Rapid detection of pathogens and antimicrobial susceptibility directly in patient samples

 描述（由申请人提供）：医院感染造成巨大的医疗和经济损失，死亡人数超过艾滋病，乳腺癌和车祸的总和，每年花费美国医疗保健系统数百亿美元。对所有类别抗生素都有抗药性的病原体的出现以及新抗生素的缺乏，可能使人容易受到感染，这让人想起前抗生素时代。快速准确的诊断检测可以及时实施正确的抗菌治疗，从而挽救生命，降低疾病的严重程度，改善抗生素管理并降低医疗成本。然而，今天的大多数测试都依赖于缓慢的世纪微生物培养方法，这些方法需要几天的时间才能让临床医生得到他们需要的答案，以最佳地治疗他们的病人。核酸检测可以快速，但价格昂贵，并且不能可靠地确定使用哪种抗生素来治疗大多数病原体。由于耐药菌基因组的快速进化和容易的种间基因转移，核酸检测受到耐药菌通常广泛的遗传多态性的限制。我们的目标是开发新的多路径平台，以填补技术空白，提供快速，准确，定量和廉价的识别 以及对引起最重要的医院感染类型的病原体的快速抗菌药物敏感性。由于该方法在抗生素存在下测试生长，因此这种快速方法对耐药病原体的遗传复杂性具有鲁棒性且不受其阻碍。简单且具有成本效益的数字成像技术可直接测试患者样本，无需样本制备、细胞破碎、移液、生化纯化、酶和洗涤步骤。在这里，我们提议开发第一个多路径面板测试最常见的医院获得性感染之一，尿路感染，这可能会导致危及生命的血液感染。该项目开发了用于直接分析尿液样本的两阶段UTI诊断方法的检测试剂盒和耗材盒。首先，MultiPath UTI ID检测将在快速（15分钟）和灵敏（<500 CFU/ml）的检测中定量检测9种最常见的UTI病原体。然后，如果有感染，将在快速（3小时）多路径UTI AST测试中分析患者尿样中的病原体对一组相关抗生素的敏感性。具体而言，我们建议（1）开发UTI靶特异性MultiPath免疫试剂，（2）开发和证明多重MultiPath UTI ID检测试剂盒，（3）开发和证明多重MultiPath UTI ID检测试剂盒的性能，（4）开发和证明多重MultiPath UTI AST检测试剂盒，以及（5）开发和证明多重MultiPath UTI AST检测试剂盒。成功的项目将提供具有集成稳定试剂的制造的检测盒耗材，其性能数据证明检测有可能满足严格的监管规范。未来的工作将完善和扩展耗材制造工艺，并将MultiPath UTI耗材与正在开发的高通量、按需、随机访问、自动化台式分析仪集成，该分析仪由其他来源提供资金。

 DESCRIPTION (provided by applicant): Hospital infections exact an enormous medical and economic toll, killing more people than AIDS, breast cancer, and auto accidents combined and costing the US healthcare system tens of billions of dollars annually. The emergence of pathogens resistant to all classes of antibiotics and the paucity of new antibiotics threaten to create vulnerability to infection reminiscent of the pre-antibiotic era. Rapid and accurate diagnostic testing enables timely implementation of the correct antimicrobial therapy which saves lives, decreases the severity of illness, improves antibiotic stewardship, and reduces healthcare costs. However, most testing today depends on slow 19th century microbiological culture methods which require days to get clinicians the answers they need to optimally treat their patients. Nucleic acid tests can be rapid but they are expensive and cannot reliably determine which antibiotics to use to treat most pathogens. Nucleic acid tests are limited by the often extensive genetic polymorphism of resistant bacteria due to their rapidly evolving genomes and facile inter-species gene transfer. We aim to develop the novel MultiPath platform to fill the technology gap, providing rapid, accurate, quantitative, and inexpensive identification and rapid antimicrobial susceptibility for panels of pathogens that cause the most important types of hospital infections. Because the method tests for growth in the presence of antibiotics, this rapid approach is robust to and unhindered by the genetic complexity of resistant pathogens. The simple and cost-effective digital imaging technology tests patient samples directly and eliminates sample preparation, cell-disruption, pipetting, biochemical purification, enzymes, and wash steps. Here we pro- pose to develop the first MultiPath panel tests for one of the most common hospital acquired infections, urinary tract infections, which can cause life-threatening blood infections. The project develops assays and consumable cartridges for a two stage UTI diagnostic approach that direct analyzes urine samples. First, the MultiPath UTI ID test will quantitatively detect 9 of the most common UTI pathogens in a rapid (15 min) and sensitive (<500 CFU/ml) assay. Then, if there is an infection, the pathogen in the patient's urine sample will be analyzed for susceptibility to a panel of relevant antibiotics in a rapid (3 hr) MultiPath UTI AST test. Specifically, we propose to (1) develop UTI target-specific MultiPath immunoreagents, (2) develop and demonstrate the multiplexed MultiPath UTI ID assay, (3) develop and demonstrate the performance of the MultiPath UTI ID cartridge, (4) develop and demonstrate the multiplexed MultiPath UTI AST assay, and (5) develop and demonstrate the MultiPath UTI AST cartridge. The successful project will deliver manufactured cartridge consumables with integrated stabilized reagents with performance data demonstrating the tests have the potential to meet rigorous regulatory specifications. Future work will refine and scale the consumable manufacturing processes and integrate the MultiPath UTI consumables with the high-throughput, on-demand, random-access, automated benchtop analyzer being developed with funding from other sources.