Pont-of-use Acute HIV Infection Diagnostic for Substance Using Populations

家长津贴：1DP2DA051910 家长奖助金标题：药物使用人群的使用点急性艾滋病毒感染诊断 副标题：扩大丙型肝炎病毒急性感染平台使用点 据估计，有230万艾滋病毒感染者与丙型肝炎病毒(丙型肝炎)合并感染。在美国，那些 使用药物(PWUD)是这两种感染的高风险。HIV加速丙型肝炎病毒引起的肝硬变，并导致 每年有350,000人死于丙型肝炎相关的肝病。不幸的是，现有的广泛使用的快速 丙型肝炎病毒的诊断试验(RDT)并不检测病毒本身，而只是检测产生的抗丙型肝炎病毒抗体 被感染者感染了。这种基于抗体的检测带来了两个挑战。首先，宿主抗体在很长一段时间后才会产生 初次接触丙型肝炎病毒，可使感染保持在未被检测到的状态，并有可能传播给其他人。 其次，即使在丙型肝炎病毒清除后，抗丙型肝炎病毒抗体仍然存在，并可能导致假阳性结果，而不是 区分当前感染和过去感染。相比之下，针对病毒本身的测试，如核酸 扩增试验(例如RT-PCR)有可能同时检测急性和慢性丙型肝炎病毒感染。然而，这些 分析通常需要冷链储存试剂、大量的样品准备和广泛的实验室 要运行的基础设施。虽然PWUD愿意在农村等非传统场所接受艾滋病毒和丙型肝炎病毒检测 药店(Duong等人，2019年)，目前基于实验室的RT-PCR检测无法接触到这些人 传统的医疗保健已经不能提供足够的服务。因此，迫切需要一种全新的方法来诊断丙型肝炎病毒。 在PWUD中，以便使感染者在感染的最早阶段参与连续护理并预防 丙型肝炎病毒的进展和传播。 在本增刊中，我们建议将我们目前的平台技术应用于急性HIV或急性丙型肝炎病毒的检测 在使用点设置中，阈值高于临床相关的3000国际单位/毫升丙型肝炎病毒(Reipold等人，2017年)。我们正在进行的DP2 是基于我们的手持式采样到回答技术，这是一种自动化的微流控快速自主分析设备 (Microraad)。我们已经证明，microraad能够直接从全血中检测出艾滋病毒病毒。 样本。我们将干燥的扩增试剂和蜡阀结合在纸基衬底上，并进行电阻加热。 元件和低功率电路。将RT-LAMP分析的反馈控制加热与蜡阀相结合 利用纸的毛细血管流动，我们组装的设备自动从人类血液中分离病毒颗粒，裂解病毒， 扩增HIV-1RNA，并将产物输送到具有熟悉的视觉侧向流动分析读数的检测区。 整个过程对用户来说只需要3个简单的步骤：首先，将一滴血添加到样本垫中，然后， 用户分配缓冲液，并且在短暂的等待时间之后，用户读取可视的是/否结果。我们有 证明了完全集成的microRAAD直接从全血中检测到低至10^5的HIV-1 VP/反应 不到90分钟。在正在进行的DP2中，我们正在与当地的PWUD人群和艾滋病毒服务提供者合作，以 评估该技术的可行性、可接受性和可用性，并进一步将检测结果降至10^3以下 通过将基于p24蛋白的靶向和RT-LAMP相结合来提高Vp/mL，并通过 集成了内部放大控制。 我们使MicroRAAD适应急性丙型肝炎检测的下一步措施将包括： 1)建立和优化针对基因组保守区的新型丙型肝炎病毒检测方法。用于丙型肝炎病毒检测 设计我们将比较5‘非编码区(5’-NCR)和3‘非编码区(3’非编码区)，它们都已经被 用于临床丙型肝炎病毒载量分析。虽然5‘-NCR用于西方分析，但众所周知，这一方法的可变性 该区域与世界各地流传的序列有些不同。因此，我们还将考虑3‘UTR用于 RT灯设计。我们将为每个区域至少产生两个引物，并比较扩增时间(TTA)， 检出限(从ATCC购买的合成RNA中添加0-10^7IU/毫升)，以及对全血和 在每个样本中加入的血浆浓度从0-20%不等。 2)扩大我们与PWUD及其服务提供商的社区参与性研究，以评估以丙型肝炎为重点的研究 在与艾滋病毒检测相一致的使用地点进行检测。这将包括执行的可行性和可接受性 评估PWUD在POU检测丙型肝炎病毒的具体操作、临床和市场需求的研究， 评估社会文化因素和系统性护理障碍对PWUD患者丙型肝炎病毒检测和整合的影响 并评估设备原型的可用性，以确定最佳操作方案 在利益相关者中。 这一点使用的丙型肝炎病毒/艾滋病毒检测平台可以改变艾滋病毒和丙型肝炎病毒的检测和治疗 通过将急性感染检测带到药物滥用治疗设施、针头交换和注射毒品 并将阐明消除护理障碍所需的针对性干预措施。 参考资料： 雷波德·IE、伊斯特布鲁克·P、特里安尼·A、潘尼尔·N、克拉科沃·D、翁加雷洛·S、罗伯茨·T、米勒·V、登金格·C。 优化丙型肝炎病毒感染的诊断：开发目标产品简介。骨髓基质细胞感染Dis。2017年 11月1日；17(补充1)：707。DOI：10.1186/s12879-0172770-5.PMID：29143620；PMC5688443。 首页--期刊主要分类--期刊细介绍--期刊题录与文摘-期刊详细文摘内容对药房丙型肝炎病毒/艾滋病毒检测的态度 肯塔基州农村地区的吸毒者。J乡村健康。2022年1月；38(1)：93-99。DOI：10.1111/jr.12564.EPub 2021年3月5日。 PMID：33666274；PMC8418619。

Parent Grant: 1DP2DA051910 Parent Grant Title: Point-of-use Acute HIV Infection Diagnostic for Substance Using Populations Supplement Title: Expansion of Point-of-use Acute HIV Infection platform for Hepatitis C Virus An estimated 2.3 million individuals with HIV infection are co-infected with hepatitis C (HCV). In the US, people who use drugs (PWUD) are at high risk for both infections. HIV accelerates liver cirrhosis caused by HCV and contributes to the 350,000 deaths that occur due to HCV-related liver disease each year. Unfortunately, existing widely used rapid diagnostic tests (RDTs) for HCV do not detect the virus itself but instead detect only the anti-HCV antibodies produced by the infected individual. This antibody-based detection presents two challenges. First, host antibodies develop long after initial exposure to HCV which can allow for infections to remain undetected and potentially spread to other individuals. Second, anti-HCV antibodies remain even after HCV clearance and can result in false positive results that do not differentiate a current infection from a past infection. In contrast, test targeting the virus itself, such as nucleic acid amplification tests (e.g. RT-PCR), have the potential to detect both acute and chronic HCV infections. However, these assays typically require cold-chain storage of reagents, significant sample preparation, and extensive laboratory infrastructure to run. While PWUD are willing to get tested for HIV and HCV at non-traditional sites such as rural pharmacies (Duong et al 2019), current laboratory-based RT-PCR tests are unable to reach these individuals who are already underserved by traditional healthcare. Thus, there is an urgent need for a radically new approach to diagnose HCV among PWUD in order to engage infected individuals in the care continuum at the earliest stages of infection and prevent progression and transmission of HCV. In this supplement, we propose to adapt our current platform technology for acute HIV or order to to detect acute HCV thresholds above the clinically relevant 3000 HCV IU/mL (Reipold et al 2017) at point-of-use settings. Our ongoing DP2 is based on our handheld sample-to-answer technology, an automated microfluidic rapid autonomous analytical device (microRAAD). We have demonstrated that microRAAD is capable of detecting HIV virons directly from a whole blood samples. We incorporated dried amplification reagents and wax valves in paper-based substrates with resistive heating elements and low-power circuitry. By combining feedback-controlled heating of the RT-LAMP assay and wax valves with paper’s capillary flow, our assembled device automatically isolates viral particles from human blood, lysis the virus, amplifies HIV-1 RNA, and transports products to a detection zone with familiar visual lateral flow assay readout. This entire process requires only 3 simple steps for the user: first, a drop of blood is added to the sample pad, next, the user dispenses a buffer solution, and after a brief waiting period, the user reads the visual yes/no result. We have demonstrated that the fully integrated microRAAD detects as few as 10^5 HIV-1 vp/reaction directly from whole blood in under 90 minutes. In the ongoing DP2, we are working with local populations of PWUD and HIV service providers to evaluate feasibility, acceptability, and usability of the technology and to further reduce this detection to below 10^3 HIV vp/mL by combining p24 protein-based targeting and RT-LAMP together as well as improving clinical relevance via integration of an internal amplification control. Our next steps to adapt MicroRAAD to acute HCV detection will include: 1) Develop and optimize a novel assay for HCV detection targeting the conserved regions of the genome. For HCV assay design we will compare the 5’non-coding region (5’-NCR) and the 3’untranslated region (3’UTR) which have both been used in clinical HCV viral load assays. While the 5’-NCR is used in western assays, it’s known that the variability in this region is somewhat divergent from the sequences circulating worldwide. Therefore, we will also consider the 3’UTR for RT-LAMP design. We will generate at least two primers to each region and will compare the time to amplification (TTA), limit of detection (spiking in 0-10^7 IU/mL of synthetic RNA purchased from ATCC), and resistance to whole blood and plasma spiked into each sample at concentrations ranging from 0-20%. 2) Expand our community-based participatory research with PWUD and their service providers to evaluate HCV-focused detection at the point-of-use in concert with HIV detection. This will include performing feasibility and acceptability studies to evaluate the operational, clinical, and market needs specific for HCV detection by and for PWUD at the POU, evaluate how socio-cultural factors and systemic barriers to care among PWUD impact their HCV testing and integration into the current HIV care continuum and assess usability of device prototypes to determine the best operational protocols among stakeholders. This platform for point-of-use HCV/HIV detection could transform both testing and treatment of HIV and HCV among PWUD by bringing acute infection detection to substance abuse treatment facilities, needle exchanges, and injection drug clinics and will elucidate the targeted interventions needed to remove barriers to care. References: Reipold IE, Easterbrook P, Trianni A, Panneer N, Krakower D, Ongarello S, Roberts T, Miller V, Denkinger C. Optimising diagnosis of viraemic hepatitis C infection: the development of a target product profile. BMC Infect Dis. 2017 Nov 1;17(Suppl 1):707. doi: 10.1186/s12879-017-2770-5. PMID: 29143620; PMCID: PMC5688443. Duong M, Delcher C, Freeman PR, Young AM, Cooper HLF. Attitudes toward pharmacy-based HCV/HIV testing among people who use drugs in rural Kentucky. J Rural Health. 2022 Jan;38(1):93-99. doi: 10.1111/jrh.12564. Epub 2021 Mar 5. PMID: 33666274; PMCID: PMC8418619.