Diagnostic and therapeutic progress of multi-drug resistance with anti-HBV nucleos(t)ide analogues

Abstract

Nucleos(t)ide analogues (NA) are a breakthrough in the treatment and management of chronic hepatitis B. NA could suppress the replication of hepatitis B virus (HBV) and control the progression of the disease. However, drug resistance caused by their long-term use becomes a practical problem, which influences the long-term outcomes in patients. Liver transplantation is the only choice for patients with HBV-related end-stage liver disease. But, the recurrence of HBV after transplantation often caused by the development of drug resistance leads to unfavorable outcomes for the recipients. Recently, the multi-drug resistance (MDR) has become a common issue raised due to the development and clinical application of a variety of NA. This may complicate the antiviral therapy and bring poorly prognostic outcomes. Although clinical evidence has suggested that combination therapy with different NA could effectively reduce the viral load in patients with MDR, the advent of new antiviral agents with high potency and high genetic barrier to resistance brings hope to antiviral therapy. The future of HBV researches relies on how to prevent the MDR occurrence and develop reasonable and effective treatment strategies. This review focuses on the diagnostic and therapeutic progress in MDR caused by the anti-HBV NA and describes some new research progress in this field.

Key Words: Hepatitis B virus; Multi-drug resistance; Nucleos(t)ide analogues; Gene mutation; Liver transplantation

INTRODUCTION

Globally, it is estimated that about 400 million are chronically infected with hepatitis B virus (HBV). The prevention and control of hepatitis B is an important public health issue. HBV infection may not only result in fulminant, subfulminant, and chronic hepatitis, but may also contribute to the development of liver cirrhosis and hepatocellular carcinoma in the long-term[1]. Decades of clinical experiences have shown that the administration of nucleos(t)ide analogues (NA) could postpone the progression of the disease. NA mainly suppresses the HBV replication and reduces the damage to hepatocytes by hindering the synthesis of reverse transcriptase (RT), which is the prerequisite for viral replication. The main disadvantage of NA is the recurrence of HBV after therapy interruption. Hence, the long-term treatment becomes a mandate. But, this paves the way for other challenges like HBV genome mutation and clinical drug resistance. The reports of multi-drug resistance (MDR) to NA in the recent years are increasing, and become an important issue for clinicians to modulate the treatment strategies during drug resistance.

MECHANISMS OF ANTIVIRAL RESISTANCE

HBV has a DNA genome. But, it replicates through an RNA intermediated reverse transcription. The lack of proofreading ability of the viral encoded, RNA-dependent DNA polymerase could result in potential mutations at each nucleotide position within the entire genome[2]. This, together with the large number of virions produced (10¹²-10¹³/d), indicates that every mutation of the 3.2 kb HBV genome can theoretically be produced daily[3,4]. Under the selective pressure of antiviral agents, such diversity is likely to give rise to drug-resistant mutants[5]. RT is the target of NA and also the chief place of gene mutations. Several factors are associated with the development of antiviral resistance, including viral fitness, potency, and genetic barrier to resistance of the antiviral agents[6]. Studies have shown that there are five pathways leading to antiviral resistance[7]. (1) The L-nucleoside pathway (rtM204V/I), leads to the resistance of lamivudine (LAM), emtricitabine, telbivudine (LdT), and clevudine. This pathway includes entecavir (ETV) resistance in LAM-experienced patients; (2) The acyclic phosphonate pathway (rtN236T), associates with the resistance to adefovir (ADV) and tenofovir disoproxil fumarate (TDF)[8,9]; (3) A shared pathway (rtA181T/V), results in the selection of HBV quasispecies when treated with L-nucleosides or acyclic phosphonates;(4) Naïve entecavir resistance pathway (rtL180M + rtM204V with either rtT184, S202, or M250 codon changes). In this pathway, three mutations are required to appear simultaneously accounting for the very low resistance profile of entecavir[10]; and (5) Multidrug resistance pathway, complex patterns and clusters of specific mutations in HBV polymerase are associated with it.

TDF is recommended since 2008, as first line drug in patients chronically-infected with HBV and led to high rates of virologic success[11,12]. Its effect on HBV DNA suppression appears similar to ETV and LdT and superior to LAM and ADV[13,14].It remains largely active even against ADV-resistant or ETV-resistant mutations[15-17]. However, the rtA194T polymerase mutation has been found in HBV/HIV co-infected patients during TDF treatment and may be associated with TDF resistance[18-20]. Whether the rtA194T mutation truly confers resistance against TDF has remained controversial, as the in vitro phenotypic assays showed variable results across laboratories[21,22]. Thus, the potential impact of this mutation on TDF susceptibility deserves further study[20]. The primary antiviral drug resistance mutations in the polymerase gene are listed in Table 1[23].

Table 1 Primary antiviral drug resistance mutations in the polymerase gene[23].

	Domain A	Domain B	Domain C	Domain D	Domain E	Numbers of mutations
Lamivudine and Telbivudine		rtV173L
	--	rtL180M	rtM204V/I	--	--	1
		rtA181T/V
Adefovir	--	rtA181T/V		rtN236T	--	1
Tenofovir	--	rtA181T/V		rtN236T	--	?
Entecavir	--	rtL180M	rtM204I/V	--	rtM250I/V	3
		rtT184	rtS202

HBV strains, resistant to at least two anti-HBV agents from different subclasses of NA without a cross-resistance profile, are defined as MDR[24]. The main reasons for MDR are the sequential monotherapy to treat primary resistance and use of agents with similar cross-resistance profiles. The development of MDR is a major challenge for antiviral therapy, and the improper administration of NA may lead to serious outcomes. Thus, more researches on the choice of antiviral agents in treating patients with MDR have been carried out and some significant solutions have been achieved.

THE CURRENT SITUATION AND STRATEGIES OF DIFFERENT TYPES OF MDR

LAM + ADV resistance

LAM, the first oral antiviral agent against HBV, is safe and well tolerated even in patients with decompensated liver cirrhosis[25]. Globally, it has been mostly used with a low genetic barrier to resistance and cumulative incidence of resistance as high as 70% after 5 years of treatment[26,27]. Early studies had suggested that, ADV monotherapy had shown similar antiviral effects to combination therapy with LAM+ADV for LAM-resistant patients in the short-term, and a strategy of switching to ADV monotherapy had widely been adopted[28]. However, recent studies have showed that ADV resistant mutations emerge more frequently during sequential ADV monotherapy in LAM resistance than in treatment-naïve patients[29,30]. The rate of ADV resistance in LAM-resistant patients was shown to be as high as 18% at 1 year, compared with 0% in LAM-naïve patients[31]. Another long-term study reported that the cumulative genotypic resistance and virologic breakthrough at 5 years of sequential ADV monotherapy in LAM-resistant patients were 65.6% and 61.8%, respectively[32]. Fung et al[33] reported that the cumulative rate of ADV resistance in LAM-resistant patients at 2 years was 18% for patients who were switched to ADV and 7% for patients who had ADV added to their treatment regimen. In another study of 42 LAM-resistant patients (HBeAg-negative), the ADV resistance rates at 15-18 mo of treatment were 21% (3/14) for patients who were switched to ADV and 0% for patients who had ADV added[34]. It can be assumed that the ADV resistance rate in LAM-resistant patients can be greatly reduced by adding rather than switching to ADV. There are more researches exploring the mechanisms of LAM + ADV dual-resistance, as these two agents were launched early. When the mutations causing resistance to LAM and ADV are not on the same viral genome, a combination therapy of these two agents will likely be effective in suppressing the mutants resistant to each of the drugs. In contrast, when the antiviral resistance mutations are on the same viral genome, the combination treatment may not be adequate[30]. In vitro analysis have shown that most of MDR mutations collocate on the same viral genome[35], but the in vivo confirmation on the same is lacking. There is no unified clinical treatment strategy for LAM + ADV dual-resistance, but different methods of mono or combination therapy have been carried out.

Due to the limited alternative of NA in the early stage, interferon (IFN) had been tried as a choice for dual-resistance to LAM and ADV. Phenotypic analysis have indicated that IFN-α suppresses equally the mutant strains and wild-type strains in vitro[36]. Furthermore, IFN-α also suppresses the replication of LAM-resistant and ADV-resistant mutants in vivo[37]. Besides that, IFN-α administration predictably have reduced the resistance to NA when combined with LAM[38,39], as IFN-α exhibits at least two HBV-specific antiviral activities independent of the viral polymerase sequence with one reducing the levels of core protein and replicative intermediates, and the other leading to posttranscriptional degradation of HBV RNA[40]. However, there are certain potential limitations with IFN therapy such as low probability of sustained response, parenteral administration, relatively poor tolerability, and frequent and potential serious adverse effects in patients with advanced liver disease[41]. These deficiencies limited the clinical application of IFN, and more researches focused on the application of oral NA in MDR.

In vitro studies show that the majority of MDR mutations to LAM and ADV collocate on the same viral genome[31]. Therefore, the combination therapy with LAM and ADV may not effectively deal with the patients, who are resistant to these two agents. The advent of ETV enabled a new choice for antiviral therapy. Since TDF is not available in many Asian countries, the 2008 updated guidelines by Asian Pacific Association for the Study of the Liver recommended ETV in LAM and ADV resistant patients[42]. But satisfactory clinical results were not acquired in these patients treated with ETV. Heo et al[30] compared the clinical efficacy of LAM + ADV combined therapy and ETV monotherapy in patients with dual-resistance to LAM and ADV. The mean reduction in serum HBV DNA concentration was significantly lower in the LAM + ADV than in the ETV group. But, the difference in mean decline in serum alanine aminotransferase (ALT) levels over 12 mo of treatment and the rate of HBeAg seroconversion at 12 mo did not differ significantly between two groups. Park et al[43] reported that ETV monotherapy could not reach an optimal clinical efficacy in ADV-refractory chronic hepatitis B patients with prior LAM resistance. In this long-term study (up to 4 years), the authors have suggested that an early virologic response is essential for a successful ETV monotherapy in this group of patients. Clinically, initial virologic response at 3 mo (IVR-3) is an independent predictor for virologic response, and it may help determine whether to maintain ETV monotherapy or not. Choe et al[44] evaluated the antiviral efficacy of ETV in patients, who had failed to achieve viral response during LAM and ADV rescue therapy. The virologic response was achieved in 1 of 18 patients with pre-existing rt204 mutations, whereas it was achieved in all 4 patients without pre-existing rt204 mutations regardless of the presence of rt181 or rt236 mutations. The poor treatment efficacy of ETV in patients with LAM and ADV dual-resistance might have resulted from the pre-existing rt204 mutations, which could further lead to ETV resistance.

As the ideal clinical efficacy was not achieved by ETV monotherapy, researches continued to seek other treatment strategies with ETV combination therapies. A study tried combination of ETV and ADV in LAM-resistant chronic hepatitis B patients with suboptimal response to LAM + ADV[45]. This strategy provided superior virologic response and favorable resistance profiles, when compared with combination therapy of LAM and ADV. But similar to ETV monotherapy, an optimal virologic response still could not be reached. This may likely due to the relatively low antiviral potency of ADV, which suggested to replace ADV with another drug with a similar resistance profile and higher potency against LAM-resistant mutants.

TDF shares the similar molecular structure with ADV. It has higher antiviral potency and lower rate of developing drug resistance. Despite its structural similarity to ADV, TDF partially suppresses ADV-resistant HBV, and it is also highly effective against LAM-resistant virus, suggesting that this drug may be an effective treatment for patients who have previously failed treatment with LAM and ADV[46]. Van Bommel et al[47] introduced TDF for LAM-Resistant patients with high HBV DNA level during ADV therapy. The administration of TDF (300 mg daily for all the patients except one) led to an undetectable HBV DNA level in 19 of 20 patients within a median of 3.5 mo. The only patient who did not become HBV DNA negative during the observation period received a reduced TDF dose (300 mg every second day) because of renal insufficiency. Patterson et al[48] reported similar efficacy with TDF in another group of similar patients. But, the virologic response to TDF in this study appeared to be inferior to that observed in treatment-naïve patients. The development of antiviral resistance to TDF in the long-term is uncertain, but the combination therapy with high potency NA without cross-resistance is still a superior strategy for MDR. Recently, Petersen et al[49] reported a multicenter study with ETV + TDF as rescue therapy in pretreated chronic hepatitis B patents. HBV DNA was undetectable in 51 out of 57 patients with different types of resistance, and the ALT levels improved in most of them, suggesting a reduction in liver inflammation. Besides, this strategy was efficient, safe, and well tolerated in patients with and without advanced liver disease. The updated 2009 American Association for the Study of the Liver Diseases (AASLD) practice guideline recommended TDF + ETV in patients with sequential LAM and ADV treatment failure[27].

LAM + ETV resistance

ETV is an effective antiviral agent with high potency and high genetic barrier to resistance. In NA-naïve patients, the 5-year cumulative probability of genotypic ETV resistance and genotypic ETV resistance associated with virologic breakthrough was only 1.2% and 0.8%, respectively[50]. However, the emergence of resistance to ETV occurs more frequently in LAM-refractory population. Based on the previous reports, the resistance to ETV in LMV-refractory patients was detected in 8% of patients after 12 mo, 43% after 48 mo, and 51% after 60 mo of treatment[51,52]. In a clinical study, the cumulative rates of ETV genotypic resistance in patients with LAM resistance are 6%, 15%, 36%, 46%, and 51% from years 1 to 5, respectively[53]. The resistance to ETV in the sequential monotherapy in LAM-refractory patients shares the same pathway (mutation of rtM204V/I) with LAM resistance. Yatsuji et al[54] reported a patient with dual-resistance to LAM and ETV, but was effectively treated with ADV. Interestingly, the typical mutation strains of ETV (rtL180M + M204V + S202G) were observed in this patient. An in vitro study indicated that the rtL180M + M204V + S202G mutant had no resistance against ADV, and this case report confirmed this view clinically. Another study included 12 patients with dual-resistance to LAM and ETV, and half of them reached complete virological response with the combination therapy of ADV and ETV after 18 mo. In addition, no enrolled patients developed virologic breakthrough and had mutations resistant to ADV at the end of follow-up. However, not all the patients realized virologic response, which may due to the low potency of ADV and high pretreatment levels of HBV DNA[55]. This combination therapy strategy may be helpful as ETV is effective to rtA181T related mutant and ADV is effective to rtM204V related mutant. The optimal strategy for such dual-resistance patients has not been determined due to less availability of literature data.

LAM + ADV + ETV resistance

Recently, MDR to three NA has been described in case reports. Liu et al[56] reported LAM + ADV could be helpful in a patient, who was resistant to LAM, ADV, and ETV. It could be speculated that the HBV DNA replication had been suppressed due to LAM and ADV. The immune response against the MDR strains might also have contributed to the clearance of these strains. The exact effect of this strategy remains to be observed due to the short follow-up period. Sayan et al[24] reported a case of another patient, who was effectively treated with ETV and TDF. Three primer drug resistance mutations in different domains of HBV viral polymerase, such as rtA181V/T, rtL180M + rtM204V mutations and the rtN236T, were characterized in the same genome, which might explain the MDR profile. In another study of MDR, amino acid changes consisting of L80V, L91I, M204I, S219A, N238D, and Y245H were found on the same dominant viral genome strain. These newly discovered mutation types may also relate to MDR[57]. This type of resistance, usually caused by sequential monotherapy, may be effectively treated by combination therapy. However, the mechanisms and preventive methods for MDR to three NA need to be studied.

Treatment strategy of MDR in liver transplant recipients

Liver transplant is an effective method for patients with HBV-related end-stage liver disease. In a liver transplant setting, three different clinical phases have to be considered: (1) treatment of HBV infection during waiting list; (2) prophylaxis of hepatitis B recurrence after liver transplant; and (3) treatment of recurrent hepatitis B when prophylactic measures have failed[58]. The main factors associated with HBV recurrence were HBeAg status at transplant listing and serum HBV-DNA level at transplant[59]. The goals of antiviral therapy in the pretransplant patients include the reduction of viral load to low or nondetectable serum HBV DNA levels[60]. The development of drug resistance for patients in the waiting list for liver transplant is a common problem. Osborn et al[61] found that the antiviral therapy failure in patients with HBV in the waiting list did not impair clinical outcomes when recognized early and also when the salvage therapy was promptly initiated and neither the survival rate with transplant nor without transplant was negatively impacted by antiviral therapy failure.

However, the recurrence of HBV after transplant is still a troublesome problem and may influence the long-term outcomes. The combination of both LAM and hepatitis B immunoglobulin (HBIg) has emerged as the most effective prophylactic strategy in HBV transplant recipients with a 3-year recurrence rate of 0-10%[58,62,63]. But, the drug resistance to this combination therapy has also emerged. In addition to the typical resistance to LAM, it was detected in 45% of the immunosuppressed patients within the first year following liver transplant[64]. The escape mutation from anti-HBs occurs in the common α determinant region of the surface gene, which is a highly conservative region of the HBsAg protein[65]. IFN is not currently used for the treatment of hepatitis B after transplantation, given the risk of graft rejection[66,67]. The availability of ADV has changed the clinical course of LAM-resistant patients, and it is effective and safe among liver transplant recipients[68-70]. Researches showed that ADV could be an effective rescue therapy for patients with LAM-resistant hepatitis B post-liver transplant[71,72]. In a retrospective review, viral recurrence was noted in 5 out of 23 liver transplant recipients, who are on combination prophylaxis of LAM and HBIg with 1 patient receiving TDF and 4 receiving ADV. Only 1 death related to HBV recurrence was reported in this population, who had been switched to ADV[73].

Apart from ADV, ETV appears to be a more attractive candidate than ADV for use in the transplant setting[74]. A study of 30 patients receiving a combination of ETV and low-dose HBIg confirmed the efficacy and safety of ETV in preventing recurrence of HBV after liver transplant[75]. Another small study of 8 patients showed that ETV was safe and effective as prophylaxis after liver transplant with no interactions observed with the immunosuppressive medications[76]. Fung et al[77] reported that although only 26% of patients had complete viral suppression at the time of transplant and 91% of patients underwent loss of HBsAg after 2 years of follow-up. Also, 98.8% of patients achieved undetectable HBV DNA levels with ETV as the sole antiviral agent after liver transplant. It can be inferred that ETV may play an effective role in recipients resistant to both LAM and HBIg.

ETV could be a choice in liver transplant recipients, and TDF may also be effective when used alone or in combination therapy. Villet et al[78] reported a recipient who was resistant to LAM, ADV, and HBIg. The combination therapy of LAM and TDF effectively suppressed the HBV DNA replication. Karlas et al[79] reported that the combination therapy with ETV and TDF may prevent post-liver transplant hepatitis B recurrence even without HBIg maintenance therapy. He illustrated that the combination oral antiviral therapy might substitute for HBIg as indefinite prophylactic regimen due to profound antiviral efficacy and low risk of viral resistance. The optimal combination method for MDR in liver transplant recipients is still uncertain, and the therapeutic experiences in non-transplant patients could be learned.

NEW PERSPECTIVES ON THE RESEARCH OF MDR

There are still some limitations on the current research on drug resistance. For instance, the genetic analysis of resistance to NA inhibitors is usually focused on the RT domain, and only infrequently takes into consideration of the whole genome of intra-host HBV variants[80-82]. Thai et al[83] reported that the rtM204I/V substitution is insufficient for establishing resistance against LAM. The analysis of 639 HBV whole-genome sequences obtained from 11 patients showed that rtM204I/V was independently acquired by more than one intra-host HBV variant, indicating the convergent nature of LAM resistance. Currently, the most commonly used methods for detecting HBV drug-resistance mutations are direct sequencing and reverse hybridization[84]. However, these methods do not enable haplotype analysis, and hence, they cannot be used to determine the collocation of mutations on the same viral genome. This limits the accurate identification of viral mutants that are resistant to drugs with high genetic barrier[85]. Recently, several next-generation sequencing technologies, such as ultra-deep pyrosequencing, are available to generate more data[85-88]. These methods may offer significant advantages in explaining and predicting the responses of HBV patients to antiviral therapy and enable quantification of HBV quasispecies variants.

Recently, some studies analyzed the mutation pattern in relation to the HBV genotypes and found that the rtL180M mutation is significantly connected to the rtM204V mutation in genotypes A, B, and C. Also, the HBV genotypes differ in their mutation pattern of LAM resistance[89]. Another study indicated the association of genotype C with higher rates of hepatitis B recurrence after transplant due to LAM resistance[90]. It can be inferred that HBV genotypes may play an important role in the progression of HBV-related liver disease and response to antiviral therapy. The assessment of HBV genotype prior to the treatment may help to individualize the antiviral therapy and reduce the incidence of treatment failures and complications[91,92]. However, the relationship between HBV genotypes and antiviral resistance is unclear. Extensive researches may provide new perspectives for the prevention and optimal rescue therapy to patients with drug resistance.

PREVENTION OF MDR

Treatment failure in anti-HBV therapy could be regarded as an iatrogenic factor, and a judicious use of NA in chronic hepatitis B patients is the most effective prophylaxis against the development of MDR. Thus, proper strategy should be applied by clinicians at the beginning of therapy. An antiviral agent with the highest potency and a high genetic barrier to resistance should be selected[93,94]. The pros and cons of initiating the treatment with combination therapy in minimizing the development of antiviral resistance are currently being investigated[95,96]. Avoiding the sequential use of NA monotherapy is an effective preventive method for MDR[24]. A roadmap suggested that HBV DNA concentration at week 12 after initial treatment should be checked to identify patients with primary treatment failure, which is defined by < 1 log copies/mL reduction of HBV DNA concentration. For patients with primary treatment failure with good drug compliance, addition of another NA is indicated. The second assessment of HBV DNA should then be done at week 24[97,98]. Treatment strategies in patients with partial virologic response are based on the potency and genetic barrier of the antiviral agent. Patients receiving NA with a high genetic barrier can remain the treatment beyond 48 wk. Patients receiving a less potent NA should continue treatment and be re-assessed at week 48, and those, who receive NA with a low genetic barrier, should add a more potent drug due to the high risk of resistance when the treatment is not adapted[26]. To avoid MDR, the combination therapy of NA with cross-resistance should be avoided. The patterns and pathways of antiviral drug resistance in chronic hepatitis B in the context of cross-resistance are listed in Table 2[99]. Patient’s adherence to antiviral therapy is another important factor in avoiding resistance. Adherence may be monitored using patient reports, dispensed medication counts, or HBV DNA detection[93]. If low response or virologic breakthrough is observed with the primary treatment, gene sequencing should be done to find out the type and location of mutations, in order to guide the optimal rescue therapy. Recently, An et al[100] reported that the family history, negative conversion time of HBV DNA, and different NA were independent risk factors of gene resistant mutation, which provided a theoretical basis for predicting drug resistance and salvage treatment.

Table 2 Patterns and pathways of antiviral drug resistance in chronic hepatitis B in the context of cross-resistance[99].

Pathway	Amino acid substitution in the rt domain	LAM	LdT	ETV	ADV	TDF
	WT	S	S	S	S	S
L-nucleoside (LAM/LdT)	M204I/V	R	R	I	S	S
Acyclic phosphonate (ADV)	N236T	S	S	S	R	I
Shared (LAM, LdT, ADV)	A181T/V	R	R	S	R	I
Double(ADV, TDF)	A181T/V + N236T	R	R	S	R	R
D-Cyclopentane (ETV)	L180M + M204V/I ± I169 ± T184 ± S202 ± M250	R	R	R	S	S

I: Intermediate sensitivity; R: Resistant; S: Sensitive based on cell culture and clinical; LAM: Lamivudine; LdT: Telbivudine; ETV: Entecavir; TDF: Tenofovir; ADV: Adefovir.

There is still no consensus statement on the management of MDR in current HBV treatment guidelines. However, the guidelines for primary treatment failure could be used in order to prevent MDR. The management of antiviral-resistant HBV in guidelines is listed in Table 3[27,101]. In these updated guidelines, the combination therapy was not recommended in all circumstances. The antiviral agents with high potency and high genetic barrier to resistance, such as ETV and TDF, could also be used alone.

Table 3 Management of antiviral-resistant hepatitis B virus in updated guidelines.

	AASLD 2009[27]	EASL 2012[101]
LAM-resistance	Add ADV or TDF	Switch to TDF or add ADV
	In patients with no prior exposure to other NA: Add LAM or ETV	In patients with no prior exposure to other NA: Switch to ETV or TDF
ADV-resistance	In patients with prior LAM resistance: Switch to TDF plus LAM or ETV	In patients with prior LAM resistance: Switch to TDF and another NA
ETV-resistance	Switch to TDF	Switch to or add TDF
TDF-resistance	--	In patients with no prior exposure to LAM: Switch to ETV
		In patients with prior LAM resistance: Add ETV

LAM: Lamivudine; LdT: Telbivudine; ETV: Entecavir; TDF: Tenofovir; ADV: Adefovir; NA: Nucleos(t)ide analogues.

CONCLUSION

In summary, MDR to NA is a thorny issue in the anti-HBV therapy. The clinical efficacy in patients has been improved to some extent by the combination therapy. ETV and TDF can be regarded as the optimal choice for patients with MDR. Further investigations on the mechanisms and optimal treatment modalities are still lacking. The progress and emergence of new genetic testing technology will probably improve the anti-HBV therapy. The newly discovered forms of gene mutations to resistance may provide useful clues to solve the problem of MDR. The efficacy of IFN in patients with MDR needs further exploration. The development of new anti-HBV agents, which act not only on RT, but also on other targets of HBV, may be a new approach to prevent MDR in NA.