A "Culture" Shift: Integrated Bacterial Screening and Antibacterial Susceptibility Test on Microfluidic Digital Array for Bloodstream Infections

项目总结 临床医生在急诊应用靶向抗菌药物有效治疗细菌感染的能力 设置取决于能够广泛识别病原体并确定其对 及时进行抗菌药物治疗。血液感染是一种特别有代表性的疾病，因为 它是由疾病快速发展的感染引起的主要死亡原因。不幸的是， 血培养用于确诊的不便延误导致了广泛的经验性应用。 广谱抗菌药与多重耐药病原菌的出现。为了解决这一关键问题 为了满足未满足的需求，我们建议开发一种新的分子诊断平台，该平台集成了细菌检测、 简化的血液样本的菌种鉴定(ID)和药敏试验(AST) 测试。预计样本到答案的周转时间ID为90分钟，AST最早为2-3小时。是这样的 建议时间范围内的集成诊断解决方案将改变急诊临床医生的能力 建立细菌感染的诊断、感染控制的需要和抗菌治疗 目的为提高临床疗效提供数据。 使用创新的微流控数字阵列芯片检测单细胞作为主干技术，我们 建议开发一个新的分子诊断平台，承诺快速ID和AST，并允许 可根据临床情况定制工作流程和化验方法，同时根据实时结果进行调整。 该阵列芯片无缝集成了细胞数字化、短暂孵育(在各种药物条件下) 单细胞聚合酶链式反应(ScPCR)或逆转录酶聚合酶链式反应(scRT-PCR)和单细胞高分辨熔融 (ScHRM)。因此，可以在单细胞水平上检测细菌病原体，并根据物种鉴定。 特定的熔体曲线，以及随后通过测量变化来评估其抗菌敏感性分布 RRNA水平作为细胞活力的生物合成标志。单链聚合酶链式反应/单链构象聚合酶链式反应能够灵敏地检测 对单个细胞rRNA的绝对定量对于快速可靠地区分活的和 没有活细胞；而scHRM克服了批量HRM的一个关键限制，无法分解多个物种 诊断多菌感染或丢弃污染物。由于ID和AST都不依赖区域性， 它们将总周转时间从几天减少到几分钟/小时。 我们已经组建了一支由多学科调查人员和行业顾问组成的精湛团队， 相互补充的专业知识和良好的团队科学记录。我们提出以下目标：1) 开发集成ID和AST的简化BSI诊断方案；2)开发微流控阵列芯片 这使得ID和AST具有单细胞分辨率；3)开发用于单细胞分辨率的仪器和分析程序 细胞ID和AST；4)为了演示单细胞诊断平台，我们将进行分析和试点 临床验证研究。

PROJECT SUMMARY The ability for clinicians to effectively treat bacterial infections with targeted antibacterials in the acute-care settings hinges on diagnostics capable of identifying the pathogen broadly and determining its susceptibility to antibacterials in a timely manner. Bloodstream infection (BSI) is a particularly representative disease because it is the leading cause of death due to infections with rapid disease progression. Unfortunately, the inconvenient delay of blood culture for definitive diagnosis contributes to widespread empiric use of broad- spectrum antibacterials and emergence of multi-drug-resistant pathogens. Toward addressing this critical unmet need, we propose to develop a new molecular diagnostic platform that integrates bacterial detection, species identification (ID), and antibacterial susceptibility testing (AST) from blood samples in a streamlined test. The expected sample-to-answer turnaround time is 90 min for ID and as early as 2-3 hr for AST. Such integrated diagnostic solution within the proposed timeframe will transform acute-care clinicians’ ability to establish diagnosis of bacterial infections, need for infection control, and antibacterial treatment based on objective data to improve clinical outcome. Using an innovative microfluidic digital array chip for assaying single cells as a backbone technology, we propose to develop a new molecular diagnostic platform which promises rapid ID and AST and allows customizable workflow and assay tailored to the clinical scenario while adjustable based on real-time results. The array chip seamlessly integrates digitization of cells, brief incubation (under various drug conditions), single-cell PCR (scPCR) or reverse transcriptase PCR (scRT-PCR) and single-cell high-resolution melt (scHRM). Thereby, bacterial pathogen can be detected at the level of single-cells, identified based on species- specific melt curves, and their antibacterial susceptibility profile subsequently assessed by measuring changes in rRNA level as a biosynthetic marker of cell viability. ScPCR/scRT-PCR enables sensitive detection and absolute quantification of rRNA of individual cells critical to rapid and reliable differentiation between viable and no-viable cells; while scHRM overcomes a key limitation of bulk HRM to resolve multiple species for diagnosing polymicrobial infections or discarding contaminations. Since both ID and AST do not rely on culture, they reduce total turnaround time from days to minutes/hours. We have assembled a superb team of multi-disciplinary investigators and industry advisors with complementary expertise and strong track record of team science. We propose the following aims:1) to develop a streamlined BSI diagnostic protocol for integrated ID and AST; 2) to develop a microfluidic array chip that enables ID and AST with single-cell resolution; 3) to develop instrument and analysis programs for single- cell ID and AST; and 4) to demonstrate the single-cell diagnostic platform, we will perform analytical and pilot clinical validation studies.