Abstract
BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data.ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p < 0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes.ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.
Highlights
Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs)
Diarrhoeagenic E. coli (DEC) can generally be divided into six pathotypes, based on specific virulence markers that are encoded on plasmids and/or chromosomal islands [7]
Multiplex real-time PCR assay for detecting diarrhoeagenic Escherichia coli We firstly validated PCR amplification for Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC)/Shigella, Enteropathogenic E. coli (EPEC), and Shiga-toxin producing E. coli (STEC) in monoplex using cloned target sequences and with genomic DNA extracted from the various E. coli pathovars
Summary
Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. Diarrhoeagenic E. coli (DEC) can generally be divided into six pathotypes (enterotoxigenic E. coli, ETEC; enteroaggregative E. coli, EAEC; enteropathogenic E. coli, EPEC, enteroinvasive E. coli, EIEC; enterohemorrhagic E. coli, EHEC and shiga-toxin producing E. coli, STEC), based on specific virulence markers that are encoded on plasmids and/or chromosomal islands [7]. ETEC, EAEC, and EPEC have all been implicated in causing diarrhoea in young children in low-middle income countries (LMICs) [8,9,10]. The epidemiology of STEC in LMICs, in children in Southeast Asia, are not well described
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