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

Vu Thuy Duong, Le Thi Phuong Tu, Ha Thanh Tuyen, Le Thi Quynh Nhi, James I. Campbell, Pham Van Minh, Hoang Le Phuc, Tran Thi Hong Chau, Nguyen Minh Ngoc, Lu Lan Vi, Claire Jenkins, Iruka Okeke, Ellen Higginson, Stephen Baker*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number192
JournalBMC Microbiology
Volume20
Issue number1
DOIs
Publication statusPublished - 3 Jul 2020

Bibliographical note

Funding Information:
This work was supported by a Wellcome senior research fellowship to Stephen Baker to (215515/Z/19/Z). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Publisher Copyright:
© 2020 The Author(s).

Keywords

  • Co-infection
  • Diarrhoea children
  • EAEC
  • EHEC
  • EIEC
  • EPEC
  • ETEC
  • Healthy children
  • Multiplex real-time PCR

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