Update 2016–2018 of the Nationwide Danish Fungaemia Surveillance Study: Epidemiologic Changes in a 15-Year Perspective

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.


Introduction
Candidaemia is the most common manifestation of fungaemia and of invasive candidiasis [1]. The overall 30-day mortality rate was 43% and even higher (54%) in the intensive care unit (ICU) in a nationwide study in Denmark in 2010-2011 [2]. Host factors include multimorbidity and gastrointestinal disease [2]. Main risk factors are prior abdominal/complicated surgery, antibiotic exposure, an indwelling central venous catheter, and Candida colonisation [2]. The recommended first-line treatment of candidaemia is echinocandin [3][4][5].
The Danish candidaemia surveillance has existed since 2003 [6,7] and has been nationwide since 2004 [8][9][10]. The highest annual incidence in Denmark was 10.05/100,000 inhabitants in 2011 [9]. During the surveillance period echinocandin resistance has emerged and fluconazole non-susceptibility increased [10]. Echinocandin resistance has emerged particularly in C. glabrata. The target enzyme for the echinocandins, the β-(1,3)-D-glucan synthase, is encoded by the FKS genes [11,12]. Mutations in specific "hotspot" regions of FKS1 for all Candida species as well as FKS2 for C. glabrata lead to MIC elevation and reduce the sensitivity of the enzyme up to several thousandfold [13]. The position of the mutation, the specific amino acid alteration, and the species in which it is inserted all affect the level of resistance [11]. Therefore, FKS sequencing is highly informative and essential for interpretation, particularly of MICs close to the echinocandin breakpoint.
Denmark has the highest fungaemia incidence among the Nordic countries [14][15][16][17][18]. Suggested causes have been a higher antibacterial drug use and a higher prevalence of haematological malignancy [19]. The impact of antibiotic use was supported by a Danish study on ICU patients, which found that exposure to ciprofloxacin-containing antibiotics increased the risk of invasive Candida infections [20]. In contrast, the importance of differences in prevalence of underlying haematologic malignancy was not supported, as only a minority of Danish candidaemia patients had underlying haematological disease (9%) [2].
Denmark has also experienced a larger shift in species proportion from Candida albicans to Candida glabrata than the other Nordic countries and has had the highest consumption of antifungal drugs in both the primary and hospital sector [19]. Prior antifungal use has been shown to lead to a higher proportion of candidaemia with non-C. albicans species, especially C. glabrata following azole and C. parapsilosis following echinocandin exposure [1]. Azole antifungal agents are recommended as prophylaxis for certain patient groups in Denmark depending on their underlying disease and risk factors-especially in the ICU, in haematological patients, in low-birth-weight neonates, and in lung and liver transplant recipients [3].
Due to the changing epidemiology, the active nationwide surveillance programme has continued. Knowledge of the local epidemiology is important for timely revision of guidelines for initial antifungal therapy and informative with respect to whether changes in antifungal stewardship approaches, infection control, or prophylaxis strategies are needed. We report the most recent national data on the epidemiology of fungaemia including antifungal susceptibility over a 15-year perspective.

Isolates, Episode Definition, and Blood-Culture Systems
Fungal blood isolates were referred to the National Reference Mycology Laboratory at Statens Serum Institut for species verification and susceptibility testing from the ten Danish clinical microbiological departments in the years 2016 to 2018. Thirteen isolates (0.9%) were not referred for confirmatory identification and susceptibility testing, but are included in the analysis according to the species identification performed locally. These included: C. glabrata n = 6, C. albicans n = 4, C. krusei n = 1, Candida parapsilosis n = 1, and Candida dubliniensis n = 1. Confirmatory species identification was performed based upon morphology and Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (Bruker, Bremen, Germany) [9] with the online available spectrum database MSI [21] or DNA sequencing as previously described when needed [10].
Isolates were considered unique despite originating from the same patient if either (a) belonging to a different species, (b) having a different susceptibility pattern, or (c) obtained more than 21 days apart. Non-unique isolates were excluded. An episode was defined as polyfungal when more than one species was isolated in blood cultures obtained the same day. The numbers of episodes were centre-based, in accordance with previous Danish candidaemia publications [7,9,10,22]. The incidences are defined as number of episodes relative to the number of inhabitants, discharges, or bed days.
Most departments served several hospitals. Four centres used BacT/ALERT (bioMérieux, Marcy l'Etoile, France), one centre used BACTEC (Becton Dickinson, Franklin Lakes, NJ, USA), and the remaining five either changed systems during the study period or used both systems concomitantly (Supplementary Table S1).

Susceptibility Testing and FKS Gene Sequence Analysis
Susceptibility testing was done prospectively for ≥98.8% of the isolates according to EUCAST E.Def 7.3 [23]. Stock solutions (5000 mg/L in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Brøndby, Denmark) were used of the following compounds: fluconazole, voriconazole and amphotericin B (Sigma-Aldrich), anidulafungin (Pfizer A/S, Ballerup, Denmark), and micafungin (Astellas Pharma Inc., Tokyo, Japan; and Molcan Corporation, Toronto, Canada from 15 May 2018). C. parapsilosis ATCC 22019, or C. krusei ATCC 6258, or both were included as quality controls. The final test concentration ranges varied over the years, but the following concentrations were included throughout: amphotericin B 0.016-4 mg/L, anidulafungin 0.008-1 mg/L (C. dubliniensis: 0.004-1 mg/L), micafungin 0.008-1 mg/L, fluconazole 0.125-16 mg/L, and voriconazole 0.03-4 mg/L. Susceptibility classification was performed adopting the current EUCAST clinical breakpoints v. 10.0 [24]. For drug-species combinations without breakpoints, MICs were interpreted as follows. For amphotericin B, the non-species-specific susceptibility breakpoint of 1 mg/L was used for all species, except C. lusitaniae (which is regarded as intrinsically resistant regardless of the MIC due to a high mutation rate and lower amphotericin B cidality [25]). For fluconazole, the EUCAST non-species-specific breakpoints were used (S: ≤2 and R: >4 mg/L) for all Candida spp. For echinocandins against C. dubliniensis specifically, single-centre 99% wildtype upper limits (WT-UL 99 ) were determined using the ECOFF finder program v. 2.1 [26] and adopted as susceptibility breakpoints (anidulafungin: ≤0.03 mg/L and micafungin: ≤0.06 mg/L). Finally, established EUCAST ECOFFs were used to determine the proportion of non-wildtype isolates according to the European Committee on Antimicrobial Susceptibility Testing [27].
FKS sequencing was performed as previously described for Candida isolates with an elevated echinocandin MIC [10]. In case of discordant susceptibility classification for anidulafungin and micafungin, the isolate was deemed resistant if FKS sequencing confirmed a known hotspot alteration. Acquired echinocandin resistance rates were determined for C. albicans, C. dubliniensis, C. glabrata, C. krusei, Candida tropicalis, and Candida kefyr, and compared with data from the previous years [10].

Population Data
Annual Danish population data from the first quartile was obtained from dst.dk accessed on 25 May 2021. The total numbers of discharges and bed days among somatic admissions were obtained and accessed on 19 December 2019 from www.esundhed.dk. Furthermore, the microbiologists at the clinical microbiological departments provided data separately from their own centre. Numbers of selected abdominal surgical procedures were available at www.esundhed.dk accessed on 25 May 2021, from the period 2005 to 2018.
In order to use Poisson regression analysis and compare numbers of episodes in patient groups relative to the population number per year, patients count individually per year and according to patient ID in the current study period.

Consumption of Antifungal Compounds
The antifungal consumption for Denmark was retrieved for primary health care sector and hospital from the website www.medstat.dk, obtained and accessed on 8 July 2020. Global antifungal use for Norway, Sweden, and Finland (DDD/1000 inhabitants/year) was acquired from Grossistbasert legemiddelstatistikk, Folkehelseinstituttet or in English: Norwegian Drug Wholesales Statistics, Norwegian Institute of Public Health-www.fhi.no, obtained and accessed on 21 August 2020; www.socialstyrelsen.se, obtained and accessed on 15 August 2017; and www.firmea.fi, obtained and accessed on 6 September 2020, respectively. Data on antifungal consumption in Sweden were not available for 2017 and 2018, thus the comparison is made only for Denmark, Norway, and Finland for these years.

Statistics
The χ 2 -test was used when comparing isolate proportions in groups, and Fischer's exact test was used when the expected counts were <5. The χ 2 -test for a trend was used when comparing isolate proportions in a four-period time interval or more than two age groups, using GraphPad Prism v. 8.3.0. (San Diego, CA, USA). A negative binominal dispersion was used when comparing numbers of episodes relative to the number of bed days in the four periods as well as numbers of episodes relative to the numbers of inhabitants for the period 2011 to 2018 using IBM SPSS Statistics v. 26 (Armonk, New York, NY, USA). A Poisson regression analysis was used for numbers of episodes in gender and age groups using IBM SPSS Statistics v. 26.
The study was approved by Compliance at Statens Serum Institut (journal number: 21/00993).

Incidence
The incidence was 8.  (Table 1, Table 2, and Figure 2). The incidence did not decrease significantly from the peak in 2011 to 2018 (p = 0.08). The numbers of episodes relative to the numbers of bed days increased significantly in the four periods (p < 0.001). The highest incidences were observed at the extremes of age ( Figure 3). Gender-specific incidences were different: there were 9.73/100,000 male inhabitants and 6.56/100,000 female inhabitants in 2016-2018, with a male/female incidence rate ratio (IRR) of 1.666 (95% CI: 1.655-1.668).       The incidence rate was significantly higher in the older age groups for both males (80-89 and ≥90 years) and for females (80-89 years) compared to all other age groups ( Figure 3, Supplementary Table S3).

Species and Gender
In 2016-2018 C. albicans was more common in males than females (44.4% and 38.8%, p = 0.03), and C. glabrata was more common in females than in males (35.3% and 29.9%, p = 0.03).

Species and Age
The proportion of C. glabrata isolates increased with increasing age group (p < 0.001).
Of note, C. parapsilosis was not detected among patients <1 years, and only a single C. parapsilosis has been found in this age group (2.4%) since 2012 as compared to 10 (16.9%) during 2004-2011 (p= 0.03) (Figure 4). The age-specific species distribution also varied among the centres. The referral hospital Rigshospitalet had the lowest proportion of C. glabrata and the highest proportion of patients with candidaemia in the age group below 50 years. Details of blood-culture systems and centre-specific incidences are presented in Supplementary Table S1.

Susceptibility
MICs for the 1439 isolates referred for susceptibility testing are shown in Table 3. Thirteen isolates were not referred, and two isolates were not susceptibility tested, as further detailed in the methods section.

Susceptibility
MICs for the 1439 isolates referred for susceptibility testing are shown in Table 3. Thirteen isolates were not referred, and two isolates were not susceptibility tested, as further detailed in the methods section.

Antifungal Consumption
From a Nordic perspective, the consumption of echinocandins was comparable to that in the other Nordic countries. The consumption of fluconazole and posaconazole remained notably larger in Denmark than in the other Nordic countries, and the consumption of voriconazole and itraconazole was larger in Denmark than in Sweden and Norway ( Figure 5 and Supplementary Figure S1). mained notably larger in Denmark than in the other Nordic countries, and the consumption of voriconazole and itraconazole was larger in Denmark than in Sweden and Norway ( Figure 5 and Supplementary Figure S1).

Discussion
We previously reported an increase in incidence up until 2011 and a slight decrease in 2012-2015 [10] but this trend did not seem to continue during 2016-2018. In contrast, the incidence appears to have stabilised. Consequently, Denmark remained a high incidence country from both Nordic and global perspectives, with an incidence similar to the one found in the CDC's Emerging Infections Program US [14][15][16][17][18]28]. The species distribution, however, continued to shift towards a higher proportion of C. glabrata and a lower proportion of C. albicans (even below 40% in 2017). A C. albicans proportion below 40% has been reported in the US and South America [28][29][30][31][32], but no other Nordic country has reported a C. albicans proportion below 50% [14,16,17,33]. This change in species distribution was the main cause of the observed decrease in overall fluconazole susceptibility, yet acquired fluconazole and voriconazole resistances were found in 4.6% and 3.7%, respectively, of the normally susceptible common Candida spp. isolates. The echinocandin resistance rate increased in a four-period perspective (where also the echinocandin use increased), but remained stable during 2012-2018 and less common than acquired resistance to fluconazole [10]. C. glabrata was confirmed as the species with the highest rates of acquired echinocandin, fluconazole, and voriconazole resistance as also found elsewhere

Discussion
We previously reported an increase in incidence up until 2011 and a slight decrease in 2012-2015 [10] but this trend did not seem to continue during 2016-2018. In contrast, the incidence appears to have stabilised. Consequently, Denmark remained a high incidence country from both Nordic and global perspectives, with an incidence similar to the one found in the CDC's Emerging Infections Program US [14][15][16][17][18]28]. The species distribution, however, continued to shift towards a higher proportion of C. glabrata and a lower proportion of C. albicans (even below 40% in 2017). A C. albicans proportion below 40% has been reported in the US and South America [28][29][30][31][32], but no other Nordic country has reported a C. albicans proportion below 50% [14,16,17,33]. This change in species distribution was the main cause of the observed decrease in overall fluconazole susceptibility, yet acquired fluconazole and voriconazole resistances were found in 4.6% and 3.7%, respectively, of the normally susceptible common Candida spp. isolates. The echinocandin resistance rate increased in a four-period perspective (where also the echinocandin use increased), but remained stable during 2012-2018 and less common than acquired resistance to fluconazole [10]. C. glabrata was confirmed as the species with the highest rates of acquired echinocandin, fluconazole, and voriconazole resistance as also found elsewhere [14,28,34]. Of note, no cases involving C. auris were detected in Denmark during the observation period.
Candidaemia remained most frequent in males in accordance with previous findings globally [2,9,10,[14][15][16]33,[35][36][37][38][39][40][41][42]. The median age (70 years) was slightly higher but nevertheless in accordance with previous studies in Denmark, other Nordic countries (64-69 years) and elsewhere (all above 60 years) [10,14,16,31,33,35,[37][38][39]42]. The incidence was highest among males in the age group ≥90 years, and thus later in life than previously [10]. Since the surveillance programme was initiated, life expectancy has increased by 3.6 and 2.7 years for Danish males and females, respectively, which may be part of the explanation for the increase in median age (life expectancy-Statistics Denmark). Moreover, an increasing number of surgical procedures and a minor increase in number of admissions to the ICU in the elderly age groups (www.esundhed.dk/Registre accessed on 31 March 2021 and Regionernes Kliniske Kvalitetsudviklingsprogram from the Danish Intensive Database provided 8 June 2021, respectively) suggest that more intensive management strategies are currently offered to the elderly population.
The proportion of C. glabrata isolates increased over time, with age, and was still most common in females [10]. Underlying drivers may be the growing elderly population and a high azole use in Denmark, which remained higher than in the other Nordic countries, and which in the primary health care sector is three times higher in females than in males. C. parapsilosis was not detected in children of less than one year of age during the current 3-year surveillance, and number of isolates was found to be significantly less common in comparing the periods 2012-2018 with 2004-2011. This was somewhat surprising as C. parapsilosis historically has been the second most common species in this age group in Denmark and elsewhere [6,9,22,43,44]. A recent European paediatric study found geographical differences in incidences of C. parapsilosis with the highest incidence in Southern Europe [44]. It is unknown whether these differences over time and between countries are potentially related to differences in infection control practices, use of prophylaxis, or composition of the normal colonising flora.
This study has strengths and limitations. The major strength is that it is population based, nationwide, and includes 15 years of continued surveillance. A limitation is the lack of clinical data and patient-specific antifungal medication. Moreover, differences over time and among centres in blood-culture practices (blood-culture system and sample volume) and antifungal prophylaxis may impact blood-culture sensitivity overall and for the individual species differentially [8]. Another potential caveat is that patients' episodes are counted twice when transferred between centres and when blood culture is positive at both sites, in order to allow centre-specific incidence comparisons of the candidaemia burden. However, since the initiation of the nationwide surveillance, the number of clinical microbiological departments have been reduced-potentially leading to fewer transfers between centres and thus fewer cases being counted twice. Moreover, the number of transfer cases was limited (19 (1.4%) episodes), resulting in a corrected nationwide incidence of 8.02 rather than 8.13/100,000 inhabitants if transferred cases were omitted, a difference that does not affect the overall findings and conclusions of the study.
In conclusion, we found a stable incidence of fungaemia since the peak in 2011 with a continued shift in species proportion towards C. glabrata, a decreasing overall azole susceptibility rate and increase in acquired echinocandin and azole resistance, especially in C. glabrata. This leads to challenges in management of candidaemia since echinocandin treatment in some cases is inappropriate and de-escalation to fluconazole less often possible. This highlights the need for antifungal stewardship and new antifungal agents with alternative targets. Notable differences were found in comparing the epidemiology between the centres, illustrating the importance of following the local epidemiology.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7060491/s1. Table S1: Centre based data. Species distribution, blood culture systems and episode rates at the ten clinical microbiological departments in 2016 to 2018. Table S2: Number of patients related to age groups. Table S3: P-values for comparisons of episodes in relation to number of inhabitants in patient groups. Figure S1: Annual consumption of itraconazole and posaconazole. Funding: This study did not receive external finding.

Institutional Review Board Statement:
The study was approved by Compliance at Statens Serum Institut journal number: 21/00993.

Informed Consent Statement:
It is a data-based surveillance study. Informed consent was not necessary for this type of study.
Data Availability Statement: Data are only available for research upon reasonable request to Statens Serum Institut and within the framework of the Danish data protection legislation.