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The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non-cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ling Lu reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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 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 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.

Background: Cystic fibrosis (CF), an inherited autosomal recessive disorder, is linked with high morbidity and mortality rates due to bacteria, filamentous, yeast and black yeast-like fungi colonisation in the upper respiratory tract. Although Candida species are the most common fungi isolated from CF patients, azole-resistant Aspergillus fumigatus (ARAf) is a big concern for invasive aspergillosis. Notably, the exact prevalences of Aspergillus species and the prevalence of ARAf isolates among Iranian CF patients have yet to be previously reported and are unknown. We aimed to investigate the prevalence of ARAf isolates in CF patients among Iranian populations by focusing on molecular mechanisms of the mutations in the target gene., Methods: The 1 year prospective study recovered 120 sputum samples from 103 CF patients. Of these, 55.1% (86/156) yielded Aspergillus species, screened for ARAf using plates containing itraconazole (4 mg/L) and voriconazole (1 mg/L). According to the CLSI-M38 guidelines, antifungal susceptibility testing was performed using the broth microdilution method. In all phenotypically resistant isolates, the target of azole agents, the cyp51A gene, was sequenced to detect any possible single nucleotide polymorphisms (SNP) mediating resistance., Results: Of 120 samples, 101 (84.2%) were positive for filamentous fungi and yeast-like relatives, with 156 fungal isolates. The most common colonising fungi were Aspergillus species (55.1%, 86/156), followed by Candida species (39.8%, 62/156), Exophiala species (3.8%, 6/156) and Scedosporium species (1.3%, 2/156). Forty out of 86 (46.5%) were identified for section Fumigati, 36 (41.9%) for section Flavi, 6 (7%) for section Nigri and 4 (4.6%) for section Terrei. Fourteen out of 40 A. fumigatus isolates were phenotypically resistant. The overall proportion of ARAf in total fungal isolates was 9% (14/156). cyp51A gene analysis in resistant isolates revealed that 13 isolates harboured G448S, G432C, T289F, D255E, M220I, M172V, G138C, G54E and F46Y mutations and one isolate carried G448S, G432C, T289F, D255E, M220I, G138C, G54E and F46Y mutations. Additionally, this study detects two novel cyp51A single-nucleotide polymorphisms (I242V and D490E)., Conclusions: This study first investigated ARAf isolates in Iranian CF patients. Due to a resistance rate of up to 9%, it is recommended that susceptibility testing of Aspergillus isolates from CF patients receiving antifungal treatment be a part of the routine diagnostic workup. However, extensive multicentre studies with a high volume of CF patients are highly warranted to determine the impact of ARAf on CF patients., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp 51A and cyp 51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp 51A and cyp 51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp 51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp 51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp 51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp 51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp 51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp 51A and cyp 51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved., (© 2024 Dhoha Ghorbel et al., published by Sciendo.)

Background: Azole-resistant Aspergillus fumigatus (ARAf), reported as a global public health concern, has been unexpectedly observed in different countries., Aim: To identify ARAf and detect azole resistance related to the CYP51A mutation in different hospital environmental samples., Methods: In this multi-centre study from Iran, surfaces of electronic equipment and appliances from different hospitals in Iran were sampled using cotton swabs. All samples were cultured using azole-containing agar plates (ACAPs). Recovered Aspergillus isolates were identified at the species level using partial DNA sequencing of the β-tubulin gene. The azole susceptibility testing of A. fumigatus isolates was performed using the Clinical and Laboratory Standards Institute M38-A3 guideline. The sequencing of the CYP51A gene was also performed to detect mutations related to resistance., Findings: Out of the 693 collected samples, 89 (12.8%) Aspergillus species were recovered from ACAPs. Aspergillus fumigatus (41.6%) was the most prevalent, followed by A. tubingensis (23.6%) and A. niger (15.6%). Among 37 isolates of A. fumigatus, 19 (51.3%) showed high minimum inhibitory concentration (MIC) values to at least one of the three azoles, voriconazole, itraconazole, and posaconazole. CYP51A polymorphisms were detected in all 19 isolates, of which 52.6% showed the TR 34 /L98H mutation. Other detected mutations were G432C, G448S, G54E/G138C, F46Y, and Y121F/M220I/D255E. T289F and G432C were the first reported mutations in ARAf., Conclusion: There was a considerable level of azole resistance in hospital environmental samples, a serious warning for patients vulnerable to aspergillosis. Our findings have also revealed a different mutation pattern in the CYP51A gene., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to different mutations in the cyp51A gene. Azole resistance has been observed in several continents and appears to be a globally distributed phenomenon. Specific mutations in cyp51A that lead to azole resistance, such as the TR34/L98H modification, have been reported., Aims: To evaluate the azole resistance in clinically isolated A. fumigatus strains., Methods: As a result of our passive surveillance strategy, a total of 23 A. fumigatus isolates from clinical origins were identified through a phylogenetic analysis using the ITS region and β-tubulin gene fragments, and typed with the CSP microsatellite. Azole susceptibility profiles were performed by disk diffusion and microdilution broth methodologies according to CLSI guidelines., Results: Here we describe, for the first time, the detection of azole-resistant A. fumigatus isolates from clinical origins in Chile with mutations in the cyp51A gene. In addition to the TR34/L98H mutation, one isolate exhibited an F46Y/M172V/E427K-type mutation. Furthermore, microsatellite typing based on cell surface protein (CSP) was performed, showing the t02 (TR34/L98H), t15 (F46Y/M172V/E427K) and t01 (susceptible clinical isolates) genotypes., Conclusions: Our study demonstrates the presence of mutations related to azole resistance in A. fumigatus strains isolated from clinical samples in Chile. In order to obtain information that may help to tackle the spread of antifungal resistance among A. fumigatus populations, and to ensure the efficacy of future treatments against aspergillosis, a further research is necessary., (Copyright © 2024 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Azole antifungals remain the "gold standard" therapy for invasive aspergillosis. The world-wide emergence of isolates resistant to this drug class, however, developed into a steadily increasing threat to human health over the past years. In Aspergillus fumigatus , major mechanisms of resistance involve increased expression of cyp51A encoding one of two isoenzymes targeted by azoles. Yet, the level of resistance caused by cyp51A upregulation , driven by either clinically relevant tandem repeat mutations within its promoter or the use of high expressing heterologous promoters, is limited. Cytochrome P450 enzymes such as Cyp51A rely on redox partners that provide electrons for their activity. A. fumigatus harbors several genes encoding putative candidate proteins including two paralogous cytochrome P450 reductases, CprA and CprB, and the cytochrome b 5 CybE. In this work, we investigated the contribution of each cprA , cprB, and cybE -mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter cyp51A -mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter PxylP , we conditionally expressed these genes in combination with cyp51A , revealing cprA as the main limiting factor. Similar to this approach, we overexpressed cprA in an azole-resistant background strain carrying a cyp51A allele with TR34 in its promoter, which led to a further increase in its resistance. Employing sterol measurements, we demonstrate an enhanced eburicol turnover during upregulation of either cprA , which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance.cyp51A , which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance., Competing Interests: The authors declare no conflict of interest.

Aspergillus lentulus was first reported in 2005 as a cryptic species of Aspergillus fumigatus, and since then, its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Although it has been reported that P450 14-α sterol demethylase (Cyp51) is involved in azole resistance in A. lentulus, the specific resistance mechanism has not been elucidated. In this study, we successfully introduced the entire A. fumigatus cyp51A gene into the cyp51A locus in A. lentulus using the CRISPR/Cas9 genome-editing system. The A. lentulus strains harboring A. fumigatus cyp51A showed reduced minimum inhibitory concentrations for itraconazole and voriconazole compared with those of the parent strain. This finding suggests that Cyp51A is involved in azole resistance in A. lentulus and may contribute to the elucidation of the mechanism of resistance to azole drugs via Cyp51A and to the development of new antifungal drugs. In addition, our successful application of the CRISPR/Cas9 system to A. lentulus opens the door to examination of other gene functions in this fungus.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) among chronic pulmonary aspergillosis (CPA) patients treated with azoles in Japan is unknown. The aim of this study was to determine the detection rate of ARAF in isolates from CPA patients who were treated with azoles for varying durations. The potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104) between February 2012 and February 2019, at National Hospital Organization Tokyo National Hospital. The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF among all isolates was 8.3% (n = 10). Of the 10 resistant isolates, eight were ITCZ-resistant and five were VRCZ-resistant. Among 47 isolates obtained from 36 CPA patients who were treated with ITCZ (for an average of 256 days) and/or VRCZ (for an average of 29 days), the resistance rates were 17.0% and 10.6%, respectively. In addition, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients, particularly in those with ongoing long-term azole treatment, at the time of azole antifungal therapy failure., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Aspergillus fumigatus is the main cause of invasive aspergillosis (IA) with a high annual global incidence and mortality rate. Recent studies have indicated an increasing prevalence of azole-resistant A. fumigatus (ARAF) strains, with agricultural use of azole fungicides as a potential contributor. China has an extensive agricultural production system and uses a wide array of fungicides for crop production, including in modern growth facilities such as greenhouses. Soils in greenhouses are among the most intensively cultivated. However, little is known about the occurrence and distribution of ARAF in greenhouse soils. Here, we investigated genetic variation and triazole drug susceptibility in A. fumigatus from greenhouses around metropolitan Kunming in Yunnan, southwest China. Abundant allelic and genotypic variations were found among 233 A. fumigatus strains isolated from nine greenhouses in this region. Significantly, ∼80% of the strains were resistant to at least one medical triazole drug, with >30% showing cross-resistance to both itraconazole and voriconazole. Several previously reported mutations associated with triazole resistance in the triazole target gene cyp51A were also found in our strains, with a strong positive correlation between the frequency of mutations at the cyp51A promoter and that of voriconazole resistance. Phylogenetic analyses of cyp51A gene sequences showed evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in these greenhouses. Evidence for multiple origins of azole resistance and the widespread distributions of genetically very diverse triazole-resistant strains of A. fumigatus in greenhouses calls for significant attention from public health agencies. IMPORTANCE The origin and prevalence of azole-resistant Aspergillus fumigatus have been attracting increasing attention from biologists, clinicians, and public health agencies. Current evidence suggests agricultural fungicide use as a major cause. In southwest China, greenhouses are used to produce large amounts of fruits, flowers, and vegetables for consumers throughout China as well as those in other countries, primarily in southeast Asia. Here, we found a very high frequency (∼80%) of triazole-resistant A. fumigatus in our sample, the highest reported so far, with a significant proportion of these strains resistant to both tested agricultural fungicides and medical triazole drugs. In addition, we found novel allelic and genotypic diversities and evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in greenhouse populations in this region. Our study calls for a systematic evaluation of the effects of azole fungicide usage in greenhouses on human health., (Copyright © 2021 Zhou et al.)

The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00 μg/ml, 4.00 μg/ml, and 8.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described., (Copyright © 2020 American Society for Microbiology.)

In a multicenter study, we determined a prevalence rate of 4% for azole-resistant Aspergillus fumigatus in Taiwan. Resistance emerged mainly from the environment (TR 34 /L98H, TR 34 /L98H/S297T/F495I, and TR 46 /Y121F/T289A mutations) but occasionally during azole treatment. A high mortality rate observed for azole-resistant aspergillosis necessitates diagnostic stewardship in healthcare and antifungal stewardship in the environment.

Reference methods used to assess the drug susceptibilities of Aspergillus fumigatus isolates consisted of EUCAST and CLSI standardized broth microdilution techniques. Considering the increasing rate and the potential impact on the clinical outcome of azole resistance in A. fumigatus , more suitable techniques for routine testing are needed. The gradient concentration strip (GCS) method has been favorably evaluated for yeast testing. The aim of this study was to compare the CGS test with EUCAST broth microdilution for amphotericin B (AMB), posaconazole (PCZ), itraconazole (ITZ), voriconazole (VRZ), and isavuconazole (ISA). A total of 121 Aspergillus section Fumigati strains were collected, including 24 A. fumigatus sensu stricto strains that were resistant to at least one azole drug. MICs were determined using GCS and EUCAST methods. Essential agreement between the 2 methods was considered when MICs fell within ±1 dilution or ±2 dilutions of the 2-fold dilution scale. Categorical agreement was defined as the percentage of strains classified in the same category (susceptible, intermediate, or resistant) with both methods. Essential agreements with ±1 dilution and ±2 dilutions were 96.7, 93.4, 90.0, 89.3, and 95% and 100, 99.2, 100, 97.5, and 100% for AMB, PCZ, ITZ, VRZ, and ISA, respectively. Categorical agreements were 94.3, 86.1, 89.3, and 88.5% for AMB, PCZ, ITZ, and VRZ, respectively. Detection of resistance was missed with the GCS for one strain (4.1%) for PCZ and for 2 strains (8.3%) for ISA. Determination of ITZ MICs using the GCS allowed the detection of 91.7% of azole-resistant strains. The GCS test appears to be a valuable method for screening azole-resistant A. fumigatus clinical isolates., (Copyright © 2020 American Society for Microbiology.)

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) in Japan is unclear. We aimed to investigate the epidemiology of clinically isolated Aspergillus species and the frequency of azole resistance in Aspergillus species, particularly Aspergillus fumigatus, in the Kyoto and Shiga regions of Japan. Strains of clinically isolated Aspergillus species were prospectively collected from nine acute care hospitals. Species identification was performed by DNA sequence analysis, and all strains were subjected to antifungal susceptibility testing. Sequencing of the Aspergillus cyp51A gene and promoter region and genotyping by short tandem repeats were performed for ARAF isolates. A total of 149 strains were collected, and 130 strains were included for the subsequent analysis after the exclusion of duplicate isolates. The most commonly isolated species was Aspergillus fumigatus, accounting for 43.1% (56 isolates) overall, and seven (12.7%) of 55 strains of A. fumigatus were azole-resistant. Azole-resistance of other Aspergillus species were also found that two (22.2%) of nine strains of A. tubingensis and two (28.6%) of seven strains of A. flavus were azole-resistant. DNA sequence analysis of the ARAF strains revealed that two carried the cyp51A TR34/L98H mutation, one carried G448S, one carried M220I, and three had no relevant mutations (wild type). Genotyping and phylogenetic analyses showed that the TR34/L98H strains were clustered with the strains from the Netherlands and France. These data suggest the emergence of ARAF with TR34/L98H in Japan, and continuous surveillance will be important to identify trends in resistance., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Aspergillus fumigatus is the species section Fumigati most frequently isolated from the respiratory tract of cystic fibrosis (CF) patients. Recent studies suggest that mutations in the Cyp51 gene, particularly TR 34 /L98H, are responsible for azole resistance., Objectives and Methods: The focus of this study was on section Fumigati isolates isolated from the respiratory tract samples of CF patients. More specifically, the goal was to detect A. fumigatus isolates, test their antifungal susceptibility to itraconazole, voriconazole and posaconazole, and finally determine the presence of TR 34 /L98H and other mutations in the isolates Cyp51A gene., Results and Conclusions: A set of 31 isolates of Aspergillus section Fumigati were obtained from the sputum samples of 6 CF patients and subsequently identified to species level by microsatellite genotyping. All isolates were determined as A. fumigatus and involved 14 different genotypes. The minimal inhibitory concentrations to the three azoles were determined by the E-test method, and the Cyp51A gene was sequenced. One of the genotypes was found to be resistant to all azoles but no mutations were detected in the Cyp51A gene, especially the TR 34 /L98H mutation. Therefore, mutations in genes other than Cyp51A or other distinct mechanisms may be responsible for this reported multiazole resistance found in a Turkish CF patient.

One of the most recently described Aspergillus fumigatus CYP51A -mediated azole resistance mechanisms is TR46 Y121F T289A. Clinical A. fumigatus strains harboring these substitutions have been reported worldwide, with the exception of South America. We describe the first clinical A. fumigatus strain with this resistance mechanism isolated from an Argentinian patient. The strain was isolated in 2009 (1 year after the first-described mutant in United States), demonstrating that these alleles were scattered worldwide earlier than previously thought., (Copyright © 2018 American Society for Microbiology.)

Tricyclazole as a common fungicide wildly used to control rice blast disease in the Asian country may induce azole resistance in Aspergillus fumigatus isolates. The main reason of the acquired azole resistance is probably environmental exposure through wide fungicide use in agriculture. The present study was conducted with the aim of investigating the current status of the azole-resistant A. fumigatus obtained from the paddy fields with exposure to tricyclazole. A total of 108 soil samples were collected from four different locations of paddy fields in Mazandaran Province, Iran. Pure fungal colonies were initially identified based on the conventional tools, and then reconfirmed by using DNA sequencing of the partial ß-tubulin gene. In addition, the in vitro antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute document (CLSI) M38-A2. The identification of the mutations in the CYP51A gene was accomplished by the implementation of the polymerase chain reaction amplification assay on the selected isolates. Overall, 31 of 108 (28.7%) isolates were identified as A. fumigatus, four (3.7%) of which were recognized as azole-resistant with MICs of itraconazole ≥8μg/ml and voriconazole ≥4μg/ml. Only two out of the four azole-resistant A. fumigatus isolates harboured TR34/L98H variant and the other two isolates were identified as azole-resistant without any CYP51A gene mutations. However, other point mutations (TR46/Y121F/T289A) were not detected in the CYP51A gene. The high molecular structure similarity between environmental and medical triazoles may result in the selection of resistance mechanisms. Nonetheless, one might conclude that tricyclazole with different molecular structures against medical azoles induces azole-resistance in A. fumigatus isolates. The behavior of such pesticides as tricyclazole in the rice paddy fields would have an effective role in the development of azole-resistance that requires detailed information., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.

Aspergillus fumigatus is a saprophytic fungus that is a major causative pathogen for aspergillosis. Only a few classes of antifungals are used for treating this life-threatening fungal infection. Azoles are the first-line drugs and are widely used for the management and prophylaxis of aspergillosis. An emerging issue is the increasing incidence of resistant isolates worldwide. In particular, environmentally derived tandem-repeat-type azole-resistant mutations, such as Cyp51A TR34/L98H, and Cyp51A TR46/Y121F/T289A, have emerged over the last decade. In particular, azole-resistant isolates were prevalent in clinical settings in European countries; many of the reports are from the Netherlands, UK, and Germany. In contrast, reports on azole-resistant A. fumigatus isolates from East Asian countries are still few and have only recently begun to increase. Herein, all literature on East Asian azole-resistant A. fumigatus isolates were reviewed, and a complete list of resistant isolates from China, Japan, Taiwan, and Korea is provided. As of this report, the total numbers of tandem-repeat-type azole-resistant isolates are 26, 3, 32, and 1 in China, Japan, Taiwan, and Korea, respectively.

Aspergillus fumigatus is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in A. fumigatus is worrisome. The aim of this study was to validate the new MycoGENIE A. fumigatus real-time PCR kit and to evaluate its performance on clinical samples for the detection of A. fumigatus and its azole resistance. This multiplex assay detects DNA from the A. fumigatus species complex by targeting the multicopy 28S rRNA gene and specific TR 34 and L98H mutations in the single-copy-number cyp51A gene of A. fumigatus The specificity of cyp51A mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical A. fumigatus isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the Aspergillus 28S rRNA gene and 6 copies for the cyp51A gene harboring the TR 34 and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR 34 and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of A. fumigatus DNA and azole resistance due to TR 34 and L98H mutations in clinical samples., (Copyright © 2017 American Society for Microbiology.)

Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus ( n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR ( n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR 34 /L98H was the most prevalent mutation (46.7%), followed by TR 46 /Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology., (Copyright © 2017 American Society for Microbiology.)

Recently, azole-resistant Aspergillus fumigatus containing a 34-bp or 46-bp tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) has appeared in the environment worldwide, including several Asian countries. In this study, we isolated the 34-bp tandem repeat-containing azole-resistant A. fumigatus strain OKH50 from a patient in Japan in May 2016. The patient had not been treated with medical azoles before the strain isolation, suggesting that the resistant property was acquired before infection. In addition, the patient had not traveled overseas. Our analysis of short tandem repeats of the strain indicates that the strain is strongly related to the 34-bp tandem repeat-containing isolates from European countries and Asia-Oceania countries but not to susceptible isolates from Japan, suggesting that the strain was introduced from overseas and might spread in Japan., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A -containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background., (Copyright © 2017 American Society for Microbiology.)

Azole-resistant strains of Aspergillus fumigatus containing a tandem repeat in the cyp51A promoter and amino acid substitution(s) have been isolated in the environment worldwide; however, this type of resistant strain had never been isolated from the environment in Japan. Our previous study indicated that an azole-resistant A. fumigatus strain OKH50 containing a 34-bp tandem repeat in cyp51A promoter with L98H substitution in Cyp51A was isolated from a patient in Obihiro of Hokkaido, Japan. In this study, we collected azole-resistant Aspergillus spp. by air sampling from the environment in Japan. One Aspergillus-like colony was isolated from one of 10 sampling sites surveyed. The strain Env1 was confirmed as A. fumigatus by nucleotide sequencing and possessed a 34-bp tandem repeat in the promoter region of cyp51A with L98H substitution in Cyp51A. A. fumigatus Env1 has the identical short tandem repeat pattern with the OKH50 strain, indicating that these strains are closely related with each other. Additionally, the short tandem repeat pattern is closely related to Danish and Iranian environmental isolates, suggesting that azole-resistant strains have crossed transnational boundaries and are now present in Japan, and therefore, further analysis throughout Japan is required to determine the distribution of this type of azole-resistant A. fumigatus.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Azole-resistant Aspergillus fumigatus originating from the environment as well as induced during therapy are continuously emerging in Danish clinical settings. We performed a laboratory-based retrospective study (2010-2014) of azole resistance and genetic relationship of A. fumigatus at the national mycology reference laboratory of Denmark. A total of 1162 clinical and 133 environmental A. fumigatus isolates were identified by morphology, thermotolerance and/or β-tubulin sequencing. Screening for azole resistance was carried out using azole agar, and resistant isolates were susceptibility tested by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) E.Def 9.2 reference method and CYP51A sequenced. Genotyping was performed for outbreak investigation and, when appropriate, short tandem repeat Aspergillus fumigatus microsatellite assay. All 133 environmental A. fumigatus isolates were azole susceptible. However, from 2010 to 2014, there was an increasing prevalence of azole resistance (from 1.4 to 6% isolates (p <0.001) and 1.8 to 4% patients (p <0.05)) among the clinical isolates, with the well-known environmental CYP51A variant TR34/L98H responsible for >50% of the azole resistance mechanisms. Among 184 Danish A. fumigatus isolates, 120 unique genotypes were identified and compared to a collection of 1822 international genotypes. Seven (5.8%) Danish genotypes were shared between isolates within Denmark but with different origin, 19 (15.8%) were shared with foreign genotypes, and two (11.8%) of 17 genotypes of isolates carrying the TR34/L98H resistance mechanisms were identical to two Dutch TR34/L98H isolates. Our findings underlines the demand for correct identification and susceptibility testing of clinical mould isolates. Furthermore, although complex, genotyping supported the hypotheses regarding clonal expansion and the potential of a single origin for the TR34/L98H clone., (Copyright © 2016. Published by Elsevier Ltd.)

The Aspergillus fumigatus cyp51A gene TR46/Y121F/T289A mutation is a new emerging resistance mechanism with high-level voriconazole (VOR) resistance, and elevated MICs to all other medical azoles. This is highly worrisome as VOR is the primary drug for the treatment of many aspergillus diseases. The 46 base pair tandem repeat (TR46) is positioned at the same location of the cyp51A gene promoter region as has been described for other tandem repeats. The exact role of the TR46 in combination with the two amino acid changes (Y121F and T289A) in the CYP51A protein is unknown. In this study this azole resistance mechanism was investigated by recombinant analysis study combined with homology modelling. MICs of the TR46/Y121F/T289A recombinant corresponded to the MICs of the original clinical isolates containing the same mutations with high-level resistance to VOR. The TR46 or Y121F by itself has only a moderate effect on azole susceptibility. The combination of TR46/Y121F, however, appears to be highly resistant not only for VOR but also for itraconazole (ITZ). The genetic change of T289A in combination with TR46 or by itself has no significant effect on the phenotype but moderates the phenotype of the ITZ resistance only in the presence of Y121F. The striking resistant phenotype of the TR46/Y121F mutant is supported by the structural analysis of the CYP51A homology model. The A. fumigatus CYP51A Y121 residue forms an H-bond with the heme centre of the enzyme. Disruption of the H-bond by the Y121F substitution destabilizes the active centre of CYP51A which appears to be essential with respect to azole resistance. In CYP51A-azole complexes, residue T289 is in close proximity of the azole moiety of VOR. Replacement of the polar amino acid threonine by the more hydrophobic amino acid alanine might promote more stable drug-protein interactions and has thereby an impact on ITZ susceptibility, which is confirmed by the MICs of the genetic recombinants., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: Several studies, especially in Europe, have recently reported the emerging phenomenon of azole resistance in Aspergillus fumigatus, but very few data are available in France. Our study aimed to determine the resistance prevalence in A. fumigatus isolates recovered from clinical samples over a 1-year period in two university hospital centers., Methods: All A. fumigatus isolates were screened for azole resistance using RPMI agar plates supplemented with itraconazole and voriconazole. Resistance was then confirmed by the EUCAST method. A part of the beta-tubulin gene was amplified for resistant isolates to confirm the A. fumigatus species, and the Cyp51A gene and its promoter were afterward sequenced to detect mutations potentially responsible for this resistance., Results: One hundred sixty-five A. fumigatus isolates were recovered from 134 patients. Three isolates recovered from three patients were found resistant with MICs of >8 mg/l, 4 mg/l, and 1 mg/l for itraconazole, voriconazole, and posaconazole, respectively. The TR34/L98H mutation, previously and largely described in other countries, was detected in the three isolates., Conclusion: Our study demonstrated the occurrence of azole resistance among unselected A. fumigatus clinical isolates, with an overall prevalence of 1.8%., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Aspergillus spp. are the most frequently isolated filamentous fungi in the sputum of patients with cystic fibrosis (CF). Resistance to the azoles, the mainstay of current antifungal therapy, has been increasingly observed worldwide, but few data are available on the resistance of Aspergillus spp. in German CF patients. This study investigated the epidemiology of Aspergillus spp. and the molecular origin of azole resistance in a large German CF centre., Methods: In total, 2677 respiratory samples from 221 CF patients collected between April 2010 and April 2013 were analysed; of these, 573 yielded Aspergillus spp., which were screened for azole resistance. Isolates with reduced susceptibility to itraconazole and/or voriconazole were tested according to the EUCAST reference procedure. Sequencing of cyp51A, the target of azole antifungals, was performed in all resistant isolates., Results: Six isolates obtained from four patients were highly resistant to itraconazole (all identified as Aspergillus fumigatus sensu stricto); five of them were pan-azole resistant. The TR34/L98H mutation was the most frequent mutation identified in azole-resistant isolates (n = 4), followed by M220L and TR46/Y121F/T289A, a mutation previously reported from Belgium and the Netherlands only. Three of four patients harbouring azole-resistant A. fumigatus had not received any prior azole treatment., Conclusions: Resistance to azoles in Aspergillus spp. is still infrequent in German CF patients and is mainly caused by the TR34/L98H mutation. Worryingly, pan-azole-resistant TR46/Y121F/T289A has spread to Germany. Azole resistance has to be considered also in azole-naive CF patients and susceptibility testing of Aspergillus spp. isolates should be performed in all patients requiring treatment., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Azole resistance in Aspergillus is emerging in European and Asian countries. As azoles are mainstay of therapy in the management of aspergillosis, azole resistance has serious implications in patient management. We report the emergence of resistance to triazoles in environmental Aspergillus fumigatus isolates in Iran. The TR34/L98H mutation was the only resistance mechanism. Overall 3.3% of the A. fumigatus isolates from hospital surroundings in Sari and Tehran had the same TR34/L98H STRAf genotype and were related to some resistant clinical and environmental TR34/L98H isolates from the Netherlands and India. It is emphasised that routine resistance surveillance studies focusing on environmental and clinical samples are warranted to yield the true prevalence of azole resistance in A. fumigatus in Iran., (© 2013 Blackwell Verlag GmbH.)

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a common causative pathogen of aspergillosis. At present, triazole resistance of A. fumigatus poses an important challenge to human health globally. In this study, the biological characteristics and mechanisms of azole resistance of five A. fumigatus strains (AF1, AF2, AF4, AF5, and AF8) were explored. There were notable differences in the sporulation and biofilm formation abilities of the five test strains as compared to the standard strain AF293. The ability of strain AF1 to avoid phagocytosis by MH-S cells was significantly decreased as compared to strain AF293, while that of strains AF2, AF4, and AF5 were significantly increased. Fungal burden analysis with Galleria mellonella larvae revealed differences in pathogenicity among the five strains. Moreover, the broth microdilution and E-test assays confirmed that strains AF1 and AF2 were resistant to itraconazole and isaconazole, while strains AF4, AF5, and AF8 were resistant to voriconazole and isaconazole. Strains AF1 and AF2 carried the cyp51A mutations TR34/L98H/V242I/S297T/F495I combined with the hmg1 mutation S541G, whereas strains AF4 and AF8 carried the cyp51A mutation TR46/Y121F/V242I/T289A, while strain AF5 had no cyp51A mutation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed differences in the expression levels of genes associated with ergosterol synthesis and efflux pumps among the five strains. In addition, transcriptomics, RT-qPCR, and the NADC/NADH ratio demonstrated that the mechanism of voriconazole resistance of strain AF5 was related to overexpression of genes associated with energy production and efflux pumps. These findings will help to further elucidate the triazole resistance mechanism in A. fumigatus. [ABSTRACT FROM AUTHOR]

Purpose of the Review: Azole-resistant Aspergillus fumigatus is an emerging clinical problem. Resistance can develop both with clinical exposure and with environmental exposure to azole and azole-like fungicides used against phytopathogens. Here, our current understanding of the epidemiology and prevalence of this emerging problem is reviewed. Recent Findings: Azole-resistant A. fumigatus is now a worldwide problem, as it has been documented in clinical and environmental samples on all but one continent. Although Europe has been the center of this issue, hotspots are now emerging in Asia. However, there are several limitations to our current understanding of the extent of this problem, including issues with surveillance strategies and our reliance on phenotypic methods for detecting resistance. Summary: Azole-resistant A. fumigatus is an emerging issue both clinically and in the environment that requires greater attention in order to preserve this very useful class of antifungal agents for the treatment of aspergillosis. [ABSTRACT FROM AUTHOR]

Background: Eumycetoma is a neglected tropical disease. It is a chronic inflammatory subcutaneous infection characterised by painless swellings which produce grains. It is currently treated with a combination of itraconazole and surgery. In an ongoing clinical study, the efficacy of fosravuconazole, the prodrug of ravuconazole, is being investigated. For both itraconazole and ravuconazole, no clinical breakpoints or epidemiological cut‐off values (ECV) to guide treatment are currently available. Objective: To determine tentative ECVs for itraconazole and ravuconazole in Madurella mycetomatis, the main causative agent of eumycetoma. Materials and Methods: Minimal inhibitory concentrations (MICs) for itraconazole and ravuconazole were determined in 131 genetically diverse clinical M. mycetomatis isolates with the modified CLSI M38 broth microdilution method. The MIC distributions were established and used to determine ECVs with the ECOFFinder software. CYP51A sequences were sequenced to determine whether mutations occurred in this azole target gene, and comparisons were made between the different CYP51A variants and the MIC distributions. Results: The MICs ranged from 0.008 to 1 mg/L for itraconazole and from 0.002 to 0.125 mg/L for ravuconazole. The M. mycetomatis ECV for itraconazole was 1 mg/L and for ravuconazole 0.064 mg/L. In the wild‐type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499 (S499G). The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. Conclusion: The proposed M. mycetomatis ECV for itraconazole is 1 mg/L and for ravuconazole 0.064 mg/L. [ABSTRACT FROM AUTHOR]

Azole resistance in Aspergillus fumigatus has increasingly been reported worldwide. Its major mechanism of resistance is mediated by mutations in cyp51A. The objective of this study was to test the antifungal susceptibilities of A. fumigatus isolates from Chang Gung Memorial Hospital (CGMH), the largest tertiary referral hospital in Taiwan, and to investigate cyp51A mutations in azole-resistant strains. A. fumigatus isolates preserved in the Research Laboratory of Medical Mycology of CGMH from 2015 to 2021 were used. Antifungal susceptibility testing was performed using the YeastOneTM method. Isolates with high minimal inhibitory concentrations (MICs) against antifungals were further tested using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Mutations in the cyp51A in azole-resistant strains were detected by Sanger sequencing. The overall prevalence of azole-resistant isolates was 1.77% (two out of 113 isolates). The two azole-resistant strains had tandem repeats (TR) in the promoter region and mutations in the cyp51A gene (TR34/L98H and TR34/L98H/S297T/F495I). One strain showed intermediate susceptibility to voriconazole, and its Cyp51A protein had five amino acid substitutions (F46Y/M172V/N248T/D255E/E427K). TR34/L98H and TR34/L98H/S297T/F495I are the most prevalent cyp51A mutations in Taiwan, mediating azole resistance based on current publications and our results. YeastOneTM was validated as a rapid tool for the antifungal susceptibility test; however, further confirmation by CLSI should be considered when MIC values of voriconazole, posaconazole, and amphotericin B are close to the clinical breakpoints or ecological cutoff values. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a common ubiquitous fungal pathogen present in the environment including soil, decaying leaves, compost, grain crops and household dust. It can turn opportunistic in immunocompromised individuals causing allergic diseases, respiratory problems and invasive blood stream infections. Being saprophytic, it propagates by producing minute grey-green conidia that can become airborne and reach humans. In majority of cases, aspergillosis due to this organism can heal with treatment by antifungals. The azole compounds, one of the most important antifungal drugs, generally target the 14-a sterol demethylase enzyme, encoded by the cyp51A gene of the fungus, causing impaired membrane ergosterol synthesis. However in recent years the situation has turned grave due to development of azole resistant fungal species both in the environment as well as in the clinics. The main mechanism of resistance in A. fumigatus involves incorporation of tandem repeats in the promoter region of cyp51A, along with point mutations in the gene sequence. Resistant isolates tend to arise either due to excessive use of azole fungicides in the agricultural fields, from where they reach human through sporulation; or by prolonged treatment of patients with azole drugs. The early diagnosis of aspergillosis is very crucial for successful treatment of a patient. The article brings forth a comprehensive study of this organism, the mechanism of development and spread of azole resistant fungal isolates, genetic basis of development of azole resistance, detection of resistant strains and finally drugs used to combat the fungal infection. [ABSTRACT FROM AUTHOR]

Azole-resistant Aspergillus fumigatus is an emerging worldwide problem with increasing reports of therapy failure cases produced by resistant isolates. A case of azole-resistant A. fumigatus hospital colonization in a patient is reported here. Investigations of the hospital environment led to the recovery of A. fumigatus strains harboring the TR34/L98H and the G448S Cyp51A azole resistance mechanisms. Isolate genotyping showed that one strain from the environment was isogenic with the patient strains. These are the first environmental A. fumigatus azole resistant strains collected in a hospital in Spain; it supports the idea of the hospital environment as a source of dissemination and colonization/infection by azole resistant A. fumigatus in patients. The isolation of an azole-resistant strain from an azole-naïve patient is an interesting finding, suggesting that an effective analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus. The emergence and spread of these resistance mechanisms in A. fumigatus is of major concern because it confers high resistance to voriconazole and is associated with treatment failure in patients with invasive aspergillosis. [ABSTRACT FROM AUTHOR]

The emergence and spread of Aspergillus fumigatus azole resistance has been acknowledged worldwide. The main problem of azole resistance is the limited therapeutic options for patients suffering aspergillosis. Azole resistance mechanisms have been mostly linked to the enzyme Cyp51A, a target of azole drugs, with a wide variety of modifications responsible for the different resistance mechanisms described to date. However, there are increasing reports of A. fumigatus strains showing azole resistance without Cyp51A modifications, and thus, novel resistance mechanisms are being explored. Here, we characterized two isogenic A. fumigatus clinical strains isolated two years apart from the same patient. Both strains were resistant to clinical azoles but showed different azole resistance mechanisms. One strain (CM8940) harbored a previously described G54A mutation in Cyp51A while the other strain (CM9640) had a novel G457S mutation in Cyp51B, the other target of azoles. In addition, this second strain had a F390L mutation in Hmg1. CM9640 showed higher levels of gene expression of cyp51A, cyp51B and hmg1 than the CM8940 strain. The role of the novel mutation found in Cyp51B together with the contribution of a mutation in Hmg1 in azole resistance is discussed. [ABSTRACT FROM AUTHOR]

Summary: Background: Triazole resistance in Aspergillus fumigatus sensu stricto due to mutations in the cyp51A gene has been widely reported. Data from Argentina, and particularly from cystic fibrosis (CF) patients, are limited. Objectives: To investigate the prevalence and molecular mechanisms of azole resistance in A. fumigatus sensu stricto recovered from this population. Methods: Ninety‐three A. fumigatus isolates from 50 CF patients were retrospectively analysed for azole resistance using the standard microbroth dilution method according to CLSI M38‐A2 guidelines. Sequencing analysis of the cyp51A gene and its promoter region was conducted in those isolates displaying high MIC values to itraconazole, voriconazole and/or posaconazole. Results: Overall, 14% of isolates displayed high MIC values to at least one azole. Of them, 30.7% had the mutation TR34‐L98H. No mutations in the cyp51A gene or its promoter were found in the remaining non–wild‐type strains. Therefore, other mechanisms associated with azole resistance can be highly prevalent in these isolates. Conclusions: To the best of our knowledge, this is the first study in Latin America reporting azole‐resistant A. fumigatus strains recovered from respiratory secretions of CF patients. Noteworthy, the prevalence of azole resistance in A. fumigatus sensu stricto in the studied Argentinean CF population is alarmingly high. [ABSTRACT FROM AUTHOR]

The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.

The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus. [ABSTRACT FROM AUTHOR]

Azole resistance in Aspergillus fumigatus is an emerging problem, especially in immunocompromised patients. It has been reported worldwide, including in Asia, but has not yet been reported in Korea. Here, we report a case of invasive pulmonary aspergillosis (IPA) caused by azole-resistant A. fumigatus that developed in a hematopoietic stem cell transplantation recipient during posaconazole prophylaxis for immunosuppressive therapy of graft-versus-host diseases. We identified TR34/L98H/S297T/F495L mutation in the CYP51A gene of A. fumigatus clinical isolate obtained from bronchial washing fluid. Minimal inhibitory concentrations for itraconazole, voriconazole, and posaconazole were > 16, 1, and 4 μg/mL, respectively. While IPA improved partially under voriconazole treatment, the patient died from carbapenemase-producing Klebsiella pneumoniae bacteremia. Further epidemiological surveillance studies are warranted. [ABSTRACT FROM AUTHOR]

Invasive aspergillosis is a life-threatening mycosis caused by the pathogenic fungus Aspergillus. The predominant causal species is Aspergillus fumigatus, and azole drugs are the treatment of choice. Azole drugs approved for clinical use include itraconazole, voriconazole, posaconazole, and the recently added isavuconazole. However, epidemiological research has indicated that the prevalence of azole-resistant A. fumigatus isolates has increased significantly over the last decade. What is worse is that azole-resistant strains are likely to have emerged not only in response to long-term drug treatment but also because of exposure to azole fungicides in the environment. Resistance mechanisms include amino acid substitutions in the target Cyp51A protein, tandem repeat sequence insertions at the cyp51A promoter, and overexpression of the ABC transporter Cdr1B. Environmental azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/T289A) have become widely disseminated across the world within a short time period. The epidemiological data also suggests that the number of Aspergillus spp. other than A. fumigatus isolated has risen. Some non-fumigatus species intrinsically show low susceptibility to azole drugs, imposing the need for accurate identification, and drug susceptibility testing in most clinical cases. Currently, our knowledge of azole resistance mechanisms in non-fumigatus Aspergillus species such as A. flavus, A. niger, A. tubingensis, A. terreus, A. fischeri, A. lentulus, A. udagawae, and A. calidoustus is limited. In this review, we present recent advances in our understanding of azole resistance mechanisms particularly in A. fumigatus. We then provide an overview of the genome sequences of non-fumigatus species, focusing on the proteins related to azole resistance mechanisms. [ABSTRACT FROM AUTHOR]

Two isolates belonging to Aspergillus section Fumigati were recovered from German soil on itraconazole containing agar media. Phylogenetic analysis and phenotypic characterization of both isolates show that they represent a novel species named Aspergillus oerlinghausenensis (holotype CBS H-22119HT, ex-type CBS 139183T = IBT 33878 = DTO 316-A3). The species is phylogenetically related to A. fischeri and A. fumigatus. Aspergillus oerlinghausenensis can be differentiated from A. fischeri by its higher growth rate at 50°C. Furthermore, A. oerlinghausenensis is protoheterothallic as only the MAT1-1 idiomorph was detected, while A. fischeri is homothallic. The species differs from A. fumigatus by a weak sporulation on malt extract agar at 25°C, a floccose colony texture on Czapek yeast extract agar and malt extract agar and subglobose instead of subclavate vesicles. The cyp51A promoter region of A. oerlinghausenensis deviates from the previously reported cyp51A promoter regions in A. fumigatus and potentially presents a novel azole resistance conferring modification. Due to the close relationship of A. oerlinghausenensis with A. fischeri and A. fumigatus, this species is placed in a good position for comparative studies involving these species. [ABSTRACT FROM AUTHOR]