Detection by GenoType MTBDR sl Test of Complex Mechanisms of Resistance to Second-Line Drugs and Ethambutol in Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates

Abstract

The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, and second-line aminoglycosides (amikacin and kanamycin) and cyclic peptide (capreomycin) in Mycobacterium tuberculosis. A set of 41 multidrug-resistant (MDR) M. tuberculosis strains, 8 extensively drug-resistant (XDR) M. tuberculosis strains, and 3 non-MDR M. tuberculosis strains were tested by the MTBDRsl test and by DNA sequencing of the resistance-determining regions in gyrA and gyrB (fluoroquinolones [FQ]), rpsL (streptomycin), rrs and tlyA (aminoglycosides and/or cyclic peptide), and embB (ethambutol). The sensitivity and specificity of the MTBDRsl test were as follows: 87% and 96%, respectively, for fluoroquinolones; 100% for both for amikacin; 77% and 100%, respectively, for kanamycin, 80% and 98%, respectively, for capreomycin; and 57% and 92%, respectively, for ethambutol. Analysis of the discrepant results indicated that three FQ-resistant strains (including one XDR strain) with mutations in gyrB were missed by the MTBDRsl test and that one FQsusceptible strain, identified as resistant by the MTBDRsl test, had a double mutation (T80A-A90G) in GyrA that did not confer resistance to FQ. Five strains (including two XDR strains) without mutations in rrs were monoresistant to aminoglycosides or cyclic peptide and were missed by the MTBDRsl test. Finally, 12/28 ethambutol-resistant strains had no mutation at codon 306 in embB, while 2/24 ethambutol-susceptible strains had such a mutation. In conclusion, the MTBDRsl test efficiently detects the most common mutations involved in resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol and accurately assesses susceptibility to amikacin. However, due to mutations not included in the test (particularly in gyrB) or resistance mechanisms not yet characterized (particularly those related to ethambutol resistance and to monoresistance to aminoglycosides or cyclic peptide), the wild-type results yielded by the MTBDRsl test should be confirmed by drug susceptibility testing.

Evaluation of Two Molecular Assays for Rapid Detection of Mycobacterium tuberculosis Resistance to Fluoroquinolones in High-Tuberculosis and -Multidrug-Resistance Settings

“…Assays for the detection of mutations in genes associated with resistance to ethambutol (EMB), aminoglycosides/cyclic peptides (AG/CP), and FQ have been developed by Hain Lifescience, GmbH (Nehren, Germany), evaluated in low-incidence settings in two recent studies. and demonstrated good sensitivity and specificity (5,17). Pyrosequencing assays have also proved effective for the detection of mutations in genes associated with the drug resistance (19).…”

Performance Assessment of The Polymerase Chain Reaction-restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of XDR Tuberculosis in Clinical Isolates of Mycobacterium Tuberculosis

“…These results are quite compatible with the results of this research. Other researchers found mutation in codon 306 in less than 10 sensitive strains (11,17).…”

“…Sensitivity and specificity of MTBDRsl test for EMB was 57% and 92%, respectively (11). Analysis showed conflicting results; among the 28 strains resistant to EMB, 16 strains were reported mutations in the embB gene.…”

“…Totally, MTBDRs1 test (57%) identified strains resistant to EMB. Also, among the 24 strains susceptible to EMB, 22 cases had no mutation in codon 306 embB gene and two strains susceptible to EMB were associated with mutations (11).…”

“…Thus, culture results do not comply fully with the molecular results (determine the occurrence of mutations with low sensitivity, besides high specificity). The precision of determining resistant strains by molecular method has been always debated in previous research (3)(4)(5)(6)11). In most of the studies conducted by other researchers, no mutation in sensitive strains was found (10).…”