Identification of mutations in genes associated with metronidazole resistance and susceptibility in Trichomonas vaginalis.

Abstract

Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen worldwide. Although, metronidazole cure rates are high for T. vaginalis infection, resistance has been reported. Mutations in the nitroreductase genes of T. vaginalis have also been implicated in metronidazole resistance. Therefore, the aim of this study was to detect mutations in the nitroreductase genes and link the mutations to metronidazole resistance patterns in T. vaginalis isolated from South African pregnant women, a currently under-researched area. Vaginal swabs were collected from 362 pregnant women recruited from the King Edward VIII hospital antenatal clinic in Durban from October 2018 to March 2019. The swabs were cultured in Diamonds TYM medium to obtain pure isolates of T. vaginalis. Pure isolates were sub-cultured and subjected to metronidazole susceptibility assays. The susceptibility assays were conducted under aerobic and anaerobic conditions. DNA was extracted from the pure isolates to perform the polymerase chain reaction (PCR) assays for the detection of the nitroreductase genes and the PFOR gene. The PCR amplicons were sequenced using the Sanger approach in order to identify mutations associated with resistance. A total of 21/362 (5.8%) pregnant women tested positive for T. vaginalis infection. Of the 21 T. vaginalis isolates tested for anaerobic metronidazole susceptibility, 9.5% (2/21) had an MIC of 4 μg/ml (resistant), 38.1% (8/21) had an MIC of 2 μg/ml (intermediate) and 52.4% (11/21) had an MIC ≤ 1 μg/ml (susceptible). For the ntr2 gene, susceptible and resistant isolates carried mutations which were absent in the intermediate isolates. The susceptible isolates carried mostly insertion mutations and the resistant isolate carried substitution mutations. Some deletion mutations were also observed in the ntr2 gene. For the ntr3 gene, two substitution mutations were observed in the intermediate isolate only. In the ntr4 gene, substitution mutations were only observed in the susceptible and resistant isolate. For the ntr5 gene, substitution and insertion mutations were observed in the resistant isolate and not in the intermediate or susceptible isolates. For the ntr6 gene, insertion and deletion mutations were observein the intermediate isolate and a single deletion mutation in the resistant isolate. For the PFOR gene, a single substitution mutation was observed in the intermediate and resistant isolate. In this study, mutations in the ntr2, ntr3, ntr4, ntr5 and ntr6 genes were observed across metronidazole susceptibility profiles. Previous studies have not identified mutations in the ntr2, ntr3 and ntr5 genes, so there is not enough data to support the functions of those genes and their association with metronidazole resistance. Future studies aimed at identifying the function of these mutations are needed.