Rapid ID and AST Directly from Whole Blood Using Single Molecule Detection

项目摘要 血液病原体的标准鉴定(ID)和药敏试验(AST)是 使用需要血液培养的缓慢方法进行。诊断结果可在三天或更长时间内获得 在诊断过程开始之后。缺乏快速诊断结果迫使医生治疗患有 广谱抗生素，对越来越多的耐药微生物无效，可以 诱导产生更多的抗菌素耐药性。在这里，我们提出了一种快速发展的 诊断平台，如果成功，将使血液感染患者能够进行有效和有针对性的治疗 在几个小时内。 新的平台将使用简单而强大的单分子检测来直接分析全血样本 这种方法被称为单分子系留或SMOLT。平台将是快速的(1小时内ID，4小时内AST- 6小时)、易用且性价比高。我们的初步结果表明，SMOLT多重分析可以检测到 血液中细菌的检测限(LOD)在1-10cfu/ml之间，周转时间约为一小时。 同样的技术也可以用于AST。初步结果显示，与抗生素短暂孵育 紧随其后的是SMOLT检测，可以用来确定菌株是敏感的、中等的还是高度抗性的 与指定类别的一致性是一种标准参考方法。在目标1中，我们提出了发展 一种能够检测和鉴定六种高度相关的病原体rRNA的SMOLT多重ID方法 直接用全血。目标2的目标是开发一种简单的表型AST检测方法，用于组合 6个ID种和14个相关药物。我们的第三个目标是开发一个用于SMOLT ID和 AST检测方法简单易用，实际操作时间最少。最后一个目的是评估ID和AST检测 使用混合了新鲜和库存临床分离株以及模式菌株的血液样本。

Project Summary Standard identification (ID) and antimicrobial susceptibility testing (AST) of bloodstream pathogens are conducted using slow methods that require blood culture. Diagnostic results are available three or more days after the diagnostic process is started. The lack of rapid diagnostic results forces physicians to treat patients with broad-spectrum antibiotics which are not effective against a growing number of resistant organisms and can induce the development of more antimicrobial resistance. Here, we propose the development of a rapid diagnostic platform that, if successful, will enable effective and targeted therapy of bloodstream infection patients within hours. The new platform will directly analyze whole blood samples using a simple and robust single molecule detection approach called Single MOlecule Tethering or SMOLT. The platform will be rapid (ID in one hour and AST in 4- 6 hours), easy-to-use, and cost-effective. Our preliminary results show that a SMOLT multiplex assay can detect bacteria in blood with limits of detection (LOD) between 1-10 CFU/mL with a turn-around-time of about an hour. The same technique can be used for AST. Preliminary results show that a brief incubation with an antibiotic follow by SMOLT detection can be used to determine if a strain is susceptible, intermediate or resistance in high agreement with the category assigned be a standard reference method. In aim 1, we propose the development of a SMOLT multiplex ID assay capable of detecting and identifying the rRNA of six highly relevant pathogens directly in whole blood. The goal of aim 2 is to develop a SMOLT phenotypic AST assay for combinations of the six ID species and 14 relevant drugs. Our third aim is to develop a prototype instrument for the SMOLT ID and AST assays that is easy-to-use with minimum hands-on-time. The last aim is to evaluate the ID and AST assays using blood samples seeded with a combination of fresh and stock clinical isolates as well as type strains.