Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDR sl Assays

Abstract

In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

Keywords: Hain GenoType MTBDRsl; Mycobacterium tuberculosis; fluoroquinolones.

FIG 1

Line probe assays consist of oligonucleotide probes that are immobilized on a nitrocellulose strip. This diagram depicts the region of gyrA targeted by the MTBDRsl assay (numbers refer to codons). The binding of a mutant probe (MUT1-3D) that targets the three codons highlighted in dark gray (90, 91, and 94; the corresponding nucleotide and amino acid changes are shown under the respective codons) and/or lack of binding of a wild-type probe (WT1-3) is interpreted as genotypic fluoroquinolone resistance, provided that all control bands of the assay, including the one for gyrA, are positive. The diagram was based on the package insert of version 1 of the assay (40). The exact design of the wild-type probes is regarded as a trade secret by Hain Lifescience, so it is unclear whether the WT3 band covers all three nucleotides of codon 92. The mutant probes cannot be depicted, as they also constitute a trade secret. Versions 1 and 2 of the assay are identical with regard to the gyrA region; thus, results from version 1, which was used for most experiments in this study, should also be valid for version 2 (4).

FIG 2

Maximum likelihood phylogeny based on 3,710 single nucleotide variants differentiating all 95 Uganda and Uganda-like M. tuberculosis strains. The numerical code shown corresponds to the lineage classification by Coll et al. (41). Phylogenetic variants in the gyrA fluoroquinolone resistance-determining region are color coded. The 28 T80A+A90G strains (or variants thereof) formed a monophyletic group and were consistently susceptible to ofloxacin and other fluoroquinolones when tested (see Table S1 in the supplemental material). This group included the novel T80A+A90C double mutant and, importantly, the T80A+A90G+D94G triple mutant, which comprised the high-confidence D94G resistance mutation that was genetically linked to the double mutations (as opposed to occurring in the same population as a mixed infection) (12). This was in line with a recent report by Pantel et al., who suggested that classical resistance mutations may not cause resistance in a T80A+A90G background, whereas a study by Brossier et al. found that this combination of mutations did correlate with ofloxacin resistance (6, 15). It is therefore possible that these triple mutants have MICs close to the epidemiological cutoff value for ofloxacin, although more data are required to confirm this hypothesis (42, 43).