RNA based diagnostics for rapid pathogen identification and drug resistance

 描述(由申请人提供)：对传染病的快速诊断既能识别病原体，又能实时提供药敏数据，这将改变患者管理和关键的公共卫生问题，如当前的抗生素耐药性危机。它们的应用将广泛地从诊所和初级保健办公室扩展到三级保健医院，为治疗干预提供即时指导，从而导致更谨慎和适当地使用抗生素，在某些情况下有利于死亡。面对不断升级的抗生素耐药性，对经验性抗生素决策构成挑战的情况下，生成实时药敏模式的需求尤为迫切。 我们建议开发一个快速、通用的基于RNA杂交的诊断平台，该平台将提供对微生物病原体的实时鉴定，并揭示它们对各种抗生素的敏感性。病原体鉴定将通过检测高度丰富的核糖体RNA中物种特有的序列变异来实现。药物敏感性将通过检测转录特征来确定，这些转录特征由抗生素暴露在敏感但不耐药的菌株中极快地诱导(在几分钟内)。重要的是，与现有的基于DNA的诊断方法不同，这种基于RNA的方法将能够在不需要核酸扩增和药物敏感性分析的情况下识别病原体，而不需要先验地了解导致耐药性的基因或突变。此外，由于对抗生素的转录反应发生得比可测量的生长变化早得多，这个平台将比基于培养的分析更早地提供表型读数。值得注意的是，这项工作与微流控技术相结合，消除了对培养的需要。原则性研究证明了这一方法的可行性。 我们现在建议扩展和优化我们用于病原体识别和高度耐药ESKAPE病原体(肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌属)的RNA签名，以解决开发诊断设备所需的个别工程挑战，并将这些元素集成到用于快速病原体识别和抗生素敏感性的微流控原型中。为此，我们已经组建了一个由科学家、工程师和行业合作伙伴组成的多学科团队，他们将合作开展拟议的研究。

 DESCRIPTION (provided by applicant): Rapid diagnostics for infectious diseases that both identify the pathogen and provide drug susceptibility data in real-time would transform patient management and critical public health issues such as the current antibiotic resistance crisis. Their application would reach broadly from clinics and primary care offices to tertiary care hospitals, providing immediate guidance for therapeutic intervention thereby resulting in more prudent and appropriate use of antibiotics, in some cases with mortality benefit. The need to generate real- time drug susceptibility patterns is particularly acute in the face of escalating antibiotic resistance that is challenging empiric antibiotic decision-making. We propose to develop a rapid, universal RNA hybridization-based diagnostic platform that would provide both real-time identification of microbial pathogens and reveal their susceptibility to diverse antibiotics. Pathogen identification will be achieved by the detection of species-specific sequence variations in highly abundant ribosomal RNAs. Drug susceptibility will be determined by detection of transcriptional signatures that are extremely rapidly induced (within minutes) by antibiotic exposure in susceptible but not resistant strains. Importantly, in contrast o existing DNA-based diagnostics, this RNA-based approach will enable pathogen identification without nucleic acid amplification and drug susceptibility profiling without a priori knowledge of the genes or mutations that confer drug resistance. Moreover, since transcriptional responses to antibiotics occur much earlier than measurable changes in growth, this platform would provide a phenotypic readout much earlier than culture-based assays. Significantly, this work is coupled with microfluidic technology to obviate the need for culture. Proof of principle studies have demonstrated the feasibility of this approach. We now propose to expand upon and optimize our RNA-signatures for pathogen identification and drug susceptibility for the high priority antibiotic resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species), to solve individual engineering challenges required for development of a diagnostic device, and to integrate these elements into a microfluidic prototype for rapid pathogen identification and antibiotic susceptibility. To that ed, we have formed a multidisciplinary team of scientists, engineers, and industry partners that will work collaboratively to carry out the proposed research.