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To assess the diagnostic reliability of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for virus-associated opportunistic diseases of the central nervous system (CNS) in HIV-infected patients.CSF samples from 500 patients with HIV infection and CNS symptoms were examined by PCR. In 219 patients the PCR results were compared with CNS histological findings.Nested PCR for detection of herpes simplex virus (HSV) type 1 or 2, varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6), and JC virus (JCV) DNA. Histopathological examination of CNS tissue obtained at autopsy or on brain biopsy.DNA of one or more viruses was found in CSF in 181 out of 500 patients (36%; HSV-1 2%, HSV-2 1%, VZV 3%, CMV 16%, EBV 12%, HHV-6 2%, and JCV 9%). Among the 219 patients with histological CNS examination, HSV-1 or 2 was detected in CSF in all six patients (100%) with HSV infection of the CNS, CMV in 37 out of 45 (82%) with CMV infection of the CNS, EBV in 35 out of 36 (97%) with primary CNS lymphoma, JCV in 28 out of 39 (72%) with progressive multifocal leukoencephalopathy. Furthermore, HSV-1 was found in one, VZV in four, CMV in three, EBV in three, HHV-6 in seven, and JCV in one patient without histological evidence of the corresponding CNS disease.CSF PCR has great relevance for diagnosis of virus-related opportunistic CNS diseases in HIV-infected patients as demonstrated by its high sensitivity, specificity, and the frequency of positive findings.