Azole resistant Aspergillus fumigatus in Denmark: a laboratory based study on resistance mechanisms and genotypes

Maiken Cavling Arendrup


Background: Azole resistant Aspergillus fumigatus was first found in Danish clinical samples in 2007.
In order to investigate the contemporary epidemiology of azole resistant A. fumigatus in Denmark a
laboratory based retrospective study was performed including all samples received at the national
mycology reference laboratory in 2010-2014. The underlying resistance mechanisms were described
and the accumulated microsatellite genotypes of Danish isolates compared with those of foreign
Material/methods: A total of 1162 A. fumigatus isolates were identified by morphology, thermotolerance
(48°C) and beta-tubulin sequencing. The majority, comprising 1098 isolates (94.5%), were
screened for azole resistance using azole agars (itraconazole 4 mg/L, voriconazole 1 mg/L and
posaconazole 0.5 mg/L) and susceptibility tested when relevant by the EUCAST E.Def 9.2 reference
method. In addition, an environmental survey was carried out during autumn of 2014 and 133 A.
fumigatus isolates were isolated from air samples and characterised as described above. Resistant
isolates were CYP51A sequenced and genotyped using the Short Tandem Repeat Aspergillus
fumigatus (STRAf) microsatellite assay. STRAf genotypes were compared to those of a representative
collection of clinical and environmental wild-type isolates as well as 1822 genotypes from A. fumigatus
isolates obtained from 15 countries around the world.
Results: Through 2010-2014 an increasing prevalence of azole resistance was observed in Denmark,
1.4%-6% isolates (P<0.001) and 1.8%-3.8% patients (P<0.05). Azole resistance mechanisms were
dominated by the CYP51A variants TR34/L98H and TR46/Y121F/T289A (57%), while the remaining
isolates harboured five different CYP51A mutations (21%) or were CYP51A wild-type (21%). All 133
environmental A. fumigatus isolates were azole susceptible. STRAf analysis revealed 120 unique
genotypes among 184 genotyped Danish A. fumigatus isolates (Fig). One potential hospital outbreak
involving six patients was examined using STRAf genotyping of six clinical and six air-filter isolates but
no shared genotypes were identified. Overall, seven (5.8%) Danish genotypes were shared between
isolates with different origin (Figure, A), 19 (15.8%) were shared with foreign genotypes and two out of
17 (11.8%) genotypes of isolates carrying the TR34/L98H resistance mechanisms were identical to two
Dutch TR34/L98H isolates (Figure, B).
Conclusions: Azole resistance among clinical A. fumigatus isolates in Denmark is increasing and
dominated by resistance mechanisms derived from the environment. This may complicate the
management of patients with invasive A. fumigatus infections and emphasises the demand for
susceptibility testing. Genotyping revealed a high degree of shared genotypes among isolates with
different origins (clinical, environmental and/or geographical), which could support previous
hypotheses on clonal expansion. The hypothesis of one single ancestor of the TR34/L98H clones may
still be relevant to pursue and would require further and more thorough genetic analyses such as
whole genome sequencing.



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26th European Congress of Clinical Microbiology and Infectious Diseases