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.

Neurocryptococcosis: diagnosis by PCR method.

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting.

We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 x 10(2) to 2 x 10(2) CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood.

A multiplex PCR-immunoassay for the rapid diagnosis of bacterial meningitis from cerebrospinal fluid (CSF) or peripheral blood was compared with conventional microbiological techniques used in the routine diagnostic laboratory. The multiplex PCR was designed to detect simultaneously a universal conserved sequence coding for bacterial 16S rRNA and the Neisseria meningitidis porA gene. The PCR-immunoassay had a detection limit of (3-5) x 10(2) cfu/ml (equivalent to 1-3 cfu/PCR) with spiked CSF or blood samples, compared with (3-5) x 10(3) cfu/ml for PCR followed by conventional agarose gel electrophoresis for detection of PCR products. Of 294 CSF samples from patients suspected on clinical grounds of suffering from meningitis, the PCR-immunoassay detected bacterial DNA in 29 CSF samples, 15 of which were also positive for N. meningitidis DNA. The 29 positive CSF samples comprised 25 samples that were also reported positive and four that were reported negative by conventional methodology; the latter four were all positive for N. meningitidis by the PCR-immunoassay. Of 173 peripheral blood samples examined, the PCR-immunoassay detected bacterial DNA in 18 samples, 14 of which were also positive for N. meningitidis DNA. In comparison, only 10 samples were reported positive for N. meningitidis by conventional methodology. All negative PCR-immunoassay results correlated with those obtained by conventional methodology for both CSF and blood samples. The sensitivity and speed of the PCR-immunoassay system indicated that it could be used as a routine diagnostic test for meningococcal meningitis, enabling a diagnosis to be made within 4 h of receipt of the specimen.