Sabino Raquel, Gonçalves Paulo, Martins Melo Aryse, Simões Daniela, Oliveira Mariana, Francisco Mariana, Viegas Carla, Carvalho Dinah, Martins Carlos, Ferreira Teresa, Toscano Cristina, Simões Helena, Veríssimo Cristina
Infectious Diseases Department, National Health Institute Dr. Ricardo Jorge, 1649-016 Lisbon, Portugal.
European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control, 16973 Solna, Sweden.
J Fungi (Basel). 2021 Jan 6;7(1):28. doi: 10.3390/jof7010028.
Identification of to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017-2018, aimed to study the molecular epidemiology of 256 isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental isolates ( = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant isolates. sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all isolates recovered from 2012-2019, the frequency of cryptic species was 5.3% (18/337), with the identification of (complex), , , , and . To determine the frequency of azole resistance of , isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine sensu stricto and six cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect mutations and sequencing of 51A gene and its promoter were performed. The overall frequency of resistance to azoles in sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant environmental isolate, collected in 2015. The TR/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation ( = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of isolates.
鉴定到种水平很重要，因为近缘种可能对多种抗真菌药物表现出不同的敏感性，而且正确鉴定有助于提高流行病学研究的认知。在葡萄牙国家真菌学参考实验室进行了两项关于监测的回顾性实验室研究。第一项研究涵盖2017 - 2018年期间，旨在研究从呼吸道、皮下或全身感染患者以及环境样本中分离出的256株曲霉菌株的分子流行病学。第二项研究使用我们在2012年至2019年期间收集的全部临床和环境曲霉菌株（n = 337），旨在确定对唑类耐药的曲霉菌株的频率。狭义烟曲霉是临床和环境样本中最常见的物种。总体而言，考虑到所有鉴定出的烟曲霉部分，基于β - 微管蛋白或钙调蛋白测序检测到高频率的隐种（临床样本中为37%，环境分离株中为51%）。关于2012 - 2019年回收的所有烟曲霉分离株，隐种的频率为5.3%（18/337），鉴定出了烟曲霉复合群、烟曲霉、土曲霉、黄曲霉、构巢曲霉和黑曲霉。为了确定烟曲霉对唑类耐药的频率，使用唑类琼脂对烟曲霉分离株进行唑类耐药筛选，并通过CLSI微量稀释参考方法对53株可能耐药的分离株进行检测。9株狭义烟曲霉和6株烟曲霉隐种分离株对伊曲康唑、伏立康唑和/或泊沙康唑表现出高最低抑菌浓度。进行了实时PCR检测烟曲霉突变以及51A基因及其启动子的测序。狭义烟曲霉对唑类耐药的总体频率为3.0%。通过这项回顾性分析，我们能够检测到一株2015年收集的对唑类耐药的G54R突变环境分离株。与唑类耐药的环境传播途径相关的TR/L98H突变是最常检测到的突变（n = 4；1.4%）。我们的研究结果强调了对烟曲霉分离株进行正确鉴定和药敏试验的必要性。
