Caracterização genotípica de amostras clínicas de Cryptococcus spp. e avaliação do perfil de virulência in vitro e em modelo de Galleria mellonella
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2021-08-02
Autores
Carmo, Fabíolla Nascimento do
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Universidade Estadual Paulista (Unesp)
Resumo
A criptococose é uma infecção oportunista comum em pacientes HIV positivos, mas também pode afetar pacientes com outras comorbidades imunológicas ou imunocompetentes. Neste trabalho, cepas clínicas de Cryptococcus spp. provenientes de hospitais de São José dos Campos (SP) foram identificadas pela amplificação do gene SRT. A seguir, foi avaliada a sensibilidades das cepas a fluconazol e anfotericina B (AMB) pela técnica de microdiluição conforme EUCAST. Também foi analisada a capacidade de formação de biofilme por cristal violeta (formação da biomassa) e XTT (viabilidade celular). Foram analisadas in vivo a formação da cápsula durante a infecção, a curva de sobrevivência e do índice de saúde em modelo invertebrado de Galleria mellonella. Seis cepas foram identificadas como C. neoformans var. grubbii e uma como C. gattii. Todas as cepas clínicas foram sensíveis a AMB e a concentração inibitória mínima para fluconazol variou entre 2 e 32 μg/mL. Todos os isolados de Cryptococcus spp. foram capazes de produzir biofilme. A sobrevida de G. mellonella infectada variou entre 6 e 7 dias para C. neoformans, e a cepa clínica 6 se mostrou menos virulenta, com 24% das larvas vivas no último dia de experimento. Para cepa de C. gattii, 20,8% das larvas infectadas permaneceram vivas ao final da análise. Em relação a análise do tamanho da cápsula após inoculação in vivo, as cepas clínicas de ambas espécies apresentaram aumento no tamanho, sendo observado o maior percentual de cápsulas ≥ 30μm na cepa 6. O índice de saúde foi capaz de agregar informações ao resultado da curva de sobrevivência de G. mellonella, uma vez que ele mostrou diferenças de saúde das larvas entre os grupos que apresentaram mesmo perfil de sobrevida. A associação do índice de saúde à curva de sobrevivência amplia a visão da vitalidade das larvas durante os dias de experimento e, ainda, auxilia na comparação de resultados entre laboratórios. O conhecimento das características genotípicas e fenotípicas de Cryptococcus spp. em uma determinada região pode ser ferramenta importante para implementação de políticas públicas de saúde eficazes.
Cryptococcosis is a common opportunistic infection in HIV-positive patients, but it can also affect patients with other immunological comorbidities or immunocompetent. In this work, clinical strains of Cryptococcus spp. from hospitals in São José dos Campos (SP) were identified by amplification of the SRT gene. Next, the sensitivities of the strains to fluconazole and amphotericin B (AMB) were evaluated by the microdilution technique according to EUCAST. Biofilm formation capacity by crystal violet (biomass formation) and XTT (cellular viability) was also analyzed. Capsule formation during infection, survival curve and health index in an invertebrate model of Galleria mellonella were analyzed in vivo. Six strains were identified as C. neoformans var. grubbii and one as C. gattii. All clinical strains were sensitive to AMB and the minimum inhibitory concentration for fluconazole ranged between 2 and 32 μg/mL. All Cryptococcus spp. were able to produce biofilm. The survival of infected G. mellonella ranged between 6 and 7 days for C. neoformans, and the clinical strain 6 was less virulent, with 24% of larvae alive on the last day of the experiment. For the C. gattii strain, 20.8% of the infected larvae remained alive at the end of the analysis. Regarding the analysis of capsule size after in vivo inoculation, the clinical strains of both species showed an increase in size, with the highest percentage of capsules ≥ 30μm being observed in strain 6. The health index was able to add information to the result of the survival curve of G. mellonella, since it showed differences in the health of larvae between the groups that presented the same survival profile. The association of the health index to the survival curve broadens the vision of the larvae's vitality during the days of the experiment and also helps in comparing the results between laboratories. The knowledge of the genotypic and phenotypic characteristics of Cryptococcus spp. in a given region, it can be an important tool for the implementation of effective public health policies.
Cryptococcosis is a common opportunistic infection in HIV-positive patients, but it can also affect patients with other immunological comorbidities or immunocompetent. In this work, clinical strains of Cryptococcus spp. from hospitals in São José dos Campos (SP) were identified by amplification of the SRT gene. Next, the sensitivities of the strains to fluconazole and amphotericin B (AMB) were evaluated by the microdilution technique according to EUCAST. Biofilm formation capacity by crystal violet (biomass formation) and XTT (cellular viability) was also analyzed. Capsule formation during infection, survival curve and health index in an invertebrate model of Galleria mellonella were analyzed in vivo. Six strains were identified as C. neoformans var. grubbii and one as C. gattii. All clinical strains were sensitive to AMB and the minimum inhibitory concentration for fluconazole ranged between 2 and 32 μg/mL. All Cryptococcus spp. were able to produce biofilm. The survival of infected G. mellonella ranged between 6 and 7 days for C. neoformans, and the clinical strain 6 was less virulent, with 24% of larvae alive on the last day of the experiment. For the C. gattii strain, 20.8% of the infected larvae remained alive at the end of the analysis. Regarding the analysis of capsule size after in vivo inoculation, the clinical strains of both species showed an increase in size, with the highest percentage of capsules ≥ 30μm being observed in strain 6. The health index was able to add information to the result of the survival curve of G. mellonella, since it showed differences in the health of larvae between the groups that presented the same survival profile. The association of the health index to the survival curve broadens the vision of the larvae's vitality during the days of the experiment and also helps in comparing the results between laboratories. The knowledge of the genotypic and phenotypic characteristics of Cryptococcus spp. in a given region, it can be an important tool for the implementation of effective public health policies.
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Galleria mellonella, Criptococcus, Reação em cadeia da polimerase (PCR), Susceptibilidade antifúngica, Polymerase Chain Reaction (PCR), Antifungal susceptibility, ASfa