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

项目摘要 临床医生在急诊护理中使用靶向抗菌药物有效治疗细菌感染的能力 设置取决于诊断能够识别病原体广泛，并确定其易感性， 及时使用抗菌药物。血流感染（BSI）是一种特别有代表性的疾病， 它是由于疾病迅速发展的感染而导致死亡的主要原因。可惜 不方便的延迟血培养的明确诊断有助于广泛的经验性使用广泛的， 广谱抗菌药物和多重耐药病原体的出现。为了解决这一关键问题， 未满足的需求，我们建议开发一种新的分子诊断平台，整合细菌检测， 种属鉴定（ID）和抗菌药物敏感性测试（AST）从血液样本中，在一个精简的 test. ID的预期样本至应答周转时间为90分钟，AST最早为2-3小时。等 在建议的时间范围内，集成诊断解决方案将改变急诊临床医生的能力， 确定细菌感染的诊断、感染控制的需要和基于以下方面的抗菌治疗： 改善临床结果的客观数据。 使用创新的微流控数字阵列芯片作为骨干技术， 我建议开发一种新的分子诊断平台，该平台承诺快速ID和AST，并允许 根据临床情况定制的可定制工作流程和分析，同时可根据实时结果进行调整。 阵列芯片无缝集成了细胞的数字化，短暂的孵育（在各种药物条件下）， 单细胞PCR（scPCR）或逆转录酶PCR（scRT-PCR）和单细胞高分辨率熔解 （scHRM）。因此，可以在单细胞水平上检测细菌病原体，基于物种进行鉴定， 特定的熔解曲线，以及随后通过测量变化评估的抗菌敏感性曲线 rRNA水平作为细胞活力的生物合成标志物。ScPCR/scRT-PCR能够实现灵敏的检测， 单个细胞rRNA的绝对定量对于快速可靠地区分活细胞和 无活细胞;而scHRM克服了批量HRM的关键限制，以解决多个物种， 诊断多种微生物感染或丢弃污染物。由于ID和AST都不依赖于培养， 它们将总周转时间从数天减少到数分钟/小时。 我们组建了一支由多学科调查人员和行业顾问组成的优秀团队， 互补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 使ID和AST具有单细胞分辨率; 3）开发用于单细胞的仪器和分析程序， 细胞ID和AST;以及4）为了演示单细胞诊断平台，我们将进行分析和试验 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.