a Division of Clinical Microbiology , Helsinki University Hospital, HUSLAB , Helsinki , Finland.
b Department of Infectious Diseases , Danderyds Hospital , Stockholm , Sweden.
Infect Dis (Lond). 2017 Sep;49(9):655-663. doi: 10.1080/23744235.2017.1320728. Epub 2017 Apr 27.
In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools.
A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea.
The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica.
Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.
在发展中国家，腹泻是 5 岁以下儿童死亡的最常见原因，贾第鞭毛虫、隐孢子虫和溶组织内阿米巴是最常见的致病寄生虫。传统的粪便寄生虫显微镜检查敏感性和特异性差，而新的分子方法可能提供更准确的诊断。在样本存储受不确定电力供应阻碍的贫困地区，研究将受益于能够分析干燥粪便的方法。
开发了一种实时多重 PCR 方法，带有内部抑制控制，可直接从粪便标本中检测贾第鞭毛虫、人隐孢子虫/微小隐孢子虫和溶组织内阿米巴。通过与小测试材料中的新鲜样本进行比较，检查了对干燥样本的适用性。最后，该检测方法应用于来自几内亚比绍腹泻儿童的干燥样本。
PCR 的分析灵敏度限值为 0.1ng/ml 的 G. lamblia DNA、0.01ng/ml 的 E. histolytica DNA 和 0.1ng/ml 的 Cryptosporidium sp。在测试材料中，新鲜粪便和干燥粪便的检测结果相似。在 52 份几内亚比绍样本中，当地显微镜检查显示有 15%的寄生虫，而 PCR 检测出至少有 62%的寄生虫呈阳性：44%的干燥样本有贾第虫，23%的隐孢子虫，0%的溶组织内阿米巴。
我们新的用于原生动物的多重实时 PCR 是一种适用于干燥样本的敏感方法。作为概念验证，它在来自几内亚比绍腹泻儿童的粪便样本中表现良好。它为分析资源匮乏地区的干燥标本提供了一种流行病学工具。
