Song Yinggai, Buil Jochem B, Rhodes Johanna, Zoll Jan, Tehupeiory-Kooreman Marlou, Ergün Mehmet, Zhang Jianhua, Li Ruoyu, Bosch Thijs, Melchers Willem J G, Verweij Paul E
Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China; Department of Medical Microbiology and Radboudumc-CWZ Center of Expertise for Mycology, Radboud University Medical Center, Nijmegen, Netherlands; National Clinical Research Center for Skin and Immune Diseases, Beijing, China; Research Center for Medical Mycology, Peking University, Beijing, China.
Department of Medical Microbiology and Radboudumc-CWZ Center of Expertise for Mycology, Radboud University Medical Center, Nijmegen, Netherlands.
Lancet Microbe. 2025 Aug;6(8):101114. doi: 10.1016/j.lanmic.2025.101114. Epub 2025 Jul 9.
Aspergillus fumigatus is the main cause of invasive aspergillosis and triazole antifungals are the primary treatment option. The effectiveness of triazole therapy is hampered by the emergence of resistance, mainly caused by mutations in the cyp51A gene and a tandem repeat (TR) of 34 bases (TR/Leu98His) and 46 bases (TR/Tyr121Phe/Thr289Ala) in the promoter region, which correspond with signature triazole resistance phenotypes. We aimed to investigate the occurrence of triazole phenotype and genotype variation over a 29-year period in the Netherlands.
In this genomic and phenotypic study, we screened all clinical A fumigatus isolates from Dutch hospitals collected between Jan 6, 1994, and Dec 31, 2022, for resistance to triazole using agar-based methods, and characterised them by sequencing the cyp51A gene and in vitro susceptibility testing using the European Committee on Antimicrobial Susceptibility Testing reference method. Whole-genome sequencing was performed on selected isolates, including those harboring TR variants, high-frequency single-nucleotide polymorphisms, and wild-type strains. Clinical information such as age, underlying disease, diagnosis, therapy, and outcomes was collected for patients who had isolates cultured at the Radboud University Medical Centre, Nijmegen, Netherlands, between Jan 1, 2017, and Dec 31, 2022.
1979 (15·6%) of the screened 12 679 A fumigatus isolates harboured cyp51A triazole resistance mutations, predominately TR/Leu98His sensu stricto in 1338 (67·6%) resistant isolates and TR/Tyr121Phe/Thr289Ala sensu stricto in 332 (16·8%) resistant isolates. Phenotype and genotype variations were observed in 325 (17·2%) triazole resistant isolates harbouring a TR-resistance mechanism, including 12 cyp51A genotype variants. Whole-genome sequencing showed that isolates with combinations of TR-based and TR-based polymorphisms seemed to be derived from separate populations, but there was some overlap. 59 cases of proven or probable invasive aspergillosis were identified, including 13 triazole-resistant cases, of which three were caused by genotype variants. Mixed genotype infection was observed in 11 (84·6%) of 13 triazole-resistant patients and the number of antifungal treatment switches was higher compared with triazole-susceptible disease (p<0·0001).
Our study showed variation in triazole genotypes and phenotypes in clinical A fumigatus isolates with cyp51A-mediated resistance, some of which were cultured from triazole-resistant invasive aspergillosis cases. Triazole resistance variation and mixed A fumigatus genotypes represent a major challenge in clinical management of Aspergillus diseases because current molecular diagnostic tools will increasingly fail to predict the resistance phenotype, underscoring the need for improved detection methods.
National Key Research and Development Program of China, National Natural Science Foundation of China, and Wellcome Trust.
烟曲霉是侵袭性曲霉病的主要病因，三唑类抗真菌药物是主要的治疗选择。三唑类治疗的有效性受到耐药性出现的阻碍，耐药性主要由cyp51A基因突变以及启动子区域中34个碱基的串联重复序列（TR/Leu98His）和46个碱基的串联重复序列（TR/Tyr121Phe/Thr289Ala）引起，这些与典型的三唑类耐药表型相对应。我们旨在调查荷兰29年间三唑类表型和基因型变异的发生情况。
在这项基因组和表型研究中，我们使用基于琼脂的方法筛选了1994年1月6日至2022年12月31日期间从荷兰医院收集的所有临床烟曲霉分离株对三唑类的耐药性，并通过对cyp51A基因进行测序以及使用欧洲抗菌药物敏感性试验委员会参考方法进行体外药敏试验对其进行表征。对选定的分离株进行全基因组测序，包括那些携带TR变异、高频单核苷酸多态性和野生型菌株的分离株。收集了2017年1月1日至2022年12月31日期间在荷兰奈梅亨拉德堡大学医学中心培养出分离株的患者的临床信息，如年龄、基础疾病、诊断、治疗和结局。
在筛选的12679株烟曲霉分离株中，1979株（15.6%）携带cyp51A三唑类耐药突变，其中1338株（67.6%）耐药分离株主要为严格意义上的TR/Leu98His，332株（16.8%）耐药分离株为严格意义上的TR/Tyr121Phe/Thr289Ala。在3个携带TR耐药机制的三唑类耐药分离株中观察到表型和基因型变异，包括12个cyp51A基因型变异。全基因组测序表明，具有基于TR和基于TR多态性组合的分离株似乎来自不同的群体，但存在一些重叠。共鉴定出59例确诊或可能的侵袭性曲霉病病例，包括13例三唑类耐药病例，其中3例由基因型变异引起。在13例三唑类耐药患者中的11例（84.6%）观察到混合基因型感染，与三唑类敏感疾病相比，抗真菌治疗转换的次数更高（p< 0.0001）。
我们的研究显示，在具有cyp51A介导耐药性的临床烟曲霉分离株中，三唑类基因型和表型存在变异，其中一些分离株来自三唑类耐药的侵袭性曲霉病病例。三唑类耐药变异和混合烟曲霉基因型代表了曲霉病临床管理中的一项重大挑战，因为当前的分子诊断工具越来越无法预测耐药表型，这突出了改进检测方法的必要性。
中国国家重点研发计划、中国国家自然科学基金和惠康信托基金会。
