Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.
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.
Saha
Samir K
SK
Department of Microbiology, Bangladesh Institute of Child Health, Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. sksaha@bangla.net
Darmstadt
Gary L
GL
Baqui
Abdullah H
AH
Hossain
Belal
B
Islam
Maksuda
M
Foster
Dona
D
Al-Emran
Hassan
H
Naheed
Aliya
A
Arifeen
Shams El
SE
Luby
Stephen P
SP
Santosham
Mathuram
M
Crook
Derrick
D
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
D017931:DNA Primers; D004269:DNA, Bacterial; D022242:Pneumococcal Vaccines