Carrasco Martin, M. | bioRxiv Science

bioRxiv2026

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

BackgroundTreatment 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. ResultsThe 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. ConclusionThis 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.

genetics↗

bioRxiv2025

Drug-Resistance Biomarkers in Leishmania infantum through Nanopore-Based Detection of Aneuploidy and Gene Copy Number Variations with LeishGenApp

BackgroundDrug-resistant strains of Leishmania infantum challenge the effectiveness of treatments for clinical leishmaniosis and may lead to more frequent relapses. Copy number variation (CNV) at specific genetic loci is associated with drug resistance and virulence, but information about its prevalence in endemic regions is limited. This study examines the drug resistance and virulence status of Leishmania strains in human and canine isolates from the Mediterranean region. MethodsForty-eight Leishmania infantum isolates were whole-genome sequenced with nanopore long reads, followed by de novo assembly. We analyzed chromosomal aneuploidies and gene copy number variation in loci linked to drug resistance and virulence in Leishmania, alongside the genomic structure and rearrangements responsible for these variations. ResultsComplete genomes were de novo assembled for 35 L. infantum isolates (22 from dogs and 13 from humans), revealing significant chromosomal variability. We assessed copy number variation for 22 potential biomarkers: 15 genes related to drug resistance to first-line drugs (METK for allopurinol; LdSMT for amphotericin B; AQP1 and H-locus for antimonials; LdMT, LdRos3, and MSL for miltefosine; PPM for paramomycin) and seven genes related to virulence (lipophosphoglycan and proteophosphoglycan biosynthesis, and the Lack protein). Drug-resistance biomarkers were identified in 80% of the isolates. Canine strains primarily showed resistance to allopurinol and antimonials, while human isolates exhibited a broader resistance spectrum, especially to antimonials and paromomycin. The co-occurrence of resistance biomarkers was common, especially for allopurinol and antimonial resistance. Distinct mechanisms underlie the observed copy number variations. Virulence-associated genes were less variable among isolates. ConclusionsThe prevalence of drug-resistance biomarkers in Leishmania infantum strains from the Mediterranean region, as revealed by this study, underscores the critical need for routine resistance surveillance in managing clinical leishmaniosis. These findings not only inform current clinical practice but also pave the way for more effective management strategies in the future.

genetics↗