Cryptococcosis is caused by capsulated yeasts of the genus Cryptococcus, covering two main species: C. neoformans and C. gattii. Some reports indicate that the genus Cryptococcus, in particular C. gattii strains, have high potential to develop resistance to fluconazole in vitro, which could explain the constant therapeutic failures and relapses in patients affected by this disease and under antifungal treatment. Strains of C. gattii seem to be more virulent since they can infect immunocompetent patients more often than C. neoformans strains, and animal studies have revealed data supporting this idea. Triazoles are the largest class of antifungal drugs in clinical settings and fluconazole is employed as a long-term therapy for the management of Cryptococcosis in AIDS patients that could lead to the emergence of resistant strains. Of note, subpopulation of cells may have variable growth at a certain in vitro concentration of fluconazole defined by the heteroresistance pattern. Few North American data demonstrate that all environmental and clinical tested strains of C. gattii and C. neoformans manifested fluconazole heteroresistance. No available data related to Latin American Cryptococcus isolates was found in literature. We aim in this study investigate the level of fluconazole heteroresistance in the Cryptococcosis agents. The heteroresistance level of 50 C. gattii clinical strains has been compared with those of the 50 C. neoformans clinical strains. The strains were maintained at the fungal culture collection of the Institute Adolfo Lutz. All strains were previously molecularly typed using a set of standard strains (provided by Dr.Wieland Meyer). All clinical and reference strains were stored in glycerol (15%) at -20°C until use and were maintained on YPD (2% glucose, 1% yeast extract, 2% peptone) agar at 25°C during this study. The minimal inhibition concentration (MIC) of fluconazole will be performed using Etest (bioMérieux, Fr) strips for all 100 strains. Then an initial screening for heteroresistance will be performed. Cell suspensions of all strains will be suspended in sterile saline and plated onto YPD plates containing various concentrations of fluconazole (4 to 128 mg/L). The growth pattern will be read after 72h of incubation at 30°C. The isolates will be regarded as possibly heteroresistant when only a fraction of the population grows on plates containing a concentration higher than the concentration which allowed 100% growth of viable cells. In this manner, the level of fluconazole heteroresistance for each isolate will be determined. To obtain the highly resistant subclones, the heteroresistant will be isolated and inoculate on YPD agar containing stepwise (twofold) increases in the concentrations of fluconazole (up to 256 mg/L). The culture plates of each passage will be incubated at 30°C for 72 to 96 h. With these experiments we will analyze if the resistant subpopulation of each resistant isolate can adapt in a stepwise manner to higher concentrations of fluconazole. Finally we will verify if the fluconazole heteroresistance is transient assessing as the strains became susceptible after serial platting in the absence of the drug. Highly resistant (>64 mg/l) subclones from strains manifesting heteroresistance at >16 mg/L were used in this part of the study. So, we can determine the extension the resistance in the subpopulation and confirm if it is a reversible adaptive response to the presence of the drug. The study on heteroresistance may reveal a novel adaptive mechanism for survival under the azole stress and can offer helpful insights into the management of long-term fluconazol therapy cases.
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