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A cervicovaginal
washing is a highly sensitive specimen collection method for
detecting HSV-2 DNA in female genital secretions.
Comparison of
washing and swabbing procedures for collecting genital fluids to
assess cervicovaginal shedding of herpes simplex virus type 2 DNA.
Ndjoyi-Mbiguino A, Ozouaka F, Legoff
J, Mbopi-Kéou F-X, Si-Mohamed A, Onas IN, Avoune E, Bélec L.
Journal of Clinical
Microbiology 2003;41:2662-2664
Summary:
Question
How do cervicovaginal lavage and cervical swab specimens compare for the
detection of HSV-2 from asymptomatic, HSV-2 seropositive women?
Design
This study describes a prospective comparison of two female genital
secretion sample collection methods, cervical swab and cervicovaginal
lavage, for detection by real time PCR of genital shedding of HSV-2 in
asymptomatic, HIV-negative African women.
Participants
One hundred eighty-eight nonpregnant, HSV-2 seropositive, HIV-seronegative
women, aged 18 to 48 years (mean age = 24.7 years) attending the
Gynecology and Obstetrical Department of the Centre Hopitalier de
Libreville and the Maternité Joséphine Bongo in Libreville, Gabon, for
family planning or gynecological evaluation were tested. Type-specific
antibodies to HSV-2 were assessed using an ELISA assay with monoclonal
antibodies to HSV-2. All women were asymptomatic for current STDs.
Description of Tests and Diagnostic
Standard
Genital secretions were collected from each woman first by gently
rotating, with slight pressure, a dry swab on the cervical os, and second
by washing for 60 s with 3 ml of PBS, using three aspiration discharges
against the vaginal walls and cervix with a plastic pipette. The lavage
was centrifuged and the presence of hemoglobin was detected in the
supernatant by spectrophotometry. Both the cell-free lavage supernatant
and the dry cervical swab specimens were frozen until testing by PCR.
Prior to testing, each swab was placed in 400 μl of lysis buffer and
the DNA extracted and diluted in 200 μl of specimen diluent using the
QiaAmp DNA minikit (Qiagen, Courtaboeuf, France) according to the
manufacturer's instructions. The DNA concentration was determined by
spectrophotometry at 260 nm. One ml of each lavage specimen was
centrifuged at 23,000 X g for 60 min and the DNA was extracted from the
pellet and diluted in 200 μl of specimen diluent using the same kit.
Contaminating semen was determined using a PCR assay to detect Y
chromosome DNA. PCR inhibitors were assessed by β-globin PCR
detection in all samples.
HSV-2 DNA was detected and quantified by adding 5 μl of each DNA
preparation to a real time PCR LightCycler assay using primers to the HSV
DNA polymerase gene. The results were expressed as the number of HSV
copies per microgram of total DNA and per ml for the swab and lavage
samples, respectively. The lower limit of detection for this assay was 400
copies/μg for cervical swabs and 400 copies/ml for lavage samples. A
woman was considered an HSV shedder if either specimen was above the limit
of detection for HSV DNA in the PCR assay. HSV typing was performed by
restriction fragment length polymorphism analysis
Main Outcome Measures
The correlations between HSV viral loads for the two specimen types were
assessed by using the Spearman rank correlations coefficient.
Main Results
None of the samples were positive for hemoglobin or Y chromosome DNA. The
results of the real time PCR assay performed on both cervical swab and
cervicovaginal lavage samples from 188 women are shown in the table. The
prevalence of HSV-2 DNA detection was higher when lavage samples were
tested than when swab samples were tested (P = 0.036). The mean
level of HSV-2 DNA was 6.31 x 107 copies/ml and 2.11 x 107
copies/μg of DNA in positive lavage and swab samples, respectively.
The levels of HSV-2 DNA in the two sample types were highly correlated (r
= 0.87, P < 0.0001).
Authors' Conclusions
Using the specimen processing procedures and
real time PCR detection thresholds described in this study, HSV-2 DNA was
detected more often in cervicovaginal lavage specimens than in paired
cervical swabs.
Source of funding: Agence Nationale de
Recherches sur le SIDA, Paris, France.
For correspondence: Laurent Bélec,
Labortoire de Virologie, Hôpital Européen Georges Pompidou, 20 Rue
Leblanc, 75908 Paris Cedex 15, France. E-mail address: Laurent.belec@egp.ap-hop-paris.fr.
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