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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The emergence of cryptococcus gattii in British Columbia : veterinary aspects

Duncan, Colleen 16 June 2005
A series of presumed or confirmed Cryptococcus gattii cases diagnosed between 1999 and 2003 was compiled through review of records from veterinary laboratories and human diagnostic services. There was a continual increase in the annual number of animal, but not human, cases diagnosed; no seasonality was observed. Animal cases exceeded human cases by almost 75% even though it was hypothesized that animal cases are more likely to go undiagnosed or unreported when compared to humans. Animal cryptococcosis cases were identified on Vancouver Island prior to 1999 suggesting the organism may have emerged in the region prior to its identification as a causative agent for human disease; therefore animals may serve as a good sentinel for human cryptococcosis infection.

There were 50% more feline than canine cases and disease appeared more commonly in middle aged cats and younger dogs. There was no sex predilection for either species. The primary system involved was most commonly respiratory, followed by central nervous system (CNS) in both cats and dogs. There was a higher proportion of CNS disease in dogs relative to cats, and cats were much more likely to have subcutaneous or dermal masses relative to dogs. Multivariate survival analysis identified only the presence of neurological symptoms as a statistically significant predictor of mortality; those animals exhibiting CNS symptoms were over four times more likely to die than those never showing neural signs. A case-control study identified host and environmental risk factors for clinical C. gattii infection in dogs and cats suggesting that where an infectious agent is not uniformly distributed, individual risk increases when the organism is re-distributed through large scale environmental disturbance, or when the animal has increased opportunities for exposure through travel or activity level.

Serum samples and material for fungal culture were collected from dogs, cats, horses and terrestrial mammal species residing within the region where clinical cases had been diagnosed. Nasal colonization was identified in squirrels ( Sciurus carolinensis), horses, dogs and cats. Most of the animals sampled had no signs of systemic infection however asymptomatic infection, defined as the presence of cryptococcal antigen in the bloodstream in the absence of clinical symptoms, was identified in a small number of dogs and cats. Fourteen months of follow-up testing of asymptomatic animals revealed that animals can progress to clinical disease, remain sub-clinically infected, or clear the organism.

2

The emergence of cryptococcus gattii in British Columbia : veterinary aspects

Duncan, Colleen 16 June 2005 (has links)
A series of presumed or confirmed Cryptococcus gattii cases diagnosed between 1999 and 2003 was compiled through review of records from veterinary laboratories and human diagnostic services. There was a continual increase in the annual number of animal, but not human, cases diagnosed; no seasonality was observed. Animal cases exceeded human cases by almost 75% even though it was hypothesized that animal cases are more likely to go undiagnosed or unreported when compared to humans. Animal cryptococcosis cases were identified on Vancouver Island prior to 1999 suggesting the organism may have emerged in the region prior to its identification as a causative agent for human disease; therefore animals may serve as a good sentinel for human cryptococcosis infection.

There were 50% more feline than canine cases and disease appeared more commonly in middle aged cats and younger dogs. There was no sex predilection for either species. The primary system involved was most commonly respiratory, followed by central nervous system (CNS) in both cats and dogs. There was a higher proportion of CNS disease in dogs relative to cats, and cats were much more likely to have subcutaneous or dermal masses relative to dogs. Multivariate survival analysis identified only the presence of neurological symptoms as a statistically significant predictor of mortality; those animals exhibiting CNS symptoms were over four times more likely to die than those never showing neural signs. A case-control study identified host and environmental risk factors for clinical C. gattii infection in dogs and cats suggesting that where an infectious agent is not uniformly distributed, individual risk increases when the organism is re-distributed through large scale environmental disturbance, or when the animal has increased opportunities for exposure through travel or activity level.

Serum samples and material for fungal culture were collected from dogs, cats, horses and terrestrial mammal species residing within the region where clinical cases had been diagnosed. Nasal colonization was identified in squirrels ( Sciurus carolinensis), horses, dogs and cats. Most of the animals sampled had no signs of systemic infection however asymptomatic infection, defined as the presence of cryptococcal antigen in the bloodstream in the absence of clinical symptoms, was identified in a small number of dogs and cats. Fourteen months of follow-up testing of asymptomatic animals revealed that animals can progress to clinical disease, remain sub-clinically infected, or clear the organism.

3

Efeito dos inibidores da bomba de prótons na sensibilidade in vitro e inibição da produção de melanina em Cryptococcus spp. Effect of proton pump inhibitors on in vitro sensitivity and inhibition of melanin production in Cryptococcus spp.

España, Jaime David Acosta 3 August 2017 (has links)
ESPAÑA, J. D. A. Efeito dos inibidores da bomba de prótons na sensibilidade in vitro e inibição da produção de melanina em Cryptococcus spp. 2017. 95 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-10-05T13:17:02Z No. of bitstreams: 1 2017_dis_jdaespaña.pdf: 2088840 bytes, checksum: d2bb1dff4ee77a727fe5f7b3b3182827 (MD5) Approved for entry into archive by denise santos (denise.santos@ufc.br) on 2017-10-05T13:29:54Z (GMT) No. of bitstreams: 1 2017_dis_jdaespaña.pdf: 2088840 bytes, checksum: d2bb1dff4ee77a727fe5f7b3b3182827 (MD5) Made available in DSpace on 2017-10-05T13:29:54Z (GMT). No. of bitstreams: 1 2017_dis_jdaespaña.pdf: 2088840 bytes, checksum: d2bb1dff4ee77a727fe5f7b3b3182827 (MD5) Previous issue date: 2017-08-03 Cryptococcus neoformans sensu latu and Cryptococcus gattii sensu latu are hyaline yeasts capable of producing melanin, being associated with a condition called cryptococcosis.For installation and its spread, several virulence factors have been associated, such as melanin production, which is produced by phenoloxidase called laccase, which oxidizes phenolic compounds. Studies have shown that proton pump inhibitors (PPI´s) have an inhibitory effect on melanin production, in melanocytes,produced by a phenoloxidase, called tyrosinase. This effect on inhibition of melanogenesis was associated with inhibition of tyrosinase.Since melanocytes and Cryptococcus spp. produce the melanin with a similar oxidation action of phenolic compounds, the objective of this work was to test the effect of PPI´s on the inhibition of melanin production in Cryptococcusneoformans/gattii lato sensu. For this, a new methodology was initially developed for the quantification of melanin, through the gray scale. In this experiment, 12 strains of Cryptococcus spp., 1 strain of Candidaparapsilosis ATCC 22019 (negative control) and 1 strain of Hortaeawerneckii (positive control) were analyzed, resulting in an adequate production and quantification of melanin in testedstrains. Next, PPIs were tested (omeprazole, rabeprazole, lansoprazole, esomeprazole and pantoprazole), classical antifungal agents (fluconazole and amphotericin B) and glyphosate (inhibitor of melanogenesis in Cryptococcus spp.) on the susceptibility of 20 strains of Cryptococcus spp.and 1 strain of C. parapsilosis ATCC 22019, based on the protocol M27-A3 of the Clinical Laboratory Standart Institute (CLSI).The following MIC50 values were observed: for omeprazole 166.7 ± 91.29 μg/ml, esomeprazole 256 ± 115.1 μg/ml, lansoprazole 226 ± 200.1 μg/ml, rabeprazole 142.6 ± 74.6 μg/ml, pantoprazole 428.6 ± 297.3 μg/ml, glyphosate 952.4 ± 150.4 μg/ml, fluconazole 4,757 ± 1,6 μg/ml and amphotericin B 1,464 ± 2.6 μg/ml. Finally, subinhibitory concentrations of PPIs were tested on melanin production in Cryptococcus spp., resulting in an inhibition of melanin for protons pump inhibitors, the best effect being obtained with omeprazole, lansoprazole and pantoprazole. Therefore, PPI´s has an important effect on the inhibition of melanin in Cryptococcus spp., and further studies are needed to understand the mechanisms of inhibition and its potential in reducing virulence, dissemination and infection of the CNS. Cryptococcus neoformans lato sensu e Cryptococcus gattii lato sensu são leveduras hialinas capazes de produzir melanina, estando associados auma doença chamada criptococose. Para sua instalação e disseminação, vários fatores de virulência têm sido associados, como a produção de melanina, que é produzida por uma fenoloxidase chamada lacase que oxida os compostos fenólicos. Estudos demostraram que os inibidores de bomba de prótons (IBP´s) têm efeitos inibitórios na produção de melanina, em melanócitos, produzidos por uma fenoloxidase chamada tirosinase. Este efeito inibitório da melanogênese foi associado ao bloqueio da tirosinase. Uma vez que os melanócitos e Cryptococcus spp. produzem melanina por uma via semelhante à oxidação de compostos fenólicos, o objetivo deste trabalho foi testar o efeito de IBP´s na inibição de produção de melanina em C.neoformans/C. gattiilatosensu. Para tanto, inicialmente foi desenvolvida uma nova metodologia para quantificação de melanina por meio da escala de cinza. Neste sentido, foram analisadas 12 cepas de Cryptococcus spp., uma cepa de Candidaparapsilosis ATCC 22019 (controle negativo) e uma cepa de Hortaeawerneckii (controle positivo), resultando em uma adequada produção e quantificação de melanina nas cepas testadas. A seguir, foram testados os IBP´s (omeprazol, rabeprazol, lansoprazol, esomeprazol e pantoprazol), os antifúngicos clássicos (fluconazol e anfotericina B) e glifosato (inibidor da melanogênese em Cryptococcus spp.) sobre a sensibilidade de 20 cepas de Cryptococcus spp. e uma cepa de C. parapsilosis ATCC 22019, baseado no documento M27-A3 do Clinical Laboratory Standart Institute (CLSI). Foram observados os seguintes valores de MIC50: para omeprazol 166,7 ± 91,29 µg/mL; esomeprazol 256 ± 115,1 µg/mL; lansoprazol 226 ± 200,1 µg/mL; rabeprazoL 142,6 ± 74,6 µg/mL; pantoprazol 428,6 ± 297,3 µg/mL; glifosato 952,4 ± 150,4 µg/mL; fluconazol 4,757 ± 1,6 µg/mLe; anfotericina B 1,464 ± 2,6 µg/mL. Finalmente, foram testadas concentrações subinibitórias dos IBP´s sobre a produção de melanina em Cryptococcus spp., obtendo como resultado uma inibição da melanina para os inibidores de bomba de prótons, sendo o melhor efeito obtido com omeprazol, lansoprazol e pantoprazol. Portanto, IBP´s têm um efeito importante na inibição de melanina em Cryptococcus spp., sendo necessário estudos mais aprofundados para compreender os mecanismos de inibição e seu possível potencial na diminuição de virulência, disseminação e infecção do SNC.
4

Real-time PCR assays for genotyping of Cryptococcus gattii in North America

Kelley, Erin, Driebe, Elizabeth, Etienne, Kizee, Brandt, Mary, Schupp, James, Gillece, John, Trujillo, Jesse, Lockhart, Shawn, Deak, Eszter, Keim, Paul, Engelthaler, David 2014 (has links)
BACKGROUND:Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen.METHODS:We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human.RESULTS:Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA.CONCLUSIONS:The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.
5

Desenvolvimento de protótipos antifúngicos contra Cryptococcus gattii utilizando modelos alternativos animais Prototype development antifungal against Cryptococcus gattii using alternative animal models

Cerrejón-Palanco, Ana 16 September 2016 (has links)
Submitted by ANA CERREJÓN PALANCO null (anacerrejon@live.com) on 2016-09-29T21:40:59Z No. of bitstreams: 1 Mestrado-Ana-Cerrejón-Palanco.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-10-04T19:11:41Z (GMT) No. of bitstreams: 1 carrejonpalanco_a_me_arafcf.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) Made available in DSpace on 2016-10-04T19:11:41Z (GMT). No. of bitstreams: 1 carrejonpalanco_a_me_arafcf.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) Previous issue date: 2016-09-16 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A criptococose é uma infecção fúngica sistêmica e oportunista, causada pelas leveduras capsuladas das espécies Cryptococcus neoformans e Cryptococcus gattii, afetando tanto pacientes imunocompetentes quanto imunocomprometidos. Uma propriedade importante do gênero Cryptococcus é a capacidade de formação de biofilmes. A resistência dos biofilmes aos agentes antifúngicos traz a necessidade da busca de novas terapias. Desta forma, o nosso grupo vem trabalhando com moléculas antifúngicas derivadas de fontes naturais, como as chalconas, onde foi verificado que C. neoformans é sensível a algumas destas moléculas. Assim, o objetivo desse trabalho foi testar a 3’- chalcona como antifúngico contra C. gattii em situação planctônica e biofilme, como também a eficiência e toxicidade em modelos animais alternativos como Danio rerio (Zebrafish) e Galleria mellonella, propondo uma nova alternativa para o tratamento. In vitro, os resultados para a forma planctônica do fungo mostraram que a 3’- chalcona foi mais eficaz para a cepa 118, com concentração inibitória mínima (CIM) de 0,96 µg/mL e para a cepa 56990 de C. gattii, a molécula mostrou ação potente com CIM entre 1,95 e 3,90 µg/mL. No ensaio de formação do biofilme com as duas cepas, observou-se uma atividade metabólica crescente até 72 horas e uma biomassa e matriz extracelular que alcançou seu máximo nas 48 horas. Em seguida, foi feita a avaliação da atividade da 3’- chalcona contra a formação do biofilme maduro após 48 horas, apresentando uma inibição a uma concentração de 62,5 µg/mL para cepa ATCC e de 31,2 µg/mL frente ao isolado 118. Também foi avaliado o crescimento do biofilme frente à anfotericina B (AmB), obtendo como resultado, inibição em todas as concentrações por parte das duas cepas e, frente ao fluconazol, apresentaram-se resistentes. In vivo, os resultados de toxicidade em embriões de Zebrafish revelaram uma concentração letal 50% (CL50) de 3,42±0,6 µg/mL em 48 horas pós-fertilização (hpf). No modelo G. mellonella as doses testadas de 3’- chalcona (2 a 160 mg/Kg) não foram tóxicas. Entretanto, nas larvas infectadas com as duas cepas de C. gattii, a molécula não apresentou eficácia antifúngica. Assim, considerando o potencial antifúngico in vitro, inclusive contra o biofilme de C. gattii, a 3’- chalcona pode passar por modificações moleculares que aumentem sua distribuição e eficácia in vivo. Criptococose is a fungal, systemic and opportunistic infection mainly caused by capsulated yeast of the species Cryptococcus neoformans e Cryptococcus gattii, affecting immunocompetent and immunocompromised patients. An important property of the genus Cryptococcus is the biofilm formation, which can be considered a virulence factor and resistance. The resistance of biofilms to antifungal agents brings the need to search new therapies. Thus, our group has been working with antifungal molecules derived from natural sources such as chalcones, where it was found that C. neoformans is sensitive to some of these molecules. For this fact, the objective of this study was to test the 3'- chalcone as antifungal against C. gattii in planktonic and biofilm forms, as well as efficiency and toxicity in alternative animal models like Danio rerio (Zebrafish) and Galleria mellonella, proposing a new alternative for treatment. In vitro, the results for the planktonic form of the fungus showed that the 3'- chalcone was more effective for the 118 strain, the minimum inhibitory concentration (MIC) was 0.96 µg/mL. For the strain 56990 of C. gattii the molecule showed potent activity with MIC values between 1.95 and 3.90 µg/mL. Biofilm formation assay for both strains had an increased metabolic activity within 72 hours and a biomass and extracellular matrix which reached its maximum in 48 hours. Then, the evaluation of the activity of the 3'-chalcone against the formation of mature biofilm was made after 48 hours, showing inhibition at a concentration of 62.5 µg/mL for ATCC strain and 31.2 µg/mL against the isolated 118. Also, the growth of biofilm against to amphotericin B (AmB) was evaluated, obtained as a result of inhibition at all concentrations for the two strains and against fluconazole were resistant. In vivo, the results of toxicity in zebrafish embryos revealed a lethal concentration 50% (LC50) of 3.42 ± 0.6 µg/mL at 48 hours post-fertilization (hpf). In the model G. mellonella tested doses of 3’- chalcona (2 to 160 mg/kg) were not toxic. However, in larvae infected with the two strains of C. gattii, the molecule did not show efficacy of antifungal agents. Thus, considering the potential antifungal in vitro, including against biofilms of C. gattii, 3'- chalcone can experiment molecular changes that increase their distribution and efficacy in vivo.
6

Suscetibilidade in vitro de Cryptococcus neoformans e Cryptococcus gattii frente a drogas antifúngicas pela citometria de fluxo

Morales, Bernardina Penarrieta 2009 (has links)
Made available in DSpace on 2014-08-11T12:37:24Z (GMT). No. of bitstreams: 4 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) bernardina_morales_ipec_mest_2009.pdf: 1163368 bytes, checksum: f202e217c54ce0cedbcb4c79e65c8190 (MD5) bernardina_morales_ipec_mest_2009.pdf.txt: 105699 bytes, checksum: d49cfc5d18502e60aefc9acd95bd3a8b (MD5) bernardina_morales_ipec_mest_2009.pdf.jpg: 1571 bytes, checksum: d645520da44b852110b75eff2c71cf71 (MD5) Previous issue date: 2014-05-06 Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil Cryptococcus neoformans (C. n) e Cryptococcus gattii (C. g) são agentes da criptococose. A carência de publicações sobre teste de suscetibilidade de C. n e principalmente de C. g é evidente. A maioria de isolados clínicos de C. g mostra-se suscetível in vitro a fluconazol e itraconazol; no entanto o surgimento de resistência a drogas antifúngicas incentivou os testes de suscetibilidade in vitro. Como conseqüência, Clinical and Laboratory Standadards Institute \2013 CLSI, anteriormente National Committee for Clinical Laboratory Standards \2013 NCCLS, publicaram metodologia padronizada M27-A2 para alcançar reprodutibilidade e permitir a comparação de resultados de suscetibilidade entre laboratórios, porém alguns problemas como o tempo de incubação e o padrão de leitura levaram à busca de técnicas alternativas, tais como a citometria de fluxo. O objetivo deste trabalho é padronizar a técnica de citometria de fluxo para o teste de suscetibilidade in vitro de C.n e C.g. visando a redução do tempo de incubação, a utilização de leitura automatizada e a obtenção de resultados rápidos e reprodutíveis A concentração inibitória mínima (CIM) de 20 isolados de C.n e 21 de C.g foi determinada por citometria de fluxo e os resultados comparados com o protocolo padrão proposto pelo CLSI/M27A-2. Após duas horas de incubação com anfotericina B utilizando FUN-1, C. gattii resultou em 100% de concordância entre as duas técnicas para diluição de 2\03BCg/mL e 95,2% para 1\03BCg/mL e C. neoformans resultou em 100% de concordância para 1 e 2\03BCg/mL. Para azólicos e flucitosina, foram obtidos resultados reprodutíveis com o fluorocromo Acridine Orange após 18 horas de incubação, que resultou em 78% de concordância entre as duas técnicas para fluconazol, 85% para itraconazol e 97% para flucitosina. Em ambas as metodologias, C. gattii foi menos suscetível do que C. neoformans frente ao itraconazol e flucitosina (p<0,05). A citometria de fluxo é uma ferramenta útil, com potencial para testes in vitro e determinação da CIM dos antifúngicos estudados, com apreciável redução do tempo mínimo para obtenção de resultados Cr yptococcus neoformans ( C.n ) and Cryptococcus gattii ( C.g ) are the agents of cryptococcosis. The lack of publications on susceptibility tests of C.n and especially C.g is evident. Most clinical isolates of C.g proved to be susceptible in vitro to fluconazol e and itraconazole and yet the emergence of resistance to antifungal drugs encouraged susceptibility testing in vitro . As a consequence, Clinical and Laboratory Standadards Institute – CLSI ( National Committee for Clinical Laboratory Standards – NCCLS ) pub lished standardized methodology to achieve reproducibility and allow comparison of susceptibility results between laboratories (M27 - A2 document) , however some problems of methodology, like incubation time and standard reading, led to the search for alterna tive techniques, such as flow cytometry. The objective of this work is to standardize the technique of flow cytometry to test the in vitro susceptibility of C . n and C . g in order to reduce the incubation time, use of automated reading and obtain fast and re producible results . The minimum inhibitory concentration (MIC) of 20 isolates of C.n and 21 of C.g was determined by flow cytometry and the results compared with the standard protocol proposed by CLSI/M27A - 2. Flow citometry showed 100% agreement with CLSI/ M27A - 2 results for 2 μ g/mL and 95.2% for 1 μ g/mL dilution when C.g isolates were tested, and 100% agreement for 1 and 2 μ g/ml dilution when C.n were tested after two hours of incubation with amphotericin B using FUN - 1 . Reproducible results were obtained with fluorochrome Acridin e Orange for azoles and flucytosine after 18 hours incubation, resulting in 78% agreement for fluconazole, 85% for itraconazole and 97% for flucytosine. C.g was less susceptible than C.n against itraconazole and flucytosine (p <0.05) in both methodologies. Flow cytometry is a useful tool, with potential for in vitro susceptibility test s of the antifungal agents studied, with appreciable reduction in the minimum time for achieving results.
7

Alterações histopatológicas iniciais pulmonares na infecção aguda experimental de camundongos Swiss webster imunocompetentes por Cryptococcus neoformans e C. gatti.

Lourenço, Manoel Gionovaldo Freire 2008 (has links)
Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-06-06T00:07:57Z No. of bitstreams: 1 manoel_gf_lourenco_ioc_bp_0041_2008.pdf: 1698623 bytes, checksum: 26dac485e0b5d05c5bbc0ba69007c66a (MD5) Made available in DSpace on 2012-06-06T00:07:57Z (GMT). No. of bitstreams: 1 manoel_gf_lourenco_ioc_bp_0041_2008.pdf: 1698623 bytes, checksum: 26dac485e0b5d05c5bbc0ba69007c66a (MD5) Previous issue date: 2008 Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil O envolvimentonto do sistema nervoso central com meningites tem sido tradicionalmente relatado em ambas as infecções por C. neoformans e C. gattii, com ou sem sinais clínicos ou radiológicos de lesão pulmonar. Quase todas as formas disseminadas por cepas de C. neoformans ocorreram em indivíduos imunocomprometidos, exceto em poucos casos com fatores predisponentes, sugerindo a existência de alguns mecanismos de escape. Em contraste criptococose por C. gattii, quase sempre afeta indivíduos sem prejuízo imune identificável, e comumente apresenta lesões pulmonares e do sistema nervoso central associado à meningite. Como os mecanismos envolvidos nos estágios precoces da infecção pulmonar por espécies de Cryptococcus ainda são poucos conhecidos, foram acompanhados separadamente o processo de internalizaçao, de duas cepas de C. neoformans e C. gattii, nos pulmões de camundongos imunocompetentes, descrevendo esta penetração e comparando possíveis diferenças. Materiais e Métodos: Camundongos Swiss Webster foram instilados intratraquealmente com: a) 10 5 leveduras de C. neoformas em 50µL de salina (30 animais); b) 10 5 leveduras de C. gattii em 50µL de salina (30 animais); ou c) 50µL de salina sem leveduras de Cryptococcus (10 animais). Após a instilação o abdômen dos camundongos foi comprimido e liberado para promover uma inalação profunda. Os animais foram mortos 2, 12, 24, 48 e 72h após a instilação.A cada período os animais foram submetidos a eutanásia por injeção intraperitonial de 1.0 mL de pentobarbital sódico a 2,5%. Resultados e Discussão: A histopatologia entre os pulmões de camundongos infectados por C. neoformans ou C. gattii revelaram peculiaridades importantes. Uma das diferenças principais foi a expressão precoce da cápsula de polissacarídeo por C. neoformans em comparação com C. gattii. Pór causa da expressão precoce da sua cápsula de polissacarídeo, C. neoformans é maior em tamanho, o que pode contribuir para seu escape dos mecanismos de defesa do hospedeiro e, maior persistência no trato respiratório. Este mecanismo de escape e a conseqüente dificuldade para difusão resultam numa grande reação inflamatória local acompanhada de brotamento precoce. Conclusões: a invasão pulmonar e os mecanismos de disseminação de Cryptococcus no trato respiratório em estágios precoces da infecção experimental não dependeram diretamente da participação de células mononucleares e neutrofilicas. Este processo aconteceu, pela interação física entre os fungos e o epitélio respiratório, contudo sendo desconhecido os mecanismos intrínsecos The envolvimentonto central nervous system with meningitis has traditionally been reported in both infections C. and C. neoformans gattii, with or without clinical or radiological signs of lung injury. Almost all forms disseminated by strains of C. neoformans occurred in immunocompromised individuals, except in a few cases with predisposing factors, suggesting the existence of some escape mechanisms. In contrast cryptococcosis by C. gattii, often affects individuals without identifiable immune injury, and commonly shows pulmonary lesions and central nervous system associated with meningitis. As the mechanisms involved in the early stages of pulmonary infection by Cryptococcus species are still little known, were tracked separately the process of internalization of two strains of C. and C. neoformans gattii, the lungs of immunocompetent mice, describing and comparing this penetration possible differences. Materials and Methods: Swiss Webster mice were intratracheally instilled with: a) 10 5 yeasts of C. 50μL neoformas in saline (30 animals), b) 10 5 yeasts C. gattii in 50μL of saline (30 animals), or c) 50μL of saline without yeast Cryptococcus (10 animals). After instillation, the abdomen of the mice was compressed and released to promote a deep inhalation. Animals were sacrificed 2, 12, 24, 48 and 72h after instilação.A each period the animals were euthanized by intraperitoneal injection of 1.0 mL of 2.5% sodium pentobarbital. Results and Discussion: The histopathology of the lungs of mice infected with C. or C. neoformans gattii revealed important peculiarities. One of the main differences is the early expression of capsule polysaccharide by C. in comparison to C. neoformans gattii. Because of their expression of early polysaccharide capsule, C. neoformans is larger in size, which may contribute to their escape from host defense mechanisms, and greater persistence in the respiratory tract. This escape mechanism and the consequent difficulty for the diffusion result in significant local inflammatory reaction accompanied by sprouting early. Conclusions: Lung invasion and the mechanisms of dissemination of Cryptococcus in the respiratory tract in the early stages of experimental infection did not directly dependent on the participation of mononuclear cells and neutrophilic. This process took place, the physical interaction between fungi and respiratory epithelium, but the mechanisms are unknown intrinsic
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Padrão taxonômico, quimiotipagem e atividade antifúngica in vitro de Anfotericina B, Ciclopirox Olamina e β-Lapachona em amostras de Cryptococcus

OLIVEIRA, Patrícia Cariolano de 31 January 2009 (has links)
Made available in DSpace on 2014-06-12T15:06:57Z (GMT). No. of bitstreams: 2 arquivo6511_1.pdf: 674340 bytes, checksum: 31ec95f94224d536898df188d91ae077 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 A criptococose é uma infecção fúngica causada por Cryptococcus neoformans, que possui tropismo pelo sistema nervoso central. O propósito deste estudo foi diagnosticar criptococose, identificar variedades de C. neoformans e avaliar a suscetibilidade a anfotericina B, ciclopirox olamina e β-lapachona em amostras da Micoteca URM-UFPE e recém isoladas de Líquor de imunocomprometidos de ambos os sexos atendidos em hospitais de Recife-PE, Brasil. Para o diagnóstico de criptococose, exame direto foi realizado com tinta da China e o semeio em ágar Sabouraud. As variedades foram detectadas com meio L-Canavanina-azul bromotimol. A concentração inibitória seguiu o Clinical and Laboratory Standards Institute. Dezenove casos de criptococose foram diagnosticados com obtenção de 16 culturas. Da Micoteca, foram obtidas 14 amostras de C. neoformans. O exame direto mostrou leveduras capsuladas. C. neoformans var. neoformans ocorreu em 50% dos casos. O CIM (μg/mL) desta variedade foi 0,25 - 4 (anfotericina B), 0,125 - 1 (ciclopirox olamina), 4 e 8 (β-lapachona). Os isolados de C. gattii apresentaram-se 0,25 - 4 (anfotericina B), 0,25-1 (ciclopirox olamina), 4 e 8 (β-lapachona). A concentração fungicida da β-lapachona foi 64 μg/mL. Criptococose predominou no sexo masculino com faixa etária de 25-46 anos e apresentando AIDS como fator de risco predominante. Detectaram-se 4 isolados resistentes a anfotericina B (3 isolados de C. gattii e 1 isolado de C. neoformans var. neoformans). A eficácia do ciclopirox olamina e da β-lapachona, comparada a resistência a anfotericina B, mostra a importância da susceptibilidade associados à variedade para monitorar desenvolvimento de resistência possibilitando descoberta de novas alternativas terapêuticas para criptococose.
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Suscetibilidade in vitro e resistência a antifúngicosestudo comparativo entre os tipos moleculares VNI de Cryptococcus neoformans e VGI, VGII de Cryptococcus gattii pela citometria de fluxo

Morales, Bernardina Penarrieta 2013 (has links)
Made available in DSpace on 2016-05-19T13:20:06Z (GMT). No. of bitstreams: 2 bernardina_morales_ipec_dout_2013.pdf: 4097768 bytes, checksum: a3e4244f0c2be9b0d8d257c5f8921c52 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil A criptococose é uma micose sistêmica adquirida pela inalação de basidiosporos ou leveduras desidratadas de Cryptococus neoformans e Cryptococus gattii, estas duas espécies podem causar criptococose oportunista e primária respectivamente. C. neoformans está constituído de tipos moleculares VNI-VNIV e C. gattii de VGI-VGIV que apresentam distribuição geográfica diferenciada, como por exemplo, o tipo VNI é cosmopolita e está associado a AIDS e VGI predominando na Austrália e EUA, o tipo VGII predominando no Brasil e America Latina. Este trabalho tem por objetivo realizar estudo comparado dos tipos moleculares VNI de C. neoformans, VGI e VGII de C. gattii analisando diferentes aspectos tais como: 1- Determinar o perfil da suscetibilidade in vitro da concentração inibitória mínima (CIM) de fluconazol (FLZ), itraconazol (ITZ), 5-fluorocitosina (5FC) e anfotericina B (AMB), isoladamente e de forma combinada de AMB com 5FC e AMB com Voriconazol (VRZ); 2- Determinar CIM pela citometria de fluxo (CMF) frente a FLZ, ITZ e AMB; 3- Definir a concentração mínima letal (CML) de AMB e 5FC, isoladamente e em combinação; 4- Avaliar a ação da melanina frente a 5FC e AMB na forma combinada e isolada de 5FC; 5- Induzir a resistência in vitro para FLZ e padronizar os fluorocromos: acetoximetil - calceína (calceina-AM), acetoximetil - 2\2019, 7\2019 -bis-(2-carboxietil)-5-(e -6)- carboxifluoresceína (BCECF-AM), rodamina 123 (Rh123) e iodeto de 3, 3\2019 \2013dipentiloxacarbocianina (DiOC5) na CMF para verificar a expressão de bombas de efluxo; 6- Comparar a expressão de bombas de efluxo Os resultados permitiram identificar diferentes fenótipos de suscetibilidade que foram analisados e comparados entre as duas espécies e os tipos moleculares, permitindo a produção de quatro artigos; sendo assim, concluímos que: 1- Na análise das CIMs o tipo molecular VGII apresentou-se menos suscetível em relação a VGI e VNI; já na combinação in vitro de AMB e VRZ foi observado 100% de indiferença, e na combinação de AMB e 5FC observou-se necessidade de padronização da concentração da glicose para obter testes que possam ser futuramente relacionados a casos clínicos; 2- O método de CMF demonstrou ser alternativa de leitura automatizada, reprodutível, para os testes de suscetibilidade antifúngica com uso de Laranja de Acridina e FUN-1; 3- Foi verificada a importância da realização do teste da CML para verificar a ação protetora da melanina frente a combinação de AMB e 5FC. 4- A expressão da melanina, na combinação de AMB e 5FC reduz a detecção do sinergismo e o efeito aditivo in vitro. 5. Os isolados induzidos à resistência ao FLZ permitiram obter resultados estatisticamente significativos na verificação de Bombas de efluxo in vitro na CMF com uso de fluorocromo Dioc5 e bloqueador de protonóforos CCCP; 6- Foi verificado que 65% de isolados não induzidos a resistência e 90% de isolados induzidos a resistência do tipo molecular VGII expulsam o FLZ com certa vantagem em relação aos tipos moleculares VGI e VNI. Os resultados deste trabalho contribuem para compreensão do comportamento in vitro de C. neoformans e C. gattii frente a drogas antifúngicas cujos resultados poderão ser aplicados em estudos clínicos de correlação in vitro e in vivo para melhor compreensão da terapêutica antifúngica da criptococose e validação dos testes de suscetibilidade para estas duas espécies Cryptococcosis is a system ic mycosis acquired by inhalation of dried yeasts or basidiospores of Cryptococus neoformans and Cryptococus gattii , these species can cause cryptococcosis opportunistic and primary respectively. C. neoformans is composed of molecular types VNI - VNIV and C. gattii VGI - VGIV they have different geographical distribution, the VNI type is cosmopolitan and is associated with AIDS, VGI type is predominant in Australia and the USA; while VGII type occurs in Brazil and Latin America. This paper aims to conduct a comparative study of the molecular types VNI C. neoformans and VGI, VGII C. gattii analyzing different aspects such as : 1 – The susceptibility profile in vitro of fluconazole ( FLZ ), itraconazole ( ITZ ), 5 - fluorocytosine ( 5FC ) and amphotericin B ( A MB ) alone and in combination with 5FC and AMB with voriconazole ( VRZ ) 2 – The minimum inhibitory concentration ( MIC ) by flow cytometry ( FCM ) comparing MIC of FLZ , ITZ and AMB with CLSI; 3 - The minimum lethal concentration ( MLC ) of AMB and 5FC, a lone and in combination; 4 - The action of melanin against 5FC and AMB alone and combined 5FC, 5 – The induced resistance in vitro to FLZ and standardize the fluorochromes: acetoximetil - calceína (calceina - AM), acetoximetil - 2’, 7’ - bis - (2 - carboxietil) - 5 - (e - 6) - carboxifluoresceína (BCECF - AM), rodamina 123 (Rh123) e iodeto de 3, 3’ – dipentiloxacarbocianina (DiOC5) in FCM to verify expression of efflux pumps; 6 - The compare the expression of efflux pumps. These results permitted the identification of dif ferent susceptibility phenotypes which were analyzed and compared between the two species and molecular types, allowing the production of four articles . T hus, we concluded that: 1 - The analysis of the molecular type VGII MICs presented less susceptibility against VGI and VNI, whereas in the combination of AMB and VRZ in vitro 100% indifference was observed, in the combination of AMB and 5FC we observed the need for standardization of the concentration of glucose for future tests which can be related to cli nical cases; 2 - the FCM method proved to be an alternative automated reproducible reading for antifungal susceptibility testing with use of Acridine Orange and FUN - 1, 3 - The importance of carrying out the MLC test to verify the protective action of mela nin when exposed to the combination of AMB and 5FC. 4 - Was also verified that expression of melanin, in combination AMB and 5FC reduces the detection of synergism and additive effect in vitro . 5 - The strains induced to resistance to fluconazole made it p ossible to obtain statistically significant results in the verification of acriviry of efflux pumps in vitro using FCM with fluorochrome Dioc5 and protonophores blocker CCCP, 6 - It was found that 65% isolates no inducted to resistance and 90% isolates ind ucted to resistance of the molecular ty pe VGII expels the FLZ with a certain advantage in relation to the molecular types VNI and VGI. The results of these studies contribute to the understanding of the behavior in vitro of C. neoformans and C. gattii agai nst antifungal drugs and can be applied in clinical correlation studies in vitro and in vivo to better understand the antifungal therapy of cryptococcosis and validation of susceptibility testing for these two species.
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Estudo microbiológico: amostragem de Cryptococcus sp em São José do Rio Preto/SP

Barboza, Lílian Stefani [UNESP] 11 April 2011 (has links)
Made available in DSpace on 2014-06-11T19:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-11Bitstream added on 2014-06-13T18:31:22Z : No. of bitstreams: 1 barboza_ls_me_sjrp_parcial.pdf: 54066 bytes, checksum: 3901016bbfd3977caa34c67746541ca7 (MD5) Bitstreams deleted on 2015-08-28T16:08:50Z: barboza_ls_me_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-08-28T16:09:56Z : No. of bitstreams: 1 000642588.pdf: 349486 bytes, checksum: eea3a5247c5cd27a996061bb9441acaa (MD5) A criptococose é uma micose sistêmica que afeta principalmente o sistema nervoso central, causando meningite. Causada pela inalação de propágulos de fungos do gênero Cryptococcus sp, C neoformans acomete hospedeiros com imunodepressão celular, cujo maior contingente é representado por indivíduos com AIDS. A fonte principal de aquisição desta levedura são fezes de aves, principalmente pombos (Columba livia). C. gattii infecta indivíduos aparentemente imunocompetentes, sendo isolado principalmente em espécies de eucaliptos. C. albidus e C. laurentii aparece em casos de meningites, fungemias, pneumonias, abscessos pulmonares e infecções cutâneas. C.uniguttulatus é encontrado no trato gastrointestinal, fezes de pássaros e contaminante de leitos de animais e habitats de roedores. Termo-tolerância, parede celular, cápsula, adesão, e produção de enzimas são fatores importantes de virulência do gênero. São José do Rio Preto é a 5ª cidade entre os 100 municípios do estado de São Paulo em de casos de AIDS. Analisou-se morfologicamente e bioquimicamente 48 amostras clínicas e 155 ambientais. Amostras de líquor resultaram em 30 isolados da espécie C. neoformans, 17 de C. gattii e um de C. luteolus. Foram identificados 26 isolados de C.albidus, 22 de C. laurentii, 9 de C. neoformans, 5 de C. gattii e um de C.uniguttulatus nas amostras ambientais. A concentração inibitória mínima para antifúngicos foi determinada para todos os isolados por meio do teste de microdiluição em caldo padronizado pelo NCCLS. Os resultados obtidos mostraram resistência à anfotericina B e itraconazol nas cepas clínicas de C. neoformans e C. gattii, e as cepas de C.albidus e C. laurentii apresentaram resistência a 5-fluorcitosina, itraconazol e anfotericina B Cryptococcosis is a systemic mycosis that mainly affects the central nervous system, causing meningitis. Caused by inhalation of seedlings of fungi of the genus Cryptococcus sp, C. neoformans affects hosts with immunosuppression cellular, whose largest contingent is represented by individuals with AIDS. The main source of acquiring this yeast is feces of birds, especially pigeons (Columba livia). C. gattii infects individuals apparently immunocompetent, being isolated mainly in species of eucalyptus. C. albidus and C. laurentii appear in cases of meningitis, nosocomial fungemia, pneumonia, lung abscesses and skin infections. C. uniguttulatus is found in the gastrointestinal tract and feces of birds and contaminant of beds of animals and habitats of rodents. Thermo-tolerance, cell wall, capsule, accession, and production of enzymes are important factors in the virulence of the genus. São José do Rio Preto is the 5th city among the 100 cities in the state of São Paulo in cases of AIDS. We analyzed morphologically and biochemically 48 clinical samples and 155 environmental. Liquor samples resulted in 30 isolates of the species C. neoformans, 17 of C. gattii and one of C. luteolus. We identified 26 isolates of C. albidus, 22 of C. laurentii, 9 of C. neoformans, 5 of C. gattii and one of C. uniguttulatus in environmental samples. The minimal inhibitory concentration for antifungal agents was determined for all isolates by means of the test of broth microdilution standardized by the NCCLS. The results obtained showed resistance to amphotericin B and itraconazole in clinical strains of C. neoformans and C. gattii, C. albidus and C. laurentii exhibited resistance to 5-fluorocytosine, itraconazole and amphotericin B

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