ERG11 Analysis among Clinical Isolates of Trichosporon asahii with Different Azole Susceptibility Profiles.

We analyzed a cohort of Trichosporon asahii strains with different MICs of fluconazole and voriconazole and evaluated the presence of ERG11 mutations. ERG11 mutation conferring an amino acid change was found and its resistance potential was evaluated by cloning into Saccharomyces cerevisiae susceptible host strain. Transformants were not resistant to either fluconazole nor voriconazole. Our results suggest that ERG11 variants exist among T. asahii isolates, but are not responsible for resistance phenotypes.

Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris.

Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

Limited ERG11 Mutations Identified in Isolates of Candida auris Directly Contribute to Reduced Azole Susceptibility.

Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. aurisERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. aurisERG11 mutations resulted directly in reduced azole susceptibility.

Significantly Improved Pharmacokinetics Enhances In Vivo Efficacy of APX001 against Echinocandin- and Multidrug-Resistant Candida Isolates in a Mouse Model of Invasive Candidiasis.

APX001 is a first-in-class, intravenous and orally available, broad-spectrum antifungal agent in clinical development for the treatment of life-threatening invasive fungal infections. The half-life of APX001A, the active moiety of APX001, is significantly shorter in mice than in humans (1.4 to 2.75 h in mice versus 2 to 2.5 days in humans), making the exploration of efficacy in mouse models difficult. After pretreatment with 1-aminobenzotriazole (ABT), a nonspecific cytochrome P450 inhibitor, greatly increased plasma APX001A exposure was observed in mice of different strains and of both genders. As a consequence, 26 mg/kg APX001 plus ABT sterilized kidneys in mice infected with Candida albicans, while APX001 alone at the same dose resulted in a modest burden reduction of only 0.2 log10 CFU/g, relative to the vehicle control. In the presence of ABT, 2 days of once-daily dosing with APX001 at 26 mg/kg also demonstrated significant in vivo efficacy in the treatment of Candida glabrata infections in mice. Potent kidney burden reduction was achieved in mice infected with susceptible, echinocandin-resistant, or multidrug-resistant strains. In contrast, the standard of care (micafungin) was ineffective in treating infections caused by the resistant C. glabrata isolates.

Changes in In Vitro Susceptibility Patterns of Aspergillus to Triazoles and Correlation With Aspergillosis Outcome in a Tertiary Care Cancer Center, 1999-2015.

BACKGROUND: Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. METHODS: We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. RESULTS: Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. CONCLUSIONS: Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort.

Emergence of Echinocandin Resistance Due to a Point Mutation in the fks1 Gene of Aspergillus fumigatus in a Patient with Chronic Pulmonary Aspergillosis.

We have identified the first case of an fks1 hot spot 1 point mutation causing echinocandin resistance in a clinical Aspergillus fumigatus isolate recovered from a chronic pulmonary aspergillosis patient with an aspergilloma who first failed azole and polyene therapy and subsequently failed micafungin treatment.

De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That both Overlap and Are Distinct from Those Conferring Echinocandin Resistance.

SCY-078 is an orally active antifungal whose target is the ß-(1,3)-d-glucan synthase (GS). We evaluated the spontaneous emergence of SCY-078-resistant Candida glabrata isolates following drug exposure in vitro Resistant isolates were analyzed using broth microdilution methodology and FKS sequencing. The kinetic inhibition parameter IC50 (50% inhibitory concentration) was also determined from GS complexes. The spectrum of resistance mutations found suggested a partially overlapping but independent binding site for SCY-078 relative to echinocandins on GS.

Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling.

Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidy-independent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

CD101: a novel long-acting echinocandin.

CD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin-susceptible and -resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin-resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild-type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long-lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory.

Enfumafungin derivative MK-3118 shows increased in vitro potency against clinical echinocandin-resistant Candida Species and Aspergillus species isolates.

MK-3118 is as an orally active new antifungal in the early stage of clinical development that inhibits the biosynthesis of ß-(1,3)-glucan. We evaluated the in vitro activity of this compound against wild-type and echinocandin-resistant (ER) isolates containing mutations in the FKS gene(s) of Candida spp. and Aspergillus spp. MK-3118 demonstrated enhanced efficacy for most C. albicans and C. glabrata ER isolates relative to caspofungin, with decreased MICs and half-maximal inhibitory concentrations (IC50s).

Target enzyme mutations confer differential echinocandin susceptibilities in Candida kefyr.

Candida kefyr is an increasingly reported pathogen in patients with hematologic malignancies. We studied a series of bloodstream isolates that exhibited reduced echinocandin susceptibilities (RES). Clinical and surveillance isolates were tested for susceptibilities to all three echinocandins, and those isolates displaying RES to one or more echinocandins were selected for molecular and biochemical studies. The isolates were analyzed for genetic similarities, and a subset was analyzed for mutations in the echinocandin target gene FKS1 and glucan synthase echinocandin sensitivities using biochemical methods. The molecular typing did not indicate strong genetic relatedness among the isolates except for a series of strains recovered from a single patient. Two unrelated isolates with RES had previously uncharacterized FKS1 mutations: R647G and deletion of amino acid 641 (F641Δ). Biochemical analysis of the semipurified R647G glucan synthase generated differential echinocandin sensitivity (resistance to micafungin only), while the deletion of F641 resulted in a glucan synthase highly insensitive to all three echinocandins. The consecutive isolates from a single patient with RES all harbored the common S645P mutation, which conferred resistance to all three echinocandins. The MIC values paralleled the glucan synthase inhibition kinetic data, although the S645P isolates displayed relatively higher susceptibility to caspofungin (2 µg/ml) than the other two echinocandins (>8 µg/ml). These findings highlight novel and common FKS1 mutations in C. kefyr isolates. The observation of differential susceptibilities to echinocandins may provide important mechanistic insights for echinocandin antifungals.

Breakthrough candidemia due to multidrug-resistant Candida glabrata during prophylaxis with a low dose of micafungin.

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-ß-D-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Set of classical PCRs for detection of mutations in Candida glabrata FKS genes linked with echinocandin resistance.

Clinical echinocandin resistance among Candida glabrata strains is increasing, especially in the United States. Antifungal susceptibility testing is considered mandatory to guide therapeutic decisions. However, these methodologies are not routinely performed in the hospital setting due to their complexity and the time needed to obtain reliable results. Echinocandin failure in C. glabrata is linked exclusively to Fks1p and Fks2p amino acid substitutions, and detection of such substitutions would serve as a surrogate marker to identify resistant isolates. In this work, we report an inexpensive, simple, and quick classical PCR set able to objectively detect the most common mechanisms of echinocandin resistance in C. glabrata within 4 h. The usefulness of this assay was assessed using a blind collection of 50 C. glabrata strains, including 16 FKS1 and/or FKS2 mutants.

Correction: Jiménez-Ortigosa et al. Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins. J. Fungi 2021, 7, 120.

The authors wish to update the article title to "Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins" [...].

Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations.

BACKGROUND: Fluconazole (FLC) resistance is common in C. glabrata and echinocandins are often used as first-line therapy. Resistance to echinocandin therapy has been associated with FKS1 and FKS2 gene alterations. METHODS: We reviewed records of all patients with C. glabrata bloodstream infection at Duke Hospital over the past decade (2001-2010) and correlated treatment outcome with minimum inhibitory concentration (MIC) results and the presence of FKS gene mutations. For each isolate, MICs to FLC and echinocandins (anidulafungin, caspofungin, and micafungin) and FKS1 and FKS2 gene sequences were determined. RESULTS: Two hundred ninety-three episodes (313 isolates) of C. glabrata bloodstream infection were analyzed. Resistance to echinocandins increased from 4.9% to 12.3% and to FLC from 18% to 30% between 2001 and 2010, respectively. Among the 78 FLC resistant isolates, 14.1% were resistant to 1 or more echinocandin. Twenty-five (7.9%) isolates harbored a FKS mutation. The predictor of a FKS mutant strain was prior echinocandin therapy (stepwise multivariable analysis, odds ratio, 19.647 [95% confidence interval, 7.19-58.1]). Eighty percent (8/10) of patients infected with FKS mutants demonstrating intermediate or resistant MICs to an echinocandin and treated with an echinocandin failed to respond or responded initially but experienced a recurrence. CONCLUSIONS: Echinocandin resistance is increasing, including among FLC-resistant isolates. The new Clinical and Laboratory Standards Institute clinical breakpoints differentiate wild-type from C. glabrata strains bearing clinically significant FKS1/FKS2 mutations. These observations underscore the importance of knowing the local epidemiology and resistance patterns for Candida within institutions and susceptibility testing of echinocandins for C. glabrata to guide therapeutic decision making.

Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins.

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells.

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target ß-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida sp., a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single-cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, of which the latter was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypotolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance. IMPORTANCE Echinocandin drugs are a first-line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen, and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here, we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypotolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.

Antifungal Drug Susceptibility and Genetic Characterization of Fungi Recovered from COVID-19 Patients.

Fungal infections are common complications of respiratory viral infections and are associated with the increased need for intensive care and elevated mortality. Data regarding microbiological and molecular characteristics of such infections in COVID-19 patients are scarce. Here, we performed a comprehensive analysis, including species identification, antifungal susceptibility testing, molecular resistance determinants analysis, typing, and retrospective clinical data review, of fungal isolates recovered from 19 COVID-19 patients, who were hospitalized at the Hackensack University Medical Center (HUMC) in Hackensack, New Jersey, USA, in the initial phase of the pandemic from April-May 2020. In total, 17 Candida albicans, two C. parapsilosis, and two Aspergillus fumigatus were analyzed. All Candida spp. isolates were susceptible to micafungin and azole drugs (fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole). A. fumigatus isolates were susceptible to micafungin and all triazole drugs except fluconazole (intrinsic resistance). Multilocus sequence typing (MLST) of C. albicans isolates revealed 15 different sequence types (STs), which clustered below the clade-defining limit of p-distance < 0.04. Pulsed-field gel electrophoresis (PFGE) karyotyping revealed no chromosomal rearrangements in these isolates. A. fumigatus isolates were of different, non-related genotypes. We speculate that virus- and drug-induced immunosuppression (94.7% of the patients received corticosteroids), together with prolonged hospital stay (median duration of 29 days) and mechanical ventilation (median duration of 24 days) likely increased the susceptibility to secondary respiratory and bloodstream infections in the studied patient population. The presence of fungi in blood or respiratory tract fluid was a prognosticator for poor clinical outcome, which presented as an 89.5% 30-day mortality in our patient cohort.