On demand blood tests for select agent diagnosis

描述（由申请人提供）：诊断包括防御选择代理感染的关键组成部分。大多数细菌选择剂的毒性需要快速检测和早期治疗，以防止严重的发病率和死亡率。大多数细菌生物恐怖主义制剂引起血流感染（BSI）作为其疾病综合征的一部分。基于血液的检测为早期BSI诊断提供了希望。然而，快速BSI检测尤其具有挑战性。化验必须非常灵敏才能发现早期疾病。所要求的灵敏度为每毫升血液中选择剂< 5 cfu的量级。然而，诊断分析也必须快速、可靠和易于执行，最好在护理点附近进行。如果在紧急情况下无法使用，即使是完美的选择代理测试也几乎没有用处。因此，最佳选择试剂必须能够在很短的时间内制造，或者非常便宜，易于储存，并且具有较长的保质期。最好的化验应该具备所有这些特征。造父变星和Alland实验室在开发传染病快速、敏感、按需检测方面有着成功的合作历史。我们最近发现了一种可能提供高灵敏度BSI检测的方法。我们的方法自动从1 - 10毫升的血液中提取细菌，然后自动执行非常小的扩增子巢式PCR。整个测试可以在一次性塑料盒和高度灵活的仪器平台内进行。作为主要证据，我们的初步研究在几乎90%的金黄色葡萄球菌BSI患者的血液中检测到金黄色葡萄球菌。在这里，我们建议进一步发展我们的方法，以创建快速的即时检测方法，在患者可能有症状但仍有低水平菌血症的早期阶段检测出选择剂bsi。我们还将制定一项战略，允许在生物恐怖袭击发生后几周内快速生产和分发适当的精选试剂，无论是否储存初始测试批次。我们的六个具体目标是：1)为每种A类选择剂细菌和每种“非常规”B类细菌选择剂开发高灵敏度，小扩增子巢式pcr检测；2)开发针对主要选择代理风险的多重分析；3)使每个检测适应造父变星试剂盒和GeneXpert(R)平台；4)优化我们的1ml血液处理方案，并开发一种使用造父变星样本处理盒从10ml血液中分离选择剂DNA的方法；5)对含有选定试剂的模拟临床样品进行单组分和多组分分析试验；6)为“按需”生产和紧急FDA使用授权制定应急计划。

DESCRIPTION (provided by applicant): Diagnostics comprise a critical component of defense against select agent infections. The virulence of most bacterial select agents requires rapid detection and early treatment to prevent serious morbidity and mortality. Most bacterial bioterrorism agents cause blood stream infections (BSI) as part of their disease syndrome. Blood-based detection offers the promise of early BSI diagnosis. However, rapid BSI detection is particularly challenging. Assays must be extremely sensitive to detect early disease. The sensitivity required is on the order of < 5 cfu of the select agent per ml of blood. Yet, the diagnostic assays must also be rapid, robust and easy to perform, preferably near the point-of-care. Even the perfect select agent test will be of little use if it is not be available when neede in an emergency. Thus, the best select agent assays must be able to be manufactured upon very short notice, or be very inexpensive, easy to stockpile, and have a prolonged shelf life. The very best assays would have all of these characteristics. Cepheid and the Alland laboratory have a history of successful collaborations devoted to developing rapid, sensitive, on-demand tests for infectious disease. We recently discovered an approach that is likely to provide highly-sensitive BSI detection. Our approach automatically extracts bacteria from 1 - 10 ml of blood and then automatically performs very-small- amplicon nested PCR. The entire test can be performed within a disposable plastic cartridge and a highly- flexible instrument platform. As proof of principal, our preliminary studies detected S. aureus in the blood of almost 90% of S. aureus BSI patients. Here, we propose to further develop our approach to create rapid point-of-care assays that will detect select agent BSIs at the early stage when a patient may be symptomatic but still have low-level bacteremia. We will also develop a strategy that permits rapid manufacture and distribution of appropriate select agent assays within weeks of a bioterrorist attack with or without stockpiling of an initial test lot. Our six specific aims are t: 1) Develop highly-sensitive, small amplicon nested-PCR assays for each category A select agent bacterium and for each "unconventional" category B bacterial select agent; 2) Develop multiplex assays for major select agent risks; 3) Adapt each assay to the Cepheid cartridge and the GeneXpert(R) platform; 4) Optimize our 1 ml blood processing protocol and develop a method to isolate select agent DNA from 10 ml of blood using Cepheid sample processing cartridges; 5) Perform analytic tests of both uniplex and multiplex assays on simulated clinical samples containing select agents; and 6) Develop an emergency plan for "on demand" manufacture and emergency FDA use authorization.