Microdilui????o em caldo para teste de susceptibilidade de Malassezia furfur, Malassezia obtusa e Malassezia sympodialis

• Author(s): Souza, Victor Costa de
• Date: 2016
• Origin: Oasisbr
• Subject(s): Testes de susceptibilidade; Pitir??ase versicolor; Infec????es f??ngicas; Dermatologia; Fungos; Micoses; CI??NCIAS DA SA??DE
• Document Type: Master thesis
• Language: Portuguese

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CNPq - Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico

In the Amazon, the tropical climate is the main factor that favors the development of superficial fungal infections and opportunistic, especially the high temperature and relative humidity, providing ideal conditions for continuity in the life cycle of pathogenic and opportunistic fungi. Among the most prevalent mycosis in this region, is pityriasis versicolor (PV), a major skin diseases treated at health care in dermatology, can affect people of both genders, age, race and social class. Different species of Malassezia spp., Some of which recently described by means of genetic studies, are responsible for PV and, despite its high prevalence in several regions of the world and in Brazil, there are few studies with more detailed approach on epidemiological, clinical and laboratory features of this mycosis in our region. Research on the susceptibility of these species to antifungal drugs is fundamental to the advancement of scientific knowledge in order to better clinical management and treatment of PV. The testing for evaluation of in vitro susceptibility to antifungal agents for yeasts and molds (vitro antifungal susceptibility), constitutes an important tool for monitoring the resistance of fungal strains and also to assist in choosing the best therapeutic regimen. Given this, the Clinical and Laboratory Standards Institute (CLSI), formerly National Committee for Clinical Laboratory Standards (NCCLS) have developed methods for the assessment of fungal susceptibility, however, due to different nutritional requirements of these species, there is still no specific protocol for testing with Malassezia spp. that has been recommended by this or any other body accredited for the regulation of clinical and laboratory procedures. The present study with the main objective to contribute to the development of a simplified protocol for assessing the susceptibility of three species of Malassezia, being isolated from 01 M. furfur, 01 and 01 M.obtusa M. sympodialis front to drugs ketoconazole, itraconazole and fluconazole. For this, bioassay were performed using the method in broth at different experimental conditions, which were defined from some methodological variants found in publications in the last 10 years, for the optimum conditions for growth, which were tested three different concentrations of inoculum, four types of culture media, incubation temperatures and two different criteria for interpretation of results. According to the results, the conditions that led to better performance during the experiments and the growth curves in bioassays with the drugs were: inoculum concentration of 0.5 to 2.5 x104 cells / mL and incubation temperature of 32 ?? C; medium modified Leeming-Notman with some changes in its composition, and spectrophotometric reading at 620 nm after 3 days of incubation. Accordingly, the MIC values obtained were: MIC ??? 0.03 mg / mL for ketoconazole and itraconazole to inhibit 50% and 80% of the growth of M. obtusa and M. furfur. For the fluconazole MICs were found for 2 and 4 mg / mL to inhibit 50% of the growth of M. sympodialis and M. obtusa respectively, and MIC of 4:08 g / mL for inhibition of 80%, while for M. furfur found an MIC ??? 0.03 mg / mL of this drug to inhibit 50% and 80% of fungal growth. Therefore, although preliminary, considering the small number of samples analyzed, the results of this study provided important information in view of developing a protocol for susceptibility testing of Malassezia species.

Na regi??o Amaz??nica, o clima tropical ?? o principal fator que favorece o desenvolvimento de infec????es f??ngicas superficiais e oportunistas, especialmente pela elevada temperatura e umidade relativa do ar, oferecendo condi????es ideais ?? continuidade no ciclo de vida dos fungos oportunistas e patog??nicos. Dentre as micoses mais prevalentes nesta regi??o, encontra-se a pitir??ase versicolor (PV), uma das principais dermatoses atendidas nos servi??os de sa??de em dermatologia, podendo acometer pessoas de ambos os g??neros, idade, ra??a e classe social. Diferentes esp??cies do g??nero Malassezia spp., algumas das quais recentemente descritas por meio de estudos gen??ticos, s??o respons??veis pela PV e, a despeito de sua alta preval??ncia em diversas regi??es do mundo e no Brasil, ainda s??o poucos os estudos com abordagem mais aprofundada acerca dos aspectos epidemiol??gicos, cl??nicos e laboratoriais desta micose em nossa regi??o. A investiga????o sobre a susceptibilidade destas esp??cies ??s drogas antif??ngicas ?? de fundamental import??ncia para o avan??o do conhecimento cient??fico na perspectiva de uma melhor abordagem cl??nica e terap??utica da PV. A realiza????o de testes para avalia????o da suscetibilidade in vitro aos agentes antif??ngicos para leveduras e fungos filamentosos (antifungigrama), se constitui em uma importante ferramenta para o monitoramento da resist??ncia de cepas f??ngicas e tamb??m para auxiliar na escolha do melhor esquema terap??utico. Diante disto, o Clinical and Laboratorial Standards Institute (CLSI), antigo National Committee for Clinical Laboratory Standards (NCCLS), t??m desenvolvido m??todos de refer??ncia para a avalia????o da susceptibilidade f??ngica, entretanto, devido ??s exig??ncias nutricionais diferenciadas dessas esp??cies, ainda n??o existe um protocolo espec??fico para testes com Malassezia spp. que tenha sido recomendado por este ou por algum outro ??rg??o credenciado para a regulamenta????o de procedimentos cl??nicos e laboratoriais. O presente estudo teve como objetivo principal contribuir para a elabora????o de um protocolo simplificado para avalia????o da susceptibilidade de tr??s esp??cies de Malassezia, sendo, 01 isolado de M. furfur, 01 de M.obtusa e 01 de M. sympodialis frente ??s drogas cetoconazol, itraconazol e fluconazol. Para isto, foram realizados bioensaios com base no m??todo de microdilui????o em caldo, em diferentes condi????es experimentais, as quais foram definidas a partir de algumas variantes metodol??gicas encontradas em publica????es nos ??ltimos 10 anos, quanto ??s condi????es ??timas de crescimento, no qual foram testadas tr??s diferentes concentra????es de inoculo, quatro tipos de meios de cultura, duas temperaturas de incuba????o e diferentes crit??rios de leitura dos resultados. De acordo com os resultados obtidos, as condi????es que permitiram melhor desempenho durante os experimentos nas curvas de crescimento e nos bioensaios com a drogas foram: concentra????o do inoculo de 0,5 a 2,5x104 c??ls/mL; temperatura de incuba????o de 32 ??C; meio de cultura Leeming-Notman modificado com algumas altera????es em sua composi????o, e leitura espectrofotom??trica em 620nm ap??s 3 dias de incuba????o. Nestas condi????es, os valores de CIMs obtidos foram: CIM ???0,03 ??g/mL, para o cetoconazol e itraconazol para inibir 50% e 80% do crescimento de M. obtusa e M. furfur. Para o fluconazol foram encontradas CIMs de 2 e de 4 ??g/mL para inibir 50% do crescimento de M. sympodialis e M. obtusa respectivamente, e CIM de 4 e 8 ??g/mL para inibi????o de 80%; enquanto que para M. furfur foi encontrada uma CIM ???0,03 ??g/mL desta droga para inibir 50% e 80% do crescimento f??ngico. Portanto, apesar de preliminares, considerando o n??mero reduzido de amostras analisadas, os resultados obtidos neste estudo forneceram informa????es importantes na perspectiva da elabora????o de um protocolo para testes de susceptibilidade ??s esp??cies de Malassezia.