Duplex CRISPR-Cas12/13 Based Detection of Schistosoma and Hookworm Coinfection

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.