Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the me thod was optimized and carried out and its sensitivity was compared to prev iously described assays. The assay has been used on a limited number of cli nical samples and must be thoroughly evaluated in the clinical context. A t otal of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were i ncluded in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of th e highest dilution positive by PCR for HSV-1 and HSV-2 respectively, wherea s for VZV, CW and EBV, 14, 18, and 160 ag of genomic DNA were detected corr esponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was de tected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%) , and CMV was also detected in one neonate (1.16%). None of the samples tes ted was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification fr om CSF samples was avoided by the inclusion in the present multiplex PCR as say of ci-tubulin primers. The present multiplex PCR assay detects simultan eously five different herpesviruses and sample suitability for PCR in a sin gle amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool a llowing the application of antiviral therapy on the basis of a specific vir al diagnosis. The results of this preliminary study should prompt a more ex haustive analysis of the clinical value of the present multiplex PCR assay.