An Integrated Diagnostic System for Rapid Antimicrobial Susceptibility Testing

描述（由申请人提供）：导致许多常见人类传染病的病原体，如尿路感染（UTI）、肠胃炎、肺炎和伤口感染，已被证明在获得抗微生物药物耐药性机制方面非常熟练。卫生保健提供者经验性使用抗生素的普遍不明智做法以及抗生素在食物链中的渗透加速了耐药病原体的选择和传播。因此，临床医生的治疗选择更少，尤其是在最需要帮助的病人身上。这一问题的一个例子是对甲氧苄啶-磺胺甲恶唑（SXT）耐药大肠杆菌的迅速出现，它占社区环境中尿路感染的85-90%。在20世纪90年代之前，氨苄西林（AMP）等β -内酰胺类药物是急性无并发症尿路感染的标准抗菌方案，但当大肠杆菌对β -内酰胺类药物的耐药性超过25%时，就被SXT所取代。然而，随着使用的增加，sst耐药性大幅增加，喹诺酮类药物如环丙沙星（CIP）成为首选抗生素。不出所料，耐喹诺酮尿路病原体呈上升趋势。在耐多药病原体问题更严重的医院，尿路致病性大肠杆菌的喹诺酮耐药率现在在某些情况下已超过50%。NIAID先进技术SBIR II期应用的目标是开发和验证RAST（快速抗菌药物敏感性测试），这是一种集成诊断紧张型系统，使临床医生能够指导护理点（POC），以证据为基础选择抗生素治疗急性细菌感染。RAST通过提供快速（90分钟vs. 2天）和分散（POC vs.基于实验室）的检测，解决了标准表型AST平台（例如，bioMerieux Vitek, BD Phoenix）的主要局限性。RAST补充了我们正在进行的NIAID合作协议，一种用于护理点病原体鉴定的集成诊断生物芯片（U01 AI082457），用于使用集成微流体的电化学生物传感器快速分子诊断尿路感染（UTI）。自I期以来，我们已经完成了几个关键的里程碑：(1)开发和临床验证了3.5小时的台式RAST方案，准确率为94%；(2) RAST与临床尿液样品的相容性，无需初始细菌分离；（3）

DESCRIPTION (provided by applicant): Pathogens responsible for many of the common human infectious diseases such as urinary tract infection (UTI), gastroenteritis, pneumonia, and wound infections have proven to be highly adept in acquiring mechanisms of antimicrobial resistance. Widespread injudicious practice of empiric antibiotic usage by healthcare providers and infiltration of antibiotics in the food chain have accelerated selection and dissemination of resistant pathogens. As a consequence, clinicians have fewer treatment options, particularly in the most needy patients. An example of the problem was the rapid emergence of trimethoprim- sulfamethoxazole (SXT) resistant E. coli, which accounts for 85-90% of the UTIs in the community setting. Prior to the 1990s, beta-lactams such as ampicillin (AMP) were the standard antimicrobial regimen for acute uncomplicated UTIs, but was replaced with SXT when E. coli resistance against beta-lactams surpassed 25%. With increasing use, however, SXT resistance increased substantially and quinolones such as ciprofloxacin (CIP) became the antibiotic of choice. Not surprisingly, quinolone-resistant uropathogens are on the rise. In hospitals where MDR pathogens are of even greater problem, the quinolone-resistance rate for uropathogenic E. coli has now exceeded 50% in some settings. The goal of this Phase II NIAID Advanced Technology SBIR application is to develop and validate RAST (rapid antimicrobial susceptibility testing), an integrated diagnostic compact system to enable clinicians to direct point-of-care (POC), evidence-based selection of antibiotics for treatment of acute bacterial infections. RAST addresses the major limitations of standard phenotypic AST platforms (e.g., bioMerieux Vitek, BD Phoenix) by providing rapid (90 minutes vs. 2 days) and decentralized (POC vs. laboratory-based) testing. RAST complements our ongoing NIAID Cooperative Agreement, An Integrated Diagnostic Biochip for Point of Care Pathogen Identification (U01 AI082457), for rapid molecular diagnosis urinary tract infections (UTI) using electrochemical biosensors integrated with microfluidics. Since Phase I, we have accomplished several critical milestones: (1) development and clinical validation of a 3.5 hour bench-top RAST protocol showing 94% accuracy; (2) compatibility of RAST with clinical urine samples without need for initial bacterial isolation; (3) feasibility of on-chip electrokinetic bacterial concentration and assay enhancement; (4) on-chip bacterial culture using microchannels; (5) integrated microfluidic cartridge for pathogen identification; and (6) preliminary feasibility of cartridge-based RAST. In the current Phase II project, we propose three Specific Aims: Specific Aim 1. Optimization and validation of electrokinetic (EK) processing modules for volume reduction and in situ assay enhancement. The goal of Aim 1 is to develop an EK volume reduction module for enriching the sample 100-fold within 10 min and to develop an in situ EK enhancement technique for improving the detection sensitivity of the electrochemical assay by 10-fold. Specific Aim 2. Development of the RAST cartridge for rapid phenotypic antimicrobial susceptibility testing. The goal of Aim 2 is to develop the process flow and fabrication process for the RAST cartridge and reader/manifold system, including sample loading, EK volume reduction, on-chip sample culturing in selective media containing different antibiotics of interest, and phenotypic AST by quantitative measurement of bacterial 16S rRNA. Specific Aim 3. Clinical translation of RAST cartridge in urine. The goal of Aim 3 is to perform analytical validation of RAST cartridge and reader/manifold system and a clinical feasibility study using 30 unknown clinical samples from patients suspected to have UTI. The development and validation of RAST will adhere to the recommended standards of Quality Management Standard for Medical Devices (ISO 13485) and federal regulations for fully automated short-term incubation cycle antimicrobial susceptibility system (21 CFR 866.1645)(see Commercialization Plan D.1.3). Successful accomplishment of our milestones in this Phase II application will be followed by FDA 510(k) submission to demonstrate the system is substantially equivalent to a predicate device. A separate Milestones and Timeline section is included at the end of the Research Strategy. PUBLIC HEALTH RELEVANCE: Development of a compact platform capable of rapid pathogen ID and AST directly from patient's samples can provide clinicians at the point of care (e.g., outpatient office, emergency department) with evidenced-based information to start patient-specific antimicrobial treatment only when necessary. Rapid susceptibility test results and alterations in the use of antibiotics, even short-term, have been found to favorably impact patient care and the antibiotic resistance profiles.