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

HIV估计每天在受感染的个体中产生10 - 100亿个新病毒颗粒。这种高病毒复制率，加上病毒逆转录酶转录酶无法纠正错误掺入的核苷酸，产生了对抗逆转录病毒药物产生耐药性的突变。虽然在1995年通过结合三种具有不同耐药机制的药物实现了对艾滋病毒复制的抑制，但当三种药物中的两种的细胞内水平下降时，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，人们注意到在获得耐药病毒的个体中发生了快速治疗失败。为了避免这种结果，在开始抗逆转录病毒疗法之前检测艾滋病毒耐药性成为常规。艾滋病毒序列分析，其成本> 500美元/标本，是首选的测试。然而，在资源贫乏的社区，这一费用令人望而却步。随着非洲传播的耐药性不断增加，需要一种经济的耐药性测试，以防止耐药性病毒破坏健康成果，并降低ART计划导致的艾滋病毒传播率。我们采用了一种廉价的点突变检测方法来检测对一线抗逆转录病毒疗法产生耐药性的突变，以用于资源匮乏的社区。该检测试剂扩增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，并在EIA平板中检测连接产物。在研究中，这种寡核苷酸连接试验（奥拉）预测了泰国人和肯尼亚人中一线ART的病毒学失败。奥拉在病人管理方面可能是有效的，但复杂的实验室程序一直是资源匮乏的实验室采用的障碍。在这里，我们建议重新设计的奥拉，使它是快速和简单的执行。我们的目标是系统地简化奥拉程序，将测定时间从8小时以上缩短至约1小时，并使经过最低限度培训的实验室人员也能使用。我们已经组建了一个团队，他们拥有通过以下修改简化奥拉所需的专业知识：使用刺激响应试剂浓缩血液标本中的核酸。 通过等温链置换扩增来扩增HIV DNA。 开发针对与一线ART最相关的HIV密码子的“一锅法”连接。 都是突变密码子 联合收割机将重新设计的方案组合成一个快速简化的工具包（OLA-Simple）。OLA-Simple工具包将促进资源贫乏地区的艾滋病毒护理，使无意中接触艾滋病毒的个人和怀孕后期的妇女能够迅速实施适当的抗逆转录病毒疗法预防。最近的数据表明，治疗急性艾滋病毒感染可能会防止持续感染，如果得到证实，将提出迫切需要一个快速检测艾滋病毒耐药性测试，以确保适当的抗逆转录病毒治疗。

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.