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Virological breakthrough and resistance in patients with chronic hepatitis B receiving nucleos(t)ide analogs in clinical practice - pdf attached
 
 
  Hepatology May 2011 advance accepted publication
 
Hepatitis B Virus Reemerges with Long-term Nucleoside Analog Treatment: 'non-adherence appears to be problem, adherence counselling & HBV DNA monitoring recommended'
 
"Medication adherence is likely to be lower in clinical practice than in phase III clinical trials where highly motivated patients are recruited and closely monitored. Differentiating between VBT due to medication non-adherence and VBT due to drug resistance is important as virological response can be restored by reinforcement of adherence in the former case while rescue therapy is needed in the latter situation......In phase III clinical trials of nucleos(t)ide analogs for CHB, 65.7% to 87.5% of patients receiving lamivudine or telbivudine and 0% of patients receiving entecavir or tenofovir, who experienced VBT during the first year had GR (4, 7-9)......follow-up data [in this study] suggest that most of the patients who experienced VBT but did not have confirmed VBT or GR were not adherent to their antiviral medication(s)."
 
"In conclusion, this study revealed that VBT was common in clinical practice. However, VBT was not always related to antiviral drug resistance. CHB patients receiving nucleos(t)ide analog therapy who experienced VBT should be counseled on medication adherence and for patients who are immunocompetent and have compensated liver disease, confirmation of VBT and/or determination of GR is prudent before the initiation of rescue therapy to avoid unnecessary changes in antiviral medications. We acknowledge that this study is limited by the small number of patients, the heterogeneity in treatment regimens, and the lack of data on medication adherence. However, the results of this study highlight the importance of HBV DNA monitoring and counseling on medication adherence throughout the course of nucleos(t)ide analog treatment and the fine balance between prompt initiation of rescue therapy versus avoidance of unnecessary changes to the treatment regimen."
 
"As expected, among NUC-naïve patients, those receiving entecavir monotherapy were less likely to experience VBT than those receiving lamivudine monotherapy. Among NUC-experienced patients, those receiving combination therapy (lamivudine + adefovir or emtricitabine + tenofovir) or tenofovir monotherapy were less likely to experience VBT than those receiving lamivudine, adefovir or entecavir monotherapy."

 
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April 27, 2011
 
Hepatitis B Virus Reemerges with Long-term Nucleoside Analog Treatment
 
A recently published study revealed that virological breakthrough (VBT) is common in patients receiving nucleoside analogs (NUCs) for chronic hepatitis B. Nearly 40% of the VBTs found were not related to antiviral drug resistance. Details of this retrospective study are published in the May issue of Hepatology, a journal published by Wiley-Blackwell on behalf of the American Association for the Study of Liver Diseases.
 
VBT is the first manifestation of antiviral drug resistance during NUC therapy of chronic hepatitis B. NUC drugs approved for treatment of chronic hepatitis B include lamivudine (LAM), adefovir (ADV), entecavir (ETV), telbivudine (TBV), and tenofovir (TDF). While the medications suppress the virus with few side effects, they do not eradicate HBV and require long-term treatment to provide clinical benefit. With long-term NUC therapy, studies have shown an increasing risk of drug resistance particularly with monotherapy regimens.
 
In the current study, Anna Lok, M.D. and colleagues from the University of Michigan Health System examined the incidence of VBT and genotypic resistance (GR) in 148 patients with chronic hepatitis B who were treated with NUCs between January 2000 and July 2010. The mean age of study participants was 45 years and 73% were male. Researchers reviewed medical records and recorded patient demographics, hepatitis B virus (HBV) markers, liver panel, blood counts and liver histology.
 
Results showed that during a mean follow-up of 38 months, 39 (26%) patients had at least one VBT, and upon retesting, 15 (38%) of these patients did not have a VBT and 10 had no evidence of GR. Researchers reported the probability of VBT, confirmed VBT, and GR at five years was 46%, 30%, and 34%, respectively. "Our analysis showed an alarmingly high rate of VBT in clinical practice and the only factor significantly linked to VBT was failure to achieve undectectable HBV DNA," said Dr. Lok.
 
HBV DNA decreased in the ten patients who initially experienced a VBT, but who did not have confirmed VBT or GR, when the same drug regimen was maintained. Nine of these patients had undetectable HBV DNA at the most recent follow-up, a mean of 7 months after the initial VBT. These data suggest that nonadherence to medication may be a common cause of VBT. "Counseling patients with chronic hepatitis B on the importance of medication adherence, and confirming reemergence of the virus and genetic mutations that cause resistance, can help to avoid unnecessary changes to antiviral treatments," advised Dr. Lok.
 
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Virological breakthrough and resistance in patients with chronic hepatitis B receiving nucleos(t)ide analogs in clinical practice

 
Chanunta Hongthanakorn, Watcharasak Chotiyaputta, Kelly Oberhelman, Robert J. Fontana, Jorge A. Marrero, Tracy Licari, and Anna S.F. Lok Division of Gastroenterology, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, USA
 
Abstract
 
Virological breakthrough (VBT) is the first manifestation of antiviral drug resistance during nucleos(t)ide analog (NUC) treatment of chronic hepatitis B (CHB) but not all VBTs are due to drug resistance. The aims of this study were to determine the incidence of virological breakthrough (VBT) and genotypic resistance (GR) in CHB patients receiving NUCs in clinical practice. Records of CHB patients receiving NUCs were reviewed. All patients with VBT were tested for drug resistance mutations. Of 148 patients included, 73% were men, mean age was 44.9 years. During a mean follow-up of 37.5±20.1 months, 39 (26%) patients had at least 1 VBT. Of these 39 patients, 15 (38%) were not confirmed to have VBT on retesting and 10 of these 15 had no evidence of GR. The cumulative probability of VBT, confirmed VBT, and GR at 5 years was 46.1%, 29.7%, and 33.9%, respectively. In multivariate analysis, failure to achieve undetectable HBV DNA was the only factor significantly associated with VBT. Among the 10 patients who had VBT but no confirmed VBT or GR and who were maintained on the same medications, serum HBV DNA decreased in all 10 and 9 had undetectable HBV DNA a mean of 6.8 months after the VBT. Four patients had persistently undetectable HBV DNA while six had transient increase in HBV DNA during follow-up but none had GR. Conclusion: VBT was common in CHB patients receiving NUCs in clinical practice, but nearly 40% of the VBTs were not related to antiviral drug resistance. Counseling of CHB patients on medication adherence and confirmation of VBT and/or GR can avoid unnecessary changes in antiviral medications.
 
INTRODUCTION
 
Five nucleos(t)ide analogs (NUCs): Lamivudine (LAM), Adefovir (ADV), Entecavir (ETV), Telbivudine (TBV), and Tenofovir (TDF) have been approved for the treatment of chronic hepatitis B (CHB). Nucleos(t)ide analogs are administered orally and have very few side effects; however, these medications suppress but do not eradicate hepatitis B virus (HBV). Therefore, most patients with CHB will require long-term treatment to derive clinical benefit. However, long- term NUC treatment is associated with increasing risk of drug resistance particularly when NUCs with low genetic barrier to resistance are used as monotherapies.
 
Virological breakthrough (VBT) is the first clinical manifestation of antiviral drug resistance and may precede biochemical breakthrough (1-3). Phase III clinical trials of nucleos(t)ide analogs in NUC-naïve patients revealed that 0 to 87.5% of patients with VBT had confirmed genotypic resistance (GR) (4-9). In the phase III trial of telbivudine vs. lamivudine, 32 of 680 (4.7%) and 99 of 687 (14.4%) patients who received telbivudine and lamivudine, respectively, experienced VBT after one year of treatment but only 28 (87.5%) and 75 (75.8%) patients with VBT were confirmed to have GR (9). In the phase III trial of entecavir vs. lamivudine, 11 of 679 (1.6%) and 88 of 668 (13.2%) patients who received entecavir and lamivudine, respectively, experienced VBT after one year of treatment but 0 (0%) and 65 (73.9%) of the patients with VBT, respectively were confirmed to have GR (7, 8). In the phase III trial of tenofovir, 10 of 426 (2.3%) patients who received tenofovir experienced VBT after one year of treatment but none of these patients were confirmed to have GR (4). These data indicate that not all VBTs are related to antiviral drug resistance.
 
Possible explanations for the discrepancy between the rates of VBT and GR include poor adherence to medications, failure to detect drug-resistance mutations due to insensitive assays, and failure to recognize new mutations associated with antiviral drug resistance. VBT during the first year of treatment was attributed to medication non-adherence in patients who received entecavir or tenofovir in the phase III trials (4, 7). Medication adherence is likely to be lower in clinical practice than in phase III clinical trials where highly motivated patients are recruited and closely monitored. Differentiating between VBT due to medication non-adherence and VBT due to drug resistance is important as virological response can be restored by reinforcement of adherence in the former case while rescue therapy is needed in the latter situation.
 
The aims of this study were (1) to determine the incidence of VBT and GR in CHB patients treated with nucleos(t)ide analogs in clinical practice, (2) to determine the factors associated with VBT in CHB patients receiving nucleos(t)ide analogs, and (3) to determine the outcomes of patients with VBTs that were not confirmed to be associated with antiviral drug resistance mutations.
 
DISCUSSION
 
This study examined the rates of VBT, confirmed VBT and GR in 148 CHB patients treated with nucleos(t)ide analogs in clinical practice. We found a high rate of VBT, 39 (26%) patients experienced at least 1 episode of VBT with a cumulative probability of 46% at 5 years. Twenty- four (16%) patients had confirmed VBT; of these, 19 (79%) had GR by direct sequencing. An additional 5 patients had GR but were not confirmed to have VBT on retesting or received rescue therapy without retesting. Thus, in total 24 (16%) patients had GR. The finding that 38% of patients who experienced VBT were not confirmed to have VBT on retesting and 38% did not have antiviral resistance mutations on direct sequencing suggests that medication non- adherence may be the cause of the VBT in these patients.
 
We acknowledge that direct sequencing is insensitive and will not detect viral variants that comprise <20% of the viral population. In this study, all patients were also tested by a line probe assay which is more sensitive and can detect viral variants that comprise >5% of the viral population. Two additional patients were found to have N236T mutation by the line probe assay. Thus, signature antiviral resistance mutations were not detected in 13 (33%) patients with VBT by both direct sequencing and line probe assay. None of these 13 patients was noted to have other substitutions in the reverse transcriptase region of the HBV polymerase gene. Only one had BBT. In phase III clinical trials of nucleos(t)ide analogs for CHB, 65.7% to 87.5% of patients receiving lamivudine or telbivudine and 0% of patients receiving entecavir or tenofovir, who experienced VBT during the first year had GR (4, 7-9).
 
Although antiviral resistance mutations may be detected if we had used more sensitive methods such as single genome sequencing or pyrosequencing, follow-up data suggest that most of the patients who experienced VBT but did not have confirmed VBT or GR were not adherent to their antiviral medication(s). Because this was a retrospective study, data on medication adherence was not available; however, follow-up data suggest that non adherence was an important cause of these unconfirmed VBTs. All 10 patients who continued treatment with the same medication(s) had further decrease in serum HBV DNA levels and all but one had undetectable HBV DNA after the VBT. Six of these 10 patients experienced ≥1 episode of transient increase in serum HBV DNA level during follow-up despite counseling on the importance of medication adherence. None of the five retested for GR was found to have antiviral resistance mutations and all had subsequent decline in serum HBV DNA during continued treatment with the same medication(s). We acknowledge that our attribution that nonadherence was an important cause of VBT is speculative. In a separate prospective study of 105 patients in whom adherence was evaluated using a self-administered questionnaire, 26% admitted to missing their medication at least once in the past 30 days and patients who had <100% adherence on serial assessments had a trend towards a higher rate of VBT (14).
 
Medication adherence has been shown to be important in maintaining response in patients receiving treatment for other conditions. Published studies showed that patients who were adherent to anti-hypertensive medications were more likely to have adequately controlled blood pressure (15-17). Several studies of antiretroviral medications in patients with HIV infection also revealed that failure to adhere to HIV treatment regimens and repeated drug holidays (defined as stopping treatment entirely for ≥ 48 hours) were associated with a higher rate of virological failure (18, 19). In one study of HIV treatment that included a protease inhibitor, 80% of patients with <80% adherence had virological failure, compared to 22% of those with ≥95% adherence (20).
 
There are very little data on adherence to nucleos(t)ide analog treatment for hepatitis B. In a previous study of pharmacy claims database including 11,100 patients receiving nucleos(t)ide analogs for CHB, we found that mean adherence 1 year after enrollment (defined as percent of days in which the patient had medications during that year) was 87.8% (21). In that study, we were not able to correlate medication adherence with virological response or occurrence of VBT. In the current study, we found that nadir response and type of medication used were significantly associated with the occurrence of VBT. Thus, patients who had rapid response with undetectable serum HBV DNA within 1 year of treatment and those who had undetectable serum HBV DNA at some point during the course of treatment were less likely to experience VBT. These data are in accord with previous studies showing that undetectable serum HBV DNA after 24 weeks of treatment is associated with significantly lower rates of antiviral resistance (22, 23). As expected, among NUC-naïve patients, those receiving entecavir monotherapy were less likely to experience VBT than those receiving lamivudine monotherapy.Among NUC-experienced patients, those receiving combination therapy (lamivudine + adefovir or emtricitabine + tenofovir) or tenofovir monotherapy were less likely to experience VBT than those receiving lamivudine, adefovir or entecavir monotherapy. This is not surprising because these patients had previous nonresponse or resistance to lamivudine or adefovir and it is now known that switching from lamivudine to adefovir or entecavir monotherapy in patients with prior lamivudine resistance is associated with a high rate of subsequent resistance to adefovir or entecavir (24-26). In this study, all the patients who received entecavir or tenofovir alone or in combination with another nucleos(t)ide analog had undetectable HBV DNA within 6 months of rescue therapy while three patients with lamivudine resistance still had detectable HBV DNA 6 months after rescue therapy with adefovir alone or in combination with lamivudine.
 
In conclusion, this study revealed that VBT was common in clinical practice. However, VBT was not always related to antiviral drug resistance. CHB patients receiving nucleos(t)ide analog therapy who experienced VBT should be counseled on medication adherence and for patients who are immunocompetent and have compensated liver disease, confirmation of VBT and/or determination of GR is prudent before the initiation of rescue therapy to avoid unnecessary changes in antiviral medications. We acknowledge that this study is limited by the small number of patients, the heterogeneity in treatment regimens, and the lack of data on medication adherence. However, the results of this study highlight the importance of HBV DNA monitoring and counseling on medication adherence throughout the course of nucleos(t)ide analog treatment and the fine balance between prompt initiation of rescue therapy versus avoidance of unnecessary changes to the treatment regimen.
 
PATIENTS AND METHODS

 
Consecutive adult patients with CHB seen at the liver clinic of the University of Michigan Health System between January 2000 and July 2010, who had received nucleos(t)ide analog treatment for at least one year with serum HBV DNA <10,000 IU/mL after one year of treatment were included. Patients who were receiving combination therapy of nucleos(t)ide analog and interferon, patients receiving nucleos(t)ide analogs to prevent reactivation of hepatitis B during immunosuppressive or cancer treatment or to prevent recurrent hepatitis B after liver transplantation, patients with human immunodeficiency virus (HIV), hepatitis C virus or hepatitis D virus co-infection, and patients with impaired renal function requiring dose adjustment of nucleos(t)ide analogs were excluded. The study was approved by the Institutional Review Board of the University of Michigan.
 
Medical records were reviewed and information on patient demographics (age, gender, race) body weight, HBV markers (hepatitis B e antigen [HBeAg], hepatitis B e antibody [anti- HBe], HBV DNA); hepatic panel (albumin, aspartate aminotransferase [AST], alanine aminotransferase [ALT], total bilirubin [TB], and alkaline phosphatase [alk phos]); complete blood count (CBC); prothrombin time (PT)/international normalized ratio (INR); and liver histology were recorded. HBV treatment history was reviewed. Index treatment was defined as the first course of nucleos(t)ide analog therapy initiated at our liver clinic. Start and stop dates of index and prior HBV treatment, medications used during each course of treatment, and serial results of HBeAg, anti-HBe, HBV DNA, AST, and ALT during treatment were recorded.
 
During treatment, serum HBV DNA and hepatic panel was tested every 3 months and HBeAg/anti-HBe every 6-12 months. Serum HBV DNA was quantified by commercial polymerase chain reaction (PCR) assays - Amplicor HBV monitor test (Roche Molecular Diagnostics, Indianapolis, IN) with a lower limit of detection of 200-1,000 copies/mL (40-200 IU/mL) between 2000 and 2005, and real-time polymerase chain reaction assays, COBAS TaqMan HBV (Roche Molecular Diagnostics) with a lower limit of detection of 29 IU/mL from July 2005 onwards.
 
Tests for antiviral drug resistance mutations
 
HBV DNA from serum samples of patients with VBT was amplified and sequenced as described previously (10). The DNA sequences were aligned with SeqmanTM II and EditSeqTM software (DNASTAR Inc, Madison, WI) and compared to consensus sequences of the respective HBV genotype. All samples were also tested for antiviral drug resistance mutations by a line probe assay, INNO-Lipa HBV DR v.2 and v.3 (Innogenetics NV, Gent, Belgium) according to the manufacturer's instructions (10, 11).
 
Definition of virological breakthrough, genotypic resistance, and biochemical breakthrough
 
Virological breakthrough (VBT) was defined as any increase in serum HBV DNA by >1 log10 from nadir or redetection of serum HBV DNA at levels ≥10-fold the lower limit of detection of the HBV DNA assay after having an undetectable result. Thus, a patient who previously had undetectable serum HBV DNA by an assay with a lower limit of detection of 29 IU/mL would be considered to have a VBT if serum HBV DNA is subsequently detected at levels ≥290 IU/mL. A cutoff ≥10-fold the lower limit of detection was chosen because the consensus definition of VBT required a 10- fold increase in HBV DNA(2, 12). In addition, HBV DNA levels slightly above the limit of detection may not be reproducible on retesting of the same serum sample. Confirmed virological breakthrough (confirmed VBT) was defined as persistence of VBT on repeat test (fulfilling same criteria) 1-3 months later (with or without further increase in serum HBV DNA). Genotypic resistance (GR) was defined as detection of signature resistance mutations by direct sequencing. Signature resistance mutations included alanine to threonine or valine at codon 181 (rtA181T/V), threonine to alanine, cysteine, glycine, isoleucine, leucine, methionine, or serine at codon 184 (rtT184A/C/G/I/L/M/S), alanine to threonine substitution at codon 194 (rtA194T), serine to cysteine, glycine, or isoleucine at codon 202 (rtS202C/G/I), methionine to valine or isoleucine substitution at codon 204 (rtM204V/I), asparagine to threonine at codon 236 (rtN236T), and methionine to isoleucine or valine at codon 250 (rtM250I/V). Compensatory mutations such as leucine to methionine substitution at codon 180 (rtL180M) were not included (13).
 
Biochemical breakthrough (BBT) was defined as ALT above the upper limit of normal (ULN) (35 IU/L) in patients who had normalized ALT and ALT >2 times nadir in those who never had normal ALT. ALT flare was defined as ALT >5 times ULN in patients who had normalized ALT and ALT >5 times nadir in those who never had normal ALT.
 
RESULT
 
Characteristics of patients at the start of the index regimen

 
A total of 148 CHB patients were included. Five patients who met other criteria but whose serum HBV DNA exceeded 10,000 IU/mL after 1 year of treatment were excluded, all five patients had >1 log10 decrease in HBV DNA after 6 months of treatment. Characteristics of the patients at the start of the index regimen are shown in table 1. Seventy-three percent of the patients were men and the mean age was 44.9 ± 12.3 years. Approximately half (48.7%) of the patients were Asian, 41.2% were Caucasian, and the remainder were of other races. Roughly half (52.7%) of the patients were HBeAg positive and the mean HBV DNA was 6.2 ± 1.8 log10 IU/mL. Most (87.2%) patients had an elevated serum ALT based on our hospital laboratory reference range and 42 (28.4%) had ALT level >5 times the upper limit of normal.
 
Index regimen and initial response
 
Table 2 lists the medications used in the index regimen. Among the 81 NUC-naïve patients, entecavir alone (n=43) and lamivudine alone (n=26) were the most common medications. Of the 67 NUC-experienced patients; 19 received combination therapy, 16 received adefovir, 15 received tenofovir, 13 received entecavir, and 4 received lamivudine. The mean duration of follow-up was 37.5 ± 20.1 months (median 31.5 (range 12-102) months). Forty-two (28.4%) patients had been receiving the index treatment regimen for more than 48 months.
 
After one year on the index regimen, 70.9% of the patients had undetectable HBV DNA (Table 2). With continued treatment, 86.5% (87.7% NUC-naïve and 85.1% NUC-experienced) of patients achieved undetectable HBV DNA. The mean interval from the start of the index treatment to nadir virological response was 9 ± 6.3 months. Of the 136 patients who had baseline elevated ALT, 105 (77.2%) achieved ALT normalization after a mean of 8.4 ± 8.3 months.
 
Virological breakthrough during treatment
 
Thirty-nine (26.4%) patients experienced at least one VBT after the first year of treatment, 24 (62%) of these patients had confirmed VBT, 24 (62%) had GR by direct sequencing, 13 (33%) had biochemical breakthrough and only one had ALT flare (Figure 1). Of the 36 patients who had repeat HBV DNA after VBT, the mean interval from VBT to confirmed VBT was 2.4 ± 1.6 months. Line probe assay revealed two additional patients had GR. Both were receiving adefovir and found to have rtN236T mutation (Figure 1). The first patient had confirmed VBT and was switched to combination of tenofovir and emtricitabine with undetectable HBV DNA 6 months after start of rescue therapy (supplementary table1, patient T). The second patient (patient 8) was not confirmed to have VBT on retesting and continued to receive adefovir monotherapy.
 
The baseline characteristics of the patients who did or did not experience VBT were similar except for lower serum albumin among the patients who subsequently experienced VBT (Table 1). The cumulative probability of VBT at 3 and 5 years was 21.5% and 46.1%, confirmed VBT was 13.7% and 29.7%, and GR was 10.7% and 33.9%, respectively (Figure 2). If the traditional definition of VBT (increase in HBV DNA by >1 log from nadir or redetection of any level of HBV DNA after becoming undetectable) was used, 45 (30%) patients would be considered to have experienced at least 1 VBT and 26 (58%) of these patients would have confirmed VBT.
 
Among the NUC-naïve patients, the cumulative probability of VBT, confirmed VBT, and GR at 3 and 5 years was 18% and 54%, 12% and 31%, and 13% and 39%, respectively. NUC- naïve patients who experienced VBT were more likely to be receiving lamivudine than other nucleos(t)ide analogs (Table 2). Fourteen of 26 (53.8%) patients receiving lamivudine monotherapy experienced at least one VBT compared to 3 of 43 (7%) patients receiving entecavir monotherapy. Antiviral drug resistance mutations were detected in 12 of the 14 patients receiving lamivudine and 1 of the 3 patients receiving entecavir who experienced VBT. The cumulative probability of VBT, confirmed VBT, and GR at 3 years was 37%, 30%, and 33%, respectively among the patients receiving lamivudine monotherapy (Figure 3A); and 13%, 3.1%, and 3.1%, respectively among the patients receiving entecavir monotherapy (Figure 3B) (P = 0.001, 0.001, and <0.001, respectively).
 
Among the NUC-experienced patients, the cumulative probability of VBT, confirmed VBT, and GR at 3 and 5 years was 22% and 33%, 14% and 28%, and 8% and 29%, respectively. Sixteen of 33 (48.5%) patients who were receiving lamivudine, adefovir or entecavir monotherapy but only 2 of 34 (5.9%) patients receiving combination therapy or tenofovir monotherapy experienced VBT. The cumulative probability of VBT, confirmed VBT, and GR at 3 years was 32.3%, 23.6%, and 13.8%, respectively among the patients receiving lamivudine, adefovir or entecavir monotherapy; and 8.7%, 3.8%, and 0%, respectively among the patients receiving combination therapy or tenofovir monotherapy (P = 0.005, 0.01, and 0.04, respectively).
 
Univariate analyses found that failure to achieve undetectable HBV DNA after 1 year of treatment (HR 2.6, 95% CI 1.39-5.0, P = 0.003) and at nadir response (HR 6.92, 95% CI 3.4- 14.1, P = <0.001) significantly increased the risk of VBT. Multivariate analyses showed that failure to achieve undetectable HBV DNA at nadir response was the only factor significantly associated with VBT in the overall population (HR 5.5, 95% CI 2.49-12.28, P=<0.001). For sub- group analyses, the predictors of VBT among NUC-naïve patients were failure to achieve undetectable HBV DNA at 1 year (HR 2.79, 95% CI 1.11-7.01, P = 0.03) and at nadir response (HR 3.95, 95% CI 1.46-10.71, P = 0.007), and receipt of lamivudine monotherapy (HR 3.19, 95% CI 1.22-8.33, P = 0.02). The predictors of VBT among the NUC-experienced patients were failure to achieve undetectable HBV DNA at nadir response (HR 9.59, 95% CI 1.64-56.07, P = 0.01), and monotherapy with drugs other than tenofovir (HR 8.15, 95% CI 1.61-41.29, P = 0.01) (Table 3).
 
Outcome of patients who had virological breakthrough
 
Of the 39 patients who had at least 1 episode of VBT, 24 (62%) had confirmed VBT, 12 were not confirmed to have VBT on retesting, and three were not available for retesting because they received rescue therapy when VBT was first detected (Figure 1). Two of the latter three patients had GR. BBT was observed in 9 (37.5%) of 24 patients with confirmed VBT, 3 (25%) of 12 not confirmed to have VBT and 1 of the 3 patients who received rescue therapy when VBT was first detected (Figure 1). Only one patient had an ALT flare. This patient had confirmed VBT, nadir ALT was 30 IU/L, and ALT at the time of VBT was 34 IU/L. This patient was non-compliant and did not have repeat HBV DNA testing until 8 months after the initial VBT. At the time of confirmed VBT, ALT was 258 IU/L.
 
Among the 24 patients who had confirmed VBT, 19 patients had GR, and all 19 received rescue therapy. The index treatment, mutations detected at the time of VBT, rescue therapy administered, and response to rescue therapy in these 19 patients are shown in supplementary table 1 (patients A-S). Seventeen of these 19 patients had undetectable HBV DNA within 6 months of initiating rescue therapy.
 
Of the 5 patients who did not have GR by direct sequencing, two (patients 1 and 2) continued on the same treatment (Figure 4) and the other three received rescue therapy (supplementary table 1, patients T-V). Patient 1 had further decrease in HBV DNA level during continued treatment with the same medication but HBV DNA remained detectable. Serum HBV DNA at the last visit was 2.2 log10 IU/mL, 12 months after the VBT. Patient 2 had a slow decline in serum HBV DNA level, retesting for GR 10 months later did not reveal any signature antiviral resistance mutations and HBV DNA became undetectable 19 months after the VBT. This patient subsequently developed two more episodes of VBT but testing for GR did not reveal any signature antiviral resistance mutations and HBV DNA became undetectable again after each episode of VBT. No BBT was observed during subsequent episodes of VBT. The three patients who received rescue therapy had undetectable HBV DNA within 6 months.
 
Among the 12 patients who had VBT that was not confirmed on retesting, 3 were found to have signature antiviral resistance mutations. All three patients were receiving lamivudine monotherapy and were found to have M204I mutation. Two patients (supplementary table 1, patients W and X) had undetectable HBV DNA within 6 months after rescue therapy while the third was lost to follow-up. The remaining 9 patients who did not have confirmed VBT had no evidence of GR, 2 had BBT with ALT of 56 IU/L and 65 IU/L. Eight of these nine patients (patients 3-10) continued on the same treatment while one patient received rescue therapy. HBV DNA levels in four patients (patients 3-6) who continued on the same treatment became consistently undetectable during follow-up. Three (patients 7-9) patients' HBV DNA levels became undetectable during continued treatment with the same medication but HBV DNA subsequently became detectable at levels below our VBT criteria. Retesting did not reveal any signature antiviral resistance mutations and HBV DNA levels of these 3 patients became undetectable again with continued treatment on the same medication. The last patient (patient 10) had an initial decline in HBV DNA during continued treatment with the same medication but developed a second VBT 33 months after the first episode of VBT. This second episode of VBT was not confirmed on retesting and no signature antiviral resistance mutation was detected. HBV DNA level subsequently became undetectable (Figure 5).
 
In total, 10 patients who experienced VBT continued on the same treatment and had been followed for a mean of 29.3 ± 20.5 months (median 24.5 (range 9-75) months) after the initial episode of VBT. All 10 patients had further decrease in serum HBV DNA and nine had undetectable HBV DNA a mean of 6.8 ± 6.3 months after the VBT. Six patients had one or more episodes of transient increase in serum HBV DNA; of these, two met our criteria for VBT but none had GR. Two of these six patients had a mild increase in ALT with peak values of 56 and 65 IU/L during subsequent episodes of HBV DNA increase.
 
Of the remaining 29 patients with VBT, 28 received rescue therapy and all but three had undetectable HBV DNA within 6 months of initiating rescue therapy while one was lost to follow- up.
 
 
 
 
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