Title: Reactivation of hepatitis B in patients of chronic hepatitis C with hepatitis B virus infection treated with direct acting antivirals Short title: HBV reactivation in DAA therapy Authorship Ming-Lun Yeh1,2,3, Chung-Feng Huang2,3, Meng-HsuanHsieh2,3,5, Yu-Min Ko2, Kuan-Yu Chen2, Ta-Wei Liu2, Yi-Hung Lin2, Po-Cheng Liang2, Ming-Yen Hsieh2,Zu-YauLin2,3, Shinn-CherngChen2,3,Ching-I Huang2, Jee-Fu Huang2,3,4, Po-Lin Kuo1, Chia-Yen Dai1,2,3,4,5*, Ming-Lung Yu2,3,4,6.7*, Wan-Long Chuang2,3,4 Affiliations: 1 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3
School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Lipid Science and Aging Research Center, and Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Health Management Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 6 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 7 Liver Center, Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA *
Correspondence: Chia-Yen Dai or Ming-Lung Yu Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Tzyou Road, Kaohsiung City 807, Taiwan. Tel: +886-7-312-1101 ext. 7475 Fax: +886-7-312-3955 Email:[email protected] / [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.13771 This article is protected by copyright. All rights reserved.
Word counts: abstract: 249, text:2485, 4 figures, 2 tables Abbreviations: HBV, hepatitis B virus; CHC, chronic hepatitis C; DAA, direct acting antivirals; Anti-HBc, hepatitis B core antibody; HBsAg, hepatitis B surface antigen; Peg-IFN, pegylated interferon; RBV, ribavirin; SVR, sustained virological response; HCV, hepatitis C virus; Anti-HCV, HCV antibodies; HBsAb, hepatitis B surface antibody; LLOQ, low limit of quantification; EOT VR, end of treatment virological response; ULN, upper limit of normal; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Statement of Interests Author's declaration of personal interests:Nothing to disclose for all the authors Declaration of funding interests: This study was funded in part by Kaohsiung Medical University Hospital (grant number KMUH103-3R03,KMUH104-4R05, KMUH104-4T02), and Ministry of Science and Technology, R.O.C (grant number MOST 105-2628-B-037-002-MY2). The present study had never been presented in any meeting Authorship Statement Guarantor of article: CY Dai Specific author contributions:ML Yeh, CY Dai, ML Yu, and WL Chuang designed the research study; ML Yeh, MH Hsieh, TW Liu, YH Lin, PC Liang, MY Hsieh, ZY Lin, SC Chen, CI Huang, JF Huang, CY Dai, ML Yu, and WL Chuang collected the data;ML Yeh, CF Huang, YM Ko, KY Chen, JF Huang, PL Kuo, CY Dai, ML Yu performed the data analysis and interpretation; ML Yehwrote the paper, JF Huang, CY Dai, ML Yu, and WL Chuang helped the manuscript drafting and critical revising. All the authors have read and approved the submitted manuscript Writing Assistance: None
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Abstract Background& Aims: Hepatitis B virus (HBV) may reactivate when treating chronic hepatitis C (CHC) with direct acting antivirals (DAA). We aim to investigate the risk of HBV reactivation during DAA therapy. Methods: CHC patients receiving pan-oral DAA therapy from Dec. 2013 to Aug. 2016 were evaluated. Fifty-seven patients that had a past HBV infection (negative hepatitis B surface antigen (HBsAg)) and positive hepatitis B core antibody (Anti-HBc)) and 7 patients that had a current HBV infection (positive HBsAg) were enrolled. Serum HBV and hepatitis C virus (HCV) markers were regularly measured. The endpoints were the HCV sustained virological response (SVR) and the HBV virological/clinical reactivation. Results: The overall SVR12 rate was 96.9%, and 2 patients, one with positive HBsAg, had a relapse of HCV. No episodes of HBV virological reactivation were observed among the patients with a past HBV infection. For the 7 patients with a current HBV infection, HBV virological reactivation was found in 4 (57.1%) of the 7 patients. Clinical reactivation of HBV was observed in one patient with pre-treatment detectable HBV DNA and recovered after entecavir administration. For the other 3 patients with HBV virological reactivation, the reappearance of low level HBV DNA without clinical reactivation was observed. HBsAg levels demonstrated only small fluctuations in all the patients. Conclusions: There was a minimal impact of Anti-HBc seropositivity on HCV efficacy and safety. For CHC patients with current HBV infection, the risk of HBV reactivation was present and monitoring the HBV DNA level during therapy is warranted. Key words: hepatitis C virus; hepatitis B virus, dual-infection, direct acting-antivirals; reactivation.
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Introduction Interferon (IFN)-free regimens with directly acting antivirals (DAA) have markedly improved the treatment efficacy for chronic hepatitis C (CHC), with advantages including shorter treatment duration and fewer adverse events, when compared to PEGylated interferon (Peg-IFN)/ribavirin (RBV) combination therapy.1, 2 Using the new DAA combination therapy, a greater than 95% sustained virological response (SVR) rate can be achieved for hepatitis C virus (HCV) genotype 1, and a 90% SVR rate can be achieved for genotype 2 infected patients.3-7 To date, IFN-free DAA combination therapy has been recommended as the first line therapy for CHC.8-10 In high hepatitis B virus (HBV) endemic areas, such as the Asia-Pacific region, evidence of HBV exposure, seropositivity for hepatitis B core antibody (Anti-HBc), exists in a large proportion of adults. Approximately 10% of HCV viremic patients were seropositive for hepatitis B surface antigen (HBsAg).11Additionally, more than 50% of subjects without an HBV vaccination were seropositive for Anti-HBc in a high HBV endemic area.12, 13 For patients with a HBV/HCV dual-infection, Peg-IFN/RBV therapy achieved a similar HCV sustained virological response (SVR) rate compared with HCV mono-infection patients.14, 15 The reactivation of HBV after HCV eradication has also been reported.14 We found that 19.1% of HBV/HCV dually infected patients, who had undetectable HBV DNA pre-treatment, had detectable HBV DNA at 24 weeks post-treatment. However, the risk of an HBV-related hepatic flare was very rare in the clinical setting with Peg-IFN/RBV. In contrast, several case reports revealed that HBV reactivation, even induced fulminant hepatitis, was found in DAA-treated HCV patients with positive HBsAg, even in patients with isolated Anti-HBc seropositivity.16-18 Since the Asia-Pacific area is hyperendemic for HBV exposure for adults who were born before the mass HBV vaccination program, the concern regarding DAA treatment resulting in HBV reactivation needs to be clarified. Herein, we aimed to explore the impact of HBV infection on the treatment efficacy and the risk of HBV reactivation for CHC patients treated with pan-oral DAA therapy. Materials and Methods The present study was conducted at one tertiary hospital in Taiwan from Dec. 2013 to Aug. 2016. A total of 132 CHC patients treated with pan-oral DAA therapy were evaluated. Forty-six patients with ongoing DAA therapy and 22 Anti-HBc negative patients were excluded. Finally, a total of 64 patients, including seven with current HBV infections (positive HBsAg) and 57 with past HBV infections (negative HBsAg/positive Anti-HBc) were enrolled (Figure 1). The key inclusion criteria were 1) seropositivity for HCV antibodies (Anti-HCV) and HCV RNA for >6 months; 2) either HBsAg or Anti-HBc seropositivity; and 3) a completed pan-oral DAA therapy schedule for CHC. The key exclusion criteria were human immunodeficiency virus co-infection and other liver diseases, except for HBV or This article is protected by copyright. All rights reserved.
HCV infections, such as autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, Wilson’s disease, and α1-antitrypsin deficiency. The study was conducted according to the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the ethics committee of Kaohsiung Medical University Hospital. Written informed consent was obtained from all of the patients. The biochemistry and serum HBV markers including HBsAg, Anti-HBc, antibodies to HBsAg (Anti-HBs), and HBV DNA level and HCV markers including anti-HCV, HCV genotype, and HCVRNA level were determined during the study period. Serum HBsAg, Anti-HBc and Anti-HBs were tested by using commercially available enzyme-linked immunosorbent assay kits (Abbott Laboratories, North Chicago, IL, USA). Anti-HCV was determined by a third-generation enzyme immunoassay (Abbott Laboratories, North Chicago, IL). Serum levels of HCV RNA and the HCV genotype were measured by commercially available real-time PCR assays (RealTime HCV, detection limit: 12 IU/ml and Abbott RealTime HCV Genotype II; Abbott Molecular, Des Plaines IL, USA).19, 20 Serum HBV DNA levels were determined using the CobasAmpliPrep/CobasTaqMan HBV assay (CAP/CTM version 2.0, Roche Diagnostics, Indianapolis, IN, USA; the dynamic range was 20 IU/mL – 1.7x108IU/mL).21 Serum HBsAg levels were quantified using the Architect HBsAg QT assay (Abbott Diagnostic, Germany). The sensitivity of the Architect assay ranged from 0.05 to 250 IU/mL. Samples with an HBsAg level higher than 250 IU/mL were further diluted to1:500 and 1:1000 to obtain a reading within the range of the calibration curve. The endpoints of the study were 1) achievement of HCV SVR12, defined as undetectable HCV RNA 12 weeks post-treatment and 2) virological and clinical reactivation of HBV. The definitions of HBV virological reactivation were, for patients with a current HBV infection, at least 1 log10 IU/mL increase from baseline for those with baseline detectable HBV DNA or a reappearance of HBV DNA for those with baseline undetectable HBV DNA22, and, for patients with a past HBV infection, a reappearance of HBsAg or an HBV DNA > 2000 IU/mL. Clinical reactivation was defined as a serum alanine aminotransferase (ALT) level ≥ 3-fold over the upper limit of normal (ULN) or > 100 U/L.23, 24 The HCV end of treatment virological response (EOTVR) was defined as undetectable HCV RNA at the end of treatment. Statistics Descriptive statistics were computed for all of the variables, including the median and range for continuous variables. For the categorical variables, the frequencies and percentages were estimated. The Mann-Whitney U test was used to compare continuous variables, and the chi-squared and Fisher’s exact tests were used to compare categorical variables. All tests were two-sided, and a P value of < 0.05 was considered to be statistically This article is protected by copyright. All rights reserved.
significant. All analyses were performed using the SPSS 19.0 statistical package (SPSS, Inc., Chicago, IL, USA). Results Demographics Table 1 shows the demographics of all patients with current or past HBV infections. Of all of the patients, 17 (26.6%) patients were male with a median age of 63.0 years. Twenty-two (34.4%) and 4 (6.3%) of the patients had liver cirrhosis and hepatocellular carcinoma before therapy, respectively. The median pre-treatment aspartate aminotransferase (AST) and ALT levels were 64.0 and 75.5 IU/L in all of the patients, respectively. Forty-six (71.9%) patients had an HCV genotype 1 infection. The median pre-treatment HCV RNA level was 6.2 log10 IU/mL. Three (4.7%) of the patients had HBV DNA that was detectable pre-treatment, and all of them had a current HBV infection. Forty-five (70.3%) patients were naïve to anti-HCV therapy. The DAA regimens included ledipasvir/sofosbuvir fixed dose combination ± ribavirin (n = 23, 35.9%), sofosbuvir+ ribavirin (n = 17, 26.6%), ombitasvir/paritaprevir/ritonavir co-formulation + dasabuvir (n = 9, 14.1%), and daclatasvir-based DAA combination (n = 15, 23.4%). Compared to patients with a past HBV infection, patients with a current HBV infection had a significantly lower pre-treatment HCV RNA level (p = 0.016) and a higher rate of detectable HBV DNA (p = 0.001).
HCV virological response The overall rates of EOTVR and SVR12 were 100% and 96.9%, respectively. Of the 2 patients without HCV SVR, one had a current HBV infection, and the other had a past HBV infection. Figure 2 shows the HCV responses at treatment week 4, the end of treatment and 12 weeks post-treatment. Reactivation of HBV No episodes of HBV virological reactivation during DAA therapy were observed among the patients with a past HBV infection. Four patients had clinical reactivation, 2 at the 4th week, 1 at the 8th week and another at EOT. All of the clinical reactivations late subsided; none were related to HBV. Three (42.9%) of the 7 patients with a current HBV infection had detectable pre-treatment HBV DNA levels of 127, 998, and 16600 IU/mL before DAA therapy. The median pre-treatment HBsAg level of the 7 patients was 228.6 (8.2, 1141.2) IU/mL. Figure 3 reveals the individual dynamics of the ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 3 patients with detectable pre-treatment HBV DNA. For the 3 This article is protected by copyright. All rights reserved.
patients with detectable HBV DNA before treatment, undetectable HBV DNA was found during DAA therapy in the patient with a baseline HBV DNA level of 127 IU/mL. However, HBV DNA reappeared (890 IU/mL) at 12 weeks post-treatment but did not reach the definition of virological reactivation (Figure 3A). Another patient with a baseline HBV DNA level of 998 IU/mL demonstrated a persistent detectable HBV DNA level but without a 1 log10 IU/mL increase during or after treatment(Figure 3B); there was no concomitant clinical reactivation in these 2 patients. An HBV virological reactivation combined with a clinical reactivation was found in the final patient (Figure 3C). In this patient, the baseline HBV DNA level was as high as 16600 IU/mL and then increased gradually after starting the treatment. In treatment week 2, the HBV DNA level had increased more than one log10 IU/mL but without a concomitant ALT elevation. In treatment week 6, a persistent increased HBV DNA level with clinical reactivation (ALT 108 U/L) was observed. Then, jaundice with a total bilirubin level >2 mg/dL was identified at treatment week 7, and 0.5 mg per day of entecavir was immediately added. After the entecavir add-on, the HBV DNA and ALT levels declined gradually. For the four patients with undetectable HBV DNA pre-treatment, one had persistent undetectable HBV DNA through the end of follow up (Figure 4A), and 3 patients had an HBV virological reactivation. Of the three patients, 2 had HBVDNA reappearance at the1st and 4th weeks of treatment that persisted until the end of follow up (Figure 4B&4C). Notably, an HBV DNA surge (reappearance with HBV DNA >1000 IU/mL) was found in the remaining patient at treatment week 8 and spontaneously declined to an undetectable level by the end of treatment (Figure 4D). However, none of the patients had clinical reactivation, even in the patient with an HBV DNA surge. Overall, 4 (57.1%) of the 7 patients had an HBV virological reactivation, and 1 (14.3%) patient had an HBV-related clinical reactivation. Mortality or liver transplantation was not observed in these patients. No obvious fluctuation of the HBsAg level was observed in any of the patients, including the one who had an HBV-related clinical reactivation. The factors associated with an HBV reactivation were not analyzed because of the small sample size; a high pre-treatment HBV DNA level might be an important risk factor.
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Discussion There is increasing concern regarding hepatitis flare related to HBV reactivation during DAA therapy in CHC patients with current or past HBV infections. The current study demonstrated that there is no impact of HBV exposure on CHC treatment with pan-oral DAA in high HBV endemic areas, such as Taiwan, in terms of HCV efficacy. However, we also found that patients still carried the risk of HBV virological reactivation with a clinical hepatitis flare-up during therapy. To date, several case reports have described that HBV reactivation may occur in HCV patients with current/past HBV infections while undergoing treatment with pan-oral DAA.16-18, 25-27 Table 2 summarizes the reported HBV reactivations during DAA therapy from the published literature. HBV reactivation might occur not only with in patients with a current HBV infection but also in patients with a past HBV infection. Of the reported cases, virological and clinical reactivation were reported; most cases recovered after HBV oral antiviral therapy administration, except one who had a fulminant hepatitis flare with eventual liver transplantation. Wang et al. reported their single cohort study on hepatitis flare because of HBV during DAA therapy for CHC. In the report, none of the 124 patients with occult HBV infection had a hepatitis flare related to HBV reactivation. However, 3 (30%) of the 10 patients with positive HBsAg experienced a hepatitis flare; all were related to HBV reactivation. Similarly, our results show that the risk of virological or clinical reactivation related to HBV was extremely low in patients with a past HBV infection. The presence of Anti-HBc neither influenced HCV efficacy nor led to HBV DNA reactivation during DAA therapy. For patients with positive HBsAg, only 1 (14.3%) of the 7 patients suffered clinical reactivation related to HBV reactivation. Our study also demonstrated the detailed dynamic changes of HCV, HBV viral load and liver enzymes that were not present in the prior study and helped further describe the viral interactions during DAA therapy. All of the results encourage screening for HBV infection prior to DAA therapy in CHC patients. The U.S. Food and Drug Administration recently released a warning for the reactivation of HBV during DAA therapy for CHC. The mechanism through which HBV reactivation occurs remains unknown. Prior studies have demonstrated that hepatitis C virus might suppress hepatitis B virus, but the mechanism remains uncertain.28-31Although IFN-based therapy was effective in the treatment of HBV/HCV dually infected patients32-34, HBV reactivation after HCV eradication has also been reported with IFN-based therapy for HBV/HCV dually infected patients.14, 32 Interestingly, HBV reactivation has not been reported in patients with an occult HBV infection with Peg-IFN-based therapy but was reported in those with pan-oral DAA therapy. The distinct mechanism that was involved was considered but requires further investigation.
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When a hepatocyte is infected with HCV and/or HBV, viral nucleic acids are released to the cytoplasm, and then, the virus exploits host machinery in order for viral replication to proceed. The viral nucleic acids also trigger host innate immune responses via pattern recognition receptors, leading to innate immune antiviral signaling cascades and orchestrating adaptive host immune responses. The immune responses play critical roles in determining the outcomes after HCV and HBV infections.35, 36 However, both viruses might have developed mechanisms to evade normal immune responses, resulting in persistence and chronic infection. Several domains of immune function have been reported to be impaired including innate responses and several domains of T cell functionality.37 Additionally, HBV reactivation has been well described, particularly in patients undergoing immunosuppressive therapy or chemotherapy.38, 39 The viral-viral interactions that take place in the context of HCV/HBV dual infection might also alter immune responses. Both in vitro and in vivo HCV infections have been shown to be the dominant infection, resulting in the suppression of HBV.40-42 Lower levels of HBV DNA have also been reported in patients with an HBV/HCV dual infection compared to in those with an HBV mono infection. This might be because of the impaired immune response in the context of HCV infection and the limited host machinery for viral replication to take place. All of this further emphasizes the importance of immune responses in viral infection and might explain the reactivation of HBV during DAA therapy. The limited case number, especially for patients with a current HBV infection, was a limitation of the present study. Because of the reimbursement policy, only a small number of CHC patients can afford the expensive regimens in Taiwan. Further large scale, prospective studies are needed to explore the critical issues for HBV/HCV dually infected patients treated with pan-oral DAA regimens. In conclusion, we observed a minimal impact of Anti-HBc seropositivity on HCV efficacy and safety in HBsAg-negative CHC patients. However, CHC patients with a current HBV infection still carried the risk of HBV reactivation during DAA therapy. Monitoring HBV DNA levels closely during therapy is warranted, especially in patients with a high pre-treatment HBV DNA level. Further investigation is needed to confirm the actual risk and mechanism.
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Table 1. Baseline characteristics of allCHC patients and patients with currentor past HBV infections. All
Current HBV infection
Past HBV infection
N = 64
N=7
N = 57
63.0 (35.0, 81.0)
57.0 (41.0, 66.0)
63.0 (35.0, 81.0)
0.109
Male gender
17 (26.6)
4 (57.1)
13 (22.8)
0.074
Liver cirrhosis
22 (34.4)
2 (28.6)
20 (35.1)
1.000
4 (6.3)
0 (0)
4 (7.0)
1.000
AST, U/L
64.0 (21.0, 284.0)
77.0 (24.0, 99.0)
63.0 (21.0, 284.0)
0.923
ALT, U/L
75.5 (16.0, 253.0)
50.0 (29.0, 153.0)
84.0 (16.0, 253.0)
0.747
46 (71.9)
4 (57.1)
42 (73.7)
0.391
6.2 (2.2, 7.4)
4.7 (2.8, 6.2)
6.3 (2.2, 7.4)
0.016
3 (4.7)
3 (42.9)
0 (0)
0.001
45 (70.3)
5 (71.4)
40 (70.2)
1.000
Age, years
Hepatocellular carcinoma
HCV genotype 1 HCV RNA, log10 IU/mL Detectable HBV DNA Treatment naive DAA regimen
p
0.932
Ledipasvir/Sofosbuvir± ribavirin
23 (35.9)
2 (28.6)
21 (36.8)
Sofosbuvir + ribavirin
17 (26.6)
2 (28.6)
15 (26.3)
Ombitasvir/paritaprevir/ritonavir +
9 (14.1)
2 (28.6)
7 (12.3)
15 (23.4)
1 (14.3)
14 (24.6)
Dasabuvir Daclatasvir based DAA combination
AST, aspartate aminotransferase; ALT, alanine aminotransferase; HCV, hepatitis C virus; HBV, hepatitis B virus; DAA, direct acting antivirals
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Table 2. Summary of reported HBV reactivation in HCV patients with current/resolved HBV infection during DAA therapy Current HBV infection: HBsAg (+); Resolved HBV infection: HBsAg (-)/Anti-HBc (+) Author/Reference
Current
Study type
The Present study
Cohort study
Numbers
7
HBV
DAA regimen
Cohort study
10
Outcome
Before DAAs
After DAAs
HBV DNA
Virological
Clinical
HBV DNA
log10 IU/mL
reactivation
reactivation
log10 IU/mL
LDV/SOF, PrOD,
Undetectable – 4
4 (57.1%)*
1 (14.3%)*
4.2 -> 5.5
DCV/SOF, SOF/RBV
Detectable – 3
patient with clinical
(2.1, 3.0, 4.2)
reactivation.
infection Wang C et al.22
HBV infection
LDV/SOF, DCV/SOF,
week
2
added ETV and recovery in
3.2 (2.0)
3 (30%)
3 (30%)†
NA
8
Hepatic failure in one
PrOD Collins JM et al.17
Case report
1
SMV/SOF
3.4
Yes
Yes
7.3
8
added TDF/FTC and recovery
Case report
1
DCV/ASV
1.7
Yes
Yes
6.2
6
added ETV and recovery
Kimura H et al.24
Case report
1
SMV/PegIFN/RBV
2.5
Yes
No
3.5
11
added ETV and recovery
The Present study
Cohort study
57
LDV/SOF, ProD,
undetectable
0 (0%)*
0 (0%)*
undetectable
NA
0 (0%)†
0 (0%)†
NA
Takayama H et al.
Resolved
18
HBV
DCV/ASV, DCV/SOF
infection
SOF/RBV Wang C et al.22
Cohort study
124
LDV/SOF, DCV/SOF, PrOD
Ende AR et al.16
Case report
1
SMV/SOF
undetectable
Yes
Yes
7.5
11
added TDF and liver transplant
Collins JM et al.17
Case report
1
SMV/SOF
2000 IU/mL in patients with a past HBV infection. Definition of clinical reactivation: serum alanine aminotransferase level ≥ 3-foldof the upper limit of normal or > 100 U/L. †Definition of virological reactivation of resolved HBV infection: HBsAg turning from negative to positive, or appearance of HBV DNA in absence of HBsAg, or HBV DNA turned fromundetectable to detectable. Definition of clinical reactivation: more than 2-fold increase of serum alanine aminotransferase on two consecutive determinations at least five days apart, from the nadir during DAA therapy and follow-up.
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Figure 1. Study allocation flow chart.
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Figure 2. Rates of negative HCV RNA at week 4, EOT VR and SVR12 in all,current and past HBV infection patients.
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Figure 3. Dynamics of ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 3 HBsAg positive patients with detectable HBV DNA levels pre-treatment during DAA therapy.
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Figure 4. Dynamics of ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 4 HBsAg positive patients with undetectable HBV DNA pre-treatment during DAA therapy.
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School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Lipid Science and Aging Research Center, and Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Health Management Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 6 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 7 Liver Center, Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA *
Correspondence: Chia-Yen Dai or Ming-Lung Yu Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Tzyou Road, Kaohsiung City 807, Taiwan. Tel: +886-7-312-1101 ext. 7475 Fax: +886-7-312-3955 Email:[email protected] / [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.13771 This article is protected by copyright. All rights reserved.
Word counts: abstract: 249, text:2485, 4 figures, 2 tables Abbreviations: HBV, hepatitis B virus; CHC, chronic hepatitis C; DAA, direct acting antivirals; Anti-HBc, hepatitis B core antibody; HBsAg, hepatitis B surface antigen; Peg-IFN, pegylated interferon; RBV, ribavirin; SVR, sustained virological response; HCV, hepatitis C virus; Anti-HCV, HCV antibodies; HBsAb, hepatitis B surface antibody; LLOQ, low limit of quantification; EOT VR, end of treatment virological response; ULN, upper limit of normal; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Statement of Interests Author's declaration of personal interests:Nothing to disclose for all the authors Declaration of funding interests: This study was funded in part by Kaohsiung Medical University Hospital (grant number KMUH103-3R03,KMUH104-4R05, KMUH104-4T02), and Ministry of Science and Technology, R.O.C (grant number MOST 105-2628-B-037-002-MY2). The present study had never been presented in any meeting Authorship Statement Guarantor of article: CY Dai Specific author contributions:ML Yeh, CY Dai, ML Yu, and WL Chuang designed the research study; ML Yeh, MH Hsieh, TW Liu, YH Lin, PC Liang, MY Hsieh, ZY Lin, SC Chen, CI Huang, JF Huang, CY Dai, ML Yu, and WL Chuang collected the data;ML Yeh, CF Huang, YM Ko, KY Chen, JF Huang, PL Kuo, CY Dai, ML Yu performed the data analysis and interpretation; ML Yehwrote the paper, JF Huang, CY Dai, ML Yu, and WL Chuang helped the manuscript drafting and critical revising. All the authors have read and approved the submitted manuscript Writing Assistance: None
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Abstract Background& Aims: Hepatitis B virus (HBV) may reactivate when treating chronic hepatitis C (CHC) with direct acting antivirals (DAA). We aim to investigate the risk of HBV reactivation during DAA therapy. Methods: CHC patients receiving pan-oral DAA therapy from Dec. 2013 to Aug. 2016 were evaluated. Fifty-seven patients that had a past HBV infection (negative hepatitis B surface antigen (HBsAg)) and positive hepatitis B core antibody (Anti-HBc)) and 7 patients that had a current HBV infection (positive HBsAg) were enrolled. Serum HBV and hepatitis C virus (HCV) markers were regularly measured. The endpoints were the HCV sustained virological response (SVR) and the HBV virological/clinical reactivation. Results: The overall SVR12 rate was 96.9%, and 2 patients, one with positive HBsAg, had a relapse of HCV. No episodes of HBV virological reactivation were observed among the patients with a past HBV infection. For the 7 patients with a current HBV infection, HBV virological reactivation was found in 4 (57.1%) of the 7 patients. Clinical reactivation of HBV was observed in one patient with pre-treatment detectable HBV DNA and recovered after entecavir administration. For the other 3 patients with HBV virological reactivation, the reappearance of low level HBV DNA without clinical reactivation was observed. HBsAg levels demonstrated only small fluctuations in all the patients. Conclusions: There was a minimal impact of Anti-HBc seropositivity on HCV efficacy and safety. For CHC patients with current HBV infection, the risk of HBV reactivation was present and monitoring the HBV DNA level during therapy is warranted. Key words: hepatitis C virus; hepatitis B virus, dual-infection, direct acting-antivirals; reactivation.
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Introduction Interferon (IFN)-free regimens with directly acting antivirals (DAA) have markedly improved the treatment efficacy for chronic hepatitis C (CHC), with advantages including shorter treatment duration and fewer adverse events, when compared to PEGylated interferon (Peg-IFN)/ribavirin (RBV) combination therapy.1, 2 Using the new DAA combination therapy, a greater than 95% sustained virological response (SVR) rate can be achieved for hepatitis C virus (HCV) genotype 1, and a 90% SVR rate can be achieved for genotype 2 infected patients.3-7 To date, IFN-free DAA combination therapy has been recommended as the first line therapy for CHC.8-10 In high hepatitis B virus (HBV) endemic areas, such as the Asia-Pacific region, evidence of HBV exposure, seropositivity for hepatitis B core antibody (Anti-HBc), exists in a large proportion of adults. Approximately 10% of HCV viremic patients were seropositive for hepatitis B surface antigen (HBsAg).11Additionally, more than 50% of subjects without an HBV vaccination were seropositive for Anti-HBc in a high HBV endemic area.12, 13 For patients with a HBV/HCV dual-infection, Peg-IFN/RBV therapy achieved a similar HCV sustained virological response (SVR) rate compared with HCV mono-infection patients.14, 15 The reactivation of HBV after HCV eradication has also been reported.14 We found that 19.1% of HBV/HCV dually infected patients, who had undetectable HBV DNA pre-treatment, had detectable HBV DNA at 24 weeks post-treatment. However, the risk of an HBV-related hepatic flare was very rare in the clinical setting with Peg-IFN/RBV. In contrast, several case reports revealed that HBV reactivation, even induced fulminant hepatitis, was found in DAA-treated HCV patients with positive HBsAg, even in patients with isolated Anti-HBc seropositivity.16-18 Since the Asia-Pacific area is hyperendemic for HBV exposure for adults who were born before the mass HBV vaccination program, the concern regarding DAA treatment resulting in HBV reactivation needs to be clarified. Herein, we aimed to explore the impact of HBV infection on the treatment efficacy and the risk of HBV reactivation for CHC patients treated with pan-oral DAA therapy. Materials and Methods The present study was conducted at one tertiary hospital in Taiwan from Dec. 2013 to Aug. 2016. A total of 132 CHC patients treated with pan-oral DAA therapy were evaluated. Forty-six patients with ongoing DAA therapy and 22 Anti-HBc negative patients were excluded. Finally, a total of 64 patients, including seven with current HBV infections (positive HBsAg) and 57 with past HBV infections (negative HBsAg/positive Anti-HBc) were enrolled (Figure 1). The key inclusion criteria were 1) seropositivity for HCV antibodies (Anti-HCV) and HCV RNA for >6 months; 2) either HBsAg or Anti-HBc seropositivity; and 3) a completed pan-oral DAA therapy schedule for CHC. The key exclusion criteria were human immunodeficiency virus co-infection and other liver diseases, except for HBV or This article is protected by copyright. All rights reserved.
HCV infections, such as autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, Wilson’s disease, and α1-antitrypsin deficiency. The study was conducted according to the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the ethics committee of Kaohsiung Medical University Hospital. Written informed consent was obtained from all of the patients. The biochemistry and serum HBV markers including HBsAg, Anti-HBc, antibodies to HBsAg (Anti-HBs), and HBV DNA level and HCV markers including anti-HCV, HCV genotype, and HCVRNA level were determined during the study period. Serum HBsAg, Anti-HBc and Anti-HBs were tested by using commercially available enzyme-linked immunosorbent assay kits (Abbott Laboratories, North Chicago, IL, USA). Anti-HCV was determined by a third-generation enzyme immunoassay (Abbott Laboratories, North Chicago, IL). Serum levels of HCV RNA and the HCV genotype were measured by commercially available real-time PCR assays (RealTime HCV, detection limit: 12 IU/ml and Abbott RealTime HCV Genotype II; Abbott Molecular, Des Plaines IL, USA).19, 20 Serum HBV DNA levels were determined using the CobasAmpliPrep/CobasTaqMan HBV assay (CAP/CTM version 2.0, Roche Diagnostics, Indianapolis, IN, USA; the dynamic range was 20 IU/mL – 1.7x108IU/mL).21 Serum HBsAg levels were quantified using the Architect HBsAg QT assay (Abbott Diagnostic, Germany). The sensitivity of the Architect assay ranged from 0.05 to 250 IU/mL. Samples with an HBsAg level higher than 250 IU/mL were further diluted to1:500 and 1:1000 to obtain a reading within the range of the calibration curve. The endpoints of the study were 1) achievement of HCV SVR12, defined as undetectable HCV RNA 12 weeks post-treatment and 2) virological and clinical reactivation of HBV. The definitions of HBV virological reactivation were, for patients with a current HBV infection, at least 1 log10 IU/mL increase from baseline for those with baseline detectable HBV DNA or a reappearance of HBV DNA for those with baseline undetectable HBV DNA22, and, for patients with a past HBV infection, a reappearance of HBsAg or an HBV DNA > 2000 IU/mL. Clinical reactivation was defined as a serum alanine aminotransferase (ALT) level ≥ 3-fold over the upper limit of normal (ULN) or > 100 U/L.23, 24 The HCV end of treatment virological response (EOTVR) was defined as undetectable HCV RNA at the end of treatment. Statistics Descriptive statistics were computed for all of the variables, including the median and range for continuous variables. For the categorical variables, the frequencies and percentages were estimated. The Mann-Whitney U test was used to compare continuous variables, and the chi-squared and Fisher’s exact tests were used to compare categorical variables. All tests were two-sided, and a P value of < 0.05 was considered to be statistically This article is protected by copyright. All rights reserved.
significant. All analyses were performed using the SPSS 19.0 statistical package (SPSS, Inc., Chicago, IL, USA). Results Demographics Table 1 shows the demographics of all patients with current or past HBV infections. Of all of the patients, 17 (26.6%) patients were male with a median age of 63.0 years. Twenty-two (34.4%) and 4 (6.3%) of the patients had liver cirrhosis and hepatocellular carcinoma before therapy, respectively. The median pre-treatment aspartate aminotransferase (AST) and ALT levels were 64.0 and 75.5 IU/L in all of the patients, respectively. Forty-six (71.9%) patients had an HCV genotype 1 infection. The median pre-treatment HCV RNA level was 6.2 log10 IU/mL. Three (4.7%) of the patients had HBV DNA that was detectable pre-treatment, and all of them had a current HBV infection. Forty-five (70.3%) patients were naïve to anti-HCV therapy. The DAA regimens included ledipasvir/sofosbuvir fixed dose combination ± ribavirin (n = 23, 35.9%), sofosbuvir+ ribavirin (n = 17, 26.6%), ombitasvir/paritaprevir/ritonavir co-formulation + dasabuvir (n = 9, 14.1%), and daclatasvir-based DAA combination (n = 15, 23.4%). Compared to patients with a past HBV infection, patients with a current HBV infection had a significantly lower pre-treatment HCV RNA level (p = 0.016) and a higher rate of detectable HBV DNA (p = 0.001).
HCV virological response The overall rates of EOTVR and SVR12 were 100% and 96.9%, respectively. Of the 2 patients without HCV SVR, one had a current HBV infection, and the other had a past HBV infection. Figure 2 shows the HCV responses at treatment week 4, the end of treatment and 12 weeks post-treatment. Reactivation of HBV No episodes of HBV virological reactivation during DAA therapy were observed among the patients with a past HBV infection. Four patients had clinical reactivation, 2 at the 4th week, 1 at the 8th week and another at EOT. All of the clinical reactivations late subsided; none were related to HBV. Three (42.9%) of the 7 patients with a current HBV infection had detectable pre-treatment HBV DNA levels of 127, 998, and 16600 IU/mL before DAA therapy. The median pre-treatment HBsAg level of the 7 patients was 228.6 (8.2, 1141.2) IU/mL. Figure 3 reveals the individual dynamics of the ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 3 patients with detectable pre-treatment HBV DNA. For the 3 This article is protected by copyright. All rights reserved.
patients with detectable HBV DNA before treatment, undetectable HBV DNA was found during DAA therapy in the patient with a baseline HBV DNA level of 127 IU/mL. However, HBV DNA reappeared (890 IU/mL) at 12 weeks post-treatment but did not reach the definition of virological reactivation (Figure 3A). Another patient with a baseline HBV DNA level of 998 IU/mL demonstrated a persistent detectable HBV DNA level but without a 1 log10 IU/mL increase during or after treatment(Figure 3B); there was no concomitant clinical reactivation in these 2 patients. An HBV virological reactivation combined with a clinical reactivation was found in the final patient (Figure 3C). In this patient, the baseline HBV DNA level was as high as 16600 IU/mL and then increased gradually after starting the treatment. In treatment week 2, the HBV DNA level had increased more than one log10 IU/mL but without a concomitant ALT elevation. In treatment week 6, a persistent increased HBV DNA level with clinical reactivation (ALT 108 U/L) was observed. Then, jaundice with a total bilirubin level >2 mg/dL was identified at treatment week 7, and 0.5 mg per day of entecavir was immediately added. After the entecavir add-on, the HBV DNA and ALT levels declined gradually. For the four patients with undetectable HBV DNA pre-treatment, one had persistent undetectable HBV DNA through the end of follow up (Figure 4A), and 3 patients had an HBV virological reactivation. Of the three patients, 2 had HBVDNA reappearance at the1st and 4th weeks of treatment that persisted until the end of follow up (Figure 4B&4C). Notably, an HBV DNA surge (reappearance with HBV DNA >1000 IU/mL) was found in the remaining patient at treatment week 8 and spontaneously declined to an undetectable level by the end of treatment (Figure 4D). However, none of the patients had clinical reactivation, even in the patient with an HBV DNA surge. Overall, 4 (57.1%) of the 7 patients had an HBV virological reactivation, and 1 (14.3%) patient had an HBV-related clinical reactivation. Mortality or liver transplantation was not observed in these patients. No obvious fluctuation of the HBsAg level was observed in any of the patients, including the one who had an HBV-related clinical reactivation. The factors associated with an HBV reactivation were not analyzed because of the small sample size; a high pre-treatment HBV DNA level might be an important risk factor.
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Discussion There is increasing concern regarding hepatitis flare related to HBV reactivation during DAA therapy in CHC patients with current or past HBV infections. The current study demonstrated that there is no impact of HBV exposure on CHC treatment with pan-oral DAA in high HBV endemic areas, such as Taiwan, in terms of HCV efficacy. However, we also found that patients still carried the risk of HBV virological reactivation with a clinical hepatitis flare-up during therapy. To date, several case reports have described that HBV reactivation may occur in HCV patients with current/past HBV infections while undergoing treatment with pan-oral DAA.16-18, 25-27 Table 2 summarizes the reported HBV reactivations during DAA therapy from the published literature. HBV reactivation might occur not only with in patients with a current HBV infection but also in patients with a past HBV infection. Of the reported cases, virological and clinical reactivation were reported; most cases recovered after HBV oral antiviral therapy administration, except one who had a fulminant hepatitis flare with eventual liver transplantation. Wang et al. reported their single cohort study on hepatitis flare because of HBV during DAA therapy for CHC. In the report, none of the 124 patients with occult HBV infection had a hepatitis flare related to HBV reactivation. However, 3 (30%) of the 10 patients with positive HBsAg experienced a hepatitis flare; all were related to HBV reactivation. Similarly, our results show that the risk of virological or clinical reactivation related to HBV was extremely low in patients with a past HBV infection. The presence of Anti-HBc neither influenced HCV efficacy nor led to HBV DNA reactivation during DAA therapy. For patients with positive HBsAg, only 1 (14.3%) of the 7 patients suffered clinical reactivation related to HBV reactivation. Our study also demonstrated the detailed dynamic changes of HCV, HBV viral load and liver enzymes that were not present in the prior study and helped further describe the viral interactions during DAA therapy. All of the results encourage screening for HBV infection prior to DAA therapy in CHC patients. The U.S. Food and Drug Administration recently released a warning for the reactivation of HBV during DAA therapy for CHC. The mechanism through which HBV reactivation occurs remains unknown. Prior studies have demonstrated that hepatitis C virus might suppress hepatitis B virus, but the mechanism remains uncertain.28-31Although IFN-based therapy was effective in the treatment of HBV/HCV dually infected patients32-34, HBV reactivation after HCV eradication has also been reported with IFN-based therapy for HBV/HCV dually infected patients.14, 32 Interestingly, HBV reactivation has not been reported in patients with an occult HBV infection with Peg-IFN-based therapy but was reported in those with pan-oral DAA therapy. The distinct mechanism that was involved was considered but requires further investigation.
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When a hepatocyte is infected with HCV and/or HBV, viral nucleic acids are released to the cytoplasm, and then, the virus exploits host machinery in order for viral replication to proceed. The viral nucleic acids also trigger host innate immune responses via pattern recognition receptors, leading to innate immune antiviral signaling cascades and orchestrating adaptive host immune responses. The immune responses play critical roles in determining the outcomes after HCV and HBV infections.35, 36 However, both viruses might have developed mechanisms to evade normal immune responses, resulting in persistence and chronic infection. Several domains of immune function have been reported to be impaired including innate responses and several domains of T cell functionality.37 Additionally, HBV reactivation has been well described, particularly in patients undergoing immunosuppressive therapy or chemotherapy.38, 39 The viral-viral interactions that take place in the context of HCV/HBV dual infection might also alter immune responses. Both in vitro and in vivo HCV infections have been shown to be the dominant infection, resulting in the suppression of HBV.40-42 Lower levels of HBV DNA have also been reported in patients with an HBV/HCV dual infection compared to in those with an HBV mono infection. This might be because of the impaired immune response in the context of HCV infection and the limited host machinery for viral replication to take place. All of this further emphasizes the importance of immune responses in viral infection and might explain the reactivation of HBV during DAA therapy. The limited case number, especially for patients with a current HBV infection, was a limitation of the present study. Because of the reimbursement policy, only a small number of CHC patients can afford the expensive regimens in Taiwan. Further large scale, prospective studies are needed to explore the critical issues for HBV/HCV dually infected patients treated with pan-oral DAA regimens. In conclusion, we observed a minimal impact of Anti-HBc seropositivity on HCV efficacy and safety in HBsAg-negative CHC patients. However, CHC patients with a current HBV infection still carried the risk of HBV reactivation during DAA therapy. Monitoring HBV DNA levels closely during therapy is warranted, especially in patients with a high pre-treatment HBV DNA level. Further investigation is needed to confirm the actual risk and mechanism.
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Table 1. Baseline characteristics of allCHC patients and patients with currentor past HBV infections. All
Current HBV infection
Past HBV infection
N = 64
N=7
N = 57
63.0 (35.0, 81.0)
57.0 (41.0, 66.0)
63.0 (35.0, 81.0)
0.109
Male gender
17 (26.6)
4 (57.1)
13 (22.8)
0.074
Liver cirrhosis
22 (34.4)
2 (28.6)
20 (35.1)
1.000
4 (6.3)
0 (0)
4 (7.0)
1.000
AST, U/L
64.0 (21.0, 284.0)
77.0 (24.0, 99.0)
63.0 (21.0, 284.0)
0.923
ALT, U/L
75.5 (16.0, 253.0)
50.0 (29.0, 153.0)
84.0 (16.0, 253.0)
0.747
46 (71.9)
4 (57.1)
42 (73.7)
0.391
6.2 (2.2, 7.4)
4.7 (2.8, 6.2)
6.3 (2.2, 7.4)
0.016
3 (4.7)
3 (42.9)
0 (0)
0.001
45 (70.3)
5 (71.4)
40 (70.2)
1.000
Age, years
Hepatocellular carcinoma
HCV genotype 1 HCV RNA, log10 IU/mL Detectable HBV DNA Treatment naive DAA regimen
p
0.932
Ledipasvir/Sofosbuvir± ribavirin
23 (35.9)
2 (28.6)
21 (36.8)
Sofosbuvir + ribavirin
17 (26.6)
2 (28.6)
15 (26.3)
Ombitasvir/paritaprevir/ritonavir +
9 (14.1)
2 (28.6)
7 (12.3)
15 (23.4)
1 (14.3)
14 (24.6)
Dasabuvir Daclatasvir based DAA combination
AST, aspartate aminotransferase; ALT, alanine aminotransferase; HCV, hepatitis C virus; HBV, hepatitis B virus; DAA, direct acting antivirals
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Table 2. Summary of reported HBV reactivation in HCV patients with current/resolved HBV infection during DAA therapy Current HBV infection: HBsAg (+); Resolved HBV infection: HBsAg (-)/Anti-HBc (+) Author/Reference
Current
Study type
The Present study
Cohort study
Numbers
7
HBV
DAA regimen
Cohort study
10
Outcome
Before DAAs
After DAAs
HBV DNA
Virological
Clinical
HBV DNA
log10 IU/mL
reactivation
reactivation
log10 IU/mL
LDV/SOF, PrOD,
Undetectable – 4
4 (57.1%)*
1 (14.3%)*
4.2 -> 5.5
DCV/SOF, SOF/RBV
Detectable – 3
patient with clinical
(2.1, 3.0, 4.2)
reactivation.
infection Wang C et al.22
HBV infection
LDV/SOF, DCV/SOF,
week
2
added ETV and recovery in
3.2 (2.0)
3 (30%)
3 (30%)†
NA
8
Hepatic failure in one
PrOD Collins JM et al.17
Case report
1
SMV/SOF
3.4
Yes
Yes
7.3
8
added TDF/FTC and recovery
Case report
1
DCV/ASV
1.7
Yes
Yes
6.2
6
added ETV and recovery
Kimura H et al.24
Case report
1
SMV/PegIFN/RBV
2.5
Yes
No
3.5
11
added ETV and recovery
The Present study
Cohort study
57
LDV/SOF, ProD,
undetectable
0 (0%)*
0 (0%)*
undetectable
NA
0 (0%)†
0 (0%)†
NA
Takayama H et al.
Resolved
18
HBV
DCV/ASV, DCV/SOF
infection
SOF/RBV Wang C et al.22
Cohort study
124
LDV/SOF, DCV/SOF, PrOD
Ende AR et al.16
Case report
1
SMV/SOF
undetectable
Yes
Yes
7.5
11
added TDF and liver transplant
Collins JM et al.17
Case report
1
SMV/SOF
2000 IU/mL in patients with a past HBV infection. Definition of clinical reactivation: serum alanine aminotransferase level ≥ 3-foldof the upper limit of normal or > 100 U/L. †Definition of virological reactivation of resolved HBV infection: HBsAg turning from negative to positive, or appearance of HBV DNA in absence of HBsAg, or HBV DNA turned fromundetectable to detectable. Definition of clinical reactivation: more than 2-fold increase of serum alanine aminotransferase on two consecutive determinations at least five days apart, from the nadir during DAA therapy and follow-up.
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Figure 1. Study allocation flow chart.
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Figure 2. Rates of negative HCV RNA at week 4, EOT VR and SVR12 in all,current and past HBV infection patients.
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Figure 3. Dynamics of ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 3 HBsAg positive patients with detectable HBV DNA levels pre-treatment during DAA therapy.
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Figure 4. Dynamics of ALT, total bilirubin, HCV RNA, HBsAg and HBV DNA levels of the 4 HBsAg positive patients with undetectable HBV DNA pre-treatment during DAA therapy.
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