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IntroductionUnderstanding mother-to-infant transmission of Group B Streptococcus (GBS) is vital to the prevention and control of GBS disease. We investigated the transmission and phylogenetic relationships of mothers colonised by GBS and their infants in a peri-urban setting in The Gambia.MethodsWe collected nasopharyngeal swabs from 35 mother-infant dyads at weekly intervals from birth until six weeks post-partum. GBS was isolated by conventional microbiology techniques. Whole-genome sequencing was performed on GBS isolates from one mother-infant dyad (dyad 17).ResultsWe recovered 85 GBS isolates from the 245 nasopharyngeal swabs. GBS was isolated from 16.33% and 18.37% of sampled mothers and infants, respectively. In 87% of cultured swabs, the culture status of an infant agreed with that of the mother (Kappa p-value ConclusionGBS colonisation in mothers presents a significant risk of colonisation in their infants. We confirm vertical transmission from mother to child in dyad 17, accompanied by within-host diversity.

Background Pediatric bacterial meningitis (PBM) remains an important cause of disease in children in Africa. We describe findings from sentinel site bacterial meningitis surveillance in children, We conducted sentinel pediatric bacterial meningitis surveillance following pneumococcal conjugate vaccine introduction in Benin during 2011–2016. A total of 194 cases were confirmed, predominantly caused by pneumococcus (50.5%). Overall, meningitis and pneumococcal meningitis cases progressively declined during surveillance.

SynopsisObjectiveWe reported a novel tetracycline-resistant gene in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.MethodsWe whole genome sequenced 12,254 pneumococcal isolates from twenty-nine countries on an Illumina HiSeq Sequencer. Serotypes, sequence types and antibiotic resistance were inferred from genomes. Phylogeny was built based on single-nucleotide variants. Temporal changes of spread were reconstructed using a birth-death model.ResultsWe identified tet(S/M) in 131 pneumococcal isolates, 97 (74%) caused invasive pneumococcal diseases among young children (59% HIV-positive, where HIV status was available) in South Africa. A majority of tet(S/M)-positive isolates (129/131) belong to clonal complex (CC)230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sub-lineage that exhibited multidrug-resistance. Using the genomic data and a birth-death model, we detected an unrecognised outbreak of this sub-lineage in South Africa between 2000 and 2004 with an expected secondary infections (R) of ~2.5. R declined to ~1.0 in 2005 and ConclusionsThe prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognised outbreak of CC230 in South Africa prior to ART and PCVs. However, capsular switching in this multidrug-resistant sub-lineage highlighted its potential to continue to cause disease in the post-PCV13 era.

The pneumococcus produces a polysaccharide capsule, encoded by the cps locus, that provides protection against phagocytosis and determines serotype. Nearly 100 serotypes have been identified with new serotypes still being discovered, especially in previously understudied regions. Here we present an analysis of the cps loci of more than 18 000 genomes from the Global Pneumococcal Sequencing (GPS) project with the aim of identifying novel cps loci with the potential to produce previously unrecognized capsule structures. Serotypes were assigned using whole genome sequence data and 66 of the approximately 100 known serotypes were included in the final dataset. Closer examination of each serotype’s sequences identified nine putative novel cps loci (9X, 11X, 16X, 18X1, 18X2, 18X3, 29X, 33X and 36X) found in ~2.6 % of the genomes. The large number and global distribution of GPS genomes provided an unprecedented opportunity to identify novel cps loci and consider their phylogenetic and geographical distribution. Nine putative novel cps loci were identified and examples of each will undergo subsequent structural and immunological analysis. Fil: van Tonder, Andries. Wellcome Genome Campus; Fil: Gladstone, Rebecca A.. Wellcome Genome Campus; Fil: Lo, Stephanie W.. Wellcome Genome Campus; Fil: Nahm, Moon H.. University Of Alabama At Birmingahm. School Of Medicine; Estados Unidos Fil: du Plessis, Mignon. National Institute For Communicable Diseases; Sudáfrica. University of the Witwatersrand; Sudáfrica Fil: Cornick, Jennifer. Malawi Liverpool Wellcome Trust; Malaui Fil: Kwambana Adams, Brenda. Vaccines And Immunity Theme; Gambia Fil: Madhi, Shabir A.. University of the Witwatersrand; Sudáfrica Fil: Hawkins, Paulina A.. University Of Emory. Rollins School Of Public Health; Estados Unidos Fil: Benisty, Rachel. Ben Gurion University of the Negev; Israel Fil: Dagan, Ron. Ben Gurion University of the Negev; Israel Fil: Everett, Dean. University of Edinburgh; Reino Unido Fil: Antonio, Martin. Vaccines And Immunity Theme; Gambia Fil: Klugman, Keith P.. University Of Emory. Rollins School Of Public Health; Estados Unidos Fil: von Gottberg, Anne. National Institute For Communicable Diseases; Sudáfrica. University of the Witwatersrand; Sudáfrica Fil: Breiman, Robert F.. University Of Emory. Rollins School Of Public Health; Estados Unidos. University of Edinburgh; Reino Unido Fil: McGee, Lesley. Centers for Disease Control and Prevention; Estados Unidos Fil: Bentley, Stephen D.. Wellcome Genome Campus; . University Of Emory. Emory College Of Arts And Sciences.; Estados Unidos Fil: Faccone, Diego Francisco. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Background: Staphylococcus aureus is a major human pathogen. Panton-Valentine leukocidin (PVL) is a virulence factor produced by some strains that causes leukocyte lysis and tissue necrosis. PVL-associated S. aureus (PVL-SA) predominantly causes skin and soft-tissue infections (SSTIs) but can also cause invasive infections such as necrotizing pneumonia. It is carried by both community-associated methicillin susceptible S. aureus (CA-MSSA) and methicillin resistant S. aureus (CA-MRSA). This study aims to determine the prevalence of PVL-SA among patients seen at an urban Gambian hospital and associated antibiotic resistance. Methods: Archived clinical S. aureus (70 invasive bacteraemia and 223 non-invasive SSTIs) from 293 patients were retrieved as well as relevant data from clinical records where available. Antibiotic susceptibility was assessed using disc diffusion according to Clinical Laboratory Standards Institute (CLSI) guidelines. Genomic DNA was extracted and the presence of lukF and lukS PVL genes was detected by conventional gel-based PCR. Result: PVL-SA strains accounted for 61.4% (180/293) of S. aureus isolates. PVL prevalence was high in both Gambian bacteraemia and SSTIs S. aureus strains. Antimicrobial resistance was low and included chloramphenicol (4.8%), cefoxitin (2.4%), ciprofloxacin (3.8%), erythromycin (8.9%), gentamicin (5.5%) penicillin (92.5%), tetracycline (41.0%), and sulfamethoxazole-trimethoprim (24.2%). There was no association of PVL with antimicrobial resistance. Conclusion: PVL expression is high among clinical S. aureus strains among Gambian patients. Reporting of PVL-SA clinical infections is necessary to enable the monitoring of the clinical impact of these strains in the population and guide prevention of the spread of virulent PVL-positive CA-MRSA strains. SUMMARY Staphylococcus aureus (S. aureus) is a major human pathogen with several virulence factors. We performed a retrospective analysis to investigate the prevalence of one such virulence factor (PVL) amongst clinical S. aureus samples. We found a high prevalence in our setting but antimicrobial resistance including methicillin resistance was low.

Iron deficiency and iron deficiency anemia are highly prevalent in low-income countries, especially among young children. Hepcidin is the major regulator of systemic iron homeostasis. It controls dietary iron absorption, dictates whether absorbed iron is made available in circulation for erythropoiesis and other iron-demanding processes, and predicts response to oral iron supplementation. Understanding how hepcidin is itself regulated is therefore important, especially in young children. We investigated how changes in iron-related parameters, inflammation and infection status, seasonality, and growth influenced plasma hepcidin and ferritin concentrations during infancy using longitudinal data from two birth cohorts of infants in rural Gambia (n=114 and n=193). This setting is characterized by extreme seasonality, prevalent childhood anemia, undernutrition, and frequent infection. Plasma was collected from infants at birth and at regular intervals, up to 12 months of age. Hepcidin, ferritin and plasma iron concentrations declined markedly during infancy, with reciprocal increases in soluble transferrin receptor and transferrin concentrations, indicating declining iron stores and increasing tissue iron demand. In cross-sectional analyses at 5 and 12 months of age, we identified expected relationships of hepcidin with iron and inflammatory markers, but also observed significant negative associations between hepcidin and antecedent weight gain. Correspondingly, longitudinal fixed effects modeling demonstrated weight gain to be the most notable dynamic predictor of decreasing hepcidin and ferritin through infancy across both cohorts. Infants who grow rapidly in this setting are at particular risk of depletion of iron stores, but since hepcidin concentrations decrease with weight gain, they may also be the most responsive to oral iron interventions.

Background: Invasive pneumococcal disease remains an important health priority owing to increasing disease incidence caused by pneumococci expressing non-vaccine serotypes. We previously defined 621 Global Pneumococcal Sequence Clusters (GPSCs) by analysing 20 027 pneumococcal isolates collected worldwide and from previously published genomic data. In this study, we aimed to investigate the pneumococcal lineages behind the predominant serotypes, the mechanism of serotype replacement in disease, as well as the major pneumococcal lineages contributing to invasive pneumococcal disease in the post-vaccine era and their antibiotic resistant traits. Methods: We whole-genome sequenced 3233 invasive pneumococcal disease isolates from laboratory-based surveillance programmes in Hong Kong (n=78), Israel (n=701), Malawi (n=226), South Africa (n=1351), The Gambia (n=203), and the USA (n=674). The genomes represented pneumococci from before and after pneumococcal conjugate vaccine (PCV) introductions and were from children younger than 3 years. We identified predominant serotypes by prevalence and their major contributing lineages in each country, and assessed any serotype replacement by comparing the incidence rate between the pre-PCV and PCV periods for Israel, South Africa, and the USA. We defined the status of a lineage as vaccine-type GPSC (≥50% 13-valent PCV [PCV13] serotypes) or non-vaccine-type GPSC (>50% non-PCV13 serotypes) on the basis of its initial serotype composition detected in the earliest vaccine period to measure their individual contribution toward serotype replacement in each country. Major pneumococcal lineages in the PCV period were identified by pooled incidence rate using a random effects model. Findings: The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. These serotypes were associated with more than one lineage, except for serotype 5 (GPSC8). Serotype replacement was mainly mediated by expansion of non-vaccine serotypes within vaccine-type GPSCs and, to a lesser extent, by increases in non-vaccine-type GPSCs. A globally spreading lineage, GPSC3, expressing invasive serotypes 8 in South Africa and 33F in the USA and Israel, was the most common lineage causing non-vaccine serotype invasive pneumococcal disease in the PCV13 period. We observed that same prevalent non-vaccine serotypes could be associated with distinctive lineages in different countries, which exhibited dissimilar antibiotic resistance profiles. In non-vaccine serotype isolates, we detected significant increases in the prevalence of resistance to penicillin (52 [21%] of 249 vs 169 [29%] of 575, p=0·0016) and erythromycin (three [1%] of 249 vs 65 [11%] of 575, p=0·0031) in the PCV13 period compared with the pre-PCV period. Interpretation: Globally spreading lineages expressing invasive serotypes have an important role in serotype replacement, and emerging non-vaccine serotypes associated with different pneumococcal lineages in different countries might be explained by local antibiotic-selective pressures. Continued genomic surveillance of the dynamics of the pneumococcal population with increased geographical representation in the post-vaccine period will generate further knowledge for optimising future vaccine design. Funding: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control. Fil: Lo, Stephanie W.. Wellcome Sanger Institute; Reino Unido Fil: Gladstone, Rebecca A.. Wellcome Sanger Institute; Reino Unido Fil: van Tonder, Andries J.. Wellcome Sanger Institute; Reino Unido Fil: Lees, John A.. University Of New York. School Of Medicine; Estados Unidos Fil: du Plessis, Mignon. National Institute For Communicable Diseases; Sudáfrica Fil: Benisty, Rachel. Ben Gurion University of the Negev; Israel Fil: Givon Lavi, Noga. Ben Gurion University of the Negev; Israel Fil: Hawkins, Paulina A.. University of Emory. Rollins School of Public Health; Estados Unidos Fil: Cornick, Jennifer E.. Malawi liverpool wellcome trust; Malaui Fil: Kwambana Adams, Brenda. University College London; Estados Unidos Fil: Law, Pierra Y.. University of Hong Kong; China Fil: Ho, Pak Leung. University of Hong Kong; China Fil: Antonio, Martin. Medical Research Council Unit The Gambia; Gambia Fil: Everett, Dean B.. University of Edinburgh; Reino Unido Fil: Dagan, Ron. Ben Gurion University of the Negev; Israel Fil: Von Gottberg, Anne. National Institute For Communicable Diseases; Sudáfrica Fil: Klugman, Keith P.. University of Emory. Rollins School of Public Health; Estados Unidos Fil: McGee, Lesley. Centers for Disease Control and Prevention; Estados Unidos Fil: Breiman, Robert F.. University of Emory. Rollins School of Public Health; Estados Unidos Fil: Bentley, Stephen D.. Wellcome Sanger Institute; Reino Unido Fil: Brooks, Abdullah W.. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Corso, Alejandra. The Global Pneumococcal Sequencing Consortium; Reino Unido. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina Fil: Davydov, Alexander. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Maguire, Alison. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Pollard, Andrew. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Kiran, Anmol. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Skoczynska, Anna. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Moiane, Benild. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Beall, Bernard. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Sigauque, Betuel. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Aanensen, David. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Lehmann, Deborah. The Global Pneumococcal Sequencing Consortium; Reino Unido Fil: Faccone, Diego Francisco. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development., Author summary Streptococcus pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and otitis media. Despite its inclusion in the most recent pneumococcal conjugate vaccine, PCV13, serotype 3 remains epidemiologically important globally. We investigated the persistence of serotype 3 using whole-genome sequencing data from 301 isolates collected among 24 countries from 1993–2014. Through phylogenetic analysis, we identified three distinct lineages within a single clonal complex, CC180, and found one that has recently emerged and grown in prevalence. We then compared genomic difference among lineages as well as variations in pneumococcal vaccine use among sampled countries. We found that the recently emerged lineage, termed Clade II, has a higher prevalence of antibiotic resistance compared to other lineages, diverse surface protein antigens, and a higher rate of recombination, a process by which bacteria can uptake and incorporate genetic material from its surroundings. Differences in vaccine use among sampled countries did not appear to be associated with the emergence of Clade II. We highlight the need for routine, representative sampling of bacterial isolates from diverse geographic areas and show the utility of genomic data in resolving epidemiological differences within a pathogen population.

After the successful roll out of MenAfriVac, Nigeria has experienced sequential meningitis outbreaks attributed to meningococcus serogroup C (NmC). Zamfara State in North-western Nigeria recently was at the epicentre of the largest NmC outbreak in the 21st Century with 7,140 suspected meningitis cases and 553 deaths reported between December 2016 and May 2017. The overall attack rate was 155 per 100,000 population and children 5–14 years accounted for 47% (3,369/7,140) of suspected cases. The case fatality rate (CFR) among children 5–9 years was 10%, double that reported among adults ≥ 30 years (5%). NmC and pneumococcus accounted for 94% (172/184) and 5% (9/184) of the laboratory-confirmed cases, respectively. The sequenced NmC belonged to the ST-10217 clonal complex (CC). All serotyped pneumococci were PCV10 serotypes. The emergence of NmC ST-10217 CC outbreaks threatens the public health gains made by MenAfriVac, which calls for an urgent strategic action against meningitis outbreaks.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG , which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage ( n = 4) and disease ( n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons ( n = 22) and an in-frame mutation ( n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.

Background There are large data gaps in the epidemiology of diseases caused by Salmonella enterica in West Africa. Regional surveillance of Salmonella infections is necessary, especially with the emergence and spread of multidrug-resistant clones. Methods Data on Salmonella isolated from various clinical specimens from patients from across The Gambia were collected and analyzed retrospectively from 2005 to April 2015. Antibiotic sensitivity testing of Salmonella isolates was performed by disk diffusion method. Serotyping and serogrouping of Salmonella isolates was performed using standard microbiology techniques. Results Two hundred three Salmonella isolates were isolated from 190 patients: 52% (106/203) from blood and 39% (79/203) from stool specimens. Salmonella was also isolated from urine, aspirates, cerebrospinal fluid, wounds, and abscesses. The prevalence of Salmonella in blood cultures was 0.8% (106/13,905). Of the serotyped salmonellae, 14% (21/152) were Salmonella enterica serovar Typhi, whereas 86% (131/152) were serovars other than Typhi (nontyphoidal Salmonella). Of the 102 typed NTS isolates, 40% (41) were Salmonella enterica serovar Typhimurium, 10% (10) were Salmonella enterica serovar Enteritidis, and 3% (3) were Salmonella enterica serovar Arizonae. Overall, 70% (142/203) of the salmonellae were pansusceptible. Multidrug resistance was found in 4% (9/203) of the isolates, 3 of which were Salmonella Enteritidis. Conclusions Salmonellae are associated with a wide spectrum of invasive and noninvasive infections across all ages in The Gambia. There is evidence of multidrug resistance in salmonellae that warrants vigilant monitoring and surveillance.

In this study we provide important evidence to show that changes in the epidemiology of pneumococcal serotype 1 sequence types in The Gambia may be a direct consequence of differences in virulence and increased ability to colonize hosts over time., Streptococcus pneumoniae serotype 1 is one of the leading causes of invasive pneumococcal disease (IPD) in West Africa, with ST618 being the dominant cause of IPD in The Gambia. Recently however, a rare example of clonal replacement was observed, where the ST3081 clone of serotype 1 replaced the predominant ST618 clone as the main cause of IPD. In the current study, we sought to find the reasons for this unusual replacement event. Using whole-genome sequence analysis and clinically relevant models of in vivo infection, we identified distinct genetic and phenotypic characteristics of the emerging ST3081 clone. We show that ST3081 is significantly more virulent than ST618 in models of invasive pneumonia, and is carried at higher densities than ST618 during nasopharyngeal carriage. We also observe sequence type–specific accessory genes and a unique sequence type–specific fixed mutation in the pneumococcal toxin pneumolysin, which is associated with increased hemolytic activity in ST3081 and may contribute to increased virulence in this clone. Our study provides evidence that, within the same serotype 1 clonal complex, biological properties differ significantly from one clone to another in terms of virulence and host invasiveness, and that these differences may be the result of key genetic differences within the genome.

There is growing concern that interventions that alter microbial ecology can adversely affect health. We characterised the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal carriage and the bacterial component of the nasopharyngeal microbiome during infancy. Newborns were recruited into three groups as follows: Group1 (n = 33) was the control group and comprised infants who received PCV7 after 6 months and came from unvaccinated communities. Group 2 (n = 30) came from unvaccinated communities and Group 3 (n = 39) came from vaccinated communities. Both group 2 and 3 received PCV7 at 2, 3 and 4 months. Culture and 16 S rRNA gene sequencing were performed on nasopharyngeal specimens collected at regular intervals from infants. Nasopharyngeal carriage of PCV7 serotypes in Group 1 was significantly higher than in Group 2 and 3 (p α-diversity (p = 0.48) across the groups. Immediate replacement of pneumococcal vaccine serotypes with non-vaccine serotypes may mitigate the impact of PCV7 on nasopharyngeal bacterial community structure and ecology.

Enteroaggregative Escherichia coli (EAEC) cause acute and persistent diarrhea, mostly in children worldwide. Outbreaks of diarrhea caused by EAEC have been described, including a large outbreak caused by a Shiga toxin expressing strain. This study investigated the association of EAEC virulence factors with diarrhea in children less than 5 years. We characterized 428 EAEC strains isolated from stool samples obtained from moderate-to-severe diarrhea cases (157) and healthy controls (217) children aged 0–59 months recruited over 3 years as part of the Global Enteric Multicenter Study (GEMS) in The Gambia. Four sets of multiplex polymerase chain reaction were applied to detect 21 EAEC-virulence genes from confirmed EAEC strains that target pCVD432 (aatA) and AAIC (aaiC). In addition, Kirby-Bauer disc diffusion antimicrobial susceptibility testing was performed on 88 EAEC strains following Clinical Laboratory Standard Institute guidelines. We observed that the plasmid-encoded enterotoxin [odds ratio (OR): 6.9, 95% confidence interval (CI): 2.06–29.20, P < 0.001], aggregative adherence fimbriae/I fimbriae (aggA) [OR: 2.2, 95% CI: 1.16–4.29, P = 0.008], and hexosyltransferase (capU) [OR: 1.9, 95% CI 1.02–3.51, P = 0.028] were associated with moderate-to-severe diarrhea among children < 12 months old but not in the older age strata (> 12 months). Our data suggest that some EAEC-virulent factors have age-specific associations with moderate-to-severe diarrhea in infants. Furthermore, our study showed that 85% and 72% of EAEC strains tested were resistant to sulphamethoxazole-trimethoprim and ampicillin, respectively. Sulphamethoxazole-trimethoprim and ampicillin are among the first-line antibiotics used for the treatment of diarrhea in The Gambia.

Rotavirus is the leading cause of diarrhea in children5 years of age. In light of the implementation of rotavirus vaccines of limited valency, it is important to characterize the genotypic diversity of circulating rotavirus in sub-Saharan Africa.We collected stool samples from children 0-59 months of age who presented at the health centres as cases with moderate-to-severe diarrhea in the Upper River Region of The Gambia. Stool samples were also collected from age, sex and area-matched healthy controls. All stool samples were assayed for rotavirus antigens by enzyme-linked immunosorbent assay and genotyping was done using reverse transcriptase polymerase chain reaction.We enrolled 1029 cases and 1569 controls during the 3-year study period (2008-2010). The detection rate of rotavirus among the cases was 20% (204/1029) and 3% (42/1569) among controls. At least 18 genotypes were found and the predominant genotypes were G2P[6] (28%), G1P[8] (26%) and G1P[10] (10%). The rare identified genotypes (1%) were G2P[14], G8P[6], G9P[6] and G4P[10]. There was also a strong positive association between rotavirus infection and the dry season (odds ratio: 9.83, 95% confidence interval: 6.18-15.63, P0.001). A significant increase in the odds of rotavirus and G1P[8] detection with the use of untreated water and the presence of cats, rodents and cows in the child's residence was also found.This study provides important baseline data for the genotypes circulating before vaccine implementation. The wide diversity of genotypes circulating in The Gambia implies the need for vigilant effectiveness surveillance following the implementation of RotaTeq in August 2013.

Background In West Africa, penicillin, macrolide and lincosamide resistance among beta-haemolytic streptococci (BHS) isolates has rarely been described. However, such data are critical to detect and track the emergence of antibiotic resistance. Methods Beta-haemolytic streptococci were cultured from clinical specimens from patients attending the clinic at the Medical Research Council Unit The Gambia (n = 217) and kept at −70 °C. Of these, 186 were revived and tested for penicillin susceptibility by disc diffusion and E-test methods, and the D-test for determination of constitutive and inducible macrolide–lincosamide (MLSB) resistance phenotypes. Results The majority of BHS isolates from infections were group A streptococci (GAS) (126/186, 67.7%). Of these, 16% were from invasive disease (30/186). Other BHS isolated included lancefield groups B (19, 10.2%); C (9/186, 4.8%), D (3/186, 1.6%), F (5/186, 2.7%), G (16/186, 8.6%) and non-typeable (8/186, 4.3%). Prevalence of BHS isolated from blood cultures ranges from 0% (2005) to 0.5% (2010). Most (85, 45.7%) of the isolates were from wound infections. Of the 186 BHS isolates, none was resistant to penicillin and 14 (6.1%) were resistant to erythromycin. Of these, 8 (4.3%) demonstrated constitutive MLSB resistance, and 5 (2.7%) were inducible MLSB resistant. All the inducible MLSB isolates were GAS, and majority of the constitutive MLSB isolates (6/8, 75.0%) were non-GAS. Conclusions Beta-haemolytic streptococci, predominantly GAS are associated with a wide range of infections in The Gambia. It is reassuring that macrolide and lincosamide resistance is relatively low. However, monitoring of MLSB resistance is necessary with the global spread of resistant BHS strains.

BACKGROUND: Drug-resistant tuberculosis (TB) is a global public health problem. Adequate management requires baseline drug-resistance prevalence data. In West Africa, due to a poor laboratory infrastructure and inadequate capacity, such data are scarce. Therefore, the true extent of drug-resistant TB was hitherto undetermined. In 2008, a new research network, the West African Network of Excellence for Tuberculosis, AIDS and Malaria (WANETAM), was founded, comprising nine study sites from eight West African countries (Burkina Faso, The Gambia, Ghana, Guinea-Bissau, Mali, Nigeria, Senegal and Togo). The goal was to establish Good Clinical Laboratory Practice (GCLP) principles and build capacity in standardised smear microscopy and mycobacterial culture across partnering laboratories to generate the first comprehensive West African drug-resistance data. METHODS: Following GCLP and laboratory training sessions, TB isolates were collected at sentinel referral sites between 2009-2013 and tested for first- and second-line drug resistance. RESULTS: From the analysis of 974 isolates, an unexpectedly high prevalence of multi-drug-resistant (MDR) strains was found in new (6 %) and retreatment patients (35 %) across all sentinel sites, with the highest prevalence amongst retreatment patients in Bamako, Mali (59 %) and the two Nigerian sites in Ibadan and Lagos (39 % and 66 %). In Lagos, MDR is already spreading actively amongst 32 % of new patients. Pre-extensively drug-resistant (pre-XDR) isolates are present in all sites, with Ghana showing the highest proportion (35 % of MDR). In Ghana and Togo, pre-XDR isolates are circulating amongst new patients. CONCLUSIONS: West African drug-resistance prevalence poses a previously underestimated, yet serious public health threat, and our estimates obtained differ significantly from previous World Health Organisation (WHO) estimates. Therefore, our data are reshaping current concepts and are essential in informing WHO and public health strategists to implement urgently needed surveillance and control interventions in West Africa.

Staphylococcus aureus is an important pathogen of humans and animals. We genome sequenced 90 S. aureus isolates from The Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates, and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys ( Chlorocebus sabaeus ) in The Gambia over different time scales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by the loss of phage-carrying genes that are known to play an important role in human colonization. We also report recent anthroponotic transmission of the well-characterized human lineages sequence type 6 (ST6) and ST15 to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact, there might be an increased risk of additional interspecies exchanges of potential pathogens. IMPORTANCE The population structures of Staphylococcus aureus in humans and monkeys in sub-Saharan Africa have been previously described using multilocus sequence typing (MLST). However, these data lack the power to accurately infer details regarding the origin and maintenance of new adaptive lineages. Here, we describe the use of whole-genome sequencing to detect transmission of S. aureus between humans and nonhuman primates and to document the genetic changes accompanying host adaptation. We note that human-to-monkey switches tend to be more common than the reverse and that a novel monkey-associated clade is likely to have emerged from such a switch approximately 2,700 years ago. Moreover, analysis of the accessory genome provides important clues as to the genetic changes underpinning host adaptation and, in particular, shows that human-to-monkey switches tend to be associated with the loss of genes known to confer adaptation to the human host.

Background An outbreak of pneumococcal meningitis among non-infant children and adults occurred in the Brong-Ahafo region of Ghana between December 2015 and April 2016 despite the recent nationwide implementation of a vaccination programme for infants with the 13-valent pneumococcal conjugate vaccine (PCV13). Methods Cerebrospinal fluid (CSF) specimens were collected from patients with suspected meningitis in the Brong-Ahafo region. CSF specimens were subjected to Gram staining, culture and rapid antigen testing. Quantitative PCR was performed to identify pneumococcus, meningococcus and Haemophilus influenzae. Latex agglutination and molecular serotyping were performed on samples. Antibiogram and whole genome sequencing were performed on pneumococcal isolates. Results Eight hundred eighty six patients were reported with suspected meningitis in the Brong-Ahafo region during the period of the outbreak. In the epicenter district, the prevalence was as high as 363 suspected cases per 100,000 people. Over 95 % of suspected cases occurred in non-infant children and adults, with a median age of 20 years. Bacterial meningitis was confirmed in just under a quarter of CSF specimens tested. Pneumococcus, meningococcus and Group B Streptococcus accounted for 77 %, 22 % and 1 % of confirmed cases respectively. The vast majority of serotyped pneumococci (80 %) belonged to serotype 1. Most of the pneumococcal isolates tested were susceptible to a broad range of antibiotics, with the exception of two pneumococcal serotype 1 strains that were resistant to both penicillin and trimethoprim-sulfamethoxazole. All sequenced pneumococcal serotype 1 strains belong to Sequence Type (ST) 303 in the hypervirulent ST217 clonal complex. Conclusion The occurrence of a pneumococcal serotype 1 meningitis outbreak three years after the introduction of PCV13 is alarming and calls for strengthening of meningitis surveillance and a re-evaluation of the current vaccination programme in high risk countries. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1914-3) contains supplementary material, which is available to authorized users.

BACKGROUND: In West Africa, the carriage of Group B Streptococcus (GBS), among infants is poorly characterised. We investigated co-carriage of GBS with other respiratory pathogens in the infants' nasopharynx in The Gambia. METHODS: We assessed the carriage, serotypes and antibiotic susceptibility of Beta-haemolytic Streptococci (BHS) groups A-G; along with the carriage of Streptococcus pneumoniae; Haemophilus influenzae; Staphylococcus aureus and Moraxella catarrhalis in 1200 two-month old infants. RESULTS: The BHS prevalence was 20.0 % and GBS dominated (13.8 %), particularly serotypes V and II; serotype V being negatively associated with H. Influenzae carriage (OR 0.41 [95 % CI: 0.18-0.93], p = 0.033). Although co-colonization of GBS and other BHS was not seen, colonization with GBS was positively associated with S. aureus (OR 1.89 [95 % CI: 1.33-2.69], P < 0.001) and negatively associated with S. pneumoniae (OR 0.47 [95 % CI: 0.33-0.67], p < 0.001) and M. catarrhalis (OR 0.61 [95 % CI: 0.40-0.92], p = 0.017). ≥ 89 % of GBS isolates were susceptible to most antibiotics tested, except for tetracycline resistance, which was 89 %. CONCLUSION: This study provides baseline data on the carriage of GBS in two month old infants from West Africa. The dominant serotypes of GBS in this setting are serotypes V and II. This may be important for future GBS vaccine development for the West African sub-region.

Summary Background Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). Methods GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. Findings We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2–96·0) at the population level, compared with 51·5% (48·0–55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6–80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. Interpretation A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. Funding Bill & Melinda Gates Foundation.

Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease.We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis.The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043).Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes.BillMelinda Gates Foundation Next Generation Molecular Diagnostics Project.

New research implicates a dysfunctional gut microbiome in the etiology of severe childhood malnutrition and confirms a role for antibiotics in its treatment.

Approximately 46% and 32% of deaths among children under five globally occur in sub-Saharan Africa and South Asia, respectively. Over 80% of the 4.2 million child deaths in Africa are caused by infectious diseases, sharply contrasted to Europe where 39% of the 0.15 million child deaths are attributable to infectious diseases (Fig. 5.1) (Black et al. 2010). Hence, despite the remarkable public health advancements in hygiene, sanitation, antimicrobial drugs and vaccine strategies of the twenty-first century, the burden of infectious diseases remains unacceptably high in the developing world.

Background Frozen storage often precedes metagenomic analysis of biological samples; however, the freezing process can have adverse effects on microbial composition. The effect of freezing on the detection of bacteria inhabiting the infant nasopharynx, a major reservoir of bacterial pathogens, was investigated. Methods 16S ribosomal RNA (rRNA) gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of nasopharyngeal (NP) swabs from twelve Gambian infants was employed. NP swabs were analysed within hours of collection and then after 30 days of storage at -70°C. Results There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected. Nevertheless, the mean number of OTUs decreased after frozen storage and the relative abundance for 72% of the OTUs changed by less than 0.5% after deep frozen storage. There were differences in the odds of detection and relative abundance of OTUs matched with Moraxella sp., Haemophilus sp./Burkholderia sp., and Pseudomonas sp. A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage. Conclusions Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked. Moreover, genetic factors such as sex may influence the integrity of nucleic acids during the freezing process.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein modifications over the course of infection have been associated with coreceptor switching and antibody neutralization resistance, but the effect of the changes on replication and host cell receptor usage remains unclear. To examine this question, unique early- and chronic-stage infection envelope V1-toV5 (V1-V5) segments from eight HIV-1 subtype A-infected subjects were incorporated into an isogenic background to construct replication-competent recombinant viruses. In all subjects, viruses with chronic-infection V1-V5 segments showed greater replication capacity than those with early-infection V1-V5 domains in cell lines with high levels of both the CD4 and the CCR5 receptors. Viruses with chronic-infection V1-V5s demonstrated a significantly increased ability to replicate in cells with low CCR5 receptor levels and greater resistance to CCR5 receptor and fusion inhibitors compared to those with early-infection V1-V5 segments. These properties were associated with sequence changes in the envelope V1-V3 segments. Viruses with the envelope segments from the two infection time points showed no significant difference in their ability to infect cells with low CD4 receptor densities, in their sensitivity to soluble CD4, or in their replication capacity in monocyte-derived macrophages. Our results suggest that envelope changes, primarily in the V1-V3 domains, increase both the ability to use the CCR5 receptor and fusion kinetics. Thus, envelope modifications over time within a host potentially enhance replication capacity.

Background Chrysomya spp are common blowflies in Africa, Asia and parts of South America and some species can reproduce in prodigious numbers in pit latrines. Because of their strong association with human feces and their synanthropic nature, we examined whether these flies are likely to be vectors of diarrheal pathogens. Methodology/Principal Findings Flies were sampled using exit traps placed over the drop holes of latrines in Gambian villages. Odor-baited fly traps were used to determine the relative attractiveness of different breeding and feeding media. The presence of bacteria on flies was confirmed by culture and bacterial DNA identified using PCR. A median of 7.00 flies/latrine/day (IQR = 0.0–25.25) was collected, of which 95% were Chrysomya spp, and of these nearly all were Chrysomya putoria (99%). More flies were collected from traps with feces from young children (median = 3.0, IQR = 1.75–10.75) and dogs (median = 1.50, IQR = 0.0–13.25) than from herbivores (median = 0.0, IQR = 0.0–0.0; goat, horse, cow and calf; p, Author Summary While it is well recognized that the house fly can transmit enteric pathogens, here we show the common African latrine fly, Chrysomya putoria, is likely to be an important vector of these pathogens, since an average latrine can produce 100,000 latrine flies each year. Our behavioral studies of flies in The Gambia show that latrine flies are attracted strongly to human feces, raw beef and fish, providing a clear mechanism for faecal pathogens to be transferred from faeces to food. We used PCR techniques to demonstrate that these flies are carrying Shigella, Salmonella and E. coli, all important causes of diarrhea. Moreover our culture work shows that these pathogens are viable. Latrine flies are likely to be important vectors of diarrheal disease, although further research is required to determine what proportion of infections are due to this fly.

Background Although Haemophilus influenzae type b (Hib), Staphylococcus aureus and Moraxella catarrhalis are important causes of invasive and mucosal bacterial disease among children, co-carriage with Streptococcus pneumoniae during infancy has not been determined in West Africa. Methods Species specific PCR was applied to detect each microbe using purified genomic DNA from 498 nasopharyngeal (NP) swabs collected from 30 Gambian neonates every two weeks from 0 to 6 months and bi-monthly up to 12 months. Results All infants carried S. pneumoniae, H. influenzae and M. catarrhalis at several time points during infancy. S.pneumoniae co-colonized the infant nasopharynx with at least one other pathogen nine out of ten times. There was early colonization of the newborns and neonates, the average times to first detection were 5, 7, 3 and 14 weeks for S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus respectively. The prevalence of S. pneumoniae, H. influenzae and M. catarrhalis increased among the neonates and exceeded 80% by 13, 15 and 23 weeks respectively. In contrast, the prevalence of S. aureus decreased from 50% among the newborns to 20% amongst nine-week old neonates. S. pneumoniae appeared to have a strong positive association with H. influenzae (OR 5.03; 95% CI 3.02, 8.39; p < 0.01) and M. catarrhalis (OR 2.20; 95% CI 1.29; p < 0.01) but it was negatively associated with S. aureus (OR 0.53; 95% CI 0.30, 0.94; p = 0.03). Conclusion This study shows early acquisition and high co-carriage of three important respiratory pathogens with S. pneumoniae in the nasopharyngeal mucosa among Gambian neonates and infants. This has important potential implications for the aetiology of respiratory polymicrobial infections, biofilm formation and vaccine strategies.