International Laboratory Branch, Division of Global HIV and Tuberculosis (TB), Center for Global Health, Centers for Disease Control and Preventiongrid.416738.f (CDC), Atlanta, Georgia, USA.
CDC Kenya, Nairobi, Kenya.
Microbiol Spectr. 2022 Jun 29;10(3):e0177621. doi: 10.1128/spectrum.01776-21. Epub 2022 May 5.
As dolutegravir (DTG)-containing HIV regimens are scaled up globally, monitoring for HIV drug resistance (HIVDR) will become increasingly important. We designed a partially multiplexed HIVDR assay using Sanger sequencing technology to monitor HIVDR mutations in the protease, reverse-transcriptase (PRRT), and integrase (INT). A total of 213 clinical and analytical plasma and dried blood spot (DBS) samples were used in the evaluation. The assay detected a wide range of known HIV-1 subtypes and circulating recombinant forms (CRFs) of group M from 139 samples. INT accuracy showed that the average nucleotide (nt) sequence concordance was 99.8% for 75 plasma samples and 99.5% for 11 DBS samples compared with the reference sequences. The PRRT accuracy also demonstrated the average nucleotide sequence concordance was 99.5% for 57 plasma samples and 99.2% for 33 DBS samples. The major PRRT and INT DR mutations of all samples tested were concordant with those of the reference sequences using the Stanford HIV database (db). Amplification sensitivity of samples with viral load (VL) >5000 copies/mL showed plasma exceeded 95% of positivity, and DBS exceeded 90% for PRRT and INT. Samples with VL (1000 to 5000 copies/mL) showed plasma exceeded 90%, and DBS reached 88% positivity for PRRT and INT. Assay precision and reproducibility showed >99% nucleotide sequence concordance in each set of replicates for PRRT and INT. In conclusion, this HIVDR assay met WHO HIVDR assay performance criteria for surveillance, worked for plasma and DBS, used minimal sample volume, was sensitive, and was a potentially cost-effective tool to monitor HIVDR mutations in PRRT and INT. This HIVDR genotyping assay works for both plasma and DBS samples, requires low sample input, and is sensitive. This assay has the potential to be a user-friendly and cost-effective HIVDR assay because of its partially multiplexed design. Application of this genotyping assay will help HIVDR monitoring in HIV high-burdened countries using a DGT-based HIV drug regimen recommended by the U.S. President's Emergency Plan for AIDS Relief and the WHO.
随着含有多替拉韦的艾滋病毒治疗方案在全球范围内扩大使用，监测艾滋病毒耐药性（HIVDR）将变得越来越重要。我们设计了一种使用 Sanger 测序技术的部分多重 HIVDR 检测方法，以监测蛋白酶、逆转录酶（PRRT）和整合酶（INT）中的 HIVDR 突变。共有 213 份临床和分析血浆及干血斑（DBS）样本用于评估。该检测方法能够检测来自 139 个样本的广泛的已知 HIV-1 亚型和 M 组的循环重组形式（CRF）。INT 准确性表明，与参考序列相比，75 份血浆样本的平均核苷酸（nt）序列一致性为 99.8%，11 份 DBS 样本的平均核苷酸序列一致性为 99.5%。PRRT 准确性也表明，57 份血浆样本的平均核苷酸序列一致性为 99.5%，33 份 DBS 样本的平均核苷酸序列一致性为 99.2%。使用斯坦福 HIV 数据库（db）对所有测试样本的主要 PRRT 和 INT DR 突变进行检测，结果与参考序列一致。对于病毒载量（VL）>5000 拷贝/mL 的样本，扩增灵敏度显示血浆的阳性率超过 95%，而 PRRT 和 INT 的 DBS 阳性率超过 90%。VL（1000 至 5000 拷贝/mL）的样本显示，血浆的阳性率超过 90%，而 PRRT 和 INT 的 DBS 阳性率达到 88%。检测精度和重现性表明 PRRT 和 INT 的每个重复集的核苷酸序列一致性均超过 99%。总之，该 HIVDR 检测方法符合世卫组织 HIVDR 检测方法性能标准，适用于监测，可用于血浆和 DBS，样本用量少，灵敏度高，是一种具有成本效益的监测 PRRT 和 INT 中 HIVDR 突变的工具。该 HIVDR 基因分型检测方法可同时用于血浆和 DBS 样本，所需样本量低，灵敏度高。由于其部分多重设计，该检测方法具有成为一种用户友好且具有成本效益的 HIVDR 检测方法的潜力。应用该基因分型检测方法将有助于使用美国总统艾滋病紧急救援计划和世卫组织推荐的基于多替拉韦的 HIV 药物方案，在艾滋病毒负担沉重的国家监测 HIVDR。
