Multilocus microsatellite typing (MLMT) reveals genetically isolated populations between and within the main endemic regions of visceral leishmaniasis

Multilocus enzyme electrophoresis (MLEE) is the gold standard for taxonomy and strain typing of Leishmania, but has some limitations. An alternative reliable and fast genotyping method for addressing population genetic and key epidemiological questions, is multilocus microsatellite typing (MLMT). MLMT using 15 markers was applied to 91 strains of L. donovani, L. archibaldi, L. infantum and L. chagasi from major endemic regions of visceral leishmaniasis. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. Six main genetically distinct populations were identified: (1) L. infantum/L. chagasi MON-1 and (2) L. infantum/L. chagasi non-MON-1, both Mediterranean region/South America; (3) L. donovani (MON-18), L. archibaldi (MON-82), L. infantum (MON-30, 81) and (4) L. donovani (MON-31, 274), L. archibaldi (MON-82, 257, 258), L. infantum (MON-267), both Sudan/Ethiopia; (5) L. donovani MON-2, India; (6) L. donovani (MON-36, 37, 38), Kenya and India. Substructures according to place and time of strain isolation were detected. The VL populations seem to be predominantly clonal with a high level of inbreeding. Allelic diversity was highest in the Mediterranean region, intermediate in Africa and lowest in India. MLMT provides a powerful tool for global taxonomic, population genetic and epidemiological studies of the L.donovani complex.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

Genetic polymorphism of Algerian Leishmania infantum strains revealed by multilocus microsatellite analysis

The present study applies multilocus microsatellite typing (MLMT) for studying the polymorphism among 55 strains of Leishmania infantum from Algeria. These strains from different Algerian foci representing different zymodemes, hosts and clinical forms were analysed using 14 microsatellite markers. All 55 strains had individual MLMT profiles and no relationship was observed between them and different host or geographical origins. Three populations of Algerian L. infantum were identified by a Bayesian clustering approach implemented in STRUCTURE software and supported by genetic distance analysis. Two populations, A and B, consisted mainly of strains belonging to zymodeme MON-1, and the third population, C, mainly of MON-24 strains isolated from cutaneous leishmaniasis cases. Interestingly, a small group of strains appeared as a mixture of different populations and might be putative hybrids. Genetic migration was noticed among the two MON-1 populations, A and B, as well as between populations A and C. Due to its high discriminatory power MLMT could be also successfully applied for differentiating relapses or re-infection for patients suffering from multiple episodes of visceral leishmaniasis.     

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

Background

Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region.

Principal Findings

KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World.

Conclusions

LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.     

Comparative microsatellite typing of new world leishmania infantum reveals low heterogeneity among populations and its recent old world origin

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe.     

Parasite identification in the surveillance of imported leishmaniasis cases in Italy

An accurate Leishmania classification was defined since 1980s by the use of isoenzyme analysis. To date, this procedure still represents the reference identification technique, despite the increasing use of molecular approaches. Studies and surveillance methods on leishmaniases are strongly conditioned by the knowledge and mapping of all the parameters characterizing each nosogeographical entity. On this respect, the identification of parasites from all the actors of the natural life cycle plays a key role. With the increasing population movements and climate changes, novel risk factors could be identified associated to Leishmania geographical distribution and spreading: a) the introduction into Italy of new populations of L. infantum from other countries; b) the introduction of new Leishmania species that may find a suitable milieau to support their life cycle in our country. The objective of this report is to present the surveillance activity on imported leishmaniases by the Leishmania Identification Reference Centre, ISS. Two different methodologies were routinely applied: a) isoenzyme electrophoretic analysis, which requires parasite culture, and b) a number of molecular techniques, used for both diagnosis and parasite identification, differently applied according to the geographical origin of the suspected leishmaniasis case. When possible, both types of methodologies were applied. From 1986 to June 2002, 38 imported cases of leishmaniases were identified: 9 visceral (VL) and 29 cutaneous (CL) cases, of which 22 from the Old and 7 from the New World. Pathology, Leishmania species/zymodeme and geographical origin features are reported in the paper. Seven out of 9 VL patients were HIV positive, of whom 5 detected in the period 1993-1995. This high importation rate can be associated to the general increase in Mediterranean Leishmania/HIV coinfections in that period. Following HAART treatment, VL imported cases became occasional; no introduction of new L. infantum populations has been detected. On the other hand, our findings show an increase of CL imported cases from different areas of the Old and New Worlds. This phenomenon, however, is so far limited to new Leishmania species that could hardly be introduced in our country, because of their strict biological requirements (i.e. vectors and/or natural reservoir hosts). Since June 2002, 10 further suspected imported cases were recorded. For these patients--whose Leishmania identification is still in course--the origin/visited geographical areas were only slight different from the previous. However, the characteristics of the patients are changing: there are more immigrants that occasionally visited their place of origin, and Italian military staff.     

The genus Leishmania in Italy

Seventy-four Leishmania isolates collected in Italy from six different Regions where leishmaniases are endemic, have been typed. Parasites have been isolated from: man (VL and CL), dog, black rat (Rattus rattus), fox (Vulpes vulpes) and geckoes (Tarentola mauritanica and Cyrtodactylus kotschyi). The isolates have been characterized by starch-gel electrophoresis for 9-16 enzymes whose mobility was compared with that of international reference strains for L. infantum, L. tropica, L. major, L. donovani, L. aethiopica and L. tarentolae. The results obtained have shown that the genus Leishmania in Italy is represented by five zymodemes which may be grouped into two taxa: L. infantum s.l. (L. infantum s.st., L. infantum NH130 variant, L. infantum NH140 variant and L. infantum GOT, MDH, NH variant), agent of mammalian leishmaniases (including human leishmaniases), and L. tarentolae, parasite of geckoes. At the moment, the absence of L. tropica in Italy as agent of CL has been revealed. Through the analysis of epidemiological data obtained from the foci where Leishmania parasites were isolated two zymodemes only, L. infantum s.st. and L. infantum NH140 variant, show to be widely distributed. However, L. infantum s.st. appears to be prevalent in Thyrrenean foci which are characterized by VL cases and by high density of Phlebotomus perniciosus, and L. infantum NH140 variant is present in Adriatic areas where CL is diffuse and P. perfiliewi is the probable vector.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

The genetic structure of Leishmania infantum populations in Brazil and its possible association with the transmission cycle of visceral leishmaniasis

Leishmania infantum is the etiologic agent of visceral leishmaniasis (VL) in the Americas, Mediterranean basin and West and Central Asia. Although the geographic structure of L. infantum populations from the Old World have been described, few studies have addressed the population structure of this parasite in the Neotropical region. We employed 14 microsatellites to analyze the population structure of the L. infantum strains isolated from humans and dogs from most of the Brazilian states endemic for VL and from Paraguay. The results indicate a low genetic diversity, high inbreeding estimates and a depletion of heterozygotes, which together indicate a predominantly clonal breeding system, but signs of sexual events are also present. Three populations were identified from the clustering analysis, and they were well supported by F statistics inferences and partially corroborated by distance-based. POP1 (111 strains) was observed in all but one endemic area. POP2 (31 strains) is also well-dispersed, but it was the predominant population in Mato Grosso (MT). POP3 (31 strains) was less dispersed, and it was observed primarily in Mato Grosso do Sul (MS). Strains originated from an outbreak of canine VL in Southern Brazil were grouped in POP1 with those from Paraguay, which corroborates the hypothesis of dispersal from Northeastern Argentina and Paraguay. The distribution of VL in MS seems to follow the west-east construction of the Bolivia-Brazil pipeline from Corumbá municipality. This may have resulted in a strong association of POP3 and Lutzomyia cruzi, which is the main VL vector in Corumbá, and a dispersion of this population in this region that was shaped by human interference. This vector also occurs in MT and may influence the structure of POP2. This paper presents significant advances in the understanding of the population structure of L. infantum in Brazil and its association with eco-epidemiological aspects of VL.     

Genetic diversity analysis of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> causing stem rot in chickpea using RAPD,ITS-RFLP,ITS sequencing and mycelial compatibility grouping

Sclerotinia sclerotiorum is one of the most devastating soil-inhabiting fungal plant pathogens infecting various crop plants including chickpea. Genetic diversity of 24 isolates of S. sclerotiorum representing 10 different states of India was determined by different molecular markers and mycelial compatibility grouping (MCG). The majority of the isolates showed more than 90% genetic similarity. Unweighted paired group method with arithmetic average cluster analysis of DNA profiles generated by 21 RAPD primers grouped the isolates into seven categories showing high magnitude of genetic homogeneity and showed partial correlation with geographical origin of the isolates. Identical ITS-RFLP profiles were generated in all the isolates. Limited variability was observed among the nucleotide sequences of ITS region of the isolates. The phylogenetic tree generated from bootstrap neighbor-joining analysis indicated that 50% of Indian populations were distinct and grouped separately. The isolates were variable in mycelial compatibility and they were grouped into seven MCGs, namely, MCG A, MCG B, MCG C, MCG D, MCG E, MCG F and MCG G.     

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.     

Multilocus microsatellite typing (MLMT) of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group

Background

New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.

Methodology/Principal Findings

The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.

Conclusion

The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.     

Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania

Molecular approaches are being used increasingly for epidemiological studies of visceral and cutaneous leishmaniases. Several molecular markers resolving genetic differences between Leishmania parasites at species and strain levels have been developed to address key epidemiological and population genetic questions. The current gold standard, multilocus enzyme typing (MLEE), needs cultured parasites and lacks discriminatory power. PCR assays identifying species directly with clinical samples have proven useful in numerous field studies. Multilocus sequence typing (MLST) is potentially the most powerful phylogenetic approach and will, most probably, replace MLEE in the future. Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. Population genetic studies by MLMT revealed geographical and hierarchic population structure in L. tropica, L. major and the L. donovani complex. The existence of hybrids and gene flow between Leishmania populations suggests that sexual recombination is more frequent than previously thought. However, typing and analytical tools need to be further improved. Accessible databases should be created and sustained for integrating data obtained by different researchers. This would allow for global analyses and help to avoid biases in analyses due to small sample sizes.     

Genetic diversity of <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>strains isolated in northeastern Brazil as revealed by DNA sequencing,PCR-based analyses and molecular karyotyping

Background  

Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

The Leishmania donovani complex: genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing

Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region

Leishmania isolates from 57 cases of human cutaneous (CL), human visceral (VL), and canine visceral (CVL) leishmaniasis in Turkey were grouped by multi-site DNA polymorphism analyses into five genotypes. The initial grouping was based on DNA heterogeneity of the faster-evolving mitochondrion (kinetoplast) minicircles and the intergenic regions of two nuclear repetitive genes. Taxonomic affiliation and phylogenetic relationships of the five genotypes were inferred by comparing them with reference species for sequence heterogeneity in a approximately 1.4 kb conserved single-copy gene, encoding N-acetylglucosamine-1-phosphate transferase (NAGT). Alignment of the available sequences revealed no gap, but up to 7% scattered base substitutions, suggesting that this functionally important gene is a suitable marker. Three genotypes are completely identical to the NAGTs of the reference species, identifying them as L. infantum, L. tropica. and L. major, respectively. The remaining two are recognized as L. major NAGT variants with one and four base substitutions, respectively. As expected, Maximum Likelihood analysis of the NAGT sequences separates them into three clades, corresponding to the three species. The majority of the isolates obtained are L. infantum and L. tropica, which have been known to cause infantile VL and anthroponotic CL in western and southeastern Turkey, respectively. Unexpected is the finding of Leishmania major variants and their dispersal, possibly as previously unrecognized clinico-epidemiologic entities of CL and VL.