Estudo sobre a participação de roedores na cadeia de transmissão de Leishmania infantum (Protozoa: Trypanosomatidae) no Rio Grande do Norte

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by the bite of the sand flies Lutzomia longipalpis.The main domestic reservoir is the dog, while foxes and opposums are the known wild reservoirs. However, identification of natural infections with L. infantum in rodents appears for need of investigating the participation of these rodents how source of infection of the parasite. In the present work the Leishmania infantum infection was investigated in rodents captured in Rio Grande do Norte, aiming at to offer subsidies to the understanding of the epidemic chains of LVA in the State. Thirteen Galea spixii were distributed in four groups, being G1 the group control with four animals and the others, G2, G3 and G4, with three animals each. Those animals were intraperitoneally inoculated with 107 promastigotas of L. infantum and accompanied for, respectively, 30, 90 and 180 days. Weekly the animals were monitored as for the corporal weight and rectal temperature. At the end of each stipulated period the animals were killed. Blood were used for determination of the parameters biochemical and haematological, PCR, ELISA, microscopic examination and cultivation in NNN medium. Liver, spleen and lymph node were used in Giemsa-stained impression and cultivation in NNN medium. Liver and spleen fragments were still used in PCR and histopathological, respectively. At the same time 79 rodents of the species Rattus rattus, Bolomys lasiurus, Oligoryzomys nigripis, Oryzomys subflavus and Trichomys apereoides were captured in the Municipal districts of Brejinho, Campo Grande, Coronel Ezequiel, Passa e Fica and Vázea for identification of natural infection with L. infantum. Evidence of infection was checked by direct examination of Giemsa-stained impression of liver, spleen and blood and culture of these tissues in NNN medium. Antibodies were researched by ELISA. They were not found differences among the weigh corporal final, rectal temperature and biochemical and haematological parameters of the Galea spixii controls and infected. The rectal temperature of the animals varied from 36OC to 40OC. For the first time values of the haematocrit (33,6% to 42,8%), hemoglobin (10,2 to 14,5g/dl), erythrocyts number (4,67x106 to 6,90x106/mm3), total leukocytes (0,9x103 to 9,2x103/mm3), platelets (49x103 to 509x103/mm3) total proteins (1,56 to 6,06 g/dl), albumin (1,34 to 3,05 g/dl) and globulins (0,20 to 3,01 g/dl) of the Galea spixii were determined. The lymphocytes were the most abundant leucocytes. Infection for L. infantum was diagnosed in two animals euthanasied 180 days after the infection. In one of the animals was also identified antibodies anti-Leishmania. The parasite was not found in none of the five other species of rodents captured. Galea spixii are resistant to the infection for L. infantum and they are not good models for the study for visceral leishmaniose, although they can act as infection sources. More studies are necessary to determine the paper of the rodents in the epidemic chain of transmission of the visceral leishmaniose in the State of Rio Grande do Norte

Perfil de memória e ativação de linfócitos T na leishmaniose visceral

Visceral leishmaniasis (VL) in Brazil is a disease caused by Leishmania infantum chagasi (L.i.chagasi). The clinical evolution post-infection depends on the vertebrate host immune response, which is genetically mediated. This study aimed to evaluate the immune response of individuals living in endemic area for VL in the state of the Rio Grande do Norte, considering individuals with VL under treatment (n = 9), recovered VL <1 year post treatment (n = 10), > 10 years posttreatment (n = 9), uninfected individuals living in endemic areas (n = 7), individuals that lost DTH response (n=6) and asymptomatic individuals for VL (n=9). Peripheral blood cells were evaluated in the presence and absence of soluble Leishmania antigens (SLA) and ex vivo, to determine activation, presence of regulatory cells and memory cells. The Leishmania parasitemia and anti-Leishmania antibodies were determined respectively by qPCR and ELISA. Cells from individuals with VL under treatment showed less cell activation after stimulation with SLA for the markers CD4/CD69, CD8/CD69 and CD8/CD25 compared with VL post treatment treatment (p <0.001). Apparently uninfected individuals have a higher cell activation than symptomatic VL (p <0.001), with the exception of CD8/CD25 marker (p = 0.6662). On the other hand, in the ex-vivo group, significant differences were observed for CD4/CD69, CD8/CD69 and CD8/CD25 between the 4 groups due to increased cell activation present in cells of individuals symptomatic LV (p <0.001). VL cells under treatment, ex vivo, have a lower percentage of memory cells (CD4/CD45RO and CD8/CD45RO) than individuals VL post-treatment or control group (p = <0.01). Likewise, individuals with symptomatic VL have fewer regulatory cells when stimulated by SLA [CD4/CD25 (p = 0.0022) and CD4/FOXP3 (p = 0.0016)] and in the ex-vivo group (p = 0.0017). Finally, DNA isolated from recovered VL contained Leishmania DNA, supporting the hypothesis of non-sterile clinical cure for Leishmania infection. Recovered VL, even 10 years after treatment have high levels of memory cells, which may be due to the presence of stimulation, either by reexposure to Leishmania or non-sterile cure

Estado nutricional e efeito da vitamina A na resposta imune frente à infecção por Leishmania Infantum

Nutritional status is an important determinant to the response against Leishmania infection, although few studies have characterized the molecular basis for the association found between malnutrition and the disease. Vitamin A supplementation has long been used in developing countries to prevent mortality by diarrheal and respiratory diseases, but there are no studies on the role of vitamin A in Leishmania infection, although we and others have found vitamin A deficiency in visceral Leishmaniasis (VL). Regulatory T cells are induced in vitro by vitamin A metabolites and are considered important cells implicated T CD4+ cell suppression in human VL. This work aimed to examine the correlation of nutritional status and the effect of vitamin A in the response against Leishmania infantum infection. A total of 179 children were studied: 31 had active VL, 33 VL history, 44 were DTH+ and 71 were DTH- and had negative antibody to Leishmania (DTH-/Ac-). Peripheral blood monuclear cells were isolated in a subgroup of 10 active VL and 16 DTH-/Ac- children and cultivated for 20h under 5 different conditions: 1) Medium, 2) Soluble promastigote L. infantum antigens (SLA), 3) All-trans retinoic acid (ATRA), 4) SLA + ATRA and 5) Concanavalin A. T CD4+CD25highFoxp3+, T CD4+CD25-Foxp3- and CD14+ monocytes were stained and studied by flow cytometry for IL-10, TGF-β and IL-17 production. Nutritional status was compromised in VL children, which presented lower BMI/Age and retinol concentrations when compared to healthy controls. We found a negative correlation between nutritional status (measured by BMI/Age and serum retinol) and anti-Leishmania antibodies and acute phase proteins. There was no correlation between nutritional status and parasite load. ATRA presented a dual effect in Treg cells and monocytes: In healthy children (DTH-/Ac-), it induced a regulatory response, increasing IL-10 and TGF-β production; in VL children it modulated the immune response, preventing increased IL-10 production after SLA stimulation. Furthermore, we found a positive correlation between BMI/Age and IL-17 production and negative correlation between serum retinol and IL-10 and TGF-β production in T CD4+CD25highFoxp3+ cells after SLA stimulus. Our results show a potential dual role of vitamin A in the immune system: improvement of regulatory profile during homeostasis and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might improve recovery from disease status in Leishmania infantum infection

Anticorpos anti-trypanosoma cruzi como preditivos de infecção por leishmania infantum

Leishmania infantum and Trypanosoma cruzi are trypanosomatids of medical importance and are, respectively, the etiologic agents of visceral leishmaniasis (VL) and Chagas disease (CD) in Brazil. People infected with L. infantum or T. cruzi may develop asymptomatically, enabling the transmission of pathogens through blood transfusion and / or organs. The assessment of the infection by T. cruzi is included among the tests performed for screening blood donors in Brazil, however, there is no availability of tests for Leishmania. Serological tests for T. cruzi are very sensitive, but not specific, and may have cross-reactions with other microorganisms. Thus, the aim of this study was to determine the prevalence of Leishmania infection in blood donors and assess whether the serological test for T. cruzi detect L. infantum. Among the 300 blood samples from donors, discarded in 2011, 61 were T. cruzi positive, 203 were from donors with other infections and 36 were from handbags with low blood volume, but without infection. We also assessed 144 samples from donors without infections and able to donate blood, totaling 444 subjects. DNA was extracted from blood samples of all to perform quantitative PCR (qPCR) to detect Leishmania DNA. The buffy coat obtained from all samples was grown in Schneider medium supplemented and NNN. All samples were evaluated for the presence of anti-Leishmania antibody. The serological results indicate a percentage of 22% of Leishmania infection in blood samples obtained from discarded bags. A total of 60% of samples positive in ELISA for T. cruzi were negative by IFI, used as confirmatory test, ie 60% false positive for Chagas. Among these samples false positive for Chagas, 72% were positive by ELISA for Leishmania characterizing the occurrence of cross reaction between serologic assays. Of the 300 cultures performed, 18 grew parasites that were typed by qPCR and specific isoenzymes, found the species Leishmania infantum crops. Among the 18 cultures, 4 were purged from scholarships for low volume and all negative serology blood bank, thus demonstrating that there is a real risk of Leishmania transmission via transfusion. It is concluded that in an area endemic for leishmaniasis in Brazil, serological diagnosis performed to detect infection by T. cruzi among blood donors can identify infection by L. infantum and although cause false positive for Chagas, this cross-reactivity reduces the risk of Leishmania infection via blood transfusion, since tests are not applied specific detection of the parasite. In this way, there remains the need to discuss the implementation of a specific serological screening test for Leishmania in endemic countries such as Brazil

Estudio de la capacidad inmunoprofil��ctica de proteínas recombinantes de Leishmania infantum en el modelo murino

En esta Tesis Doctoral, cuatro proteínas que están sobre-expresadas en las fases infectivas del ciclo celular de L. infantum (STPKA, STPK, PP2B and PABP3) fueron purificadas para inmunizar ratones BALB/c, con el interés de estudiar su posible capacidad protectora contra la VL. Tres de ellas indujeron una fuerte respuesta humoral en los ratones, rLiSTPKA, rLiSTPK y rLiPABP3, a diferencia de la proteína rLiPP2B. Además, la rLiPABP3 fue la única que indujo una disminución significativa de la carga parasitara tanto en bazo como en hígado 2 meses después de la infección experimental con el parásito. Con el objetivo de determinar los efectos que la inmunización con rLiPABP3 tenía sobre el sistema inmune murino, se determinó la expresión génica de 106 genes relacionados con el sistema inmune en ratones control, inmunizados e infectados. Los niveles de expresión génica fueron comparados con los obtenidos para los grupos control. Los resultados mostraron que durante todo el experimento la proteína rLiPABP3 promueve la inhibición de la respuesta inmune inflamatoria en el bazo de los ratones infectados. Además, no se observa una respuesta adaptiva claramente polarizada hacia un tipo concreto. Un mes después de la infeccion, en los animales previamente inmunizados se observa la sobre-expresion de Il2rb lo que nos hace pensar que rLiPABP3 podría estimular la presencia de linfocitos T memoria. En fases más avanzadas de la infección, a pesar de que no observamos una clara diferenciación de una población concreta de linfocitos T efectores, sí observamos la infra-expresión del gen codificante del TNF-alfa, lo que unido a la sobre-expresión del gen Cxcr4 y la ausencia de cambios de la expresión del gen Ccr7 nos hace pensar que la inmunización no sólo mantiene la micro-arquitectura del bazo, sino que también promueve la correcta migración de las DCs desde la MZ hasta el PALS.

Evidence of Leishmania infantum infection in rabbits (Oryctolagus cuniculus) in a natural area in Madrid, Spain

Leishmaniasis is one of most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world-wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Estudo dos flebótomos (Diptera, Phlebotominae), vectores de Leishmania sp. No Concelho de Torres Novas, Portugal

Estudo dos flebótomos (Diptera, Phlebotominae), vectores de Leishmania sp. no Concelho de Torres Novas, Portugal. Sofia Isabel Martins Branco PALAVRAS-CHAVE: flebótomos, bioecologia, Leishmania, Torres Novas, Portugal. Os flebótomos são insectos vectores de vários agentes patogénicos, dos quais se destacam os protozoários do Género Leishmania. Em Portugal, as leishmanioses, canina e humana, são causadas por L. infantum, sendo o cão o principal reservatório e Phlebotomus perniciosus e P. ariasi os vectores comprovados do parasita. São conhecidos três focos de doença, mas casos de leishmaniose canina têm sido reportados em outras regiões nas quais se desconhecem as espécies flebotomínicas presentes e respectivas taxas de infecção. Neste trabalho, efectuou-se a primeira prospecção flebotomínica no Concelho de Torres Novas, Distrito de Santarém, localizado na região Centro de Portugal. Os principais objectivos foram determinar a fauna flebotomínica do Concelho, os aspectos bioecol��gicos, as taxas de infecção por Leishmania e os factores de risco para a transmissão vectorial. De Junho a Novembro de 2010, 275 biótopos foram prospectados com armadilhas CDC. As capturas foram realizadas em 91 localidades, nas 17 freguesias do Concelho, e incluíram habitats domésticos, peridomésticos e silváticos. Os exemplares capturados foram identificados morfologicamente, as fêmeas utilizadas para detecção molecular de DNA de Leishmania e identificação das refeições sanguíneas. Análises de regressão simples e múltipla foram utilizadas para avaliação dos factores de risco para a presença das várias espécies flebotomínicas. Testes não paramétricos foram usados para comparar densidades. Dos 1262 flebótomos capturados, quatro espécies foram assinaladas com as seguintes abundâncias relativas: P. perniciosus 73,69%, P. ariasi 8,16%, P. sergenti 6,58% e Sergentomyia minuta 11,57%. Em 82% das localidades prospectadas foi detectada pelo menos uma espécie flebotomínica e em 71,4% destas foi capturada pelo menos uma das espécies comprovadamente vectoras de L. infantum. P. perniciosus foi assinalado em todas as 17 freguesias do Concelho. Os factores de risco identificados foram: temperaturas elevadas e humidades relativas baixas, locais abrigados e ausência de vento forte, presença de pinheiros como vegetação dominante, biótopos peridomésticos, particularmente currais de ovelhas e coelheiras, ou na proximidade de ovelhas, aves de capoeira e ninhos com andorinhas. A taxa de infecção flebotomínica por L. infantum foi de 4% para P. ariasi e de 0,32% para o total de fêmeas capturadas. A maioria das fêmeas para as quais se identificou a origem da refeição sanguínea pertencia a P. perniciosus. Esta espécie apresentou um comportamento oportunista, alimentando-se numa grande variedade de hospedeiros vertebrados. A elevada abundância e distribuição das espécies vectoras, juntamente com a seropreval��ncia de Leishmania nos cães do Distrito (5-10%), e a captura de uma fêmea grávida de P. ariasi (infectante), sugerem que o Concelho de Torres Novas é um foco de leishmaniose no país. A maior abundância relativa de P. sergenti, comparando com prospecções realizadas noutras áreas da região Centro de Portugal, sugere que este potencial vector esteja a expandir-se para latitudes mais elevadas, aumentando o risco de introdução de L. tropica no território, por contacto com imigrantes ou viajantes infectados de áreas endémicas. A monitorização flebotomínica, e dos hospedeiros vertebrados, deverá ser continuada no Concelho para que medidas eficazes de controlo possam ser definidas e implementadas.

Comportamento biol��gico "in vitro" de "novas espécies" de Leishmania no Velho Mundo

As leishmanioses são um grupo de doenças causadas pelo parasita protozoário Leishmania sp. Na Bacia mediterrânica, Leishmania infantum, é a principal espécie causadora de leishmaniose visceral, a forma mais severa da doença, sendo L. major um dos agentes etiol��gicos da leishmaniose cutânea. Apesar de se considerar que estes parasitas têm uma reprodução essencialmente clonal, nos últimos 20 anos tem vindo a ser descrita a recombinação genética entre diferentes estirpes e espécies, com ocorrência de híbridos naturais, quer no Velho quer no Novo Mundo. Recentemente, em Portugal, foram isoladas e identificadas pela primeira vez, estirpes híbridas de L. infantum/L. major. O presente estudo teve como principais objetivos, a pesquisa de “novas espécies” de Leishmania e a análise do comportamento “in vitro” de estirpes parentais e híbridas de L. infantum e L. major. Numa primeira parte do trabalho efetuou-se a cultura e pesquisa de DNA de Leishmania sp., em amostras de sangue medular de 229 cães provenientes de uma região endémica de Portugal, utilizando diferentes marcadores moleculares (kDNA, ITS1 e SSU rRNA) e protocolos de PCR. Não foi encontrado DNA de espécies híbridas, tendo-se no entanto, identificado DNA de Leishmania sp. em 45,85% (105/229) das amostras, incluindo cães sem sinais cl��nicos. Na segunda parte do trabalho, realizaram-se diversos ensaios “in vitro” com estirpes híbridas naturais L. infantum/L. major e parentais L. infantum e L. major. Em condições normais de crescimento, observou-se um padrão de crescimento distinto para cada estirpe estudada. Em condições de “stress” oxidativo, destacou-se uma diferença significativa entre as duas estirpes híbridas estudadas. Em condições de “stress” nutricional, as estirpes não apresentaram diferenças entre si. Após avaliação da suscetibilidade das estirpes na presença de Anfotericina B, todas se mostraram suscetíveis, com concentrações inibitórias (CI50) entre 0.21 e 1.15 μg/mL. Após infeção em linhas celulares monocíticas, não se verificaram diferenças estatisticamente significativas na taxa e intensidade de infeção das estirpes híbridas em comparação às putativas parentais. Os resultados obtidos, contribuíram para um melhor conhecimento sobre o comportamento biol��gico destas estirpes híbridas naturais L. infantum/L. major. Estas demonstraram um comportamento “in vitro” intermédio, relativamente às estirpes parentais. Estes resultados poderão servir de base para o desenvolvimento de outros estudos com estas “novas espécies”, nomeadamente estudos de patogenicidade “in vivo” e o papel de biomarcadores de virul��ncia, que permitam um potencial prognóstico da infeção e avaliação do seu risco epidemiol��gico.

Identificação de motivos relevantes para a função do fator de iniciação da tradução EIF4E3 de Leishmania sp

Os tripanossomatídeos são organismos caracterizados pelo controle póstranscricional da expressão gênica, principalmente em nível de tradução. Na tradução em eucariotos, tem grande destaque o complexo eIF4F, sendo um de seus principais componentes o fator de iniciação da tradução eIF4E. Já foram descritos em tripanossomatídeos seis homólogos para o eIF4E, nomeados EIF4E1 a 6. Em um estudo com Leishmania amazonensis, focado no EIF4E3, percebeu-se que seu perfil de expressão se alterava rapidamente numa curva de crescimento, com este apresentando ao menos duas bandas. As mudanças observadas sugeriam modificações pós-traducionais do tipo fosforilação, algo posteriormente confirmado. Analisando-se a sequência do EIF4E3 de Leishmania, foi possível identificar a presença de possíveis sítios de fosforilação e de ligação a parceiros funcionais como homólogos do eIF4G, outro componente do complexo eIF4F, e da proteína de ligação á cauda poli-A (PABP). No presente estudo foi analisado o perfil de expressão e a capacidade de ligação a parceiros funcionais do EIF4E3 de Leishmania superexpresso em células transfectadas e no qual foram introduzidas mutações em motivos específicos. Os resultados mostraram um perfil de expressão de ao menos três bandas para o EIF4E3 de L. amazonensis e duas para L. infantum, com o sítio S75, presente apenas na primeira, sendo o responsável por esta diferença. Em ensaios de imunoprecipitação, foi identificado um motivo que, quando mutado, aboliu a ligação do EIF4E3 com a PABP3, sugerindo este como o sítio de interação entre os fatores. Com a análise do efeito de mutações no EIF4E3 de L. amazonensis, foi percebido que ao se mutar três motivos implicados na à PABP e o possível sítio de ligação ao EIF4G, sua fosforilação diminuiu drasticamente, sugerindo a necessidade destas interações para que a fosforilação ocorra. Estes resultados indicam um complexo mecanismo de modificações pós-traducionais responsáveis pela regulação do EIF4E3 e contribuem a para a caracterização da sua função em Leishmania

Estratégias para o aprimoramento do diagnóstico molecular na leishmaniose visceral

A leishmaniose visceral (LV) é uma doença infecto-parasitária causada por protozoários do gênero Leishmania. O trabalho teve como objetivo avaliar estratégias para o aprimoramento do diagnóstico molecular na LV, que compreenderam a avaliação da eficiência de diferentes métodos de extração de DNA em amostras de urina; a análise do uso da Reação em Cadeia da Polimerase quantitativa em tempo real (qPCR) como ferramenta para a detecção do DNA de Leishmania infantum na referida amostra; e a padronização de uma reação duplex qPCR para a detecção simultânea do DNA de L. infantum e do gene G3PD (controle endógeno). Depois da escolha do protocolo de extração de DNA mais apropriado, e após a otimização e a análise de reprodutibilidade, uma qPCR em urina foi padronizada. Em paralelo, após o desenho e a síntese de sondas TaqMan® compatíveis com os sistemas LINF 1B e G3PD1, após otimização e análise de reprodutibilidade, uma duplex qPCR em sangue também foi padronizada. Para avaliação dos protocolos desenvolvidos foram utilizadas técnicas de estatística descritiva. Para análise comparativa com técnicas cl��ssicas de diagnóstico da LV utilizou-se Teste Qui Quadrado de independência ou Teste Exato de Fisher (p<0,05 e p<0,01, respectivamente). Como resultados, após otimização, o limite de detecção alcançado pela qPCR em urina utilizando o protocolo de extração selecionado (kit comercial) foi de 5 fg/microlitros de amostra 0,034 parasitos) A duplex qPCR em sangue alcançou um limite de detecção de 2x102 fg/microlitros de amostra 1,4 (ou ~ 1,4 parasito), após a otimização. A partir dos dados estatísticos obtidos, pôde-se analisar alta concordância percentual entre a qPCR e urina e o conjunto de critérios diagnósticos (sorologia rK39 + qPCR em sangue), bem como entre a duplex qPCR em sangue e a qPCR em sangue, para os Grupos 01 (pacientes com suspeita de LV) e 02 (pacientes HIV positivos co-infectados ou não). Como um conjunto de critérios, os dois novos ensaios obtiveram excelentes concordâncias com o conjunto de técnicas cl��ssicas: 88,89 por cento e 94,74 por cento para os Grupos 01 e 02, respectivamente. Não houve diferenças estatísticas significativas entre os testes. Pôde-se concluir que ambos os ensaios mostraram bom potencial para a incorporação, após validação, ao diagnóstico da LV; em conjunto ou individualmente (quando necessário), trazendo mais conforto, praticidade, confiabilidade e rapidez ao diagnóstico definitivo da patologia

Genetic and Functional Evidence Implicating DLL1 as the Gene That Influences Susceptibility to Visceral Leishmaniasis at Chromosome 6q27

Background. Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27. Methods. Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample. Results. Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 x 10(-4) at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 x 10(-7)) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 x 10(-6) < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1. Conclusions. DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

O papel modulatório do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-γ, TNF-α, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 μM supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-γ, TNF-α and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-α, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-γ, TNF-α e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-γ, TNF-α and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs

VISCERAL LEISHMANIASIS IN A CAPTIVE CRAB-EATING FOX CERDOCYON THOUS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia, and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs. (c) 2006 Elsevier Ltd. All rights reserved.