title: Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.
abstract: BACKGROUND
  PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases.
  
  
  METHODS
  Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases.
  
  
  CONCLUSIONS
  This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.
authors.affiliation: Department of Microbiology, Bangladesh Institute of Child Health, Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. sksaha@bangla.net
authors.forename: Samir K
authors.initials: SK
authors.lastname: Saha
authors.forename: Gary L
authors.initials: GL
authors.lastname: Darmstadt
authors.forename: Abdullah H
authors.initials: AH
authors.lastname: Baqui
authors.forename: Belal
authors.initials: B
authors.lastname: Hossain
authors.forename: Maksuda
authors.initials: M
authors.lastname: Islam
authors.forename: Dona
authors.initials: D
authors.lastname: Foster
authors.forename: Hassan
authors.initials: H
authors.lastname: Al-Emran
authors.forename: Aliya
authors.initials: A
authors.lastname: Naheed
authors.forename: Shams El
authors.initials: SE
authors.lastname: Arifeen
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Luby
authors.forename: Mathuram
authors.initials: M
authors.lastname: Santosham
authors.forename: Derrick
authors.initials: D
authors.lastname: Crook
mesh_terms: D000465:Algorithms; D001459:Bangladesh; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000138:chemical synthesis; D004269:DNA, Bacterial / Q000032:analysis; D015195:Drug Design; D006801:Humans; D008586:Meningitis, Pneumococcal / Q000134:cerebrospinal fluid / Q000145:classification* / Q000235:genetics / Q000382:microbiology; D022242:Pneumococcal Vaccines / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011159:Population Surveillance / Q000379:methods*; D011786:Quality Control; D012703:Serotyping / Q000191:economics / Q000379:methods; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D022242:Pneumococcal Vaccines