Hybrid Molecular Lateral Flow Assay Targeting the lipL32 Gene for Rapid and Highly Sensitive Detection of Pathogenic Leptospira

Leptospirosis remains a major neglected zoonotic disease in tropical and subtropical regions, where delayed diagnosis contributes to severe clinical outcomes and continued underrecognition. Although real-time PCR provides high diagnostic accuracy, its dependence on specialized instrumentation limits its use in resource-constrained endemic settings. This study developed and clinically evaluated a lipL32-targeted hybrid molecular lateral flow assay (Hybrid LFA) for visual detection of pathogenic Leptospira DNA in urine, plasma, and whole blood specimens. Thirty-five clinical samples, comprising 17 urine, 14 plasma, and 4 whole blood specimens, were processed using automated magnetic bead-based DNA extraction, followed by PCR amplification and lateral flow-based amplicon detection. Assay performance was compared with culture, real-time PCR, and conventional PCR. Real-time PCR showed complete agreement with culture, correctly identifying all 23 culture-positive and 12 culture-negative specimens. The Hybrid LFA detected 18 of 23 positive specimens, yielding an overall sensitivity of 78.26%, specificity of 100%, and diagnostic accuracy of 85.71%. Matrix-specific analysis showed the highest sensitivity in urine specimens (84.62%), followed by whole blood (75.00%) and plasma (57.14%), indicating that specimen type substantially influenced assay performance. In contrast, conventional PCR detected only 4 of 23 positive specimens, corresponding to a sensitivity of 17.39%. Analytical sensitivity testing demonstrated that the Hybrid LFA achieved a limit of detection of 1.13 × 10−7 ng/µL, approximately three orders of magnitude lower than agarose gel electrophoresis. These findings indicate that the lipL32-targeted Hybrid LFA is a highly specific PCR-coupled visual detection platform with promising potential for rapid leptospirosis screening, particularly using urine specimens. Larger prospective validation studies are required to confirm its clinical applicability in endemic and resource-limited settings.