Significant changes in the management of fungaemia have occurred over the last decade with increased use of fluconazole prophylaxis, of empirical treatment and of echinocandins as first-line agents for documented disease. These changes may impact the epidemiology of fungaemia. We present nationwide data for Denmark from 2010 to 2011. A total of 1081 isolates from 1047 episodes were recorded in 995 patients. The numbers of patients, episodes and recovered isolates increased by 13.1%, 14.5% and 14.1%, respectively, from 2010 to 2011. The incidence rate was significantly higher in 2011 (10.05/100 000) than in 2010 (8.82/100 000), but remained constant in the age groups 0-79 years. The incidence rate was highest at the extremes of age and in males. Candida albicans accounted for 52.1% but declined during 2004-11 (p 0.0155). Candida glabrata accounted for 28% and increased during 2004-2011 (p <0.0001). Candida krusei, Candida tropicalis and Candida parapsilosis remained rare (3.3-4.2%). The species distribution changed with increasing age (fewer C. parapsilosis and more C. glabrata) and by study centre. Overall, the susceptibility rates were: amphotericin B 97.3%, anidulafungin 93.8%, fluconazole 66.7%, itraconazole 69.6%, posaconazole 64.2% and voriconazole 85.0%. Acquired echinocandin resistance was molecularly confirmed in three isolates. The use of systemic antifungals doubled over the last decade (2002-2011) (from 717 000 to 1 450 000 defined daily doses/year) of which the vast majority (96.9%) were azoles. The incidence of fungaemia continues to increase in Denmark and is associated with a decreasing proportion being susceptible to fluconazole. Changes in demography, higher incidence in the elderly and higher antifungal consumption can at least in part explain the changes.

e Azole-resistant Aspergillus fumigatus harboring the TR 34 /L98H or TR 46 /Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR 46 /Y121/T289A and three cases of TR 34 /L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11 A. fumigatus and 4 Rhizomucor pusillus isolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113 A. fumigatus isolates were examined. Aspergillus isolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevant A. fumigatus isolates, CYP51A sequencing and microsatellite genotyping were performed. Three patients harbored TR 34 /L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-type A. fumigatus or R. pusillus isolates, complicating and delaying diagnosis. The TR 46 /Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistant Aspergillus isolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR 34 /L98H and TR 46 /Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistant A. fumigatus in the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmental A. fumigatus.

Candida palmioleophila has previously been misidentified as C. famata or C. guilliermondii. We have investigated traditional and modern identification methods for the identification of this and related species. Forty-one clinical isolates previously identified as C. famata or C. guilliermondii and 8 reference strains were included. Color development on CHROMagar, growth temperature ranges, micromorphologies, carbon assimilation (ID32C), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles, and susceptibility profiles (mica-and anidulafungin and itra-, vori-, posa-, and fluconazole MICs were determined by EUCAST method EDef 7.1, and caspofungin MICs were determined by Etest) were determined, and results were compared to those of molecular identification (ITS1 and ITS2 sequencing). The following five different species were identified among the clinical isolates by sequencing, but no C. famata isolates were found: C. guilliermondii (22 isolates), C. palmioleophila (8 isolates), C. fermentati (6 isolates), C. lusitaniae (3 isolates), and C. intermedia (2 isolates). C. palmioleophila developed a distinct scintillating color of turquoise to rose, grew at 40°C, and failed to produce pseudohyphae within 14 days. The ID32C profile for 7/9 C. palmioleophila isolates was 5367352315, and all were unable to hydrolyze esculin (Esc). The six related species were well discriminated by MALDI-TOF MS. The susceptibility pattern for C. palmioleophila was unique, as the echinocandin MICs were low (range, 0.008 to 0.125 g/ml) and fluconazole MICs were high (range, 8 to >16 g/ml). Correct identification of C. palmioleophila is important due to its unique susceptibility profile. Identification is possible yet laborious with conventional techniques, whereas MALDI-TOF MS easily separated the related species.

A three-month laboratory-based prospective survey was conducted at four major university hospitals covering one-third of the Danish population in order to determine the prevalence, significance, and susceptibility pattern of aspergilli in airway samples. Samples received in January-March 2007 for routine microbiologic investigation were examined for Aspergillus following routine procedures and with extended incubation (5 days). Identification was done by morphologic criteria and susceptibility testing using EUCAST method for azoles and amphotericin B E-test. Invasive aspergillosis (IA) was evaluated using modified EORTC/MSG criteria. A total of 11,368 airway samples were received. Growth of Aspergillus spp. was found in 129 and 151 patients using routine and extended incubation, respectively. Three patients had proven IA (2%), 11 probable (7%), four had allergic bronchopulmonary aspergillosis (ABPA) (3%), but the majority was colonised (88%). Underlying conditions were cystic fibrosis in 82 patients (55%), chronic obstructive pulmonary disease in 19 (13%) and haematological disorder in 11 (7%). Twenty-six patients (18%) were at intensive care unit and 69 (47%) received steroid treatment. Azole MICs were elevated for five isolates as follows (itraconazole, posaconazole, voriconazole MICs [mg/L]): two A. fumigatus isolates (>4; >4; 2 and >4; 0.125; 1), one A. lentulus isolate (2; 2; 0.5) and two A. terreus isolates (2; 2; 2 and 2; 0.125; 1). For four isolates the amphotericin B MIC was >1 μg/ml (3/112 A. fumigatus, 1/2 A. terreus). In conclusion, Aspergillus appears to be an important pathogen in Denmark. Elevated itraconazole MICs were detected in 4% of the isolates including a multi-azole resistant isolate.

Current molecular methods are on the verge of overcoming most of the early challenges regarding dermatophyte taxonomy, DNA extraction procedures and species specificity, and thus may lead to an increased adoption of such methods. This may point towards a novel consensus in which molecular methods supplement or even replace classical diagnosis of dermatophytosis.