Abstract
The InPouch Trichomonas vaginalis test is the gold standard for clinical culture for Trichomonas vaginalis screening. The current package insert recommends an examination period of 3 days. After review of 2,499 InPouch tests spanning 13 years, we observed that examination up to 3 days will detect only 82.8% (95% confidence interval [CI], 79.0% to 86.2%) of positive specimens.
TEXT
Trichomonas vaginalis is the most prevalent nonviral sexually transmitted infection (STI) worldwide (1, 2) and is a recognized risk factor for the acquisition and transmission of the human immunodeficiency virus (HIV), herpes simplex virus (HSV), and human papillomavirus (HPV) (3–5). Furthermore, increasing evidence implicates T. vaginalis infection as a risk factor in men for prostate cancer (6). Prior to the recent introduction of the highly sensitive (95.2 to 100%) T. vaginalis nucleic acid amplification test (NAAT) (7), the diagnosis of trichomoniasis has relied primarily on vaginal wet prep and culture using the InPouch T. vaginalis test (Biomed Diagnostics, White City, OR). The sensitivity of vaginal wet prep compared to T. vaginalis culture is approximately 50.0% to 66.0% (8), while the sensitivity of T. vaginalis culture compared to T. vaginalis NAAT is 75.0% to 95.7% in women and 28.6% to 100% in men at 100% specificity (9). Nevertheless, many clinics do not currently have access to T. vaginalis NAAT, and neither it nor the vaginal wet prep allows for the organism to be grown in culture for drug susceptibility testing and/or genotyping, if necessary.
The validation practices of next-generation molecular testing platforms use the InPouch as the comparison gold standard and follow the current instructional guidelines of an incubation and observational cutoff of 3 days postinoculation (Biomed Diagnostics document no. 100-001, revision K). The older revisions of the package insert (for example, Biomed Diagnostics document no. 100-001, revision D) indicate an examination range of 1 to 5 days. Our laboratory, one of the testing laboratories during the design and implementation of the InPouch system, uses a read range of five daily readings spanning 5 to 7 days, which is a realistic observation range for many laboratories not staffed on weekends. In actual laboratory practice, two of five collection days have a concurrent three-day observation period: Monday and Tuesday collections. Specimens collected on a Wednesday or Thursday have their third reads on day 5. Friday collections have their first observational read on day 3 (Table 1). The optimal timing for incubation and observation of the T. vaginalis InPouch culture is currently unclear. The objective of this study was to qualify the newer T. vaginalis InPouch package insert instructions with actual laboratory scheduling. We hypothesized that a cutoff of 3 days will fail to capture a substantial number of T. vaginalis-positive specimens.
Table 1.
Collection and observation matrix, five readings over seven daysa
| Collection day | No. of days postinoculation |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Tuesday | Wednesday | Thursday | Friday | Monday | Tuesday | Wednesday | Thursday | Friday | |
| Monday | 1 | 2 | 3 | 4 | 7 | ||||
| Tuesday | 1 | 2 | 3 | 6 | 7 | ||||
| Wednesday | 1 | 2 | 5 | 6 | 7 | ||||
| Thursday | 1 | 4 | 5 | 6 | 7 | ||||
| Friday | 3 | 4 | 5 | 6 | 7 | ||||
Saturday and Sunday are included in the seven days.
T. vaginalis InPouch specimens were collected from women undergoing gynecological care at The University of Alabama at Birmingham (UAB) and the Jefferson County Department of Health (JCDH) sexually transmitted diseases (STD) clinic and from men clinically suspected to have T. vaginalis as a cause of nongonococcal urethritis (NGU). We subsequently reviewed all InPouch testing results at these institutions (UAB and the JCDH STD clinic) processed by our laboratory. InPouch cultures were inoculated in the clinic exam room for outpatients or in the patient's hospital room for hospitalized patients, where they were kept at room temperature for less than 8 h (in clinics or hospital areas without incubators) or incubated immediately at 35°C to 37°C (in clinics or hospital areas with incubators) until transfer to the laboratory. In the laboratory, the InPouch was incubated at 35°C to 37°C during the testing period. Day 1 was defined as the next day after specimen collection. InPouch cultures were monitored daily for a total of five readings until positive, up to 7 days postinoculation, as part of our laboratory's standard protocol. InPouch cultures that did not grow at the end of the total of five daily readings were recorded as negative for live T. vaginalis. This study was approved by the UAB Institutional Review Board. Positive InPouch prevalence rates are presented in two formats: positive prevalence among the total population by day and positive prevalence per day of examination period. Statistics were calculated using OpenEpi version 2.3.1 (Open Source Epidemiologic Statistics for Public Health, version 2.3.1) and SPSS version 21 (IBM Corporation, Armonk, NY). Alpha values were set at 5%.
A total of 2,499 T. vaginalis InPouch specimens were collected from patients between May 2000 and April 2013. Of 2,499 InPouch specimens tested, 419 (16.8%; 95% confidence interval [CI], 15.3% to 18.3%) were positive. The day of positive culture determination for these 419 T. vaginalis InPouch specimens is stratified in Table 2. During the first 3 days, approximately 82.8% (95% CI, 79.0% to 86.2%) of positive T. vaginalis cultures were detected. Of the remaining positive T. vaginalis cultures, 17.2% (95% CI, 13.8% to 21.0%) were detected in four to seven days (Table 3). To test our hypothesis, the observed number of positive specimens (347) in the first 3 days was compared to the expected number of positive specimens (419), the actual total of positive T. vaginalis specimens; the difference between expected and observed was statistically significant (Fisher's exact 2-tailed P = 0.005). The average proportion of positives detected daily was approximately 4.5% of the total number of unresolved InPouches. By the final, fifth daily reading, all positive InPouch cultures had been observed.
Table 2.
Positive Trichomonas vaginalis InPouch findings matrix by days postinoculation (n = 419)
| Collection day | No. of positive cultures by day postinoculation |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Tuesday | Wednesday | Thursday | Friday | Monday | Tuesday | Wednesday | Thursday | Friday | |
| Monday | 31 | 21 | 6 | 1 | 0 | ||||
| Tuesday | 41 | 27 | 9 | 1 | 0 | ||||
| Wednesday | 31 | 29 | 4 | 0 | 0 | ||||
| Thursday | 68 | 33 | 2 | 0 | 0 | ||||
| Friday | 84 | 19 | 11 | 1 | 0 | ||||
Table 3.
Positive Trichomonas vaginalis InPouch findings by day postinoculation
| No. of days postinoculation | No. of positive cultures observed | % positive among total | Daily % positive rate |
|---|---|---|---|
| 1 | 171 | 40.8 | 6.8 |
| 2 | 77 | 18.4 | 3.3 |
| 3 | 99 | 23.6 | 4.4 |
| 4–7 | 72 | 17.2 | 3.3 |
| Total | 419 | 100 |
A comparison of the observed distribution of positive InPouch specimens indicates that approximately 17.2% of positive InPouch cultures will be observed beyond the first three read days and that this differential is statistically significant. This finding has several important public health implications. The first has to do with the three-day cutoff affecting treatment management. By prematurely ending a test, T. vaginalis that is slow to grow for various reasons may not make it to visually critical levels, as the InPouch does require careful scanning of the pouch in three dimensions and not just along the edges of the viewing field. These false negatives represent a potential failure to treat an infected patient and a reservoir for ongoing infection. A second implication is the cutoff of 3 days in the development of next-generation molecular typing methods. There is no argument that the new molecular platforms, i.e., the APTIMA T. vaginalis assay (Hologic Gen-Probe, San Diego, CA), are much more sensitive; however, they cannot differentiate active colonization of live T. vaginalis from dead T. vaginalis. From a purist viewpoint, by using shorter cutoff T. vaginalis InPouch read times, and thereby potentially failing to identify 13.8% to 21.0% additional positive specimens, the validation calculations used by developers of NAAT procedures may not be correct, although one could argue that they probably would not change the outcome. By failing to capture additional positive cultures, the specificity and positive predictive values for the performance of the tests would be imprecise. In conclusion, our data support the recommendation that the InPouch be examined daily for 5 days over a seven-day period to reduce the possibility of a false negative for an appropriately handled culture.
Footnotes
Published ahead of print 4 September 2013
REFERENCES
- 1.World Health Organization 2001. Global prevalence and incidence of selected curable sexually transmitted infections: overviews and estimates. WHO/HIV_AIDS/2001.02 World Health Organization, Geneva, Switzerland [Google Scholar]
- 2.Hobbs MM, Seña AC, Swygard H, Schwebke JR. 2008. Trichomonas vaginalis and trichomoniasis, p 771–794 In Holmes K, Sparling P, Stamm W, Piot P, Wasserheit J, Corey L, Cohen M, Sexually transmitted diseases, 4th ed. McGraw-Hill Companies, Inc., New York, NY [Google Scholar]
- 3.Kissinger P, Adamski A. 20 April 2013. Trichomoniasis and HIV interactions: a review. Sex. Transm. Infect. 89:426–433 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Gottlieb SL, Douglas JM, Jr, Foster M, Schmid DS, Newman DR, Baron AE, Bolan G, Iatesta M, Malotte CK, Zenilman J, Fishbein M, Peterman TA, Kamb ML. 2004. Incidence of herpes simplex virus type 2 infection in 5 sexually transmitted disease (STD) clinics and the effect of HIV/STD risk-reduction counseling. J. Infect. Dis. 190:1059–1067 [DOI] [PubMed] [Google Scholar]
- 5.Shew ML, Fortenberry JD, Tu W, Juliar BE, Batteiger BE, Qadadri B, Brown DR. 2006. Association of condom use, sexual behaviors, and sexually transmitted infections with the duration of genital human papillomavirus infection among adolescent women. Arch. Pediatr. Adolesc. Med. 160:151–156 [DOI] [PubMed] [Google Scholar]
- 6.Stark JR, Judson G, Alderete JF, Mundodi V, Kucknoor AS, Giovannucci EL, Platz EA, Sutcliffe S, Fall K, Kurth T, Ma J, Stampfer MJ, Mucci LA. 2009. Prospective study of Trichomonas vaginalis infection and prostate cancer incidence and mortality: Physicians' Health study. J. Natl. Cancer Inst. 101:1406–1411 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Schwebke JR, Hobbs MM, Taylor SN, Sena AC, Catania MG, Weinbaum BS, Johnson AD, Getman DK, Gaydos CA. 2011. Molecular testing for Trichomonas vaginalis in women: results from a prospective U.S. clinical trial. J. Clin. Microbiol. 49:4106–4111 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Wiese W, Patel SR, Patel SC, Ohl CA, Estrada CA. 2000. A meta-analysis of the Papanicolaou smear and wet mount for the diagnosis of vaginal trichomoniasis. Am. J. Med. 108:301–308 [DOI] [PubMed] [Google Scholar]
- 9.Nye MB, Schwebke JR, Body BA. 2009. Comparison of APTIMA Trichomonas vaginalis transcription-mediated amplification to wet mount microscopy, culture, and polymerase chain reaction for diagnosis of trichomoniasis in men and women. Am. J. Obstet. Gynecol. 200:188.e1–188.e7 [DOI] [PubMed] [Google Scholar]