Rapid Visual Detection of Pythium insidiosum by LAMP-LFD

Pythium insidiosum, the causative agent of pythiosis, remains an important pathogen in tropical and subtropical regions, where delayed diagnosis is associated with severe disease and poor clinical outcomes. We developed a loop-mediated isothermal amplification coupled with lateral flow dipstick (LAMP-LFD) assay for the rapid detection of P. insidiosum. The assay employs dual-labeled LAMP amplicons that are directly detected by a lateral flow dipstick, enabling simple visual interpretation without agarose gel electrophoresis. Analytical performance was evaluated using genomic DNA from 102 clinically relevant isolates, comprising 51 P. insidiosum isolates and 51 non-target organisms. The LAMP-LFD assay correctly detected all P. insidiosum isolates, achieving an inclusivity of 100.0% (95% CI: 93.0-100.0%), and correctly excluded 48 of 51 non-target organisms, yielding an exclusivity of 94.1% (95% CI: 83.8-98.8%). The assay demonstrated a detection limit of 10 fg of genomic DNA per reaction, representing at least 1000-fold greater analytical sensitivity than the reference multiplex PCR (m-PCR) assay. Clinical validation using 47 blinded animal tissue specimens showed a sensitivity of 90.3%, specificity of 87.5%, accuracy of 89.4%, and good agreement with the reference standard (culture and ITS sequence analysis) (κ = 0.76). In comparison, the m-PCR assay achieved a sensitivity of only 41.9%, indicating substantially lower clinical detection performance. In conclusion, direct LFD-based detection preserves the analytical performance of the established P. insidiosum LAMP assay while providing a clear and easily interpretable visual endpoint. The LAMP-LFD assay represents a rapid, sensitive, and practical tool for screening pythiosis, offering improved diagnostic performance and more reliable result interpretation.