Zoonotic parasites infecting free-living armadillos from Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild-caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen's disease). Forty-three free-living armadillos from Pantanal and seven road-killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti-T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

[Paracoccidioidomycosis: chronicle of a neglected disease]. / Paracoccidioidomicosis: crónica de una enfermedad olvidada.

Paracoccidioidomycosis (PCM) is among the systemic mycoses which are endemic only in Latin America. In Argentina, the vast majority of the cases are reported at north of latitude 34.5° S. The disease is produced by thermodimorphic fungi of the genus Paracoccidoides: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), P. venezuelensis (PS4) y P. lutzii. The natural habitat of members of this genus is the soil, where they produce infectious conidia. Little is known, however, about their specific ecologic niche(s), and this knowledge gap hampers the design of measures to control the infection. Rural male workers are the group most at risk of developing PCM. Infection occurs by inhalation of aerosolized conidia and may either be asymptomatic or cause mild respiratory symptoms. In turn, this primary infection may be self-limited or progress to severe pulmonary or disseminated disease. The disease has two clinical presentations: (i) acute or subacute (juvenile), frequent in children, adolescents and people with immunodeficiencies; and (ii) chronic progressive, in adults. Active lesions often resolve into fibrotic scars which can cause dysphagia, dysphonia, adrenal insufficiency, and intestinal obstruction. Although efficient tools are available for diagnosis and treatment, the nonspecific nature of PCM clinical manifestations frequently delay the diagnosis. In addition, the poor adherence to long antifungal treatments allows the advance of the disease and the development of extensive fibrosis compromising severely and permanently respiratory and adrenal functions, thus altering the patient"s quality of life and even causing his/her death.

Paracoccidioidomicosis: crónica de una enfermedad olvidada / Paracoccidioidomycosis: chronicle of a neglected disease

La paracoccidioidomicosis (PCM) es una de las micosis sistémicas que son endémicas exclusivamente en América Latina. Está causada por hongos termodimorfos del género Paracoccidoides: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), P. venezuelensis (PS4) y P. lutzii. Paracoccidioides habita y produce conidios infecciosos en suelos de zonas subtropicales húmedas. En Argentina está presente al norte del paralelo 34.5° S. Poco se sabe sobre su nicho ecológico específico, lo que dificulta el diseño de medidas de control de la PCM. La infección ocurre en hospederos susceptibles cuando inhalan conidios aerosolizados. Los trabajadores rurales varones son el grupo más expuesto a contraer PCM. La primoinfección puede ser asintomática o causar un cuadro respiratorio leve; este, a su vez, puede autolimitarse o progresar a enfermedad, ya sea pulmonar o diseminada. Existen dos formas de presentación: (i) aguda/subaguda, en niños, adolescentes y personas con sistemas inmunes comprometidos; y (ii) crónica progresiva, en adultos. La cicatrización de las lesiones resulta en secuelas fibróticas que pueden causar disfagia, disfonía, insuficiencia suprarrenal y obstrucción intestinal. Aunque existen herramientas para su diagnóstico, la PCM es rara vez sospechada precozmente porque sus manifestaciones clínicas iniciales son inespecíficas. Sumados, el diagnóstico tardío y la baja adherencia a los efectivos pero largos tratamientos antimicóticos permiten el avance de la enfermedad y el desarrollo de fibrosis tisular extensa, lo que compromete gravemente la función respiratoria y suprarrenal, altera permanentemente la calidad de vida del paciente y puede causar su muerte.

Paracoccidioidomycosis (PCM) is among the systemic mycoses which are endemic only in Latin America. In Argentina, the vast majority of the cases are reported at north of latitude 34.5° S. The disease is produced by thermodimorphic fungi of the genus Paracoccidoides: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), P. venezuelensis (PS4) y P. lutzii. The natural habitat of members of this genus is the soil, where they produce infectious conidia. Little is known, however, about their specific ecologic niche(s), and this knowledge gap hampers the design of measures to control the infection. Rural male workers are the group most at risk of developing PCM. Infection occurs by inhalation of aerosolized conidia and may either be asymptomatic or cause mild respiratory symptoms. In turn, this primary infection may be self-limited or progress to severe pulmonary or disseminated disease. The disease has two clinical presentations: (i) acute or subacute (juvenile), frequent in children, adolescents and people with immunodeficiencies; and (ii) chronic progressive, in adults. Active lesions often resolve into fibrotic scars which can cause dysphagia, dysphonia, adrenal insufficiency, and intestinal obstruction. Although efficient tools are available for diagnosis and treatment, the nonspecific nature of PCM clinical manifestations frequently delay the diagnosis. In addition, the poor adherence to long antifungal treatments allows the advance of the disease and the development of extensive fibrosis compromising severely and permanently respiratory and adrenal functions, thus altering the patient´s quality of life and even causing his/her death.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis.

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366 kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366 kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366 kDa) in sera from PCM patients.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis / Fração de alta massa molecular em isolados clínicos de Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

INTRODUÇÃO: Diferentes níveis sorológicos de IgG/IgE contra a fração de alta massa molecular (hMM) (~366kDa) de Paracoccidioides brasiliensis têm sido encontrados na PCM aguda e crônica. Considerando a inexistência de investigação sobre esta fração em isolados clínicos de P. brasiliensis, o objetivo deste estudo foi investigar a presença da fração hMM (~366kDa) no preparado livre de células (CFA) de P. brasiliensis obtidos de isolados clínicos. MÉTODOS: CFA de 10 isolados e de cepa de referência (Pb18) foram submetidas à eletroforese em gel de SDS-poliacrilamida (SDS-PAGE) seguida de captura de imagem e análise por densitometria. Adicionalmente, CFA de 20 isolados e de Pb18 foram analisados por ELISA captura (cELISA) utilizando anticorpos policlonal (polAb) ou monoclonal (mAb) para fração hMM. RESULTADOS: A presença do componente de hMM foi observada em todas as amostras analisadas por SDS-PAGE/densitometria e por cELISA. Adicionalmente, o teste de correlação de Pearson demonstrou forte relação entre os níveis de fração hMM usando pAb e mAb (R = 0.853) no cELISA. CONCLUSÕES: Conclui-se que a fração hMM está presente em todos os isolados clínicos de P. brasiliensis analisados e no isolado referencial, sugerindo a possibilidade dos mesmos serem utilizados como fonte desta fração antigênica. Este trabalho também introduz pela primeira vez o método de cELISA para detecção da fração hMM de P. brasiliensis, sugerindo que detecção utilizando anticorpos polAb ou mAb é viável e essa metodologia poderá ser útil para investigação futura desta fração solúvel (~366kDa) em soros de pacientes com PCM.

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus.

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Cytokines and lymphocyte proliferation in juvenile and adult forms of paracoccidioidomycosis: comparison with infected and non-infected controls.

Cellular immune response to Paracoccidiodes brasiliensis antigens (PbAg) was evaluated in patients with the juvenile (JF) and adult (AF) forms of paracoccidioidomycosis as well as in a group of infected individuals living in the endemic area but without any clinical manifestation of the disease. The immune profile of this group of paracoccidioidomycosis-infected individuals was characterized by: 1) a positive skin test to P. brasiliensis antigen; 2) absence of specific antibodies; 3) a vigorous lymphoproliferative response to PbAg; and 4) a typical Th1 pattern of cytokines, with production of IFN-gamma and basal levels of IL-4, IL-5 and IL-10. At the opposite end of the spectrum were the JF patients whose proliferative response to PbAg was significantly impaired and whose cytokine pattern was characteristically Th2, i.e. lower IFN-gamma secretion and significantly higher levels of IL-4, IL-5 and IL-10. These profiles are compatible with forms of higher and lower resistance, respectively. Intermediate immune responses were observed in AF patients, whose specific lymphoproliferative response was lower than in the paracoccidioidomycosis-infected group but higher than in the JF patients. The secretion of IFN-gamma and IL-10 did not differ from the JF group, although IL-4 and IL-5 levels were significantly lower. Since AF patients are able to control fungal dissemination for decades, they can be considered more resistant than JF patients, who manifest the disease soon after infection.

Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis.

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Experimental paracoccidioidomycosis in hamster: transmission electron microscopy of inoculation site lesion.

Interaction between Paracoccidioides brasiliensis (Pb) and inflammatory cells in hamster testis was studied sequentially by transmission electron microscopy. In early lesions (six hours after inoculation), polymorphonuclear neutrophils (PMNs) were the major and mononuclear cells and eosinophils were the minor constituents of the inflammatory cells. PMNs were later replaced by mononuclear cells. Viable Pb cells were phagocytosed or surrounded by inflammatory cells. Preserved Pb cells usually had broad host-parasite interphases, whereas dying ones had narrow interphases. The outer layer of the fungus wall was sometimes broken by PMN in some focal points, broken pieces being peeled off and phagocytosed. Small Pb cells were uninuclear, and were often related to broad interphase. Large Pb cells were multinucleated with irregularly shaped wall, and sometimes had lomasome and/or myelin like structures. Different interaction patterns of Pb with inflammatory cells may be due to functionally different host cell flow to the inoculation site or due to the age of Pb cells or both.

Contribuiçäo para o estudo clínico-laboratorial e terapêutico da formaçäo juvenil da paracoccidioidomicose / Paracoccidioidomycosis: clinical and terapeutic study of the juvenil form of the disease

A paracoccidioidomicose é a micose sistêmica mais frequente na regiäo do Triângulo, no Estado de Minas Gerais. A maioria dos casos diagnosticados nesta localidade enquadra-se na forma adulta da moléstia (idade 30 anos), mas cerca de um terço (36,5 por cento ) do total de doentes corresponde a forma juvenil desta infecçåo (idade 30 anos), modalidade sabidamente de maior gravidade e com feiçöes clínico-laboratoriais bastante peculiares. No presente trabalho, realizou-se um estudo prospectivo com o objetivo de analisar detalhadamente os aspectos clínicos, laboratoriais e terapêuticos da forma juvenil desta micose, em 24 pacientes observados no período de fevereiro de 1982 a dezembro de 1987, no Hospital de Clínicas da Universidade Federal de Uberlândia. Este estudo permitiu ampla revisäo da literatura latinoamericana sobre o assunto e, com detalhes, destacou as características peculiares, como já referido, desta forma clínica, em particular: a) a raridade da doença na infância, notadamente, na primeira década de vida; b) a baixa proporçäo na incidência entre homens e mulheres (5/1) comparado com a relatada na forma adulta da moléstia (até 125/1); c) o elevado acometimento pelo fungo do sistema macrofágico-linfóide manifestado principalmente por linfadenopatia, localizada ou generalizada, hepatoesplenomegalia e envolvimento do tubo digestivo e do sistema ganglionar-mesentérico; d) a ocorrência menos frequente de lesöes cutâneo-mucosas, comumente vistas na forma adulta da micose; e) a presença de icterícia, ascite e massa abdominal palpável, alteraçÆes decorrentes do envolvimento quase constante do sistema linfático-abdominal; f) a raridade do acometimento pulmonar, habitualmente visto em pacientes de faixas etárias mais elevadas. O trabalho ainda salientou as alteraçöes laboratoriais decorrentes da doença, em particular a eosinofilia, e discutiu os resultados controversos das reaçÆes sorológicas específicas realizadas no decurso desta moléstia. O papel dos métodos de imagem, em particular, da ultrasonografia foi destacado, pelas importantes informaçÆes que trouxe em relaçåo ao acometimento extremamente frequente dos orgåos intra-abdominais pelo P. brasilienses, tendo sido recomendado sua utilizaçåo rotineira na avaliaçäo inicial e evolutiva destes pacientes. Finalmente, este estudo mostrou os resultados terapêuticos obtidos com os sulfamídicos, com a Anfotericina B e, particularmente com o Ketoconazol nesta modalidade da micose, enfatizando a excelente resposta observada com este derivado imidazólico, que permitiu alcançar remissÆes em curtos períodos de tempo e com mínimos efeitos colaterais, mesmo em forma relativamente graves desta doença

Relaçäo parasito-hospedeiro na paracoccidioidomicose: revisäo da literatura / Parasite-host relation in paracoccidioidomycosis: review of the literature

Realizou-se revisäo da literatura dos estudos sobre a relaçäo parasitahospedeiro na paracoccidioidomicose, visando correlacionar estes dados com a patogenia da doença. Inicialmente, foram revistas mecanismos de resistência do hospedeiro ao nível da porta de entrada; açäo dos estrógenos, interagindo com receptores do fungo, bloqueando a transformaçäo dos micélios as formas leveduriformes parasitárias; papel dos fagócitos e do complemento; açäo das células citotóxicas naturais. A seguir, discutiram-se os dados experimentais e humanos indicativos do papel do controle genético sobre resistência e suscetibilidade ao P. brasiliensis. Por fim, foi apresentado o papel da imunidade humoral e celular anti-P, brasiliensis nas defesas do hospedeiro, visando bloquear a divisäo e a disseminaçäo do fungo, e sua interrelaçäo com a morfogênese do granuloma