DETECTION OF BACTERIAL-DNA IN CEREBROSPINAL-FLUID BY AN ASSAY FOR SIMULTANEOUS DETECTION OF NEISSERIA-MENINGITIDIS, HAEMOPHILUS-INFLUENZAE,AND STREPTOCOCCI USING A SEMINESTED PCR STRATEGY

Primers specific to conserved and variable regions in the 16S rRNA seq uence were selected from the partially sequenced 16S rRNA genes of Nei sseria ia meningitidis, Haemophilus influenzae, Streptococcus pneumoni ae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was d ivided into two DNA amplifications. The first resulted in a general ba cterial amplicon, and the second resulted in a species specific amplic on. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinic al cerebrospinal fluid samples, including 125 samples from patients wi th bacterial meningitis, were assayed to investigate the diagnostic se nsitivity and specificity for bacterial meningitis. The assay showed h igh sensitivity (0.94) and specificity (0.96) with the clinical sample s, although some false results were obtained, the reasons for which ar e discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.