Azole-Resistant Aspergillus Emerges in Chinese Hospitals at 8.6 Percent

Researchers surveying fungal infections in two Chinese cities have mapped the local landscape of antifungal resistance in clinical Aspergillus isolates, finding that 8.6 percent of Aspergillus fumigatus strains recovered from patients were resistant to azole antifungals, the first-line drugs for aspergillosis. The surveillance study, conducted at Zhejiang Provincial People’s Hospital in Hangzhou and the Fourth Affiliated Hospital of Zhejiang University in Yiwu, also uncovered a resistant strain whose resistance could not be explained by any known resistance mutation, pointing instead to a transcriptional adaptation that boosts ergosterol biosynthesis and drug efflux.

The team analyzed 110 Aspergillus clinical isolates collected from patients at the two hospitals. Bronchoalveolar lavage fluid accounted for the majority of specimens at 65.5 percent, followed by sputum at 23.6 percent, with the remainder drawn from sources including blood, serum, cerebrospinal fluid, deep tissue aspirates, pus, urine and skin scrapings. Species identification combined microscopy, culture, internal transcribed spacer sequencing and MALDI-TOF mass spectrometry. Eight distinct Aspergillus species emerged from the collection, with A. fumigatus predominating at 66.4 percent of isolates and A. flavus second at 16.4 percent.

Antifungal susceptibility testing followed the European Committee on Antimicrobial Susceptibility Testing guideline AFST_BP_v.10.2, with confirmatory minimum inhibitory concentration measurements using gradient test strips and three biological replicates per strain. Among 70 clinical A. fumigatus strains, six, or 8.6 percent, showed resistance to azole treatment, and the same proportion, 8.6 percent, grew on agar containing amphotericin B at 2 micrograms per milliliter, classifying them as amphotericin B resistant. Among the azole-resistant isolates, 83 percent showed markedly high itraconazole MIC values exceeding 32 micrograms per milliliter, and all tested resistant strains showed resistance to posaconazole, although only two strains showed similarly high MIC values for voriconazole and three strains remained susceptible to that drug.

Molecular characterization by PCR screening of the cyp51A gene and its promoter region revealed the classical resistance signatures in most resistant isolates. Three strains carried the TR34/L98H mutation, a 34-nucleotide tandem repeat in the cyp51A promoter combined with a leucine-to-histidine substitution, two of them also carrying S297T and F495I substitutions. Another strain carried the TR46 tandem repeat with the Y121F/T289A mutation combination. Both genotypes are associated with pan-azole resistance and are considered characteristic of environmental acquisition, linked to azole fungicide use in agriculture rather than selection during patient therapy. Half of the resistant isolates in this study therefore harbored the environmentally associated TR34/L98H mutation.

One resistant isolate, designated Afu9073 X 678, showed no known resistance mutation on PCR screening. Whole-genome sequencing revealed the F46Y/M172V/E427K mutation combination in cyp51A when aligned to the Af1163 reference genome, a combination previously reported mainly in environmental isolates and rarely in clinical strains. This strain’s itraconazole MIC exceeded 32 micrograms per milliliter, substantially higher than the approximately 16 micrograms per milliliter reported for environmental isolates carrying the same mutations, and it was also resistant to voriconazole and posaconazole, suggesting additional resistance mechanisms beyond cyp51A alterations. Phylogenetic analysis based on whole-genome sequencing placed this strain in a distinct clade, separate from the other resistant clinical isolates.

To probe the unexplained resistance, the researchers performed variant calling on cyp51A, cyp51B including promoter regions, hmg1 and hapE, using the Af293 reference genome. Two previously unreported mutations emerged: S437F in cyp51A and P298T in hmg1. The team then used CRISPR-Cas9 genome editing to introduce each variant into the CEA17 ΔakuBKU80 wild-type background and tested azole susceptibility by MIC measurement. The mutants’ MIC values remained consistent with wild type, and a control strain carrying the known resistance-conferring G54W cyp51A mutation behaved as expected, excluding S437F and P298T as direct resistance drivers.

Transcriptomic analysis then redirected the investigation. The researchers performed RNA sequencing on the wild-type control strain, on Afu9073 X 678, and on the clinical resistant strain Afu1358 X 581 carrying known mutations, all exposed to itraconazole at 8 micrograms per milliliter in liquid culture for 30 minutes, with three biological replicates per strain. Differentially expressed genes were identified with DESeq2 using an adjusted p-value below 0.05 and a log2 fold change above 1. The resistant strain Afu9073 X 678 showed extensive transcriptional reprogramming compared with both controls, with significant upregulation of ergosterol biosynthesis genes including erg5, erg24B, erg3 and erg25, and upregulation of the abcD transporter gene. The authors interpret this dual pattern as enhanced flux through the ergosterol biosynthetic pathway compensating for azole-induced inhibition, combined with active drug export reducing intracellular drug concentrations.

The surveillance also documented resistance patterns in non-A. fumigatus species that carry clinical weight. All A. niger isolates were resistant to both itraconazole and posaconazole, consistent with the known intrinsic resistance of that species, while all remained susceptible to amphotericin B. Among A. flavus isolates, itraconazole MICs ranged from 0.38 to 2 micrograms per milliliter with 3 of 18 isolates, or 17 percent, classified as resistant, and posaconazole MICs spanned 0.19 to 1 microgram per milliliter with 9 isolates, or 50 percent, resistant; all A. flavus isolates were fully susceptible to voriconazole and amphotericin B. For A. terreus, all isolates were susceptible to itraconazole and amphotericin B, but 3 of 8 isolates, or 37.5 percent, were classified as posaconazole resistant.

The 8.6 percent azole resistance rate in Zhejiang sits within a strikingly variable national picture. A Shanghai study covering 2019 to 2021 reported a non-susceptible rate of 7.1 percent, a nationwide multicenter study across 12 provinces found a clinical resistance rate of 2.5 percent, and a Ningxia Province study reported 1.2 percent among 307 clinical isolates. In agricultural environments the picture is far worse: one Yunnan study found approximately 80 percent of greenhouse soil isolates were azole resistant. The authors note that the prevalence is concerning because azole antifungals are the first-line therapy for aspergillosis and resistance is associated with significantly increased mortality, with studies demonstrating up to a 25 percent increase in day-90 mortality in patients with drug-resistant invasive aspergillosis compared with wild-type infections.

The amphotericin B finding adds a further layer of concern, because that drug serves as a critical therapeutic alternative for azole-resistant infections. The authors describe the 8.6 percent amphotericin B resistance rate among A. fumigatus isolates as particularly alarming, noting that previous studies have associated amphotericin B MIC values of 2 micrograms per milliliter or higher with treatment failure, and that in one pivotal study 22 of 23 patients infected with amphotericin B-resistant Aspergillus isolates died while all patients with susceptible isolates survived. The mechanisms of amphotericin B resistance in A. fumigatus remain poorly understood, though some studies suggest catalase activity may play a role. The study’s broader context is sobering: recent estimates indicate over 6.5 million invasive fungal infection cases annually worldwide with approximately 3.8 million deaths, and A. fumigatus was designated a critical priority pathogen by the World Health Organization in 2022. The authors argue that resistance surveillance cannot rely solely on detecting known mutations and must incorporate comprehensive molecular characterization, since strains carrying identical cyp51A mutations can display substantially different resistance phenotypes depending on additional transcriptional adaptations, and they call for continued regional surveillance and rapid resistance detection methods to inform clinical treatment strategies.

Subject of Research: Antifungal resistance surveillance and molecular mechanisms in clinical Aspergillus isolates from Zhejiang Province, China

Article Title: Antifungal resistance landscapes in major fungal pathogens in Zhejiang, China

Article References: Wang, H., Zhang, J., Zhang, N., Ji, Y., Hong, X., Sámano-Sánchez, H., Xu, F., Han, Y., Xiong, X., Welburn, S. C., Schulze, H., Bachmann, T. T., Ge, Y., & Scharf, D. H. (2026). Antifungal resistance landscapes in major fungal pathogens in Zhejiang, China. Fungal Biology and Biotechnology, 13(1), Article 19. https://doi.org/10.1186/s40694-026-00223-1

Image Credits: AI Generated

DOI: 10.1186/s40694-026-00223-1

Keywords: Aspergillus fumigatus, azole resistance, cyp51A, TR34/L98H, amphotericin B resistance, ergosterol biosynthesis, efflux transporters, CRISPR-Cas9, transcriptomics, antifungal surveillance, Zhejiang China, EUCAST