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

项目摘要 艾滋病毒仍然是一个全球性的健康危机：2014年有120多万人死于这种疾病。虽然已经 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 抗逆转录病毒疗法（ART）在全球范围内-我们监测和提供高质量的艾滋病毒/艾滋病护理的能力 患者仍然部分地受到可用诊断工具的限制。 美国和发达国家都有高质量的诊断工具。然而，尽管 快速艾滋病毒诊断测试（RDTs）促进了病例发现率的提高， 在接受抗逆转录病毒治疗的中低收入国家， 通过测量循环病毒的数量来监测治疗效果的诊断测试。确定病毒 载量-血液中病毒粒子的计数-对于确定治疗失败至关重要， 耐药病毒株或患者不依从。病毒载量检测对诊断也至关重要 婴儿，对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PCR） 迄今为止，在大多数全球卫生环境中，这些问题都很复杂，而且遥不可及。 Daktari HIV VL检测将建立在Daktari现有的POC诊断检测平台之上， 适用于全球健康环境：坚固、便携、电池供电，并具有内置连接， 数据传输具体而言，Daktari HIV VL检测将基于Daktari的ViPr平台，旨在 直接从全血中检测循环蛋白质和病毒体。该测试需要少量的整体 血液，这可以通过手指针刺由一个最低限度的培训医护人员，并返回结果在30 几分钟，让临床决策立即作出。所有必要的样品制备步骤如下： 将其并入自动化测定中，消除了用户的动手步骤。中的第一产品 Daktari ViPr平台是一种丙型肝炎（HCV）病毒载量检测，已进入开发阶段。 HIV Viral Load的工作原理与HCV产品类似。首先，HIV病毒体是 使用磁性颗粒从全血中提取，其将纯化的病毒体浓缩成小的 音量.接下来，用裂解缓冲液处理病毒体以释放靶分析物（p24蛋白），并将靶分析物（p24蛋白）与裂解缓冲液混合。 使用采用银纳米颗粒的夹心免疫测定来定量释放的p24蛋白的量 作为标签。最后，使用阳极溶出伏安法测量结合的银的量。此时， 我们已经完成了概念验证工作，以降低该计划的风险（从 血液在高效率和1 fM的检测限为p24），并已证明的可行性，检测 和定量来自HIV样品的HIV病毒体。HIV VL计划已准备好进入开发阶段。 在这个直接到第二阶段的建议中，我们将首先优化艾滋病毒特异性试剂，并将联合收割机的所有检测步骤 以表明当从整个样品开始时，该检测试剂盒可提供1，000拷贝/mL的所需检测限 血液样本接下来，我们将HIV VL检测试剂盒整合到Daktari ViPr平台（检测盒和 仪器）。最后，我们将在小范围内评价集成HIV ViPr Viral Load系统的性能 在我们的肯尼亚网站学习。第二阶段项目的最终成果是一个HIV病毒载量系统的原型 能够直接从50 µL全血中定量HIV病毒载量，灵敏度> 99%， 检测病毒载量> 1，000拷贝/mL，5，000拷贝/mL时CV> 5%。

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.