Molecular Detection of Invasive Fungal Infections

描述（由申请人提供）：真菌感染的发病率，特别是念珠菌感染，第四大最常见的医院病原体，在过去的20年里持续增加。这一增长是由于移植手术、化疗方案、医疗和外科疗法的进步以及诸如血管内中央静脉导管等侵入性装置的使用增加，导致越来越多的患者遭受严重的免疫抑制。虽然大多数侵袭性真菌病（IFD）是由念珠菌和曲霉菌引起的，但其他罕见的真菌病原体在最近几十年变得更加普遍，并与极高的死亡率相关。由于目前诊断方法的周转时间较慢，对IFD的适当治疗经常被推迟，导致与该疾病相关的高死亡率。微生物培养是目前的诊断标准，平均需要48-72小时才能完成，并且无法检测到大约50%的真菌种类。由于IFD的适当治疗需要及时和准确的诊断，真菌诊断领域的一个未满足的需求是一个能够提供1)早期发现2)敏感结果3)物种水平区分和4)多重功能的平台。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。我们的反向线印迹（RLB）为基础的分析使用PCR，然后与物种特异性探针杂交，提供了一个解决方案，通过提供一个敏感和快速（少于8小时）测试几种真菌病原体在多重格式的未满足的需求。在这个I期申请中，我们建议开发基于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物种，包括荚膜菌、镰刀菌、合子菌、隐球菌和球虫。I期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分析，这些样本来自几位具有真菌学专业知识的合作者，包括圣裘德医学中心的Randall Hayden，阿拉巴马大学的Peter Pappas，德克萨斯大学的Luis Ostrosky，塔夫茨大学的David Snydman和疾病控制中心的Mary Brandt。我们的最终产品将在第二阶段进一步开发，届时我们将把我们的开放系统转变为封闭系统，在专用仪器中进行PCR和杂交反应，与我们的工程合作伙伴Wi engineering合作设计和测试。该产品提供了一种快速、敏感和多路复用的方法，满足了IFD理想诊断平台的要求。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。

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.