Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis

Background Treatment response in canine leishmaniosis is driven by the dog host, the Leishmania parasite, and pharmacological factors, with drug resistance increasingly undermining the effectiveness of therapy. A direct quantitative PCR test (LeishGenR™) was applied to 104 clinical samples from 95 dogs in the Mediterranean area diagnosed with leishmaniosis in veterinary clinical settings and testing positive for Leishmania infantum by PCR. The assay enabled rapid detection of genetic drug-resistance biomarkers for allopurinol ( metk ), meglumine antimoniate ( mrpa ), and miltefosine ( LdMT ), providing a clinically relevant, timely alternative to culture-based approaches by directly analyzing circulating Leishmania infantum amastigotes. Results The assay (LeishGenR™) showed high specificity (100%) and sensitivity (> 87.5%) for genetic drug-resistance profile assignment and a strong correlation with whole-genome sequencing for gene copy number assessment ( metk : r = 0.878; mrpa : r = 0.943 and LdMT  = 0.691). Genetic drug-resistance biomarkers were detected in 24.3% of L. infantum DNA from clinical samples analyzed (20/82; 95% CI 16.3–34.6)), most commonly for allopurinol (13.4%; 95% CI 7.6–22.4), then meglumine antimoniate (9.4%; 95% CI 4.6–18.2), and for miltefosine (5.4%; 95% CI 1.8–14.8). Prevalence was higher in dogs previously treated for leishmaniosis. Conclusion This study demonstrates the ability to detect genetic biomarkers of drug resistance in L. infantum directly from clinical samples of dogs with leishmaniosis. This method enables rapid, precise detection of genomic biomarkers, circumventing delays associated with culture-based methods and supporting more effective clinical management and surveillance. Among dogs with high parasitemia referred to clinics in Mediterranean regions sampled in this study, the findings reveal a significant prevalence of circulating L. infantum strains carrying genomic drug resistance biomarkers to standard treatments for canine leishmaniosis.