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BackgroundThe introduction in many countries of conjugate vaccines against Haemophilus influenzae type-b, Streptococcus pneumoniae, and Neisseria meningitidis has led to significant reductions in acute bacterial meningitis (ABM) in children. However, recent population-based data on ABM in sub-Saharan Africa are limited.MethodsPopulation-based surveillance for meningitis was carried out in a rural area of The Gambia under demographic surveillance from 2008 to 2017, using standardised criteria for referral, diagnosis and investigation. We calculated incidence using population denominators.ResultsWe diagnosed 1,599 patients with suspected meningitis and collected cerebrospinal fluid (n=1,121) and/or blood (n=1,070) from 1,427 (88%) of cases. We detected 169 cases of ABM, 209 cases of non-bacterial meningitis and 1,049 cases of clinically suspected meningitis. The estimated average annual incidence of ABM was high at 145 per 100,000 population in the Streptococcus pneumoniae (n=44), Neisseria meningitidis (n=42), and Gram-negative coliform bacteria (n=26). Eighteen of 22 cases caused by pneumococcal serotypes included in PCV13 occurred prior to vaccine introduction and four afterwards. The overall case fatality ratio for ABM was 29% (49/169) and highest in the ConclusionsGambian children continue to experience substantial morbidity and mortality associated with suspected meningitis, especially acute bacterial meningitis. Such severely ill children in sub-Saharan Africa require improved diagnostics and clinical care.Summary of the articles main pointPopulation-based surveillance in a health demographic surveillance area in Gambia showed a high incidence and mortality in clinically suspected, acute-bacterial, and non-bacterial meningitis among children 14-years of age. Findings revealed potential gaps in the diagnosis of meningitis in The Gambia requiring urgent attention.

Staphylococcus aureus bacteremia is a substantial cause of childhood disease and death, but few studies have described its epidemiology in developing countries. Using a population-based surveillance system for pneumonia, sepsis, and meningitis, we estimated S. aureus bacteremia incidence and the case-fatality ratio in children

Invasive non-typhoidal Salmonella (iNTS) disease continues to be a significant public health problem in sub-Saharan Africa. Common clinical misdiagnosis, antimicrobial resistance, high case fatality and lack of a vaccine make iNTS a priority for global health research. Using whole genome sequence analysis of 164 invasive Salmonella isolates obtained through population-based surveillance between 2008 and 2016, we conducted genomic analysis of the serovars causing invasive Salmonella diseases in rural Gambia. The incidence of iNTS varied over time. The proportion of atypical serovars causing disease increased over time from 40 to 65 % compared to the typical serovars Enteritidis and Typhimurium that decreased from 30 to 12 %. Overall iNTS case fatality was 10%, but case fatality associated with atypical iNTS alone was 10 %. Genetic virulence factors were identified in 14/70 (20 %) typical serovars and 45/68 (66 %) of the atypical serovars and were associated with: invasion, proliferation and/or translocation (Clade A); and host colonization and immune modulation (Clade G). Among Enteritidis isolates, 33/40 were resistant to four or more of the antimicrobials tested, except ciprofloxacin, to which all isolates were susceptible. Resistance was low in Typhimurium isolates, but all 16 isolates were resistant to gentamicin. The increase in incidence and proportion of iNTS disease caused by atypical serovars is concerning. The increased proportion of atypical serovars and the high associated case fatality may be related to acquisition of specific genetic virulence factors. These factors may provide a selective advantage to the atypical serovars. Investigations should be conducted elsewhere in Africa to identify potential changes in the distribution of iNTS serovars and the extent of these virulence elements.

Little is known about the genomic diversity of Escherichia coli in healthy children from sub-Saharan Africa, even though this is pertinent to understanding bacterial evolution and ecology and their role in infection. We isolated and whole-genome sequenced up to five colonies of faecal E. coli from 66 asymptomatic children aged three-to-five years in rural Gambia (n = 88 isolates from 21 positive stools). We identified 56 genotypes, with an average of 2.7 genotypes per host. These were spread over 37 seven-allele sequence types and the E. coli phylogroups A, B1, B2, C, D, E, F and Escherichia cryptic clade I. Immigration events accounted for three-quarters of the diversity within our study population, while one-quarter of variants appeared to have arisen from within-host evolution. Several isolates encode putative virulence factors commonly found in Enteropathogenic and Enteroaggregative E. coli, and 53% of the isolates encode resistance to three or more classes of antimicrobials. Thus, resident E. coli in these children may constitute reservoirs of virulence- and resistance-associated genes. Moreover, several study strains were closely related to isolates that caused disease in humans or originated from livestock. Our results suggest that within-host evolution plays a minor role in the generation of diversity compared to independent immigration and the establishment of strains among our study population. Also, this study adds significantly to the number of commensal E. coli genomes, a group that has been traditionally underrepresented in the sequencing of this species.

Little is known about the genomic diversity of Escherichia coli in healthy children from sub-Saharan Africa, even though this is pertinent to understanding bacterial evolution and ecology and their role in infection. We isolated and whole-genome sequenced up to five colonies of faecal E. coli from 66 asymptomatic children aged three-to-five years in rural Gambia (n=88 isolates from 21 positive stools). We identified 56 genotypes, with an average of 2.7 genotypes per host. These were spread over 37 seven-allele sequence types and the E. coli phylogroups A, B1, B2, C, D, E, F and Escherichia cryptic clade I. Immigration events accounted for three-quarters of the diversity within our study population, while one-quarter of variants appeared to have arisen from within-host evolution. Several study strains were closely related to isolates that caused disease in humans or originated from livestock. Our results suggest that within-host evolution plays a minor role in the generation of diversity than independent immigration and the establishment of strains among our study population. Also, this study adds significantly to the number of commensal E. coli genomes, a group that has been traditionally underrepresented in the sequencing of this species.

Summary Background Little information is available about the effect of pneumococcal conjugate vaccines (PCVs) in low-income countries. We measured the effect of these vaccines on invasive pneumococcal disease in The Gambia where the 7-valent vaccine (PCV7) was introduced in August, 2009, followed by the 13-valent vaccine (PCV13) in May, 2011. Methods We conducted population-based surveillance for invasive pneumococcal disease in individuals aged 2 months and older who were residents of the Basse Health and Demographic Surveillance System (BHDSS) in the Upper River Region, The Gambia, using standardised criteria to identify and investigate patients. Surveillance was done between May, 2008, and December, 2014. We compared the incidence of invasive pneumococcal disease between baseline (May 12, 2008–May 11, 2010) and after the introduction of PCV13 (Jan 1, 2013–Dec 31, 2014), adjusting for changes in case ascertainment over time. Findings We investigated 14 650 patients, in whom we identified 320 cases of invasive pneumococcal disease. Compared with baseline, after the introduction of the PCV programme, the incidence of invasive pneumococcal disease decreased by 55% (95% CI 30–71) in the 2–23 months age group, from 253 to 113 per 100 000 population. This decrease was due to an 82% (95% CI 64–91) reduction in serotypes covered by the PCV13 vaccine. In the 2–4 years age group, the incidence of invasive pneumococcal disease decreased by 56% (95% CI 25–75), from 113 to 49 cases per 100 000, with a 68% (95% CI 39–83) reduction in PCV13 serotypes. The incidence of non-PCV13 serotypes in children aged 2–59 months increased by 47% (−21 to 275) from 28 to 41 per 100 000, with a broad range of serotypes. The incidence of non-pneumococcal bacteraemia varied little over time. Interpretation The Gambian PCV programme reduced the incidence of invasive pneumococcal disease in children aged 2–59 months by around 55%. Further surveillance is needed to ascertain the maximum effect of the vaccine in the 2–4 years and older age groups, and to monitor serotype replacement. Low-income and middle-income countries that introduce PCV13 can expect substantial reductions in invasive pneumococcal disease. Funding GAVI's Pneumococcal vaccines Accelerated Development and Introduction Plan (PneumoADIP), Bill & Melinda Gates Foundation, and the UK Medical Research Council.

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.

Objective To evaluate pneumococcal colonisation before and after the introduction of pneumococcal conjugate vaccine (PCV) in eastern Gambia. Methods Population-based cross-sectional survey of pneumococcal carriage between May and August 2009 before the introduction of PCV into the Expanded Program on Immunization. Nasopharyngeal swabs were collected from all household members, but in selected households, only children aged 6–10 years were swabbed. This age group participated in an earlier trial of a nine-valent PCV between 2000 and 2004. Results The prevalence of nasopharyngeal pneumococcal carriage in 2933 individuals was 72.0% in underfives (N = 515), 41.6% in children aged 5–17 (N = 1508) and 13.0% in adults ≥18 (N = 910) years. The age-specific prevalence of serotypes included in PCV7, PCV10 and PCV13 was 24.7%, 26.6% and 46.8% among children

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.

Background Cryptosporidium is a major pathogen associated with diarrheal disease in young children. We studied Cryptosporidium diarrhea in children enrolled in the Global Enteric Multicenter Study (GEMS) in rural Gambia. Methods We recruited children, Author summary Cryptosporidium, a protozoan parasite, is one of the most common diarrheal pathogens in young children living in developing countries. We describe the prevalence and risk factors for Cryptosporidium diarrhea in under-five children in The Gambia using data from the Global Enteric Study (GEMS), conducted in seven developing countries in Asia and Africa (2008–2012). We enrolled 1938 diarrhea cases and 2969 matched controls. We found that 12.0% of diarrhea cases and 4.8% controls were positive for Cryptosporidium. Most (85.7%) Cryptosporidium diarrhea cases were aged 6–23 months, and most (81.4%) occurred during the rainy season. Cryptosporidium hominis was the predominant species (82.6%). We found that consumption of stored drinking water and animals (cow, cat, rodents) living in the compound are potential risk factors for Cryptosporidium diarrhea. Improved drinking water storage may reduce the burden of Cryptosporidium diarrhea in a resource poor hygienic and sanitation setting.

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.

In 2012, an outbreak of Neisseria meningitidis serogroup W135 occurred in The Gambia. The attack rate was highest among young children. The associated risk factors were male sex, contact with meningitis patients, and difficult breathing. Enhanced surveillance facilitates early epidemic detection, and multiserogroup conjugate vaccine could reduce meningococcal epidemics in The Gambia.

The family Polyomaviridae is comprised of circular double-stranded DNA viruses, several of which are associated with diseases, including cancer, in immunocompromised patients. Here we describe a novel polyomavirus recovered from the fecal microbiota of a child in Malawi, provisionally named STL polyomavirus (STLPyV). We detected STLPyV in clinical stool specimens from USA and The Gambia at up to 1% frequency. Complete genome comparisons of two STLPyV strains demonstrated 5.2% nucleotide divergence. Alternative splicing of the STLPyV early region yielded a unique form of T antigen, which we named 229T, in addition to the expected large and small T antigens. STLPyV has a mosaic genome and shares an ancestral recombinant origin with MWPyV. The discovery of STLPyV highlights a novel alternative splicing strategy and advances our understanding of the complex evolutionary history of polyomaviruses.

BACKGROUND: Streptococcus pneumoniae serotype 5 is among the most common serotypes causing invasive pneumococcal disease (IPD) in The Gambia. We anticipate that introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13) into routine vaccination in The Gambia will reduce serotype 5 IPD. However, the emergence of new clones that have altered their genetic repertoire through capsular switching or genetic recombination after vaccination with PCV-13 poses a threat to this public health effort. In order to monitor for potential genetic changes post-PCV-13 vaccination, we established the baseline population structure, epidemiology, and antibiotic resistance patterns of serotype 5 before the introduction of PCV-13. METHODS: Fifty-five invasive S. pneumoniae serotype 5 isolates were recovered from January 2009 to August 2011 in a population-based study in the Upper River Region of The Gambia. Serotyping was done by latex agglutination and confirmed by serotype-specific Polymerase Chain Reaction (PCR). Genotyping was undertaken using Multilocus Sequence Typing (MLST). Antimicrobial sensitivity was done using disc diffusion. Contingency table analyses were conducted using Pearson's Chi(2) and Fisher's exact test. Clustering was performed using Bionumerics version 6.5. RESULTS: MLST resolved S. pneumoniae serotype 5 isolates into 3 sequence types (ST), namely ST 289(6/55), ST 3339(19/55) and ST 3404(30/55). ST 289 was identified as the major clonal complex. ST 3339, the prevalent genotype in 2009 [84.6% (11/13)], was replaced by ST 3404 [70.4% (19/27)] in 2010 as the dominant ST. Interestingly, ST 3404 showed lower resistance to tetracycline and oxacillin (P

Sixty-two invasive non-typhoidal Salmonella (NTS) isolates from children aged 2–29 months in rural Gambia were examined for serovar prevalence and antimicrobial susceptibility, and characterized using multilocus sequence typing (MLST) of seven genes, aroC, dnaN, hemD, hisD, purE, sucA and thrA. Salmonella enterica serovar Enteritidis was the most common serovar (80.6 %), followed by S. enterica serovar Typhimurium (8.0 %). Thirty-three per cent of the isolates were resistant to all eight antimicrobials tested, including ampicillin (74.2 %), cotrimoxazole (64.5 %) and tetracycline (63 %). A total of 40.3 % of the NTS cases had an initial clinical diagnosis of malaria, whilst 27.3 % had a diagnosis of clinical pneumonia and 18 % had a diagnosis of septicaemia. MLST of NTS resulted in ten different sequence types (STs), of which five were novel, representing five different NTS serovars. In general, STs were restricted to the same serovar. One type (ST11) encompassed 80.6 % of the NTSs. A new NTS serovar named S. enterica serovar Dingiri was discovered. S. Dingiri was isolated from a 6-month-old male with an initial clinical diagnosis of malaria but a final clinical diagnosis of anaemia and septicaemia. S. Dingiri, which possesses an antigenic formula of 17:z:1,6, was sensitive to ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, cotrimoxazole and tetracycline but resistant to gentamicin, and was ST338.

Introduction: Healthcare-associated infections (HAIs) are better documented in developed than in developing countries. There are emerging reports regarding the high frequency of HAIs in developing countries. We aimed to report an outbreak of an HAI caused by Serratia liquefaciens at a rural health center in The Gambia. Methodology: Following an abrupt increase in the isolation of S. liquefaciens in clinical samples, laboratory and clinical consumables, as well as staff, were screened for contamination with S. liquefaciens. Conventional microbiological techniques and biochemical identification tests were used. A phenotypic typing was achieved using the Kirby-Bauer antibiotic susceptibility method. Strategies to control the outbreak were implemented. Results: A total of 794 samples were processed during the outbreak; 44 (6%) grew S. liquefaciens. Five (25%) of the 20 suspected contaminated materials (hospital consumables and equipment) screened yielded growth of the organism. The primary source of the outbreak was hospital consumables. Three (7%) of the 44 infected children died with no other known cause than S. liquefaciens infection. Ninety-nine percent similarity of the antibiogram phenotypic typing suggests the isolates were from the same clonal origin. The outbreak was successfully controlled after the removal and sterilization of the respective contaminated fluids and equipment. Conclusions: This HAI was caused by poor practice in the preparation of medications for nebulization and intravenous infusion, hygiene practices, and a lack of awareness among staff about infection control. We recommend further studies to delineate the role played by HAIs in the developing world.

BACKGROUND: In 1997, The Gambia became the first African country to introduce conjugate Haemophilus influenzae type b (Hib) vaccine with good disease control through to 2010. METHODS: Culture-based surveillance for invasive bacterial disease in eastern Gambia, specifically the Basse Health and Demographic Surveillance System (BHDSS) area, was conducted from 12 May 2008 and in Fuladu West district from 12 September 2011 until 31 December 2013. In 2011, Hib serology was measured in 5-34-year-olds. RESULTS: In all, 16,735 of 17,932 (93%) eligible patients were investigated. We detected 57 cases of invasive H. influenzae disease; 24 (42%) were type b. No cases of Hib disease were detected in the BHDSS area in 2008-2009; 1 was detected in 2010, 2 in 2011, 4 in 2012 and 7 in 2013. In 2013, the incidence of Hib disease in those aged 2-11 and 2-59 months in the BHDSS area was 88 [95% confidence interval (CI): 29-207] and 22 (95% CI: 9-45) cases per 10⁵ person-years, respectively. In 2013, disease incidence in Fuladu West among those aged 0-59 months was 26 (95% CI: 7-67) cases per 10⁵ person-years. Nine of 24 Hib cases were vaccine failures (2 HIV positive) and 9 were too young to have been vaccinated. The proportion of children aged 5-6 years (n = 223) with anti-Hib IgG ≥1.0 μg/mL was 67%; the antibody nadir was in 9-14-year-olds (n = 58) with 55% above threshold. CONCLUSIONS: Hib disease in eastern Gambia has increased in recent years. Surveillance in developing countries should remain alert to detect such changes.

Co-infection with Shigella spp. and other microbes modifies diarrhea risk., Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p

Background/aim: Trachomatous trichiasis frequently returns following surgery. Several factors may promote recurrence: preoperative disease severity, surgeon ability, surgical procedure, healing responses, and infection. This study investigates whether enhanced control of infection, both of Chlamydia trachomatis and other bacteria, with azithromycin can improve surgical outcome in a trachoma control programme. Methods: Individuals with trachomatous trichiasis were examined and operated. After surgery patients were randomised to the azithromycin or control group. The azithromycin group and children in their household were given a dose of azithromycin. Antibiotic treatment was repeated at 6 months. All patients were reassessed at 6 months and 12 months. Samples were collected for C trachomatis polymerase chain reaction and general microbiology at each examination. Results: 451 patients were enrolled. 426 (94%) were reassessed at 1 year, of whom 176 (41.3%) had one or more lashes touching the eye and 84 (19.7%) had five or more lashes. There was no difference in trichiasis recurrence between the azithromycin and control group. Recurrent trichiasis was significantly associated with more severe preoperative trichiasis, bacterial infection, and severe conjunctival inflammation at 12 months. Significant variability in outcome was found between surgeons. Visual acuity and symptoms significantly improved following surgery. Conclusion: In this setting, with a low prevalence of active trachoma, azithromycin did not improve the outcome of trichiasis surgery conducted by a trachoma control programme. Audit of trichiasis surgery should be routine.

Background: Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease. Results: We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age. Conclusions: Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni , Salmonella enterica , and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10 4 ipaH copies per 100 ng of stool DNA for set 1 ( n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10 4 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella . Individuals with ≥2.9 × 10 4 ipaH copies have 5.6-times-higher odds of having diarrhea than those with 4 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH , a 90% increase from the 172 cases detected by culture in both samples. Among a subset ( n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% ( n = 129) for culture to 17.6% ( n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10 4 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella .

Routine use of pneumococcal conjugate vaccines (PCVs) in developing countries is expected to lead to a significant reduction in childhood deaths. However, PCVs have been associated with replacement disease with non-vaccine serotypes. We established a population-based surveillance system to document the direct and indirect impact of PCVs on the incidence of invasive pneumococcal disease (IPD) and radiological pneumonia in those aged 2 months and older in The Gambia, and to monitor changes in serotype-specific IPD. Here we describe how this surveillance system was set up and is being operated as a partnership between the Medical Research Council Unit and the Gambian Government. This surveillance system is expected to provide crucial information for immunisation policy and serves as a potential model for those introducing routine PCV vaccination in diverse settings.

Background Non-Typhoidal Salmonella (NTS) is an important cause of invasive bacterial disease and associated with mortality in Africa. However, little is known about the environmental reservoirs and predominant modes of transmission. Our study aimed to study the role of domestic animals in the transmission of NTS to humans in rural area of The Gambia. Methodology Human NTS isolates were obtained through an active population-based case-control surveillance study designated to determine the aetiology and epidemiology of enteric infections covering 27,567 Gambian children less than five years of age in the surveillance area. Fourteen children infected with NTS were traced back to their family compounds and anal swabs collected from 210 domestic animals present in their households. Identified NTSs were serotyped and genotyped by multi-locus sequencing typing. Principal Findings NTS was identified from 21/210 animal sources in the households of the 14 infected children. Chickens carried NTS more frequently than sheep and goats; 66.6%, 28.6% and 4.8% respectively. The most common NTS serovars were S. Colindale in humans (21.42%) and S. Poona in animals (14.28%). MLST on the 35 NTS revealed four new alleles and 24 sequence types (ST) of which 18 (75%) STs were novel. There was no overlap in serovars or genotypes of NTS recovered from humans or animal sources in the same household. Conclusion Our results do not support the hypothesis that humans and animals in close contact in the same household carry genotypically similar Salmonella serovars. These findings form an important baseline for future studies of transmission of NTS in humans and animals in Africa., Author Summary Salmonellosis is a neglected tropical disease causing serious dysentery and septicaemia particularly in young infants, elderly and immunocompromised individuals such as HIV patients and associated with substantial mortality in developing countries. Salmonellosis also constitutes a major public health problem as it is considered the most widespread bacterial zoonosis of food origin throughout the world. Many epidemiological data exist from developed countries concerning transmission of Non-Typhoidal Salmonella (NTS) but few are available from developing countries. In addition few studies in sub-Saharan Africa have considered the interface between humans and their environment in relation to animals present in the household and food hygiene. This study describes the prevalence of NTS among fourteen Gambian children and 210 domestic animals living in close proximity (household) to the children in a rural setting in The Gambia. We found that the domestic animals living in the same household as patients carried different NTS serovar and genotypes; indicating that zoonotic transmission does not occur in our setting. This study provides baseline data for future studies of transmission of NTS in rural Africa.

Introduction: The prevalence of virulence genes in non-typhoidal Salmonella (NTS) and its association with commonly used antibiotics in West Africa is unknown . Methodology: We tested 185 NTS isolates from children, animals, and food products for the presence of twelve virulence genes by PCR. Ten of the virulence genes tested belonged to the five Salmonella pathogenicity islands implicated in its pathogenesis. Results: Ten of twelve virulence genes except sopE and pefA were present in at least 70% of the isolates tested; sopE and pefA were observed in 33% and 44% of the isolates, respectively. The most prevalent gene was invA (99.5%), which is an invasion gene conserved within the Salmonella enterica . pipD and sopB genes, which were associated with serovar Enteritidis, were detected in 92.4% and 94.1% of isolates respectively. S. Istanbul and S. Javiana, which were isolated from chicken-serving restaurants, carried all the virulence genes of the five pathogenicity islands. There was significant association between sopB , sitC , orfLC , pipD and pefA virulence genes and resistance to commonly used antibiotics in Senegal and The Gambia, namely amoxicillin, ticarcillin, trimethoprim plus sulfamethoxazole, tetracycline, trimethoprim, spectinomycin, streptomycin, sulfonamides and nitrofurantoin. Conclusions: This study shows that virulence genes are present in NTS strains isolated from various sources . The significant association between some virulence genes and antibiotic resistance may have important implications with regard to the spread and persistence of resistance and virulence genes in Salmonella and to the prudent use of antimicrobial agents in humans and animals in West Africa.

Background. Establishing the pattern of infection and antimicrobial sensitivities in the local environment is critical to rational use of antibiotics and the development of management algorithms.Methods. Morbidity history and physical examination of 140 children with severe acute malnutrition were recorded. Their blood, stool, and urine samples were cultured and antibiotic sensitivity patterns determined for any bacterial pathogens isolated.Results. Thirty-eight children had a pathogen isolated from blood culture, 60% of which were considered contaminants. Coagulase negative staphylococcus was the predominant contaminant, while the major causes of bacteraemia were nontyphoidalSalmonella(13%),S. pneumoniae(10%), andE. coli(8%).E. coliaccounted for 58% of the urinary isolates. No pathogen was isolated from stool. In vitro sensitivity by disk diffusion showed that 87.5% of the isolates were sensitive to ampicillin and/or gentamicin and 84.4% (27/32) to penicillin and/or gentamicin.Conclusions. A combination of ampicillin and gentamicin provides adequate antibiotic cover for severely malnourished children in The Gambia.

BACKGROUND: Malaria is a risk factor for invasive non-typhoidal Salmonella (NTS) infection in children. In the last 10 years, indices of malaria infection in The Gambia have fallen substantially. METHODS: We compared temporal trends of childhood malaria and NTS infection in two Gambian locations. In Fajara, on the coast, the incidence of NTS infection at three time points between 1979 and 2005 was compared to the percentage of malaria positive outpatient thick blood films and the percentage of admissions associated with malaria over time. In Basse, in the eastern part of the country, the incidence of NTS infection at three time points between 1989 and 2008 was compared to the prevalence of malaria parasitaemia at four time points between 1992 and 2008. RESULTS: The estimated incidence of NTS infection in Fajara fell from 60 (1979-1984) to 10 (2003-05) cases per 100,000 person years. The proportion of outpatients in Fajara with suspected malaria who were parasitaemic fell from 33% (1999) to 6% (2007) while the proportion of admissions associated with malaria fell from 14.5% (1999) to 5% (2007). In Basse, the estimated incidence of NTS infection fell from 105 (1989-1991) to 29 (2008) cases per 100,000 person years while the prevalence of malaria parasitaemia fell from 45% (1992) to 10% (2008). The incidence of pneumococcal bacteraemia in Fajara and Basse did not fall over the study period. CONCLUSIONS: These data support an association between malaria and NTS infection. Reductions in malaria infection may be associated with reduced rates of invasive childhood NTS infection.

The introduction of molecular diagnostic methods is crucial for improved understanding of the aetiology and epidemiology of bacterial infections in communities in resource poor settings. A blood sample from a 7 month old patient diagnosed with malaria in 2001 in a Gambian outpatient clinic was reported as culture negative after it was subjected to traditional bacterial culture protocols. We re-addressed the analysis of the blood sample from this case more recently (after 6.5 years in archival storage) in pilot work establishing 16S rRNA PCR in our molecular laboratory. Initial 16S rRNA PCR results confirmed the presence of bacterial DNA in the sample. 16S rRNA sequence analysis identified the organism as Campylobacter spp. In light of the molecular evidence we successfully grew the organism using appropriate culture conditions and subsequently biochemically confirmed that the isolate was Campylobacter jejuni. PCR and DNA sequencing of a set of seven C. jejuni housekeeping genes and in silico Multilocus Sequence Typing (MLST) analysis revealed that the isolate exhibits a novel sequence type (ST) of C. jejuni (ST 2928) and belongs to ST-443 clonal complex. This study demonstrates the potential for molecular tools to enhance the diagnosis of bacterial infections, which remain a major killer globally, not least in children in the developing world. Improvements in diagnostics are needed, and will be important not only for sick individuals but also for populations, where better measures of disease burden will contribute significantly to the improvement of public health policy.

Background Few studies on bacteraemia in Africa have been published. We aimed to prospectively identify the causative organisms of bacteraemia in The Gambia and their relation to clinical diagnoses, outcome and antimicrobial susceptibility. Methods Between November 2003 and February 2005 we studied those admitted to the Medical Research Council hospital who were suspected of having bacteraemia. We documented clinical features, outcome, pathogens identified and their susceptibility patterns, and searched for factors associated with bacteraemia. Results 871 patients were admitted and had a blood culture taken. The median age was 2 years (range 2 months to 80 years) and 36 of 119 tested were HIV positive; 54.5% were male. 297 (34%) had a positive result and 93 (10.7% overall) were considered a genuine pathogen. Those with bacteraemia were more likely to die in hospital (OR 2.79; 1.17–6.65, p = 0.017) and to have a high white cell count (WCC; OR 1.81;95% CI 1.09–3.02; p = 0.022). Three organisms accounted for 73% of bacteraemias: Streptococcus pneumoniae (45.2%), Staphylococcus aureus (18.3%) and Escherichia coli (9.7%) while non-typhoidal salmonellae (NTS) accounted for 8.6%. Antimicrobial susceptibility of S. pneumoniae was very high to penicillin (97.5%); high resistance was found to co-trimoxazole. S. aureus was generally highly susceptible to cloxacillin, gentamicin and chloramphenicol. E. coli and NTS were all susceptible to ciprofloxacin and mostly susceptible to gentamicin. Thirteen (33%) S. pneumoniae isolates were of serotypes contained in a 7-valent pneumococcal conjugate vaccine and 20 (51.3%) were of the same serogroup. Conclusion In The Gambia, those with bacteraemia are more likely than those without to die in hospital and to have a raised peripheral blood WCC. S. pneumoniae is the most common organism isolated. Introduction of a pneumococcal conjugate vaccine can be expected to lead to a reduction in disease incidence.

OBJECTIVES To describe the characteristics of pneumococcal isolates obtained from patients with invasive pneumococcal disease in The Gambia. METHODS Pneumococcal isolates were obtained from children aged

Background: Trichiasis surgery is believed to reduce the risk of losing vision from trachoma. There are limited data on the long term outcome of surgery and its effect on vision and corneal opacification. Similarly, the determinants of failure are not well understood. Methods: A cohort of people in the Gambia who had undergone surgery for trachomatous trichiasis 3–4 years earlier was re-assessed. They were examined clinically and the conjunctiva was sampled for Chlamydia trachomatis polymerase chain reaction (PCR) and general bacterial culture. Results: In total, 141/162 people were re-examined. Recurrent trichiasis was found in 89/214 (41.6%) operated eyes and 52 (24.3%) eyes had five or more lashes touching the globe. Corneal opacification improved in 36 of 78 previously affected eyes. There was a general deterioration in visual acuity between surgery and follow up, which was greater if new corneal opacification developed or trichiasis returned. Recurrent trichiasis was associated with severe conjunctival inflammation and bacterial infection. C trachomatis was detected in only one individual. Conclusions: Recurrent trichiasis following surgery is a common potentially sight threatening problem. Some improvement in the cornea can occur following surgery and the rate of visual loss tended to be less in those without recurrent trichiasis. The role of conjunctival inflammation and bacterial infection needs to be investigated further. Follow up of patients is advised to identify individuals needing additional surgical treatment.

PURPOSE: Trachoma is the leading infectious cause of blindness. However, there are few data on the natural history of trachomatous trichiasis to guide program planning or that investigate its pathogenesis. METHODS: A cohort of Gambians with trichiasis in one or both eyes who had declined surgery was observed. Clinical examinations were performed at baseline and 4 years later. Conjunctival swab samples were collected for Chlamydia trachomatis PCR and bacteriology. RESULTS: One hundred fifty-four people were examined at baseline and 4 years later (241 nonsurgical eyes). At baseline 124 (52%) eyes had major trichiasis (5+ lashes), 75 (31%) minor trichiasis (1-4 lashes), and 42 (17%) no trichiasis. By 4 years, trichiasis had developed in 12 (29%) of 42 previously unaffected eyes. Minor trichiasis progressed to major in 28 (37%) of 75 eyes. New corneal opacification more commonly developed in eyes that had major (10%) compared to minor (5%) trichiasis at baseline. Bacterial infection was common (23%), becoming more frequent with increasing trichiasis. C. trachomatis infection was rare (1%). Conjunctival inflammation was common (29%) and was associated with progressive trichiasis and corneal opacification. CONCLUSIONS: Trichiasis progressed in the long-term in this environment, despite a low prevalence of C. trachomatis. Blinding corneal opacification develops infrequently, unless major trichiasis is present. Epilation and early surgery need to be formally compared for the management of minor trichiasis. The pathologic correlates and promoters of conjunctival inflammation need to be investigated.