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

“文化”转变：针对血流感染的微流控数字阵列的综合细菌筛查和抗菌药敏测试

• 批准号: 10321227
• 负责人: Tza-Huei Jeff Wang
• 金额: $ 71.43万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-23 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321227
• 关键词: Acute; Address; Anti-Bacterial Agents; Bacterial Infections; Biological Assay; Blood; Blood Cells; Blood Tests; Blood specimen; Cause of Death; Cell Separation; Cell Survival; Cells; Clinical; Cytolysis; Data; Detection; Devices; Diagnosis; Diagnostic; Disease; Disease Progression; Goals; Gold; Hour; Individual; Industry; Infection; Infection Control; Libraries; Life; Machine Learning; Measures; Methods; Microbiology; Microfluidics; Nutritional; Outcome; Patients; Pharmaceutical Preparations; Predisposition; Preparation; Protocols documentation; RNA-Directed DNA Polymerase; Research Personnel; Research Project Grants; Resolution; Ribosomal DNA; Ribosomal RNA; Sampling; Science; Sepsis; Technology; Testing; Time; Transcript; Validation; Vertebral column; Whole Blood; acute care; automated algorithm; base; chromatin immunoprecipitation; classification algorithm; clinical decision-making; design; diagnostic platform; diagnostic technologies; digital; empowered; imager; improved; innovation; instrument; melting; molecular diagnostics; mortality; multi-drug resistant pathogen; multidisciplinary; novel; pathogen; pathogenic bacteria; programs; prospective; prototype; rRNA Precursor; screening; validation studies

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.

项目概要 在急症护理中使用靶向抗菌药物有效治疗细菌感染的能力 设置取决于能够广泛识别病原体并确定其易感性的诊断 及时使用抗菌药物 血流感染（BSI）是一种特别具有代表性的疾病，因为。 不幸的是，它是由于疾病快速进展而导致死亡的主要原因。 为明确诊断而进行血培养的不便延迟导致了广泛的经验性使用 谱抗菌药物和多重耐药病原体的出现，以解决这一关键问题。 未满足的需求，我们建议开发一个集成细菌检测的新分子诊断平台， 简化的血液样本物种鉴定 (ID) 和抗菌药敏试验 (AST) ID 的预期样本到答案的周转时间为 90 分钟，AST 的预期周期为 2-3 小时。 在提议的时间范围内的综合诊断解决方案将改变急症护理老板的能力 建立细菌感染的诊断、感染控制的必要性和抗菌治疗的基础 客观数据以改善临床结果。 使用创新的微流控数字阵列芯片作为单细胞分析的骨干技术，我们 提议开发一种新的分子诊断平台，该平台有望实现快速 ID 和 AST 并允许 可根据临床情况定制的可定制工作流程和测定，同时可根据实时结果进行调整。 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 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，并根据物种进行识别。 特定的熔解曲线，以及随后通过测量变化评估其抗菌敏感性曲线 rRNA 水平作为细胞活力的生物合成标记物可以实现灵敏的检测和检测。 单个细胞 rRNA 的绝对定量对于快速可靠区分活细胞和活细胞至关重要 无法存活的细胞；而 scHRM 克服了批量 HRM 的一个关键限制，可以解决多个物种的问题 由于 ID 和 AST 都不依赖于培养，因此诊断多种微生物感染或丢弃污染物。 它们将总周转时间从几天缩短到几分钟/小时。 我们组建了一支由多学科研究人员和行业顾问组成的优秀团队 我们提出以下目标：1) 互补的专业知识和良好的团队科学记录。 开发集成 ID 和 AST 的简化 BSI 诊断协议 2) 开发微流控阵列芯片 使 ID 和 AST 具有单细胞分辨率；3) 开发单细胞仪器和分析程序 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。

项目成果

Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument.

使用无电源血浆分离和便携式磁流控聚合酶链式反应仪器对血液中的 HIV 病毒载量进行灵敏和定量的护理点定量。

• DOI: 10.1021/acs.analchem.2c03897
• 发表时间: 2022-12-23
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: H. T. Ngo;Mei Jin;Ale;er Y. Trick;er;Fan;Liben Chen;K. Hsieh;Tza
• 通讯作者: Tza

RNA markers for ultra-rapid molecular antimicrobial susceptibility testing in fluoroquinolone-treated Klebsiella pneumoniae.

用于对氟喹诺酮治疗的肺炎克雷伯菌进行超快速分子抗菌药物敏感性测试的 RNA 标记。

• DOI: 10.1093/jac/dkaa078
• 发表时间: 2020-03-19
• 期刊: The Journal of antimicrobial chemotherapy
• 作者: Xi Yang;Marjan M. Hashemi;Nadya Andini;Michelle M. Li;Shuzhen Kuang;K. Carroll;Tza;Samuel Yang
• 通讯作者: Samuel Yang

A Novel Platform Using RNA Signatures To Accelerate Antimicrobial Susceptibility Testing in Neisseria gonorrhoeae.

一种利用 RNA 特征加速淋病奈瑟菌抗菌药物敏感性测试的新平台。

• 发表时间: 2020
• 影响因子: 9.4
• 作者: Hashemi, Marjan M;Ram;Yang, Xi;Andini, Nadya;Gessner, Nicholas R;Carroll, Karen C;Wang, Tza;Yang, Samuel
• 通讯作者: Yang, Samuel

Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis.

用于数字核酸扩增测试和分析的液滴磁流体自动样品制备的轻松耦合。

• 发表时间: 2020-10-06
• 影响因子: 7.4
• 作者: Gaddes, David E;Lee, Pei;Trick, Alexander Y;Athamanolap, Pornpat;O'Keefe, Christine M;Puleo, Chris;Hsieh, Kuangwen;Wang, Tza
• 通讯作者: Wang, Tza

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19.

用于对抗 COVID-19 的新兴多重核酸诊断测试。

• 发表时间: 2022-11-07
• 作者: Akarapipad, Patarajarin;Bertelson, Elizabeth;Pessell, Alexander;Wang, Tza;Hsieh, Kuangwen
• 通讯作者: Hsieh, Kuangwen