Rapid Antibiotic Susceptibility Testing for Neonatal Intensive Units

新生儿重症监护病房的快速抗生素敏感性测试

• 批准号: 8063070
• 负责人: Vincent Jen-Jr Gau
• 金额: $ 21.59万
• 依托单位: GENEFLUIDICS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063070
• 关键词: Acute; Address; Agreement; Ampicillin; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Arizona; Automation; Award; Bacteremia; Bacterial Infections; Biological Assay; Biosensor; Biotechnology; Blood; Blood Cells; Blood Tests; Blood specimen; Body Fluids; California; Cefotaxime; Centrifugation; Childhood; Clinical; Clinical Microbiology; Clinical Research; Collaborations; Communicable Diseases; Development; Devices; Diagnosis; Diagnostic; Doctor of Medicine; Doctor of Philosophy; Engineering; Enrollment; Ensure; Escherichia coli; Escherichia coli Infections; Excision; Filtration; Gases; Gentamicins; Goals; Growth; Hospitals; Hour; Imipenem; Infection; Laboratories; Lead; Libraries; Los Angeles; Maps; Measures; Microfluidics; Molecular; Molecular Analysis; Molecular Diagnosis; Multi-Drug Resistance; Neonatal; Neonatal Intensive Care Units; Organism; Outcome; Outsourcing; Patients; Phase; Preparation; Prevalence; Process; Protocols documentation; Reporting; Reproducibility; Research; Research Proposals; Resistance profile; Ribosomal RNA; Saliva; Sample Size; Sampling; Screening procedure; Small Business Innovation Research Grant; Specimen; System; Systems Development; Technical Expertise; Techniques; Technology; Testing; Time; Universities; Urine; Validation; Whole Blood; Work; antimicrobial; base; blood filter; clinical practice; clinically relevant; design; electric impedance; evidence base; improved; innovation; instrument; multidisciplinary; novel; pathogen; phase 1 study; point of care; point-of-care diagnostics; product development; public health relevance; sample collection; validation studies

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.

描述（由申请人提供）：基于菌血症的标准培养诊断，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。护理时缺乏明确的微生物学诊断在很大程度上导致了新生儿重症监护病房 (NICU) 中抗生素的过度和滥用，导致抗生素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出现，微生物诊断得到了改善，但该过程仍然耗时且需要大量的技术专业知识。标准自动化仪器体积庞大，通常需要在 AST 之前从体液样本中预先分离病原体。现代临床微生物学实验室的巨大工作负担导致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，以便仅在必要时开始针对患者的抗菌治疗。研究发现，即使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，这样的平台可能会加快新型抗生素的筛选。在本提案中，我们将利用我们在尿液和唾液检测即时诊断平台（U01 AI082457 和 U01 DE017790）上的持续开发，通过结合互补的快速血细胞去除技术，创建专门用于快速血液检测的集成诊断盒错流过滤器和电动 (EK) 浓缩器。拟议的研究将利用错流过滤代替离心，采用高纵横比透气微通道以获得细菌生长快速表型评估的最佳条件，利用 EK 样品制备技术进行片上基质管理，并开发电化学基于流体盒，可在 90 分钟内从受感染的血液样本中进行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用于诊断大肠杆菌感染的微流体盒，但在第一阶段完成后，该平台将扩展到诊断第二阶段新生儿重症监护病房中发现的其他流行病原体引起的感染。 具体目标 1 和 2该项目的目的是研究和开发用于基质管理的错流过滤和 EK 操作，以及流体通道中的快速抗生素敏感性测试。拟议目标 1 的结果将是使用基于 PDMS 的两层微通道设计错流过滤器去除 95% 的血细胞。具体目标 2 的重点是测量错流过滤血液的阻抗，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 Specific Aim 3 中构建的流体卡盒相当。Specific Aim 1 和 2 的设计输入将被纳入抗生素敏感性测试 (RAST) 卡盒中。具体目标 3 的目标是使用添加了已知浓度大肠杆菌的标准血液样本和新鲜血液样本来开发和验证 RAST 流体盒。具体目标 3 的通过标准是，在具体目标 1 和 2 中获得的最佳 RAST 检测条件下，将血培养结果与微流控盒和相关控制系统在 90 分钟内获得的结果进行比较时，达到 100% 一致。我们将验证 RAST装有 10 个加标全血样本的试剂盒，以证明能够在 90 分钟内利用洛杉矶儿童医院在 I 期研究中提供的已知抗生素耐药性大肠杆菌获得抗生素敏感性。在第二阶段，我们计划将病原体识别和 RAST 纳入集成流体盒中，以进行多中心验证研究，并扩大其他常见病原体的范围。 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。 公共卫生相关性：基于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，从采集临床样本到报告结果需要 2-3 天。由于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快速菌血症诊断的集成诊断盒，利用高纵横比透气微通道获得细菌生长快速表型评估的最佳条件，采用错流过滤和动电操纵技术进行片上矩阵管理，并开发一种基于电化学的流体盒，以在 90 分钟内实现病原体识别并从受感染的血液样本中获得抗菌药物敏感性评估。