Changing Cultures in Sepsis: Rapid single-cell pathogen identification and antibiotic susceptibility testing directly from whole blood

脓毒症中培养物的改变：直接从全血中进行快速单细胞病原体鉴定和抗生素敏感性测试

• 批准号: 10411988
• 负责人: Pak Kin Wong
• 金额: $ 72.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10411988
• 关键词: Adopted; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Bacteria; Bar Codes; Biological Assay; Blood Cells; Blood specimen; Cells; Characteristics; Classification; Clinical; Collaborations; Computer software; Cytology; Cytolysis; Data; Data Analyses; Detection; Diagnosis; Diagnostic; Disease; Drug Controls; Electroporation; Evolution; Goals; Growth; Hour; Individual; Industry; Kinetics; Label; Life; Measures; Membrane; Metabolism; Methods; Microbe; Microbiology; Microfluidic Microchips; Microfluidics; Microscopy; Minimum Inhibitory Concentration measurement; Molecular; Molecular Probes; Monitor; Morbidity - disease rate; Nanotubes; Pathogen detection; Patients; Performance; Phenotype; Predisposition; Process; Protocols documentation; RNA, Ribosomal, 18S; Research Personnel; Research Project Grants; Resistance; Resolution; Ribosomal RNA; Sampling; Scheme; Science; Sepsis; Sorting - Cell Movement; Speed; System; Techniques; Technology; Temperature; Testing; Time; Translating; Triage; Validation; Vertebral column; Whole Blood; base; commensal microbes; commercialization; computerized data processing; design; diagnostic platform; drug resistant pathogen; fungus; imaging system; improved; innovation; microbial; microscopic imaging; microwave electromagnetic radiation; mortality; multidisciplinary; multimodality; novel; optical imaging; pathogen; precision medicine; product development; prospective; rapid diagnosis; response; single cell analysis; time use; validation studies; Adopted; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Bacteria; Bar Codes; Biological Assay; Blood Cells; Blood specimen; Cells; Characteristics; Classification; Clinical; Collaborations; Computer software; Cytology; Cytolysis; Data; Data Analyses; Detection; Diagnosis; Diagnostic; Disease; Drug Controls; Electroporation; Evolution; Goals; Growth; Hour; Individual; Industry; Kinetics; Label; Life; Measures; Membrane; Metabolism; Methods; Microbe; Microbiology; Microfluidic Microchips; Microfluidics; Microscopy; Minimum Inhibitory Concentration measurement; Molecular; Molecular Probes; Monitor; Morbidity - disease rate; Nanotubes; Pathogen detection; Patients; Performance; Phenotype; Predisposition; Process; Protocols documentation; RNA, Ribosomal, 18S; Research Personnel; Research Project Grants; Resistance; Resolution; Ribosomal RNA; Sampling; Scheme; Science; Sepsis; Sorting - Cell Movement; Speed; System; Techniques; Technology; Temperature; Testing; Time; Translating; Triage; Validation; Vertebral column; Whole Blood; base; commensal microbes; commercialization; computerized data processing; design; diagnostic platform; drug resistant pathogen; fungus; imaging system; improved; innovation; microbial; microscopic imaging; microwave electromagnetic radiation; mortality; multidisciplinary; multimodality; novel; optical imaging; pathogen; precision medicine; product development; prospective; rapid diagnosis; response; single cell analysis; time use; validation studies

PROJECT SUMMARY Sepsis, commonly caused by bloodstream infections (BSI), is a rapidly progressive and life-threatening disease. Unfortunately, prolonged delay in microbiological diagnosis increases patient mortality, promotes the misuse of antibiotics, and consequently, the evolution of antibiotic-resistant pathogens. Herein, we aim to deliver an amplification-free, microfluidic system for pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) directly from whole blood. To achieve our goal, we propose a platform based on microfluidic- assisted microscopy to sort, trap, detect, and monitor pathogens at single cell resolution. For pathogen ID, we will adopt a multispectral barcoding scheme to differentially label molecular probes for direct multiplex ribosomal RNA (rRNA) detection to classify and speciate pathogens, along with a nanotube assisted microwave electroporation (NAME) technique to efficiently deliver the probes intracellularly for amplification-free single microbe detection. Positive pathogen ID will guide quantitative multimodal phenotypic AST (mPhAST), in which we will monitor early changes in microbial growth kinetics with cytological measures of viability in response to relevant antibiotic conditions at the single cell level to determine susceptibility/resistance with improved speed and reliability. Combined with upstream whole blood pre-processing for pathogen isolation and concentration followed by ID then AST, we aim to deliver sample to answer within 90 min for BSI triage and as early as 30 minutes more for antibiotic minimum inhibitory concentration (MIC) determination. We have assembled a superb team of multi-disciplinary investigators and industry-leading advisors with complementary expertise and a strong track record of collaboration. We propose the following aims: 1) to develop a rapid BSI triage protocol for broad pathogen detection, classification, and ID; 2) to develop a quantitative mPhAST; 3) to develop an integrated ID- mPhAST platform; 4) to perform analytical and clinical validation of our ID-mPhAST platform. Our short-term goal is to obtain the necessary preliminary data to plan for product development and commercialization, with the long-term goal of translating our diagnostic platform to reduce sepsis-related morbidity and mortality.

项目摘要 败血症通常是由血液感染引起的（BSI），是一种快速进行性和威胁生命的疾病。 不幸的是，微生物诊断的延迟延迟会增加患者死亡率，促进滥用 抗生素，因此是抗生素耐药病原体的演变。在此，我们旨在提供 无扩增，微流体系统，用于病原体检测，鉴定（ID）和抗菌易感性 直接从全血中进行测试（AST）。为了实现我们的目标，我们提出了一个基于微流体的平台 辅助显微镜以单细胞分辨率对病原体进行排序，捕获，检测和监测病原体。对于病原体ID，我们 将采用多光谱条形码方案来差异标记分子探针的直接多重核糖体 RNA（RRNA）检测以对病原体进行分类和规定，以及纳米管辅助微波炉 电穿孔（名称）技术，可有效地在细胞内输送探针以进行无扩增的单个 微生物检测。正病原体ID将指导定量多模式表型AST（mphast），其中 我们将使用细胞学测量的生存力来监测微生物生长动力学的早期变化 在单细胞水平上相关的抗生素条件，以确定速度提高的敏感性/电阻 和可靠性。结合上游全血的预处理，以进行病原体隔离和浓度 然后是ID，然后是AST，我们的目标是在90分钟内为BSI Triage提供样本，最早为30 抗生素最小抑制浓度（MIC）测定的时间更高。我们组装了一流 具有补充专业知识的多学科研究人员和行业领先的顾问团队 协作的记录。我们提出以下目的：1）为广泛开发快速的BSI分流协议 病原体检测，分类和ID； 2）开发定量的mphast； 3）开发一个集成的ID- mphast平台； 4）对我们的ID-MPHAST平台进行分析和临床验证。我们的短期 目标是获取必要的初步数据，以计划产品开发和商业化，并使用 将我们的诊断平台转换以降低败血症相关的发病率和死亡率的长期目标。