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Journal of Clinical Microbiology, September 2009, p. 2818-2825, Vol. 47, No. 9
0095-1137/09/$08.00+0 doi:10.1128/JCM.00645-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Development of a Multilocus Microsatellite Typing Approach for Discriminating Strains of Leishmania (Viannia) Species
Rolando Oddone,1,2
Carola Schweynoch,1
Gabriele Schönian,1*
Cintia dos Santos de Sousa,3
Elisa Cupolillo,3
Diego Espinosa,4
Jorge Arevalo,4
Harry Noyes,5
Isabel Mauricio,6 and
Katrin Kuhls1
Institut für Mikrobiologie und Hygiene, Universitätsmedizin Berlin, Dorotheenstr. 96, Berlin 10117, Germany,1 Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Rio de la Plata y Lagerenza, Asunción, Paraguay,2 Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365-Pav Leonidas Deane, Rio de Janeiro 21040-900, Brazil,3 Instituto de Medicina Tropical Alexander von Humboldt, Av. Honorio Delgado 430, San Martin de Porres, Lima 100, Peru,4 Biosciences Department, University of Liverpool, Crown St., Liverpool L69 7ZB, United Kingdom,5 London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 E7HT, United Kingdom6
Received 31 March 2009/ Returned for modification 20 May 2009/ Accepted 2 July 2009
A multilocus microsatellite typing (MLMT) approach based on the analysis of 15 independent loci has been developed for the discrimination of strains belonging to different Viannia species. Thirteen microsatellite loci were isolated de novo from microsatellite-enriched libraries for both Leishmania braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Markers were designed to contain simple dinucleotide repeats flanked by the minimal possible number of nucleotides in order to allow variations in repeat numbers to be scored as size variations of the PCR products. The 15 markers in total were amplified for almost all of the strains of Viannia tested; one marker did not amplify from the two L. peruviana strains included in the study. When 30 strains of L. braziliensis, 21 strains of L. guyanensis, and 2 strains of L. peruviana were tested for polymorphisms, all strains except two strains of L. guyanensis had individual MLMT types. Distance-based analysis identified three main clusters. All strains except one strain of L. guyanensis grouped together. Two clusters consisted of strains of L. braziliensis according to their geographical origins. The two strains of L. peruviana grouped together with strains of L. braziliensis from Peru and the adjacent Brazilian state of Acre. MLMT has proven capable of individualizing strains even from the same areas of endemicity and of detecting genetic structures at different levels. MLMT is thus applicable for epidemiological and population genetic studies of strains within the subgenus Viannia.
* Corresponding author. Mailing address: Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin Berlin, Dorotheenstr. 96, Berlin D-10117, Germany. Phone: 49-30-450524028. Fax: 49-30-450524902. E-mail: gabriele.schoenian{at}charite.de
Published ahead of print on 8 July 2009.
Journal of Clinical Microbiology, September 2009, p. 2818-2825, Vol. 47, No. 9
0095-1137/09/$08.00+0 doi:10.1128/JCM.00645-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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