PANDAA for rapid genotyping of HIV-1 infected patients failing protease inhibitor ART in resource-limited settings

2015年世卫组织指南建议对所有艾滋病毒感染者进行抗逆转录病毒治疗，并大量 到2020年，扩大抗逆转录病毒治疗的努力将导致3000万成年人和170万儿童接受抗逆转录病毒治疗，成本几乎为 530亿美元。因此，使用基于蛋白酶抑制剂(PI)的ART的替代疗法的患者数量 在一线治疗失败后，治疗方案也将增加。资源有限条件下基于PI的抗逆转录病毒治疗患者的研究 各国在治疗12个月和24个月时显示出很高的病毒学失败率，患者保持 失败的PI方案积累了阻碍当前和未来的耐药突变(DRM) 治疗方案。资源的限制往往限制了收集基因信息的能力，而且不受基因的影响 与根据耐药性对患者进行分层相比，药物转换已被证明是低成本的 基因分型。因此，迫切需要一种负担得起的艾滋病毒基因分型方案，供PI失败的患者选择。 以艺术为基础。集中基因分型可以通过以下方式显著加强患者护理：1)优化NRTI主干 在仅有NRTI抵抗的基于PI的方案失败的患者中；或2)在失败的患者中预防ART切换 没有抵抗，直到坚持的问题得到纠正。阿尔达图的泛简并放大与改编 (PANDAA™)技术是一种新型的点突变检测技术，能够实现廉价和高通量的聚焦 基因耐药检测，这样的方法可以为国家ART项目节省成本。PANDAA 通过消除次级多态来补偿患者内和患者间艾滋病毒基因组的高度变异性， 将它们对qPCR敏感性/特异性的影响降至最低，并首次启用qPCR进行艾滋病毒基因分型。 可行性研究表明，PANDAA：1)通过以下方式检测NNRTI和NRTI耐药HIV变种 &gt；99%的敏感度；2)不依赖于HIV亚型；3)可多路复用以同时量化耐药性 在多个基因组位置。Aldatu是PANDAA商业开发的先驱，并建立了 一种试剂配方，允许在耐热的样品中生产基于PANDAA的诊断方法. 准备好格式。在这个二期项目中，阿尔达图将把PANDAA技术应用到PANDAA的开发中 PIDR+，一种快速、低成本、耐热的检测方法，用于检测PI-ART失败患者的耐药性 方案，这可以从根本上改善低收入和中等收入国家的临床决策。通过 在这里提出的目标，我们将1)实验验证PANDAA试剂的设计，以量化突变 与蛋白酶抑制剂抗性有关，占病毒准种的10%；2)建立广泛的， PANDAA PIDR+验证的耐药和药物敏感HIV-1分离株协作熟练程度小组； 3)使用艾滋病毒耐药基因分型的既定业绩标准评估PANDAA PIDR+ 在GMP条件下生产PANDAA PIDR+；以及4)验证最终用户、多站点实施 PANDAA PIDR+具有高度的重复性。第一个此类产品是经过验证的、由GMP生产的PANDAA PIDR+检测试剂盒 来自Aldatu的公司将准备在这一快速增长的诊断市场机会中占据相当大的份额。

The 2015 WHO guidelines recommend anti-retroviral therapy (ART) for all HIV-infected people, and massive efforts to expand ART access will result in >30M adults and >1.7M children on ART by 2020, at a cost of almost US$53B. Consequently, the number of patients prescribed alternative, protease-inhibitor (PI)-based ART following failure of first-line regimens will also increase. Studies of patients on PI-based ART in resource-limited countries have shown high rates of virologic failure at 12 and 24 months on treatment, and patients maintained on failing PI-based regimens accumulate drug resistance mutations (DRMs) that hamper current and future treatment options. Resource constraints often limit the ability to collect genotype information, and genotype-blind drug switching has been shown to be cost-ineffective when compared to stratifying patients based on resistance genotype. Thus, there is an urgent unmet need for an affordable HIV genotyping option for patients failing PI- based ART. Focused genotyping could significantly enhance patient care by: 1) optimizing the NRTI backbone in patients failing a PI-based regimen with only NRTI resistance; or 2) preventing ART switches in patients failing without resistance until adherence issues are rectified. Aldatu’s Pan-Degenerate Amplification and Adaptation (PANDAA™) technology is a novel point mutation assay that enables inexpensive and high-throughput focused genotypic resistance testing, and such an approach could be cost-saving for national ART programs. PANDAA compensates for high intra- and inter-patient HIV genomic variability by removing secondary polymorphisms, minimizing their impact on qPCR sensitivity/specificity, and enabling qPCR for HIV genotyping for the first time. Feasibility studies have demonstrated that PANDAA: 1) detects NNRTI and NRTI-resistant HIV variants with >99% sensitivity; 2) is HIV subtype-independent; and 3) can be multiplexed to simultaneously quantify resistance at multiple genomic positions. Aldatu has pioneered the commercial development of PANDAA and established a reagent formulation that allows for the production of PANDAA-based diagnostics in a thermostable, sample- ready format. In this Phase II project, Aldatu will apply the PANDAA technology to the development of PANDAA PIDR+, a rapid, low-cost, thermostable test for detection of drug resistance in patients failing a PI-based ART regimen, which can radically improve clinical decision-making in low- and middle-income countries. Through the aims proposed here, we will 1) experimentally validate the design of PANDAA reagents to quantify mutations associated with protease inhibitor resistance comprising 10% of the viral quasispecies; 2) establish an extensive, collaborative proficiency panel of drug resistant and drug sensitive HIV-1 isolates for PANDAA PIDR+ validation; 3) assess PANDAA PIDR+ using established performance criteria for HIV drug resistance genotyping and produce PANDAA PIDR+ under GMP conditions; and 4) verify that end-user, multi-site implementation of PANDAA PIDR+ is highly reproducible. The first of its kind, a validated, GMP-produced PANDAA PIDR+ test kit from Aldatu will be poised to capture a significant share of this rapidly growing diagnostic market opportunity.