Rapid, Highly Multiplexed and Sensitive Blood Test for Drug-resistant Bacteria

描述（由申请人提供）：迫切需要快速、高灵敏度、特异性、易于使用和具有成本效益的医疗诊断测试，以诊断暴露于和/或感染有症状前、症状性或非特异性症状的医疗相关病原体的个体，并准确识别临床样本中的传染性病原体或毒素，以便执行适当的治疗干预。由于抗生素耐药性在世界各地迅速蔓延，导致人类生命和医疗保健支出的成本不断增加，因此确定日益复杂的微生物耐药性模式至关重要。然而，由于患者样本有限，病原体浓度通常极低（<10个生物体/ml），串行分析是不切实际的，因此需要高度多路复用的系统，而不是单个病原体特异性测试。Nesher Technologies, Inc. （NTI）的长期目标是开发一种快速，高度多路复用（每个标准患者样本的容量为100个分析物），超敏感和特异性，定量，经济高效，全自动，基于核酸和蛋白质的细菌感染诊断系统，以便在临床样品中快速准确地识别病原体，包括获得的遗传特征，如抗生素耐药性或增强的毒力。NTI已经获得了一项革命性的超灵敏生物检测技术的知识产权，该技术具有精致的单孔复用潜力，该技术是由加州大学洛杉矶分校单分子生物物理实验室（由Shimon Weiss教授领导）开发的。它是基于3色交替激光激发（3c-ALEX）单分子荧光光谱，其中两个（或三个）识别分子被不同颜色的荧光染料标记。两种或三种颜色的同步共聚焦检测构成一个积极的目标检测事件，可以对溶液中自由扩散的分子进行分子鉴定，同时检测多个目标。在第一阶段资助期内，我们将通过开发一种同时区分11种细菌菌株的测试来证明可行性，这些菌株是导致医院和社区获得性感染的主要病原体。具体目标是：1.；利用三色alexi荧光光谱技术，对MSSA、MRSA、VRSA、MSSE、MRSE、VSEfaecalis、vanA VREfaecalis、vanB VREfaecium、VSEfaecium、vanA VREfaecium、vanB VREfaecium和S. oneidensis纯化DNA中的8个遗传标记（金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、E. faecium、屎肠球菌和谢氏希瓦氏菌作为阳性对照，对甲氧西林、万古霉素A和万古霉素B耐药）进行分离检测和定量。2. 多路（单孔）鉴别和定量人血细菌。3. 基于alexs的分析来自UCLA医学中心的350个存档临床分离株（除罕见的VRSA外，11种病原体类型中每种都有25个阳性）和统计分析。公共卫生相关性：Nesher Technologies， Inc.提议开发一种高度多路复用、超灵敏、定量、低成本的自动化医疗诊断测试系统，能够从单个患者样本的众多可能性中快速识别特定的医疗相关细菌菌株和耐药性模式，从根本上推动了当前技术的极限。改进后的诊断信息将使医生能够更好地为患者开出适当的抗生素，并使医院能够实施感染控制程序以降低感染率，从而挽救生命并降低医疗保健费用。这将限制广谱抗生素的使用，鼓励发展针对性较窄的治疗方法，并有助于遏制抗生素耐药性日益增加的明显全球趋势，这是传染病治疗和管理的一个主要问题，影响到美国和全世界数百万人的健康。

DESCRIPTION (provided by applicant): There is an urgent need for rapid, highly sensitive, specific, easy to use, and cost-effective medical diagnostic tests to diagnose individuals exposed to and/or infected with healthcare-associated pathogens that are pre-symptomatic, symptomatic, or have non-specific symptoms, and to accurately identify infectious agents or toxins in clinical samples so appropriate therapeutic intervention can be executed. As antibiotic resistance is spreading rapidly throughout the world causing growing costs in terms of human lives and healthcare expenditures, concomitant determination of increasingly complex microbial resistance patterns is of critical importance. However, as patient samples are limited and pathogen concentrations often extremely low (<10 organisms/ml), serial analysis is not practical, thus requiring highly multiplexed systems instead of individual pathogen-specific tests. Nesher Technologies, Inc.'s (NTI) long-term goal is to develop a rapid, highly multiplexed (with a capacity of >100 analytes per standard patient sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, nucleic acid- and protein-based diagnostic system for bacterial infections, to allow quick and accurate pathogen identification in clinical samples, including acquired genetic traits such as antibiotic resistance or enhanced virulence. NTI has licensed the intellectual property for a revolutionary ultrasensitive biodetection technology with exquisite single well multiplexing potential, which was developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss). It is based on 3- color alternating laser excitation (3c-ALEX) single molecule fluorescence spectroscopy, whereby two (or three) recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two or three colors constitutes a positive target detection event, allowing molecular identification of freely diffusing molecules in solution and detection of numerous targets simultaneously. Over the Phase I funding period, we will demonstrate feasibility by developing a test to simultaneously distinguish between 11 bacterial strains which are major pathogens responsible for nosocomial and community-acquired infections. Specific aims are: 1. Separate detection and quantification of eight genetic markers (species-specific for Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and Shewanella oneidensis as positive control; resistance-specific for methicillin, vancomycin A, and vancomycin B) in purified DNA derived from MSSA, MRSA, VRSA, MSSE, MRSE, VSEfaecalis, vanA VREfaecalis, vanB VREfaecalis, VSEfaecium, vanA VREfaecium, vanB VREfaecium, and S. oneidensis by 3-color ALEX-based fluorescence spectroscopy. 2. Multiplexed (single-well) discrimination and quantification of bacteria spiked into human blood. 3. ALEX-based analysis of 350 archived clinical isolates from UCLA's Medical Center (including 25 positive for each of the 11 pathogen types, except for rare VRSA) and statistical analysis. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc.'s proposed development of a highly multiplexed, ultrasensitive, quantitative, low- cost automated medical diagnostic test system, capable of quickly identifying specific healthcare-associated bacterial strains and drug resistance patterns among the multitude of possibilities from a single patient sample, radically pushes the limits of current technologies. The improved diagnostic information will enable physicians to better prescribe the appropriate antibiotics to patients and enable hospitals to implement infection control procedures to lower their infection rates, thereby saving human lives and reducing healthcare costs. This will allow limiting the use of broad-spectrum antibiotics, encourage the development of narrowly targeted therapeutics, and help curtailing the marked global trend towards increasing antibiotic drug resistance which is a major concern for treatment and management of infectious diseases, affecting the health of millions of people in the United States and around the world.