title: A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco. diawaraidris@gmail.com.
authors.forename: Idrissa
authors.initials: I
authors.lastname: Diawara
authors.forename: Khalid
authors.initials: K
authors.lastname: Katfy
authors.forename: Khalid
authors.initials: K
authors.lastname: Zerouali
authors.forename: Houria
authors.initials: H
authors.lastname: Belabbes
authors.forename: Naima
authors.initials: N
authors.lastname: Elmdaghri
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D052160:Benzothiazoles; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D002648:Child; D002675:Child, Preschool; D003365:Costs and Cost Analysis; D003959:Diamines; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D008585:Meningitis, Meningococcal / Q000175:diagnosis*; D008586:Meningitis, Pneumococcal / Q000175:diagnosis*; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009345:Neisseria meningitidis / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors; D044366:Transition Temperature; D055815:Young Adult
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I