title: Application of 16S rDNA based seminested PCR for diagnosis of acute bacterial meningitis.
abstract: OBJECTIVE
  The diagnosis of bacterial meningitis remains a challenge to the clinician because of its rapid lethal course lacking the consistency to particular clinical signs and symptoms. Moreover, in many clinical settings use of rampant and short course antibiotic therapy prior to lumbar puncture reduces the chance of isolation of bacteria in CSF culture making the diagnosis difficult. The present study was done to evaluate a multiplex seminested PCR based method for rapid diagnosis of bacterial meningitis even after initiation of antibiotics.
  
  
  METHODS
  A 16S rDNA based PCR technique was evaluated using universal bacterial primers to detect any bacterial pathogen in CSF samples. The simultaneous use of three species-specific primers in a multiplex and seminested PCR format was done to identify Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis within 4 h.
  
  
  RESULTS
  Analysis of 267 CSF samples obtained from suspected cases of acute bacterial meningitis revealed 94 per cent concordance in results for conventional (Gram stain and culture) and molecular methods. Conventional techniques failed to detect five PCR positive samples where clinical diagnosis, cell count and biochemical findings of CSF supported the evidence of infection. The overall sensitivity, specificity, positive predictive value and negative predictive value of 16S rDNA PCR were 79.24, 97.6, 89.36 and 94.88 per cent respectively when culture was considered as gold standard. The detection limit of 16S rDNA PCR was determined to be 1000 cfu/ml of E. coli and 4000 cfu/ml of S. pneumoniae.
  
  
  CONCLUSIONS
  The results suggest that 16S rDNA PCR can be used as a valuable supplementary test in routine clinical practice for diagnosis of acute bacterial meningitis in hospital setting.
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India. prithwi4@gmail.com
authors.forename: P
authors.initials: P
authors.lastname: Chakrabarti
authors.forename: B K
authors.initials: BK
authors.lastname: Das
authors.forename: Arti
authors.initials: A
authors.lastname: Kapil
mesh_terms: D000704:Analysis of Variance; D017931:DNA Primers / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D006801:Humans; D007194:India; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal