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.

Fig 1

A selection of multiplex PCR gels (a) Multiplex PCR1 with lane L, 1-kb size ladder, Lane 2, EAEC 042 reference control, Lane N, negative control, lane 3–4 E. coli strains INOLLD97B and INOLLH029A positive for eae gene, lane 6, 11, 12, positive isolated E. coli strains with bfp gene, Lane 7, isolated E. coli strains with the bfp and eae genes, lane 10, EPEC E2348/69 reference control with the bfp and eae genes, lane 13 and 14 E. coli strains INOLWH056C and INOLLH297C positive for CVD432 gene (b) Multiplex PCR3 with lane L, 1-kb size ladder, Lane N, negative control, Lane 1, EIEC E137 reference control with ipaH gene, lane 2 ETEC H10407 reference control with the elt and est genes, lane 3–4 isolates INOMNH199C and INOLLD018D E. coli strains positive for est gene. (c) Multiplex PCR4 with EHEC EDL933 reference controls (Lane C). Lane 1–5, strains INOMND61D, INOLLH045A, INOLLH030F, INOMNH077E and INOLKH217A with stx2 genes, Lane L, 1-kb size ladder.

Fig 2. Maximum likelihood SNP tree of genomes used in this evaluation showing sequence type, results for stx1, stx2, stx1+2 PCR positivity; elt, est and elt+est PCR and WGS positivity, and CVD432 PCR and WGS positivity.

The figure can be engaged interactively at https://microreact.org/project/pWbLpY3qhV6Npa9q8Rpgrk-wgs-n-pcr. While there was some concordance for CVD432, there was little for the STEC and ETEC toxins. Nodes for the ST720 and ST69 complexes in which elt false positives are over-represented are indicated with arrows. The matrix to the left of the tree is colored for positivity to specific specific targets indicated in the figure key or negativity (N) for those targets. (b) False and true positives, and negatives, for elt overall and for ST720 and ST69 complexes.

Fig 3

(a) stx mis-prining amplicon sequence with matches >6 contiguous base pairs on any stx primer highlighted. (b) Corresponding highlighted regions in stx primers, indicated in the same colors.