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Candidíase De Orofaringe É a Infecção Fúngica Oportunística Mais Comum (Vargas & Joly 2002)

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Candidíase De Orofaringe É a Infecção Fúngica Oportunística Mais Comum (Vargas & Joly 2002) UNIVERSIDADE FEDERAL DE GOIÁS INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA CAROLINA RODRIGUES COSTA Identificação e suscetibilidade in vitro de espécies de Candida isoladas de pacientes HIV positivos. Orientador: Profa Dra Maria do Rosário Rodrigues Silva Dissertação de Mestrado Goiânia-GO, 2005 UNIVERSIDADE FEDERAL DE GOIÁS INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA CAROLINA RODRIGUES COSTA Identificação e suscetibilidade in vitro de espécies de Candida isoladas de pacientes HIV positivos. Orientador: Profa Dra Maria do Rosário Rodrigues Silva Dissertação apresentada ao curso de Mestrado em Medicina Tropical e Saúde Pública da Universidade Federal de Goiás,como requisito parcial à obtenção do grau de Mestre em Medicina Tropical, na área de concentração de Microbiologia. Goiânia-GO, 2005 2 “Se nós pudéssemos ver como os outros nos vêem, compreenderíamos até que ponto as aparências são enganosas”. Franklin Jones 3 Aos meus pais Theobaldo Silva Costa e Maria do Rosário Rodrigues Silva, e aos meus irmãos Márcio e Théo pelo estímulo, carinho, amizade e ainda pelo incentivo e apoio dados nesse momento. AGRADECIMENTOS Agradeço a Deus por me fazer tão perseverante mesmo diante de seus desígnios . 4 A minha orientadora, profa. Dra. Maria do Rosário Rodrigues Silva, minha maior amiga que me deu apoio e dedicou se a mim nesse período que convivi no laboratório. Obrigada pelo incentivo e palavras de carinho que recebi. A professora Orionalda de Fátima Lisboa Fernandes pelo carinho e amor com que sempre me tratou fazendo-me enxergar mais longe Aos amigos do laboratório de Micologia nas pessoas de Karla Carvalho Miranda, Crystiane Rodrigues Araújo, Claudine Hassan Abbas Krais, Xisto Sena Passos, Lúcia Kioko Hasimoto e Souza, Janine de Aquino Lemos pelo auxilio técnico, solidariedade e constantes colaborações. .Ao professor Evandro Leão Ribeiro e funcionários do laboratório de Micologia do IPTSP – UFG, Hildene Menezes e Cícero Júnior, pelo auxílio prestado durante o processamento experimental da pesquisa. Ao professor Gercino Monteiro Filho, pela forma eficiente com que realizou a análise estatística deste trabalho. Aos integrantes do serviço de ambulatório do Hospital de Doenças Tropicais de Goiânia-GO, em especial à Dra. Ana Joaquina Cohen, que se dispôs a examinar os pacientes e coletar o material biológico. Aos pacientes que mesmo na dor provocadas por suas enfermidades, contribuíram em prol da ciência. A todos que direta ou indiretamente contribuíram para realização deste trabalho, minha sincera gratidão. 5 Resumo A candidíase está entre as doenças cuja incidência tem aumentado nos últimos 20 anos, principalmente nos pacientes HIV (+). Candida albicans encontrada na mucosa bucal como comensal é uma das espécies mais comuns isoladas desta cavidade. Há vários fatores que podem contribuir para que a levedura seja patogênica, incluindo alterações do hospedeiro ou virulência do microrganismo. Entre os fatores que contribuem para alta patogenicidade do microrganismo estão: o dimorfismo, o aumento da adesão, a produção de proteinase aspartil secretora e resistência aos fármacos disponíveis para o tratamento. Além disso vários outros fatores relacionados ao hospedeiro tais como alteração da barreira epitelial, microbiota e imunidade celular contribuem para a proliferação da levedura bucal. A grande maioria dos pacientes infectados pelo HIV desenvolverão lesões de candidíase esofageana como progresso de doença viral. A carga viral e contagem de célula T CD4 são marcadores para a progressão da AIDS. Para tratamento das infecções causadas por Candida tem sido usado fluconazol, itraconazol, voriconazol e anfotericina B. O desenvolvimento de resistência aos antifúngicos, observados em isolados de leveduras sensíveis e em candidíase de orofaringe recorrente seguindo tratamento com diferentes antifúngicos, são fatores importantes nos quais devem ser usado teste de suscetibilidade in vitro. Este trabalho teve o objetivo de investigar a etiologia de carreadores de Candida bucal nos pacientes infectados pelo HIV e verificar se houve associação entre contagem de célula T CD4 e carga viral nos pacientes carreadores de Candida e determinar o padrão de suscetibilidade dos isolados frente ao fluconazol, itraconazol, voriconazol e anfotericina B. Amostras da cavidade bucal foram coletadas em 99 pacientes infectados pelo “HIV tratados” no Hospital de Doenças Tropicais de Goiânia, Goiás, Brasil. Em todos os pacientes foram feitos 6 contagem de célula T CD4 usando citometria de fluxo e medida de carga viral usando Versant HIV-1 RNA 3.0 branched DNA. Os isolados de Candida foram identificados por tubo germinativo, produção de clamidoconídio em ágar Corn Meal suplementado com 1% de tween 80 e por testes de assimilação de açúcar e de nitrogênio. Para diferenciar C. albicans e C. dubliniensis foi usado teste de formação de clamidoconídeo em agar Staib, crescimento à 45oC, caldo Sabouraud hipertônico. Teste de suscetibilidade foi feito usando microdiluição em caldo de acordo com a metodologia recomendada pelo Comitê Nacional para normas de Laboratório Clínico (NCCLS) documento M-27 A2. Dos 99 pacientes HIV positivos, 62 (62,6%) tinham cultura positiva para Candida. O agente etiológico mais comum foi C. albicans (50,0%) e C. tropicalis (21,0%). Não identificamos C. dubliniensis pelos métodos fenotípicos usados. Dos 62 pacientes carreadores a média de CD4 foi de 402,2 células mm3, enquanto a média de carga viral foi de 14069 cópias/ml, para os não carreadores a média de CD4 foi de 341,1 células mm3, enquanto a média de carga viral foi de 9956 cópias/ml. O teste de Mann-Whitney foi usado para determinar se havia associação de contagen de célula T CD4+ e carga viral entre os pacientes carreadores de Candida. Um valor de P < 0,05 foi considerado estatisticamente significante. Nossos resultados mostraram que não houve diferença significativa das variáveis: contagem de célula T CD4 e carga viral entre os pacientes carreadores e não carreadores de Candida. O teste de suscetibilidade in vitro mostrou que alguns isolados de Candida foram resistentes aos derivados azólicos, sendo 8,1%, 12,9% e 3,2% resistentes respectivamente ao itraconazol, fluconazol, e voriconazol. Nenhum isolado foi resistente a anfotericina B. Em conclusão, colonização de espécies de Candida na cavidade bucal, principalmente C. albicans foi detectada com alta freqüência em pacientes infectados pelo HIV. Os nossos resultados permitiram também verificar que carreadores de Candida não estão associados com número de células T CD4+ ou carga viral. O desenvolvimento de resistência aos agentes antifúngicos, observados nos isolados, 7 é um fator importante para determinar o uso de teste de suscetibilidade in vitro em rotina de laboratório. Palavras chave: AIDS; carreadores de Candida bucal; suscetibilidade; carga viral; célula T CD4+. 8 Abstract Candidiasis is among those diseases whose incidence has increased over the past 20 years, particularly in AIDS patients. Candida albicans frequently, recovered from oral mucosa as comensal is by far the most common species isolated from oral candidiasis. There are several factors that may contribute for the yeast to be pathogen, including changes in the host or the virulence of the organism. Among the factors that contribute to higher pathogenicity of the microrganism include dimorphism, increased adhesion, production of secretory aspartyl proteases, and drug resistance. Besides other several factors relationship to host such as changes of epithelial barrier, normal flora and cell immunity contributed for oral yeast proliferation. So, a majority of HIV-positive patients could develop clinical lesions of oropharyngeal or esophageal candidosis as the viral disease progresses, according to quantitative HIV-1 RNA in plasma and CD4+ cells count. For treating the infection caused by Candida has been used fluconazole, voriconazole, itraconazole and amphotericin B. The increased use of fluconazole has given rise to the development of resistance among Candida spp, mainly C. glabrata, C. krusei and C. dubliniensis isolates. The development of resistance to antifungal agents, observed in previously susceptible yeast isolates, as well as recurrence of oropharyngeal candidiasis following treatment with different antifungal drugs, are highly relevant factors which should be taken into account to determine the use of in vitro susceptibility tests. Thus the present study had the aim of 9 investigating the etiology of oral Candidal carriage in HIV-infected patients, of verifying whether there was association of CD4+ cell count, of HIV-1 RNA measurement in plasma with oral candida carriage patients and to determine the in vitro susceptibility pattern of the isolates against fluconazole, voriconazole, itraconazole and amphotericin B. Samples from the oral mucosa were collected of 99 HIV-infected patients treated at the Hospital de Doenças Tropicais, Goiânia, Goiás State, Brazil. CD4+ cells count by using flow cytometer and HIV-1 RNA measument in plasma by using Versant HIV-1 RNA 3.0 branched DNA assay were determined for all patients at the moment of collect. The yeast isolates were identified by germ tube test, chlamydoconidia production on cornmeal agar supplemented with 1% tween 80, and by sugar and nitrogen assimilation tests. For differentiation between C. albicans and C. dubliniensis we used chlamydospore formation on Staib agar, growth at 45oC and at hypertonic Sabouraud broth. Antifungal susceptibility tests were performed by using the broth microdilution assay, according
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