Pyosequencing And Analysis Of Nucleos(t)ide Analogues-Resistance Mutation Of The P Gene Of Hepatitis B Virus

BACKGROUND The key treatment for chronic hepatitis B (CHB) patients is antiviral treatment. Two categories of anti-HBV drugs have been approved by State Food and Drug Administration (SFDA):Interferon (IFN) and nucleos(t)ide analogues which include lamivudine (LAM),adefovir dipivoxil (ADV),entecavir (ETV) and telbivudine (LdT). Nucleos(t)ide analogues demonstrate clinically significant antiviral activity, however, mutations associated with nucleos(t)ide analogues-resistance may develop during nucleos(t)ide analogues therapy; and viral breakthrough, biochemical breakthrough, hepatic flare and hepatic decompensation may rebound as a result. Regular monitoring, early detection of resistant mutations, prompt and effective rescue therapy are important to successful treatment.Pyrosequencing was reported to be used in LAM resistance as a new sequencing method, which has a high accuracy with the method of direct sequencing and we can detect mutations when 5%～10% mutant species are present.OBJECTIVE To analyze the nucleos(t)ide analogues-resistant mutations at 10 sites of the P gene of hepatitis B virus (HBV).METHODS The 10 sites (rtI169T,rtV173L,rtL180M,rtA181V/T,rtT184G,rtA194T,rtS202I,rtM204V/I,rtN236T,M205V) of HBV P gene in CHB patients who had virologic breakthrough or inadequate virologic response during taking nucleos(t)ide analogues were detected by pyrosequencing. The HBV-DNA levels among the different mutation modes when LAM-resistance and ADV-resistance were compared respectively.RESULTS①Eight modes at 4 sites resistance mutation associated with lamivudine were found in 48 cases, in which rtM204 was the most common; adefovir dipivoxil-resistance of 4 modes at 2 sites in 10 cases, in which rtA181 was the most common; entacavir- resistance of 4 modes at 7 sites in 6 cases, in which rtT184 was the most common; telbivudine-resistance of 2 modes at 2 sites in 4 cases, in which all exist rtM204I.②The HBV-DNA levels among different mutation modes of LAM-resistance were not statistically significant difference (F=0.427, P=0.881), however, in comparison with mutation modes of ADV-resistance, the difference was statistically significant,and the serum HBV DNA loads in patients with the rtA181V+rtA236T combination mutation were higher than that with single rtA181V mutation(P=0.01).CONCLUSIONS①For CHB patients who developed virologic breakthrough, mutations in HBV P gene can be detected during nucleos(t)ide analogues treatment and tailored therapies should be taken.②The HBV-DNA levels may differ in patients with different mutations when ADV-resistance, and the serum HBV-DNA loads in patients with the rtA181V+rtA236T combination mutation were higher than that with single rtA181V mutation.