We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid.

We previously conducted a multicenter surveillance study on Candida epidemiology and antifungal resistance in Madrid (CANDIMAD study; 2019–2021), detecting an increase in fluconazole-resistant Candida parapsilosis . We here present data on isolates collected in 2022. Furthermore, we report the epidemiology and antifungal resistance trends during the entire period, including an analysis per ward of admission. Candida spp. incident isolates from blood cultures and intra-abdominal samples from patients cared for at 16 hospitals in Madrid, Spain, were tested with the EUCAST E.Def 7.3.2 method against amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp and were molecularly characterized. In 2022, we collected 766 Candida sp. isolates (686 patients; blood cultures, 48.8%). Candida albicans was the most common species found, and Candida auris was undetected. No resistance to amphotericin B was found. Overall, resistance to echinocandins was low (0.7%), whereas fluconazole resistance was 12.0%, being higher in blood cultures (16.0%) mainly due to fluconazole-resistant C. parapsilosis clones harboring the Y132F-R398I ERG11p substitutions. Ibrexafungerp showed in vitro activity against the isolates tested. Whereas C. albicans was the dominant species in most hospital wards, we observed increasing C. parapsilosis proportions in blood. During the entire period, echinocandin resistance rates remained steadily low, while fluconazole resistance increased in blood from 6.8% (2019) to 16% (2022), mainly due to fluconazole-resistant C. parapsilosis (2.6% in 2019 to 36.6% in 2022). Up to 7 out of 16 hospitals were affected by fluconazole-resistant C. parapsilosis . In conclusion, rampant clonal spreading of C. parapsilosis fluconazole-resistant genotypes is taking place in Madrid.

Updated analysis on yeast isolates causing fungaemia in patients admitted to a tertiary hospital in Madrid, Spain, over a 13-year period. We studied 896 isolates associated to 872 episodes of fungaemia in 857 hospitalized patients between January 2007 and December 2019. Antifungal susceptibility was assessed by EUCAST EDef 7.3.2. Mutations conferring azole and echinocandin resistance were further studied, and genotyping of resistant clones performed with species-specific microsatellite markers. Candida albicans (45.8%) was the most frequently identified species, followed by the Candida parapsilosis complex (26.4%), Candida glabrata (12.3%), Candida tropicalis (7.3%), Candida krusei (2.3%), other Candida spp. (3.1%), and non-Candida yeasts (2.8%). Rate of fluconazole resistance in Candida spp. was 4.7%, ranging from 0% (C. parapsilosis) to 9.1% (C. glabrata). The overall rate of echinocandin resistance was 3.1%. Resistance was highly influenced by the presence of intrinsically resistant species. Although the number of isolates between 2007 and 2013 was almost two-fold higher than that of the 2014-2019 period (566 vs. 330), fluconazole resistance in Candida spp. was greater in the second period (3.5% vs. 6.8%; P < 0.05), while overall resistance to echinocandins remained stable (3.5% vs. 2.4%; P > 0.05). Resistant clones were collected from different wards and/or time points, suggesting no epidemiological links. The number of fungaemia episodes has been decreasing over the last 13 years, with a slight increase in the rate of fluconazole resistance and stable echinocandin resistance. Antifungal resistance is not the cause of the spread of resistant clones.

To identify unrecognized niches of resistant Candida isolates and compartmentalization, we retrospectively studied the antifungal susceptibility of 1,103 Candida spp. isolates from blood cultures, nonblood sterile samples, and nonsterile samples. Antifungal susceptibility was assessed by EUCAST E.Def 7.3.2; sequencing and genotyping of the FKS1-2 and ERG11 genes were carried out for non-wild-type isolates.

Several institutions reported a rise not only in fungemia incidence but also in the number of cases caused by Candida auris or fluconazole-resistant C. parapsilosis during the COVID-19 pandemic. Since the pandemic broke out in early 2020, we studied its impact on fungemia incidence, species epidemiology, potential patient-to-patient transmission, and antifungal resistance in 166 incident yeast isolates collected from January 2020 to December 2022. Isolates were molecularly identified, and their antifungal susceptibilities to amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp were studied following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method, and genotyped. The fungemia incidence (episodes per 1000 admissions) tended to decrease over time (2020 = 1.60, 2021 = 1.36, 2022 = 1.16); P > .05). Species distribution was C. albicans (50.6%, n = 84), C. parapsilosis (18.7%, n = 31), C. glabrata (12.0%, n = 20), C. tropicalis (11.4%, n = 19), C. krusei (3.0%, n = 5), other Candida spp. (1.2%, n = 2), and non-Candida yeasts (3.0%, n = 5). The highest and lowest proportions of C. albicans and C. parapsilosis were detected in 2020. The proportion of isolates between 2020 and 2022 decreased in C. albicans (60.3% vs. 36.7%) and increased in C. parapsilosis (10.3% vs. 28.6%; P < .05) and C. tropicalis (8.8% vs. 16.3%; P > .05). Only three C. albicans intra-ward clusters involving two patients each were detected, and the percentages of patients involved in intra-ward clusters reached 9.8% and 8.0% in 2020 and 2021, respectively, suggesting that clonal spreading was not uncontrolled. Fluconazole resistance (5%) exhibited a decreasing trend (P > .05) over time (2020 = 7.6%; 2021 = 4.2%; and 2022 = 2.1%). Ibrexafungerp showed high in vitro activity. Fungemia incidence increased during the COVID-19 pandemic in our hospital, however, clonal spreading was not uncontrolled. The proportion of C. parapsilosis and C. tropicalis cases constantly increased. Antifungal resistance remained very low, and fluconazole-resistant C. parapsilosis was undetected.

Echinocandins, which target the fungal β-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus ( Candida glabrata ) in which combined mutations in ERG3 and FKS2 , but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude Erg3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic cross-talk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes. IMPORTANCE: A clinical case in which the combination of variation in a β‑1,3‑glucan synthase-encoding gene ( FKS2 ) and the sterol desaturase-encoding gene ERG3 seems to underlie echinocandin resistance, prompted us to hypothesize that membrane sterol changes may modulate, rather than independently cause, Fks‑linked resistance. We were able to explore this hypothesis due to recent developments in the field, such as the release of the FungAMR database, which enables global co‑occurrence analyses; AI‑driven variant effect predictors such as Evolutionary Scale Modeling (ESM) that can explore the impact of thousands of ERG3 alleles; the cryo‑EM resolution of the Fks1 protein; and the first mechanistic model of echinocandin‑Fks1 binding. Together, these advances provide the structural and computational framework needed to delineate our hypothesis that specific sterol variants might influence β‑1,3‑glucan synthase function and drug binding. Further surveillance of this potentially epistatic interaction can be of significant clinical importance amid rising multidrug‑resistant infections, as overlooking such interactions could lead to under‑calling resistance and misguided therapy.