Cutaneous Leishmaniasis In Brazil Research Articles

In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis.

Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.

Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.

Assessment of drug resistance related genes as candidate markers for treatment outcome prediction of cutaneous leishmaniasis in Brazil

The great public health problem posed by leishmaniasis has substantially worsened in recent years by the emergence of clinical failure. In Brazil, the poor prognosis observed for patients infected by Leishmania braziliensis (Lb) or L. guyanensis (Lg) may be related to parasite drug resistance. In the present study, 19 Lb and 29 Lg isolates were obtained from infected patients with different treatment outcomes. Translated amino acid sequence polymorphisms from four described antimony resistance related genes (AQP1, hsp70, MRPA and TRYR) were tested as candidate markers for antimonial treatment failure prediction. Possibly due to the low intraspecific variability observed in Lg samples, none of the prediction models had good prognosis values. Most strikingly, one mutation (T579A) found in hsp70 of Lb samples could predict 75% of the antimonial treatment failure clinical cases. Moreover, a multiple logistic regression model showed that the change from adenine to guanine at position 1735 of the hsp70 gene, which is responsible for the T579A mutation, significantly increased the chance of Lb clinical isolates to be associated with treatment failure (OR=7.29; CI 95%=[1.17, 45.25]; p=0.0331). The use of molecular markers to predict treatment outcome presents practical and economic advantages as it allows the development of rapid assays to monitor the emergence of drug resistant parasites that can be clinically applied to aid the prognosis of cutaneous leishmaniasis in Brazil.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

BackgroundLeishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.MethodsFlagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.ResultsThe success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.ConclusionsThe data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.

Comparative analysis of the geographic distribution of the histopathological spectrum and Leishmania species of American cutaneous leishmaniasis in Brazil

Knowledge of the relationship between specific geographical differences and histopathology of the American cutaneous leishmaniasis is limited because host-parasite interactions in space and time are complex. To describe the geographic analysis of the histopathological pattern of localized American cutaneous leishmaniasis and the relationship with Leishmania species. A cross-sectional, descriptive and analytical study of a sample consisting of 55 patients of municipal districts of Montes Claros and 32 of Caratinga, Minas Gerais, by comparing with other endemic regions in Brazil, assessing historical and microregional data and developing confidence intervals. A preponderance of cellular exudative reactions in the cutaneous form of American cutaneous leishmaniasis was found in the municipal districts of Caratinga, east of Minas Gerais, Montes Claros, north of Minas Gerais and Uberlândia, Triangulo Mineiro, and also in the states of Mato Grosso and Ceara. Necrotic and exudative granulomatous reactions were found to be more frequent in the northern areas, mainly in the states of Amazonas and Para. Necrotic and exudative reactions were the most frequent form of presentation found throughout Brazil when the predominance of L. braziliensis was below 90% in relation to other species. There is a geographic variation of the immune inflammatory response in American cutaneous leishmaniasis. Geographical correlations between the predominant species of Leishmania and the most frequent forms of histopathological presentation of American cutaneous leishmaniasis, which until the present study had not been performed, enables new approaches to the study of Leishmania subpopulations in areas with a predominance of one species and provides evidence of variations in the biological behavior of different species.

Wound healing genes and susceptibility to cutaneous leishmaniasis in Brazil

Leishmania braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. In the mouse, Fli1 was identified as a gene influencing enhanced wound healing and resistance to CL caused by Leishmania major. Polymorphism at FLI1 is associated with CL caused by L. braziliensis in humans, with an inverse association observed for ML disease. Here we extend the analysis to look at other wound healing genes, including CTGF, TGFB1, TGFBR1/2, SMADS 2/3/4/7 and FLII, all functionally linked along with FLI1 in the TGF beta pathway. Haplotype tagging single nucleotide polymorphisms (tag-SNPs) were genotyped using Taqman technology in 325 nuclear families (652 CL cases; 126 ML cases) from Brazil. Robust case-pseudocontrol (CPC) conditional logistic regression analysis showed associations between CL and SNPs at CTGF (SNP rs6918698; CC genotype; OR 1.67; 95%CI 1.10–2.54; P=0.016), TGFBR2 (rs1962859; OR 1.50; 95%CI 1.12–1.99; P=0.005), SMAD2 (rs1792658; OR 1.57; 95%CI 1.04–2.38; P=0.03), SMAD7 (rs4464148; AA genotype; OR 2.80; 95%CI 1.00–7.87; P=0.05) and FLII (rs2071242; OR 1.60; 95%CI 1.14–2.24; P=0.005), and between ML and SNPs at SMAD3 (rs1465841; OR 2.15; 95%CI 1.13–4.07; P=0.018) and SMAD7 (rs2337107; TT genotype; OR 3.70; 95%CI 1.27–10.7; P=0.016). Stepwise logistic regression analysis showed that all SNPs associated with CL at FLI1, CTGF, TGFBR2, and FLII showed independent effects from each other, but SNPs at SMAD2 and SMAD7 did not add independent effects to SNPs from other genes. These results suggest that TGFβ signalling via SMAD2 is important in directing events that contribute to CL, whereas signalling via SMAD3 is important in ML. Both are modulated by the inhibitory SMAD7 that acts upstream of SMAD2 and SMAD3 in this signalling pathway. Along with the published FLI1 association, these data further contribute to the hypothesis that wound healing processes are important determinants of pathology associated with cutaneous forms of leishmaniasis.

Molecular detection of Leishmania braziliensis in Rattus norvegicus in an area endemic for cutaneous leishmaniasis in Brazil

Leishmania nested PCR (LnPCR) targeted to the SSUrRNA gene and DNA sequencing were used to analyze 315 tissue samples from 80 Rattus norvegicus specimens trapped in an area endemic for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil. Of the samples analyzed, 17.46% (55/315) of all tissues, 10% (8/80) of skin, 26.92% (21/78) of blood, 30.76% (24/78) of bone marrow and 2.53% (2/79) of spleen were positive for Leishmania. The overall infection prevalence was 36.25% (29/80) The DNA sequencing showed that 65.51% (19/29) of the positive animals were infected by parasites belonging to the Leishmania braziliensis complex. The identification of L. braziliensis DNA in R. norvegicus in an area with a high prevalence of leishmaniasis might imply a zoonotic role of this species. The rodent control programs and health education may represent important measures toward the control of leishmaniasis.

FLI1 polymorphism affects susceptibility to cutaneous leishmaniasis in Brazil

Mapping murine genes controlling cutaneous leishmaniasis (CL) identified Fli1 as a candidate influencing resistance to L. major and enhanced wound healing. We examine FLI1 as a gene controlling CL and mucosal leishmaniasis (ML) caused by L. braziliensis in humans. Intron 1 single nucleotide polymorphisms tagging promoter and enhancer elements were analysed in 168 nuclear families (250 CL; 87 ML cases) and replicated in 157 families (402 CL; 39 ML cases). Robust case-pseudocontrol logistic regression analysis showed association between allele C (odds ratio (OR) 1.65; 95% confidence interval 1.18-2.29; P=0.003) of FLI1_rs7930515 and CL in the primary sample that was confirmed (OR 1.60; 95% confidence interval 1.10-2.33; P=0.014) in the replication set (combined P=1.8 × 10(-4)). FLI1_rs7930515 is in linkage disequilibrium with the functional GAn microsatellite in the proximal promoter. Haplotype associations extended across the enhancer, which was not polymorphic. ML associated with inverse haplotypes compared with CL. Wound healing is therefore important in CL, providing potential for therapies modulating FLI1.

Avanços no tratamento da leishmaniose tegumentar do novo mundo nos últimos dez anos: uma revisão sistemática da literatura

The therapeutic arsenal against cutaneous leishmaniasis is very limited. Pentavalent antimonial compounds have been the drugs of choice for treatment of this disease for over 50 years. Despite their effectiveness, these drugs require daily injections, have many side effects and present prolonged healing time. To carry out a systematic literature review on the advances in the treatment of cutaneous leishmaniasis in the new world in the last ten years. We conducted an electronic search on the Pubmed and LILACS database as well as on the SciELO electronic library in June 2009. The search words in English were: "cutaneous", "leishmaniasis" and "treatment". We included only randomized, double-blind and placebo controlled trials. We used the Jadad scale to assess the quality of the selected studies. According to the inclusion and exclusion criteria, only eight articles were selected. The drugs evaluated in the selected studies were glucantime®, miltefosine, immunotherapy, imiquimod, rhGM-CSF and pentoxifylline, and paromomycin. Although cutaneous leishmaniasis is a major public health issue, the published data on the use of new drugs for the treatment of cutaneous leishmaniasis in Brazil are still quite limited.

BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

Background Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult.Methodology/Principal FindingsHere, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S).Conclusion/SignificanceWe conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.

Dogs as Reservoirs forLeishmania braziliensis

To the Editor: I have read the review by Sousa and Pearson (1), which provides a fascinating historical account of the Great Drought and the smallpox epidemic of the 1870s and their association with the emergence of cutaneous leishmaniasis in Ceara, Brazil. In their review, the authors went back to the 19th century, remembering the hard years experienced by those who faced the Great Drought, which prompted the immigration of thousands of persons from Ceara to the Amazon region, and a devastating smallpox epidemic, which resulted in the death of >100,000 persons. Later, they returned to the present situation of cutaneous leishmaniasis in Brazil.

The −2518 bp promoter polymorphism at CCL2/ MCP1 influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil

Mucosal leishmaniasis (ML) follows localized cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. Proinflammatory responses mediate CL self-healing but are exaggerated in ML. Proinflammatory monocyte chemoattractant protein 1 (MCP-1; encoded by CCL2) is associated with CL. We explore its role in CL/ML through analysis of the regulatory CCL2 −2518 bp promoter polymorphism in CL/ML population samples and families from Brazil. Genotype frequencies were compared among ML/CL cases and control groups using logistic regression and the family-based association test (FBAT). MCP-1 was measured in plasma and macrophages. The GG recessive genotype at CCL2 −2518 bp was more common in patients with ML ( N = 67) than in neighborhood control (NC; N = 60) subjects (OR 1.78; 95% CI 1.01–3.14; P = 0.045), than in NC combined with leishmanin skin-test positive ( N = 60) controls (OR 4.40; 95% CI 1.42–13.65; P = 0.010), and than in controls combined with CL ( N = 60) patients (OR 2.78; 95% CI 1.13–6.85; P = 0.045). No associations were observed for CL compared to any groups. FBAT (91 ML and 223 CL cases in families) confirmed recessive association of ML with allele G ( Z = 2.679; P = 0.007). Higher levels of MCP-1 occurred in plasma ( P = 0.03) and macrophages ( P < 0.0001) from GG compared to AA individuals. These results suggest that high MCP-1 increases risk of ML.

CXCR1 and SLC11A1polymorphisms affect susceptibility to cutaneous leishmaniasis in Brazil: a case-control and family-based study

BackgroundL. braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. Wound healing neutrophil (PMN) and macrophage responses made following the bite of the vector sand fly contribute to disease progression in mice. To look at the interplay between PMN and macrophages in disease progression in humans we asked whether polymorphisms at genes that regulate their infiltration or function are associated with different clinical phenotypes. Specifically, CXCR1 (IL8RA) and CXCR2 (IL8RB) are receptors for chemokines that attract PMN to inflammatory sites. They lie 30-260 kb upstream of SLC11A1, a gene known primarily for its role in regulating macrophage activation, resistance to leishmaniasis, and wound healing responses in mice, but also known to be expressed in PMN, macrophages and dendritic cells.MethodsPolymorphic variants at CXCR1, CXCR2 and SLC11A1 were analysed using Taqman or ABI fragment separation technologies in cases (60 CL; 60 ML), unrelated controls (n = 120), and multicase families (104 nuclear families; 88 ML, 250 CL cases) from Brazil. Logistic regression analysis, family-based association testing (FBAT) and haplotype analysis (TRANSMIT) were performed.ResultsCase-control analysis showed association between the common C allele (OR 2.38; 95% CI 1.23-4.57; P = 0.009) of CXCR1_rs2854386 and CL, supported by family-based (FBAT; Z score 2.002; P = 0.045) analysis (104 nuclear families; 88 ML, 250 CL cases). ML associated with the rarer G allele (Z score 1.999; P = 0.046). CL associated with a 3' insertion/deletion polymorphism at SLC11A1 (Z score 2.549; P = 0.011).ConclusionsThe study supports roles for CXCR1 and SLC11A1 in the outcome of L. braziliensis infection in humans. Slc11a1 does not influence cutaneous lesion development following needle injection of Leishmania in mice, suggesting that its role here might relate to the action of PMN, macrophage and/or dendritic cells in the wound healing response to the sand fly bite. Together with the CXCR1 association, the data are consistent with hypotheses relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele suggests that PMN also have an important positive role to play in preventing this form of the disease.

Proven and putative vectors of American cutaneous leishmaniasis in Brazil: aspects of their biology and vectorial competence

The aim of the present review is to give relevant information on aspects of the biology and ecology, including the vectorial competence of Lutzomyia sand fly species suggested as vectors of American cutaneous leishmaniasis in Brazil. The disease, due to Leishmania (Viannia) braziliensis, has been registered in most municipalities in all the Brazilian states and its transmission is associated with more than one sand fly species in each geographical region. A variety of Leishmania species can be found in the Amazon basin, where different epidemiological chains have been detected with the participation of different phlebotomine vectors. Finally, a discussion is presented on some sand fly species found naturally infected by Leishmania, but for which there is as yet no evidence regarding their epidemiological importance.

Species structure of sand fly (Diptera: Psychodidae) fauna in the Brazilian western Amazon

We surveyed areas of the state of Rondônia in western Amazon for phlebotomine, which are potential vectors of leishmaniasis. A total of 5,998 specimens were captured, resulting in the identification of 48 species within the Lutzomyia (99.98%) and Brumptomyia (0.02%) genera. The predominant species was Lutzomyia davisi, followed by Lutzomyia umbratilis, Lutzomyia llanosmartinsi, Lutzomyia c. carrerai, Lutzomyia dendrophyla, Lutzomyia nevesi and Lutzomyia whitmani. All sand flies identified as vectors for cutaneous leishmaniasis in Brazil, i.e., Lu. davisi, Lu. umbratilis, Lu. c. carrerai and Lu. whitmani, were found in the surveyed areas.

Leishmania sp. isolated from human cases of cutaneous leishmaniasis in Brazil characterized as Leishmaniamajor-like

The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.

Data on sand fly fauna (Diptera, Psychodidae, Phlebotominae) in Itatiaia National Park, Rio de Janeiro State, Brazil

The sand fly fauna in Itatiaia National Park, Rio de Janeiro, Brazil, was investigated in different habitats ranging from sylvatic areas to those altered by human activity related to ecotourism, specifically identifying species that have been suggested as potential leishmaniasis vectors. Sand flies were captured from June 2002 to March 2004, using CDC light traps and Shannon traps. A total of 1,256 sand fly specimens were captured, from species belonging to genera Lutzomyia and Brumptomyia: Brumptomyia guimaraesi, B. troglodytes, Lutzomyia (Lutzomyia) amarali, L. lanei, L. migonei, L. sallesi, L. edwardsi, L. tupynambai, L. (Pintomyia) pessoai, L. (P.) bianchigalatie, L. rupicola, L. (Psathyromyia) shannoni, L. pascalei, L. (Psychodopygus) matosi, L. (P.) davisi, L. (P.) hirsuta hirsuta, L. (P.) ayrozai, L. peresi, L. monticola, and L. misionensis. Worthy of special attention were four species that are considered potential vectors for cutaneous leishmaniasis in Brazil: L. ayrozai, L. hirsuta hirsuta, L. migonei, and L. davisi, representing 19.19% of the specimens captured in this study.

Molecular polymorphism, differentiation and introgression in the period gene between Lutzomyia intermedia and Lutzomyia whitmani

BackgroundLutzomyia intermedia and Lutzomyia whitmani (Diptera: Psychodidae) are important and very closely related vector species of cutaneous leishmaniasis in Brazil, which are distinguishable by a few morphological differences. There is evidence of mitochondrial introgression between the two species but it is not clear whether gene flow also occurs in nuclear genes.ResultsWe analyzed the molecular variation within the clock gene period (per) of these two species in five different localities in Eastern Brazil. AMOVA and Fst estimates showed no evidence for geographical differentiation within species. On the other hand, the values were highly significant for both analyses between species. The two species show no fixed differences and a higher number of shared polymorphisms compared to exclusive mutations. In addition, some haplotypes that are "typical" of one species were found in some individuals of the other species suggesting either the persistence of old polymorphisms or the occurrence of introgression. Two tests of gene flow, one based on linkage disequilibrium and a MCMC analysis based on coalescence, suggest that the two species might be exchanging alleles at the per locus.ConclusionIntrogression might be occurring between L. intermedia and L. whitmani in period, a gene controlling behavioral rhythms in Drosophila. This result raises the question of whether similar phenomena are occurring at other loci controlling important aspects of behavior and vectorial capacity.

IL6−174 G/C Promoter Polymorphism Influences Susceptibility to Mucosal but Not Localized Cutaneous Leishmaniasis in Brazil

Mucosal leishmaniasis (ML) is associated with exaggerated tumor necrosis factor- alpha and interferon- gamma responses and tissue destruction. ML follows localized cutaneous leishmaniasis (CL) caused by Leishmania braziliensis infection. Interleukin (IL)-6 down-regulates T helper (Th) cell type 1 differentiation and drives Th2 cell differentiation. The IL6 -174 G/C polymorphism is associated with proinflammatory diseases and IL-6 regulation. The -174 G/C polymorphism was genotyped in population samples and families with CL and ML from Brazil. Genotype frequencies were compared among patients with ML, patients with CL, and 2 control groups by logistic regression and family-based association test (FBAT) analysis. IL-6 levels were measured in macrophages. The C allele was more common in patients with ML than in patients with CL (odds ratio [OR], 2.55 [95% confidence interval {CI}, 1.32-4.91]; P=.005), than in patients who were leishmanin skin-test positive (OR, 2.23 [95% CI, 1.23-4.05]; P=.009), and than in neighborhood control subjects (OR, 2.47 [95% CI, 1.24-4.90]; P=.01). FBAT analysis confirmed an association between allele C and ML under both additive (z=4.295; P=.000017) and dominant (z=4.325; P=.000015) models. Significantly lower levels of IL-6 were measured in unstimulated macrophages from CC individuals than from GG individuals (P=.003) as well as after stimulation with soluble leishmania antigen (P=.009). IL-6 may regulate type 1 proinflammatory responses, putting individuals with low macrophage IL-6 levels at increased risk for ML.