Multiplexed, Non-Amplified, Nucleic Acid-Based Identification of Multidrug Resistant Pathogens Using an Integrated Optofluidic Platform

使用集成光流控平台对多重耐药病原体进行多重、非扩增、基于核酸的鉴定

• 批准号: 9221242
• 负责人: Aaron R. Hawkins
• 金额: $ 100.28万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9221242
• 关键词: Antibiotic Resistance; Antibiotics; Bacteremia; Bacteria; Bacterial Infections; Biological Assay; Blood; Blood Circulation; Blood Tests; Blood specimen; Cells; Cessation of life; Clinical; Complex; Cytolysis; DNA; Dangerousness; Detection; Development; Devices; Diagnosis; Diagnostic Procedure; Diagnostic tests; Drug resistance; Enterobacter; Enterobacteriaceae; Enzymes; Escherichia coli; FDA approved; Filtration; Fluorescence; Genes; Goals; Health; Healthcare; Hospitalization; Hospitals; Hour; Human; Hybrids; Industrialization; Infection; Klebsiella pneumonia bacterium; Label; Lead; Length; Medical; Methods; Microfluidics; Molecular; Morbidity - disease rate; Nucleic Acids; Organism; Outcome; Pathogen detection; Patients; Pharmaceutical Preparations; Predisposition; Prevalence; Process; Public Health; Reproducibility; Resistance; Resistance profile; Resistance to infection; Sampling; Sepsis; Solid; System; Technology; Testing; Time; Treatment Cost; Whole Blood; antimicrobial; bacterial resistance; base; blood filtration; carbapenem-resistant Enterobacteriaceae; carbapenemase; design; diagnostic assay; drug resistant bacteria; effective therapy; fluorophore; global health; improved; inhibitor/antagonist; interest; mortality; multi-drug resistant pathogen; multiplex detection; novel; pathogen; public health relevance; resistance gene; single molecule; success; treatment strategy

 DESCRIPTION (provided by applicant): Antibiotic resistance has emerged as a major public health threat. Patients infected with drug- resistant pathogens suffer significantly higher rates o morbidity and mortality, most often due to delays in the administration of effective antimicrobial therapies. In particular for bloodstream infections, the need to rapidly identify both pathogen and resistance profile is crucial, as treatment with antibiotics to which the organism is sensitive is essential and time-critical. Indeed, sepsis is involved in up to half of all hospital deaths. Drug susceptibility information for a pathogen is typically not received by clinicians until at least 24 hours post-sampling, because of reliance on culture-based diagnostic methods. Recently, bloodstream infections caused by carbapenem-resistant Enterobacteriaceae (CRE) have become increasingly problematic. A rapid diagnostic assay for the detection and resistance determination of these pathogens is urgently needed. Although PCR-based assays are rapid, specific, and amenable to multiplexing, they have largely failed to perform in blood samples. Our industrial partner, Great Basin Corporation, has developed a fully disposable cartridge system for pathogen detection in cultured blood. We propose major improvements to this platform through the development of a multiplexed, non-amplified, non-cultured, nucleic acid-based assay for the detection and identification of multidrug resistant pathogens using a novel integrated optofluidic device. Bacteria will be concentrated directly from a blood sample by cross-flow filtration, and then delivered to a lysis and DNA-shearing chamber. Target DNAs containing the genes of interest will be captured on a solid substrate by hybridization. Molecular beacons will be hybridized to specific targets on the captured nucleic acids. These complexes will be released and specific beacons detected by an advanced optofluidics system capable of detecting single molecule fluorescence. We will demonstrate identification within one hour of bacteria in blood at levels as low as 10 CFU/mL. Initially, the focus will be to detect and characterize CRE isolated directly from a blood sample. The KPC, NDM, VIM, and IMP carbapenemase genes will be identified along with the simultaneous detection of specific markers for Klebsiella pneumoniae, Escherichia coli, and Enterobacter species. This platform is readily expandable to additional pathogens and their relevant antibiotic resistance genes. This technology has the potential to significantly reduce time to diagnosis and improve clinical outcomes.

 描述（由申请人提供）：抗生素耐药性已成为主要的公共卫生威胁，感染耐药病原体的患者发病率和死亡率显着升高，这通常是由于有效抗菌疗法的延迟施用所致。血流感染，需要快速识别病原体和 耐药性至关重要，因为使用对微生物敏感的抗生素进行治疗至关重要且时间紧迫。事实上，多达一半的医院死亡病例涉及病原体的药物敏感性信息。至少 24 由于对基于培养的诊断方法的依赖，最近，由碳青霉烯类耐药肠杆菌科细菌 (CRE) 引起的血流感染变得越来越成问题，迫切需要一种快速诊断方法来检测和确定这些病原体的耐药性。尽管基于 PCR 的检测快速、特异且易于多重检测，但它们在很大程度上无法在血液样本中执行。我们的工业合作伙伴 Great Basin Corporation 已开发出一种完全一次性的试剂盒系统，用于培养血液中的病原体检测。我们建议通过开发一种多重、非扩增、非培养、基于核酸的检测方法来对该平台进行重大改进，用于使用新型集成光流控装置来检测和鉴定多重耐药病原体，细菌将直接从浓缩物中浓缩。血液样本通过错流过滤，然后输送到裂解和 DNA 剪切室，通过杂交将含有感兴趣基因的目标 DNA 捕获在固体基质上。这些复合物将被释放，并由能够检测单分子荧光的先进光流控系统检测到特定的信标，我们将在一小时内证明血液中低至 10 CFU 水平的细菌的识别。最初，重点是检测和表征直接从血液样本中分离的 CRE，同时检测 KPC、NDM、VIM 和 IMP 碳青霉烯酶基因。该平台可轻松扩展到其他病原体及其相关抗生素耐药基因。该技术有可能显着缩短诊断时间并改善临床结果。