Rapid identification and antibiotic susceptibility testing of sepsis pathogens

描述(申请人提供)：有效治疗败血症的关键是血液病原体的快速鉴定和药敏试验。我们的实验室开发了一种创新的电化学传感器检测方法，用于快速(d30分钟)鉴定细菌病原体的基因型别，并已得到独立验证。电化学传感器分析包括细菌rRNA与特定物种捕获和检测器探针的“夹心”杂交。捕获探针用于将目标rRNA分子锚定在电化学传感器的表面，而检测器探针则通过它们与辣根过氧化物酶的链接来产生电化学信号。我们的传感器分析可以在室温下进行，灵敏度低至100个细菌/毫升。对尿路感染患者的尿样进行的临床研究证明了这一概念。最近，我们证明了电化学传感器试验可以通过测定前体rRNA(Pre-rRNA)来快速确定药物敏感性。由于前rRNA是成熟rRNA形成的一个不稳定的中间阶段，因此前rRNA水平对抗生素的生长抑制反应迅速。成熟rRNA水平对抗生素的反应相对较慢，但前rRNA水平下降10倍。 在敏感的大肠杆菌暴露于环丙沙星后15分钟内。最近，我们发现细菌对β-内酰胺类抗生素也有类似的反应。我们相信，这种方法可以在做出有关抗生素治疗的知情决定所需的时间框架内提供有关血流病原体的可操作信息。这项研究应用的目标是开发一种快速细菌鉴定和药敏(RBIS)测试系统，从获得阳性血培养到细菌鉴定在D30分钟内，抗菌药物敏感性测试结果在D60分钟内。《研究计划》有两个具体目标。在具体目标1中，我们将确定前rRNA水平的早期变化是否准确地预测抗生素敏感性。在具体目标2中，我们将对盲法阳性血培养进行临床研究，以确定我们方法的准确性。这些研究需要为随后的临床试验产生初步数据，以衡量RBIS测试系统对临床结果和患者护理成本的影响。

DESCRIPTION (provided by applicant): The key to effective treatment of sepsis is rapid identification and antibiotic susceptibility testing of bloodstream pathogens. Our laboratory has developed an innovative electrochemical sensor assay for rapid (d30 min) genotypic identification of bacterial pathogens, which has been validated independently. The electrochemical sensor assay involves "sandwich" hybridization of bacterial rRNA to species-specific capture and detector probes. Capture probes are used to anchor target rRNA molecules to the surface of electrochemical sensors, while the detector probes generate the electrochemical signal through their link to horseradish peroxidase. Our sensor assay can be performed at room temperature and has the sensitivity to detect as few as 100 bacteria/ml. Proof of concept has been demonstrated in a clinical study of urine specimens from patients with urinary tract infection. Recently, we demonstrated that the electrochemical sensor assay can be employed to rapidly determine antimicrobial susceptibility through measurement of precursor rRNA (pre-rRNA). Because pre-rRNA is a labile, intermediate stage in the formation of mature rRNA, pre-rRNA levels respond rapidly to growth inhibition by antibiotics. Mature rRNA levels fall relatively slowly in response to antibiotics, but pre-rRNA levels drop by >10-fold within 15 min after exposure of susceptible E. coli to ciprofloxacin. More recently, we have found similar responses of bacteria to beta-lactam antibiotics. We believe that this approach can provide actionable information about bloodstream pathogens in the time frame needed to make informed decisions about antibiotic therapy. The goal of this research application is to develop a Rapid Bacterial Identification and Susceptibility (RBIS) Testing System with a time from acquisition of a positive blood culture to bacterial identification of d30 minutes and antimicrobia susceptibility testing results in d60 minutes. The Research Plan has two Specific Aims. In Specific Aim 1 we will determine whether early changes in pre-rRNA levels accurately predict antibiotic susceptibility. In Specific Aim 2 we will perform a clinical study of blinded positive blood cultures to determine the accuracy of our approach. These studies are required to generate preliminary data for a subsequent clinical trial to measure the impact of RBIS Testing System on clinical outcomes and patient care costs.