ABSTRACTAs a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQr) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance inMycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context.gyrAandgyrBsequencing, performed prospectively onM. tuberculosisclinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQrstrains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates withgyrAmutations were FQr, whereas only 36% of those withgyrBmutations were FQr. Only 12% of the FQrisolates had a single mutation ingyrB. CombinedgyrAandgyrBsequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results ofgyrAandgyrBsequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combinedgyrAandgyrBsequencing in the investigation of FQ susceptibility inM. tuberculosisand challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.
Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique