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Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Cryptococcus spp., the causative agents of cryptococcosis, are responsible for deaths of hundreds of thousands of people every year worldwide. The drawbacks of available therapeutic options are aggravated by the increased resistance of yeast to the drugs, resulting in inefficient therapy. Also, the antifungal 5FC is not available in many countries. Therefore, a combination of antifungal drugs may be an interesting option, but in vitro and theoretical data point to the possible antagonism between the main antifungals used to treat cryptococcosis, i.e., fluconazole (FLC), and amphotericin B (AMB). Therefore, in vivo studies are necessary to test the above hypothesis. In this study, the efficacy of FLC and AMB at controlling C. gattii infection was evaluated in a murine model of cryptococcosis caused by C. gattii. The infected mice were treated with FLC + AMB combinations and showed a significant improvement in survival as well as reduced morbidity, reduced lung fungal burden, and the absence of yeast in the brain when FLC was used at higher doses, according to the Tukey test and principal component analysis. Altogether, these results indicate that combinatorial optimization of antifungal therapy can be an option for effective control of cryptococcosis.

The emergence of fluconazole-resistant Cryptococcus gattii is a global concern, since this azole is the main antifungal used worldwide to treat patients with cryptococcosis. Although pharmacokinetic (PK) and pharmacodynamic (PD) indices are useful predictive factors for therapeutic outcomes, there is a scarcity of data regarding PK/PD analysis of antifungals in cryptococcosis caused by resistant strains. In this study, PK/PD parameters were determined in a murine model of cryptococcosis caused by resistant C. gattii. We developed and validated a suitable liquid chromatography-electrospray ionization tandem mass spectrometry method for PK studies of fluconazole in the serum, lungs, and brain of uninfected mice. Mice were infected with susceptible or resistant C. gattii, and the effects of different doses of fluconazole on the pulmonary and central nervous system fungal burden were determined. The peak levels in the serum, lungs, and brain were achieved within 0.5h. The AUC/MIC index (area under the curve/minimum inhibitory concentration) was associated with the outcome of anti-cryptococcal therapy. Interestingly, the maximum concentration of fluconazole in the brain was lower than the MIC for both strains. In addition, the treatment of mice infected with the resistant strain was ineffective even when high doses of fluconazole were used or when amphotericin B was tested, confirming the cross-resistance between these drugs. Altogether, our novel data provide the correlation of PK/PD parameters with antifungal therapy during cryptococcosis caused by resistant C. gattii., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.