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

Figure 1. Trichomonas Assay Procedure

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.*

ABSTRACT

MATERIALS AND METHODS

Table 3. Test Comparison for Detection of T. vaginalis

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%

CONCLUSIONS

*Gen-Probe’s Trichomonas vaginalis assay is in research.

C-120C-120

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.

Table 2. Total Positives for Each Test Method Wet Affirm In-Pouch PAP Gen-Probe Rpt.Gen-Probe

N=152 Mount Trich. Culture Smear TMA TMA Pos.Total Positives: 22 23 32 28 55 49

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HPAHPAHybridization Protection AssayHybridization Protection Assay

Wet Affirm In-Pouch PAP Gen-ProbeNo. Mount Trich. Culture Smear RLU

1 NEG NEG NEG NEG 2,5352 NEG NEG NEG NEG 3,519,5183 NEG NEG NEG NEG 2,0414 NEG NEG NEG NEG 3,0405 NEG NEG NEG NEG 2,0966 NEG NEG MISSED NEG 2,7297 NEG NEG MISSED NEG 4,091,9628 NEG NEG NEG NEG 38,6189 NEG NEG NEG NEG 2,07710 NEG NEG POS NEG 3,944,60711 POS POS POS POS 4,428,65012 NEG NEG NEG NEG 2,00213 NEG NEG NEG NEG 2,59614 NEG POS POS NEG 4,499,51015 NEG NEG NEG NEG 1,53916 NEG NEG NEG NEG 1,38117 NEG NEG POS NEG 4,443,20818 NEG NEG NEG NEG 1,91819 NEG NEG NEG NEG 3,029,18620 NEG NEG NEG NEG 1,89221 NEG NEG NEG NEG 1,55222 NEG NEG NEG NEG 1,74423 NEG NEG NEG NEG 1,95924 NEG NEG MISSED NEG 2,28125 NEG NEG NEG NEG 1,87226 NEG NEG NEG NEG 1,95327 NEG NEG NEG NEG 1,86328 NEG NEG NEG NEG 1,76329 NEG NEG NEG NEG 1,42530 POS NEG POS POS 4,375,03731 NEG NEG NEG NEG 987,99432 NEG NEG NEG NEG 1,33833 NEG NEG NEG NEG 1,68134 NEG NEG NEG NEG 1,69035 NEG NEG NEG NEG 1,93336 NEG NEG NEG NEG 2,41237 NEG NEG NEG NEG 2,61938 NEG NEG NEG NEG 3,29339 NEG NEG NEG NEG 2,61940 NEG NEG NEG NEG 3,29341 NEG NEG NEG NEG 2,29242 NEG NEG NEG NEG 4,473,83543 NEG NEG POS NEG 4,540,39144 POS POS POS POS 4,086,51645 NEG NEG NEG NEG 4,262,45746 NEG NEG NEG NEG 2,32747 POS POS POS POS 4,308,03748 NEG NEG NEG NEG 1,59249 NEG NEG NEG NEG 1,33850 NEG NEG NEG NEG 1,67751 NEG NEG NEG NEG 1,71952 NEG NEG NEG NEG 1,51653 NEG NEG NEG NEG 1,33754 NEG NEG NEG NEG 3,968,61555 NEG POS POS POS 3,874,95856 NEG NEG NEG NO R 1,42657 POS POS POS POS 3,761,45558 NEG NEG NEG NEG 1,65559 NEG NEG NEG NEG 1,31360 NEG NEG NEG NEG 1,23561 NEG POS POS NEG 3,719,18762 NEG NEG NEG NEG 243,26763 NEG NEG NEG NEG 1,61164 NEG NEG NEG NEG 1,33465 NEG NEG NEG NEG 4,96666 NEG NEG NEG NEG 1,30467 POS POS POS POS 4,046,95768 NEG NEG POS NEG 3,199,97869 NEG NEG NEG NEG 3,862,83570 NEG NEG NEG NEG 1,17071 NEG NEG NEG NEG 1,36772 NEG NEG NEG NEG 1,315

73 NEG NEG NEG NEG 7,43374 NEG NEG NEG NEG 1,78675 NEG NEG NEG NEG 1,70276 NEG NEG NEG NEG 1,45577 NEG NEG NEG NEG 1,56178 NEG NEG NEG NEG 1,73479 NEG NEG NEG NEG 1,79180 NEG NEG NEG NEG 5,141,86881 NEG NEG NEG NEG 1,36382 NEG NEG NEG NEG 5,839,03983 NEG NEG NEG NEG 1,16684 NEG NEG NEG NEG 1,70185 NEG NEG NEG NEG 1,58186 NEG NEG NEG NEG 1,37387 NEG NEG POS POS 5,958,67588 NEG NEG NEG POS 5,746,26789 POS POS POS POS 5,974,23090 POS POS POS POS 6,137,38491 POS POS POS POS 5,425,52292 NEG NEG NEG NEG 1,54593 NEG NEG NEG NEG 1,64094 NEG NEG NEG NEG 1,77595 NEG NEG NEG NEG 1,41796 NEG NEG NEG NEG 1,37697 POS POS POS POS 5,767,99398 NEG NEG POS NEG 5,573,16499 POS POS POS POS 9,106,003100 NEG NEG POS NEG 5,589,897101 POS POS POS POS 5,559,030102 NEG NEG NEG NEG 1,551103 NEG NEG NEG NEG 1,764104 NEG NEG NEG NEG 1,729105 NEG NEG NEG NEG 1,767106 POS NEG POS POS 5,655,568107 POS POS POS POS 6,149,147108 NEG NEG NEG NEG 5,698,527109 NEG NEG NEG NEG 1,650110 POS POS POS POS 6,250,286111 POS POS POS POS 5,835,878112 NEG NEG NEG NEG 1,521113 NEG NEG NEG NEG 1,673114 POS POS POS POS 7,709,815115 NEG NEG NEG NEG 1,604116 NEG NEG NEG NEG 8,478117 NEG NEG NEG NEG 4,037,866118 NEG NEG NEG NEG 1,767119 NEG NEG NEG NEG 4,150,859120 NEG NEG NEG NEG 4,071121 NEG NEG NEG NO R 2,351122 NEG NEG NEG NEG 2,452123 POS POS POS POS 4,418,451124 NEG NEG NEG NEG 4,199,744125 NEG NEG NEG NEG 3,511,952126 NEG NEG NEG NEG 3,820,877127 NEG NEG NEG NEG 226128 NEG NEG NEG NEG 2,154129 NEG NEG NEG POS 2,486130 NEG NEG NEG POS 1,996131 NEG NEG NEG NEG 1,958132 NEG NEG NEG NEG 2,069133 NEG NEG NEG NEG 2,335134 NEG NEG NEG NEG 2,431135 NEG NEG NEG NEG 1,944136 NEG NEG NEG NEG 3,704,381137 NEG NEG NEG NEG 4,210,858138 POS POS POS POS 4,329,830139 NEG NEG NEG NEG 2,361140 NEG NEG NEG NEG 2,184141 NEG NEG NEG NEG 3,443,951142 NEG NEG NEG NEG 1,978143 NEG NEG NEG NEG 2,209144 POS POS POS POS 4,089,380145 POS POS POS POS 3,994,075146 NEG NEG NEG NEG 1,896

147 NEG NEG NEG POS 3,979,386148 NEG NEG NEG NO R 2,048149 NEG NEG NEG NEG 3,089150 NEG NEG NEG NEG 2,214151 NEG NEG NEG NEG 2,117152 POS POS POS POS 4,419,082

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Figure 2. Target Capture StepFigure 3. TMA Step

• 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.

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.

Figure 4. HPA Step

Table 1. Trichomonas vaginalis Test Results

Gen-ProbeWet Mount Affirm Trich. In-Pouch Culture PAP Smear TMA Number

Positive Positive Positive Positive Positive 20Negative Negative Negative Negative Positive 21*Negative Negative Positive Negative Positive 6Negative Positive Positive Negative Positive 2Positive Negative Positive Positive Positive 2Negative Negative Negative Positive Positive 2Negative Negative Negative Positive Negative 2Negative Positive Positive Positive Positive 1Negative Negative Positive Positive Positive 1Negative Negative Negative Negative Negative 95

* 15 of these 21 were positive on repeat testing; one was QNS.