Rapid Antibiotic Susceptibility Testing for Neonatal Intensive Units

描述（由申请方提供）：标准的基于培养的菌血症诊断，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新生儿重症监护室（NICU）中抗生素的过度和滥用，导致耐药性微生物的比例和患病率增加。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了临床实验室检验外包实践的增加。能够快速鉴定病原体和AST的床旁（POC）平台的开发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，这样的平台可能会加快新型抗生素的筛选。在本提案中，我们将利用我们正在进行的尿液和唾液检测床旁诊断平台（U 01 AI 082457和U 01 DE 017790）的开发，通过整合互补的快速血细胞去除交叉流过滤器和电动（EK）浓缩器，创建一个专门用于快速血液检测的集成诊断盒。拟议的研究将利用交叉流过滤来代替离心，采用高纵横比透气微通道来获得快速细菌生长表型评估的最佳条件，利用EK样品制备技术进行芯片基质管理，并开发基于电化学的流体盒以在90分钟内从感染的血液样品中获得病原体鉴定和抗菌药物敏感性评估。该项目的最终目标是利用已建立的微流控盒技术和表型测定的进步，开发用于诊断NICU菌血症的POC平台。第一阶段的目标是开发一种用于诊断大肠杆菌的微流体盒。大肠杆菌感染，该平台将在第1阶段完成后扩展到诊断由第2阶段NICU中发现的其他流行病原体引起的感染。本项目的具体目标1和2是研究和开发用于基质管理的交叉流过滤和EK操作，以及流体通道中的快速抗生素敏感性测试。所提出的目标1的结果将使用具有双层微通道设计的基于PDMS的错流过滤器去除95%的血细胞。具体目标2的重点是测量交叉流过滤血液的阻抗，并根据阻抗分析将最佳EK操作条件应用于每个血液样本。流动通道几何形状、材料和制造细节将与特定目标3中构建的射流卡盘相当。Specific Aim 1和2的设计输入将被纳入抗生素敏感性测试（RAST）测试卡片中。Specific Aim 3的目标是开发和验证RAST射流盒，其具有加标已知大肠杆菌的标准和新鲜血液样本。大肠杆菌浓度。特定目标3的通过标准是在特定目标1和2中获得的最佳RAST测定条件下，在90分钟内将血液培养结果与微流控检测盒和相关对照系统获得的结果进行比较时，达到100%一致。我们将使用10份加标全血样本验证RAST测试卡片，以证明在90分钟内获得已知耐药大肠埃希菌抗生素敏感性的能力。大肠杆菌，由洛杉矶儿童医院提供。在第二阶段，我们计划将病原体鉴定和RAST整合到集成的射流卡盘中，用于多中心验证研究，其中包括其他常见病原体的扩展组。第二阶段的临床研究将由洛杉矶儿童医院的Grace Aldrovani博士领导。样本量和入组计划将在I期研究结束时最终确定。 公共卫生相关性：细菌感染的标准培养诊断，包括病原体鉴定和抗菌药物敏感性测试，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，导致新生儿重症监护室过度和滥用抗生素。我们提出开发一种专门用于快速菌血症诊断的集成诊断盒，通过利用高纵横比的透气微通道来获得用于细菌生长的快速表型评估的最佳条件，采用错流过滤和电动操作技术用于芯片上基质管理，并开发出一种电化学-基于射流卡盘的检测系统可在90分钟内从感染的血液样本中实现病原体鉴定并获得抗菌药物敏感性评估。

DESCRIPTION (provided by applicant): Standard culture-based diagnosis of bacteremia, including pathogen identification (ID) and antimicrobial susceptibility testing (AST), requires 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 in the neonatal intensive care unit (NICU), resulting in an increase in proportion and prevalence of antibiotic-resistance organisms. 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 to an increase in outsourcing practice of clinical laboratory tests. Development of a point-of-care (POC) platform capable of rapid pathogen identification and AST can provide clinicians with evidence-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 could potentially expedite the screening of novel class of antibiotics. In this proposal, we will leverage our ongoing development on the point-of-care diagnostic platform for urine and saliva testing (U01 AI082457 and U01 DE017790) to create an integrated diagnostic cartridge specifically for rapid blood testing by incorporating a complementary rapid blood cell removal cross-flow filter and an electrokinetic (EK) concentrator. The proposed study will utilize cross-flow filtration to replace centrifugation, employ high aspect-ratio gas-permeable microchannels to obtain optimal conditions for rapid phenotypic assessment of bacterial growth, exploit EK sample preparation techniques for on-chip matrix management, and develop an electrochemical-based fluidic cartridge to obtain pathogen identification and antimicrobial susceptibility assessment from infected blood samples in 90 minutes. The ultimate goal of this project is to leverage the advancement of the established microfluidic cartridge technology and the phenotypic assay to develop a POC platform for diagnosing bacteremia in the NICU. While the goal for Phase 1 is to develop a microfluidic cartridge for diagnosing E. coli infection, this platform will be extended to diagnose infections caused by other prevalent pathogens found in the NICU in Phase 2 upon completion of Phase 1. Specific Aims 1 and 2 of this project is to investigate and develop cross-flow filtration and EK manipulation for matrix management, and rapid antibiotic susceptibility testing in fluidic channels. The outcome of the proposed Aim 1 will remove 95% of blood cells with a PDMS-based cross-flow filter with a two-tier micro-channel design. The focus of Specific Aim 2 is to measure the impedance of the cross-flow filtered blood and apply the optimal EK manipulation conditions to each blood specimen based on the impedance analysis. The flow channel geometry, materials and fabrication details will be comparable with the fluidic cartridge to be built in Specific Aim 3. The design inputs from the Specific Aim 1 and 2 will be incorporated into the antibiotic susceptibility testing (RAST) cartridge. The goal of Specific Aim 3 is to develop and validate the RAST fluidic cartridge with standard and fresh blood samples spiked with known E. coli concentrations. The passing criteria of Specific Aim 3 is to achieve 100% agreement when comparing blood culture results with results acquired by the microfluidic cartridge and associated control system in 90 minutes under optimal RAST assay conditions obtained in Specific Aim 1 and 2. We will validate the RAST cartridge with 10 spiked whole blood samples to demonstrate the ability to obtain antibiotic susceptibility in 90 minutes with known antibiotic-resistant E. coli provided by Childrens Hospital Los Angeles in Phase I study. In Phase II, we plan to incorporate the pathogen identification and RAST into an integrated fluidic cartridge for a multi-center validation study with an expanded panel of other common pathogens. The clinical study in Phase II will be led by Dr. Grace Aldrovani at Childrens Hospitals Los Angeles. The sample size and enrollment plan will be finalized toward the end of the Phase I study. PUBLIC HEALTH RELEVANCE: Standard culture-based diagnosis of bacterial infections, including pathogen identification and antimicrobial susceptibility testing require 2-3 days for clinical sample acquisition to result reporting. The absence of definitive microbiological diagnosis at the point of care has led to over- and misuse of antibiotics in neonatal intensive care units. We proposed to develop an integrated diagnostic cartridge specifically for rapid bacteremia diagnosis by utilizing high aspect-ratio gas-permeable microchannels to obtain optimal conditions for rapid phenotypic assessment of bacterial growth, employing cross-flow filtration and electrokinetic manipulation techniques for on-chip matrix management, and developing an electrochemical- based fluidic cartridge to achieve pathogen identification and obtain antimicrobial susceptibility assessment from infected blood samples in 90 minutes.