Combined assay for two-hour identification of Streptococcus pneumoniae and Neisseria meningitidis and concomitant detection of 16S ribosomal DNA in cerebrospinal fluid by real-time PCR.

The main object was to examine the diagnostic performance of a novel combination of a specific real-time PCR (combined real-time PCR) for immediate and simultaneous detection of Streptococcus pneumoniae and Neisseria meningitidis and of a real-time PCR of the 16S rRNA gene (16S DNA). During 12 months, 1015 routine CSF samples were consecutively collected from patients in the County of Aarhus, Denmark. The samples were cultured, examined by microscopy, and, in parallel, CSF DNA was automatically purified and subjected to real-time PCR. Melting curve analysis discriminated between the 2 specific pathogens and 16S DNA positive samples were sequenced. Clinical data were extracted from patients having positive samples. Clinically, 35 of 46 (76%) patients with positive samples had bacterial meningitis. 18 of these 35 patients had a concomitant culture and real-time PCR-positive sample. The remaining 17 patients were either culture positive (n =7) or real-time PCR-positive (n = 10). The aetiology of bacterial meningitis was revealed by microscopy in 18/35 (51.4%), culture in 24/35 (68.6%) and combined real-time PCR in 27/35 (77.1%) patients, respectively. In conclusion, the combined real-time PCR strategy is superior to microscopy and a valuable supplement to routine culture to establish the aetiology of bacterial meningitis.