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. 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. 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.

中文翻译：

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新型多重实时 PCR 检测揭示了越南南部健康和腹泻儿童中致泻大肠杆菌致病型的高患病率。


致泻性大肠杆菌 (DEC) 感染在中低收入国家 (LMIC) 的儿童中很常见。然而，检测各种 DEC 致病型很复杂，因为它们无法通过经典微生物学来区分。我们开发了四种多重实时 PCR 检测方法来检测六种 DEC 致病型的毒力标记物；使用 DEC 对照和其他肠道病原体测试特异性。来自六种大肠杆菌致病型的 PCR 扩增子经过纯化和扩增，用于优化 PCR 反应并计算重现性。经过验证后，这些测定被应用于来自健康和腹泻的越南儿童的临床样本，并与临床数据相关联。研究发现多重实时 PCR 具有可重复性和特异性。 34.7% (978/2815) 的腹泻儿童粪便样本中至少检测到一种 DEC 变异； EAEC、EIEC 和非典型 EPEC 最常见。值得注意的是，41.2% (205/498) 的非腹泻儿童样本呈 DEC 病理型阳性。在该人群中，只有 EIEC 与腹泻显着相关，其在 34.3% (99/289) 的腹泻样本中检测到，而在非腹泻样本中检测到 0.8% (4/498) (p < 0.001)。当应用于临床样本时，多重实时 PCR 是一种针对 DEC 病理型的高效且高通量的方法。这种方法揭示了越南儿童中 DEC 致病型的高携带率。我们描述了一种新的 DEC 诊断方法，为未来评估中低收入国家 DEC 负担的监测研究提供基线数据。