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

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，它是一个主要的健康问题，每年困扰着数百万人。革兰氏阴性 细菌是大多数感染的原因，其中大肠埃希菌是主要病原体，容易引起 抗生素耐药性。菌种诊断和药敏测定的标准方法是培养- 因此，尿路感染是使用抗菌药最常见的原因之一 在没有确诊的情况下，在医疗机构开具处方。因此，现代快车 促进快速识别和抗菌管理的诊断方法至关重要。 这项研发的长期目标是为快速(≤3 h)开发一个易于使用的平台 尿路感染病原菌的鉴定及药敏试验(ID/AST)。一个 主要目标是开发一种诊断方法，它可以绕过细菌扩增和分离的需要，从而 克服当前诊断的主要时间限制步骤。该平台将由特定物种组成 设计表达异源标记蛋白的噬菌体(噬菌体)，侧向流动免疫分析， 由我们的Quidel合作者生产和销售的荧光分析读取器Sofia进行分析。vt.在.的基础上 细菌细胞感染后，重组噬菌体会产生大量的外源标志蛋白， 在噬菌体介导的细胞裂解后，可以通过免疫分析快速和灵敏地检测到 由索菲亚客观地阅读。由于标记蛋白的产生与细胞的健康状况相关，因此该平台称为 噬菌体加速试验(PHACT)，可以快速确定一种分离物对特定病毒的敏感性或抵抗力 抗生素，这是这个“后抗生素”时代的一个关键属性。第一阶段的原则证明研究将侧重于 证明了对大肠杆菌的可行性，因为该物种导致了70%的简单尿路感染。特定目标 1将产生含有外源标记蛋白的基因工程大肠杆菌噬菌体的鸡尾酒。 特异性目标2将发展噬菌体介导的侧向流动免疫分析并确定其性能 尿样直接ID/AST的特征。这项研究具有重要意义，因为这项技术使 无需培养扩增和分离即可同时检测和表型药敏分析， 从而绕过了诊断中的主要时间限制步骤。这是关键，因为减少 诊断和适当治疗的实施会对患者的预后产生积极影响，并促进 抗菌管理。

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.