Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia).

BACKGROUND/OBJECTIVES: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. METHODOLOGY/PRINCIPAL FINDINGS: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. CONCLUSIONS/SIGNIFICANCE: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains

PCR diagnosis and characterization of Leishmania in local and imported clinical samples

Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slide

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

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

Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia

International audienceTunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome

Geographical distribution of the three main populations inferred by STRUCTURE.

<p>According to the Bayesian clustering algorithm (STRUCTURE) the Brazilian strains of <i>L.</i> (<i>Viannia</i>) were assigned to three different populations, shown in green, red and blue. Pie-charts show the proportion of each population sampled in the respective geographical region. The distribution of the main populations correlates, at least partially, with the geographical origin of the strains.</p

Numbers of strains per species, region, clinical picture and host of the 120 strains studied.

<p>VL – visceral leishmaniasis, CL – cutaneous leishmaniasis, MCL – mucocutaneous leishmaniasis, DL – disseminated cutaneous leishmaniasis, nd – not defined, IOC/Z –zymodemes according to the CLIOC system <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002490#pntd.0002490-Cupolillo1" target="_blank">[11]</a>.</p

Populations and subpopulations of Brazilian strains of the subgenus of <i>Leishmania</i> (<i>Viannia</i>).

<p>A midpoint rooted Neighbour-joining (NJ) tree (radial version, rectangular version in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002490#pntd.0002490.s001" target="_blank">Figure S1</a>) was calculated for the MLMT profiles of 120 strains of different species of subgenus <i>L.</i> (<i>Viannia</i>), based on 15 microsatellite markers and using the Chord distance measure. The assignment of these strains to three main populations by the Bayesian model-based clustering approach implemented in STRUCTURE is indicated by coloured circles: population 1 (green), population 2 (red) and population 3 (blue). Strains belonging to these populations are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002490#pntd.0002490.s007" target="_blank">Table S1</a>. Population 1 comprises all but one strain of <i>L. (V.) guyanensis</i> analysed in this study. Population 2 consists of 43 strains of <i>L. (V.) braziliensis</i> mainly from east Brazil. Population 3 is very diverse and includes all investigated strains of <i>L. (V.) lainsoni</i>, <i>L. (V.) naiffi</i>, <i>L. (V.) shawi</i>, <i>L. (V.) utingensis</i>, <i>L. (V.) lindenbergi</i>, 20 strains of <i>L. (V.) braziliensis</i> mainly from the north of Brazil as well as one strain of <i>L. (V.) guyanensis</i> from Acre. Putative hybrids are indicated by red or blue circles, according to their population assignment. Strain origins are indicated in the window alongside.</p

Factorial correspondence analysis (FCA) of 120 Brazilian strains of the subgenus <i>L. (Viannia)</i>.

<p>The strains labelled in green, red and blue correspond to those that were assigned, by STRUCTURE, to populations 1, 2 and 3, respectively. The two strains of Population 2, L2516 and L2446 that grouped within the blue cloud are indicated by a pink arrow. Four strains of mixed population membership, with predominating traits of population 3, are indicated by blue arrows.</p

NeighborNet network based on the MLMT profiles of 120 Brazilian <i>L. (Viannia)</i> strains.

<p>The network was obtained using SplitsTree4 software and calculation of Chord distances for the 15 microsatellite markers used. The assignment of the strains to the sub-populations as inferred by STRUCTURE is indicated.</p

<i>F</i>_ST values and corresponding <i>p</i>-values for the main three populations found by STRUCTURE.

<p><i>F</i>_ST values are in the upper triangle, <i>p</i>-values in the lower triangle. Number of strains belonging to each population is given in brackets.</p