Bartolini Barbara, Selleri Marina, Garbuglia Anna Rosa, Giombini Emanuela, Taibi Chiara, Lionetti Raffaella, D'Offizi Gianpiero, Capobianchi Maria R
"L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
"L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
J Clin Virol. 2015 Nov;72:60-5. doi: 10.1016/j.jcv.2015.07.310. Epub 2015 Jul 30.
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.
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.
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.
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.
丙型肝炎病毒（HCV）准种中预先存在的携带耐药相关突变（RAMs）的变异对直接作用抗病毒药物（DAA）治疗结果的影响存在争议，并且由于缺乏对病毒储存库（肝脏）和循环部分之间准种分布的了解而变得复杂。
评估基线血浆和肝活检样本中NS3蛋白酶的异质性以及RAMs的存在情况。还在治疗期间及其暂停后分析了血浆动力学。研究设计对两名接受基于特拉匹韦（TVR）治疗并因TVR耐药而出现治疗失败的HCV 1a基因型患者进行了超深度焦磷酸测序（UDPS）。
在两名患者中，血浆中NS3准种的基线多样性均高于肝脏（分别为183.6×10⁻⁴对47.8×10⁻⁴以及246.0×10⁻⁴对55.0×10⁻⁴核苷酸替换/位点，p<0.0001），但系统发育树未显示两个部分之间的分隔。在基线时，两种样本类型中均检测到极低水平的RAMs（即V36A、T54A，范围：0.31 - 0.52%）。然而，系统发育分析显示，基线时携带这些突变的病毒变异与突破时固定的变异不同，突破时观察到组合的V36M + R155K，赋予对TVR的高水平耐药性。停药后，耐药相关变异的频率下降。
UDPS允许对TVR停药后的准种分隔及其动态进行广泛评估。血浆和肝脏NS3准种，包括低水平的RAMs，没有显示出显著差异。
