Lateral flow assay for efficient detection of IgM antibodies in human leptospirosis

Introduction: Diagnosing leptospirosis using the Microscopic Agglutination Test (MAT) is a laborious and time-consuming process.PCR and ELISA-based assays require specialized equipment and trained technicians.Lateral flow assays (LFA) offer a point-of-care alternative, with advantages such as low cost, simplicity, rapid results, and suitability for field execution.Objectives: Development of a LFA for Human Leptospirosis Based on IgM Detection Using Recombinant Proteins from L. interrogans Serovar Copenhageni.Methodology: For the prototype, recombinant proteins produced at LATER/Bio-Manguinhos were tested both individually and in combination.MDI8, MDI 10, CN180 nitrocellulose membranes were evaluated with an anti-IgM colloidal gold conjugate and a control protein (protein A).Various reaction buffers, urea concentrations 0.5 M to 0.75 M and reaction times of 15, 20, 30 min were evaluated.Sera from leptospirosis-positive and -negative patients, together with sera positive for Chagas, hepatitis B and C, syphilis, toxoplasmosis, dengue, zika and chikungunya, were used to assess sensitivity, specificity, interrater reliability (kappa index) and cross-reactivity.Results: Optimum performance was achieved using three recombinant proteins, the MDI8 membrane, the following concentrations (recombinant proteins: 1.7 mg/mL; protein A: 0.5 mg/mL), the anti-IgM colloidal gold conjugate, and the impregnation flows (test line = 0.1 L/mm, control line = 0.06 L/mm, conjugate = 0.4 L/mm).Buffers containing 0.6 M to 0.75 M urea showed no significant differences (p = 0.99).The Friedman test indicated no significant differences in reaction times.No cross-reactivity was observed with eight different diseases.A kappa index of 0.88 (based on 162 positive sera) confirmed high reliability.The test demonstrated a sensitivity of 96% and a specificity of 100%, based on 89 acute positive sera and 71 negative sera. Conclusion:Although these preliminary results suggest the potential for developing a diagnostic tool with high sensitivity and specificity for the rapid detection of acute leptospirosis cases, further evaluations are needed.These should include panels of characterized sera from patients in the acute phase to validate the performance of the TR-Leptospirosis-IgM prototype.