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      Molecular investigation of resistance to second line injectable drugs in multidrug-resistant clinical isolates of Mycobacterium tuberculosis in France

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          Abstract

          The second line injectable drugs (SLID, i.e. amikacin, kanamycin, capreomycin) are key drugs for the treatment of multidrug-resistant tuberculosis. Mutations in rrs region 1400, tlyA and eis promoter are associated with resistance to SLID, to capreomycin and to kanamycin respectively. In this study, the sequencing data of SLID resistance-associated genes were compared to the results of phenotypic drug susceptibility testing by the proportion method for the SLID in 206 multidrug-resistant clinical isolates of Mycobacterium tuberculosis collected in France. Among the 153 isolates susceptible to the 3 SLID, 145 showed no mutation, 1 harbored T1404C plus G1473A mutations in rrs and 7 had an eis promoter mutation. Among the 53 strains resistant to at least 1 of the SLID, mutations in rrs accounted for resistance to amikacin, capreomycin and kanamycin for 81%, 75% and 44% isolates, respectively, while mutations in eis promoter were detected in 44% of the isolates resistant to kanamycin. By contrast, no mutations in tlyA were observed in the isolates resistant to capreomycin. The discrepancies observed between the genotypic (on the primary culture) and phenotypic drug susceptibility testing were explained by i) resistance to SLID with MICs close to the critical concentration used for routine DST and not detected by phenotypic testing (n=8, 15% of SLID-resistant strains), ii) low-frequency heteroresistance not detected by sequencing of drug resistance-associated genes on the primary culture (n=8, 15% of SLID-resistant strains), and iii) to other resistance mechanisms not yet characterized (n=7, 13% of SLID-resistant strains).

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          Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.

          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.
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            Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

            The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)
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              Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to Amikacin, Kanamycin and Capreomycin: A Systematic Review

              Background Rapid molecular diagnostics for detecting multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) primarily identify mutations in Mycobacterium tuberculosis (Mtb) genes associated with drug resistance. Their accuracy, however, is dependent largely on the strength of the association between a specific mutation and the phenotypic resistance of the isolate with that mutation, which is not always 100%. While this relationship is well established and reliable for first-line anti-TB drugs, rifampin and isoniazid, it is less well-studied and understood for second-line, injectable drugs, amikacin (AMK), kanamycin (KAN) and capreomycin (CAP). Methodology/Principal Findings We conducted a systematic review of all published studies evaluating Mtb mutations associated with resistance to AMK, KAN, CAP in order to characterize the diversity and frequency of mutations as well as describe the strength of the association between specific mutations and phenotypic resistance in global populations. Our objective was to determine the potential utility and reliability of these mutations as diagnostic markers for detecting AMK, KAN and CAP resistance. Mutation data was reviewed for 1,585 unique clinical isolates from four continents and over 18 countries. Mutations in the rrs, tlyA, eis promoter and gidB genes were associated with AMK, KAN and/or CAP resistance. Conclusions/Significance The rrs A1401G mutation was present in the majority of AMK, KAN and CAP resistant Mtb strains reviewed, but was also found in 7% of CAP susceptible strains. The 1401 mutation alone, however, was not found with sufficient frequency to detect more than 70–80% of global Mtb strains resistant to AMK and CAP, and 60% of strains resistant to KAN. Additional mutations in the rrs, eis promoter, tlyA and gidB genes appear to be associated with resistance and could improve sensitivity and specificity of future diagnostics.
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                Author and article information

                Journal
                Antimicrobial Agents and Chemotherapy
                Antimicrob. Agents Chemother.
                American Society for Microbiology
                0066-4804
                1098-6596
                November 28 2016
                : AAC.01299-16
                Article
                10.1128/AAC.01299-16
                5278698
                27895017
                298ab063-e3f9-4e26-8c0c-78f1a33401e0
                © 2016
                History

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