Consensus multilocus sequence typing (MLST) scheme for worldwide leishmania species classification

LAUTHIER, J; RUYBAL, P; HOYOS, C; BARROSO, PA; HASHIGUCHI, Y; MARCO, JD; KORENAGA, M

Toledo

Congreso; Worldleish 6; 2017

Worldleish 6

Abstract:Background:In leishmaniasis the differentiation of the parasites is relevant to eco-epidemiology, clinical diagnosis, and management of patients. Molecular identification of Leishmania parasites has been addressed using different kinds of techniques, where DNA based approaches offer multiple possibilities such as distinction among different isolates, their assignment to taxa, detection of Leishmania parasites, and detection of intra-specific diversity, and so on.Methods:Twenty-seven gene fragments, previously published by other authors, were analyzed by in silico approaches for 14 Leishmania spp. A total of 35.2% of polymorphisms were observed. A scheme optimization was carried out by testing all possible combinations among the genes (MLSTest 1.0). Monophyly of the groups and branch support were maximized in order to select the best 15 gene combination. Primers were designed and amplified over 25 reference strains of the main Leishmania spp. for the final selection of 3 and 5 gene combination. Maximum likelihood and Neighbor-Joining trees were constructed in order to identify variation among different strains (MEGA 6). Congruence among trees was evaluated by using Templeton test.Results:From the in silico approaches a global DP was calculated (0,994) and 15 gene candidates were selected: ASAT, GPI, 6PGD, PPA, MKK, ICD, MPI, G6PD, PGM, HX, ACON, ALAT, ENO, PMM and Ch36-1190. Those genes exhibited the best conditions for monophyly and branch support for every complex and species. All the genes were successfully amplified and two different MLST schemes were selected. The first scheme consists on: ENOL, PPA, GPI, 6PGD and HX. Meanwhile, the second MLST scheme was based on: ACO, ENOL and PPA. Both schemes exhibited good bootstrap values and showed the same phylogenetic relationships than the tree constructed with the 15 analyzed loci. Conclusions:To our knowledge, this is the first attempt to obtain a consensus MLST scheme from all the proposed gene targets used until nowadays. These two levels of MLST proposed in the current work will be called as a nested Multilocus Sequence Typing. Further analysis including more strains will be done.