A fully integrated CentriFluidic system for direct bloodstream infection PID/AST

用于直接血流感染 PID/AST 的完全集成 CentriFluidic 系统

• 批准号: 9241943
• 负责人: Vincent Jen-Jr Gau
• 金额: $ 70.42万
• 依托单位: GENEFLUIDICS, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9241943
• 关键词: Acinetobacter baumannii; Acute; Address; Agreement; Algorithmic Analysis; Antibiotic Resistance; Antibiotics; Antimicrobial susceptibility; Automation; Bacteremia; Bacteria; Bacterial Infections; Biological Assay; Blood; Blood Cells; Blood specimen; Blood-Borne Pathogens; Categories; Centrifugation; Characteristics; Clinical; Clinical Management; Clinical Sensitivity; Culture Techniques; Cytolysis; Detection; Devices; Diagnosis; Diagnostic; Dose; Enterobacter; Escherichia coli; Etiology; Feasibility Studies; Genotype; Goals; Gravitation; Guidelines; Hospitals; Hour; Hybrids; Incidence; Infection; Inflammatory Response; Intestines; Klebsiella pneumonia bacterium; Libraries; Liquid substance; Mass Spectrum Analysis; Minimum Inhibitory Concentration measurement; Modality; Modification; Molecular; Molecular Analysis; Monitor; Multi-Drug Resistance; Observational Study; Organism; Patients; Phenotype; Physicians; Predisposition; Procedures; Process; Protocols documentation; Pseudomonas aeruginosa; Public Health; Publishing; Recovery; Regimen; Reporting; Reproducibility; Research; Research Project Grants; Resistance profile; Sampling; Sensitivity and Specificity; Sepsis; Septic Shock; Specificity; Specimen; Survival Rate; System; Systems Integration; Technology; Testing; Time; Ultracentrifugation; Urinary tract infection; Urine; Whole Blood; antimicrobial; base; blind; clinically relevant; evidence base; improved; innovation; melting; microorganism; mortality; pathogen; precision medicine; prospective; prototype; public health relevance; routine Bacterial stain; time use; validation studies

 DESCRIPTION (provided by applicant): There is a pressing need for a technology platform that can aid rapid AST profiling where MIC results are presented within a narrow time window of less than 3 hours. This acquires tremendous significance during bloodstream infections and rapid onset sepsis; the choice of antibiotics to surmount emergent antibiotic resistance and associated treatment modality in reduction of host inflammatory response is crucial for patient survival and recovery. Such a platform will finally usher in the "Precision medicine" approach for treating bacterial infections, whereby physicians are able to track antibiotic resistance profiles f pathogens in real time and adjust their antibiotic dosing regimens. It is highly unlikely that culture techniques can still be improved to significantly decrease total assay time (TAT). Therefore, this proposed system focuses on improving TAT by using molecular technologies for identification and characterization of microorganism susceptibility profile directly from fresh whole blood specimen of BSI patients. A first step forward, to decrease time-to-result, can be achieved by utilizing blood lysis centrifugation. Moreover, eliminating steps for obtaining clinica isolates will lead to the most optimal turnaround time. The overall hypotheses of this application are that a CentriFluidic system can be used to perform a rapid PID and AST directly from whole blood without a need of blood cultivation or PCR amplification based on our successful clinical feasibility studies on urinary tract infection (UTI) directly from raw urine specimen with reproducible demonstration of polymicrobial infection detection and multi-drug resistance profiling. We developed, demonstrated and published an innovative molecular-based genotypic-phenotypic-hybrid approach for multiplexed bacterial PID and AST profiling with 100% clinical sensitivity, 96% clinical specificity, 98% minimum inhibitory concentration (MIC) and 97% categorical agreement in our most recent ongoing clinical feasibility study on 73 raw clinical urine samples. However, critical limitations of the current UTI platform exist, including inabilityto detect much lower abundance of bloodborne pathogens, the lack of system integration with an ultracentrifugation module and inability to cover all clinical relevant pathogens for BSIs. The main goal of this RO1 research project is to combine the advantages of lab automation, rapid molecular analysis, melt-curve signature analysis, genotypic pathogen quantification and phenotypic antibiotic conditions to dramatically improve the sensitivity and specificity of rapid, evidence-based PID and AST directly from patient blood samples. We propose to test the following hypotheses: Specific Aim 1: Transition the current electrochemical-based molecular analysis PID/AST platform technology from UTI to BSIs - Hypothesis: Lysis centrifugation can address the change of matrix effect from raw urine to whole blood samples and the issue of low abundant pathogen for BSIs. Specific Aim 2: Develop a dual-mode electrochemical-based dynamic hybridization analysis algorithm to expand the BSI PID/AST panel - Hypothesis: Dynamic hybridization analysis can be utilized to expand the species-specific identification of common and emerging pathogens for BSIs. Specific Aim 3: Prototype, validate and manufacture the CentriFluidic cartridge and system - Hypothesis: Ultracentrifugation (up to 50,000 g, gravitational force) can be incorporated into a multiplexed fluidic cartridge for a fully automated BSIs PID/AST from whole blood samples in 3 hours. Specific Aim 4: Clinically validate the rapid BSI PID/AST CentriFluidic system according to CLSI guidelines - Hypothesis: Blood samples spiked with ATCC strain bacteria used in the analytical validation studies represent critical matrix characteristics of fresh whole blood samples from patients.

 描述（由申请人提供）：迫切需要一种技术平台，可以帮助快速AST分析，其中MIC结果在不到3小时的狭窄时间窗内呈现。这在血流感染和快速发作的脓毒症期间获得了巨大的意义;抗生素的选择以克服紧急的抗生素耐药性和减少宿主炎症反应的相关治疗方式对于患者的生存和恢复至关重要。这样的平台最终将迎来治疗细菌感染的"精准医学"方法，医生能够真实的跟踪病原体的抗生素耐药性，并调整他们的抗生素给药方案。仍然可以改进培养技术以显着减少总测定时间（TAT）的可能性极小。因此，该系统的重点是通过使用分子技术直接从BSI患者的新鲜全血标本中鉴定和表征微生物药敏谱来改善TAT。向前迈出的第一步是减少获得结果的时间，这可以通过利用血液裂解离心来实现。此外，消除获得临床分离株的步骤将导致最佳的周转时间。 本申请的总体假设 基于我们对直接来自原始尿液标本的尿路感染（UTI）的成功临床可行性研究，CentriFluorescent系统可用于直接从全血中进行快速PID和AST，而无需血液培养或PCR扩增，并可重复证明多微生物感染检测和多药耐药谱。我们开发、展示并发表了一种创新的基于分子的基因型-表型-杂交方法，用于多重细菌PID和AST分析，在我们最近对73份原始临床尿液样本进行的临床可行性研究中，该方法具有100%的临床灵敏度、96%的临床特异性、98%的最小抑菌浓度（MIC）和97%的分类一致性。然而，当前UTI平台存在严重局限性，包括无法检测丰度低得多的血源性病原体，缺乏与超离心模块的系统集成，以及无法覆盖BSI的所有临床相关病原体。RO1研究项目的主要目标是将实验室自动化、快速分子分析、熔解曲线特征分析、基因型病原体定量和表型抗生素条件的优势联合收割机结合起来，以显著提高直接从患者血液样本中快速、循证PID和AST的灵敏度和特异性。我们建议测试以下假设：具体目标1：将当前基于电化学的分子分析PID/AST平台技术从UTI转换为BSI-假设：裂解离心可以解决从原始尿液到全血样本的基质效应变化以及BSI的低丰度病原体问题。具体目标二：开发一种基于双模式电化学的动态杂交分析算法，以扩展BSI PID/AST面板-假设：动态杂交分析可用于扩展BSI常见和新兴病原体的种属特异性鉴定。具体目标3：原型设计、验证和制造CentriFluorescent检测盒和系统-假设：超离心（高达50，000 g，重力）可并入多路复用射流检测盒中，用于全自动检测 在3小时内从全血样本中获得的BSI PID/AST。具体目标4：根据CLSI指南对快速BSI PID/AST CentriFluorescent系统进行临床验证-假设：分析验证研究中使用的加标ATCC菌株细菌的血液样本代表患者新鲜全血样本的关键基质特征。