Detection And Analysis Of Adefovir Genotypic Resistance In Chronic Hepatitis B Patients

Background and ObjectiveAntiviral therapy is of paramount importance to chronic hepatitis B(CHB) patients.Two categories of anti-HBV drugs have been approved by State Food and Drug Administration(SFDA):Interferons(IFN) and nucleoside(tide) analogues which include lamivudine(LAM),adefovir dipivoxil(ADV),telbivudine(LdT) and entecavir(ETV).Since approved,ADV has been widely used in CHB patients, especially in LAM refractory patients.Many mutations in HBV reverse transcriptase (rt) region had been reported to be associated with ADV resistance,which include rtV84M,rtS85A,rtV214A,rtQ215S,rtI233V and rtN238T/D,et al.But only rtA181V/T and rtN236T are generally accepted.According to ADV pivotal trials in HBeAg negative CHB patients,the cumulative incidences of resistance at 1,2,3,4 and 5 years were 0%,3%,11%,18%and 29%.Antiviral resistance mutation may be followed by virological breakthrough,biochemical breakthrough,hepatic flare and hepatic decompensation.Regular monitoring,early detection of resistant mutations and prompt and effective rescue therapy are important to successful treatment.Serum HBV DNA assay is key to ADV resistance monitoring.But serum HBV DNA may also be influenced by patient compliance,drug metabolism and virus natural fluctuation besides resistance mutation.Specific ADV resistance assay is necessary to confirm ADV genotypic resistance.Currently reported ADV genotypic resistance assays include:direct sequencing of polymerase chain reaction(PCR) product,cloning sequencing,line probe reverse hybridization,PCR-restriction fragment length polymorphism(RFLP),real time fluorescent quantitation PCR and resitriction fragment mass polymorphism.Real time fluorescent quantitation PCR has not been used in clinic.Line probe reverse hybridization kit is now commercially available.But it is not cost-effective,as is RFMP assay.Cloning sequencing is both expensive and laborious.In China,PCR product direct sequencing and PCR-RFLP have been widely used.As in PCR product direct sequencing,di-deoxy sequencing is the most common used method for sequencing.But it has the disadvantage of low sensentivity and can detect mutations only when at least 20%～30%mutant species are present.PCR-RFLP assay is cost-effective,but specific primers and restriction sites are need for each mutation. During PCR-RFLP assay,restriction followed PCR amplification may be influenced by personnel error and restriction system.HBV resistance assays with adequate sensitivity and specificity,cost-effective and high automatization are needed for clinical ADV resistance monitoring.Gene chip assay has high throughput and high sensitivity.Reports on gene chip for LAM resistance assay showed the low limit of detection is 2～3 log₁₀ copies/ml and could detect the mutation even when only 10%mutants is present.But no gene chip assay for ADV resistance has been reported.Also pyrosequencing was reported in LAM resistance assay with high sensitivity and the ability to detect at least 10% mutants.And there is no report about pyrosequencing for ADV resistance assay.In our study,we collected serum samples and clinical data from ADV treated patients who once underwent virological breakthrough(VB group) or could not achieve virological response after at least 48 weeks therapy(VF group).ADV resistance profiles of these patients were detected with the gene chip,PCR products di-deoxy sequencing and pyrosequencing.Results from different assays were compared.Besides,resistance profiles were compared between VB group and VF group.Correlation between ADV resistance and HBV genotype were also analyzed.Partâ… :Analysis of assays for ADV genotypic resistance in chronic hepatitis B patientsIn this part,we collected serum samples and clinical data from ADV treated patients who once underwent viral breakthrough or could not achieve virological response after at least 48 weeks therapy.We analyze the ADV resistance profiles of these patients with the gene chip,PCR products di-deoxy sequencing and pyrosequencing.Results of different assays were compared and possible causes for undetected mutations in each assay were analyzed.Also the mutants ratios of different mutants were compared.Materials and Methods:1.Serum samples from ADV treated patients(10mg/d) who underwent virologic breakthrough(virologic breakthrough group,VB group) or could not achieve virologic response after at least 48 weeks treatment(virologic failure group,VF group) were collected for extraction of HBV DNA.HBV rt regions were amplified with nested PCR.PCR products were used for di-deoxy sequencing.The results were analyzed by NTI Vector 9 and Chromas software.2.Serum samples were used for extraction of HBV DNA.After PCR amplification,ADV resistance was detected with Jingxin^? HBV resistance gene chip kit.The results were analyzed by the software of Jingxin^? HBV resistance gene chip assay system.3.Serum samples were used for extraction of HBV DNA.After PCR amplification,ADV resistance was detected with Genetech(Shanghai) HBV ADV resistance pyrosequencing kit.Resistance and ratio of mutant were analyzed with software of sequencer.4.The t test,t' test,Wilcoxon test,Pearson test and Yates test were used for statistic analysis with SPSS 11.5 software.Results:1.Serum samples were collected from 106 VB group patients and 104 VF group patients.There were no statistical differences in sex ratio and mean age between the two groups.2.ADV resistance in two groups:(1).PCR product di-deoxy sequencing results:All patients got positive amplification after nested PCR and products were used for sequencing.38 patients in VB group and 14 in VF group were positive for ADV resistance.(2).Gene chip assay results:21 cases showed no amplification after PCR(12 in VB group and 9 in VF group).33 cases in VB group and 14 in VF group were ADV resistance positive.(3).PCR product pyroseqencing results:10 cases showed no amplification after PCR(5 in VB group and 5 in VF group).42 cases in VB group and 22 in VF group were ADV resistance positive.(4).Totally 67 ADV resistance paitents(45 cases in VB group and 22 in VF group) and 92 mutants were detected.3.Comparing results from different assays:(1).The accordance rate between the 3 assays was 74.8%.The accordance rate between the di-deoxy sequencing and pyrosequencing was 83.8%.The accordance rate between the gene chip assay and pyrosequencing was 81.4%.The accordance rate between the di-deoxy sequencing and gene chip assay was 83.3%.(2).Positive resistance rates of different assays in confirmed resistance patients: Pyrosequencing assay detected more resistance patients(P=0.002) and more mutants (P=0.002) than di-deoxy sequencing.Also pyrosequencing assay detected more resistance patients(P＜0.001) and more mutants(P＜0.001) than gene chip.There was no statistical difference in resistance patient ratio(P＞0.05) and mutant ratio(P＞0.05) between gene chip assay and di-deoxy sequencing.(3).Positive predictive values of different assays:PCR di-deoxy sequencing and pyrosequencing are both assays based sequencing and the positive predictive values were both 100%.In this study,positive predictive value of gene chip assay is also 100%.4.Analysis of undetected mutants of different assays(1).PCR product di-deoxy sequencing had 21 false-negative ADV resistance muations and the causes for undetected mutations can be attributed to low mutant ratio and serum HBV DNA levels.(2).Gene chip had 29 false-negative ADV resistance muations and the cause for undetected mutations can be attributed to low mutant copies in serum.(3).PCR product pyrosequencing had 6 false-negative ADV resistance mutations.5.Analysis of ratio of mutants:(1).Mean mutant ratio in virus population of rtA181T,rtA181V and rtN236T when detected were 38.96%,54.89%and 54.01%.Mean mutant ratio of rtA181V is higher than that of rtA181T(P=0.005).No statistical difference was found in mean mutant ratio between rtA181V and rtN236T(P＞0.05).(2).In VB group,24 patients had single mutation resistance and the ratio of mutants in 14/24 patients＜50%.In VF group,20 patients had single mutation resistance and the ratio of mutants in 12/20 patients＜50%.Conclusion:1.Initial study shows the positive resistance patients and mutant rates of pyrosequencing are higher than both di-deoxy sequencing and gene chip assay.There are no statistical differences in positive resistance patients and mutant rate between di-deoxy sequencing and gene chip assay.2.For PCR product di-deoxy sequencing,the causes for undetected mutations can be attributed to low mutant ratio and serum HBV DNA level.And for gene chip assay,the cause for undetected mutations can mainly be attributed to low mutant copy numbers in serum.3.In both VB and VF patients,the mutant when detected may not be the dominant species in the virus population.The correlation between ratio of mutant and HBV DNA needs further study.Partâ…¡:Correlation factors to ADV resistance in CHB patientsFrom partâ… ,it has been demonstrated that in both VB and VF groups,only partial patients had ADV resistance.So we further analyzed the possible correlation factors to ADV resistance in both groups and the correlation between HBV genotype and ADV resistance. Materials and Methods:1.Analysis of HBV genotype:nested PCR were used to amplification HBV HBsAg region.The PCR products were used for sequencing and the results were used for genoytyping with the NCBI HBV genotyping software.2.The procedures for detecting ADV resistance see partâ… .3.The t test,t' test,Wilcoxon test,Pearson test and Yates test were used for statistic analysis with SPSS 11.5 software.Results:1.In VB group,18 patients had genotype B HBV and 88 patients had genotype C. In VF group,21 patients had genotype B HBV and 83 patients had genotype C.No other genotypes were detected in both groups.2.Totally 67 patients were ADV resistance(45 in VB group and 22 in VF group) and 92 mutants were found(rtA181V/T:63 mutants;rtN236T:29 mutants).The resistance rate in VB group is higher than that in VF group(P＜0.001).VB group had more combination mutant patients(P=0.013) and less single rtA181T mutant patients(P=0.017) than VF group did.3.In VB group,there were no statistical differences in the rising level of HBV DNA(P＞0.05) and biochemical breakthrough rates(P＞0.05) between patients with single mutant and combination mutants.But patients with rtA181T mutant had lower rising level of HBV DNA(P=0.014) and higher rate of biochemical breakthrough(P =0.023) than resistance patients without rtA181T mutant.4.There was no statistical difference(P＞0.05) in resistance rate between genotype B and C patients.Single rtN236T mutant patients are more common in genotype B ADV resistant CHB patients than in genotype C(P=0.011).5.Both in VB group and VF group,ADV resistance patients are more common in LAM refractory patients than in nucleoside-na(i|¨)ve patients(VB group:P=0.010; VF group:P=0.020).Conclusion:1.In ADV resistance mutants,rtA181V/T is more common than rtN236T.Both ADV treated patients underwent virological breakthrough and those who did not achieve virological response after at least 48 weeks therapy should take ADV resistance assay for possible treatment modification.2.In virological breakthrough patients,rtA181T mutant can decrease the HBV DNA rising level,but increase the possibility of biochemical breakthrough.3.Initial study showed single rtN236T mutant is more common in genotype B ADV resistance patients.Further validation and possible mechanism studies are needed.4.In both ADV treated patients underwent virological breakthrough and those who did not achieve virological response after 48 weeks treatment,ADV resistances are more common in LAM refractory patients than in nucleoside-na(i|¨)ve patients.