Figure 1. Trichomonas Assay Procedure Rapid Detection of Trichomonas vaginalis from Vaginal Specimens by Transcription-Mediated Amplification A. Sitay1, J. Bungo1, K. Dickey1, W. Weisburg1, T. Aguirre2, D. Fuller2, L. Jasper2, T. Davis2; 1Gen-Probe Incorporated, San Diego, CA; 2Wishard Memorial Hospital, Indianapolis, IN C-120 ABSTRACT Background: Trichomonas vaginalis (Tvag) is a common cause of sexually transmitted disease (STD), with an estimated 5 million new cases occurring annually in the U.S. Ten to 50% of infections are asymptomatic. Diagnosis of Tvag infection is problematic. The commonly used wet mount, while rapid, has low sensitivity. Culture and Pap stain are lengthy procedures and technically challenging. A rapid, amplified assay system is described here for detection of Tvag. Methods: The test includes target capture, Transcription-Mediated Amplification (TMA) and a Hybridization Protection Assay (HPA). Target capture uses specific DNA capture oligos and magnetic beads for separation of target rRNA from clinical specimens. TMA amplifies a specific region of the target rRNA. HPA uses a chemiluminescent probe in a homogenous assay format whereby probe binds specifically to Tvag amplicon and is induced to emit light. Results: A total of 152 vaginal swabs from patients attending STD clinics were tested in the Tvag assay system at Gen-Probe Incorporated and compared with wet mount, InPouch culture, BD Affirm, and Pap stain performed at Wishard Memorial Hospital. Thirty-six specimens were positive by any one of the 4 comparator methods; 34 of these were positive by TMA. One hundred sixteen specimens were negative for T. vaginalis by all 4 comparator methods; 95 of these were negative by TMA and 21 were positive by TMA. The apparent sensitivity and specificity of the TMA assay were 94% and 82%, respectively. It is unclear if the TMA+, comparator- specimens are TMA false positive results or reflect the greater sensitivity of target amplification. Fifteen of the 21 TMA+, comparator- specimens were positive on repeat TMA testing, suggesting that they may be true positives. If this is the case, the sensitivity and specificity would be 96% and 94%, respectively. The remaining six may be false positives or contain such low concentrations of T. vaginalis as to be subject to sampling variation. Conclusions: Our results suggest that target amplification may be a more rapid and sensitive method to detect T. vaginalis than alternative methods, including culture.* The Trichomonas vaginalis assay procedure is illustrated in Figure 1. In brief: Target rRNA is separated from the other specimen components and the transport media by the addition of Target Capture Reagent, magnetic bead separation, and washing using a target capture system (Figure 2). Amplification Reagent, Oil and Enzyme Reagent are added to the rRNA target on the magnetic beads. Isothermal TMA amplification occurs at 42oC (Figure 3). Detection occurs by HPA (Figure 4) and the reaction is read as Relative Light Units (RLU) in a LEADERR HC+ luminometer. RESULTS Table 1. Trichomonas vaginalis Test Results A total of 152 vaginal swab specimens were tested at Wishard and at Gen-Probe to help determine the feasibility of a TMA-based method for the detection of T. vaginalis from vaginal swabs (Table 1). A preliminary cut-off of 30,000 RLU was used to evaluate the TMA test data. Thirty-six specimens (24% prevalence) were positive by any one of the 4 comparator methods; 34 of these were positive by TMA. Two specimens that were positive only by the PAP smear method (only by one of the 3 cytologists in one case and by 2 of the 3 in the other case) were negative by the TMA method. Of the 116 specimens that were negative by all 4 comparator methods, 95 were negative by TMA and 21 were positive by TMA. Fifteen of the 21 TMA+/comparator- specimens were positive on repeat TMA testing, and one was unavailable for a repeat test. This suggests that the 15 may be true positives. The remaining 6 may be false positives or contain such low concentrations of T. vaginalis as to be subject to sampling errors. If the 15 TMA+/comparator- specimens were considered to be true positives, then the TMA test sensitivity would be 96% and the specificity 94%. A summary of the total positive tests for each method for the 152 swab specimens is shown in Table 2. A summary of a selected set of test comparisons is shown in Table 3. Assuming that the total number of true positive results is 51 (49 repeat + TMA tests, and 2 + by PAP only), then the actual prevalence of T. vaginalis infection in this population was 34%, and the sensitivity of each test method was: Wet Mount = 43% Affirm VP = 44% PAP Smear = 55% In-Pouch Culture = 63% Gen-Probe TMA = 96% Figure 1. Trichomonas Assay Procedure Manual sample pipetting Addition of target capture reagent Washing with wash buffer using target capture system Incubation for TMA Addition of probe , selection and detection reagents Reading using luminometer Addition of oil, amplification and enzyme reagents Figure 2. Target Capture Step Figure 3. TMA Step Table 2. Total Positives for Each Test Method *Gen-Probe’s Trichomonas vaginalis assay is in research. CONCLUSIONS MATERIALS AND METHODS The T. vaginalis assay method is easy to perform, could be automated or semi-automated, and the steps are identical to the Gen-Probe APTIMAR Combo 2 method used in many clinical laboratories to detect Chlamydia trachomatis and Neisseria gonorrhoeae from genital swab and urine specimens. While this study only included female vaginal swabs, future work with male urethral swabs and male and female urine specimens is planned. The commonly-used wet mount was the least sensitive method of the 4 comparator methods, in spite of an experienced staff in a STD clinic. In order of sensitivity: TMA test >culture >PAP smear >DNA probe >Wet Mount. Our results suggest that target amplification may be a more rapid and sensitive method to detect T. vaginalis than alternative methods, including culture. Vaginal swabs (n=152) were collected from women attending an STD clinic at Wishard Memorial Hospital (Wishard) in Indianapolis, IN. The following tests were performed to detect Trichomonas vaginalis at Wishard, using their standard laboratory methods: Culture (In-PouchTM TV, BioMed Diagnostics, Inc., San Jose, CA) for 96 hours, with microscopic examination every ~24 hours DNA Probe (AffirmTM VP III Test, BD Diagnostics Systems, Sparks, Md) Wet Mount PAP smear, examined by 3 different cytologists The specimens were shipped to Gen-Probe Incorporated in swab transport media tubes and stored at 2-8oC until testing. The TMA tests were performed at Gen-Probe. Table 3. Test Comparison for Detection of T. vaginalis Figure 4. HPA Step