Molecular Detection of Invasive Fungal Infections

描述（由申请人提供）：真菌感染的发生率，特别是念珠菌感染（第四大常见医院病原体），在过去20年中持续增加。这一增加是由于移植手术、化疗方案、内科和外科治疗的进步以及侵入性器械（如血管内中心静脉导管）的使用增加导致严重免疫抑制的患者数量增加。虽然大多数侵袭性真菌病（IFD）是由念珠菌属和曲霉属引起的，但其他罕见的真菌病原体在近几十年来变得更加普遍，并与极高的死亡率相关。由于目前诊断方法的周转时间缓慢，IFD的适当治疗往往被延迟，导致与这种疾病相关的高死亡率。目前的诊断标准微生物培养平均需要48-72小时才能完成，并且无法检测到大约50%的真菌物种。由于IFD的适当治疗需要迅速和准确的诊断，真菌诊断领域中未满足的需求是可以提供1）早期检测2）敏感结果，3）物种水平区分和4）多重能力的平台。已经开发了几种分子方法来解决这一需求，但未能满足所有这些要求。我们的基于反向线印迹（RLB）的检测使用PCR，然后与物种特异性探针杂交，通过以多重形式为多种真菌病原体提供灵敏快速（不到8小时）的检测，为这一未满足的需求提供了解决方案。在本I期申请中，我们提出开发基于RLB的检测方法，用于检测9种念珠菌、5种曲霉和几种更罕见的IFD，包括H。荚膜镰刀菌属（Fusarium spp.）、接合菌属（Zygomycetes）、隐球菌属（Cryptococcus spp.）和球孢子菌属（Coccidioides spp.）第一阶段的一个重要里程碑将是开发和验证一种基于RLB的多重PCR检测方法，用于检测IFD中涉及的大多数真菌病原体。我们将在临床血液和BAL样本中验证我们的RLB检测试剂盒，这些样本来自具有真菌学专业知识的几位合作者，包括圣犹达医疗中心的Randall Hayden、亚拉巴马大学的Peter Pappas、德克萨斯大学的Luis Ostrosky、塔夫茨大学的大卫斯奈德曼和疾病控制中心的玛丽勃兰特。我们的最终产品将在第二阶段进一步开发，在第二阶段，我们将把我们的开放系统过渡到一个封闭系统，在一个专用的仪器中进行PCR和杂交反应，该仪器是与我们的工程合作伙伴Wi Engineering合作设计和测试的。该产品提供了一种快速、灵敏和多路复用的方法，满足了理想的IFD诊断平台的要求。通过显著缩短检测时间，这一创新平台将导致IFD治疗的变化，最值得注意的是，使用靶向抗真菌药物进行更快的治疗。 公共卫生相关性：真菌感染的发病率，其中许多与高死亡率有关，在过去20年中持续增加。目前的诊断方法受到周转时间和低灵敏度的限制。我们建议开发一种快速的分子检测方法，这将显着提高侵袭性真菌病的检测和治疗。

DESCRIPTION (provided by applicant): The incidence of fungal infections and, in particular, Candida infections, the fourth most common nosocomial pathogen, has continued to increase over the past 20 years. This increase is due to the increasing numbers of patients subjected to severe immunosuppression as a result of transplant procedures, chemotherapy regimens, advances in medical and surgical therapies, and the increased use of invasive devices such as intravascular central lines. While the majority of invasive fungal disease (IFD) is caused by species of Candida and Aspergillus, other rare fungal pathogens have become more prevalent in recent decades and are associated with exceedingly high mortalities. Due to the slow turnaround time of current diagnostic methods, proper treatment of IFD is often delayed, contributing to the high mortality rates associated with this disease. Microbiologic culture, the current diagnostic standard, requires, on average, 48-72 hours for completion, and fails to detect roughly 50% of fungal species. As proper treatment of IFD calls for prompt and accurate diagnosis, an unmet need in the area of fungal diagnostics is a platform that can provide 1) early detection 2) sensitive results, 3) species level discrimination and 4) multiplex capability. Several molecular approaches have been developed to address this need but have failed to meet all these requirements. Our Reverse Line Blot (RLB) based assay uses PCR followed by hybridization with species specific probes, offering a solution to this unmet need by providing a sensitive and rapid (less than 8 hour) test for several fungal pathogens in a multiplex format. In this Phase I application we propose development of RLB based assays for the detection of 9 species of Candida, 5 species of Aspergillus, and several more rare species of IFD, including H. capsulatum, Fusarium spp, Zygomycetes, Cryptococcus spp, and Coccidioides spp. A major milestone of Phase I will be the development and validation of a multiplexed RLB-based, PCR assay for the detection of the majority of fungal pathogens implicated in IFD. We will validate our RLB assays in clinical blood and BAL samples obtained from several collaborators with expertise in mycology including Randall Hayden of St. Jude Medical Center, Peter Pappas of the University of Alabama, Luis Ostrosky of the University of Texas, David Snydman of Tufts University and Mary Brandt of the Centers for Disease Control. Our end product will be developed further in Phase II, where we will transition our open system to a closed system that performs the PCR and hybridization reactions in a dedicated instrument, designed and tested in collaboration with our engineering partner, Wi Engineering. This product offers a rapid, sensitive and multiplexed approach that meets the requirements of an ideal diagnostic platform for IFD. By significantly reducing time to detection, this innovative platform will lead to changes in the treatment of IFD, most notably, faster treatment with a targeted anti-fungal. PUBLIC HEALTH RELEVANCE: Project Narrative The incidence of fungal infections, many of which are associated with high mortality, has continued to increase over the past 20 years. Current diagnostic methods are limited by turnaround time and low sensitivity. We propose development of a rapid, molecular test that will significantly improve the detection and treatment of invasive fungal disease.