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- ItemAcesso aberto (Open Access)Análises de glicoesfingolipídeos em diferentes espécies do fungo patogênico do gênero Aspergillus(Universidade Federal de São Paulo (UNIFESP), 2018-06-28) Rodrigues, Karolina Marques [UNIFESP]; Takahashi, Helio Kiyoshi [UNIFESP]; http://lattes.cnpq.br/8090231917635170; Universidade Federal de São Paulo (UNIFESP)incidence of serious fungal infections has increased in the last decade, mainly due to the increase in the number of immunocompromised patients, representing a serious cause of morbidity and mortality in this population. Recent works show that fungi are vulnerable to inhibitors of sphingolipid biosynthesis, which led us to study the structures of fungal glycosphingolipids, in order to obtain relevant information on the biosynthesis and functional roles of these molecules and to propose new potential targets for antifungal treatment. Glycosylinositol phosphorilceramides (GIPCs), inositolphosphoryl ceramides (IPCs), and monohexosyl ceramides (CMHs) from A. fumigatus, A. flavus, A. niger and A. nidulans, filamentous fungi causing aspergillosis, were extracted, purified and characterized using methods chromatography, biochemistry and mass spectrometry. By mass spectrometry using electrospray ionization (EISMS) in positive mode, two major peaks corresponding to CMHs of 754 m/z and 756 m/z were identified for all species analyzed. By collisioninduced dissociation (CID), m/z fragments of m/z: 736, 574, 556 and 276 were identified for CMH 754 m/z. Similarly for CMH m/z 756 fragments of m/z 738, 576, 558 and 276. The fragmentation pattern for both CMHs corresponds respectively to [M+H1H2O]+, [M+H1H2OHex]+, [M+H2H2OHex]+, [M+H2H2Oacyl]+, the fragments associated with ceramidecontaining CMH, with structures not described in mammalian CMH, d19:2/h18:1 and d19:2/18:0, respectively. Glucosylceramide and galactosylceramide were characterized by high resolution thin layer chromatography (HPTLC). It was possible to identify 2 different species of IPCs [M+H]+, 926 m/z, and 954 m/z, presenting 282 m/z and 310 m/z fragments for sphingosine t18:0 and t20:0, respectively. The presence of inositol was confirmed using 241 m/z precursor ion mass spectrometry, referring to the inositol phosphate group of these structures. For the characterization of the GIPCs in the different Aspergillus species, we used collision induced dissociation (DIC) in tandem MS with eletrotron ionization (ESI), in positive mode [M+H]+. In this way we were able to detect the 1088 m/z and 1116 m/z ions corresponding to GIPCs with 1 hexose; 1250 m/z and 1278 m/z, GIPCs with 2 hexoses; 1412 m/z and 1440 m/z, corresponding to GIPCs containing 3 hexoses. The GIPCs present A. fumigatus, A. flavus and A. niger were derived from the IPC 926 m/z (containing ceramide 666 m/z) and the IPC 954 m/z (containing ceramid 694 m/z), on the other hand, the A. nidulans GIPCs are derived only from the IPC 926 m/z. The presence of galactofuranose residues in GIPCs was confirmed using the monoclonal antibody MEST1 which recognizes terminal residues of galactofuranose. By immunostaining the HPTLC plates with MEST1 the A. fumigatus, A. flavus and A. niger GIPCs, which had a component recognized by mAb MEST1, have the same chromatographic migration as GIPC called Pb1 of Paracoccidioides brasiliensis. As for A. nidulans, only small reactivity with mAb MEST1 was detected. These results indicate that in the four species studied, both CMHs, IPCs and GIPCs have structures not expressed in mammals, indicating that these molecules as well as the specific enzymes involved in the metabolism of these sphingolipids can be considered as targets for antifungal treatment .
- ItemAcesso aberto (Open Access)Características clínicas de pacientes de aspergilose pulmonar associada à COVID-19(Universidade Federal de São Paulo, 2021-12-15) Estevam, Vinícius de Oliveira [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; Melo, Daniela de Oliveira [UNIFESP]; http://lattes.cnpq.br/5052823551616937; http://lattes.cnpq.br/4373797404389169A pandemia de COVID-19 se mostrou um grande problema para o mundo todo, não só por causa do vírus e a doença causada em si, mas também por causa de infecções oportunistas que podem piorar ainda mais o quadro já crítico dos pacientes. Uma dessas infecções é a aspergilose pulmonar causada por fungos do gênero Aspergillus. Esse trabalho de conclusão de curso baseia-se em artigos contendo dados retroativos obtidos em hospitais de diferentes localizações do mundo que mostram algumas características gerais da COVID-19, da aspergilose pulmonar e da associação entre essas doenças conhecida como “aspergilose pulmonar associada a COVID-19” ou APAC. O objetivo é analisar esses dados de forma crítica e tentar mapear um perfil comum dos pacientes acometidos por essa condição. Esse perfil foi construído por idade, sexo, condição imunológica, espécies de Aspergillus spp. mais frequentes, comorbidades ocorrentes, classificação da doença e o desfecho clínico. Os dados apresentados mostram que o perfil dos pacientes APAC são em sua maioria do sexo masculino, com hipertensão, diabetes, comorbidades cardiorespiratórias e/ou obesidade. O agente causador da aspergilose mais provável é o Aspergillus fumigatus e a doença na maioria dos casos é classificada como “provável”. O paciente tem grandes chances de ir a óbito, demonstrando a gravidade da doença. A maioria dos pacientes é imunocompetente, mas não se pode descartar que imunossupressões podem aumentar a ocorrência e piorar o quadro. Este estudo trouxe interessantes dados sobre o perfil do paciente APAC, mas ainda existem perguntas mais específicas a serem respondidas, como por exemplo a relação entre severidade da doença e condição imune dos pacientes.
- ItemAcesso aberto (Open Access)Caracterização da produção de biofilme por isolados de Aspergillus spp. e o seu impacto biológico(Universidade Federal de São Paulo (UNIFESP), 2017-03-15) Trindade, Mario Roberto de Sousa [UNIFESP]; Colombo, Arnaldo Lopes [UNIFESP]; Gonçalves, Sarah Santos [UNIFESP]; http://lattes.cnpq.br/1018708169217296; http://lattes.cnpq.br/4512261018429681; http://lattes.cnpq.br/2416431642602733; Universidade Federal de São Paulo (UNIFESP)In the last decade, the biomedical literature has described that Aspergillus spp. isolates are able to grow adhered to biotic and abiotic surfaces, surrounded by a polysaccharide extracellular matrix that provides protection from physical and chemical agents, such as the immune system and antifungal drugs. This study aims to characterize the biofilm formation by 18 isolates of Aspergillus section Flavi and section Fumigati, also to evaluate its impact in the antifungal susceptibility “in vitro” and in the virulence. The identification of Aspergillus spp. isolates was realized through a polyphasic approach, including phenotypic tests and molecular identification. The in vitro susceptibility profile was evaluated using the broth microdilution method (CLSI, document M38-A2). The biofilm formed by isolates of Aspergillus spp. was quantified using the crystal violet technique and the impact of this biofilm in the antifungals activity was evaluated by the XTT tetrazolium salt. Isolates with higher biofilm formation capacity were selected for micromorphological characterization by confocal microscopy before and after treatment with amphotericin B and voriconazole. The virulence of the strains with higher and lower biofilm formation capacity were evaluated using the experimental model of infection in Galleria mellonella, generating a survival curve. The identification using a polyphasic approach, allowed the accurate identification of all 18 isolates, namely: six A. tamarii, six A. flavus and six A. fumigatus. All isolates presented MIC values against the antifungals tested in accordance to data described in the literature. The A. fumigatus isolates produced more biofilm than A. tamarii and A. Flavus isolates, with no difference between the later two. Biofilm cells from all isolates of Aspergillus spp. were resistant to triazoles and presented varied susceptibility to amphoterin B. The confocal microscopy analyses demonstrated that the biofilm is composed of well delineated and metabolically active hyphae, however, this aspect was greatly altered after exposure to antifungals. Using the G. mellonella invertebrate model, we observed that all isolates were capable of 100% mortality, in 24 hours for invertebrates infected with A. tamarii and A. flavus and in 96 hours for invertebrates infected with A. fumigatus. The high biofilm-forming phenotype did not affect the survival of the invertebrates Our results demonstrate that the antifungals tested have some level of action on the biofilm formed by Aspergillus spp. even at concentrations lower than the MIC value for these isolates. It was not possible to establish a relationship between the biofilm formation capacity and the survival time.
- ItemAcesso aberto (Open Access)Estudo da micobiota em conjuntiva sadia de diabéticos, residentes na área urbana da cidade de São Paulo - Brasil(Conselho Brasileiro de Oftalmologia, 2006-02-01) Andrade, Alfredo José Muniz de [UNIFESP]; Hofling-Lima, Ana Luisa [UNIFESP]; Yu, Maria Cecília Zorat [UNIFESP]; Godoy, Patricio [UNIFESP]; Gompertz, Olga Fischman [UNIFESP]; Bonfim, Sabrina de Souza [UNIFESP]; Andrade, Francisco Eudes Muniz de; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de PernambucoPURPOSE: To determine the mycobiota of the healthy conjunctiva in diabetic individuals, according to diabetes type, age, sex, disease time, type of treatment, and stage of diabetic retinopathy of the individuals. To identify the anemophilus mycobiota in the sampling rooms. METHODS: A cross-sectional study was carried out on 803 diabetics who reside in the urban area of São Paulo-SP/Brazil. Sabouraud's dextrose agar culture with chloramphenicol was used for primoisolation, and the key of De Hoog was used to identify filamentous fungi. RESULTS: Of the evaluated diabetics, 6.6% (53/803) presented type 1 diabetes and 93.4% (750/803) type 2. The positive cultures for fungi in the conjunctiva of diabetics was 4.2% (34/803), with 1.9% (1/53) in type 1 diabetics and 4.4% (33/740) in type 2 diabetics (p=0.720). With respect to the presence or not of isolated fungi, there was no statistically significant association regarding age (p=0.575), sex (p=0.517), disease time (p=0.633), type of treatment (p=0.422), and diabetic retinopathy stage (p=0.655) of the tested individuals. The identified fungi were all filamentous: Aspergillus spp. represented 59.5% (25/42) of isolations and 47.6% (20/42) of isolated species were Aspergillus niger. Growth of anemophilus fungi occurred in the air of the room and coincidences were observed between the isolated species from the air and those from the conjunctiva. CONCLUSIONS: Presence of mycobiota in healthy conjunctivas of diabetics was identified, with no significant association between the greater number of positive fungi isolations and the type of diabetes, age, sex, disease type, type of treatment, and stage of diabetic retinopathy. In the collection rooms, anemophilus mycobiota was identified.
- ItemSomente MetadadadosMycobiota and mycotoxins in Brazil nut samples from different states of the Brazilian Amazon region(Elsevier B.V., 2012-10-01) Reis, T. A.; Oliveira, T. D.; Baquiao, A. C.; Goncalves, S. S. [UNIFESP]; Zorzete, P.; Correa, B.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)The objective of this study was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in Brazil nut samples collected in different states of the Brazilian Amazon region: Acre, Amazonas, Amapa, and Para. A total of 200 husk samples and 200 almond samples were inoculated onto Aspergillus flavus-parasiticus agar for the detection of fungi. Mycotoxins were analyzed by high-performance liquid chromatography. the mycobiota comprised the following fungi, in decreasing order of frequency: almonds - Phialemonium spp. (54%), Penicillium spp. (16%), Fusarium spp. (13%), Phaeoacremonium spp. (11%), and Aspergillus spp. (4%), husks - Phialemonium spp. (62%), Phaeoacremonium spp. (11%), Penicillium spp. (10%), Fusarium spp. (9%), and Aspergillus spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins were detected in 22 (11%) of the 200 almond samples, with 21 samples presenting aflatoxin B-1 levels above 8 mu g/kg, the limit established by the European Commission for Brazil nuts for further processing. Nineteen (9.5%) of the 200 husk samples contained aflatoxins, but at levels lower than those seen in almonds. Cyclopiazonic acid (CPA) was detected in 44 (22%) almond samples, with levels ranging from 98.65 to 1612 mu g/kg. Aspergillus nomius and A. flavus were the most frequent Aspergillus species. the presence of fungi does not necessarily imply mycotoxin contamination, but almonds of the Brazil nut seem to be a good substrate for fungal growth. (C) 2012 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Ocorrência de fungos em sistema de distribuição de água e sua correlação com isolados do ar em uma unidade pediátrica de transplante de células tronco hematopoiéticas(Universidade Federal de São Paulo (UNIFESP), 2009-07-29) Rocha, Sabrina Mesquita [UNIFESP]; Colombo, Arnaldo Lopes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: The expansion of the immunocompromised patient population in the last two decades has led to an increased incidence of invasive fungal infections, especially by filamentous fungi. Although external sources of contamination are well known related to airconditioning in units of risk, recent investigations have shown that water distribution systems or linked to tanks of hospitals have been also identified as an important source of infection when it is colonized by pathogenic fungi. Objectives: Quantify and identify samples of fungi isolated from water and air of a unit of HSCT, and evaluate them by using molecular techniques looking for their genetic relationship among strains of A. fumigatus, A. flavus and F. solani. Material and Methods: The current study was performed in a unit of HSCT, collecting samples monthly over 12-month period. Air samples were recovered from outdoor air, bathrooms, and rooms in hospital unit, the air sample was collected with six-faced Andersen collector, with Petri dishes containing Sabouraud-dextrose agar. All water samples were collected in sterile polystyrene bottles and passed through sterile 0.45um filters using a filtration apparatus. Using sterile forceps, the filters were place directly on Sabouraud-dextrose agar plates. The phenotypic identification was made using keys for identification of Hoog (2000). In the genotypic analysis, the isolates of A. fumigatus, A. flavus and F. solani were typing using oligonucleotide and microsatellite minisatellite. Results: A total of 164 samples of water where collected. We observed higher amount of conidia in the months corresponding to the autumn and summer. The average conidia were 6.07 CFU / l. The most prevalent genera were Paecilomyces, Penicillium and Cladosporium. Fusarium was more frequent in the water of the unit. The unique parameter correlated to the increasing isolation of fungi was the water temperature. In the microbiological analysis of air from the hospital unit, a total of 264 samples were collected recovering larger quantities of conidia during the fall. The individual average of conidia was 11.09 CFU/m3. The most prevalent genera were Cladosporium and Penicillium. The genus Aspergillus was isolated in the air mostly indoor. In the molecular analysis, it was observed poor similarity among F. solani strains from air and water, however, we reported that two isolates remained in the hydraulic system of the unit for a period over 30 days. Two samples of air, showed 2 distinct environment strains of A. fumigatus showing high similarity through molecular analysis. In our molecular analysis, conflicting profiles of A. flavus strains were obtained with different oligonucleotide, it was not possible to establish any direct relationship between samples of air and water. Conclusions: environmental surveillance in the units of risk is the highest importance not only applying to outbreaks, as well as to evaluate potential sources of nosocomial infection. Pathogenic fungi to humans can be often isolated, and persist in the water for months in the hydraulic system as a reservoir of infection. We did not identified genetic relationship among samples from air and water, propagules of A. fumigatus, A. flavus and F. solani.
- ItemAcesso aberto (Open Access)Prevalência de espécies de Aspergillus em amostras de origem clínica/ambiental e análise de seu perfil de susceptibilidade(Universidade Federal de São Paulo (UNIFESP), 2013-09-25) Negri, Clara Ezequiel [UNIFESP]; Colombo, Arnaldo Lopes [UNIFESP]; Gonçalves, Sarah Santos [UNIFESP]; http://lattes.cnpq.br/4512261018429681; http://lattes.cnpq.br/9300997857265211; Universidade Federal de São Paulo (UNIFESP)Espécies do gênero Aspergillus são responsáveis pela segunda e/ou terceira maior ocorrência de infecções fúngicas invasivas (IFIs) em hospitais terciários. Na prática clínica, essas infecções são difíceis de diagnosticar e o tratamento tardio é muito frequente. Poucos centros médicos têm capacitação para realizar a identificação acurada de espécies deste gênero de fungos, visto que a formação em micologia clássica, bem como o domínio e disponibilidade de técnicas moleculares são requeridas para este diagnóstico. Os casos de resistência a antifúngicos não são comuns na medicina contemporânea, mas o número de isolados resistentes e fracasso terapêutico têm aumentado significativamente nas últimas décadas. São escassos os dados no Brasil sobre a prevalência de espécies de Aspergillus e seu perfil de susceptibilidade a antifúngicos. O presente estudo teve como objetivos analisar a distribuição das espécies do gênero Aspergillus em 150 amostras clínicas/ambientais e avaliar a ocorrência de cepas com valores de CIMs superiores ao cut-off epidemiológico proposto aos antifúngicos comumente utilizados. A identificação de espécies do gênero Aspergillus foi realizada pela abordagem polifásica, a qual inclui análise macro e micromorfológica, termotolerância, sequenciamento e filogenia se necessário, (região ITS do rDNA e dos genes calmodulina e β-tubulina). Os isolados também foram avaliados quanto à tolerância a cloreto de sódio (5% e 10%), cicloheximida (0,1% e 0,5 %) e produção de ácidos orgânicos em CREA e ácido aspergílico em AFPA. Foi avaliado o perfil de susceptibilidade in vitro dos 150 isolados frente a triazólicos (itraconazol, voriconazol e posaconazol) utilizando 2 diferentes métodos: microdiluição em caldo e E-test. Os isolados com valores de cut-off superiores aos estabelecidos, também foram testados frente a anfotericina B, caspofungina e anidulafungina. A caracterização fenotípica, assim como o sequenciamento da região ITS separou os isolados em nove seções, a saber: Circumdati, Clavati, Flavi, Flavipedes, Fumigati, Nidulantes, Nigri, Terrei e Usti. Utilizando a abordagem polifásica, 141/150 isolados foram identificados em nível de espécie. Com a abordagem realizada, não foi possível identificar 3/43 isolados pertencentes à seção Flavi e 6/17 da seção Nigri. Com relação aos triazólicos testados, 141/150 isolados apresentaram valores de CIMs menores ou iguais ao cut-off epidemiológico estabelecido utilizando a metodologia padronizada. Dentre os isolados, 1/9 Circumdati, 1/9 Usti, 1/9 Nigri, 4/9 Fumigati e 2/9 Flavi apresentaram valores mais elevados frente a estes antifúngicos; 2/9 apresentaram valores de CIM elevados também frente à anfotericina B (N. pseudofischeri e A. ochraceus). Somente o isolado de A. calidoustus apresentou valor de CIM elevado frente à caspofungina. Todos os nove isolados testados frente à anidulafungina apresentaram como valor de CIM a menor concentração testada deste fármaco, demonstrando sua ótima atividade in vitro. A falta de padronização da metodologia de E-test®, dificultou sua execução e avaliação do teste, principalmente no que diz respeito à leitura, prejudicando, assim, o estabelecimento de taxas de correlação entre as duas metodologias testadas. Esta taxa foi classificada como marginal quando consideramos como equivalentes valores de ± uma diluição e as taxas tornam-se aceitáveis quando consideramos como equivalentes valores de ± duas diluições. Os dados observados reforçam a importância na identificação acurada de espécies de Aspergillus, visto que encontramos um número substancial de espécies crípticas que apresentam variações no perfil de susceptibilidade entre os isolados aos antifúngicos comumente utilizados. Neste contexto, demonstramos a importância na padronização da metodologia de E-test® para aumentar a confiabilidade do teste.
- ItemSomente MetadadadosPurification and biochemical characterization of an extracellular serine peptidase from aspergillus terreus(Taylor & francis inc, 2016) Biaggio, Rafael Tage; da Silva, Ronivaldo Rodrigues; da Rosa, Nathalia Gonsales; Ribeiro Leite, Rodrigo Simoes; Arantes, Eliane Candiani; de Freitas Cabral, Tatiana Pereira; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Cabral, HamiltonPeptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45 degrees C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S-2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S-1 showed higher catalytic efficiency than the S-2 and S-3 subsites.
- ItemAcesso aberto (Open Access)Respiratory epithelial cells: more than just a physical barrier to fungal infections(MDPI, 2022-05-24) Barros, Bianca Carla Silva Campitelli; Almeida, Bruna Rocha; Barros, Débora Tereza Lucas [UNIFESP]; Toledo, Marcos Sergio [UNIFESP]; Suzuki, Erika [UNIFESP]; http://lattes.cnpq.br/3545915337077337; http://lattes.cnpq.br/1449454874275064; http://lattes.cnpq.br/0670710968392309; http://lattes.cnpq.br/3304493420272173; http://lattes.cnpq.br/3308810633774571The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.