Highlight
- Longitudinal genomic analysis of Aspergillus fumigatus isolates in the Netherlands shows a persistent and increasing diversity of triazole-resistant genotypes from 1994 to 2022.
- Key resistance mutations—particularly in the cyp51A gene—are driving both genotype and phenotype variation, complicating diagnosis and treatment.
- High mortality rates among patients with azole-resistant A. fumigatus and the emergence of multi-genotype infections demand urgent improvements in surveillance and therapeutic strategies.
- Dual use of azole antifungals in medicine and agriculture is a major driver of resistance, raising the stakes for stewardship efforts and new antifungal development.
Background
Aspergillus fumigatus is a ubiquitous environmental mold, recognized as the leading cause of invasive aspergillosis (IA)—a life-threatening infection primarily affecting immunocompromised patients, including those with hematologic malignancies, organ transplants, or prolonged corticosteroid therapy. Triazole antifungals (e.g., voriconazole, itraconazole, posaconazole) are the mainstay of IA treatment and prophylaxis. However, the emergence of triazole-resistant A. fumigatus (TRAF), frequently linked to the use of azoles in both clinical and agricultural sectors, poses a grave threat to patient outcomes. According to the World Health Organization (WHO), azole-resistant A. fumigatus now ranks in the highest-priority group of fungal pathogens due to its significant public health impact and high mortality rates, often exceeding 50% in affected patients.
Despite an increasing number of reports, the evolutionary dynamics, molecular epidemiology, and clinical implications of triazole resistance in A. fumigatus have been underexplored at a population level. Addressing this gap, Song et al. (Lancet Microbe, 2025) present a comprehensive, 29-year analysis of A. fumigatus isolates from Dutch hospitals, shedding light on the genetic underpinnings and clinical correlations of antifungal resistance.
Study Overview and Methodological Design
This retrospective genomic and phenotypic study screened all A. fumigatus isolates collected from hospitalized patients across the Netherlands between January 1994 and December 2022. The authors performed whole-genome sequencing on 12,679 representative isolates, including those with known triazole resistance markers, high-frequency single-nucleotide polymorphisms (SNPs), and wild-type controls. The principal focus was on mutations in the cyp51A gene—a well-established determinant of azole resistance.
Clinical data, including confirmed and possible cases of invasive aspergillosis, were integrated to assess the clinical impact of triazole resistance. The primary endpoints included the prevalence of triazole resistance, the spectrum of cyp51A mutations, clonal diversity, and the occurrence of multi-genotype infections.
Limitations acknowledged by the authors include the reliance on culture-based identification (excluding non-cultured cases), variable resistance testing protocols over the study period, and the potential confounding from analyzing multiple isolates per patient.
Key Findings
The study yielded several pivotal observations:
- Of the screened isolates, 15.6% harbored cyp51A mutations conferring triazole resistance—an increase over prior decades. The two most prevalent mutations were tandem repeats (TRs) of 34 and 46 base pairs, specifically TR34/Leu98His (67.6%) and TR46/Tyr121Phe/Thr289Ala (16.8%).
- Substantial heterogeneity was observed: 17.2% of triazole-resistant isolates with TR-based mechanisms displayed phenotypic and genotypic variation, encompassing 12 distinct cyp51A genotypic variants.
- Among 59 cases of confirmed or possible invasive aspergillosis, 13 were caused by triazole-resistant strains—many involving mixed-genotype infections within a single patient.
- Mortality rates for azole-resistant cases, consistent with global reports, ranged from 47% to 88%, underscoring the urgent clinical threat posed by resistance.
Mechanistic Insights and Pathophysiological Context
The cyp51A gene encodes the fungal 14-α sterol demethylase, a key enzyme in ergosterol biosynthesis targeted by triazole antifungals. Mutations, especially tandem repeats in the cyp51A promoter region, enhance gene expression or alter enzyme structure, reducing triazole binding and efficacy. The study’s identification of multiple TR-based and point mutations highlights the adaptive capacity of A. fumigatus under antifungal selection pressure.
The simultaneous presence of multiple genotypes within single infections suggests either polyclonal infection (from diverse environmental sources) or in-host microevolution, complicating both diagnosis and resistance surveillance. The dual use of azoles in agriculture and medicine further accelerates selection for resistant strains in the environment, contributing to their spread into clinical settings.
Clinical Implications
For clinicians, the findings reinforce the importance of:
- Heightened vigilance for azole resistance in patients with suspected or confirmed invasive aspergillosis, especially those with prior azole exposure or in regions with high environmental resistance rates.
- Incorporating molecular resistance testing (e.g., cyp51A mutation screening) into diagnostic algorithms, as phenotypic susceptibility alone may underestimate the true burden of resistance.
- Considering non-azole antifungal agents (e.g., liposomal amphotericin B or novel agents like olorofim) for empiric or salvage therapy in resistant cases.
- Advocating for antifungal stewardship across healthcare and agriculture to limit further emergence of resistance.
Limitations and Controversies
The study’s strengths—a large, longitudinal cohort and robust genomic analysis—are tempered by several limitations:
- Cultural methods may underrepresent the burden of disease, as many cases of invasive aspergillosis are diagnosed by non-culture-based methods (e.g., galactomannan antigen, PCR).
- Temporal changes in laboratory methodologies could introduce bias or affect genotypic/phenotypic identification over time.
- Multiple isolates from the same patient may skew resistance prevalence and diversity estimates.
Controversy remains regarding the optimal approach to surveillance and the threshold for switching from azole to non-azole therapy, particularly in resource-limited settings. The environmental-occupational interface—especially the impact of agricultural azole use—also remains a contentious policy area.
Expert Commentary or Guideline Positioning
Paul E. Verweij, MD, a leading authority in clinical mycology, underscores the overlooked but mounting threat of fungal antimicrobial resistance and advocates for the integration of molecular diagnostics into routine practice. He also warns that the lessons of azole resistance must be heeded as new antifungal classes (e.g., dihydroorotate dehydrogenase inhibitors like olorofim) are introduced in both clinical and agricultural spheres.
Current guidelines (e.g., Infectious Diseases Society of America, European Society for Clinical Microbiology and Infectious Diseases) recommend susceptibility testing for all clinically significant Aspergillus isolates and consideration of non-azole agents in regions or patient populations with elevated resistance rates.
Conclusion
The 29-year Dutch cohort study provides compelling evidence of the enduring and evolving challenge of triazole-resistant Aspergillus fumigatus. The increasing genetic diversity, prevalence of resistance mutations, and occurrence of multi-genotype infections demand a paradigm shift in clinical management, diagnostics, and stewardship. Future research should prioritize rapid molecular diagnostics, the clinical impact of genotype diversity, and stewardship strategies to safeguard both existing and emerging antifungal agents.
References
1. Song Y, Buil JB, Rhodes J, Zoll J, Tehupeiory-Kooreman M, Ergün M, Zhang J, Li R, Bosch T, Melchers WJG, Verweij PE. Triazole-resistant Aspergillus fumigatus in the Netherlands between 1994 and 2022: a genomic and phenotypic study. Lancet Microbe. 2025 Jun 20:101114. doi: 10.1016/j.lanmic.2025.101114.
2. World Health Organization. Fungal Priority Pathogens List to Guide Research, Development and Public Health Action. Geneva: WHO; 2022.
3. Patterson TF, Thompson GR 3rd, Denning DW, et al. Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2016 Aug 15;63(4):e1-e60. doi:10.1093/cid/ciw326.
4. Verweij PE, et al. Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis. 2009 Dec;9(12):789-95. doi:10.1016/S1473-3099(09)70265-8.
