Hijjawi Nawal, Zahedi Alizera, Ryan Una
Department of Medical Laboratory Sciences, Faculty of Applied Health Sciences, The Hashemite University, Zarqa, Jordan.
The Centre of Biosecurity and One Health, Harry Butler Institute, Murdoch University, Perth, Western Australia, Australia.
Curr Opin Gastroenterol. 2023 Jan 1;39(1):3-8. doi: 10.1097/MOG.0000000000000895. Epub 2022 Nov 10.
Although Cryptosporidium detection and typing techniques have improved dramatically in recent years, relatively little research has been conducted on point of care (POC) detection and typing tools. Therefore, the main purpose of the present review is to summarize and evaluate recent and emerging POC diagnostic methods for Cryptosporidium spp.
Microscopy techniques such as light-emitting diode fluorescence microscopy with auramine-phenol staining (LED-AP), still have utility for (POC) diagnostics but require fluorescent microscopes and along with immunological-based techniques, suffer from lack of specificity and sensitivity. Molecular detection and typing tools offer higher sensitivity, specificity and speciation, but are currently too expensive for routine POC diagnostics. Isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) including a commercially available LAMP kit have been developed for Cryptosporidium but are prone to false positives. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas diagnostic technologies (CRISPRDx) have recently been combined with isothermal amplification to increase its specificity and sensitivity for detection and typing. Other emerging technologies including amplification-free CRISPR detection methods are currently being developed for Cryptosporidium using a smartphone to read the results.
Many challenges are still exist in the development of POC diagnostics for Cryptosporidium. The ideal POC tool would be able to concentrate the pathogen prior to detection and typing, which is complicated and research in this area is still very limited. In the short-term, CRISPR-powered isothermal amplification lateral flow tools offer the best opportunity for POC Cryptosporidium species and subtype detection, with a fully integrated autonomous biosensor for the long-term goal.
尽管近年来隐孢子虫的检测和分型技术有了显著改进，但针对即时检测（POC）工具的研究相对较少。因此，本综述的主要目的是总结和评估隐孢子虫属的近期及新兴即时诊断方法。
诸如采用金胺酚染色的发光二极管荧光显微镜（LED-AP）等显微镜技术，在即时检测诊断中仍有应用价值，但需要荧光显微镜，并且与基于免疫的技术一样，存在特异性和敏感性不足的问题。分子检测和分型工具具有更高的敏感性、特异性和物种鉴定能力，但目前对于常规即时检测诊断来说成本过高。已经开发出用于隐孢子虫检测的等温扩增方法，如环介导等温扩增（LAMP）或重组酶聚合酶扩增（RPA），包括一种市售的LAMP试剂盒，但容易出现假阳性。成簇规律间隔短回文重复序列（CRISPR）-Cas诊断技术（CRISPRDx）最近已与等温扩增相结合，以提高其检测和分型的特异性和敏感性。目前正在开发其他新兴技术，包括无需扩增的CRISPR检测方法，用于隐孢子虫检测，并使用智能手机读取结果。
隐孢子虫即时检测诊断的开发仍存在许多挑战。理想的即时检测工具应能够在检测和分型之前浓缩病原体，这一过程很复杂，且该领域的研究仍然非常有限。短期内，由CRISPR驱动的等温扩增侧流工具为即时检测隐孢子虫物种和亚型提供了最佳机会，而长期目标是开发完全集成的自主生物传感器。
