Rapid Antibiotic Susceptibility Testing for Neonatal Intensive Units

描述(由申请人提供)：菌血症的标准培养诊断，包括病原体鉴定(ID)和药敏试验(AST)，临床样本采集需要2-3天才能报告结果。在护理时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病房(NICU)抗生素的过度和滥用，导致耐药微生物的比例和流行率增加。虽然随着大型临床微生物学实验室的高通量、自动化仪器的出现，微生物学诊断得到了改善，但这一过程仍然很耗时，需要大量的技术专长。标准的自动化仪器体积庞大，通常需要在AST之前从体液样本中事先分离出病原体。现代临床微生物学实验室的巨大工作负担导致了临床实验室检测外包实践的增加。开发能够快速识别病原体和AST的护理点(POC)平台可以为临床医生提供循证信息，以便只有在必要时才开始针对患者的抗菌治疗。甚至在抗生素使用方面的短期改变也被发现对抗生素耐药性图谱产生了有利的影响。此外，这样的平台可能会加快新型抗生素的筛选。在这项计划中，我们将利用我们正在开发的用于尿液和唾液检测的护理点式诊断平台(U01 AI082457和U01 DE017790)，通过结合补充的快速血细胞去除横流过滤器和电动(EK)浓缩器，创建专门用于快速血液检测的集成诊断试剂盒。这项拟议的研究将利用错流过滤取代离心法，使用高纵横比气体渗透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上基质管理，并开发基于电化学的流体盒，在90分钟内从受感染的血液样本中获得病原体鉴定和抗菌素敏感性评估。该项目的最终目标是利用已建立的微流控芯片技术和表型分析的进步来开发用于诊断NICU菌血症的POC平台。虽然第一阶段的目标是开发一种用于诊断大肠杆菌感染的微流控试剂盒，但该平台将在第一阶段完成后扩展到第二阶段诊断由NICU中发现的其他流行病原体引起的感染。该项目的具体目标1和2是研究和开发用于基质管理的错流过滤和EK操作，以及在流体通道中进行快速抗生素敏感性测试。拟议的AIM 1的结果将使用具有两层微通道设计的基于PDMS的错流过滤器来去除95%的血细胞。具体目标2的重点是测量横流过滤血液的阻抗，并在阻抗分析的基础上将最佳的EK操作条件应用于每个血液样本。流道几何形状、材料和制造细节将与将在特定目标3中建造的射流药筒相媲美。来自特定目标1和2的设计输入将被纳入抗生素敏感性测试(RAST)药盒。具体目标3的目标是开发和验证RAST流体色谱柱，使用标准和新鲜血液样本添加已知浓度的大肠杆菌。具体目标3的通过标准是，在特定目标1和2获得的最佳RAST检测条件下，在90分钟内将血培养结果与微流控试剂盒和相关控制系统获得的结果进行比较时，达到100%的符合率。在第一阶段研究中，我们将用10个添加的全血样本验证RAST试剂盒，以展示在90分钟内获得抗生素敏感性的能力，该细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整合到一个集成的流控色谱柱中，用于与其他常见病原体的扩展小组进行多中心验证研究。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。样本量和招生计划将在第一阶段研究结束时敲定。 公共卫生相关性：细菌感染的标准培养诊断，包括病原体鉴定和药敏试验，需要2-3天的临床样本采集才能报告结果。由于在护理时缺乏明确的微生物学诊断，导致新生儿重症监护病房过度和滥用抗生素。我们建议开发一种专门用于菌血症快速诊断的集成诊断试剂盒，通过利用高纵横比气体渗透微通道获得快速细菌生长表型评估的最佳条件，使用错流过滤和电动操纵技术进行芯片基质管理，并开发基于电化学的流控试剂盒，在90分钟内实现对感染血液样本的病原体鉴定和药敏评估。

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.