Multicentre Surveillance of Candida Species from Blood Cultures during the SARS-CoV-2 Pandemic in Southern Europe (CANCoVEU Project)

Abstract Introduction: Surveillance of Candida species isolates from blood cultures (BCs) in Europe is considered fragmented, unable to allow the definition of targets of antifungal stewardship recommendations especially during the SARS-CoV-2 pandemic. Methods: We performed a multicentric retrospective study including all consecutive BC Candida isolates from six Southern European tertiary hospitals (1st January 2020 to 31st December 2021). Etiology, antifungal susceptibility patterns, and clinical setting were analyzed and compared. Results: C. albicans was the dominant species (45.1%), while C. auris was undetected. Candida species positive BC events increased significantly in COVID-19 ICUs in 2021 but decreased in other ICUs. Resistance to azole increased significantly and remained very high in C. albicans (fluconazole from 0.7% to 4.5%, p = 0.03) and C. parapsilosis complex (fluconazole up to 24.5% and voriconazole up to 8.9%), respectively. Resistance to caspofungin was remarkable in C. tropicalis (10%) and C. krusei (20%), while resistance to at least one echinocandin increased in 2021, especially in C. parapsilosis complex (from 0.8% to 5.1%, p = 0.05). Although no significant differences were observed over the study period, fluconazole and echinocandin resistance increased in COVID-19 ICUs by up to 14% and 5.8%, respectively, but remained undetected in non-intensive COVID-19 wards. Conclusions: Antifungal stewardship activities aimed at monitoring resistance to echinocandin in C. tropicalis and C. krusei, and against the spread of fluconazole resistant C. parapsilosis complex isolates are highly desirable. In COVID-19 patients, antifungal resistance was mostly present when the illness had a critical course.


Introduction
Candidemia is one of the most frequent health care-associated bloodstream infections (BSIs) and represents a global clinical challenge, especially given the burden of associated morbidity and mortality [1,2]. Several authors have reported an increase in the incidence of Candida species BSIs during the SARS-CoV-2 pandemic, highlighting the need for active surveillance especially in patients with severe COVID-19 [3][4][5][6][7]. Antibiotic therapy, corticosteroids, immunosuppressive therapy, intravascular devices, long hospital stays, and direct disruption of the intestinal barrier caused by SARS-CoV-2 have been deemed to pave the way to Candida species BSIs [4,5]. Despite the implementation of infection control measures during the pandemic, several authors reported cases of infections due to the emerging multidrug-resistant C. auris [8][9][10], highlighting flaws in antimicrobial stewardship programs probably due to hospital reorganization, use of broad-spectrum antimicrobials and horizontal spread of resistant strains [11].
Despite their suboptimal sensitivity, blood cultures (BCs) remain the diagnostic reference standard for Candida species BSIs, as they allow for both the identification of Candida at the species level and susceptibility testing. Candida species identification is of paramount importance to promptly optimize antifungal therapy, safeguard the use of more expensive antifungals, de-escalate treatment whenever possible and steer antifungal stewardship efforts. In fact, in addition to C. krusei being intrinsically resistant, resistance to fluconazole was also reported to be remarkable only for C. glabrata [12], C. guilliermondii [13], C. auris [14], and, more recently, in C. parapsilosis complex [6,7,15].
Data on the impact of SARS-CoV-2 pandemic on the epidemiology of Candida species BSIs and resistance to antifungal agents in multicenter surveillance studies are limited. Likewise, surveillance of antifungal resistance in Candida species BC isolates in Europe is considered fragmented, unable to define the burden and proper targets of antifungal stewardship recommendations [16]. This study was aimed at monitoring and comparing the epidemiology and antifungal susceptibility of Candida species isolated from BCs collected from patients admitted in six Southern European tertiary hospitals during the first two years of the COVID-19 pandemic.

Study Design
We performed a multicentric retrospective observational study including all consecutive BC Candida isolates from six European tertiary hospitals located in five countries (Northern Italy, Southern Italy, Portugal, Spain, Greece, and Cyprus; 6225 hospital beds overall) collected from 1st January 2020 to 31st December 2021. BCs yielding the same Candida species and subsequent samples obtained within 20 days of each other were regarded as representing a continuing positive BC event and were therefore excluded from the analysis.

Aims of the Study
The primary aim was to depict the epidemiology of the Candida species isolated from BCs of a European cohort of patients hospitalized during the first two years of the SARS-CoV-2 pandemic. The secondary aim was to evaluate the antifungal susceptibility patterns of the Candida species included.

Candida Species Identification and Susceptibility
For each Candida species positive BC event, the following data were recorded: clinical setting in which the pathogen was isolated (emergency, medical, surgical, or COVID-19 wards, ICU, COVID-19 ICU) and susceptibility testing results. Species identification method, antifungal susceptibility testing, and clinical breakpoints used by the respective institution during the study period were considered (Table S1).
Isolation of the Candida species was mostly performed using RPMI 1640 and/or BBLTM CHROMagarTM Candida medium (Becton Dickinson GmbH, Heildelberg, Germany). Resistance to azole was defined as resistance to at least one antifungal agent among fluconazole and voriconazole. Resistance to echinocandin was defined as resistance to at least one antifungal agent among anidulafungin, micafungin, and caspofungin.

Statistics
Descriptive data were shown as absolute (n) and relative (%) frequencies for categorical data. The chi-square test or Fisher exact test were used to compare the distribution of the categorical variables. Summary statistics used for MIC values included the MIC50 and MIC90. For all tests, a p value ≤ 0.05 was considered significant. All analyses will be performed with SPSS v. 25.0 (IBM Corp., Armonk, NY, USA).

Results
In the two-year period, 250,591 BCs were processed. Among these, 1451 were positive for Candida species and 745 deemed to be Candida species positive BC events and suitable to be analyzed for the aims of the study ( Table 1). The most frequently identified Candida species were C. albicans (45.1%), C. parapsilosis complex (31.8%), C. glabrata (14.1%), and C. tropicalis (5.4%). The comparison of the Candida species distributions over the study period showed a statistically significant increase in C. albicans (p = 0.02) and C. krusei (p = 0.03) and a decrease in C. parapsilosis complex (p < 0.01) in 2021.
The analysis of the yearly distribution of Candida species according to hospital ward ( Table 2) showed that Candida species BC events occurred more frequently in medical wards (34.9%), followed by ICUs (23.6%), and surgical wards (21.6%). Moreover, Candida species BC events decreased in non-COVID-19 ICUs (p = 0.02) and increased significantly in COVID-19 ICUs (p = 0.02) in 2021. The comparison of the distribution of Candida species over the two years according to hospital ward (Table S2) showed that in 2021 (1) C. albicans increased in both surgical wards (p = 0.03) and ICUs (p = 0.05), while C. parapsilosis complex decreased in the same departments (p = 0.02 and p < 0.01, respectively); (2) C. glabrata increased in medical wards.

Discussion
This study investigated the epidemiology of Candida species isolated from BCs in a European cohort of patients hospitalized during the first two years of the SARS-CoV-2 pandemic, a period of incessant hospitals reorganization, high patient care load and huge antimicrobial prescription. Our results showed that Candida species BC events most frequently occurred in medical wards, C. albicans was the dominant species, C. auris was undetected, and while C. parapsilosis complex decreased in 2021, C. krusei increased significantly. Overall, Candida species positive BC events increased significantly in COVID-19 ICUs and decreased in the other ICUs in 2021. Overall antifungal resistance was low among the Candida species isolates tested, except for C. parapsilosis complex (fluconazole and EUCAST voriconazole), C. glabrata (fluconazole), C. tropicalis (anidulafungin and EUCAST caspofungin), C. krusei (EUCAST caspofungin and EUCAST anidulafungin), and C. guilliermondii (CLSI fluconazole, CLSI caspofungin, and CLSI micafungin) where resistance ≥10% (range 10-33.3%) to one or more antifungal agents was observed. Resistance rate to azole over the study period increased significantly in C. albicans (fluconazole) and remained very high in C. parapsilosis complex. Resistance to echinocandin increased in 2021, especially in C. parapsilosis complex, while it was undetected in both C. albicans and C. glabrata during the same period. Although no significant differences were shown over the study period, azole and echinocandin resistance increased in COVID-19 ICUs while these remained undetected in non-intensive COVID-19 wards.
Although the distribution of Candida species varies according to geographic areas and may be related to age, level of care intensity, and prior use of antifungals, C. albicans is the most frequently isolated from the BCs and, together with C. parapsilosis complex and C. glabrata, have been reported as the etiological agents of more than 90% of Candida species BSIs [16][17][18]. Our findings are consistent with this evidence and, as well as highlight the burden of Candida species positive BC events in medical wards [19], confirm that C. auris is currently identified from BCs only sporadically or during nosocomial outbreaks [8][9][10][11]. However, it must be emphasized that for ecological reasons related to the colonization of patients and hospital environments, a longer study time may be necessary to document possible future positive BC events caused by C. auris. Therefore, our data suggest that, at present, the SARS-CoV-2 pandemic does not appear to have resulted in dramatic epidemiological changes in Candida species, although a marginal increase in C. krusei is worth monitoring. Of note, the 2021 increase in Candida species positive BC events in COVID-19 ICUs, with a matching decrease in other ICUs. This finding could be linked, in addition to the increased incidence of Candida species BSI in patients with severe COVID-19, to hospital reorganization, which was undertaken to increase availability of COVID-19 ICU beds, frequently by converting pre-existing ICU beds or using other non-state-of-the-art-ICU facilities.
Although the agreement between the commercial and reference methods for Candida species susceptibility has been described as variable, since it may depend on the antifungals, the species, and the incubation time [22][23][24][25], our results provided relevant indications. In fact, the remarkable resistance rates to: 1) fluconazole in C. parapsilosis complex (up to 31.2%), C. glabrata (up to 13.5%), and C. guilliermondii (up to 33.3%); 2) EUCAST voriconazole in C. parapsilosis complex (up to 11%); 3) EUCAST and/or CLSI echinocandin in C. tropicalis (up to 12.5%), C. krusei (up to 25%), and C. guilliermondii (up to 33.3%) were consistent with those of both the ARIA project and recent reports analyzing clinical samples from Greece and the Madrid region that highlighted the emergence of fluconazole resistance in C. parapsilosis complex (up to 23%) and a low rate of resistance to echinocandins (up to 1-3%) [7,15]. Our results went into further detail by also highlighting an increase in resistance to fluconazole in C. albicans (up to 4.5%), and fluconazole (up to 24.5%) and echinocandin (up to 5.1%) in C. parapsilosis complex, respectively, despite the reduced frequency of C. parapsilosis complex in 2021. It is also important to point out that a difference in resistance rates to certain antifungals has been found in some Candida species depending on the type of breakpoints used. In fact, in our study, this was more evident especially for fluconazole (EUCAST 31.2% vs. CLSI 11.7%) and voriconazole (EUCAST 11% vs. CLSI 0%) resistance in C. parapsilosis complex, and caspofungin (EUCAST 20% vs. CLSI 0%) and anidulafungin (EUCAST 25% vs. CLSI 0%) resistance in C. krusei. Even though in need of harmonization, EUCAST and CLSI methods are described to produce comparable results for testing these agents against the five most common species of Candida [26,27]. One possible explanation for this discrepant data could be that the dissemination of fluconazole-and/or voriconazole-resistant C. parapsilosis complex is still restricted to certain geographical areas and occurred predominantly and completely by chance in areas belonging to centers using the EUCAST method. The same speculation could be made for C. krusei, but the limited number of isolates tested prevents this. Of note, the fact that azole and echinocandin resistance increased in COVID-19 ICUs and remained undetected in non-intensive COVID-19 wards might emphasize that while Candida species BSIs may have many predisposing factors, antifungal resistance is mostly present when the illness has a critical course [28].
The main strength of this study is its large number of BC isolates across different countries, which allowed better assessment of the epidemiology of Candida species and antifungal resistance in Southern Europe.
The limited number of isolates limits the generalizability of results regarding C. tropicalis, C. krusei, and C. guilliermondii and should be considered a study limitation. In addition, the heterogeneity of the SARS-CoV-2 pandemic and the relative diagnostic capacities in the different centers even belonging to the same countries in the early 2020s and the continuous hospital reorganizations may have had a small influence on some of the results presented regarding the 2020 vs. 2021 epidemiological comparisons.

Conclusions
Our investigation showed an epidemiological picture in which C. albicans remained the main Candida species in positive BC events during the SARS-CoV-2 pandemic. No C. auris positive BCs were detected. Most Candida species BC events occurred in medical wards. The most remarkable finding was the resistance rate to fluconazole that increased significantly by up to 4.5% and remained >22% in C. albicans and C. parapsilosis complex, respectively. Resistance to echinocandin, which was generally low, increased in 2021, especially in C. parapsilosis complex. Hospital reorganization and conversion of ICUs into COVID-19 ICUs might have contributed to the numerical shift in Candida species positive BC events in these units. However, the increase of these events in COVID-19 ICUs was accompanied by an increase in resistance to azole and echinocandin, which was not the case in non-intensive COVID-19 wards. Further surveillance studies are warranted to confirm these findings and design antifungal stewardship activities, especially against the spread of fluconazole resistant C. parapsilosis complex isolates.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11030560/s1, Table S1: Candida species identification and susceptibility results methods according to hospital center; Table S2: Proportion per year and comparison of the distribution of Candida species isolates according to hospital ward; Table S3: Azole and echinocandin resistance per year according to hospital ward.  Informed Consent Statement: Informed consent was waived due to the retrospective nature of the study.

Data Availability Statement:
The dataset analyzed during the current study is available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.