The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.
Children's Hospital 1, Ho Chi Minh City, Vietnam.
BMC Microbiol. 2020 Jul 3;20(1):192. doi: 10.1186/s12866-020-01878-5.
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.
产毒性大肠杆菌（DEC）感染在中低收入国家（LMICs）的儿童中很常见。然而，由于它们不能通过经典微生物学区分，因此检测各种 DEC 病原体型非常复杂。我们开发了四种多重实时 PCR 检测方法，用于检测六种 DEC 病原体型的毒力标记物；使用 DEC 对照和其他肠病原体对特异性进行了测试。从六种大肠杆菌病原体型的 PCR 扩增子中纯化并扩增 PCR 扩增子，以优化 PCR 反应并计算重现性。经过验证后，将这些检测方法应用于来自健康和腹泻越南儿童的临床样本，并与临床数据相关联。
发现多重实时 PCR 具有可重复性和特异性。在腹泻儿童的粪便样本中，至少有一种 DEC 变体的检出率为 34.7%（978/2815）；EAEC、EIEC 和非典型 EPEC 最为常见。值得注意的是，41.2%（498/1208）非腹泻儿童的样本呈 DEC 病原体型阳性。在该人群中，只有 EIEC 在 34.3%（289/842）腹泻样本中检出，而非腹泻样本中为 0.8%（4/498）（p<0.001），与腹泻显著相关。当将多重实时 PCR 应用于临床样本时，是一种高效且高通量的 DEC 病原体型检测方法。
该方法揭示了越南儿童中 DEC 病原体型的高携带率。我们描述了一种 DEC 的新诊断方法，为未来在 LMICs 中评估 DEC 负担的 DEC 监测研究提供了基线数据。
