title: Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples.
abstract: BACKGROUND
  Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans.
  
  
  METHODS
  A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae.
  
  
  RESULTS
  All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains.
  
  
  CONCLUSIONS
  The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.
authors.affiliation: Fundação Oswaldo Cruz (FIOCRUZ), Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Rio de Janeiro, RJ, Brazil. Electronic address: ivano.defilippis@incqs.fiocruz.br.
authors.forename: Ivano
authors.initials: I
authors.lastname: de Filippis
authors.affiliation: Fundação Oswaldo Cruz (FIOCRUZ), Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Rio de Janeiro, RJ, Brazil.
authors.forename: Claudia Ferreira
authors.initials: CF
authors.lastname: de Andrade
authors.affiliation: Fundação Oswaldo Cruz (FIOCRUZ), Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Rio de Janeiro, RJ, Brazil.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Caldeira
authors.affiliation: Fundação Oswaldo Cruz (FIOCRUZ), Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Rio de Janeiro, RJ, Brazil.
authors.forename: Aline Carvalho
authors.initials: AC
authors.lastname: de Azevedo
authors.affiliation: Fundação Oswaldo Cruz (FIOCRUZ), Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Rio de Janeiro, RJ, Brazil.
authors.forename: Antonio Eugenio
authors.initials: AE
authors.lastname: de Almeida
mesh_terms: D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008583:Meningitis, Haemophilus / Q000175:diagnosis* / Q000382:microbiology; D008585:Meningitis, Meningococcal / Q000175:diagnosis* / Q000382:microbiology; D008586:Meningitis, Pneumococcal / Q000175:diagnosis* / Q000382:microbiology; D009345:Neisseria meningitidis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Diagnostic; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pneumoniae