Comparative evaluation and analytical performance of four lipL32-based qPCR assays for detection of pathogenic Leptospira spp

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BACKGROUND: Leptospira spp. cause leptospirosis, a globally distributed zoonosis with significant morbidity in tropical regions. Early diagnosis is challenging, particularly in the acute phase when serological markers may not yet be detectable. Molecular approaches such as real-time PCR (qPCR) enable direct pathogen detection; however, several lipL32-targeting assays have been described and rarely compared under equivalent conditions. METHODS: Four previously published qPCR assays (A-D) targeting the lipL32 gene were comparatively evaluated using serial dilutions of Leptospira interrogans culture to assess assay performance across decreasing relative bacterial loads. The assay showing the most consistent performance was further analyzed using genome equivalent (GEq)-based quantification to determine amplification efficiency, linearity, limit of detection (LoD), and reproducibility. Its performance was also evaluated across different experimentally inoculated human clinical matrices, including whole blood, serum, plasma, urine, and cerebrospinal fluid. RESULTS: Assay C demonstrated the most consistent performance, with an amplification efficiency of 95% and R² of 0.97. The LoD was established at 8.9 × 10⁰ GEq/µL, with consistent detection across replicates at this concentration. Whole blood, serum, CSF, and urine showed consistent detection across dilutions, whereas plasma exhibited reduced detection consistency at the low target levels. CONCLUSIONS: Among the evaluated assays, Assay C demonstrated robust and reproducible performance across multiple experimentally inoculated human clinical matrices. These findings support its potential application for molecular detection of Leptospira spp., while highlighting the importance of assay selection and pre-analytical optimization to improve molecular detection performance.