Antimony is the first line drug for treating American tegumentary leishmaniasis (ATL) in Brazil. In this country, Leishmania braziliensis causes at least three distinct forms of disease: localized cutaneous (CL), mucosal (ML) and disseminated leishmaniasis (DL). All forms can be found in Corte de Pedra, Northeast Brazil. ML and DL respond poorly to antimony, in contrast to CL. The L. braziliensis population causing ATL in Corte de Pedra is genetically very diverse, with strains of the parasite associating with the clinical form of leishmaniasis. We tested the hypotheses that antimony refractoriness is associated with L. braziliensis genotypes, and that parasites from ML and DL present greater in vitro resistance to antimony than L. braziliensis from CL. Comparison of geographic coordinates of living sites between antimony responders and non-responders by Cusick and Edward́s test showed that refractoriness and responsiveness to the drug were similarly wide spread in the region (p>0.05). Parasites were then genotyped by sequencing a locus starting at position 425,451 on chromosome 28, which is polymorphic among L. braziliensis of Corte de Pedra. Haplotype CC- in CHR28/425,451 was associated with risk of treatment failure among CL patients (Fisheŕs exact test, p=0.03, odds ratio=4.65). This haplotype could not be found among parasites from ML or DL. Finally, sensitivity to antimony was evaluated exposing L. braziliensis promastigotes to increasing concentrations of meglumine antimoniate in vitro. Parasites from ML and DL were more resistant to antimony at doses of 2mg/100μL and beyond than those isolated from CL (Fisher's exact test, p=0.02 and p=0.004, respectively). The intrinsically lower susceptibility of L. brazliensis from ML and DL to antimony parallels what is observed for patients' responsiveness in the field. This finding reinforces that ML and DL patients would benefit from initiating treatment with drugs currently considered as second line, like amphotericin B.

GP63 or leishmanolysin is the major surface protease of Leishmania spp. involved in parasite virulence and host cell interaction. As such, GP63 is a potential target of eventual vaccines against these protozoa. In the current study we evaluate the polymorphism of gp63 in Leishmania (Viannia) braziliensis isolated from two sets of American tegumentary leishmaniasis (ATL) cases from Corte de Pedra, Brazil, including 35 cases diagnosed between 1994 and 2001 and 6 cases diagnosed between 2008 and 2011. Parasites were obtained from lesions by needle aspiration and cultivation. Genomic DNA was extracted, and 405 bp fragments, including sequences encoding the putative macrophage interacting sites, were amplified from gp63 genes of all isolates. DNA amplicons were cloned into plasmid vectors and ten clones per L. (V.) braziliensis isolate were sequenced. Alignment of cloned sequences showed extensive polymorphism among gp63 genes within, and between parasite isolates. Overall, 45 different polymorphic alleles were detected in all samples, which could be segregated into two clusters. Cluster one included 25, and cluster two included 20 such genotypes. The predicted peptides showed overall conservation below 50%. In marked contrast, the conservation at segments with putative functional domains approached 90% (Fisher’s exact test p<0.0001). These findings show that gp63 is very polymorphic even among parasites from a same endemic focus, but the functional domains interacting with the mammalian host environment are conserved.

Summary Aims The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils ( PMN s) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis ( CL ) or disseminated leishmaniasis ( DL ) patients. Methods and results Neutrophils from 30 healthy subjects ( HS ) were infected with isolates of L . ( V .) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine ( DHR ) were evaluated by flow cytometry and cytokine production by ELISA . The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates ( P < 0.05). The DHR and CD 66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production. Conclusion The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

Background Leishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil. Methodology/Principal findings Two temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008–2011) primary sample, N = 120; (1999–2001) validation sample, N = 35. Parasites were genotyped by Sanger’s sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward’s comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software. Conclusions/Significance These findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite’s lifecycle.

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