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A urine-based
PCR-ELISA assay may be useful for the detection of T. vaginalis
in some clinical research settings where vaginal specimens are not
available.
Development and validation of a PCR-based enzyme-linked
immunosorbent assay with urine for use in clinical research settings
to detect Trichomonas vaginalis in women.
Kaydos SC, Swygard H, Wise SL, Sena
AC, Leone PA, Miller WC, Cohen MS, Hobbs MM.
Journal of Clinical Microbiology 2002;40:89-95.
Summary:
Question
What is the performance of a urine-based PCR amplification method for the
detection of T. vaginalis in women compared to detection in vaginal
specimens by conventional wet mount and culture?
Design
This paper describes a cross-sectional, blinded study to directly compare
a PCR amplification assay for the detection of T. vaginalis in
urine samples from women versus the combined reference standard of wet
mount and T. vaginalis culture of vaginal swab specimens.
Participants
Urine specimens were collected from 2,930 women presenting to the sexually
transmitted disease clinics of two county health departments in North
Carolina. Participants were between 18 and 65 years old, spoke English,
and had not used oral or topical metronidazole for four weeks prior to
specimen collection. The women were predominantly African-American (81%)
and Caucasian (16%). Sixty-five percent were "symptomatic",
defined as having a reported or observed abnormal vaginal discharge. The
final study population comprised 2,147 women for whom the time and
temperature limits for urine storage and transportation were maintained.
Of these, 780 (549 from "symptomatic" women and 231 from
"asymptomatic" women) had urine samples that were tested by the
PCR-ELISA assay and by the reference standard methods.
Description of Tests and Diagnostic
Standard
From 2 to 100 mL of first-void urine was collected in sterile containers
for PCR analysis. The samples were stored at 4oC and transported on ice
within three days of collection to the testing laboratory. One mL of urine
was processed using the Amplicor CT Urine Specimen Prep kit (Roche
Diagnostic Systems, Indianapolis, IN), and an aliquot was added to the T.
vaginalis PCR reaction, which contained one primer labeled with
digoxigenin. PCR amplicons were detected using a biotinylated probe and
the PCR ELISA DIG detection kit (Roche Diagnostic Systems). The limit of
detection for this assay was eight T. vaginalis organisms per PCR
reaction. Two vaginal swabs were collected for a conventional wet mount
microscopy and for a T. vaginalis culture using the InPouch TV culture
system (Biomed, San Jose, CA), processed according to the manufacturer's
instructions. The reference standard was based on the combined results of
wet mount and culture, with a positive sample defined as positive for
either wet mount or culture, and a negative sample as negative for both
wet mount and culture.
Main Outcome Measures
The sensitivity and specificity of the PCR-ELISA assay for the detection
of T. vaginalis in urine samples compared to the detection in
vaginal swabs by the combined wet mount and culture reference standard
were determined. The specificity of the PCR-ELISA was then adjusted using
a formula that assumed the true sensitivity of the combined reference
standard to be 0.70 and the true specificity of the standard to be 1.0 (Staquet
M., et al., J. Chron. Dis., 1981;34:599-610).
Main Results
The performance of the PCR-ELISA compared to the reference standard is
shown in the table. The test performed with equivalent sensitivity and
specificity in patients with or without symptoms or signs of abnormal
vaginal discharge, a history of douching, or the presence of chlamydial
infection, gonorrhea, or bacterial vaginosis. The assay was slightly more
sensitive with urine specimens with smaller volumes (<20 ml) and when
more than 1.5 h had elapsed since the previous void.
| T.
vaginalis PCR-ELISA performance compared to combined wet mount
and culture. |
| Subject
group |
n |
%
Sensitivity
(95% CI) |
%
Specificity
(95% CI) |
%
Adjusted specificity |
| Asymptomatic |
231 |
89.5
(66.5, 98.2) |
83.5
(73.9, 90.2) |
90.7 |
| Symptomatic |
549 |
91.8
(84.1, 96.2) |
87.2
(83.6-90.0) |
95.3 |
| Overall |
780 |
90.8
(84.2, 95.0) |
86.1
(83.2, 88.7) |
93.4 |
Authors' Conclusions
PCR combined with ELISA for detection of T.
vaginalis in women's urine performed well compared to wet mount
microscopy and culture from vaginal swabs. The T. vaginalis
PCR-ELISA on urine samples should be useful for the detection of this
organism in settings where collection of vaginal specimens for microscopy
and culture is not practical.
Source of funding: NIH Sexually
Transmitted Diseases Clinical Trails Unit, NIH Sexually Transmitted
Diseases Cooperative Research Centers, and NIH National Study of
Adolescent Health: Survey 2000.
For correspondence: Marcia Hobbs,
Department of Medicine, Campus Box 7030, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599. E-mail address: mmhobbs@med.unc.edu.
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