Rapid identification and antibiotic susceptibility testing of sepsis pathogens

描述（由申请人提供）：有效治疗脓毒症的关键是快速识别血流病原体并进行抗生素敏感性测试。我们的实验室开发了一种创新的电化学传感器检测方法，用于细菌病原体的快速（d30 min）基因型鉴定，该方法已独立验证。电化学传感器分析涉及细菌rRNA与物种特异性捕获和检测探针的“夹心”杂交。捕获探针用于将目标rRNA分子锚到电化学传感器的表面，而检测器探针通过其与辣根过氧化物酶的连接产生电化学信号。我们的传感器检测可以在室温下进行，并且具有检测低至100个细菌/ml的灵敏度。在一项对尿路感染患者尿液标本进行的临床研究中，已证明了概念的可行性。最近，我们证明了电化学传感器测定法可用于通过测量前体rRNA（pre-rRNA）来快速确定抗菌药物敏感性。由于前rRNA是成熟rRNA形成的不稳定中间阶段，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缓慢，但前rRNA水平下降>10倍 敏感E.对环丙沙星的敏感性。最近，我们发现细菌对β-内酰胺抗生素有类似的反应。我们相信，这种方法可以在做出抗生素治疗明智决定所需的时间范围内提供有关血流病原体的可操作信息。本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的时间为30分钟，抗菌药物敏感性测试结果为60分钟。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能准确预测抗生素敏感性。在特定目标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。这些研究需要为后续临床试验生成初步数据，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.