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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

Distribution of capsular types among A) Isolates and B) Culture negative meningitis cases.
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pone-0003576-g004: Distribution of capsular types among A) Isolates and B) Culture negative meningitis cases.

Mentions: During January 2004 to December 2007, there were 358 (20%; 358/1,828) cases of pneumococcal meningitis enrolled at DSH; however, an isolate was grown from only 136 (38%; 136/358) cases. For the other 222 (62%; 222/358) culture-negative cases, the etiology was determined either by LAT (41%; 92/222) or Binax (59%; 130/222). Of these 222 cases, 127 (48 and 79 positives based on LAT and ICT testing, respectively) had sufficient remaining CSF for performing sequential multiplex PCR. PCR yielded the capsular serotype for an additional 51 (40%; 51/127) cases, although the cps was positive for all 127 cases. The yield of positive capsular serotype identity by PCR was higher (p<0.001) from the LAT positive cases (77%, 37/48) than from LAT-negative ICT-positive cases (18%, 14/79) (Figure 1). The proportional distribution of PCR-detected serotypes from culture negative cases was similar to the serotype distribution of isolates identified from CSF cultures, except for serotypes 1 and 24, which ranked 2nd and 23rd, respectively, among the cultured isolates, compared to 6th and 3rd, respectively, among the serotypes detected from culture negative cases by PCR (Figure 4A, 4B). Vaccine coverage for serotypes of culture positive meningitis cases by the existing PCV-7 and the upcoming 10 and 13-valent vaccines were 18%, 33% and 42%, respectively. On the other hand, coverage for PCR detected serotypes from culture negative cases was 18%, 33% and 33%, in that order. Coverage of the 3 vaccine formulations for all meningitis-causing serotypes (detected by any method) was 18%, 37% and 40%, respectively.


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)

Distribution of capsular types among A) Isolates and B) Culture negative meningitis cases.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003576-g004: Distribution of capsular types among A) Isolates and B) Culture negative meningitis cases.
Mentions: During January 2004 to December 2007, there were 358 (20%; 358/1,828) cases of pneumococcal meningitis enrolled at DSH; however, an isolate was grown from only 136 (38%; 136/358) cases. For the other 222 (62%; 222/358) culture-negative cases, the etiology was determined either by LAT (41%; 92/222) or Binax (59%; 130/222). Of these 222 cases, 127 (48 and 79 positives based on LAT and ICT testing, respectively) had sufficient remaining CSF for performing sequential multiplex PCR. PCR yielded the capsular serotype for an additional 51 (40%; 51/127) cases, although the cps was positive for all 127 cases. The yield of positive capsular serotype identity by PCR was higher (p<0.001) from the LAT positive cases (77%, 37/48) than from LAT-negative ICT-positive cases (18%, 14/79) (Figure 1). The proportional distribution of PCR-detected serotypes from culture negative cases was similar to the serotype distribution of isolates identified from CSF cultures, except for serotypes 1 and 24, which ranked 2nd and 23rd, respectively, among the cultured isolates, compared to 6th and 3rd, respectively, among the serotypes detected from culture negative cases by PCR (Figure 4A, 4B). Vaccine coverage for serotypes of culture positive meningitis cases by the existing PCV-7 and the upcoming 10 and 13-valent vaccines were 18%, 33% and 42%, respectively. On the other hand, coverage for PCR detected serotypes from culture negative cases was 18%, 33% and 33%, in that order. Coverage of the 3 vaccine formulations for all meningitis-causing serotypes (detected by any method) was 18%, 37% and 40%, respectively.

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