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

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: 2014-05-06Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, BrasilCryptococcus 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 resultadosCr 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

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

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: 2013Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, BrasilA 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 espéciesCryptococcosis 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

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density &#91;in colony forming units per gram of wood (CFU.g-1)&#93; compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique

Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry

Submitted by Rodrigo Senorans ([email protected]) on 2015-06-16T17:59:15Z No. of bitstreams: 1 Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.pdf: 1021538 bytes, checksum: 5c1f9a814cf5dc80e92b43d66efc8174 (MD5)Approved for entry into archive by Anderson Silva ([email protected]) on 2015-06-17T12:02:26Z (GMT) No. of bitstreams: 1 Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.pdf: 1021538 bytes, checksum: 5c1f9a814cf5dc80e92b43d66efc8174 (MD5)Approved for entry into archive by Anderson Silva ([email protected]) on 2015-06-17T12:02:37Z (GMT) No. of bitstreams: 1 Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.pdf: 1021538 bytes, checksum: 5c1f9a814cf5dc80e92b43d66efc8174 (MD5)Made available in DSpace on 2015-06-18T17:02:14Z (GMT). No. of bitstreams: 1 Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.pdf: 1021538 bytes, checksum: 5c1f9a814cf5dc80e92b43d66efc8174 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Recent studies have used flow cytometry (FCM) as an important alternative method to determine the antifungal susceptibility of yeasts compared to the broth microdilution Clinical and Laboratory Standards Institute (CLSI) reference procedure. We present a comparative study of the broth microdilution method and flow cytometry to assess the in vitro antifungal susceptibility of Cryptococcus neoformans (n = 16) and C. gattii (n = 24) to fluconazole. The minimum inhibitory concentration (MIC) assays by flow cytometry were defined as the lowest drug concentration that showed ∼50% of the count of acridine orange negative cells compared to that of the growth control. Categorical classification showed all C. neoformans isolates were susceptible to fluconazole. Three isolates of C. gattii were susceptible dose-dependent and the remaining 21 isolates were classified as susceptible. MICs comparison of both methodologies demonstrated 100% categorical agreement of the results obtained for C. neoformans and C. gattii. The MICs obtained with the CLSI-approved method and flow cytometry were compared by the Spearman correlation test and a significant Pv = 0.001. The flow cytometric method has the advantage of analyzing a large and constant number of cells in less time, i.e., 9 h incubation for fluconazole using acridine orange versus 72 h for broth microdilution method. In conclusion, the two methods were comparable and flow cytometry method can expedite and improve the results of in vitro susceptibility tests of C. neoformans and C. gattii against fluconazole and also allows comparative studies in vitro/in vivo more rapidly, which along with clinical data, could assist in selecting the most appropriate treatment choice

Cryptococcus gattii tipo molecular VGII como agente causador de meningoencefalite em criança saudável no Rio de Janeiro, Brasil: relato de um caso autóctone

Submitted by Repositório Arca ([email protected]) on 2019-04-24T16:33:05Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-12-18T13:00:44Z (GMT) No. of bitstreams: 2 ve_Pinto_Junior_Vitor_etal_INI_2019.pdf: 804119 bytes, checksum: 9c60d6a75d9edf8cb59280263cf4da0e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-12-18T13:00:44Z (GMT). No. of bitstreams: 2 ve_Pinto_Junior_Vitor_etal_INI_2019.pdf: 804119 bytes, checksum: 9c60d6a75d9edf8cb59280263cf4da0e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Brasília, DF, Brasil / Catholic University of Brasília. School of Medicine. Brasília, DF, Brazil.Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Cryptococcus gattii é causa de meningoencefalite em hospedeiros imunocompetentes, ocorrendo endemicamente em regiões tropicais e subtropicais. Recentemente foi causador de surtos na Ilha de Vancouver e na Columbia Britânica (Canadá). Nesta região de clima temperado, o tipo VGII é predominante. Relatamos um caso de meningoencefalite pelo C.gattii tipo VGII acometendo criança previamente saudável autóctone do Rio de Janeiro, região não endêmica do Brasil. O agente foi identificado por testes bioquímicos e o tipo molecular determinado através de URA5-RFLP. O presente relato enfatiza a necessidade de vigilância clínica para a meningite criptocóccica primária em áreas não endêmicas.Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil