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Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.

Saha SK, Darmstadt GL, Baqui AH, Hossain B, Islam M, Foster D, Al-Emran H, Naheed A, Arifeen SE, Luby SP, Santosham M, Crook D - PLoS ONE (2008)

Bottom Line: 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.Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers.Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bangladesh Institute of Child Health, Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. sksaha@bangla.net

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.

Methodology: 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.

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Related in: MedlinePlus

Amplified serotype (1, 2, 14 & 18) genome from culture negative CSF specimens by 1st reaction of multiplex PCR scheme.Lane 1–10 & 14–17: Culture negative latex Positive CSF, Lane 11–13: Culture negative ICT Positive CSF, Lane 18: Positive Control Containing amplified cps gene and genome of 4 known isolates of reaction 1, L 19: Reagent control–reagents devoid template, L 20: 100bp ladder.
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pone-0003576-g003: Amplified serotype (1, 2, 14 & 18) genome from culture negative CSF specimens by 1st reaction of multiplex PCR scheme.Lane 1–10 & 14–17: Culture negative latex Positive CSF, Lane 11–13: Culture negative ICT Positive CSF, Lane 18: Positive Control Containing amplified cps gene and genome of 4 known isolates of reaction 1, L 19: Reagent control–reagents devoid template, L 20: 100bp ladder.

Mentions: The 5 newly designed primers showed 100% specificity when tested using individual isolates of each of 56 different serotypes; there were no discrepant results. The sequential multiplex PCR scheme (Figures 2 and 3), as expected, identified the capsular serotype of isolates in decreasing order of frequency. The first of 7 multiplex reactions yielded the capsular serotype of 40% of strains and, by the end of reaction 4, it reached to 85%. The capsular serotype of 94% of isolates could be determined when using all 7 multiplex reactions (Figure 2A). The initial multiplex design identified the serotype of 31% (N = 39/125) and 48% (N = 64/132) of pneumonia/sepsis and meningitis isolates, respectively (p = 0.007). By redesigning the multiplex scheme specifically for pneumonia/sepsis or meningitis, the capsular serotype of 43% and 48% of isolates could be identified with the 1st reaction, and 86% and 88% of isolates by the 4th reaction, respectively (Figure 2B, 2C).


Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.

Saha SK, Darmstadt GL, Baqui AH, Hossain B, Islam M, Foster D, Al-Emran H, Naheed A, Arifeen SE, Luby SP, Santosham M, Crook D - PLoS ONE (2008)

Amplified serotype (1, 2, 14 & 18) genome from culture negative CSF specimens by 1st reaction of multiplex PCR scheme.Lane 1–10 & 14–17: Culture negative latex Positive CSF, Lane 11–13: Culture negative ICT Positive CSF, Lane 18: Positive Control Containing amplified cps gene and genome of 4 known isolates of reaction 1, L 19: Reagent control–reagents devoid template, L 20: 100bp ladder.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571985&req=5

pone-0003576-g003: Amplified serotype (1, 2, 14 & 18) genome from culture negative CSF specimens by 1st reaction of multiplex PCR scheme.Lane 1–10 & 14–17: Culture negative latex Positive CSF, Lane 11–13: Culture negative ICT Positive CSF, Lane 18: Positive Control Containing amplified cps gene and genome of 4 known isolates of reaction 1, L 19: Reagent control–reagents devoid template, L 20: 100bp ladder.
Mentions: The 5 newly designed primers showed 100% specificity when tested using individual isolates of each of 56 different serotypes; there were no discrepant results. The sequential multiplex PCR scheme (Figures 2 and 3), as expected, identified the capsular serotype of isolates in decreasing order of frequency. The first of 7 multiplex reactions yielded the capsular serotype of 40% of strains and, by the end of reaction 4, it reached to 85%. The capsular serotype of 94% of isolates could be determined when using all 7 multiplex reactions (Figure 2A). The initial multiplex design identified the serotype of 31% (N = 39/125) and 48% (N = 64/132) of pneumonia/sepsis and meningitis isolates, respectively (p = 0.007). By redesigning the multiplex scheme specifically for pneumonia/sepsis or meningitis, the capsular serotype of 43% and 48% of isolates could be identified with the 1st reaction, and 86% and 88% of isolates by the 4th reaction, respectively (Figure 2B, 2C).

Bottom Line: 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.Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers.Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bangladesh Institute of Child Health, Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. sksaha@bangla.net

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.

Methodology: 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.

Show MeSH
Related in: MedlinePlus