Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the south of Vietnam

Abstract

Background: Diarrhoeagenic 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. Results: The 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. Conclusions: This 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.

Assessing the impact of a novel house design on the incidence of malaria in children in rural Africa: study protocol for a household-cluster randomized controlled superiority trial

“…In addition, a swab will be collected from a child without symptoms residing in a control house. The swabs will be transported in an appropriate transport medium at ambient temperature to the study hub in Mtwara town where the specimens will be stored at − 20 °C until final processing using multiplex PCR [ 22 ]. An air sampler will be used to collect standardized air samples to detect pathogens in air samples [ 23 ].…”

The Effect of Oral Iron Supplementation on Gut Microbial Composition: a Secondary Analysis of a Double-Blind, Randomized Controlled Trial among Cambodian Women of Reproductive Age

“…In our study, we used 16S rRNA gene sequencing to examine changes in the gut microbiome following 12 weeks of daily supplementation of either ferrous sulfate or ferrous bisglycinate in non-pregnant Cambodian women of reproductive age (WRA). We then used real-time PCR (qPCR) to assess the presence and abundance of bfpA and eae , virulence genes from enteropathogenic E. coli (EPEC), a common culprit of diarrheal disease worldwide ( 17 – 19 ). Due to the differences in bioavailability among the 2 iron interventions, we hypothesized that the women who received ferrous sulfate would have an increased relative abundance of pathogenic bacteria compared to those who received ferrous bisglycinate.…”

Genomic characterization of endemic diarrheagenic Escherichia coli and Escherichia albertii from infants with diarrhea in Vietnam

“…Some studies of DEC in Vietnamese children have been conducted so far [6][7][8]. Nguyen et al [6] reported that one or more marker genes of DEC were detected in 22.5% of fecal samples (132/587) collected from young children with diarrhea from 2001 to 2002.…”

“…Meanwhile, Hien et al [7] isolated DEC strains from 25.7% (64/249) of stool samples of diarrhea patients who were less than five years of age from 2001 to 2002 and classified them into pathotypes. Recently, Duong et al [8] developed new multiplex real-time polymerase chain reactions (PCRs), which revealed that 34.7% and 41.2% of stool samples from children with and without diarrhea from 2014 to 2016, respectively, were positive for the DEC pathotypes. Thus, Vietnamese children are widely contaminated with various DEC strains possibly associated with diarrhea.…”