title: Use of quantitative broad-based polymerase chain reaction for detection and identification of common bacterial pathogens in cerebrospinal fluid.
abstract: BACKGROUND
  Conventional laboratory diagnosis of bacterial meningitis based on microscopy followed by culture is time-consuming and has only moderate sensitivity.
  
  
  OBJECTIVE
  The objective was to define the limit of detection (LOD), analytic specificity, and performance characteristics of a broad-based quantitative multiprobe polymerase chain reaction (PCR) assay for rapid bacterial detection and simultaneous pathogen-specific identification in patients with suspected meningitis.
  
  
  METHODS
  A PCR algorithm consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by pathogen identification using either pathogen-specific probes or Gram-typing probes, was employed to detect pathogens. The 16S rRNA gene, which contains both conserved and variable regions, was chosen as the target. Pathogen-specific probes were designed for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. Gram-positive and -negative typing probes were designed based on conserved regions across all eubacteria. The LOD and time to detection were assessed by dilutional mocked-up samples. A total of 108 convenience cerebrospinal fluid (CSF) clinical samples obtained from the Johns Hopkins Hospital (JHH) microbiology laboratory were tested, and results were compared with hospital microbiologic culture reports.
  
  
  RESULTS
  The LOD of the assay ranged from 10(1) to 10(2) colony-forming units (CFU)/mL. Pathogen-specific probes showed no cross-reactivity with other organisms. Time to detection was 3 hours. In clinical specimens, the universal probe correctly detected 16 of 22 culture-positive clinical specimens (sensitivity = 72.7%; 95% confidence interval [CI] = 49.8% to 89.3%), which were all correctly characterized by either pathogen-specific or Gram-typing probes. Adjusted sensitivity after removing probable microbiologic laboratory contaminants was 88.9% (95% CI = 65.3% to 98.6%). The universal probe was negative for 86 of 86 culture-negative specimens.
  
  
  CONCLUSIONS
  A broad-based multiprobe PCR assay demonstrated strong analytic performance characteristics. Findings from a pilot clinical study showed promise in translation to human subjects, supporting potential utility of the assay as an adjunct to traditional diagnostics for early identification of bacterial meningitis.
authors.affiliation: Department of Emergency Medicine, Johns Hopkins University, Baltimore, MD, USA. rrothman@jhmi.edu
authors.forename: Richard
authors.initials: R
authors.lastname: Rothman
authors.forename: Padmini
authors.initials: P
authors.lastname: Ramachandran
authors.forename: Samuel
authors.initials: S
authors.lastname: Yang
authors.forename: Andrew
authors.initials: A
authors.lastname: Hardick
authors.forename: Helen
authors.initials: H
authors.lastname: Won
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Kecojevic
authors.forename: Celeste
authors.initials: C
authors.lastname: Quianzon
authors.forename: Yu-Hsiang
authors.initials: YH
authors.lastname: Hsieh
authors.forename: Charlotte
authors.initials: C
authors.lastname: Gaydos
mesh_terms: D000465:Algorithms; D001419:Bacteria; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D057230:Limit of Detection; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial