University of Bern, Institute for Infectious Diseases, Bern, Switzerland.
University of Bern, Institute for Infectious Diseases, Bern, Switzerland.
Clin Microbiol Infect. 2023 Nov;29(11):1451.e1-1451.e5. doi: 10.1016/j.cmi.2023.07.032. Epub 2023 Aug 5.
The diagnosis of larval cestodiases in humans primarily depends on using imaging techniques in combination with serological tests. However, in case of atypical imaging results, negative serology results due to immunosuppression, or infection with rare taeniid species, traditional diagnostic tools may not provide a definitive species-level diagnosis. We aimed to validate a rapid, reliable, and cost-effective single-step real-time PCR method that can identify and differentiate larval cestodiases from biopsy material.
We validated a real-time PCR technique able to distinguish Echinococcus multilocularis, E. granulosus sensu lato (s.l.), and Taenia spp. from biopsy or cytology material in a single-step analysis. Further Sanger sequencing of E. granulosus s.l. and Taenia spp. amplicons enables differentiation of various Echinococcus and Taenia species. The assay was validated on (a) a reference sample collection of 69 clinical and veterinary cases confirmed by imaging, serology, and morphological analysis, (b) 38 routine human patient samples confirmed for aforementioned pathogens by a conventional end-point PCR, and (c) 127 samples from patients with suspected echinococcosis that were submitted to our laboratory for diagnostic analysis.
Compared to a conventional reference end-point PCR approach, the quadruplex real-time PCR exhibited a lower limit of detection in a serial dilution with 5-log dilutions for all three targets (2 log for E. multilocularis, 1 log for E. granulosus s.s., and 1 log for T. saginata). We were able to detect DNA from E. multilocularis, E. granulosus s.l. (E. granulosus s.s., E. canadensis, E. ortleppi, and E. felidis), a wide range of Taenia spp., as well as from non-echinococcal metacestodes such as Hydatigera taeniaformis, Hymenolepis spp., Versteria sp., and Spirometra erinaceieuropaei.
We suggest that the presented real-time PCR method is a suitable tool to be routinely used in a clinical microbiology laboratory to rapidly detect and identify larval cestodiases in human tissue.
人体幼虫性包虫病的诊断主要依赖于影像学技术与血清学检测联合应用。然而，在影像学结果不典型、由于免疫抑制而出现血清学阴性结果，或感染罕见带绦虫物种的情况下，传统的诊断工具可能无法提供明确的种属水平诊断。本研究旨在验证一种快速、可靠且经济有效的单步实时聚合酶链反应（PCR）方法，该方法可从活检材料中识别和区分幼虫性包虫病。
我们验证了一种实时 PCR 技术，该技术能够在单步分析中从活检或细胞学材料中区分细粒棘球绦虫、细粒棘球绦虫亚种（Echinococcus granulosus s.l.）和带绦虫属。进一步对 E.granulosus s.l.和带绦虫属的扩增子进行 Sanger 测序，可区分各种细粒棘球绦虫和带绦虫物种。该方法在以下方面进行了验证：（a）通过影像学、血清学和形态学分析确认的 69 例临床和兽医参考样本集；（b）通过传统终点 PCR 确认的 38 例人类常规患者样本；（c）提交给我们实验室进行诊断分析的 127 例疑似包虫病患者样本。
与传统的参考终点 PCR 方法相比，四重实时 PCR 在所有三个靶标的连续稀释中具有更低的检测下限（E.multilocularis 为 5 个对数稀释度，E.granulosus s.s.和 T.saginata 为 1 个对数稀释度）。我们能够检测到细粒棘球绦虫、E.granulosus s.l.（E.granulosus s.s.、E.canadensis、E.ortleppi 和 E.felidis）、广泛的带绦虫属、以及非细粒棘球蚴性包虫病的幼虫，例如Hydatigera taeniaformis、Hymenolepis spp.、Versteria sp. 和 Spirometra erinaceieuropaei。
我们建议，所提出的实时 PCR 方法是一种适合在临床微生物学实验室常规使用的工具，可快速检测和识别人类组织中的幼虫性包虫病。
