Rapid Diagnosis of Plasmodium knowlesi Infection Utilizing the Loop-Mediated Isothermal Amplification Technique Coupled with a Lateral Flow Assay

Objective: To develop a highly sensitive and specific molecular diagnostic assay for detecting P. knowlesi by combining LAMP with lateral flow assay (LFA).Material and Methods: Six LAMP primers targeting the P. knowlesi 18S rRNA gene were designed, incorporating FITC and biotin labels into the FIP and LoopF primers. Specificity and sensitivity were evaluated using DNA from various Plasmodium species. Clinical samples were tested, and the results were visualized on LFA.Results: The PkLAMP-LFA assay demonstrated a sensitivity of 95.4% (95% CI: 84.5–99.4%) and a specificity of 100% (95% CI: 15.8–100%) compared to nested PCR. The limit of detection was 10 copies/µL. The assay produced results within 60 minutes and required only minimal equipment.Conclusion: The PkLAMP-LFA assay is a rapid, accurate, and field-deployable diagnostic tool for P. knowlesi, offering a practical solution for improved malaria detection and control in endemic regions.