A cross-sectional study was conducted during April to August 2012 at Khartoum, Sudan. After signing an informed consent and recording the socio-demographic characteristics and symptomatic history (vaginal discharge, itching, dysuria, dyspareunia, and foul odor) women who attended the gynecological clinic in Khartoum were approached to participate in the study.
Cotton-tipped applicators were used to obtain vaginal fluid swabs from each woman using a vaginal speculum. At least three swabs were collected in a sterile test tube that contained 0.2 ml of 5% glucose saline. This tube was maintained at body temperature for wet mount examination, which was performed within 10 minutes of collection of the specimen. The applicator was gently agitated in the saline, and a wet mount on a clean slide was prepared and observed under a microscope for motile trichomonads (400×) to confirm flagellar movement, morphological features, and the number of T. vaginalis.
The wet preparation was then inoculated into Diamond’s media, the latex agglutination test was applied, and T vaginalis DNA was detected by real-time PCR. An Amplicor swab (Roche Molecular) was used to collect specimens for PCR amplification, while Dacron-tipped swabs were used to collect materials for microscopic examination, the latex agglutination test, and culturing of the organism.
A total of 30 g of Diamond’s media was suspended in 900 ml of purified filtered water. This was then heated with frequent agitation, boiled for 1 minute, and then sterilized at 121°C for 15 minutes. After cooling to 45–50°C, 100 ml of horse serum and antibiotics were added and mixed thoroughly, and were dispensed into sterile culture tubes. The prepared media was stored at 2–8°C and protected from direct light, and dehydrated powder was stored in a dry place in tightly-sealed containers at 2–25°C. Prior to inoculation, the media was brought up to 35–37°C in an aerobic incubator for approximately 1–2 hours. Specimens were examined for T. vaginalis prior to inoculation into culture media. Tubes were incubated aerobically in a slanted position (45° angle) at 35–37°C. Cultures were examined by wet mount for motile trophozoites after 24 or 48 hours. Centrifugation of the culture was used to detect low-level growth, and then examined microscopically daily by adding one drop on a slide, and covering with a cover slip. Negative cultures were incubated for at least 96 hours and examined before reporting the culture.
Quality control of diamond’s media
An internal quality control for Diamond’s media was included by incubating one tube of Diamond’s media per batch to monitor the sterility of reagents. One tube of Diamond’s media was inoculated with a known culture of T. vaginalis. To check the quality of the batch of Diamond’s media, another tube of Diamond’s media was inoculated with a known culture.
Latex agglutination test
All reagents were brought to room temperature. The test latex was shaken well immediately before use. A volume of 50 μL of the vaginal swab suspension was eluted by agitation in phosphate-buffered saline. One drop of test latex was added. Both liquids were stirred to a completely homogenous mixture that covered the whole surface of the reaction zone. The glass slide was tilted with a rotating action continuously for 3 minutes. After 3 minutes, the degree of agglutination was obtained.
The degree of agglutination was recorded as follows. Latex that had agglutinated with a lot of accumulation around the edge of the reaction zone was recoded as positive+++. Agglutinated particles that were clearly seen against a background of granular latex were recorded as positive++. Agglutination that was just able to be discerned compared with a negative control was recorded as positive+. No agglutination compared with a negative control was negative.
A negative control was used in an adjacent reaction zone in parallel with a test sample to distinguish between a weak positive and negative result. A positive control was used to monitor the performance of the test latex. Running a positive control the first time the kit was used was recommended, as well as when removed from storage.
Positive and negative controls were run every time the latex agglutination test was performed. A negative test was repeated to ensure the results.
The target for the real-time PCR assay was a 67-base pair region of a repeated sequence of the T. vaginalis genome (Gene Bank Accession Number L23861). The primers and probes were designed using Primer Express (Applied Biosystems, Foster City, CA, USA). The PCR reaction contained 25 μl of 2× TaqMan1 Universal Master Mix (Applied Biosystems, manufactured by Roche, Branchburg, NJ, USA), with 5 ml each (final concentration of 900 nM) of TV forward primer (Tv3, 5′-ATTGTCGAACATTGGTCTTACCTC-3) and TV reverse primer (Tv7, 5′-TCTGTGCCGTCTTCAAGTATGC-3′), 5 ml (final concentration of 225 nM) of probe (5′FAM-TCA146 A.M. . TTT CGG ATG GTC AAG CAG CCA-TAMRA-3′), and 5 μl of sample DNA. A volume of 5 μl of eluted DNA was used as a template in a final volume of 25 μl with PCR buffer (Hotstar Taq Mastermix; QIN gene), 25 μg of bovine serum albumin, 5 Mm Mg C12, and 2.0 pmol of each primer. The thermocycler used was set to provide amplification consisting of 15 minutes at 95°C followed by 50 cycles of 15 seconds at 95°C, and 1 minute at 60°C. Amplification detection and data analysis were performed with the Applied Biosystems 7500 Real Time PCR System. The PCR output from this system consisted of a cycle threshold (Ct) value, representing the amplification cycle in which the signal exceeded the background fluorescence, and indicating the parasite-specific DNA load in the sample tested.
A sample size of 295 subjects was calculated using the OpenEpi-Epidemiological calculator with 80% power and a confidence interval of 95% to detect a difference of 5% at α = 0.05, with 10% of non-respondents/incomplete data.
Ethical approval for the study was obtained from the Khartoum Hospital Ethical Board.
Data were analyzed using SPSS software version 19.0. Sensitivity, specificity, and positive and negative predictive values were determined as described previously .