Selection and persistence of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 in patients starting and stopping non-nucleoside therapy
Selection and persistence of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 in patients starting and stopping non-nucleoside therapy
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DOI：
10.1097/01.aids.0000216370.69066.7f
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发表时间：
2006-03-21
期刊：
影响因子：
3.8
通讯作者：
Coffin, JM
中科院分区：
文献类型：
--
作者：
Palmer, S;Boltz, V;Coffin, JM
背景资料：了解耐药HIV-1变异体的选择和衰减对于设计最佳抗逆转录病毒治疗非常重要。目的：开发一种高通量、实时逆转录酶（RT）聚合酶链反应（PCR）检测方法，用于定量非核苷类逆转录酶抑制剂（NNRTI）耐药突变体K103 N（AAT或AAC等位基因）的频率低至0.1%，并应用此监测这些变异之前，期间和之后NNRTI治疗。方法：将从开始和停止NNRTI治疗的患者中获得的纵向血浆样本中的HIV-1 RNA转化为cDNA，并通过实时PCR扩增和定量靶序列区域。约10（7）个拷贝/反应提供了使用区分突变体和野生型等位基因的引物进行第二轮PCR的模板。结果：12例初治患者中，9例患者的103 N频率与检测背景值相近（0.029%），3例患者的103 N频率瞬时升高至0.21- 0.48%，是检测背景值的7 ~ 16.5倍。对6名NNRTI患者的纵向血浆样本分析显示了三种模式：停止NNRTI治疗后103 N变异体的持续存在，NNRTI治疗期间AAC和AAT之间103 N的密码子转换，以及停止NNRTI治疗后103 N变异体衰减至低于测定背景。等位基因特异性RT-PCR在很宽的频率范围内（0.1-100%）定量了患者中耐药变异体的出现和衰减。停止NNRTI治疗后K103 N变异体的衰减率是高度可变的。(C)2006年利平科特威廉姆斯&威尔金斯。
Background: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy.Objective: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy.Methods: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10(7) copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis.Results: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy.Conclusions: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable. (C) 2006 Lippincott Williams & Wilkins.