ESwab as an Optional Collection Device for Use with the Affirm VPIII Microbial Test System

The ESwab collection device was compared to the collection swab provided as part of the Affirm VPIII microbial identification test kit for testing vaginal specimens with the Affirm test system. There was excellent agreement between the two sampling devices for Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis.     

Comparison of Affirm VPIII and Papanicolaou tests in the detection of infectious vaginitis

To compare the Affirm VPIII molecular test (Becton Dickinson, Burlington, NC) with morphologic identification used in routine Papanicolaou (Pap) test screening in the detection and identification of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis, we identified 431 cases with a concomitant Pap test and Affirm VPIII assay performed from the archives of a large academic institution. The study population consisted of women ranging in age from 17 to 79 years (mean and median ages, 33 and 31 years, respectively). With a routine Pap test, 60 patients (13.9%) were found to have bacterial vaginosis, 60 (13.9%) candidiasis, and 3 (0.7%) Trichomonas infection. With the Affirm VPIII assay, 183 (42.5%) patients tested positive for G vaginalis, 70 (16.2%) positive for Candida species, and 10 (2.3%) positive for T vaginalis. The differences were statistically significant. The results demonstrate that our patient population had a high incidence of bacterial vaginosis/Candida vaginitis; however, the Affirm VPIII was a more sensitive diagnostic test for the detection and identification of all 3 organisms compared with the Pap test.     

Evaluation of the Affirm Ambient Temperature Transport System for the detection and identification of Trichomonas vaginalis, Gardnerella vaginalis, and Candida species from vaginal fluid specimens

The objective of this study was to measure the performance of the Affirm Ambient Temperature Transport System (ATTS) over time and to estimate the length of time the system can preserve a vaginal specimen containing the three common organisms causing vaginitis: Trichomonas vaginalis, Candida species, and Gardnerella vaginalis (one of the causative agents of bacterial vaginosis). Women with symptoms of vaginitis presenting to one of three clinical centers were evaluated over a 4- to 8-week period. Four simultaneously obtained swabs were collected and tested by the Affirm VPIII assay at time zero with and without a preservative reagent, at 24 h with reagent, and at either 48 or 72 h with reagent. For each of the three organisms, Trichomonas, Gardnerella, and Candida, positivity at each time point was evaluated and compared to that at reference time zero with and without the ATTS. A total of 940 specimens were obtained from the three clinical sites. Eight hundred three were positive for one or more of the three organisms. Gardnerella had the highest overall positive rate (62%), followed by Candida with 18% and Trichomonas at 9%. The percent sensitivity versus control for Trichomonas ranged from 100% at time zero with and without reagent to 91% by 72 h. Gardnerella and Candida sensitivity remained at 100% for each time period. The Affirm VPIII ATTS system performed within 10% of the control swab (no transport reagent) at all four time points (0, 24, 48, and 72 h) for Trichomonas, Gardnerella, and Candida.     

Detection of Trichomonas vaginalis DNA by Use of Self-Obtained Vaginal Swabs with the BD ProbeTec Qx Assay on the BD Viper System

Trichomonas vaginalis is the most prevalent nonviral sexually transmitted infection worldwide, and improved diagnostic methods are critical for controlling this pathogen. Diagnostic assays that can be used in conjunction with routine chlamydia/gonorrhea nucleic acid-based screening are likely to have the most impact on disease control. Here we describe the performance of the new BD T. vaginalis Q^x (TVQ) amplified DNA assay, which can be performed on the automated BD Viper system. We focus on data from vaginal swab samples, since this is the specimen type routinely used for traditional trichomonas testing and the recommended specimen type for chlamydia/gonorrhea screening. Vaginal swabs were obtained from women attending sexually transmitted disease or family planning clinics at 7 sites. Patient-collected vaginal swabs were tested by the TVQ assay, and the Aptima T. vaginalis (ATV) assay was performed using clinician-collected vaginal swabs. Additional clinician-collected vaginal swabs were used for the wet mount and culture methods. Analyses included comparisons versus the patient infection status (PIS) defined by positive results with the wet mount method or culture, direct comparisons assessed with κ scores, and latent class analysis (LCA) as an unbiased estimator of test accuracy. Data from 838 women, 116 of whom were infected with T. vaginalis, were analyzed. The TVQ assay sensitivity and specificity estimates based on the PIS were 98.3% and 99.0%, respectively. The TVQ assay was similar to the ATV assay (κ = 0.938) in direct analysis. LCA estimated the TVQ sensitivity and specificity as 98.3 and 99.6%, respectively. The TVQ assay performed well using self-collected vaginal swabs, the optimal sample type, as recommended by the CDC for chlamydia/gonorrhea screening among women.     

Evaluation of the OSOM Trichomonas rapid test versus wet preparation examination for detection of Trichomonas vaginalis vaginitis in specimens from women with a low prevalence of infection

The OSOM Trichomonas rapid test (OSOM Trich) was compared to the wet preparation examination (WP) for the detection of Trichomonas vaginalis vaginitis in women with a low prevalence of infection. A total of 19/1,009 (2%) women had T. vaginalis infection. OSOM Trich had very good performance, with sensitivity, specificity, efficiency, positive predictive value, and negative predictive value of 94.7, 100, 99.9, 100, and 99.9%, respectively. The implementation of OSOM Trich would decrease labor costs.     

Correlation of Leukorrhea and Trichomonas vaginalis Infection

Trichomonas vaginalis is a common sexually transmitted infection (STI) causing vaginitis. Microscopy has poor sensitivity but is used for diagnosis of trichomoniasis in resource-poor settings. We aimed to provide a more reliable diagnosis of trichomoniasis by investigating an association with leukorrhea. Women presenting for evaluation of vaginal discharge, STI exposure, or preventative gynecologic examination were evaluated for Trichomonas infection. Vaginal pH was determined and microscopy was performed by the provider, who recorded the number of polymorphonuclear leukocytes (PMNLs) per epithelial cell and the presence of clue cells, yeast, and/or motile trichomonads. Leukorrhea was defined as greater than one PMNL per epithelial cell. Culture and a nucleic acid amplification test (NAAT) were used to detect T. vaginalis. Patients were evaluated for Chlamydia trachomatis and Neisseria gonorrhoeae using NAATs and bacterial vaginosis using Gram stains. Two hundred ninety-four women were enrolled, and 16% were found to have Trichomonas (46/294). Trichomonas infection was more common in parous non-Hispanic, black women, who reported low rates of contraceptive use (33% versus 17%; P = 0.02) and a STI history (85% versus 55%; P = 0.002). These women were more likely to report vaginal discharge (76% versus 59%; P = 0.02) and have an elevated vaginal pH (87% versus 48%; P < 0.001) and gonorrhea infection (15% versus 4%; P = 0.002). Leukorrhea was associated with a 4-fold-increased risk of Trichomonas infection. Leukorrhea on microscopy was associated with Trichomonas vaginitis. Patients with leukorrhea should be evaluated with more-sensitive tests for T. vaginalis, preferably NAATs, if microscopy is negative.     

Evaluation and subsequent optimizations of the quantitative AmpliSens Florocenosis/Bacterial vaginosis‐FRT multiplex real‐time PCR assay for diagnosis of bacterial vaginosis

Traditional microscopy‐based methods for diagnosis of bacterial vaginosis (BV) are underutilized in many settings, and molecular techniques may provide opportunities for rapid, objective, and accurate BV diagnosis. This study evaluated the quantitative AmpliSens Florocenosis/Bacterial vaginosis‐FRT multiplex real‐time PCR (Florocenosis–BV) assay. Vaginal samples from a previous study including unselected female subjects (n = 163) and using Amsel criteria and 454 pyrosequencing for BV diagnosis were examined with the Florocenosis–BV test and additionally tested for the presence and quantity of Gardnerella vaginalis clades 3 and 4. The Florocenosis–BV assay demonstrated 100% and 98% sensitivity compared with the Amsel criteria and 454 pyrosequencing, respectively, with 91% specificity. The modified Florocenosis–BV assay (detecting also G. vaginalis clades 3 and 4) resulted in 100% sensitivity vs the Amsel criteria and 454 pyrosequencing with specificity of 86% and 88%, respectively. Further optimizations of thresholds for the quantitative parameters used in the kit resulted in 99–100% accuracy vs Amsel criteria and 454 pyrosequencing for selected parameters. The Florocenosis–BV assay is an objective, accurate, sensitive, and specific method for BV diagnosis; however, the performance of the test can be further improved with some minor optimizations.     

Diagnosis of Trichomonas vaginalis Infection by PCR Using Vaginal Swab Samples

Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.     

Diagnostic Rates Differ on the Basis of the Number of Read Days with the Use of the InPouch Culture System for Trichomonas vaginalis Screening

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.     

Relationship of the virulence of Trichomonas vaginalis and the major histocompatibility complex in murine trichomonad infection

 A biological assay based upon the induction of abscesses in mice injected subcutaneously with Trichomonas vaginalis was shown to be a valid method for comparing the virulence of two isolates of T. vaginalis cultured from two patients, one suffering from severe vaginitis and the other exhibiting only mild disease. The data showed excellent correlation between the physical dimensions of abscesses in mice injected with each trichomonad isolate and the severity of vaginitis produced in the women from whom the isolates were obtained. The assay employed in our study incorporated measurement of the mean abscess volumes from day 1 to day 6 post-inoculation with T. vaginalis. We found that the abscess assay was clearly superior to a murine intraperitoneal assay for virulence evaluation of trichomonad isolates. We then used the murine abscess assay to determine the susceptibility of different strains of mice to infection with a virulent T. vaginalis isolate so as to test whether the genetic constitution of the host would influence the pathogenesis of the disease. BALB/c (H-2^d) mice were susceptible to infection with T. vaginalis, but both CBA/CaH (H-2^k) and BALB/c-H-2^k mice were shown to be resistant. The quantitation of abscess formation in these inbred and congeneic resistant mouse strains demonstrates that the severity of infection with T. vaginalis is governed by genes mapping within the major histocompatibility complex. Received: 16 October, 1995 / Accepted: 15 March, 1996     

Use of DNA hybridization test for diagnosing bacterial vaginosis in women with symptoms suggestive of infection

The purpose of this study was to evaluate a DNA hybridization test (Affirm VPIII) as an alternative to Gram stain for the rapid diagnosis of bacterial vaginosis in women with clinical signs of vaginal infection. Vaginal specimens were collected from 321 symptomatic women, and analyzed for bacterial vaginosis by both Gram stain using Nugent criteria and DNA hybridization test. Sensitivity, specificity, positive predictive value, and negative predictive value of the DNA hybridization test were determined using the Gram staining as the standard for diagnosis of bacterial vaginosis. Of the 321 patients, 115 (35.8%) were Gram positive for bacterial vaginosis and 126 (39.2%) were negative. 80 patients (25.0%) demonstrated intermediate Gram staining that was also considered negative. The Affirm system detected G. vaginalis in 107 (93.0%) of 115 vaginal specimens positive for bacterial vaginosis diagnosed by Gram stain. Compared to the Gram stain, DNA hybridization test had a sensitivity of 87.7% and a specificity of 96.0%. Positive and negative predictive values of the DNA hybridization test were 93.0% and 92.7%, respectively. In conclusion, Affirm VPIII hybridization test correlated well with Gram stain and may be used as a rapid diagnostic tool to exclude bacterial vaginosis in women with genital complaints.     

Development of PCR Assays for Detection of Trichomonas vaginalis in Urine Specimens

Trichomonas vaginalis infections are usually asymptomatic or can result in nonspecific clinical symptoms, which makes laboratory-based detection of this protozoan parasite essential for diagnosis and treatment. We report the development of a battery of highly sensitive and specific PCR assays for detection of T. vaginalis in urine, a noninvasive specimen, and development of a protocol for differentiating among Trichomonas species that commonly infect humans.     

Bacterial vaginosis,aerobic vaginitis,vaginal inflammation and major Pap smear abnormalities

The purpose of this investigation was to evaluate the impact of the vaginal milieu on the presence of abnormal Pap smears and a positive human papilloma virus (HPV) test. A cross-sectional study was conducted between June 2014 and May 2015, evaluating the vaginal discharge by fresh wet mount microscopy and comparing these data with Pap smear findings. Wet mount slides were scored for bacterial vaginosis (BV), aerobic vaginitis (AV), presence of Candida and Trichomonas vaginalis. Cytologic evaluation was done on all Pap smears according to the Bethesda criteria. The cobas© HPV Test (Roche) was performed for HPV detection. A total of 622 cases were evaluated. The mean age of the patients was 41.6?±?10.65 years (range 21–75). Eighty-three women (13.3 %) had a cytology result worse than low-grade squamous intraepithelial lesion (LSIL). When comparing this group with the one with normal or minor [atypical squamous cells of undetermined significance (ASC-US) or LSIL] Pap smear abnormalities, there were no differences in the presence of Candida (32.5 % vs. 33.2 %, p?=?1.0), absence of lactobacilli (38.6 % vs. 32.5 %, p?=?0.32) or BV (20.5 % vs. 13.2 %, p?=?0.09). On the other hand, moderate or severe inflammation (msI) (41.0 % vs. 28.8 %, p?=?0,04), moderate or severe AV (msAV) (16.9 % vs. 7.2 %, p?=?0.009) and msAV/BV (37.3 % vs. 20.0 %, p?=?0.001) were more common in women with such major cervical abnormalities. No significant association was found between deviations of the vaginal milieu and high-risk HPV infection. The presence of msI or msAV, but not BV, is independently associated with an increased risk of major cervical cytological abnormalities, but not with HPV infection.     

The presence of dsRNA virus in Trichomonas vaginalis isolates from symptomatic and asymptomatic Indian women and its correlation with in vitro metronidazole sensitivity

Purpose: Trichomonas vaginalis, a protozoan parasite, is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted disease. The infection encompasses from a complete asymptomatic presentation to severe sequelae; yet, the virulence markers have been poorly understood. It is suggested that the presence of Trichomonas vaginalis virus (TVV) in T. vaginalis may have an impact on its virulence, and its relatedness to in vitro metronidazole resistance has been reported. The aim of the study was to assess the presence of TVV in fresh and Long –Term Cultivated (LTC) maintained T. vaginalis isolates from symptomatic (S) and asymptomatic (AS) Indian women and its relatedness, if any, with symptomatology and in vitro drug sensitivity. Materials and Methods: One thousand women (537 S and 463 AS) were screened for the presence of T. vaginalis by wet smear and culture examination of vaginal swab and urine sample. Fresh and LTC (6 months–2 years) maintained 15 isolates each from 15 S and 15 AS women were subjected to agarose gel electrophoresis following total cellular RNA extraction to evaluate the presence of double stranded (ds) RNA viral infection. The susceptibility of isolates to metronidazole was determined in vitro. Results: On agarose gel electrophoresis, three bands (5.5, 2.5 and 1.5 kb) were observed in all the 30 fresh isolates from 15 S and 15 AS women and only in 7 LTC isolates from 3 S and 4 AS women. All the fresh isolates harbouring TVV were found to be sensitive to metronidazole in vitro irrespective of the symptomatology of subjects, and out of seven LTC isolates harbouring TVV, six were sensitive to metronidazole and one showed borderline resistance. Conclusions: The results suggest that the presence of TVV alone may not be a virulence marker and loss of TVV on LTC appears to be related to drug resistance. The T. vaginalis Indian isolates are sensitive to metronidazole.     

Efficient Diagnosis of Vulvovaginal Candidiasis by Use of a New Rapid Immunochromatography Test

The clinical symptoms of vulvovaginal candidiasis (VVC) are nonspecific, and misdiagnosis is common, leading to a delay in the initiation of antifungal treatment. We evaluated a new immunochromatography test (ICT), the CandiVagi assay (SR2B, Avrille, France), for the rapid diagnosis of VVC. This test, which employs an immunoglobulin M antibody directed against the β-1,2-mannopyranosyl epitopes found in the yeast cell wall, was compared with direct microscopic examination and culture of vaginal swabs. Two-hundred five women were investigated, including 130 women with symptomatic vaginitis and 75 asymptomatic controls. Two vaginal swabs were obtained from each woman: one was used to prepare a wet mount and Gram-stained preparations for direct microscopic examination and was also cultured on Sabouraud dextrose agar for the isolation of Candida spp., and the second swab was used for ICT. The sensitivities of microscopic examination, culture, and ICT for the diagnosis of VVC were 61%, 100%, and 96.6%, respectively, while the specificities of the three methods were 100%, 82%, and 98.6%, respectively. ICT had a negative predictive value of 98.6%, a positive predictive value of 96.6%, and an efficiency of 98%. ICT provided a rapid result and a better compromise between sensitivity and specificity than conventional microscopy and culture for the diagnosis of VVC. This easy-to-perform diagnostic test will be useful to practitioners treating women with symptoms of vaginitis.Vaginitis is the commonest reason for gynecological consultation in women of childbearing age. Anaerobic bacteria are the most prevalent cause of vaginal infection in the United States and Europe, followed closely by Candida spp. (34, 37). It is estimated that at least 75% of healthy adult women will suffer one episode of Candida vulvovaginitis during their reproductive lives and that 5% will have recurrent infectious episodes (20, 30). Candida albicans is responsible for infection in 80 to 90% of cases, although the incidence of vulvovaginal candidiasis (VVC) due to non-C. albicans species such as C. glabrata has increased steadily over the past few decades (21, 36).The main symptoms of VVC have been widely described and include vulvar and vaginal pruritus, pain or a burning sensation, and external dysuria (8). Physical examination may reveal perineal edema, vulvar and vaginal erythema, fissures, and a thick curdy discharge (8). However, these symptoms are nonspecific and do not enable clinicians to distinguish confidently between VVC, bacterial vaginosis, and Trichomonas vaginalis infection (2, 22, 23, 31), leading to subsequent suboptimal care. The accurate diagnosis of VVC currently depends on the demonstration of a Candida sp. in vaginal swabs by direct microscopic examination and/or culture. A positive Gram stain, the absence of a watery discharge, and patient self-diagnosis of “another yeast infection” have been identified as the best predictors of a positive culture for patients with VVC (1).Several rapid diagnostic tests have been developed over the past 25 years in an attempt to speed up the diagnosis of VVC (17, 29). Latex particle agglutination (LPA) was found to be more sensitive than KOH microscopy (19, 28, 29) and was more specific than other diagnostic criteria (10). However, a few studies reported false-positive reactions with this test (39) or a sensitivity that was lower than that of KOH microscopic examination carried out by experienced clinical practitioners (38).We have developed a sensitive immunochromatography test (ICT), the CandiVagi assay (SR2B, Avrille, France), for the rapid diagnosis of VVC using an immunoglobulin M (IgM) monoclonal antibody (MAb) directed against the Candida mannan (18, 40). Here, we present the results of a preliminary evaluation of this test and a comparison of the results obtained by ICT with those obtained by conventional microscopy and culture. Specific attention was focused on the ability of ICT to discriminate between Candida carriage and Candida infection and its specificity for women with bacterial/trichomonal vaginitis.     

Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel''s criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel''s criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.     

Reciprocal Interference between Lactobacillus spp. and Gardnerella vaginalis on Initial Adherence to Epithelial Cells

Bacterial vaginosis (BV) is the most common vaginal disorder in women of child-bearing age. It is widely accepted that the microbial switch from normal microflora to the flora commonly associated with BV is characterized by a decrease in vaginal colonization by specific Lactobacillus species together with an increase of G. vaginalis and other anaerobes. However, the order of events leading to the development of BV remains poorly characterized and it is unclear whether the decrease in lactobacilli is a cause or a consequence of the increase in the population density of anaerobes. Our goal was to characterize the interaction between two Gardnerella vaginalis strains, one of which was isolated from a healthy woman (strain 5-1) and the other from a woman diagnosed with BV (strain 101), and vaginal lactobacilli on the adherence to cervical epithelial cells. In order to simulate the transition from vaginal health to BV, the lactobacilli were cultured with the epithelial cells first, and then the G. vaginalis strain was introduced. We quantified the inhibition of G. vaginalis adherence by the lactobacilli and displacement of adherent lactobacilli by G. vaginalis. Our results confirmed that pathogenic G vaginalis 101 had a higher capacity for adhesion to the cervical epithelial cells than strain 5-1. Interestingly, strain 101 displaced L. crispatus but not L. iners whereas strain 5-1 had less of an effect and did not affect the two species differently. Furthermore, L. iners actually enhanced adhesion of strain 101 but not of strain 5-1. These results suggest that BV-causing G. vaginalis and L. iners do not interfere with one another, which may help to explain previous reports that women who are colonized with L. iners are more likely to develop BV.     

Stepwise diagnosis of Trichomonas vaginalis infection in adolescent women

The objective of this study was to examine the effects of clinical factors and of the type and timing of a secondary test in improving the sensitivity of Trichomonas vaginalis detection in young women over that of a wet mount alone. For this purpose, sexually active adolescent women (n = 345) were recruited from a hospital teen clinic or emergency department. Following an interview and a pelvic exam, four primary T. vaginalis tests (wet mount, culture, a rapid test, and a nucleic acid amplification test [NAAT]) were performed on vaginal swabs. If the wet-mount result was negative, two secondary tests (culture and a rapid test) were performed on the used wet-mount swab and saline. A positive result by any of the four primary tests was considered a true T. vaginalis-positive result. The prevalence of T. vaginalis was 18.8% overall and 8.8% in the 307 wet-mount-negative women. There was 100% concordance between primary and secondary rapid tests. Secondary culture was 80% sensitive compared to primary culture. The likelihood of a positive rapid test increased with increasing time between specimen collection and testing. A wet mount followed by a rapid test was the most sensitive strategy using two tests (86.4%; confidence interval [CI], 75.3 to 93.4%). Limiting secondary testing to those with multiple partners resulted in a lower sensitivity (73.9%; CI, 61.5 to 84%) that was not significantly better than that of the wet mount alone (58.5%; CI, 45.6 to 70.6%). We conclude that a rapid test can be delayed or performed on a used swab with no loss of sensitivity. Until a NAAT for T. vaginalis is commercially available, a stepwise approach using an additional rapid test for wet-mount-negative women is recommended for adolescent women regardless of clinical factors.