Point of care diagnostics for Cryptosporidium: new and emerging technologies

摘要

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.