18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis

Abstract

Trichomonas vaginalis remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of T. vaginalis was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to T. vaginalis culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with HaeIII. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of T. vaginalis infection.

FIG. 1

Agarose gel electrophoresis of PCR products and REA. Lanes 1 and 2, amplification products of two strains of T. vaginalis. Lanes 3 and 4, REA of amplified products of two different strains of T. vaginalis. Lane 5, REA of amplified product of T. tenax. Lane 6, REA of PCR product performed using water as the sample. Lane M, 100-bp DNA ladder.

FIG. 2

PCR sensitivity based on T. vaginalis genomic DNA quantity, Lanes 1 to 7, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg, respectively. Lane 8, water control. Lane M, 100-bp DNA ladder.