Jureen P. Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing. Journal of clinical microbiology. 2012; 50(6):2026–33. Epub 2012/03/31. doi

Conventional phenotypic drug susceptibility testing (DST) methods forMycobacterium 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 fol-lowing 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 ofM. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistantM. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistantM. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screen-ing inM. tuberculosis. Tuberculosis (TB), caused by Mycobacterium tuberculosis, per-sists as a global public health problem.Drug-resistant bacteria, especially multidrug-resistant (MDR) M. tuberculosis and exten-sively drug-resistant (XDR) M. tuberculosis, pose challenges for the prevention and control of this deadly disease (64). MDR M. tuberculosis is defined as an isolate resistant to at least the twomai