Correlation of Leukorrhea and Trichomonas vaginalis Infection

Gweneth B. Lazenby,a David E. Soper,a Frederick S. Nolteb

Department of Obstetrics and Gynecologya and Department of Pathology and Laboratory Medicine,b Medical University of South Carolina, Charleston, South Carolina,
USA

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.

he 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 (25). 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 (1012). 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 nonpreg-

July 2013 Volume 51 Number 7

nant 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%) (1316). 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

Received 12 February 2013 Returned for modification 6 March 2013

Accepted 5 May 2013
Published ahead of print 15 May 2013
Address correspondence to Gweneth B. Lazenby, lazenbgb@musc.edu.
Copyright 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00416-13

Journal of Clinical Microbiology

p. 23232327

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Lazenby et al.

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
Nugents 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 37C 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 manufacturers recommendations using the semiautomated
direct tube sampling (DTS) system. The Aptima T. vaginalis analyticspecific 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 70C 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 Students t test to compare means and the Wilcoxon rank sum
test to compare medians. For dichotomous variables, chi-square and Fishers 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.

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TABLE 1 Patient characteristics

Characteristica

Value

Age (yr) (mean SD)

Parity, median (interquartile range)

28 9
1.4 (08)

% of patients (no. of patients/total) with characteristic

Non-Hispanic, black race
Hispanic race
Single marital status
Exclusively heterosexual partner preference
Vaginal douching
Regular menstrual cyclesb
Currently not using contraception (including condoms)
Report condoms only for contraception
Hormonal contraception
Surgical contraceptionc
History of abnormal cervical cytology
Multiple sexual partnersd
Patient reported vaginal discharge
History of one prior sexually transmitted infection (STI)
History of more than one STI
History of Trichomonas infection
History of Chlamydia infection
History of gonorrhea infection
History of genital herpes

74 (216/293)
0.6 (2/293)
90 (260/290)
98 (287/292)
16 (46/286)
63 (170/272)
20 (58/292)
7 (22/294)
58 (169/292)
14 (41/292)
17 (47/275)
24 (68/278)
61 (179/292)
60 (175/292)
49 (84/170)
40 (66/170)
62 (106/170)
37 (61/166)
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.
c
Surgical contraception may include female tubal ligation or male vasectomy.
d
Defined as 1 partner in the 12 months preceding evaluation.
b

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.
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).
According to Nugents 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 Nugents 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 stain-

Journal of Clinical Microbiology

Leukorrhea and Trichomonas vaginalis Infection

TABLE 2 Comparison of women with Trichomonas infection and those not infected
Value for groupa
Characteristic

Trichomonas infection

No Trichomonas

P valuee

Age (yr), mean ( SD)

Parity, median (interquartile range)

29.3 (10.8)
2 (06)

27.7 (8.6)
1 (08)

0.34
0.001

% with characteristic (no. of women/total)

Non-Hispanic, black race
Single marital status
Exclusively heterosexual
Douching
Regular menstrual cycles
No contraception, including condoms
Reported condom use
History of abnormal cervical cytology
Multiple sexual partners
History of at least one STI
Reported vaginal discharge
Abnormal vaginal pHb
PMNLs on microscopy
PMNLs on Gram staining
Bacterial vaginosisc
Chlamydia infectiond
Gonorrhea infection

93 (43/46)
91 (41/45)
98 (45/46)
20 (9/45)
49 (19/39)
33 (15/46)
4 (2/46)
15 (7/46)
26 (11/42)
85 (39/46)
76 (35/46)
87 (40/46)
78 (36/46)
87 (40/46)
43 (20/46)
7 (3/46)
15 (7/46)

70 (173/247)
89 (219/245)
98 (242/246)
15 (37/241)
65 (151/233)
17 (43/246)
8 (20/248)
17 (40/229)
24 (57/236)
55 (136/246)
59 (144/246)
48 (120/248)
49 (118/240)
69 (167/243)
41 (99/243)
4 (11/248)
4 (9/248)

0.0004
0.7
0.8
0.4
0.06
0.02
0.5*
0.7
0.7
0.002
0.02
0.0001
0.0003
0.01
0.7
0.5*
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.
c
Bacterial vaginosis is defined as a Nugent score of 7 (17).
d
Determination of Chlamydia and gonorrhea infection was based on positive NAATs.
e
, result of Fishers exact test for small sample size. Otherwise, P values were calculated using chi-square tests.
a
b

ing 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).
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.

July 2013 Volume 51 Number 7

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).
DISCUSSION

The incidence of Trichomonas vaginalis infection among the

women in this study population (16%) is five times higher than

TABLE 3 Factors associated with Trichomonas vaginalis infection

Regression value (95% CI)
Characteristic

Univariate

Multivariatea

Parous
Non-Hispanic, black race
Not currently using contraception
History of at least one STI
Reported vaginal discharge
Abnormal vaginal pHb
PMNLs on microscopy
PMNLs on Gram staining
Gonorrhea infection

3.8 (1.69.3)
6.1 (1.820.4)
2.3 (1.14.6)
4.5 (1.910.5)
2.3 (1.14.6)
7.1 (2.917.4)
3.7 (1.87.8)
3.2 (1.37.8)
4.8 (1.713.5)

2.2 (0.85.8)*
3.0 (0.811.2)*
2.2 (1.14.7)*
3.1 (1.37.4)*
1.5 (0.73.2)
6.0 (2.415.1)
1.8 (0.84.2)
2.7 (1.06.7)
3.6 (1.111.6)

a
, 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.
b
Abnormal vaginal pH as defined by a pH of 4.5.

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TABLE 4 Diagnostic techniques for the detection of Trichomonas compared to nucleic acid amplification testsa
Method or target
b

Culture
Motile trichomonads
Leukorrhea on microscopyc
Motile trichomonads or leukorrhea
PMNLs on Gram stainingd

% Sensitivity

% Specificity

% PPV

% NPV

93 (38/41)
51 (21/41)
78 (32/41)
78 (32/41)
90 (37/41)

100 (205/205)
98 (200/204)
51 (102/199)
53 (109/207)
33 (68/207)

100 (38/38)
84 (21/25)
25 (32/129)
25 (32/130)
21 (37/176)

99 (205/208)
91 (200/220)
92 (102/111)
92 (109/118)
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).
d
PMNLs on Gram staining, 1 PMNL per hpf (1,000).
b
c

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 womens 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

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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 moresensitive 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, 2123). 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.

Journal of Clinical Microbiology

Leukorrhea and Trichomonas vaginalis 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 GenProbe Scientific Advisory Board from 2007 to the present. The annual
compensation associated with this activity is approximately $10,000. GenProbe products were used to determine the Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis infection status of the patients described in our article.

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