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，HIV 在感染者体内每天会产生 1-100 亿个新病毒颗粒。这种高病毒复制率，加上病毒逆转录酶无法纠正错误掺入的核苷酸，产生了抗逆转录病毒药物耐药性的突变。虽然 1995 年通过结合具有不同耐药机制的三种药物实现了对 HIV 复制的抑制，但当三种药物中的两种药物的细胞内水平减弱时，很容易选择耐药病毒。在成功的抗逆转录病毒治疗（ART）出现后不久，人们发现在获得耐药病毒的个体中会出现快速治疗失败。为了避免这种结果，在开始抗逆转录病毒治疗之前进行艾滋病毒耐药性检测已成为常规。 HIV 序列分析是检测的首选方法，每个样本的成本 > 500 美元。然而，在资源匮乏的社区，这一成本却令人望而却步。随着非洲传播的耐药性不断增加，需要进行经济的耐药性测试，以防止耐药性病毒破坏抗逆转录病毒治疗项目带来的健康成果并降低艾滋病毒传播率。我们采用了一种廉价的点突变测定法来检测赋予第一线抗逆转录病毒治疗艾滋病毒耐药性的突变，以便在资源匮乏的社区中使用。该检测扩增 HIV 聚合酶基因，连接寡核苷酸探针以特异性检测赋予耐药性的点突变，并以 EIA 板形式检测连接产物。在研究中，这种寡核苷酸连接测定 (OLA) 预测了泰国人和肯尼亚人第一线 ART 的病毒学失败。 OLA 在患者管理方面可能有效，但复杂的实验室程序一直是资源匮乏实验室采用的障碍。在这里，我们建议重新设计 OLA，使其执行起来快速且简单。我们的目标是系统地简化 OLA 程序，将测定时间从 8 个多小时减少到约 1 小时，并使其可供经过最低限度培训的实验室人员使用。我们组建了一个拥有所需专业知识的团队，通过以下修改简化 OLA：使用刺激响应试剂浓缩血液样本中的核酸。通过等温链置换扩增来扩增 HIV DNA。开发针对与第一线 ART 最相关的 HIV 密码子的“一锅”连接。开发一种简单快速的纸条测试，用于多重检测所有突变密码子。将重新设计的方案组合成快速且简化的套件（OLA-Simple）。 OLA-Simple 套件将通过对无意中接触 HIV 的个人以及妊娠晚期的妇女快速实施适当的 ART 预防措施，促进资源匮乏地区的 HIV 护理。最近的数据表明，治疗急性艾滋病毒感染可以预防持续感染，如果得到证实，将迫切需要一种快速检测方法来检测艾滋病毒耐药性，以确保采取适当的抗逆转录病毒疗法。