Phage-accelerated test system for ID/AST of urinary tract infections

尿路感染 ID/AST 噬菌体加速检测系统

• 批准号: 9407512
• 负责人: DAVID A SCHOFIELD
• 金额: $ 29.11万
• 依托单位: GUILD ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9407512
• 关键词: Accident and Emergency department; Accounting; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Bacteria; Bacterial Infections; Bacteriophages; Biological Assay; Bypass; Cells; Characteristics; Cytolysis; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Disease; Drug resistance; Engineering; Escherichia coli; Genes; Genetic Engineering; Genotype; Goals; Gram-Negative Bacteria; Health; Health Care Costs; Health care facility; High Prevalence; Hospitals; Human; Immunoassay; Infection; Lateral; Length; Libraries; Longevity; Measures; Mediating; Metabolic; Methods; Modernization; Molecular; Patient-Focused Outcomes; Patients; Performance; Pharmaceutical Preparations; Phase; Phenotype; Physicians' Offices; Plasmids; Predisposition; Production; Proteins; Protocols documentation; Public Health; Reader; Recombinants; Research; Resistance; Sampling; Signal Transduction; System; Technology; Testing; Time; Urinary tract infection; Urine; Visit; antimicrobial; antimicrobial drug; base; clinical diagnostics; cost; extracellular; fighting; fitness; instrument; pathogen; protein biomarkers; research and development; response; tool

Urinary tract infections (UTIs) are one of the most common types of bacterial infection encountered by humans throughout their lifespan and are a major health problem afflicting millions of people each year. Gram-negative bacteria cause the majority of infections of which Escherichia coli is the primary pathogen and prone to antibiotic resistance. The standard methods for species diagnosis and susceptibility determination are culture- based protocols that take up to 48 h. As a result, UTIs are one of the most frequent reasons for antimicrobial prescriptions in healthcare facilities, without the benefit of a confirmed diagnosis. Therefore, modern day rapid diagnostic methods that promote rapid identification and antimicrobial stewardship are crucial. The long-term objective of this R&D is to develop an easy to use platform for the rapid (≤3 h) identification and antimicrobial susceptibility testing (ID/AST) of UTI pathogens directly from urine. A major goal is to develop a diagnostic that can bypass the need for bacterial amplification and isolation and thus overcome the major time-limiting step of current diagnostics. The platform will consist of species-specific bacteriophages (phages) engineered to express heterologous marker proteins, a lateral flow immunoassay, and analysis by Sofia, a fluorescent assay reader produced and marketed by our Quidel collaborators. Upon bacterial cell infection, the recombinant phages will produce large amounts of a foreign marker protein, which following phage-mediated cell lysis, can be rapidly and sensitively detected by an immunoassay and objectively read by Sofia. As the marker protein production is correlated to cell fitness, the platform termed Phage-accelerated Test (PhACT), can rapidly determine an isolates sensitivity, or resistance to a particular antibiotic, a key attribute in this 'post-antibiotic' era. The Phase I proof-of-principle studies will focus on demonstrating feasibility for E. coli as this species is responsible for >70% of uncomplicated UTIs. Specific Aim 1 will generate a cocktail of genetically engineered E. coli phages harboring the foreign marker protein. Specific Aim 2 will develop phage-mediated lateral-flow immunoassay and determine its performance characteristics for direct ID/AST from urine samples. The research is significant as the technology enables simultaneous detection and phenotypic drug susceptibility analysis without culture amplification and isolation, thus bypassing the major time limiting step in diagnostics. This is key as decreasing length of time for diagnosis and the administration of appropriate therapy positively impacts patient outcomes and promotes antimicrobial stewardship.

尿路感染是人类最常见的细菌感染类型之一 在他们的一生中，是一个主要的健康问题，每年困扰着数百万人。兰阴性 细菌引起大多数感染，其中大肠杆菌是主要病原体， 抗生素耐药性用于物种诊断和敏感性测定的标准方法是培养- 基于需要长达48小时的协议。因此，UTI是抗生素治疗的最常见原因之一。 医疗机构的处方，没有确诊的好处。因此，现代快速 促进快速识别和抗菌药物管理的诊断方法至关重要。 该研发的长期目标是开发一种易于使用的平台，用于快速（≤3小时） 直接从尿液中进行UTI病原体的鉴定和抗菌药物敏感性测试（ID/AST）。一 主要目标是开发一种诊断方法，可以绕过细菌扩增和分离的需要， 克服了当前诊断的主要时间限制步骤。该平台将包括特定物种的 工程化以表达异源标记蛋白质的噬菌体，侧流免疫测定， 并由索菲亚进行分析，这是一种由我们的Quidel合作者生产和销售的荧光测定读数器。后 当细菌细胞感染时，重组的大肠杆菌将产生大量的外源标记蛋白， 在噬菌体介导的细胞裂解后，可以通过免疫测定快速和灵敏地检测， 索菲亚客观地阅读了。由于标记蛋白的产生与细胞适应性相关，因此该平台被称为 噬菌体加速试验（PhACT），可以快速确定一个分离株的敏感性，或对特定的 抗生素，这是“后抗生素”时代的一个关键属性。第一阶段的原理验证研究将侧重于 证明了E.大肠杆菌，因为该物种负责>70%的简单UTI。具体目标 1将产生一种基因工程E.大肠杆菌表达外源标记蛋白。 Specific Aim 2将开发噬菌体介导的侧流免疫分析并确定其性能 尿液样本的直接ID/AST特征。这项研究意义重大，因为这项技术使 无需培养扩增和分离， 从而绕过诊断中的主要时间限制步骤。这是关键，因为减少时间长度， 诊断和适当的治疗管理积极影响患者的结果，并促进 抗菌管理。