also low. The prevalence of resistance to isoniazid among rifampicin-susceptible patients was higher, at 6.6% (95% CI: 4.4–9.8) among new and 8.7% (95% : 3.2–21.2) among previously treated patients. Diagnosing and treating isoniazid-resistant patients remains a challenge, given that many will be missed by the current national diagnostic algorithm that is driven by detecting rifampicin resistance by Xpert MTB/RIF. This is the first nationwide survey incorporating targeted sequencing directly on sputum. It serves as a proof-of-concept for other settings that do yet have rapid specimen transport networks or capacity to conduct culture. [ABSTRACT FROM AUTHOR]