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: 10190825
• 负责人: Pak Kin Wong
• 金额: $ 73.95万
• 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/10190825
• 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

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.

项目概要 脓毒症通常由血流感染 (BSI) 引起，是一种进展迅速且危及生命的疾病。 不幸的是，微生物学诊断的长期延误增加了患者死亡率，促进了误用 抗生素，以及抗生素耐药病原体的进化。在此，我们的目标是提供一个 用于病原体检测、识别 (ID) 和抗菌敏感性的无扩增微流体系统 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分类、捕获、检测和监测。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，用于直接多重核糖体 RNA (rRNA) 检测可结合纳米管辅助微波对病原体进行分类和分类 电穿孔 (NAME) 技术可有效地将探针递送至细胞内，以实现无扩增单 微生物检测。阳性病原体 ID 将指导定量多模式表型 AST (mPhAST)，其中 我们将通过细胞学的活力测量来监测微生物生长动力学的早期变化，以应对 单细胞水平的相关抗生素条件，以提高速度确定敏感性/耐药性 和可靠性。结合上游全血预处理进行病原体分离和浓缩 然后是 ID，然后是 AST，我们的目标是在 90 分钟内提供样本以进行 BSI 分类，最早可在 30 分钟内提供答案 测定抗生素最低抑菌浓度 (MIC) 需多花几分钟。我们集结了一支精湛的 由多学科研究人员和行业领先的顾问组成的团队，具有互补的专业知识和强大的 合作记录。我们提出以下目标：1）为广泛的人群开发快速 BSI 分类协议 病原体检测、分类和识别； 2) 开发定量 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 目标是获得必要的初步数据来规划产品开发和商业化， 长期目标是转化我们的诊断平台以降低脓毒症相关的发病率和死亡率。