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

描述：据估计，HIV每天在一个感染者体内产生10 - 100亿个新的病毒颗粒。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1995年通过联合使用具有不同耐药机制的三种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出现后不久，注意到在获得耐药病毒的个人中出现了治疗迅速失败的情况。为了避免这一结果，在开始抗逆转录病毒治疗之前进行艾滋病毒耐药性检测成为常规做法。HIV序列分析是检测的首选方法，每个样本需要500美元。然而，在资源贫乏的社区，这个成本是令人望而却步的。由于非洲传播的耐药性正在增加，需要一种经济的耐药性测试，以防止耐药病毒破坏抗逆转录病毒治疗项目带来的健康成果和降低艾滋病毒传播率。我们采用了一种廉价的点突变检测方法，用于资源贫乏的社区检测使艾滋病毒对一线抗逆转录病毒药物产生耐药性的突变。该检测扩增HIV聚合酶基因，连接寡核苷酸探针特异性检测赋予耐药性的点突变，并在EIA平板格式中检测连接产物。在研究中，这种寡核苷酸连接试验（OLA）预测了泰国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资源不足的实验室采用的障碍。在此，我们建议重新设计OLA，使其能够快速和简单地执行。我们的目标是系统地简化OLA程序，将分析时间从8小时以上减少到约1小时，并使受过最低培训的实验室人员也能使用。我们组建了一个专业团队，通过以下修改简化OLA所需的专业知识：使用刺激反应试剂从血液标本中浓缩核酸。用等温链置换扩增扩增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”结扎。开发一种简单快速的纸条测试，用于多路检测

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.