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
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.
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Posted 20 May, 2020
On 03 Jul, 2020
On 19 May, 2020
Received 19 May, 2020
Invitations sent on 14 May, 2020
On 11 May, 2020
On 10 May, 2020
On 20 Mar, 2020
On 30 Apr, 2020
Received 26 Apr, 2020
On 03 Apr, 2020
Received 02 Apr, 2020
On 01 Apr, 2020
Invitations sent on 31 Mar, 2020
On 19 Mar, 2020
On 18 Mar, 2020
On 18 Mar, 2020
On 18 Mar, 2020
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
Posted 20 May, 2020
On 03 Jul, 2020
On 19 May, 2020
Received 19 May, 2020
Invitations sent on 14 May, 2020
On 11 May, 2020
On 10 May, 2020
On 20 Mar, 2020
On 30 Apr, 2020
Received 26 Apr, 2020
On 03 Apr, 2020
Received 02 Apr, 2020
On 01 Apr, 2020
Invitations sent on 31 Mar, 2020
On 19 Mar, 2020
On 18 Mar, 2020
On 18 Mar, 2020
On 18 Mar, 2020
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