title: Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis.
abstract: BACKGROUND
  Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard.
  
  
  RESULTS
  CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously.
  
  
  RESULTS
  Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF.
  
  
  CONCLUSIONS
  Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.
authors: Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, CDC, Atlanta, GA 30333, USA. hmwu@emory.edu
authors: Henry M
authors: HM
authors: Wu
authors: Soraia M
authors: SM
authors: Cordeiro
authors: Brian H
authors: BH
authors: Harcourt
authors: Mariadaglorias
authors: M
authors: Carvalho
authors: Jailton
authors: J
authors: Azevedo
authors: Tainara Q
authors: TQ
authors: Oliveira
authors: Mariela C
authors: MC
authors: Leite
authors: Katia
authors: K
authors: Salgado
authors: Mitermayer G
authors: MG
authors: Reis
authors: Brian D
authors: BD
authors: Plikaytis
authors: Thomas A
authors: TA
authors: Clark
authors: Leonard W
authors: LW
authors: Mayer
authors: Albert I
authors: AI
authors: Ko
authors: Stacey W
authors: SW
authors: Martin
authors: Joice N
authors: JN
authors: Reis
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002555:Cerebrospinal Fluid / Q000382:microbiology; D002648:Child; D002675:Child, Preschool; D005840:Gentian Violet; D006193:Haemophilus influenzae / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016920:Meningitis, Bacterial / Q000175:diagnosis*; D008875:Middle Aged; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification; D010619:Phenazines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D055815:Young Adult
chemical_list: C035863:Gram's stain; D010619:Phenazines; D005840:Gentian Violet