Background

Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples.

Methodology/Principal findings

A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R² = 0.74–0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65–88%), compared to the sensitivity (91–100%) of the new molecular diagnostic workflow.

Conclusions/Significance

Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.

中文翻译：

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炒鸡蛋：用于粪便样品中特定的Fasciola物种检测的高度灵敏的分子诊断工作流程

背景

由于肝片吸虫和巨大片吸虫而引起的片虫病是一种重新出现的人畜共患寄生虫病，在世界范围内具有重要意义。人和动物的感染通常通过传统的沉积和粪便卵计数技术来诊断。但是，这种技术很耗时，并且在必须处理大量样品或操作人员缺乏足够经验的情况下容易出现灵敏度错误。此外，只有在经过12周的专利申请期之后，才能进行诊断。最近，商业上可买到的coprologic抗原ELISA使检测F成为可能。肝尽管在寄生虫共生体领域不可能进行物种区分，但可以在专利期结束之前进行诊断，从而提供更早的诊断和更高的通量。实时PCR为中型到大型样品提供了高度敏感的物种分化的综合优势。但是，目前尚不存在用于鉴定Fasciola spp的分子诊断工作流程。在粪便样本中。

方法/主要发现

一种新的分子诊断工作流程，用于高灵敏度检测和鉴定Fasciola spp。在粪便样本中被开发出来。该技术包括在DNA分离之前沉淀和沉淀样品以浓缩卵，然后在台式均质器中通过敲打珠子来破坏卵以确保获得DNA。尽管新的分子工作流程和传统的沉降技术都非常敏感和特定，但新的分子工作流程在中型到大型流行病学研究中实现了更快的样品通量，并提供了物种形成的额外好处。此外，良好的相关性（R ²使用新的分子工作流程，在所有牛群和采样期间，实时PCR值与粪便卵数（FEC）之间的差异为0.74-0.76）。最终，没有发现在70％的乙醇中储存对沉淀和DNA分离结果的影响。无需完整的冷链就能将样品从流行国家运送到非流行国家。与新分子诊断工作流程的灵敏度（91–100％）相比，即使在调整阳性阈值（65–88％）之后，市售的ELISA仍显示出较差的灵敏度。

结论/意义

特定种类的测定可用于Fasciola spp的灵敏检测。可以在人和动物环境中进行事前诊断。这包括东南亚，那里可能有许多未记录的人类病例，并且对生产动物进行尸检可能很困难。新的分子工作流程提供了一种灵敏的定量诊断方法，可快速测试中到大型样品，有可能取代传统的沉淀和FEC技术，并在动物和人类健康资金有限的地区启用监视程序。