CRISPR/Cas-Based Molecular Diagnostics for Intestinal Parasites: From Proof-of-Concept to Point-of-Care Testing

Intestinal parasitic infections remain a significant global public health concern, particularly in low- and middle-income countries, where timely and accurate diagnosis is essential for effective disease management and control. Although conventional diagnostic methods, including microscopy, antigen detection, and polymerase chain reaction (PCR), are widely used, each possesses inherent limitations related to sensitivity, turnaround time, laboratory infrastructure, or point-of-care applicability. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems have led to the development of highly sensitive and rapid molecular diagnostic platforms suitable for decentralized testing. This review summarizes current applications of CRISPR/Cas-based diagnostics for intestinal and foodborne parasitic infections, with particular emphasis on assays developed for Giardia duodenalis, Cryptosporidium parvum, Clonorchis sinensis, Fasciola hepatica, and Schistosoma spp. Published studies have predominantly employed Cas12a, with additional applications of Cas12b and Cas13a and, more recently, a type I-A Cascade-Cas3 system, generally coupled with isothermal amplification methods such as recombinase polymerase amplification (RPA) or recombinase-aided amplification (RAA), enabling highly sensitive detection within 60-90 min using fluorescence or lateral-flow strip readouts. The available evidence indicates high analytical sensitivity and specificity, with several assays demonstrating substantial agreement with conventional molecular methods and potential for point-of-care development. However, current CRISPR-based assays remain limited by dependence on nucleic acid extraction, relatively small clinical validation cohorts, lack of standardized protocols, and the persistence of false-negative results associated with low parasite burdens and pre-analytical variables. Emerging strategies, including simplified or extraction-free sample preparation, one-pot sample-to-answer platforms, and detection of parasite-derived cell-free DNA, may further enhance the clinical utility of CRISPR diagnostics. Overall, CRISPR/Cas-based technologies represent a promising next-generation approach for intestinal parasite diagnosis, although large-scale clinical validation and workflow optimization are still required before widespread implementation in routine clinical and field settings.