Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa; Department of Molecular Medicine and Haematology, National Health Laboratory Services, Charlotte Maxeke Johannesburg Academic Hospital, South Africa.
J Virol Methods. 2013 Dec;194(1-2):300-7. doi: 10.1016/j.jviromet.2013.08.015. Epub 2013 Aug 28.
HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDR(ultralight)). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS EasyQ v1.2, Roche) with an R(2) of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDR(ultralight) assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays' compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.
HIV-1 RNA 病毒载量是监测抗逆转录病毒治疗 (ART) 期间病毒学失败的首选工具。及时发现病毒学失败可以降低 HIV-1 耐药性的流行率和复杂性。这项现场评估进一步描述了一种两步法来识别病毒学失败，作为评估 ART 依从性的一种手段，并检测 HIV-1 逆转录酶 (RT) 基因中的 HIVDR 突变。对 248 份样本进行了测试；其中 225 份来自南非参加 PharmAccess 非洲研究评估耐药性 (PASER) 队列的 HIV-1 参与者，其中 40 份有配对的干血斑 (DBS) 样本和 23 份 HIV-1 阴性样本。所有样本均使用新开发的病毒学失败检测法 (ARTA-VFA) 进行检测，病毒载量>5000 RNA 拷贝/ml 的样本使用缩短的 RT 方案进行基因分型，以检测 HIVDR（ARTA-HIVDR(ultralight)）。与商业参考检测法（NucliSENS EasyQ v1.2，罗氏）相比，ARTA-VFA 显示出良好的精密度和线性度，R(2)为 0.99。与参考方法相比，对血浆和 DBS ARTA-VFA 结果的准确性研究表明，标准偏差<1 log RNA 拷贝/ml。ARTA-VFA 的预期用途是提供定性结果，结果要么<或>5000 RNA 拷贝/ml。结果表明，对于血浆，无论使用 5000 RNA 拷贝/ml 还是 1000 RNA 拷贝/ml 作为截止值，结果<或>两者的比例均无显著差异，表明这两个截止值都有用。当使用 DBS 时，这些比例存在显著差异（P=0.0002），5000 RNA 拷贝/ml 作为截止值更合适。使用 5000 RNA 拷贝/ml 截止值时，ARTA-VFA 对血浆的灵敏度和特异性分别为 95%和 93%，对 DBS 的灵敏度和特异性分别为 91%和 95%。ARTA HIVDR(ultralight) 检测法对病毒载量>5000 RNA 拷贝/ml 的血浆和 DBS 样本可靠，扩增和测序成功率分别为 91%和 92%（血浆），80%和 95%（DBS）。血浆和 DBS 的 HIVDR 图谱与参考检测法完全一致。本研究评估了先前描述的两种检测法的组合，这些检测法在评估 HIV-1 病毒学失败和耐药性方面可能有用，与参考检测法具有良好的一致性。这些检测法操作简单，价格合理，是在某些资源有限的环境中检测病毒学失败的可行选择。这些检测法与 DBS 采样的兼容性扩展了 HIV-1 病毒学监测在更偏远环境中的应用。
