Volume 30, Number 8—August 2024
Research
Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020–2023
, Rasmus Krøger Hare, Karin Meinike Jørgensen, Ulla E. Bollmann, Tina B. Bech, Cecilie Cetti Hansen, Thies M. Heick, and Lise Nistrup Jørgensen
Abstract
Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018–2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat–related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.
Azole resistance in Aspergillus fumigatus fungi has increased during the past 25 years. Increasing evidence documents that selection of azole-resistant A. fumigatus (ARAf) takes place in the environment (1,2). Investigations have been performed or initiated in several countries to investigate the relative contributions of various environmental azole fungicide applications to selection for ARAf (3–5).
In Denmark during June–August 2009, ARAf was first found in 1/17 A. fumigatus isolates from hospital surroundings and 3/21 from a park in Copenhagen (6), but subsequent environmental soil and air samples collected during September–October 2013 were negative for ARAf (7). That finding is somewhat in contrast to findings in clinical samples from Denmark. After the first isolation of TR34/L98H mutants in late 2007 and TR46/Y121F/T289A in 2012 (7–9), an increasing rate of ARAf of environmental origin from 1.5% (2/133) in 2007–2009 to 3.6% (5/137) in 2018 has been found in patients with cystic fibrosis (8,10). Moreover, during 2018–2020, the nationwide surveillance of ARAf revealed a rate of 3.6% environmental ARAf among 1,083 patients (11).
Which environmental azole fungicide uses are potentially safe and which contribute mostly to the increasing proportion of ARAf is not clear. However, because selection of resistance through either emergence of resistance in a susceptible isolate or favored growth of an already existing ARAf subpopulation requires A. fumigatus multiplication, azole residues in soils or plant debris where A. fumigatus fungi thrives are probably the biggest source for dissemination of ARAf. Prior studies have suggested that hot spots for ARAf include azole-treated flower bulb production (1), plant waste piles, and composting heaps (1,12), whereas cold spots probably include animal manure and grain (1,13) and arable farming (14,15), including potato fields (3). However, variable findings have been reported for several settings, including greenhouses and strawberry crops (3,5,16).
On the basis of those findings, the Danish Ministry of Environment supported a research project about the presence and selection of ARAf in Denmark. The project included extensive environmental sampling with determination of azole-susceptible and -resistant A. fumigatus and of azole concentrations; characterization of resistance mechanisms and molecular genotypes to determine if resistant genotypes come from outside (by wind and goods) or multiply and expand in Denmark; and microcosmos and field experiments investigating the potential of various azole fungicides to select for ARAf.
Environmental Hot Spot and Field-Experiment Sampling
We collected 366 samples (Appendix Table 1): agricultural fields (air and soil; n = 167, including 40 samples obtained before/between/after azole spraying); park and private garden soil (n = 60); flower and potatoes (n = 100); compost soil (from garden waste) and compost heaps from vegetable waste and garden waste (n = 20); animal manure heaps with straw or peat and associated stable bedding (n = 25); and wood paint–associated soil (n = 14). We sampled air (1 m3/sample) through a gelatin filter by using a Sartorius MD8 Airport Portable Sampler (https://shop.sartorius.com). We placed the gelatin filter on yeast glucose chloramphenicol (YGC) agar and incubated it 1 day at 37°C, 1 day at 50°C, and 1 day at 37°C, inspecting it daily. That procedure favored growth of A. fumigatus fungi over other molds, thereby enhancing A. fumigatus isolation in a pilot study. Solid samples (e.g., soil top 5 cm [5], compost, manure heap) were suspended in sterile water with 0.1% Tween 20 (2.5 mL/g sample), vortexed, and allowed to settle for 10–15 minutes. We transferred ≈10 mL top fluid to a new tube, vortexed it, and cultured 500 µL or 250 µL on YGC and on YGC supplemented with tebuconazole (3 mg/g agar [YGC-Teb]). We centrifuged the remaining fluid (3,000 rpm, ≈1,942 g, 10 minutes), discarded ≈8 mL supernatant, and resuspended the pellet in the remaining liquid followed by plating of 500 µL on YGC and YGC-Teb. For air samples, we incubated all plates as described above.
We isolated A. fumigatus fungi (maximum 30 isolates/sample), subcultured, and identified by using macro- and micro-morphology and thermotolerance of 50°C supplemented with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker, https://www.bruker.com) and the online available spectrum database mass spectrometry imaging when needed (17,18). When we identified mixed TR34/L98H and TR46/Y121F/T289A mutations, we attempted isolation from susceptibility plate wells containing voriconazole (favoring TR46/Y121F/T289A) and posaconazole (favoring TR34/L98H).
Susceptibility Testing
Initially, A. fumigatus colonies on YGC-Teb underwent azole-resistance screening (EUCAST E.Def 10.1), followed by determination of MICs of itraconazole, posaconazole, isavuconazole, and voriconazole (EUCAST E.Def 9.3) if screening positive. Because of equal performance of YGC-TEB and E.Def 10.1, we subsequently omitted the E.Def 10.1 screening step (19,20). We compared individual proportions of ARAf pairwise by using a χ2 or Fisher exact test with the GraphPad Prism 9.3.1 program (https://www.graphpad.com).
Extraction and Concentration Determination of Azoles
We analyzed azole content as previously described for soil samples by using sonication/shaking-extraction and high-performance liquid chromatography–tandem mass spectrometry analysis (21) with minor modifications: soil samples were sieved (2 mm) and homogenized manually; potted plant soil/root mix and freeze-dried potato peels were homogenized in a blender (Appendix Table 2). We prepared blank and control samples as well as calibration standards in a reference matrix (organically farmed soil or potato peel), extracted, and analyzed together with each set of samples. When no matching reference matrix was available (potted plants, compost), we used standard addition.
Molecular Characterization of Azole Resistance Mechanisms
We sequenced the cyp51A gene, including promoter, as previously described for ARAf isolates and selected susceptible A. fumigatus isolates (8,22) (Appendix Table 3). Azole-resistant isolates that were cyp51A wild-type underwent full-length hmg1 sequencing as previously reported (23), with some modifications (Appendix Table 4). We assembled sequences and compared them with appropriate reference sequences (cyp51A, GenBank accession no. AF338659; hmg1, GenBank accession no. Afu2g03700) by using CLC Main Workbench versions 22 and 23 (QIAGEN, https://www.qiagen.com). We reported only tandem repeats in the promoter region (cyp51A only) and mutations leading to amino acid changes.
Genotyping
We conducted genotyping by using the short tandem repeat A. fumigatus (STRAf) method with all 9 microsatellite markers as previously described (24) (Appendix Table 5). We performed genotype analyses by using BioNumerics versions 7 and 8 (bioMérieux, https://www.biomerieux.com), illustrated as minimum spanning trees with default settings. We compared the genotype to worldwide genotypes from the Czech Republic (n = 1), Australia (n = 2), China (n = 8), the United Kingdom (n = 10), Cuba (n = 14), Switzerland (n = 71), Germany (n = 100), the United States (n = 102), Belgium (n = 108), Norway (n = 209), Spain (n = 219), and the Netherlands (n = 615) (9), as well as addition genotypes not previously reported from Finland (n = 1), Austria (n = 3), and Sweden (n = 5).
Microcosmos Selection Experiments
For microcosmos experiments, we placed 4 g dry sterile soil and 1 mL of 2–5 × 102 CFU/mL A. fumigatus solution (wild type, TR34/L98H, and TR46/Y121F/T289A) in 0.85% NaCl in 25 mL glass vials. We included sandy soil (total organic carbon content 0.92%) and a soil with high organic content (total organic carbon content 5.68%). The soils originated from fields organically farmed for 40 years (Svanholm Gods, Denmark). The microcosmos vials were initially incubated at 10°C, 15°C, and 20°C and consecutively sampled for A. fumigatus and ARAf quantification (Appendix Figure 1). For selection experiments, we chose incubation at 20°C and added 100 µL azole fungicide solution (tebuconazole [Folicur EW-250, 250 g/L; Bayer]), prothioconazole [Proline EC-250, 250 g/L; Bayer], mefentrifluconazole [Revysol, 100 g/L; BASF, https://agriculture.basf.com], or MilliQ water [control; Sigma Aldrich, https://www.sigmaaldrich.com]) 2 days after inoculation in application concentrations of 2.5–2,500 mg/L and homogenized the content with an inoculation loop. Final wet-weight concentrations were 0.049–49 mg/kg (spike solution concentration × applied volume)/dry weight) (Appendix Table 6).
A. fumigatus and ARAf Quantification in Microcosmos by PCR
We extracted DNA from the microcosmos samples (≈250 mg) and the collected soil samples by using DNeasy PowerLyzer PowerSoil Kit (QIAGEN) and 50 μL elution buffer. To quantify, we used quantitative PCR or droplet digital PCR (Appendix Table 7). For the first microcosmos experiments, the target was a multicopy internal transcribed spacer, and for subsequent experiments, we used primers and probes targeting the cyp51A promoter able to distinguish TR34/L98H and TR46/Y121F/T289A (Appendix Table 6). We ran controls for the standard curve and samples in triplicate.
Environmental Sampling
Environmental sampling consisted of 366 samples and 4,538 A. fumigatus isolates (Table 1). In 2020, ARAf harboring TR34/L98H or associated variants (TR34/T-67G/L98H or TR34/L98H/S297T/F495I), specifically, were found in all sample types and years, except 1 potato field. In 2021 and 2022, A. fumigatus fungi harboring TR46/Y121F/T289A or associated variants (TR46/Y121F/T289A/S363P/I364V/G448S or TR463/Y121F/M172I/T289A/G448S) were found in samples from fields, flowers/flower beds, compost, and stable bedding.
Agricultural Fields
ARAf was less common during 2020 (2.3%–7.7% of soil and air samples and 0.3%–1.7% of isolates) than during 2021–2022 (20%–21.6% of soil and air samples and 2.8%–6.6% of isolates). Most ARAf harbored TR34/L98H (25/32, 78%), whereas 1 harbored TR46/Y121F/T289A and 1 harbored TR46/Y121F/T289A/S363P/I364V/G448S (6% of ARAf). Air sampling was performed before (15 samples), during (29 samples), and after (19 samples) harvesting. The A. fumigatus counts were highest in samples taken during harvest (380 [13.1/sample]), compared with before harvest (28 [1.9/sample)] and after harvest (46 [2.4/sample]). Ten air samples (10/63 [15.9%]) contained ARAf, 8 of which were taken during harvest (8/29 [27.6%]). Among 454 A. fumigatus air isolates, 4.6% were ARAf (including 3.7% TR34/L98H and 0.2% TR46/Y121F/T289A).
Produce
Potatoes from supermarket potatoes (washed and bagged) contained very little A. fumigatus and no ARAf (Table 1). Potatoes from the farm shop and fields had some soil on the surface. All potato samples were positive for A. fumigatus fungi (2.4–15.9/sample), and 25% (4/16) samples contained ARAf harboring TR34/L98H (3.1%–10.5% of isolates). Flowerpot soil samples from 3 flower types and nurseries contained high amounts of A. fumigatus fungi. ARAf was absent in cactus pot soil, whereas 25% (10/40) of samples from poinsettia and campanula contained ARAf (2.5%–4.8% of isolates), including TR34/L98H or TR34/L98H/S297T/F495I (21/27 ARAf isolates during 2020–2021) and TR46/Y121F/T289A or TR46/Y121F/T289A/S363P/I364V/G448S (3/14 ARAf isolates during 2021). One ARAf harbored an F262 deletion within the sterol-sensing domain of Hmg1, which has previously been associated with azole MIC elevation (23). Last, air samples from a plant nursery contained few A. fumigatus fungi and no ARAf.
Flower beds
From flower beds sampled in 3 public parks and 2 private gardens, 59/60 samples contained A. fumigatus isolates (mean 24.6 isolates/sample). ARAf was found at all sites and in 30% of samples, ranging from 5% (1/20) to 47% (7/15) among public parks and 50% (5/10) of samples from private gardens. TR34/L98H and TR34/T-67G/L98H were found in 85% of ARAf isolates and 3% of A. fumigatus isolates. TR46/Y121F/T289A, TR46/Y121F/T289A/S363P/I364V/G448S, and TR463/Y121F/M172I/T289A/G448S found in 1 park and both gardens constituted 0.5% of A. fumigatus isolates and accounted for most ARAf (6/7 ARAf isolates) in the 2 private gardens.
Soil
Soil near painted allotment houses/terraces was sampled because runoff water from painted surfaces might contain azoles. All samples contained A. fumigatus isolates (mean 25.6 isolates/sample). Two samples were positive for ARAf (14.3% samples and 1.1% A. fumigatus isolates); 3/4 ARAf isolates harbored TR34/L98H, and 1 harbored an Hmg1 alteration E306K in the sterol-sensing domain.
Compost
All compost soil samples contained A. fumigatus isolates (mean 43.8 isolates/sample), and 4/5 samples contained ARAf isolates harboring TR34/L98H (2.7% of isolates). Investigation of garden waste heap samples was complicated by high contents of Mucorales interfering with A. fumigatus isolation. Consequently, it was only possible to isolate 12 individual A. fumigatus isolates, 11 of which harbored TR34/L98H (n = 7), TR46/Y121F/T289A or TR46/Y121F/T289A/S363P/I364V/G448S (n = 3), or F46Y/M172V/E427K (n = 1) Cyp51A alterations. The samples from a vegetable composting heap all grew A. fumigatus fungi, ARAf, and TR34/L98H; and 3/5 samples also grew TR46/Y121F/T289A or TR46/Y121F/T289A/S363P/I364V/G448S. Moreover, many samples grew >200 colonies/plate. Isolation from voriconazole/posaconazole susceptibility plate wells yielded 21 single ARAf isolates, of which 81% harbored TR34/L98H- and 19% TR46/Y121F/T289A-related mechanisms. However, the true number of resistant isolates was probably higher because genotyping suggested mixed genotypes in isolates with a single resistance mechanism.
Manure Heaps and Stable Bedding
Of 12 manure heap samples, 8 contained A. fumigatus isolates; the highest isolate numbers were in the 4–5-month-old manure heap at the center 1 (7.7 isolates/sample vs. 3.8 isolates/sample at center 2 with frequent emptying). ARAf isolates were found at center 1 (3/5 samples and 25.9% of isolates, all harboring TR34/L98H or TR34/T-67G/L98H) but not at center 2. Sampling of used stable bedding and the same unused material documented ARAf (TR34/L98H and TR46/Y121F/T289A) in stable bedding but not in unused straw or peat (Table 1).
Azole Fungicide
Concentrations in environmental samples were determined for 8 azole fungicides (Appendix Table 8). Levels were generally low and without correlation to ARAf detection. Hypothesizing, that a selective fungicide concentration should be at least 1 tenth of the mean MIC against wild-type A. fumigatus fungi, we found such concentrations for prothioconazole-desthio in 18 field soil samples (range 9.8–42.9 μg/kg), one of which was ARAf positive; for metconazole (38.4–135 μg /kg) in 4 potted plant samples, 3 of which contained ARAf; and for difenoconazole (367–717 μg /kg) in 4 field samples, none of which contained ARAf. In contrast, ARAf was found in 4 potato samples, 1 cactus pot soil, 8 flower bed samples, 5 compost, and 2 wood paint–associated samples with no or very low azole fungicide concentrations (25).
Molecular Characterization of A. fumigatus Isolates
Molecular analyses of the 194 resistant and 38 comparator study isolates demonstrated 139 microsatellite genotypes (Table 2). A total of 103 genotypes were found among the resistant isolates and 37 genotypes among susceptible isolates. One genotype was shared among a susceptible and a nonsusceptible isolate (both wild-type cyp51A).
Figure 1
Figure 1. Minimum spanning tree of 232 Aspergillus fumigatusgenotyped study isolates including 741 Denmark background isolates (627 isolates from 326 patients and 114 isolates from the environment) for study of...
Figure 2
Figure 2. Minimum spanning tree of 1,042 Aspergillus fumigatusgenotypes from Denmark (green, red, and purple) compared with 1,468 genotypes from other countries (gray) as part of study of environmental hot...
The TR34/L98H study isolates included 1 main cluster-1 of 34 TR34/L98H identical or closely related isolates from 14 different sampling sites (Figure 1). Other isolate clusters primarily reflect multiple isolates cultured from the same sites. Among the 22 TR46-ARAf study isolates, 13 unique genotypes were found. One final comparison introduced 1,468 worldwide genotypes from 16 countries (Figure 2). The genotypes from Denmark were widely distributed, corresponding to the worldwide diversity of genotypes. Most ARAf isolates were gathered in the top, except for all cluster-1 ARAf isolates, which were placed to the right.
Microcosmos Experiments for ARAf Selection
Figure 3
Figure 3. Selective pressure of TBZ on Aspergillus fumigatus wild type (A, B), TR34/L98H (TR34) (C, D), and TR46/Y121F/T289A (TR46) (E, F) in sandy soil (n...
Wild-type A. fumigatus, TR34/L98H, and TR46/Y121F/T289A failed to grow at 10°C but grew equally well to a maximum of 106–107 CFU/g in heat-sterilized organic rich and sandy soil at 15°C and 20°C (Appendix Figures 1, 2). Sustained complete inhibition was found for wild-type A. fumigatus fungi but not ARAf at the highest tebuconazole concentration (≈49 mg/kg wet weight) (Figure 3). Prothioconazole conferred initial growth inhibition for all strains, but growth appeared on day 5 or 8 after application and reached the levels of the untreated controls for the TR34/L98H and TR46/Y121F/T289A strains but not for the wild type (Appendix Figure 3). In contrast, treatment with mefentrifluconazole inhibited growth during the entire microcosmos experiment except for a single replicate with TR34/L98H day 27 (Appendix Figure 3).
Field Experiment for ARAf Selection
Of the prespraying and postspraying samples obtained from untreated and azole-treated field sites (Table 3), all samples contained A. fumigatus isolates (n = 360, mean 9 isolates/sample), but for all fields, numbers declined 2-fold over time. Ten (2.8%) ARAf isolates were found, 4 in unsprayed soil (4/167 = 2.4%) and 6 in treated soil (6/193 = 3.1%, p = 0.757). Seven harbored TR34/L98H, 4 found in untreated soil and 3 found after the first prothioconazole spraying in Flakkebjerg. One harbored TR46/Y121F/T289A and was found after the third tebuconazole spraying. Two isolates harbored Hmg1 alterations, of which the W272L alteration is situated within the sterol-sensing domain. Those 2 isolates were found after treatment with mefentrifluconazole and prothioconazole. Overall, the resistance percentage increased numerically (p>0.05) from 2.5% before spraying to 6.3% in the first postspraying samples and then declined by 2.5%, 1.9%, and 0 in the remaining postspraying samples. The percentages of ARAf harboring tandem repeat mechanisms followed the same pattern.
Our study demonstrated that ARAf is extensively distributed in the environment in Denmark. ARAf was found in 20% of 366 samples, and 4.2% of 4,538 investigated isolates were azole resistant, dominated by TR34/L98H-related and, to a lesser extent, TR46/Y121F/T289A-related mechanisms. Although the study was not designed to capture longitudinal changes, 3 observations suggest that ARAf is increasing in Denmark. First, although ARAf percentages were relatively low (0.3%–2.8%) among A. fumigatus isolates in agricultural soil samples from Denmark, they were higher than in studies conducted in 2010 and 2013, where no ARAf was found among 113 A. fumigatus isolates from flower beds, potted plants, and conventional and organic fields (7). Second, the ARAf proportion was higher in field air in 2021 than in 2020 (p = 0.0202) and higher in field soil in 2022 than in 2020 (p = 0.0127). Third, TR46/Y121F/T289A was found in multiple settings during 2021–2022 but not in 2020, despite a comparable number of samples. Those findings coincide with the first of several isolations of TR46/Y121F/T289A from patients in Denmark in 2021 (M.C. Arendrup, unpub. data) since the initial finding of this genotype in a single patient in 2014 (7). Genotyping identified a nationwide cluster of TR34/L98H with wide geographic distribution across Denmark, including clinical and environmental isolates. That particular clone has remained dominant among azole-resistant clinical isolates from Denmark since 2018. Whether that trait of augmented mutation rate is a virulence factor and responsible for the relatively high prevalence among Denmark ARAf warrants further investigation, but it aligns with the observed increasing incidence. It is also of interest that that cluster is located quite distant from most other tandem repeat isolates, possibly indicating that that clone has appeared through sexual recombination of unrelated strains.
The ARAf isolates were more closely related than the A. fumigatus–susceptible isolates. That finding suggests more recent ancestors and that the increasing environmental resistance rates are driven mainly by factors favoring propagation of TR34/L98H and TR46/Y121F/T289A genotypes already present over the susceptible population rather than induction of resistance in susceptible isolates from outside.
Besides characterizing the prevalence of ARAf and relevant hot spots in Denmark, it was our intention to investigate potential links between the presence of azoles in the samples and ARAf. Azoles were found at low concentrations in most soil samples, indicating persistence of azoles in the soils and a measurable carryover concentration from season to season (data not shown). We saw no association between ARAf findings and azole concentration in any specific sample or across sample types, nor did we verify increasing resistance after azole spraying in wheat field trials, potentially because A. fumigatus growth was absent. In contrast, our microcosmos experiments suggested that azole fungicides may favor ARAf growth over wild-type A. fumigatus in soil. Few studies have been able to confirm a link between specific azole use and resistance in A. fumigatus (3). A study in China indicated a link between use of azoles in paddy rice and resistance development, whereas a recent study in Switzerland found that azole resistance was neither associated with any specific agricultural practice nor with the presence of azole fungicides (26,27). The concentration of azoles in an environmental sample is a snapshot, which fails to provide information about previous exposures, potentially relevant for resistance selection. Other factors may influence selection and presence of ARAf at the time of sampling (e.g., soil type, temperature, humidity, competition from the indigenous microbial community, azole application concentration and subsequent kinetics of free and soil bound fractions, liquid manure application, and amount of organic matter). Those factors complicate identification of safe and unsafe procedures, particularly as the annual increase in ARAf appears to be well below 1% in Denmark, suggesting a slow and potentially fluctuating increase that is difficult to capture in light of the heterogeneity of environmental samples.
We confirmed that the hot spots for ARAf are compost, flower beds, and flower production; but we also found ARAf in stables and horse manure heaps (1,3,28). Azoles are not used in parks, gardens, or stables. However, planting azole-treated bulbs and using compost soil based on azole-containing plant material can turn flower beds and garden waste heaps into hot spots (29). Of note, the tulip cultivars found in the private gardens were old cultivars and the azole contents were very low, suggesting that the ARAf found could reflect the general background ARAf population combined with good growth conditions for A. fumigatus rather than a direct link to azole-treated bulb planting. In addition, our study findings suggest that it is plausible that use of azole-containing conventional straw for stable bedding similarly can turn stable bedding and manure heaps into hot spots and thus reflect collateral damage associated with azole use elsewhere.
One limitation of our study is that the sensitivity for ARAf detection in a given sample and sample type will vary because of the variable number of A. fumigatus in the environmental samples. Consequently, we cannot exclude that ARAf may be found in negative samples, in which A. fumigatus numbers were low. Yet it is plausible that such settings, because of the overall lower A. fumigatus prevalence, may contribute less to resistance selection and human exposure. Another limitation is that we did not have funding for whole-genome sequencing. However, microsatellite typing has been widely used and has a high discriminative power, enabling us to compare with already published data.
In conclusion, our study and the available literature strongly suggest that the dual use of azoles in clinical medicine and for crop and material protection has introduced azole resistance in A. fumigatus, which challenges patient management. Isolates harboring environmental resistance mechanisms were found in every setting explored and expand the numbers, genotypes, and target gene variants found in earlier studies. Because of a lack of fitness cost (30), the ARAf variants will remain even if use of azoles active against A. fumigatus is terminated. It seems advisable to avoid future dual use of agents used in human medicine, such as drug candidates olorofim and fosmanogepix, which are threatened by new compounds developed for plant protection (31). Prioritizing the use of A. fumigatus active azole fungicides might potentially slow the rise in rates of resistance.
Dr. Arendrup is a professor at Copenhagen University and the head of the national reference laboratory for mycology and the EUCAST development laboratory at the Statens Serum Institute in Copenhagen, Denmark. Her main research interests are the epidemiology, susceptibility, breakpoint development, diagnostics, and treatment of fungal infections.
Acknowledgments
The study was funded by the Danish Ministry of Environment's Research Program for Pesticides (Journal number 2019-15114).
The authors declare no conflicts of interest related to this study. Outside this study, the authors declare that M.C.A. has over the past 5 years received research grants/contract work (paid to the SSI) from Cidara, F2G, Gilead, and Scynexis and speaker honoraria (personal fee) from Astellas, Chiesi, Gilead, and F2G. She is the current chairman of the EUCAST-AFST. L.N.J has over the past 5 years received research grants/contract work (paid to the AU) from BASF, BAYER CropScience, Syngenta Nordic, Corteva Agriscience, ADAMA, and UPL Limited. and speaker honoraria (personal fee) from BASF, Syngenta, and Corteva. R.K.H. has over the past 5 years received a travel grant and an unrestricted research grant from Gilead.
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Cite This ArticleOriginal Publication Date: June 27, 2024
Table of Contents – Volume 30, Number 8—August 2024
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Maiken Cavling Arendrup, Unit for Mycology Building 43/317, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
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