Antifungal Resistance Trends of Candida auris Clinical Isolates, New York-New Jersey, 2016-2020

About 55% of U.S. Candida auris clinical cases were reported from New York and New Jersey from 2016 through 2020. Nearly all New York-New Jersey clinical isolates (99.8%) were fluconazole resistant, and 50% were amphotericin B resistant. Echinocandin resistance increased from 0% to 4% and pan-resistance increased from 0 to <1% for New York C. auris clinical isolates but not for New Jersey, highlighting the regional differences.

Changing Trends of Candida Species and Antifungal Susceptibility Profile of Candida Bloodstream Isolates: A 5-Year Retrospective Survey

Background: Candida species have emerged as one of the most common causes of bloodstream infections (BSIs). There are limited data on the distribution of Candida spp. and susceptibility by year. Objectives: In this study, we analyzed changes in the distribution of Candida spp. and their antifungal susceptibility profiles from blood cultures. Methods: Records from January 2016 to December 2020 were obtained from the microbiology laboratory in Istanbul. Antifungal susceptibility tests were performed using the VITEK 2 compact system and evaluated according to EUCAST breakpoints. A total of 241 unique candidemia episodes were included in this study. Results: Candida albicans was the predominant pathogen (n = 95, 39.42%), followed by C. parapsilosis (n = 82, 34.02%), C. glabrata (n = 18, 7.47%), C. tropicalis (n = 17, 7.05%), C. krusei (n = 15, 6.22%), and other Candida spp. (n = 14, 5.79%). There was no statistically significant difference in the percentage of episodes of Candida spp. After data analysis, a tendency to shift from C. albicans to C. parapsilosis was observed in the period analyzed in this study. Candida albicans was the most common species in intensive care units (ICUs), hematology and hemopoietic stem cell transplantation units, and surgical clinics, with C. parapsilosis predominant in medical clinics. In general, micafungin susceptibility was the highest, and fluconazole was the lowest. There was reduced sensitivity to fluconazole and voriconazole for C. albicans and C. parapsilosis over 5 years. Conclusions: Detecting changes in the distribution of Candida spp. and antifungal susceptibility over time will lead to the selection of appropriate empirical therapy and monitor phenomena of antifungal resistance. Empirical treatment with antifungal agents is associated with high costs, toxicities, and risk of antifungal resistance. Therefore, it is mandatory to determine and monitor Candida spp. and antifungal susceptibility testing to select appropriate antifungal agents.

Tools for detecting a “superbug”: updates on Candida auris testing

Candida auris is an emerging yeast species that has the unique characteristics of patient skin colonization and rapid transmission within healthcare facilities and the ability to rapidly develop antifungal resistance. When C. auris first started appearing in clinical microbiology laboratories, it could only be identified using DNA sequencing. In the decade since its first identification outside of Japan there have been many improvements in the detection of C. auris . These include the expansion of MALDI-TOF databases to include C. auris , the development of both laboratory-developed tests and commercially available kits for its detection, and special CHROMagar for identification from laboratory specimens. Here we discuss the current tools and resources that are available for C. auris identification and detection.

Antifungal Resistance in Clinical Isolates of Candida glabrata in Ibero-America

In different regions worldwide, there exists an intra-and inter-regional variability in the rates of resistance to antifungal agents in Candida glabrata, highlighting the importance of understanding the epidemiology and antifungal susceptibility profiles of C. glabrata in each region. However, in some regions, such as Ibero-America, limited data are available in this context. Therefore, in the present study, a systematic review was conducted to determine the antifungal resistance in C. glabrata in Ibero-America over the last five years. A literature search for articles published between January 2015 and December 2020 was conducted without language restrictions, using the PubMed, Embase, Cochrane Library, and LILACS databases. The search terms that were used were “Candida glabrata” AND “antifungal resistance” AND “Country”, and 22 publications were retrieved from different countries. The use of azoles (fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, ketoconazole, and miconazole) varied between 4.0% and 100%, and that of echinocandins (micafungin, caspofungin, and anidulafungin) between 1.1% and 10.0%. The limited information on this subject in the region of Ibero-America emphasizes the need to identify the pathogens at the species level and perform antifungal susceptibility tests that may lead to the appropriate use of these drugs and the optimal doses in order to avoid the development of antifungal resistance or multi-resistance.

The effect of acquired triazole resistance on abiotic stress tolerance and virulence in Candida auris micro evolved strains

Recently, C. auris become one of the most prominent members of the genus Candida. Since its occurrence, several C. auris outbreaks have been reported worldwide. These outbreaks were associated with isolates displaying decreased susceptibility towards fluconazole, the first-line agent for prophylaxis. Fluconazole is the most frequently used antifungal drug to treat bloodstream Candida infections. The physiological effects of acquired antifungal resistance was investigated in this species using fluconazole, posaconazole and voriconazole resistant mutant strains generated by the in vitro microevolution method. Alterations in antifungal susceptibility and cross resistance were determined by the microdilution method, utilizing azoles (fluconazole, voriconazole, posaconazole), echinocandins (caspofungin, micafungin, anidulafungin) and a polyene (amphotericin B). Changes in the abiotic stress tolerance was examined by spotting assay, using osmotic stressors, cell wall perturbants and a membrane detergent. To evaluate the impact of the acquired resistance on sterol biosynthesis, ergosterol composition of all generated mutant strains were examined. A potential relationship between virulence and acquired antifungal resistance was also studied both in vitro and in vivo. Phagocytosis of the generated strains by J774.2 mouse macrophage-like cells was measured and analyzed by flow cytometry. In the murine infection model fungal burden of the triazole evolved strains was determined in spleen, kidney, liver and brain and compared to the fungal burden associated with the initial azole susceptible strain. Significant differences in virulence of the initial and the generated strains was observed suggesting a potential connection between the virulence and antifungal susceptibility of the emerging fungal pathogen, C. auris.

Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis

Introduction Candida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions. Materials and Methods Six clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®. Results/Discussion The impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.

Combined Pan-, Population-, and Phylo-Genomic Analysis of Aspergillus fumigatus Reveals Population Structure and Lineage-Specific Diversity

Aspergillus fumigatus is a deadly agent of human fungal disease, where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics, with the potential to address the full genetic repertoire of a species. Here, we use a combination of population genomics, phylogenomics, and pan-genomics to assess population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes in A. fumigatus. We provide evidence for three distinct populations of A. fumigatus, structured by both gene variation (SNPs and indels) and distinct gene presence-absence variation with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism, hinting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Despite low levels of outcrossing, A. fumigatus demonstrated a large pan-genome including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome based approach for evaluating intraspecific variation, and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.

Antifungal susceptibility profile of invasive Candida glabrata isolates (2009–2020) from a tertiary care hospital laboratory in Pakistan

Introduction. Invasive infections with Candida glabrata are a global concern due to poor clinical outcomes and propensity to acquire resistance to antifungal agents. Hypothesis/Gap Statement. Monitoring emerging resistance and trends in Candida glabrata, an important agent of candidemia in Pakistan, is critical for patient management; data that is missing from Pakistan. Aim. Thus, this study evaluated antifungal resistance and MICs) distribution in invasive C. glabrata isolates from Pakistan. Methods. This cross-sectional and retrospective study was conducted from January 2009 to March 2020 at a clinical laboratory in Pakistan that has a nation-wide network. Antifungal susceptibility data of 277 candidemia, deep organ and soft tissue (invasive) C. glabrata sensu lato isolates against fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, micafungin, caspofungin and amphotericin B was retrieved. Susceptibility testing was performed using colorimetric broth microdilution and interpreted using CLSI criteria. Demographics, clinical history and outcome were studied. Chi-square test was used to demonstrate association between antifungal resistance and clinical characteristics of the patients. Results. We identified 277 patients with invasive C. glabrata infection. Of which 48 (18.4%) isolates were resistant to fluconazole (MIC ≥64 mg l−1), one isolate each was resistant to amphotericin (MIC=2 mg l−1), anidulafungin (MIC=1 mg l−1) and micafungin (MIC=0.5 mg l−1). MIC90 for fluconazole was 64 mg l−1 and other triazoles 2 mg l−1, caspofungin 0.12 mg l−1, anidulafungin 0.06 mg l−1, micafungin 0.03 mg l−1 and amphotericin 0.5 mg l−1. Fluconazole MIC ≥64 mg l−1, caspofungin MIC >0.06 mg l−1 and amphotericin MIC >0.25 mg l−1 (above MIC50) were significantly associated with patient being alive at the time of reporting, no use of healthcare devices, nor infection with other fungi. Fluconazole resistance was significantly associated with prior antifungal use by the patient. Conclusion. Surveillance data of antifungal resistance among common Candida species should be monitored closely for identification of resistant strains.