Abstract
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.
INTRODUCTION
The prevalence of trichomoniasis in U.S. women is more than the combined prevalences of Neisseria gonorrhoeae and Chlamydia trachomatis infections (1). Infection with Trichomonas is associated with a significant risk of morbidity in women, including pelvic inflammatory disease, adverse pregnancy outcomes (e.g., preterm labor and delivery), cervical dysplasia, infertility, increased risk of postoperative infection, and HIV acquisition and transmission (2–5). U.S. health care costs related to trichomoniasis approach 34 million dollars per year (1).
Trichomonas vaginalis is a parasitic flagellate that adheres to and engulfs vaginal epithelial cells through phagocytosis. The resulting death of epithelial cells leads to inflammation within the genital tract and an increased number of polymorphonuclear leukocytes (PMNLs) in vaginal fluid (6). An increase in PMNLs noted on microscopy of vaginal secretions indicates an infectious or inflammatory process and may be a useful screening tool when evaluating vaginal discharge.
Leukorrhea refers to the presence of PMNLs in vaginal fluid, which can be quantified as the number of PMNLs seen on microscopy. Previous studies have defined leukorrhea based on the number of PMNLs per high-power field (hpf). Leukorrhea is considered present if there are more than one PMNL per oil immersion field (×1,000) or more than 10 PMNLs per hpf (×400) (7, 8). Leukorrhea has also been described according to the ratio of PMNLs to vaginal epithelial cells; more than 1 PMNL per epithelial cell is consistent with leukorrhea (9).
Noninfectious conditions, such as pregnancy, menstruation, or infertility, are associated with an increased number of PMNLs in vaginal fluid (10–12). Genital tract infections, such as C. trachomatis cervicitis and endometritis, are strongly associated with leukorrhea (8). This association of leukorrhea and C. trachomatis infection is true in pregnant and nonpregnant women. Leukorrhea is a strong predictor for C. trachomatis infection in nonpregnant and pregnant women (relative risk [RR], 59; 95% confidence interval [CI], 8 to 413; versus RR, 16; 95% CI, 7 to 32) (7). The absence of leukorrhea has a high negative predictive value (95%) for the diagnosis of upper genital tract infection, i.e., endometritis (8).
There are currently no studies in the English literature for which a correlation between leukorrhea in vaginal secretions and Trichomonas infection is reported. This information would be clinically useful when evaluating vaginal discharge and lower genital tract symptoms. Microscopy is the most common method used to diagnose trichomoniasis, but the sensitivity is poor (55%; 35 to 77%) (13–16). The presence of leukorrhea in the absence of motile trichomonads could potentially be presumed to be cervicitis and empirically treated without further evaluation for Trichomonas. A known significant relationship between leukorrhea on microscopy and Trichomonas might prompt clinicians to screen women in this clinical scenario for this common infection using more-sensitive methods.
The objective of this study was to determine an association between the presence of leukorrhea on microscopy of vaginal secretions and Trichomonas infection.
MATERIALS AND METHODS
The study was approved by the Medical University of South Carolina Institutional Review Board (HR no. 19180). Two hundred ninety-four women presenting to our urban academic practice for a gynecologic exam were enrolled in the study. Patients were eligible for the study if they had undergone an Aptima Combo 2 assay for N. gonorrhoeae and C. trachomatis (Gen-Probe Inc., San Diego, CA) as part of a routine exam or an evaluation for vaginal discharge or sexually transmitted infection (STI) exposure. Women were excluded if they were pregnant, HIV positive, and/or had a gynecologic malignancy, vaginal bleeding, or an active genital herpes infection. Women were also excluded if they had undergone surgery or were treated for pelvic inflammatory disease, appendicitis, vaginitis, or a STI within 28 days of presentation. The following patient information was collected: age, race, marital status, parity, first day of last menstrual period, contraceptive use, condom use, douching practices, partner gender preference, number of partners in the last year, history of STIs, and history of abnormal cervical cytology.
During a gynecologic examination, the following tests were performed: vaginal pH, Gram stain, T. vaginalis culture (InPouch, Biomed diagnostics, White City, OR), nucleic acid amplification tests (NAATs) for N. gonorrhoeae, C. trachomatis, and T. vaginalis (Aptima Combo 2 and TV analyte specific reagent; Gen-Probe Inc., San Diego, CA), and microscopy of vaginal secretions. The microscopy specimen was prepared by placing a vaginal swab into a test tube containing a small amount of normal saline (approximately 3 drops). A drop of this solution was then placed on a glass slide and reviewed under an hpf (×400). When reviewing microscopy of vaginal secretions, examiners noted the presence of PMNLs, clue cells, yeast, and/or motile trichomonads. Leukorrhea on microscopy was defined as >1 PMNL per epithelial cell per hpf (×400). Clinicians performing microscopy were Clinical Laboratory Improvement Amendments (CLIA) certified and participated in competency assessment and proficiency testing with guidance from the clinical laboratories' point-of-care testing team.
Gram staining of vaginal secretions, T. vaginalis cultures, and NAATs were performed by the Medical University of South Carolina Microbiology and Molecular Pathology labs. Gram stains were interpreted using Nugent's criteria to diagnose bacterial vaginosis (BV) (17). The number of PMNLs per hpf on Gram staining was rated using the following scale: 0 PMNLs/hpf, 1 to 5 PMNLs/hpf, and more than 5 PMNLs/hpf. T. vaginalis cultures were incubated at 37°C and inspected every other day up to 5 days for the presence of motile trichomonads.
Vaginal swab samples were transported to the laboratory for NAATs using the Aptima transport medium. The Aptima Combo 2 assays for N. gonorrhoeae and C. trachomatis were performed and interpreted according to the manufacturer's recommendations using the semiautomated direct tube sampling (DTS) system. The Aptima T. vaginalis analytic-specific reagent was used to develop a NAAT for T. vaginalis on the DTS system (4). Aliquots of the Aptima vaginal swab transport medium were stored for up to 12 months at −70°C before testing for T. vaginalis. Specimens with relative-light-unit values of <100,000 were considered negative, and those with ≥100,000 relative-light-unit values were considered positive.
Statistical analyses were performed using the SAS 9.3 software program (SAS, Cary, NC). Continuous variables (age and parity) were analyzed using Student's t test to compare means and the Wilcoxon rank sum test to compare medians. For dichotomous variables, chi-square and Fisher's exact tests were used to compare women with and without Trichomonas infection. Univariate logistic regression analyses were used to determine factors associated with Trichomonas infection. Variables with a P value of ≤0.2 were included in multivariate logistic regression analyses. Kappa statistics were calculated to determine the agreement between Trichomonas culture and NAATs. Chi-square tests were used to calculate the sensitivity, specificity, and positive and negative predictive values of culture, visualization of motile trichomonads, and leukorrhea on microscopy for the diagnosis of trichomoniasis.
RESULTS
Two hundred ninety-four women, ages 16 to 67, were enrolled in this study. More than 60 percent of the subjects presented for evaluation of a vaginal discharge (179). The majority of subjects were non-Hispanic, black (216) and currently unmarried (260). Ninety-eight percent of women reported being exclusively heterosexual (287), and more than half had a history of an STI (175). Most women reported using a method of contraception, including condoms (234). Other patient characteristics are listed in Table 1.
Table 1.
Patient characteristics
| Characteristica | Value |
|---|---|
| Age (yr) (mean ± SD) | 28 ± 9 |
| Parity, median (interquartile range) | 1.4 (0–8) |
| % of patients (no. of patients/total) with characteristic | |
| Non-Hispanic, black race | 74 (216/293) |
| Hispanic race | 0.6 (2/293) |
| Single marital status | 90 (260/290) |
| Exclusively heterosexual partner preference | 98 (287/292) |
| Vaginal douching | 16 (46/286) |
| Regular menstrual cyclesb | 63 (170/272) |
| Currently not using contraception (including condoms) | 20 (58/292) |
| Report condoms only for contraception | 7 (22/294) |
| Hormonal contraception | 58 (169/292) |
| Surgical contraceptionc | 14 (41/292) |
| History of abnormal cervical cytology | 17 (47/275) |
| Multiple sexual partnersd | 24 (68/278) |
| Patient reported vaginal discharge | 61 (179/292) |
| History of one prior sexually transmitted infection (STI) | 60 (175/292) |
| History of more than one STI | 49 (84/170) |
| History of Trichomonas infection | 40 (66/170) |
| History of Chlamydia infection | 62 (106/170) |
| History of gonorrhea infection | 37 (61/166) |
| History of genital herpes | 12 (21/170) |
Some patient characteristic data were not available for all participants.
Regular menstrual cycles defined as monthly menses every 21 to 35 days.
Surgical contraception may include female tubal ligation or male vasectomy.
Defined as >1 partner in the 12 months preceding evaluation.
Trichomonas testing results were available for almost all 294 women enrolled in the study: 292 T. vaginalis cultures and 247 nucleic acid amplification tests. Sixteen percent (46) of the study subjects had a positive Trichomonas test result. Women with a Trichomonas infection were more likely to be parous (88% versus 64%; P = 0.002) and non-Hispanic black (93% versus 70%; P = 0.0004). They were less likely to use any form of contraception (33% versus 17%; P = 0.02), but rates of condom use were similar in the two groups (4% versus 8%; P = 0.5). Women with Trichomonas infection were more likely to report a history of STIs (85% versus 55%; P = 0.002) (Table 2).
Table 2.
Comparison of women with Trichomonas infection and those not infected
| Characteristic | Value for groupa |
P valuee | |
|---|---|---|---|
| Trichomonas infection | No Trichomonas | ||
| Age (yr), mean (± SD) | 29.3 (±10.8) | 27.7 (±8.6) | 0.34 |
| Parity, median (interquartile range) | 2 (0–6) | 1 (0–8) | 0.001 |
| % with characteristic (no. of women/total) | |||
| Non-Hispanic, black race | 93 (43/46) | 70 (173/247) | 0.0004 |
| Single marital status | 91 (41/45) | 89 (219/245) | 0.7 |
| Exclusively heterosexual | 98 (45/46) | 98 (242/246) | 0.8 |
| Douching | 20 (9/45) | 15 (37/241) | 0.4 |
| Regular menstrual cycles | 49 (19/39) | 65 (151/233) | 0.06 |
| No contraception, including condoms | 33 (15/46) | 17 (43/246) | 0.02 |
| Reported condom use | 4 (2/46) | 8 (20/248) | 0.5* |
| History of abnormal cervical cytology | 15 (7/46) | 17 (40/229) | 0.7 |
| Multiple sexual partners | 26 (11/42) | 24 (57/236) | 0.7 |
| History of at least one STI | 85 (39/46) | 55 (136/246) | 0.002 |
| Reported vaginal discharge | 76 (35/46) | 59 (144/246) | 0.02 |
| Abnormal vaginal pHb | 87 (40/46) | 48 (120/248) | <0.0001 |
| PMNLs on microscopy | 78 (36/46) | 49 (118/240) | 0.0003 |
| PMNLs on Gram staining | 87 (40/46) | 69 (167/243) | 0.01 |
| Bacterial vaginosisc | 43 (20/46) | 41 (99/243) | 0.7 |
| Chlamydia infectiond | 7 (3/46) | 4 (11/248) | 0.5* |
| Gonorrhea infection | 15 (7/46) | 4 (9/248) | 0.002 |
For the Trichomonas-infected group, n = 46; for the uninfected group, n = 253.
Abnormal vaginal pH is defined as a pH of >4.5.
Bacterial vaginosis is defined as a Nugent score of ≥7 (17).
Determination of Chlamydia and gonorrhea infection was based on positive NAATs.
*, result of Fisher's exact test for small sample size. Otherwise, P values were calculated using chi-square tests.
According to Nugent's criteria, 41% of women in the study had BV (119/289). Five Gram stains could not be scored due to inadequate specimen collection. The rates of BV determined by Nugent's criteria and high-power microscopy findings of clue cells were equal (both 41%), but the agreement between these tests was low (kappa statistic = 0.5). The rates of BV were similar among the women infected with Trichomonas versus those not infected (43% versus 41%; P = 0.7). PMNLs were noted on 71% of all Gram stain specimens. Compared to uninfected women, women with T. vaginalis were more likely to have PMNLs on Gram staining and leukorrhea (>1 PMNL per hpf) noted on microscopy (87% versus 69%; P = 0.01; and 78% versus 49%; P = 0.0003). Women with Trichomonas were 3 times as likely to have gonorrhea (15% versus 4%; P = 0.002), but the rates of C. trachomatis infection were not different between groups (7% versus 4%; P = 0.5) (Table 2).
Factors associated with Trichomonas infection in univariate logistic regression analysis were parity, race, lack of contraceptive use, history of STIs, vaginal discharge on presentation, an elevated vaginal pH (>4.5), leukorrhea, and concurrent gonorrhea. When controlling for race and parity, both a history of STIs (odds ratio [OR], 3.1; 95% CI, 1.3 to 7.4) and lack of contraceptive use (OR, 2.2; 95% CI, 1.1 to 4.7) were associated with Trichomonas infection. When controlling for abnormal vaginal pH, leukorrhea on microscopy, and patient-reported vaginal discharge at presentation, women with a gonorrhea infection were 4 times more likely to have trichomoniasis (OR, 3.6; 95% CI, 1.1 to 11.6) (Table 3).
Table 3.
Factors associated with Trichomonas vaginalis infection
| Characteristic | Regression value (95% CI) |
|
|---|---|---|
| Univariate | Multivariatea | |
| Parous | 3.8 (1.6–9.3) | 2.2 (0.8–5.8)* |
| Non-Hispanic, black race | 6.1 (1.8–20.4) | 3.0 (0.8–11.2)* |
| Not currently using contraception | 2.3 (1.1–4.6) | 2.2 (1.1–4.7)* |
| History of at least one STI | 4.5 (1.9–10.5) | 3.1 (1.3–7.4)* |
| Reported vaginal discharge | 2.3 (1.1–4.6) | 1.5 (0.7–3.2) |
| Abnormal vaginal pHb | 7.1 (2.9–17.4) | 6.0 (2.4–15.1) |
| PMNLs on microscopy | 3.7 (1.8–7.8) | 1.8 (0.8–4.2) |
| PMNLs on Gram staining | 3.2 (1.3–7.8) | 2.7 (1.0–6.7) |
| Gonorrhea infection | 4.8 (1.7–13.5) | 3.6 (1.1–11.6) |
*, multivariate logistic regression model controlling for patient characteristics associated with Trichomonas infection: parity, race, contraceptive use, and history of STIs. The remaining variables were used in a separate multivariate logistic regression model to determine clinical findings associated with Trichomonas infection.
Abnormal vaginal pH as defined by a pH of >4.5.
NAATs and T. vaginalis culture were highly correlated (kappa statistic = 95%). NAATs detected an additional 3 cases of Trichomonas infection. Compared to NAATs, the sensitivity and specificity of culture were 93% and 100%. Using NAATs as the gold standard for the diagnosis of Trichomonas infection, the sensitivity of motile trichomonads on microscopy was very poor (51%). Leukorrhea (>1 PMNL per epithelial cell) on microscopy had an increased sensitivity for Trichomonas infection compared to visualization of motile trichomonads (78% versus 51%), but the specificity and positive predictive values (PPV) were poor. When combining the findings of motile trichomonads and/or leukorrhea on microscopy compared to leukorrhea on microscopy alone, the sensitivity for diagnosis of Trichomonas was unchanged. The visualization of any PMNLs on Gram staining (hpf, ×1,000) was a more-sensitive test for Trichomonas infection than leukorrhea on microscopy (hpf, ×400) (90% versus 78%). However, the specificity of Gram staining was lower than that of leukorrhea on microscopy. The PPV for leukorrhea assessed by Gram staining versus the PPV for assessment by microscopy were similar, 21 and 25%, respectively (Table 4).
Table 4.
Diagnostic techniques for the detection of Trichomonas compared to nucleic acid amplification testsa
| Method or target | % Sensitivity | % Specificity | % PPV | % NPV |
|---|---|---|---|---|
| Cultureb | 93 (38/41) | 100 (205/205) | 100 (38/38) | 99 (205/208) |
| Motile trichomonads | 51 (21/41) | 98 (200/204) | 84 (21/25) | 91 (200/220) |
| Leukorrhea on microscopyc | 78 (32/41) | 51 (102/199) | 25 (32/129) | 92 (102/111) |
| Motile trichomonads or leukorrhea | 78 (32/41) | 53 (109/207) | 25 (32/130) | 92 (109/118) |
| PMNLs on Gram stainingd | 90 (37/41) | 33 (68/207) | 21 (37/176) | 94 (68/72) |
Parenthetical numbers are no. of correct results/no. tested. PPV, positive predictive value; NPV, negative predictive value.
InPouch Trichomonas culture was more sensitive and specific than microscopy for the diagnosis of Trichomonas.
Leukorrhea, ≥1 PMNL per epithelial cell per hpf (×400).
PMNLs on Gram staining, ≥1 PMNL per hpf (×1,000).
DISCUSSION
The incidence of Trichomonas vaginalis infection among the women in this study population (16%) is five times higher than the reported prevalence of Trichomonas infection in American women (1). In our population, Trichomonas vaginitis was associated with leukorrhea on microscopy (>1 PMNL per epithelial cell per hpf, ×400) and PMNLs on Gram staining (>1 PMNL per hpf, ×1,000). Both of these findings have lower sensitivity for Trichomonas infection than for culture (93% versus 78% and 90%, respectively) but improved sensitivity over that for motile trichomonads on microscopy (51%). Negative predictive values for motile trichomonads, leukorrhea, PMNLs on Gram staining, and culture were comparable. In comparison to NAATs for the diagnosis of trichomoniasis, neither leukorrhea on microscopy nor Gram staining was specific, and both had low positive predictive values (Table 4).
The incidence of Trichomonas infection in this group of women is likely not reflective of the general population. Our obstetrics-gynecology (OBGYN) residency women's health practice provides care to mostly young, minority women with a high rate of STIs. The mean age of these study subjects was 28 (±9) years, the majority of women (74%) were non-Hispanic black, and more than half (60%) reported a history of at least one STI (Table 1).
In a national survey of American women, low level of education (high school or less), poverty, increasing number of sexual partners, early age of first sexual intercourse, and douching were associated with Trichomonas infection (1). In our population, douching and reporting more than one sexual partner in the past 12 months was not associated with trichomoniasis. These findings are likely due to the high proportion of patients reporting douching and multiple partners in the overall study group. Risk factors associated with Trichomonas infection among the women in this study were parity, non-Hispanic black race, lack of contraceptive use, including condoms, and a history of a STI. These factors associated with increased risk of STIs are consistent with those in previous reports with the exception of an increased number of sexual partners (Table 2) (18).
Exam findings associated with Trichomonas vaginitis in our population were an elevated vaginal pH (>4.5), leukorrhea on microscopy, PMNLs on Gram staining, and gonorrhea infection. Patient report of vaginal discharge was associated with trichomoniasis in univariate analysis, but this association was attenuated in multivariate analysis when accounting for other exam findings: pH, PMNLs on Gram staining, and cervicitis (Table 3). The poor association between symptomatic vaginal discharge is consistent with other population-based studies of Trichomonas infection (1). The high rate of asymptomatic infections in women supports the notion that clinicians should not limit evaluations for Trichomonas vaginitis to microscopy in those reporting vaginal discharge (19). We suggest that clinicians consider the factors associated with Trichomonas infection in order to identify patients at risk and use tests more sensitive than microscopy, such as culture or NAATs, to diagnose Trichomonas infection.
Similar to studies of other genital tract infections, leukorrhea in vaginal secretions was associated with Trichomonas infection. This is physiologically plausible, because Trichomonas causes increased inflammation within the vaginal environment and has been found to “attract” PMNLs (6). Specifically, CD4 lymphocytes are recruited to the site of Trichomonas infection (20).
In the absence of leukorrhea on microscopy (>1 PMNL per epithelial cell), Trichomonas infection was unlikely (OR, 0.3; 95% CI, 0.1 to 0.8). Although gonorrhea cervicitis was associated with an increased risk of Trichomonas infection, this did not impact the association between leukorrhea and Trichomonas vaginitis. When controlling for the presence of gonorrhea, C. trachomatis infection, and bacterial vaginosis, patients were 4 times more likely to have Trichomonas if leukorrhea was noted on microscopy (OR, 3.6; 95% CI, 1.6 to 7.6). Our findings suggest that the presence of leukorrhea on microscopy should increase clinicians' suspicion of Trichomonas infection and lead to further evaluation with more-sensitive tests if microscopy is negative for motile trichomonads.
The Aptima T. vaginalis analyte-specific reagent used in this study has emerged as the most sensitive test for detection of Trichomonas infections (4, 21–23). Because concomitant C. trachomatis and N. gonorrhoeae infections are common in patients with trichomoniasis, a panel of NAATs for these three infectious agents would be ideal for comprehensive STI diagnosis. The Aptima T. vaginalis assay was recently cleared by the FDA for diagnostic use. This should greatly increase availability of the test, with cost and turnaround time for results similar to those of the Aptima C. trachomatis and N. gonorrhoeae assay.
The association between leukorrhea and Trichomonas vaginitis in our population is both physiologically plausible and likely applicable to other populations seeking evaluation for vaginal discharge or gynecologic exam. Our study subjects are not representative of a general population given the young age of participants, high rates of reported and concurrent STIs, and disproportionate representation of minorities. However, this population is similar to others studied in urban centers, STI clinics, and family planning centers. It is in these low-resource settings that microscopy is often used for the diagnosis of Trichomonas. Our findings suggest that the presence of leukorrhea on microscopy should increase clinical suspicion for Trichomonas vaginitis, prompting additional tests, and may enhance identification of this common and morbid infection.
ACKNOWLEDGMENTS
We acknowledge Gen-Probe, Inc., for their support and funding of this research.
We thank the staff of the MUSC Microbiology and Molecular Pathology labs for their tireless work in analyzing the additional specimens required for this study. We especially recognize April Kegl.
The coinvestigator Fredrick Nolte has been a member of the Gen-Probe Scientific Advisory Board from 2007 to the present. The annual compensation associated with this activity is approximately $10,000. Gen-Probe products were used to determine the Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis infection status of the patients described in our article.
Footnotes
Published ahead of print 15 May 2013
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