Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens

Abstract

The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.

FIG. 1.

Representative patterns of a pansusceptible strain (lane 2), resistant strains (lanes 3 to 6), and mixtures of strains (lanes 7 to 11), as well as a negative control (lane 1). The positions of the oligonucleotides are given. The targeted genes and specificity are shown from the bottom to the top as follows. The MTBDRsl assay: CC, conjugate control; AC, amplification control; TUB, MTBC-specific control; gyrA amplification control; gyrA wild-type probes WT1 to -3 located at codons 85 to 97; gyrA mutant probes MUT1, -2, and -3A to -D with mutations in gyrA codons A90V (GCG-GTG), S91P (TCG-CCG), D94A (GAC-GCC), D94N/Y (GAC-AAC/TAC), D94G (GAC-GGC), and D94H (GAC-CAC); rrs amplification control; rrs wild-type probes WT1 and WT2; rrs mutation probes MUT1 with a A1401G exchange and MUT2 with a G1484T exchange; embB amplification control; embB gene wild-type probe WT1 spanning the region around codon 306; embB mutant probes MUT1A with mutation M306I (ATG-ATA) and MUT1B with mutation M306V (ATG-GTG). The pansusceptible isolate (lane 2) was positive with all WT gyrA, rrs, and embB probes of the MTBDRsl assay. The isolates in lane 3 to 6 showed FLQ resistance by different positive gyrA MUT probes. Two isolates (lanes 4 and 5) show further EMB resistance, and the isolate in lane 6 shows resistance to EMB, AM, and CM. The latter is an XDR strain, since rifampin and isoniazid resistance was also found (data not shown). In lanes 7 to 11, mixtures of strains are shown as follows: lane 7, gyrA WT and gyrA MUT3C; lane 8, gyrA WT, gyrA MUT2, and gyrA MUT3C; lane 9, gyrA WT, gyrA MUT3B, and gyrA MUT3C; lane 10, rrs WT1 and rrs MUT1; lane 11, rrs WT1 and rrs MUT1B.