Evaluation of Multi-Target Genotyping (ITS-hsp70-cpb) for Detecting Population Heterogeneity Within Mediterranean Leishmania infantum, with a Focus on Zymodeme MON-24

<i>L. infantum</i> and <i>L. donovani</i>, distinct species in the <i>L. donovani</i> complex, show high phenotypic and genotypic polymorphism and share molecular traits. Therefore, genotyping by a single molecular target can give uncertain results. This study focuses on genotyping a set of <i>L. donovani</i> complex strains, including 18 zymodemes classified according to Montpellier nomenclature (ZMONs) and different clinical forms, by internal transcribed spacer (ITS), heat shock protein 70 (<i>hsp70</i>), and cysteine proteinase b (<i>cpb</i>) sequencing to evaluate their ability in species discrimination. We found an unexpected <i>L. infantum hsp70</i> variability, with 8 sequence variants. <i>Cpb</i>-PCR could not distinguish <i>L. donovani</i> complex species, due to a <i>L. infantum</i> intraspecific allelic (<i>cpbEF</i>) polymorphism. By combining <i>ITS</i>-<i>hsp70-cpb</i> sequence variants, we obtained different genotypes. <i>ITS(A)-hsp70inf(2)-cpbE</i> identified 69.9% of <i>L. infantum</i> strains representing 12 ZMONs from Mediterranean visceral and cutaneous cases, <i>ITS(A)-hsp70inf(2)-cpbF</i> identified a non-ZMON-1 cluster. Four genotypes represented ZMON-24: <i>ITS(A, B)-hsp70(Y)-cpbF</i> identified a cutaneous cluster from Italy and North Africa, <i>ITS(A, Lombardi)-hsp70(2)-cpbE</i> identified cutaneous and visceral cases from Mediterranean areas. We believe this study contributes to an overview of <i>L. infantum</i> variant populations, and to the discussion on diagnostic targets, in single or multi -target-based approaches, to identify <i>Leishmania</i> populations in the Mediterranean area.