Multilocus Microsatellite Typing as a New Tool for Discrimination of Leishmania infantum MON-1 Strains

Abstract

The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.Leishmania is a genus of protozoan flagellates that cause a broad spectrum of diseases, ranging from self-limiting localized cutaneous lesions to visceral leishmaniasis with fatal spontaneous evolution (2). The majority of visceral manifestations are caused by parasites of the Leishmania donovani complex (26), which consists of L. donovani Ross, 1903; L. infantum Nicolle, 1908-L. chagasi Cunha and Chagas, 1937; and L. archibaldi Castellani and Chalmers, 1919. Currently, multilocus enzyme electrophoresis (MLEE) is the generally accepted "gold standard" for the identification and classification of isolates of Leishmania. By this method, strains are divided into groups with identical enzyme patterns, called "zymodemes." The main criticism of this approach is that genotypes are assayed indirectly, with the consequence that nucleotide substitutions may not be observed in synonymous sites or in nonsynonymous sites, if it is assumed that subsequent changes in the amino acid composition do not lead to different electrophoretic mobilities. In contrast, posttranslational modifications may change the electrophoretic mobilities, despite identical genotypes. Furthermore, the method is quite slow, laborious, and costly. Growth in vitro is inevitable, and the data sets of the few laboratories in which these analyses are performed are difficult to compare.Unlike, e.g., L. donovani or L. tropica, which present extended genetic and enzymatic polymorphisms (27, 53), L. infantum is a relatively uniform species (46). More than 80% of the strains of L. infantum isolated thus far belong to the predominant zymodeme, 42). The alternative methods for ...

Atypical Lesions as a Sign of Cutaneous Dissemination of Visceral Leishmaniasis in a Human Immunodeficiency Virus–Positive Patient Simultaneously Infected by Two Viscerotropic Leishmania Species

“…Two Leishmania isolates were obtained and characterized by enzyme electrophoresis (MON-1, type A), 11 and IOC/L2930 had an L. donovani MON-2 sequence (Genbank accession numbers FN398343 and FN398344 for IOC/L3020 and IOC/L2930, respectively). Multilocus microsatellite typing 12,13 confirmed this data. When cutaneous leishmaniasis was diagnosed, Montenegro's skin test was negative, and results of immunofluorescent assay was positive at a dilution of 1/720.…”

An Assessment of the Genetic Diversity of Leishmania infantum Isolates from Infected Dogs in Brazil

“…Five of these seven markers were shown to be monomorphic. Using the same seven microsatellite markers, other works have detected locus polymorphism among L. donovani complex isolates from several locations in the Old World (India, East Africa, the Mediterranean Basin, 30 Portugal, Spain, and Greece…”

“…Seven microsatellite markers were selected for the present study: Li22-35, Li23-41, Li45-24, Li71-33, Lm2TG, Lm4TA, and TubCA. 30 These markers were previously developed for the L. donovani complex and showed polymorphism within the MON-1 zymodeme, the predominant L. infantum zymodeme in the Old World and New World. 17,30 Each polymerase chain reaction (PCR) was performed using fluorescent primers, 3 ng DNA target, and a stepdown protocol as described earlier.…”

“…30 These markers were previously developed for the L. donovani complex and showed polymorphism within the MON-1 zymodeme, the predominant L. infantum zymodeme in the Old World and New World. 17,30 Each polymerase chain reaction (PCR) was performed using fluorescent primers, 3 ng DNA target, and a stepdown protocol as described earlier. 16 The allele sizes were determined in a 6% denaturing polyacrylamide gel using an Automatic Laser Fluorescent (ALF) sequencer (GE Healthcare, Milwaukee, WI).…”

Development of a Multilocus Microsatellite Typing Approach for Discriminating Strains of Leishmania ( Viannia ) Species

“…After differential stringency washes, the DNA was recovered, amplified by PCR, and ligated into plasmid vector pCR 2.1-TOPO (Invitrogen). Plasmids containing a microsatellite insert were identified by the presence of two or more amplified products after PCR with the linker and the (GT) 12 oligonucleotides and subsequently by hybridization with a (GT) 12 probe labeled with [ guyanensis MHOM/SR/87/TRUUS1 was prepared by a modification of the method described above (27).…”