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

项目总结 败血症通常由血液感染(BSI)引起，是一种进展迅速、危及生命的疾病。 不幸的是，微生物诊断的长期延误增加了患者的死亡率，促进了滥用 抗生素，因此，抗药性病原体的进化。在此，我们的目标是提供一个 用于病原体检测、鉴定(ID)和抗菌药敏感性的无放大微流控系统 直接从全血中检测(AST)。为了实现我们的目标，我们提出了一个基于微流控的平台-- 辅助显微镜在单细胞分辨率下对病原体进行分类、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对直接多重核糖体的分子探针进行差异标记 核糖核酸(RRNA)检测用于病原体的分类和分类，以及纳米管辅助微波 电穿孔(NAME)技术有效地将探针输送到细胞内，实现无扩增的单细胞 微生物检测。病原体ID阳性将指导定量多模式表型AST(MPhAST)，其中 我们将通过细胞学方法监测微生物生长动力学的早期变化，以应对 在单细胞水平上的相关抗生素条件，以提高速度确定敏感性/耐药性 和可靠性。结合上游全血前处理的病原菌分离浓缩 接下来是ID，然后是AST，我们的目标是在BSI分诊的90分钟内提供样本到答案，最早30分钟 抗生素最小抑菌浓度(MIC)测定的时间延长。我们已经组装了一流的 由多学科调查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 协作的跟踪记录。我们提出了以下目标：1)开发一种快速的BSI分诊协议 病原体检测、分类和ID；2)建立mPhAST的定量；3)建立一体化的ID- MPhAST平台；4)对我们的ID-mPhAST平台进行分析和临床验证。我们的短期计划 目标是获得必要的初步数据，以计划产品开发和商业化， 翻译我们的诊断平台以减少败血症相关的发病率和死亡率的长期目标。

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.