Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst.

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.

The use of standard methodology for determination of antifungal activity of natural products against medical yeasts Candida sp and Cryptococcus sp

Methodology for testing natural compounds for determination of antifungal activity had been developed with adaptations. The most used are bioautography and agar diffusion with a complex and no defined media. In this study, different methods for determination of antifungal activity of natural products are discussed and the use of M27-A2 microdilution test from CLSI (Clinical and Laboratory Standards Institute, 2002), as general standard methodology for testing plant extracts activity is recommended.

Metodologias para determinar atividade antifúngica foram desenvolvidas com adaptações para avaliar produtos naturais. As mais usadas são bioautografia e o método de difusão em agar, que empregam meios de cultura complexos, não definidos. Neste estudo são discutidos os métodos para determinação de atividade antifúngica de produtos naturais e é recomendado o uso do micrométodo modificado segundo o documento M27A2 do CLSI.

Prevalência e susceptibilidade de leveduras vaginais / Prevalence and susceptibility of vaginal yeast

Entre as vulvovaginites, a candidíase é apontada como a causa mais freqüente em mulheres na idade fértil. Atualmente, várias pesquisas mostram aumento na freqüência das espécies não-albicans e grande preocupação com episódios de repetição, assim como sua relação com a resistência ao tratamento. OBJETIVO: O presente estudo teve como objetivo verificar a distribuição de gêneros e espécies de leveduras causadoras de vaginite e analisar o perfil de sensibilidade das leveduras frente às drogas antifúngicas. MATERIAL E MÉTODO: Foram colhidas amostras de fluido vaginal de 250 pacientes para cultura, realizados identificação e antifungigrama dos isolados. RESULTADOS: Leveduras do gênero Candida estavam presentes em 27,6% das amostras. Candida albicans foi a levedura mais isolada em 74% dos casos, seguida de Candida glabrata, em 14,5%; Candida tropicalis, em 7,3%; e Candida parapsilosis, em 4,3%. Todos os isolados Candida albicans foram sensíveis à anfotericina B, e apenas um isolado da espécie não-albicans apresentou concentração inibitória mínima (CIM) mais elevada (2'g/ml). Em Candida albicans, 5,9% das amostras mostraram-se sensíveis, dependendo da dose de fluconazol, e 9,8%, resistentes. Apenas um isolado mostrou-se resistente, com CIM de 8'g/ml, para itraconazol. Nas espécies não-albicans, 11,7% dos isolados foram considerados resistentes ao fluconazol e 23,5, ao itraconazol. CONCLUSÃO: Candida albicans foi a espécie mais freqüentemente encontrada na microbiota vaginal; no entanto, outras espécies foram também comuns nessa população. Porcentual importante de isolados de Candida albicans e não-albicans foi resistente a fluconazol e itraconazol, mostrando a importância de realização de testes de identificação e antifungigrama para os episódios de candidíase vaginal.

Among the vulvovaginitis, candidiasis is the most frequent cause in women at reprodutive age of and several researches have shownd an increase in the frequency of the species non-albicans, and a great concern with repetitive episodes, as well as its relationship with the resistance to the treatment. OBJECTIVE: The purpose of this study was to verify the yeast species that cause vaginitis and their susceptibility to antifungal agents. MATERIAL AND METHOD: Vaginal swabs were colleted from 250 patients and were evaluated for culture, species identification and susceptibility to antifungal agents. RESULTS: Candida was present in 27.6% of the samples. Candida albicans was the predominant species (74%), followed by Candida glabrata (14.5%), Candida tropicalis (7,3%) and Candida parapsilosis (4,3%). All the C. albicans isolates were susceptible to anfotericin B and only one sample of non-albicans showed greater MIC (2mg/mL). 5.9% of C. albicans strains were susceptible-dose dependent to FLU and 9,8% resistant. Only one sample showed resistance to itraconazol, with CIM 8mg/mL. In the species non-albicans, 11.8% of the isolates were considered resistant to fluconazol and 23.5% to itraconazol. CONCLUSION: Candida albicans is the most frequent in the vaginal microenviroment, however non-albicans are common vaginal isolates even in a primary care population. The species isolated are less susceptible to fluconazole and itraconazole than most C. albicans, showing the importance of the performance of the identification tests and susceptibility to antifungal agents of vulvovaginal candidiasis.