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Development and evaluation of different PCR-based typing methods for discrimination of Leishmania donovani isolates from Nepal

Published online by Cambridge University Press:  29 January 2010

N. R. BHATTARAI ,
J. C. DUJARDIN ,
S. RIJAL ,
S. DE DONCKER ,
M. BOELAERT  and
G. VAN DER AUWERA
N. R. BHATTARAI
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium B.P. Koirala Institute of Health Sciences, Dharan, Nepal Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
J. C. DUJARDIN
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
S. RIJAL
Affiliation:
B.P. Koirala Institute of Health Sciences, Dharan, Nepal
S. DE DONCKER
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium
M. BOELAERT
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium
G. VAN DER AUWERA*
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium
*
*Corresponding author: Department of Parasitology, Institute of Tropical Medicine Antwerp, Nationalestraat 155, 2000 Antwerp, Belgium. Tel: +32 3 2476586. Fax: +32 3 2476359. E-mail: [email protected]
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Summary

Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

Keywords

visceral leishmaniasisLeishmania donovaniPCR-RFLPkinetoplast DNANepalTeraimicrosatellitecysteine proteinaseglycoproteinhydrophilic acylated surface protein
Type
Research Article
Information
Parasitology , Volume 137 , Issue 6 , May 2010 , pp. 947 - 957
Copyright
Copyright © Cambridge University Press 2010

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