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HSV was detected at
a higher frequency by PCR compared to virus isolation in samples
obtained from mucosal surfaces.
Polymerase chain reaction for
detection of herpes simplex virus (HSV) DNA on mucosal surfaces:
comparison with HSV isolation in cell culture.
Wald A, Huang M-L, Carrell D, Selke S, Corey
L.
Journal
of Infectious Diseases 2003;188:1345-51.
Summary:
Question
What is the frequency of detection of HSV by PCR compared to viral culture
in mucosal swab specimens from different patient groups?
Design
This paper describes a comparison of the rate of isolation of HSV in
culture versus the detection of HSV DNA by real-time quantitative PCR in
mucosal swab specimens obtained from a variety of persons, clinical
settings, and anatomic sites.
Participants
Paired specimens (n = 36,471) were collected at the University of
Washington Virology Research Clinic from 296 (137 women, 159 men) subjects
enrolled in a variety of studies of HSV infection, including natural
history, therapeutic vaccine, and treatment evaluation. The median age was
34 years. Thirty percent were HIV positive. All subjects had HSV
infections confirmed by Western blot: 45% were only HSV-2 antibody
positive, 43% were both HSV-1 and HSV-2 antibody positive, and 13% were
only HSV-1 antibody positive. The mean number of sample pairs obtained per
subject was 123. Samples obtained on days during which antiviral
medication was administered were excluded from the analyses.
Description of Tests and Diagnostic
Standard
Paired swab samples, held together, were obtained daily by participants at
home or by clinicians in the clinic. Women collected cervicovaginal,
vulvar, and perianal swab samples. Men collected penile and perianal swab
samples. Separate swabs were obtained of lesions, when present. Some
subjects collected swab samples from oral and nasal mucosa. One swab of
each pair was placed into viral transport medium for viral culture. The
other swab, for HSV DNA PCR, was placed into PCR digestion buffer. All
samples were refrigerated until transported to the laboratory. In the
laboratory, swabs for PCR were frozen until processing.
Specimens for virus isolation were
inoculated onto human diploid fibroblasts. All isolates were typed by
monoclonal antibodies. DNA was extracted from the specimens for PCR and
amplified using primers that target the HSV glycoprotein B gene. Each
specimen was analyzed in duplicate using a real-time quantitative PCR
assay (ABI Prism 7700 Sequence Detection System, Applied Biosystems).
Specimens were considered positive if both replicates were above the
cutoff value for the assay (>10 copies of HSV DNA/reaction or 500
copies/ml of transport buffer).
Main Outcome Measures
The rates of HSV detection by the two methods and between the groups of
subjects were compared. Comparisons were limited to subjects who
contributed at least 10 specimens.
Main Results
Overall, 1087 (3.0%) and 4415 (12%) of 36,471 specimens were positive for
HSV by viral isolation and PCR, respectively. Of 4464 samples that were
positive by either test, 3377 (75.7%) were positive by PCR only, and 49
(1.1%) were positive by viral culture only. There was a linear
relationship between the frequency of HSV isolation in culture and the
number of HSV DNA copies detected, regardless of lesion status, HIV
serostatus, or gender of the subject. The rates of virus isolation and HSV
DNA detection by PCR among the paired specimens, stratified by gender,
presence of lesions, and HIV serostatus, are shown in the table. The ratio
of PCR positivity to viral culture positivity varied from 3.1:1 on days
when lesions were present, to 5.1:1 on days when lesions were absent.
However, for cervical swabs, HSV was detected by PCR in 42% and by culture
in 3.8% on days when lesions were present. The ratio of PCR-positive to
viral culture positive specimens was 4.5:1 for swabs collected at home
during January and February compared with 8.8:1 for swabs collected during
July and August. In contrast, the rate of HSV DNA detection was 8.8% in
the winter months compared to 8.9% in the summer months. A 50% reduction
in virus isolation appears attributable to a seasonal effect on specimen
viability.
Authors' Conclusions
HSV was detected more frequently by PCR than
by viral culture regardless of whether samples were obtained from HSV
lesions, or from genital or oral secretions during a period of subclinical
shedding. Yield of virus positivity is 4 times greater by PCR than by
culture, and the results are more reliable, especially in settings in
which transport or climate may interfere with the yield from viral
culture. Health planners and regional reference laboratories should
consider using their resources for PCR-based detection methods rather than
for virus isolation.
Source of funding: National Institutes
of Health
For correspondence:
Anna Wald, University of Washington Virology Research Clinic, 600
Broadway, Suite 400, Seattle, WA 98122. E-mail address: annawald@u.washington.edu
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