Development of the Pneumococcal Genome Library, a core genome multilocus sequence typing scheme, and a taxonomic life identification number barcoding system to investigate and define pneumococcal population structure.

Investigating the genomic epidemiology of major bacterial pathogens is integral to understanding transmission, evolution, colonization, disease, antimicrobial resistance and vaccine impact. Furthermore, the recent accumulation of large numbers of whole genome sequences for many bacterial species enhances the development of robust genome-wide typing schemes to define the overall bacterial population structure and lineages within it. Using the previously published data, we developed the Pneumococcal Genome Library (PGL), a curated dataset of 30 976 genomes and contextual data for carriage and disease pneumococci recovered between 1916 and 2018 in 82 countries. We leveraged the size and diversity of the PGL to develop a core genome multilocus sequence typing (cgMLST) scheme comprised of 1222 loci. Finally, using multilevel single-linkage clustering, we stratified pneumococci into hierarchical clusters based on allelic similarity thresholds and defined these with a taxonomic life identification number (LIN) barcoding system. The PGL, cgMLST scheme and LIN barcodes represent a high-quality genomic resource and fine-scale clustering approaches for the analysis of pneumococcal populations, which support the genomic epidemiology and surveillance of this leading global pathogen.

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

Changes in the incidence of invasive disease due to Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis during the COVID-19 pandemic in 26 countries and territories in the Invasive Respiratory Infection Surveillance Initiative: a prospective analysis of surveillance data.

BACKGROUND: Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, which are typically transmitted via respiratory droplets, are leading causes of invasive diseases, including bacteraemic pneumonia and meningitis, and of secondary infections subsequent to post-viral respiratory disease. The aim of this study was to investigate the incidence of invasive disease due to these pathogens during the early months of the COVID-19 pandemic. METHODS: In this prospective analysis of surveillance data, laboratories in 26 countries and territories across six continents submitted data on cases of invasive disease due to S pneumoniae, H influenzae, and N meningitidis from Jan 1, 2018, to May, 31, 2020, as part of the Invasive Respiratory Infection Surveillance (IRIS) Initiative. Numbers of weekly cases in 2020 were compared with corresponding data for 2018 and 2019. Data for invasive disease due to Streptococcus agalactiae, a non-respiratory pathogen, were collected from nine laboratories for comparison. The stringency of COVID-19 containment measures was quantified using the Oxford COVID-19 Government Response Tracker. Changes in population movements were assessed using Google COVID-19 Community Mobility Reports. Interrupted time-series modelling quantified changes in the incidence of invasive disease due to S pneumoniae, H influenzae, and N meningitidis in 2020 relative to when containment measures were imposed. FINDINGS: 27 laboratories from 26 countries and territories submitted data to the IRIS Initiative for S pneumoniae (62 837 total cases), 24 laboratories from 24 countries submitted data for H influenzae (7796 total cases), and 21 laboratories from 21 countries submitted data for N meningitidis (5877 total cases). All countries and territories had experienced a significant and sustained reduction in invasive diseases due to S pneumoniae, H influenzae, and N meningitidis in early 2020 (Jan 1 to May 31, 2020), coinciding with the introduction of COVID-19 containment measures in each country. By contrast, no significant changes in the incidence of invasive S agalactiae infections were observed. Similar trends were observed across most countries and territories despite differing stringency in COVID-19 control policies. The incidence of reported S pneumoniae infections decreased by 68% at 4 weeks (incidence rate ratio 0·32 [95% CI 0·27-0·37]) and 82% at 8 weeks (0·18 [0·14-0·23]) following the week in which significant changes in population movements were recorded. INTERPRETATION: The introduction of COVID-19 containment policies and public information campaigns likely reduced transmission of S pneumoniae, H influenzae, and N meningitidis, leading to a significant reduction in life-threatening invasive diseases in many countries worldwide. FUNDING: Wellcome Trust (UK), Robert Koch Institute (Germany), Federal Ministry of Health (Germany), Pfizer, Merck, Health Protection Surveillance Centre (Ireland), SpID-Net project (Ireland), European Centre for Disease Prevention and Control (European Union), Horizon 2020 (European Commission), Ministry of Health (Poland), National Programme of Antibiotic Protection (Poland), Ministry of Science and Higher Education (Poland), Agencia de Salut Pública de Catalunya (Spain), Sant Joan de Deu Foundation (Spain), Knut and Alice Wallenberg Foundation (Sweden), Swedish Research Council (Sweden), Region Stockholm (Sweden), Federal Office of Public Health of Switzerland (Switzerland), and French Public Health Agency (France).

Molecular diagnostic assays for the detection of common bacterial meningitis pathogens: A narrative review.

Bacterial meningitis is a major global cause of morbidity and mortality. Rapid identification of the aetiological agent of meningitis is essential for clinical and public health management and disease prevention given the wide range of pathogens that cause the clinical syndrome and the availability of vaccines that protect against some, but not all, of these. Since microbiological culture is complex, slow, and often impacted by prior antimicrobial treatment of the patient, molecular diagnostic assays have been developed for bacterial detection. Distinguishing between meningitis caused by Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, and Streptococcus agalactiae and identifying their polysaccharide capsules is especially important. Here, we review methods used in the identification of these bacteria, providing an up-to-date account of available assays, allowing clinicians and diagnostic laboratories to make informed decisions about which assays to use.

The global meningitis genome partnership.

Genomic surveillance of bacterial meningitis pathogens is essential for effective disease control globally, enabling identification of emerging and expanding strains and consequent public health interventions. While there has been a rise in the use of whole genome sequencing, this has been driven predominately by a subset of countries with adequate capacity and resources. Global capacity to participate in surveillance needs to be expanded, particularly in low and middle-income countries with high disease burdens. In light of this, the WHO-led collaboration, Defeating Meningitis by 2030 Global Roadmap, has called for the establishment of a Global Meningitis Genome Partnership that links resources for: N. meningitidis (Nm), S. pneumoniae (Sp), H. influenzae (Hi) and S. agalactiae (Sa) to improve worldwide co-ordination of strain identification and tracking. Existing platforms containing relevant genomes include: PubMLST: Nm (31,622), Sp (15,132), Hi (1935), Sa (9026); The Wellcome Sanger Institute: Nm (13,711), Sp (> 24,000), Sa (6200), Hi (1738); and BMGAP: Nm (8785), Hi (2030). A steering group is being established to coordinate the initiative and encourage high-quality data curation. Next steps include: developing guidelines on open-access sharing of genomic data; defining a core set of metadata; and facilitating development of user-friendly interfaces that represent publicly available data.

Prophages and satellite prophages are widespread in Streptococcus and may play a role in pneumococcal pathogenesis.

Prophages (viral genomes integrated within a host bacterial genome) can confer various phenotypic traits to their hosts, such as enhanced pathogenicity. Here we analyse >1300 genomes of 70 different Streptococcus species and identify nearly 800 prophages and satellite prophages (prophages that do not encode their own structural components but rely on the bacterial host and another helper prophage for survival). We show that prophages and satellite prophages are widely distributed among streptococci in a structured manner, and constitute two distinct entities with little effective genetic exchange between them. Cross-species transmission of prophages is not uncommon. Furthermore, a satellite prophage is associated with virulence in a mouse model of Streptococcus pneumoniae infection. Our findings highlight the potential importance of prophages in streptococcal biology and pathogenesis.

Genomic Analyses of >3,100 Nasopharyngeal Pneumococci Revealed Significant Differences Between Pneumococci Recovered in Four Different Geographical Regions.

Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a "supercore" genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population.

Vaccination of Icelandic Children with the 10-Valent Pneumococcal Vaccine Leads to a Significant Herd Effect among Adults in Iceland.

The introduction of pneumococcal conjugate vaccines (PCVs) into childhood vaccination programs has reduced carriage of vaccine serotypes and pneumococcal disease. The 10-valent PCV was introduced in Iceland in 2011. The aim of this study was to determine PCV impact on the prevalence of serotypes, genetic lineages, and antimicrobial-resistant pneumococci isolated from the lower respiratory tract (LRT) of adults. Pneumococci isolated between 2009 and 2017 at the Landspitali University Hospital were included (n = 797). The hospital serves almost three-quarters of the Icelandic population. Isolates were serotyped and tested for antimicrobial susceptibility, and the genome of every other isolate collected between 2009 and 2014 was sequenced (n = 275). Serotypes and multilocus sequence types (STs) were extracted from the genome data. Three study periods were defined, 2009 to 2011 (PreVac), 2012 to 2014 (PostVac-I), and 2015 to 2017 (PostVac-II). The total number of isolates and vaccine-type (VT) pneumococci decreased from PreVac to PostVac-II (n = 314 versus n = 230 [p = 0.002] and n = 170 versus n = 33 [p < 0.001], respectively), but non-vaccine-type (NVT) pneumococci increased among adults 18 to 64 years old (n = 56 versus n = 114 [p = 0.008]). Serotype 19F decreased in the PostVac-II period; these isolates were all multidrug resistant (MDR) and were members of the Taiwan19F-14 PMEN lineage. Serotype 6A decreased among adults ≥65 years old in the PostVac-II period (p = 0.037), while serotype 6C increased (p = 0.021) and most serotype 6C isolates were MDR. Nonencapsulated Streptococcus pneumoniae (NESp) isolates increased among adults 18 to 64 years old in the PostVac-II period, and the majority were MDR (p = 0.028). An overall reduction in the number of LRT samples and pneumococcus-positive cultures and significant changes in the serotype distribution became evident within 4 years, thereby demonstrating a significant herd effect.

Genome Sequencing Reveals a Large and Diverse Repertoire of Antimicrobial Peptides.

Competition among bacterial members of the same ecological niche is mediated by bacteriocins: antimicrobial peptides produced by bacterial species to kill other bacteria. Bacteriocins are also promising candidates for novel antimicrobials. Streptococcus pneumoniae (the "pneumococcus") is a leading cause of morbidity and mortality worldwide and a frequent colonizer of the human nasopharynx. Here, 14 newly discovered bacteriocin gene clusters were identified among >6,200 pneumococcal genomes. The molecular epidemiology of the bacteriocin clusters was investigated using a large global and historical pneumococcal dataset dating from 1916. These analyses revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also found in other streptococcal species. Genomic hotspots for the integration of different bacteriocin gene clusters were discovered. Experimentally, bacteriocin genes were transcriptionally active when the pneumococcus was under stress and when two strains were co-cultured in broth. These findings reveal much more diversity among bacterial defense mechanisms than previously appreciated, which fundamentally broaden our understanding of bacteriocins relative to intraspecies and interspecies nasopharyngeal competition and bacterial population structure.

Effect of Vaccination on Pneumococci Isolated from the Nasopharynx of Healthy Children and the Middle Ear of Children with Otitis Media in Iceland.

Vaccination with pneumococcal conjugate vaccines (PCVs) disrupts the pneumococcal population. Our aim was to determine the impact of the 10-valent PCV on the serotypes, genetic lineages, and antimicrobial susceptibility of pneumococci isolated from children in Iceland. Pneumococci were collected between 2009 and 2017 from the nasopharynges of healthy children attending 15 day care centers and from the middle ears (MEs) of children with acute otitis media from the greater Reykjavik capital area. Isolates were serotyped and tested for antimicrobial susceptibility. Whole-genome sequencing (WGS) was performed on alternate isolates from 2009 to 2014, and serotypes and multilocus sequence types (STs) were extracted from the WGS data. Two study periods were defined: 2009 to 2011 (PreVac) and 2012 to 2017 (PostVac). The overall nasopharyngeal carriage rate was similar between the two periods (67.3% PreVac and 61.5% PostVac, P = 0.090). Vaccine-type (VT) pneumococci decreased and nonvaccine-type (NVT) pneumococci (serotypes 6C, 15A, 15B/C, 21, 22F, 23A, 23B, 35F, and 35B) significantly increased in different age strata post-PCV introduction. The total number of pneumococci recovered from ME samples significantly decreased as did the proportion that were VTs, although NVT pneumococci (6C, 15B/C, 23A, and 23B) increased significantly. Most serotype 6C pneumococci were multidrug resistant (MDR). Serotype 19F was the predominant serotype associated with MEs, and it significantly decreased post-PCV introduction: these isolates were predominantly MDR and of the Taiwan19F-14 PMEN lineage. Overall, the nasopharyngeal carriage rate remained constant and the number of ME-associated pneumococci decreased significantly post-PCV introduction; however, there was a concomitant and statistically significant shift from VTs to NVTs in both collections of pneumococci.

Diverse Streptococcus pneumoniae Strains Drive a Mucosal-Associated Invariant T-Cell Response Through Major Histocompatibility Complex class I-Related Molecule-Dependent and Cytokine-Driven Pathways.

Mucosal-associated invariant T (MAIT) cells represent an innate T-cell population that can recognize ligands generated by the microbial riboflavin synthesis pathway, presented via the major histocompatibility complex class I-related molecule (MR1). Streptococcus pneumoniae is a major human pathogen that is also associated with commensal carriage; thus, host control at the mucosal interface is critical. The recognition of pneumococci by MAIT cells has not been defined nor have the genomics and transcriptomics of the riboflavin operon. We observed robust recognition of pneumococci by MAIT cells, using both MR1-dependent and MR1-independent pathways. The pathway used was dependent on the antigen-presenting cell. The riboflavin operon was highly conserved across a range of 571 pneumococci from 39 countries, dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and pneumococci.

Pneumococcal prophages are diverse, but not without structure or history.

Bacteriophages (phages) infect many bacterial species, but little is known about the diversity of phages among the pneumococcus, a leading global pathogen. The objectives of this study were to determine the prevalence, diversity and molecular epidemiology of prophages (phage DNA integrated within the bacterial genome) among pneumococci isolated over the past 90 years. Nearly 500 pneumococcal genomes were investigated and RNA sequencing was used to explore prophage gene expression. We revealed that every pneumococcal genome contained prophage DNA. 286 full-length/putatively full-length pneumococcal prophages were identified, of which 163 have not previously been reported. Full-length prophages clustered into four major groups and every group dated from the 1930-40 s onward. There was limited evidence for genes shared between prophage clusters. Prophages typically integrated in one of five different sites within the pneumococcal genome. 72% of prophages possessed the virulence genes pblA and/or pblB. Individual prophages and the host pneumococcal genetic lineage were strongly associated and some prophages persisted for many decades. RNA sequencing provided clear evidence of prophage gene expression. Overall, pneumococcal prophages were highly prevalent, demonstrated a structured population, possessed genes associated with virulence, and were expressed under experimental conditions. Pneumococcal prophages are likely to play a more important role in pneumococcal biology and evolution than previously recognised.

Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes.

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

Genomic analyses of pneumococci reveal a wide diversity of bacteriocins - including pneumocyclicin, a novel circular bacteriocin.

BACKGROUND: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the 'pneumococcus'). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure. RESULTS: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the 'bacteriocin/immunity region' of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes. CONCLUSIONS: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci.

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

Defining the estimated core genome of bacterial populations using a Bayesian decision model.

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance.

Streptococcus pneumoniae carriage prevalence in Nepal: evaluation of a method for delayed transport of samples from remote regions and implications for vaccine implementation.

BACKGROUND: Pneumococcal disease is a significant cause of morbidity and mortality in young children in Nepal, and currently available pneumococcal conjugate vaccines offer moderate coverage of invasive disease isolates. METHODS: A prevalence study of children aged 1.5 to 24 months in urban and rural Nepal was conducted. In the urban group, nasopharyngeal swabs (NPS) were transported using silica desiccant packages (SDP) with delayed processing (2 weeks), or skim-milk-tryptone-glucose-glycerin (STGG) with immediate processing (within 8 hours). Pneumococcal nasopharyngeal carriage prevalence, serogroup/type distribution and isolate genotypes (as defined by multilocus sequence typing) were determined. RESULTS: 1101 children were enrolled into the study: 574 in the urban group and 527 in the rural group. Overall carriage prevalence based on culture from specimens transported and stored in STGG was 58.7% (337/574), compared to 40.9% (235/574) in SDP. There was concordance of detection of pneumococcus in 67% of samples. Using the SDP method, pneumococcal carriage prevalence was higher in the rural population (69.2%; 364/526) compared to the urban population (40.9%; 235/574). Serogroup/type distribution varied with geographical location. Over half of the genotypes identified in both the urban and rural pneumococcal populations were novel. CONCLUSION: The combination of delayed culture and transport using SDP underestimates the prevalence of pneumococcal carriage; however, in remote areas, this method could still provide a useful estimate of carriage prevalence and serogroup/type distribution. Vaccine impact is unpredictable in a setting with novel genotypes and limited serotype coverage as described here. Consequently, continued surveillance of pneumococcal isolates from carriage and disease in Nepali children following the planned introduction of pneumococcal conjugate vaccines introduction will be essential.