Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus isolates from Denmark

Ferry Hagen, Rasmus Hare Jensen, Jacques F. Meis, Maiken Cavling Arendrup


Background: Cryptococcosis is the encompassing description of disease symptoms caused by species within the basidiomycetous yeast genus Cryptococcus. The majority of cryptococcal infections are caused by members of the recently taxonomically revised C. gattii/C. neoformans species complexes. Here we report the molecular characterization and in vitro antifungal susceptibility testing of 108 clinical cryptococcal isolates from Denmark, collected during the period 1973-2013.

Material/methods: The 108 clinical Danish Cryptococcus spp. isolates were subjected to qPCR to determine the species and serotype, and amplified fragment length polymorphism (AFLP) fingerprinting was performed to determine the species and genotype. In vitro antifungal susceptibility testing was performed for the compounds amphotericin B, 5-flucytosine, fluconazole, voriconazole and isavuconazole according to the EUCAST E.Def 7.2 reference method using incubation at 30 °C for 2 days.

Results: The qPCR-based species identification was confirmed by AFLP fingerprinting, the majority of the isolates were C. neoformans (formerly known as var. grubii; serotype A; n=66) and could be split into genotype AFLP1 (n=61) and AFLP1B (n=5). Twenty isolates were found to be C. deneoformans (formerly var. neoformans; serotype D; genotype AFLP2), and 13 were genotyped as being C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3). Seven isolates were typed as C. gattii sensu lato by qPCR, but AFLP fingerprinting revealed that one was a C. deneoformans × C. gattii interspecies hybrid (genotype AFLP8). Two isolates were molecularly identified as C. curvatus. All isolates were susceptible to amphotericin B (MICs ≤ 1 mg/L) with only discrete differences among the species. Cryptococcus neoformans was slightly less susceptible than C. deneoformans (MIC50 0.125 vs. 0.06 mg/L, and GM 0.149 vs. 0.096 mg/L, respectively). Similarly, flucytosine susceptibility was uniform with an MIC50 of 4-8 mg/L for all species with the exception of C. curvatus that was less susceptible (MICs >32 mg/L) (Figure). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles than the other species. MICs for Fluconazole ( >32 mg/L), voriconazole (≥ 0.5 mg/L) and isavuconazole (0.06 and 0.25 mg/L, respectively) were elevated for 1/19 C. deneoformans isolate and 1/2 C. curvatus isolate (Fig). Similarly, flucytosine MIC was elevated for 1/61 C. neoformans AFLP1 isolate (>32 mg/L).

Conclusions: The epidemiology of cryptococcal isolates from Denmark reflects that of other Western European countries, with the majority of clinical isolates identified as C. neoformans followed by C. deneoformans. Antifungal susceptibility testing according to the EUCAST E.Def 7.2 reference method revealed species specific differential susceptibility but suggested that acquired resistance was an infrequent phenomenon.


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26th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 26th (2016)