摘要目的 分析慢性乙型肝炎病毒(HBV)感染者核苷(酸)类药物(NAs)HBV逆转录酶区相关耐药突变位点的异质性,为规范抗病毒治疗和耐药管理提供参考价值.方法 收集2011年4月至2018年3月宁波市第二医院及宁波市第四医院慢性HBV感染者NAs治疗发生病毒学突破或应答不佳2765例的血液标本,采用实时荧光定量PCR方法对血清HBV多聚酶区进行扩增,对PCR产物进行直接测序并分析测序结果.根据用药情况分为拉米夫定(LAM)单药耐药组(n=603)、替比夫定(LdT)单药耐药组(n=147)、阿德福韦酯(ADV)单药耐药组(n=68)、恩替卡韦(ETV)单药耐药组(n=10)和NAs序贯或联合耐药组(n=365),分析各组耐药突变位点及耐药模式(通路).采用SPSS 19.0统计软件对数据进行分析.结果 在2765例慢性HBV感染者的血液标本中,检测出NAs相关HBV-RT区耐药突变1193例,总突变率为43.15％.LAM单药耐药组的突变率为62.62％(603/963),共19种突变类型,以单位点突变rtM204I/V最多见(40.30％,243/603).LdT单药耐药组的突变率为45.51％(147/323),共3种突变类型,以单位点突变rtM204I/V最多见(59.86％,88/147).ADV单药耐药组的突变率为17.80％(68/382),主要为rtA181T单点突变(64.71％,44/68).ETV单药耐药组的突变率为4.06％(10/246),以rtT184A/G/S/I/L/F单点突变最多见(80.00％,8/10).序贯或联合治疗耐药组的突变率为41.91％(365/871),其中LAM/LdT应答不佳或耐药序贯ADV组突变率为63.39％(142/224),以rtA181V/T最多见(35.21％,50/142);LAM/LdT应答不佳或耐药联合ADV组突变率为42.19％(54/128),以rtA181V/T最多见(46.30％,25/54);LAM/LdT应答不佳或耐药后序贯ETV 1.0 mg组突变率为44.66％(117/262),以rtL180M+M204I/V+S202G/I最多见(31.62％,37/117);LAM/LdT应答不佳或耐药ETV联合ADV组突变率为7.14％(5/70),均为多位点突变;ADV应答不佳或耐药序贯ETV 0.5 mg组突变率为28.14％(47/167),均为多位点突变.rtV173L、rtL180M、rtV214A等次要(补偿)位点及rtV207I/L/G、rtS213T、rtN238T等尚未完全明确位点的单位点突变均被检测出.NAs单药治疗的耐药模式(通路)较为单一,NAs经治患者(序贯或联合治疗组)的耐药模式(通路)复杂多样,存在多个耐药模式(通路),并随着NAs种类的增加而增加.非一线NAs相关耐药模式(通路)的构成比基本呈总体下降趋势,而ETV相关耐药的构成比基本呈总体上升趋势.结论 NAs相关HBV耐药突变位点(模式)复杂多样,特别是多位点突变、难治性耐药突变、多药耐药突变和交叉耐药突变.因此,优选、优化抗病毒治疗策略和耐药管理观念需不断更新.

abstractsObjective To analyze the heterogeneities of hepatitis B virus ( HBV ) reverse transcriptase domain (RT) gene mutations related to nucleos (t)ide analogues (NAs) resistance.Methods Blood samples from 2765 chronic hepatitis B patients with virological breakthrough or poor drug response treated in Ningbo No .2 Hospital and Ningbo Fourth Hospital from April 2011 to March 2018 were collected . According to the medication status , it was divided into LAM monotherapy group ( n =603 ) , LdT monotherapy group (n=147), ADV monotherapy group (n=68), ETV monotherapy group (n=10) and the sequential or combined drug resistance of NAs group (n=365).The resistance mutation sites and drug resistance patterns (pathways) of each group were analyzed .The SPSS 19.0 software was used to analyze the data.Results Among 2765 serum samples, the NAs-related HBV-RT resistance mutations were detected in 1193 cases with an overall mutation rate of 43.15％.The mutation rate of LAM monoclonal resistance group was 62.62％ (603/963) with 19 mutation types, the most common single point mutation was rtM204I/V (40.30％, 243/603).The mutation rate of LdT monoclonal resistance group was 45.51％(147/323), and there were 3 mutation types, with the single point mutation rtM204I/V being the most common (59.86％, 88/147).The mutation rate of the ADV monoclonal resistance group was 17.80％(68/382), mainly rtA181T single point mutation (64.71％, 44/68).The mutation rate of the ETV monoclonal resistance group was 4.06％(10/246), and the single point mutation of rtT184A/G/S/I/L/F was the most common one (80.00％, 8/10).The mutation rate of the sequential or combination therapy group was 41.91％ (365/871), among which the mutation rate of the LAM/LdT poor response or the resistance with the sequential ADV group was 63.39％(142/224), and the most single mutation point was rtA181V/T ( 35.21％, 50/142 );the mutation rate of LAM/LdT poor response or drug-resistant with combined ADV group was 42.19％ (54/128), and the most common mutation point was rtA181V/T (46.30％, 25/54);the mutation rate of LAM/LdT with poor response or resistance after sequential ETV 1.0 mg was 44.66％(117/262), and the most common mutation point was rtL180M+M204I/V+S202G/I (31.62％, 37/117);the LAM/LdT poor response or the drug-resistant ETV combined with ADV group had a mutation rate of 7.14％(5/70), all of which were multi-site mutations;the mutation rate of poor response to ADV or resistant with sequential ETV 0.5 mg group was 28.14％(47/167), all of which were multi-site mutations.Secondary ( compensation ) sites such as rtV173L, rtL180M, and rtV214A, and single-point mutations such as rtV207I/L/G, rtS213Tand rtN238T, which were not fully defined , were detected.The resistance patterns ( pathways ) of NAs monotherapy were relatively simple , and the resistance patterns ( pathways ) of NAs experienced patients ( sequential or combined treatment group ) were complex and diverse, and multiple resistance patterns (pathways) existed, along with NAs increasing in species.Non-first-line NAs-related resistance patterns ( pathways ) showed an overall downward trend sand ETV-related drug-resistant mutation showed an overall upward trend .Conclusion The NAs-related HBV resistance mutation sites ( patterns ) are complex and diverse , especially multi-site mutations , refractory drug resistance mutations, multidrug resistance mutations and cross-resistance mutations.Therefore, the optimization of antiviral treatment strategies and drug resistance management concepts need to be continuously updated .