Consensus Multilocus Sequence Typing (MLST) scheme for worldwide Leishmania species classification

LAUTHIER JUAN JOSÉ; RUYBAL PAULA; HOYOS CARLOS; BARROSO PAOLA; HASHIGUCHI YOSHIHISA; MARCO JORGE DIEGO; KORENAGA MASATAKA

Toledo

Conferencia; Wordleish 6, 6th World Conference on Leishmaniasis; 2017

BackgroundIn leishmaniasis the differentiation of the parasites is relevant to eco-epidemiology, clinical diagnosis, andmanagement of patients. Molecular identification of Leishmania parasites has been addressed using different kinds oftechniques, where DNA based approaches offer multiple possibilities such as distinction among different isolates, theirassignment to taxa, detection of Leishmania parasites, and detection of intra-specific diversity, and so on.MethodsTwenty-seven gene fragments, previously published by other authors, were analyzed by in silico approaches for 14Leishmania spp. A total of 35.2% of polymorphisms were observed. A scheme optimization was carried out by testingall possible combinations among the genes (MLSTest 1.0). Monophyly of the groups and branch support weremaximized in order to select the best 15 gene combination. Primers were designed and amplified over 25 referencestrains of the main Leishmania spp. for the final selection of 3 and 5 gene combination. Maximum likelihood andNeighbor-Joining trees were constructed in order to identify variation among different strains (MEGA 6). Congruenceamong trees was evaluated by using Templeton test.ResultsFrom 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 exhibitedthe best conditions for monophyly and branch support for every complex and species. All the genes were successfullyamplified and two different MLST schemes were selected. The first scheme consists on: ENOL, PPA, GPI, 6PGD andHX. Meanwhile, the second MLST scheme was based on: ACO, ENOL and PPA. Both schemes exhibited goodbootstrap values and showed the same phylogenetic relationships than the tree constructed with the 15 analyzed loci.ConclusionsTo our knowledge, this is the first attempt to obtain a consensus MLST scheme from all the proposed gene targets useduntil nowadays. These two levels of MLST proposed in the current work will be called as a nested MultilocusSequence Typing. Further analysis including more strains will be done.