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Neutrophil granulocytes (NGs) are among the key players in the defense against Aspergillus fumigatus ( A. fumigatus ). To better elucidate a pathophysiological understanding of their role and functions, we applied a human cell model using NGs from healthy participants and septic patients to evaluate their inhibitory effects on the growth of A. fumigatus ex vivo. Conidia of A. fumigatus (ATCC ® 204305) were co-incubated with NGs from healthy volunteers or septic patients for 16 h. A. fumigatus growth was measured by XTT assays with a plate reader. The inhibitory effect of NGs on 18 healthy volunteers revealed great heterogeneity. Additionally, growth inhibition was significantly stronger in the afternoon than the morning, due to potentially different cortisol levels. It is particularly interesting that the inhibitory effect of NGs was reduced in patients with sepsis compared to healthy controls. In addition, the magnitude of the NG-driven defense against A. fumigatus was highly variable among healthy volunteers. Moreover, daytime and corresponding cortisol levels also seem to have a strong influence. Most interestingly, preliminary experiments with NGs from septic patients point to a strongly diminished granulocytic defense against Aspergillus spp.

Objectives: Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents., Methods: Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed., Results: Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation., Conclusions: This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n = 20), Scedosporium aurantiacum (n = 2), Scedosporium apiospermum (n = 6), Rasamsonia argillacea species complex (n = 23), Exophiala dermatitidis (n = 10) and azole-resistant Aspergillus fumigatus (ARAF) (n = 25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

In this study a commercially available multiplex real-time PCR (AsperGenius®) was evaluated for its efficacy in detecting Aspergillus fumigatus and azole resistance markers in comparison with conventional culture methods and galactomannan (GM) testing from BAL fluids in allogeneic HSCT recipients. Between January 2015 and May 2017 100 allogeneic HSCT recipients with pulmonary infiltrates and suspicion of invasive fungal infection were recruited to the study from a tertiary care center in Germany. BAL fluid was routinely assessed using the following diagnostic tests: AsperGenius® PCR assay, GM testing (cut-off: 1.0) and conventional culture. Susceptibility testing of azoles was performed by using Etest and, in case presenting elevated MICs, PCR for mutations in the cyp51A gene was carried out. Criteria of EORTC/MSG were used to classify the patients for invasive fungal disease. According to the EORTC/MSG criteria 23 patients presented with probable invasive aspergillosis (IA). Aspergillus PCR showed a sensitivity of 65% for probable IA cases. A combination of PCR and GM results in BAL displayed a sensitivity of 96% (22/23) and 100% specificity. Mutations in the cyp51A gene were detected by PCR in three cases (3/23; 13%) which were also found resistant with the culture method. In one case a Y121F/T289A mutation and in two cases a L98H were found. The combination of a commercial Aspergillus PCR assay and GM testing from BAL demonstrated a high sensitivity and specificity for diagnosing IA in allogeneic HSCT recipients. The Aspergillus PCR assay was not superior in detecting azole resistant A. fumigatus compared to culture., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Invasive aspergillosis (IA) in immunocompromised patients has been associated with demolition in or adjacent to hospitals. In recent years, azole-resistant clinical isolates of Aspergillus fumigatus, the most common agent of IA, have emerged in Western Europe and are spreading globally., Aim: To determine the potential risk of IA, including azole resistance, in patients caused by demolition of a hospital building., Methods: Air sampling before, during and after demolition, screening for azole resistance, genotyping of non-susceptible isolates, and comparing those with strains from patients with azole-resistant IA during demolition., Findings: Mean concentrations of A. fumigatus spores did not differ significantly between the three periods before [17.5 colony-forming units (cfu)/m³], during (20.8 cfu/m³) (P=0.26) and after (17.7 cfu/m³) demolition (P=0.33). No significant difference in IA cases documented by clinicians was found when comparing the timeframe of demolition with the previous year (44 vs 42 cases). Thirty of 200 A. fumigatus isolates (15%) showed azole resistance. Genotyping by microsatellite polymerase chain reaction of the azole-resistant environmental and clinical isolates showed a polyclonal distribution., Conclusions: The results suggest that with implemented preventive measures, there is no increased risk for IA, including azole resistance, in immunocompromised patients during outdoor demolition work. Further prospective studies are needed to confirm these findings., (Copyright © 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Objectives: Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods., Methods: Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed., Results: Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Münster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy., Conclusions: This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up.

Therapy of invasive aspergillosis is becoming more difficult due to the emergence of azole resistance in Aspergillus fumigatus. A majority of resistant strains carries mutations in the CYP51A gene. Due to a lack of sensitivity of culture-based methods, molecular detection of A. fumigatus has become an important diagnostic tool. We set up the database FunResDB (www.nrz-myk.de/funresdb) to gather all available information about CYP51A-dependent azole resistance from published literature. In summary, the screening resulted in 79 CYP51A variants, which are linked to 59 nonsynonymous mutations. A tailor-made online sequence analysis tool allows for genotypic susceptibility testing of A. fumigatus., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

We retrospectively analyzed the performance and relevance of the SeptiFast assay in detecting Aspergillus fumigatus DNA in whole blood samples from 38 critically ill intensive care unit (ICU) patients with probable or proven invasive aspergillosis (IA) and 100 ICU patients without IA. The assay exhibited 66% sensitivity, 98% specificity, a 93% positive predictive value, and an 88% negative predictive value. A. fumigatus DNAemia was associated with poor outcome., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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.)

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

We report the clinical features, diagnosis, and monitoring findings of invasive aspergillosis (IA) in 2 liver transplant recipients. Blood/serum samples and bronchoalveolar lavage (BAL) fluids were analyzed by a novel commercial polymerase chain reaction (PCR) assay (SeptiFast) and an Aspergillus galactomannan (GM) enzyme-linked immunoassay (EIA). The diagnosis of IA could be performed in <6 h with the detection of Aspergillus fumigatus DNA in blood and BAL fluid. High GM values (mean: 9.1, range: 7.3-10.8) in serum and BAL fluid confirmed the SeptiFast result. Follow-up of the SeptiFast findings and GM index correlated with the clinical course. The molecular detection of A. fumigatus-specific DNA and GM test in blood/serum and BAL samples appears to be a useful tool for prompt diagnosis of IA. Further prospective clinical trials are necessary to evaluate the accuracy of SeptiFast and the GM test in diagnosing IA.

Strains of Aspergillus fumigatus (n = 24) were isolated from the sputa of six patients with cystic fibrosis during periods from 3 to 11 months. The genetic polymorphisms of the strains were studied using the random amplified polymorphic DNA (RAPD) assay with three single oligonucleotides and pairwise combined primers. The analysis of RAPD patterns resulted in 15 different RAPD types. In four patients, the colonizing type changed, whereas in two others the same types were detected over periods between 3 and 11 months. The genetic diversity as well as the shift of the colonizing strains found in some patients might be important for the epidemiology of Aspergillus infections in patients with cystic fibrosis.

Aspergillus fumigatus isolates (n = 6) from a lung transplant recipient, one isolate from a patient who had been on the same ward and a reference strain (NCPF 2140) were compared using three typing methods: SDS-PAGE, immunoblotting with serum from the transplant patient and random amplified polymorphic DNA (RAPD) assay. Neither the SDS-PAGE, immunoblot nor RAPD assay with single primers revealed differences between the eight isolates. Digestion of one primer product with the endonuclease EcoRI discriminated between the six patient isolates and the other two strains. The RAPD assay using pairwise combined primers showed identical patterns for the patient's strains but differentiated between the two other strains. It is concluded that any single technique may fail to detect strain differences and that a spectrum of typing methods is necessary in order to reveal or to exclude cross-infections with Aspergillus fumigatus.

A decrease of the susceptibility of Aspergillus fumigatus strains to amphotericin B was found when tested in combination with high concentrations (128-2048 mg/l) of the antibacterial agent imipenem by checkerboard titration and agar diffusion assay. Only bacteriologically active imipenem showed the antagonism. The mechanism of action is unknown. However, imipenem and amphotericin B did not react directly, as shown by checkerboard titration with bacterial strains as well as by HPLC analysis. It is concluded that imipenem medication may influence the efficacy of amphotericin B treatment in aspergillosis.