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Background: Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by Mycobacterium tuberculosis. RIF resistance is caused by mutations in rpoB, at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in rpoABC genes have been shown to alleviate the fitness cost of rpoB:S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations. Objectives: We performed an association study on a globally representative set of 4,309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations. Results and conclusions: Of the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in rpoABC for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to rpoB:S450L. However, an outbreak of 22 closely related isolates each carried three rpoB mutations, the rare RIFR markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of rpoABC mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional rpoABC mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant M. tuberculosis strains with restored fitness, which pose a greater risk of causing resistant outbreaks.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Drug-resistant tuberculosis poses a persistent public health threat. The ReSeqTB platform is a collaborative, curated knowledgebase, designed to standardize and aggregate global Mycobacterium tuberculosis complex (MTBC) variant data from whole genome sequencing (WGS) with phenotypic drug susceptibility testing (DST) and clinical data. We developed a unified analysis variant pipeline (UVP) ( https://github.com/CPTR-ReSeqTB/UVP ) to identify variants and assign lineage from MTBC sequence data. Stringent thresholds and quality control measures were incorporated in this open source tool. The pipeline was validated using a well-characterized dataset of 90 diverse MTBC isolates with conventional DST and DNA Sanger sequencing data. The UVP exhibited 98.9% agreement with the variants identified using Sanger sequencing and was 100% concordant with conventional methods of assigning lineage. We analyzed 4636 publicly available MTBC isolates in the ReSeqTB platform representing all seven major MTBC lineages. The variants detected have an above 94% accuracy of predicting drug based on the accompanying DST results in the platform. The aggregation of variants over time in the platform will establish confidence-graded mutations statistically associated with phenotypic drug resistance. These tools serve as critical reference standards for future molecular diagnostic assay developers, researchers, public health agencies and clinicians working towards the control of drug-resistant tuberculosis.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.

Background: Recent pandemic of coronavirus SARS-CoV-2 (COVID-19) caused limitations in the country's strategies to fight against mycobacterial infections. The aim of this study was to compare the suspected tuberculosis (TB) pulmonary patients before and during the COVID-19 pandemic (January 2018-December 2021) who were referred to the National Reference TB Laboratory (NRL TB), Tehran, Iran. The mycobacterial isolated strains were identified and compared with previous data. Methods: A total of 16,899 clinical samples collected from 7041 suspected pulmonary TB patients were received from 2018 to 2021. Primary isolation of Mycobacterium isolates was done on Löwenstein-Jensen medium. Then, the DNA was extracted from acid-fast bacillus culture-positive samples and identification was performed by IS6110, Hsp65, and 16S-23S rRNA genes using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and nested PCR methods. Results: A total of 11679 specimens (69.1%) from 4866 suspected TB patients were collected in 2018-2019 and 5220 specimens (30.8%; from 2175 patients) in 2020-2021. Out of 11679 specimens, 2046 samples that belong to 852 patients were infected with Mycobacterium tuberculosis, and the remaining were non-TB Mycobacterium (NTM) species (n = 244) isolated from 102 patients. The cultures for 12894 specimens were either negative (76.3%) or contaminated (845/16899; 5%). A comparison of the total number of patients who were referred for diagnosis and treatment (954/666 patients, P > 0.05) showed a 30.1% reduction during the COVID-19 pandemic. Although, with these low number of patients, the significant increases of NTM species (P < 0.05) among suspected TB pulmonary patients were observed. Besides, new species of NTM, for example, Mycobacterium peregrinum and Mycobacterium montefiorense, were detected. For the past 20 years, these two species were not reported from pulmonary patients in Iran. Conclusions: During the pandemic of COVID-19, the TB diagnosis network became irregular, as a consequence, many patients could not reach the treatment center, and this could increase the circulation of mycobacterial diseases (TB and NTM). The study shows the emergence of new opportunistic NTM species also. [ABSTRACT FROM AUTHOR]

Objective To assess the problem of multidrug-resistant tuberculosis (MDR-TB) throughout Belarus and investigate the associated risk factors. Methods In a nationwide survey in 2010-2011, 1420 tuberculosis (TB) patients were screened and 934 new and 410 previously treated cases ofTB were found to meet the inclusion criteria. Isolates of Mycobacterium tuberculosis from each eligible patient were tested for susceptibility to anti-TB drugs. Sociobehavioural information was gathered in interviews based on a structured questionnaire. Findings MDR-TB was found in 32.3% and 75.6% of the new and previously treated patients, respectively, and, 11.9% of the 612 patients found to have MDR-TB had extensively drug-resistant TB (XDR-TB). A history of previous treatment for TB was the strongest independent risk factor for MDR-TB (odds ratio, OR: 6.1; 95% confidence interval, CI: 4.8-7.7). The other independent risk factors were human immunodeficiency virus (HIV) infection (OR: 2.2; 95% CI: 1.4-3.5), age < 35 years (OR: 1.4; 95% CI: 1.0-1.8), history of imprisonment (OR: 1.5; 95% CI: 1.1-2.0), disability sufficient to prevent work (OR: 1.9; 95% CI: 1.2-3.0), alcohol abuse (OR: 1.3; 95% CI: 1.0-1.8) and smoking (OR: 1.5; 95% CI: 1.1-2.0). Conclusion MDR-TB is very common among TB patients throughout Belarus. The numerous risk factors identified for MDR-TB and the convergence of the epidemics of MDR-TB and HIV infection call not only for stronger collaboration between TB and HIV control programmes, but also for the implementation of innovative measures to accelerate the detection of TB resistance and improve treatment adherence. Objectif Evaluer le probleme de la tuberculose multiresistante (TB-MR) sur le territoire bielorusse et explorer les facteurs de risque associes. Methodes Au cours d'une enquete nationale menee en 2010-2011, 1420 cas de tuberculose (TB) ont ete depistes et 934 cas nouveaux ainsi que 410 cas precedemment traites ont ete juges conformes aux criteres d'inclusion. Des isolats de Mycobacterium tuberculosis provenant de chaque patient admissible ont ete testes pour leur sensibilite envers les medicaments antituberculeux. Des informations sociocomportementales ont ete recueillies lors d'entretiens bases sur un questionnaire structure. Resultats La TB-MR a ete detectee dans respectivement 32,3% et 75,6% des cas nouveaux et des cas traites anterieurement, et 11,9% des 612 patients porteurs de la TB-MR presentaient une forme de tuberculose ultraresistante (TB-UR). Un historique de traitement anterieur pour la TB representait le principal facteur de risque independant pour la TB-MR (rapport des cotes, RC: 6,1; intervalle de confiance a 95%, IC: 4,8 a 7,7). Les autres facteurs de risque independants comprenaient l'infection par le virus d'immunodeficience humaine (VIH) (RC: 2,2; IC a 95%: 1,4 a 3,5), l'age Conclusion La TB-MR est tres frequente chez les patients atteints de tuberculose en Belarus. Les nombreux facteurs de risque identifies pour la TB-MR et la convergence entre l'epidemie de TB-MR et l'infection par le VIH exigent non seulement de renforcer la collaboration entre les programmes antituberculeux et de lutte contre le VIH, mais aussi la mise en ceuvre de mesures innovantes pour accelerer la detection de la resistance a la tuberculose et ameliorer l'observance du traitement. Objetivo Evaluar el problema de la tuberculosis multirresistente (TB-MR) en Bielorrusia e investigar los factores de riesgo asociados. Metodos En una encuesta a nivel nacional llevada a cabo entre 2010 y 2011, se evaluo a 1420 pacientes con tuberculosis (TB) y se considero que 934 nuevos casos de TB y 410 casos de TB previamente tratados reunian los criterios de inclusion. Se analizaron cepas de Mycobacterium tuberculosis de cada paciente elegible con el fin de determinar la susceptibilidad a los farmacos antituberculosos. Se recopilo informacion socioconductual mediante entrevistas basadas en un cuestionario estructurado. Resultados Se detecto TB-MR en el 32,3% y el 75,6% de los pacientes de nuevo diagnostico y tratados previamente, respectivamente, y se observo que el 11,9% de los 612 pacientes con TB-MR presentaba tuberculosis ultrarresistente (TB-XR). Los antecedentes de tratamiento previo de la TB resultaron ser el factor de riesgo independiente que mas predispone a sufrir TB-MR (razon de posibilidades, OR: 6,1; intervalo de confianza del 95%, IC: 4,8-7,7). Los demas factores de riesgo independientes fueron el virus de la inmunodeficiencia humana (VIH) (OR: 2,2; IC del 95%: 1,4-3,5), edad < 35 anos (OR: 1,4; IC del 95%: 1,0-1,8), antecedentes de encarcelamiento (OR: 1,5; IC del 95%: 1,1-2,0), incapacidad suficiente para impedir el trabajo (OR: 1,9; IC del 95%: 1,2-3,0), alcoholismo (OR: 1,3; IC del 95%: 1,0-1,8) y tabaquismo (OR: 1,5; IC del 95%: 1,1-2,0). Conclusion La TB.MR es muy frecuente entre los pacientes con tuberculosis en Bielorrusia. Los numerosos factores de riesgo identificados para la TB-MR, unidos a la convergencia de las epidemias de TB-MR y la infeccion por el VIH, exigen no solo una mayor colaboracion entre los programas de control de la TB y del VIH, sino tambien la aplicacion de medidas innovadoras destinadas a acelerar la deteccion de la resistencia a la TB y mejorar el cumplimiento terapeutico., Introduction The increasing prevalence of infection with drug-resistant Mycobacterium tuberculosis represents a global public health emergency: At any given time, about 630 000 people in the world are thought to [...]

The rise in multidrug-resistant tuberculosis makes it increasingly important that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce clinically meaningful and technically reproducible results. Unfortunately, this is not always the case because mycobacteriology specialists have not followed generally accepted modern principles for the establishment of susceptibility breakpoints for bacterial and fungal pathogens. These principles specifically call for a definition of the minimum inhibitory concentrations (MICs) applicable to organisms without resistance mechanisms (also known as wild-type MIC distributions), to be used in combination with data on clinical outcomes, pharmacokinetics and pharmacodynamics, in a series of papers the authors have defined tentative wild-type MIC distributions for M. tuberculosis and hope that other researchers will follow their example and provide confirmatory data. They suggest that some breakpoints are in need of revision because they either (i) bisect the wild-type distribution, which leads to poor reproducibility in antimicrobial susceptibility testing, or (ii) are substantially higher than the MICs of wild-type organisms without supporting clinical evidence, which may result in some strains being falsely reported as susceptible. The authors recommend, in short, that susceptibility breakpoints for antituberculosis agents be systematically reviewed and revised, if necessary, using the same modern tools now accepted for all other bacteria and fungi by the scientific community and by the European Medicines Agency and the European Centre for Disease Prevention and Control. For several agents this would greatly improve the accuracy and reproducibility of antimicrobial susceptibility testing of M. tuberculosis. [TEXT NOT REPRODUCIBLE IN ASCII.] Remise en question d'un dogme: points de rupture des tests de sensibilite aux antimicrobiens de la Mycobacterium tuberculosis En raison de l'augmentation de la tuberculose multiresistante, il est de plus en plus important que les tests de sensibilite aux antimicrobiens de la Mycobacterium tuberculosis produisent des resultats cliniquement significatifs et techniquement reproductibles. Malheureusement, ce n'est pas toujours le cas, les specialistes en mycobacteriologie n'appliquant pas les principes modernes generalement acceptes de raise en place de points de rupture de la sensibilite aux pathogenes bacteriens et fongiques. Ces principes requierent plus particulierement une definition des concentrations minimales inhibitrices (CMI) applicables aux organismes sans mecanismes de resistance (egalement appeles distributions de CMI de souche sauvage), devant etre utilises en combinaison avec les donnees sur les resultats cliniques, la pharmacocinetique et la pharmacodynamique. Dans plusieurs publications, les auteurs ont defini les distributions experimentales de CMI de souche sauvage pour la M. tuberculosis et esperent que d'autres chercheurs suivront leur exemple et fourniront des donnees de confirmation. Ils suggerent que certains points de rupture doivent etre revises, car ils (i) divisent la distribution de souche sauvage, ce qui entraine une mauvaise reproductibilite des tests de sensibilite aux antimicrobiens, ou (ii) sont sensiblement plus eleves que les CMI des organismes de souche sauvage sans confirmer les preuves cliniques, certaines souches pouvant etre e tort reporteees comme sensibles. En resume, les auteurs recommandent que les points de rupture de la sensibilite aux agents antituberculeux soient eiystematiquement examines et revises, en utilisant si necessaire les memes outils modernes qui sont desormais acceptes pour tous les autres bacteries et champignons par la communaute scientifique, l'Agence europeenne des medicaments et le Centre europeen de prevention et de controle des maladies. Pour plusieurs agents, cela ameliorerait considerablement la precision et la reproductibilite des tests de sensibilite aux antimicrobiens de la M. tuberculosis. [TEXT NOT REPRODUCIBLE IN ASCII.] Desafiar un dogma: valores limite en pruebas de sensibilidad antimicrobiana para Mycobacterium tuberculosis El aumento en la tuberculosis resistente a la medicacion nace que resulte cada vez mes importante que las pruebas de sensibilidad antimicrobiana de Mycobactefium tuberculosis produzcan resultados clinicamente significativos y tecnicamente reproducibles. Desgraciadamente, este no es siempre el caso, porque los especialistas en micobacteriologia no hart seguido los principios modernos generalizados para establecer los valores limite de la sensibilidad para patogenos bacterianos y micoticos. Estos principios requieren especificamente una definicion de las concentraciones inhibitorias minimas (ClM) aplicables a los organismos sin mecanismos de resistencia (tambien conocidos como distribuciones de CIM naturales), para usarla en combinacion con los datos sobre resultados clinicos, farmacocineticos y farmacodinemicos. En una serie de articulos, los autores han definido de manera provisional las distribuciones de ClM naturales para M. tuberculosis y esperan que otros investigadores sigan en el futuro su ejemplo y proporcionen datos que lo confirmen. Sugieren que algunos valores limite necesitan una revision porque o (i) bisecan la distribucion natural, lo que provoca una reproducibilidad pobre en las pruebas de sensibilidad antimicrobiana, o (ii) son considerablemente mes elevados que los CIM de los organismos naturales sin aportar pruebas clinicas, lo que puede provocar que algunas cepas sean consideradas de manera equivocada como susceptibles. En resumen, los autores recomiendan que se estudien y se revisen sistemeticamente los valores limite de la sensibilidad de los agentes antituberculosos, si fuera necesario, usando las mismas herramientas modernas aceptadas actualmente por la comunidad cientifica, la Agencia Europea de Medicamentos y el Centro Europeo para la Prevencion y Control de Enfermedades para el resto de bacterias y hongos. Segun varios agentes, esto mejoraria considerablemente la precision y reproductibilidad de las pruebas de sensibilidad antimicrobiana de M. tuberculosis., Introduction The emergence of multidrug-resistant (MDR) and extensively drug-resistant tuberculosis makes it necessary to ensure that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce results that are clinically meaningful and technically [...]

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen. [ABSTRACT FROM AUTHOR]