Point of care diagnostics for Cryptosporidium: new and emerging technologies

Abstract

Purpose of reviewAlthough 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. Recent findingsMicroscopy 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. SummaryMany 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.

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4)

“…Previous studies have demonstrated that the combination of RPA and CRISPR-Cas-based detection systems can improve detection sensitivity and specificity (Hijjawi et al 2023 ). We then tried to employ RPA in the CRISPR-Cas12b-based detection systems to enhance the efficacy of the systems for mcr-1 and tet (X4) detection.…”

“…Notably, the CRISPR-Cas system can be combined with isothermal amplification methods, such as RPA, which offer the advantages of simplicity and rapidity, to further enhance the specificity and sensitivity of detection (Hijjawi et al 2023 ; Li et al 2022a , b , c , d , e ). However, this approach involves nucleic acid amplification before detection using the CRISPR-Cas system, which may result in aerosol contamination (Lin et al 2022 ).…”

“…Currently, Cas12b has been used to rapidly and accurately detect pathogenic bacteria, such as Campylobacter jejuni (sample-to-answer time < 40 min) (Huang et al 2021 ), Mycobacterium tuberculosis complex (< 70 min) (Yang et al 2023 ), Pseudomonas aeruginosa (< 1 h) (Qiu et al 2023 ), and Klebsiella pneumoniae (Qiu et al 2022 ). Moreover, nucleic acid amplification technologies, such as recombinase polymerase amplification (RPA), which is a rapid amplification technology that can exponentially amplify the target sequence within 30 min, can be applied to the CRISPR-Cas-based detection systems to further enhance the specificity and sensitivity of detection (Hijjawi et al 2023 ). The combination of RPA and CRISPR-Cas12a can enable the rapid detection of methicillin-resistant Staphylococcus aureus , achieving the detection of as low as 10 copies of the target gene within only 20 min (Li et al 2022a , b , c , d , e ).…”

Medical prospects of cryptosporidiosis in vivo control using biofabricated nanoparticles loaded with Cinnamomum camphora extracts by Ulva fasciata

“…Cryptosporidium, a single-celled parasite, has broad host specificity [1]. Cryptosporidium parvum is the most common infectious species, particularly in neonates and/or young ruminants [2,3].…”

One-pot virus detection based on isothermal amplification coupled with temperature-activated argonaute

“…Yet, INAs have some limitations, e.g. low precision and restrictions in multiplex detection (8)(9)(10)(11)(12)(13). Nevertheless, the ease of use of INAs provides a valuable diagnostic tool for pandemic response.…”