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The aim was to develop an RT-qPCR targeting Aspergillus fumigatus and compare its performance to that of Aspergillus fumigatus qPCR for the diagnosis of invasive aspergillosis (IA). Samples from patients of the Lyon University hospitals for whom a suspicion of IA led to the realization of an Aspergillus fumigatus qPCR molecular diagnostic test over a 2-year period were included. The patients were classified according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSGERC) criteria for suspected IA; RT-qPCR and qPCR assays were performed on all included samples. The sensitivities and specificities of RT-qPCR and qPCR were calculated and compared using the results of the EORTC-MSGERC classification as reference. The cycle threshold (Ct) results were compared according to IA classification and sample type. Among the 193 samples analyzed, 91 were classified as IA excluded, 46 as possible IA, 53 as probable IA, and 3 as proven IA. For all sample types, RT-qPCR was significantly more sensitive than qPCR for all IA classifications with an additional 17/102 samples detected ( P -value < 0.01). For plasma samples, sensitivity was significantly higher and specificity significantly lower using RT-qPCR for all IA classifications ( P -value < 0.001). The mean Ct obtained with RT-qPCR were significantly lower than those obtained with qPCR for all IA classifications and all sample types ( P -value < 0.001 and P -value < 0.0001, respectively). RT-qPCR presents a higher sensitivity than qPCR for the diagnosis of IA due to Aspergillus fumigatus , particularly in samples with an intrinsically low fungal load.IMPORTANCE Aspergillus fumigatus belongs to the critical priority group of the World Health Organization fungal priority pathogens list. Invasive aspergillosis (IA) is a life-threatening infection with poor prognosis and challenging diagnosis. PCR has been integrated into the 2020 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions for IA diagnosis. However, due to frequent low fungal burdens, its sensitivity needs to be improved. This work presents an innovative method for detecting total nucleic acids, corresponding to both ribosomal RNA and DNA, that enables IA diagnosis with greater sensitivity than conventional techniques, especially in non-invasive samples such as blood, enhancing the monitoring of this infection in high-risk patients., Competing Interests: The authors declare no conflict of interest.

Aspergillus fumigatus is a common opportunistic pathogen in different animals, including birds such as penguins. For the first time, a fungal strain identified as A. fumigatus was isolated from soil in the nests of gentoo penguins, Pygoscelis papua, on Livingston Island, South Shetland Islands (maritime Antarctica). This isolate (A. fumigatus UFMGCB 11829) displayed a series of potentially pathogenic characteristics in vitro. We evaluated its detailed molecular taxonomy and submitted the A. fumigatus UFMGCB 11829 Antarctic strain to in vivo pathogenic modelling. The isolate was confirmed to represent A. fumigatus morphological and phylogenetic analysis showed that it was closely related to A. fumigatus sequences reported from animals, immunosuppressed humans, storage grains, plants and soils. The strain displayed the best mycelial growth and conidia production at 37 ºC; however, it was also able to grow and produce conidia at 15º, demonstrating its capability to survive and colonize penguin nest at least in the summer season in maritime Antarctica. In pathogenicity tests, healthy mice did not showed symptoms of infection; however, 50% lethality was observed in immunosuppressed mice that were inoculated with 10 6 and 10 7 spores. Lethality increased to 100% when inoculated with 10 8 spores. Our data highlight the potential pathogenicity of opportunistic A. fumigatus that may be present in the Antarctic, and the risks of both their further transfer within Antarctica and outwards to other continents, risks which may be exacerbated due global climatic changes., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains., Competing Interests: The authors have declared that no competing interests exist.

Background: Mutations in cyp51A gene are known as main mechanisms of azole resistance in Aspergillus fumigatus, whereas azole-susceptible strains also carry cyp51A mutations (polymorphisms). The polymorphisms found in Europe mainly consist of two combinations of mutations, that is combinations of five single-nucleotide polymorphisms (SNPs) of cyp51A, referred to as cyp51A-5SNPs, and combinations of three SNPs of cyp51A, referred to as cyp51A-3SNPs. Few studies have compared the distributions of cyp51A polymorphisms between different regions., Objectives: The aim of this study was to investigate the regional differences of cyp51A polymorphisms., Methods: We compared the proportions of cyp51A polymorphisms in clinical and environmental strains isolated in various countries, and analysed the strains phylogenetically using short tandem repeats (STRs) and whole-genome sequence (WGS)., Results: Among the Japanese strains, 15 out of 98 (15.3%) clinical strains and 8 out of 95 (8.4%) environmental strains had cyp51A polymorphisms. A mutation of cyp51A N248K was the most prevalent polymorphism in both clinical (n = 14, 14.3%) and environmental strains (n = 3, 3.2%). Only one environmental strain harboured cyp51A-5SNPs, which was reported to be the most prevalent in Europe. For phylogenetic analyses using STRs and WGS, 183 and 134 strains, respectively, were employed. They showed that most of the strains with cyp51A N248K clustered in the clades different from those of the strains with cyp51A-5SNPs and cyp51A-3SNPs as well as from those with TR 34 /L98H mutations., Conclusions: This study suggests that there are genetic differences between cyp51A polymorphisms of A. fumigatus in Japan and Europe., (© 2021 Wiley-VCH GmbH.)

The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), first described in Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as opportunistic fungal pathogens from the genus Aspergillus. To gain insight into COVID-19-associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit significant differences from the genome of the Af293 reference strain. By examining a number of factors, including virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, secondary metabolite biosynthesis, and the MIC of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss-of-function mutations in genes known to increase virulence when deleted. Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains Af293 and CEA17, but similarly virulent to two other clinical strains of A. fumigatus. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA. IMPORTANCE The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has already killed millions of people. COVID-19 patient outcome can be further complicated by secondary infections, such as COVID-19-associated pulmonary aspergillosis (CAPA). CAPA is caused by Aspergillus fungal pathogens, but there is little information about the genomic and phenotypic characteristics of CAPA isolates. We conducted genome sequencing and extensive phenotyping of four CAPA isolates of Aspergillus fumigatus from Germany. We found that CAPA isolates were often, but not always, similar to other reference strains of A. fumigatus across 206 genetic determinants of infection-relevant phenotypes, including virulence. For example, CAPA isolate D was more virulent than other CAPA isolates and reference strains in an invertebrate model of fungal disease, but similarly virulent to two other clinical strains. These results expand our understanding of COVID-19-associated pulmonary aspergillosis.

The main objective of the study is to characterize two new strains of Aspergillus fumigatus through morphometric, biochemical, molecular methods, and to evaluate their antimicrobial potentiality. The micro-morphotaxonomy, growth, and metabolic behavior of the strains, nHF-01 and PPR-01, were studied in different growth conditions and compared with standard strain. The molecular characterization was done by sequencing the ncrDNA ITS1-5.8S-ITS2 and D1-D2 domains of the nc 28S rDNA region and compared with a secondary structure-based phylogenetic tree. The secretory antimicrobials and pigments were characterized by TLC, UV-Vis, and FT-IR spectroscopy. Both the strains showed distinct growth patterns in different nutritional media and could assimilate a wide range of carbohydrates with distinctive biochemical properties. The molecular characterization revealed the strains, nHF-01 and PPR-01, as Aspergillus fumigatus (GenBank Accession No. MN190286 and MN190284, respectively). It was observed that the strain nHF-01 produces red to brownish pigments having mild antimicrobial activity while the strain PPR-01 does not represent such transformations. The extractable compounds had a significant antimicrobial potentiality against the human pathogenic bacteria. From this analysis, it can be concluded that the nHF-01 and PPR-01 strains are distinct from other A. fumigatus by their unique characters. Large-scale production and detailed molecular elucidation of the antimicrobial compounds may lead to the discovery of new antimicrobial compounds from these strains.

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 origin of isolates routinely used by the community of Aspergillus fumigatus researchers is periodically a matter of intense discussion at our centre, as the construction of recombinant isolates have sometimes followed convoluted routes, the documentation describing their lineages is fragmented, and the nomenclature is confusing. As an aide memoir, not least for our own benefit, we submit the following account and tabulated list of strains (Table 1) in an effort to collate all of the relevant information in a single, easily accessible document. To maximise the accuracy of this record we have consulted widely amongst the community of Medical Mycologists using these strains. All the strains described are currently available from one of these organisations, namely the Fungal Genetics Stock Centre (FGSC), FungiDB, Ensembl Fungi and The National Collection of Pathogenic Fungi (NCPF) at Public Health England. Display items from this manuscript are also featured on FungiDB., Lay Abstract: We present a concise overview on the definition, origin and unique genetic makeup of the Aspergillus fumigatus isolates routinely in use by the fungal research community, to aid researchers to describe past and new strains and the experimental differences observed more accurately., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus , which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context. IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus , which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus ., (Copyright © 2020 Barber et al.)

Aspergillus fumigatus is a pathogenic fungus responsible for invasive aspergillosis (IA). Typically, it can produce abundant conidia to survive and spread. The infection by A. fumigatus usually occurs in immunocompromised patients due to failed clearance of inhaled conidia. However, the incidence of aspergillosis in immunocompetent hosts has been increasing, the pathogenesis of which is still unknown. Our team previously obtained two clinical nonsporulating A. fumigatus isolates from non-immunocompromised patients, which only have the form of hyphae. This present study demonstrated the in vitro and in vivo characteristics of the two nonsporulating A. fumigatus isolates and verified that their conidiation defects are associated to abolished expression of the sporulation-related central regulatory pathway brlA gene. In addition, we confirmed the mutation site of brlA gene (c.657&#95;660delTCCT) contributes to the nonsporulating phenotype in one clinical isolate. Plate assay showed that the two nonsporulating isolates have a similar resistance to antifungal drugs, cell wall disturbing substances, and oxidative stress compared with the wild-type reference Af293. Most important of all, we employed an immunocompetent mouse model to mimic the pathogenesis of pulmonary aspergillosis in non-immunocompromised patients. It revealed that the hyphae of two nonsporulating isolates and Af293 have similar virulence in immunocompetent hosts. Interestingly, the hyphae fragments of Af293 but not conidia are able to induce invasive aspergillosis in immunocompetent mice. In conclusion, our study indicate that the form of hyphae may play a dominant causative role in pulmonary aspergillosis of immunocompetent hosts rather than conidia., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Azole antifungal resistance in Aspergillus fumigatus is a worldwide concern. As in most public hospitals in Brazil, antifungal susceptibility tests are not routinely performed for filamentous fungi at our institution. A 4-year retrospective azole antifungal resistance screening revealed two azole-resistant A. fumigatus clinical isolates carrying the CYP51A TR34 (34-bp tandem repeat)/L98H (change of L to H at position 98)/S297T/F495I resistance mechanism mutations, obtained from two unrelated patients. Broth microdilution antifungal susceptibility testing showed high MICs for itraconazole, posaconazole, and miconazole. Short tandem repeat (STR) typing analysis presented high levels of similarity between these two isolates and clinical isolates with the same mutations reported from the Netherlands, Denmark, and China, as well as environmental isolates from Taiwan. Our findings might indicate that active searching for resistant A. fumigatus is necessary. They also represent a concern considering that our hospital provides tertiary care assistance to immunocompromised patients who may be exposed to resistant environmental isolates. We also serve patients who receive prophylactic antifungal therapy or treatment for invasive fungal infections for years. In these two situations, isolates resistant to the antifungal in use may be selected within the patients themselves. We do not know the potential of this azole-resistant A. fumigatus strain to spread throughout our country. In this scenario, the impact on the epidemiology and use of antifungal drugs will significantly alter patient care, as in other parts of the world. In summary, this finding is an important contribution to alert hospital laboratories conducting routine microbiological testing to perform azole resistance surveillance and antifungal susceptibility tests of A. fumigatus isolates causing infection or colonization in patients at high risk for systemic aspergillosis., (Copyright © 2020 American Society for Microbiology.)

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.

Aspergillus fumigatus is a globally distributed opportunistic fungal pathogen capable of causing highly lethal invasive aspergillosis in immunocompromised individuals. Recent studies have indicated that the global population consists of multiple, divergent genetic clusters that are geographically broadly distributed. However, most of the analyzed samples have come from continental Eurasia and the Americas where the effects of ancient versus recent factors are difficult to distinguish. Here, we investigated environmental A. fumigatus isolates from Auckland, New Zealand, a geographically isolated population, and compared them with those from other parts of the world to determine the relative roles of historical differentiation and recent gene flow in shaping A. fumigatus populations. Our data suggest that the Auckland A. fumigatus population contains both unique indigenous genetic elements as well as genetic elements that are similar to those from other regions such as Europe, Africa, and North America. Though the hypothesis of random recombination was rejected, we found abundant evidence for phylogenetic incompatibility and recombination within the Auckland A. fumigatus population. Additionally, susceptibility testing identified two triazole-resistant strains, one of which contained the globally distributed mutation TR 34 /L98H in the cyp51A gene. Our results suggest that contemporary gene flow, likely due to anthropogenic factors, is a major force shaping the New Zealand A. fumigatus population.

Azole resistance is a major concern for treatment of infections with Aspergillus fumigatus. Environmental resistance selection is a main route for Aspergillus spp. to acquire azole resistance. We investigated the presence of environmental hotspots for resistance selection in the Netherlands on the basis of the ability of A. fumigatus to grow and reproduce in the presence of azole fungicide residues. We identified 3 hotspots: flower bulb waste, green waste material, and wood chippings. We recovered azole-resistant A. fumigatus from these sites; all fungi contained cyp51A tandem repeat-mediated resistance mechanisms identical to those found in clinical isolates. Tebuconazole, epoxiconazole, and prothioconazole were the most frequently found fungicide residues. Stockpiles of plant waste contained the highest levels of azole-resistant A. fumigatus, and active aerobic composting reduced Aspergillus colony counts. Preventing plant waste stockpiling or creating unfavorable conditions for A. fumigatus to grow in stockpiles might reduce environmental resistance burden.

Background: Studies on Aspergillus fumigatus azole resistance in cystic fibrosis patients are scarce despite the fact that it is the most frequently isolated fungus from respiratory samples from these individuals., Objectives: To evaluate resistance prevalence, investigate mechanisms of resistance and explore the relationship between resistant isolates by genotyping., Methods: We conducted a prospective 1 year study (from 1 January to 31 December 2015), based on the investigation of up to five colonies per sample from cystic fibrosis patients., Results: Twenty-three (6.5%) isolates among the 355 tested were resistant to at least one triazole drug, using the EUCAST reference method, leading to a prevalence of 6.8% (6/88 patients). Analysis of resistance mechanisms highlighted TR34/L98H (n = 10), TR46/Y121F/T289A (n = 1), WT cyp51A (n = 11) and F46Y/M172V/N248T/D255E/E427K (n = 1). No genotype was shared between patients., Conclusions: This study showed a relatively stable resistance prevalence in comparison with the previous study conducted in 2010-11 (8%), although resistance mechanisms varied between the two studies., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR 34 )/L98H and TR 46 /Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STR Af ), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR 34 /L98H and TR 46 /Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus. IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection., (Copyright © 2019 Sewell et al.)

Aims: Aspergillus sp. are ubiquitous saprophytic fungi and their conidia easily inhaled. This is particularly important in immunocompromised patients, more susceptible to developing invasive aspergillosis. In addition to A. fumigatus sensu stricto, cryptic species, many resistant to antifungal drugs, have been associated with invasive infections, making it important to assess their presence and diversity in different environments. Therefore, the aim of this study was to assess the presence, diversity and susceptibility to antifungal drugs of airborne fungi. Moreover, in azole-resistant A. fumigatus sensu stricto isolates the presence of underlying molecular mechanisms of resistance was investigated., Methods and Results: Eighty-four Aspergillus isolates were collected from the environment air in hospitals and the Water Treatment Plant. The use of molecular tools allowed to detect 12 different cryptic species, showing a prevalence of 21·4%. The majority of isolates (69·0%) belonged to A. fumigatus complex and 65·4% were A. fumigatus sensu stricto. Among these, 21·8% were resistant to itraconazole (ITZ), 38·2% to posaconazole and 87·3% to isavuconazole; none of them were resistant to voriconazole or amphotericin B. Sequencing of the cyp51A gene on the 12 A. fumigatus sensu stricto ITZ-resistant isolates revealed the presence of mutations., Conclusion: Our study reports a large number of environmental-resistant Aspergillus species, including A. fumigatus sensu stricto that display an important role in invasive fungal infections. None of the environmental isolates showed mutations on cyp51A gene related to azole resistance., Significant and Impact of the Study: This study is the first assessment of molecular resistance mechanisms in A. fumigatus sensu stricto environmental isolates, in Portugal. Since TR34/L98H and TR46/Y121F/T289A cyp51A mutations were already reported in the clinical setting in Portugal (Monteiro et al. J Glob Antimicron Resist 13: 190-191, 2018; Pinto et al. Front Microbiol 9: 1656, 2018), and have been linked to environmental route, it is utmost importance to perform surveillance network for azole-resistant A. fumigatus., (© 2019 The Society for Applied Microbiology.)

We collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.

More than a decade ago a short tandem repeat-based typing method was developed for the fungus Aspergillus fumigatus . This STR Af assay is based on the analysis of nine short tandem repeat markers. Interpretation of this STR Af assay is complicated when there are only one or two differences in tandem repeat markers between isolates, as the stability of these markers is unknown. To determine the stability of these nine markers, a STR Af assay was performed on 73-100 successive generations of five clonally expanded A. fumigatus isolates. In a total of 473 generations we found five times an increase of one tandem repeat unit. Three changes were found in the trinucleotide repeat marker STR Af 3A, while the other two were found in the trinucleotide repeat marker STR Af 3C. The di- or tetranucleotide repeats were not altered. The altered STR Af markers 3A and 3C demonstrated the highest number of repeat units (≥50) as compared to the other markers (≤26). Altogether, we demonstrated that 7 of 9 STR Af markers remain stable for 473 generations and that the frequency of alterations in tandem repeats is positively correlated with the number of repeats. The potential low level instability of STR Af markers 3A and 3C should be taken into account when interpreting STR Af data during an outbreak.

Aspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR 34 /L98H and TR 46 /Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the β-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR 46 /Y121F/T289A (n = 8), TR 34 /L98H, and TR 53 . Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.

Invasive aspergillosis (IA) is a deep tissue infection with a high mortality occurring mostly in immunocompromised patients. To investigate the pathology of patients with IA it may be important to determine the genotype of the invasive isolate of Aspergillus , however available tissues for study are often formalin fixed paraffin embedded (FFPE). Although DNA has been successfully isolated from such tissues for species identification, genotyping of Aspergillus species on such tissues has not yet been performed. In this study we aimed to determine the genotype of Aspergillus fumigatus in FFPE tissue and serum samples from five patients with invasive aspergillosis using nine highly polymorphic short tandem repeat (STR Af ) loci. FFPE lung and bronchial biopsies from all patients were successfully typed. By comparing the latter result with non-FFPE materials from non-sterile samples such as sputum, bronchoalveolar lavage and lung abscess, we found identical genotypes within three patients, while the two other patients had a dominant genotype shared among all sample types. Genotyping of serum samples was successful in two serum samples with galactomannan ratios of 4 and 5.6, but failed in serum samples with galactomannan levels <0.5. In addition, testing a subset of these materials with the AsperGenius multiplex qPCR assay, we did not find azole resistance mutations. With this STR Af assay, A. fumigatus from FFPE tissue and serum was successfully genotyped, allowing retrospective examination of A. fumigatus in culture negative patients with IA.

Background: Aspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs., Results: In this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR 34 /L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment., Conclusions: 1. A. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. 2. Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations. 3. Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.

Fungal spores are one of several environmental factors responsible for causing respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), and aspergillosis. These spores also are able to trigger exacerbations during chronic forms of disease. Different fungal spores may contain different allergens and mycotoxins, therefore the health hazards are varying between the species. Thus, it is highly important quickly to identify the composition of fungal spores in the air. In this study, UV-Raman spectroscopy with an excitation wavelength of 244 nm was applied to investigate eight different fungal species implicated in respiratory diseases worldwide. Here, we demonstrate that darkly colored spores can be directly examined, and UV-Raman spectroscopy provides the information sufficient for classifying fungal spores. Classification models on the genus, species, and strain levels were built using a combination of principal component analysis and linear discriminant analysis followed by evaluation with leave-one-batch-out-cross-validation. At the genus level an accuracy of 97.5% was achieved, whereas on the species level four different Aspergillus species were classified with 100% accuracy. Finally, classifying three strains of Aspergillus fumigatus an accuracy of 89.4% was reached. These results demonstrate that UV-Raman spectroscopy in combination with innovative chemometrics allows for fast identification of fungal spores and can be a potential alternative to currently used time-consuming cultivation.

Hospital-acquired aspergillosis is usually associated with environmental contamination. In 2015, continuous monitoring of airborne fungi and multilocus variable-number tandem-repeat analysis identified the source of Aspergillus fumigatus as the airway of a patient. Therefore, patients colonized with Aspergillus spp. should be treated in airborne infection isolation rooms.

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a gliotoxinogenic species into section Fumigati commonly found in contaminated animal environments. In dairy herds, exposed areas of lactating cows, as mammalian glandule, can be easily contaminated by them. This study was aimed to identify A. fumigatus sensu lato strains (identified based on morphology) isolated from raw cow milk at species level, by morphological and molecular techniques, and to estimate their genetic variability. Forty-five A. fumigatus strains showed similar RAPD profiles (generated with PELF and URP1F primers) to each other and to A. fumigatus sensu stricto reference strains; also, they were almost identical to clinical human and feed-borne A. fumigatus strains included in the assay, since their similarity coefficient ranged from 0.7 to 1.00. Therefore, all strains were characterized as belonging to A. fumigatus sensu stricto species. This result was supported by sequencing the benA gene of selected strains and by maximum parsimony analysis. In addition, RAPD fingerprinting demonstrated intra-specific genetic variability into the A. fumigatus sensu stricto cluster. The results found in this study strengthen the fact that A. fumigatus sensu stricto is the predominant species in the Aspergillus section Fumigati found in animal environments such as dairy herd environments, while other species such as A. novofumigatus, A. fumigatiaffinis, A. udagawae and A. lentulus may be rarely isolated. Since no differences between animal and human strains were observed they can become pathogenic also for farm handlers'. Moreover, the presence of A. fumigatus sensu stricto in raw cow milk is probably a very important risk factor since milk and its by-products are generally indented for human consumption, then gliotoxin could be transferred to them., (Copyright © 2018. Published by Elsevier B.V.)

Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. Although the first naturally occurring isocyanide (xanthocillin) was isolated from the fungus Penicillium notatum over half a century ago, the biosynthetic origins of fungal isocyanides remain unknown. Here we report the identification of a family of isocyanide synthases (ICSs) from the opportunistic human pathogen Aspergillus fumigatus Comparative metabolomics of overexpression or knockout mutants of ICS candidate genes led to the discovery of a fungal biosynthetic gene cluster (BGC) that produces xanthocillin ( xan ). Detailed analysis of xanthocillin biosynthesis in A. fumigatus revealed several previously undescribed compounds produced by the xan BGC, including two novel members of the melanocin family of compounds. We found both the xan BGC and a second ICS-containing cluster, named the copper-responsive metabolite ( crm ) BGC, to be transcriptionally responsive to external copper levels and further demonstrated that production of metabolites from the xan BGC is increased during copper starvation. The crm BGC includes a novel type of fungus-specific ICS-nonribosomal peptide synthase (NRPS) hybrid enzyme, CrmA. This family of ICS-NRPS hybrid enzymes is highly enriched in fungal pathogens of humans, insects, and plants. Phylogenetic assessment of all ICSs spanning the tree of life shows not only high prevalence throughout the fungal kingdom but also distribution in species not previously known to harbor BGCs, indicating an untapped resource of fungal secondary metabolism. IMPORTANCE Fungal ICSs are an untapped resource in fungal natural product research. Their isocyanide products have been implicated in plant and insect pathogenesis due to their ability to coordinate transition metals and disable host metalloenzymes. The discovery of a novel isocyanide-producing family of hybrid ICS-NRPS enzymes enriched in medically and agriculturally important fungal pathogens may reveal mechanisms underlying pathogenicity and afford opportunities to discover additional families of isocyanides. Furthermore, the identification of noncanonical ICS BGCs will enable refinement of BGC prediction algorithms to expand on the secondary metabolic potential of fungal and bacterial species. The identification of genes related to ICS BGCs in fungal species not previously known for secondary metabolite-producing capabilities (e.g., Saccharomyces spp.) contributes to our understanding of the evolution of BGC in fungi., (Copyright © 2018 Lim et al.)

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.

Aspergillus fumigatus is the leading cause of mortality in severely immunocompromised individuals. Understanding pathogen dispersion and relatedness is essential for determining the epidemiology of nosocomial infections. Therefore, the aim of this study was to investigate the diversity and putative origins of clinical and environmental azole-susceptible and -resistant A. fumigatus isolates from Iran. In all, 79 isolates, including 64 azole-susceptible and 15 -resistant isolates, were genotyped using the cell surface protein (CSP) gene. Seven distinct repeat types (r01, r02, r03, r04, r05, r06 and r07) and 11 different CSP variants (t01, t02, t03, t04A, t06A, t06B, t08, t10, t18A, t18B and t22) were observed. Interestingly, t06B, t18A and t18B were exclusively present in azole-resistant isolates. The Simpson's index of diversity (D) was calculated at 0.78. Resistant isolates were genetically less diverse than azole-susceptible isolates. However, azole-resistant A. fumigatus without TR 34 /L98H were more diverse than with TR 34 /L98H. The limited CSP type diversity of the TR 34 /L98H isolates versus azole-susceptible isolates suggests that repeated independent emergence of the TR 34 /L98H mechanism is unlikely. It has been suggested that CSP types might have a common ancestor that developed locally and subsequently migrated worldwide., (© 2017 Blackwell Verlag GmbH.)

Background: Aspergillus fumigatus is a widespread fungus that colonizes dead organic substrates but it can also cause fatal human diseases. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides; however, resistant isolates appeared recently in the medical and also environmental area. The present study aims at molecular characterizing and quantifying A. fumigatus in major environmental habitats and determining its sensitivity to medical and agricultural DMI fungicides., Results: A. fumigatus was isolated only rarely from soil and meadow/forest organic matter but high concentrations (10 3 to 10 7  cfu/g) were detected in substrates subjected to elevated temperatures, such as compost and silage. High genetic diversity of A. fumigatus from compost was found based on SSR markers, distinguishing among fungal isolates even when coming from the same substrate sample, while subclustering was observed based on mutations in cyp51A gene. Several cyp51A amino acid substitutions were found in 15 isolates, although all isolates were fully sensitive to the tested DMI fungicides, with exception of one isolate in combination with one fungicide., Conclusion: This study suggests that the tested A. fumigatus isolates collected in Italy, Spain and Hungary from the fungus' major living habitats (compost) and commercial growing substrates are not potential carriers for DMI resistance in the environment. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Aspergillosis affects all types of birds; it causes the loss of specimens with high ecologic value and also leads to significant economic losses within the poultry industry. The main etiologic agent is Aspergillus fumigatus , a filamentary fungus with multiple virulence factors, such as gliotoxin (GT), which is an immunosuppressive epipolythiodioxopiperazine molecule. Necropsy was performed on 73 poultry from different provenances, all of which presented with a respiratory semiology compatible with aspergillosis. A mycological culture was performed on the injured lungs of diseased birds, as was chloroform extraction of the GT, a thin-layer chromatography analysis (TLC), and a histopathology analysis with hematoxylin-eosin and Grocott stainings. The A. fumigatus identification was confirmed by PCR, where the ITS 1 5.1-5.8S-ITS 2 fragment of the rDNA complex was amplified. The in vitro GT production was studied by TLC in the recovered isolates from A. fumigatus . Seven isolates of A. fumigatus were obtained and in six of them, GT-like compounds were detected. In a lung sample, a compound with the same retention time (RF) as the reference GT was detected; whereas RF compounds different from the GT standard were observed in three lung samples.

The AsperGenius ® assay detects several Aspergillus species and the A. fumigatus Cyp51A mutations TR 34 /L98H/T289A/Y121F that are associated with azole resistance. We evaluated its contribution in identifying A. lentulus and A. felis, 2 rare but intrinsically azole-resistant sibling species within the Aspergillus section Fumigati. Identification of these species with conventional culture techniques is difficult and time-consuming. The assay was tested on (i) 2 A. lentulus and A. felis strains obtained from biopsy proven invasive aspergillosis and (ii) control A. fumigatus (n=3), A. lentulus (n=6) and A. felis species complex (n=12) strains. The AsperGenius ® resistance PCR did not detect the TR 34 target in A. lentulus and A. felis in contrast to A. fumigatus. Melting peaks for L98H and Y121F markers differed and those of the Y121F marker were particularly suitable to discriminate the 3 species. In conclusion, the assay can be used to rapidly discriminate A. fumigatus, A. lentulus and A. felis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is a ubiquitous environmental mold and the leading cause of diverse human diseases ranging from allergenic bronchopulmonary aspergillosis (ABPA) to invasive pulmonary aspergillosis (IPA). Experimental investigations of the biology and virulence of this opportunistic pathogen have historically used a few type strains; however, it is increasingly observed with this fungus that heterogeneity among isolates potentially confounds the use of these reference isolates. Illustrating this point, Kowalski et al. (mBio 7:e01515-16, 2016, https://doi.org/10.1128/mBio.01515-16) demonstrated that variation in 16 environmental and clinical isolates of A. fumigatus correlated virulence with fitness in low oxygen, whereas Fuller et al. (mBio 7:e01517-16, 2016, https://doi.org/10.1128/mBio.01517-16) showed wide variation in light responses at a physiological and protein functionality level in 15 A. fumigatus isolates. In both studies, two commonly used type strains, Af293 and CEA10, displayed significant differences in physiological responses to abiotic stimuli and virulence in a murine model of IPA., (Copyright © 2017 Keller.)

Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson's index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks., Competing Interests: The authors have declared that no competing interests exist.

Introduction and Objective: The aim was to make an exact diagnosis of 20 strains using molecular biological methods which were isolated from the atmosphere of the inpatient rooms in the Oncology and other departments of the Ege University Medical Faculty Hospital, and identified as Aspergillus fumigatus through phenotypic tests, and to determine their antibiotic susceptibility patterns., Materials and Method: It was confirmed that the 20 phenotypically-identified A. fumigatus strains belonged to the section Fumigati after they were tested by the ITS-PCR method. Their sequence analysis was performed and the results sent to the NCBI GenBank, and their accession numbers were obtained. For their exact diagnosis at the species level, the β-tub (β-tubulin) and rodA (RodletA) genes were examined with the multiplex PCR. Anti-fungal susceptibility of the 20 strains was determined according to the M38-A2 standards., Results: As a result of ITS-PCR, it was confirmed that 19 of the 20 strains identified as A. fumigatus through the phenotypic methods belonged to the section Fumigati. However, after the detection of β-tub and rodA genes, all 20 strains were identified as A. fumigatus., Conclusion: Although the results of the phenotypic and molecular biological tests applied to filamentous fungi do not often overlap, in this study, the results obtained from the molecular analysis confirmed the results of the phenotypic tests. However, 1 of the 20 strains phenotypically-identified as A. fumigatus was identified as Penicillium spp. as a result of ITS-PCR and sequence analysis. On the other hand, the profile obtained from β-tub and rodA tests indicated that the strain was A. fumigatus. Based on these results, this strain is thought to belong to the Aspergilloides genus which has the features of both genera.

Unlabelled: This study used the Illumina MiSeq to analyse compositions and diversities of Aspergillus species in outdoor air. The seasonal air samplings were performed at two locations in Seoul, South Korea. The results showed the relative abundances of all Aspergillus species combined ranging from 0·20 to 18% and from 0·19 to 21% based on the number of the internal transcribed spacer 1 (ITS1) and β-tubulin (BenA) gene sequences respectively. Aspergillus fumigatus was the most dominant species with the mean relative abundances of 1·2 and 5·5% based on the number of the ITS1 and BenA sequences respectively. A total of 29 Aspergillus species were detected and identified down to the species rank, among which nine species were known opportunistic pathogens. Remarkably, eight of the nine pathogenic species were detected by either one of the two markers, suggesting the need of using multiple markers and/or primer pairs when the assessments are made based on the high-throughput sequencing. Due to diversity of species within the genus Aspergillus, the high-throughput sequencing was useful to characterize their compositions and diversities in outdoor air, which are thought to be difficult to be accurately characterized by conventional culture and/or Sanger sequencing-based techniques., Significance and Impact of the Study: Aspergillus is a diverse genus of fungi with more than 300 species reported in literature. Aspergillus is important since some species are known allergens and opportunistic human pathogens. Traditionally, growth-dependent methods have been used to detect Aspergillus species in air. However, these methods are limited in the number of isolates that can be analysed for their identities, resulting in inaccurate characterizations of Aspergillus diversities. This study used the high-throughput sequencing to explore Aspergillus diversities in outdoor, which are thought to be difficult to be accurately characterized by traditional growth-dependent techniques., (© 2016 The Society for Applied Microbiology.)

The prevalence of invasive aspergillosis (IA) at the intensive care unit (ICU) is unknown and difficult to assess since IA also develops in patients lacking specific host factors. In the Netherlands, increasing azole-resistance in Aspergillus fumigatus complicates treatment of patients with IA. The aim of this study was to determine the prevalence of IA by azole-resistant A. fumigatus at the ICU among patients receiving antifungal treatment and to follow their clinical outcome and prognosis. A retrospective cohort study was conducted in a university hospital ICU from January 2010 to December 2013. From all patients who received antifungal treatment for suspected IA, relevant clinical and microbiological data were collected using a standardised questionnaire. Of 9,121 admitted ICU-patients, 136 had received antifungal treatment for suspected IA, of which 38 had a positive A. fumigatus culture. Ten of the 38 patients harboured at least one azole-resistant isolate. Resistance mechanisms consisted of alterations in the cyp51A gene, more specific TR34/L98H and TR46/T289A/Y121F. Microsatellite typing did not show clonal relatedness, though isolates from two patients were genetically related. The overall 90-day mortality of patients with IA by azole-resistant A. fumigatus and patients with suspicion of IA by azole-susceptible isolates in the ICU was 100% (10/10) vs 82% (23/28) respectively. We conclude that the changing pattern of IA in ICU patients requires appropriate criteria for recognition, diagnosis and rapid resistance tests. The increase in azole resistance rates also challenges a reconsideration of empirical antifungal therapy., (This article is copyright of The Authors, 2016.)

Aspergillus fumigatus is a widespread opportunistic fungal pathogen causing an alarmingly high mortality rate in immunocompromised patients. Nosocomial infections by drug-resistant A. fumigatus strains are of particular concern, and there is a pressing need to understand the origin, dispersal and long-term evolution of drug resistance in this organism. The objective of this study was to investigate the diversity and putative origins of triazole resistance of A. fumigatus from India. Eighty-nine isolates, including 51 multiple triazole resistant (MTR) isolates and 38 azole-susceptible isolates, were genotyped using multilocus sequence typing (MLST), mating typing and PCR fingerprinting. MLST resolved the 51 MTR isolates into three genotypes, two of which have susceptible counterparts, suggesting that MTR isolates originated multiple times in India. The multiple-origin hypothesis was further supported by the diversity of sequences at the triazole target gene CYP51A among the MTR isolates, and by PCR fingerprints. Interestingly, there is abundant evidence for mating and recombination in natural population of A. fumigatus in India, suggesting that sexual spread of TR34 /L98H, the dominant MTR allele, is possible. Our results call for greater attention to MTR in A. fumigatus and for better management of antifungal drug use., (© 2016 Blackwell Verlag GmbH.)

Filamentous mycoses are often associated with significant morbidity and mortality. Prompt diagnosis and aggressive treatment are essential for good clinical outcomes in immunocompromised patients. The host immune response plays an essential role in determining the course of exposure to potential fungal pathogens. Depending on the effectiveness of immune response and the burden of organism exposure, fungi can either be cleared or infection can occur and progress to a potentially fatal invasive disease. Nonspecific cellular immunity (i.e., neutrophils, natural killer [NK] cells, and macrophages) combined with T-cell responses are the main immunologic mechanisms of protection. The most common potential mold pathogens include certain hyaline hyphomycetes, endemic fungi, the Mucorales, and some dematiaceous fungi. Laboratory diagnostics aimed at detecting and differentiating these organisms are crucial to helping clinicians make informed decisions about treatment. The purpose of this chapter is to provide an overview of the medically important fungal pathogens, as well as to discuss the patient characteristics, antifungal-therapy considerations, and laboratory tests used in current clinical practice for the immunocompromised host.

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a toxigenic fungus largely regarded as a single species by macroscopic and microscopic features. However, molecular studies have demonstrated that several morphologically identified A. fumigatus strains might be genetically distinct. This work was aimed to apply PCR-restriction length fragment polymorphisms (PCR-RFLP) and random amplification of polymorphic DNA (RAPD) molecular markers to characterize a set of feed-borne and clinical A. fumigatus sensu lato strains isolated from Argentina and Brazil and to determine and compare their genetic variability. All A. fumigatus strains had the same band profile and those typical of A. fumigatus sensu stricto positive controls by PCR-RFLP. Moreover, all Argentinian and Brazilian strains typified by RAPD showed similar band patterns to each other and to A. fumigatus sensu stricto reference strains regardless of their isolation source (animal feeds or human/animal clinical cases) and geographic origin. Genetic similarity coefficients ranged from 0.61 to 1.00, but almost all isolates showed 78% of genetic similarly suggesting that genetic variability was found at intraspecific level. Finally, benA sequencing confirmed its identification as A. fumigatus sensu stricto species. These results suggest that A. fumigatus sensu stricto is a predominant species into Aspergillus section Fumigati found in animal environments as well as in human/animal clinical cases, while other species may be rarely isolated. The strains involved in human and animal aspergillosis could come from the environment where this fungus is frequently found. Rural workers and animals would be constantly exposed., (© 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.)

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus., (© 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 fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated., Methods: The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats., Results: In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France., Conclusions: This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus., (© The Author 2015. 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.)

We studied whether short tandem repeats of Aspergillus fumigatus (STRAf) can differentiate between invasive and colonizing genotypes of A. fumigatus. Of the 395 genotypes detected (n = 1,373 isolates), 50 were clusters and 24 (6% of all genotypes) involved the patients with invasive aspergillosis and those colonized with A. fumigatus, indicating that genotyping cannot discriminate between invasive and colonizing isolates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: Resistance to triazoles in Aspergillus fumigatus has emerged in several countries and is usually mediated by mutations in the cyp51A gene. We determined the presence of both itraconazole-susceptible and -resistant A. fumigatus in routine cultures isolated from environmental and clinical samples., Methods: A total of 50 environmental and 16 clinical A. fumigatus isolates obtained from single colonies were tested for itraconazole susceptibility by Etest. Serial dilution and plating for selected A. fumigatus cultures were performed to separate triazole-susceptible and -resistant phenotypes. Antifungal drug susceptibility of subcultures to itraconazole, posaconazole and voriconazole was determined by the broth microdilution method. Itraconazole resistance was determined by the presence of tandem repeats in the promoter region and other resistance-conferring mutations by PCR and/or direct DNA sequencing of cyp51A. Genotyping was performed with a panel of nine microsatellite loci., Results: PCR amplification of the promoter region identified 1 of 50 environmental and 2 of 16 clinical A. fumigatus isolates as mixed (itraconazole-resistant and -susceptible) cultures, while the remaining isolates yielded patterns that were consistent with their data for susceptibility to itraconazole. Purified subcultures yielded distinct susceptibility profiles, concomitant with genetic determinants of triazole susceptibility/resistance in cyp51A and different microsatellite patterns., Conclusions: Concomitant presence of triazole-susceptible and -resistant strains in single colonies of routine A. fumigatus cultures, obtained from environmental and clinical samples, has been conclusively demonstrated., (© 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.)

Fibrosis is generally characterized by an excessive accumulation and deposition of extracellular matrix (ECM) that progressively leads to the destruction of functional nephrons of the kidney. Current study is aimed to propose a probable antifibrotic mechanism of gliotoxin, isolated from marine Aspergillus fumigatus strain, in proximal tubule renal epithelial cells by inhibiting HIF-1α, Ang-II and NF-κB in a dose dependent manner. GTX at a concentration of 1.6 ng/ml also showed more than 80% cell viability. Scanning Electron Microscopy, fluorescence microscopy, FACS, mRNA and protein profiles further confirmed that gliotoxin reverses the epithelial to mesenchymal transition in HK-2 cells by scavenging the production of reactive oxygen species (g=69.7) when compared to negative control (g=467.74). Under the light of obtained results, the present study holds immense promise for the biomedical implication of the fungal metabolite, Gliotoxin in medicine for treating various clinical cases of renal fibrosis., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The genus Aspergillus contains etiologic agents of aspergillosis. The clinical manifestations of the disease range from allergic reaction to invasive pulmonary infection. Among the pathogenic aspergilli, Aspergillus fumigatus is most ubiquitous in the environment and is the major cause of the disease, followed by Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, and several species in the section Fumigati that morphologically resemble A. fumigatus. Patients that are at risk for acquiring aspergillosis are those with an altered immune system. Early diagnosis, species identification, and adequate antifungal therapy are key elements for treatment of the disease, especially in cases of pulmonary invasive aspergillosis that often advance very rapidly. Incorporating knowledge of the basic biology of Aspergillus species to that of the diseases that they cause is fundamental for further progress in the field., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

One hundred isolates of Aspergillus fumigatus sensu lato mainly from China, as well as from Australia, France, India, Indonesia, Ireland, UK, and USA were analyzed to infer their sequence types (STs) and population diversity based on partial calmodulin, calcineurin regulatory subunit B, beta-tubulin, cytochrome C and calcineurin catalytic subunit A genes as well as their mating types, using ClonalFrame, Structure and MEGA software. Our results inferred 48 STs and showed that most of the STs or lineages evolved independently and without clear population structure among them. Whereas one lineage was recognized that could be a true population and in which one clade might diverge into another distinct lineage, namely, a cryptic species, A. neoellipticus. In addition, we found that mutation, parasexual, and sexual recombination could, respectively, play specific roles in the evolution of these fungi. Our results also showed that MAT1-1/MAT1-2 mating type ratios of A. fumigatus sensu lato was biased to nearly 1:1.4 (20/28) when clone-corrected, but when not clone-corrected, the ratio of MAT1-1/MAT1-2 was so biased as near 1:2 (35/65), which might mean that isolates with MAT1-2 are in the process of losing sexual ability preceding those with MAT1-1.