Fluconazole-susceptible Isolates Research Articles

Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme

ObjectivesWe propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured C. parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes. MethodsA total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n=94; harbouring the Y132F ERG11p substitution [n=85], the G458S substitution [n=6], the R398I substitution [n=2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n=129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers). ResultsThe screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n=129/129) and sensitivity (Y132F isolates; n=85/85) values, however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n=98; mean 23, range 13-38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5x10-6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. ConclusionBoth proposed PCR formats allow to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates.

Genotypic and phenotypic characteristics of Candida parapsilosis bloodstream isolates: Health Care Associated Infections in a teaching Hospital in Italy

BackgroundCandidemia is the most common healthcare associated invasive fungal infection. Over the last few decades, candidemia caused by Candida species other than Candida albicans, particularly the Candida parapsilosis complex, has emerged worldwide. The aims of this study were: to analyze the genotypic and phenotypic characteristics of C. parapsilosis strains isolated from blood cultures and the environment in a hospital in southern Italy, to study the possible source of infection and to correlate the isolated strains. MethodsFrom April to October 2022, cases of candidemia due to C. parapsilosis in patients admitted to a hospital in the Apulia region were investigated. However, 119 environmental samples from the intensive care unit were collected for identification of the likely environmental reservoir of infection. Routine antifungal (amphotericin B, anidulafungin, fluconazole) susceptibility was performed on all isolates. Whole genome sequencing was performed to study the genotypic correlation of the isolates. Biofilm biomass and metabolic activity were also quantified for all isolates. ResultsA total of 43 C. parapsilosis isolates were cultured from the bloodstream of each patient in different departments, and seven surface samples were positive for C. parapsilosis. Most of the isolated yeasts (41/50; 85 %) were resistant to fluconazole and were genetically related to each other, suggesting an ongoing clonal outbreak of this pathogen. The fluconazole-susceptible isolates produced significantly more biofilm than did the resistant isolates. Metabolic activity was also higher for fluconazole-susceptible than resistant isolates. ConclusionCross-transmission of the microorganisms is suggested by the phenotypic similarity and genetic correlation between clinical and environmental strains observed in our study.

Risk factors associated with fluconazole resistance in Candida parapsilosis candidemia: A case-case study.

Candida species are among the most important invasive pathogens in intensive care units (ICUs). Non-albicans species including Candida parapsilosis (C. parapsilosis) has increased in recent years. Fluconazole is the leading antifungal agent but resistance is a concern among C. parapsilosis species. The aim of this study was to determine the factors associated with fluconazole resistance in patients with candidemia due to C. parapsilosis in ICUs. This case-case study was conducted in a 750-bed, tertiary hospital between 2015 and 2021. Patients with fluconazole-resistant C. parapsilosis candidemia constituted the 'cases of interest' group and patients with fluconazole-susceptible C. parapsilosis candidemia constituted the 'comparison cases' group. Demographic and clinical data of the patients were recorded. Logistic regression analysis was performed using the backward elimination method to determine the independent predictors of fluconazole-resistant C. parapsilosis bloodstream infections. The study included 177 patients. In the cultures of these patients, 76 (43%) fluconazole-resistant, 13 (7.3%) fluconazole-reduced susceptible, and 88 (49.7%) fluconazole-susceptible isolates were found. In the regression analysis the risk factors for fluconazole-resistant C. parapsilosis bloodstream infection, malignancy, immunosuppressive treatment, history of intra-abdominal surgery, hypoalbunemia, previous fluconazole use, and SOFA score were found to be associated in univariate analysis. In multivariate regression analysis, history of intra-abdominal surgery (OR: 2.16; 95% CI: 1.05-4.44), hypoalbuminemia (OR: 2.56; 95% CI: 1.06-6.17) and previous fluconazole use (OR: 3.35; 95% CI: 1.02-11) were found to be independent predictors. In this study, a significant correlation was found between candidemia due to fluconazole-resistant C. parapsilosis in ICUs and intra-abdominal surgery, hypoalbuminemia, and previous fluconazole use. C. parapsilosis isolates and fluconazole resistance should be continuously monitored, strict infection control measures should be taken and antifungal stewardship programs should be implemented.

Sphingolipid diversity in Candida auris: unraveling interclade and drug resistance fingerprints.

In this study, we explored the sphingolipid (SL) landscape in Candida auris, which plays pivotal roles in fungal biology and drug susceptibility. The composition of SLs exhibited substantial variations at both the SL class and molecular species levels among clade isolates. Utilizing principal component analysis, we successfully differentiated the five clades based on their SL class composition. While phytoceramide (PCer) was uniformly the most abundant SL class in all the isolates, other classes showed significant variations. These variations were not limited to SL class level only as the proportion of different molecular species containing variable number of carbons in fatty acid chains also differed between the isolates. Also a comparative analysis revealed abundance of ceramides and glucosylceramides in fluconazole susceptible isolates. Furthermore, by comparing drug-resistant and susceptible isolates within clade IV, we uncovered significant intraclade differences in key SL classes such as high PCer and low long chain base (LCB) content in resistant strains, underscoring the impact of SL heterogeneity on drug resistance development in C. auris. These findings shed light on the multifaceted interplay between genomic diversity, SLs, and drug resistance in this emerging fungal pathogen.

Clonal aggregation of fluconazole-resistant Candida tropicalis isolated from sterile body fluid specimens from patients in Hefei, China.

Candida tropicalis, a human conditionally pathogenic yeast, is distributed globally, especially in Asia-Pacific. The increasing morbidity and azole resistance of C. tropicalis have made clinical treatment difficult. The correlation between clonality and antifungal susceptibility of clinical C. tropicalis isolates has been reported. To study the putative correlation in C. tropicalis isolated from normally sterile body fluid specimens and explore the distinct clonal complex (CC) in Hefei, 256 clinical C. tropicalis isolates were collected from four teaching hospitals during 2016-2019, of which 30 were fluconazole-resistant (FR). Genetic profiles of 63 isolates, including 30 FR isolates and 33 fluconazole-susceptible (FS) isolates, were characterized using multilocus sequence typing (MLST). Phylogenetic analysis of the data was conducted using UPGMA (unweighted pair group method with arithmetic averages) and the minimum spanning tree algorithm. MLST clonal complexes (CCs) were analyzed using the goeBURST package. Among 35 differentiated diploid sequence types (DSTs), 16 DSTs and 1 genotype were identified as novel. A total of 35 DSTs were assigned to five major CCs based on goeBURST analysis. CC1 (containing DST376, 505, 507, 1221, 1222, 1223, 1226, and 1229) accounted for 86.7% (26/30) of the FR isolates. However, the genetic relationships among the FS isolates were relatively decentralized. The local FR CC1 belongs to a large fluconazole non-susceptible CC8 in global isolates, of which the putative founder genotype was DST225. The putative correlation between MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Hefei showed that DSTs are closely related to FR clones.

The Fourier-transform infrared spectroscopy-based method as a new typing tool for Candida parapsilosis clinical isolates.

The Fourier-transform infrared spectroscopy-based IR Biotyper is a straightforward typing tool for bacterial species, but its use with Candida species is limited. We applied IR Biotyper to Candida parapsilosis, a common cause of nosocomial bloodstream infection (BSI), which is aggravated by the intra-hospital spread of fluconazole-resistant isolates. Of 59 C. parapsilosis isolates studied, n = 56 (48 fluconazole-resistant and 8 fluconazole-susceptible) and n = 3 (2 fluconazole-resistant and 1 fluconazole-susceptible) isolates, respectively, had been recovered from BSI episodes in 2 spatially distant Italian hospitals. The latter isolates served as an outgroup. Of fluconazole-resistant isolates, n = 40 (including one outgroup) harbored the Y132F mutation alone and n = 10 (including one outgroup) harbored both Y132F and R398I mutations in the ERG11-encoded azole-target enzyme. Using a microsatellite typing method, which relies on the amplification of genomic short tandem repeats (STR), two major clusters were obtained based on the mutation(s) (Y132F or Y132F/R398I) present in the isolates. Regarding IR Biotyper, each isolate was analyzed in quintuplicate using an automatic (i.e., proposed by the manufacturer's software) or tentative (i.e., proposed by us) cutoff value. In the first case, four clusters were identified, with clusters I and II formed by Y132F or Y132F/R398I isolates, respectively. In the second case, six subclusters (derived by the split of clusters I and II) were identified. This allowed to separate the outgroup isolates from other isolates and to increase the IR Biotyper typeability. The agreement of IR Biotyper with STR ranged from 47% to 74%, depending on type of cutoff value used in the analysis. IMPORTANCE Establishing relatedness between clinical isolates of Candida parapsilosis is important for implementing rapid measures to control and prevent nosocomial transmission of this Candida species. We evaluated the FTIR-based IR Biotyper, a new typing method in the Candida field, using a collection of fluconazole-resistant C. parapsilosis isolates supposed to be genetically related due to the presence of the Y132F mutation. We showed that IR Biotyper was discriminatory but not as much as the STR method, which is still considered the method of choice. Further studies on larger series of C. parapsilosis isolates or closely related Candida species will be necessary to confirm and/or extend the results from this study.

Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt

IntroductionCandida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates.MaterialsFifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene.ResultsAntifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S).ConclusionsThis study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

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.

357. Risk Factors and Antifungal Susceptibility Pattern of Candida parapsilosis Candidemia: A Single centre Observation study from Central India.

Abstract Background Candida parapsilosis infection has recently emerged as an important antifungal-resistant nosocomial pathogen having the unique ability to grow on inanimate objects and surfaces. The aim of this study was to analyze the predisposing conditions, outcome, and antifungal susceptibility pattern of candidemia due to C. parapsilsosis complex. Methods A single-center retrospective observational study from January 2019 to December 2021 of all cases of candidemia was carried out at an 890-bedded University Hospital in central India. Data regarding demographic characteristics and clinical risk factors were collected from the patient’s medical records. Antifungal susceptibility testing was performed; MIC results were interpreted according to CLSI breakpoints (M27-A3). Risk factors and outcome association at the species level were analyzed by using Fisher’s exact test. Results Of 211 patients diagnosed with Candidemia during the study period, 53 (25.1%) were infected with C. parapsilosis which represented the second most frequently isolated yeast after C. albicans (n=98; 46.4%). A total of 26 (49%) C. parapsilosis isolates were non-susceptible to fluconazole (NSF), which included resistant (n = 20) and susceptible dose-dependent (n = 06) isolates. The median patient age was 63 years.15.3% were neonates. The majority of patients (90%) suffered from multiple comorbidities, diabetes mellitus (43%) being the commonest. A total of 55 % of patients underwent surgical intervention within 30 days from the onset of candidemia. Univariate logistic regression revealed that ICU admission (odds ratio [OR] 2.45), central venous catheter use (OR 2.46), renal impairment (OR 1.687) were more common among FNS isolates than fluconazole-susceptible (FS) isolates (all P < 0.05). The overall crude mortality at 30 days was 36%; higher in patients infected with FNS isolates than FS isolates. Conclusion There is an increase in the absolute number of invasive infections by C. parapsilosis observed over the past 2 years. At this moment, the percentage of fluconazole non-susceptible C. parapsilosis is very high and poses a threat to infected patients and has a clinical impact in our hospital. Being able to identify and treat infections caused by this pathogen is important to prevent clinical outbreaks. Disclosures All Authors: No reported disclosures.

Expression pattern of drug-resistance genes ERG11 and TAC1 in Candida albicans Clinical isolates.

Candida albicans (C. albicans) is an opportunistic fungus and the most common cause of vulvovaginal candidiasis (VVC). In recent years, the use of antifungal drugs has led to the incidence of drug-resistant C. albicans strains. The purpose of this study is twofold: to determine the pattern of drug susceptibility and the relationship between demographic factors and the incidence of drug resistance among C. albicans isolates and to investigate the expression pattern of drug-resistance genes ERG11 and TAC1 in C. albicans isolates. This descriptive cross-sectional study was conducted on 50C. albicans isolates from women with VVC. Antifungal susceptibility of the isolates was tested by M27-A3/S4 broth micro dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. High susceptibility rates were recorded for itraconazole and voriconazole (68%), followed by ketoconazole (46%). Fluconazole had the lowest susceptibility to C. albicans with susceptibility of 36%. The change in ERG11 and TAC1 genes expression was determined by qPCR. The mean ∆Ct values of ERG11 and TAC1genes were significantly different between fluconazole-resistant and susceptible groups (p < 0.001). Interestingly, we found that 77% of fluconazole-susceptible isolates had significantly upregulated ERG11 gene (2.9-99.0 fold). In addition, the expression of TAC1 was upregulated in 44% of fluconazole-susceptible isolates (3.86-89.8 fold). Our finding revealed that incidence of drug resistance in C. albicans is not simply controlled by genes but is a multi-factorial phenomenon, where several factors and mechanisms are involved in the process of drug resistance.

P195 Factors related to outcome of bloodstream infections due to Candida parapsilosis complex: A single center observation study from Central India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Candida parapsilosis infection has recently emerged as an important antifungal-resistant nosocomial pathogen having the unique ability to grow on inanimate objects and surfaces. Very limited studies from low and middle-income countries are available on the association of risk factors and antifungal susceptibility testing (AFST) of this species. The aim of this study was to analyze the predisposing conditions, outcome, and antifungal susceptibility pattern of candidemia due to C. parapsilsosis complex.MethodsA single-center retrospective observational study from January 2019 to December 2021 of all cases of candidemia was carried out at an 890-bedded University Hospital in central India. Data regarding demographic characteristics and clinical risk factors were collected from the patient's medical records. Antifungal susceptibility testing was performed; MIC results were interpreted according to CLSI breakpoints (M27-A3). Risk factors and outcome association at the species level were analyzed by using Fisher's exact test. Variables with a P ≤ .05 at the descriptive analysis were analyzed by Cox regression. A P-value of ≤ .05 was considered to represent the statistical significance and all statistical tests were two-tailed.ResultsOf 211 patients diagnosed with Candidemia during the study period, 53 (25.1%) were infected with C. parapsilosis which represented the second most frequently isolated yeast after C. albicans (n = 98; 46.4%). A total of 26 (49%) C. parapsilosis isolates were non-susceptible to fluconazole (NSF), which included resistant (n = 20) and susceptible dose-dependent (n = 06) isolates. The median patient age was 63 years.15.3% were neonates. The majority of patients (90%) suffered from multiple comorbidities, diabetes mellitus (43%) being the commonest. A total of 55% of patients underwent surgical intervention within 30 days from the onset of candidemia. Univariate logistic regression revealed that ICU admission [odds ratio (OR) 2.45], central venous catheter use (OR 2.46), renal impairment (OR 1.687) were more common among NSF isolates than fluconazole-susceptible (FS) isolates (all P <.05). The overall crude mortality at 30 days was 36%; higher in patients infected with FNS isolates than FS isolates.ConclusionThere is an increase in the absolute number of invasive infections by C.parapsilosis observed over the past 2 years. At this moment, the percentage of fluconazole non-susceptible C. parapsilosis is very high and poses a threat to infected patients and has a clinical impact in our hospital. Being able to identify and treat infections caused by this pathogen is important to prevent clinical outbreaks.

Whole Genome Sequencing Shows Genetic Diversity, as Well as Clonal Complex and Gene Polymorphisms Associated with Fluconazole Non-Susceptible Isolates of Candida tropicalis.

Resistance to azoles in Candida tropicalis is increasing and may be mediated by genetic characteristics. Using whole genome sequencing (WGS), we examined the genetic diversity of 82 bloodstream C. tropicalis isolates from two countries and one ATCC strain in a global context. Multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogenies were generated. Minimum inhibitory concentrations (MIC) for antifungal agents were determined using Sensititre YeastOne YO10. Eleven (13.2%) isolates were fluconazole-resistant and 17 (20.5%) were classified as fluconazole-non susceptible (FNS). Together with four Canadian isolates, the genomes of 12 fluconazole-resistant (18 FNS) and 69 fluconazole-susceptible strains were examined for gene mutations associated with drug resistance. Fluconazole-resistant isolates contained a mean of 56 non-synonymous SNPs per isolate in contrast to 36 SNPs in fluconazole-susceptible isolates (interquartile range [IQR] 46–59 vs. 31–48 respectively; p < 0.001). Ten of 18 FNS isolates contained missense ERG11 mutations (amino acid substitutions S154F, Y132F, Y257H). Two echinocandin-non susceptible isolates had homozygous FKS1 mutations (S30P). MLST identified high genetic diversity with 61 diploid sequence types (DSTs), including 53 new DSTs. All four isolates in DST 773 were fluconazole-resistant within clonal complex 2. WGS showed high genetic variation in invasive C. tropicalis; azole resistance was distributed across different lineages but with DST 773 associated with in vitro fluconazole resistance.

Genetic relatedness among azole-resistant Candida tropicalis clinical strains in Taiwan from 2014 to 2018

To monitor trends in the distribution of yeast species and the susceptibilities of these species to commonly prescribed antifungal drugs, we conduct the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) every 4 years. We found that 25 of 294 Candida tropicalis isolates from TSARY 2014 and 31 of 314 C. tropicalis isolates from TSARY 2018 were resistant to fluconazole. We determined the genetic relatedness among fluconazole-resistant C. tropicalis isolates by multilocus sequence typing (MLST). Among 174 C. tropicalis isolates, including all 56 fluconazole-resistant, all 26 susceptible-dose dependent and 92 selected fluconazole-susceptible isolates, 59 diploid sequence types (DSTs) were identified. We found that 22 of the 25 fluconazole-resistant C. tropicalis from TSARY 2014 and 29 of the 31 fluconazole-resistant C. tropicalis from TSARY 2018 were genetically related and belonged to the same cluster (clade 4). A combination of mutation and overexpression of ERG11, encoding the target of azole drugs, was the major mechanism contributing to drug resistance. Approximately two-thirds of reviewed patients infected or colonised by fluconazole-resistant C. tropicalis were azole-naïve. Furthermore, there was no evidence of patient-to-patient transmission. Because the clade 4 fluconazole-resistant C. tropicalis strain persists in Taiwan, it is important to identify the source of azole-resistant C. tropicalis to prevent the spread of this resistant strain.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

ABSTRACTResistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris.IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

A High Rate of Recurrent Vulvovaginal Candidiasis and Therapeutic Failure of Azole Derivatives Among Iranian Women.

Recurrent vulvovaginal candidiasis (RVVC) is one of the most prevalent fungal infections in humans, especially in developing countries; however, it is underestimated and regarded as an easy-to-treat condition. RVVC may be caused by dysbiosis of the microbiome and other host-, pathogen-, and antifungal drug-related factors. Although multiple studies on host-related factors affecting the outcome have been conducted, such studies on Candida-derived factors and their association with RVVC are lacking. Thus, fluconazole-tolerant (FLZT) isolates may cause fluconazole therapeutic failure (FTF), but this concept has not been assessed in the context of Candida-associated vaginitis. Iran is among the countries with the highest burden of RVVC; however, comprehensive studies detailing the clinical and microbiological features of this complication are scarce. Therefore, we conducted a 1-year prospective study with the aim to determine the RVVC burden among women referred to a gynecology hospital in Tehran, the association of the previous exposure to clotrimazole and fluconazole with the emergence of FLZT and fluconazole-resistant (FLZR) Candida isolates, and the relevance of these phenotypes to FTF. The results indicated that about 53% of the patients (43/81) experienced RVVC. Candida albicans and C. glabrata constituted approximately 90% of the yeast isolates (72 patients). Except for one FLZT C. tropicalis isolate, FLZR and FLZT phenotypes were detected exclusively in patients with RVVC; among them, 27.9% (12/43) harbored FLZR strains. C. albicans constituted 81.2% of FLZR (13/16) and 100% of the FLZT (13/13) isolates, respectively, and both phenotypes were likely responsible for FTF, which was also observed among patients with RVVC infected with fluconazole-susceptible isolates. Thus, FTF could be due to host-, drug-, and pathogen-related characteristics. Our study indicates that FLZT and FLZR isolates may arise following the exposure to over-the-counter (OTC) topical azole (clotrimazole) and that both phenotypes can cause FTF. Therefore, the widespread use of OTC azoles can influence fluconazole therapeutic success, highlighting the necessity of controlling the use of weak topical antifungals among Iranian women.

Fluconazole-Resistant Candida glabrata Bloodstream Isolates, South Korea, 2008-2018.

We investigated the clinical outcomes and molecular mechanisms of fluconazole-resistant (FR) Candida glabrata bloodstream infections. Among 1,158 isolates collected during multicenter studies in South Korea during 2008–2018, 5.7% were FR. For 64 patients with FR bloodstream infection isolates, the 30-day mortality rate was 60.9% and the 90-day mortality rate 78.2%; these rates were significantly higher than in patients with fluconazole-susceptible dose-dependent isolates (30-day mortality rate 36.4%, 90-day mortality rate 43.8%; p<0.05). For patients with FR isolates, appropriate antifungal therapy was the only independent protective factor associated with 30-day (hazard ratio 0.304) and 90-day (hazard ratio 0.310) mortality. Sequencing of pleiotropic drug-resistance transcription factor revealed that 1–2 additional Pdr1p amino acid substitutions (except genotype-specific Pdr1p amino acid substitutions) occurred in 98.5% of FR isolates but in only 0.9% of fluconazole-susceptible dose-dependent isolates. These results highlight the high mortality rate of patients infected with FR C. glabrata BSI isolates harboring Pdr1p mutations.

Experimental Evolution Identifies Adaptive Aneuploidy as a Mechanism of Fluconazole Resistance in Candida auris.

Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increasing concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of chromosome V. Genomic and transcriptome sequencing (RNA-Seq) analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally upregulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in the genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

Pola Kepekaan Candida krusei Isolat Jakarta terhadap Flukonazol

Pendahuluan. Candida krusei menghuni tubuh manusia sebagai saprofit namun dapat berubah menjadi patogen dan menyebabkan mikosis. Candida krusei umumnya bersifat resisten primer terhadap flukonazol, namun data laboratorium di Jakarta menemukan adanya C. krusei yang bersifat sensitif terhadap flukonazol sehinga dilakukan studi prospektif mengenai hasil uji kepekaan C. krusei isolat Jakarta, memakai isolat C. krusei yang berbeda.&#x0D; Metode: Penelitian dilaksanakan tahun 2019-2020 di laboratorium parasitologi Fakultas Kedokteran Universitas Indonesia. Sebanyak 15 isolat C. krusei diuji dengan metode difusi cakram. Sebelum digunakan isolat uji disegarkan dan dipurifikasi sehingga diperoleh koloni tunggal.&#x0D; Hasil: Isolat yang digunakan berasal dari feses, sputum, broncho-alveolar lavage (BAL) dan satu isolat dari abses paru. Hasil uji kepekaan didapat sembilan isolat resisten, satu isolat intermediate dan lima isolat yang bersifat sensitif terhadap flukonazol.&#x0D; Kesimpulan: Secara in vitro, C. krusei isolat Jakarta tidak seluruhnya bersifat resisten primer terhadap flukonazol, namun sebagian peka.

Dynamics of in vitro development of azole resistance in Candida tropicalis

ObjectivesIncreasing incidence of azole resistance in Candida tropicalis, especially to fluconazole, has been seen in Asian countries including India. Limited knowledge is available on the molecular mechanisms associated with the development of azole resistance in C. tropicalis. The present study examined the dynamics of in vitro azole resistance in C. tropicalis after prolonged treatment with fluconazole. MethodsNine fluconazole-susceptible isolates of C. tropicalis were used in this study. Fluconazole resistance was induced experimentally in C. tropicalis isolates. The stability of induced resistance and cross-resistance to other azoles was examined. The molecular mechanisms of azole resistance were assessed by measuring the expression and mutation analysis of different genes. ResultsVarying degrees of resistance [five with minimum inhibitory concentrations (MICs) ≤32 mg/L and four with MICs ≥128 mg/L] were noticed, and the resistance was developed in 3 months. Of the nine resistant isolates, four induced resistant isolates with MICs ≥128 mg/L presented temporal resistance stability up to 10 subcultures. These four isolates presented cross-resistance to other azoles and also an inducible overexpression of transporters (CDR1, CDR2, CDR3 and MDR1), ergosterol biosynthesis pathway genes (ERG1, ERG2, ERG3 and ERG11), transcription factors (TAC1 and UPC2) and stress-responsive genes (HSP90 and MKC1) was noticed. No mutations were seen in any of the four genes (ERG1, ERG3, ERG11 and UPC2) tested. ConclusionsCandida tropicalis isolates adapt themselves in the presence of continuous drug exposure and switch back to being susceptible in the absence of the drug. The acquisition of resistance in C. tropicalis is mediated by the overexpression of different resistance-related genes without any molecular alterations.

Gene Expression Analysis of Key Players Associated with Fluconazole Resistance in Candida albicans

Background: Fluconazole resistance in Candida albicans has become a serious public health problem. Most previous studies have focused on deciphering the relationship between fluconazole resistance and amino acid substitutions in ERG11, which encodes cytochrome P450 lanosterol 14α-demethylase whose enzymatic activity is inhibited by azoles. However, azole resistance in C. albicans is a multifactorial phenomenon and several lines of evidence indicate that other genes and mechanisms may contribute to the development of fluconazole resistance. Objectives: The present study aimed to investigate the underlying role of six genes in fluconazole-resistant clinical strains of C. albicans, including ERG11, RTA2, and the efflux pump genes CDR1, CDR2, MDR1, and FLU1. Methods: We collected 40 fluconazole-resistant isolates and 40 susceptible isolates from patients with Candida infections in the First Affiliated Hospital of Nanchang University in China from 2005 to 2008. The susceptibility of the isolates to antifungal agents was tested by the M27-A3 broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Then, the gene expression levels of several key players in azole resistance were quantified. Results: Most fluconazole-resistant strains analyzed in this study were found to be cross-resistant to ketoconazole, itraconazole, and clotrimazole. We observed that the FLU1 gene expression significantly increased (P < 0.05) and exhibited major changes in most of the fluconazole-resistant isolates (75.0%). In addition, the expression of a novel gene, RTA2, was remarkably upregulated (P < 0.05). Interestingly, we found that 10% of the fluconazole-resistant isolates were simultaneously associated with ERG11 mutation and overexpression of RTA2, CDR1, and FLU1 genes. Unlike other studies, we did not find any difference in the expression of CDR2, MDR1, and ERG11 genes between the fluconazole-susceptible and resistant isolates. Conclusions: Our findings suggested that the overexpression of FLU1 and RTA2 genes may cause azole resistance; this finding had not been reported previously in clinical isolates of C. albicans. The upregulation of FLU1 and RTA2 genes was the predominant mechanism of fluconazole resistance in C. albicans in China.