Approach Considerations
Given the poor reliability of history and physical findings, diagnosis of trichomoniasis depends on laboratory testing. According to the CDC, providers should perform laboratory tests for trichomoniasis in all women seeking care for vaginal discharge and all women at high risk of STIs. [48]
Tests for trichomoniasis are quick and can be performed in the medical office. [73] In the past, saline wet mount testing was commonly used to test for trichomoniasis, although these tests have low sensitivity. The CDC now recommends molecular diagnostic tests, when available, to evaluate patients at risk for trichomoniasis. [11] Self-testing has also been proposed and studied but is not currently approved by the US Food and Drug Administration (FDA). [74, 75]
The basic office evaluation includes tests to exclude other possible causes of the patient’s complaints. Because T vaginalis infection is strongly associated with the presence of other STIs, [52] providers should test for other STIs, including gonorrhea, chlamydia, syphilis, HIV infection, hepatitis B, and hepatitis C. In multiple studies, the majority of women with T vaginalis infections were also found to have bacterial vaginosis. [76, 77, 52]
Standard Laboratory Studies
Molecular Techniques for Detecting Antigen, DNA, or RNA
The following six FDA-approved molecular tests are currently available for diagnosing trichomoniasis in women:
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Affirm VPIII Microbial Identification Test (a DNA probe; BD Diagnostic Systems, Sparks, Maryland)
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APTIMA T vaginalis assay (a nucleic acid hybridization test, Hologic Gen-Probe, San Diego, California)
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BD ProbeTec TV Qx Amplified DNA Assay (a DNA probe, Becton Dickinson, Franklin Lakes, New Jersey)
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OSOM Trichomonas Rapid Test (an antigen-based test; Genzyme Diagnostics, Cambridge, Massachusetts)
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Solana Trichomonas Assay (a DNA probe, Quidel, San Diego, California)
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Xpert TV Assay (a DNA probe, Cepheid, Sunnyvale, California)
All the above tests have a sensitivity greater than 83% (except for the Affirm VPIII, at 63%) and a specificity greater than 97% for detecting T vaginalis in vaginal secretions. Of the above tests, only Xpert TV has been FDA-approved for the diagnosis of trichomoniasis in men. [78] However, according to the CDC, APTIMA may also be used with male urine or urethral swabs from men. [11]
Affirm VPIII Microbial Identification Test
The Affirm VPIII Microbial Identification Test detects the presence of Trichomonas, Gardnerella, and Candida species by using direct hybridization technology. Its sensitivity is 90%-100%, [79] and the detection threshold is reported to be 5,000 trichomonads/mL. Results from the Affirm VPIII test are available within about 45 minutes.
A bulletin on proper preparation and testing of specimens is available from the manufacturer. Specimens for which testing is expected to be delayed for more than 1 hour at ambient temperature or 4 hours with refrigeration should be stored in the Affirm VPIII Ambient Temperature Transport System (ATTS) for up to 72 hours. [80] In a study by Hollman et al, no difference was noted in detection of T vaginalis in urine samples compared with vaginal swabs. [81]
APTIMA Trichomonas Vaginalis Assay
The APTIMA Trichomonas vaginalis Assay (Gen-Probe, San Diego, Calif) uses nucleic acid hybridization technology to detect the presence of T vaginalis. [57, 82] Sensitivity is 74%-98%, and specificity is 87%-98%. [38] One study reported decreased sensitivity for tests performed on first-void urine specimens in male patients. [79] The sample is run on a proprietary processing system capable of running about 1,000 samples per day. Expected turnaround time is about 1-2 days. Currently, the APTIMA Combo2 assay is FDA-approved for the diagnosis of chlamydia and gonorrhea, so laboratories using this technology may wish to add the T vaginalis assay to their existing technology.
BD ProbeTec TV Qx Amplified DNA Assay
The BD ProbeTec TV Qx Amplified DNA Assay is a DNA-amplification test developed for the BD Viper system, a proprietary automated platform capable of testing for T vaginalis, Chlamydia trachomatis, N gonorrhoeae, HSV-1, and HSV-2. [83] Sensitivity for the assay using self-collected vaginal swabs is 98.3% and specificity is 99%, compared with a composite reference standard of wet mount microscopy and culture. [78] The system can run 96 samples per batch. [83] An independent evaluation of the system found that samples can be run with between 78.7 and 81.2 minutes of total processing time and only 29.8 and 34.2 minutes of “hands-on” laboratory work. [84]
OSOM Trichomonas Rapid Test
The OSOM Trichomonas Rapid Test uses color immunochromatographic “dipstick” technology with murine monoclonal antibodies. Results are read within 10 minutes. Freezing and transportation of specimens do not appreciably alter the test results. In a comparison with a composite reference standard of wet mount microscopy and culture, Huppert et al found the sensitivity of the OSOM test to be 83.3% and the specificity 98.8%. [85] A second study by Huppert et al found a sensitivity of 82% in comparison with a composite reference standard of wet mount, culture, rapid antigen testing, and polymerase chain reaction (PCR). [86] A third study compared the OSOM test to a composite reference standard for which a positive sample was defined as one that was positive by any combination of wet mount, Aptima ATV assay, or OSOM test. The prevalence of infection in the population tested was low, at 2%. The sensitivity was 94.7%, and the specificity was 100%. [87]
Solana Trichomonas Assay
The Solana Trichomonas Assay is a relatively rapid point-of-care assay that uses isothermal helicase-dependent amplification to amplify and identify T vaginalis DNA present in vaginal swabs and urine samples of female patients. [88] Results can be read within 40 minutes. Gaydos et al found that the sensitivity of the Solana assay compared to the Aptima-TV assay was 89.7% for swabs and 100% for urine samples. [89] The same study found that the specificity of the Solana assay was 99% for swabs and 98.9% for urine samples. [89]
Xpert TV Assay
The Cepheid Xpert TV Assay is a DNA amplification assay developed for the Cepheid GeneXpert System, a proprietary automated cartridge platform capable of performing diagnostic tests for a wide variety of infectious agents, including T vaginalis, C trachomatis, N gonorrhoeae, influenza, group B Streptococcus, and Clostridium difficile. [90] The assay is unique among currently available nuclear amplification assays in that it is validated by the FDA for use with male urine samples. [91] An independent evaluation of the Xpert TV Assay compared the test to a combination of APTIMA TV Assay and InPouch TV broth culture. This clinical review of the Xpert TV Assay found that the test had a sensitivity ranging between 99.5% and 100% for all female samples (including swabs, urine samples, and self-collected vaginal swabs) and a specificity ranging between 99.4% and 99.9%. [91] For male urine samples, the assay had a sensitivity of 97.2% and a specificity of 99.9%. [91]
According to the manufacturer, the Xpert TV Assay has a total “hands-on” time of 1 minute, and first results are available within 40 minutes. [90]
Other Molecular Techniques
PCR methods yield a high sensitivity (84%) and specificity (94%). [92, 93] PCR is based on DNA amplification and detection using known primers to TV genes. Because no approved PCR tests are available for general use, this approach is limited to research studies. Sensitivities of PCR tests for T vaginalis have been reported at 85-100%. [2, 94, 95] Amplicor, an FDA-approved PCR assay for gonorrhea and chlamydia modified to detect T vaginalis, was found to have a sensitivity of 88%-97% and specificity of 98%-99%. [96] Some researchers have suggested that PCR has great diagnostic potential, [30] particularly in men, while others maintain it offers little advantage over culture. In men, performing PCR on urine sediment rather than urethral swabs may improve detection rates. [97]
Direct fluorescent antibody (DFA) staining is more sensitive than saline wet mount but less sensitive than culture. DFA allows rapid diagnosis but requires a trained microscopist and a fluorescent microscope.
Saline wet mount evaluation
In women, vaginal trichomoniasis has historically been diagnosed via wet mount microscopy, which has very low sensitivity (25%) for detecting T vaginalis compared with most molecular tests. [98] Although it is not the criterion standard technique for trichomoniasis diagnosis, wet mount is frequently used because it is quick, inexpensive, and easy to perform. [57, 12, 13] The absence of trichomonads on microscopy does not rule out a diagnosis of vaginal trichomoniasis. Because they involve the direct visualization of the trichomonads, wet mounts are more likely to return positive results in women with high organism loads. [2] Wet mount microscopy is not an effective test for the diagnosis of trichomoniasis in men. Saline wet mount evaluation is performed by placing a small amount of vaginal discharge on a microscope slide and mixing with a few drops of saline solution. The slide is then examined under a microscope at low or medium power. See the video below.
The presence of flagellated pyriform protozoa, or trichomonads, indicates a positive test result. These ovoid-shaped parasites are slightly larger than polymorphonuclear leukocytes (PMNs), a type of white blood cell, and may be identified by their ameboid mobility. Trichomonads cause an inflammatory reaction; therefore, a large number of PMNs are usually present and correlate with the severity of infection.
Slides must be read immediately after collection. [57] Kingston et al looked at samples that were positive for trichomonads on initial reading and then reevaluated them every 10 minutes. [99] At 10 minutes, 20% of samples became negative; by 30 minutes, 35% were negative; and by 2 hours, 78% had become negative.
The relatively poor sensitivity of saline wet mount evaluation may be increased somewhat by using cervical vaginal lavage. [100] In one study, sensitivity was increased to 74.4% using the cervical vaginal lavage technique versus 54.7% vaginal swab alone. [100]
A short downloadable video illustrating this test is available from the Seattle STD/HIV Prevention Training Center. [101]
Standard culture
Culture was the criterion standard for trichomoniasis diagnosis before the introduction of molecular testing techniques. Culture is more sensitive and specific than microscopy but similar in both respects to molecular testing techniques. [57, 102] Providers may perform T vaginalis cultures when the suspicion of trichomoniasis is high but other tests do not reveal the protozoan. Culture has also been demonstrated to be useful in individuals with suspected resistant trichomoniasis, as providers can obtain the drug susceptibility of the strain. In addition, culture may be useful as diagnostic screening in high-risk populations. [16]
Disadvantages of culture include testing time and availability. [30] The CDC does not recommend oral and rectal testing, as infection rates at these sites appear to be low. [48]
In order to perform culture, a swab is put in broth and incubated anaerobically at 37°C. Growth is usually detected within 48 hours, and samples without growth after 7 days are considered negative for trichomoniasis. [102]
InPouch TV Culture System
The InPouch TV Culture System (Biomed, White City, Oregon), a combined wet mount and culture kit, is commonly used and readily available. This test kit has a sensitivity of 81%-100% and may detect as little as 1 parasite in the sample. [103]
The clinician inoculates the upper chamber of the pouch with a cotton swab. The pouch can be kept at room temperature for up to 18 hours without significant alteration of sensitivity. In the laboratory, a viewing clamp is placed across the upper chamber and examined under a microscope at a magnification of 100. If no trichomonads are viewed, the bottom chamber is inoculated by using the medium from the upper chamber. The InPouch is incubated at 37°C and viewed at regular intervals.
Nonspecific Laboratory Tests
Papanicolaou Smear
Trichomonads may be viewed on Pap smear, but this test yields low sensitivity and should not be relied on for diagnosis of T vaginalis infection. The sensitivity of Pap smear for detecting trichomonads is 40-60%. [104, 105] Specificity approaches 95% in the hands of trained technicians. [106] False-positive results are also common with this technique. [30]
Whiff Test (amine odor test)
A whiff test is performed by adding several drops of 10% potassium hydroxide to a sample of vaginal discharge. A strong fishy odor is indicative of a positive test result. Such a result may suggest either trichomoniasis or bacterial vaginosis. Thus, the whiff test should not be considered an accurate means of diagnosing trichomoniasis. It is 1 of the 4 parts of the Amsel criteria used to diagnose bacterial vaginosis. [107]
The whiff test is now combined with vaginal pH on a single card; the FemExam pH and Amines TestCard. On this card, the pH paper color change and the odor test are replaced with plus or minus signs.
pH Testing
Vaginal pH may be determined by touching a swab containing vaginal secretions to pH indicator paper. A normal pH practically excludes the diagnosis of symptomatic trichomoniasis. A pH greater than 4.5 is usually found with trichomoniasis. [61] However, an elevation in pH is not specific for trichomoniasis. [57]
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Trichomonas vaginalis on a saline wet mount at 40X on the microscope. Several motile parasites transit through the field, surrounded by white blood cells and squamous epithelial cells.
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Life cycle of Trichomonas vaginalis. T vaginalis trophozoite resides in female lower genital tract and in male urethra and prostate (1), where it replicates by binary fission (2). The parasite does not appear to have a cyst form and does not survive well in the external environment. T vaginalis is transmitted among humans, the only known host, primarily via sexual intercourse (3). Image courtesy of Centers for Disease Control and Prevention.
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Trichomonas vaginalis. (A) Two trophozoites of T vaginalis obtained from in vitro culture, stained with Giemsa. (B) Trophozoite of T vaginalis in vaginal smear, stained with Giemsa. Images courtesy of Centers for Disease Control and Prevention.
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Trichomoniasis overview.
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Trichomoniasis life cycle.
