Rapid identification and antibiotic susceptibility testing of sepsis pathogens

描述（由申请人提供）：血液病原体的快速鉴定和药敏试验是有效治疗脓毒症的关键。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（30分钟）基因型鉴定细菌病原体，并已独立验证。电化学传感器试验涉及细菌rRNA与物种特异性捕获和检测器探针的“三明治”杂交。捕获探针用于将目标rRNA分子锚定在电化学传感器表面，而探测探针通过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，灵敏度低至100个细菌/ml。概念的证明已在尿路感染患者尿液标本的临床研究中得到证实。最近，我们证明了电化学传感器试验可以通过测量前体rRNA （pre-rRNA）来快速确定抗菌药物的敏感性。由于pre-rRNA是成熟rRNA形成的一个不稳定的中间阶段，因此pre-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下降相对缓慢，但前rRNA水平下降了10倍

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.