Advanced Techniques in Diagnostic Microbiology

368 A. C. T. Lo and K. M. Kam
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.
PCR
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