A rapid point-of-treatment diagnostic assay for HIV-resistance to 1st-line ART

HIV 对第一线 ART 耐药性的快速治疗点诊断测定

基本信息

批准号：
9266304
负责人：
Lisa M Frenkel
金额：
$ 45.33万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9266304
关键词：
AIDS prevention Acute Address Adoption Adult Advocate Africa Anti-Retroviral Agents Antiretroviral drug resistance Area Asia Biological Assay Blood Blood specimen Caring Cessation of life Chronic Client Clinic Clinic Visits Codon Nucleotides Communities Complex Consensus Sequence DNA Data Detection Drug resistance Drug usage Early treatment Engineering Ensure Failure Goals HIV HIV Infections HIV drug resistance HIV resistance Health Health Professional High Prevalence Hour Human immunodeficiency virus test Incidence Individual Infection Intervention Laboratories Laboratory Personnel Laboratory Procedures Laboratory Technicians Ligation Medical Methods Modification Mutation Nature New York City Nucleic Acids Nucleotides Oligonucleotide Probes Oligonucleotides Outcome Paper Patients Persons Pharmaceutical Preparations Point Mutation Polymerase Gene Population Pregnancy Procedures Prophylactic treatment Protocols documentation Reagent Resistance Resources Risk Rural Scientist Sequence Analysis Specimen Stimulus Technical Expertise Testing Time Transcriptase Treatment Failure Treatment outcome Variant Viral Viral Load result Virus Replication Woman aged clinically relevant cost diagnostic assay dideoxynucleotide drug resistant virus drug testing improved innovation laboratory experience multiplex detection mutant pandemic disease particle point of care prevent programs public health relevance rapid detection reproductive tract research study resistance mechanism screening standard of care transmission process treatment program virology

项目摘要

DESCRIPTION: HIV is estimated to produce 1-10 billion new viral particles per day in an infected individual. This high rate of viral replication, paired with an inability of the viral revrse transcriptase to correct misincorporated nucleotides, generates mutations that confer resistance to antiretroviral drugs. While suppression of HIV replication was achieved in 1995 by combining three drugs with different resistance mechanisms, drug-resistant viruses are readily selected when intracellular levels of two of the three drugs wane. Not long after the advent of successful antiretroviral treatment (ART), rapid treatment failure was noted to occur in individuals who acquired drug-resistant viruses. To avoid this outcome, testing for HIV-drug-resistance prior to initiation of ART became routine. HIV sequence analysis, which costs >$500/specimen, is preferred for testing. However, in the resource-poor communities, this cost is prohibitive. As transmitted drug resistance is increasing in Africa, an economical drug resistance test is needed to prevent drug-resistant viruses from undermining the health gains and reduced HIV transmission rates that have resulted from ART programs. We have adapted an inexpensive point mutation assay to detect mutations conferring HIV-drug-resistance to 1st-line ART for use in resource-poor communities. The assay amplifies the HIV polymerase gene, ligates oligonucleotide probes to specifically detect point mutations that confer drug resistance, and ligation products are detected in an EIA plate format. In research studies, this oligonucleotide ligation assay (OLA) has predicted virologic failure of 1st-line ART among Thais and Kenyans. OLA could be effective in patient management, but the complex laboratory procedures have been an obstacle to adoption in low-resource laboratories. Here, we propose to re-engineer the OLA so that it is rapid and simple to perform. Our goal is to systematically simplify the OLA procedure to reduce the assay time from 8+ hours to about an hour and make it accessible to minimally-trained laboratory personnel. We have assembled a team with the expertise needed to simplify OLA through the following modifications: Concentrate nucleic acids from blood specimens using stimuli-responsive reagents. Amplify HIV DNA by isothermal strand displacement amplification. Develop a "one-pot" ligation that targets the HIV codons most relevant to 1st-line ART. Develop a simple and rapid paper strip test for multiplexed detection of all mutant codons. Combine the re-engineered protocols into a rapid and simplified kit (OLA-Simple). The OLA-Simple kit will advance HIV care in resource-poor areas by allowing rapid implementation of appropriate ART prophylaxis for individuals inadvertently exposed to HIV, and to women in late pregnancy. Recent data suggest that treatment of acute HIV infection may prevent persistent infection, which if confirmed, would present compelling and urgent need for a rapid assay to detect HIV-drug-resistance testing to ensure the appropriate ART.

描述：据估计，艾滋病毒每天在感染者中每天产生1-1亿新的病毒颗粒。这种高的病毒复制速率与病毒RevRSE转录酶无法纠正核苷酸核苷酸的不足，产生了赋予对抗逆转录病毒药物的耐药性的突变。虽然1995年通过将三种具有不同耐药机制的药物结合使用，但在三种药物的细胞内水平减弱时，很容易选择抗HIV复制的抑制。成功的抗逆转录病毒治疗（ART）出现后不久，在获得耐药性病毒的个体中会发现快速治疗失败。为了避免这种结果，在启动艺术之前对HIV毒品抗性进行测试成为常规。 HIV序列分析的费用> $ 500/样品，是测试的首选。但是，在资源贫乏的社区中，这一成本是艰巨的。随着非洲传播耐药性的增加，需要进行经济的耐药性测试，以防止抗药性病毒破坏健康增长，并降低由艺术计划导致的HIV传播率。我们已经适应了廉价的点突变测定法，以检测突变，将HIV-Prug抗性赋予一线艺术，以用于资源贫乏的社区。该测定方法放大了HIV聚合酶基因，将寡核苷酸探针绑扎以特异性检测赋予耐药性的点突变，并以EIA板格式检测到结扎产物。在研究中，这种寡核苷酸连接测定法（OLA）预测了泰国人和肯尼亚人中一线艺术的病毒学衰竭。 OLA可能在患者管理方面有效，但是复杂的实验室程序是在低资源实验室中采用的障碍。在这里，我们建议重新设计OLA，以使其快速且易于执行。我们的目标是系统地简化OLA程序，以将测定时间从8个小时减少到大约一个小时，并使最低限度训练的实验室人员可以使用。我们已经组装了一个通过以下修饰来简化OLA所需的专业知识的团队：使用刺激性反应试剂从血液样本中浓缩核酸。通过等温链位移扩增扩增HIV DNA。开发针对与一线艺术最相关的艾滋病毒密码子的“一锅”连接。开发简单而快速的纸带测试，以进行多路复用检测所有突变密码子。将重新设计的协议合并为快速而简化的套件（OLA-SIMPLE）。 Ola-simple套件将通过允许对无意间暴露于HIV的个体以及怀孕后期妇女的个人快速实施适当的ART预防，以促进资源贫乏的地区的艾滋病毒护理。最近的数据表明，急性艾滋病毒感染的治疗可能会阻止持续性感染，如果得到证实，将出现迫切且迫切需要进行快速测定以检测HIV-Crug耐药性测试以确保适当的ART。