title: Single locus genotyping to track Leishmania donovani in the Indian subcontinent: Application in Nepal.
abstract: We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010.
  
  
  
  As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002-2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution.
  
  
  
  Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region.
authors.affiliation: Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.|Department of Zoology, West Bengal State University, Barasat, West Bengal, India.
authors.forename: Keshav
authors.initials: K
authors.lastname: Rai
authors.affiliation: Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.
authors.forename: Narayan Raj
authors.initials: NR
authors.lastname: Bhattarai
authors.affiliation: Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, New York, NY, United States of America.
authors.forename: Manu
authors.initials: M
authors.lastname: Vanaerschot
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
authors.forename: Hideo
authors.initials: H
authors.lastname: Imamura
authors.affiliation: Department of Animal Science, College of Agriculture, Aksum University, Aksum, Ethiopia.
authors.forename: Gebreyohans
authors.initials: G
authors.lastname: Gebru
authors.affiliation: Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.
authors.forename: Basudha
authors.initials: B
authors.lastname: Khanal
authors.affiliation: Department of Internal Medicine, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.
authors.forename: Suman
authors.initials: S
authors.lastname: Rijal
authors.affiliation: Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium.
authors.forename: Marleen
authors.initials: M
authors.lastname: Boelaert
authors.affiliation: Department of Zoology, West Bengal State University, Barasat, West Bengal, India.
authors.forename: Chiranjib
authors.initials: C
authors.lastname: Pal
authors.affiliation: Department of Internal Medicine, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.
authors.forename: Prahlad
authors.initials: P
authors.lastname: Karki
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.|Department of Biomedical Sciences, University of Antwerp, Belgium.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Dujardin
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
authors.forename: Gert
authors.identifier: 0000-0002-3742-7046
authors.initials: G
authors.lastname: Van der Auwera
mesh_terms: D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007893:Leishmania donovani / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000453:epidemiology / Q000469:parasitology*; D017720:Molecular Epidemiology / Q000379:methods*; D009390:Nepal / Q000453:epidemiology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D062211:Spatio-Temporal Analysis