Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. Cryptosporidium spp., Entamoeba histolytica, Giardia intestinalis, (and, arguably, Dientamoeba fragilis and Blastocystis spp.) are among the most important and common parasitic protozoans causing diarrhea. Several multiplex real-time PCR assays have been developed for the synchronous detection of these parasites. However, most assays include the use of hydrolysis probes, increasing the cost of stool examination. In this study, we designed and evaluated a real-time PCR protocol, based on high-resolution melting (HRM) curve analysis, to simultaneously detect and differentiate five gastrointestinal parasites. Using a blinded panel of 143 clinical samples with laboratory diagnostic data to evaluate the method, we obtained a 95.8% concordance with conventional methods. Moreover, 4.2% of the samples were positive for D. fragilis and 2.8% additional Cryptosporidium infections were found with our multiplex assay. Our method is sensitive and specific for the selected parasites with the additional possibility of being run in single-plex as a backup control for mixed infections. The assay is a convenient and cost-effective method that could contribute to a quicker and accurate diagnosis as well as to more targeted therapies of parasite-derived diarrhea. Finally, this new multiplex PCR assay could also be instrumental in epidemiology studies on these parasites.

中文翻译：

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一种检测人类腹泻寄生虫的新型 5-Plex qPCR-HRM 检测方法。

肠道寄生虫病在世界范围内发生，其诊断提出了相当大的挑战。隐孢子虫属、溶组织内阿米巴、肠贾第虫（以及，可以说，脆弱丁阿米巴和芽囊虫属）是最重要和最常见的引起腹泻的寄生虫原虫。已经开发了几种多重实时 PCR 检测方法来同步检测这些寄生虫。然而，大多数检测包括使用水解探针，增加了粪便检查的成本。在本研究中，我们设计并评估了基于高分辨率熔解 (HRM) 曲线分析的实时 PCR 方案，以同时检测和区分五种胃肠道寄生虫。使用具有实验室诊断数据的 143 个临床样本的盲组来评估该方法，我们获得了 95。与传统方法的一致性为 8%。此外，4.2% 的样本对脆弱 D. 呈阳性，并且在我们的多重检测中发现了 2.8% 的额外隐孢子虫感染。我们的方法对选定的寄生虫具有敏感性和特异性，并有可能在单重中运行作为混合感染的备用控制。该测定是一种方便且具有成本效益的方法，有助于更快、更准确地诊断以及对寄生虫源性腹泻进行更有针对性的治疗。最后，这种新的多重 PCR 检测也可能有助于这些寄生虫的流行病学研究。我们的方法对选定的寄生虫具有敏感性和特异性，并有可能在单重中运行作为混合感染的备用控制。该测定是一种方便且具有成本效益的方法，有助于更快、更准确地诊断以及对寄生虫源性腹泻进行更有针对性的治疗。最后，这种新的多重 PCR 检测也可能有助于这些寄生虫的流行病学研究。我们的方法对选定的寄生虫具有敏感性和特异性，并有可能在单重中运行作为混合感染的备用控制。该测定是一种方便且具有成本效益的方法，有助于更快、更准确地诊断以及对寄生虫源性腹泻进行更有针对性的治疗。最后，这种新的多重 PCR 检测也可能有助于这些寄生虫的流行病学研究。