| BACKGROUND Infection of hepatitis B virus(HBV) is a worldwide public healthy problem. HBV is the most important factor for chronic hepatitis B(CHB)and liver cirrhosis progression, so antiviral treatment is the key treatment. Now the two kinds of effective antiviral drug approved internationally are interferon(IFN) and nucleos(t)ide analogues(NUCs). The effective, which NUCs inhibit HBV replication is dependable, could reduce or prevent the incidece of liver cirrhosis and primary carcinoma of the liver(HCC). Mutations may develop with prolonged treatment, which can bring about hepatitis attack, so it is great significance for monitoring regularly, early detection of NUCs-resisitant mutations and effective salvage therapy for disease control. Pyrosequencing, as a new sequencing method, can detect fast multiple drug-resistence mutations with high accuracy and sensitivity. It was in line with the high rate of direct sequencing.OBJECTIVE To analyze the NUCs-resistant mutations at10sites of the reverse transcription gene of HBV, explore mutation sites, mutation modes and correlations in patients infected by HBV during NUCs treatment. To investigate the previous history of medication, clinical characteristics and the effect of related individualized treatment secondly in chronic hepatitis B (CHB) patients with rtA181mutation.METHODS The10sites (rtI169T, rtV173L, rtL180M, rtA181V/T, rtT184Q rtA194T, rtS202I, rtM204V/I, rtN236T, rtM250V) of HBV reverse transcription gene were detected quantitatively by pyrosequencing in the180patients infected by HBV who occur virological breakthrough during NUCs treatment. To investigate the correlations of between the resistance mutation discipline and lamivudine(LAM), adefovir dipivoxil(ADV), entecavir(ETV), telbivudine(LdT), respectively. The serum level of HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotrotransferase (ALT) was tested for the54patients, who were confirmed rtA181mutation by pyrosequencing during nucleos(t)ide analogues (NUCs) treatment when viological breakthrough happened. Analyze retrospectively the mutation pattern among the patients with different medical history. Compare the HBV DNA level between baseline and virological breakthrough, one and a few mutation sites contained rtA181. Investgate serologically index of the patients with rtA181T or V mutation. Effect of individual treatment was analysed by prospective cohort study. T test and Mann-Whitney U test were used for continuous variables with normal or skewed distributions. To compare the values between two groups, x2or Fisher’s exact tests were applied to analyse categorical variable, respectively.RESULTS①10mutation modes at4sites of LAM-resistant in71cases,6modes at2sites of ADV-resistent in35cases,6modes at4sites of LdT-resistent in35cases and6modes at7sites of ETV-resistent in11cases were detected respecyively, in which rtM204, rtA181, rtM204I, rtT184L was the most common respecyively.②RtA181T mutation was found in35of54patients with rtA181mutations. Previous medication history mainly contained ADV and LAM. The multi-site mutations occurred more frequently in the patients with multi-NUCs history (57.6%) than in those with single NUCs (28.6%)(x2=4.342, P＜0.05).③Serum HBV-DNA level at virological breakthrough is lower than that at baseline of first antiviral treatment [(5.66±1.011og10copies/mL) vs (6.75±0.811og10copies/mL)(t=-4.210, P＜0.01)]. The suerum HBsAg level of the mutation sites including rtA181T is higher than rtA181V [(3.80±0.451og10IU/mL) vs (3.46±0.601og10IU/mL,(t=2.109, P<0.05)].④Two methods of intervention, i.e. adding/switching to entecavir (group A) and adding telbivudine (group B), were adopted when drug resistance was confirmed. In patients with serum HBV DNA>6logiocopies/mL at viral breakthrough,8of8patients in group A and3of4patients in group B occurred virological response at week24after intervention,3and1patients with undetectable HBV DNA were found in group A and B, respectively. In patients with serum HBV DNA<6log10copies/mL at viral breakthrough,14of14and5of7cases respectively in group A and B occured viological response at week24after intervention, serum HBV DNA decreased to undetectable level in12and4patients in group A and B respectively.CONCLUSIONS①NUCs related mutations of HBV reverse transcription gene can be detected during prolonged NUCs-treatment for the patients infected HBV; RtM204was the main mutation site related to LAM-resistance, rtM204V always appeared accompanied with rtL180M, rtM204I existed alone; RtA181and rtT184was the most common mutation related to ADV-resistance and ETV-resistance respectively, rtM204I existed in all cases developed LdT-resistance, rtA181T related to the use of LAM and ADV.②RtA181mutation pattern correlates with previous medical history, and multi-site mutation occur more frequently in the patients with multi-NUCs in previous medication history.③The effect of adding or switching to ETV is better than that of adding LdT in patients with rtA181mutations. |
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