Rapid Antimicrobial Susceptibility Test Cartridge for Resource-Limited Settings

项目描述（由申请人提供）：项目主任/首席研究员（最后，第一，中）：Gau， Vincent项目总结和摘要基于培养的标准细菌感染诊断，包括病原体鉴定（ID）和抗菌药物敏感性试验（AST），从临床样品采集到结果报告需要2-3天。在护理点缺乏明确的微生物学诊断在很大程度上推动了抗生素的过度和滥用。虽然微生物学诊断随着大型临床微生物学实验室中高通量自动化仪器的可用性而得到改善，但该过程仍然耗时且需要大量的技术专长。标准自动化仪器体积庞大，通常需要在AST之前从体液样本中先验地分离病原体。现代临床微生物实验室巨大的工作负担，导致越来越多的临床实验室检测外包的做法。开发能够快速识别病原体和AST的护理点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针对患者的抗菌治疗。即使是短期改变抗生素的使用也会对抗生素耐药性产生有利的影响。此外，该平台可能有助于加快新型抗生素的筛选。在本提案中，我们将通过开发互补的快速抗菌药敏试验（RAST）生物芯片，利用我们正在进行的开发用于快速病原体鉴定的集成诊断生物芯片（U01 AI082457）的努力。本研究将利用高宽高比、可透气微通道获得最佳条件，用于细菌生长的快速表型评估和芯片上基质管理的电动力学（EK）样品制备技术，并开发基于电化学的RAST流体盒，在90分钟内对确诊阳性患者群体进行抗菌药物敏感性评估。AST发展的最终目标是利用已建立的微流控药盒技术的进步和在组装团队的所有关键人员中开发的表型分析来建立对尿液标本和其他体液的全面了解。Aim 1的总体重点是通过各种阻抗分析来表征样品基质的物理特性，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品的阻抗映射，将应用定制的电动样品制备来获得最大的提取效率，而不是使用广泛的样品条件的通用条件。拟议的目标1的结果将通过阻抗分析提供98%尿液标本的全面阻抗映射，该信息将与目标1中开发的最佳EK操作条件相关联。Specific Aim 2的通过标准是在90分钟内与尿液培养相比，与微流体墨盒和相关控制系统达到100%的一致性。PHS 398/2590 （Rev. 11/07）第1页延续格式页

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