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ABSTRACT Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

BACKGROUND: Triazole resistance in the human fungal pathogen Aspergillus fumigatus has been a growing challenge in clinic treatment with triazole drugs such as itraconazole. The fast evolvement of triazole resistance in A. fumigatus in the ecosystem has drawn great attention, and there has been a possible link between the application of triazole fungicides in agriculture and triazole resistance in A. fumigatus. The change in susceptibility of A. fumigatus exposed to the new chiral triazole fungicide mefentrifluconazole was investigated in this study. RESULTS: The results indicated that triazole resistance in A. fumigatus was acquired with exposure to mefentrifluconazole at a level of greater than or equal to 2 mg L−1 in liquid medium and soil (not at 0.4 nor 1 mg L−1). Interestingly, stereoselectivity was found in the acquisition of triazole resistance in A. fumigatus when exposed to mefentrifluconazole. R-mefentrifluconazole, which is very active on plant pathogens, exhibited stronger possibility in the development of the resistance in A. fumigatus than its antipode. Overexpression of cyp51A, AtrF, AfuMDR1 and AfuMDR4 were associated with the acquired resistance in A. fumigatus with hereditary stability. CONCLUSION: The results suggest that triazole resistance in A. fumigatus could be resulted from the selection of mefentrifluconazole at concentrations larger than 2 mg L−1. Mefentrifluconazole should be applied within the dosage recommended by good agricultural practice to avoid the resistance in A. fumigatus in soil. This also may be applicable to other triazole fungicides. [ABSTRACT FROM AUTHOR]

The activity of isavuconazole and other triazoles against non- fumigatus (non-AFM) Aspergillus causing invasive aspergillosis was evaluated. A total of 390 non-AFM isolates were collected (1/patient) in 2017-2021 from 41 hospitals. Isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and/or internal spacer region/β-tubulin sequencing and tested by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. CLSI epidemiological cutoff values were applied, where available. Isavuconazole showed activity against Aspergillus sections Flavi (n = 122; minimum inhibitory concentration [MIC] 50/90 , 0.5/1 mg/L), Terrei (n = 57; MIC 50/90 , 0.5/0.5 mg/L), Nidulantes (n = 34; MIC 50/90 , 0.12/0.25 mg/L), Versicolores (n = 7; MIC 50 , 1 mg/L), and Circumdati (n = 2; MIC range, 0.12-2 mg/L). Similar activity was displayed by other triazoles against those Aspergillus sections. Most of the isolates from Aspergillus sections Fumigati (n = 9), Nigri (n = 146), and Usti (n = 12) exhibited elevated MIC values to isavuconazole (MIC 50/90 , 2/-, 2/4, and 2/8 mg/L), voriconazole (MIC 50/90 , 2/-, 1/2, and 4/8 mg/L), itraconazole (MIC 50/90 , 2/-, 2/4, and 8/>8 mg/L), and posaconazole (MIC 50/90 , 0.5/-, 0.5/1, and >8/>8 mg/L), respectively. Isavuconazole was active (MIC values, ≤1 mg/L) against Aspergillus parasiticus , Aspergillus tamarii , Aspergillus nomius , Aspergillus nidulans , Aspergillus unguis , Aspergillus terreus , Aspergillus alabamensis , and Aspergillus hortai , while isavuconazole MIC values between 2 and 8 mg/L were observed against cryptic isolates from Aspergillus section Fumigati . Isavuconazole inhibited 96.1% of Aspergillus niger and 80.0% of Aspergillus tubingensis at ≤4 mg/L, the CLSI wild-type cutoff value for A niger . Voriconazole, itraconazole, and posaconazole showed similar activity to isavuconazole against most cryptic species. Isavuconazole exhibited potent in vitro activity against non-AFM; however, the activity of triazoles varies among and within cryptic species., Competing Interests: Potential conflicts of interest. JMI Laboratories was contracted to perform services in 2023 for AbbVie, Inc, Adamed Pharma S.A., AimMax Therapeutics, Allecra Therapeutics, Amicrobe, Inc, AN2 Therapeutics, Inc, Apnimed, Astellas Pharma, Inc, Basilea Pharmaceutica AG, Baxis Pharmaceuticals, Inc, Beckman Coulter, Inc, bioMérieux, Biosergen AB, Blacksmith Medicines, Bugworks, Carnegie Mellon University, Center for Discovery and Innovation, Cerba Research NV, Cidara Therapeutics, Cipla USA Inc, ContraFect Corporation, CorMedix Inc, Crestone, Inc, Discoveric Bio Beta Ltd, Dr. Falk Pharma GmbH, Entasis Therapeutics, Evopoint Biosciences, Fedora Pharmaceuticals, Forge Therapeutics, GARDP Foundation, Genentech, Gilead Sciences, Inc, GlaxoSmithKline plc, Harvard University, Institute for Clinical Pharmacodynamics, Iterum Therapeutics plc, Janssen Biopharma, Johnson & Johnson, Kbio, Inc, Lakewood-Amedex Inc, Locus Biosciences, Inc, McGill University, Medpace, Inc, Meiji Seika Pharma, Melinta Therapeutics, Menarini Group, Merck & Co, MetCura Pharmaceuticals, Inc, MicuRx Pharmaceutical Inc, Mundipharma International Ltd., Nabriva Therapeutics, National Cancer Institute, National Institutes of Health, NovoBiotic Pharmaceuticals, LLC, Ohio State University, Omnix Medical Ltd, Paratek Pharmaceuticals, Pfizer, Inc, PPD Global Central Labs, LLC, Pulmocide Ltd, Qpex Biopharma, Inc, Revagenix, Inc, Roche Holding AG, Scynexis, Inc, Seed Health, SeLux Diagnostics, Shionogi & Co, Ltd, Spero Therapeutics, Sumitovant Biopharma, Inc, Swedish National Reference Laboratory, TenNor Therapeutics, ThermoFisher Scientific, US Food and Drug Administration, University of Wisconsin, VenatoRx Pharmaceuticals, Wockhardt, and Zoetis, Inc. The authors report no individual conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non- cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus. [ABSTRACT FROM AUTHOR]

Spot blotch, caused by Cochliobolus sativus is an economical disease of barley worldwide. Resistance C. sativus to Triadimefon (TDM) a triazole compound fungicide has emerged due to its high genetic diversity, short life cycle and plentiful spore production; however, mechanism(s) underlying such TDM-resistance remains unclear. In the current work, expression of four well known genes involved in fungicide resistance mechanisms cytb, MFS, ABC1 and ABC2 were monitored in a highly virulent C. sativus strain (Cs16) at early points of TDM treatments using quantitative real-time PCR (qPCR). Data showed 50% mycelial growth inhibition (EC50) was recorded after 48h at 0.25 μg mL-1 TDM treatment. The qRT-PCR revealed significant variance in the expression patterns of the four genes at 24 hours post TDM treatments as compared to the non-inoculated controls. It is also noteworthy that maximum expressions for cytb (7.69 -fold), MFS (6.11 -fold), ABC1 (3.4 -fold) and ABC2 (1.7-fold) were recorded at different time points of TDM treatments. The obtained results suggest that these genes, positively activated in C. sativus during TDM applications, which can provide testable hypotheses that will need direct future tests to decide how these changes may be specified in the genome of the virulent C. sativus pathotype. [ABSTRACT FROM AUTHOR]

World-wide, emerging triazole resistance increasingly complicates treatment of invasive aspergillosis (IA). In settings with substantial (>10%) prevalence of triazole resistance, empiric combination therapy with both a triazole and liposomal amphotericin B (LAmB) can be considered because of the low yields of susceptibility testing. To avoid toxicity while optimizing outcome, a strategy with monotherapy would be preferable. A newly designed treatment algorithm based on literature and expert consensus provided guidance for empiric monotherapy with either voriconazole or LAmB. Over a four and a half year period, all adult patients in our hospital treated for IA were included and patient data were collected. An independent committee reviewed the attributability of death to IA for each patient. Primary outcomes were 30- and 100-day crude mortality and attributable mortality. In total, 110 patients were treated according to the treatment algorithm. Fifty-six patients (51%) were initially treated with voriconazole and 54 patients (49%) with LAmB. Combined attributable and contributable mortality was 13% within 30 days and 20% within 100 days. Treatment switch to LAmB was made in 24/56 (43%) of patients who were initially treated with voriconazole. Combined contributable and attributable 100-day mortality in this subgroup was 21% and was not increased when compared with patients initially treated with LAmB (P  = 0.38). By applying a comprehensive clinical decision algorithm, an antifungal-sparing regime was successfully introduced. Further research is warranted to explore antifungal treatment strategies that account for triazole-resistance. Lay summary Due to resistance of Aspergillus against triazoles, combination therapy with liposomal amphotericin B (LAmB) is applied more often as primary therapy against invasive aspergillosis. This study presents the results of a decision tool which differentiated between triazole or LAmB monotherapy. [ABSTRACT FROM AUTHOR]

Triazole-resistant Aspergillus fumigatus is a global health concern. In general, each triazole resistance pattern caused by the specified amino acid substitution of Cyp51A has a typical pattern depending on the mutation site. We evaluated the contribution of both Cyp51A and Hmg1 mutations to atypical triazole resistance in A. fumigatus. We used clinical triazole-resistant A. fumigatus strains collected in Japan and investigated the sequences of cyp51A and hmg1 genes. To delineate the association between the hmg1 mutation and atypical triazole resistance, the mutant hmg1 alleles in clinical multi-azole resistant strains were replaced with the wild-type hmg1 allele by CRISPR/Cas9 system. In our study, the combination of Cyp51A mutation and Hmg1 mutation was shown to additively contribute to triazole resistance. We also demonstrated that the triazole resistance conferred by the Hmg1 mutation showed a different pattern depending on the mutation site, similar to the Cyp51A mutation. Our results indicate that focusing on the phenotypes of multiple genes is essential to clarify the overall picture of the triazole resistance mechanism of A. fumigatus. Lay Summary The number of triazole-resistant Aspergillus fumigatus is increasing. We confirmed thatmutation in a hydroxymethylglutaryl-CoA reductase (Hmg1) in the fungus contributesto the resistance separately from Cyp51A mutation, and that susceptibility patterns aredifferent based on mutation site. [ABSTRACT FROM AUTHOR]

Cochliobolus sativus is the causal agent of barley spot blotch (SB), a disease responsible for serious crop losses. Triadimefon (TDM) a triazole compound is used for controlling SB worldwide. The resistance of C. sativus to TDM fungicide has been well documented, but its mechanisms are still poorly understood. ATP-binding cassette (ABC) transporters are believed to be crucial in the adaptation of fungal pathogens to a range of fungicides. In this work, TDM sensitivity of two virulent and avirulent C. sativus pathotypes was measured by the relative growth rate at four concentrations and the effective concentration for 50% inhibition (EC50) was determined. ABC1 expression was verified using quantitative reverse transcriptase (qRT-PCR). Results demonstrated that 50% mycelial growth inhibition (EC50) was achieved for both C. sativus pathotypes following TDM treatment after 96h at 0.25 μg mL-1. Moreover, qRT-PCR showed intensification of ABC1 transporter in both pathotypes 24h post TDM treatments as compared to controls. Interestingly, the ABC1 expression was greater in the virulent pathotype Pt4 as compared to the avirulent one, Pt1. Together, changes in ABC1 levels after TDM short-term treatment in both C. sativus pathotypes suggested its importance in the triazole resistance. [ABSTRACT FROM AUTHOR]

Background: The emergence of azole resistance in non‐fumigatus Aspergillus strains is on the raise. Objectives: To study the susceptibility profiles and the molecular mechanisms of azole resistance of environmental and clinical strains of Aspergillus flavus from Argentina. Methods: Thirty‐five A flavus isolates (18 from soybean seeds and chickpea seeds and 17 from the clinic) were analysed for amphotericin B and azole resistance using the standard microbroth dilution method according to CLSI M38‐A2 guidelines. Sequencing analysis of the cyp51 genes was conducted in those isolates displaying high MICs values to itraconazole, voriconazole and/or posaconazole. Results: Among the environmental isolates, 33.3% of them showed high MIC values for at least one triazole whereas 23.5% of the clinical isolates displayed high MIC values for amphotericin B. Point mutations in the Cyp51C gene were recorded in most environmental isolates with non‐wild‐type MIC values. Conclusions: Susceptibility differences among environmental A flavus isolates might suggest the possibility of native resistance to certain triazole antifungals used in the clinic. To the best of our knowledge, this is the first report of antifungal screening of environmental strains of A flavus in soybean seeds and chickpea seeds from Argentina that showed increased resistance to voriconazole and itraconazole in comparison to clinical strains. [ABSTRACT FROM AUTHOR]

Triazole resistant A. fumigatus has been documented in many parts of the world. In the Netherlands, incidence is now above 10% and results in the need for long-term parenteral therapy with liposomal amphotericin B (LAmB). The long terminal half-life of LAmB suggests that intermittent dosing could be effective, making the application of outpatient antifungal therapy (OPAT) possible. Here, we report our experience with the use of OPAT for Invasive Fungal Infections (IFI). All adult patients treated with LAmB with a 2 or 3 times weekly administration via the outpatient departments in four academic tertiary care centers in the Netherlands and Belgium since January 2010 were included in our analysis. Patient characteristics were collected, as well as information about diagnostics, therapy dose and duration, toxicity, treatment history and outcome of the IFI. In total, 18 patients were included. The most frequently used regimen (67%) was 5 mg/kg 3 times weekly. A partial response to the daily treatment prior to discharge was confirmed by CT-scan in 17 (94%) of patients. A favorable outcome was achieved in 13 (72%) patients. Decrease in renal function occurred in 10 (56%) cases but was reversible in all and was treatment limiting in one patient only. The 100-day mortality and 1-year mortality after initiation of OPAT were 0% and 6%, respectively. In a selected population, and after confirmation of initial response to treatment, our data support the use of OPAT with LAmB for treatment of IFI in an intermittent dosing regimen. [ABSTRACT FROM AUTHOR]

Antibiotic resistance in fungal isolates is increasing on a global scale. Despite knowledge that pig farmers are occupationally exposed to infectious species of fungi, such as Aspergillus spp., little is known regarding their potential exposure to antifungal-resistant Aspergillus spp. The aim of this study is to obtain knowledge regarding the antifungal resistance profiles of isolates of Aspergillus species taken from different source materials—including airborne dust, surface dust, faeces, and straw—within a pig farm. The EUCAST broth microdilution method was used for testing antifungal resistance from 43 isolates of Aspergillus sampled in 3 periods inside a Danish pig farm. Seven species of Aspergillus were obtained, including A. candidus (n = 5), A. fumigatus (n = 5), A. glaucus (n = 13), A. nidulans (n = 2), A. niger (n = 15), A. terreus (n = 1), and A. versicolor (n = 2). Overall, 27.9% of the Aspergillus isolates displayed resistance against at least one antifungal, and 11.6% of Aspergillus isolates displayed resistance against multiple antifungals. The most abundant group exhibiting antifungal resistance was affiliated with the species A. niger, with isolates exhibiting resistance to itraconazole, voriconazole, and caspofungin. One isolate of A. glaucus and two isolates of A. versicolor were resistant to amphotericin B (MIC ≥ 2 mg/L amphotericin B). Antibiotic-resistant fungi were found on all three sampling days.

Background: During the past decades, the incidence of invasive aspergillosis (IA) caused by Aspergillus fumigatus has increased dramatically. The aims of this study were to investigate the susceptibility of clinical isolates of A. fumigatus to triazole and the underlying cyp51A mutations in triazole-resistant A. fumigatus. Methods: A total of 126 A. fumigatus clinical isolates from 126 patients with proven or probable IA were obtained from four large tertiary hospitals in Nanjing, China, between August 2012 and July 2015. The determination of minimal inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole was performed by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing reference method. Results: A total of 4 A. fumigatus isolates (3.17%) were confirmed to be itraconazole resistant, with MICs of ≥8 mg/L, and one isolate (0.8%) was confirmed to be voriconazole resistant and posaconazole resistant, with MICs of 4 mg/L and 0.5 mg/L, respectively. We found that two of the 4 isolates of triazole-resistant A. fumigatus had the L98H amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region, one isolate had an M220I mutation, and another itraconazole-resistant isolate did not have a substitution in the cyp51A gene. Conclusions: This study shows that triazole-resistant A. fumigatus clinical isolates are present in Nanjing, China, which is a new challenge to the clinical management of IA.

The association between healthcare-associated invasive aspergillosis and hospital construction/building works is well recognized. This infection can cause significant morbidity and mortality and imposes a substantial burden on the healthcare system. The population of patients at risk for this opportunistic infection has expanded and multi-triazole drug resistance has emerged globally. Hence the need for a multi-faceted approach to prevent acquisition of invasive aspergillosis in acute care settings. This article is a summary of the Irish National Guidelines for the prevention of healthcare-associated aspergillosis which is based on published reports, international clinical guidelines, official engineering standards, and technical guidelines. We discuss the key recommendations and strategies for the prevention of invasive aspergillosis from the planning/pre-construction, construction, and post-construction phases. The importance of multi-disciplinary team involvement, education, and communication is emphasized. [ABSTRACT FROM AUTHOR]

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 TR34/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. [ABSTRACT FROM AUTHOR]

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

• Azole-resistant Aspergillus fumigatus isolated in the air of the hospital. • Increasing number of resistant isolates colonizing cystic fibrosis patients. • Pots of tulips (from the Netherlands) with azole-resistant A fumigatus. • No resistant strains imported by prevailing winds. Screening has been performed for azole-resistant Aspergillus fumigatus in the indoor air of the hospital since 2015 and in soil and dust samples since January 2019. In total, 83 azole-resistant A fumigatus isolates with a TR 34 /L98H mutation have been obtained: 1 from the air of the intensive care unit, 16 from the main corridors, 59 from pots of tulips imported from the Netherlands, and 5 from the soil of trees grown in pots. [ABSTRACT FROM AUTHOR]

Summary: Influenza virus infection is now recognised as a risk factor for invasive pulmonary aspergillosis (IPA). Delays in diagnosis contribute to delayed commencement of antifungal therapy. In addition, the emergence of resistance to first‐line triazole antifungal agents puts emphasis on early detection to prevent adverse outcomes. We present 2 allogeneic stem cell transplant patients who developed IPA due to triazole‐resistant Aspergillus fumigatus following influenza infection. We underline the challenges faced in the management of these cases, the importance of early diagnosis and need for surveillance given the emergence of triazole resistance. [ABSTRACT FROM AUTHOR]

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and there from 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies. [ABSTRACT FROM AUTHOR]

Invasive candidiasis and aspergillosis are amongst major medical concerns with high mortality and morbidity in immunocompromised patients. Management of these infections is dependent on early and efficient antifungal therapy, as well as drug resistance monitoring. Decreased sensitivity of these pathogens to antifungal drugs during recent decades, rapid detection of drug resistance in pathogenic fungi is highly recomended. Generally, identification of drug resistance associated mutations requires complicated and expensive methods such as DNA sequencing. However, nowadays application of accurate, fast and highly sensitive techniques, including Rolling Circle Amplification (RCA), PCR-RFLP, Real-Time PCR, and ARMS-PCR provide the possibility for detection of target sequence containing nucleotide polymorphisms even at the one base pair level. In this review we aimed to discuss the usage, advantages and disadvantages of these techniques in order to identify the mutations of the azole-resistant strains. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies., Competing Interests: The authors declare a conflict of interest. Katrien Lagrou received consultancy fees from MRM Health and MSD, speaker fees from Pfizer and Gilead and a service fee from Thermo Fisher Scientific and TECOmedical. The remaining authors report no conflict of interest.

Azole resistance in Aspergillus fumigatus is a One Health resistance threat, where azole fungicide exposure compromises the efficacy of medical azoles. The use of the recently authorized fungicide ipflufenoquin, which shares its mode-of-action with a new antifungal olorofim, underscores the need for risk assessment for dual use of antifungals. • Azole resistance in Aspergillus fumigatus is a One Health resistance threat. • Azole fungicide exposure compromises the efficacy of medical azoles. • The fungicide Iplufenoquin shares its mode of action with a new antifungal olorofim. • Dual use of antifungal compounds requires risk assessment for human pathogens. [ABSTRACT FROM AUTHOR]

Background: Triazole resistance in the human fungal pathogen Aspergillus fumigatus has been a growing challenge in clinic treatment with triazole drugs such as itraconazole. The fast evolvement of triazole resistance in A. fumigatus in the ecosystem has drawn great attention, and there has been a possible link between the application of triazole fungicides in agriculture and triazole resistance in A. fumigatus. The change in susceptibility of A. fumigatus exposed to the new chiral triazole fungicide mefentrifluconazole was investigated in this study., Results: The results indicated that triazole resistance in A. fumigatus was acquired with exposure to mefentrifluconazole at a level of greater than or equal to 2 mg L -1 in liquid medium and soil (not at 0.4 nor 1 mg L -1 ). Interestingly, stereoselectivity was found in the acquisition of triazole resistance in A. fumigatus when exposed to mefentrifluconazole. R-mefentrifluconazole, which is very active on plant pathogens, exhibited stronger possibility in the development of the resistance in A. fumigatus than its antipode. Overexpression of cyp51A, AtrF, AfuMDR1 and AfuMDR4 were associated with the acquired resistance in A. fumigatus with hereditary stability., Conclusion: The results suggest that triazole resistance in A. fumigatus could be resulted from the selection of mefentrifluconazole at concentrations larger than 2 mg L -1 . Mefentrifluconazole should be applied within the dosage recommended by good agricultural practice to avoid the resistance in A. fumigatus in soil. This also may be applicable to other triazole fungicides. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Aim: Triazole resistance in Aspergillus fumigatus is associated with mutations in cyp51 genes, therefore, a single-tube multiplex PCR was proposed for rapid detection of such mutations. Methods: Relevant markers (n = 21) located in cyp51A and cyp51B were amplified in a multiplex reaction and subsequently analyzed by mini-sequencing. Results: A set of nonresistant A. fumigatus were tested. The markers F46, G89, M172, D255, L358, E427 and C454 located in cyp51A, as well as P394 and S35 from cyp51B, were found to be modified. Conclusions: A. fumigatus triazole resistance in Portugal is rare, nevertheless, some isolates showed alterations in the cyp51 genes. Multicenter studies with more isolates should better evaluate and validate the potential use of this method in clinical laboratories. The new methodology allows the addition of extra markers if described as relevant for A. fumigatus susceptibility to triazoles. First draft submitted: 27 March 2015; Accepted: 24 August 2015; Online: 30 October 2015 [ABSTRACT FROM AUTHOR]

This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans ( n = 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GC–MS chromatogram, and no missense mutations were found in ERG3 . Furthermore, seven amino acid substitutions in ERG11 , A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole. [ABSTRACT FROM AUTHOR]

We investigated whether plants imported to Ireland from the Netherlands might harbor triazole-resistant Aspergillus fumigatus. Samples of plant bulbs were positive for triazole-resistant A. fumigatus with CYP51A mutations. We hypothesize that this represents a route for intercountry transfer of an emerging resistance mechanism in a major opportunistic mold pathogen. [ABSTRACT FROM AUTHOR]

Alternaria alternata is an opportunistic human fungal pathogen and a ubiquitous phytopathogen capable of causing diseases to >100 agricultural crops and ornamental plants. To control plant diseases caused by A. alternata, triazole fungicides have been widely used both in open crop and vegetable fields and in indoor growth facilities such as greenhouses. At present, the effect of fungicide use on triazole resistance development in A. alternata populations is not known. Here, we isolated 237 A. alternata strains from nine greenhouses around metropolitan Kunming in Yunnan, southwest China, determined their genotypes using 10 short tandem repeat markers, and quantified their susceptibility to four triazoles (difenoconazole, tebuconazole, itraconazole, and voriconazole). Abundant allelic and genotypic diversities were detected among these A. alternata strains. Significantly, over 17% of the strains were resistant to difenoconazole, and both known and new drug-resistance mutations were found in the triazole target gene cyp51 . Our findings of high-level genetic variation of A. alternata in greenhouses coupled with high-frequency fungicide resistance call for greater attention to continued monitoring and to developing alternative plant fungal disease management strategies in greenhouses. IMPORTANCE Alternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants. Furthermore, it can cause human infections. However, our understanding of its genetic diversity and antifungal susceptibility is very limited. Indeed, the critical threshold values for resistance have not been defined for most antifungal drugs in this species. Greenhouses are known to have heavy applications of agricultural fungicides. In this study, we analyzed strains of A. alternata from nine greenhouses near metropolitan Kunming in southwestern China. Our study revealed very high genetic diversity and identified strains with high MIC values against two agricultural and two medical triazole antifungals within each of the nine greenhouses. Our study calls for greater attention to this emerging threat to food security and human health.

Aspergillus fumigatus is a saprophytic mold and an opportunistic pathogen with a broad geographic and ecological distribution. A. fumigatus is the most common etiological agent of aspergillosis, affecting over 8,000,000 individuals worldwide. Due to the rising number of infections and increasing reports of resistance to antifungal therapy, there is an urgent need to understand A. fumigatus populations from local to global levels. However, many geographic locations and ecological niches remain understudied, including soil environments from arctic regions. In this study, we isolated 32 and 52 A. fumigatus strains from soils in Iceland and the Northwest Territories of Canada (NWT), respectively. These isolates were genotyped at nine microsatellite loci and the genotypes were compared with each other and with those in other parts of the world. Though significantly differentiated from each other, our analyses revealed that A. fumigatus populations from Iceland and NWT contained evidence for both clonal and sexual reproductions, and shared many alleles with each other and with those collected from across Europe, Asia, and the Americas. Interestingly, we found one triazole-resistant strain containing the TR34 /L98H mutation in the cyp51A gene from NWT. This strain is closely related to a triazole-resistant genotype broadly distributed in India. Together, our results suggest that the northern soil populations of A. fumigatus are significantly influenced by those from other geographic regions. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a saprophytic mold and an opportunistic pathogen with a broad geographic and ecological distribution. A. fumigatus is the most common etiological agent of aspergillosis, affecting over 8,000,000 individuals worldwide. Due to the rising number of infections and increasing reports of resistance to antifungal therapy, there is an urgent need to understand A. fumigatus populations from local to global levels. However, many geographic locations and ecological niches remain understudied, including soil environments from arctic regions. In this study, we isolated 32 and 52 A. fumigatus strains from soils in Iceland and the Northwest Territories of Canada (NWT), respectively. These isolates were genotyped at nine microsatellite loci and the genotypes were compared with each other and with those in other parts of the world. Though significantly differentiated from each other, our analyses revealed that A. fumigatus populations from Iceland and NWT contained evidence for both clonal and sexual reproductions, and shared many alleles with each other and with those collected from across Europe, Asia, and the Americas. Interestingly, we found one triazole-resistant strain containing the TR 34 /L98H mutation in the cyp51A gene from NWT. This strain is closely related to a triazole-resistant genotype broadly distributed in India. Together, our results suggest that the northern soil populations of A. fumigatus are significantly influenced by those from other geographic regions.

Antibiotic resistance in fungal isolates is increasing on a global scale. Despite knowledge that pig farmers are occupationally exposed to infectious species of fungi, such as Aspergillus spp., little is known regarding their potential exposure to antifungal-resistant Aspergillus spp. The aim of this study is to obtain knowledge regarding the antifungal resistance profiles of isolates of Aspergillus species taken from different source materials—including airborne dust, surface dust, faeces, and straw—within a pig farm. The EUCAST broth microdilution method was used for testing antifungal resistance from 43 isolates of Aspergillus sampled in 3 periods inside a Danish pig farm. Seven species of Aspergillus were obtained, including A. candidus (n = 5), A. fumigatus (n = 5), A. glaucus (n = 13), A. nidulans (n = 2), A. niger (n = 15), A. terreus (n = 1), and A. versicolor (n = 2). Overall, 27.9% of the Aspergillus isolates displayed resistance against at least one antifungal, and 11.6% of Aspergillus isolates displayed resistance against multiple antifungals. The most abundant group exhibiting antifungal resistance was affiliated with the species A. niger, with isolates exhibiting resistance to itraconazole, voriconazole, and caspofungin. One isolate of A. glaucus and two isolates of A. versicolor were resistant to amphotericin B (MIC ≥ 2 mg/L amphotericin B). Antibiotic-resistant fungi were found on all three sampling days. [ABSTRACT FROM AUTHOR]

Sterol overproduction is shown to be the biochemical basis of resistance to the triazole LAB 170250F (2-(4-chlorophenyl)-3-phenyl-1-(1H-1,2,4-triazol-1-yl)-2,3-oxidopropane), an inhibitor of cytochrome P450-dependent-obtusifoliol-14α-demethylase, in the tobacco ( Nicotiana tabacum L. cv. Xanthi) mutant LAB 1-4. Genetic analysis at the callus level indicates that the resistance and the biochemical phenotypes co-segregate during meiotic recombination and therefore result most probably from the same mutation. Analysis of the intracellular distribution of sterols shows that in LAB 1-4 calli containing tenfold the sterol amount of the wild type, the overproduced metabolites, mainly obtusifoliol, are esterified by fatty acids and stored in hyaloplasmic lipid droplets. Thus, the mutant calli maintain a concentration of free sterols in cell membranes, mainly end-products of the sterol-biosynthesis pathway, which corresponds to physiological requirements, whereas the level of free sterols in the wild-type calli treated with the triazole is too low to ensure viability. We also show that sterol overproduction confers resistance to other phytotoxic sterolbiosynthesis inhibitors. [ABSTRACT FROM AUTHOR]

Triazole resistance in the pathogenic mold Aspergillus fumigatus has increased worldwide, posing a growing therapeutic challenge. Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene ( hmg1 ) have been associated with triazole resistance. Here, we describe a novel E306K triazole resistance-conferring mutation in the HMG-CoA reductase gene from an Israeli patient with chronic cavitary pulmonary aspergillosis (CCPA).

Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene ( hmg1 ) have been identified to be associated with triazole resistance in Aspergillus fumigatus. Here, we describe the first case of the G929C mutation in the hmg1 gene, leading to the W272C amino acid substitution, in a triazole-resistant isolate of A. fumigatus recovered from a chronic cavitary pulmonary aspergillosis patient who failed voriconazole therapy in China.