| BACKGROUND Chronic hepatitis B (CHB) and liver cirrhosis are common infections diseases which have serious damage to human health. Information of evidence-based medicine have proved, hepatitis B virus (HBV) is the most important factor for disease progression, so antiviral treatment is the key treatment for CHB. Now the two kinds of effective antiviral drugs appoved internationally are Interferon (IFN) and nucleos (t) ide analogues (NA), the latter include lamivudine(LAM), adefovir dipivoxil(ADV), entecavir(ETV), telbivudine (LdT) and tenofovir disoproxil (TDF) (former four have been approved by State Food and Drug Administration-SFDA). NA have a significant role in inhibition of HBV replication, however, persistent existence of covalently closed circular DNA (cccDNA) in liver cells determines prolonged treatment with CHB. Mutations associated with NA-resistant may develop with prolonged theatment, which can lead to hepatitis attack, even liver failure and death. Thus NA-resistant mutation is an important clinical question. Monitoring regularly, early detection of resistant mutations, prompt and effective salvage therapy for disease control are of great significance.Serum HBV DNA assay is the most important indicator for NA-resistance monitoring, but it may also be influenced by patient compliance, drug metabolism and virus natural fluctuation besides resistance mutation. Therefore, specific genetic resistance detection is the necessary conditions to determine NA-resistance mutations. Pyrosequencing as a new sequencing method with high accuracy and sensitivity, can detect mutations when above 5% mutant species are present, and can detect multiple drug-resistence sites quantitively to meet the need of the study—to detect the common sites of HBV P gene region associated with NA-resistence. It detected fast and was in line with the high rate of direct sequencing reported to be used for LAM-resistance and ADV- resistance detection.OBJECTIVE To analyze the NA-resistant mutations at 10 sites of the P gene of HBV by pyrosequencing, explore the detectablerate and mutation modes in patients with CHB before NA treatment, explore HBV-related mutation sites,mutation patterns and relationships occered during NA treatment, explore the development of mutant resistants of HBV reverse transcriptase area in patients who had inadequate virologic response after ADV treatment, intervene individually and observate vertically.METHODS The 10 sites (rtI169,rtV173L,rtL180M,rtA181V/T,rtT184G,rtA194T,rtS202I,rtM204V/I,rtN236T,rtM250V) of HBV P gene in 281 cases with CHB were detected quantitatively by pyrosequencing. Individual treatment and vertical observation were implementated according to the different mutational states of patients.RESULTS①Nature mutations were detected in 3.66%(3/82) of patients with CHB before NA treatment, single rtA181T mutation in 2 cases and single rtV173L mutation in 1 case respectively, they existed as non-dominant strains.②11 mutation patterns at 4 sites of LAM-resistant in 95 cases,6 modes at 2 sites of ADV-resistent in 26 cases,6 modes at 5 sites related to LAM and ADV in 20 cases,5 modes at 4 sites of LdT-resistent in 20 cases and 6 modes at 7 sites of ETV-resistent in 10 cases were detected respecyively, in which rtM204,rtA181V,rtA181T,rtM204I,rtT184 was the most common respecyively.③The differences of HBV DNA level among different mutation patterns after ADV-resistance or LAM-resistance had no statistical significance (P>0.05), the HBV DNA level of LAM-resistance group was higher than that of ADV-resistance group (P<0.05).④In 28 cases who appeared incomplete virologic response after ADV treatment, mutation was not detected in 25 cases, HBV was not detected in another 3 cases.⑤ADV was added to patients when mutations related to LAM, LdT or ETV were detected, LAM was added to patients with ADV-resistance (N236T), patients with rtAl 81 mutation were rescued by changing to or adding ETV. these measures were effective, IFN was effective for some patients with NA-resistance.⑥After intervention, resistance mutations in 25% (12/48) of sufferers would decrease even disappeared in 3 months,35.42%(17/48) had no change with before intervention, resistance mutations would exist for a long term (≥6 months) in 16.67%(8/48) of sufferers, new drug resistance mutations would be chosen in 22.92%(11/48) of sufferers.CONCLUSIONS①Natural mutations as non-dominant strains existed rarely in patients with CHB before NA treatment.②Mutations of HBV P gene can be detected during prolonged NA-treatment; RtM204 was the main mutation rite related to LAM-resistance, rtM204V always appeared accompanied with rtL180M, rtM204I always existed alone, rtV173L always appeared based on rtM204+rtL180M mutation; RtA181 and rtT184 was 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.③The differences of HBV DNA level in patients with different mutations after ADV-resistance or LAM-resistance had no statistical significance, the HBV DNA level of LAM-resistance group was higher than that of ADV-resistance group.④Genotype drug-resistant was not the reason to get inadequate virologic response for patients with CHB after ADV treatment.⑤Once drug-resistance happened, individual theatment of adding or changing drugs with different drug-resistant sites or IFN timely and detecting virological response regularlly are needed.⑥Dynamic evolution of HBV population was detected after drug interention. |
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