Point of care diagnosis of HIV1 viral load using nano reagents and isothermal PCR

使用纳米试剂和等温 PCR 进行 HIV1 病毒载量的护理诊断

• 批准号: 8060485
• 负责人: Rebecca R. Richards-Kortum
• 金额: $ 21.48万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-13 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8060485
• 关键词: Anti-Retroviral Agents; Biological Assay; Blood; Childhood; Clinical; Collaborations; Country; Cryptosporidium parvum; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Failure; Goals; Gold; HIV; HIV-1; Health Services Accessibility; Hospitals; Human Resources; Income; Learning; Malawi; Messenger RNA; Molecular Biology; Monitor; Nucleic Acid Amplification Tests; Nucleic Acids; Oligonucleotides; Optics; Parasites; Patients; Performance; Perinatal; Pilot Projects; Plasma; Population; RNA; Reading; Reagent; Relative (related person); Reporting; Reproducibility; Research Infrastructure; Resources; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Sensitivity and Specificity; Specimen; Spottings; Target Populations; Technology; Test Result; Testing; Therapeutic; Travel; United States National Institutes of Health; Universities; Viral; Viral Load result; Work; antiretroviral therapy; base; clinical Diagnosis; instrumentation; medical schools; member; nano; nanoparticle; particle; point of care; public health relevance; scale up; trend

DESCRIPTION (provided by applicant): Our goal is to develop an integrated diagnostic test for HIV-1 viral load with high sensitivity, specificity, reliability, and reproducibility for use in minimal infrastructure settings. Such a test, applied in perinatal HIV-1 diagnosis, could save 180,000 DALYs each year if 5% of the targeted population has early access to therapy, and up to 2.5 million DALYs each year if 100% of the population has access to treatment [2]. It would also help to overcome one of the major challenges to universal access to therapy: the lack of adequate diagnosis and treatment of pediatric HIV-1 disease (WHO) [1]. Currently the most sensitive and reliable assays to quantify HIV-1 viral load rely on nucleic acid amplification and detection, but these tests often require sophisticated instrumentation and expensive reagents. Alternatively, the high analytical sensitivity of oligonucleotide-coated gold nanoparticles as targeting/reporting agents in nucleic acid tests has been demonstrated [2, 3]. To achieve the required sensitivity, we will develop an HIV-1 viral load assay which integrates isothermal PCR amplification of HIV-1 RNA with the use of targeted gold nanoparticles and colorimetric quantification of test results. We will develop this assay for use in low - resource settings, with minimal infrastructure requirements and a sensitivity and specificity comparable to that of commercial viral load assays available in the developed world. The specific aims of the proposal are to: (1) Develop an inexpensive, sensitive, and specific diagnostic test for determining type 1 HIV viral loads of seropositive patients in low-resource settings. The assay will combine: target isolation and isothermal amplification technologies to yield at least 104 fold amplification from samples containing a minimum of 1000 viral copies/ml. An oligonucleotide-targeted gold nanoparticle detection assay and a quantitative read-out will be then used to detect to achieve a dynamic detection range from 103 to 106 HIV-1 viral copies/ml at the POC. (2) Validate the performance of this assay for HIV-1 viral load determination in clinical specimens. In collaboration with Dr. Richard Sutton from Yale School of Medicine who has expertise in molecular biology of HIV-1, we will test the ability of the assay to detect RNA from whole viral particles, and from group M clades on an NIH/UNAIDS reference panel, comparing the assay to RT-PCR. In collaboration with Dr. Elizabeth Molyneux from Queen Elizabeth Central Hospital, Blantyre, Malawi who has expertise in clinical diagnosis of HIV-1, we will carry out a pilot study to determine the sensitivity and specificity of this new assay. Personnel from Dr. Molyneux's team will travel to Houston to learn the assay, and members of the Richards-Kortum lab will travel to Malawi to work with her group to evaluate the assay in pediatric clinical samples relative to the gold standard of dried blood spot RT-PCR. PUBLIC HEALTH RELEVANCE: Our goal is to develop an integrated diagnostic test for HIV viral load with high sensitivity, specificity, reliability, and reproducibility for use in minimal infrastructure settings. Viral load determination is needed to determine when to initiate therapy, monitor compliance, and most importantly, as an early indicator of therapeutic failure. Despite the encouraging trends in the scale-up of antiretroviral treatment in low- and middle-income countries reported by the WHO, reliable and accurate HIV load testing has yet to be introduced into the management of infected patients in low resource settings and remains one of the major challenges to universal access to therapy [1]. Such a diagnostic test, applied in perinatal diagnosis, could save 180,000 DALYs each year if 5% of the targeted population has early access to therapy, and up to 2.5 million DALYs if 100% of the population has access to treatment [2].

描述（由申请人提供）：我们的目标是开发一种针对 HIV-1 病毒载量的综合诊断测试，具有高灵敏度、特异性、可靠性和可重复性，可在最低限度的基础设施环境中使用。这种应用于围产期 HIV-1 诊断的测试，如果 5% 的目标人群能够及早获得治疗，每年可以节省 180,000 DALY，如果 100% 的人群能够获得治疗，每年可以节省高达 250 万 DALYs [2]。它还将有助于克服普遍获得治疗的主要挑战之一：儿童 HIV-1 疾病缺乏充分的诊断和治疗 (WHO) [1]。目前，量化 HIV-1 病毒载量的最灵敏、最可靠的检测方法依赖于核酸扩增和检测，但这些检测通常需要复杂的仪器和昂贵的试剂。另外，寡核苷酸包被的金纳米粒子作为核酸测试中的靶向/报告剂的高分析灵敏度已得到证实 [2, 3]。为了达到所需的灵敏度，我们将开发一种 HIV-1 病毒载量测定方法，该方法将 HIV-1 RNA 的等温 PCR 扩增与使用靶向金纳米粒子和测试结果的比色定量相结合。我们将开发这种检测方法，用于资源匮乏的环境，具有最低的基础设施要求，其灵敏度和特异性与发达国家可用的商业病毒载量检测方法相当。该提案的具体目标是： (1) 开发一种廉价、灵敏且特异的诊断测试，用于确定资源匮乏地区血清反应阳性患者的 1 型 HIV 病毒载量。该检测将结合：目标分离和等温扩增技术，使至少含有 1000 个病毒拷贝/ml 的样品产生至少 104 倍的扩增。然后，将使用寡核苷酸靶向金纳米颗粒检测分析和定量读数进行检测，以在 POC 处实现 103 至 106 HIV-1 病毒拷贝/ml 的动态检测范围。 (2) 验证该测定法在临床样本中测定 HIV-1 病毒载量的性能。我们将与耶鲁大学医学院的 Richard Sutton 博士（他在 HIV-1 分子生物学方面具有专业知识）合作，测试该检测方法检测整个病毒颗粒以及 NIH/UNAIDS 参考小组中 M 组进化枝中 RNA 的能力，并将该检测方法与 RT-PCR 进行比较。我们将与马拉维布兰太尔伊丽莎白女王中心医院的 Elizabeth Molyneux 博士合作开展一项试点研究，以确定这种新检测方法的敏感性和特异性。Elizabeth Molyneux 博士拥有 HIV-1 临床诊断方面的专业知识。 Molyneux 博士团队的人员将前往休斯顿学习该检测方法，Richards-Kortum 实验室的成员将前往马拉维与她的团队合作，根据干血斑 RT-PCR 的黄金标准评估儿科临床样本中的检测方法。 公共卫生相关性：我们的目标是开发一种针对 HIV 病毒载量的综合诊断测试，具有高灵敏度、特异性、可靠性和可重复性，可在最低限度的基础设施环境中使用。需要确定病毒载量来确定何时开始治疗、监测依从性，最重要的是，作为治疗失败的早期指标。尽管世界卫生组织报告称，低收入和中等收入国家在扩大抗逆转录病毒治疗方面出现了令人鼓舞的趋势，但可靠、准确的艾滋病毒载量检测尚未引入资源匮乏地区的感染患者管理中，这仍然是普遍获得治疗的主要挑战之一[1]。这种应用于围产期诊断的诊断测试，如果 5% 的目标人群能够及早获得治疗，每年可以节省 180,000 DALY，如果 100% 的人群能够获得治疗，则可以节省高达 250 万 DALYs [2]。