Phenotypic resistance is an in vitro demonstration of decrease susceptibility of an HBV polymerase to an antiviral treatment, confirmed by the demonstration of a given mutation amino acid substitution.

Viral resistance or viral breakthrough indicates that the HBV DNA level has increased after initial suppression during antiviral therapy. The clinical criterion generally adopted is greater than one log₁₀ increase from nadir in a patient who is compliant and is still on treatment.

Diagnosis of viral resistance

Genotypic resistance is diagnosed by sequence analysis on the HBV genome in the viral polymerase domain by direct sequencing assays or line probe assays. Direct sequencing of PCR products may not be sufficiently sensitive when viral load is minimal. Although it involves tedious laboratory work, it allows full-length reverse transcriptase sequencing for the detection of major or consensus sequence, and new mutations and is vital for research analysis. Line probe assays, such as INNO-LiPA DR v2®, are user friendly and more sensitive, but can only detect known mutations designed for the probes, thus more suitable for clinical practice.[3] The advantage of cloning and sequencing of the viral genome clones is that it allows viral dynamics to be studied in vivo. The replication fitness of the mutants can be studied. There are a number of technologies under development which facilitate the quick and accurate diagnosis of genotypic resistance, e.g. DNA chip technology, and mass spectrometry even in the presence of overwhelming wild-type HBV;[4] and there is also a report from Taiwan on a single-tube PCR reaction containing two consecutive steps to detect lamivudine resistance quantitatively, and then, using a novel annealing curve analysis to differentiate YMDD, YIDD and YVDD mutants by their distinct Tm values even when they exist in less that 10% of the total viral population.[5] In most clinical practices in Asia, genotypic analysis is not readily available. Diagnosis of resistance by viral breakthough requires close monitoring of the serum HBV DNA level to detect increase in serum HBV DNA by one log₁₀or more from nadir after confirmation of drug compliance.

Clinical significance of viral resistance

The crucial goal of antiviral therapy for CHB is persistent and potent suppression of viral replication. NAs are evaluated by speed and efficacy in log reduction of the serum HBV DNA level. In HBeAg negative CHB patients, this is evaluated by the proportion of treated patients reaching undetectable serum HBV DNA by PCR assays. In HBeAg positive patients, it is HBeAg loss or seroconversion together with undetectable serum HBV DNA by PCR assays. A requirement of long-term or even life-long therapy is expected as virologic response is not often sustained when off therapy. This theory is controversial however, due to lack of clinical data and surrogate markers for the clinically significant reduction, or better still, elimination of intrahepatic cccDNA. Furthermore, long-term NA therapy will be acceptable only if it is safe, cheap, and has a minimal resistance risk. The latter is vitally important since the emergence of resistant mutants is usually followed by viral breakthrough, relapse of hepatitis, and reversion of clinical benefit; making it neither safe nor cheap.[6]
 

Lamivudine resistance

Lamivudine resistant mutants at domains B and C of the polymerase YMDD loci, namely rtM204I/V and rtL180M, can be detected genotypically as early as month six of therapy. On extended therapy, the incidence increases as demonstrated in the Asian Lamivudine Study on HBeAg positive CHB patients, at 17%, 40%, 55%, 67% and 69% of the treated patients at the end of each successive years of therapy[7] and 23% and 31% of patients after one and two years of therapy, respectively in HBeAg negative CHB.[8] YMDD mutants initially appear to be less competent in replicating but viral breakthrough and elevation of serum ALT occurs in a majority of patients. Integrated analysis of all multicentre international clinical trials showed that the possibility of encountering ALT elevation and hepatitis flare increased with the duration of harbouring YMDD mutants. 40.6% of patients with YMDD mutants experienced acute exacerbation 4 to 94 weeks after its emergence. Occasionally hepatitis flare and even hepatic decompensation occurs. HBeAg seroconversion may occur in some patients after relapse of hepatitis.[ 9] Worsening liver histology was observed after 3 years of therapy amongst those harbouring MYDD resistance.[10] Deterioration in fibrosis occurred among 57%, 44% and 50% of patients who harboured YMDD mutants for <1 year, 1-2 years and >2 years respectively compared to 28% among patients without YMDD.[11] Progression of liver disease and development of HCC occurred more frequently among patients harbouring YMDD mutants. The benefit of longterm therapy in preventing disease progression in patients with advanced fibrosis or cirrhosis was negated by the emergence of YMDD mutants.

Only 5% of patients without genotypic resistance experienced disease progression, compared to 15% amongst those who harboured resistant mutants. This occurred in 49% after 3 years of lamivudine therapy.[12] Lamivudine therapy for patients presenting with severe acute exacerbation induced higher HBeAg seroconversion rates (78% versus 52% control; p=0.02). However, 33% developed lamivudine resistance and virological breakthrough by year 5.[13] Lamivudine resistant mutants are susceptible to adefovir or tenofovir therapy, but have cross resistance with emtricitabine, telbibudine and entecavir, possible clevudine.

Adefovir resistance

rtN236T and rtA181V mutations are associated with decreased susceptibility to adefovir. The overall incidence of adefovirresistance mutation is low. In HBeAg-positive CHB treated with adefovir dipivoxil 10 mg daily, no resistance was reported after the first year of therapy. 65 patients (74% Asian, 23% Caucasian, median baseline serum HBV DNA 8.45 log ₁₀ copies/mL were enrolled for the Long Term Safety and Efficacy Study. At 5 years, 58% achieved HBeAg loss and 48% seroconversion. Adefovir resistance mutations A181V or N236T developed in 13 (20%) of treated patients; the first observation was at study week 195.[14] In HBeAg-negative CHB patients, genotypic resistance was detected at 0 %, 3%, 11%, 18% and 29% at the end of each successive year of therapy.[15] Adefovir resistance may be associated with significant viral rebound and fatal hepatic decompensation has been reported.[16] Adefovir dipivoxil-resistant mutants remain susceptible to lamivudine, emtricitabine, telbivudine, and entecavir in vitro. The rtA181T/V HBV is resistant to adefovir and all the L-nucleosides, but sensitive to entecavir. [17] There are reports of primary non-responders to adefovir therapy which involved a rare HBV variant with valine at position 233 of the reverse-transcriptase domain instead of isoleucine (rtI233V). This HBV variant displayed sensitivity to tenofovir in vitro[18] and also sensitivity has been demonstrated with entecavir by both in vitro and in vivo studies.[19]

Entecavir resistance

Entecavir has a high genetic barrier and drug resistance emergence requires at least 3 mutations, namely rtL180M + rtM204V + rtT184, rtS202 and/or rtM250. In treatment naïve patients, less than 1% of treated patients showed viral breakthrough with genotypic resistance after the first three years. By years 4 and 5, 1-2% showed resistance profile. Controversy exists as to whether these patients harboured lamivudine resistance mutations at baseline, either naturally or lamivudine-induced resistance due to previous exposure. In the pivotal clinical trial of HBeAg positive and HBeAg negative CHB patients, genotyping all patients with PCR-detectable HBV DNA at weeks 48, 96, or end of dosing identified seven additional patients with lamivudine resistance mutations, including one with simultaneous emergence of resistance to both lamivudine and entecavir. Eight of the ten patients had lamivudine resistance detectable at baseline, but seven of them subsequently achieved undetectable HBV DNA levels on ETV therapy. It was suggested that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to entecavir resistance in nucleoside naïive patients. However, the cumulative probability of a virologic breakthrough due to entecavir resistance through 4 years is less than 1% in naïve and 6%, 15% 35% and 40-43% after each successive year of therapy in lamivudine refractory patients.[20] In a detailed analysis of a patient who developed resistance to entecavir following lamivudine breakthrough, a mixture of  lamivudineresistant HBV strains coexisted following viral breakthrough to lamivudine. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped transiently. Three years later, the viral level rose again with a complex mixture of entecavir-resistant strains, all harbouring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation. This mutant did not show the highest viral genome replication capacity but it conferred one of the strongest resistance levels to entecavir.[21]

Telbivudine resistance

The c-domain mutation rtM204I and the B-domain mutation rtA181T/V are the common mutations associated with telbivudine resistance. In the GLOBE randomised control trial for HBeAg-positive and HBeAg negative CHB, telbivudine was shown asto be more potent than lamivudine. However, among those treated with telbivudine, resistance emergence is not satisfactory. In HBeAg positive CHB, after one year therapy, 5.0% of the telbivudine treated experience viral breakthrough with genotypic resistance (vs. 11% among lamivudine-treated) and in HBeAg negative CHB patients, the resistance rate at year one was 2.3% (vs. 10.7% among lamivudine treated).[22] After 2 years, this increased to 22% in HBeAg positive and 9% in HBeAg negative CHB patients treated with telbivudine.[23]

Tenofovir resistance

Tenofovir is active against HBV in patients with or without lamivudine-associated mutations. Development of clinical or virological breakthrough during tenofovir therapy has not been reported so far.[24] Phenotypic analyses revealed that constructs harbouring rtA194T combined with rtL180M and rtM204V displayed an over 10-fold increase in the IC50 for TDF compared with the wild type.[25] Tenofovir was approved by the FDA for the treatment of CHB in 2008 and more clinical data is anticipated to confirm the promising response and low resistance risk.

Management of patients with viral resistance

The approach and strategy for managing patients with treatment induced resistance have been evolving. Before the availability of effective antiviral therapy for lamivudine-resistant mutants, discontinuation of lamivudine therapy was one option since it was no longer effective. The viral level may rebound as wild type HBV re-emerges. However, the ALT level remains normal or minimally raised and serious relapse of hepatitis rarely occurs. Standard and pegylated interferon therapy is another option with response seen in those with ALT elevation. Controlled clinical trials to evaluate pegylated interferons for lamivudine refractory CHB patients are going on. Switching to, or adding of, another NA(s) such as adefovir, entecavir, tenofovir with or without emtricitabine are possible options with initial response, pending confirmation from further studies. The long-term efficacy awaits confirmation to determine the appropriate rescue regimen that can minimise the risk of emergence of multiple drug resistance.

Resistance mutations

Switching to adefovir monotherapy for patients with lamivudine resistance may fail as adefovir is not very potent and subsequent emergence of adefovir resistance may occur. A higher adefovir dosage of 20 mg daily has been reported to be of benefit without the added renal toxicity. In compensated or decompensated liver cirrhosis associated with lamivudine resistance, switch to both adefovir monotherapy and adefovir add-on lamivudine therapy significantly improved the Child- Pugh’s score, serum ALT, and HBV-DNA levels. The switch therapy was considered a reasonably safe and cost-effective approach.[26] The initial recommendation of “switch” was soon replaced by “overlap” regimen, then by the current recommendation of “add-on” combination therapy. It is now evident that prompt “add-on” lamivudine and adefovir combination therapy [27,28,29,30,31,32,33,34] combination therapy is more effective in suppressing HBV replication and minimising viral breakthrough with rtN236T and reA181T mutations. Response is less favourable if rescue combination therapy is commenced after serum HBV-DNA level has risen over 7log ₁₀ copies/ml. Adefovir therapy suppresses viral replication in more than 70% of lamivudine resistant case. Factors associated with virologic response are female gender, HBeAgnegative status, AST level and low baseline serum HBV-DNA levels. Genotype D HBV infection and low baseline HBV-DNA levels independently predict HBeAg loss. At the beginning of ADV therapy, substitutions at rtA181 (rtA181T and rtA181S) were identified in 3 patients (2.3%). Two (1.6%) of the 129 patients developed new ADV-resistant mutants; one was rtA181S and another was rtA181T plus rtN236T mutation. Therefore, the emergence of adefovir-resistant mutants is rare, at least over a period of 2 years, in patients with combination therapy.[35,36] In transplant candidates with lamivudine resistance, the combination of adefovir and lamivudine therapy without HBIg use is safe and efficacious in preventing posttransplant graft re-infection.[37] Serum HBV-DNA levels became undetectable in 40% and 65% at weeks 48 and 96 respectively. The cumulative probabilities of emergence of adefovir resistance were 0%, 2%, and 2% at weeks 48, 96, and 144, respectively. Only 4% of patients discontinued adefovir for treatment-related adverse events.[38] Incomplete virological response to adefovir dipivoxil in patients with lamivudineresistant infection may be at risk for the development of multiple resistance and disease progression. By replacing adefovir with tenofovir disoproxil fumarate, 19 of 20 patients achieved undetectable serum HBV-DNA (below the detection limit of 400 copies/mL) after a median of 3.5 months. ALT normalisation, HBeAg loss and even seroconversion to anti- HBs have been reported. Lamivudine-associated mutations (rtV173L, rtL180M, rtM204V/I) were detected in 6 patients at baseline of tenofovir treatment, but these did not influence the response.[39] Lamivudine refractory CHB patients responded initially to entecavir monotherapy at dosage of 1.0 mg daily. Histologic improvement occurred in 55% of entecavir-treated vs. 28% (32/116) of lamivudine-treated patients (p< .0001).[40] However the presence of lamivudine resistance mutations (M204V and& L180M) permits the rapid emergence of entecavir resistance (T184, S202 and/or M250 substitutions). The incidence of entecavir resistance was unacceptably high at 1%, 11%, 22%, and 29% by the end of years 1, 2, 3, and 4, respectively.[41,42,43] In a two-year assessment in lamivudinerefractory CHB patients, isolates from 192 entecavir-treated patients were sequenced and phenotypically analysed. The T184, S202, or M250 substitutions were found in  lamivudine resistant HBV at baseline in 6% of patients and emerged in isolates from another 6% and 8% of entecavir -treated patients by weeks 48 and 96, respectively. Only a subset of the changes in entecavir resistant isolates altered their susceptibilities, and virtually all isolates had significant replication impairment in vitro. Consequently, only two of 187 (1%) patients experienced entecavir resistant rebounds in year 1. An additional 14 of 151 (9%) patients experienced entecavir resistance rebounds in year 2. Isolates from all 16 patients with rebounds were lamivudine resistant and harboured the T184 and/or S202 change. Seventeen other novel substitutions emerged during entecavir therapy, but none reduced the susceptibility to ETV or resulted in a rebound. Four years after switch to entecavir, viral load rose with a complex mixture of entecavir-resistant strains harbouring the lamivudineresistance signature rtL180M+M204V and the rtS202G mutation. The rtL180M+S202G+M204V variant conferred one of the strongest resistance levels to entecavir.[44,45] Case report and retrospective analysis revealed that during lamivudine treatment three other mutations had been selected as well, namely rtE1D, rtV207L, and rtI220L. On the basis of the data from cross-resistance and sensitivity testing in vitro, tenofovir proved to be a good treatment option for entecavir-resistant patients.[46] There have been few reports on the management of adefovir resistance in the absence of lamivudine resistance but this mutant has been shown to be susceptible to lamivduine treatment.