Characterization of Leishmania infantum strains in blood samples from infected dogs and humans by PCR-RFLP

Abstract

Characterization of Leishmania infantum is based on zymodeme analysis, which requires parasite isolation and therefore is not routinely employed. Moreover, the majority of strains in the Mediterranean Basin belong to zymodeme MON-1, and this is a major limitation for this technique in epidemiological studies in this region. We developed a PCR-RFLP method based on kDNA amplification, which was able to discriminate L. infantum strains directly from peripheral blood. Twenty-eight samples were tested with this technique: four obtained from promastigote cultures, and 24 collected from dogs (18) and human donors (six) from traditionally endemic and newly endemic areas of northwestern Italy. Extracted DNAs were amplified using RV1-RV2 primers and PCR products were digested using two restriction enzymes separately: BsiY I and Mlun NI. Some patterns were specific to certain areas. In particular, the identity of PCR-RFLP patterns from a human patient from a newly endemic area and three dogs allow the confirmation of the autochthonous origin of this case. This approach could be applied to epidemiological studies in order to trace the diffusion of L. infantum within dog populations, as well as its transmission to humans.