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A duplex real-time PCR is a sensitive and convenient method for diagnosing and typing genital herpes.
Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions.
Filen F, Strand A, Allard A, Blomberg J, Herrmann B.
Sexually Transmitted Diseases 2004;31:331-36.
Summary:
Question
How does a real-time quantitative PCR perform in comparison to viral culture for the diagnosis of genital and cutaneous HSV-1 and HSV-2?
Design
A duplex, real-time quantitative PCR assay was compared to culture and clinical diagnosis for the detection of HSV-1 and HSV-2 in mucocutaneous swab samples.
Participants
Eighty-nine outpatients attending STD and dermatology clinics who presented with symptoms of HSV infection (vesicles, ulcers, crusting lesions) and atypical symptoms suspected for HSV were tested, including 81 genital and 8 cutaneous specimens. The patients included 44 males (mean age = 25 years) and 45 females (mean age = 22
years).
Description of Tests and Diagnostic Standard
Two swab specimens were collected from each patient in rotating order for PCR and culture.For virus isolation, cotton-tipped swabs were transported at room temperature, and cultured according to standard procedures.For PCR, swabs intended for chlamydia nucleic acid detection (ProbeTec, Becton Dickinson, Sparks, MD) were frozen in 0.4 mL of buffer until analysis.Thirty additional genital samples from patients with symptoms were collected using the Virocult tube (Medical Wire Equipment, Corsham, UK), which was kept at 4oC, and a cotton-tipped swab kept in a dry collection tube at room temperature to which 0.4 mL of buffer was added prior to DNA extraction.DNA was extracted from 0.2 mL of each swab specimen using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the
manufacturer 's instructions.One water control was extracted for every 3 clinical samples to monitor for contamination.The duplex real time PCR assay included a primer set that targeted the HSV-1 glycoprotein G/J gene junction and a primer set that targeted the HSV-2 glycoprotein G gene.The TaqMan probes were
5' labeled with the fluorescent dyes 6FAM for HSV-1 and HEX for HSV-2.Primer and probe concentrations were optimized.Ten μL of each DNA sample was added to the reaction mix and amplified for 45 cycles.Quantification was achieved using standard curves prepared from samples containing 1000, 100, 30, and 10 copies/reaction of a purified plasmid containing a 330 bp insert covering the target sequences of the HSV-1 and HSV-2 primers.
Main Outcome Measures
The results of the PCR assay and culture for detection of HSV-1 and HSV-2 in swabs from patients with first episode or recurrent infections with HSV were compared.
Main Results
The detection limit of the PCR assay was between 1 and 5 copies per reaction in the duplex format.Of 30 water controls from 8 different runs, none was positive in the PCR assay.Of positive control samples from 25 consecutive runs, the mean value was 151+65 copies/reaction for HSV-1 and 97+46 copies/reaction for HSV-2.All samples with less than 103 copies/reaction were confirmed with repeated extraction and amplification of DNA.Among the 30 samples obtained on Virocult and cotton-tipped swabs, both swabs gave concordant results of 16 positive tests.HSV was detected in 57 (64%) of 89 samples by PCR compared with 45 (51%) by virus isolation.The PCR assay increased the detection rate by 27% compared with virus culture (P = 0.006).The results of PCR and culture by HSV virus type are shown in the table.Of 57 PCR positive samples, 48 (84%) contained at least 1000 copies/reaction of HSV (median = 9.8 X 104).There was no significant difference in viral copies/reaction between samples positive for HSV-1 and HSV-2.
Patients with first-episode infections had significantly higher viral loads (median = 4.2 X 105 copies/reaction) than did patients with recurrent infections (median = 1.0 X 104 copies/reaction) (P = 0.0002), for both HSV-1 and HSV-2 infections.The copy number per reaction related to duration of symptoms was significant for recurrent infection but not for first-episode infections (P = 0.01).
Results of real-time PCR assay and viral culture for detection of HSV-1 and HSV-2 in 89 clinical swab specimens
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Culture result
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PCR assay result
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HSV-1
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HSV-2
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Negative
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Total
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HSV-1
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22
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0
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2
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24
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HSV-2
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0
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21
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0
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21
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Negative
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11
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3
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30
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44
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Total
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33
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24
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32
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89
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Authors ' Conclusions
The duplex format quantitative PCR assay for HSV-1 and HSV-2 using swab samples was shown to be more sensitive than the current gold standard method for diagnosing mucocutaneous HSV infections, raising the detection rate by 27%. The improved test performance was observed across all clinical presentations.By using quantitative PCR, we could gain more knowledge of the course of infections and the effect of antiviral
treatment.
Source of funding:Uppsala University Hospital
For correspondence:Finn Filen, Department of Dermatology and Venereology, University Hospital, SE-75185 Uppsala,
Sweden.
E-mail address: finn.filen@medsci.uu.se.
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