García Lerma J, Schinazi R F, Juodawlkis A S, Soriano V, Lin Y, Tatti K, Rimland D, Folks T M, Heneine W
HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Antimicrob Agents Chemother. 1999 Feb;43(2):264-70. doi: 10.1128/AAC.43.2.264.
Monitoring for lamivudine (3TC) resistance is important both for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of transmission of 3TC-resistant HIV-1. We developed a novel non-culture-based assay for the rapid analysis of phenotypic resistance to 3TC of HIV-1 in plasma. The assay measures the susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT assay, an ultrasensitive PCR-based RT assay. Under our assay conditions, we found that 5 microM 3TC-TP inhibited RT activity from wild-type (WT), zidovudine-resistant, or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184V mutation or multidrug (MD) resistance mutations (77L/116Y/151M or 62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold of 10% 3TC-resistant virus (M184V) in a background of WT HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC was tested for evidence of phenotypic resistance by the Amp-RT assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 samples had evidence of phenotypic resistance. All 12 samples with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved before treatment with 3TC. In contrast, all 18 specimens with 3TC-resistant RT were posttherapy samples. This assay provides a simple, rapid, and reliable method for the detection of phenotypic resistance of HIV-1 to 3TC in plasma.
监测拉米夫定（3TC）耐药性对于接受3TC治疗的1型人类免疫缺陷病毒（HIV-1）感染患者的临床管理以及监测3TC耐药HIV-1的传播都很重要。我们开发了一种新型的非培养检测方法，用于快速分析血浆中HIV-1对3TC的表型耐药性。该检测方法测量血浆中HIV-1逆转录酶（RT）活性对三磷酸3TC（3TC-TP）的敏感性。RT检测通过Amp-RT检测法进行，这是一种基于超灵敏PCR的RT检测法。在我们的检测条件下，我们发现5 microM 3TC-TP可抑制野生型（WT）、齐多夫定耐药或奈韦拉平耐药HIV-1的RT活性，但不能抑制携带M184V突变或多药（MD）耐药突变（77L/116Y/151M或62V/75I/77L/116Y/151M）的HIV-1的RT活性。混合实验显示，在WT HIV-1背景下，检测3TC耐药病毒（M184V）的阈值为10%。为了验证该检测方法用于检测血浆样本中HIV-1对3TC的表型耐药性，通过Amp-RT检测法对15例患者在接受3TC治疗前和治疗期间采集的30份血浆标本中的HIV-1 RT进行了表型耐药性检测。将结果与基因型分析结果进行比较。发现12份样本中的RT对3TC敏感，而18份样本中的RT有表型耐药的证据。所有12份RT对3TC敏感的样本在密码子184处具有WT基因型，且是在接受3TC治疗前采集的。相比之下，所有18份RT对3TC耐药的标本均为治疗后样本。该检测方法为检测血浆中HIV-1对3TC的表型耐药性提供了一种简单、快速且可靠的方法。
