Multiplex LAMP-LFA for rapid field-compatible detection of environmental DNA from Schistosoma japonicum and Oncomelania hupensis quadrasi

Abstract This study developed a field-adaptable multiplex Loop-Mediated Isothermal Amplification combined with Lateral Flow Assay (mLAMP-LFA) for simultaneous detection of Schistosoma japonicum and its intermediate host, Oncomelania hupensis quadrasi , in freshwater environments to improve schistosomiasis environmental surveillance. The mLAMP-LFA was evaluated using environmental DNA (eDNA) samples collected from 30 sites across major water bodies in Ekiran village, municipality of Alangalang, Leyte Province, Philippines, during four field surveys conducted between July 2023 and March 2024. The assay demonstrated probit-derived limits of detection (LOD 95 ) ranging from 57.2 to 194.9 copies per reaction across different sample matrices and conditions, with corresponding 95% confidence intervals ranging from 7.7–427.3 to 35.2–1,077.9 copies per reaction. No cross-reactivity observed to selected non-target species. Compared with malacological surveys, the mLAMP-LFA showed higher detection rates for both S. japonicum (18.5% vs. 1.7%) and O. h. quadrasi (15.1% vs. 10.9%). Agreement between mLAMP-LFA and reference methods, assessed using Gwet’s AC1, was substantial for S. japonicum (AC1 = 0.774–0.779) and moderate to substantial for O. h. quadrasi (AC1 = 0.538–0.766), with all comparisons showing statistically significant agreement ( p < 0.001). The corresponding 95% confidence intervals were 0.672–0.881 for S. japonicum and 0.380–0.870 for O. h. quadrasi , indicating generally good agreement across molecular and malacological methods despite differences in detection approaches and sensitivity. Overall, the mLAMP-LFA provides a rapid, potentially field-adaptable, and equipment-minimal platform for multiplex eDNA surveillance that may strengthen schistosomiasis monitoring and support targeted control efforts in resource-limited endemic settings.