Department of Parasitology-Mycology, National Reference Centre for Echinococcoses, University Hospital of Besançon, 25030 Besançon, France - UMR CNRS 6249 Laboratoire Chrono-environnement, University of Franche-Comté, 16 Route de Gray, 25030 Besançon, France.
UMR CNRS 6249 Laboratoire Chrono-environnement, University of Franche-Comté, 16 Route de Gray, 25030 Besançon, France.
Parasite. 2023;30:3. doi: 10.1051/parasite/2023003. Epub 2023 Jan 25.
Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10 ng/μL (seven PCR assays positive), corresponding to 37-42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.
对罕见的人类感染性病原体进行分子鉴定，似乎是目前用于快速诊断和治疗患者的最相关方法之一。聚合酶链反应（PCR）技术，特别是实时定量 PCR，最适合检测病原体的 DNA，即使在低浓度下也是如此。包虫病感染是由细粒棘球绦虫属的寄生虫引起的，其密切相关的物种会导致寄生性病变，影响动物，偶尔也会影响人类。我们开发了一种多重 qPCR（MLX qPCR）检测方法，该方法可以检测欧洲与泡型包虫病（AE）和囊型包虫病（CE）相关的四种棘球蚴（多房棘球绦虫、细粒棘球绦虫、石渠棘球绦虫和加拿大棘球绦虫），该方法基于短的线粒体靶点。收集了 81 份新鲜和福尔马林固定石蜡包埋（FFPE）的 AE 和 CE 病变组织。qPCR 检测在三重体系中进行，用于棘球属的检测，同时还进行了 qPCR 抑制剂的控制。还设计了一种双重 qPCR 检测方法，用于诊断另外两种无进展性寄生虫病（囊尾蚴病（猪带绦虫）和犬弓首线虫病（犬弓首线虫和猫弓首线虫））。评估了 qPCR 的灵敏度，范围从 1 到 5×10 ng/μL（七个 PCR 检测为阳性），对应于可定量 DNA 的 37-42 个循环。所有目标的特异性均为 100%。这种适应于少量 DNA 的多重 qPCR 可以在实验室中快速进行棘球属物种的分子诊断。
