摘要
目的研究核苷(酸)类似物(NAs)治疗慢性乙型肝炎(CHB)过程中多重耐药乙型肝炎病毒(HBV)株的演变规律及多重耐药形成机制。
方法1)706例接受NAs抗病毒治疗过程中疗效不佳的CHB患者,对其血清HBV DNA进行PCR扩增后直接测序,分析其RT区序列特征;2)准种分析采用“PCR-克隆-测序”方法,对5例临床资料完整、具有代表性意义的患者的15份血清样本进行研究。并对其中4例患者连续血清样本进行动态监测,分析不同时间点血清标本HBV毒株准种特征。
结果1)在不同NAs单药、序贯或联合治疗CHB过程中疗效不佳的706例患者中发现多重耐药25例。总的多重耐药检出率为3.5%,其中,LAM、ADV双重耐药检出率为72%,LAM、ADV、ETV三重耐药检出率为28%;2)多重耐药的出现不仅可导致病毒学反弹、生化学突破、肝炎复发,还可引起肝衰竭导致患者死亡;3)克隆分析发现准种的进化过程可分为以下三种模式:①在序贯治疗过程中,患者体内准种可由对单药耐药克隆进化到单药耐药克隆和多重耐药克隆的混合克隆;②序贯治疗过程中准种从单药耐药克隆全部演变为多重耐药克隆;③在药物的选择压力下,患者体内准种可由多重耐药克隆为主联合单药耐药克隆的混合克隆进化到以复制能力更强的单药耐药克隆为优势准种。4)根据准种特征,3例多重耐药患者采取ETV+ADV挽救治疗,血清HBV DNA水平于12周内降至检测水平以下。
结论1)NAs单药、序贯或联合治疗过程中均可诱导多重耐药株的出现;2)多重耐药株的出现不但可导致病毒学突破,还可引起肝衰竭导致患者死亡;3)克隆分析可发现各种耐药类型的HBV毒株,多位点联合变异可出现在同一HBV DNA基因组上;4)HBV准种动态变化过程可能与药物选择压力、病毒复制能力、复制空间有关;5) ETV+ADV挽救治疗方案可能对部分多重耐药患者有效。
Objective
The aim of this study was to determine the evolution patterns and formation mechanisms of multi-drug resistant hepatitis B virus during nucleos(t)ide analog therapy in patients with chronic hepatitis B.
Methods
1)706 patients diagnosed as chronic hepatitis B and with unsatisfactory curative effect during nucleos(t)ide analog therapy were enrolled in this study. Amino acid sequence of HBV RT domain which amplified by PCR and then directly sequenced was analysized. 15 serum samples from five representative patients with complete clinical data were selected out for clonal analysis using‘PCR-cloning-sequencing’method. Additionally, dynamic detection of consecutive serum samples was carried out in four patients to determine the evolution of HBV strains.
Results
1) The total multi-drug resistance detection rate of the patients who received nucleos(t)ide analogue monotherapy, sequential therapy or combination therapy was 3.5%. Among which, double resistance to LAM and ADV was 72%; triple resistance to LAM, ADV and ETV was 28%. 2) Multi-drug resistance can not only lead to viral rebound, biochemical breakthrough, hepatitis B recurrence but also liver failure or even death. 3) Clonal analysis revealed that the evolution of quasispecies can be divided into the following three models.①During sequential therapy, progressive evolution was from single drug resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have single drug resistant HBV mutations only.②Progressive evolution was from single drug resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations during sequential therapy.③Within drug selective pressure, progressive evolution of the variants was from mixtures of clones that have multi-drug resistant mutations and clones that have single drug resistant HBV mutations only, ultimately to the single drug resistant HBV mutations as dominant variant for its high replication ability. 4) Combination therapy with ETV and ADV reduced HBV DNA to the detection limit of 500copies/ml within 12 weeks in 3 patients.
Conclusion
1) Multi-drug resistance can be induced during nucleos(t)ide analogue monotherapy, sequential therapy or combination therapy. 2) Multi-drug resistance can not only lead to viral breakthrough, but also liver failure or even death. 3) Clonal analysis showed mutations conferring multi-drug resistance colocate on the same viral genome. 4) Dynamic changes of HBV quasispecies had relation to the selective pressure of NAs therapy, distinct replicative advantage of the variants and replication space. 5) Combination therapy with ETV and ADV may be effective for some multi-drug resistant patients.
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