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.