Detection of Trichomonas vaginalis (TV) in Female Urine and Vaginal Specimens Using a Transcription-Mediated Amplification (TMA) Assay

Martin DH1, Lillis RA1, Nsuami M1, Smith B1, Cammarata C1, Diodene D1, and Dicky K2 1LSU Health Sciences Center and the Gulf South STI TM Cooperative Research Center, New Orleans, LA 2Gen-Probe Incorporated, San Diego, CA U.S.A.

TMA Positive/Culture Positive

Urine Vaginal Swab

Sensitivity 81/87 (93.1%) 87/87 (100%)

Specificity 298/313 (95.2%) 152/311 (48.9%)

396 Total Cases* Wet Preparation Culture TMA Urine

51 Positive Positive Positive

4 Positive Positive Negative

1+ Positive Negative Positive

30 Negative Positive Positive

7 Positive Negative Negative

2 Negative Positive Negative

14§ Negative Negative Positive

287 Negative Negative Negative

Figure 1. Trichomonas vaginalis TMA Assay Procedure

Objectives: To determine the sensitivity and specificity of a new, research-use TMA assay for the detection of TV in female urine and vaginal swab (VS) specimens.Methods: Women greater than 17 years old attending the New Orleans STD clinic were enrolled. A first voided urine specimen was obtained initially. At the time of the vaginal speculum examination, swab specimens were obtained for InPouch TV culture (BioMed Diagnostics, Inc., CA) and the TMA assay. The TMA assay includes a magnetic bead-based target capture step, TMA, and a Hybridization Protection Assay (HPA) for detection following the standard APTIMA (Gen-Probe Incorporated) assay protocols. Culture was used to determine the infection status of each subject.Results: Four hundred women were enrolled, of whom 95% were African American. TV cultures of vaginal swabs were positive in 87 (21.8%) of the subjects. The sensitivity of the TMA assay in urine and VS specimens was 93.6% and 100%, respectively, while the specificity was 97.4% and 48.9%, respectively. As there appeared to be a contamination problem with VS specimens for the TMA assay, all clinical and laboratory procedures were reviewed. This investigation revealed that a common container used to temporarily hold vaginal swabs while the investigator completed the pelvic examination was the likely source even though it appeared that only the shafts of the swabs directly touched the container walls.Conclusions: The data from this study suggests that TMA testing of female urine specimens could be a useful, non-invasive TV screening assay. A urine specimen placed in the TMA assay processing medium for the purpose of C. trachomatis and N. gonorrhoeae testing also could be tested for TV, thus efficiently adding this organism to the TMA STD screening repertoire. Additionally, clinicians should be aware of the potential for contaminating specimens intended for nucleic acid amplification testing.

ABSTRACT

MATERIALS AND METHODS

RESULTS

The sensitivity and specificity of the vaginal swab TMA test were 100% and 48.9% respectively. Given the very high false positive rate compared to the urine TMA test results a contamination problem was suspected. Relative light unit (RLU) readings from the TMA luminometer provide a crude estimate of the quantity of Tvag ribosome targets in each sample. Median RLUs was 3.5 X 106 for the culture positive/urine TMA positive cases compared to 3.3 X 106 for the culture negative/urine TMA positive cases. In contrast the median RLUs for the culture negative/vaginal swab TMA positive cases was only 757,000 compared to 4.0 X 106 for the culture positive/vaginal swab TMA positive cases. This result along with the low specificity of vaginal swab TMA compared to urine TMA suggested to us that the majority of vaginal swab positive results were false positive. Culture was positive in 87/400 (21.8%) of women. As shown in Table 1, the sensitivity and specificity of the urine TMA tests were 93.1% and 95.2% respectively. As can be seen in Table 2, of the six TMA urine false negative results, four were both wet preparation and culture positive. Of the 15 “false positive” urine TMA results, one was wet preparation positive and, therefore, probably represented a false negative culture. A repeat urine TMA test result was positive in this case. There were 14 wet preparation/culture negative cases that were urine TMA positive (Table 2). Of these 14 cases, 8 were negative on repeat testing. To determine the source of contamination all clinical and laboratory procedures were reviewed. The investigation revealed that a common container was used by clinical research personnel to temporarily hold vaginal swabs while they completed the pelvic examination (Figure 5). This procedure seemed reasonable as it appeared that only the shafts of the swabs directly touched the container walls. However, there was no other step in the specimen collection or handling protocols that could have accounted for the large number of vaginal swab false positives.

CONCLUSIONS

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

The Tvag TMA assay method is easy to perform. The test could be automated or semi-automated, and the steps are identical to the GEN-PROBE® APTIMA® COMBO 2 Assay method currently used in many clinical laboratories. Overall, urine specimens tested by TMA appears to have promise for screening for trichomoniasis in women and could be a useful tool for population based studies of this infection where pelvic examinations are not practical. A prospective clinical trial utilizing an alternative rRNA region as a target for a confirmation assay is needed to better understand the true perfomance characteristics of the Tvag TMA assay for urine specimens. We experienced a significant contamination problem with vaginal swabs tested by TMA. The organism load in women with trichomoniasis often is very high. Furthermore, Tvag contains multiple copies of the rRNA gene. These two factors no doubt increase the risk of cross contamination of specimens during the collection process. Clinicians should be aware of the potential for contaminating specimens intended for nucleic acid amplification testing in the course of collecting female genital tract specimens for all organisms but especially for Tvag.

HPAHPAHybridization Protection AssayHybridization Protection Assay

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

• Vaginal swabs and matching urine specimens were collected from 400 women attending the New Orelans Delgado STD clinic. Ninety-five percent of the women were African American.• The following tests were performed to detect Tvag at the LSU Health Sciences Center STD Pathogens Laboratory, using vaginal swabs specimens:

•Culture (In-PouchTM TV, BioMed Diagnostics, Inc., San Jose, CA) with microscopic examination on days 1, 3 and 5 post innoculation.•Wet Mount

• The specimens were placed in Gen-Probe Incorporated swab and urine transport media tubes and stored at 2-8oC until testing. The TMA tests were performed at the LSU laboratory.

• The Tvag TMA assay procedure, including target capture, TMA and HPA steps is illustrated in Figure 1. The following is a summary of each step:

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 LEADER® HC+ luminometer.A Tvag TMA result over 50,000 RLU was considered positive.

Figure 4. HPA Step

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Gen-Probe Proprietary Target Capture Technology

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Bead Bead OligoOligo

Capture Capture OligoOligo "Tail" "Tail"

Target Target SequenceSequence

Capture Capture SequenceSequence

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ProteinProteinCell debrisCell debris

PlasmaPlasma

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INTRODUCTION

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. From 10 to 50% of infections are asymptomatic. Diagnosis of Tvag infection is problematic. The commonly used wet mount, while rapid, has low sensitivity as does the Pap’s smear. Culture has greater sensitivity but takes up to five days. A rapid, amplified assay system is described here for the detection of Tvag from female vaginal swabs and matching urine specimens. An important goal of this study was to determine if female urine specimens would have adequate sensitivity to be used as screening samples for Tvag detection.

REFERENCES

Table 1. Sensitivity and Specificity of TMA vs Culture

Table 2. Test Results by Wet Prep, Culture and Urine TMA

*Wet preparation results missing in 4 cases.+A repeat TMA test using the remnant original specimen was positive in this case.§6/14 repeat TMA tests were positive.

Figure 5. Holding Cup for Vaginal Swabs. Same cup was used for multiple patients probably resulting in contamination.