EVALUATION OF FOUR COMMERCIAL REAL-TIME PCR ANALYTE-SPECIFIC REAGENTS (ASRs) FOR THE DETECTION OF HERPES SIMPLEX VIRUS IN CEREBROSPINAL FLUID AND BLOOD

            Herpes simplex virus (HSV)  types 1 and 2 are the cause of important central nervous system infections and can also cause life threatening disseminated infections in newborns and immunocompromised individuals.  Polymerase chain reaction (PCR) detection of HSV DNA in cerebrospinal fluid (CSF) and blood is the preferred method for the laboratory diagnosis of these infections.  Our laboratory has performed a conventional home brew PCR assay for HSV from CSF and whole blood since 1994.  Because real-time PCR offers convenience and labor-savings we decided to evaluate four commercially available real-time HSV PCR ASRs as potential replacements for our conventional assay for CSF and blood specimens.

            The real-time ASRs included the Roche Lightcycler HSV1/2 Primer/Hybridization Probes, the Cepheid HSV Typing Primer Probe Set, the Nanogen MGB Alert HSV Typer Mix, and the Eragen MultiCode-RTx HSV 1/2 Primer Mix.  For instrumentation, the Roche Lightcycler was used for the Roche, Nanogen, and Eragen ASRs and the Cepheid Smartcycler was used for the Cepheid ASR.  The Eragen ASR was also run on the Smartcycler.  The Roche, Eragen and Nanogen ASRs used melting curve analysis to differentiate between HSV types 1 and 2 whereas the Cepheid ASR used separately labeled probes.

            We used 60 specimens previously tested with our conventional assay, including 54 CSF specimens (20 positive for HSV 2; 16 positive for HSV 1; 18 negative) and 6 whole blood specimens (3 positive for HSV 1; 1 positive for HSV 2; 2 negative) to evaluate the real-time ASRs.  The testing algorithm was to first test all the original nucleic acid extracts.  To resolve discrepancies, for any original extract yielding a discrepancy with any of the ASRs, original specimen was re-extracted and retested by all of the assays, including the original conventional assay.  Resolved CSF results were as follows:  All of the ASRs detected 16/16 HSV1 positive CSFs; the Eragen and Cepheid ASRs detected 20/20 HSV2 positive CSFs whereas the Nanogen ASR detected 19/20 and the Roche ASR detected 18/20.  All 18 of previously negative CSF specimens tested negative by all of the ASRs.  Resolved results of testing whole blood specimens were as follows:  the Eragen ASR detected 3/3 HSV1 positive blood specimens; the Cepheid ASR detected 1/3 and the Nanogen and Roche ASRs detected 0/3 HSV1 positive blood specimens; all of the ASRs detected the single HSV2 positive blood specimen.  In several instances the typing results of the ASRs differed from the original conventional PCR result.  The Nanogen ASR typed one HSV1 positive CSF specimen as an HSV2 and the Roche ASR failed to provide typing results on 3 HSV1 positive CSF specimens.  One blood specimen which was positive for HSV1 using the conventional PCR assay was found to contain both HSV1 and 2 by the Eragen ASR and HSV2 by the Cepheid ASR.

In conclusion, with the exception of 3 discrepancies in typing, all of the ASRs performed well in detecting HSV1 in CSF.  The Eragen and Cepheid ASRs detected all 20 of the HSV2 positive CSF specimens while the Nanogen and Roche ASRs each failed to identify two of the HSV2 positive CSFs.  Although the number of blood specimens was small, it appeared that blood specimens proved problematic for the detection HSV, with only the Eragen ASR being positive for all 4 of the previously positive specimens.