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It is feasible to
use real time PCR to detect and quantify the numbers of bacterial
vaginosis-related organisms in cervicovaginal lavage samples.
Detection of bacterial
vaginosis-related organisms by real-time PCR for lactobacilli, Gardnerella
vaginalis, and Mycoplasma hominis.
Zariffard MR, Saifuddin M, Sha BE, Spear GT.
FEMS Immunol Med Microbiol. 2002;34:277-281.
Summary:
Question
Can PCR be used to detect and quantify bacterial vaginosis (BV)-related
organisms in cervicovaginal lavage samples and how do the results compare
with the clinical diagnosis of BV?
Design
The feasibility of using real-time PCR to detect and quantify BV-related
organisms (G. vaginalis, lactobacilli, and M. hominis) in
the genital tract of women was determined by testing stored genital tract
samples. The results were compared with the clinical diagnosis of BV
identified by Amsel criteria.
Participants
Stored, frozen, cell pellets prepared from 10 mL of cervicovaginal lavage
sample were tested in the PCR assays. The samples were from 21 HIV
seropositive and 10 HIV seronegative women enrolled in the Chicago
Consortium of the Women's Interagency HIV Study, a prospective
epidemiological and natural history study of HIV-infected and high risk
uninfected women, who were evaluated at Rush-Presbyterian-St. Luke's
Medical Center, in Chicago, IL.
Description of Tests and Diagnostic
Standard
The lavage sample pellets were lysed in lysosyme and detergent and treated
to remove RNA and protein. The DNA was ethanol precipitated, quantified by
UV spectrophotometry, and 50 ng was added to each of three real-time,
syber green PCR amplification reactions. The PCR primers for detection of G.
vaginalis, and M. hominis were designed from the respective 16S
rRNA genes. The lactobacillus primer sequences were selected from regions
of 16S rRNA that were homologous between L. jenseneii and L.
crispatus and detected both organisms. All three primer sets resulted
in amplification of DNA from the appropriate type of bacteria and showed
no cross reactivity with DNA from the other two organisms, E. coli,
or 14 other organisms commonly found in the genital tract. To quantify the
number of organisms, seven standards consisting of 102 to 107 copies of
DNA from the appropriate bacterium were amplified to generate a standard
curve. Amsel clinical criteria were used to diagnose BVs.
Main Outcome Measures
The number of lactobacilli, G. vaginalis, and M. hominis
detected in the lavage samples by PCR compared to the clinical diagnosis
of BV identified by Amsel criteria was determined.
Main Results
Lactobacilli were detected in 21, G. vaginalis in 20, and M.
hominis in 6 of 21 samples from HIV seropositive women. Using Amsel
criteria, 16 women among the 21 who were HIV seropositive, and all 10 of
the samples tested from HIV seronegative women were BV negative. The mean
number of bacteria per lavage sample detected in each diagnosis group is
shown in the table. The number of lactobacilli was significantly higher in
the BV-negative group than in the BV-positive group (P=0.013, Mann-Whitney
U-test), while the number of G. vaginalis was significantly higher
in the BV-positive group than in the BV-negative (P=0.004). The number of
lactobacilli was not different between the HIV seropositive, BV-negative
and the HIV seronegative, BV-negative groups (P=0.17); however, the number
of G. vaginalis was significantly different between the two groups
(P=0.01).
| Number of organisms
based on clinical diagnosis of BV |
| Organism |
BV
positive (n=5) |
BV
negative |
| HIV
positive (n=16) |
HIV
negative (n=10) |
| Lactobacillus |
8.5 X 106 |
1.1 X 109 |
1.5 X 109 |
| G.
vaginalis |
1.3 X 1010 |
3.35 X 107 |
<104 |
| M.
hominis |
<104 |
<104 |
Not
reported |
Authors' Conclusions
Real-time PCR can be used to detect G.
vaginalis, M. hominis, and lactobacilli in genital tract
samples. Samples from women with BV that was diagnosed clinically have
significantly higher numbers of G. vaginalis, but significantly
lower numbers of lactobacilli.
Source of funding: NIH and Women's
Interagency HIV Study.
For correspondence: Gregory T. Spear,
Departments of Immunology/Microbiology and Medicine, Section of Infectious
Diseases, Rush University, 1653 West Congress Parkway, Chicago, IL 60612.
E-mail address: gspear@rush.edu.
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