Dual use of antifungals in medicine and agriculture: How do we help prevent resistance developing in human pathogens?

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.

Emergence of W272C Substitution in Hmg1 in a Triazole-Resistant Isolate of Aspergillus fumigatus from a Chinese Patient with Chronic Cavitary Pulmonary Aspergillosis.

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.

Triazole-Resistant Aspergillus fumigatus in an Israeli Patient with Chronic Cavitary Pulmonary Aspergillosis Due to a Novel E306K Substitution in Hmg1.

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).

Implementation of a clinical decision rule for selecting empiric treatment for invasive aspergillosis in a setting with high triazole resistance.

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.

Hmg1 mutations in Aspergillus fumigatus and their contribution to triazole susceptibility.

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.

Stable prevalence of triazole-resistance in Aspergillus fumigatus complex clinical isolates in a Belgian tertiary care center from 2016 to 2020.

BACKGROUND: Prevalence reports of triazole-resistance in Aspergillus fumigatus differ between countries and centers and may likewise vary over time. Continuous local surveillance programs to establish the evolving epidemiology of triazole-resistance in A. fumigatus are crucial to guide therapeutic recommendations. Here, we determined the prevalence of triazole-resistance in A. fumigatus complex culture-positive patients at the tertiary care center University Hospitals Leuven in Belgium in clinical isolates from 2016 to 2020. METHODS: All A. fumigatus complex isolates cultured from UZ Leuven patients between 2016 and 2020 were screened for triazole-resistance. Confirmation of resistance to voriconazole, posaconazole and itraconazole was performed with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. Mutations in the cyp51A gene in triazole-resistant isolates were determined by sequencing. Patients were classified as susceptible or resistant cases based on their isolate's susceptibility phenotype. RESULTS: We screened 2494 A. fumigatus complex isolates from 1600 patients (320 ± 38 [SD] patients per year). The prevalence of triazole-resistance in patients was 8.3% (28/337), 6.7% (26/386), 7.0% (21/301), 7.1% (21/294) and 7.4% (21/282) in 2016, 2017, 2018, 2019 and 2020 respectively, with an overall triazole-resistance prevalence of 7.1% (85/1192; 95% CI 6.6-7.7%). The TR34/L98H mutation was the most prevalent (83.0%, 78/94) with most isolates displaying resistance to all triazole antifungals tested (94.8%, 74/78). CONCLUSION: The prevalence of triazole-resistance in A. fumigatus has remained stable from 2016 to 2020 in our center ranging between 6.7 and 8.3%, with an overall five-year prevalence of 7.1%. The environmentally associated cyp51A gene mutations were most prevalent amongst triazole-resistant isolates and conferred resistance to all antifungals tested in 73% of the isolates.

Antifungal susceptibility profile and molecular identification of Cyp51C mutations in clinical and environmental isolates of Aspergillus flavus from Argentina.

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.

Outpatient parenteral antifungal therapy (OPAT) for invasive fungal infections with intermittent dosing of liposomal amphotericin B.

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.

A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells.

Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

A simple and low cost tetra-primer ARMS-PCR method for detection triazole-resistant Aspergillus fumigatus.

The mutation at codon L98 accompanied by a tandem repeat of 34 base pairs (TR34/L98H) in the 5´upstream region of cyp51A is the principal mechanism of triazole resistance of Aspergillus fumigatus. We aimed to evaluate a simple and low-cost tetra-primer amplification refractory mutation system (ARMS)-PCR technique for detection of TR34/L98H mutations in the cyp51A gene of azole-resistant A. fumigatus. The tetra-primer ARMS-PCR assay optimized by four primers in one reaction consists of external primers for detection of tandem repeats in the promoter region and internal primers for detection of a point mutation in codon 98 (L98H) in the cyp51A gene of azole-resistant A. fumigatus. The specificity of TR34/L98H mutation detection was assessed by testing 36 clinical and environmental A. fumigatus strains. The tetra-primer ARMS-PCR assay from A. fumigatus, containing wild-type sequence (T allele) and L98H mutation at cyp51A (A allele), yielded two DNA fragments of 908 bp and 740 bp and two of 942 bp and 212 bp, respectively. None of the A. fumigatus isolates without the TR34/L98H mutation yielded false-positive results. The ARMS-PCR assay was 100% concordant with DNA sequencing results. Prevalence and screening of the TR34/L98H mutation in the cyp51A gene in A. fumigatus isolates may now be determined by a fast, low-cost, and simple method in resource-poor settings.

Contemporary Gene Flow is a Major Force Shaping the Aspergillus fumigatus Population in Auckland, New Zealand.

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.

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 µg/ml for the CLSI method and 0.25 µg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 µg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 µg/ml. The tentative ECOFFinder ECV with SYO was 0.06 µg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 µg/ml (CLSI, EUCAST, Etest) or 0.06 µg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

Post-influenzal triazole-resistant aspergillosis following allogeneic stem cell transplantation.

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.

Intercountry Transfer of Triazole-Resistant Aspergillus fumigatus on Plant Bulbs.

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.

Triazole Resistance Is Still Not Emerging in Aspergillus fumigatus Isolates Causing Invasive Aspergillosis in Brazilian Patients.

Aspergillus fumigatus azole resistance has emerged as a global health problem. We evaluated the in vitro antifungal susceptibility of 221 clinical A. fumigatus isolates according to CLSI guidelines. Sixty-one isolates exhibiting MICs at the epidemiological cutoff value (ECV) for itraconazole or above the ECV for any triazole were checked for CYP51A mutations. No mutations were documented, even for the isolates (1.8%) with high voriconazole MICs, indicating that triazoles may be used safely to treat aspergillosis in Brazil.

Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides.

Resistance to medical triazoles in Aspergillus fumigatus is an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages of A. fumigatus from previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes.

Diversity and origins of Indian multi-triazole resistant strains of Aspergillus fumigatus.

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

Occurrence of triazole-resistant Aspergillus fumigatus with TR34/L98H mutations in outdoor and hospital environment in Kuwait.

BACKGROUND: Invasive aspergillosis due to triazole-resistant Aspergillus fumigatus, a common airborne fungal pathogen, has emerged in some European and Asian countries, likely due to exposure to azole fungicides in the environment. This study determined the occurrence of triazole-resistant A. fumigatus in outdoor and hospital environment in Kuwait, a semi-arid desert country in Arabian Peninsula. METHODS: Outdoor/indoor air, water and cotton swab samples were cultured on malt extract agar and A. fumigatus colonies were identified by phenotypic and molecular methods. Drug susceptibility of A. fumigatus isolates to itraconazole, posaconazole and voriconazole was carried out by an Etest and a broth microdilution method. Resistance mechanisms involving cyp51A mutations were probed by mixed-format real-time (MF-rt)-PCR assays. Triazole-resistant isolates were typed by nine-locus microsatellite analysis. A multiplex allele-specific (MAS)-PCR assay was developed for detection of L98H mutation in cyp51A. RESULTS: Of 115 A. fumigatus isolates obtained from 362 environmental samples from across Kuwait, 8 isolates were resistant to itraconazole, posaconazole and voriconazole. All itraconazole-resistant isolates contained a 34-bp tandem repeat (TR34) in the promoter region and a L98H mutation at codon 98 (TR34/L98H) in cyp51A. These mutations were absent in all itraconazole-susceptible isolates. MAS-PCR accurately detected L98H mutation in all triazole-resistant isolates. Three microsatellite patterns were observed among resistant isolates with one pattern clustering with Indian clinical and environmental isolates. CONCLUSIONS: Triazole-resistant A. fumigatus with TR34/L98H mutations in cyp51A is prevalent in Kuwait. Although triazole-resistant A. fumigatus has not yet been isolated from clinical specimens, its presence in the environment suggests that the possibility of susceptible individuals getting infected with such strains exists and may pose therapeutic challenges in its management.

Triazole resistance in Aspergillus fumigatus exposed to new chiral fungicide mefentrifluconazole.

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.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

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.