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

描述：据估计，艾滋病毒感染者每天会产生10-100亿个新的病毒颗粒。这种高病毒复制速度，再加上病毒逆转录酶无法纠正错误结合的核苷酸，产生了对抗逆转录病毒药物产生耐药性的突变。虽然抑制艾滋病毒复制是在1995年通过结合三种不同耐药机制的药物实现的，但当三种药物中的两种细胞内水平下降时，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，获得抗药性病毒的个人很快就会出现治疗失败。为了避免这一结果，在开始抗逆转录病毒治疗前进行艾滋病毒耐药性测试成为常规。艾滋病毒序列分析是检测的首选方法，每个样本的费用为500美元。然而，在资源匮乏的社区，这一成本令人望而却步。由于非洲传播的抗药性正在增加，需要一种经济的抗药性测试，以防止抗药性病毒破坏因抗逆转录病毒疗法计划而获得的健康成果和降低艾滋病毒传播率。我们已经改进了一种廉价的点突变检测方法，以检测对一线抗逆转录病毒药物具有抗药性的突变，用于资源匮乏的社区。该方法扩增HIV聚合酶基因，连接寡核苷酸探针以特异性检测导致耐药性的点突变，连接产物以EIA平板格式检测。在研究中，这种寡核苷酸连接分析(OLA)预测了泰国人和肯尼亚人一线抗逆转录病毒治疗的病毒学失败。OLA在患者管理中可能是有效的，但复杂的实验室程序一直是在低资源实验室采用的障碍。在这里，我们建议对OLA进行重新设计，使其执行起来既快速又简单。我们的目标是系统地简化OLA程序，将分析时间从8个多小时减少到大约一个小时，并使受过最低培训的实验室人员能够接触到它。我们已经组建了一个团队，拥有通过以下改进简化OLA所需的专业知识：使用刺激反应试剂从血液样本中浓缩核酸。等温链置换扩增HIV DNA。开发一种针对与一线抗逆转录病毒治疗最相关的艾滋病毒密码子的“一锅法”结扎。建立一种简便、快速的纸条检测方法，用于多重检测 都是突变密码子。将重新设计的协议合并到一个快速简化的套件中(OLA-SIMPLE)。OLA-SIMPLE试剂盒将通过允许对无意中接触艾滋病毒的个人和怀孕晚期妇女迅速实施适当的ART预防，促进资源匮乏地区的艾滋病毒护理。最近的数据表明，对急性艾滋病毒感染的治疗可以防止持续感染，如果得到证实，将迫切需要一种快速检测艾滋病毒耐药性的方法，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.