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Real-time PCR was a highly reproducible, rapid, and labor efficient method for HSV detection in genital swabs.
Diagnosis of genital herpes by real-time PCR in routine clinical practice
Ramaswamy M, McDonald C, Smith M, Thomas D, Maxwell S, Tenant-Flowers M, Geretti AM.
Sex Transmitted Infect 2004;80:406-10.
Summary:
Question
Can a real-time PCR assay replace viral culture for the diagnosis of genital herpes in genitourinary medicine attendees?
Design
The performance of a real-time PCR assay was compared to virus culture for the diagnosis and typing of genital herpes in a population of genitourinary medicine (GUM) clinic
attendees. Three specimen preparation methods for the PCR assay were evaluated.
Participants
Two hundred thirty-three consecutive GUM clinic attendees who presented with clinical features suggestive of genital herpes at Kings College Hospital in London were
tested. Of the first 145 patients, 85 were men and 60 were women. Genital swabs were collected from penile skin in 74% of the men and from the vulva in 80% of the females.
Description of Tests and Diagnostic Standard
Genital swabs were collected from each patient and placed into 2.5 mL of viral
transport medium for viral culture and
PCR. For virus isolation, Vero monkey kidney cells were inoculated with 200 μL of each specimen, incubated at 37oC and examined daily for cytopathic
effect. When a cytopathic effect was observed, the cells were stained with fluorescein
labeled murine monoclonal antibodies against HSV-1 and HSV-2 (Syva Microtrak, Palo Alto, CA) according to the
manufacturer's
instructions. Cultures were held for 10 days before being reported as
negative. For PCR, three methods were used to prepare DNA from 200 μL of 140 specimens, including 1) the QIAamp DNA Mini kit (Qiagen, UK), modified by adding poly dA (5
μg/mL) during lysis and 230 μL of ethanol at the first wash step, 2) the MagNA Pure LC instrument (Roche Diagnostics, Germany) according to the
manufacturer's instructions, and 3) polyethylene glycol (PEG)/NaCl DNA
precipitation. The final volumes of the specimen after extraction by QIAamp, MagNA Pure, and
PEG precipitation were 60, 100, and 20 μL, respectively. Real-time PCR was performed for 50 cycles as previously described in a LightCycler instrument (Roche) using 5
μL of each DNA specimen, the primers HSV POL F and HSV POL A, which amplify a 215 bp region of the HSV DNA polymerase gene, and the FRET hybridization probes HSV-1 FLU and HSV-2
LCR. Melting curve analysis was performed after amplification. The probe Tms were 59oC and 64oC and/or 71oC for HSV-1 and HSV-2,
respectively. True positive specimens were defined as either positive by culture and PCR or positive by PCR for at least 2 specimen preparation methods and at least 2 separate
assays. The RealArt HSV1/2 LC PCR kit (Artus Biotech, Germany) was used according to the
manufacturer's instructions to confirm specimens positive for HSV by the in-house real-time PCR
method. DNA sequencing was performed on the Trugene HIV-1 Opengene sequencing system (Visible Genetics, Ontario, Canada) under standard
conditions. Sequence data were compared to other HSV sequences to confirm the HSV type obtained by the in-house real-time PCR assay.
Main Outcome Measures
HSV detection by virus culture and real time PCR was compared among 233
specimens. Specimen preparation methods for the PCR assay were compared to the reference
"true positive" result as defined above for 140 specimens.
Main Results
HSV was detected in 79 (34%) and 132 (57%) of 233 specimens by culture and PCR,
respectively. One hundred twenty (91%) of the 132 PCR positive specimens were typed as
HSV-2. All specimens positive by culture were also positive by PCR. Thirty-two specimens that were negative by culture but positive by PCR were positive and the same type in at least 2 separate
assays. Fifteen specimens that were negative by culture but positive by PCR were positive and the same type by the RealArt PCR
assay. Fourteen HSV-2 positive specimens were sequenced and confirmed as HSV-2, although 4 had sequence variation in the FLU probe binding
site. The real time PCR increased HSV detection by 67%. The average time for a positive culture was 3
days. PCR results were available in less than 4 h. The rates of HSV detection were similar in men and
women. Culture but not PCR was more likely to be positive in patients presenting with lesions less than 5 days after onset compared to later.
Seventy-two of the 140 specimens prepared by 3 methods prior to PCR analysis were positive, including 61 (85%), 62 (86%), and 65 (90%) by the QIAamp, MagNA Pure, and PEG precipitation methods,
respectively. The PEG precipitation method was more rapid and less expensive than the other two methods.
Authors' Conclusions
The real time PCR significantly increased the rate of HSV detection in genital swabs compared to virus culture and its implementation is feasible for routine diagnostic
settings. The real time PCR assay was reliable, reproducible, and provided HSV typing without additional post-PCR
steps. Although the cost of consumables is higher for PCR compared to culture, the PCR is cost effective by significantly reducing labor costs.
Source of
funding: None given.
For
correspondence: A. M. Geretti, Royal Free and University College Medical School, Department of Virology, Hampstead Site, Rowland Hill Street, London NW3 2PF,
UK. E-mail address: a.geretti@rfc.ucl.ac.uk
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