sybr-green-i and Meningitis--Meningococcal

Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis.. We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity.. A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4-100) for N. meningitidis and 100% (95% CI = 85.1-100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6-100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods.. Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations.

Topics: Adolescent; Adult; Aged; Benzothiazoles; Cerebrospinal Fluid; Child; Child, Preschool; Costs and Cost Analysis; Diamines; Female; Humans; Infant; Male; Meningitis, Meningococcal; Meningitis, Pneumococcal; Middle Aged; Molecular Diagnostic Techniques; Multiplex Polymerase Chain Reaction; Neisseria meningitidis; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling; Streptococcus pneumoniae; Time Factors; Transition Temperature; Young Adult

Invasive meningococcal infections are usually diagnosed by culture. This approach is far from optimal due to, e.g., treatment with precollection antibiotics. Molecular-genetics methods are therefore recognized as important tools for optimal laboratory confirmation of meningococcal infections as well as characterization of meningococci (Mc). The aims of the present study were to develop real-time PCRs for identification and genogrouping (A, B, C, Y, and W-135) of Mc and porA amplification that further can be used for subsequent genosubtyping. In a first run Mc were identified. In a second run they were genogrouped and porA genes were amplified. All the Mc isolates (n = 71) but one and cerebrospinal fluid samples (n = 11) tested gave the expected positive results. The specificity, inter- and intra-assay variations, and effects of different amounts of DNA on the melting temperatures were also explored. The LightCycler PCR system was found to effectively detect and characterize Mc in a few hours. This testing, including the DNA sequencing of the porA gene to reveal the genosubtype, does not take more than a working day, and the results can be compared to those from culturing with phenotypic analysis, which takes at least a few days.

Topics: Bacterial Typing Techniques; Benzothiazoles; Diamines; Fluorescent Dyes; Genotype; Humans; Meningitis, Meningococcal; Meningococcal Infections; Neisseria meningitidis; Organic Chemicals; Polymerase Chain Reaction; Porins; Quinolines; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA; Temperature; Time Factors