Antifungal Susceptibility Pattern of Candida glabrata from a Referral Center and Reference Laboratory: 2012–2022

The prevalence of invasive candidiasis caused by non-Candida albicans has rapidly increased. Candida glabrata (Nakaseomyces glabrata) is an important pathogen associated with substantial mortality. Our study examined the antifungal temporal susceptibility of C. glabrata and cross-resistance/non-wild-type patterns with other azoles and echinocandins. Laboratory data of all adult patients with C. glabrata isolated from clinical specimens at the Mayo Clinic, Rochester, from 2012 to 2022 were collected. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used. We obtained 1046 C. glabrata isolates from 877 patients. Using CLSI and EUCAST breakpoints, 187 (17.9%) isolates and 256 (24.5%) isolates were fluconazole-resistant, respectively. Focusing on C. glabrata bloodstream infections, fluconazole-resistance ranged from 16 to 22%. Among those 187 fluconazole-resistant isolates, 187 (100%) and 184 (98.4%) isolates were also voriconazole and posaconazole non-wild-type, respectively, with 97 (51.9%) isolates deemed non-wild type for itraconazole. The fluconazole susceptibility pattern has not changed over the past decade. The proportion of fluconazole-resistant C. glabrata is relatively high, which could be due to the complexity of patients and fluconazole exposure. Itraconazole appears to be a compelling step-down therapy for fluconazole-resistant C. glabrata, given the high proportion of wild-type isolates. Further research to examine clinical outcomes is warranted.


Introduction
Candida species are typical human microbiota that can be found on the skin and gastrointestinal mucosal surface of healthy individuals [1].Invasive candidiasis is a deepseated organ infection with Candida species that can occur with or without bloodstream infection or candidemia [2].Invasive candidiasis leads to significant morbidity and mortality worldwide, especially among critically ill and immunocompromised patients.The estimated mortality of invasive candidiasis ranged from 20 to 40%, with a significant healthcare burden [3][4][5].Important risk factors for invasive candidiasis include broad-spectrum antibiotic exposure, the colonization of Candida, the presence of a central venous catheter, and total parenteral nutrition [6].
Candida glabrata, which has an alternate taxonomic name of Nakaseomyces glabrata [7], is the second most common cause of invasive candidiasis, following Candida albicans.The prevalence of invasive C. glabrata is increasing, especially in North America [8,9].In 2022, the World Health Organization issued the fungal priority pathogens list to guide research, development, and public health action.This list categorized C. glabrata into the highpriority group for two main reasons: First, it is the most common cause of non-C.albicans invasive candidiasis, which is associated with a high mortality, especially in those with hematologic malignancy, prior fluconazole exposure, or neutropenia.Second, data for resistance patterns, pathogenicity, and preventative measures are lacking [10,11].
The clinical management of invasive candidiasis from C. glabrata is challenging.It is well-established that C. glabrata has reduced susceptibility to several antifungal agents, and its susceptibility pattern has changed over time [12].The first-line empiric therapy for invasive C. glabrata infection is an echinocandin, with high-dose fluconazole or voriconazole used as step-down options [13].However, previous studies have demonstrated that the prevalence of fluconazole-resistant C. glabrata has gradually increased in the United States [9,14].Additionally, some fluconazole-resistant C. glabrata isolates were resistant to voriconazole; however, the pattern of cross-resistance for echinocandin and non-wild type for itraconazole and posaconazole were not well-delineated, partly due to a lack of established breakpoints and differences in antifungal susceptibility techniques [15].Given the geographic and temporal variability in the C. glabrata susceptibility profile, this current study aimed to describe the trend in the susceptibility pattern of C. glabrata and the rate of cross-resistance/non-wild type among the fluconazole-resistant isolates at our institution over the past decade.

Data Collection and Objectives
A retrospective review of laboratory data was conducted.All adult patients who received care at the Mayo Clinic, Rochester, Minnesota, USA, with C. glabrata isolated from clinical specimens from 1 January 2012 to 31 May 2022 were included in the study.The patients who had more than one isolate of C. glabrata from an anatomically contiguous site were counted as the same isolate.The patients who had more than one isolate from the same anatomical site were considered a different isolate if the subsequent cultures were obtained more than two weeks from when the first isolate was.Baseline demographic data, including age, sex, and race, were obtained from Mayo Clinic Data Explorer software, which provided demographic data from the Mayo Clinic's electronic medical records.This study was approved by the Mayo Clinic Institutional Review Board (study number . The primary objective of the study was to examine the antifungal susceptibility pattern of C. glabrata via the use of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints.The secondary objectives were (1) to determine the prevalence of fluconazole-resistant isolates over time and (2) to determine the prevalence of cross-resistance/non-wild type of fluconazoleresistant isolates to other antifungals.Cross-resistance was defined as fluconazole-resistant C. glabrata isolates with a minimal inhibitory concentration (MIC) above the breakpoint of echinocandins.Non-wild type was defined as fluconazole-resistant C. glabrata isolates with a minimal inhibitory concentration (MIC) above the epidemiological cut-off value (ECV or ECOFF) of other azoles.

C. glabrata Identification and Antifungal Susceptibility Testing
Blood was collected and inoculated into Becton Dickinson BD BACTEC™ blood culture media bottles (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA).Blood culture bottles were incubated at 35 • C in the BD BACTEC™ FX blood culture system for five days.Blood culture bottles that showed yeast from the Gram stain were subcultured into a Sabouraud Dextrose Agar plate at 28-30 • C in room air and examined daily for growth.The pure growth of yeast was further identified via matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) using a Bruker Daltonics MALDI Biotyper ® System (Bruker Daltonics, Bremen, Germany).Clinical specimens other than blood were cultured to agar plates including inhibitory mold agar and brain heart infusion agar containing chloramphenicol, gentamycin, and, for respiratory specimen sources, cycloheximide.Plates were incubated at 30 ± 1 • C in ambient air for up to 28 days.Isolates were identified as C. glabrata using either MALDI-TOF MS or the D2 region of the large subunit ribosomal ribonucleic acid (D2 rRNA) gene sequencing, as previously described [16,17].Two cryptic species, including C. nivariensis or C. bracarensis, were not included in this study.

Statistical Analysis
Descriptive statistics were used, including the median and interquartile range (IQR) for continuous variables in addition to frequency and percentage for categorical variables.All analyses were performed using BlueSky Statistics version 7.40 (BlueSky Statistics LLC, Chicago, IL, USA).
Figure 1: Fluconazole susceptibility pattern based on year.

Discussion
Our study examined 1046 isolates of C. glabrata over the past decade.The proportion of fluconazole-resistant isolates was 18% and 25% based on the CLSI and EUCAST breakpoints, respectively.The highest proportion of fluconazole-resistant isolates was from the skin and mucosa, although the clinical significance of susceptibility testing in this setting remains unclear.There was no appreciable change in the prevalence of fluconazoleresistant C. glabrata during the past ten years.Additionally, the overall rate of echinocandin and amphotericin resistance/non-wild-type isolates remains low.Focusing on C. glabrata bloodstream infections, fluconazole resistance ranged from 16% to 22%, significantly higher than previously reported.Pfaller et al. reported the first multicenter Candida bloodstream infection surveillance from 1992 to 2001 after the introduction of fluconazole in 1990, with a C. glabrata fluconazole resistance rate of approximately 9% [21,22].A recent study updated the susceptibility pattern of 2860 C. glabrata isolates worldwide from 1997 to 2016 via the use of the SENTRY Antifungal Surveillance Program.The proportion of fluconazole-resistant C. glabrata increased from 8.6% in 1997 to 10.1% in 2014.The highest rate of fluconazoleresistance in North America was 11% [14].Previous studies have postulated that the increasing trend towards fluconazole-resistant C. glabrata may be due to a rise in patient complexity, prior fluconazole exposure, and aging populations [23,24].Additionally, owing to selection pressure, prior fluconazole exposure increased the risk of invasive non-C.albicans infections, especially in patients with hematologic malignancies, bone marrow transplants, and critical illnesses [25][26][27].Although detailed clinical characteristics were not examined in our study, we speculate that these risk factors likely remain applicable among our complex patient population.
The variability in the CLSI and EUCAST breakpoints for C. glabrata is notable.Both CLSI and EUCAST use a broth microdilution technique to obtain MIC data.Although some processes were different between the CLSI and EUCAST, such as inoculum density, incubation time, type of well, glucose content, and endpoint, the breakpoints for fluconazole are comparable between the two standard-setting organizations [28].The clinical breakpoint was established by taking the clinical outcome and pharmacology of the drug into account.There is no established CLSI and EUCAST clinical breakpoint of C. glabrata for itraconazole, voriconazole, and posaconazole.Therefore, the ECV (for the CLSI) and ECOFF (for the EUCAST) are utilized.The CLSI and EUCAST cutoffs for posaconazole are the same, while itraconazole has a one-dilution difference and voriconazole has a two-dilution difference.This difference in the cutoff does not impact the proportion of non-wild-type isolates for itraconazole and posaconazole, yet it made the interpretation of voriconazole non-wildtype challenging with 43.5% based on the CLSI ECV versus 14.0% based on the EUCAST ECOFF.It would be interesting to examine further how this difference affects the clinical decision on using voriconazole when considering both the ECV and ECOFF.
There are numerous proposed resistance mechanisms for fluconazole with C. glabrata [29].The primary mechanism is a "gain of function" of several genes, such as C. glabrata CDR1, CDR2, and SNQ2.Increased activity of these genes leads to an overexpression of the fluconazole binding target and a decrease in intracellular fluconazole concentration due to efflux pump enhancement [30][31][32].Both in vitro and clinical studies have shown that C. glabrata also demonstrated a significant cross-resistance to other azoles [33,34].Our study demonstrated that almost 100% of fluconazole-resistant isolates were voriconazole and posaconazole non-wild-type based on the CLSI breakpoint.Interestingly, only half of fluconazole-resistant isolates were itraconazole non-wild-type.A previous study from Castanheira et al. noted a similar observation [35].The explanation for why itraconazole preserved the highest rate of wild-type isolates among other azoles remains unclear.The cross-resistance to echinocandins and amphotericin among fluconazole-resistant isolates was low, at < 10%, except for anidulafungin, which has a lower EUCAST breakpoint compared with the CLSI breakpoint.This observation is essential in clinical practice, primarily in the setting of highly fluconazole-resistant C. glabrata.Echinocandins are effective drugs against fluconazole-resistant C. glabrata, and remain a mainstay of therapy [13].However, intravenous administration sometimes creates logistical challenges.Itraconazole may be a compelling option for the step-down strategy, given its lower probability of being a non-wild type.Nevertheless, more clinical data on itraconazole use in this scenario are needed.
Our study has several limitations.First, C. glabrata MIC data were obtained from laboratory data without a clinical course nor antifungal treatment correlation.Thus, it is impossible to examine the association between fluconazole resistance and clinical outcomes.Second, some isolates were obtained from the same patient within a different timeframe.Hence, some might develop emerging resistance from antifungal exposure related to the first episode, yet we could not clearly demonstrate a pattern in our study.Third, the MIC breakpoint for the CLSI and EUCAST have changed over time.However, we applied the current breakpoint to all of the isolates; therefore, it would not affect the trend of a resistance pattern.Additionally, although there are some differences between the CLSI and EUCAST methods, a previous study has shown that the MICs/ECVs/ECOFFs are comparable [36].Fourth, multilocus genome sequencing was not performed in the resistant isolates to determine the resistance mechanism.Fifth, our hospital is a tertiary referral center, and these results may not be generalizable to other patient populations.Lastly, selection bias may especially occur with the isolates from a non-blood source.For example, one may request the identification of species level and susceptibility when the patient did not have a good clinical response.Otherwise, the isolate may not be identified at the species level.

Conclusions
In conclusion, our study demonstrated a relatively high proportion of fluconazoleresistant C. glabrata, including 16-22% of blood isolates.Non-wild-type voriconazole and posaconazole were also noted, but the in vitro activity of itraconazole seems to be preserved.Further studies focusing on the clinical correlation and utility of itraconazole as a step-down therapy in fluconazole-resistant C. glabrata is warranted.

Figure 1 .
Figure 1.The proportion of fluconazole-resistant isolates was not different between years.A: based on CLSI breakpoint and B: based on EUCAST breakpoint.

Figure 1 .
Figure 1.The proportion of fluconazole-resistant isolates was not different between years.A: based on CLSI breakpoint and B: based on EUCAST breakpoint.

Table 3 .
Fluconazole susceptibility based on source.

Table A2 .
Cross-resistance/non-wild type for blood isolates.: CLSI = Clinical and Laboratory Standards Institute; ECV and ECOFF = epidemiologic cutoff value; EUCAST = European Committee on Antimicrobial Susceptibility Testing; N/A = not applicable; and WT= wild type. Abbreviations