Chagas disease in an area of recent occupation in Cochabamba, Bolivia

INTRODUCTION: A descriptive, entomological and seroepidemiological study on Chagas disease was conducted in a place of recent occupation on the outskirts of Cochabamba, Bolivia: Avaroa/Primer de Mayo (population:3,000), where the socio-economic level is low and no control measures have been made available. METHODS: The immunofluorescent antibody test (IFAT) was used for IgG and IgM anti-Trypanosoma cruzi antibodies in filter paper bloodspot eluates from 128 subjects (73 females, 55 males) selected by systematic sampling. Concerning each subject age, gender, birthplace, occupation, duration of residence and building materials used in their houses were recorded. Vectors were captured both in domestic and peridomestic environments. RESULTS: Seropositive, 12.5% (16/128): females, 15.1% (11/73); males, 9.1% (5/55). Average time of residence: 6.1 years for the whole population sample and 7.4 years for the seropositive subjects. Most houses had adobe walls (76.7% , n= 30), galvanized iron rooves (86.7%) and earthen floors (53.4%) 80% of the walls had crevices. One hundred forty seven specimens of Triatoma infestans were captured, of which 104 (70.7%) were domestic, and 1 peridomestic Triatoma sordida. Precipitin host identification: birds, 67.5%; humans, 27.8%; rodents, 11.9%; dogs, 8.7%; cats, 1.6%. House infestation and density indices were 53.3 and 493.0 respectively. We found 21 (14.3%) specimens of T. infestans infected with trypanosomes, 18 (85.7%) of which in domestic environments. DISCUSSION: The elements for the vector transmission of Chagas disease are present in Avaroa/Primer de Mayo and the ancient custom of keeping guinea pigs indoors adds to the risk of human infection. In neighboring Cochabamba, due to substandard quality control, contaminated blood transfusions are not infrequent, which further aggravates the spread of Chagas disease. Prompt action to check the transmission of this infection, involving additionally the congenital and transfusional modes of acquisition, is required.

Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

CHRONIC CHAGASIC CARDIOPATHY: THE PRODUCT OF A TURBULENT HOST-PARASITE RELATIONSHIP

The pathogenesis of chronic chagasic cardiopathy is still a debated matter. In this review, the main theories raised about it since the first description of the disease in 1909 by Carlos Chagas, are considered. The scarcity of T.cruzi parasites into the myocardium and the apparent lack of correlation between their presence and the occurrence of myocardial inflammatory infiltrate, have originated many theories indicating that chronic Chagas' cardiopathy is an autoimmune disease. Recently however, papers using immunohistochemical technique or PCR have demonstrated a strong association between moderate or severe myocarditis and presence of T.cruzi Ags, indicating a direct participation of the parasite in the genesis of chronic chagasic myocarditis. Different patterns of cytokine production seem to have important role in the outcome of the disease. Participation of the microcirculatory alterations and fibrosis as well as the relationship with the parasite are also emphasized. Finally, the author suggests that the indeterminate form of the disease occurs when the host immunological response against the parasite is more efficient while the chronic cardiopathy occurs in patients with hyperergic and inefficient immune response

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.

Long term evaluation of etiological treatment of Chagas disease with benznidazole

The aim of this article is to present an investigation of cure rate, after long follow up, of specific chemotherapy with benznidazole in patients with both acute and chronic Chagas disease, applying quantitative conventional serological tests as the base of the criterion of cure. Twenty one patients with the acute form and 113 with one or other of the various chronic clinical forms of the disease were evaluated, after a follow up period of 13 to 21 years, for the acute, and 6 to 18 years, for the chronic patients. The duration of the acute as well as the chronic disease, a condition which influences the results of the treatment, was determined. The therapeutic schedule was presented, with emphasis on the correlation between adverse reactions and the total dose of 18 grams, approximately, as well as taking into consideration precautions to assure the safety of the treatment. Quantitative serological reactions consisting of complement fixation, indirect immunofluorescence, indirect hemagglutination, and, occasionally, ELISA, were used. Cure was found in 76 per cent of the acute patients but only in 8 per cent of those with chronic forms of the disease. In the light of such contrasting results, fundamentals of the etiological therapy of Chagas disease were discussed, like the criterion of cure, the pathogenesis and the role of immunosuppression showing tissue parasitism in long standing chronic disease, in support of the concept that post-therapeutic consistently positive serological reactions mean the presence of the parasite in the patient's tissues. In relation to the life-cycle of T. cruzi in vertebrate host, there are still some obscure and controversial points, though there is no proof of the existence of resistant or latent forms. However, the finding over the last 15 years, that immunosuppression brings about the reappearance of acute disease in long stand chronic patients justifies a revision of the matter. Facts were quoted in favor of the treatment of chronic patients.

dogs may be a reservoir host for Angiostrongylus costaricensis

Angiostrongylus costaricensis is a parasitic nematode of wild rodents. Several other vertebrate species including man may become infected by ingestion of the third stage larvae produced by the intermediate hosts, usually slugs from the family Veronicellidae. There is a report of the diagnosis of abdominal angiostrongyliasis in Canis familiaris with lesions resembling those found in human disease. As a preliminar evaluation of the adequacy of a canine model for pathogenetic studies, a dog was inoculated with 75 L3 of A. costaricensis. Infection was established and fist stage larvae were found in feces up to 88 days post infection, sometimes in very large numbers (9.5 x 10(4) L1/g). No clinical manifestations or significant lesions were detected. These are indications that dog may play a role as a reservoir host for A. costaricensis.

Hepatitis C viral load does not predict disease outcome: going beyond numbers

The analysis of 58 patients with chronic hepatitis C without cirrhosis and treated with interferon-alpha demonstrated that hepatitis C viral (HCV) load does not correlate with the histological evolution of the disease (p = 0.6559 for architectural alterations and p = 0.6271 for the histological activity index). Therefore, the use of viral RNA quantification as an evolutive predictor or determinant of the severity of hepatitis C is incorrect and of relative value. A review of the literature provided fundamental and interdependent HCV (genotype, heterogeneity and mutants, specific proteins), host (sex, age, weight, etc) and treatment variables (dosage, time of treatment, type of interferon) within the broader context of viral kinetics, interferon-mediated immunological response (in addition to natural immunity against HCV) and the role of interferon as a modulator of fibrogenesis. Therefore, viral load implies much more than numbers and the correct interpretation of these data should consider a broader context depending on multiple factors that are more complex than the simple value obtained upon quantification.

A Triatoma maculata (Hemiptera, Reduviidae, Triatominae) population from Roraima, Amazon region, Brazil, has some bionomic characteristics of a potential Chagas disease vector

Even though Chagas disease is rare in the Brazilian Amazon, the conditions for the establishment of domiciliated cycles prevail in many areas where triatomines are of frequent occurrence. In Roraima, a previous serological and entomological survey in three agricultural settlements showed the existence of all transmission cycle elements, i.e., individuals infected by Trypanosoma cruzi, triatomine species previously found harboring T. cruzi in the broader Amazon region of neighboring countries and, domicile/ peridomicile conditions favorable to triatomine colonization. Triatoma maculata was the most frequent species, found in chicken houses in the peridomicile and sporadically within residences. Aiming to investigate the possibility of T. maculata to possess the potentiality to transmit T. cruzi in the area, bionomic characteristics were studied under laboratory conditions. These were feeding frequency, time for defecation after a blood meal, time elapsed in voluntary fasting pre- and pos-ecdysis, moulting time periods, pre-oviposition and oviposition periods and index of oviposition, incubation period, egg viability, longevity and mortality rate. Results show that the Passarão population of T. maculata should be considered a potential vector of T. cruzi since it shows a capacity to infest artificial ecotopes in the peridomicile, to carry out large number of meals during the nymphal cycle, to have a relatively short developmental cycle capable of producing 2.9 generations/year, to blood source eclecticism, to defecate immediately after the blood meal while still on the host and to the fact that has been previously found naturally infected by T.cruzi.

Feeding sources and trypanosome infection index of Rhodnius pallescens in a Chagas disease endemic area of Amador County, Panama

The sylvatic triatomine Rhodnius pallescens is considered to be the most important and widespread vector of Trypanosoma cruzi and Trypanosoma rangeli in Panama. However, its behavior and biological characteristics have only been partially investigated. Thus, to achieve sustainable and efficient control over Chagas disease in Panama, a better understanding of the ecology and biology of R. pallescens is essential. In this study we evaluated R. pallescens host feeding sources using a dot-blot assay, and the trypanosome infection index by PCR analysis in a Chagas disease endemic area of central Panama. It was found that in peridomestic palm trees, 20.3% of the examined bugs had fed on opossums (Didelphis marsupialis). However, we observed an increased anthropophagy (25.4%) for those bugs collected inside houses. Considering the domestic and peridomestic habitats as a whole, the proportion of collected R. pallescens infected with trypanosomes was 87.4%. In the two habitats the predominant infection was with T. cruzi (80-90%). Between 47-51% of the analyzed triatomines were infected with T. rangeli. Mixed infections (40-51%) were also detected. These findings provide a better basis for the implementation of a rational control and surveillance program for Chagas disease in regions where R. pallescens is endemic.

Effects of vitamin C supplementation on acute phase Chagas disease in experimentally infected mice with Trypanosoma cruzi QM1 strain

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

Host-induced changes in the cell surface N-linked glycoproteins, from Aspergillus fumigatus. Search for specific targets with potential for clinical therapy and/or diagnosis.

This dissertation is presented to obtain a Master degree in Structural and Functional Biochemistry

OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST

SUMMARYSchistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively).

SPATIAL DISTRIBUTION OF LYMNAEIDAE (MOLLUSCA, BASOMMATOPHORA), INTERMEDIATE HOST OF Fasciola hepatica LINNAEUS, 1758 (TREMATODA, DIGENEA) IN BRAZIL

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

Evaluation of the phagocytic activity of peripheral blood monocytes of patients with Jorge Lobo's disease

Studies on host-parasite interaction in Jorge Lobo's disease are scarce, with no report in the literature on the phagocytosis of Lacazia loboi by phagocytic mononuclear cells. Thus, the objective of the present study was to assess the phagocytic activity of blood monocytes in the presence of L. loboi in patients with the disease and in healthy subjects (controls) over 3 and 24 hours of incubation. Statistical analyses of the results showed no significant difference in percent phagocytosis of the fungus between patient and control monocytes. With respect to incubation time, however, there was a significant difference, in that percent phagocytosis was higher at 3 hours than at 24 hours (p <0.01). These results suggest that monocytes from patients with the mycosis are able to phagocyte the fungus, as also observed in control individuals.