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: 7772573
• 负责人: Rebecca R. Richards-Kortum
• 金额: $ 18.67万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7772573
• 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; Universities; Viral; Viral Load result; Work; antiretroviral therapy; base; clinical Diagnosis; instrumentation; medical schools; member; nano; nanoparticle; particle; pediatric human immunodeficiency virus; 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用于HIV-1病毒载量的综合诊断测试，具有高灵敏度，特异性，可靠性和重现性，可用于最小的基础设施设置。这种检测应用于围产期HIV-1诊断，如果5%的目标人群能够抢先体验治疗，每年可节省180，000个DALY，如果100%的人口能够获得治疗，每年可节省高达250万个DALY [2]。它还将有助于克服普遍获得治疗的主要挑战之一：缺乏对儿科HIV-1疾病的充分诊断和治疗（世卫组织）[1]。目前，最敏感和可靠的测定，以量化HIV-1病毒载量依赖于核酸扩增和检测，但这些测试往往需要复杂的仪器和昂贵的试剂。或者，已经证明了在核酸测试中作为靶向/报告剂的雷帕霉素包被的金纳米颗粒的高分析灵敏度[2，3]。为了达到所需的灵敏度，我们将开发一种HIV-1病毒载量检测方法，该方法将HIV-1 RNA的等温PCR扩增与靶向金纳米颗粒的使用和检测结果的比色定量相结合。我们将开发这种检测方法用于低资源环境，具有最低的基础设施要求，灵敏度和特异性与发达国家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、敏感和特异的诊断测试，用于在资源匮乏的环境中确定血清阳性患者的1型艾滋病毒载量。该试验将结合联合收割机：靶向分离和等温扩增技术，以从含有最少1000个病毒拷贝/ml的样品中产生至少104倍扩增。然后，将使用靶向抗逆转录病毒肽的金纳米颗粒检测试验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)验证该检测试剂盒用于临床标本中HIV-1病毒载量测定的性能。在与耶鲁大学医学院的Richard萨顿博士（他在HIV-1分子生物学方面具有专长）的合作中，我们将测试该检测试剂盒检测整个病毒颗粒以及NIH/UNAIDS参考面板上M组分支RNA的能力，并将该检测试剂盒与RT-PCR进行比较。我们将与马拉维布兰太尔伊丽莎白女王中央医院的伊丽莎白·莫利纽克斯博士合作，开展一项试点研究，以确定这种新检测方法的灵敏度和特异性。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一起评估儿科临床样本中相对于干血斑RT-PCR金标准的检测方法。 公共卫生关系：我们的目标是开发一种用于HIV病毒载量的综合诊断测试，具有高灵敏度，特异性，可靠性和重现性，可用于最小的基础设施环境。需要测定病毒载量，以确定何时开始治疗，监测依从性，最重要的是，作为治疗失败的早期指标。尽管世卫组织报告说，在中低收入国家扩大抗逆转录病毒治疗的趋势令人鼓舞，但在资源匮乏的环境中，尚未将可靠和准确的艾滋病毒载量检测引入受感染患者的管理，这仍然是普及治疗的主要挑战之一[1]。如果5%的目标人群能够抢先体验治疗，这种应用于围产期诊断的诊断测试每年可以节省180，000丹麦克朗，如果100%的人群能够获得治疗，则可以节省多达250万丹麦克朗[2]。