Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species

In Leishmania, DPPIII plays a significant role in parasite growth and survival. A previous study used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay specific to dermotropic Old World (OW) Leishmania species. This study aimed to further assess the use of the DPPIII gene for Leishmania species identification and phylogeny, and thus for diagnostic and molecular epidemiology studies of OW Leishmania species. Orthologous DPPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assay was developed and 72 Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples. Sequence analysis showed 478 variable sites, with 302 being parsimony informative. A test of natural selection (dN-dS) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. The study demonstrated that the DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica, and L. turanica, and the L. (Sauroleishmania) L. tarentolae. The species L. donovani and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples. The DPPIII gene is an essential gene conserved in all Leishmania species. Comparative sequence analyses of the DPPIII gene fragments and the development of DPPIII-based PCR RFLP assays showed its potential for molecular diagnosis, epidemiology, and taxonomy. The study concluded that this molecular marker can be used to complement other molecular methods for OW Leishmania species identification and discrimination and can contribute to evidence-based disease control and surveillance. Leishmaniases are a group of neglected tropical diseases caused by protozoan parasites members of more than 20 Leishmania species. Leishmaniases are epidemiologically complex diseases, with their complexity relating to the diversity of species and transmission cycle components, often poorly elucidated. Risk factors for changing eco-epidemiological profiles include environmental and climate changes, migrations, and conflicts, contributing to leishmaniases emergence in disease-free areas, leading to co-sym-patry of the major pathogens in the same transmission areas and even foci. Taxonomical identification of the parasites is a central issue for patient management, molecular epidemiology, and thus disease control and surveillance.