Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. The S. japonicum SHERLOCK assay achieved 93–100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. Australian Infectious Diseases Research Centre seed grant (2022) and 10.13039/501100000925 National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

Schistosomiasis and hookworm affect hundreds of millions, particularly in low-resource settings where coinfections contribute to substantial morbidity and severe clinical outcomes. We developed a portable duplex CRISPR assay for simultaneous detection of Necator americanus and Schistosoma mansoni. Building on our established S. mansoni CRISPR-Cas13 assay, we designed a CRISPR-Cas12 assay for N. americanus combining recombinase polymerase amplification (RPA) with Cas12 detection. Analytical sensitivity was assessed using serially diluted DNA from third-stage larvae, and specificity was evaluated using DNA from multiple helminth species. The N. americanus Cas12 and S. mansoni Cas13 assays were integrated into a duplex format following optimisation of Cas12/Cas13 detection conditions. Fluorescence was measured using a portable multichannel reader. Diagnostic performance was evaluated in 208 stool-derived DNA samples from Uganda using qPCR as the reference method. The N. americanus Cas12 assay had a detection limit of 0.1 pg/µL and showed no cross-reactivity with the other helminth species tested. In duplex format, N. americanus detection achieved 91.4% sensitivity and 100% specificity, whereas S. mansoni detection achieved 99.1% sensitivity and 99.0% specificity. This portable duplex RPA-CRISPR platform enables sensitive, highly specific simultaneous detection of N. americanus and S. mansoni, supporting integrated surveillance of helminth coinfections in resource-limited settings.

Background: Disease surveillance is critical for WHO’s Global Programme to Eliminate Lymphatic Filariasis (LF), but current field-ready diagnostics exhibit insufficient sensitivity and/or specificity in low-endemic settings. We aim to develop an ultra-sensitive and field-friendly CRISPR/Cas13-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) tool for point-of-care detection of Wuchereria bancrofti infection. Methods: The SHERLOCK assay was performed in two-step and one-pot formats with sensitivity compared to qPCR. Assays were validated using 149 pooled mosquito and 185 human blood samples from Samoa, which were previously tested for LF antigen using WHO-recommended Filariasis Test Strips (FTS) and microscopically examined for microfilariae (Mf). We also used lyophilised one-pot SHERLOCK master mix and assessed its thermostability after storage at 25°C, 4°C and -20°C.Findings: The W. bancrofti SHERLOCK assays achieved a detection limit of 0.1 fg/μL gBlock DNA and showed no cross-reaction with five other filarial species. For both mosquito and human samples, the two-step SHERLOCK assay reached 100% sensitivity and specificity, while one-pot SHERLOCK showed 94.0-96.4% sensitivity and 100% specificity. The lyophilised one-pot SHERLOCK mix maintained a detection limit of 10 fg/μL W. bancrofti gBlock after 10 days at 25°C, indicating reduced reliance on cold-chain for reagent storage and transport. However, when testing human blood samples with lyophilised SHERLOCK, reduced sensitivity (59.7%) was observed.Interpretation: This CRISPR-based platform offers a promising field-ready next-generation approach to LF diagnostics, potentially addressing current limitations, enhancing surveillance and elimination efforts.