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

在资源有限的环境中筛查 HIV 耐药性的简单方法

• 批准号: 10384759
• 负责人: Mario Stevenson
• 金额: $ 30万
• 依托单位: DISCIDIUM BIOSCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-17 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384759
• 关键词: Address; Alleles; Anti-Retroviral Agents; Antiretroviral drug resistance; Antiretroviral resistance; Biological Assay; Blood; Caring; Clinical; Cold Chains; Complementary DNA; Data; Detection; Development; Encapsulated; False Negative Reactions; Fluorescence; Fluorescent Probes; Freeze Drying; Genes; Genetic Polymorphism; Genome; Genotype; Goals; Gold; HIV; HIV Infections; HIV drug resistance; HIV resistance; HIV therapy; HIV-1; HIV-1 drug resistance; HIV-1 protease; Health Personnel; Human immunodeficiency virus test; Individual; Infection; Infrastructure; Interruption; Label; Laboratories; Lamivudine; Light; Methods; Modification; Mutation; Nucleosides; Peptide Hydrolases; Performance; Persons; Pharmaceutical Preparations; Phase; Plasma; Point Mutation; Polymerase; Polymers; RNA; RNA purification; RNA-Directed DNA Polymerase; Reaction; Reagent; Recommendation; Regimen; Research Priority; Resistance; Resource-limited setting; Reverse Transcription; Sampling; Screening procedure; Source; Testing; Treatment outcome; Tube; United States National Institutes of Health; Viral; Visualization; Vulnerable Populations; Whole Blood; base; commercialization; cost; cost effective; design; efavirenz; improved; inhibitor; low and middle-income countries; mutant; non-nucleoside reverse transcriptase inhibitors; novel strategies; particle; reconstitution; resistance mutation; resistance to lamivudine; screening; tool; treatment optimization; viral RNA

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.

抽象的 对HIV感染的抗逆转录病毒疗法的耐药性是一个严重的临床问题，没有成本效益 标准基因型抗性测定的低收入和中等收入国家（LMIC）的解决方案 负担不起。原先的目标是开发使用冻干反应物，标准PCR的单管测定法 和管中的读数，可以用作筛查测试，在实验室中 基础设施。我们已经探索了一种绝对需要3端匹配的TAQ聚合酶， 与抗性相关的多态性相匹配的适当引物构成了低成本等位基因的基础 特定的PCR抗性测定法。该测定法揭示了第一线阻力突变的存在 在LMICS中绝大多数人失败的个体中强调抗性的常见NRTI。我们有 还开发了一种荧光方法，允许在蓝光下直接可视化PCR产品 来源。因此，该测定是单步，在同一管中发生放大和读数。我们的 初步数据支持使用该测定法来针对会议阻力的最常见突变 Arts（用于NNRTIS的K103N和NRTIS的M184V），我们建议优化该测定法 未经处理的血液，并使用HIV-1携带K103N和 M184V突变。该测定有可能发展成一个完整的套件，医疗保健工作者 可以用来在LMIC中执行HIV-1耐药性测试，以指导HIV-1感染的最佳管理 个人。