CRISPR-assisted test for Schistosoma haematobium

Abstract

Schistosomiasis is a major neglected tropical disease targeted for elimination as a public health issue by 2030, however there is an urgent need for more sensitive and specific diagnostic tests suitable to resource-limited settings. Here we developed CATSH, a CRISPR-assisted diagnostic test for Schistosoma haematobium, utilising recombinase polymerase amplification, Cas12a-targeted cleavage and portable real-time fluorescence detection. CATSH showed high analytical sensitivity, consistent detection of a single parasitic egg and specificity for urogenital Schistosoma species. Thanks to a novel CRISPR-compatible sample preparation developed using simulated urine samples containing parasitic eggs, CATSH had a sample-to-result within 2 h. The components of CATSH can be lyophilised, reducing cold chain dependence and widening access to lower and middle-income countries. This work presents a new application of CRISPR diagnostics for highly sensitive and specific detection of parasitic pathogens in remote areas and could have a significant impact on the elimination of neglected tropical diseases.

Figure 1

CATSH design and optimisation. (a) Visualisation of the targeted S. haematobium Dra1 repeat region with RPA primers, amplicon and crRNAs. (b) Schematic of the proposed CATSH workflow. Preparation of urine samples with in-house CATSH-compatible protocol, followed by CATSH testing. Real-time fluorescence is recorded on a portable reader. (c) Optimisation of ssDNA-FQ concentration. (d) Comparison of three crRNAs. (e) Optimisation of RPA input. (f) Optimisation of Cas12a to crRNA ratio. All reactions were run in five repeats. Bars represent the average endpoint fluorescence after background subtraction and error bars represent the standard deviation between repeats.

Figure 2

Evaluation of CATSH. (a) Calculation of analytical sensitivity (EC₉₅) with genomic DNA using the fraction positive (dots) calculated with five replicates for each concentration tested. The Probit regression was plotted with the 95% confidence interval (dashed lines). (b) Time to cut-off for positive reactions. Bars represent the average time to cut-off and error bars represent the standard deviation between positive reactions. (c) Detection of a single parasitic egg in spiked buffer. Three individual repeats are plotted and dashed line show the fluorescent cut-off (= 30). (d) Species specificity tested with S. haematobium, S. mansoni, S. bovis and S. curassoni. Individual reactions are plotted. (e) Detection of parasitic eggs after in-house extraction from simulated urine sample. Individual reactions are plotted. (f) Thermostability of freeze-dried CATSH reactions. For each condition, three repeats were carried out. Bars represent the average endpoint fluorescence and error bars represent the standard deviation between repeats.