Molecular epidemiology of Leishmania infantum on the island of Majorca: a comparison of phenotypic and genotypic tools

Abstract

In the Mediterranean basin, Leishmania infantum is the causative agent of both visceral and cutaneous leishmaniasis, and is an important opportunistic parasite in patients infected with human immunodeficiency virus (HIV). The commonest method used to study the variability of Leishmania spp. is isoenzyme analysis. In addition to this, we employed 3 assays based on the polymerase chain reaction (PCR): random amplified polymorphic deoxyribonucleic acid (RAPD), intergenic region typing (IRT), based on the amplification of ribosomal ribonucleic acid internal transcribed spacers and restriction fragment length polymorphism (PCR-RFLP). We used 54 L. infantum stocks isolated from HIV co-infected patients, 38 isolated from dogs, 3 isolated from immunocompetent patients and 3 isolated from 1826 sand files in the island of Majorca (Spain), a closed ecological niche. Zymodemes MON-1 (70%), MON-24 (11%) and MON-34 (18%) were found among the human isolates, and MON-1 (95%) and MON-108 (5%) among those from dogs. RAPD and IRT could not discriminate among the strains as they all gave the same pattern, even when different zymodemes were examined. In contrast, PCR-RFLP was able to distinguish the strains and, furthermore, a dendrogram (unweighted pair group method with arithmetic average [UPGMA]) was constructed from the genetic distances derived from RFLP data. The Leishmania isolates from HIV-infected subjects formed a single cluster, supporting the existence of an artificial anthroponotic cycle previously proposed by our group, in which syringes have been substituted for sand flies, and in which certain clones have been spread among intravenous drug users. This contrasts with the clusters representing a zoonotic cycle, involving dogs, sand flies and both immunocompetent and immunocompromised humans.