Molecular detection of Leishmania spp. in feline squamous cell carcinoma in Portugal.

Cutaneous manifestations of feline leishmaniosis may occur at anatomical sites commonly affected by squamous cell carcinoma (SCC), creating potential diagnostic overlap. Although concurrent Leishmania infection and SCC have been described in individual cats, the presence of parasite DNA in feline SCC tissue has not been systematically investigated. This retrospective cross-sectional study evaluated 219 formalin-fixed, paraffin-embedded feline SCC samples submitted to a veterinary diagnostic laboratory in Portugal between 2020 and 2023. Samples were screened using a Leishmania-genus-specific nested PCR targeting SSU rDNA. PCR-positive cases were subsequently analyzed at four additional loci (cytB, g6pdh, hsp70, and ITS rDNA) and reassessed histopathologically. Leishmania spp. SSU rDNA was detected in 3/219 samples (1.4%; 95% CI: 0.5-3.9%), corresponding to SCC lesions on the pinna, nasal planum, and eyelid. Molecular characterization was possible in one case, involving an SCC of the nasal planum. Phylogenetic analysis showed that the hsp70 sequence clustered with reference sequences of L. infantum, whereas the cytB sequence grouped within the L. donovani complex without species-level resolution. In the same lesion, structures morphologically compatible with amastigotes were observed within macrophages in the tumor-associated inflammatory infiltrate. Overall, Leishmania DNA was detected in only a small proportion of feline SCCs, and the findings support coexistence rather than an association between infection and tumor development. These findings have direct diagnostic relevance in endemic areas, where histopathologic confirmation of SCC should not preclude investigation for concurrent Leishmania infection when clinical or epidemiologic suspicion persists.