Dataset Information

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing.

ABSTRACT: Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.

SUBMITTER: Engstrom A

PROVIDER: S-EPMC3372151 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing.

Engström Anna A Morcillo Nora N Imperiale Belen B Hoffner Sven E SE Juréen Pontus P

Journal of clinical microbiology 20120329 6

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following l ...[more]

PMID: 22461677

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Project description:BackgroundDrug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls.ResultsThe majority of isoniazid and rifampin resistant strains had mutations in katG315 (71.9%) and rpoB531 (50%). However, rpoB mutations in codons 511, 516 and 533 were also detected in five rifampin susceptible strains. MIC determinations revealed low-level rifampin resistance for those strains. Thus, the sensitivity and specificity of sequencing of katG for detection of drug resistance were 86.7% and 100% and for sequencing of rpoB 100% and 93.8%, respectively.Strikingly, none of the streptomycin resistant strains had mutations in rrs, but 47.5% harboured mutations in rpsL. Further changes were detected in gidB. Among ethambutol resistant strains 46.7% had mutations at embB306. Pyrazinamide resistant strains displayed a variety of mutations throughout pncA. The specificities of sequencing of rpsL, embB and pncA for resistance detection were high (96-100%), whereas sensitivities were lower (48.8%, 73.3%, 70%).ConclusionsOur study reveals a good correlation between data from molecular and phenotypic resistance testing in this high-incidence setting. However, the fact that particular mutations in rpoB are not linked to high-level resistance is challenging and demonstrates that careful interpretation of molecular resistance assays is mandatory. In addition, certain variations, especially in gidB, appear to be phylogenetically informative polymorphisms rather than markers for drug resistance.

Dataset Information

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing.

Publications

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing.

Similar Datasets

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