title: 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.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. anna.engstrom@ki.se
authors.forename: Anna
authors.initials: A
authors.lastname: Engström
authors.forename: Nora
authors.initials: N
authors.lastname: Morcillo
authors.forename: Belen
authors.initials: B
authors.lastname: Imperiale
authors.forename: Sven E
authors.initials: SE
authors.lastname: Hoffner
authors.forename: Pontus
authors.initials: P
authors.lastname: Juréen
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins