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A multiplex PCR
assay is less sensitive than Gram stain examination for the
diagnosis of bacterial vaginosis.
A multiplex
polymerase chain reaction-based diagnostic method for bacterial
vaginosis.
Obata-Yasuoka M, Ba-Thein W, Hamada H,
Hayashi H.
Obstet Gynecol. 2002;100:759-64
Summary:
Question
How well does a PCR assay that detects multiple anaerobic bacteria compare
to Gram- stained slides scored according to the Nugent method for the
diagnosis of bacterial vaginosis (BV)?
Design
This study evaluates a multiplex PCR assay that detects multiple anaerobes
implicated in bacterial vaginosis and compares its performance for the
diagnosis of BV to that of Gram-stained slides scored according to the
Nugent method.
Participants
One thousand women presenting to the Obstetrics and Gynecology Department
in the Mito Saiseikai General Hospital, Ibaraki, Japan were tested for BV.
Women younger than 15 and older than 76 years, and those who had
antimicrobial therapy within 2 weeks of sampling were excluded. Swab
samples from 172 women were randomly selected for testing by PCR from
among 853 with Gram stain-interpretable slides. One hundred thirty-eight
women (mean age = 29.9 years) were pregnant and 34 (mean age = 39.8 years)
were not.
Description of Tests and Diagnostic
Standard
One vaginal swab collected from each patient was used to make a slide and
inoculate a culture, then placed into 2 mL of saline. Bacteria in the
saline sample were pelleted by centrifugation, suspended in water, boiled,
and centrifuged, and the supernatant was added to a multiplex PCR
reaction. The PCR reaction mix contained three primer pairs that amplified
the 16S rDNA gene from Mobiluncus species (M. mulieris and M.
curtisii), the nanH gene from Bacteroides fragilis, and
the internal spacer region of rDNA from Gardnerella vaginalis. PCR
amplicons were detected on agarose gels. To determine the sensitivity of
the assay, dilutions of each target organism with a known number of colony
forming units in 2 mL of saline were tested. The specificity of the assay
was confirmed by sequencing the amplicons produced by amplification of the
target organisms, and by testing 13 other bacterial and one yeast clinical
isolate found as normal vaginal flora. Vaginal swab samples were
considered BV positive when one or more species of targeted bacteria were
detected by multiplex PCR. Vaginal slides were Gram stained and evaluated
using the Nugent scoring system. Samples with a score greater than 6 were
considered BV positive.
Main Outcome Measures
The sensitivity, specificity, positive predictive value (PPV), and
negative predictive value (NPV) of the PCR assay for the diagnosis of BV
as determined by a Gram stain Nugent score were calculated.
Main Results
The sensitivity of the multiplex PCR assay was 6 X 103 to 2 X 104
colony-forming units per 2 mL for the four target organisms. No amplified
products were detected in samples containing other bacteria or yeast. The
results of the PCR assay and the Gram stain Nugent score for 172 vaginal
samples are shown in the table. The sensitivity, specificity, PPV, and NPV
of the multiplex PCR assay compared to Gram stain examination for the
diagnosis of BV were 78.4%, 95.6%, 82.9%, and 94.2%, respectively.
| Results of BV
diagnosis by multiplex PCR and Gram stain evaluation |
|
Multiplex PCR |
Gram stain using
Nugent score |
|
Positive |
Negative |
Total |
|
Positive |
29 |
6 |
35 |
|
Negative |
8 |
129 |
137 |
|
Total |
37 |
135 |
172 |
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Authors' Conclusions
Although Gram staining remains an
indispensable tool for the diagnosis of BV, the multiplex PCR method, in
combination with clinical diagnosis, can be used as an alternative
screening test, especially for women who are undergoing other screening.
Source of funding:
Ministry of Education, Culture, Sports, Science and Technology of Japan
For correspondence: Mana
Obata-Yasuoka, Institute of Clinical Medicine, University of Tsukuba,
Department of Obstetrics and Gynecology, Tsukuba, 305-8575, Japan. E-mail
address: md995455@md.tsukuba.ac.jp.
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