On demand blood tests for select agent diagnosis

按需血液测试用于选择药物诊断

• 批准号: 8606394
• 负责人: David Alland
• 金额: $ 107.34万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-07 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8606394
• 关键词: Address; Authorization documentation; Bacteremia; Bacteria; Beauty; Biological Assay; Bioterrorism; Blood; Blood Tests; Body Fluids; Buffers; Categories; Characteristics; Clinical; Clinical Sensitivity; Collaborations; Communicable Diseases; Complex; Coupled; DNA; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostics Research; Disease; Early treatment; Emergency Situation; Ensure; Goals; Hour; Indium; Infection; Influenza; Laboratories; Licensing; Life; Marketing; Methods; Molecular; Morbidity - disease rate; Nested PCR; New Agents; Patients; Plastics; Polymerase Chain Reaction; Preparation; Process; Protocols documentation; Recording of previous events; Risk; ST14 gene; Sampling; Science; Shipping; Ships; Simulate; Specificity; Specimen; Staging; Staphylococcus aureus; Stream; Symptoms; Syndrome; System; Techniques; Testing; Virulence; Work; base; cost; cost effective; design; flexibility; inhibitor/antagonist; innovation; instrument; mortality; pathogen; point of care; prevent; rapid detection; reagent testing

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.

描述(由申请人提供)：诊断是防御特定病原体感染的关键组成部分。大多数细菌选择剂的毒力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分子都会导致血流感染(BSI)，作为其疾病综合征的一部分。基于血液的检测提供了早期BSI诊断的希望。然而，BSI的快速检测尤其具有挑战性。化验必须极其灵敏，才能发现早期疾病。所需的灵敏度约为每毫升血液中选择试剂的5 cfu。然而，诊断化验也必须是快速、可靠和易于执行的，最好是在护理地点附近。即使是完美的选择试剂测试，如果在紧急情况下无法使用，也将毫无用处。因此，最好的选择试剂分析必须能够在非常短的时间内生产，或者非常便宜，易于储存，并具有较长的保质期。最好的化验方法应该具备所有这些特征。造父变种和Alland实验室有成功合作的历史，致力于开发快速、敏感、按需的传染病测试。我们最近发现了一种可能提供高度敏感的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扩增的套式聚合酶链式反应。整个测试可以在一次性塑料墨盒和高度灵活的仪器平台中进行。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来创建快速护理点分析，在患者可能有症状但仍有低水平菌血症的早期阶段检测选择试剂BSI。我们还将制定一项战略，允许在生物恐怖袭击发生后的几周内快速制造和分发适当的精选试剂分析，无论是否储存了初始测试批次。我们的六个具体目标是：1)为每一种A类选择试剂细菌和每一种“非常规”B类细菌选择试剂开发高灵敏、小扩增子套式聚合酶链式反应分析；2)开发针对主要选择药物风险的多重分析方法；3)使每种分析方法适用于Cepheid试剂盒和GeneXpert(R)平台；4)优化我们的1毫升血液处理程序，开发一种使用Cepheid样本处理试剂盒从10毫升血液中分离选择试剂DNA的方法；5)对含有选择试剂的模拟临床样本进行Uniplex和多重分析测试；以及6)为“按需”生产和FDA紧急使用授权制定应急计划。