New data from the years 2012 to 2015 from the Danish National Fungemia Surveillance are reported, and epidemiological trends are investigated in a 12-year perspective (2004 to 2015). During 2012 to 2015, 1,900 of 1,939 (98%) fungal bloodstream isolates were included. The average incidence was 8.4/100,000 inhabitants, and this appears to represent a stabilizing trend after the increase to 10.1/100,000 in 2011. The incidence was higher in males than females (10.0 versus 6.8) and in patients above 50 years, and those changes were mainly driven by an increasing incidence among 80-to-89-year-old males (65.3/100,000 in 2014 to 2015). The proportion of isolates decreased from 2004 to 2015 (64.4% to 42.4%) in parallel with a doubling of the proportion of isolates (16.5% to 34.6%, < 0.0001). was more common among females (34.0% versus 30.4% in males). Following an increase in 2004 to 2011, the annual drug use stabilized during the last 2 to 3 years of that time period but remained higher than in other Nordic countries. This was particularly true for the fluconazole and itraconazole use in the primary health care sector, which exceeded the combined national levels of use of these compounds in each of the other Nordic countries. Fluconazole susceptibility decreased (68.5%, 65.2%, and 60.6% in 2004 to 2007, 2008 to 2011, and 2012 to 2015, respectively, < 0.0001), and echinocandin resistance emerged in (0%, 0.6%, and 1.7%, respectively, < 0.001). Amphotericin B susceptibility remained high (98.7%). Among 16 (2.7%) echinocandin-resistant isolates (2012 to 2015), 13 harbored FKS mutations and 5 (31%) were multidrug resistant. The epidemiological changes and the increased incidence of intrinsic and acquired resistance emphasize the importance of continued surveillance and of strengthened focus on antifungal stewardship.

We report a fatal aspergillosis case in which STR Af typing and whole-genome sequencing substantiated in vivo emergence of an azole-resistant Aspergillus fumigatus with a 120-bp tandem repeat in the promoter region of cyp51A . This event, previously restricted to the environment, challenges current understanding of azole resistance development in A. fumigatus .

Azole-resistant (azole-R) Aspergillus is an increasing challenge worldwide. Patients with cystic fibrosis (CF) are at risk of Aspergillus colonization and disease due to a favorable lung environment for microorganisms. We performed a nationwide study in 2018 of azole-non-susceptible Aspergillus in CF patients and compared with data from two prior studies. All airway samples with mold isolates from patients monitored at the two CF centers in Denmark (RH, Jan-Sept and AUH, Jan-Jun) were included. Classical species identification (morphology and thermo-tolerance) was performed and MALDI-TOF/β-tubulin sequencing was performed if needed. Susceptibility was determined using EUCAST E.Def 10.1, and E.Def 9.3.2. cyp51A sequencing and STRAf genotyping were performed for azole-non-susceptible isolates and relevant sequential isolates. In total, 340 mold isolates from 159 CF patients were obtained. The most frequent species were Aspergillus fumigatus (266/340, 78.2%) and Aspergillus terreus (26/340, 7.6%). Azole-R A. fumigatus was cultured from 7.3% (10/137) of patients, including 9.5% (9/95) of patients at RH and 2.4% at AUH (1/42), respectively. In a 10-year perspective, azole-non-susceptibility increased numerically among patients at RH (10.5% in 2018 vs 4.5% in 2007-2009). Cyp51A resistance mechanisms were found in nine azole-R A. fumigatus from eight CF patients. Five were of environmental origin (TR 34 /L98H), three were human medicine-driven (two M220K and one M220R), and one was novel (TR 34 3 /L98H) and found in a patient who also harbored a TR 34 /L98H isolate. STRAf genotyping identified 27 unique genotypes among 45 isolates and ≥2 genotypes in 8 of 12 patients. This included one patient carrying two unique TR 34 /L98H isolates, a rare phenomenon. Genotyping of sequential TR 34 3 /L98H and TR 34 /L98H isolates from the same patient showed only minor differences in 1/9 markers. Finally, azole-R A. terreus

As part of a national surveillance programme initiated in 2004, fungal blood isolates from 2016–2018 underwent species identification and EUCAST susceptibility testing. The epidemiology was described and compared to data from previous years. In 2016–2018, 1454 unique isolates were included. The fungaemia rate was 8.13/100,000 inhabitants compared to 8.64, 9.03, and 8.38 in 2004–2007, 2008–2011, and 2012–2015, respectively. Half of the cases (52.8%) involved patients 60–79 years old and the incidence was highest in males ≥70 years old. Candida albicans accounted for 42.1% of all isolates and Candida glabrata for 32.1%. C. albicans was more frequent in males (p = 0.03) and C. glabrata in females (p = 0.03). During the four periods, the proportion of C. albicans decreased (p < 0.001), and C. glabrata increased (p < 0.001). Consequently, fluconazole susceptibility gradually decreased from 68.5% to 59.0% (p < 0.001). Acquired fluconazole resistance was found in 4.6% Candida isolates in 2016–2018. Acquired echinocandin resistance increased during the four periods 0.0%, 0.6%, 1.7% to 1.5% (p < 0.0001). Sixteen echinocandin-resistant isolates from 2016–2018 harboured well-known FKS resistance-mutations and one echinocandin-resistant C. albicans had an FKS mutation outside the hotspot (P1354P/S) of unknown importance. In C. glabrata specifically, echinocandin resistance was detected in 12/460 (2.6%) in 2016–2018 whereas multidrug-class resistance was rare (1/460 isolates (0.2%)). Since the increase in incidence during 2004–2011, the incidence has stabilised. In contrast, the species distribution has changed gradually over the 15 years, with increased C. glabrata at the expense of C. albicans. The consequent decreased fluconazole susceptibility and the emergence of acquired echinocandin resistance complicates the management of fungaemia and calls for antifungal drug development.

Background Azole resistance complicates treatment of patients with invasive aspergillosis with an increased mortality. Azole resistance in Aspergillus fumigatus is a growing problem and associated with human and environmental azole use. Denmark has a considerable and highly efficient agricultural sector. Following reports on environmental azole resistance in A. fumigatus from Danish patients, the ministry of health requested a prospective national surveillance of azole‐resistant A. fumigatus and particularly that of environmental origin. Objectives To present the data from the first 2 years of the surveillance programme. Methods Unique isolates regarded as clinically relevant and any A. fumigatus isolated on a preferred weekday (background samples) were included. EUCAST susceptibility testing was performed and azole‐resistant isolates underwent cyp51A gene sequencing. Results The azole resistance prevalence was 6.1% (66/1083) at patient level. The TR34/L98H prevalence was 3.6% (39/1083) and included the variants TR34/L98H, TR343/L98H and TR34/L98H/S297T/F495I. Resistance caused by other Cyp51A variants accounted for 1.3% (14/1083) and included G54R, P216S, F219L, G54W, M220I, M220K, M220R, G432S, G448S and Y121F alterations. Non‐Cyp51A‐mediated resistance accounted for 1.2% (13/1083). Proportionally, TR34/L98H, other Cyp51A variants and non‐Cyp51A‐mediated resistance accounted for 59.1% (39/66), 21.2% (14/66) and 19.7% (13/66), respectively, of all resistance. Azole resistance was detected in all five regions in Denmark, and TR34/L98H specifically, in four of five regions during the surveillance period. Conclusion The azole resistance prevalence does not lead to a change in the initial treatment of aspergillosis at this point, but causes concern and leads to therapeutic challenges in the affected patients.