Virulence Traits and Azole Resistance in Korean Candida auris Isolates.

We analyzed the virulence traits and azole resistance mechanisms of 104 Candida auris isolates collected from 13 Korean hospitals from 1996 to 2022. Of these 104 isolates, 96 (5 blood and 91 ear isolates) belonged to clade II, and 8 (6 blood and 2 other isolates) belonged to clade I. Fluconazole resistance (minimum inhibitory concentration ≥32 mg/L) was observed in 68.8% of clade II and 25.0% of clade I isolates. All 104 isolates were susceptible to amphotericin B and three echinocandins. In 2022, six clade I isolates indicated the first nosocomial C. auris cluster in Korea. Clade II C. auris isolates exhibited reduced thermotolerance at 42 °C, with diminished in vitro competitive growth and lower virulence in the Galleria mellonella model compared to non-clade II isolates. Of the 66 fluconazole-resistant clade II isolates, several amino acid substitutions were identified: Erg11p in 14 (21.2%), Tac1Ap in 2 (3.0%), Tac1Bp in 62 (93.9%), and Tac1Bp F214S in 33 (50.0%). Although there were a limited number of non-clade II isolates studied, our results suggest that clade II C. auris isolates from Korean hospitals might display lower virulence traits than non-clade II isolates, and their primary fluconazole resistance mechanism is linked to Tac1Bp mutations.

Molecular and Clinical Features of Fluconazole Non-susceptible Candida albicans Bloodstream Isolates Recovered in Korean Multicenter Surveillance Studies.

Acquired fluconazole resistance (FR) in bloodstream infection (BSI) isolates of Candida albicans is rare. We investigated the FR mechanisms and clinical features of 14 fluconazole non-susceptible (FNS; FR and fluconazole-susceptible dose-dependent) BSI isolates of C. albicans recovered from Korean multicenter surveillance studies during 2006-2021. Mutations causing amino acid substitutions (AASs) in the drug-target gene ERG11 and the FR-associated transcription factor genes TAC1, MRR1, and UPC2 of the 14 FNS isolates were compared with those of 12 fluconazole-susceptible isolates. Of the 14 FNS isolates, eight and seven had Erg11p (K143R, F145L, or G464S) and Tac1p (T225A, R673L, A736T, or A736V) AASs, respectively, which were previously described in FR isolates. Novel Erg11p, Tac1p, and Mrr1p AASs were observed in two, four, and one FNS isolates, respectively. Combined Erg11p and Tac1p AASs were observed in seven FNS isolates. None of the FR-associated Upc2p AASs were detected. Of the 14 patients, only one had previous azole exposure, and the 30-day mortality rate was 57.1% (8/14). Our data show that Erg11p and Tac1p AASs are likely to contribute to FR in C. albicans BSI isolates in Korea and that most FNS C. albicans BSIs develop without azole exposure.

Whole-Genome Sequence Analysis of Candida glabrata Isolates from a Patient with Persistent Fungemia and Determination of the Molecular Mechanisms of Multidrug Resistance.

Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2-3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4-8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25-2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.

Detection and Characterization of Two Phenotypes of Candida parapsilosis in South Korea: Clinical Features and Microbiological Findings.

We newly detected two (sinking and floating) phenotypes of Candida parapsilosis among bloodstream infection (BSI) isolates from Korean hospitals and assessed their microbiological and clinical characteristics. During the performance of a Clinical and Laboratory Standards Institute (CLSI) broth microdilution antifungal susceptibility testing, the sinking phenotype had a characteristic smaller button-like appearance because all yeast cells sank to the bottoms of the CLSI U-shaped round-bottom wells, whereas the floating phenotype comprised dispersed cells. Phenotypic analysis, antifungal susceptibility testing, ERG11 sequencing, microsatellite genotyping, and clinical analysis were performed on C. parapsilosis isolates from 197 patients with BSI at a university hospital during 2006 to 2018. The sinking phenotype was detected in 86.7% (65/75) of the fluconazole-nonsusceptible (FNS) isolates, 92.9% (65/70) of the isolates harboring the Y132F ERG11 gene substitution, and 49.7% (98/197) of all isolates. Clonality was more frequently observed for the Y132F-sinking isolates (84.6% [55/65]) than for all other isolates (26.5% [35/132]; P < 0.0001). Annual incidence of Y132F-sinking isolates increased 4.5-fold after 2014, and two dominant genotypes, persistently recovered for 6 and 10 years, accounted for 69.2% of all Y132F-sinking isolates. Azole breakthrough fungemia (odds ratio [OR], 6.540), admission to the intensive care unit (OR, 5.044), and urinary catheter placement (OR, 6.918) were independent risk factors for BSIs with Y132F-sinking isolates. The Y132F-sinking isolates exhibited fewer pseudohyphae, a higher chitin content, and lower virulence in the Galleria mellonella model than the floating isolates. These long-term results illustrate the increasing BSIs caused by clonal transmission of the Y132F-sinking isolates of C. parapsilosis. IMPORTANCE We believe that this is the first study describe the microbiological and molecular characteristics of bloodstream isolates of C. parapsilosis in Korea exhibiting two phenotypes (sinking and floating). An important aspect of our findings is that the sinking phenotype was observed predominantly in isolates harboring a Y132F substitution in the ERG11 gene (92.9%), fluconazole-nonsusceptible (FNS) isolates (86.7%), and clonal BSI isolates (74.4%) of C. parapsilosis. Although the increase in the prevalence of FNS C. parapsilosis isolates has been a major threat in developing countries, in which the vast majority of candidemia cases are treated with fluconazole, our long-term results show increasing numbers of BSIs caused by clonal transmission of Y132F-sinking isolates of C. parapsilosis in the period with an increased echinocandin use for candidemia treatment in Korea, which suggests that C. parapsilosis isolates with the sinking phenotype continue to be a nosocomial threat in the era of echinocandin therapy.

Nationwide Surveillance of Antifungal Resistance of Candida Bloodstream Isolates in South Korean Hospitals: Two Year Report from Kor-GLASS.

We incorporated nationwide Candida antifungal surveillance into the Korea Global Antimicrobial Resistance Surveillance System (Kor-GLASS) for bacterial pathogens. We prospectively collected and analyzed complete non-duplicate blood isolates and information from nine sentinel hospitals during 2020−2021, based on GLASS early implementation protocol for the inclusion of Candida species. Candida species ranked fourth among 10,758 target blood pathogens and second among 4050 hospital-origin blood pathogens. Among 766 Candida blood isolates, 87.6% were of hospital origin, and 41.3% occurred in intensive care unit patients. Adults > 60 years of age accounted for 75.7% of cases. Based on species-specific clinical breakpoints, non-susceptibility to fluconazole, voriconazole, caspofungin, micafungin, and anidulafungin was found in 21.1% (154/729), 4.0% (24/596), 0.1% (1/741), 0.0% (0/741), and 0.1% (1/741) of the isolates, respectively. Fluconazole resistance was determined in 0% (0/348), 2.2% (3/135, 1 Erg11 mutant), 5.3% (7/133, 6 Pdr1 mutants), and 5.6% (6/108, 4 Erg11 and 1 Cdr1 mutants) of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis isolates, respectively. An echinocandin-resistant C. glabrata isolate harbored an F659Y mutation in Fks2p. The inclusion of Candida species in the Kor-GLASS system generated well-curated surveillance data and may encourage global Candida surveillance efforts using a harmonized GLASS system.

Dynamics and Predictors of Mortality Due to Candidemia Caused by Different Candida Species: Comparison of Intensive Care Unit-Associated Candidemia (ICUAC) and Non-ICUAC.

We investigated mortality and predictors of mortality due to intensive care unit-associated candidemia (ICUAC) versus non-ICUAC by Candida species. This study included all candidemia cases in 11 hospitals from 2017 to 2018 in South Korea. The all-cause mortality rates in all 370 patients with ICUAC were approximately twofold higher than those in all 437 patients with non-ICUAC at 7 days (2.3-fold, 31.1%/13.3%), 30 days (1.9-fold, 49.5%/25.4%), and 90 days (1.9-fold, 57.8%/30.9%). Significant species-specific associations with 7- and 30-day ICUAC-associated mortality were not observed. Multivariate analysis revealed that ICU admission was an independent predictor of Candida glabrata (OR, 2.07-2.48) and Candida parapsilosis-associated mortality (OR, 6.06-11.54). Fluconazole resistance was a predictor of C. glabrata-associated mortality (OR, 2.80-5.14). Lack (less than 3 days) of antifungal therapy was the strongest predictor of 7-day mortality due to ICUAC caused by Candida albicans (OR, 18.33), Candida tropicalis (OR, 10.52), and C. glabrata (OR, 21.30) compared with 30- and 90-day mortality (OR, 2.72-6.90). C. glabrata ICUAC had a stronger association with lack of antifungal therapy (55.2%) than ICUAC caused by other species (30.6-36.7%, all p < 0.05). Most predictors of mortality associated with ICUAC were distinct from those associated with non-ICUAC and were mediated by Candida species.

Disk Diffusion Susceptibility Testing for the Rapid Detection of Fluconazole Resistance in Candida Isolates.

BACKGROUND: Given the increased fluconazole resistance (FR) among Candida isolates, we assessed the suitability of disk diffusion susceptibility testing (DDT) for the early detection of FR using well-characterized Candida isolates. METHODS: In total, 188 Candida isolates, including 66 C. albicans (seven Erg11 mutants), 69 C. glabrata (33 Pdr1 mutants), 29 C. parapsilosis (15 Erg11 mutants), and 24 C. tropicalis (eight Erg11 mutants) isolates, were tested in this study. FR was assessed using DDT according to the standard CLSI M44-ED3 method, except that two cell suspensions, McFarland 0.5 (standard inoculum) and 2.5 (large inoculum), were used, and the inhibition zones were read at 2-hour intervals from 10 hours to 24 hours. RESULTS: DDT results for the standard inoculum were readable after 14 hours (C. albicans, C. glabrata, and C. tropicalis) and 20 hours (C. parapsilosis) for >95% of the isolates, whereas the results for the large inoculum were readable after 12 hours (C. glabrata and C. tropicalis), 14 hours (C. albicans), and 16 hours (C. parapsilosis) for >95% of the isolates. Compared with the results produced using the CLSI M27-ED4 broth microdilution method, the first readable results from the DDT method for each isolate exhibited an agreement of 97.0%, 98.6%, 72.4%, and 91.7% for the standard inoculum and 100%, 98.6%, 96.6%, and 95.8% for the large inoculum for C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, respectively. CONCLUSIONS: DDT using large inoculum may detect FR rapidly and reliably in the four most common Candida species.

Evaluation of Two Commercial Broth Microdilution Methods Using Different Interpretive Criteria for the Detection of Molecular Mechanisms of Acquired Azole and Echinocandin Resistance in Four Common Candida Species.

The abilities of the new Vitek 2 AST-YS08 (YS08) and Sensititre YeastOne (SYO) systems to detect the resistances of Candida isolates to azoles and echinocandins were evaluated. In total, 292 isolates, including 28 Candida albicans (6 Erg11 and 2 Fks mutants), 57 Candida parapsilosis (26 Erg11 mutants), 24 Candida tropicalis (10 Erg11 and 1 Fks mutants), and 183 Candida glabrata (39 Pdr1 and 13 Fks mutants) isolates, were tested. The categorical agreements (CAs) between the Clinical and Laboratory Standards Institute (CLSI) method and YS08 fluconazole MICs obtained using clinical breakpoints were 92.4% (C. albicans), 96.5% (C. parapsilosis), and 87.0% (C. tropicalis), and the CAs between the CLSI and SYO MICs were 92.3% (C. albicans), 77.2% (C. parapsilosis), 100% (C. tropicalis), and 98.9% (C. glabrata). For C. glabrata, the CAs with the CLSI micafungin MICs were 92.4% and 55.5% for the YS08 micafungin and caspofungin MICs, respectively; they were 100%, 95.6%, and 98.9% for the SYO micafungin, caspofungin, and anidulafungin MICs, respectively. YS08 does not provide fluconazole data for C. glabrata; the CA with the CLSI fluconazole MIC was 97.8% for the YS08 voriconazole MIC, using an epidemiological cutoff value (ECV) of 0.5 µg/ml. Increased CAs with the CLSI MIC were observed for the YS08 MIC using CLSI ECVs (for fluconazole and C. tropicalis, 100%; for micafungin and C. glabrata, 98.9%) and for the SYO MIC using method-specific ECVs (for fluconazole and C. parapsilosis, 91.2%; for caspofungin and C. glabrata, 98.9%). Therefore, the YS08 and SYO systems may have different abilities to detect mechanisms of azole and echinocandin resistance in four Candida species; the use of method-specific ECVs may improve the performance of both systems.

Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping.

Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

Multilocus Sequence Typing (MLST) Genotypes of Candida glabrata Bloodstream Isolates in Korea: Association With Antifungal Resistance, Mutations in Mismatch Repair Gene (Msh2), and Clinical Outcomes.

Candida glabrata bloodstream infection (BSI) isolates from a particular geographic area have been reported to comprise a relatively small number of the major sequence types (STs) by multilocus sequence typing (MLST) analysis. Yet little is known about the characteristics of major ST strains of C. glabrata. To address this question in Korea, we investigated antifungal resistance and non-synonymous mutations of the mismatch repair gene (msh2 mutations) in C. glabrata BSI isolates, as well as associated clinical characteristics, and compared the results according to MLST genotype. We assessed a total of 209 C. glabrata BSI isolates from seven hospitals in Korea for 2 years (2009 and 2014). Clinical features of candidemia and their outcomes were analyzed for 185 available cases. According to MLST, ST7 (47.8%) was the most common type, followed by ST3 (22.5%); the remainder represented 28 types of minor STs (29.7%). Fluconazole-resistance (FR) rates for ST7, ST3, and other strains were 9.0% (9/100), 8.5% (4/47), and 4.8% (3/62), respectively, and all were susceptible to amphotericin B and micafungin. All ST7 isolates harbored the V239L mutation in msh2, known to confer hypermutability, while 91.5% of ST3 isolates did not harbor the msh2 mutation. Overall, isolates of the same ST had identical msh2 mutations, with the exception of nine isolates. The msh2 mutations were identified in 68.8% (11/16) of the FR isolates and 67.4% (130/193) of the fluconazole susceptible-dose dependent isolates. There was no significant difference in all clinical characteristics between ST3 and ST7. However, the 30-day mortality of C. glabrata candidemia due to the two major ST (ST3 or ST7) strains was significantly higher than that of candidemia due to other minor ST strains (45.1 vs. 25.0%, p < 0.05). Multivariate logistic regression analysis also showed that two major STs (ST3 and ST7) were independent predictors of 30-day mortality. This study showed for the first time that two STs (ST7 and ST3) were predominant among BSI isolates in Korea, and that C. glabrata BSI isolates belonging to two major MLST genotypes are characterized by higher mortality. In addition, most msh2 mutations align with MLST genotype, irrespective of FR.

Antifungal susceptibilities to amphotericin B, triazoles and echinocandins of 77 clinical isolates of cryptic Aspergillus species in multicenter surveillance in Korea.

We investigated the in vitro antifungal susceptibilities of cryptic Aspergillus species from nine Korean hospitals. Based on the CLSI epidemiological cutoff values, resistance rates to amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin were as follows: A. awamori (34 isolates; all 0%), A. tubingensis (22; 0%, 4.5%, 0%, 0%, and 0%, respectively), A. sydowii (16; 0%, 6.3%, 0%, 0%, and 6.3%), A. lentulus (2; 50%, 0%, 100%, 50%, and 0%), and A. tamarii (2; all 0%). A. calidoustus (one isolate) showed resistance to multiple drugs. Thus, cryptic species identification can be mandatory for clinically important Aspergillus isolates, with their susceptibility data.

Diversity of clinical isolates of Aspergillus terreus in antifungal susceptibilities, genotypes and virulence in Galleria mellonella model: Comparison between respiratory and ear isolates.

We analyzed the antifungal susceptibility profiles, genotypes, and virulence of clinical Aspergillus terreus isolates from six university hospitals in South Korea. Thirty one isolates of A. terreus, comprising 15 respiratory and 16 ear isolates were assessed. Microsatellite genotyping was performed, and genetic similarity was assessed by calculating the Jaccard index. Virulence was evaluated by Galleria mellonella survival assay. All 31 isolates were susceptible to itraconazole, posaconazole, and voriconazole, while 23 (74.2%) and 6 (19.4%) showed amphotericin B (AMB) minimum inhibitory concentrations (MICs) of ≤ 1 mg/L and > 4 mg/L, respectively. Notably, respiratory isolates showed significantly higher geometric mean MICs than ear isolates to AMB (2.41 vs. 0.48 mg/L), itraconazole (0.40 vs. 0.19 mg/L), posaconazole (0.16 vs. 0.08 mg/L), and voriconazole (0.76 vs. 0.31 mg/L) (all, P <0.05). Microsatellite genotyping separated the 31 isolates into 27 types, but the dendrogram demonstrated a closer genotypic relatedness among isolates from the same body site (ear or respiratory tract); in particular, the majority of ear isolates clustered together. Individual isolates varied markedly in their ability to kill infected G. mellonella after 72 h, but virulence did not show significant differences according to source (ear or respiratory tract), genotype, or antifungal susceptibility. The current study shows the marked diversity of clinical isolates of A. terreus in terms of antifungal susceptibilities, genotypes and virulence in the G. mellonella model, and ear isolates from Korean hospitals may have lower AMB or triazole MICs than respiratory isolates.