Time distributions of <i>L</i>. <i>braziliensis</i> isolations for the primary (2008–2011) and validation (1999–2001) parasite samples used in the study.

(A) Primary sample of parasite isolates obtained from ATL patients enrolled during six consecutive semesters from July/2008 to June/2011 in Corte de Pedra, Northeast Brazil. (B) Validation sample obtained from ATL patients enrolled during five consecutive semesters from July/1999 to December/2001 in the same region. Columns depict the number of isolates obtained per semester, according to the left Y axes. Lines consist in percent contribution of each semester to the total number of isolates in the corresponding sample, according to right Y axes.</p

<i>L</i>. <i>braziliensis</i> individuals contribute similarly to putative subpopulations predicted by Structure, based on their genotypes in CHR24/3074 and CHR28/425451 <i>loci</i>, in Corte de Pedra.

Structuralization within Corte de Pedra’s L. braziliensis population into discrete subpopulations was assessed using the software STRUCTURE [21]. The analyses were performed using combined data of CHR24/3074 and CHR28/425451 loci. Two conditions were tested: (A) structuralization into two subpopulations, i.e. K = 2; and (B) structuralization into 20 subpopulations, i.e. K = 20. Histograms show the proportional contributions of each individual in the overall sample to each theoretical subpopulation ordered by sampling period (i.e. 1999–2001 and 2008–2011) after a burn-in step of 10,000 runs followed by 100,000 MCMC replications of the data.</p

Haplotypes of nucleotides found in biallelic polymorphic positions at chromosomal <i>loci</i> CHR24/3074 and CHR28/425451 of at least two different isolates in the collection of <i>L</i>. <i>braziliensis</i> used in the study.

For each locus, DNA sequences of four to six clones per L. braziliensis isolate of the study collection (i.e. primary plus validation samples) were aligned using MEGA X. Multiple alignment across all study isolates permitted identify polymorphic nucleotides at biallelic positions 31, 52, 68, 129, 469 and 604 in locus CHR24/3074, and 30, 286 and 545 in CHR28/425451. (A) Haplotypes of nucleotides detected within polymorphic positions at CHR24/3074; (B) Haplotypes of polymorphic nucleotides detected in CHR28/425451. The proportional representation of each haplotype in the sample is within parenthesis at the tips of the dendrograms.</p

Time distributions of homozygous and heterozygous genotypes in CHR24/3074 and CHR28/425451.

Each L. braziliensis isolate in the collection was genotyped at each locus according to their haplotype contents, considering diploid genomes. (A and C) Proportions of CTTCAG: CTTCAG (green), CTTCAG: TCATGA (red) and TCATGA: TCATGA (blue) in CHR24/3074 for the primary (A; i.e. 2008–2011) and validation (C; i.e. 1999–2001) parasite samples, respectively. (B and D) Proportions of CCT:CCT (green), CCT:TT- (red) and TT-:TT- (blue) in CHR28/425451 for the primary (B) and validation (D) samples, respectively. Overall fluctuations of genotype frequencies within each locus in each sample were not statistically significant (Chi-square p>0.05).</p

Clinical variables compared between 51 atypical (ACL) and 51 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.

<p>Clinical variables compared between 51 atypical (ACL) and 51 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.</p

Comparison of SNP allele frequencies in locus CHR28/425451^* between <i>L. (V.) braziliensis</i> isolated from 16 atypical (ACL) and 38 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.

<p>SNPs in this table correspond to individual SNPs in haplotypes delineated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005100#pntd.0005100.t002" target="_blank">Table 2</a>.</p

Comparison of SNP haplotypes frequencies in locus CHR28/425451 between <i>L</i>. <i>(V</i>.<i>) braziliensis</i> isolated from 16 atypical (ACL) and 38 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.

<p>Comparison of SNP haplotypes frequencies in locus CHR28/425451 between <i>L</i>. <i>(V</i>.<i>) braziliensis</i> isolated from 16 atypical (ACL) and 38 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.</p

Homozygous and heterozygous genotypes in <i>L</i>. <i>braziliensis loci</i> greatly overlap in Corte de Pedra.

Homozygous and heterozygous genotypes observed in L. braziliensis loci CHR24/3074 and CHR28/425451 of parasites obtained during 1999–2001 (A and B) and 2008–2011 (C and D) sampling periods were mapped and the resulting sets of geographic events were statistically compared. CHR24/3074 (A and C): CTTCAG: CTTCAG (yellow), CTTCAG: TCATGA (red) and TCATGA: TCATGA (blue). CHR28/425451 (B and D): CCT:CCT (yellow), CCT:TT- (red) and TT-:TT- (blue). Cuzick and Edward´s comparisons of combined homozygous versus heterozygous spatial distributions rendered non-significant (p>0.05) for data depicted in all four maps of the panel. Total number of dots plotted in each map may be smaller than the number of corresponding parasite isolates described in text due to overlap of some ATL patients’ geographic coordinates. Link to the United States Geological Survey (USGS) Landsat satellite photograph used in the figure [39]: https://earthexplorer.usgs.gov/scene/metadata/full/5e83d1193824e4fc/LT52160691994219CUB00/.</p

Atypical Manifestations of Cutaneous Leishmaniasis in a Region Endemic for <i>Leishmania braziliensis</i>: Clinical, Immunological and Parasitological Aspects

<div><p>Background</p><p>Atypical cutaneous leishmaniasis (ACL) has become progressively more frequent in Corte de Pedra, Northeast Brazil. Herein we characterize clinical presentation, antimony response, cytokine production and parasite strains prevailing in ACL.</p><p>Methodology/Principal Findings</p><p>Between 2005 and 2012, 51 ACL (cases) and 51 temporally matched cutaneous leishmaniasis (CL) subjects (controls) were enrolled and followed over time in Corte de Pedra. Clinical and therapeutic data were recorded for all subjects. Cytokine secretion by patients’ peripheral blood mononuclear cells (PBMC) stimulated with soluble parasite antigen in vitro, and genotypes in a 600 base-pair locus in chromosome 28 (CHR28/425451) of the infecting <i>L</i>. <i>(V</i>.<i>) braziliensis</i> were compared between the two groups. ACL presented significantly more lesions in head and neck, and higher rate of antimony failure than CL. Cytosine–Adenine substitutions at CHR28/425451 positions 254 and 321 were highly associated with ACL (p<0.0001). In vitro stimulated ACL PBMCs produced lower levels of IFN-γ (p = 0.0002) and TNF (p <0.0001), and higher levels of IL-10 (p = 0.0006) and IL-17 (p = 0.0008) than CL PBMCs.</p><p>Conclusions/Significance</p><p>ACL found in Northeast Brazil is caused by distinct genotypes of <i>L</i>. <i>(V</i>.<i>) braziliensis</i> and presents a cytokine profile that departs from that in classical CL patients. We think that differences in antigenic contents among parasites may be in part responsible for the variation in cytokine responses and possibly immunopathology between CL and ACL.</p></div

Hardy-Weinberg equilibrium testing of genotypes in two temporally distinct <i>L</i>. <i>braziliensis</i> populations endemic in Corte de Pedra, Northeast Brasil: 1999–2001 and 2008–2011.

Hardy-Weinberg equilibrium testing of genotypes in two temporally distinct L. braziliensis populations endemic in Corte de Pedra, Northeast Brasil: 1999–2001 and 2008–2011.</p