Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory
Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory
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DOI：
10.1128/jcm.02652-12
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发表时间：
2013-03-01
影响因子：
9.4
通讯作者：
Turner, Dan
中科院分区：
文献类型：
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作者：
Avidor, Boaz;Girshengorn, Shirley;Turner, Dan
检测HIV-1的低丰度耐药突变（DRM）是临床实践中不断发展的方法。超深度焦磷酸测序已被证明在检测此类突变方面是有效的。缺乏标准化的商业化检测限制了该方法在临床环境中的广泛使用。454 Life Sciences（Roche）正在为其台式测序仪开发HIV超深度焦磷酸测序测定。我们在临床实验室评估了原型接骨板。通过标准化TruGene试剂盒进行基因分型的血浆样品通过该测定法进行回顾性检测。包括治疗失败的药物治疗受试者和药物初治患者。DRM分析基于国际艾滋病协会美国DRM列表和斯坦福大学算法。原型测定法检测了TruGene检测到的所有DRM和另外50种低丰度DRM。一些患者具有低丰度的D 67 N、K70 R和M184 V逆转录酶抑制剂突变，这些突变在引起这些突变的药物停药后持续很长时间。其他患者携带低丰度V32 I主要蛋白酶抑制剂突变，其在地瑞那韦选择下进化，后来被TruGene检测到。斯坦福大学的分析表明，一些低丰度的DRM可能会影响这些受试者的耐药负担。原型检测至少与TruGene一样好，并且具有检测TruGene未检测到的低丰度耐药突变的优势。它的易用性和实验室规模的平台可能会促进其在临床实验室中的使用。低丰度DRM的检测将在多大程度上影响患者管理仍是未知的，但希望在临床实践中使用这种测定将有助于解决这个重要问题。
Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had low-abundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.