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.

The SARS‐CoV‐2 coronavirus is responsible for the current COVID‐19 pandemic, with an ongoing toll of over 5 million infections and 333 thousand deaths worldwide within the first 5 months. Insight into the phylodynamics and mutation variants of this virus is vital to understanding the nature of its spread in different climate conditions. The incidence rate of COVID‐19 is increasing at an alarming pace within subtropical Southeast Asian nations with high temperatures and humidity. To understand this spread, we analyzed 444 genome sequences of SARS‐CoV‐2 available on the GISAID platform from 6 Southeast Asian countries. Multiple sequence alignments and maximum likelihood phylogenetic analyses were performed to analyze and characterize the non‐synonymous mutant variants circulating in this region. Global mutation distribution analysis showed that the majority of the mutations found in this region are also prevalent in Europe and North America, and the concurrent presence of these mutations at a high frequency in other countries indicate possible transmission routes. Unique spike protein and non‐structural protein mutations were observed circulating within confined area of a given country. We divided the circulating viral strains into 4 major groups and 3 sub‐groups on the basis of the most frequent non‐synonymous (NS) mutations. Strains with a unique set of 4 co‐evolving mutations were found to be circulating at a high frequency within India, specifically. Group 2 strains characterized by two co‐evolving NS mutants which alter in RdRp (P323L) and spike (S) protein (D614G), were found to be common in Europe and North America. These European and North American variants have rapidly emerged as dominant strains within Southeast Asia, increasing from a 0% prevalence in January to an 81% by May 2020. These variants may have an evolutionary advantage over their ancestral types and could present a large threat to Southeast Asia for the coming winter.

High levels of nonsusceptibility to cotrimoxazole and susceptibility to penicillin suggest that penicillin may be a drug of choice for treatment of invasive pneumococcal disease. Although serotype distribution is diverse, with changes over time and differences between syndromes observed, implementation of use of the currently available 10- or 13-valent vaccines would have a substantial impact on pneumococcal disease in Bangladesh.

IPD contributes substantially to childhood morbidity in rural Bangladesh. S. pneumoniae can cause invasive but nonsevere disease in children, and IPD incidence can be seriously under reported if such cases are overlooked. The emerging high resistance to trimethoprim-sulfamethoxazole should be addressed. Data on serotype distribution would help to guide appropriate pneumococcal conjugate vaccine formulation.