An HIV Viral Load Assay for the Point-Of-Care

用于护理点的 HIV 病毒载量测定

• 批准号: 9200559
• 负责人: Marta Fernandez-Suarez
• 金额: $ 64.96万
• 依托单位: DAKTARI DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-14 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200559
• 关键词: AIDS/HIV problem; Address; Africa; Antibodies; Binding; Biological Assay; Blood; Blood specimen; Buffers; CD4 Lymphocyte Count; Caring; Clinical; Communicable Diseases; Complex; Computer software; Country; Cytolysis; Data; Detection; Developed Countries; Developing Countries; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug resistance; Environment; Evaluation; Feasibility Studies; Freezing; Geometry; Gold; HIV; HIV Core Protein p24; HIV diagnosis; Health Personnel; Hepatitis C; Hepatitis C virus; Immunoassay; Individual; Infant; Label; Life; Magnetism; Marketing; Measures; Methods; Molecular; Molecular Biology; Monitor; Mothers; Patient Noncompliance; Patients; Performance; Persons; Phase; Point-of-Care Systems; Preparation; Production; Proteins; Protocols documentation; Quality of Care; Reagent; Resources; Risk; Sampling; Secure; Shipping; Ships; Silver; Site; System; Technology; Temperature; Testing; Time; Training; Treatment Efficacy; Treatment Failure; United States; Venous blood sampling; Viral Load result; Viral Proteins; Virion; Virus; Whole Blood; Work; antiretroviral therapy; base; case finding; cohort; cost; data exchange; design; field study; global health; innovation; instrument; low and middle-income countries; meetings; nanoparticle; operation; particle; point of care; point-of-care diagnostics; programs; prototype; resistant strain; tool; viral detection

Project Summary HIV remains a global health crisis: more than 1.2 million people died from the disease in 2014. While there has been significant progress in the last ten years—more than 15 million people currently receive effective antiretroviral therapy (ART) worldwide—our ability to monitor and provide high-quality care to HIV/AIDS patients is still limited, in part, by the available diagnostics tools. High-quality diagnostic tools are available in the U.S. and developed nations. However, while the advent of rapid HIV diagnostic tests (RDTs) has catalyzed an increase in case finding rates—ultimately increasing the number of people on ART—, low- and middle- income countries still suffer from the lack of simple, affordable diagnostic tests to monitor treatment efficacy, by measuring the amount of circulating virus. Determining viral load – the count of virions in the blood – is critical to the determination of treatment failure, due to either drug-resistant strains of virus or patient non-compliance. Viral load testing is also critical to diagnosing infants, for whom antibody tests are not sufficient. Most molecular biology-based tests (generally using PCR) are, to-date, complex and out of reach in most global-health settings. The Daktari HIV VL test will build upon Daktari's existing platform of POC diagnostic tests, which are designed for global health environments: rugged, portable, battery-powered, and with built-in connectivity for secure data transmission. Specifically, the Daktari HIV VL test will be based on Daktari's ViPr platform, designed to detect circulating proteins and virions directly from whole blood. The test requires a small volume of whole blood, which can be drawn via fingerstick by a minimally-trained healthcare worker, and returns results in 30 minutes, allowing clinical decisions to be made immediately. All the necessary sample preparation steps are incorporated into the automated assay, eliminating hands-on steps for the user. The first product in the Daktari ViPr platform is a Hepatitis C (HCV) Viral Load Test, which is well into the development phase. The principle of operation of the HIV Viral Load is analogous to that of the HCV product. First, HIV virions are extracted from whole blood using magnetic particles, which concentrate the purified virions into a small volume. Next, the virions are treated with lysis buffer to release the target analyte (p24 protein), and the amount of released p24 protein is quantified using a sandwich immunoassay that employs silver nanoparticles as the label. Finally, the amount of silver bound is measured using anodic stripping voltammetry. At this time, we have finished the proof-of-concept work to de-risk the program (ability to pull-down HIV virions from blood at high efficiency and a 1 fM limit of detection for p24) and have demonstrated feasibility of detecting and quantifying HIV virions from HIV samples. The HIV VL program is ready to enter the development phase. In this Direct-to-Phase-II proposal, we will first optimize HIV-specific reagents and will combine all assay steps to show that the assay provides the required limit of detection of 1,000 copies/mL when starting from whole blood samples. Next, we will integrate the HIV VL assay into the Daktari ViPr platform (cartridge and instrument). Finally, we will evaluate performance of the integrated HIV ViPr Viral Load system in a small field study at our Kenyan site. The final deliverable of this Phase II program is a prototype HIV Viral Load system capable of quantification of HIV viral loads directly from 50 µL of whole blood and with > 99% sensitivity to detect viral loads > 1,000 copies/mL, and a > 5% CV at 5,000 copies/mL.

项目摘要 艾滋病毒仍然是一种全球健康危机：2014年有超过120万人死于这种疾病。在那里有 在过去十年中取得了重大进展--目前有1500多万人获得有效的 全球抗逆转录病毒疗法(ART)-我们监测艾滋病毒/艾滋病并提供高质量护理的能力 在一定程度上，患者仍然受到现有诊断工具的限制。 美国和发达国家都有高质量的诊断工具。然而，虽然它的出现 艾滋病毒快速诊断测试(RDT)促进了病例发现率的上升-最终增加了 在艺术、低收入和中等收入国家，许多人仍然缺乏简单、负担得起的 通过测量传播的病毒量来监测治疗效果的诊断试验。确定病毒 负荷--血液中病毒粒子的计数--对于确定治疗失败至关重要，这是由于 病毒耐药株或患者不依从。病毒载量测试也是诊断 婴儿，他们的抗体测试是不够的。大多数基于分子生物学的检测(通常使用聚合酶链式反应) 到目前为止，在大多数全球健康环境中，这些问题都很复杂，而且遥不可及。 达克塔里艾滋病毒VL检测将建立在达克塔里现有的POC诊断测试平台的基础上，这些平台是 适用于全球健康环境：坚固耐用、便携、电池供电，并具有安全的内置连接 数据传输。具体地说，达克塔里艾滋病毒VL检测将基于达克塔里的VIPR平台，旨在 直接从全血中检测循环蛋白质和病毒粒子。这项测试需要一个小体积的整体 血液，可以由受过最低限度培训的医护人员通过手指提取，并在30分钟内返回结果 几分钟，允许立即做出临床决定。所有必要的样品制备步骤都是 整合到自动分析中，为用户省去了实际操作步骤。世界上第一个产品 达克塔里VIPR平台是一种丙型肝炎病毒(HCV)病毒负载测试，目前正处于开发阶段。 HIV病毒载量的工作原理类似于丙型肝炎病毒产品。首先，艾滋病毒病毒粒子是 使用磁性颗粒从全血中提取，将纯化的病毒粒子浓缩成一小部分 音量。接下来，用裂解缓冲液处理病毒粒子以释放目标分析物(p24蛋白)，并 P24蛋白的释放量是通过使用银纳米颗粒的三明治免疫分析来定量的 作为标签。最后，用阳极溶出伏安法测定了银键合量。在这个时候， 我们已经完成了降低该计划风险的概念验证工作(能够从 高效率的血液和对p24的检测极限为1 fM)，并证明了检测的可行性 以及从HIV样本中量化HIV病毒粒子。艾滋病毒VL方案已准备好进入开发阶段。 在这个直接到第二阶段的提案中，我们将首先优化HIV特异性试剂，并将所有检测步骤结合在一起 为了表明该检测方法提供了从整体开始时所需的1000拷贝/毫升的检测限值 血样。接下来，我们将把艾滋病毒VL检测整合到达克塔里VIPR平台(试剂盒和 仪器)。最后，我们将评估集成的HIV VIPR病毒负载系统在小范围内的性能 在我们的肯尼亚网站学习。这一第二阶段计划的最终交付成果是艾滋病毒病毒加载系统的原型 能够直接从50微米L的全血中定量检测艾滋病毒病毒载量，并具有99%的敏感性 检测病毒载量1,000拷贝/毫升，以5,000拷贝/毫升检测5%变异系数。