A RAPID ASSAY FOR DETECTION OF DETECTION OF CARBAPENEMASE PRODUCING ORGANISMS

 描述(由申请人提供)：罗纳德·里根加州大学洛杉矶分校医学中心最近爆发了与受污染的十二指肠镜相关的碳青霉烯耐药肠杆菌科(CRE)，突显了对这些临床重要病原体进行早期检测的重要性。巧合的是，奥巴马政府发布了《国家抗击抗药性细菌行动计划》，针对该计划的步骤之一是减缓抗药性细菌的出现，防止抗药性感染的传播。在卫生保健中心检测和鉴定碳青霉烯酶产生菌(CPO)是限制来源和防止这些多重耐药病原体潜在传播的第一步。然而，在临床实验室检测铜绿假单胞菌是具有挑战性的，因为分离株对碳青霉烯类抗生素的敏感性可能只有适度的降低，耐药可能由其他机制(即染色体和质粒介导的头孢菌素酶或膜通透性降低的超广谱β-内酰胺酶)介导。检测碳青霉烯酶基因的分子方法受到检测的碳青霉烯酶靶标数量的限制，此外还有其他限制。目前检测产碳青霉烯酶肠杆菌的参考表型方法--改良霍奇法，存在检测周期长、缺乏特异性、敏感性差等缺点。 碳青霉烯酶类型(即金属β-内酰胺酶)。该项目旨在开发一种低成本、易使用(基本上是一个手动步骤)和快速(10-15分钟)的方法来鉴定碳青霉烯类耐药革兰氏阴性菌的碳青霉烯酶产生，适合于监测和临床实验室使用。该方法使用荧光素衍生的β-内酰胺酶底物来检测β-内酰胺酶活性。所有试剂都将在主混合物(试剂I)中配制，这样化验基本上只需一个手动步骤：将样品加入主混合物。为了将碳青霉烯类酶与其他β-内酰胺酶区分开来，将配制含有碳青霉烯类抗生素的第二种主混合物(试剂II)。试剂I的信号指示β-内酰胺酶的存在或不存在，试剂II与试剂I的信号比率指示β-内酰胺酶是否是碳青霉烯酶。拟议的第一阶段项目将优化化验，并使用特征良好的分离株以及预期的临床应用。 以评估该检测方法。这一第一阶段项目的成功完成将为第二阶段研究铺平道路，第二阶段研究将涉及多个地点和更大的样本数量。一种简便、快速的检测铜绿假单胞菌的方法将在监测和指导治疗和感染控制措施中发挥重要作用。

 DESCRIPTION (provided by applicant): A recent outbreak of carbapenem resistant Enterobacteriaceae (CRE) associated with contaminated duodenoscopes at the Ronald Reagan UCLA Medical Center highlights the importance of early detection of these clinically significant pathogens. Coincidentally, the Obama Administration released The National Action Plan to Combat Antibiotic-Resistant Bacteria for which one of the steps of the action plan is to slow the emergence of resistant bacteria and prevent the spread of resistant infections. Detection and identification of carbapenemase producing organisms (CPO) in health-care centers is the first step to confining the source and preventing the potential spread of these multidrug resistant pathogens. However, detection of CPO in clinical laboratories is challenging, as isolates may only have moderate reductions in susceptibilities to carbapenems and resistance may be mediated by other mechanisms (i.e., chromosomal and plasmid-mediated cephalosporinases or extended-spectrum β-lactamase (ESBL) producers with decreased membrane permeability). Molecular methods for the detection of carbapenemase genes are limited by the number of carbapenemase targets assayed in addition to other limitations. The reference phenotypic method for detection of carbapenemase-producing Enterobacteriaceae, the Modified Hodge test, suffers from a long turn- around time, lacks specificity and has poor sensitivity for certain types of carbapenemase enzymes (i.e., metallo-β-lactamase enzymes). This project aims to develop a low cost, easy-to-use (essentially one manual step), and rapid (10-15 min) assay to identify carbapenemase production in carbapenem resistant Gram-negative bacteria suitable for surveillance and clinical laboratory use. The proposed assay uses a luciferin derivatized β-lactamase substrate for detection of β-lactamase activity. All reagents will be formulated in a master mix (Reagent I) such that the assay involves essentially one manual step: addition of the sample to the master mix. To differentiate carbapenemases from other β-lactamases, a second master mix containing a carbapenem will be formulated (Reagent II). The signal of Reagent I indicates the presence or absence of β-lactamase while the signal ratio of Reagent II to Reagent I indicates whether the β-lactamase is a carbapenemase. The proposed Phase I project will optimize the assay and use well-characterized isolates as well as prospective clinical isolates to evaluate the assay. Successful completion of this Phase I project will pave the way for a Phase II study, which will involve multiple sites and substantially larger sample numbers. A simple and rapid assay for detection of CPO will play a significant role in surveillance and help guide therapy and infection control measures.