Hepatitis B Virus Drug Resistance Research Articles

HBV Drug Resistance Development, Testing, and Prevention

Chronic infection by hepatitis B virus (HBV) can result in cirrhosis, hepatic failure, and hepatocellular carcinoma (HCC) if left untreated. The extent of liver injury and the risk of HCC closely correlate with active viral replication, as indicated by high levels of circulating HBV DNA. Five oral antiviral agents, each targeting the viral DNA polymerase, are currently available in the United States to inhibit viral replication. Their effective use requires proper monitoring of drug resistance development. The current clinical guidelines recommend measuring “total” circulating HBV DNA to evaluate the efficacy of treatment and to serve as an indirect measure of drug-resistant mutant viruses. This may result in delayed identification of drug-resistant mutants that can cause hepatic injuries and decompensation. This review discusses the virologic mechanisms of drug resistance, the need and strategies for early detection of mutant viruses, and the recent studies on the prevention of drug resistance.

Acute hepatitis B infection associated with drug-resistant hepatitis B virus

Background Pre-existing antiviral-resistant hepatitis B virus (HBV) has been associated with primary non-response to lamivudine treatment in patients with chronic hepatitis B, but little is known of the capacity for resistant HBV to cause primary infection. Objective The study was to investigate if Beijing patients with acute hepatitis B (AHB) are infected with drug-resistant HBV. Study design Sera were collected from 201 NA-untreated patients with AHB. Direct polymerase chain reaction (PCR) sequencing was used to screen HBV reverse-transcriptase (RT) domain and clonal sequencing were performed for all resistance-positive samples. Results Direct PCR sequencing showed that 14 samples (7.0%) were positive for drug-resistant HBV variants, comprised of 11 with the lamivudine-resistant pattern rtM204I and/or rtM204V in the presence and absence of compensatory mutations rtL80I, rtV173L, and rtL180M; two with the adefovir-resistant pattern rtA181V; and one with the entecavir-resistant pattern rtL180M + rtS202G + rtM204V. Concomitance of resistance variants with wild-type HBV was observed in samples from 13 patients. Clonal sequencing verified direct sequencing results. Furthermore, variants associated with resistance to adefovir or entecavir were found in 3 samples. Conclusions Drug-resistant HBV strains, including those not resistant to lamivudine, are transmissible and can cause acute hepatitis B in China.

Hepatitis B virus: From diagnosis to treatment

During the next few decades, vaccination against hepatitis B virus (HBV) will dramatically change the epidemiological profile of this worldwide infection especially when Heath Policies encourage including HBV vaccination program for the newborns. However, it is still estimated that more than 2000 millions living people have met HBV. Symptomatic hepatitis with jaundice is less frequent than asymptomatic infection; however, as much as 350 millions of individuals remain chronically infected by HBV. In these cases, the need for efficient antiviral therapy remains clear when a viral replication is observed to control the risk of progression and the need for liver transplantation, which represents the only end-stage treatment. Indeed, patients having chronic hepatitis B (CHB) can now be successfully treated using nucleos(t)ide analogs (NA) or pegylated interferon (PEG-IFN). Therefore, beside vaccination, prevention of the progression of the disease to cirrhosis and liver decompensation, leading to end-stage liver disease and/or to hepatocellular carcinoma, by inhibiting viral replication seems to represent the best approach to improve survival. At last but not least, co-morbidities and other viral infections, leading also to chronic liver cirrhosis or liver inflammation such as the specific satellite delta virus (HDV), human immunodeficency virus (HIV) and/or hepatitis C (HCV) virus, are able to accelerate the progression and have to be taken in account. Interestingly, in treated infection, the dogma of the irreversibility of the liver fibrosis, when the cirrhosis is constituted, is tumbling down. In this review, we will focus on the clinical, virological and therapeutic aspects of hepatitis B infection in order to expose the proposals to follow-up and treat HBV-infected patients and the prevention of drug-resistant HBV mutants that frequently arise, leading to treatment failure and progression to liver disease.

On‐treatment monitoring of chronic hepatitis B virus infection: An Asian–Pacific perspective

Chronic infection with hepatitis B virus (HBV) is a major global health problem and an important cause of morbidity and mortality from sequelae of liver cirrhosis and hepatocellular carcinoma. In the past decades, better understanding of the natural history and immunopathogenesis of chronic HBV infection and of the development of many powerful antiviral agents has allowed us to improve therapeutic efficacy. Among these agents, nucleos(t)ide analogs are important and potent viral suppressors. However, when administered alone, they are not able to permanently eradicate HBV, and long-term maintenance therapy is required for therapeutic efficacy. Additionally, prolonged treatment is frequently associated with the emergence of drug-resistant HBV mutants. Before an 'ideal' drug(s), or drug combination, with optimal antiviral efficacy and negligible rates of drug resistance becomes available, the on-treatment monitoring approach using serum HBV DNA level as a predictor for therapeutic efficacy and drug resistance is useful. However, most countries in the Asia-Pacific region have low income economies, insufficient medical care systems, and low awareness of the disease among the general population and government officers. The easy approach of the road-map concept using an affordable drug to treat chronic HBV infection is more important in this region. There is already evidence that the long-term outcomes of chronic HBV infection can be improved under well-managed antiviral therapy. Profound and long-lasting suppression of HBV replication, either maintained on-therapy or sustained after stopping therapy, has been identified as the key determinant for achieving the goals of therapy, for reducing liver damage, and for preventing development of cirrhosis and/ or hepatocellular carcinoma.

Hepatitis B Virus Drug Resistance in HIV-1–Infected Patients Taking Lamivudine-Containing Antiretroviral Therapy

A cross-sectional study was conducted in HIV-1-infected patients receiving lamivudine-containing antiretroviral therapy (ART) to determine the prevalence and risk factors of hepatitis B virus drug resistance (HBV-DR). HBV DNA and HBV genotypic resistance test were performed. Patients were categorized into two groups: with and without HBV-DR. There were 84 patients with a mean age (standard deviation [SD]) of 42.2 (10.2) years and 77% were males. Median (range) duration of ART and lamivudine use was 46 (3-177) and 40 (3-140) months, respectively. Median (range) CD4 cell count was 352 (49-790) cells/mm(3). Of all, 19 (23%) had HBV-DR with a median (range) HBV DNA of 2.56 x 10(7) (2.54 x 10(3)-11 x 10(7)) IU/mL. In univariate analysis, there were no differences in age, gender, ART regimen, liver function test, anti-HBc antibody, anti-HCV antibody between the two groups. Patients with HBV-DR had a higher proportion of positive HBeAg (68.4% versus 3.8%, p < 0.001). In multivariate analysis, positive HBeAg (odds ratio [OR) 16.64; 95% confidence interval [CI], 3.31-83.60] and duration of lamivudine use [per 6-month increment, OR 1.24; 95% CI, 1.06-1.36] were significant risk factors for HBV-DR. All 19 patients with HBV-DR had lamivudine resistance with the mutations as follows: M204V/I (95%), L180M/A181T (95%), L80V/I (47%), V173L (32%), and N236T (21%). Of these, 95%, 84%, 84%, and 0% of patients had HBV-DR to telbivudine, entecavir, adefovir, and tenofovir, respectively. HBV-DR is common in HBV/HIV-1 coinfected patients receiving lamivudine-containing ART without tenofovir. Positive HBeAg and longer duration of lamivudine use are risk factors for HBV-DR. In addition to lamivudine resistance, cross-resistance to other anti-HBV drugs is also frequently observed.

Prevalence and clinical characterization of patients with acute hepatitis B induced by lamivudine‐resistant strains

Acute hepatitis caused by lamivudine (LMV)-resistant strains has not been reported, and the clinical impact of LMV-resistant strains on acute hepatitis is not known. The aim of this study was to investigate the molecular and clinical characteristics of patients with acute hepatitis B caused by LMV-resistant strains. Forty-five patients with acute hepatitis B were studied. Hepatitis B virus (HBV) subgenotypes and LMV-resistance mutations were determined by direct sequencing of the preS and polymerase regions, respectively. HBV subgenotypes A2 (n = 18), B1 (n = 1), B2 (n = 3), B3 (n = 2), C1 (n = 1), C2 (n = 19) and C6 (n = 1) were detected in patients with acute hepatitis. LMV-resistance mutations were detected in two patients. LMV-resistance mutations (L180M, M204I) were detected in a patient with subgenotype C2 who had acute self-limited hepatitis. The other patient with LMV-resistance mutations (L180M, M204V) was infected with subgenotype A2 and had severe hepatitis. LMV-resistant strains are rare, but they are starting to be found in patients with acute hepatitis B. Surveillance for detecting drug-resistant HBV strains would be important for clinical practice.

RtE218G, a novel hepatitis B virus mutation with resistance to adefovir dipivoxil in patients with chronic hepatitis B

Antiviral therapy is a potentially successful approach for the treatment of patients with Hepatitis B virus (HBV) infection. One antiviral agent is the nucleoside analogue adefovir dipivoxil (ADV). Its efficiency is compromised by the emergence of drug-resistant HBV mutants. Although three major ADV-resistant mutations of HBV are known, rtA181T/V and rtN236T, HBV mutations associated with ADV resistance have not been fully identified. We analyzed DNA sequences that covered a 244 base pair region of the HBV polymerase gene from patients with clinical manifestations of ADV resistance. A novel pattern of amino acid substitutions in HBV polymerase was detected in 26 out of 86 patients. This mutant exhibited a substitution of glycine for glutamic acid at residue 218 (rtE218G). Transient transfection of the HBV replication-competent construct including the rtE218G mutation was performed in HepG2 cells in order to determine the relevance of this mutation to ADV resistance. Phenotypic analyses demonstrated that the rtE218G mutation could independently confer resistance to ADV in vitro, with a 50% inhibitory concentration (IC(50)) 5.5-fold higher than wild-type HBV. RtE218G-mutated HBV also showed a decreased replication capacity in vitro, equal to 87% of wild-type HBV. The present study showed that the rtE218G mutation may be a novel ADV-resistant mutation. Further work will focus on resistance surveillance and cross-resistance analyses, and the molecular mechanisms involved.

Hepatitis B virus inhibition in mice by lentiviral vector mediated short hairpin RNA

BackgroundChronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.MethodsHepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.ResultsCo-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.ConclusionLentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.

Current trends in chronic hepatitis B management: results of a questionnaire

Diagnosis and treatment of chronic hepatitis B (CHB) have improved in recent years with introduction of new oral antiviral drugs. Aim of this study was to analyze general knowledge, therapeutic and diagnostic trends of physicians treating CHB in Spain. A questionnaire was distributed to 692 physicians of the Spanish Association for the Study of the Liver. The questionnaire assessed habits in CHB management and approach to a pair of hypothetical CHB cases: HBeAg positive and HBeAg negative. One hundred and nineteen (17%) physicians answered the questionnaire. Most of them correctly identified indications for CHB treatment (95%), and had experience with lamivudine (89%), adefovir dipivoxil (84%) and interferon (76%), but only 45% knew the efficacy rate of these drugs. Seventy-six percent recommended pegylated interferon as initial treatment for HBeAg-positive patients, and 86% would treat HBeAg-negative patients with oral antiviral drugs (42% with lamivudine, 36% with adefovir dipivoxil, and 8% with entecavir). Hepatitis B virus (HBV)-DNA determination was used to monitor therapy by 74%, and only 24% used HBV drug resistance test. The only independent factor associated with adequate use of drugs and correct monitoring was experience of treating more than five patients per year. Antiviral prophylaxis for HBV carriers receiving immunosuppression was indicated by 78% of physicians. Physicians working at university hospitals were significantly more likely to recommend antiviral prophylaxis than physicians working at nonuniversity hospitals (86 vs. 65%; P=0.009). CHB management decisions are not always based on scientific evidence. Continuous medical education could improve knowledge and management of CHB.

Lamivudine and adefovir resistance in children and young adults with chronic hepatitis B

Objective Long-term lamivudine (LAM) and adefovir (ADV) treatment has been found to induce the emergence of drug-resistant hepatitis B virus (HBV) in a significant number of patients with chronic hepatitis B (CHB) infection. The aim of our study was to evaluate the LAM and ADV mutations detected in our patient group. Materials and methods Twenty-four patients diagnosed with CHB were enrolled in this study. The patient group consisted of those who had received 6 months of treatment with interferon-α and who did not response to this therapy. Patients were evaluated based on virologic and serologic response to therapy, and were classified as responders or non-responders. The treatment of non-responders continued with LAM (3 mg/kg/d, maximum 100 mg/d). Due to a lack of response to treatment, ADV (10 mg/g) was added to the treatment regimen of eight young adult patients. The mutations associated with HBV drug resistance were investigated using reverse hybridization methods and PCR. Results The mutation studies indicated that 14 (58.4%) of the patients had resistance. Three patients developed ADV-associated mutations (A181T), one after 18 months of ADV; the other two had undergone 18 and 36 months of LAM therapy without ADV exposure. Although the average LAM treatment period of the patients with LAM resistance was longer than for those in whom no resistance was detected, no statistically significant difference was found. Conclusions HBV treatment with nucleoside analogues results in the development of mutant strains, leading to drug resistance. Therefore genotypic resistance testing is important in planning and monitoring HBV treatment.

Two types of drug-resistant hepatitis B viral strains emerging alternately and their susceptibility to combination therapy with entecavir and adefovir

The most serious problem of nucleoside/nucleotide analogue therapy for hepatitis B virus (HBV) infection is the emergence of drug-resistant mutant virus. Here, we describe a patient with chronic hepatitis B infection with a complex drug-resistant mutant virus during sequential therapy with lamivudine (3TC), entecavir (ETV) and adefovir dipivoxil (ADV). The patient was a 52-year-old male with positive hepatitis B e antigen and high HBV DNA (>7.6 log(10) copies/ml). Initial 3TC monotherapy offered little benefit and 3TC resistance was established by the virus with rtA181T and not rtM204V/I. HBV DNA was reduced slightly by replacement with ETV monotherapy and was followed by virological breakthrough. At that time, rtA181T was undetectable and the virus with rtM204V and rtL180M became predominant. ETV resistance was established by an additional rtS202G mutation. Efficacy of subsequent combination therapy with ADV and 3TC was limited because of reappearance of the virus with rtA181T, which might confer cross-resistance to 3TC and ADV. Final combination therapy with ETV and ADV reduced HBV DNA to 3.7 log(10) copies/ml for 5 months, which was the most effective therapy for this patient. Thus, two kinds of mutant viruses (rtM204V-related and rtA181T-related) appeared alternately in this patient. Combination therapy with ETV and ADV might have been effective because these drugs share therapeutic roles, that is, ETV affects the rtA181T-related virus and ADV affects the rtM204V-related virus. This is the first report suggesting clinical significance of combination therapy with ETV and ADV for controlling replication of the complex drug-resistant mutant HBV.

Benefits and risks of combination therapy for hepatitis B #

In successful antiviral therapy of hepatitis B, drug combinations, particularly combinations without cross-resistance, can delay or prevent the emergence of drug-resistant mutants. Because drug-resistant mutants are archived and may limit future therapeutic options, prevention is important for long-term therapeutic efficacy. Additionally, combining drugs may achieve synergistic or additive antiviral effects compared with single drug therapy. Undesirable aspects of combination therapy include higher treatment costs and possibly lower adherence rates (due to pill number or complexity of regimen). Potentially harmful effects of combination therapy include higher rates of side effects, reduced efficacy due to drug competition, and the risk of multidrug-resistant hepatitis B virus (HBV) if combination therapy is insufficient to prevent resistance. Combination therapy has been shown to reduce the rate of drug resistance in chronic hepatitis B, but only when drugs with a low barrier to resistance are used (lamivudine, adefovir). Combination therapies may achieve greater degrees of HBV DNA suppression, but this has not been associated with higher rates of seroconversion (hepatitis B e antigen or hepatitis B surface antigen) compared to single drug therapy. The benefit of combination therapy has yet to be demonstrated with agents that are associated with a high barrier to resistance (tenofovir, entecavir). The use of combination therapy is recommended in specific patient groups: those with decompensated cirrhosis, those coinfected with human immunodeficiency virus and HBV who are on antiretroviral therapy, those who have undergone liver transplantation, and those with drug-resistant HBV infection. There is insufficient evidence to recommend combination therapy as first-line therapy for all patients with chronic hepatitis B.

Clinical efficacy of adefovir dipivoxil combined with anti-tuberculosis drugs inn treatment for chronic hepatitis B complicated with pulmonary tuberculosis

Objective It is purposed to evaluate clinical efficacy of adefovir dipivoxil combined with anti-tuberculosis (TB) drugs in treatment for chronic hepatitis B (HB) complicated with pulmonary TB. Methods Ninety-four eases of pulmonary TB complicated with chronic HB receiving anti-TB drug treatment at Liaocheng Hospital for Infectious Disease during September 2006 to September 2008 were divided into treatment and control groups matched on their pulmonary TB lesion, damage in liver function and HB virus (HBV) DNA load. Forty-seven cases in the treatment group were administered with anti-TB drugs, i.e., isoniazid (INH) 0. 3 g daily, rifapentine (RFT) 0.45 g twice a week, pyrazinamide (PZA) 1.5 g daily, streptomycin (SM) 0. 75 g daily or ethambutol (EMB) 0. 75 g daily, and regular liver-protection drags, as well as oral adefovir dipivoxil 10 mg daily for eight months, and another 47 cases in the control group were administered with the same anti-TB drugs and regular liver-protection drugs as the treatment group. Two to eight weeks after treatment, clinical symptoms, liver function, HBV DNA load, negative conversion of acid-fast staining bacteria in sputum smear, and absorption of pulmonary TB lesion were evaluated for all the patients in both groups. Data were analyzed with U-test and χ2-test, respectively. Results After treatment, damage in liver function worsened in three cases (6.4%), as compared to that in 47.3 percent of the control group (P<0.05). Clinical symptoms, including tiredness, loss of appetite, abdominal distension, uncomfortable in upper abdomen, and so on, appeared in three cases of the treatment group (6.4%) during anti-TB treatment, as compared to those in 18 cases of the control group (47.3%) (P<0.05). HBV DNA conversed to negative (less than 1.0×103 lg copies/ml) in 24 cases of the treatment group (51.1%), HBV load went down to 1.0×103-1.0×104 lg copies/ml in 23 cases (48. 9%), sputum smear bacteria conversed to negative in 41 cases (87. 2%) and pulmonary lesion absorbed in 45 cases (95.7%), all with statistically significance as compared to those in the control group (47. 3% , 0. 0% , 2. 6% , 39. 5% and 50. 0% , respectively, P <0. 05). Renal function was all in normal in both groups and no patient with drug resistant variant HBV was found during and after treatment, and no obviously adverse reaction was found in patients with adefovir dipivoxil. Conclusion Adefovir dipivoxil has a good safety and can reduce laver damage in anfi-TB drug treatment for patients of chronic hepatitis B complicated with pulmonary TB. Key words: Hepatitis B, chronic; Tuberculosis,pulmonary; Antitubercular agents

Heterologous Replacement of the Supposed Host Determining Region of Avihepadnaviruses: High In Vivo Infectivity Despite Low Infectivity for Hepatocytes

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22–90 (Du-He4) or its subsegments 22–37 and 37–90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 108 viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants.

Dynamics of lamivudine‐resistant hepatitis B virus during adefovir monotherapy versus lamivudine plus adefovir combination therapy

Adefovir dipivoxil has been used alone or together with lamivudine to suppress lamivudine-resistant hepatitis B virus (HBV). However, the dynamics of HBV populations under different selection pressures and their impact on treatment outcome are poorly understood. Pyrosequencing was applied to quantify longitudinally the evolution of wild type and lamivudine/adefovir-resistant HBV. Eight patients, with lamivudine-resistant HBV, were randomized to receive adefovir monotherapy or adefovir/lamivudine combination therapy for a median of 79 and 71 weeks, respectively. Pyrosequencing proved highly sensitive with a lower limit of quantitation of minor HBV populations of 2% irrespective of viraemia levels. Adefovir/lamivudine treatment resulted in greater viraemia reduction than adefovir monotherapy. During combination therapy, lamivudine-resistant HBV populations (codons 180 and 204) remained dominant (>90%) and no adefovir-resistance developed. During adefovir monotherapy, reversion to wild-type HBV was detected in two patients with one patient accumulating rapidly adefovir-resistant HBV along with increased viraemia. In conclusion, the dynamics of drug-resistant HBV strains vary under different selection pressures which have a significant impact on the success of rescue therapy, as well as for the selection of new mutations. The use of techniques such as Pyrosequencing provides an evidence-based approach for successful management of drug-resistant HBV.

The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound†

The hepatitis B virus (HBV) mutation that encodes rtA181T is selected in the viral polymerase during antiviral drug therapy and can also encode a stop codon in the overlapping surface gene at amino acid 172 (sW172*) resulting in truncation of the last 55 amino acids of the C-terminal hydrophobic region of the surface proteins. This mutation is usually detected as a mixed population with wild-type HBV. In vitro analysis revealed that the rtA181T/sW172* variant is not only defective in secretion of viral particles causing intracellular retention of surface proteins, it also has a dominant negative effect on virion but not subviral particle secretion when coexpressed with the wild type. This dominant negative effect was attributed to the truncated S protein alone. Furthermore, these truncated surface proteins were less glycosylated, and the truncated L protein was able to support virion secretion. Examination of sequential HBV DNA levels in patients failing lamivudine or adefovir therapy where only the rtA181T change was detected via polymerase chain reaction sequencing revealed that viral load rebound did not occur or was not as large as usually observed with drug-resistant HBV. The rtA181T/sW172* variant has a secretory defect and exerts a dominant negative effect on wild-type HBV virion secretion. The selection of rtA181T/sW172* reduced the typical extent of virological breakthrough, resulting in a missed diagnosis of drug resistance if viral load was used as the only criterion for drug failure, necessitating HBV polymerase chain reaction sequencing or other genotypic methods to diagnose antiviral drug resistance in these cases.

Evolution of Primary and Compensatory Lamivudine Resistance Mutations in Chronic Hepatitis B Virus-Infected Patients during Long-Term Lamivudine Treatment, Assessed by a Line Probe Assay

With the availability of more potent nucleotide/nucleoside analogues, the early detection of drug-resistant mutants of hepatitis B virus (HBV) is important for the strategic treatment of chronic hepatitis B. We studied 336 serum samples from 80 patients chronically infected with HBV who were receiving lamivudine treatment for the presence of lamivudine resistance mutations at codons 80, 173, 180, and 204 of the HBV polymerase. The sequencing data were compared with the results generated with the INNO-LiPA HBV DR (drug resistance) v2 strip, a line probe assay (LiPA) covering wild-type and mutant motifs, for resistance mutations to lamivudine and adefovir dipivoxil. This method provided at least the same information as sequencing for 99.1% of all codons analyzed. On the basis of the LiPA results, 20 of 80 patients developed a lamivudine resistance mutation after 1 year. In all 20 patients, the mutation occurred in the YMDD motif at reverse transcriptase position 204 (rt204; M204V/I) either with or without the compensatory mutation at position rt180 (L180M). A compensatory mutation at position rt80 (L80V/I) was detected in half of these patients. After 36 months, a compensatory mutation was seen at position rt173 (V173L) in 3/15 patients. Time-to-event survival analysis indicated a 2.8 times greater chance for LiPA to detect a given mutation than sequencing at any moment in time (hazard ratio, 2.8, 95% confidence interval, 1.79, 4.41; P < 0.0001). These results demonstrate that a highly sensitive and specific assay such as the INNO-LiPA HBV DR v2 can precociously detect and monitor the emergence of primary and compensatory lamivudine resistance mutations in patients chronically infected with HBV and is more sensitive than sequencing.

In Vitro Drug Susceptibility Analysis of Hepatitis B Virus Clinical Quasispecies Populations

Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC(50)) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC(50). In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.

Prediction of treatment outcomes for lamivudine

Prediction of treatment outcomes for lamivudine

YMDD motif variants in inactive hepatitis B carriers detected by Inno‐Lipa HBV DR assay

Mutations of hepatitis B virus (HBV) polymerase, especially occurring at the highly conserved YMDD region, are related to resistance to lamivudine. Although these mutations are frequently secondary to lamivudine use, they can also occur naturally. The aim of the present study was to determine the prevalence of YMDD variants that exist naturally in patients who are inactive HBV carriers. Seventy-one adult inactive HBV carriers were studied. All of the patients were confirmed to have maintained normal alanine aminotransferase (ALT) values for one or more years by monitoring serum ALT levels at 3-monthly intervals. None of the patients received interferon or antiviral agents. YMDD variants were analyzed by the HBV Drug Resistance Line Probe assay (Inno-Lipa HBV-DR). YMDD variants were detected in 13 (18.3%) of the 71 anti-HBe positive inactive HBV carriers. Of the 13 patients, 10 (76.9%) also had accompanying L180M mutation. The combination of wild type and YMDD variant HBV was present in 11 of 13 patients. In two patients, only YIDD and/or YVDD variants plus L180M were detected without the presence of wild YMDD motif. Naturally occurring YMDD motif variants were detected at a high rate in a group of lamivudine-untreated inactive HBV carriers.