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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.

Objectives: To evaluate a genome-based surveillance of all Danish third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec ) from bloodstream infections between 2014 and 2015, focusing on horizontally transferable resistance mechanisms.Methods: A collection of 552 3GC-R Ec isolates were whole-genome sequenced and characterized by using the batch uploader from the Center for Genomic Epidemiology (CGE) and automatically analysed using the CGE tools according to resistance profile, MLST, serotype and fimH subtype. Additionally, the phylogenetic relationship of the isolates was analysed by SNP analysis.Results: The majority of the 552 isolates were ESBL producers (89%), with bla CTX-M-15 being the most prevalent (50%) gene, followed by bla CTX-M-14 (14%), bla CTX-M-27 (11%) and bla CTX-M-101 (5%). ST131 was detected in 50% of the E. coli isolates, with the remaining isolates belonging to 73 other STs, including globally disseminated STs (e.g. ST10, ST38, ST58, ST69 and ST410). Five of the bloodstream isolates were carbapenemase producers, carrying bla OXA-181 (3) and bla OXA-48 (2). Phylogenetic analysis revealed 15 possible national outbreaks during the 2 year period, one caused by a novel ST131/ bla CTX-M-101 clone, here observed for the first time in Denmark. Additionally, the analysis revealed three individual cases with possible persistence of closely related clones collected more than 13 months apart.Conclusions: Continuous WGS-based national surveillance of 3GC-R Ec , in combination with more detailed epidemiological information, can improve the ability to follow the population dynamics of 3GC-R Ec , thus allowing for the detection of potential outbreaks and the effects of changing treatment regimens in the future.

Objectives: The aim of this study was to identify clonally-related carbapenemase-producing Klebsiella pneumoniae complex members that could be involved in outbreaks among hospitalized patients in Denmark, and to identify possible epidemiological links. Methods: From January 2014 to June 2018, 103 isolates belonging to the K. pneumoniae complex were collected from 102 patients. From the whole-genome sequencing (WGS) data, presence of genes encoding carbapenemase and multilocal sequence typing (MLST) data were extracted. Core genome MLST (cgMLST) cluster analysis was performed. Using data from the Danish National Patient Registry (DNPR) and reported travel history, presumptive outbreaks were investigated for possible epidemiological links. Results: The most common detected carbapenemase gene was bla OXA-48, followed by bla NDM-1. The 103 K. pneumoniae complex isolates belonged to 47 sequence types (STs) and cgMLST subdivided the isolates into 80 different complex types. cgMLST identified 13 clusters with 2-4 isolates per cluster. For five of the 13 clusters, a direct link (the patients stayed at the same ward on the same day) could be detected between at least some of the patients. In two clusters, the patients resided simultaneously at the same hospital, but not the same ward. A possible link (same ward within 1-13 days) was detected for the patients in one cluster. For five clusters detected by cgMLST, no epidemiological link could be detected using data from DNPR. Conclusion: In this study, cgMLST combined with patient hospital admission data and travel information was found to be a reliable and detailed approach to detect possible clonal transmission of carbapenemase-producing K. pneumoniae complex members.

OBJECTIVES: To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm).METHODS: Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme.RESULTS: Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain.CONCLUSIONS: Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Background: Denmark has a high incidence rate of candidaemia. A Nordic study suggested a higher Danish prevalence of haematological malignancies as an underlying reason. This nationwide study ascertained clinical characteristics of Danish candidaemia patients and investigated potential factors contributing to the high incidence and mortality. Methods: Microbiological and clinical data for candidaemia patients in 2010-2011 were retrieved. 30-day mortality was estimated by hazard ratios (HR) with 95% confidence intervals (CI, Cox regression). Results: Data were available for 912/973 candidaemia episodes (93.7%). Intensive care unit (ICU) held the largest share of patients (43.2%). Prevalent host factors were multi-morbidity (≥2 underlying diseases, 74.2%) and gastrointestinal disease (52.5%). Haematological disease was infrequent (7.8%). Risk factors included antibiotic exposure (90.5%), CVC (71.9%) and Candida colonisation (66.7%). 30-day mortality was 43.4%, and 53.6% in ICU. Mortality was lower for patients with recent abdominal surgery (HR 0.70, 95% CI: 0.54-0.92). Conclusion: A substantial prevalence of multi-morbidity and a high 30-day mortality was found. We hypothesise, that an increasing population of severely ill patients with prolonged supportive treatment and microbiological testing may in part explain the high candidaemia incidence in Denmark. Nationwide studies are warranted to clarify this issue. Keywords: Candidaemia, Candida, Epidemiology, Outcome, Mortality

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 Candida albicans isolates decreased from 2004 to 2015 (64.4% to 42.4%) in parallel with a doubling of the proportion of Candida glabrata isolates (16.5% to 34.6%, P < 0.0001). C. glabrata 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, P < 0.0001), and echinocandin resistance emerged in Candida (0%, 0.6%, and 1.7%, respectively, P < 0.001). Amphotericin B susceptibility remained high (98.7%). Among 16 (2.7%) echinocandin-resistant C. glabrata 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.

Karen Rokkedal Lausch,1 Mette Søgaard,2,3 Flemming Schønning Rosenvinge,4,5 Helle Krogh Johansen,6 Trine Boysen,7 Bent Løwe Røder,8 Klaus Leth Mortensen,1,9Lene Nielsen,10 Lars Lemming,9 Bente Olesen,10 Christine Leitz,11 Lise Kristensen,9,12 Esad Dzajic,13 Lars Jørgen Østergaard,1 Henrik Carl Schønheyder,14,15 Maiken Cavling Arendrup6,16,17 1Department of Infectious Disease, Aarhus University Hospital, 8200 Aarhus, Denmark; 2Department of Cardiology, Aalborg University Hospital, 9000 Aalborg, Denmark; 3Aalborg Thrombosis Research Unit, Department of Clinical Medicine, Faculty of Health, Aalborg University, 9000 Aalborg, Denmark; 4Department of Clinical Microbiology, Odense University Hospital, 5000 Odence C, Denmark; 5Department of Clinical Microbiology, Lillebaelt Hospital, 5500 Middelfart, Denmark; 6Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark; 7Department of Clinical Microbiology, Hvidovre Hospital, 2650 Hvidovre, Denmark; 8Department of Clinical Microbiology, Hospital of Slagelse, Slagelse Sygehus, 4200 Slagelse, Denmark; 9Department of Clinical Microbiology, Aarhus University Hospital, 8200 Aarhus, Denmark; 10Department of Clinical Microbiology, Herlev and Gentofte Hospital, University of Copenhagen, 2730 Herlev, Denmark; 11Department of Clinical Microbiology, Viborg Regionshospital, 8800 Viborg, Denmark; 12Department of Clinical Microbiology, Herning Regionshospital, 7400 Herning, Denmark; 13Department of Clinical Microbiology, Sydvestjysk Sygehus, 6700 Esbjerg, Denmark; 14Department of Clinical Microbiology, Aalborg University Hospital, 9000 Aalborg, Denmark; 15Department of Clinical Medicine, University of Aalborg, 9000 Aalborg, Denmark; 16Unit of Mycology, Statens Serum Institute, 2300 København, Denmark; 17Department of Clinical Medicine, University of Copenhagen, 2200 Copenhagen, Denmark Background: In accordance with international guidelines, primary antifungal treatment (AFT) of candidemia with echinocandins has been nationally recommended in Denmark since 2009. Our nationwide cohort study describes the management of candidemia treatment focusing on the impact of prophylactic AFT on species distribution, the rate of adherence to the recommended national guidelines for AFT, and the effect of AFT on patient outcomes.Materials and methods: Incident candidemia cases from a 2-year period, 2010–2011, were included. Information on AFT was retrospectively collected from patient charts. Vital status was obtained from the Danish Civil Registration System. HRs of mortality were reported with 95% CIs using Cox regression.Results: A total of 841 candidemia patients was identified. Prior to candidemia diagnosis, 19.3% of patients received AFT (162/841). The risk of non-albicans candidemia increased after prior AFT (59.3% vs 45.5% among nontreated). Echinocandins as primary AFT were given for 44.2% (302/683) of patients. Primary treatment with echinocandins resulted in adequate treatment in a higher proportion of patients (97.7% vs 72.1%) and was associated with lower 0- to 14-day mortality compared with azole treatment (adj. HR 0.76, 95% CI: 0.55–1.06). Significantly lower 0- to 14-day mortality was observed for patients with Candida glabrata and Candida krusei with echinocandin treatment compared with azole treatment (adj. HR 0.50, 95% CI: 0.28–0.89), but not for patients with Candida albicans or Candida tropicalis.Conclusion: The association shown between prior AFT and non-albicans species underlines the importance of treatment history when selecting treatment for candidemia. Compliance with national recommendations was low, but similar to previously reported international rates. Primary treatment of candidemia with echinocandins compared with azoles yielded both a higher proportion of adequately treated patients and improved mortality rates. This real-life setting supports guidelines recommendation, and further focus on compliance with these seems warranted. Keywords: candidemia, candida, antifungal treatment, echinocandin, azole, Candida albicans, Candida glabrata

Objectives: To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015.Methods: During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates.Results: During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015.Conclusions: VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones.

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.

The prevalence of intrinsic and acquired resistance among colonizing Candida isolates from patients after candidemia was investigated systematically in a 1-year nationwide study. Patients were treated at the discretion of the treating physician. Oral swabs were obtained after treatment. Species distributions and MIC data were investigated for blood and posttreatment oral isolates from patients exposed to either azoles or echinocandins for = 7 days. Species identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing, susceptibility was examined by EUCAST EDef 7.2 methodology, echinocandin resistance was examined by FKS sequencing, and genetic relatedness was examined by multilocus sequence typing (MLST). One hundred ninety-three episodes provided 205 blood and 220 oral isolates. MLST analysis demonstrated a genetic relationship for 90% of all paired blood and oral isolates. Patients exposed to azoles for >= 7 days (n = 93) had a significantly larger proportion of species intrinsically less susceptible to azoles (particularly Candida glabrata) among oral isolates than among initial blood isolates (36.6% versus 12.9%; P < 0.001). A similar shift toward species less susceptible to echinocandins among 85 patients exposed to echinocandins for >= 7 days was not observed (4.8% of oral isolates versus 3.2% of blood isolates; P > 0.5). Acquired resistance in Candida albicans was rare (= 7 days. Species identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing, susceptibility was examined by EUCAST EDef 7.2 methodology, echinocandin resistance was examined by FKS sequencing, and genetic relatedness was examined by multilocus sequence typing (MLST). One hundred ninety-three episodes provided 205 blood and 220 oral isolates. MLST analysis demonstrated a genetic relationship for 90% of all paired blood and oral isolates. Patients exposed to azoles for >= 7 days (n = 93) had a significantly larger proportion of species intrinsically less susceptible to azoles (particularly Candida glabrata) among oral isolates than among initial blood isolates (36.6% versus 12.9%; P < 0.001). A similar shift toward species less susceptible to echinocandins among 85 patients exposed to echinocandins for >= 7 days was not observed (4.8% of oral isolates versus 3.2% of blood isolates; P > 0.5). Acquired resistance in Candida albicans was rare (

OBJECTIVES: In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm.METHODS: A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE.RESULTS: One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by CONCLUSIONS: This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.