Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Rome, Italy.
Hepatology Unit, University Hospital of Rome 'Tor Vergata', Rome, Italy.
J Antimicrob Chemother. 2016 Mar;71(3):739-50. doi: 10.1093/jac/dkv403. Epub 2015 Dec 17.
This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen.
NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays.
Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11).
HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.
本研究旨在评估 NS3 测序在候选接受含蛋白酶抑制剂（PI）方案治疗的 HCV1 感染患者中的可靠性和临床实用性。
采用自行开发的 HCV1 亚型特异性方案进行 NS3 蛋白酶测序。通过系统发生分析来检验测序的可靠性以及与商业基因型/亚型检测方法的一致性。
2011 年至 2014 年间，共采集了 326 例 HCV 感染患者 567 份 HCV 血浆样本，其 HCV-RNA 可定量。总体上，NS3 测序成功率为 88.9%。对于 HCV-RNA >3 log IU/mL 的样本（成功率 >92%），两种亚型方案（HCV-1a/HCV-1b）的成功率相似，而对于 HCV-RNA ≤3 log IU/mL 的 HCV-1a 样本，成功率略低于 HCV-1b 样本。系统发生分析确认了商业基因型检测方法给出的基因型/亚型在 92.9%（303/326）的分析病例中。在其余 23 例（7.1%）中，1 例为 HCV-1g（先前定义为 1a 亚型），1 例为 HCV-4d（先前定义为基因型 1b），1 例为 HCV-1b（先前定义为基因型 2a/2c）。在其他病例中，NS3 测序精确地解决了先前未确定/不一致的 1 型亚型或商业基因型检测方法的双重基因型/亚型赋值。在 31.0%的样本中检测到对 PI 的耐药相关变异（RAVs）。这种流行率根据 PI 经验而变化（初治患者为 17.1%，boceprevir/telaprevir/simeprevir 失败患者为 79.2%）。在 96 例接受 boceprevir/telaprevir 治疗后有可用病毒学结果的患者中，观察到基线 NS3 RAVs 与病毒学失败之间存在关联的趋势（特别是 HCV-1a 感染患者：21 例失败患者中有 3 例，22 例持续病毒学应答患者中无 1 例；P=0.11）。
HCV-NS3 测序提供了可靠的结果，同时提供了两个具有临床意义的信息：正确的亚型/基因型赋值和检测可能影响 PI 疗效的变异。
