Rapid Antibiotic Susceptibility Testing for Neonatal Intensive Units

描述（由申请人提供）：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学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，导致抗生素耐药生物的比例和流行率增加。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。标准的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的重大工作负担导致了临床实验室测试外包实践的增加。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影响。此外，该平台有可能加快新型抗生素的筛选。在该提案中，我们将利用我们正在进行的尿液和唾液检测即时诊断平台（U01 AI082457和U01 DE017790）的开发，通过结合互补的快速血细胞去除交叉流过滤器和电动（EK）浓缩器，创建专门用于快速血液检测的集成诊断盒。本研究将利用横流过滤代替离心，采用高纵横比气透微通道获得快速评估细菌生长表型的最佳条件，利用EK样品制备技术进行片上基质管理，并开发基于电化学的流控药瓶，在90分钟内完成感染血液样本的病原体鉴定和抗菌药敏评估。该项目的最终目标是利用已建立的微流控药盒技术和表型分析的进步，开发一个POC平台，用于诊断新生儿重症监护室的菌血症。虽然第一阶段的目标是开发用于诊断大肠杆菌感染的微流控药筒，但在第一阶段完成后，该平台将扩展到诊断NICU中其他常见病原体引起的感染。该项目的具体目标1和2是研究和开发用于基质管理的交叉流过滤和EK操作，以及在流体通道中快速进行抗生素敏感性测试。拟议的Aim 1的结果将通过基于pdms的两层微通道设计的交叉流过滤器去除95%的血细胞。具体目标2的重点是测量交叉流过滤后血液的阻抗，并根据阻抗分析对每个血液样本应用最佳EK操作条件。流道几何形状，材料和制造细节将与在特定目标3中构建的流体墨盒相当。来自Specific Aim 1和2的设计输入将被纳入抗生素敏感性测试（RAST）药筒。特异性目标3的目标是开发和验证RAST流体盒与标准和新鲜血液样品加入已知的大肠杆菌浓度。Specific Aim 3的合格标准是在Specific Aim 1和2中获得的最佳RAST测定条件下，将90分钟内的血培养结果与微流控试剂盒及相关控制系统获得的结果进行比较，结果达到100%一致。我们将用10个加标全血样本验证RAST试剂盒，以证明能够在90分钟内对洛杉矶儿童医院提供的已知耐药大肠杆菌获得抗生素敏感性。在第二阶段，我们计划将病原体鉴定和RAST纳入一个集成的流体盒中，与其他常见病原体的扩展面板一起进行多中心验证研究。II期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第一阶段研究结束时最终确定。

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.