Diarrhea is a leading cause of childhood morbidity in Africa. Outside of multi-country sentinels, of which there are none in Nigeria, few studies focus on bacterial etiology. We performed a case-control study among children under five years of age. Stool specimens were collected from 120 children with, and 357 without, diarrhea attending primary health clinics on the northern outskirts of Ibadan between November 2015 and August 2019. Up to ten E. coli isolates were obtained per specimen and at least three were whole genome-sequenced using Illumina technology. Genomes were assembled using SPAdes, quality evaluated using QUAST, and Virulencefinder was used to identify virulence genes. The microbiological quality of water from 14 wells within the study area was assessed using total and coliform counts. Diarrhoeagenic Escherichia coli (DEC) were isolated from 79 (65.8%) of cases and 217 (60.8%) control children. All DEC pathotypes except Shiga toxin-producing E. coli, a number of hybrid DEC pathotypes, Salmonella and Yersina spp. were detected but no pathogen showed association with disease (p>0.05). Enterotoxigenic E. coli were more commonly recovered from younger controls but exclusively detected in cases aged over nine months. Temporally-linked, highly similar enteroaggregative E. coli were isolated from children in different households in eight instances. No well water sample drawn in the study qualified as potable. Children in northern Ibadan are commonly colonized with DEC. Access to water and sanitation, and vaccines targeting the most abundant pathogens may be critical for protecting children from the less overt consequences of enteric pathogen carriage.
Understanding the contribution of different diarrhoeagenic Escherichia coli pathotypes to disease burden is critical to mapping risk and informing vaccine development. Targeting select virulence genes by PCR is the diagnostic approach of choice in high-burden, least-resourced African settings. We compared the performance of a commonly-used multiplex protocol to whole genome sequencing (WGS). PCR was applied to 3,815 E. coli isolates from 120 children with diarrhoea and 357 healthy controls. Three or more isolates per specimen were also Illumina-sequenced. Following quality assurance, ARIBA and Virulencefinder database were used to identify virulence targets. Root cause analysis of deviant PCR results was performed by examining target sensitivity using BLAST, Sanger sequencing false-positive amplicons, and identifying lineages prone to false-positivity using in-silico multilocus sequence typing and a Single Nucleotide Polymorphism phylogeny constructed using IQTree. The sensitivity and positive predictive value of PCR compared to WGS ranged from 0-77.8% while specificity ranged from 74.5-94.7% for different pathotypes. WGS identified more enteroaggregative E. coli (EAEC), fewer enterotoxigenic E. coli (ETEC) and none of the Shiga toxin-producing E. coli detected by PCR, painting a considerably different epidemiological picture. Use of the CVD432 target resulted in EAEC under-detection, and enteropathogenic E. colieae primers mismatched more recently described intimin alleles common in our setting. False positive ETEC were over-represented among West Africa-predominant ST8746 complex strains. PCR precision varies with pathogen genome so primers optimized for use in one part of the world may have noticeably lower sensitivity and specificity in settings where different pathogen lineages predominate.
Feco-orally transmitted infectious diseases are common in Nigeria where the potable water access is poor. In the south-western Nigerian Ibadan metropolis, supply of municipal water is meagre as residents depend on household wells and boreholes. The likelihood of fecal contamination of household water sources in Ibadan was examined longitudinally to quantify and understand its impact. Well and borehole water samples aseptically collected from 96 households in Ibadan were assessed for total heterotrophic counts (THCs), total coliform counts (TCCs) and total Escherichia coli counts (TECs) using a pour plate technique. E. coli were identified by uidA and whole-genome sequencing using Illumina technology, whereas virulence factors were predicted using VirulenceFinder. There was season-independent abundance of THC and TCC in the well and borehole with a significant recovery of E. coli in the wells during the wet season compared to the dry season (P=0.0001). Virulence genes associated with pathogenic E. coli were identified in 13 (52%) strains with one E. coli each classified as extra-intestinal E. coli, avian pathogenic E. coli and enteroaggregative E. coli. High heterotrophic and coliform counts, with rainfall-driven E. coli contamination revealed that the water sources evaluated in this study are unfit for consumption.
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