Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples

Abstract

A seminested polymerase chain reaction (PCR)-based diagnostic assay was evaluated for detection and verification ofNeisseria meningitidis,Haemophilus influenzae,Streptococcus pneumoniae,Steptococcus agalactiaeandListeria monocytogenesin cerebrospinal fluid (CSF) and other biological samples. A general bacterial amplicon from the 16S rRNA gene was amplified in a first step, and species-specific regions in a second. The detection level was 4 fg DNA/reaction, corresponding to about one bacterial genome per reaction tube. Sample preparations (Dynabeads DNA DIRECT kit) were assayed from 140 bacterial strains suspended in saline. In CSF the detection level for bacteria was 103CFU ml−1forN. meningitidis,H. influenzaeandS. pneumoniae, 104CFU ml−1forEscherichia coliand 105CFU ml−1forS. agalactiaeandL. monocytogenes. The detection levels for these bacteria were the same in the other tested biological samples, like blood with or without culture media. Clinical CSF samples were evaluated from 71 patients with proven bacterial meningitis, as were 61 CSF samples from individuals without bacterial meningitis. The diagnostic sensitivity of the assay in detecting bacteria in general was 0·97, and for the specific species in the clinical CSF samples 0·87–0·94. The specificity was 1·0 for detecting bacteria in general. Some cross-reactions were noted within the streptococcus group. The PCR results were verified by banding patterns ofHaeIII digested PCR products.