Rapid detection of pathogens and antimicrobial susceptibility directly in patient samples

 描述(由申请人提供)：医院感染造成了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，每年给美国医疗体系造成数百亿美元的损失。对所有类别抗生素具有抗药性的病原体的出现和新抗生素的缺乏可能会造成感染的脆弱性，这让人想起抗生素之前的时代。快速准确的诊断检测能够及时实施正确的抗菌治疗，从而挽救生命、降低疾病严重程度、改善抗生素管理并降低医疗成本。然而，今天的大多数测试依赖于19世纪缓慢的微生物培养方法，这种方法需要数天时间才能获得临床医生最佳治疗患者所需的答案。核酸检测可以快速，但价格昂贵，而且不能可靠地确定使用哪种抗生素来治疗大多数病原体。由于耐药细菌基因组的快速进化和方便的跨物种基因转移，耐药细菌往往具有广泛的遗传多态，这限制了核酸测试。我们的目标是开发新的多路径平台来填补技术空白，提供快速、准确、定量和廉价的识别 以及对导致最重要类型医院感染的病原体的快速抗菌药敏感性。因为这种方法是在抗生素存在的情况下测试生长情况，所以这种快速方法对耐药病原体的遗传复杂性是强大的，并且不受阻碍。简单且经济实惠的数字成像技术直接检测患者样本，省去了样本准备、细胞破坏、移液、生化提纯、酶和洗涤步骤。在这里，我们提议为最常见的医院获得性感染之一尿路感染开发第一个多路径小组测试，这种感染可能会导致危及生命的血液感染。该项目为直接分析尿样的两阶段UTI诊断方法开发了化验和消耗盒。首先，多路径UTI ID检测将在快速(15分钟)和灵敏(&lt；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。具体地说，我们建议(1)开发针对UTI靶标的多路径免疫反应物，(2)开发和演示多路径UTI ID分析，(3)开发和验证多路径UTI ID试剂盒，(4)开发和演示多路径UTI AST分析，以及(5)开发和演示多路径UTI AST试剂盒。成功的项目将提供带有集成稳定试剂的制造弹壳消耗品，性能数据表明测试具有满足严格法规规范的潜力。未来的工作将改进和扩大消耗品的制造流程，并将多路径UTI消耗品与正在利用其他来源的资金开发的高通量、按需、随机访问的自动化台式分析仪相结合。

 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.