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Objectives: Mycobacterium tuberculosis (Mtb) infections result in a wide spectrum of clinical presentations but without proven Mtb genetic determinants. Herein, we hypothesized that the genetic features of Mtb clinical isolates, such as specific polymorphisms or microdiversity, may be linked to tuberculosis (TB) severity., Methods: A total of 234 patients with pulmonary TB (including 193 drug-susceptible and 14 monoresistant cases diagnosed between 2017 and 2020 and 27 multidrug-resistant cases diagnosed between 2010 and 2020) were stratified according to TB disease severity, and Mtb genetic features were explored using whole genome sequencing, including heterologous single-nucleotide polymorphism (SNP), calling to explore microdiversity. Finally, we performed a structural equation modeling analysis to relate TB severity to Mtb genetic features., Results: The clinical isolates from patients with mild TB carried mutations in genes associated with host-pathogen interaction, whereas those from patients with moderate/severe TB carried mutations associated with regulatory mechanisms. Genome-wide association study identified an SNP in the promoter of the gene coding for the virulence regulator espR, statistically associated with moderate/severe disease. Structural equation modeling and model comparisons indicated that TB severity was associated with the detection of Mtb microdiversity within clinical isolates and to the espR SNP., Conclusion: Taken together, these results provide a new insight to better understand TB pathophysiology and could provide a new prognosis tool for pulmonary TB severity., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tuberculosis (TB) is a difficult-to-treat infection because of multidrug regimen requirements based on drug susceptibility profiles and treatment observance issues. TB cure is defined by mycobacterial sterilization, technically complex to systematically assess. We hypothesized that microbiological outcome was associated with stage-specific immune changes in peripheral whole blood during TB treatment. The T-cell phenotypes of treated TB patients were prospectively characterized in a blinded fashion using mass cytometry after Mycobacterium tuberculosis ( Mtb ) antigen stimulation with QuantiFERON-TB Gold Plus, and then correlated to sputum culture status. At two months of treatment, cytotoxic and terminally differentiated CD8 + T-cells were under-represented and naïve CD4 + T-cells were over-represented in positive- versus negative-sputum culture patients, regardless of Mtb drug susceptibility. At treatment completion, a T-cell immune shift towards differentiated subpopulations was associated with TB cure. Overall, we identified specific T-cell profiles associated with slow sputum converters, which brings new insights in TB prognostic biomarker research designed for clinical application., Competing Interests: DG reports personal fees from Biomérieux (consulting), Qiagen (consulting, lectures), and Diasorin (lectures) outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chedid, Andrieu, Kokhreidze, Tukvadze, Biswas, Ather, Uddin, Banu, De Maio, Delogu, Endtz, Goletti, Vocanson, Dumitrescu, Hoffmann and Ader.)

There is a crucial need for non-sputum-based TB tests. Here, we evaluate the performance of RISK6, a human-blood transcriptomic signature, for TB screening, triage and treatment monitoring. RISK6 performance was also compared to that of two IGRAs: one based on RD1 antigens (QuantiFERON-TB Gold Plus, QFT-P, Qiagen) and one on recombinant M. tuberculosis HBHA expressed in Mycobacterium smegmatis (IGRA-rmsHBHA). In this multicenter prospective nested case-control study conducted in Bangladesh, Georgia, Lebanon and Madagascar, adult non-immunocompromised patients with bacteriologically confirmed active pulmonary TB (ATB), latent TB infection (LTBI) and healthy donors (HD) were enrolled. ATB patients were followed-up during and after treatment. Blood RISK6 scores were assessed using quantitative real-time PCR and evaluated by area under the receiver-operating characteristic curve (ROC AUC). RISK6 performance to discriminate ATB from HD reached an AUC of 0.94 (95% CI 0.89-0.99), with 90.9% sensitivity and 87.8% specificity, thus achieving the minimal WHO target product profile for a non-sputum-based TB screening test. Besides, RISK6 yielded an AUC of 0.93 (95% CI 0.85-1) with 90.9% sensitivity and 88.5% specificity for discriminating ATB from LTBI. Moreover, RISK6 showed higher performance (AUC 0.90, 95% CI 0.85-0.94) than IGRA-rmsHBHA (AUC 0.75, 95% CI 0.69-0.82) to differentiate TB infection stages. Finally, RISK6 signature scores significantly decreased after 2 months of TB treatment and continued to decrease gradually until the end of treatment reaching scores obtained in HD. We confirmed the performance of RISK6 signature as a triage TB test and its utility for treatment monitoring.

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management., Competing Interests: The authors have declared that no competing interests exist.

Drug-resistant tuberculosis (TB) represents a substantial threat to the global efforts to control this disease. After decades of stagnation, the treatment of drug-resistant TB is undergoing major changes: two drugs with a new mechanism of action, bedaquiline and delamanid, have been approved by stringent regulatory authorities and are recommended by the WHO. This narrative review summarizes the evidence, originating from both observational studies and clinical trials, which is available to support the use of these drugs, with a focus on special populations. Areas of uncertainty, including the use of the two drugs together or for prolonged duration, are discussed. Ongoing clinical trials are aiming to optimize the use of bedaquiline and delamanid to shorten the treatment of drug-resistant TB.

Rapid and correct determination of Mycobacterium tuberculosis (MTB) drug susceptibility is a challenge for tuberculosis (TB) management. Phenotypic drug susceptibility testing (DST) remains the reference method but is time consuming. In this study, genotypic prediction of the first-line drug susceptibility profile obtained by whole-genome sequencing (WGS) was compared with that obtained by phenotypic DST and the line probe assay (LPA). All MTB strains isolated from patients during routine practice at the mycobacteria laboratory of Lyon University Hospital, France, between November 2016 and July 2019 were included (n = 274). Isolates were tested for the first-line drugs using phenotypic DST (Mycobacteria Growth Indicator Tube) and for genotypic prediction of the susceptibility profile with LPA and WGS. Considering phenotypic DST as the reference, WGS predicted resistance to rifampicin, isoniazid, ethambutol and pyrazinamide with sensitivities of 100%, 100%, 100% and 93.8%, respectively, and susceptibility to these drugs with specificities of 99.6%, 100%, 98.5% and 100%, respectively. Performance of the LPA was poorer, with sensitivity of 83.3% for rifampicin and 85.7% for isoniazid resistance. Five isolates were classified as susceptible according to phenotypic DST (1 for rifampicin, 4 for ethambutol) while WGS detected resistance mutations in rpoB and embB genes. WGS, used under appropriate quality-control conditions, has good performance to predict the resistance profile for the four first-line drugs and can correct phenotypic DST results. This study highlights the need for future guidelines recommending WGS as the initial tool in routine practice in areas where the prevalences of TB and drug-resistant MTB are low., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb) whole genome sequencing (WGS) plays an increasingly important role in tuberculosis diagnosis and research. WGS is typically performed on biobanked isolates obtained by subculture during diagnosis. Genetic variation upon culturing is known to occur in other bacterial species. However, little is understood regarding the impact of different subculture media on genome-wide diversity and variant selection in Mtb. Here we compared WGS derived from direct sequencing of sputa samples to WGS sequences from isolates subcultured on 3 different media. Based on analysis of single nucleotide polymorphisms (SNPs), there was no evidence of variant selection caused by the different culture media used, indicating that subcultured clinical strains can be reliably used to explore genetic determinants of Mtb pathogenesis and epidemiological features., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During June 2017-April 2018, active tuberculosis with Beijing SIT1 isolates was diagnosed in 14 persons living in 4 distant cities in France. Whole-genome sequencing indicated that these patients belonged to a single transmission chain. Whole-genome sequencing-based laboratory investigations enabled prompt tracing of linked cases to improve tuberculosis control.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) complex remains a deadly infectious disease worldwide. Mtb is an intracellular pathogen, and autophagy is an essential component of the immune response leading to TB clearance. Anti-TB treatment is based on classical isoniazid (INH) and rifampicin (RIF), but also new drugs, such as linezolid (LNZ) and bedaquiline (BDQ). However, little is known about these antibiotics' impact on Mtb intra-macrophagic behavior independent of their impact on host cells. We explored the effect of mycobacterial pre-treatment with these four antibiotics on the intra-macrophagic Mtb survival and trafficking, thanks to bacterial counts and microscopy confocal imaging. Our results showed that INH and BDQ impaired Mtb phagosome escape, RIF increased autolysosome formation, and LNZ and BDQ improved autophagy activation and efficacy. These data suggest that antibiotics favoring autophagy activation (LNZ and BDQ) may allowed better Mtb clearance by macrophages and could provide basis for future anti-TB strategies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb) exhibits a structured phylogeographic distribution worldwide linked with human migrations. We sought to infer how the interactions between distinct human populations shape the global population structure of Mtb on a regional scale. We applied the recently described timescaled haplotypic density (THD) technique on 638 minisatellite-based Mtb genotypes from French tuberculosis patients. THD with a long-term (200 y) timescale indicated that Mtb population in France had been mostly influenced by interactions with Eastern and Southern Europe and, to a lesser extent, Northern and Middle Africa, consistent with historical migrations favored by geographic proximity or commercial exchanges with former French colonies. Restricting the timescale to 20 y, THD identified a sustained influence of Northern Africa, but not Europe where tuberculosis incidence decreased sharply. Evolving interactions between human populations, thus, measurably influence the local population structure of Mtb. Relevant information on such interactions can be inferred using THD from Mtb genotypes.

While isoniazid and rifampin have been the cornerstone of tuberculosis therapy caused by drug-susceptible Mycobacterium tuberculosis for more than 40 years, their combined action has never been thoroughly assessed by modern quantitative pharmacology approaches. The aims of this work were to perform in vitro experiments and mathematical modeling of the antibacterial effect of isoniazid and rifampin alone and in combination against various strains of Mycobacterium tuberculosis After MIC determination of H37Rv and three strains belonging to the Beijing, Euro-American, and Indo-Oceanic lineages, the antibacterial effects of isoniazid and rifampin alone and in combination were studied in static time-kill experiments. A sigmoidal maximum effect model (Hill equation) and a response-surface model were used to describe the effect of the drugs alone and in combination, respectively. The killing effect of isoniazid and rifampin alone were well described by the Hill equation. Rifampin displayed a more concentration-dependent effect than isoniazid around the MIC. The pharmacodynamics parameters of each drug (maximal effect, median effect concentration, and coefficient of sigmoidicity) were quite similar between the four strains. The response-surface model from Minto et al. fit data of combined effect very well with low bias and imprecision (C. F. Minto, T. W. Schnider, T. G. Short, K. M. Gregg, A. Gentilini, Anesthesiology 92: 1603-1616, 2000, https://doi.org/10.1097/00000542-200006000-00017). Response-surface modeling showed that the combined action of isoniazid and rifampin was synergistic for the H37Rv, Beijing, and Euro-American strains but only additive for the Indo-Oceanic strain. This study can serve as a motivating example for preclinical evaluation of combined action of antituberculous drugs., (Copyright © 2017 American Society for Microbiology.)

The transmission dynamics of tuberculosis involves complex interactions of socio-economic and, possibly, microbiological factors. We describe an analytical framework to infer factors of epidemic success based on the joint analysis of epidemiological, clinical and pathogen genetic data. We derive isolate-specific, genetic distance-based estimates of epidemic success, and we represent success-related time-dependent concepts, namely epidemicity and endemicity, by restricting analysis to specific time scales. The method is applied to analyze a surveillance-based cohort of 1,641 tuberculosis patients with minisatellite-based isolate genotypes. Known predictors of isolate endemicity (older age, native status) and epidemicity (younger age, sputum smear positivity) were identified with high confidence (P < 0.001). Long-term epidemic success also correlated with the ability of Euro-American and Beijing MTBC lineages to cause active pulmonary infection, independent of patient age and country of origin. Our results demonstrate how important insights into the transmission dynamics of tuberculosis can be gained from active surveillance data.