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Comparison of Six Antifungal Susceptibilities of 11 Candida Species Using the VITEK2 AST–YS08 Card and Broth Microdilution Method

ABSTRACT We used a Vitek 2 AST–YS08 (YS08) system and the broth microdilution method (BMD) adopted by the Clinical and Laboratory Standards Institute (CLSI) to compare the susceptibility of 184 isolates of 11 Candida species to fluconazole, voriconazole, micafungin, caspofungin, amphotericin B, and flucytosine. In Candida albicans, the categorical agreement (CA) was 79.2%, 91.7%, 95.8%, and 95.8% for fluconazole, voriconazole, micafungin, and caspofungin, respectively. About 12.5% and 4.2% of very major errors were detected for fluconazole and voriconazole, respectively. C. glabrata showed excellent essential agreements (EAs) (>90%) for azoles but different MIC distributions for fluconazole and caspofungin. The CA between BMD fluconazole MICs and YS08 voriconazole MICs by the method-specific clinical breakpoint (CBP) was 90% in C. glabrata. Over 80% of C. glabrata and C. krusei isolates identified as micafungin–susceptible were labeled intermediate or resistant to caspofungin in YS08. In C. parapsilosis, 5.3% of very major errors and 10.5% of minor errors were found, whereas 33.3% of minor errors were observed in C. tropicalis for fluconazole. For C. tropicalis, 13 (61.9%) non-wild type (WT) isolates of fluconazole and 7 (33.3%) non-WTs of voriconazole were classified in YS08 as WT. For C. auris, the EAs were 93.3%, 100%, 82.2%, 97.8%, and 97.8% for fluconazole, voriconazole, micafungin, caspofungin, and amphotericin B, respectively. YS08 showed comparable results to the BMD. However, considering the lower YS08 fluconazole MIC results compared with BMD in Candida species and YS08 caspofungin results in C. glabrata and C. krusei, improvements are needed. IMPORTANCE The new Vitek 2 AST–YS08 (YS08) card has been updated to reflect the recently revised Clinical and Laboratory Standards Institute (CLSI) guideline. In this study, antifungal drug susceptibility tests were performed using the YS08 card and compared with the CLSI broth microdilution (BMD) method. In conclusion, YS08 showed similar results to BMD, including with C. auris. However, about 12.5% and 4.2% of major errors were detected for fluconazole and voriconazole, respectively, in C. albicans. More than 80% of C. glabrata and C. krusei isolates identified as susceptible to micafungin were labeled moderate or resistant to caspofungin in YS08. The categorical agreement between BMD fluconazole MICs and YS08 voriconazole MICs was 90% by the method-specific CBP of voriconazole, 80% by the current epidemiological cutoff value (ECV) (0.25 μg/mL) of voriconazole, and 85% by the previous ECV (0.5 μg/mL) of voriconazole. Further improvements in YS08 for the detection of fluconazole and echinocandin resistance are thus needed.

C andida species are normal commensals that localize on the skin and mucosal membranes of genitals and the gastrointestinal tract. However, they can cause various infections in vulnerable patients, such as the elderly, hospitalized, or immunosuppressed (1). Invasive fungal infection due to Candida species is widely recognized as a leading cause of morbidity and mortality in medical environments (2). The mortality rate from invasive candidiasis is estimated to be around 19-40% (3), and is much higher in intensive care unit patients, approaching 70% (4). The distribution of species has changed over the past decades (3). Candida albicans was previously the predominant pathogen (3,5), but in recent years, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and C. lusitaniae have emerged as important pathogens (6). According to epidemiologic surveillance data from Korea, C. tropicalis (36.4%) is the most common non-albicans Candida, followed by C. glabrata (28.5%), C. parapsilosis (24.7%), and C. krusei (2.6%) (7). Cyberlindnera fabianii (Candida fabianii) is an uncommon opportunistic yeast species, but it has the ability to cause septicemia and rapidly acquire resistance to fluconazole and voriconazole (8).
Antifungal agents target various biosynthetic pathways of pathogens. Echinocandins target biosynthesis of the cell wall. Azoles target the important enzyme 14a-demethylase in ergosterol biosynthesis. Flucytosine (5-FC) interferes with nucleic acid biosynthesis. Polyene drugs, including amphotericin B, bind with ergosterol to form pores and are fungicidal (1,9).
An important factor that may contribute to therapeutic failure is antifungal agent resistance (1). Increased MICs and other specific mechanisms of resistance are associated with treatment failure and mortality, making empirical treatment choices difficult for clinicians (10). The molecular mechanisms involved in antifungal resistance include overexpression of membrane transporters, changes in the biosynthesis of the cell wall and ergosterol, mutations in the transcription factors that regulate membrane transporters, and ergosterol biosynthesis (9).
In addition, the emergence of multidrug-resistant C. glabrata and C. auris, the increase of fluconazole-resistant C. tropicalis and C. parapsilosis, and the existence of the intrinsically resistant C. krusei are more problematic (11). C. auris has emerged as a new multidrug-resistant species that causes a wide range of infections, especially in intensive care units (12). In 2016, the Infectious Diseases Society of America recommended antifungal susceptibility testing for azoles in clinically relevant Candida isolates and all bloodstream infections (2).
Broth microdilution (BMD) is considered the most reliable reference method for the evaluation of antifungal susceptibility in Candida species (13). The Clinical and Laboratory Standards Institute (CLSI) has developed a standard method and established clinical breakpoints (CBPs) for the most common Candida species (14). The epidemiological cutoff value (ECV) identifies isolates that have a non-wild type (WT) profile, and CLSI has updated its documentation to provide ECVs for the less prevalent Candida species (15).
Vitek 2 (bioMérieux, Marcy l'Etoile, France) is a fully automated system capable of performing microbial identification and susceptibility testing. The system can be applied to bacteria and yeast and is used by many laboratories because of its rapidity and ease of use (16). The new Vitek 2 antimicrobial susceptibility testing (AST) system for yeast, AST-YS08 card, has been updated to reflect the recently revised CLSI CBP for common Candida species (17).
The aim of this study was to evaluate the clinical applicability of the new Vitek 2 AST-YS08 (YS08) card by comparing it with the results of the BMD method by CLSI.
In C. glabrata, the CA was 100% for micafungin but only 15% for caspofungin between the BMD and YS08. A major error of 10% (2/20) and minor error of 75% (15/20) for caspofungin were detected. In YS08, 85% (17/20) of isolates were interpreted as caspofungin-intermediate (15 isolates) or -resistant (two isolates), but all showed micafungin susceptibility. Excellent EAs for micafungin (100%), fluconazole (90.0%), and voriconazole (95.0%) were found for C. glabrata. Although fluconazole and caspofungin in C. glabrata are not recommended to be read with YS08, we compared the MIC distributions between the two methods ( Fig. 1). For fluconazole, the MICs by BMD were more widely distributed than those by YS08, and for caspofungin, YS08 generated at least a 2-fold higher MIC compared with BMD.
The EAs of flucytosine were 100% for all strains except C. albicans (87.5%, 21/24). Because the minimum MIC value that can be reported by the YS08 for flucytosine is 1 mg/mL, MICs less than 1 mg/mL could not be reported in YS08 (Table S1 in the supplemental material).
We compared the fluconazole MICs and voriconazole MICs in C. glabrata to predict the fluconazole susceptibility using different criteria ( Table 2). The CAs between BMD fluconazole MICs and YS08 voriconazole MICs were 90% (18/20) by the method-specific CBP of voriconazole, 80% (16/20) by the current ECV (0.25 mg/mL) of voriconazole, and 85% (17/20) by the previous ECV (0.5 mg/mL) of voriconazole. Only 10% of very major errors were detected by the method-specific CBP. About 5% of very major errors and 15% of major errors were detected by the current ECV criteria, whereas 10% of very major errors and 5% of major errors were found by the previous ECV criteria. However, the CAs between fluconazole ECV (8 mg/mL) and YS08 voriconazole ECV were 65% (13/ 20) by the current ECV criteria and 60% (12/20) by the previous ECV criteria.
In Table 3, the proportion of WT versus non-WT and CAs were analyzed according to the CLSI ECV criteria. Agreement between voriconazole and micafungin could not be determined in C. albicans because the minimum MIC reported by YS08 was higher than the ECV concentration (ECV: 0.03 for voriconazole and micafungin). Micafungin for C. glabrata could also not be interpreted for the same reason. The CA of C. glabrata for voriconazole was 80% (16/20), of which two non-WT isolates were classified as WT in YS08. In C. tropicalis, the CA for fluconazole was 38.1% and that for voriconazole was 66.7%. Thirteen (61.9%) non-WT isolates for fluconazole and seven (33.3%) for voriconazole were classified in YS08 as WT, respectively.
c Because CBPs for C. lusitaniae, C. orthopsilosis, C. pelliculosa and C. fabianii were not available, the value from C. albicans was used to identify isolates/species with elevated MICs.

DISCUSSION
In this study, we report six antifungal susceptibility profiles for 184 clinical isolates recovered in Korea. The CBPs were used to define susceptible or resistant isolates. ECVs   Fluconazole BMD ECV: 1 6 (100) 6 (100) YS08 6 (100 Voriconazole BMD NA YS08 Micafungin BMD ECV: 0.5 6 (100 6 (100) YS08 6 (100 Caspofungin BMD ECV: 1 6 (100 6 (100) YS08 6 (100 Amphotericin B BMD ECV: 2 6 (100 6 (100) YS08 6 (100 Flucytosine BMD ECV: 1 6 (100 6 (100) YS08 6 (100 (Continued on next page) are useful in distinguishing between WT without resistance mechanisms and non-WT with resistance mechanisms (15). Both criteria can be used to obtain the CA between the two methods (17). Currently, two commercial BMD-based systems, the Vitek 2 system (bioMérieux, France) and the Sensititre YeastOne system (Thermo Scientific, Cleveland, OH, USA), are widely used in clinical microbiological laboratories for antifungal susceptibility testing (17). The previous version of the YS08, YS07, had an FDA-accredited fluconazole formulation that has been validated against C. glabrata. However, the fluconazole formulation was modified and no validation for C. glabrata has been made; therefore, the MIC for the fluconazole of C. glabrata is not recorded in YS08 (18). The azoles are the most commonly used agents for invasive fungal infections, in particular, fluconazole for the treatment of candidemia (2). Regardless of the Candida species tested, the overall EA and CA of fluconazole between BMD and YS08 ranged from 40%-100% and from 66.7%-100%, respectively. Notably, YS08 classified five fluconazole-resistant isolates as susceptible, representing a very major error. C. albicans, C. parapsilosis, and C. tropicalis showed less than 90% EA and CA for fluconazole MIC. C. albicans showed 79.2% CA and 83.3% EA, and 12.5% of very major errors in fluconazole were detected by YS08. C. parapsilosis showed 84.2% CA and 89.5% EA with 5.3% of very major errors for fluconazole. In C. tropicalis, 66.7% CA, 66.7% EA, and 33.3% of minor errors were detected, and the median MIC (range) for fluconazole was 2 (0.5-4) using the CLSI method and 0.5 (0.5-1) by YS08. These findings led to the conclusion that the fluconazole MIC of YS08 was lower than that of the BMD method. Until recently, only two studies had assessed the clinical performance of YS08 cards compared with Sensititre YeastOne and/or BMD using clinical isolates. Comparing BMD and YS08, Lim et al. found excellent (.90%) EA and CA for fluconazole in C. albicans and C. parapsilosis, but 3.8% of very major errors and 13% of minor errors were observed in C. albicans and C tropicalis (17). However, in another study comparing YS08 and Sensititre YeastOne, Wong et al. revealed poor agreement for fluconazole in C. albicans, with 70% EA and 85% CA (19). The better and more consistent EA and CA results compared with our study might be due to the Candida species tested.
In our study, the EA of C. glabrata against fluconazole and voriconazole was excellent (.90%), but a different MIC distribution for fluconazole was observed between BMD and YS08. The CLSI guideline does not provide the voriconazole MIC value because of insufficient data on the relationship between C. glabrata and voriconazole resistance in clinical outcomes (14). However, the cross-resistance between fluconazole and voriconazole has been reported (20). In our study, the CAs with BMD fluconazole and YS08 voriconazole were 90%, 85%, and 80% using the method-specific CBP, ECV of 0.5 mg/mL, and ECV of 0.25 mg/mL, respectively. Lim et al. reported 97.8% and 82.5% CA between BMD fluconazole MIC and ECV of 0.5 mg/mL and 0.25 mg/mL, respectively (17). Therefore, YS08 voriconazole testing using the CLSI ECV might provide reliable discrimination of fluconazole-resistant C. glabrata. The CLSI recommends caution in interpretation because antifungal susceptibility tests for caspofungin have high interlaboratory variability (14). Caspofungin can show "eagle effects," which may cause variability in MICs, as described previously (21). When susceptibility tests are performed under standardized conditions, a surprising echinocandin-specific paradoxical effect can be observed. This effect refers to a phenomenon in which certain strains grow in higher concentrations of echinocandin while being completely susceptible at lower concentrations. This has been observed in several species of Candida and Aspergillus (22).
C. glabrata and C. krusei showed excellent CA for micafungin (100% and 100%, respectively) but poor CA (15% and 46.7%, respectively) for caspofungin. In C. krusei, 26.7% (4/15) of isolates in BMD showed intermediate susceptibility to caspofungin but were susceptible to micafungin. Although the reference method used was the CLSI BMD, caspofungin susceptibility testing in vitro might have contributed to reports of false resistance (23). Therefore, our results of a low EA for caspofungin of C. glabrata and C. krusei might be an unavoidable result. Lim et al. reported that YS08 caspofungin testing appeared unreliable for C. glabrata and that the YS08 micafungin result was more reliable than the caspofungin result (17). Clinical echinocandin resistance is generally associated with amino acid substitutions in specific hot spot regions of FKS1 (all Candida species) and FKS2 (C. glabrata only) (24). CLSI guidelines recommend that if the caspofungin result is susceptible, it can be reported as susceptible, but the possibility of "intermediate" or "resistance" should be confirmed through a micafungin or anidulafungin test or DNA sequence analysis of FKS genes (14). Similar to previous research using the EUCAST method, we found that antifungal susceptibility to other echinocandins or an FKS genetic study should be performed and interpreted comprehensively when interpreting echinocandin resistance (23,25).
We performed antifungal susceptibility tests on 45 C. auris strains collected in Korea from 2017-2020, and they showed excellent EA except for micafungin (82.2%). A previous study in Korea that used 61 C. auris isolates collected from 1996-2018 showed excellent EAs (.96.7%) and CAs (.93.4%) for fluconazole, amphotericin B, caspofungin, and micafungin between the BMD and the previous version of YS07 (26). However, several surveillance reports have revealed consistently high fluconazole MICs and variable resistance to echinocandins and amphotericin B and the echinocandins in C. auris (12,(27)(28)(29)(30). Moreover, multidrug-resistant strains have emerged in the Americas, Africa, Asia, and Europe (27,28,30). Higher MIC50 values of YS07 for amphotericin B, caspofungin, and voriconazole were previously reported compared with our results of 8 mg/mL, 0.5 mg/mL, and 1 mg/mL (12). However, there were no cases of echinocandin, amphotericin B, or multidrug-resistant C. auris strains in Korea (26). We observed a lower level of resistance to fluconazole and resistance to amphotericin B at a level of 24.4%. Similar to a previous study (26), our results found that C. auris in Korea had relatively low resistance to antifungal agents compared with those isolated from other regions. Multidrug-resistant strains of C. auris have been mainly identified among the South American clade (clade IV) or South Asia clade (clade I). We did not identify the clade of C. auris in this study, but previous studies have identified that Korean isolates mainly belonged to the East Asia clade (clade II) (26).
As in a previous paper (6), we used ECV/CBP of C. albicans to identify rare yeast species. In fact, cutoffs are species-specific, so it may be unwise to apply them to other species arbitrarily. Since the WT of C. albicans was universally susceptible to the antifungal agents in this study, we posit that a method to evaluate the aberrant MIC distribution based on C. albicans ECV/CBP is a meaningful approach (6).
In conclusion, YS08 showed comparable results with the BMD, including for C. auris. Voriconazole testing of YS08 with the method-specific CBP or by applying an ECV of 0.5 mg/mL may allow for reliable discrimination of fluconazole-resistant C. glabrata. However, interpreting the results of this method using the CBPs/ECVs determined for other methods may lead to erroneous results. Considering the lower YS08 fluconazole MIC results compared with BMD in Candida species and the YS08 caspofungin results in C. glabrata and C. krusei, further improvements for the detection of fluconazole and echinocandin resistance are needed.
Data analysis. MICs that were high off-scale were converted to the next highest level concentrations, and the low off-scale MICs remained unchanged (33). The EA was defined as the difference between MIC dilutions of less than two. The BMD low off-scale MICs of flucytosine, caspofungin, and micafungin, which were lower than the 2-fold dilution scale of YS08, were rounded up to the next highest 2-log dilution to simplify comparisons. The CA was defined as when the MIC results belonged to the same categories according to the CLSI CBP or/and ECV. Very major errors, major errors, and minor errors were defined with BMD as the reference method. Very major errors were those where the reference method classified an isolate as resistant and YS08 classified it as susceptible. If the reference method classified an isolate as susceptible and YS08 classified it as resistant, it was classified as a major error. Minor errors were defined as when one method classified an isolate as susceptible or resistant and the other assay classified it as intermediate or susceptible depending on the dose (19).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.2 MB. We have no conflicts of interest.