Condições experimentais para a determinação da susceptibilidade de Paracoccidioides brasiliensis e seleção de subpopulações menos sensíveis a antifúngicos

Abstract

Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis (PCM), is a dimorphic fungus, growing mycelial (M) at room temperature and yeast-like (L) at 37°C. Several drugs are available for the treatment of PCM, however, the standard methods proposed by the Clinical Laboratory and Standards Institute (CLSI) for determination of the "in vitro" antifungal susceptibility of filamentous fungi and yeasts did not include P. brasiliensis. In this study, on the basis of parameters indicated in the document M27-A2 (CLSI), experimental conditions such as size of the inoculum, reading periods, culture media, and incubation temperatures, were assayed employing twenty-one clinical and environmental isolates by the microdilution method to amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, combination sulfamethoxazole/trimetropim and terbinafine. We evaluated the fungistatic or fungicidal activities of the drugs tested. The best results were obtained after 15 days of incubation. For a given isolate under the same temperature and time of reading, it was observed that the minimum inhibitory concentrations (MICs) were significantly different in all three media tested (P<0.05) (except for fluconazole, in MHM and MVM media, at 37°C). In RPMI, 81% of the isolates showed the yeast to mycelium transition at 37C, regardless of the presence of the antifungal agents. The temperature of incubation did significantly influence the MIC values (P<0.05). The MVM culture medium proved to be the most effective for the observation of antifungal activity and determination of the MIC of sulfamethoxazole and the sulfamethoxazole/trimetropim combination (MICs of 1, 17-18,75 g/mL to both at 37C). The values recorded in RPMI and MHM media were 150-300 g/mL e 75-300 g/mL to sulfamethoxazole e 9,37-150 g/mL e 18,75-300 g/mL to combination sulfamethoxazole /trimetropim, respectively. Amphotericin B showed fungicidal against all isolates. For the other antifungal agents, the same isolate showed either fungicidal or fungistatic profiles in cultures coming either from RPMI or MHM media. Isolates that have shown the higher MIC values were chosen for the selection of fungal subpopulations tolerant to increasing concentrations of antifungal agents, at 37C. Drugs have been incorporated into YPD agar. Two clones selected from the parental Pb4 isolate and two from Pb09 grew at 700 g/mL of sulfamethoxazole, keeping the stability of the phenotype; two clones from the parental isolate Tatu and one of Pb03 grew at 600 μg/mL of the drug, one clone from the parental Pb2 and two from Pb18 grew at 500 μg/mL of sulfamethoxazole. For fluconazole, so far, four clones of the parental ED01 grew at 6 μg/mL. One clone from the parental isolate Tatu grew at 7 μg/mL and a second at 9 μg/mL. These values correspond, respectively, to three, seven and nine times the original MIC values of the parentals. At room temperature, some colonies selected from the parental isolate B339 grew in the presence of 300μg/mL of sulfamethoxazole. After adapted at increasing concentration of the antifungal agent, three clones (BCL1, BCL2 and BCL4) developed in 400 μg/mL. All these clones exhibited atypical morphology, showing cerebriform colonies and at the microscope, yeast cells. This phenotype remained stable at room temperature, even in the absence of antifungal. These of P. brasiliensis sub-populations either those tolerant in vitro to higher concentrations of sulfamethoxazole and fluconazole as well as that displaying a blockage in dimorphic transition would be of interest in studying these biological processes.