Drug Resistance During Sequential Therapy Of Nucleos(t)ide Analogue In The Treatment Of Chronic Hepatitis B

Hepatitis B virus(HBV) infection is a major health concern,more than 350 million people now chronically infected with HBV.Without therapeutic intervention, at least 60 million will die prematurely due to the infection,approximately 1 million deaths annually worldwide Lamivudine(LVD) was the first Nucleoside Reverse Transcriptase Inhibitor(NRTI) which demonstrated clinically significant antiviral activity in the treatment of chronic hepatitis B(CHB).Adefovir dipivoxil(ADV) is an oral prodrug of adefovir,a nucleotide analogue,which is proved effective in both HBeAg-positive and HBeAg-negative CHB patients,and in both treatment naive patients and lamivudine refractory patients.Entecavir(ETV),a carbocyclic analogue, which is more potent than lamivudine and adefovir and is effective against lamivudine-resistant HBV mutants.Incidence of virologic response and drug resistance mutation reported by different research centres has been various.Factors associated with NAs resistance, conclusions drawed by different researchers are various and even contradictory.The primary aim of this study is to describe the virologic response and ADV resistance profile during adefovir monotherapy at a tertiary clinical centre in Southern China. Secondary aim was to determine possible factors associated with virologic characteristics,HBeAg loss and seroconversion and ADV-related mutation.Adult CHB Patients referred to Nan fang hospital liver center clinic from 2004 to 2007 who received adefovir more than 12 months were enrolled in the study.Both HBeAg-positive and HBeAg-negative CHB patients,both treatment naive patients and lamivudine refractory patients were eligible if their serum HBV DNA level = 4 log₁₀copies/mL.Virologic response and serologic response were monitored every three months,and laboratory data of liver function and renal function of each patient enrolled were detected every three months.Drug resistant mutations were detected by direct sequencing of HBV reverse transcriptase region which is amplified by semi-nested polymerase chain reaction(PCR).HBV sequences for different genotypes obtained from GenBank were aligned to identify drug resistant substitutions.Baseline characteristics of patients included:mean age was 35±10 years,13.7%of patients were women,20.6%were liver cirrhosis,and 71.6%were HBeAg-positive.58(56.9%) patients were LVDr patients with genotypic resistance,44(42.7%) received adefovir as de novo treatment,HBV genotype were B(49/48.0%),C(53/52.0%).Mean ALT level was 126±112IU/L(range,25-597 IU/L) and mean HBV DNA was 6.1±1.3 log₁₀copies/mL(range,4-91og₁₀copies/mL).LVDr patients showed significant lower HBV DNA level(P=0.004)and ALT level(P=0.017) than patients took adefovir as de novo treatment.Liver cirrhosis was diagnosed by histology in fourteen patients and by clinical evidence including image findings together with thrombocytopenia and/or esophageal varices in seven patients.In the study,EVR was achieved in 45(44.1%) patients,IVR was achieved in 60(58.8%) patients.There was no significant difference in terms of age,gender,HBV genotype,baseline ALT and HBV DNA level and liver cirrhosis between patients with and without IVR.IVR was achieved more frequently among HBeAg negative patients rather than HBeAg positive patients(88.1 vs.60.0%,P=0.018 ).Patients who achieved EVR(P=0.000) were more likely to achieved IVR,and patients with IVR were more likely to achieved CVR(P=0.000).In the whole study population,39 patients(38.2%) achieved complete virologic response,24(23.5%) at month 3, 35(34.3%) at month 6.Patients with EVR and/or IVR displayed higher CVR rate than patients without EVR and/or IVR(P=0.000).Baseline characteristics including age, HBeAg status,ALT and HBV DNA level,HBV genotype,liver cirrhosis did not show to be related to CVR.In multivariate analysis,IVR independently predict CVR, however,male and lamivudine resistance serve as independent negative predictive value of CVR.65(63.7%) and 81(79.4%) patients had normal alanine aminotransferase(ALT) values at month 6 and month 12,respectively.Patients with IVR were more likely to achieved ALT normalization at both month 6 and month 12. No renal laboratory abnormalities or other signs of renal dysfunction associated with adefovir dipivoxil occurred.Seventeen out of seventy-three HBeAg-positive patients(23.3%) accomplished HBeAg loss from month 7 to month 24.Cumulative incidence of HBeAg loss at month 12,18 and 24 were 10%,27%and 41%,respectively.Fourteen out of seventeen patients achieved sustained HBeAg seroconversion after absence of HBeAg with cumulative incidence at month 12,18 and 24 were 7%,18%and 28%. HBsAg loss was not observed during the study.CVR served as independent predictors for absence of HBeAg.ADV resistant mutation occurred in 16(15.7%) patients who experienced either secondary treatment failure or suboptimal virologic response,among which fourteen were Lamivudine refractory patients,two were treatment naive patients.The cumulative probability at month 12 and month 24 were 9%,38%and 3%,9% respectively(P=0.008).The likelihood of developing ADV related mutation is related to older age,HBeAg negative status,liver cirrhosis and LVD resistance based on Cox univariate analysis.Importantly,liver cirrhosis was found to be the unique independent predictor for emergence of ADV-related mutation(P=0.018)According to direct sequencing,rtN236T was detected in 8 patients,rtA181V in 3,rtA181T+rtN236T in 1 and rtA181T+rtV214A in 1.Two novel substitutions rtV2141 and rtN236I was observed in 3 patients.On clonal analysis,rtN236T was detected in these three patients,rtV214I and rtN236V which serve as additional putative ADV resistant mutations was found to present either alone or in conjunction in a separate HBV genome.However,despite of showing by direct sequencing, rtN236I was not found in any clones of these patients.In conclusion,our data demonstrated 44.1%of early virologic response(EVR), 58.8%of initial virologic response(IVR) and 38.2%of complete virologic response(CVR) in chronic hepatitis B patients during adefovir monotherapy.Early virologic suppression at month 3 and/or initial virologic response at month 6 represented as crucial turning point to predict profound and sustained virologic suppression beyond 12 months as well as absence of HBeAg and seroconversion. 38%and 9%of cumulative incidence at month 24 was found in LVDr patients and treatment naive patients,respectively.Older age,HBeAg negative status,liver cirrhosis and lamivudine resistance were associated with an increased risk of genotypic resistance to adefovir.Drug resistant mutations confer to different nucleos(t)ide analogues does not show to present at the same HBV DNA isolate clone which support the combination therapy of adefovir and lamivudine.In order to explore the characteristics of mutation patterns in HBV P gene reverse transcriptase region(RT region) in lamivudine-refractory chronic hepatitis B (CHB) patients.One hundred and fifteen CHB patients who developed clinical resistance to lamivudine were enrolled,and direct sequencing of PCR products was applied to detect lamivudine genotypic resistance.And lamivudine resistant mutation was observed in 103 patients,the main mutation patterns were rtL180M+rtM204V and rtM204I,accounting for 58.3%and 22.3%,other resistant substitutions included rtL80V/I,rtT184S,rtA200V,and combined mutation of triple resistant substitutions was detected in RT region of 5 patients by direct sequencing.In lamivudine-treated patients,not only mutations at sites of rtL180 and rtM204,but also combined mutation at other sites in HBV P gene should be monitored in the drug resistant detection.Entecavir,as one of the options of rescue therapy for lamivudine refractory CHB patients is reported to have rapid,sustained suppression of HBV replication, combined with a requirement for multiple substitutions,creates a high genetic barrier to ETV resistance in nucleoside naive patients.In order to characterize virologic response,serological response and drug resistance profile of entecavir treatment for lamivudine-refractory CHB patients,we enrolled patients of chronic hepatitis B who had prior lamivudine treatment and developed lamivudine drug resistance before switched to oral administration of entecavir 1.0mg/d monotherapy at our clinical centre were prospectively observed.All patients enrolled had baseline serum HBV DNA = 4 log₁₀copies/mL and received entecavir 1.0mg/d monotherapy more than 12 months.Forty-one patients met the inclusion criteria,after 12 months of entecavir therapy at the dose of 1.0mg/d for lamivudine refractory chronic hepatitis B patients, 39%of patients enrolled achieved HBV DNA level undetectability,64%of 28 patients who had abnormal ALT level at baseline achieved ALT level normalization. Two(7.7%) of HBeAg-positive patients achieved HBeAg seroconversion at month 10 and month12 respectively.Entecavir genotypic resistance was detected in one patient with resistant substitutions at rt180M+rtM204V+rtS202G of HBV reverse transcriptase region in whom pre-existing lamivudine genotypic resistant mutant (rtL180M+rtM204V) could be detected from baseline serum.Entecavir therapy at the dose of 1.0mg/d is effective in treating lamivudine-refractory chronic hepatitis B patients,and careful monitoring for drug resistance should be performed.In the third part of our study,we enrolled three treatment naive patient received sequential therapy of nucleos(t)ide analognes at our clinic who started to received lamivudine for the treatment of chronic hepatitis B at 2001,2002 and 2004 respectively.Two of them switched to adefovir and one to entecavir for the emergence and prevention of drug resistance of lamivudine.Entecavir resistance was observed in all the three patients.Sera of at least every three months of each patients were collected for the detection of HBV DNA quantition,HBV maker,drug resistance detection,liver and renal function.Furthermore,serum samples at the commence,switching and cease of nucleos(t)ide analogue treatment underwent clonal analysis,other time point included virologic breakthrough,virologic rebound, virologic failure,10-25 clones were selected from each serum sample.No multi-drug resistant mutants conferring resistance to both lamivudine and adefovir present on the same viral genome was found in this study which also support combination therapy of nucleos(t)ide analogues.Two of the three patients presented pre-existing ETV resistant mutations before the commence:of entecavir,one of them was detected at 34^th month of lamivudine treatment,the other was detected after 26 months of lamivudine treatment and 24 months of adefovir(quit lamivudine),both patients developed genotypic and clinic drug iesistance which indicated that lamivudine treatment could possiblely select entecavir resistant mutant providing clinical challenge for the rescue therapy of lamivudine refractory chronic hepatitis B.