First isolation of cryptococcus neoformans genotype VNI MAT-alpha from wood hollow trunks of hymenaea courbaril

Cryptococcal infection is transmitted by the inhalation of Cryptococcus spp. propagules. Information about the Cryptococcus species inhabiting plants might be clinically relevant due to the epidemiological role of these habitats as possible sources of human infection. The aim of this study was to increase the knowledge about the environmental occurrence of cryptococcosis agents. Hollow tree vegetal debris of nine plant species was sampled quarterly over a 12-month period. Melanized colonies were screened for Cryptococcus neoformans and C. gattii by biochemical tests, followed by URA5-RFLP molecular analysis, M13 fingerprinting assays, and mating-typing with the specific a and α primers. The susceptibility to fluconazole of all of the confirmed species colonies was determined using the AFST-EUCAST broth dilution method. We found that the typical Brazilian flora tree Hymenaea courbaril yielded a high cryptococcal burden (median, 10(2) CFU/g) during the summer, autumn and winter seasons. C. neoformans VNI molecular type MAT alpha was identified in all of the samples. The fingerprinting analyses showed great molecular variability with no correlation with the susceptibility profile to fluconazole (MIC range 4 to ≥64 mg/l). To our knowledge, this study is the first describing the association between C. neoformans and Hymenaea courbaril. These observations extend the known geographic distribution of and substantiate a new urban environmental niche for C. neoformans and also emphasize the genetic diversity of the environmental C. neoformans VNI molecular type isolates.

Estudo comparativo da susceptibilidade de isolados clínicos de Cryptococcus neoformans (Sanfelice, 1895) frente a alguns antifúngicos de uso hospitalar e extratos vegetais obtidos de plantas medicinais da região semiárida sergipana / Comparative study of the susceptibility of clinical isolates of Cryptococcus neoformans (Sanfelice) against some antifungal agents of hospital use and plant extracts obtained from medicinal plants of the semiarid Sergipe region, Brazil

O uso de plantas medicinais no combate as micoses é uma prática comum nas comunidades rurais e, neste contexto, o objetivo desse trabalho foi analisar o perfil de sensibilidade de isolados clínicos de C. neoformans frente a extratos brutos aquosos e antifúngicos de uso hospitalar utilizando a técnica de difusão em disco. Esses produtos naturais foram obtidos de plantas medicinais popularmente utilizadas por comunidades do sertão sergipano. Foram analisados os extratos brutos aquosos de juazeiro (Ziziphus joazeiro Mart.), catingueira (Caesalpinia pyramidalis Tul.), quixabeira (Bumelia sartorum Mart.) e jatobá (Hymenaea courbaril L.). Do juazeiro foi testado o extrato preparado a partir do infuso da entrecasca e da decocção da folha. Da catingueira foi utilizado o extrato preparado a partir do infuso das folhas. Da quixabeira o extrato utilizado foi preparado a partir da decocção da entrecasca e do jatobá maceração da entrecasca. Todos esses produtos naturais foram submetidos aos seguintes tratamentos: exposição ou não a luz ultravioleta e autoclavagem (121ºC por 10minutos). Em paralelo as leveduras patogênicas foram testadas frente aos seguintes antifúngicos de uso hospitalar: anfotericina B, fluconazol, Itraconazol, Miconazol e cetoconazol. Todos os extratos brutos aquosos apresentaram ação antifúngica frente a todas as linhagens clínicas de Cryptococcus neoformans. O tratamento de submeter à autoclavagem e exposição à luz ultravioleta apresentaram melhores resultados de ação antifúngica. Sendo que o tratamento de autoclavar o extrato bruto aquoso prevaleceu estatisticamente com os melhores resultados. Outros estudos de atividade antimicrobiano são necessários para corroborar a ação antimicótica dos produtos natuais testados, como pro exemplo killer-time e fracionamento do teste de micodiluição. No teste de sensibilidade dos antifúngicos realizado foi demonstrado que as leveduras apresentaram resistência preocupante ao fluconazol e a itraconazol pelo método de difusão em disco. Para novos conhecimentos desse perfil de resistência há necessidade de testes de melhor acurácia, como por exemplo, microdiluição. Para tanto, deve-se padronizar os testes de sensibilidade quando há uso de produtos naturais oriundos de plantas medicinais, além de fornecer alternativas de uso aos pacientes cometidos por essa levedura com fornecimento de dados científicos que comprovem a ação dos produtos naturais e plantas medicinais de largo uso no sertão sergipano.

The use of medicinal plants to combat mycoses is a common practice in rural communities, and, in this context, the aim of this study was to analyze the sensitivity of clinical isolates of C. neoformans in relation to aqueous crude extracts and antifungal agents of hospital use using the disc diffusion technique. These natural products were obtained from medicinal plants popularly used empirically by backland communities in Sergipe, Brazil. The aqueous crude extracts of Ziziphus joazeiro Mart., Caesalpinia pyramidalis Tul., Bumelia sartorum Mart. and Hymenaea courbaril L. were analyzed. From the Ziziphus joazeiro Mart., we tested the extract prepared from the infusion of the inner bark and the decoction of the leaf. From the Caesalpinia pyramidalis Tul., the extract used was prepared from the infusion of the leaves. From Bumelia sartorum Mart., the extract used was prepared from the decoction of the bark, and from the Hymenaea courbaril L. we prepared the extract from the maceration of the inner bark. All these natural products were subjected to the following treatments: exposure to ultraviolet light or not and autoclaving (121°C for 10minutes). Amphotericin B, fluconazole, itraconazole, miconazole and ketoconazole were tested. All aqueous crude extracts showed antifungal activity against all clinical strains of Cryptococcus neoformans. The treatment that underwent autoclaving and exposure to ultraviolet light showed better results for antifungal action. The treatment with autoclaving of the aqueous crude extract statistically prevailed with the best results. There is the need to perform some treatments using these natural products based on the tested medicinal plants for better antifungal activity against this pathogenic yeast so they become more effective. In the antifungal susceptibility test performed, it was demonstrated that the yeast had worrying resistance to fluconazole and itraconazole by the disc diffusion method. FOr further knowledge on this resistance profile, there is the need of tests with greater accuracy, such as the microdilution test. To do so, susceptibility testing must be standardized when there is the use of natural products derived from medicinal plants, in addition to the provision of alternative uses by patients affected by this yeast, providing scientific data demonstrating the use and action of the natural products and medicinal plants of wide use in Sergipe, Brazil.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

La Criptococosis y su Agente Etiológico

Se presenta una revisión sobre la criptococosis con especial énfasis en su agente etiológico el Cryptococcus neoformans. La incidencia de la criptococosis se ha incrementado mundialmente en la última década, debido a la aparición del síndrome de inmunodeficiencia adquirida, en donde la entidad ha sido diagnosticada del 2 al 13 por ciento de los pacientes. Se ha descrito el estado anamorfo y el teleomorfo de C. neoformans y se conocen cuatro serotipos del hongo (A, B, C y D) y dos variedades: C. neoformans var. neoformans y C. neoformans var. gattii. Las variedades difieren en numerosas características, incluyendo la distribución geográfica. El hábitad de C. neoformans var. neoformans ha sido ampliamente estudiado, en contraste, C. neoformans var. gattii sólo ha sido aislado recientemente de la naturaleza en asociación con el Eucalyptus camaldulensis. El conocimiento de la ecología del hongo permitirá entender mejor su epidemiología