Abstract

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease, which has two clinical forms: subacute (juvenile type) and chronic (adult type), the leading cause of death from systemic mycoses in Brazil. PCM is caused by the dimorphic fungus Paracoccidioides brasiliensis which grows mycelium form at 25°C and yeast form at 37°C. In general, the fungus shows sensitivity to antifungal agents used in PCM treatment although there are records on in vitro resistance to sulfonamides, and clinical resistance. The objective of the work was to select subpopulations of the fungus tolerant to increasing concentrations of fluconazole. Preliminary tests were conducted to determine the minimal inhibitory concentration (MIC) of antifungal agents against 21 clinical and environmental isolates, choosing four with the higher MIC values corresponding to distinct phylogenetic species: isolates ED01 ("Pb01-like") (MIC = 2μg/mL), Pb04 (MIC = 8 μg/mL) (PS2), Tatu and Pb11 (S1) (MIC = 1μg/mL and 8 μg/mL, respectively). It was initially employed the technique of Single Step (PU): isolates were plated on YPD agar with selected antifungal concentrations and incubated at 37°C for 30 days. The colonies that grew were subjected to successive plating in increasing concentrations of the antifungal agent (Multistep-MP) and incubated under the same conditions. We obtained initially by PU seven colonies of the sample ED01 and six colonies of the sample Tatu in 2μg/mL of the antifungal agent. No colony was obtained from isolates Pb04 and Pb11. After the second MP plating, 4 clones of the isolate ED01 grew at 3μg/mL and four clones of the isolate Tatu grew at 6μg/mL (6 x the MIC value). Clones of the isolate Tatu were also cultivated at room temperature by successive plating (MP). At the end of the 2nd plating, three of the four original clones grew in 7μg/mL of fluconazole. At present, ED01 clones were challenged to 4μg/mL of fluconazole and Tatu clones at 7μg/mL of the antifungal agent. At room temperature clones derived from the isolate Tatu were challenged at 8μg/mL.਀ Supported by CNPq, CAPES e FAPEMIG.਀㰀⼀昀漀渀琀㸀㰀⼀瀀㸀㰀戀爀㸀㰀戀㸀䬀攀礀眀漀爀搀㨀 㰀⼀戀㸀☀渀戀猀瀀㬀昀氀甀挀漀渀愀稀漀氀攀Ⰰ 倀愀爀愀挀漀挀挀椀搀椀漀椀搀漀洀礀挀漀猀椀猀Ⰰ 猀攀氀攀挀琀椀漀渀 漀昀 琀漀氀攀爀愀渀琀㰀⼀琀搀㸀㰀⼀琀爀㸀㰀⼀琀愀戀氀攀㸀㰀⼀琀爀㸀㰀⼀琀搀㸀㰀⼀琀愀戀氀攀㸀㰀⼀戀漀搀礀㸀㰀⼀栀琀洀氀㸀