Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS One. 2011;6(7):e22019. doi: 10.1371/journal.pone.0022019. Epub 2011 Jul 20.
Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC-resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive "Amp-RT" assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.
对于开始接受抗逆转录病毒治疗（ART）的不断扩大的 HIV-1 感染者人群，特别是在资源有限的环境中，简单且具有成本效益的 HIV 耐药性检测方法是非常需要的。以核苷逆转录酶抑制剂（NRTI）为基础的方案，含有拉米夫定（3TC）或恩曲他滨（FTC），是首选的初始 ART 方案。这些药物组合的失败通常涉及到选择 NNRTI 和/或 3TC/FTC 耐药病毒。因此，获得简单的检测方法来测量这两种类型的耐药性是至关重要的。我们已经开发了一种高通量筛选试验，用于评估 HIV-1 RT 在血浆中对 3TC/FTC 和 NNRTIs 的酶抗性。该试验使用灵敏的“ Amp-RT”测定法，并采用新开发的实时 PCR 格式，在单次反应中同时筛选 3TC-三磷酸（3TC-TP）或奈韦拉平（NVP）的生化耐药性。根据对具有已知基因型特征的大量亚 B 型和非亚 B 型临床样本的检测，定义了检测的临界值。酶学 3TC 耐药性与 M184I/V 的存在密切相关，而 NVP 敏感性降低与 K103N、Y181C/I、Y188L 和 G190A/Q 的存在密切相关。在 M184V 样本中，检测耐药性的敏感性和特异性分别为 97.0%和 96.0%，在含有 NNRTI 突变的样本中，敏感性和特异性分别为 97.4%和 96.2%。我们还通过使用涂有 CD44 抗体的磁性珠进一步证明了 HIV 捕获方法在血浆中的实用性，该方法消除了对超速离心的需求。因此，我们的结果支持在临床样本中使用这种简单的方法来区分 WT 与 NNRTI 或 3TC/FTC 耐药病毒。这种酶学检测与亚型无关，可协助对不同人群，特别是资源有限环境中的人群进行临床管理。
