Azole Resistance Research Articles

Association of multilocus sequence typing, MSH2 gene mutations, and antifungal resistance in Candida glabrata: implications for clinical outcomes in Chinese hospitals

BackgroundCandida glabrata is the second most common cause of invasive candidiasis worldwide. In this study, we determined the clinical characteristics and drug sensitivity of C. glabrata isolates and investigated the associations between MSH2 gene mutations, sequence types (ST), and drug resistance.MethodsA total of 154 C. glabrata isolates were collected from patients being treated in three hospitals in China. The antifungal sensitivity of the strains was assessed using the broth microdilution method. Multilocus sequence typing (MLST) was also performed, followed by MSH2 sequencing. The clinical features and outcomes of C. glabrata infection were analysed for a total of 49 strains, which were collected from patients with invasive Candida infection at Longhua Hospital.ResultsAll 154 isolates were found to be susceptible to amphotericin, 5-fluorocytosine, anidulafungin, caspofungin, and micafungin, whereas 11.7% were fluconazole-resistant, 18.8% were itraconazole non-wild type, and 35.7% were voriconazole non-wild type. ST7 (62.34%) was the most common ST genotype, followed by ST10 (16.88%) and ST15 (7.79%). The total azole resistance rates for all isolates, ST7, ST10, and other STs were 36.4, 42.7, 34.6, and 18.8%, respectively. The ST7 and ST10 isolates were characterised by a higher drug resistance rate than the other minor ST isolates. Moreover, 59.09% of isolates had one or more MSH2 non-synonymous mutations, with V239L being the most commonly detected mutation. The frequency of MSH2 mutations was significantly higher in azole-resistant isolates than in other isolates, whereas P6L or L87P mutations were associated with the highest azole resistance rates of up to 87.5% and 80%, respectively. Our results indicated that ST7 and ST15 are independent predictors of mortality caused by C. glabrata infection and revealed a higher 30-day mortality in patients infected with these strains than in those infected with other ST isolates.ConclusionsOur findings revealed the relationships between MLST, MSH2 gene mutations, and drug resistance in the common pathogenic fungus C. glabrata, and thereby enabled us to identify strains that are associated with higher rates of mortality. These findings will contribute to enhancing our understanding of the pathogenesis of C. glabrata infection.

Candida species covered from Traditional Cheeses: Characterization of C. albicans Regarding Virulence Factors, Biofilm Formation, Caseinase Activity, Antifungal Resistance and Phylogeny

This study has provided characterization data (carriage of virulence, antifungal resistance, caseinase activity, biofilm-forming ability and genotyping) of Candida albicans isolates and the occurrence of Candida species in traditional cheeses collected from Kayseri, Türkiye. Phenotypic (E-test, Congo red agar and microtiter plate tests) and molecular tests (identification, virulence factors, biofilm-formation, antifungal susceptibility) were carried out. The phylogenetic relatedness of C. albicans isolates was obtained by constructing the PCA dendrogram from the mass spectra data. Of 102 samples, 13 (12.7%) were found to be contaminated with C. albicans, 15 (14.7%), 10 (9.8%) and five (4.9%) were found to be contaminated with C. krusei, C. lusitane and C. paraplosis, respectively. While seven (16.2%) of 43 Candida spp. isolates were obtained from cheese collected from villages, 36 (83.7%) belonged to cheeses collected from traditional retail stores. The carriage rate of C. albicans isolates belonging to virulence factors HSP90 and PLB1 genes was 30.7%. ALST1, ALST3, BCR, ECE, and HWP (virulence and biofilm-associated) genes were harbored by 30.7%, 23%, 38.4%, 53.8%, and 38.4% of the 13 isolates. According to the microplate test, eight (61.5%) of 13 isolates had strong biofilm production. ERG11 and FKS1 (antifungal resistance genes) were found in 46.1% and 23% of 13 isolates, respectively. Due to missense mutations, K128T, E266D and V488I amino acid changes were detected for some isolates regarding azole resistance. As a result of the E-test, of the 13 isolates, one (7.6%) was resistant to flucytosine, four (30.7%) were resistant to caspofungin, and nine (69.2%) were resistant to fluconazole. The PCA analysis clustered the studied isolates into two major clades. C. albicans isolates of traditional cheese collected from villages were grouped in the same cluster. Among the C. albicans isolates from village cheese, there were those obtained from the same dairy milk at different times. Samples from the same sales points produced at different dairy farms were also contaminated with C. albicans. Concerning food safety standards applied from farm to fork, in order to prevent these pathogenic agents from contaminating cheeses, attention to the hygiene conditions of the sale points, conscious personnel, prevention of cross contamination will greatly reduce public health threats in addition to the application of animal health control, milking hygiene, pasteurization parameters in traditional cheese production.

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene. In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S). We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

Detection in Orchards of Predominant Azole-Resistant Candida tropicalis Genotype Causing Human Candidemia, Taiwan.

Fluconazole-resistant clade 4 Candida tropicalis causing candidemia in humans has been detected in tropical/subtropical areas, including those in China, Singapore, and Australia. We analyzed 704 individual yeasts isolated from fruits, soil, water, and farmers at 80 orchards in Taiwan. The most common pathogenic yeast species among 251 isolates recovered from farmers were Candida albicans (14.7%) and C. parapsilosis (11.6%). In contrast, C. tropicalis (13.0%), C. palmioleophila (6.6%), and Pichia kudriavzevii (6.0%) were prevalent among 453 environmental isolates. Approximately 18.6% (11/59) of C. tropicalis from the environment were resistant to fluconazole, and 81.8% (9/11) of those belonged to the clade 4 genotype. C. tropicalis susceptibility to fluconazole correlated with susceptibilities to the agricultural azole fungicides, difenoconazole, tebuconazole, and triadimenol. Tandem gene duplications of mutated ERG11 contributed to azole resistance. Agriculture environments are a reservoir for azole-resistant C. tropicalis; discontinuing agricultural use of azoles might reduce emergence of azole-resistant Candida spp. strains in humans.

Exploration of novel mechanisms of azole resistance in Candida auris.

Candida auris is a pathogenic yeast of particular concern because of its ability to cause nosocomial outbreaks of invasive candidiasis (IC) and to develop resistance to all current antifungal drug classes. Most C. auris clinical isolates are resistant to fluconazole, an azole drug that is used for the treatment of IC. Azole resistance may arise from diverse mechanisms, such as mutations of the target gene (ERG11) or upregulation of efflux pumps via gain of function mutations of the transcription factors TAC1 and/or MRR1. To explore novel mechanisms of azole resistance in C. auris, we applied an in vitro evolutionary protocol to induce azole resistance in a TAC1A/TAC1B/MRR1 triple-deletion strain. Azole-resistant isolates without ERG11 mutations were further analyzed. In addition to a whole chromosome aneuploidy of chromosome 5, amino acid substitutions were recovered in the transcription factor Upc2 (N592S, L499F), the ubiquitin ligase complex consisting of Ubr2 (P708T, H1275P) and Mub1 (Y765*), and the mitochondrial protein Mrs7 (D293H). Genetic introduction of these mutations in an azole-susceptible wild-type C. auris isolate of clade IV resulted in significantly decreased azole susceptibility. Real-time reverse transcription PCR analyses were performed to assess the impact of these mutations on the expression of genes involved in azole resistance, such as ERG11, the efflux pumps CDR1 and MDR1 or the transcription factor RPN4. In conclusion, this work provides further insights in the complex and multiple pathways of azole resistance of C. auris. Further analyses would be warranted to assess their respective role in azole resistance of clinical isolates.

Routine laboratory screening for cyp51a-related azole resistance in Aspergillus fumigatus using Etest: Concordance with gene sequencing.

We evaluated the combined performance of itraconazole and voriconazole Etest® gradient concentration strips for the detection of Aspergillus fumigatus azole resistance associated with cyp51a mutations confirmed by gene sequencing. Among 118 A. fumigatus clinical isolates collected in a French center, 6 (5%) had azole resistance mutations, 5 of which were probably of environmental origin. Using recent method-dependent epidemiological cutoff values as thresholds, the combination's sensitivity and specificity were 100% [95% confidence interval 61-100] and 99% [95-100]. Our results support itraconazole and voriconazole Etest® combined use as a promising self-sufficient method for simple, efficient and reliable cyp51a-related azole-resistant A. fumigatus detection.

Unveiling environmental transmission risks: comparative analysis of azole resistance in Aspergillus fumigatus clinical and environmental isolates from Yunnan, China.

Azole resistance in Aspergillus fumigatus poses a significant clinical challenge globally. Our previous epidemiological analysis revealed a remarkably high frequency (~80%) of azole-resistant A. fumigatus in Yunnan's greenhouse environments, prompting increased local and regional research for targeted control strategies. In this study, we analyzed 94 clinical A. fumigatus isolates from Yunnan, comparing their susceptibility profiles and genotypic characteristics with environmental strains previously isolated. While the overall frequency of azole resistance in clinical isolates was lower than that in environmental samples, a significant prevalence of cross-resistance, with varying resistance patterns based on minimum inhibitory concentration (MIC) levels was observed, which exceeded rates in other regions of China. Specific mutation combinations in the cyp51A gene were linked to elevated MIC values in clinical and/or environmental samples, while some resistant strains with wild-type cyp51A remain unexplained, indicating a need for further investigation into their resistance mechanisms. The differences in unique genetic elements and the distinct genetic differentiation observed between clinical and environmental isolates can be attributed to Yunnan's unique geomorphology and potential genotype importation from other provinces and abroad. Extensive allele exchanges and sharing contributed to the selection of azole-resistant clinical isolates, suggesting a common environmental origin, and the transmission routes of local drug-resistant strains cannot be excluded. These findings emphasize the imperative for regional and targeted surveillance to monitor resistance trends and guide effective antifungal therapy, and management strategies to mitigate invasive aspergillosis risk in this region.IMPORTANCEAzole resistance in Aspergillus fumigatus is a major global health concern, with particularly high rates (~80%) observed in Yunnan's greenhouse environments. This study compares azole resistance in 94 clinical isolates from Yunnan with environmental strains, revealing lower clinical resistance but significant cross-resistance and distinct resistance patterns. Specific mutations in the cyp51A gene were associated with elevated minimum inhibitory concentration values, though some resistant strains had wild-type cyp51A, highlighting the need for further research. The unique genetic profiles and potential external genotype influences in Yunnan emphasize the need for targeted regional surveillance. Effective monitoring and control strategies are essential to manage and mitigate the risk of invasive aspergillosis.

Candida glabrata maintains two HAP1 ohnologs, HAP1A and HAP1B, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata, a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Hap1A and Hap1B, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance.

Trichophyton indotineae Erg1Ala448Thr Strain Expressed Constitutively High Levels of Sterol 14-α Demethylase Erg11B mRNA, While Transporter MDR3 and Erg11A mRNA Expression Was Induced After Addition of Short Chain Azoles

Trichophyton indotineae is an emerging pathogen causing recalcitrant skin infections and exhibiting multiple resistances to azoles and allylamines. Squalene epoxidase erg1Ala448Thr mutants often show association with azole resistance. RT-PCR gene expression analysis helps to elucidate the connection between ergosterol biosynthesis regulation and efflux control through the activation of multidrug resistance (MDR) and major facilitator superfamily (MFS1) transporters as well as heat shock proteins (HSP). Several T. indotineae isolates demonstrated a heat-dependent increase of Erg11B transcripts combined with downregulation of Erg1, suggesting a protective role for Erg11B. They also showed persistent upregulation of MFS1. The addition of fluconazole or voriconazole induced the expression of Erg11A, MDR3 and, to a lesser extent, Erg11B and Erg1. The azole-resistant erg1Ala448Thr mutant UKJ 476/21 exhibited exceptionally high transcript levels of sterol 14-αdemethylase Erg11B, combined with the inability of HSP60 and HSP90 to respond to increasing growth temperatures. Itraconazole demonstrated similar effects in a few T. indotineae isolates, but terbinafine did not enhance Erg1 transcription at all. Overexpression of Erg11B may explain the multiple azole resistance phenotype, whereas Erg11B point mutations are not associated with resistance to azoles used for medical treatment.

Aspergillus fumigatus sensu stricto genetic diversity from cystic fibrosis patients

We aimed to access the genetic diversity of Apergillus fumigatus strains obtained from cystic fibrosis (CF) patients from southern Brazil. A. fumigatus sensu stricto isolates from respiratory clinical specimens were genotyped by microsatellite markers and azole resistance was evaluated by azole-agar screening. Twenty-seven isolates from twenty-seven patients showed a high genetic diversity, with the differentiation of 25 different genotypes (25 unique and one common to two isolates). All isolates were susceptible to the azoles tested. We believe that prospectively monitoring A. fumigatus genetic diversity is essential to identify interpatient transmission and outbreaks, as is the identification of resistant strains.

Investigating role of positively selected genes and mutation sites of ERG11 in drug resistance of Candida albicans.

The steep increase in acquired drug resistance in Candida isolates has posed a great challenge in the clinical management of candidiasis globally. Information of genes and codon sites that are positively selected during evolution can provide insights into the mechanisms driving antifungal resistance in Candida. This study aimed to create a manually curated list of genes of Candida spp. reported to be associated with antifungal resistance in literature, and further investigate the structure-function implications of positively selected genes and mutation sites. Sequence analysis of antifungal drug resistance associated gene sequences from various species and strains of Candida revealed that ERG11 and MRR1 of C. albicans were positively selected during evolution. Four sites in ERG11 and two sites in MRR1 of C. albicans were positively selected and associated with drug resistance. These four sites (132, 405, 450, and 464) of ERG11 are predictive markers for azole resistance and have evolved over time. A well-characterized crystal structure of sterol-14-α-demethylase (CYP51) encoded by ERG11 is available in PDB. Therefore, the stability of CYP51 in complex with fluconazole was evaluated using MD simulations and molecular docking studies for two mutations (Y132F and Y132H) reported to be associated with azole resistance in literature. These mutations induced high flexibility in functional motifs of CYP51. It was also observed that residues such as I304, G308, and I379 of CYP51 play a critical role in fluconazole binding affinity. The insights gained from this study can further guide drug design strategies addressing antimicrobial resistance.

Unveiling the roles of CaSDH8 in Candida albicans: Implications for virulence and azole resistance

ABSTRACT Candida albicans is the most common pathogen in systemic fungal diseases, exhibits a complex pathogenic mechanism, and is increasingly becoming drug tolerant. Therefore, it is particularly important to study the genes associated with virulence and resistance of C. albicans. Here, we identified a gene (orf19.1588) that encodes a conserved mitochondrial protein known as CaSDH8, upon deletion of CaSdh8, the deleted strain (Casdh8Δ/Δ) experienced impaired growth, hyphal development, and virulence. Casdh8Δ/Δ displayed a reduced capacity to utilize alternative carbon sources, along with detrimental alterations in reactive oxygen species (ROS), mitochondrial membrane potential (MMP) depolarization, and adenosine triphosphate (ATP) levels. Interestingly, Casdh8Δ/Δ demonstrated resistance to azole drugs, and under the influence of fluconazole, the cell membrane permeability and mitochondrial function of Casdh8Δ/Δ were less compromised than those of the wild type, indicating a reduction in the detrimental effects of fluconazole on Casdh8Δ/Δ. These findings highlight the significance of CaSDH8 as a crucial gene for the maintenance of cellular homoeostasis. Our study is the first to document the effects of the CaSDH8 gene on the virulence and azole resistance of C. albicans at both the molecular and animal levels, providing new clues and directions for the antifungal infection and the discovery of antifungal drug targets.

Superiority trials in invasive aspergillosis: a harsh reality check with the IA-DUET (HOVON502) trial.

The IA-DUET study aimed to compare azole-echinocandin combination with azole monotherapy for invasive aspergillosis. Recruitment was hindered by patient ineligibility, competing studies, and guidelines favoring combination therapy when azole resistance was unknown. The low IA-attributable mortality suggests future trials may benefit from cluster randomization or composite endpoints to enhance efficiency.

Most azole resistance mutations in the Candida albicans drug target confer cross-resistance without intrinsic fitness cost.

Azole antifungals are the main drugs used to treat fungal infections. Amino acid substitutions in the drug target Erg11 (Cyp51) are a common resistance mechanism in pathogenic yeasts. How many and which mutations confer resistance is, however, largely unknown. Here we measure the impact of nearly 4,000 amino acid variants of Candida albicans Erg11 on the susceptibility to six clinical azoles. This was achieved by deep mutational scanning of CaErg11 expressed in Saccharomyces cerevisiae. We find that a large fraction of mutations lead to resistance (33%), most resistance mutations confer cross-resistance (88%) and only a handful of resistance mutations show a significant fitness cost (9%). Our results reveal that resistance to azoles can arise through a large set of mutations and this will probably lead to azole pan-resistance, with little evolutionary compromise. This resource will help inform treatment choices in clinical settings and guide the development of new drugs.

SPECIES IDENTIFICATION AND IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF ASPERGILLUS ISOLATED FROM VARIOUS CLINICAL SAMPLES

Objectives: Aspergillus is ubiquitous mold species present in environment in the form of spores. Infection due to Aspergillus is uncommon in immunocompetent individuals unless they possess any abnormality or have undergone any treatment with corticosteroids. In immunocompromised individuals, infection by this fungus is common among people with prolonged neutropenia, transplants, and human immunodeficiency virus infection. The symptoms are diverse, ranging from allergic reactions to invasive aspergillosis and life-threatening complications. The aim of this study is to isolate and identify Aspergillus species obtained from various clinical specimens and perform antifungal susceptibility testing (AFST). Methods: A total of 200 isolates in which positive direct microscopic findings correlated well with culture growth for Aspergillus were included. Antifungal susceptibility was performed by micro broth dilution technique according to clinical laboratory standard institute M38-A2 guidelines. Results: Aspergillus flavus was found to be predominant species followed by Aspergillus fumigatus. AFST was performed, where all Aspergillus strains exhibited low minimum inhibitory concentration (MIC) and minimum effective concentration (MEC) values for most of antifungal drugs tested except for one strain of A. flavus, which exhibited high MIC for voriconazole and high MEC for caspofungin. Conclusion: Polyenes, azoles, and echinocandin resistance are emerging in many parts of the world. Therefore, continued application of AFST combined with morphological characterization for species identification is critical in detecting the emergence of resistance among various Aspergillus species to reduce mortality, morbidity, and overcome treatment failure.

Whole-Genome Sequencing of Newly Emerged Fungal Pathogen Aspergillus Lentulus and Its Azole Resistance Gene Prediction.

Aims: Aspergillus lentulus is an important newly recorded species in the A. fumigatus complex and its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Comprehensive understanding of the A. lentulus is limited due to lack of genome-wide fine mapping data. The aim of this study was to investigate the A. lentulus signature at the molecular level, analyze the genome-wide profile of this strain, and predict its possible genes that execute azole resistance. Methods: In this study, a whole-genome sequencing of a clinically isolated A. lentulus strain (named A. lentulus PWCAL1) was studied by PacBio Sequel sequencing platform. Azole resistance genes were predicted based on whole-genome sequencing data analysis, gene function annotation, comparative genomic analysis, and BLASTP alignment using the Mycology Antifungal Resistance Database to comprehensively understanding the genome-wide features, pathogenicity, and resistance mechanisms of A. lentulus. Results: The results of whole-genome sequencing demonstrated that the total length of A. lentulus PWCAL1 genome was 31255105 bp, the GC content was 49.24%, and 6883 coding genes were predicted. A total of 4565, 1824, and 6405 genes were annotated in the Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases, respectively. In the Pathogen Host Interactions Database and the Database of Fungal Virulence Factors, 949 and 259 interacting virulence factors were identified, respectively, with the main virulence factor-mutant virulence phenotype, being enriched in reduced virulence. Comparative genomic analysis showed that there were 5456 consensus core genes in this strain and four closely related strains of A. fumigatus complex, which were mainly involved in human diseases, metabolism, organismal systems, etc. Among the three aligned A. lentulus strains, the number of unique genes of this bacterium was the highest with a number of 171, and these genes were mainly associated with carbohydrate metabolism and cell growth and death. Resistance gene prediction demonstrated that the A5653 gene of this bacterium had F46Y/N248T double point mutations on the CYP51A gene, but no tandem repeat mutations in the promoter region were detected. Furthermore, 12 genes belonging to the fungal multidrug resistance ATP-binding cassette (ABC) transporters were identified based on the complete genome sequence and phylogenetic analysis of A. lentulus, which belonged to the ALDp subfamily, the PDR subfamily (AtrB, CDR1, and CDR2), and the MDR subfamily (MDR1), respectively, and there were four genes that are annotated to the major facilitator superfamily multidrug transporter. Further phylogenetic tree classification of the ABC transporter subfamilies predicted in the nine selected A. fumigatus complex strains showed that these putative ABC proteins were divided into two main clusters, which belonged to the PDR (CDR1, CDR2, AtrB, and AtrF) and MDR subfamilies (MDR1, MDR2, and MDR3). The distribution of these ABC proteins varies among different species of the A. fumigatus complex. Conclusions: The main result obtained from this study for the whole genome of A. lentulus provide new insights into better understanding the biological characteristics, pathogenicity, and resistance mechanisms of this bacterium. In this study, two resistance mechanisms, which include CYP51A gene mutation and multidrug-resistant ABC transporter, were predicted in a single isolate. Based on the predicted CYP51A-F46Y/N248T site mutation combination, we speculate that the CYP51A gene of A. lentulus may be partially responsible for azole resistance. Based on the predicted ABC transporter family genes, we hypothesize that resistance to multiple azoles in A. lentulus is mediated, at least in part, by these ABC transporters with resistance.

In vitro and silico activity of piperlongumine against azole-susceptible/resistant Aspergillus fumigatus and terbinafine-susceptible/resistant Trichophyton species

In recent years, the widespread emergence of drug resistance in yeasts and filamentous fungi to existing antifungal armamentariums has become a severe threat to global health. There is also concern regarding increased rates of azole resistance in Aspergillus fumigatus and Terbinafine resistance in Trichophyton species. To overcome this concern of resistance to regular therapies, new antifungal drugs with novel and effective mechanisms are crucially needed. Herbal remedies may be promising strategies for the treatment of resistant infections. We aimed to investigate the in vitro and silico activity of piperlongumine against clinical azole susceptible/resistant A. fumigatus and terbinafine-susceptible/resistant Trichophyton species. In the current study, piperlongumine demonstrated potent antifungal activity, with minimum inhibitory concentrations (MICs) ranging from 0.016-4 μg/mL against Trichophyton isolates and 0.25-2 μg/mL for A. fumigatus isolates. Additionally, molecular docking studies indicated that piperlongumine has a strong binding affinity to the active sites of squalene epoxidase and sterol 14-alpha demethylase. However, further studies are warranted to correlate these findings with clinical outcomes and provide the basis for further investigations to pave the way for developing novel antifungal agents.

Tracking Candidemia Trends and Antifungal Resistance Patterns across Europe: An In-Depth Analysis of Surveillance Systems and Surveillance Studies.

The WHO fungal priority list classifies Candida species as critical and high-priority pathogens, and the WHO GLASS fungi initiative seeks to establish a standardised global framework for antifungal resistance monitoring. We aimed to review resistance rates and antifungal resistance patterns across European surveillance systems and studies in response to these recent calls for action. A systematic review of national and international surveillance systems and peer-reviewed surveillance studies available up to June 2024 was conducted. Descriptive and trend analyses were performed on surveillance data reporting resistance to different antifungals in Candida spp. In total, 6 national surveillance systems and 28 studies from 13 countries provided candidemia resistance data, mostly about the C. albicans, C. glabrata and C. parapsilosis complex. Azole resistance was most frequently reported (6/6 surveillance systems and 27/28 studies) with the highest resistance rate, especially for C. glabrata, in Croatia (100%, 28/28 isolates) and Slovenia (85.7%, 82/96) and C. parapsilosis in Croatia (80.6%, 54/67) and Italy (72.6%, 106/146). Echinocandin and polyene resistance rates were nearly zero. The number of isolates included in the surveillance systems increased over the years, particularly for C. albicans (+40-60 isolates/year), C. glabrata, and C. parapsilosis (+15-30 isolates/year). No surveillance system or study reported resistance data for C. auris. Pooled data from national surveillance revealed a decreasing trend in azole resistance in C. albicans and C. glabrata. The increasing azole-resistance trend in C. parapsilosis disappeared after adjusting for between-country heterogeneity. Overall, echinocandin and polyene resistance trends appeared relatively stable. Awareness of antifungal resistance is growing, but further actions are needed to strengthen surveillance capacity and knowledge-sharing networks across Europe.

Prevalence of azole-resistant Aspergillus fumigatus and other aspergilli in the environment from Argentina.

Azole resistance has emerged as a new therapeutic challenge in patients with aspergillosis. Various resistance mutations are attributed to the widespread use of triazole-based fungicides in agriculture. This study explored the prevalence of azole-resistant Aspergillus fumigatus (ARAF) and other aspergilli in the Argentine environment. A collection of A. fumigatus and other aspergilli strains isolated from soil of growing crops, compost, corn, different animal feedstuffs, and soybean and chickpea seeds were screened for azole resistance. No ARAF was detected in any of the environmental samples studied. However, five A. flavus, one A. ostianus, one A. niger and one A. tamarii recovered from soybean and chickpea seeds showed reduced susceptibility to medical azole antifungals (MAA). The susceptibility profiles of five A. flavus isolates, showing reduced susceptibility to demethylase inhibitors (DMIs), were compared with those of 10 isolates that exhibited susceptibility to MAA. Aspergillus flavus isolates that showed reduced MAA susceptibility exhibited different susceptibility profiles to DMIs. Prothioconazole and tebuconazole were the only DMIs significantly less active against isolates with reduced susceptibility to MAA. Although no ARAF isolates were found in the samples analysed, other aspergilli with reduced susceptibility profile to MAA being also important human pathogens causing allergic, chronic and invasive aspergillosis, are present in the environment in Argentina. Although a definitive link between triazole-based fungicide use and isolation of azole-resistant human pathogenic aspergilli from agricultural fields in Argentina remains elusive, this study unequivocally highlights the magnitude of the environmental spread of azole resistance among other Aspergillus species.

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs.

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates is a growing issue in clinics. The pleiotropic drug response (PDR) network in C. glabrata confers azole resistance and is defined primarily by the Zn2Cys6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1, that encodes an ATP-binding cassette transporter protein thought to act as a FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity.