Detection by GenoType MTBDRsl 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 FQ-susceptible 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.

FIG. 1.

Hybridization patterns obtained with the GenoType MTBDRsl assay. The controls, targeted genes, and mutations are given to the left of the figure: CC, conjugate control; AC, amplification control (23S rRNA); TUB, M. tuberculosis complex-specific control (23S rRNA); Control gyrA, control for gyrA amplification; gyrA WT1 to WT3, gyrA wild-type probes located in regions from codons 85 to 97; gyrA MUT1 to MUT3D, gyrA mutant probes testing mutations for codons A90V, S91P, D94A, D94N/Y, D94G, and D94H; Control rrs, control for rrs amplification; rrs WT1 and WT2, rrs wild-type probes located in regions for nucleotides 1401 and 1402 and nucleotide 1484; rrs MUT1 and MUT2, rrs mutant probes testing mutations for A1401G and G1484T; Control embB, amplification control for embB; embB WT, embB wild-type probe located in codon 306; embB MUT1A and MUT1B, mutant probes testing mutations M306V and M306I (base exchange at codon 306 [ATG→ATA]); CM, colored marker. Typical hybridization patterns were obtained and are shown in the figure as follows: lane 1, H37Rv (wild type); lane 2, gyrA D94G; lane 3, rrs A1401G; lane 4, embB M306V; lane 5, embB M306I with base exchange ATG→ATC; lane 6, gyrA D94G+wt (gyrA D94G and wild type) and rrs A1401G and embB M306V; lane 7, gyrA A90V plus gyrA D94G (mixture of two mutants) and rrs A1401G; lane 8, MDR with wild-type gyrA, rrs, and embB genes showing a weak signal intensity for embB wild-type probe, and a weak false-positive hybridization signal for rrs MUT2 (G1484T) mutation probe.