Molecular testing for Trichomonas vaginalis in women: results from a prospective U.S. clinical trial.

Abstract

Trichomoniasis is a common sexually transmitted disease associated with preterm birth, low birth weight, and increased susceptibility to infection with other pathogenic sexually transmitted microorganisms. Nucleic acid amplification tests for Trichomonas vaginalis have improved sensitivity for detecting infected individuals compared to existing culture-based methods. This prospective, multicenter U.S. clinical trial evaluated the performance of the automated Aptima T. vaginalis assay for detecting T. vaginalis in 1,025 asymptomatic and symptomatic women. Vaginal swab, endocervical swab, ThinPrep PreservCyt, and urine specimens were collected. Subject infection status was determined by wet-mount microscopy and culture. Aptima T. vaginalis assay performance was determined for each specimen type by comparison to subject infection status. Of 933 subjects analyzed, 59.9% were symptomatic. Aptima T. vaginalis clinical sensitivity and specificity were, respectively, 100% and 99.0% for vaginal swabs, 100% and 99.4% for endocervical swabs, 100% and 99.6% in ThinPrep samples, and 95.2% and 98.9% in urine specimens. Aptima T. vaginalis performance levels were similar in asymptomatic and symptomatic subjects. This study validates the clinical performance of the Aptima T. vaginalis assay for screening asymptomatic and symptomatic women for T. vaginalis infection.

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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 4106–4111
0095-1137/11/$12.00doi:10.1128/JCM.01291-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 12
Molecular Testing for Trichomonas vaginalis in Women: Results from a
Prospective U.S. Clinical Trial?†
Jane R. Schwebke,1Marcia M. Hobbs,2Stephanie N. Taylor,3Arlene C. Sena,2Michael G. Catania,4
Barbara S. Weinbaum,4Ann D. Johnson,4Damon K. Getman,4* and Charlotte A. Gaydos5
Departments of Medicine/Infectious Diseases, University of Alabama at Birmingham, Birmingham, Alabama1; Department of
Medicine, University of North Carolina, Chapel Hill, North Carolina2; Department of Medicine/Section of Infectious Diseases,
Louisiana State University Health Sciences Center, New Orleans, Louisiana3; Research and Development, Gen-Probe Inc.,
San Diego, California4; and Division of Infectious Diseases, Johns Hopkins University, Baltimore, Maryland5
Received 28 June 2011/Returned for modification 25 July 2011/Accepted 1 September 2011
Trichomoniasis is a common sexually transmitted disease associated with preterm birth, low birth
weight, and increased susceptibility to infection with other pathogenic sexually transmitted microorgan-
isms. Nucleic acid amplification tests for Trichomonas vaginalis have improved sensitivity for detecting
infected individuals compared to existing culture-based methods. This prospective, multicenter U.S.
clinical trial evaluated the performance of the automated Aptima T. vaginalis assay for detecting T.
vaginalis in 1,025 asymptomatic and symptomatic women. Vaginal swab, endocervical swab, ThinPrep
PreservCyt, and urine specimens were collected. Subject infection status was determined by wet-mount
microscopy and culture. Aptima T. vaginalis assay performance was determined for each specimen type by
comparison to subject infection status. Of 933 subjects analyzed, 59.9% were symptomatic. Aptima T.
vaginalis clinical sensitivity and specificity were, respectively, 100% and 99.0% for vaginal swabs, 100% and
99.4% for endocervical swabs, 100% and 99.6% in ThinPrep samples, and 95.2% and 98.9% in urine
specimens. Aptima T. vaginalis performance levels were similar in asymptomatic and symptomatic sub-
jects. This study validates the clinical performance of the Aptima T. vaginalis assay for screening asymp-
tomatic and symptomatic women for T. vaginalis infection.
Trichomoniasis, a common sexually transmitted disease
(STD) caused by the protozoan Trichomonas vaginalis, affects
approximately 180 million persons per year worldwide, making
it the most common nonviral STD agent in the world. An
estimated 7.4 million new cases occur annually in the United
States (1), and the disease has an overall prevalence of 3.1%
(24). Both women and men can be infected, although symp-
toms are more common in women. Symptomatic women have
a diffuse, malodorous, yellow-green vaginal discharge with vul-
var irritation which may be confused with bacterial vaginosis.
Infected men may temporarily have urethral irritation, mild
discharge, or slight burning after urination or ejaculation (5).
Many infections do not produce symptoms and when left un-
treated may lead to preterm birth, low birth weight, and pelvic
inflammatory disease (5). T. vaginalis infection also increases
susceptibility to infection with HIV (14, 22, 23). Effective and
inexpensive antibiotic therapy for T. vaginalis infection is read-
ily available, and detection and treatment of T. vaginalis in
symptomatic or asymptomatic women with a high risk of STD
are important to prevent disease acquisition, transmission, and
associated complications.
Currently, the gold standard for the diagnosis of T. vaginalis
infection is culture; however, the sensitivity of commercially
available culture has been reported to be 75% to 89% com-
pared to amplified methods (13, 20). Tests with improved sen-
sitivity are needed to diagnose this prevalent STD. The Aptima
Trichomonas vaginalis assay, an FDA-cleared, fully automated
nucleic acid amplification test, has demonstrated high sensitiv-
ity and specificity compared to culture (20). Herein, we report
the results of a large prospective multicenter trial designed to
determine the sensitivity and specificity of this new assay in
multiple specimen types from women.
MATERIALS AND METHODS
Subjects and study conduct. This clinical trial enrolled 1,025 women attend-
ing participating U.S. obstetrics and gynecology (OB/GYN), family planning,
or STD clinics (collection sites). Subjects 14 years of age or older were
enrolled in the study if they demonstrated symptoms consistent with a sus-
pected STD, such as vaginal odor, vaginal discharge, vaginal/vulvar itching,
pain/discomfort during sexual intercourse or urination, and/or lower abdom-
inal discomfort; were asymptomatic and had sexual contacts with persons with
confirmed or suspected STD(s); were asymptomatic and undergoing screen-
ing evaluation for possible STDs; or were undergoing routine Papanicolaou
(Pap) screening. Subjects were excluded if they took antibiotic medications
within 14 days of enrollment. Subjects signed Institutional Review Board
(IRB)-approved informed consent before being enrolled in the study. The
protocol of this study was approved by IRBs from all participating sites.
Specimen collection and processing. At each of the 9 collection sites, 6 spec-
imens were collected from each subject as follows (in order of collection): 1
first-catch urine, 3 vaginal swabs, 1 endocervical swab, and 1 ThinPrep Preserv-
Cyt liquid cytology cervical specimen (collected with a broom-like device or an
endocervical brush and spatula). Per the study protocol, a 20- to 30-ml urine
specimen was self-collected by each subject; all other specimens were clinician
collected. One vaginal swab specimen was tested using wet-mount microscopic
examination, and another vaginal swab specimen was cultured with the InPouch
T. vaginalis test (BioMed Diagnostics, White City, OR) at the collection site’s
laboratory or a designated laboratory (if the site did not routinely perform
culture with the InPouch T. vaginalis test) to determine infection status. The
* Corresponding author. Mailing address: Gen-Probe Inc., 10210
Genetic Center Dr., San Diego, CA 92121. Phone: (858) 410-8537.
Fax: (858) 410-8871. E-mail: damon.getman@gen-probe.com.
?Published ahead of print on 21 September 2011.
† The authors have paid a fee to allow immediate free access to this
article.
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order of the vaginal sample collected for use in the Aptima T. vaginalis assay, wet
mount, or culture was randomized for each patient.
One first-catch urine, 1 vaginal swab, 1 endocervical swab, and 1 ThinPrep spec-
imen were processed for Aptima T. vaginalis assay testing by the collection site in
accordance with the appropriate package insert instructions as follows. An aliquot of
the urine specimen was transferred into the Aptima urine specimen transport tube
and stored at 2°C to 30°C. The vaginal swab specimen was placed into an Aptima
vaginal swab specimen collection kit tube and stored at 2°C to 30°C. The endocer-
vical swab was placed into an Aptima unisex swab specimen collection kit tube and
stored at 2°C to 30°C. The ThinPrep endocervical specimen was processed by
immersing and rinsing the collection device (broom-like device or endocervical
brush and spatula) in PreservCyt solution in the ThinPrep Pap test vial; an aliquot
was transferred into an Aptima specimen transfer kit tube, and the processed
ThinPrep sample was stored at 2°C to 8°C. Approximately one-third of all processed
samples were then shipped to 1 of 3 designated testing sites.
Sample evaluation. (i) Wet-mount microscopic examination and InPouch T.
vaginalis test culture. Collection site laboratories performed wet-mount micro-
scopic examination using a vaginal swab specimen. In general, the specimen on
the swab was treated with a saline solution and examined under a microscope for
motile trichomonads. The collection site’s laboratory performed culture with the
InPouch T. vaginalis test for all except three sites, for which the InPouch T.
vaginalis test was performed at an outside laboratory. The pouch was examined
daily for the presence of motile trichomonads in accordance with the package
insert instructions (2) or for up to 5 days. Technicians performing these proce-
dures were blind to molecular test results.
(ii) Aptima T. vaginalis assay. The Aptima T. vaginalis assay is a nucleic acid
amplification test that utilizes target capture, transcription-mediated amplifica-
tion (TMA), chemiluminescent probe hybridization, and the automated Tigris
DTS system to detect T. vaginalis 18s rRNA. Processed samples were tested in
accordance with the investigational Aptima T. vaginalis assay package insert
instructions (9). Each Aptima T. vaginalis assay testing site tested approximately
one-third of the study samples and used 3 lots of Aptima T. vaginalis assay
reagent kits over the course of the study. Operators performing the Aptima T.
vaginalis assay were blind to the subjects’ clinical data and wet-mount micro-
scopic examination and culture results. Aptima T. vaginalis assay results were not
used for determining treatment or patient care.
(iii) Discordant analysis. Supplemental testing of discordant specimens was
conducted using (i) a characterized real-time T. vaginalis PCR assay for the
detection of T. vaginalis DNA (10), and (ii) an alternate TMA (Alt-TMA) T.
vaginalis assay developed at Gen-Probe which has been previously used to re-
solve T. vaginalis test discordant results (18–20). This alternate assay uses unique
primer, probe, and target capture oligomers to target a different region of the T.
vaginalis 18s rRNA (20). A cutoff for T. vaginalis positivity of 100,000 RLU was
used with the Alt-TMA T. vaginalis assay for consistency with the Aptima T.
vaginalis assay. Both the PCR and Alt-TMA T. vaginalis assays exhibit analytical
sensitivity of ?1 organism/reaction.
Infection status. The infection status was established by testing 2 vaginal swab
specimens with wet-mount microscopic examination and by culturing with the
InPouch T. vaginalis test. The infection status was considered positive if the
InPouch T. vaginalis test and/or the wet-mount microscopic examination result
was positive. The infection status was considered negative if both InPouch T.
vaginalis test and wet-mount microscopic examination results were negative. The
infection status was considered indeterminate if either the InPouch T. vaginalis
test or the wet-mount microscopic evaluation result was missing and the other
test result was negative.
Data analyses: Aptima T. vaginalis assay performance evaluation. Samples that
were not handled in accordance with the Aptima T. vaginalis assay package insert
instructions or the clinical protocol and samples with invalid Aptima T. vaginalis
assay results or indeterminate infection status were excluded from the analyses (n ?
354); this allowed estimation of the sensitivity, specificity, positive predictive value
(PPV), and negative predictive value (NPV) of the Aptima T. vaginalis assay. The
score method was used for calculating the 2-sided 95% confidence intervals (CIs) for
sensitivity and specificity. The exact method was used to compute the 2-sided 95%
CIs for PPV and NPV based on the positive and negative likelihood ratios. Analyses
were also stratified by collection site, symptom status, and age group (women 14 to
17 years of age versus women 18 years or older).
RESULTS
Subject disposition. A total of 1,025 women were enrolled at
9 sites; of these women, 26 were withdrawn because they did
not meet study eligibility criteria, and 66 were excluded
because the samples exceeded the recommended storage
period before testing (n ? 48) or did not have a conclusive
infection status (n ? 18). Thus, 933 of 1,025 (91.0%) sub-
jects were included in the evaluable analysis set. These sub-
jects produced 3,343 samples (735 urine, 875 vaginal swab,
920 endocervical swab, and 813 ThinPrep samples) with
valid Aptima T. vaginalis assay results, which were included
in the final analysis. A detailed subject disposition is pre-
sented in Fig. 1.
Subject demographic and disease characteristics. Subjects’
demographic and clinical characteristics are presented in Table
1. The median age of the 933 evaluable subjects was 24.0 years
(range: 14 to 67 years). Overall, 59.6% of the subjects were
black/African American and 33.8% were white. The majority
of subjects were symptomatic (59.9%). The most frequently
reported symptom was vaginal discharge (75.1%), followed by
vaginal odor (43.3%) and vaginal/vulvar itch (32.9%). Overall,
36.0% of the subjects were diagnosed with vaginosis, 15.9%
with vaginitis, and 7.1% with cervicitis. The prevalence of T.
vaginalis infection in this population ranged from 11.4% in
both urine (84/735) and ThinPrep (93/813) samples to 12.7%
(111/875) in vaginal swab samples (Table 2).
Aptima T. vaginalis assay performance in different specimen
types in all women. The sensitivity, specificity, NPV, and PPV
of the Aptima T. vaginalis assay in the different specimen types
are presented in Table 2. Sensitivity values for the detection of
T. vaginalis ranged from 95.2% using urine samples to 100%
using vaginal swab, endocervical swab, or ThinPrep samples;
the negative predictive values for T. vaginalis detection in these
sample types ranged from 99.4% (urine) to 100% (vaginal
swab, endocervical swab, ThinPrep). The specificity of the Ap-
tima T. vaginalis assay for the detection of T. vaginalis in these
samples was also high, ranging from 98.9% (urine) to ?99%
for vaginal swab, endocervical swab, or ThinPrep samples; the
positive predictive values for these samples ranged from 92.0%
to 96.9%. There was no significant difference between the
sensitivity and specificity of detecting T. vaginalis in urine sam-
ples and those for other sample types (P ? 0.05).
Analysis of false-negative and false-positive Aptima T. vagi-
nalis assay results. Twenty-seven samples (0.8%) had discor-
dant results in the Aptima T. vaginalis assay compared with the
infection status; of these, 4 were false negatives (all urine) and
23 were classified as false positives. The PCR and Alt-TMA T.
vaginalis assays were used to provide supplemental information
on the 27 samples with discordant Aptima T. vaginalis assay
results relative to the established infection status. The testing
results were not used to modify the calculated performance
characteristics of the Aptima T. vaginalis assay.
(i) Analysis of the 4 false-negative samples. Four urine spec-
imens were negative by the Aptima T. vaginalis assay despite a
positive infection status for these 4 subjects. All 4 subjects had
positive Aptima T. vaginalis assay results for the other 3 spec-
imen types. These 4 urine samples were also negative when
tested with the Alt-TMA T. vaginalis assay and with PCR
(Table 3), suggesting that these samples lacked T. vaginalis
nucleic acids.
(ii) Analysis of false-positive samples. Twenty-three samples
(7 urine, 8 vaginal swab, 5 endocervical swab, and 3 ThinPrep
samples) with false-positive results were observed in 10 sub-
jects with negative infection status. Nine of these 10 subjects
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had positive Aptima T. vaginalis assay results in at least 2
sample types. The T. vaginalis PCR assay also produced 5
positive results for the specimens considered false positive (1/7
for urine, 1/8 for vaginal swab, 2/4 for endocervical swab, and
1/3 for ThinPrep samples) (Table 3). Alt-TMA T. vaginalis
assay test results were positive for all vaginal swab, endocervi-
cal swab, and ThinPrep samples and for 5 of the 7 urine
samples with false-positive Aptima T. vaginalis assay results.
Overall, these results indicate the presence of the T. vaginalis
rRNA target in those specimens (Table 3).
Aptima T. vaginalis assay performance by symptom status.
Overall, the sensitivities, specificities, and negative predic-
tive values of the Aptima T. vaginalis assay were similar in
asymptomatic and symptomatic subjects, despite the preva-
lence of T. vaginalis infection in symptomatic women (15.1%
to 16.4%) being higher than its prevalence in asymptomatic
women (6.5% to 7.0%) (Table 2). The PPV for urine and
vaginal swabs was slightly lower (by approximately 6 to 10
percentage points) in asymptomatic subjects than in symp-
tomatic subjects (Table 2). The performance of the Aptima
T. vaginalis assay levels were very similar for samples col-
lected from adolescent women and adult women and for
samples collected from different geographic locations (data
not shown).
DISCUSSION
This study was conducted to validate Aptima T. vaginalis
assay performance characteristics for the detection of T. vagi-
nalis in self-collected first-catch urine specimens and clinician-
collected endocervical swab, vaginal swab, and endocervical
ThinPrep samples, with standard wet-mount and culture meth-
ods used as the reference. T. vaginalis is widely prevalent in the
U.S. population and is associated with important public health
sequelae such as preterm birth and acquisition/transmission of
HIV. Control of trichomoniasis has been hampered by the lack
of sufficiently sensitive diagnostic tests. The most commonly
used method is visualization of motile trichomonads in a saline
preparation from the vaginal swab (wet-mount microscopic
examination). Although this test is rapid and inexpensive, it
has two important limitations: it must be performed within 10
to 20 min of collection of the specimen (or the organisms lose
viability), and it has a low sensitivity (ranging from 60% to
70%) as compared to culture (5). A commercial culture
FIG. 1. Of 1,025 subjects enrolled at 9 sites, there were 933 evaluable subjects (91.0%), from which we planned to collect 3,732 samples
(933 subjects with 4 samples each). Of these, 382 (10.2%) samples were either missing or considered nonevaluable (expired). Of the 3,350
evaluable samples (738 urine, 877 vaginal swab, 922 endocervical swab, and 813 ThinPrep samples), 3,343 (99.8%) had final valid results;
seven (0.2%; 3 urine, 2 vaginal swab, and 2 endocervical swab) samples had final invalid results and were not included in the analyses. ATV,
Aptima T. vaginalis.
4108SCHWEBKE ET AL.J. CLIN. MICROBIOL.

Page 4

method (InPouch T. vaginalis test) has a reported sensitivity of
approximately 80% and a specificity of 100% compared with
wet mount (21) or with Diamond’s modified medium (17).
Trichomonads may be visualized on the Pap smear, but this
also has a limited sensitivity (approximately 60%) (15). Other
tests for T. vaginalis include the OSOM Trichomonas rapid test
(Genzyme Diagnostics, Cambridge, MA) and the Affirm VP
III (Becton Dickenson, San Jose, CA). Both tests are per-
formed on vaginal secretions with a sensitivity of ?83% and a
specificity of ?97% compared with culture or wet mount (3, 4,
8, 12, 13).
In this study, the use of the highly sensitive Aptima T. vagi-
nalis assay found that the prevalence of T. vaginalis infection
ranged from 11.4% in urine and ThinPrep specimens to 12.7%
in vaginal swabs. These values are higher than the national
average of 3.6% (24), possibly because of the high proportion
of symptomatic subjects enrolled in this study (55.1%) who
presented for screening for a possible STD or who had a
partner with an STD. The prevalence values reported here also
agree with previously reported T. vaginalis molecular test prev-
alence rates of 11.3% to 28.7% (11, 13, 19, 20).
The Aptima T. vaginalis assay sensitivity (95.2% in urine
samples and 100% in endocervical swab, vaginal swab, and
ThinPrep samples) and specificity (?99%) values reported
herein (Table 2) are similar to those reported in previous
publications (11, 13, 18, 20). In the present study, the overall
Aptima T. vaginalis assay sensitivity (?95.2% in any specimen
type) was higher than published sensitivity estimates for the
wet-mount microscopic examination (60% to 70% [6]) and
culture using the InPouch T. vaginalis test (81% and 82.4% [17,
21]). This is in agreement with studies that evaluated the ana-
lyte-specific-reagent-formatted Aptima T. vaginalis assay and
these two reference assays in side-by-side comparisons (13, 20).
The performance levels of the Aptima T. vaginalis assay
were nearly identical when testing endocervical ThinPrep spec-
imens, endocervical swab samples, and vaginal swab samples.
Performance of the Aptima T. vaginalis assay was somewhat
lower in self-collected urine samples; however, the difference
in assay sensitivity between vaginal or cervical (100%) and
urine samples (95.2%) was not statistically significant. This
finding is in agreement with results from Nye et al. (20), who
reported a slightly but not significantly lower sensitivity for
TABLE 1. Subject demographic and clinical characteristics
CharacteristicValue
Total no............................................................................................933
Age, yr
Mean (SD)................................................................................... 27.0 (9.50)
Median.......................................................................................... 24.0
Min-max .......................................................................................14–67
Age groups, no. (%)
14–17............................................................................................. 81 (8.7)
18–20.............................................................................................177 (19.0)
21–25.............................................................................................284 (30.4)
26–30.............................................................................................136 (14.6)
31–35............................................................................................. 88 (9.4)
36–40............................................................................................. 76 (8.1)
?40................................................................................................ 91 (9.8)
Ethnicity, no. (%)
Hispanic or Latino...................................................................... 93 (10.0)
Not Hispanic or Latino..............................................................836 (89.6)
Unknown/refused ........................................................................4 (0.4)
Race, no. (%)a
White ............................................................................................315 (33.8)
Black or African American........................................................556 (59.6)
Asian.............................................................................................
American Indian/Alaska native................................................. 10 (1.1)
Unknown/refused ........................................................................ 58 (6.2)
8 (0.9)
Symptom status, no. (%)
Asymptomatic..............................................................................374 (40.1)
Symptomatic.................................................................................559 (59.9)
Reported symptom (symptomatic subjects), no. (%)b
Vaginal odor................................................................................242 (43.3)
Vaginal discharge........................................................................420 (75.1)
Vaginal/vulvar itch ......................................................................184 (32.9)
Pain/discomfort during sexual intercourse............................... 33 (5.9)
Lower abdominal discomfort..................................................... 99 (17.7)
Pain/discomfort during urination.............................................. 54 (9.7)
Other............................................................................................. 89 (15.9)
Clinical diagnosis, no. (%)
Vaginosis ......................................................................................336 (36.0)
Vaginitis........................................................................................148 (15.9)
Cervicitis....................................................................................... 66 (7.1)
Urethritis/cystitis.......................................................................... 12 (1.3)
Pelvic inflammatory disease.......................................................
Unknown...................................................................................... 73 (7.8)
Otherc...........................................................................................446 (47.8)
8 (0.9)
aSubjects may have reported multiple races.
bSubjects may have reported multiple symptoms.
cIncludes diagnosis of “normal.”
TABLE 2. Performance of Aptima T. vaginalis assay in the different specimens by symptom status
Aptima T.
vaginalis assay
specimen
Symptom
status
No. of samplesa
%
Prevalenceb
% Sensitivity
(95% CI)
% Specificity
(95% CI)
% PPV
(95% CI)
% NPV
(95% CI)
Total TP FP TNFN
Urine Asymptomatic
Symptomatic
All women
Asymptomatic
Symptomatic
All women
Asymptomatic
Symptomatic
All women
Asymptomatic
Symptomatic
All women
324
411
735
345
530
875
372
548
920
353
460
813
21
59
80
24
87
111
26
88
114
23
70
93
3
4
7
4
4
8
1
4
5
0
3
3
299
345
644
317
439
756
345
456
801
330
387
717
1
3
4
0
0
0
0
0
0
0
0
0
6.8
15.1
11.4
7.0
16.4
12.7
7.0
16.1
12.4
6.5
15.2
11.4
95.5 (78.2–99.2)
95.2 (86.7–98.3)
95.2 (88.4–98.1)
100 (86.2–100)
100 (95.8–100)
100 (96.7–100)
100 (87.1–100)
100 (95.8–100)
100 (96.7–100)
100 (85.7–100)
100 (94.8–100)
100 (96.0–100)
99.0 (97.1–99.7)
98.9 (97.1–99.6)
98.9 (97.8–99.5)
98.8 (96.8–99.5)
99.1 (97.7–99.6)
99.0 (97.9–99.5)
99.7 (98.4–99.9)
99.1 (97.8–99.7)
99.4 (98.6–99.7)
100 (98.8–100)
99.2 (97.8–99.7)
99.6 (98.8–99.9)
87.5 (71.4–96.9)
93.7 (85.7–98.1)
92.0 (85.1–96.4)
85.7 (70.3–95.6)
95.6 (89.5–98.8)
93.3 (87.6–97.0)
96.3 (82.4–99.9)
95.7 (89.6–98.8)
95.8 (90.7–98.6)
100 (86.2–undc)
95.9 (88.9–99.1)
96.9 (91.4–99.3)
99.7 (98.4–100)
99.1 (97.7–99.8)
99.4 (98.5–99.8)
100 (98.9–100)
100 (99.2–100)
100 (99.5–100)
100 (99.0–100)
100 (99.2–100)
100 (99.6–100)
100 (99.0–100)
100 (99.1–100)
100 (99.5–100)
Vaginal swab
Endocervical swab
ThinPrep
aTP, true positive; FP, false positive; TN, true negative; FN, false negative.
b(TP ? FN)/n.
cund, undefined; the upper boundary of the confidence interval could not be calculated.
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Page 5

urine samples (87.5%) than for vaginal and endocervical swab
samples (96.6% and 89.8%, respectively) by use of the Aptima
T. vaginalis assay in conjunction with a molecular test-resolved
algorithm.
In the present study, 4 (0.43%) subjects had urine samples
with negative Aptima T. vaginalis assay results and positive
results for the other specimen types, indicating that these urine
results were false negatives. The rate of urethral colonization
in women with a vaginal T. vaginalis infection has been esti-
mated at ?75% (16). Thus, it is possible that these 4 subjects
had T. vaginalis infections in the vagina (they had positive
vaginal samples) but were not colonized in the urethra (nega-
tive urine samples). Alternatively, it is possible that the pres-
ence of only a few trichomonad cells in the urethra or urinary
meatus at the time of voiding could have resulted in very low
cell concentrations in the urine sample, thus leading to sam-
pling error upon addition of the 2-ml urine aliquot to the urine
transport tube. However, in a separate study, we performed
quantitative molecular testing for T. vaginalis in 38 urine sam-
ples obtained in this trial and found that the median
trichomonad cell load was 311 cells/ml of urine, with a mean of
2,040 cells/ml of urine (data not shown). Since the analytical
sensitivity of the Aptima T. vaginalis assay is less than 0.1
cell/ml in urine samples, we conclude that these 4 women likely
were infected with T. vaginalis in the vagina but not in the
urinary tract.
In spite of the slightly lower sensitivity of the Aptima T.
vaginalis assay in detecting T. vaginalis in urine specimens,
testing of urine is still of clinical benefit due to the high clinical
sensitivity shown and the ease of collection of this sample type.
Because first-catch urine is a noninvasive, self-collected spec-
imen that does not require a pelvic examination, it represents
a convenient specimen collection method for screening large
populations, especially in STD clinics and institutional settings.
The specificity of the Aptima T. vaginalis assay was high,
ranging from 98.9% in urine samples to 99.6% in ThinPrep
samples. False-positive Aptima T. vaginalis assay results were
observed in 10 of 933 (1.1%) subjects. However, 9 of these 10
subjects had positive Aptima T. vaginalis assay results in at
least 2 other specimen types, suggesting that these 9 subjects
were actually infected with trichomonads. Some of these spec-
imens were also found positive by the T. vaginalis PCR assay
and most were positive with the Alt-TMA T. vaginalis assay,
confirming the presence of T. vaginalis rRNA target and indi-
cating that these samples were likely true positives. These
findings imply that the true clinical specificity of the Aptima T.
vaginalis assay is actually higher than reported here (98.9% to
99.6%). This issue typically occurs when the performance of
the evaluated assay is calculated based on reference assays with
inherently lower sensitivity and has been previously reported
for TMA-based assays for Chlamydia trachomatis detection (7).
Importantly, the performance levels were of the Aptima T.
vaginalis assay were similar in symptomatic and asymptomatic
subjects, in adolescent and adult women, and in samples ob-
tained from different collection sites, indicating that the Ap-
tima T. vaginalis assay has broad clinical utility and consistent
performance for the detection of T. vaginalis and diagnosis of
trichomoniasis in many female populations. In addition, the
ability to use multiple specimen types in the Aptima T. vagi-
nalis assay provides clinicians with more options for T. vaginalis
testing, which is an important advantage over wet-mount ex-
amination or culture methods that use vaginal swabs only.
Moreover, the ability to use this highly accurate molecular test
on a fully automated instrumentation system represents an
effective solution for large-scale screening for T. vaginalis in-
fections in certain populations.
In summary, the results from this study validate the clinical
performance characteristics of the Aptima T. vaginalis assay
using self-collected first-catch urine specimens and clinician-
collected endocervical swab, vaginal swab, and endocervical
ThinPrep specimens from symptomatic and asymptomatic
women. The superior performance of this method compared to
that of the reference tests (wet-mount microscopic examina-
tion and culture) should improve the screening, diagnosis, and
treatment of T. vaginalis infection.
ACKNOWLEDGMENTS
We thank Rangaraj Selvarangan, Jill Huppert, Neil B. Quigley,
Edward Hook, Paul Fine, Kimberle Chapin, Stephen Kasparian, and
Mark Martens for assistance with conducting the clinical trial and
Rebecca Gunnill and Florence Paillard for assistance with preparation
of the manuscript.
Michael G. Catania, Barbara S. Weinbaum, Ann D. Johnson, and
Damon K. Getman are employed by Gen-Probe Inc. and hold stock
options in that company. Marcia M. Hobbs has received research
support from Gen-Probe Inc. Jane R. Schwebke has consulted for
Gen-Probe Inc., BioReference Laboratories, Cepheid, and Graceway
Pharmaceuticals. Stephanie N. Taylor has supported clinical trials for
Gen-Probe, Cepheid, and Roche.
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TABLE 3. Discordant test result analysis with PCR and Alt-TMA T. vaginalis assays
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4110 SCHWEBKE ET AL.J. CLIN. MICROBIOL.

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