Baseline of miltefosine resistance markers in zoonotic Leishmania infantum from Eastern Mediterranean dogs prior to widespread drug use

Miltefosine, the only oral antileishmanial drug approved for human use, is increasingly administered in canine leishmaniasis. Reduced susceptibility in Leishmania infantum has been linked to alterations in the miltefosine transporter gene (LMT; LINF_130020800) and deletion of the Miltefosine Sensitivity Locus (MSL) on chromosome 31. We analysed LMT presence and MSL integrity by PCR in 66 L. infantum isolates collected between 1996 and 2012 from dogs with clinical leishmaniasis in endemic areas of Greece, Cyprus and Türkiye. All isolates carried LMT, indicating presence of the PCR target corresponding to the miltefosine transporter gene. In contrast, 13.6% exhibited MSL deletion (targeting LinJ.31.2370), detected in Greek (33.3%) and Cypriot (9.7%) strains, whereas all Turkish strains retained an intact MSL locus. Given the strong association between MSL deletion and miltefosine treatment failure, these pre-drug-pressure findings provide critical baseline data for the Eastern Mediterranean and underscore the need for molecular surveillance within a One Health framework.