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

项目摘要 败血症，通常由血流感染（BSI）引起，是一种快速进展和危及生命的疾病。 不幸的是，微生物诊断的长期延迟增加了患者死亡率，促进了微生物诊断的滥用。 抗生素，并因此，抗药性病原体的演变。在此，我们的目标是提供一个 用于病原体检测、鉴定（ID）和抗菌剂敏感性的免扩增微流体系统 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体的平台- 辅助显微镜以单细胞分辨率分选、捕获、检测和监测病原体。对于病原体识别，我们 将采用多光谱条形码方案来差异标记用于直接多重核糖体的分子探针， RNA（rRNA）检测，用于病原体分类和物种鉴定，沿着纳米管辅助微波 电穿孔（NAME）技术，以有效地将探针递送到细胞内，用于无扩增的单克隆抗体。 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 我们将用细胞学方法监测微生物生长动力学的早期变化， 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，以便BSI分类，最早在30分钟内提供样本， 测定抗生素最小抑菌浓度（MIC）。我们召集了一个 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 合作的跟踪记录。我们提出以下目标：1）制定一个快速的BSI分诊协议， 病原体检测、分类和ID; 2）开发定量mPhAST; 3）开发集成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，以规划产品开发和商业化， 长期目标是将我们的诊断平台转化为降低脓毒症相关的发病率和死亡率。

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.