Abstract

Background

Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited.

Objectives

To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates.

Methods

Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing.

Results

The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens.

Conclusions

The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

中文翻译：

---

通过呼吸道标本的焦磷酸测序破译烟曲霉cyp51A介导的三唑抗性。

摘要

背景

由三唑耐药的烟曲霉引起的感染是一个日益严重的问题。标准培养的灵敏度很差，废除了药敏试验。早期发现耐药性可以改善患者的预后，但用于此目的的工具有限。

目标

开发和验证焦磷酸测序技术，以检测临床呼吸道标本和烟曲霉菌株中赋予抗性的cyp51A多态性。

方法

通过Sanger测序和焦磷酸测序50株具有一定三唑敏感性模式的烟曲霉菌株进行方法验证。然后，通过cyp51A焦磷酸测序评估了27个月（2017年1月至2019年3月）在英国国家曲霉病中心收集的326例曲霉定量PCR（qPCR）阳性呼吸道样本。将Sanger测序和焦磷酸测序结果与来自大量培养​​和标准药敏试验的结果进行了比较。

结果

通过焦磷酸测序和Sanger测序分析的50个分离株的cyp51A基因型匹配。在326株曲霉qPCR阳性呼吸道标本中，报告有71.2％的烟曲霉没有生长。其中56.9％（132/232）表现出WT cyp51A基因型，而31.5％（73/232）通过焦磷酸测序显示出抗性基因型。焦磷酸测序鉴定了18.7％（61/326）样品中的环境TR34 / L98H突变，而培养物中检测到的泛唑耐药性为6.4％（21/326）。重要的是，焦磷酸测序在23.3％的样本中比培养早发现了抗性。

结论

所描述的焦磷酸测序测定法可以检测到与三唑抗性相关的广泛cyp51A多态性，包括那些未通过商业测定法鉴定的多态性。当培养阴性时，该方法可迅速识别耐药性并选择适当的抗真菌治疗。