Caracterização de cepas de Exophiala armazenadas no Banco de Fungos Filamentosos do Laboratório Especial de Micologia da UNIFESP/EPM
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Data
2014-06-30
Autores
Silva, Wendy Colalto Rodrigues da [UNIFESP]
Orientadores
Melo, Analy Salles de Azevedo [UNIFESP]
Tipo
Dissertação de mestrado
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Introdução: Leveduras negras do gênero Exophiala respondem por número substancial de infecções humanas superficiais e invasivas causadas por fungos negros. A identificação acurada de espécies de Exophiala baseia-se em critérios morfológicos complementados por tipagem molecular. A infecção em humanos causada por Exophiala depende de três fatores principais: porta de entrada, estado imunológico do paciente e tolerância térmica do agente. A maioria destas infecções são cutâneas e superficiais, entretanto, infecções sistêmicas fatais já foram relatadas. Objetivos: Estudar as características fenotípicas, genotípicas e susceptibilidade a antifúngicos de 23 isolados clínicos, identificados fenotipicamente como Exophiala sp. armazenados no Banco de Fungos Filamentosos do LEMI. Métodos: Para a identificação molecular nos baseamos no
sequenciamento da região ITS do rDNA, comparamos as sequências obtidas com sequências já depositadas em bancos genômicos (NCBI e CBS) e analizamos as sequências através da ferramenta BLASTn. Para a caracterização da macromorfologia e micromorfologia os isolados foram semeados em meio PDA e incubados a 25°C. Para análise de termotolerância, cada isolado foi semeado em 3 meios de cultura: PDA, SGA e MEA, e incubados em 4 temperaturas diferentes: 15°C, 25°C, 37°C e 42°C. Para cada um dos testes fenotípicos citados, a incubação foi de 14 dias nas devidas temperaturas para a realização das leituras. Os testes de susceptibilidade aos antifúngicos, anfotericina B (AMB), itraconazol (ITC), voriconazol (VRC), posaconazol (PSC), 5-fluorocitosina (5-FC), caspofungina (CFG) e terbinafina (TRB) foram realizados por microdiluição em caldo de acordo com o documento CLSI M38-A2. Resultados: A tipagem molecular teve sucesso na identificação de 100% das amostras que foram assim reconhecidas: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) e E. mesophila (1). A macromorfologia mostrou que as espécies de Exophiala são pleomórficas com relação ao aspecto e cor das colônias, mesmo considerando isolados da mesma espécie. Em relação à micromorfologia, observamos estruturas similares entre diferentes espécies do gênero, não possibilitando assim a identificação em nível de espécie, sendo necessário confirmação por método molecular. Na análise de termotolerância, todas as espécies cresceram a 25 e 37ºC com exceção de E. mesophila, que cresceu somente a 25oC. A 42ºC apenas as espécies E. dermatitidis, E. oligosperma e E. xenobiotica apresentaram crescimento, sendo as duas últimas com crescimento reduzido, não possibilitando caracterização. Em relação à susceptibilidade, as CIMs mais baixas foram verificadas para os azólicos, enquanto que as maiores foram
observadas para 5-FC, CFG e TRB. Conclusões: (i) O sequenciamento da região ITS permitiu a correta identificação de espécie para os 23 isolados clínicos; (ii) As características observadas nos testes de macromorfologia, micromorfologia e termotolerância para os isolados de Exophiala spp. analisados, mostraram que não são suficientes para a identificação acurada de espécie; (iii) O teste de susceptibilidade para Exophiala spp, utilizando o método de microdiluição em caldo padronizado pelo CLSI (2008) documento M38-A2 para fungos negros, mostrou-se consistente e reprodutível; (iv) De forma geral, a grande maioria dos isolados testados “in vitro” apresentou CIM ≤0,5 μg/mL exceto para 5-FC e CFG; (v) Apesar das diretrizes não indicarem AMB para tratamento contra Exophiala sp., esta mostrou-se ativa com CIMs ≤1 μg/mL para todas as espécies deste estudo, com exceção de E. mesophila.
Introduction: Black yeasts of Exophiala genus are responsible for a large number of superficial and invasive i infections. The identification of Exophiala at species level is based on morphological observation complemented by molecular typing. The human infection caused by Exophiala sp. depends on 3 main factors: fungus inoculation, patient’s immunologic status and thermotolerance of the agent. Most of these infections are superficial, but fatal systemic infections have been reported. Objective: The aim of this study was to identify Exophiala spp. by molecular method using internal transcript sequences (ITS) of rDNA gene of 23 clinical isolates of Exophiala deposited in our culture collection (LEMI). We evaluated macro and micromorphological characteristics, thermotolerance, and antifungal susceptibility to 7 different agents. Methods: Molecular identification was based on sequencing the ITS region and analyzed by BLASTn tool on GenBank (NCBI and CBS). For the macromorphology and micromorphology characterization, each isolate was streaked on PDA medium and incubated at 25ºC. For the thermotolerance analysis, each isolate was streaked in 3 different media: PDA, SGA and MEA, and incubated at 4 temperatures 15ºC, 25ºC, 37ºC and 42ºC, for 14 days. We used the CLSI M38-A2 broth microdilution method to test the 23 clinical isolates against 7 antifungal agents: amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), caspofungin (CAS), terbinafine (TER) and 5-fluorocitosina (5-FC). Results: The BLASTn analysis of ITS sequences was able to identify 100% of the clinical isolates as follows: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1), E. mesophila (1). The macromorphology showed that the Exophiala species have pleomorphic characteristics considering the colony aspect and color, even for isolates of the same species. On micromorphology analysis, we observed similar structures among different species of the genus, not allowing accurate species identification. The thermotolerance analysis showed that except for E. mesophila, all the isolates of different species grew at 25 and 37ºC. Only E. dermatitidis, E. oligosperma and E. xenobiotica grew as colonies at 42ºC, while the last two presented reduced growth, not allowing characterization. The antifungal susceptibility tests showed that triazols were the most active agents in vitro against Exophiala, while higher MICs were observed for 5-FC, CFG and TRB. Conclusions: (i) ITS sequencing allowed accurate identification of all the 23 clinical isolates tested; (ii) The characteristics observed on macromorphology, micromorphology and thermotolerance analysis for all the isolates showed that these tests are not sufficient for accurate species identification; (iii) The susceptibility tests using the microdilution method standardized by the CLSI broth (2008) document M38-A2 for black fungi, was consistent and reproducible for Exophiala spp; (iv) In general, the vast majority of isolates tested in vitro presented MIC ≤ 0.5 μg/mL for most of the antifungal agents, except for 5-FC and CFG; (v) Although the guidelines do not indicate AMB for treatment against Exophiala sp., this study showed activity with MICs ≤ 1 μg/mL for all the species tested, excepted for E. mesophila.
Introduction: Black yeasts of Exophiala genus are responsible for a large number of superficial and invasive i infections. The identification of Exophiala at species level is based on morphological observation complemented by molecular typing. The human infection caused by Exophiala sp. depends on 3 main factors: fungus inoculation, patient’s immunologic status and thermotolerance of the agent. Most of these infections are superficial, but fatal systemic infections have been reported. Objective: The aim of this study was to identify Exophiala spp. by molecular method using internal transcript sequences (ITS) of rDNA gene of 23 clinical isolates of Exophiala deposited in our culture collection (LEMI). We evaluated macro and micromorphological characteristics, thermotolerance, and antifungal susceptibility to 7 different agents. Methods: Molecular identification was based on sequencing the ITS region and analyzed by BLASTn tool on GenBank (NCBI and CBS). For the macromorphology and micromorphology characterization, each isolate was streaked on PDA medium and incubated at 25ºC. For the thermotolerance analysis, each isolate was streaked in 3 different media: PDA, SGA and MEA, and incubated at 4 temperatures 15ºC, 25ºC, 37ºC and 42ºC, for 14 days. We used the CLSI M38-A2 broth microdilution method to test the 23 clinical isolates against 7 antifungal agents: amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), caspofungin (CAS), terbinafine (TER) and 5-fluorocitosina (5-FC). Results: The BLASTn analysis of ITS sequences was able to identify 100% of the clinical isolates as follows: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1), E. mesophila (1). The macromorphology showed that the Exophiala species have pleomorphic characteristics considering the colony aspect and color, even for isolates of the same species. On micromorphology analysis, we observed similar structures among different species of the genus, not allowing accurate species identification. The thermotolerance analysis showed that except for E. mesophila, all the isolates of different species grew at 25 and 37ºC. Only E. dermatitidis, E. oligosperma and E. xenobiotica grew as colonies at 42ºC, while the last two presented reduced growth, not allowing characterization. The antifungal susceptibility tests showed that triazols were the most active agents in vitro against Exophiala, while higher MICs were observed for 5-FC, CFG and TRB. Conclusions: (i) ITS sequencing allowed accurate identification of all the 23 clinical isolates tested; (ii) The characteristics observed on macromorphology, micromorphology and thermotolerance analysis for all the isolates showed that these tests are not sufficient for accurate species identification; (iii) The susceptibility tests using the microdilution method standardized by the CLSI broth (2008) document M38-A2 for black fungi, was consistent and reproducible for Exophiala spp; (iv) In general, the vast majority of isolates tested in vitro presented MIC ≤ 0.5 μg/mL for most of the antifungal agents, except for 5-FC and CFG; (v) Although the guidelines do not indicate AMB for treatment against Exophiala sp., this study showed activity with MICs ≤ 1 μg/mL for all the species tested, excepted for E. mesophila.
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Citação
SILVA, Wendy Colalto Rodrigues da. Caracterização de cepas de Exophiala armazenadas no Banco de Fungos Filamentosos do Laboratório Especial de Micologia da UNIFESP/EPM. São Paulo, 2014. 112 f. Dissertação (Mestrado em Medicina Translacional) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2014.