PERFORMANCE OF THREE DIFFERENT HSV ID/TYPING ASSAYS FOR DETECTION AND TYPING OF HERPES SIMPLEX VIRUS IN CEREBROSPINAL FLUID

Background: Human Simplex Virus (HSV) is the most common cause of non-epidemic encephalitis in the US. Cerebrospinal fluid (CSF) culture techniques are insensitive due to the low viral burden typically found associated with encephalitis. Alternatively, nucleic acid amplification testing for HSV encephalitis has become the standard of care.

Aim: The aim of this study was to evaluate the performance characteristics of three different HSV identification/typing real-time PCR methods for specific detection of HSV DNA in CSF.

Methods: CSF specimens (n=68), both positive and negative for HSV, were used to evaluate Analyte Specific reagents, Cepheid HSV Typing kit (CHT), Eragen HSV-1/2 kit (EHT) and LightCycler-HSV-1/2 detection kit (LC-HT). CHT and EHT were analyzed using Cepheid Smart Cycler^® and LC-HT was analyzed using LightCycler^® real-time PCR system and the results were compared for efficiency in detection, typing, sensitivity, specificity, accuracy and precision of the assays. HSV-DNA was extracted from 200 µl of CSF specimens using Qiagen Viral MinElute kit. HSV-1 and 2 viral cultures from ATCC, quantitated HSV DNA from Advanced Biotechnologies Inc. (ABI) and low positive controls from AcroMetrix were used to determine the analytical sensitivity.

Results: Out of 68 specimens, 24 were detected positive by CHT, 25 by EHT and 23 by LC-HT. Specimens positive by only one or two of the three assays were repeated and confirmed to be positive. HSV typing results with all positive specimens using three different ASRs were in complete agreement. The analytical sensitivity of 300 HSV-1 copies/ml and 475 HSV-2 copies/ml was observed for CHT and EHT assays with ABI-quantitated DNA. AcroMetrix low positive controls showed sensitivity of detecting 100 HSV-1 copies/ml and 250 HSV-2 copies/ml for both CHT and EHT assays. Analysis with 10-fold serial dilutions of ATCC viral cultures revealed that EHT detected two dilutions lower when compared to CHT for both HSV-1 and 2. The accuracy at 1000 and 100 copies/ml (ABI) for HSV-1 and 2 with CHT was in the range of 0.6 through 2.1% CV and 0.8 through 3.6% CV with EHT. Similarly, the precision analysis using 10,000 and 1000 copies/ml of ATCC specimens revealed CV of 1.2 through 6.0% for inter assay variation (3-runs) and 0.2  through 5.3% for intra assay variation (triplicates) for both CHT and EHT assays with HSV-1 and 2.

Conclusions: The performance characteristics of all the three ASRs were comparable. Over all, the assays were easy to set-up and simple to interpret.