Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge

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Abstract
A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.