STABILITY AND CLINICAL-APPLICATION OF A MULTIPLEX NESTED PCR ASSAY FOR THE DIAGNOSIS OF HERPES-SIMPLEX VIRUS-INFECTIONS

A novel multiplex nested polymerase chain reaction (PCR) assay was des igned and evaluated for routine diagnosis of herpes simplex virus (HSV ) infections in patients with either putative HSV infection of the cen tral nervous system or suspected HSV keratitis. Single-tube amplificat ion of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebro spinal fluid (CSF) or from keratectomy specimens was followed by diffe rentiation of the virus type-specific PCR products either by agarose g el analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obta ined from 395 consecutive patients with clinically suspected HSV infec tion, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes wer e positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstr ucted CSF samples. Inhibition could be removed following extraction wi th a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corn eal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of th is test, which has met the specific requirements of a diagnostic labor atory. (C) 1996 Wiley-Liss, Inc.