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

Co-infecção pelo vírus da hepatite G em pacientes infectados pelo Leihsmania chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Estudo clínico-laboratorial e histopatológico de cães naturalmente infectados por Leishmania chagasi com diferentes graus de manifestação física

Canine Visceral Leishmania (CVL) is an important zoonotic disease that has a world wide distribution and has a large impact on public health on the American Continent, especially in Brazil, where the nature of endemic diseases in humans affects a large part of the nation. The influence of the prevalence of CVL in the increased rate of human cases in endemic areas and in the unleashing of epidemic outbreaks shows the need for a more profound understanding, that would generate significant advances in the current measures used to control the reservoirs of sickness that are practiced by the Programa Nacional de Vigilância e Controle da Leishmaniose Visceral. The present work describes and compares the clinical-laboratorial and histopathological findings of twenty-three dogs that were naturally infected by Leishmania chagasi, from endemic areas in metropolitan Natal, Rio Grande do Norte, Brazil. These animals, that were selected and given physical and serological exams (IFI and ELISA rK-39), were classified according to the degree of clinical severity and had blood samples drawn (whole blood and serum) for a complete hemogram and a coagulogram to be done as well as biochemical tests for kidney and liver function. The confirmation of infection by L. chagasi was done after the euthanasia of the animals, through the direct demonstration of the parasite in the impression of the spleen and liver crowned with GIEMSA and through a cultivation by means of NNN/Schneider. According to the clinical evaluation, the animals were classified as asymptomatic (7), oligosymptomatic (7) and polysymptomatic (9). Among the animals that were chosen to be autopsied, there were 2 asymptomatic, 3 oligosymptomatic and 3 polysymptomatic, for the purpose of studying their histopathology, having collected fragments of the spleen, liver, kidneys and skin and were fixed in 10% tamponed formol. The comparison between the average parameters of the clinical-laboratory tested animals in the groups was done through the Student t test (a<0.05). The main clinical signals observed were lymphadenomegaly, alopecy, dermatitis, exfoliation, cutaneous ulcers, onicogriphosis and emaciation. The main clinical-laboratorial alterations established, mainly in the polysymptomatic group, were anemia, hyperproteinemia, hyperglobulinemia, alterations in the albumin/globulin ratio and increased ALT activity. Renal alterations were not verified (urea and creatinine levels were normal). Thrombocytopenia was observed in three clinical groups. However, the other indicators of coagulation function (TAP and TTPA) did not have abnormal variations. There were inflammatory infiltrations and leishmania amastigotes in the skin of polysymptomatic dogs, however, they were not found in the skin of asymptomatic animals. Hypertrophy and hyperplasia of the phagocyte mononuclear system, leishmania amastigote parasites were found in the macrophages, extramedullary hematopoiesis and degenerative alterations were detected in the spleen and liver of 8 of the animals submitted to histopathological exams. In accord with these results, it was demonstrated that the expected alterations in the hematological and biochemical parameters in function of their viscerotropic nature of CVL are mainly observed in the more advanced stages of the disease. The absence of inflammatory infiltration and parasite load in the skin suggest that infected animals without symptoms may have an importance irrelevant to the infectiousness of the vector

Flebotomíneos (Diptera: Psychodidae) em um sistema agroflorestal da Região Metropolitana de Natal, Rio Grande do Norte, Brasil

Sandflies stand out as important vectors of leishmaniasis. The females need to ingest blood meals, enabling them to transmit protozoa of the genus Leishmania, which may give rise to visceral leishmaniasis (VL) or American tegumentary leishmaniasis (ATL), in addition to transmitting other parasites. Leishmaniasis are important infirmities, distributed worldwide, whose infection results from the interaction of reservoir animals, the vector insect, parasitic protozoa and the healthy host. In the state of Rio Grande do Norte (RN), Brazil, these insects are important transmitters of VL, which usually presents in the most serious form. It occurs mainly in metropolitan areas, with the dog as its main reservoir and Lutzomyia longipalpis as the vector. ATL is most present in the highland areas of the state. In addition to hematophagia, engaged in by the females, both sexes need to ingest carbohydrates, which are essential to the sand flies energy requirements and may interfere in the development of Leishmania. The aim of this study was to determine the occurrence and abundance of sand flies in different environments on the farm belonging to the Empresa de Pesquisas Agropecuárias do RN (Institute of Agricultural Research of RN), in the municipality of Parnamirim, in order to relate this occurrence with climatological and biological references and eating habits. Three consecutive monthly collections were carried out with CDC traps in a fragment of the Atlantic Forest, in a residence, on a goat breeding farm and on cashew, dwarf and giant coconut, mango, banana, eucalyptus, acacia and bean plantations. A total of 1241 sandflies from eight species (Lutzomyia evandroi, Lutzomyia longipalpis, Lutzomyia shannoni, Lutzomyia sordellii Lutzomyia walkeri, Lutzomyia wellcomei, Lutzomyia whitmani, and Lutzomyia intermedia) were collected, most in the forest environment. L. longipalpis, the main VL transmitter, was confirmed as a species adapted to anthropic environments, whereas others such as L. wellcomei, the vector of ATL, occurred predominantly in forests. Carbohydrate characterization of the sand flies and plants of the region demonstrated that a number of exotic plants such as hay and eucalyptus may play some role in the adaptation of these species to modified environments. Breeding in laboratory showed a mean biological cycle of 53.5 days from egg to adulthood for L. shannoni and the possibility of diapause behavior in L. wellcomei. This study serves as a source of information that may contribute to the epidemiological vigilance of tegumentary and visceral leishmaniasis in the state, given that it analyzes the bioecology of transmitting species, as well as their potential to adapt to new environments

Comportamento adaptativo de Lutzomyia longipalpis (Diptera: Psychodidae) às áreas urbanas da cidade de Teresina-PI.

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae) is currently appointed as the main vector of visceral leishmaniasis in the Americas. The growth of cities in areas originally endemics to American Visceral Leishmaniasis (AVL) resulted in the spread of the disease at the same time that observed the adaptation of this species to the urban environment.Changes in behavior of L.longipalpis that enabled the adapt to increasing losings of biodiversity, as well as the frequent exposure of the vector to insecticides evident in urban areas, could justify the increasing population of the species and consequently the spread of disease for these environments .Thus, we selected sixty houses spread among three areas with increasing stages of occupation of an area endemic for AVL in Teresina-PI. We evaluated the correlation between the density of L.longipalpis captured and different aspects, such as population density of animals, vegetation cover and socio-economic aspects in each house. In addition to the correlations, the feeding preference of the vector between the predominant plant species in the neighborhoods, as well as the presence of metabolic mechanisms of resistance among the captured insects were tested. The results showed that over the growing occupations, represented by three areas, L.longipalpis demonstrate its adaptive nature through an apparent opportunistic behavior in relation to sources of carbohydrates and blood. On the evolutionary point of view, this behavior may have favored its vector competence in urban areas among the limited presence of food sources, as well as in various environments encountered.

Antígenos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Associação entre fatores nutricionais e a resposta frente à infecção por Leishmania chagasi

Leishmania chagasi infection presents a wide spectrum of clinical outcomes, ranging from asymptomatic self resolving infection to disease, visceral leishmaniasis (VL). The exact mechanisms that lead the evolution of infection to disease are not understood. It is believed that malnutrition is a risk factor associated with VL development, although there are few human studies in the area. We aimed to assess the nutritional factors associated with the response to L. chagasi infection in Rio Grande do Norte. The study was conducted from December 2006 to January 2008. 149 children were assessed: 20 active VL cases, 33 children with VL history, 40 DTH+ asymptomatic children and 56 DTH-. Nutritional status was assessed using z scores for Weight/Age, Weight/Height, Height/Age, Body Mass Index (BMI), and mid-upper arm circumference/height (MUAC/height). Vitamin A status was determined by serum retinol concentrations and the modified-relative-dose-esponse test (MRDR). Breastfeeding time and birth weight were also evaluated. VL children presented compromised nutritional status when compared to the other groups using BMI and MUAC/age, with means -1,53 ± 1,10 and -1,48 ± 1,28 z scores, respectively (ANOVA, p < 0,05). VL children also showed lower vitamin A levels: 43% presented serum retinol < 20 µg/dL and 15% MRDR > 0,060. Birth weight was inverserly associated with the risk to belong the VL group (β = -0,00; OR = 0,84; 95% CI 0,73 - 0,99; p = 0,047), whereas more breastfeeding time was directly associated with the risk to belong to the DTH+ group (β = 0,02; OD = 1,16; 95% CI 1,01 - 1,33; p = 0,036). The nutritional variables evaluated were associated with the response to the L. chagasi infection, with malnutrition and compromised vitamin A status as markers of children who present with VL. Higher birth weight was associated with protection to disease, and higher breastfeeding time was associated with increased likelihood of an asymptomatic infection. The results show that modifiable nutritional aspects in the study population are associated with the response to the L. chagasi infection

Fatores genéticos e étnicos relacionados à evolução da infecção por Leishmania infantum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Determinantes envolvidos na resposta imune celular humana à infecção por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients

Aspectos clínicos e imunológicos da infecção por Leishmania Infantum Chagasi em cães do Rio Grande do Norte / Paula Vivianne Souza de Queiroz. Natal/RN

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Aspectos ambientais e sociais envolvidos na transmissão da L. chagasi no município de Parnamirim/RN

Visceral leishmaniasis hás adapted in the past 20 years to periurban and urban areas, and in Natal, Rio Grande do Norte, it became endemic. Thid study aimed to evaluate the environmental and social aspectsof Leishmania chagasi infection and its epidemiologic transmission chain in an urban, periurban and rural area of Parnamirim-RN. A study with three sections was conducted: Section 1: Sectional study of the human and canine infection by L. chagasi and its environmental and social determinants. Section 2: Observational longitudinal cohort to evaluate the dynamics of the canine infection. Section 3: Longitudinal study to evaluate the behavior of Lu. Longipalpis vector and the seasonal factors related to its dynamics. To include in the study the hauses were randomly selected and georreferenciated. Montenegro skin test was done in the human population and blood samples were collected for anti-Leishmania antibody detection. The canine population was examinated for L. chagasi infection by RIFI, ELISA and ELISA for rK39. An entomologic surveillance was monthly done with CDC light trapsin 10 houses of each locality. Quantitative and qualitative analyses was done using STATISC 6.0. Probality and prediction maps were done using ArcGis 9.0 model. In the human population L. chagasi infection was associated with the area of the hause, age, sex, population densyti, vegetation, kind of the floor of thr hause, water and resides destiny. In the canine population L. chagasi infection was associated with the breed, size, time of living in the hause, presence of dogs in the neighborhood, presence of horses and donkeys in the neighborhood, vegetation, kind of the floor and walls of the hause. The human infection was associated with canine infction only when analyzed taking into account the locality. In the prospective study, serum conversion and antibody lost observed in 30,8% and 22% of the animals examined, respectively. The human infection rate by L. chagasi was 24,6%, by the presence of anti-Leishmania antibody and 38,6% by the Montenegro skin test. The canine infection rate 32,5% by the presence of anti-Leishmania antibody. The vector Lu longipalpis showed an atypical behavior. These results indicate that environmental and social factors are important variables associated with L. chagasi infection in humans and canines, with punctual association of thr last two. Control measures of the infection on the studied points are necessary, in the aim to reduce the endemic focus of the disease in the study area. This research was carried out in a multidisciplinary involving the categories of: doctor, biologist, veterinarian, statistical, pharmaceutical and biochemical

IL-10 na patogênese da infecção por Leishmania infantum

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Estudo do Cultivo de dois clones de Escherichia coli recombinantes (eIF, LACK) para a Expressão de Antígenos da Leishmania chagasi

With advent of the technology of the recombinant DNA, the recombinant protein expression becomes an important tool in the studies of the structure, function and identification of new proteins, mainly with therapeutical purposes. The Escherichia coli has been procarioto predominant in the studies of genetic engineering due to wealth of information regarding its metabolism. Despite the expressivo advance of the studies of molecular biology and the immunology of the infections, it does not exist, currently, no prophylactic drug capable to prevent calazar. Of this form, it exists a great necessity of specific antigen identification for the vaccine development and kits for disgnostic against the visceral Leishmaniose. In this context, this work objectified to study the recombinant antigen expression of the Leishmania chagasi during the culture of Escherichia coli in shaker. A first set of assays was carried through with the objective of if knowing the kinetic behavior of the growth of two clones recombinant proteins (eIF, LACK) in two different compositions of culture medium (2xTY, TB) supplemented by antibiotics, without IPTG addition. In the second stage of the assays, the procedure of induction for IPTG was carried through, in order to verify the influence of the composition of the ways tested in the expression them recombinant proteins. On the basis of the gotten results, can be observed that the high complexity of culture medium favored the kinetic one of growth of clones recombinant (eIF, LACK), however, to if to deal with the assays submitted to the procedure of induction for IPTG, the raised complexity of culture medium did not favor the expression of recombinant proteins. On the other hand, they had been gotten resulted positive for all clones recombinant (eIF, LACK) tested, confirmed through the eletroforético profile

Avaliação de dois clones de Escherichia coli recombinante quanto ao crescimento e expressão de antígenos de Leishmania chagasi (kmp11 e P36)

Visceral leishmaniosis caused by Leishmania chagasi, also known as calazar, presented, in the period from 1990 to 2005, tax of incidence in Brazil varying between 1 and 3 cases for 100 000 inhabitants. The Northeast region that up to the year of 2000 contributed with almost 90% of the registered cases is reducing his participation in the current decade, reaching 56% in 2005. Conventional leishmaniasis treatment is costly and it shows high toxicity, demanding more research for alternative treatments, with special interest in development of vaccines and diagnosis kits which include production of recombinant antigens by host cells. Escherichia coli has been the microorganism most studied and used as a host for recombinant protein production. Therefore, the aim of this work was to study the influence of induction on cellular growth and to verify the type of Leishmania chagasi antigens expression (intra or extracellular) during two recombinant E. coli clones (kmp11 and P36) cultivation in rotary incubator (shaker) using three different media (2xTY, TB, FASS+EL). For that, tests were carried out using conditions established in the literature for E. coli (37°C and 200 rpm) and media supplemented with antibiotics to guarantee that only competent cells grows. First, tests were carried out without induction in order to verify the two microorganisms kinetic behavior (growth and substrate consumption) in different media. Next, the induction was carried out through the addition of IPTG (1mM as final concentration), at the first hour of cultivation. It was observed that protein expression were intracellular for all clones and media tested, however the highest level of expression was clearly observed by the electrophoresis band density (intensity) for 2xTY medium and kmp11 protein. Although it contains the lowest substrate concentration, consequently, a reduced cellular concentration when compared to other media, it appeared that this medium and clone combination is the most indicated for recombinant protein production. Therefore, the objective of this work was achieved, since the interested proteins were produced. Consequently, this result motivates new studies for production optimization using different cultivation strategies

Influência das condições de cultivo na produção de antígenos recombinantes de Leishmania i. chagasi utilizando Escherichia coli M15 cultivada em incubador rotativo e biorreator

Escherichia coli has been one of the most widely used hosts in recombinant protein production, in both laboratory and industrial scale since the advent of recombinant DNA technology. Despite the substantial progress of studies on the molecular biology and immunology of infections, there is currently no medication-based prophylaxis capable of preventing leishmaniasis. As such, there is a great need to identify specific antigens for the development of vaccines and diagnostic kits against visceral leishmaniasis. Thus, the primary goal of the present study is to assess the influence of cultivation conditions on the production of Leishmania chagasi antigens, carried out in a rotating incubator and bioreactor. To that end, several assays were conducted to evaluate the kinetic behavior of antigens (648, 503) of Leishmania. i. chagasi in two different compositions of media (2xTY, TB), with and without an inducer. In order to improve expression, assays were performed in a benchtop bioreactor using the best conditions obtained in a rotating incubator, in addition to assessing the influence of stirring speed. Results show that high complexity of the cultivation medium favored kinetic growth of clones (648, 503). However, in assays submitted to induction by IPTG, this elevated complexity did not promote the expression of recombinant proteins. Expression of antigens 648 and 503 exhibited behavior associated with growth and, in terms of location, proteins 648 and 503 are intracellularly stored. Lactose may be the most adequate inducer in protein expression, when considering factors, cost, toxicity and stability. Elevated stirring may increase cell growth in clone 53, although it may not result in high concentrations for the protein of interest. On the other hand, positive results were obtained for all recombinant clones (648, 503) tested, confirmed by the electrophoretic profile