实时荧光定量PCR (qPCR)检测腹泻患儿与无症状感染患儿肠聚集性大肠杆菌(EAEC)菌量比较

David Durand, Joaquim Ruíz, Theresa J Ochoa
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引用次数: 0

摘要

肠聚集性大肠杆菌(EAEC)是致泻性大肠杆菌(DEC)的一种致病型,可在儿童、艾滋病毒感染者和旅行者中引起急性和持续性腹泻。然而,EAEC也可以在没有腹泻的人群中分离出来。本研究的目的是评估与无症状感染相比,粪便样本中的EAEC细菌负荷是否与腹泻有关。方法:我们选择165份粪便样本(腹泻患儿80份,无症状儿童85份),这些粪便样本之前通过Real-Time PCR鉴定为EAEC,用于从粪便培养物中分离的大肠杆菌中诊断DEC。我们使用十六烷基三甲基溴化铵(CTAB)法从粪便样本中提取DNA,并使用EAEC菌株042标准化qPCR来评估细菌负荷。结果:qPCR检测限为10个aggr基因拷贝(5个细菌)/mg粪便,反应效率≥93%。我们发现腹泻患者的细菌负荷[2781,95% CI(688-6875)细菌/mg粪便]高于无症状儿童[138,95% CI(75-306)细菌/mg粪便],包括单一病原体感染和合并感染的样本。在腹泻样本中,男孩的细菌载量高于女孩,18个月以上的儿童的细菌载量高于18个月以下的儿童。结论:腹泻患儿粪便样本中的EAEC载量高于无症状感染患儿。该qPCR可用于研究定植在流行地区儿童中的作用,这些地区的儿童在两组中分离出的病原体相似。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Bacterial load comparison of enteroaggregative Escherichia coli (EAEC) by real-time PCR (qPCR) between children with diarrhea and asymptomatic infection.

Introduction: Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic E. coli (DEC) that causes acute and persistent diarrhoea in children, HIV-infected patients and travelers. However, EAEC can also be isolated in people without diarrhoea. The aim of this study was to evaluated if the EAEC bacterial load in stool samples is associated with diarrhoea in comparison with asymptomatic infection.

Methods: We selected 165 stool samples (children with diarrhoea: 80 and asymptomatic children: 85) that previously were identified as EAEC by Real-Time PCR for diagnostic of DEC from E. coli isolated from stool cultures. We extracted DNA from fecal samples using the cetyltrimethylammonium bromide (CTAB) method and standardised a qPCR to evaluate the bacterial load using EAEC strain 042.

Results: The detection limit of the qPCR was 10 copies ofaggR gene (5 bacterias)/mg stool and the reaction efficiency was ≥ 93%. We found a higher bacterial load in patients with diarrhoea [2781, 95% CI (688-6875) bacteria/mg feces] than in asymptomatic children [138, 95% CI (75-306) bacteria/mg feces], including samples with single pathogen infection and co-infections. Among diarrheal samples, the bacterial load was higher in boys than girls and in children older than 18 months than younger.

Conclusions: EAEC load in stool samples are higher in children with diarrhoea than in asymptomatic infection. This qPCR could be useful for studying the role of colonisation in children living in endemic areas where the isolation of this pathogen is similar in both groups.

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