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Journal ArticleDOI

Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

TL;DR: The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex.
Abstract: The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.
Citations
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Journal ArticleDOI
TL;DR: This article presents an updated review of the mechanisms and molecular basis of drug resistance in M. tuberculosis and comments on the several gaps in current knowledge of the molecular mechanisms of drug Resistance to the main classical and new anti-TB drugs.
Abstract: Tuberculosis (TB) remains one of the leading public health problems worldwide. Declared as a global emergency in 1993 by the WHO, its control is hampered by the emergence of multidrug resistance (MDR), defined as resistance to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. More recently, severe forms of drug resistance such as extensively drug-resistant (XDR) TB have been described. After the discovery of several drugs with anti-TB activity, multidrug therapy became fundamental for control of the disease. Major advances in molecular biology and the availability of new information generated after sequencing the genome of Mycobacterium tuberculosis increased our knowledge of the mechanisms of resistance to the main anti-TB drugs. Better knowledge of the mechanisms of drug resistance in TB and the molecular mechanisms involved will help us to improve current techniques for rapid detection and will also stimulate the exploration of new targets for drug activity and drug development. This article presents an updated review of the mechanisms and molecular basis of drug resistance in M. tuberculosis. It also comments on the several gaps in our current knowledge of the molecular mechanisms of drug resistance to the main classical and new anti-TB drugs and briefly discusses some implications of the development of drug resistance and fitness, transmission and pathogenicity of M. tuberculosis.

472 citations

Journal ArticleDOI
TL;DR: Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin, and evidence of positive selection in an additional 39 genomic regions in resistant isolates was found.
Abstract: M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

385 citations

01 Sep 2013
TL;DR: In this article, the authors identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains of M. tuberculosis and identified positive selection in 39 genomic regions in resistant isolates, encoding components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways.
Abstract: M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

315 citations

Journal ArticleDOI
TL;DR: This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug- resistant phenotypes and has implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.
Abstract: 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.

266 citations

Journal ArticleDOI
TL;DR: The mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis are discussed, which will help to explore new targets for drug activity and development.
Abstract: Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis.

264 citations


Cites background from "Molecular Detection of Mutations As..."

  • ...These mutations were found in up to 80% of clinical isolates showing low-level resistance to kanamycin [88,89]....

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References
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Journal ArticleDOI
14 Jan 1994-Science
TL;DR: Results suggest that InhA is likely a primary target of action for INH and ETH and that it may be involved in mycolic acid biosynthesis.
Abstract: Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.

1,383 citations


"Molecular Detection of Mutations As..." refers background in this paper

  • ...Nineteen of the INH-resistant isolates were wild type at both these locations and also in the region of the inhA structural gene where resistance-associated mutations have been reported (data not shown) (3, 4)....

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01 Mar 2011
TL;DR: This standard includes susceptibility testing procedures for Mycobacterium tuberculosis complex (MTBC), clinically significant slowly and rapidly growing mycobacterial species, Nocardia spp.
Abstract: Clinical and Laboratory Standards Institute standard M24—Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes includes susceptibility testing procedures for Mycobacterium tuberculosis complex (MTBC), clinically significant slowly and rapidly growing mycobacterial species, Nocardia spp., and other aerobic actinomycetes. Also included in this standard are recommendations for selecting agents for first-line and second-line drug testing, organism group–specific methodologies, reporting recommendations, and organism quality control criteria. Recommendations regarding agent selection for testing mycobacteria are based primarily on published guidelines. For testing MTBC, M24 recognizes agar proportion as the reference methodology on which all other methodologies are based. In addition, this standard includes recommendations for using commercial broth susceptibility methods with shorter incubation times, which are now in widespread use for MTBC susceptibility testing, and information on molecular methods for detecting drug resistance and their integration with culture-based methods. Clinical and Laboratory Standards Institute (CLSI). Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes. 3rd ed. CLSI standard M24 (ISBN 978-1-68440-025-6 [Print]; 978-1-68440-026-3 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2018. The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to become one, or to request a copy of the catalog, contact us at: Telephone: +1.610.688.0100; Fax: +1.610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org. This is a preview of "CLSI M24-3E". Click here to purchase the full version from the ANSI store.

1,341 citations

Journal ArticleDOI

608 citations


"Molecular Detection of Mutations As..." refers background in this paper

  • ...Known mechanisms of KAN resistance are caused by mutations in rrs or the promoter region of eis which prevent the drug from inhibiting translation (15, 45)....

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  • ...Alterations within the rrs locus also play a significant role in KAN resistance, with the 65 detected RRDR mutations accounting for 60% of substitutions within KAN-resistant isolates (1, 15, 34)....

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Journal ArticleDOI
TL;DR: Sandgren et al. as mentioned in this paper described a new comprehensive resource on drug resistance mutations in M. tuberculosis, which they used to study drug-resistant mutations in drug-free tuberculosis.
Abstract: Andreas Sandgren and colleagues describe a new comprehensive resource on drug resistance mutations inM. tuberculosis.

523 citations

Journal ArticleDOI
TL;DR: The cloned and sequenced wild-type gyrA and gyrB genes will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.
Abstract: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.

469 citations


"Molecular Detection of Mutations As..." refers background or result in this paper

  • ...The most common resistance-associated mutations within gyrA are found within the QRDR at codons 94 and 90, as was the case with our study (35, 40)....

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  • ...It has been well established that mutations within gyrA, which encodes subunit A of the DNA gyrase, are often associated with resistance (33, 35, 40)....

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