Comparison of Conventional Methods with Molecular Methods in the Diagnosis of Trichomonas vaginalis and Investigation of Metronidazole Resistance

Abstract

Trichomoniasis is a sexually transmitted parasitic infection caused by Trichomonas vaginalis. In the diagnosis of trichomoniasis, direct microscopy (DM) is preferred, which is a cheap and fast method, although it has low sensitivity. Culture methods, which are accepted as the gold standard, can only be applied in certain centers due to the need for experienced personnel and the ability to get results within 2-7 days, despite their high sensitivity. In this study, it was aimed to compare conventional microscopic and culture methods used in the routine diagnosis of T.vaginalis with polymerase chain reaction (PCR) method and to investigate ntr4 and/or ntr6 gene polymorphism in the nitroreductase gene region, which are thought to be associated with metronidazole resistance in T.vaginalis strains isolated from clinical specimens. Vaginal swab specimens were collected from the posterior fornix of the vagina with two sterile ecuvion sticks during the gynecological examinations of 200 patients who applied to the Balıkesir University Health Practice and Research Hospital, Obstetrics and Gynecology Polyclinic between March 2019 and August 2021. The first swab sample was used for direct microscopic examination, Giemsa staining and conventional PCR analysis, while the second swab specimen was taken into trypticase-yeast-extract-maltose (TYM) medium for T.vaginalis culture and followed for eight days at 37 °C. All specimens were screened for the presence of T.vaginalis using primers specific to the β-tubulin (btub1) gene region and clinical isolates grown in TYM medium were examined for metronidazole resistance using primers specific for the nitroreductase gene region by using conventional PCR. Drug resistance test was also performed for the isolates in which polymorphism associated with metronidazole resistance was detected. Eight (4%) of 200 patient specimens were found positive by both culture/staining and PCR methods. The mean age of the patients included in the study was 39.9, while the mean age of the patients with positive T.vaginalis was 41.8. The most common clinical findings in the patients were foul-smelling vaginal discharge (36%), groin pain (21%), vaginal itching (19%), and burning sensation during urination (18%). In three out of eight T.vaginalis strains isolated from clinical samples, the presence of polymorphism in the ntr6 gene, which is thought to be associated with metronidazole resistance, was demonstrated by PCR. It was observed that three isolates with ntr6 gene polymorphism were phenotypically resistant to metronidazole (MLK= 390 µM). In this study, the fact that three of eight clinical isolates that were resistant to metronidazole by the broth microdilution method and as well as showing ntr6 gene polymorphism supported the thesis that there might be a close relationship between metronidazole resistance and ntr6 gene polymorphism. As a result, the use of culture and molecular methods in the diagnosis of T.vaginalis, in addition to the microscopy method, may contribute to a more accurate laboratory diagnosis of the agent, to detect metronidazole resistance molecularly and phenotypically, to determine metronidazole resistance rates in our country and to update treatment protocols within the framework of these data.