Characterization and drug resistance of Trichomonas vaginalis clinical isolates

Abstract

Sexually transmitted infections (STIs) are a major cause of acute illnesses, infertility, long term disability and death with far reaching health, social and economic consequences. Trichomonas vaginalis is the causative organism of trichomoniasis which classically presents in women as a malodorous green-yellowish discharge accompanied by itching and burning. In men infection can cause non-gonococcocal urethritis and chronic prostatitis. Complications of T. vaginalis include preterm delivery, low birth weight, predisposition to HIV infection and cervical cancer. Previous studies in South Africa have focused mostly on T. vaginalis detection with reported rates of prevalence of T. vaginalis infections ranging between 20% to 49%. Despite estimates showing T. vaginalis being the most prevalent sexually transmitted disease worldwide, very little is known about the genetic diversity of T. vaginalis clinical isolates. Furthermore, the degree of metronidazole resistance in a particular setting needs to be investigated, since this has implications on the treatment regimen of trichomoniasis. The purpose of this study was: i) To detect T. vaginalis in HIV positive female patients from the Anti-Retroviral clinic of the Tshwane District Hospital, Pretoria using three methods, namely microscopy, culture and PCR; ii) To characterize T. vaginalis isolates using both the random amplified polymorphic DNA (RAPD) assay and the intergenic spacer regionpolymerase chain reaction-restriction fragment length polymorphism (IGS-PCR RFLP) assay and iii) To phenotypically determine metronidazole resistance of the T. vaginalis isolates and to compare the results with random amplified polymorphic DNA (RAPD) assay results. Self-collected vaginal swabs from 380 women were included in the first part of the study. Trichomonas vaginalis was detected using: wet mount microscopy, culture (modified Diamond’s medium) and molecular detection using a commercial kit, Trichomonas vaginalis 240/250 IC (Sacace Biotechnology, Italy). The genetic relatedness of 92 culture positive T. vaginalis isolates was determined. Five primers (TV1, TV2, TV3, TV5 and TV6) were used for the RAPD assay. The PCR-IGS-RFLP products were digested with five enzymes, namely: AluI, HinfI, RsaI, Sau3AI and Tsp509. A 24 h and 48 h interval microtiter assay was used to detemine the metronidazole antimicrobial susceptibility of 30 T. vaginalis isolates. A total of 8% (30/380) of specimens were positive for T. vaginalis using microscopy, 24% (92/380) of specimens were positive using culture and 31% (118/380) of the specimens were positive using the commercial PCR kit Trichomonas vaginalis 240/250 IC (Sacace Biotechnology, Italy). RAPD assay analysis showed a high level of genetic diversity between the different T. vaginalis isolates. The dendrogrammes obtained from the RAPD markers grouped the 92 T.vaginalis isolates into between nine to 24 clusters with a 70% similarity, while the PCR-IGS RFLP assay results for the isolates were genetically indistinguishable. The minimal inhibitory concentration (MIC) for metronidazole was between 0.06 to 25 μg/ml. Only 6% (2/30) of the T. vaginalis isolates were resistant. The dendrogrammes constructed in the second part of the study did not group the metronidazole resistant isolates together in one cluster. No link between resistance and a specific T. vaginalis genotype could be indicated. This study proved that PCR is a more sensitive diagnostic tool for the detection of T. vaginalis to improve the diagnosis of trichomoniasis. A high prevalence of T. vaginalis in HIV positive women in South Africa was observed. The RAPD assay proved to be useful in discriminating between the different T. vaginalis isolates, while the IGS-PCR RFLP assay was not a suitable marker. In future, other T. vaginalis genes, such as the ferredoxin and beta-tubulin genes could be investigated to determine the genetic variability of T. vaginalis isolates. Although metronidazole is the only antimicrobial drug used for treatment of trichomoniasis in South Africa, a low prevalence of in vitro resistance was found. This study emphasized the importance of in vitro antimicrobial drug susceptibility testing to ensure continunous screening for possible cases of metronidazole resistance and to monitor MIC changes.