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Research article
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
Stephen Baker
University of Cambridge
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1308-5755
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at BMC Microbiology.
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% (978/2,815) 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.
Keywords
ETEC, EAEC, EIEC, EPEC, EHEC, multiplex real-time PCR, diarrhoea children, healthy children, co-infection
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CURRENT STATUS: Published
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Posted 20 May, 2020
Published
On 03 Jul, 2020
No community comments so far
Reviewer #1 agreed
On 19 May, 2020
Review #1 received
Received 19 May, 2020
Reviewers invited
Invitations sent on 14 May, 2020
Editor assigned
On 11 May, 2020
Editor invited
On 10 May, 2020
Submission checks complete
On 20 Mar, 2020
No comments provided
Editorial decision: Major revision
On 30 Apr, 2020
Review #2 received
Received 26 Apr, 2020
Reviewer #2 agreed
On 03 Apr, 2020
Review #1 received
Received 02 Apr, 2020
Reviewer #1 agreed
On 01 Apr, 2020
Reviewers invited
Invitations sent on 31 Mar, 2020
Editor assigned
On 19 Mar, 2020
First submitted
On 18 Mar, 2020
Submission checks complete
On 18 Mar, 2020
Editor invited
On 18 Mar, 2020
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This preprint is under consideration at BMC Microbiology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
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
Stephen Baker
University of Cambridge
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1308-5755
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 20 May, 2020
Published
On 03 Jul, 2020
No community comments so far
Reviewer #1 agreed
On 19 May, 2020
Review #1 received
Received 19 May, 2020
Reviewers invited
Invitations sent on 14 May, 2020
Editor assigned
On 11 May, 2020
Editor invited
On 10 May, 2020
Submission checks complete
On 20 Mar, 2020
No comments provided
Editorial decision: Major revision
On 30 Apr, 2020
Review #2 received
Received 26 Apr, 2020
Reviewer #2 agreed
On 03 Apr, 2020
Review #1 received
Received 02 Apr, 2020
Reviewer #1 agreed
On 01 Apr, 2020
Reviewers invited
Invitations sent on 31 Mar, 2020
Editor assigned
On 19 Mar, 2020
First submitted
On 18 Mar, 2020
Submission checks complete
On 18 Mar, 2020
Editor invited
On 18 Mar, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at BMC Microbiology.
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% (978/2,815) 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.
Figure 1
Figure 2