The Mating Type (MAT) and Virulence of Sporothrix schenckii sensu stricto Isolates Maintained in Culture Collection.

In an attempt to determine the mating type of different Sporothrix schenckii sensu stricto isolates that remained viable after a long period of preservation in a culture collection and to correlate them with the degree of virulence/pathogenicity, a PCR technique using primers designed for the sequences of MAT1-1-1 and MAT1-2-1 genes and a murine experimental model were used. The results showed that there was no correlation between the mating type and virulence among the isolates. Furthermore, different degrees of virulence/pathogenicity, ranging from high to low, were found among them based on different virulence parameters. It was assumed that the long period of preservation favored the changes, yielding the isolation of variants. Thus, we believe that new technologies for studies on factors can improve our knowledge of the pathogenesis of sporotrichosis.

Soil samples from sporotrichosis transmission belt area: Searching for fungal species and their antagonistic activity against Sporothrix brasiliensis.

Since 1998, the state of Rio de Janeiro, Brazil, has become a public health problem regarding sporotrichosis, a disease caused by Sporothrix spp. involving contact with infected cats. Efforts to isolate these species from environmental sources are not always successful. In our study, soil from residences situated in cities of Rio de Janeiro where cats with sporotrichosis live was collected and cultured an attempt to isolate Sporothrix spp. but it was not successful. However, other saprophytic fungal species were isolated from soil and identified and among them Purpureocillium lilacinum was the most frequent. From there, we decided to study the in vitro interaction of this species with S. brasiliensis, the principal agent that causes sporotrichosis in this state. The results showed that ten isolates of P. lilacinum inhibited the radial mycelial growth of S. brasiliensis with different percentage of inhibition. The interaction between them revealed the pattern described as overgrowth by antagonist. In conclusion, our data suggest that fungal species with very fast growth and capable of producing metabolites could hinder the growth of Sporothrix spp., it also opens the way for the identification of secondary metabolites with biological activity that could be tested against pathogenic fungi.

Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil.

Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial ß-tubulin gene sequencing and phylogenetic analysis.

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.

Tregs in the immune response of BALB/c mice experimentally infected with species of the Sporothrix genus.

Background: Sporotrichosis occurs through contact with contaminated soil and plant. However, the incidence of sporotrichosis as a zoonotic epidemic has increased, particularly in Rio de Janeiro. Aim: In this work, we decided to evaluate some T-cell phenotypes involved in the immune response. Materials & methods: We used flow cytometry to quantify TCD4+ and TCD8+ and Treg from immunocompetent and immunosuppressed mice infected with Sporothrix species with different levels of virulence and pathogenicity. Results: It was demonstrated the predominance of TCD4+ over the TCD8+ cells in both groups, inoculated with all the species, and percentages of Treg observed in infected immunocompetent mice. Conclusion: This regulatory phenotype can be associated with a protective immunity in the initial periods of infection.

A case of sporotrichosis caused by different Sporothrix brasiliensis strains: mycological, molecular, and virulence analyses.

BACKGROUND: Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES: The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS: Genotypic analyses by partial sequencing of the calmodulin, ß-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS: These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.

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.

Clinical features, fungal load, coinfections, histological skin changes, and itraconazole treatment response of cats with sporotrichosis caused by Sporothrix brasiliensis.

Zoonotic sporotrichosis caused by the fungus Sporothrix brasiliensis is usually severe in cats. This study investigated the associations between clinical features, fungal load, coinfections, histological skin changes, and response to itraconazole in cats with sporotrichosis caused by S. brasiliensis. Fifty-two cats with skin lesions and a definitive diagnosis of sporotrichosis were treated with itraconazole for a maximum period of 36 weeks. The animals were submitted to clinical examination and two subsequent collections of samples from the same skin lesion for fungal diagnosis and histopathology, as well as serology for feline immunodeficiency (FIV) and leukaemia (FeLV) viruses. Thirty-seven (71%) cats were clinically cured. Nasal mucosa lesions and respiratory signs were associated with treatment failure. Cats coinfected with FIV/FeLV (n = 12) had a lower neutrophil count in the lesion. A high fungal load in skin lesions was linked to young age and treatment failure, as well as to a longer time of wound healing, poorly formed granulomas and fewer neutrophils, macrophages and lymphocytes in these lesions. These results indicate that itraconazole is effective, but nasal mucosal involvement, respiratory signs and high fungal loads in skin lesions are predictors of treatment failure that will assist in the development of better treatment protocols for cats.

Experimental Hyalohyphomycosis by Purpureocillium lilacinum: Outcome of the Infection in C57BL/6 Murine Models.

Purpureocillium lilacinum is a filamentous, hyaline fungus considered an emerging pathogen in humans. The aim of our study was to evaluate the outcome of hyalohyphomycosis in C57BL/6 murine models inoculated with two clinical P. lilacinum isolates (S1 and S2). Each isolate was inoculated in mice randomly distributed in immunocompetent (CPT) and immunosuppressed (SPS) groups. Mice were evaluated at day 7, 21, and 45 after inoculation for histopathological analysis, recovery of fungal cells, and immunological studies. Histological analysis showed scarce conidia-like structures in lung tissue from CPT mice and a lot of fungal cells in SPS mice inoculated with S2 compared to mice inoculated with S1. The maximum recovery of fungal cells was seen in CPT mice inoculated with both isolates at day 7, but with mean significantly higher in those inoculated with S2 isolate. Phenotypical characterization of T cells showed TCD8+ lymphocytes predominance over TCD4+ in immunosuppressed mice infected and control groups. We also observed higher percentages of the central and effector memory/effector phenotype in CPT mice infected with S2 strain, especially in TCD8+ in the initial period of infection. Regulatory T cells showed higher percentages in immunosuppressed, predominantly after the acute phase. Our results showed that the P. lilacinum is a fungus capable to cause damages in competent and immunosuppressed experimental hosts. Furthermore, S2 isolate seems to cause more damage to the experimental host and it was possible to identify different cellular subsets involved in the mice immune response.

Reflexões sobre o Ensino online de Biossegurança à luz da Teoria da Aprendizagem Significativa / Reflections on the online learning of Biosafety based in the meaningful learning

Organizar eventos educacionais, com foco na aprendizagem, exige articulação entre planejamento, processo de ensino e avaliação. O presente manuscrito objetiva avaliar a estruturação pedagógica e os resultados no QBA/Online – curso de Biossegurança a distância, inserido no Moodle, oferecido aos ingressos do Instituto Oswaldo Cruz – à luz da Teoria da Aprendizagem Significativa (TAS), para identificar oportunidades de melhorias. Na pesquisa qualitativa, foram considerados os dados de 2760 participantes, de 2008 a 2014. A média de participação anual foi de 460, com predominância de alunos (40%). No planejamento, não foi utilizada uma fundamentação teórica-metodológica, mas considerados os aspectos contextuais do ensino e a natureza do conhecimento a ser ensinado. O conteúdo está em 12 textos. O maior índice de acertos nos testes foi de 18 questões, no único instrumento de avaliação da aprendizagem, com 20 perguntas, centradas nos temas dos textos. O curso foi bem avaliado pelos alunos. A flexibilidade do tempo/local está dentre os pontos positivos e a quantidade/tamanho dos textos, nos negativos. O diagnóstico ratificou a relevância de um curso online; necessidade de reorganização do material instrucional; e de oferta de estratégias de ensino para estimular o interesse dos alunos (AU)

Organizing educational events, focusing on learning, requires disposition between planning, teaching process and evaluation. This manuscrip tintent to evaluate pedagogical format and results in QBA/Online – distance learning course on Biosafety, accessed in Moodle platform, off ered to graduates from Oswaldo Cruz Institute – based in the Meaningful Learning Theory as to enlighten opportunities for improvement. A qualitative inquiry was performed, with 2760 participants and registered in the system, from 2008 to 2014. The average annual contribution is 460, with a predominance of students (40%). With regard toplanning, theoretical-methodological were not considered, only contextual aspects of education and the concepts of knowledge to be taught/instructed. Twelve texts were assigned. Thehighest scores rates in tests reached 18 questions, the only instrument for assessing learning, whichhas 20 questions, focusing on themes of current texts. The course was well rated by those who completed the evaluation form. Participants highlighted as positive aspects; flexibility as to time/place and as negative the large number of texts and its sizes. The analysis confirmed the benefits of online tool; the need to rearrange institutional material; and providing teaching strategies to encourage students’ interest (AU)

Reflexões sobre o ensino online de Biossegurança à luz da Teoria da Aprendizagem Significativa / Reflections on the online learning of Biosafety based in the meaningful learning

Organizar eventos educacionais, com foco na aprendizagem, exige articulação entre planejamento, processo de ensino e avaliação. O presente manuscrito objetiva avaliar a estruturação pedagógica e os resultados no QBA/Online ­ curso de Biossegurança a distância, inserido no Moodle, oferecido aos ingressos do Instituto Oswaldo Cruz ­ à luz da Teoria da Aprendizagem Significativa (TAS), para identificar oportunidades de melhorias. Na pesquisa qualitativa, foram considerados os dados de 2760 participantes, de 2008 a 2014. A média de participação anual foi de 460, com predominância de alunos (40%). No planejamento, não foi utilizada uma fundamentação teórica-metodológica, mas considerados os aspectos contextuais do ensino e a natureza do conhecimento a ser ensinado. O conteúdo está em 12 textos. O maior índice de acertos nos testes foi de 18 questões, no único instrumento de avaliação da aprendizagem, com 20 perguntas, centradas nos temas dos textos. O curso foi bem avaliado pelos alunos. A flexibilidade do tempo/local está dentre os pontos positivos e a quantidade/tamanho dos textos, nos negativos. O diagnóstico ratificou a relevância de um curso online; necessidade de reorganização do material instrucional; e de oferta de estratégias de ensino para estimular o interesse dos alunos

Organizing educational events, focusing on learning, requires disposition between planning, teaching process and evaluation. This manuscrip tintent to evaluate pedagogical format and results in QBA/Online ­ distance learning course on Biosafety, accessed in Moodle platform, off ered to graduates from Oswaldo Cruz Institute ­ based in the Meaningful Learning Theory as to enlighten opportunities for improvement. A qualitative inquiry was performed, with 2760 participants and registered in the system, from 2008 to 2014. The average annual contribution is 460, with a predominance of students (40%). With regard toplanning, theoretical-methodological were not considered, only contextual aspects of education and the concepts of knowledge to be taught/instructed. Twelve texts were assigned. Thehighest scores rates in tests reached 18 questions, the only instrument for assessing learning, whichhas 20 questions, focusing on themes of current texts. The course was well rated by those who completed the evaluation form. Participants highlighted as positive aspects; flexibility as to time/place and as negative the large number of texts and its sizes. The analysis confirmed the benefits of online tool; the need to rearrange institutional material; and providing teaching strategies to encourage students' interest

Feline histoplasmosis: a case of supraorbital involvement / Histoplasmose felina: relato de caso com envolvimento supraorbital

Histoplasmosis is a systemic fungal infection caused by the dimorphic fungus Histoplasma capsulatum that infects humans and a wide variety of mammalian species, including cats. In Brazil only four cases of this disease were reported in domestic cats. The present study reports a case of an indoor cat, rescued from street three years ago, that developed histoplasmosis with supraorbital involvement. The cat presented sneezing, dyspnea and increased respiratory sounds and the diagnosis suspected was allergic rhinitis. Prednisolone and amoxicillin were prescribed. The patient returned to clinic after clinical signs worsened with a swelling of the right supraorbital region, unilateral left side conjunctivitis and enlarged submandibular lymph nodes. Cytology and culture revealed Histoplasma capsulatum. Initially, itraconazole (10 mg/Kg SID orally) was prescribed and due to the worsening of clinical signs, was combined amphotericin B (0.5 mg/kg SC) for two weeks. The therapy with amphotericin was discontinued and itraconazole continued to be administered for six months. The clinical signs were resolved and no recrudescence was reported. It is important that veterinary practitioners to be aware of the clinical features of feline histoplasmosis and consider the possibility of a subclinical infection.

Histoplasmose é uma infecção sistêmica causada pelo fungo dimórfico Histoplasma capsulatum, que infecta humanos e uma ampla variedade de mamíferos, incluindo os gatos. No Brasil, somente quatro casos dessa doença foram relatados em gatos domésticos. O presente estudo relata um caso de um gato, vivendo em ambiente domiciliar sem acesso à rua, mas que tinha sido resgatado das ruas havia três anos, que desenvolveu histoplasmose com envolvimento supraorbital. O paciente apresentava espirros, dispneia e aumento dos sons respiratórios e a suspeita clínica foi rinite alérgica. O animal foi tratado com prednisolona e amoxicilina. Com a piora dos sinais clínicos o paciente retornou apresentando um edema na região supraorbital direita, conjuntivite unilateral do olho esquerdo e linfoadenomegalia na região submandibular. A citologia e a cultura revelaram a presença de Histoplasma capsulatum. Inicialmente, itraconazol (10 mg/kg via oral, uma vez ao dia) foi prescrito e devido à piora dos sinais clínicos, anfotericina B (0,5 mg/Kg via subcutânea) foi administrada por duas semanas associada ao triazólico. A terapia com anfotericina B foi descontinuada e o itraconazol mantido por seis meses até a cura clínica e nenhuma recrudescência foi registrada. É importante ressaltar que veterinários devem estar atentos para os sinais clínicos da histoplasmose e que devem considerar a possibilidade de uma infecção subclínica.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells.

INTRODUCTION: Purpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro. METHODS: Spores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy. RESULTS: After 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinum conidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells. CONCLUSIONS: P. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

Introduction Purpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro. Methods Spores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy. Results After 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinum conidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells. Conclusions P. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells. .

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.

[The importance of the contextual approach in the teaching of biosafety]. / A importância da abordagem contextual no ensino de biossegurança.

Biosafety is a field of knowledge that raises questions geared to genetically modified organisms that are linked to social and job-related employee protection. The educational process involves seeking to create a participative and transforming agent and must therefore transcend the simple concept of teaching. Thus, it is important to contextualize biosafety within a constructive teaching strategy by identification of its core concepts - risk, hazard and accident - which allows each individual to understand how risk is perceived within society and dealt with in academia in order to add multiple skills to tackle the situation. Understanding how the relationship between work and health and its consequences and effects are constructed over the course of time, makes it possible to train more critical and well prepared citizens to participate in decisions of a political and social nature that can influence their future.