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A real-time PCR
assay was more accurate than a traditional PCR assay for detection
of T. vaginalis in urine samples.
Use of the LightCycler instrument in a
real-time PCR for Trichomonas vaginalis in urine samples from
females and males.
Hardick J, Yang S, Lin S, Duncan D, Gaydos
C.
Journal of Clinical
Microbiology 2003;41:5619-5622.
Summary:
Question
What are the sensitivities of a traditional PCR assay and a real-time PCR
assay for the detection of T. vaginalis DNA in urine specimens
compared to a resolved standard?
Design
An existing PCR method for detection of T. vaginalis DNA was
adapted into a rapid real-time PCR assay based on fluorescence resonance
energy transfer (FRET) probe technology. The results of urine samples
analyzed by both methods were compared to true positive results obtained
by testing discrepant samples by PCR using a third primer set.
Participants
Urine samples were randomly collected prospectively from 253 young (<25
years old), male and female, sexually active high school students
participating in an ongoing clinical study for C. trachomatis, N.
gonorrhoeae, and T. vaginalis.
Description of Tests and Diagnostic
Standard
Clinical T. vaginalis strains were used as positive controls and
standards for the PCR assays. The organisms were stained with Evans blue,
counted in a hemocytometer, and the DNA extracted by the Chelex method.
Urine specimens and serially diluted control DNA were extracted using the
automated MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN)
according to the instructions supplied. DNA samples were analyzed in a
touchdown enzyme time release (TETR) PCR assay using a previously
published primer set, BTUB9 and BTUB2, that targets a 112 bp fragment of
the β-tubulin gene of T. vaginalis.
The amplicons were analyzed by gel electrophoresis. Samples were also
analyzed in a LightCycler (Roche) real-time PCR assay that used primers
BTUB9 and BTUB B, a modified BTUB2 primer, and two FRET probes labeled
with fluorescent dyes. Amplification was carried out for 50 cycles.
Samples having a Ct of less than 40 cycles were considered positive.
Samples which tested positive by only one of the two PCRs were adjudicated
by PCR testing with a third primer set, TVK3 and TVK4, utilizing gel
electrophoresis to detect amplicons. Samples that were positive by two of
the three PCRs were considered true positives.
Main Outcome Measures
The analytical sensitivity, specificity, and intra- and interassay
reproducibility of the FRET PCR assay were determined. The sensitivity,
specificity, positive predictive value (PPV), and negative predictive
value (NPV) of the FRET PCR assay were compared to the TETR PCR assay, and
the performances of both PCRs, compared to adjudicated true positive
results, were determined.
Main Results
The number of T. vaginalis organisms that could be consistently
detected was 4 per PCR reaction. No signal was detected during
amplification of DNA from Trichomonas species other than vaginalis. The
average coefficient of variation for five replicate analyses of six
different concentrations of T. vaginalis DNA (88 to 1 organisms/PCR)
was 18.5 and 29.4 for two PCR runs performed by different technicians on
different days.
Fifteen samples were positive and 229
samples negative by both TETR and FRET PCR. Five samples were positive by
TETR and negative by FRET, while 4 samples were positive by FRET and
negative by TETR. Discrepant analysis using a third T. vaginalis
PCR assay indicated 21 true positive urine specimens. The sensitivity,
specificity, PPV, and NPV of the FRET PCR compared to the TETR PCR were
75.0%, 98.3%, 78.9%, and 97.8%, respectively. The performances of both
assays compared to the adjudicated true positive results are shown in the
table.
Authors' Conclusions
Compared to the TETR PCR, the BTUB FRET PCR
assay was more sensitive and specific and required less time and labor to
perform.
Source of funding:
Not given
For correspondence:
Charlotte Gaydos, 1159 Ross Bldg, 720 Rutland Ave., Baltimore, MD 21205.
E-mail address: cgaydos@jhmi.edu
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