Clinical and Anatomopathological Evaluation of BALB/c Murine Models Infected with Isolates of Seven Pathogenic Sporothrix Species.

BACKGROUND: Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by seven pathogenic species of Sporothrix genus: S. schenckii sensu stricto, S. brasiliensis, S. globosa and S. luriei (clinical clade), and the species S. mexicana, S. pallida and S. chilensis (environmental clade). Isolates of the same species of Sporothrix may have different pathogenicities; however, few isolates of this fungus have been studied. Thus, the aim of this work was to analyze the clinical and anatomopathological changes in immunocompetent and immunosuppressed BALB/c mice infected with clinical and environmental isolates of seven different species of Sporothrix, from both clades. One human clinical isolate of S. schenckii sensu stricto, S. brasiliensis, S. globosa, S. luriei, S. mexicana and S. chilensis species and one environmental isolate of S. pallida were inoculated subcutaneously in immunocompetent mice and the same isolates of S. brasiliensis and S.schenckii sensu stricto were inoculated in immunossupressed mice. Clinical manifestations as external lesions, apathy, and alopecia were observed. At 21, 35, and 49 days after fungal inoculation, four mice from each group were weighed, euthanized and necropsied for evaluation of splenic index, recovery of fungal cells, macroscopic and histopathological analysis of livers, lungs, kidneys, and hearts. The survival assessment was observed for 50 days following inoculation. Our results demonstrated that, clinical S. schenckii isolate, followed by clinical S. mexicana, and environmental S. pallida isolates, the last two, species grouped in the environmental clade, were capable of inducing greater anatomopathological changes in mice, which was reflected in the severity of the clinical signs of these animals. Thus, we reinforce the hypothesis that the pathogenicity of Sporothrix is not only related to the species of this fungus, but also shows variation between different isolates of the same species.

Skin Immune Response of Immunocompetent and Immunosuppressed C57BL/6 Mice After Experimental Subcutaneous Infection Caused by Purpureocillium lilacinum.

Hyalohyphomycosis is a fungal infection characterized by the presence of a hyaline mycelium in the host. It is caused by several agents, such as Purpureocillium lilacinum. Our study aimed to evaluate some cell subsets and inflammatory markers involved in the in situ immune response to subcutaneous hyalohyphomycosis by P. lilacinum in C57BL/6 murine models. The fungal isolate was inoculated in mice randomly distributed in immunocompetent/infected (CI) and immunosuppressed/infected (SI) groups. Mice were evaluated on days 1, 3, 5, and 7 after inoculation. Histopathological studies showed several lesions in the site of infection as well as the formation of multifocal and mixed inflammatory infiltrates, which differed between the CI and SI groups. This analysis also revealed conidia and hypha-like structures in subcutaneous tissues of mice of both groups. The immunohistochemical analysis showed lower percentages of macrophages and neutrophils in the SI group compared to those in the CI group. Moreover, the intensity of interleukin (IL)-1ß and nitric oxide synthase 2 production by cells of immunosuppressed mice was discreet, compared to immunocompetent mice that ranged from moderate to intense over time. The quantitative interference of dexamethasone in the response to the fungus was also demonstrated. We concluded that our results can be useful not only to broaden the knowledge on P. lilacinum but also, based on this host-parasite relationship, to contribute to the understanding of the mechanisms of infection.

Human Pathogenic Paecilomyces from Food.

Paecilomyces spp. and Byssochlamys spp. are heat-resistant fungi important to industry because they can cause food and beverage spoilage, incurring economic loss. The consequences of food or beverage fungal colonization is the loss of nutritional value, structure and taste, and the possibility of producing toxic secondary metabolites that may result in medical problems. Furthermore, these fungi can infect animals and humans and it is unknown if contaminated foods may be fomites. P. variotii is the principal agent of food spoilage or contamination and it is most frequently associated with human hyalohyphomycosis with clinical manifestations including peritonitis, cutaneous and disseminated infections, among others. Byssochlamys spp. had not been identified as a cause of systemic infection until the case of a dog with a fungal infection, after immunosuppressive therapy. P. variotii has clinical importance because it causes severe infection in immunosuppressed patients and also because the number of immunocompetent infected patients is increasing. This review draws attention to the ability of these species to grow at high temperatures, to colonize food products, and to cause human disease.

Detection of antibody to Purpureocillium lilacinum by immunofluorescent assay and flow cytometry in serum of infected C57BL/6 mice.

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques.

Neglected and emerging fungal infections: review of hyalohyphomycosis by Paecilomyces lilacinus focusing in disease burden, in vitro antifungal susceptibility and management.

Paecilomyces lilacinus is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. This review discusses infections caused by P. lilacinus, as well as their symptoms and correlates of immune responses, morphological characteristics of the fungus, therapies, in vitro susceptibility tests, laboratory diagnosis and the experimental models available.

A estruturação do programa de capacitação profissional de biossegurança no contexto do projeto de modernização da gestão científica do Instituto Oswaldo Cruz / Development of a biosafety training program aligned with the scientific management modernization project of the Oswaldo Cruz Institute

O presente artigo apresenta as etapas de estruturação do Programa de Capacitação Profissional de Biossegurança (PCPB), em consonância com o Projeto de Modernização da Gestão Científica do Instituto Oswaldo Cruz (IOC), detalhando o ciclo planejamento-desenvolvimento-avaliação, em especial do Curso de Biossegurança em Laboratório de Pesquisa Biomédica. Inicialmente, para o ciclo diagnóstico foram aplicados questionários aos interlocutores dos laboratórios do IOC, os quais revelaram interesse de participação no PCPB para ambas as categorias profissionais (níveis médio e superior), indicando como temáticas preferenciais biossegurança e boas práticas de laboratório. Na fase de planejamento foi definido que o PCPB seria subdivido em dois projetos (Boas Práticas de Laboratório de Saúde Pública para os profissionais de nível médio e Curso de Biossegurança para Laboratórios de Pesquisa Biomédica para profissionais de nível superior). A seguir, na fase de estruturação do curso, os módulos contemplados incluíram: introdutório; riscos químico, físico e biológico; gestão da qualidade e experimentação animal. Assim, no período 2006-2008, foram capacitados 315 profissionais e realizadas avaliações segundo o modelo de David Kirkpatrick. O primeiro nível, chamado de reação, foi aferido e demonstrou que 54,03 por cento dos profissionais declararam que o curso foi excelente; 39,59 por cento classificaram como bom e os demais 6,38 por cento acharam que foi regular ou não opinaram. Para a avaliação do aprendizado foram realizados, a cada módulo, pré e pós-testes. Foi verificado que todos os módulos tiveram acréscimos nas médias do pós-teste em relação ao pré-teste. Os resultados obtidos apontaram estratégias a serem seguidas no aperfeiçoamento desse modelo de educação continuada em biossegurança.

Brazilian propolis: correlation between chemical composition and antimicrobial activity.

The chemical composition of ethanol extracts from samples of Brazilian propolis (EEPs) determined by HPLC and their activity against Trypanosoma cruzi, Staphylococcus aureus, Streptococcus pneumoniae, Klebisiella pneumoniae, Candida albicans, Sporothrix schenckii and Paracoccidioides brasiliensis were determined. Based on the predominant botanical origin in the region of samples' collection, the 10 extracts were separated into three groups: A (B. dracunculifolia + Auraucaria spp), B (B. dracunculifolia) and C (Araucaria spp). Analysis by the multiple regression of all the extracts together showed a positive correlation, higher concentrations leading to higher biological effect, of S. aureus with p-coumaric acid (PCUM) and 3-(4-hydroxy-3-(oxo-butenyl)-phenylacrylic acid (DHCA1) and of trypomastigotes of T. cruzi with 3,5-diprenyl-4-hydroxycinnamic acid derivative 4 (DHCA4) and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran (DCBEN). When the same approach was employed for each group, due to the small number of observations, the statistical test gave unreliable results. However, an overall analysis revealed for group A an association of S. aureus with caffeic acid (CAF) and dicaffeoylquinic acid 3 (CAFQ3), of S. pneumoniae with CAFQ3 and monocaffeoylquinic acid 2 (CAFQ2) and of T. cruzi also with CAFQ3. For group B, a higher activity against S. pneumoniae was associated DCBEN and for T. cruzi with CAF. For group C no association was observed between the anitmicrobial effect and any component of the extracts. The present study reinforces the relevance of PCUM and derivatives, especially prenylated ones and also of caffeolyquinic acids, on the biological activity of Brazilian propolis.

Comparison of virulence of different Sporothrix schenckii clinical isolates using experimental murine model.

The virulence of two strains of Sporothrix schenckii isolated from patients with lymphocutaneous or disseminated sporotrichosis were examined in BALB/c mice (Group 1 and 2, respectively). The mice were inoculated subcutaneously into the left hind footpad with 4 x 10(6) S. schenckii yeast cells in order to evaluate (i) the development of cutaneous lesions, (ii) signs of inactivity, (iii) weight loss, (iv) survival rates, (v) number of viable yeast cells in the lungs and spleen, (vi) splenic index, (vii) extent of organ lesions, and (viii) immunological responses. Comparison of the two groups showed more severe disease in Group 2 mice that developed significant weight and hair loss associated with inactivity and left hind footpad lesions that extended close to the testicular area. The histopathology and large number of viable microorganisms isolated from the spleen confirmed the higher invasive ability of this strain. Moreover, a decrease of an in vitro specific lymphoproliferative response and IFN-gamma production were observed over time in Group 2 mice. As a result, at the end of the experiment, the S. schenckii-antigen (Ss-Ag) response was considered negative with a stimulation index (SI) = 2. In contrast, Group 1 mice presented a positive response to Ss-Ag (SI = 14.1). These results confirm the existence of different virulence profiles in S. schenckii strains. In addition, the use of subcutaneous inoculation as a suitable route for verification of the pathogenicity of this fungus in the murine model was confirmed.

Viability and molecular authentication of Coccidioides immitis strains from culture collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.

Twenty Coccidioides immitis strains were evaluated. Only 5 of the 20 strains kept under mineral oil maintained their viability while all 5 subcultures preserved in water remained viable and none of the 13 subcultures kept in soil were viable. A 519 bp PCR product from the csa gene confirmed the identity of the strains.

Viability and molecular authentication of Coccidioides immitis strains from culture collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil

Vinte cepas de Coccidioides immitis foram avaliadas.Cinco das 20 cepas preservadas sob óleo mineral mantiveram-se viáveis, todas as 5 subculturas preservadas em água permaneceram viáveis e nenhuma das 13 subculturas mantidas em solo foi viável. Um produto de PCR de 519 pb do gene csa confirmou a identidade das cepas.

Morphological, biochemical and molecular approaches for comparing typical and atypical Paracoccidioides brasiliensis strains.

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.