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

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、可靠性和重复性，用于最低限度的基础设施环境。这种检测应用于围产期HIV-1诊断，如果5%的目标人群能够提前获得治疗，每年可以节省18万个DALY，如果100%的人口能够获得治疗，每年可以节省多达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：缺乏对儿童艾滋病毒-1疾病(世卫组织)[1]的适当诊断和治疗。目前，量化HIV-1病毒载量的最敏感和最可靠的检测依赖于核酸扩增和检测，但这些检测往往需要复杂的仪器和昂贵的试剂。或者，寡核苷酸包裹的金纳米颗粒作为核酸测试的靶向/报告剂具有很高的分析灵敏度[2，3]。为了达到所需的灵敏度，我们将开发一种HIV-1病毒载量分析方法，该方法将HIV-1 RNA的等温PCR扩增与靶向金纳米颗粒的使用以及检测结果的比色量化相结合。我们将开发这种检测方法，用于低资源环境，对基础设施的要求最低，其灵敏度和特异性可与发达国家现有的商业病毒载量检测方法相媲美。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，用于在低资源环境下确定血清阳性患者的1型艾滋病毒病毒载量。该检测将结合靶标分离和等温扩增技术，从至少包含1000个病毒拷贝/毫升的样本中获得至少104倍的扩增。然后，将使用寡核苷酸靶向金纳米颗粒检测和定量读出进行检测，以在POC实现从103到106个HIV-1病毒拷贝/毫升的动态检测范围。(2)验证该方法用于临床标本中HIV-1病毒载量检测的有效性。与耶鲁医学院在HIV-1分子生物学方面拥有专业知识的Richard Sutton博士合作，我们将在NIH/联合国艾滋病规划署的参考小组上测试该检测方法检测整个病毒颗粒和M组分支的RNA的能力，并将该检测与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在艾滋病毒-1临床诊断方面拥有专业知识的伊丽莎白·莫利纽克斯博士合作，进行一项试点研究，以确定这种新检测方法的敏感性和特异性。莫利纽克斯博士团队的工作人员将前往休斯顿学习该检测方法，理查兹-科尔图姆实验室的成员将前往马拉维，与她的团队合作，对照干血斑点RT-PCR的黄金标准，对儿科临床样本中的检测方法进行评估。 公共卫生相关性：我们的目标是开发一种具有高灵敏度、特异性、可靠性和重复性的艾滋病毒病毒载量综合诊断测试，用于最低限度的基础设施环境中。需要病毒载量测定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。尽管世卫组织报告的低收入和中等收入国家扩大抗逆转录病毒治疗的趋势令人鼓舞，但在资源匮乏的情况下，尚未将可靠和准确的艾滋病毒负荷检测引入感染患者的管理，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18万个DALY，如果100%的人口能够获得治疗，每年可以节省多达250万个DALY[2]。

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].