| ObjectiveTo detect and screen virus-positive Trichomonas vaginalis samples isolated from female individuals in Henan, China and to analyze wether the prencence of Trichomonas vaginalis virus has an influence on clinical symptoms of patients, Mycoplasma hominis synbiosis in T. vaginalis, and also metronidazole resistance, and genetic polymorphisms of T. vaginalis as well.Methods1. Genomic DNA and RNA were extracted simultaneously from 30 Trichomonas vaginalis isolates after sxenic cultures were conducted in trypticase-yeast extract-maltose (TYM) media. Then the nucleotides were run on 1% agarose gels to detect the characteristic 5.5kb bands on gels. And T vagnialis virus (TVV) were confirmed by another electrophoresis on 1% agarose gel after nuclease digestions.2. Based on the symptoms, the 30 patients viewed were grouped into four: asymptomatic, mild, mediate and severe symptoms. T.vaginalis smples isolated from these patients were subjected to methods above to determine the prensence of virus. The clinical symptoms of patients infected by TVV-positive and -negative T. vaginalis strains were compared, and the two-sample rank sum test was used to study the influences of TVV on the clinical pathogenicity of Trichomonas vaginalis isolates.3. The minimal lethal concentration (MLC) of metronidazole against Trichomonas vaginalis isolates were determined by serial dilution method. The results of TVV-positive and negative T.vaginalis strains were collected and analyzed statistically in SPSS 16.0 software pack. Thus, the impacts which TVV have metronidazole resistance against Trichomonas vaginalis isolates.4. Mycoplasma symbiosis was detected in the 30 Trichomonas vaginalis isolates respectively by using PCR with specific primers. The concurrent conditions of TVV and M.hominis were studied.5. MGE-PCR was applied to 30 Trichomonas vaginalis isolates to detected the diversities of length and location of Tvmarl gene. Genetic polymorphisms due to presence of TVV were analyzed and the phylogenetic trees, based on the results, were constructed using different algorithmic methods.Results1. Among the 30 Trichomonas vaginalis isolates examined,6 strains (Tv5, Tv10, Tv13, Tv17, Tv22, and Tv23) exhibited characteristic 5.5kbp bands on electrophoresis gels, and these bands were disappeared after RNAase digestion. Thus, these 6 strains were regarded as TVV-positive.2. The clinical symptoms of patients infected by TVV-positive T.vaginalis strains were less severe than the ones infected by TVV-negative strains (Zc=-3.428, P <0.05).3. MLC of metronidazole against the TVV-negative T.vaginalis strains in vitro was (24.27±20.899)μg/ml. It was significantly higher than that of the TVV-positive strains((5.68±3.588)μg/ml), and the difference between them was statistically significant (t=2.143, P<0.05). Thus, TVV-positive isolates were more sensitive to metronidazole4. Mycoplasma hominis was not likely to concurr with the Trichomonas vaginalis virus in same T. vgainalis isolate (χ2=0.000,P<0.05).5. The variable length and location of Tvmarl genes from 30 Trichomonas vaginalis isolates showed that the protozoan isolates have significant genetic polymorphism. And in the phylogenetic tree contructed based the results of MGE-PCR to Tvmarl genes, the 6 Trichomonas vaginalis virus-positive isolates are in the same cluster. Conclusions1. Trichomonas vaginalis virus were detected in Trichomonas vaginalis isolates from Henan, China. The proportion of infection with TVV is 20%.2. The presence of Trichomonas vaginalis virus have negative influence on virulence of T.vaginalis, metronidazole resistance and concurrence of Mycoplasma hominis, and also has an affect on the genetic polymorphisms of parasites. |
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