适用于资源有限环境的 HIV-1 逆转录酶、蛋白酶和整合酶基因的经济耐药性基因分型。
Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings.
机构信息
Department of Virology, School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal and National Health Laboratory Service, 800 Vusi Mzimela Road, Durban, 4058, South Africa.
Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa.
出版信息
AIDS Res Ther. 2023 Feb 9;20(1):9. doi: 10.1186/s12981-023-00505-3.
BACKGROUND
As use of dolutegravir (DTG) becomes more common in resource limited settings (RLS), the demand for integrase resistance testing is increasing. Affordable methods for genotyping all relevant HIV-1 pol genes (i.e., protease (PR), reverse transcriptase (RT) and integrase (IN)) are required to guide choice of future antiretroviral therapy (ART). We designed an in-house HIV-1 drug resistance (HIVDR) genotyping method that is affordable and suitable for use in RLS.
METHODS
We obtained remnant plasma samples from CAPRISA 103 study and amplified HIV-1 PR, RT and IN genes, using an innovative PCR assay. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. We genotyped samples by Sanger sequencing and assessed HIVDR mutations using the Stanford HIV drug resistance database. We compared drug resistance mutations with previous genotypes and calculated method cost-estimates.
RESULTS
From 96 samples processed, we obtained sequence data for 78 (81%), of which 75 (96%) had a least one HIVDR mutation, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, 18/78 (23%) had at least one discordant mutation, but only 2/78 (3%) resulted in different phenotypic predictions that could affect choice of subsequent regimen. All CAPRISA 103 study sequences were HIV-1C as confirmed by phylogenetic analysis. Of the 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method were 100% concordant with EQA panel results. Overall genotyping cost per sample was estimated at ~ US$43-$US49, with a processing time of ~ 2 working days.
CONCLUSIONS
We successfully designed an in-house HIVDR method that is suitable for genotyping HIV-1 PR, RT and IN genes, at an affordable cost and shorter turnaround time. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.
背景
随着多拉韦林（DTG）在资源有限环境（RLS）中的应用越来越普遍，对整合酶耐药检测的需求也在增加。需要能够负担得起的方法对所有相关的 HIV-1 pol 基因（即蛋白酶（PR）、逆转录酶（RT）和整合酶（IN））进行基因分型，以指导未来抗逆转录病毒治疗（ART）的选择。我们设计了一种内部 HIV-1 耐药性（HIVDR）基因分型方法，价格实惠，适用于 RLS。
方法
我们使用一种创新的 PCR 检测法，从 CAPRISA 103 研究中获得了残余血浆样本，并扩增了 HIV-1 PR、RT 和 IN 基因。我们使用外部质量评估（EQA）计划的残余血浆样本对该检测法进行了验证。我们通过 Sanger 测序对样本进行基因分型，并使用斯坦福 HIV 耐药性数据库评估 HIVDR 突变。我们将耐药性突变与之前的基因型进行比较，并计算了方法成本估算。
结果
从 96 个处理的样本中，我们获得了 78 个（81%）的序列数据，其中 75 个（96%）至少有一个 HIVDR 突变，未观察到主要的 IN 突变。只有一个样本有一个 E157Q INSTI 辅助突变。与之前的基因型相比，18/78（23%）有至少一个不一致的突变，但只有 2/78（3%）导致不同的表型预测，这可能影响随后治疗方案的选择。通过系统发生分析，所有 CAPRISA 103 研究序列均为 HIV-1C。在 7 个 EQA 样本中，4 个为 HIV-1C，2 个为 HIV-1D，1 个为 HIV-1A。使用 IDR 方法生成的基因型耐药数据与 EQA 面板结果 100%一致。每个样本的总体基因分型成本估计为 43-49 美元，处理时间约为 2 个工作日。
结论
我们成功设计了一种内部 HIVDR 方法，能够以负担得起的成本和更短的周转时间对 HIV-1 PR、RT 和 IN 基因进行基因分型。这种 HIVDR 基因分型方法适应了抗逆转录病毒治疗方案的变化，并将有助于指导 RLS 中的 HIV-1 治疗决策。
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