A newly developed rapid K39-based LeishmaCHECK Ab ELISA for high-throughput serological screening of canine leishmaniasis.

Canine leishmaniasis (CanL) is a zoonotic vector-borne disease caused by Leishmania infantum, with dogs acting as the main reservoir for human infection. Reliable serological assessment is essential for disease surveillance and control. This study describes the development and validation of LeishmaCHECK Ab ELISA, a new rapid qualitative and semi-quantitative assay for the detection of anti-L. infantum antibodies in canine serum. Cut-off values were established using multiple statistical approaches, including Youden's index and receiver operating characteristic (ROC) curve analysis. The assay was evaluated on 552 canine samples (313 positives, 206 negatives, 33 borderline), using IFAT as the reference serological method. LeishmaCHECK Ab ELISA showed excellent agreement with IFAT (Cohen's kappa of 0.93), achieving a relative sensitivity of 95.2% and a relative specificity of up to 99.5%. Comparative analyses also demonstrated superior diagnostic performance compared with other commercially available ELISA tests. These findings indicate that LeishmaCHECK Ab ELISA represents a reliable alternative tool for the serological screening of canine leishmaniasis.