Evolutive History of Leishmania Genus and Differential Diagnosis of Clinical Important Species Based on A Unique Kinetoplastida Chitinase

Leishmaniasis, a neglected infectious disease affecting humans, domestic and wild animals, caused by 20 from 53 Leishmania genus species, is transmitted by sandflies. Leishmania genus, belonging to Trypanosomatide Family and Kinetoplastida Order, are grouped in five subgroups according to biogeographic and evolutive history of parasites and hosts, which has led to incongruences and paraphyly. The GH18 Leishmania chitinase, encoded by a specie-specific single copy gene, conserved in basal groups of trypanosomatids, and absent in the genus Trypanosoma, was evaluated as a phylogenetic marker and a diagnostic target. Primers were designed to detect Leishmania in its host biological samples and to obtain the chitinase sequence of species not available in public databanks. The GH18 chitinase gene and its genomic context was evaluated phylogenetically. A protocol to discriminate among Leishmania subgenera by PCR and restriction fragment length polymorphism (RFLP) was developed using in silico tools. A PCR method to detect a partial 953 bp GH18 quitinase encoding gene presented high sensibility and specificity on isolated parasites DNA and using as negative controls, Trypanosoma cruzi, and DNA from Leishmania hosts. Preservation of the chitinase locus in the aquatic free-living protozoan Bodo saltans, disclose a primitive common origin. GH18 trypanosomatide chitinase amino acid sequence comparative analysis revealed high similarity to chitinase from marine prokaryotes and protozoan. Phylogenetic reconstruction based on chitinase corroborates the Supercontinent Origins theory for Leishmania. The chitinase encoding gene was effectively detected in biological samples and for differential molecular diagnosis among Leishmania clinical important species worldwide.