Literature reviews  >  Articles for review > Crucitti et al. Comparison of culture and different PCR... 

Not all PCR assays for T. vaginalis detection perform equally.

Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens.
Crucitti T, Van Dyck E, Tehe A, Abdellati S, Vuylsteke B, Buve A, Laga M.
Sexually Transmitted Infections 2003;79:393-398.
 
Summary:

Question
What are the sensitivities and specificities of 5 published PCR primer sets and culture for the detection of T. vaginalis in self-collected vaginal swab specimens?

Design
The sensitivity and specificity of culture and of 5 PCR assays employing primer sets targeting different T. vaginalis genes were compared for the detection of T. vaginalis in self-collected vaginal swabs from female sex workers attending a clinic in Côte d'Ivoire.

Participants
Four hundred twenty-five consecutive women, with or without genital complaints, who attended a female sex worker clinic in Abidjan, Côte d'Ivoire, as part of a study on the validation of clinical algorithms were tested.

Description of Tests and Diagnostic Standard
Two vaginal swabs were collected by each woman. The first swab was inoculated into Dobbel-Laidlaw culture medium (Pasteur Diagnostics, Marnes-la-Coquette, France) and incubated for 4 days, according to the procedures provided by the manufacturer. The medium was examined microscopically on days 2 and 4. The presence of motile trichomonads determined a positive test.

The second swab was placed into a dry tube and frozen until analysis. DNA was extracted prior to PCR analysis by adding buffer to the swab after thawing. Half of each specimen was then centrifuged and the cell pellet was suspended in 0.2 ml of lysis buffer containing detergent and proteinase K. The specimens were incubated at 56^oC for 60 minutes and boiled for 10 minutes. For each PCR assay, the primer set, gene target, amplicon size, and amount of sample used to amplify T. vaginalis are shown in Table 1. All primers were labeled with biotin. Amplification procedures were performed as described in the original publications except primer set IP1/2, for which only the inner primers of a nested PCR assay were used. Amplicons were detected by electrophoresis in agarose gels followed by ethidium bromide staining and by an enzyme immunoassay (EIA). Microtiter plate wells were coated with 500 ng/100μl (for assays using TVK3/7, TVA5/6, and BTUB9/2 primer sets) or 300 ng/μl (for assays using IP1/2 and TV1/2 primer sets) of oligonucleotide capture probes designed within the target sequences of the PCR amplicons. Biotin-labeled amplicons bound to probe were detected using avidin-horse radish peroxidase and a colorimetric substrate. Optical density was measured at 450 nm on a spectrophotometer. The cut-off for each assay was calculated based on the mean OD value of 30 true negative samples plus 3 standard deviations.

For each clinical specimen, a PCR for the human β₂ microglobulin gene was performed as a control for the presence of inhibitors. The 200 bp amplicon was detected by gel electrophoresis.

Main Outcome Measures
The sensitivity and specificity of culture and of each PCR assay were determined using an expanded gold standard by which specimens were considered true positive for T. vaginalis if they were positive by culture or by any two PCRs with EIA amplicon detection.

Main Results
Inhibition was detected in nine of 425 specimens tested, leaving 416 for analysis. The prevalence of trichomonas infection using the expanded gold standard was 20.0% (83/416). The prevalence of T. vaginalis by culture was 7.0% (29/416). Of the 29 specimens positive by culture, 24 were positive by at least two PCR assays. The sensitivities and specificities, as determined by the expanded gold standard, of T. vaginalis culture and of five PCR assays using gel electrophoresis or EIA for amplicon detection are shown in Table 2. The sensitivities of all the PCR assays increased with the use of the EIA amplicon detection method compared to gel electrophoresis.

Authors' Conclusions
In this study, the performance of different primer sets was different from what has been described in the literature. There were also important performance differences between the primer sets, which may be explained by T. vaginalis strain variability, specimen type, probe design, and choice of gold standard.

Source of funding: Not given

For correspondence: T. Crucitti, STD/HIV Research and Intervention Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000, Antwerp, Belgium. E-mail address: tcrucitti@itg.be

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