CLINICAL RESEARCH STUDY OF ARCHIVED CSF SAMPLES FROM PATIENTS SUSPECTED HERPES SIMPLEX ENCEPHALITIS USING INTELLIGENT MEDICAL DEVICES, INC. HERPES SIMPLEX VIRUS REAL-TIME PCR REAGENTS

Background:  Herpes simplex virus (HSV) is the most frequently diagnosed cause of sporadic viral encephalitis in the U.S., affecting both neonatal and adult populations. Early intervention is critical for minimizing the associated morbidity/mortality, and has necessitated the development of rapid, sensitive molecular diagnostic assays. Intelligent Medical Devices (IMD), Inc.’s novel bioinformatics software, PriMD®, provides a superior approach to designing diagnostic reagents for the detection of clinically relevant pathogens. By accounting for inter- and intra-species genomic variability and incorporating new algorithms that advance our understanding of nucleotide position effects on primer-probe behavior, PriMD simplifies and speeds the process of identifying optimal reagents for singleplex and multiplex real-time PCR assays. In this study, we compared real-time PCR (TaqMan) reagents designed by PriMD with clinically validated PCR-based tests for typing and non-typing of HSV. Archived cerebrospinal fluid (CSF) samples were obtained from two leading centers for HSV testing; the Viral Encephalitis Laboratory at the Wadsworth Center, New York State Department of Health (NYS Lab), and the University of Alabama Children’s Hospital (UAB).

Methods:  The HSV glycoproteins B, D and G were targeted by PriMD to identify TaqMan primer and probe candidates for HSV typing and non-typing tests. Primer and probe sets were selected that offered the highest predicted specificity and sensitivity. A total of 176 CSF samples from the two study sites were coded, randomized, and tested using IMD’s reagents on ABI 7300/7500 Cyclers. An HSV synthetic sequence was used as a positive control for sample preparation at UAB, while NYS Lab used GFP plasmid and HSV genomic DNA as positive controls for sample preparation and TaqMan assays, respectively. Tests were considered positive when the TaqMan fluorescent signal exceeded a threshold over background between cycles 15 and 40. Test results were then compared to those generated with the centers’ established protocols.

Results:  For the HSV non-typing test, all 90 samples previously testing positive for HSV at the two centers were confirmed positive by IMD's HSV non-typing TaqMan set. Similarly for the HSV typing test (performed only at NYS Lab), all 26 samples of HSV-1 and all 31 HSV-2 samples were correctly identified by IMD’s test. Of the 86 negative samples, all were negative by IMD’s test.

Conclusions:  The TaqMan reagents designed by PriMD correctly predicted the clinical outcome of all 176 CSF samples. These results support previous clinical studies that show IMD reagents are as good as, if not better than, current diagnostic tests and highlight the utility of PriMD®, IMD’s powerful new design software.