Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples

Abstract

Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.

FIG. 1

PCR primers BTUB 9 and BTUB 2 for the detection of T. vaginalis with the beta-tubulin genes. Primers were selected from well-conserved and specific regions of the genes (btub1, -2, and -3). Sequences of the beta-tubulin genes of T. vaginalis were compared to the beta-tubulin gene sequences of humans and to those of other pathogens. A dot indicates the same base, and a letter indicates a different base compared to the T. vaginalis btub1 gene sequence.

FIG. 2

Detection of T. vaginalis strains F to N by PCR with primer sets BTUB 9/2 (A) and primer set TVA 5-1/6 (B). Strains F to N are representative of 14 strains of T. vaginalis (designated A to N) isolated from vaginal secretions of patients attending a city STD clinic in Baltimore, Md.

FIG. 3

Analytical sensitivity testing of T. vaginalis PCR with primer set BTUB 9/2 (A) and primer set TVA 5-1/6 (B). Twofold dilutions of strain M of T. vaginalis were processed separately to extract the DNA and were tested by PCR.