Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE.

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism's pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

Comparative analysis of Etest and broth microdilution method (AFST-EUCAST) for trends in antifungal drug susceptibility testing of Brazilian Cryptococcus neoformans isolates.

A prospective study was performed to evaluate the correlation between the proposed standard of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) (document 7.1) and the commercial system Etest for determining the MICs of flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole for a collection of 100 clinical and environmental isolates of Cryptococcus neoformans. The agreements among Etest MICs within +/-2 log2 dilutions of AFST-EUCAST standard MICs were greater for flucytosine, fluconazole and voriconazole (76, 78 and 88 %, respectively) than for amphotericin B (5 %), the lowest agreement, and itraconazole (67 %). Overall, the correlation coefficients were statistically significant (P<0.05), and it is suggested that the Etest and AFST-EUCAST method are reliable alternatives and present good correlation for all drugs evaluated except amphotericin B. However, the observed differences related to MICs for susceptible, susceptible dose dependent and resistant strains between the methods suggest that it will be necessary to carry out further studies, including assessment of interlaboratory agreement and correlation of MICs by different methods with in vivo response.