Prevalência de espécies de Aspergillus em amostras de origem clínica/ambiental e análise de seu perfil de susceptibilidade
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Data
2013-09-25
Autores
Negri, Clara Ezequiel [UNIFESP]
Orientadores
Colombo, Arnaldo Lopes [UNIFESP]
Tipo
Dissertação de mestrado
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Resumo
Espécies do gênero Aspergillus são responsáveis pela segunda e/ou terceira maior ocorrência de infecções fúngicas invasivas (IFIs) em hospitais terciários. Na prática clínica, essas infecções são difíceis de diagnosticar e o tratamento tardio é muito
frequente. Poucos centros médicos têm capacitação para realizar a identificação acurada de espécies deste gênero de fungos, visto que a formação em micologia clássica, bem como o domínio e disponibilidade de técnicas moleculares são requeridas para este diagnóstico. Os casos de resistência a antifúngicos não são comuns na
medicina contemporânea, mas o número de isolados resistentes e fracasso terapêutico
têm aumentado significativamente nas últimas décadas. São escassos os dados no Brasil sobre a prevalência de espécies de Aspergillus e seu perfil de susceptibilidade a antifúngicos. O presente estudo teve como objetivos analisar a distribuição das espécies do gênero Aspergillus em 150 amostras clínicas/ambientais e avaliar a ocorrência de cepas com valores de CIMs superiores ao cut-off epidemiológico proposto aos antifúngicos comumente utilizados. A identificação de espécies do gênero Aspergillus foi realizada pela abordagem polifásica, a qual inclui análise macro e
micromorfológica, termotolerância, sequenciamento e filogenia se necessário, (região ITS do rDNA e dos genes calmodulina e β-tubulina). Os isolados também foram avaliados quanto à tolerância a cloreto de sódio (5% e 10%), cicloheximida (0,1% e 0,5 %) e produção de ácidos orgânicos em CREA e ácido aspergílico em AFPA. Foi avaliado o perfil de susceptibilidade in vitro dos 150 isolados frente a triazólicos (itraconazol, voriconazol e posaconazol) utilizando 2 diferentes métodos: microdiluição
em caldo e E-test. Os isolados com valores de cut-off superiores aos estabelecidos, também foram testados frente a anfotericina B, caspofungina e anidulafungina. A caracterização fenotípica, assim como o sequenciamento da região ITS separou os
isolados em nove seções, a saber: Circumdati, Clavati, Flavi, Flavipedes, Fumigati, Nidulantes, Nigri, Terrei e Usti. Utilizando a abordagem polifásica, 141/150 isolados foram identificados em nível de espécie. Com a abordagem realizada, não foi possível identificar 3/43 isolados pertencentes à seção Flavi e 6/17 da seção Nigri. Com relação
aos triazólicos testados, 141/150 isolados apresentaram valores de CIMs menores ou
iguais ao cut-off epidemiológico estabelecido utilizando a metodologia padronizada. Dentre os isolados, 1/9 Circumdati, 1/9 Usti, 1/9 Nigri, 4/9 Fumigati e 2/9 Flavi apresentaram valores mais elevados frente a estes antifúngicos; 2/9 apresentaram valores de CIM elevados também frente à anfotericina B (N. pseudofischeri e A.
ochraceus). Somente o isolado de A. calidoustus apresentou valor de CIM elevado frente à caspofungina. Todos os nove isolados testados frente à anidulafungina apresentaram como valor de CIM a menor concentração testada deste fármaco,
demonstrando sua ótima atividade in vitro. A falta de padronização da metodologia de E-test®, dificultou sua execução e avaliação do teste, principalmente no que diz respeito à leitura, prejudicando, assim, o estabelecimento de taxas de correlação entre as duas metodologias testadas. Esta taxa foi classificada como marginal quando consideramos como equivalentes valores de ± uma diluição e as taxas tornam-se aceitáveis quando consideramos como equivalentes valores de ± duas diluições. Os
dados observados reforçam a importância na identificação acurada de espécies de Aspergillus, visto que encontramos um número substancial de espécies crípticas que apresentam variações no perfil de susceptibilidade entre os isolados aos antifúngicos comumente utilizados. Neste contexto, demonstramos a importância na padronização
da metodologia de E-test® para aumentar a confiabilidade do teste.
The species of Apergillus genus are responsible for the second or third largest occurrence of invasive fungal infection in tertiary hospitals. In clinical practice, those kinds of infections are difficult to diagnose commonly resulting in the late treatment of the infection. Few medical centers are prepared to perform accurate identification of species of this particular genus of Fungi, mainly due to the lack of classical training in mycology as well as the limited availability of molecular techniques required for this diagnosis. Cases of resistance to antifungal drugs are uncommon in contemporary medicine; however the number of cases of resistance and therapeutic failures has risen significantly in recent decades. There is limited prevalence of Aspergillus species and their antifungal susceptibility in Brazil. This study aims to analyze the distribution of the species of Aspergillus genus in 150 clinical and environment samples and evaluate the occurrence of strains with MIC values above the cut-off protocols commonly used antifungal drugs. The identification of Aspergillus species was performed through a polyphasic approach, which comprised macro and micromorphology, thermotolerance, sequencing and phylogeny if necessary (ITS region, calmodulin and β-tubulin genes). The isolates were also evaluated for their sodium chloride (5% and 10%) and cicloheximide (0.1% and 0.5%) tolerance, organic acids production in CREA and aspergillic acid production with AFPA medium. The in vitro susceptibility profile of 150 isolates against triazoles (itraconazole, voriconazole and posaconazole) was evaluated using two different methods: broth microdilution and E-test®. The isolates with MICs values above the epidemiological cut-off, has also been tested against amphotericin B, caspofungin and anidulafungin. The phenotypic characterization, and the ITS region sequencing discriminated the isolates in nine sections: Circumdati, Clavati, Flavi, Flavipedes, Fumigati, Nidulantes, Nigri, Terrei and Usti. Using polyphasic approach, 141/150 isolates were identified to the species level. Based on the results, it was not possible to identify 3/43 isolates from Flavi section and 6/17 from Nigri section. Regarding to the triazoles tested, 141/150 isolates presented MIC values equal to or lowers than the value established epidemiological cut-off using the standardized methodology. Among the isolates, 1/9 Circumdati, 1/9 Usti, 1/9 Nigri, 4/9 Fumigati and 2/9 Flavi presented MIC values above the established epidemiological cut-off of these antifungal drugs; 2/9 present MIC values higher also against amphotericin B (N. pseudofischeri and A. ochraceus). Only the A. calidoustus isolate present MIC values higher also against caspofungin. All of the nine isolates were tested against anidulafungin exhibited lower concentration of drug tested as MIC values demonstrating great in vitro activity. The lack of E-test methodology standardization is a factor that can affect and compromise the results, damaging the establishment of rates of correlation between the two tested methodologies; in that case this rate has been classified as marginal, when considered as equivalent values within ± one dilution and became acceptable when considered as equivalent values within ± two-fold dilution. According to the results, the observed data reinforce the importance of accurate identification of Aspergillus species, since the study found that a substantial number of cryptic species that show variations in susceptibility profile among isolates against the commonly used antifungal drugs. This context demonstrated the importance of standardization in the Etest® methodology to increase the reliability of the test.
The species of Apergillus genus are responsible for the second or third largest occurrence of invasive fungal infection in tertiary hospitals. In clinical practice, those kinds of infections are difficult to diagnose commonly resulting in the late treatment of the infection. Few medical centers are prepared to perform accurate identification of species of this particular genus of Fungi, mainly due to the lack of classical training in mycology as well as the limited availability of molecular techniques required for this diagnosis. Cases of resistance to antifungal drugs are uncommon in contemporary medicine; however the number of cases of resistance and therapeutic failures has risen significantly in recent decades. There is limited prevalence of Aspergillus species and their antifungal susceptibility in Brazil. This study aims to analyze the distribution of the species of Aspergillus genus in 150 clinical and environment samples and evaluate the occurrence of strains with MIC values above the cut-off protocols commonly used antifungal drugs. The identification of Aspergillus species was performed through a polyphasic approach, which comprised macro and micromorphology, thermotolerance, sequencing and phylogeny if necessary (ITS region, calmodulin and β-tubulin genes). The isolates were also evaluated for their sodium chloride (5% and 10%) and cicloheximide (0.1% and 0.5%) tolerance, organic acids production in CREA and aspergillic acid production with AFPA medium. The in vitro susceptibility profile of 150 isolates against triazoles (itraconazole, voriconazole and posaconazole) was evaluated using two different methods: broth microdilution and E-test®. The isolates with MICs values above the epidemiological cut-off, has also been tested against amphotericin B, caspofungin and anidulafungin. The phenotypic characterization, and the ITS region sequencing discriminated the isolates in nine sections: Circumdati, Clavati, Flavi, Flavipedes, Fumigati, Nidulantes, Nigri, Terrei and Usti. Using polyphasic approach, 141/150 isolates were identified to the species level. Based on the results, it was not possible to identify 3/43 isolates from Flavi section and 6/17 from Nigri section. Regarding to the triazoles tested, 141/150 isolates presented MIC values equal to or lowers than the value established epidemiological cut-off using the standardized methodology. Among the isolates, 1/9 Circumdati, 1/9 Usti, 1/9 Nigri, 4/9 Fumigati and 2/9 Flavi presented MIC values above the established epidemiological cut-off of these antifungal drugs; 2/9 present MIC values higher also against amphotericin B (N. pseudofischeri and A. ochraceus). Only the A. calidoustus isolate present MIC values higher also against caspofungin. All of the nine isolates were tested against anidulafungin exhibited lower concentration of drug tested as MIC values demonstrating great in vitro activity. The lack of E-test methodology standardization is a factor that can affect and compromise the results, damaging the establishment of rates of correlation between the two tested methodologies; in that case this rate has been classified as marginal, when considered as equivalent values within ± one dilution and became acceptable when considered as equivalent values within ± two-fold dilution. According to the results, the observed data reinforce the importance of accurate identification of Aspergillus species, since the study found that a substantial number of cryptic species that show variations in susceptibility profile among isolates against the commonly used antifungal drugs. This context demonstrated the importance of standardization in the Etest® methodology to increase the reliability of the test.
Descrição
Citação
NEGRI, Clara Ezequiel. Prevalência de espécies de Aspergillus em amostras de origem clínica/ambiental e análise de seu perfil de susceptibilidade. São Paulo, 2013. 161 f. Dissertação (Mestrado em Medicina Translacional) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2013.