A Droplet-based single cell platform for pathogen identification and AST

用于病原体识别和 AST 的基于 Droplet 的单细胞平台

基本信息

批准号：
8875827
负责人：
JOSEPH C LIAO
金额：
$ 125.64万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8875827
关键词：
Address Affect Age Antibiotic susceptibility Antibiotics Arizona Bacterial Infections Biological Assay Bypass Cell Culture Techniques Cells Clinical Culture Media Cytolysis Data Detection Development Plans Device or Instrument Development Devices Diagnosis Diagnostic Diffusion Encapsulated Escherichia coli Future Generations Goals Growth Guidelines Health Healthcare Hour Human Individual Industry Infection Institutes Laboratories Life Measurement Methods Microfluidic Microchips Microfluidics Minimum Inhibitory Concentration measurement Molecular Molecular Diagnosis Molecular Probes Nucleic Acids Optics Peptide Nucleic Acids Predisposition Process Reagent Reporting Research Research Project Grants Research Subjects Ribosomal RNA Sampling Sepsis Source Specificity System Systems Integration Technology Testing Time Translations Universities Urinary tract Urinary tract infection Urine Validation Vertebral column base combinatorial community-acquired UTI design effective therapy evidence base improved industry partner innovation instrument mortality multi-drug resistant pathogen pathogen product development prospective public health relevance sample collection urinary validation studies

项目摘要

DESCRIPTION (provided by applicant): The ability for clinicians to provide effective treatments to bacterial infections with targeted antibiotics hinges on molecular diagnostics capable of identifying the pathogen and determining its susceptibility to antibiotics in a timely manner. Urinary tract infection (UTI) is a particularly representative infection because it one of the most common bacterial infections that affect all ages but is currently only diagnosed in centralized laboratories via bacterial culture, which typically takes 2-3 days for definitive diagnosis. The significant time delay between sample collection and result reporting contributes to widespread empiric use of broad-spectrum antibiotics, which has contributed towards emergence of multidrug-resistant pathogen. Toward addressing this important unmet need, our overall goal is to develop and validate an integrated diagnostic platform for bacterial pathogen identification (ID) and antibiotic susceptibility testing (AST) in a "sample-to-answer" manner in under 3 hours. Such rapid molecular diagnostics will transform the clinicians' ability to provide evidence-based diagnosis of bacterial infections, expedite treatments based on objective data, promote effective utilization of antibiotics. Specifically, we propose to develop an innovative droplet microfluidic network capable of combinatorially generating millions of picoliter (pL)-sized droplets of different compositions, i.e. mixtures of samples and probes or antibiotics at varying concentration levels, as the backbone technology. The microfluidic chip enables a streamlined approach for sample-reagent mixing, compartmentalization of mixtures into a massive number of droplets, and serial dilutions to simultaneously carry out pathogen ID and AST. In the pathogen ID module, single bacterial cells are encapsulated in droplets, achieving an effective concentration equivalent to 108-109 cfu/ml and thereby enabling rapid identification via the hybridization of molecular beacon probes in an amplification-free approach. In the AST module, individual bacterial cells are encapsulated and cultured in droplets that enhance local culture condition for bacterial growth and enable direct measurements of single bacterial doublings, thereby facilitating direct phenotypic AST from urine samples. We have assembled an academic-industry partnership consisted of Johns Hopkins University (droplet microfluidics and diagnostics), Stanford University (UTI, molecular probes, validation studies), University of Arizona (microfluidic AST), and GE Global Research (manufacturing and system integration). We propose the following aims: 1) to achieve single cell, amplification-free pathogen ID in a droplet-based microfluidic chip using a panel of peptide nucleic acid molecular beacons that target bacterial 16S rRNA; 2) to develop a droplet-based single cell AST capable of determining the minimum inhibitory concentration (MIC) for commonly used antibiotics for UTI; 3) to perform system integration and instrument development through partnership with our industry partner; and 4) to perform analytical and clinical validation with the integrated device. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.

描述（由适用提供）：临床医生为细菌感染提供有效治疗的靶向抗生素铰链的能力，该抗生素取决于分子诊断，能够及时确定病原体并确定其对抗生素的易感性。尿路感染（UTI）是一种特别具有代表性的感染，因为它是影响所有年龄段的最常见细菌感染之一，但目前仅通过细菌培养在集中式实验室中诊断，这通常需要2-3天的确定性诊断。样品收集和结果报告之间的显着时间延迟有助于广泛的经验性使用广谱抗生素，这有助于出现多药耐药性病原体。为了满足这一重要的未满足需求，我们的总体目标是在3小时内以“样本到撤离”方式开发和验证细菌病原体鉴定（ID）和抗生素易感性测试（AST）的综合诊断平台。这种快速的分子诊断将改变临床医生提供基于证据的细菌感染诊断，基于客观数据的加速治疗，促进抗生素的有效利用的能力。具体而言，我们建议开发一个创新的液滴微流体网络，能够结合产生数百万的Picoliter（PL）大小不同组成的液滴，即样品的混合物，问题或抗生素在不同浓度水平上，作为骨干技术。微流体芯片可以简单地进行样品混合，将混合物分隔为大量液滴，以及串行稀释液，以简单地执行病原体ID和AST。在病原体ID模块中，单个细菌细胞被封装在液滴中，达到了相当于108-109 CFU/mL的有效浓度，从而通过在无扩增方法中杂交分子信标问题杂交来快速识别。在AST模块中，将单个细菌细胞封装并培养在液滴中，从而增强了细菌生长的局部培养条件，并可以直接测量单细菌加倍，从而支持尿液样品中的直接表型AST。我们组建了一个学术行业伙伴关系，由约翰·霍普金斯大学（Johns Hopkins University）（液滴微流体和诊断），斯坦福大学（UTI，分子问题，验证研究），亚利桑那大学（Microfluidic AST）和GE全球研究（制造和系统整合）组成。我们提出以下目的：1）使用一组靶向细菌16S rRNA的胡椒核酸分子信标，在基于液滴的微流体芯片中实现单细胞，无扩增的病原体ID； 2）开发一个基于液滴的单细胞AST，能够确定用于UTI的常用抗生素的最小抑制浓度（MIC）； 3）通过与我们的行业合作伙伴的合作伙伴关系来执行系统集成和仪器开发； 4）使用集成装置进行分析和临床验证。为了促进技术翻译，提出了针对未来临床部署的产品开发计划。