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

项目摘要/摘要：侵袭性曲霉病(IA)是#年发病率和死亡率的主要原因。 免疫功能低下的患者，全球每年有20多万例。虽然诊断检测的进展 对于IA以及有效、毒性较低的三氮唑抗真菌药物的开发，已将IA相关死亡率从 68%至25%，耐唑烟曲霉菌的发病率和地理分布惊人地上升 (ARAF)在过去十年中稳步抵消了这些成果，并损害了三氮唑抗真菌药物的使用 作为IA的一线治疗。房颤在环境和患者中迅速爆炸的罪魁祸首 肺部似乎在全球范围内越来越多地使用结构类似于医用三氮唑的唑类杀菌剂 世界各地的农业，特别是在欧洲和亚洲--这些杀菌剂在土壤和水中持续存在数月， 施加广泛的、系统的选择压力有利于ARAF的出现，而ARAF又迅速蔓延 通过营养生长和空气传播的分生孢子大量传播。ARAF现在负责高达30%的 在欧洲，IA病例的相关死亡率为70%-100%，而世界其他地区的发病率稳步上升。 由于缺乏临床危险因素，缺乏诊断，管理ARAFIA患者具有难以置信的挑战性 能够及时区分ARAFIA和唑敏性IA的方法，以及需要使用 与唑类药物相比，抗真菌药物的疗效要低得多，毒性也更大。我们已经开发了方法 通过检测呼吸中的曲霉挥发性倍半萜次级代谢物来识别IA患者， 以及通过对这些真菌呼气代谢物的系列评估来检查体内对抗真菌治疗的反应 在抗真菌治疗的过程中。与唑类药物中次生代谢物释放的衰减相反- 使用唑类抗真菌治疗的敏感烟曲霉菌，这些代谢物反而会在使用唑类抗真菌药物的ARAF中增加 曝光。我们将检验倍半萜次生代谢物对三氮唑抗真菌作用的假设 对唑类敏感和耐唑烟曲霉菌的治疗是不同的，通过(1)比较 ARAF(包括最常见的cyp51a突变TR34/L98H、TR46/Y121F/T289A、M220和G54以及ARAF 尽管野生型cyp51a)和对唑敏感的烟熏假单胞菌菌株仍具有表型多氮唑耐药性 唑类抗真菌治疗以及(2)比较ARAF和对唑类敏感的烟曲霉菌对 使用中性粒细胞减少的小鼠IA模型进行呼吸中的唑类抗真菌治疗。最终，我们希望描绘出 在挥发性倍半萜次生代谢物的释放动力学方面，ARAF与唑类有显著差异。 在体外和体内都是敏感的烟曲霉菌。这些目标的成功实现将使 一种新的快速呼吸测试的基础工作，该测试可以区分ARAFIA患者和唑敏感患者 IA，提供迫在眉睫的治疗失败的体内指标，指导适当的抗真菌药物的选择 治疗，并降低与ARAFIA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.