title: Molecular diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis.
abstract: As 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 (FQ(r)) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance in Mycobacterium 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. gyrA and gyrB sequencing, performed prospectively on M. tuberculosis clinical 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 FQ(r) strains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates with gyrA mutations were FQ(r), whereas only 36% of those with gyrB mutations were FQ(r). Only 12% of the FQ(r) isolates had a single mutation in gyrB. Combined gyrA and gyrB sequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results of gyrA and gyrB sequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combined gyrA and gyrB sequencing in the investigation of FQ susceptibility in M. tuberculosis and challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Bernard
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Veziris
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Brossier
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Wladimir
authors.initials: W
authors.lastname: Sougakoff
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Jarlier
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Robert
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, Team E13 (Bacteriology), Paris, France AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, Paris, France alexandra.aubry@upmc.fr.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Aubry
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D024881:Drug Resistance, Bacterial; D024901:Drug Resistance, Multiple, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D027081:DNA Gyrase