焦磷酸法检测171例慢性乙型肝炎患者HBV多位点耐药突变株结果分析

Analysis of Detection of drug-resistant HBV mutants by pyrosequencing in 171 patients with chronic hepatitis

目的分析慢性乙型肝炎患者HBV常见核苷(酸)类似物耐药相关10个位点的突变对早期发现耐药变异的临床意义。方法收集171例慢性乙型肝炎患者,分为治疗组和未治疗组,提取血清对HBV反转录酶(RT)基因区核苷(酸)类似物变异耐药突变10个位点进行检测,采用焦磷酸测序技术进行DNA测序,数据分析采用PSQ-96MA型焦磷酸测序仪配套的分析软件进行;同时进行HBV DNA载量、HBeAg、HBV基因型、ALT和AST检测。结果治疗组122例中53例发生变异,检出率为43.44%,未治疗组49例中3例发生变异,检出率为6.12%;拉米夫定耐药变异形式多为M204V/I单独出现或伴随L180M或/和V173L突变,阿德福韦酯耐药变异以A181T/V位碱基替换为主,恩替卡韦耐药以S202G和M250V位点的碱基替换最为常见,均伴随拉米夫定和阿德福韦酯耐药变异。未治疗组3例耐药突变分别为M204I、M204V+V173L和A181V。两组间发生变异者与未发生变异者比较,HBV DNA载量、ALT、AST、HBeAg阴性率及基因型差异均无统计学意义(P>0.05)。结论治疗或未经核苷(酸)类似物抗病毒治疗的慢性乙型肝炎患者均存在耐药变异相关突变,对抗病毒治疗不同时期进行多位点耐药突变检测,并对变异相关因素进行分析,能使核苷(酸)类似物抗病毒治疗更加规范、科学和合理。

Objective To detect the 10 sites of reverse-transcriptase inhibitor （NRTI）-resistance mutations in hepatitis B virus （HBV）, and analyze early detection of antiviral drug-resistant mutations enabling prompt initiation of rescue therapy. Methods Pyrosequencing was used to test the NRTI-resistance mutations in HBV obtained from 171 patients, including 122 （NRTI）-treated patients and 49 NRTI- untreated patients. Drug-resistance variations in 10 sites of HBV reverse transcriptase （RT） gene region were analyzed, while HBV DNA level, HBeAg status, alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels were detected. The RT genom region of HBV was amplified by PCR, PCR products were sequenced by pyrosequencing, and data were analyzed by the analysis software PSQ-96MA type pyrosequencing instrument. Results Pyrosequencing showed that 53 cases （43.44%） were positive for drug-resistant variants among 122 NRTI-treated patients, and 3 cases （6; 12 ~/00） of 49 NRTI-untreated patients. The mutant sites of lamivudine-resistance was M204V/I alone or combined with L180M and/or V173L. Adefovir-resistance mutations mainly showed the base substitution of A181T/V. Most common base substitution sites of entecavir-resistance were $202G and M250V,and almost all of them combined with the drug resistance mutation of lamivudine and adefovir. The mutant sites of the 3 untreated patients were M204I, M204V ＋ V173L and A181V, respectively. No significant correlation between the two groups was observed inHBV DNA level, HBeAg statue, total bilirubin ALT and AST level （P~〉 0.05）. Conclusion Drug-resistance mutationexist in both NRTI-treated and NRTI-untreated patients with chronic hepatitis B （CHB）. More standardized, scientific and reasonable anti-virus treatment with nucleoside analogues （acid） will be received if antiviral drug-resistant mutations in different periods is detected early and mutation factors are analyzed.