Molecular Detection Methods

For the molecular detection of T. vaginalis, recent methods include the use of probe hybridization and PCR assays. These assays have been devised to detect various regions or genes of the genome including 2.3-kb T vaginalis fragment (Rubino et al., 1991), the feredoxin gene (Riley et al., 1992), beta-tubulin gene (Madico et al., 1998), highly repeated DNA sequences (Kengne et al., 1994), and 18S ribosomal gene (Mayta et al., 2000).

Probe Hybridization

A commercially available kit, Affirm VP system (MicroProbe, Bothweel, WA, USA), is currently available and uses synthetic DNA probes to directly detect Gardnerella vaginalis and T. vaginalis from a single vaginal swab (Briselden and Hillier, 1994). This Affirm VP deoxyribonucleic acid probe test was found to be better than wet-mount preparation and has a sensitivity of 90% and a specificity of 99.8% for the identification of T vaginalis organisms in women with a high prevalence of trichomoniasis (DeMeo et al., 1996). However, false-negative results have been encountered compared with culture technique (Briselden and Hillier, 1994). A dot blot hybridization technique has also been developed by using a 2.3-kb T vaginalis DNA fragment as a probe to detect T. vaginalis DNA from vaginal exudates (Rubino et al., 1991). However, the drawbacks of this technique are the instability of the probe and the necessity to handle and dispose of radioactive materials. To overcome these limitations, fluorescence-labeled DNA probe can be used for identification of T. vaginalis by DNA in situ hybridization technique. For asymptomatic carriers, the usefulness of these techniques still requires more definitive evaluation.

Several PCR systems have been developed to detect T. vaginalis from clinical samples. Specific TVA5-TVA6 primers targeting the unique sequences of the genome of T vaginalis have been designed. A 102-bp genomic fragment was amplified and termed as A6p sequence, which appears highly selective for a broad range of T vaginalis isolates (Riley et al., 1992). Beta-tubulin gene of T. vaginalis is a well-conserved region that has been used to develop a PCR assay (Madico et al., 1998). The sensitivity and specificity of the beta-tubulin gene PCR assay were 97% and 98%, respectively, while the sensitivities of culture and wet preparation were 70% and 36%. Another target region of T. vaginalis that has been used for PCR amplification was 2000-bp repeated fragment of T vaginalis (Kengne et al., 1994). Two sets of primers (TVK3-TVK4 and TVK3-TVK7) were used and have been shown to be highly specific for T. vaginalis without reacting with human DNA or other infectious agents tested. Another PCR assay that used primers targeting a specific region of the 18S rRNA gene of T vaginalis has also been developed (Mayta et al., 2000). The PCR amplification product was subsequently confirmed by enzyme digestion with HaeIII. Overall sensitivity and specificity of the 18S

rRNA PCR assay of vaginal swab samples were 100% and 98%, respectively, which compared favorably to T. vaginalis culture.

Recently, two real-time PCR assays (TaqMan-based and FRET-based) for T. vaginalis DNA detection were developed using double-labeled fluorescent probes (Jordan et al., 2001; Hardick et al., 2003). High levels of agreement between these real-time PCR assays and culture have been obtained for detecting T. vaginalis. The real-time process has effectively eliminated the need for post-PCR processing for PCR product detection. This has resulted in significant decrease in turn-around time and increased throughput. The closed system detection format also would allow prevention of laboratory cross-contamination. Thus, the real-time PCR assay has advantages over the conventional PCR assays. Recently, a urine-based PCR-EIA assay has also been developed and validated for the detection of trichomoniasis in men and women (Kaydos et al., 2002). Sensitivity and specificity of this assay ranged from 86.4% to 92.7% and 88.6% to 95.2%, respectively.

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