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.

摘要 抗逆转录病毒治疗HIV感染的耐药性是一个严重的临床问题，没有成本效益 在低收入和中等收入国家（LMIC），标准基因型耐药检测 负担不起Applodium的目标是开发一种使用冻干反应物的单管检测方法， 和管内读数，可用作筛选抗性测试，并在实验室中使用最小 基础设施演进我们已经开发了一种Taq聚合酶，它绝对需要末端3 '碱基匹配， 与抗性相关多态性匹配的合适引物，形成了低成本等位基因的基础， 特异性PCR抗性测定。该检测揭示了一线耐药突变的存在， 常见的NRTI，强调了LMIC中绝大多数治疗失败的个体的耐药性。我们有 我还开发了一种荧光方法，可以在蓝光下直接观察PCR产物 源头因此，该测定是单步的，因为扩增和读出发生在同一管中。我们 初步数据支持使用该测定来靶向最常见的突变， ART（K103 N用于NNRTI，M184 V用于NRTI），我们建议优化该检测方法在 未经处理的血液，并严格验证检测性能使用HIV-1携带K103 N和 M184 V突变。这种检测方法有可能被开发成一个完整的试剂盒， 可用于在中低收入国家进行HIV-1耐药性检测，以指导HIV-1感染者的最佳管理 个体