Simple Method for Screening of HIV Drug Resistance in Resource-Limited Settings

摘要 艾滋病毒感染的抗逆转录病毒治疗的耐药性是一个严重的临床问题，没有成本效益 低收入和中等收入国家(LMIC)的解决方案，这些国家的标准基因耐药分析 买不起。Discidium的目标是开发一种使用冷冻干燥反应物的单管检测方法，即标准PCR 和管内读数，可用作筛选阻力测试，并在最低限度的实验室 基础设施。我们已经开发了一种Taq聚合酶，它绝对需要终端3的碱基匹配，与 与抗性相关的多态相匹配的合适的引物，形成了低成本等位基因的基础-- 特异性聚合酶链式反应耐药性检测。这项检测最大限度地揭示了一线抗性突变的存在。 常见的NRTI，强调了在LMICs治疗失败的绝大多数个体中的耐药性。我们有 我还开发了一种荧光方法，允许在蓝光下直接显示PCR产物 消息来源。因此，该分析是单步进行的，因为扩增和读出在同一试管中进行。我们的 初步数据支持使用这种检测方法来定位最常见的导致抗药性的突变。 ARTS(K103N用于NNRTI和M184V用于NRTI)，我们建议优化该检测方法在 未处理的血液，并严格验证使用携带K103N和 M184V突变。这种检测方法有可能被开发成一套完整的试剂盒，供医护人员使用 可用于在LMIC中进行HIV-1耐药性测试，以指导对HIV-1感染者的最佳管理 个人。

Abstract Drug resistance to antiretroviral therapy for HIV infection is a serious clinical problem without cost-effective solutions in low and middle-income countries (LMICs) where standard genotypic resistance assays are unaffordable. Discidium's goal is to develop a single tube assay that uses lyophilized reactants, standard PCR and in-tube readout, that can be used as a screening resistance test and in laboratories with minimal infrastructure. We have exploited a Taq polymerase that absolutely requires a terminal 3´ base match that, with appropriate primers matched to resistance-associated polymorphisms, forms the basis for a low-cost allele- specific PCR resistance assay. This assay reveals the presence of first line resistance mutations to the most common NRTI that underscore resistance in the vast majority of individuals failing therapy in LMICs. We have also developed a fluorescence approach that allows direct visualization of PCR products under a blue light source. As such, the assay is single-step in that amplification and readout occur in the same tube. Our preliminary data supports the use of this assay to target the most common mutations that confer resistance to ARTs (K103N for NNRTIs and M184V for NRTIs), and we propose to optimize the use of this assay in unprocessed blood, and rigorously validate the assay performance using HIV-1 harboring the K103N and M184V mutations. This assay has the potential to be developed into a complete kit that health care workers can use to perform HIV-1 resistance testing in LMICs to guide optimal management of HIV-1-infected individuals.