背景:丙型肝炎病毒(HCV)准种中预先存在的变异的影响,携带抗性相关突变(RAM),关于直接作用抗病毒药物(DAA)治疗的结果存在争议,并且由于缺乏病毒库(肝脏)和循环室之间准种分布的知识而变得复杂。
目的:评估基线血浆和肝活检样品中NS3蛋白酶的异质性和RAM的存在。还在治疗期间和其悬浮之后分析了血浆动力学。研究设计在两名接受基于telaprevir(TVR)的治疗并由于TVR抵抗而导致治疗失败的HCV基因型1a患者中进行了超深度焦磷酸测序(UDPS)。
结果:在两名患者中,血浆中NS3准种的基线多样性高于肝脏(183.6×10(-4)vs47.8×10(-4)和246.0×10(-4)vs55.0×10(-4)nt置换/位点,分别,p<0.0001),但是系统发育树没有证据表明两个区室之间存在区隔。在基线RAM(即V36A,T54A)在两种样本类型中均检测到非常低的水平(范围:0.31-0.52%)。然而,系统发育分析显示,在基线时携带这些突变的病毒变体不同于在突破时固定的病毒变体,当组合V36M+R155K时,赋予TVR高电阻,被观察到。停用药物选择压后,耐药相关变异的频率下降。
结论:UDPS允许对准物种划分及其撤除TVR后的动力学进行广泛评估。血浆和肝脏NS3准种,包括低级RAM,不显示显著差异。
BACKGROUND: The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment.
OBJECTIVE: To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance.
RESULTS: In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p<0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure.
CONCLUSIONS: UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.