Multilocus sequence typing of seven genetic loci to discriminate strains of L. donovani isolated from Bangladesh.

Background: Leishmania (L.) donovani, a species of the L. donovani complex, causes fatal visceral leishmaniasis (VL) disease in the Indian Subcontinent, parts of Asia and a large part of Africa. Genetic variations in Leishmania cause heterogeneity, spread of virulent strains, resistance to chemotherapeutics and exploitation of different hosts and vectors. Previously, L. donovani strains in the Indian subcontinent were detected genetically homogeneous by MLMT. Later study using genome-wide single nucleotide polymorphism (SNP) markers showed that genetic variation existed in L. donovani strains isolated from Nepal and India. We aimed to reveal intraspecies genetic variation between strains of L. donovani derived from Bangladesh. Methods & Materials: Twenty two newly isolated L. donovani strains from VL cases were investigated in a cross sectional study by MLST of seven functionally independent housekeeping genes located in different chromosomes. Target loci were rhomboid like serine protease, fatty acid/sphingolipid δ-4 desaturase, serine/threonine-protein kinase, 5' A2rel-related gene, ubiquitinactivating enzyme e1, mannosyltransferase and splicing factor 3B subunit1 gene. Sequences of respective genes of L. donovani retrieved from GenBank and that obtained after sequencing of reference strains of L. donovani and L. infantum were used for comparison. Results: Unambiguous sites of SNPs were detected in analysed sequences across all loci. The 5' a2rel-related locus was the most diverge region with 26 SNPs, followed by mannosyltransferase with 10, splicing factor 3B subunit1 with 6, ubiquitin-activating enzyme e1 with 5, rhomboid like serine protease with 4; fatty acid/sphingolipid δ-4 desaturase and serine/threonine-protein kinase possessed 1 SNP each. Two large closely related clusters (one contained 10 and the other 7 isolates) and five separate individual groups among 22 Bangladeshi isolates were revealed by phylogenetic analysis showing the presence of 7 genotypes among L. donovani strains in the country. Conclusion: The study illustrates that intra-species genetic diversity between strains of L. donovani does exist in Bangladesh. A combination of sequencing of 5' a2rel-related gene and mannosyltransferase gene are sufficient to differentiate strains of the L. donovani. The MLST can be a future tool to determine intra- and inter-species genetic variation of Leishmania. [ABSTRACT FROM AUTHOR]