Identification of Serotype in Culture Negative Pneumococcal Meningitis Using Sequential Multiplex PCR: Implication for Surveillance and Vaccine Design

Samir K Saha,Shams El Arifeen,Hassan Al-Emran,Gary L Darmstadt,Aliya Naheed,Maksuda Islam,Derrick Crook,Abdullah H Baqui,Dona Foster,Belal Hossain,Stephen P Luby,Mathuram Santosham

doi:10.1371/journal.pone.0003576

Abstract

BackgroundPCR-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.MethodologyThirty 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.ConclusionsThis PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Highlights

Streptococcus pneumoniae is the predominant cause of bacterial pneumonia, a leading cause of meningitis, and results in more than 800,000 deaths yearly among children under–5 worldwide [1]
In this study we aimed to a) modify the design of the sequential multiplex polymerase chain reaction (PCR) method of Pai et al [23] to optimize the scheme for the capsular serotype distribution likely to occur in Bangladesh, b) validate the scheme in Bangladeshi strains isolated from meningitis and non-meningitis cases, c) apply the scheme to detect capsular serotype directly from cerebrospinal fluid (CSF) specimens of culture positive and culture negative cases of pneumococcal meningitis and d) to compare the cost and laboriousness of two serotyping methods
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)

Summary

Introduction

Streptococcus pneumoniae is the predominant cause of bacterial pneumonia, a leading cause of meningitis, and results in more than 800,000 deaths yearly among children under–5 worldwide [1]. Pneumococcal meningitis in developing countries is often recognized late, resulting in high mortality and a substantial burden of long-term disability among survivors. Resource poor countries, implementing an effective vaccine programme is challenging both in terms of cost and for determining the true burden of diseases, because many pneumococcal meningitis cases are culture negative [7,8]. Of the 90 different possible serotypes, disease is caused by a restricted set that can differ markedly between countries. The currently licensed vaccine, a 7-valent pneumococcal conjugate vaccine (PCV7), is based on the predominant serotypes causing invasive disease in North America, and, while it affords protection to .80% of invasive pneumococcal cases in these settings, it is predicted to protect against only 60% and 40% of cases in Africa and Asia, respectively [9]. PCR has not been used to determine serotype distribution in culture-negative meningitis cases

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