南京市204例慢性乙型肝炎患者乙型肝炎病毒基因分型及耐药突变特征分析

Analysis of genotype and characteristics of drug resistance-associated mutations of hepatitis B virus in 204 chronic hepatitis B patients in Nanjing city

目的分析乙型肝炎患者乙型肝炎病毒(HBV)的基因型、耐药基因突变特征及其与恶性肝病的相关性,为HBV患者的个体化治疗提供依据。方法收集2020年1月―2022年7月到南京大学医学院附属鼓楼医院就诊的慢性乙型肝炎(CHB)患者血清样本204人份,应用PCR-Sanger测序法检测其乙肝病毒基因分型及耐药基因突变情况,同时检测其血清中甲胎蛋白(AFP)、乙型肝炎e抗原(HBeAg)、乙肝表面抗原(HBsAg)、HBV DNA、谷丙转氨酶(GPT)和谷草转氨酶(GOT)水平,对基因型及突变特征进行分析。结果入组的204例CHB患者中,共有164例检出HBV,其中HBV B型68例(41.46%),HBV C型96例(58.54%)。HBV B型和C型感染者在肝脏病变(22.22%vs 77.78%,χ^(2)=4.930、P=0.026)、NAs耐药突变(54.81%vs45.19%,χ^(2)=20.861、P<0.01)、AFP水平[(3.97±0.40)ng/mlvs(6.92±1.28)ng/ml,t=2.186、P=0.033]、HBeAg(S/CO)水平[(502.9±84.91)vs(917.5±139.10),t=2.545、P=0.013]差异有统计学意义。164例患者中NAs相关耐药突变检出率为63.41%(104/164),221位点检出率最高,为53.85%(56/104),其次为204位点(36.54%,38/104)、180位点(29.81%,31/104)、184位点(12.50%,13/104)。HBV C型感染者在184位点(84.62%vs 15.38%,χ^(2)=3.956、P=0.047)、191位点(100.00%vs 0,P=0.011)突变率高于HBV B型,而在221位点突变率显著低于HBV B型(10.71%vs 89.29%,χ^(2)=80.130、P<0.01)。混合位点突变以L180+M204为主,其中C型占比70.97%(22/31),高于B型(29.03%,9/31),但差异无统计学意义(P>0.05)。结论本地CHB患者基因型以B型和C型为主,HBV C型患者较B型更易发生肝脏病变;拉米夫定和替比夫定耐药基因突变最常见,突变模式复杂。因此,在抗病毒治疗前或治疗过程中及时监测基因型及耐药基因突变情况,可以提升疾病治疗质量。

Objective To study the genotype and the characteristics of drug resistance-associated gene mutations of hepatitis B virus(HBV),and to explore their correlation with malignant liver disease in patients with chronic hepatitis B(CHB),so as to provide evidence for individualized treatment of CHB patients.Methods Serum samples were selected from 204 CHB patients who visited Nanjing Drum Tower Hospital(the Affiliated Hospital of Nanjing University Medical School)between January 2020 and July 2022.The genotype of HBV and drug resistance-associated gene mutations were detected by PCR-Sanger sequencing.The serum levels of alpha-fetoprotein(AFP),hepatitis Be antigen(HBeAg),hepatitis B surface antigen(HBsAg),HBV DNA,glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase were detected.The genotype and mutation characteristics were analyzed.Results Of the 204 CHB patients enrolled,HBV types were detected in 164 cases,including 68 cases of HBV type B(41.46%)and 96 cases of HBV type C(58.54%).There were statistically significant differences in liver disease(22.22%vs 77.78%,χ^(2)=4.930,P=0.026),nucleos(t)ide analogues(NAs)resistance mutations(54.81%vs 45.19%,χ^(2)=20.861,P<0.01),AFP level[(3.97±0.40)ng/ml vs(6.92±1.28)ng/ml,t=2.186,P=0.033]and HBeAg level(502.9±84.91 vs 917.5±139.10,t=2.545,P=0.013)between HBV B and C patients.Among the 164 patients,the detection rate of NAs drug resistance mutations was 63.41%(104/164),with the highest frequency at site 221(53.85%,56/104),followed by 204(36.54%,38/104),180(29.81%,31/104),and 184(12.50%,13/104).Compared with patients with HBV type B infection,the patients with HBV type C infection showed statistically significantly higher mutation rates at sites 184(84.62%vs 15.38%,χ^(2)=3.956,P=0.047)and 191(100.00%vs 0,P=0.011),but statistically significantly lower mutation rate at site 221(10.71%vs 89.29%,χ^(2)=80.130,P<0.01).L180+M204 was the dominant pattern of co-occurrence of drug-resistance mutation,with type C accounting for 70.97%(22/31),higher than the proportion of type B(29.03%,9/31),but the difference was not statistically significant(P>0.05).Conclusions HBV genotype in CHB patients detected in Nanjing are mainly type B and type C,and HBV patients with type C are more likely to develop liver diseases than patients with type B.Gene mutations related to resistance to lamivudine and tibivudine are the most common,and the mutation pattern is complex.Therefore,timely monitoring of genotype and drug-resistance gene mutations before or during antiviral therapy can improve the quality of treatment for CHB.