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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：
9038992
负责人：
JOSEPH C LIAO
金额：
$ 118.49万
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/9038992
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 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 molecular diagnostics mortality multi-drug resistant pathogen pathogen product development prospective 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.