Feline leishmaniosis: New insights into serological and molecular detection of Leishmania spp

Feline leishmaniosis (FeL) is increasingly recognized in geographical areas where leishmaniosis is endemic. Despite advances in understanding FeL, its diagnosis remains poorly investigated, and the methods currently employed are adapted from canine protocols, overlooking feline-specific biological and pathological features. Here, we evaluated the performance of serological and molecular diagnostic approaches for FeL detection using invasive and non-invasive samples. Based on the estimated minimum sample size, 51 cats were enrolled through convenience sampling. The cats underwent clinical examinations and were tested using parasitological (cytology, histology, and culture), serological Dual Path Platform (DPP®) and Indirect Fluorescent Antibody Test (IFAT), and molecular techniques targeting Leishmania spp. Kinetoplast DNA (kDNA-PCR) and the Internal Transcribed Spacer 1 of ribosomal DNA of Trypanosomatidae (ITS1-PCR). Parasitological methods confirmed Leishmania spp. infection in 7.8% of cats. All parasitologically positive cats exhibited clinical signs consistent with FeL, and infection was confirmed as L. infantum by sequencing. Using parasitological methods combined with clinical signs as the reference standard for confirmed FeL cases, IFAT showed 100% (CI: 40%-100%) sensitivity (Se) and 40% (CI: 26%-57%) specificity (Sp) at a 1:40 cut-off. At a 1:80 cut-off, Se remained at 100% (CI: 40%-100%), and Sp increased to 62% (CI: 46%-76%). DPP® showed higher Sp (95%; CI: 84%-99%). Molecular analysis showed 100% Se and Sp among invasive samples (lymph node, bone marrow, skin, and blood) for both kDNA-PCR and ITS1-PCR, with excellent kappa agreement between assays (κ: 0.898). Among non-invasive samples, conjunctival swabs demonstrated better diagnostic accuracy for both PCR assays, with kDNA-PCR showing improved detection [100% (CI: 40%-100%) Se and 98% (CI: 88%-100%) Sp]. In contrast, oral swabs showed reduced detection capability for both PCR methods, particularly for ITS1-PCR. Our results support the need to standardize serological tools for use in cats. Additionally, the findings support the inclusion of blood and conjunctival swabs as practical alternatives for molecular diagnosis, particularly under field conditions. This study contributes to optimizing diagnostic strategies for FeL in endemic areas and provides practical insights for veterinary diagnosis and epidemiological surveillance.