Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Sixteen years o Pityriasis Versicolor in metropolitan area of Porto Alegre, Southern Brazil / Dezesseis anos de Pitiríase Versicolor na Região Metropolitana de Porto Alegre, Sul do Brasil

SUMMARYPityriasis versicolor is the most common of the diseases caused by Malasseziayeasts. The aim of this study is to determine the prevalence of pityriasis versicolor and its etiological aspects in the metropolitan area of Porto Alegre, Brazil. A retrospective crosssectional study with data from patients of a reference hospital from 1996 to 2011 was performed. Collected data included: date, age, gender, ethnicity, anatomical region of lesion and the direct mycological examination results. Among the positive results in the direct mycological examination, 5.8% (2,239) were positive for pityriasis versicolor. The angular coefficient (B) was -0.3%/year, showing a decrease over the years. The disease was more prevalent in men (7.1% of men versus 5.1% of women that underwent the direct mycological examination); younger age (median 31 years old); "pardo" and black people (3.7% more than expected in the sample); trunk (73.44% of the affected anatomic sites). Lesions in rare sites (groin, genitals, legs, feet and hands) were also observed in this study. In conclusion, due to the decrease in the prevalence of pityriasis versicolor, long-term epidemiological studies in the metropolitan area of Porto Alegre, Brazil, are needed to continue the monitoring of this disease.

RESUMOPitiríase versicolor é a mais comum das doenças causadas pelas leveduras do gênero Malassezia. O objetivo do trabalho foi determinar a prevalência de pitiríase versicolor e seus aspectos etiológicos na região metropolitana de Porto Alegre, Brasil. Foi realizado um estudo retrospectivo transversal com dados de pacientes de hospital de referência entre 1996 a 2011. Os dados coletados foram: data, idade, gênero, etnia, local anatômico da lesão e resultados do exame micológico direto. Dentre os resultados positivos do exame micológico direto, 5,8% (2.239) foram positivos para pitiríase versicolor. O coeficiente angular (B) foi -0,3%/ ano, mostrando uma diminuição ao longo dos anos. A doença foi mais prevalente em homens (7,1% dos homens versus 5,1% das mulheres que se submeteram ao exame micológico direto); jovens (mediana de 31 anos); pardos e pretos (3,7% acima do esperado para a amostra); tronco (73,4% dos locais anatômicos afetados). Lesões em locais raros (virilha, genitálias, pernas, pés e mãos) também foram observadas no estudo. Em conclusão, devido à diminuição da prevalência de pitiríase versicolor, estudos epidemiológicos de longo prazo são necessários para continuar o monitoramento da doença na região metropolitana de Porto Alegre, Brasil.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii / Aplicação de 6-nitrocumarina como substrato fluorescente para detecção de atividade nitroredutásica em Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection. .

Introdução Sporothrix schenckii é um fungo dimórfico térmico, agente etiológico de micose subcutânea, a esporotricose. Nitrocumarina representa classe de substratos fluorogênicos em que a atividade nitroredutásica microbiana produz vários derivados, já utilizados em vários outros ensaios enzimáticos. O objetivo deste estudo foi analisar 6-nitrocumarina (6-NC) como substrato para estudo da atividade nitroredutásica em Sporothrix schenckii. Métodos Trinta e cinco isolados de S. schenckii foram cultivados por sete, 14 e 21 dias a 35 °C em um microcultivo contendo 6-nitrocumarina ou 6-aminocumarina (6-AC) solubilizada em dimetilsulfóxido ou dimetilsulfóxido como controle negativo, para posterior análise em microscópio de epifluorescência. A fase orgânica da cultura de sete, 14 e 21 dias foi analisada por meio de iluminação direta com luz UV de 365 nm e por eluição em placas de sílica gel G com hexano:acetato de etila 1:4 e revelada com luz UV. Resultados Todos os isolados mostraram a presença de 6-AC (fluorescência amarela) e 6-hidroxilaminocumarina (fluorescência azul) em cromatografia em camada delgada, que explica a fluorescência verde observada na estrutura dos fungos. Conclusão A atividade nitroredutásica é amplamente distribuída no complexo S. schenckii e 6-NC é um substrato fluorogênico de fácil obtenção e aplicabilidade para detecção da atividade nitroredutásica. .