Aspergillus fumigatus Volatile Secondary Metabolite Dynamics for the Identification of Azole-resistant Aspergillosis

项目摘要/摘要：侵袭性曲霉菌病（IA）是一个主要的发病率和死亡原因， 免疫功能低下的患者，全球每年超过20万例。虽然诊断测试的进步 以及强效、毒性较小的三唑类抗真菌药物的开发降低了IA相关的死亡率， 68%至25%，耐唑烟曲霉的发病率和地理分布出现了惊人的上升 在过去的十年中，ARAF正在稳步抵消这些收益，并影响三唑类抗真菌药物的使用 作为IA的一线治疗ARAF在环境和患者中迅速爆炸的主要原因 肺似乎是越来越多的全球使用唑类杀真菌剂类似的结构，以医疗三唑， 全球农业，特别是在欧洲和亚洲-这些杀真菌剂在土壤和水中持续数月， 施加广泛的、系统的选择压力，有利于ARAF的出现，而ARAF又迅速传播 通过营养生长和大量的气载分生孢子传播。ARAF现在负责高达30%的 在欧洲，IA病例的相关死亡率为70- 100%，世界其他地区的发病率稳步上升。 由于缺乏临床风险因素，缺乏诊断标准， 能够及时区分ARAF IA和唑类敏感IA的方法，以及采用 与唑类药物相比，抗真菌药物的功效明显较低，但毒性较大。我们已经开发出了 通过检测呼吸中曲霉挥发性倍半萜次级代谢产物来鉴定IA患者， 以及通过对这些真菌呼吸代谢物的系列评估来检查抗真菌治疗的体内反应 在抗真菌治疗过程中。与唑类药物中次级代谢产物释放的衰减相反， 易感A.烟曲霉用唑类抗真菌药治疗后，这些代谢产物反而增加了ARAF与唑类 exposure.我们将检验倍半萜次级代谢产物对三唑类抗真菌药物的反应的假设 对唑类药物敏感和耐药的A.通过（1）比较以下物质的体外反应： ARAF（包括最常见的cyp 51 A突变TR 34/L98 H、TR 46/Y121 F/T289 A、M220和G54，以及ARAF） 尽管野生型cyp 51 A具有表型多唑抗性）和唑敏感A.烟曲霉菌株， 比较ARAF和唑类敏感A.烟熏， 唑类抗真菌治疗的呼吸使用血小板减少的小鼠IA模型。最终，我们希望能描绘出 ARAF与唑类中挥发性倍半萜次级代谢产物释放动力学的显著差异- 易感A.烟曲霉，在体外和体内。成功实现这些目标将为 一种新的快速呼吸试验的基础，可以区分ARAF IA患者和唑敏感患者。 IA，提供即将发生治疗失败的体内指标，指导选择适当的抗真菌药物 治疗，并降低与ARAF IA相关的极高发病率和死亡率。

Project Summary/Abstract: Invasive aspergillosis (IA) is a leading cause of morbidity and death in immunocompromised patients, with over 200,000 cases worldwide per year. While advances in diagnostic testing for IA and the development of potent, less toxic triazole antifungal drugs have reduced IA-associated mortality from 68% to 25%, an alarming rise in the incidence and geographic distribution of azole-resistant Aspergillus fumigatus (ARAF) over the past decade is steadily offsetting these gains and compromising the use of triazole antifungal drugs as first-line therapy for IA. The major culprit in the rapid explosion of ARAF in the environment and in patient lungs appears to be the increasing global use of azole fungicides similar in structure to medical triazoles in agriculture worldwide, especially in Europe and Asia – these fungicides persist in the soil and water for months, applying widespread, systematic selection pressure favoring the emergence of ARAF, which in turn spreads quickly through vegetative growth and abundant dissemination of airborne conidia. ARAF is now responsible for up to 30% of IA cases in Europe with associated mortality of 70-100%, with steadily rising incidence elsewhere in the world. Managing patients with ARAF IA is incredibly challenging due to the lack of clinical risk factors, lack of diagnostic methods that can differentiate ARAF IA from azole-susceptible IA in a timely manner, and the need to employ antifungals with substantially lower efficacy and greater toxicity than the azole drugs. We have developed methods of identifying patients with IA via detection of Aspergillus volatile sesquiterpene secondary metabolites in breath, and of examining in vivo responses to antifungal treatment via serial assessment of these fungal breath metabolites over the course of antifungal therapy. In contrast to the attenuation of secondary metabolite release in azole- susceptible A. fumigatus with azole antifungal treatment, these metabolites instead increase in ARAF with azole exposure. We will test the hypothesis that the sesquiterpene secondary metabolite response to triazole antifungal therapy is distinct in azole-susceptible and azole-resistant A. fumigatus by (1) comparing the in vitro response of ARAF (including the most common cyp51A mutations TR34/L98H, TR46/Y121F/T289A, M220, and G54, and ARAF with phenotypic multi-azole resistance despite wild-type cyp51A) and azole-susceptible A. fumigatus strains to azole antifungal therapy and (2) comparing the in vivo response of ARAF and azole-susceptible A. fumigatus to azole antifungal therapy in breath using a neutropenic murine IA model. Ultimately, we expect to delineate marked differences in the dynamics of volatile sesquiterpene secondary metabolite release in ARAF vs. azole- susceptible A. fumigatus, both in vitro and in vivo. Successful completion of these aims would set the groundwork for a novel, rapid breath assay that can distinguish patients with ARAF IA from azole-susceptible IA, providing an in vivo indicator of imminent therapeutic failure, guiding selection of appropriate antifungal therapy, and reducing the extremely high morbidity and mortality associated with ARAF IA.