RAPID DETECTION OF ENTEROVIRAL RNA IN CEREBROSPINAL-FLUID (CSF) FROM PATIENTS WITH ASEPTIC-MENINGITIS BY REVERSE TRANSCRIPTION NESTED POLYMERASE-CHAIN-REACTION

The aim of this study was to compare conventional enterovirus isolatio n with rapid detection of enteroviral RNA by a reverse transcription-n ested polymerase chain reaction (RT-nPCR) method amplifying the 5' non translated region of the enteroviral genome in specimens from patients with aseptic meningitis. Reference enterovirus strains and clinical e nterovirus isolates were analyzed to evaluate assay sensitivity and sp ecificity. All known enteroviral serotypes tested, but one (echovirus type 22), were detected by RT-nPCR. A series of unrelated viral isolat es as well as CSF samples from patients with meningitis/encephalitis o r neurological syndromes unrelated to enterovirus-infection were inclu ded as controls. A total of 47 specimens (31 CSF, 12 rectal swabs, 4 t hroat swabs) from 30 patients with aseptic meningitis were available f or the study. Of the 31 CSF samples tested from 30 patients, 17 from 1 7 patients (54.8%) were positive by RT-nPCR, while only 10 from 10 pat ients (32.2%) were positive by culture. Thus, RT-nPCR allowed diagnosi s of enterovirus meningitis in 7 additional patients compared to cell culture. The cytopathic effect was observed 5-15 days after inoculatio n of CSF specimens onto cell cultures, while direct detection of viral RNA in CSF samples by RT-nPCR permitted diagnosis of enteroviral meni ngitis within 1-2 days. On the whole, viral isolation was positive in 12/47 (25.5%) specimens, whereas viral RNA was detected by RT-nPCR in 11 additional samples (23/47, 48.9%). Specimens of the control group w ere consistently negative by both viral isolation and RT-nPCR. Restric tion endonuclease analysis of PCR products (RFLP) was applied to diffe rentiate poliovirus (PV) from non-polio enteroviruses (NPEV). All ente rovirus strains detected in clinical samples (n=23) were identified as NPEV by RFLP. Clinical isolates were typed by neutralization as echov irus type 30 (n=6), while 6 were not typed. In conclusion, detection o f enteroviral RNA in CSF by RT-nPCR allows: i) rapid diagnosis of ente roviral meningitis: ii) increased sensitivity with respect to virus is olation: iii) differentiation between PV and NPEV infections of the ce ntral nervous system.