A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of "extra-target" RAS suggests the need for RAS screening in all three DAA target regions.

中文翻译：

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深度测序揭示了与治疗失败相关的广泛的亚型特异性HCV耐药性突变。

一定比例的丙型肝炎病毒（HCV）感染患者未能通过基于直接作用抗病毒（DAA）的治疗方案，这通常是由于与耐药性相关的替代（RAS）。这项研究的目的是表征未通过DAA治疗的一大批患者的耐药情况，并研究HCV亚型和失败之间的关系，以帮助优化这些患者的治疗。设计了一种基于深度测序的新的，标准化的HCV-RAS测试规程，并将其应用于220例先前在DAA治疗失败的患者中亚型的样品中，这些样品在西班牙的39家医院中收集。大多数接受了无DAA干扰素（IFN）α治疗方案。79％的患者接受含sofosbuvir的治疗失败。编码非结构蛋白（NS）3，NS5A，使用亚型特异性引物分析NS5B和NS5B（DAA靶区域）。病毒亚型分布如下：基因型（G）1，62.7％；G3a，21.4％；G4d，12.3％；G2，1.8％；和混合感染1.8％。总体而言，在突变谱中，有88.6％的患者携带至少1种RAS，而19％的患者携带RAS的频率低于20％。有和没有利巴韦林的治疗之间的RAS选择没有差异。无论接受哪种治疗，每种HCV亚型均显示特定类型的RAS。值得注意的是，在治疗失败的患者中，有18.6％的患者的靶蛋白中未检测到RAS，并且有30.4％的患者的RAS蛋白不是其所接受抑制剂的靶标。未能通过DAA治疗的HCV患者表现出较高的RAS多样性。利巴韦林的使用不会影响失败时RAS的类型或数量。RAS出现的亚型特定模式强调了准确的HCV亚型的重要性。“额外目标” RAS的频率表明在所有三个DAA目标区域都需要进行RAS筛查。