High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples

Abstract
Background and Objectives: Leishmania (L.) infantum is the primary causative agent of visceral leishmaniasis (VL) in the Mediterranean and Americas. Various molecular techniques, including high resolution melting (HRM) analysis, have been developed to detect and identify L. infantum infections. HRM is an automated molecular method that can detect and differentiate various genera and species of infectious agents. The objective of this study was to diagnose and identify L. infantum infection in canines from northeastern Iran using real-time PCR combined with HRM analysis. The results were compared to anti-L. infantum antibodies detected using the direct agglutination test (DAT) in both domestic and wild canines.

Methods: Serum samples were collected from 15 foxes, 14 jackals, 7 domestic dogs, and 3 wolves in villages around Shirvan and Bojnourd districts in northeastern Iran during 2014-2015. Initially, all serum samples were tested for anti-L. infantum antibodies using DAT. Genomic DNA was then extracted from the samples, and real-time PCR-HRM analysis was performed, targeting the hsp70, ITS1, and gp63 genes. Statistical analysis was used to evaluate the agreement between DAT and HRM assay results.

Results: Of the 39 serum samples, 8 were positive for anti-L. infantum antibodies at a 1:80 titer, and 1 sample had a titer of 1:160. All 9 seropositive samples tested positive by HRM analysis. Additionally, 3 DAT-negative samples were found to be positive by HRM. In total, 12 out of 39 DNA samples showed positive HRM results. Among the three gene sequences tested, gp63 provided the best species separation and identification.

Interpretation and Conclusion: HRM analysis targeting the hsp70, ITS1, and gp63 genes is a highly sensitive method for screening and early detection of L. infantum infection in both wild and domestic canines. It JG98 outperforms DAT in terms of accuracy and enables the detection and differentiation of various Leishmania species responsible for leishmaniasis.