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一种用于检测烟曲霉唑类耐药性的改良琼脂筛选方法。

An expanded agar-based screening method for azole-resistant Aspergillus fumigatus.

机构信息

Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Majadahonda, Spain.

Microbiology Department, Hospital Severo Ochoa, Leganés, Spain.

出版信息

Mycoses. 2022 Feb;65(2):178-185. doi: 10.1111/myc.13400. Epub 2021 Nov 29.

Abstract

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A. fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A. fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR , TR /L98H, TR /Y121F/T289A, TR /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A. fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A. fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation.

摘要

抗真菌药敏试验是指导抗真菌治疗的重要工具。参考方法复杂,通常仅在专门的实验室中可用。我们设计了一种扩展的琼脂基础筛选方法,用于检测唑类耐药烟曲霉分离株。通常,只有在测序 cyp51A 基因和启动子后,才能确定耐药机制。然而,我们的筛选方法提供了唑类耐药检测和推定的耐药机制鉴定。以前的一种基于琼脂的方法,包含四个含有伏立康唑、伊曲康唑、泊沙康唑和生长对照的孔,可检测到对临床唑类药物的耐药性。在这里,我们已经修改了伏立康唑和泊沙康唑的浓度,以适应针对烟曲霉的更新 EUCAST 折点。我们还扩展了该方法,包括环境唑类药物,以评估唑类耐药性和涉及的唑类耐药机制。我们使用了包括 54 株 Cyp51A 修饰(G54、M220、G448S、TR 、TR/L98H、TR/Y121F/T289A、TR/L98H/S297T/F495I)的唑类耐药分离株和 50 株野生型 Cyp51A 的唑类敏感分离株的烟曲霉收集物。当在 48 小时后任何含唑孔中有生长时,筛选方法可检测到唑类耐药烟曲霉分离株。在测试的七种唑类药物中的生长模式有助于确定潜在的唑类耐药机制。这种方法旨在监测烟曲霉唑类耐药分离株,并在抗真菌药敏试验确认之前,可用于患者的临床管理。

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