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

Abstract

To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia. 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. 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>2.5years. For C. deneoformans, the in vitro antifungal MIC90 and susceptibility ranges were for amphotericin B 0.25µg/ml (0.031-0.25µg/ml), 5-fluorocytosine 0.25µg/ml (0.063-4µg/ml), fluconazole 8µg/ml (0.5-16µg/ml), voriconazole 0.063µg/ml (0.008-0.125µg/ml), posaconazole 0.063µg/ml (0.008-0.063µg/ml) and itraconazole 0.063µg/ml (0.031-0.125µg/ml). For C. neoformans, these values were for amphotericin B 0.25µg/ml (0.063-0.5µg/ml), 5-fluorocytosine 1µg/ml (0.063-1µg/ml), fluconazole 16µg/ml (0.5-64µg/ml), voriconazole 0.125µg/ml (0.008-0.25µg/ml), posaconazole 0.063µg/ml (0.008-0.063µg/ml) and itraconazole 0.063µg/ml (0.031-0.125µg/ml). 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.