Rapid Antimicrobial Susceptibility Test Cartridge for Resource-Limited Settings

描述（由申请人提供）：项目负责人/主要研究者（最后，第一，中间）：Gau，Vincent项目总结和摘要细菌感染的标准培养诊断，包括病原体鉴定（ID）和抗菌药物敏感性测试（AST），从临床样本采集到结果报告需要2-3天。在护理点缺乏明确的微生物诊断在很大程度上推动了抗生素的过度和滥用。虽然随着大型临床微生物实验室中高通量自动化仪器的可用性，微生物诊断得到了改善，但该过程仍然耗时，需要大量的技术专长。标准自动化仪器体积庞大，并且通常需要在AST之前从体液样品中先验分离病原体。现代临床微生物实验室的重大工作负担导致越来越多的临床实验室检测外包的做法。开发能够快速进行病原体ID和AST检测的床旁平台可以为临床医生提供基于证据的信息，以便仅在必要时开始患者特异性抗菌治疗。即使是抗生素使用的短期改变也会对抗生素耐药性产生有利影响。此外，这种平台可能有助于加快筛选新型抗生素。在本提案中，我们将利用我们正在进行的努力，通过开发互补的快速抗菌药物敏感性测试（RAST）生物芯片，开发用于快速病原体鉴定的集成诊断生物芯片（U 01 AI 082457）。这项拟议的研究将利用高纵横比、透气性微通道来获得用于细菌生长的快速表型评估和用于芯片上基质管理的电动（EK）样品制备技术的最佳条件，并开发基于电化学的RAST流体盒以在90分钟内从确认的阳性患者群体获得抗菌药物敏感性评估。AST开发的最终目标是利用已建立的微流控盒技术的进步和在组装团队中的所有关键人员中开发的表型测定，以建立不仅对尿液标本而且对其他体液的全面了解。目标1的总体重点是用各种阻抗分析来表征样品基质的物理性质，并提供一种可控的方法来最大限度地减少电动捕获的基质效应。通过评估每个待测样本的阻抗图，将应用定制的电动样品制备来获得最大提取效率，而不是使用适用于各种样本条件的通用条件。拟议目标1的结果将提供98%尿液样本的全面阻抗标测和阻抗分析，该信息将与目标1中开发的最佳EK操作条件相关联。特定目标2的通过标准是与尿液培养相比，在90分钟内使用微流控盒和相关对照系统实现与尿液培养100%一致。PHS 398/2590（Rev. 11/07）第1页 公共卫生相关性：在护理点缺乏明确的微生物诊断导致抗生素的过度和滥用。我们建议开发一种基于生物芯片的快速AST，通过利用高纵横比，气体可渗透的微通道进行细菌生长的快速表型评估，电动（EK）样品制备技术用于芯片上的基质管理，并开发一种基于电化学的RAST流体盒，以获得90分钟内从感染的尿液样品的抗菌药物敏感性评估。PHS 398/2590（Rev. 11/07）第1页

DESCRIPTION (provided by applicant): Program Director/Principal Investigator (Last, First, Middle): Gau, Vincent Project summary and abstract Standard culture-based diagnosis of bacterial infections, including pathogen identification (ID) and antimicrobial susceptibility testing (AST), require 2-3 days for clinical sample acquisition to result reporting. The absence of definitive microbiological diagnosis at the point of care has largely driven the over- and misuse of antibiotics. While microbiological diagnosis has improved with the availability of high throughput, automated instruments in the larger clinical microbiology laboratories, the process remains time-consuming and requires significant technical expertise. Standard automation instruments are bulky and typically require a priori isolation of the pathogens from the body fluid samples prior to AST . The significant work burden of a modern clinical microbiology laboratory has led increasingly to the practice of outsourcing of clinical laboratory tests. Development of a point-of-care platform capable of rapid pathogen ID and AST can provide clinicians with evidenced-based information to start patient-specific antimicrobial treatment only when necessary. Even short- term alterations in the use of antibiotics have been found to favorably impact the antibiotic resistance profiles. Furthermore, such platform may be useful to expedite screening of novel class of antibiotics. In this proposal, we will leverage our ongoing efforts to develop an integrated diagnostic biochip for rapid pathogen identification (U01 AI082457) by developing a complementary rapid antimicrobial susceptibility testing (RAST) biochip. This proposed study will utilize high aspect ratio, gas permeable microchannels to obtain optimal conditions for rapid phenotypic assessment of bacterial growth and electrokinetic (EK) sample preparation techniques for on chip matrix management, and develop an electrochemical-based RAST fluidic cartridge to obtain antimicrobial susceptibility assessment from confirmed positive patient populations in 90 minutes. The ultimate goal of the AST development is to leverage the advancement of the established microfluidic cartridge technology and the phenotypic assays developed among all key personnel in the assembled team to establish a comprehensive understanding of not only just the urine specimen but also other body fluids. The overall focus of Aim 1 is to characterize the physical properties of the specimen matrix with various impedance analyses and provide a controllable approach to minimize the matrix effect with electrokinetic trapping. By assessing the impedance mapping of each specimen to be tested, a customized electrokinetic sample preparation will be applied to obtain the maximum extraction efficiency instead of using a universal condition for a wide range of specimen conditions. The outcome of the proposed Aim 1 will provide comprehensive impedance mapping of 98% of urine specimens with impedance analysis and this information will be linked to the optimal EK manipulation conditions to be developed in Aim 1. The passing criteria of Specific Aim 2 is to achieve 100% agreement comparing to urine culture with the microfluidic cartridge and associated control system in 90 minutes compared to urine culture. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: The absence of definitive microbiological diagnosis at the point of care has led to over- and misuse of antibiotics. We propose to develop a biochip-based rapid AST by utilizing high aspect ratio, gas permeable microchannels for rapid phenotypic assessment of bacterial growth, electrokinetic (EK) sample preparation techniques for on chip matrix management, and develop an electrochemical-based RAST fluidic cartridge to obtain antimicrobial susceptibility assessment from infected urine samples in 90 minutes. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page