Trichomonas vaginalis, the most common curable sexually transmitted infection among women worldwide,1 is associated with vaginitis, cervicitis, urethritis, low birth weight, preterm delivery, endometritis and may increase the risk of herpes simplex virus and human immunodeficiency virus acquisition and transmission.1–4 Repeat infections are common, ranging from 5% to 31%, and share similar sequelae to primary infections.5–9
While wet prep has been used for decades to detect T. vaginalis, it has been found to have low sensitivity ranging from 40% to 60%.10 Culture has been considered the gold standard and has a sensitivity of 75% to 96% and a specificity of up to 100%.11 Nucleic acid amplification tests (NAAT) for T. vaginalis are more sensitive than culture (92–97% sensitivity)11 and have been commercially available since 2011. Several highly sensitive T. vaginalis NAATs have been approved by the US and Food and Drug Administration (FDA) for use in women on urine, vaginal swab (including self-collected), and endocervical specimens.12–14 These include the Aptima TV (Hologic Gen-Probe), the BD ProbeTec Qx (BD Viper System; Becton-Dickinson), and GeneXpert TV (Cepheid) assay. Other FDA-approved NAAT tests that are close to becoming point of care assays include the Solana Trichomonas Assay15 and the AmpliVue Trichomonas Assay.16 The NAAT technology is based on DNA or rRNA extraction from specimens, followed by amplification of captured nucleic acids.
One issue in retesting after treatment is that remnant nucleic acid can still exist in vaginal fluids even if no viable organism persists, resulting in a positive test that may not have clinical meaning. Given high T. vaginalis repeat infection rates, the Centers for Disease Control and Prevention recommends rescreening women for T. vaginalis 3 months after completing treatment.17 However, there is some indication that most repeat infections occur early7 and that they may be due to treatment failure.18 There may be, therefore, a need to test sooner than 3 months. The Centers for Disease Control and Prevention STD Treatment Guidelines17 suggests that NAAT testing can occur as early as 2 weeks after treatment based on 2 studies among adolescent women who were mostly asymptomatic.19,20 Those studies did not take sexual exposure or treatment failure into consideration. The purpose of this present study was to determine when T. vaginalis nucleic acid clears among women who were successfully treated with metronidazole for trichomoniasis and who were not sexually exposed between diagnosis and rescreening.
This study was a substudy of a multicentered randomized trial of 2 different doses of metronidazole for the treatment of trichomoniasis. Methods have been described elsewhere,21 but briefly, study participants were recruited from 3 clinics: Delgado Personal Health Clinic, Crescent Care Health and Wellness Center in New Orleans, LA and the Jefferson County Department of Health (JCDH)—STD Specialty Clinic in Birmingham, AL. These clinics serve mostly African American, low income women, the demographic group most highly affected by T. vaginalis infections.16
Women were eligible to enroll into the study if they attended any of the clinics, were human immunodeficiency virus-uninfected, tested positive for T. vaginalis by clinical testing (wet prep or NAAT testing) with confirmation by another study test (culture or NAAT), and were willing to return to the clinic for follow-up visits. At enrollment, subjects' clinical diagnosis was confirmed with InPouch Culture or Aptima T. vaginalis (ATV) NAAT. Eligible participants were treated with oral metronidazole (MTZ) in either a 2 g single-dose (4 × 500 mg tablets) or a 7-day 500 mg twice-daily regimen.
Women were asked to complete a total of 5 study visits: the enrollment visit and 4 weekly follow-up visits. The first weekly follow-up visit was scheduled 7 days after the completion of the metronidazole treatment. For women taking the 2 g single dose this was 7 days after the enrollment visit and for women on the 7-day 500 mg twice-daily regimen this was 14 days after the enrollment visit.
At each of the 4 follow-up visits, subjects self-collected a vaginal swab for T. vaginalis NAAT testing. At visit 5 (test of cure visit), subjects also self-collected a vaginal swab for T. vaginalis testing using culture. In addition to biological specimens, study participants were either interviewed by study staff or asked to complete a brief computer-assisted survey at each visit eliciting information on medication adherence, sexual exposure and other clinical and behavioral factors.
The NAAT specimens were tested in batches at either the LSUHSC or UAB laboratories using ATV. Results for NAAT tests were not available to provider or subjects during the study period and did not affect their study participation or treatment.
To be included in the analysis, subjects needed to: be adherent to treatment, self-report no sexual exposure during follow-up, not miss more than 2 consecutive follow-up visits, and have a T. vaginalis negative culture at their TOC visit. Sexual exposure was defined as vaginal sex without the use of a condom before both partners were treated.
Kaplan-Meier survival analysis was used to assess time to first NAAT-negative test after treatment completion. The log-rank test was used to test for differences in survival functions between groups to favor larger time values. The 95% Hall-Wellner confidence bands are shown in Figures 1 and 2 to provide an estimate of confidence across the entire survival curve. SAS 9.4 was used for this analysis.
The protocol was reviewed and received ethical approval by Institutional Review Boards at Tulane University, Louisiana State University Health Sciences Center (LSUHSC), the University of Alabama at Birmingham (UAB) and the JCDH Research Review Committee.
Of 98 women participating in the study, 9 were excluded due to the following reasons: missed more than 2 consecutive visits (n = 4), positive or unknown culture result at TOC (n = 4), and sexual exposure to untreated partner during follow-up (n = 1).
The 89 women included in the analyses were predominantly African American (95.5%) and younger than 30 years (mean, 26; range, 19–70). Most (60.7%) received single dose metronidazole and most (60.7%) were enrolled at the JCDH clinic. Additional demographics, behavioral risk factors, bacterial vaginosis, past TV infection, and birth control are described in Table 1.
Median time to first ATV-negative test was 8 days (range, 6–28 days) postcompletion of treatment. Among all women, the percentage testing negative at 7 days was 41.6% (95% confidence interval [CI], 32.1%–52.5%), 91.0% (95% CI, 84.0%–95.8%) at 14 days, and 96.6% (95% CI, 91.3%–99.1%) at 21 days. By 28 days, all women had at least 1 negative test result by NAAT. By 1 week after completion of treatment, 19.8% had engaged in sexual intercourse, which increased to 60.7% by 4 weeks. All reported that they always used condoms during vaginal sexual intercourse or both the participant and sexual partner(s) had completed treatment (Table 2).
The median time to clearance for women receiving the single dose versus multidose was 9 days (range, 6–28 days) versus 7 days (range, 7–20 days) (log-rank P = 0.04). By day 21 postcompletion of treatment, all women on the multidose had tested NAAT-negative, and by day 28, all women on the single-dose tested NAAT-negative (Fig. 2).
Time to first negative NAAT is defined as time between treatment completion and first ATV-negative test. Time to negative NAAT was shorter for those receiving multidose compared with single-dose (P = 0.04) but were similar by baseline vaginal douching status, baseline symptoms, history of T. vaginalis, and BV at enrollment (Table 3).
Six (6.7%) of the 89 women, after having a negative NAAT result, had at least 1 positive NAAT result at a subsequent visit. Two (2.2%) of these women were NAAT positive at TOC (Table 4). These 2 women were retested 8 weeks later and 1 woman was culture and NAAT-negative and the other women was culture positive but NAAT-negative.
We found that trichomonal nucleic acid was no longer detectable for all women receiving multidose MTZ by 3 weeks and all women receiving single-dose MTZ by 4 weeks posttreatment completion indicating that T. vaginalis NAAT retesting should not occur before those times. Other studies where only single-dose MTZ was used found that at 3 weeks, clearance rates are between 85%20 (which was significantly lower than our study) and 93%19 (which was within the same range we found). The lower rates found in these studies could have been attributed to small sample sizes, misclassification due to treatment failure or reexposure, or the higher organism load needed for DNA based testing.
Unlike the other studies, we removed women who were either potentially reexposed or failed treatment (as detected by a positive culture at 4 weeks), thus reducing the potential for misclassification. By 3 weeks posttreatment completion, nearly half of the women had engaged in vaginal sex, though all stated they used condoms or had condomless sex only after index and partner(s) were treated. While it is possible that self-reported sexual behavior is not accurate, weekly interviews should have reduced recall bias. The majority of the participants were interviewed using computer-assisted self-administered interviews which have been shown to reduce social desirability bias.22,23 It should be noted that sexual activity increased each week and by 3 weeks nearly half of the cohort had reinitiated vaginal intercourse. Waiting to test at 3 weeks does have some potential to mix treatment failure with reinfection.
Whereas other studies had smaller sample sizes, our study had a power greater than 0.95 to detect the clearance rates at 2, 3, and 4 weeks. While our sample size was largely symptomatic, analysis stratified by symptoms did not indicate a trend by symptom status.
The first negative NAAT test was sooner for women on multidose therapy compared to single-dose therapy. It is not clear why this occurred, but it should be noted that women who were on the multidose treatment had 7 days of treatment compared to 1 day of treatment for single-dose before follow-up started, thus this difference could have been attributed to lead time bias. It is important evaluate the difference in time to negative NAAT for women receiving multidose MTZ since, whereas single dose is presently the first line of treatment, there is mounting evidence that multidose is superior to single-dose.18,21,24
It is also interesting that 6 of 89 women had a negative ATV result and then had a subsequent positive result, despite a negative 4 week culture. Some of this could have been false-negative testing from the test itself. Another possibility is that sampling error could have occurred since these were self-collected vaginal swabs. It is also possible that T. vaginalis had not been fully cured and was in a persistent undetectable state, as has been found by others.25,26 With the widespread use of NAAT for T. vaginalis testing, it is essential to understand the optimal timing for testing so that unnecessary retreatment can be avoided. Retesting using T. vaginalis NAAT should occur at 3 weeks or greater after treatment completion for those on multidose and 4 weeks for those on single-dose. Clinical application of this time estimate must account for the time needed to complete the prescribed treatment regimen, suspected adherence, and sexual activity.
1. Silver BJ, Guy RJ, Kaldor JM, et al. Trichomonas vaginalis
as a cause of perinatal morbidity: A systematic review and meta-analysis. Sex Transm Dis 2014; 41:369–376.
2. McClelland RS, Sangare L, Hassan WM, et al. Infection with Trichomonas vaginalis
increases the risk of HIV-1 acquisition. J Infect Dis 2007; 195:698–702.
3. Kissinger P, Adamski A. Trichomoniasis and HIV interactions: A review. Sex Transm Infect 2013; 89:426–433.
4. Masha SC, Cools P, Sanders EJ, et al. Trichomonas vaginalis
and HIV infection acquisition: A systematic review and meta-analysis. Sex Transm Infect 2018.
5. Krashin JW, Koumans EH, Bradshaw-Sydnor AC, et al. Trichomonas vaginalis
prevalence, incidence, risk factors and antibiotic-resistance in an adolescent population. Sex Transm Dis 2010.
6. Kissinger P, Schmidt N, Mohammed H, et al. Patient-delivered partner treatment for Trichomonas vaginalis
infection: A randomized controlled trial. Sex Transm Dis 2006; 33:445–450.
7. Kissinger P, Secor WE, Leichliter JS, et al. Early repeated infections with Trichomonas vaginalis among HIV-positive and HIV-negative women. Clin Infect Dis 2008; 46:994–999.
8. Spence MR, Harwell TS, Davies MC, et al. The minimum single oral metronidazole dose for treating trichomoniasis: A randomized, blinded study. Obstet Gynecol 1997; 89:699–703.
9. Forna F, Gulmezoglu AM. Interventions for treating trichomoniasis in women. Cochrane Libr 2003.
10. Nathan B, Appiah J, Saunders P, et al. Microscopy outperformed in a comparison of five methods for detecting Trichomonas vaginalis
in symptomatic women. Int J STD AIDS 2015; 26:251–256.
11. Nye MB, Schwebke JR, Body BA. 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 2009; 200:188 e1–188 e7.
12. Van Der Pol B, Williams JA, Taylor SN, et al. Detection of Trichomonas vaginalis
DNA by use of self-obtained vaginal swabs with the BD ProbeTec Qx assay on the BD viper system. J Clin Microbiol 2014; 52:885–889.
13. Schwebke JR, Gaydos CA, Davis T, et al. Clinical evaluation of the Cepheid Xpert TV assay for detection of Trichomonas vaginalis
with prospectively collected specimens from men and women. J Clin Microbiol 2018; 56:e01091–e01017.
14. Schwebke JR, Hobbs MM, Taylor SN, et al. Molecular testing for Trichomonas vaginalis
in women: Results from a prospective U.S. clinical trial. J Clin Microbiol 2011; 49:4106–4111.
15. Gaydos CA, Schwebke J, Dombrowski J, et al. Clinical performance of the Solana® point-of-care Trichomonas assay from clinician-collected vaginal swabs and urine specimens from symptomatic and asymptomatic women. Expert Rev Mol Diagn 2017; 17:303–306.
16. Gaydos CA, Hobbs M, Marrazzo J, et al. Rapid diagnosis of Trichomonas vaginalis
by testing vaginal swabs in an isothermal helicase-dependent AmpliVue assay. Sex Transm Dis 2016; 43:369–373.
17. Workowski KA, Bolan GA; Centers for Disease C, Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recommendations and reports : Morbidity and mortality weekly report Recommendations and reports / Centers for Disease Control 2015; 64:1–137.
18. Howe K, Kissinger PJ. Single-dose compared with multidose metronidazole for the treatment of Trichomoniasis in women: A meta-analysis. Sex Transm Dis 2017; 44:29–34.
19. Van Der Pol B, Williams JA, Orr DP, et al. Prevalence, incidence, natural history, and response to treatment of Trichomonas vaginalis
infection among adolescent women. J Infect Dis 2005; 192:2039–2044.
20. Williams JA, Ofner S, Batteiger BE, et al. Duration of polymerase chain reaction-detectable DNA after treatment of Chlamydia trachomatis
, Neisseria gonorrhoeae
, and Trichomonas vaginalis
infections in women. Sex Transm Dis 2014; 41:215–219.
21. Kissinger P, Muzny CA, Mena LA, et al. Single-dose versus 7-day-dose metronidazole for the treatment of trichomoniasis in women: An open-label, randomised controlled trial. Lancet Infect Dis 2018.
22. Kissinger P, Rice J, Farley T, et al. Application of computer-assisted interviews to sexual behavior research. Am J Epidemiol 1999; 149:950–954.
23. Richman WL, Kiesler S, Weisband S, et al. A meta-analytic study of social desirability distortion in computer-administered questionnaires, traditional questionnaires, and interviews. J Appl Psychol 1999; 84:754–775.
24. Kissinger P, Mena L, Levison J, et al. A randomized treatment trial: Single versus 7-day dose of metronidazole for the treatment of Trichomonas vaginalis
among HIV-infected women. J Acquir Immune Defic Syndr 2010; 55:565–571.
25. Peterman TA, Tian LH, Metcalf CA, et al. Persistent, undetected Trichomonas vaginalis
infections? Clin Infect Dis 2009; 48:259–260.
26. Gatski M, Kissinger P. Observation of probable persistent, undetected Trichomonas vaginalis
infection among HIV-positive women. Clin Infect Dis 2010; 51:114–115.