PCR diagnostics are insufficient for the detection of Diarrhoeagenic Escherichia coli in Ibadan, Nigeria

Abstract

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. coli eae 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.

adhesiomeR: a tool for Escherichia coli adhesin classification and analysis

“…These differences between in silico and experimental analyses might be explained by known problems associated with PCR and dot-blot analyses. Negative results of the PCR may be caused by mutations in fragments targeted by the primers [ 35 ], whereas dot-blot analyses would not identify fimbriae that are currently not being expressed. On the other hand, the presence of the full operon indicated by adhesiomeR is very unlikely to be a result of false positives as it requires hits to all genes across the system, which usually comprises at least four genes.…”

“…E. coli genomes used to create pangenomes are available from ECOR [ 32 ]. Metagenomic dataset was obtained from Hildebrand et al [ 35 ]. All code used for data analysis is available at https://github.com/ksidorczuk/adhesiomeR-paper .…”