Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical Cryptococcus deneoformans and Cryptococcus neoformans Isolates from Ljubljana, Slovenia.

Aim: To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia.

Methodology: Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing.

Results: Majority of the isolates were Cryptococcus deneoformans (n = 29/46; 63%) followed by Cryptococcus neoformans (n = 16/46; 34.8%) and their interspecies hybrid (n = 1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for > 2.5 years. For C. deneoformans, the in vitro antifungal MIC and susceptibility ranges were for amphotericin B 0.25 µg/ml (0.031-0.25 µg/ml), 5-fluorocytosine 0.25 µg/ml (0.063-4 µg/ml), fluconazole 8 µg/ml (0.5-16 µg/ml), voriconazole 0.063 µg/ml (0.008-0.125 µg/ml), posaconazole 0.063 µg/ml (0.008-0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031-0.125 µg/ml). For C. neoformans, these values were for amphotericin B 0.25 µg/ml (0.063-0.5 µg/ml), 5-fluorocytosine 1 µg/ml (0.063-1 µg/ml), fluconazole 16 µg/ml (0.5-64 µg/ml), voriconazole 0.125 µg/ml (0.008-0.25 µg/ml), posaconazole 0.063 µg/ml (0.008-0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031-0.125 µg/ml).

Conclusions: Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.