18S ribosomal DNA based PCR diagnostic assay for Trichomonas vaginalis infection in symptomatic and asymptomatic women in India

Abstract Objective To identify the cases of trichomoniasis in symptomatic and asymptomatic Trichomonas vaginalis ( T. vaginalis ) infected patients by PCR amplification of hypervariable 18S rRNA gene and to assess the sensitivity of restriction fragment length polymorphism (RFLP) technique for their diagnosis. Methods We enrolled 498 women of child bearing age groups, with their pre-informed consent, attending OPD for their routine checkups and STI related problems. Trichomoniasis was diagnosed on the basis of wet mount preparations and PCR with a primer set targeting a well-conserved region in the 18S rRNA genes of T. vaginalis , respectively. Sequencing was done for differentiating the symptomatic and asymptomatic strains of axenic and clinical isolates. Results After PCR diagnosis T. vaginalis infection was detected in 17 (3.42%) out of 498 clinical isolates. Seventeen axenic and sixteen clinical strains of T. vaginalis tested were successfully detected by PCR yielding a single predicted product of 312 bp in gel electrophoresis followed by restriction digestion with restriction endonuclease HaeIII. After restriction digestion they gave two bands, one of 101 and the other of 211 bp, while there was negative response with DNA from Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica . An optimal analytical sensitivity and specificity of one T. vaginalis organism per PCR was achieved. Sequence of symptomatic and asymptomatic strains of axenic and clinical isolates are somewhat differentiated on the basis of point mutations in their 18S rRNA gene. Conclusions Only few factors are known to predict symptoms of T. vaginalis infection, although the majority of women are infected with trichomoniasis are reported. Therefore the application of sensitive PCR based diagnosis may be quite useful for routine diagnosis of T. vaginalis strains.