Urine-based detection of Leishmania donovani antibodies using recombinant aurora kinase and otubain cysteine peptidase for noninvasive diagnosis of visceral leishmaniasis

ABSTRACT Visceral leishmaniasis (VL) is a fatal neglected tropical disease that requires early and accurate diagnosis to reduce mortality and interrupt transmission. Although parasitological confirmation using splenic aspirates is the gold standard, it is invasive and hazardous, while blood-based serological tests are often impractical in children and field settings. Consequently, noninvasive urine-based diagnostics represent an important unmet need. In the present study, we investigated the diagnostic utility of two novel Leishmania donovani antigens, otubain cysteine peptidase (LdOCP) and aurora I-related kinase (LdAIRK), using urine samples from VL-endemic regions of India. Both antigens play essential roles in parasite cell cycle progression and survival and are conserved among visceralizing Leishmania species. Urine specimens were collected from 135 individuals aged 10–60 years from Bihar and West Bengal between 2016 and 2024. Following preliminary immunoblot screening, antigen performance was validated using enzyme-linked immunosorbent assay and dipstick formats. Diagnostic accuracy was assessed using rK39 rapid diagnostic test results as the reference standard, and receiver operating characteristic analysis was performed. Both LdOCP and LdAIRK demonstrated 100% sensitivity (95% CI : 93.69–100%) and 100% specificity (95% CI: 86–100%). Mean optical density values were significantly higher in VL patients (0.491 ± 0.266 for LdOCP; 0.749 ± 0.200 for LdAIRK) than in controls (0.092 ± 0.046). Longitudinal analysis revealed a marked decline in antigen-specific antibody levels 6 months after treatment, indicating potential utility as test-of-cure markers. Concordant performance in dipstick assays supports feasibility for urine-based point-of-care diagnosis in resource-limited endemic settings and may aid treatment decisions where laboratory infrastructure is limited. IMPORTANCE This study demonstrates that urine-based detection of Leishmania donovani -specific antibodies using LdOCP and LdAIRK provides a highly accurate, noninvasive approach for visceral leishmaniasis diagnosis and prognosis. The excellent sensitivity and specificity, together with a clear post-treatment antibody decline, support application in field and rural settings. Importantly, the proposed urine biomarker panel offers a scalable framework for future development of multiplex, instrument-free diagnostic platforms, facilitating simultaneous detection of multiple pathogens. These findings lay the groundwork for prospective clinical validation, integration into point-of-care workflows, and broader deployment for infectious disease surveillance in resource-limited endemic regions with significant translational public health impact potential.