Chronic hepatitis B virus (HBV) infection remains a major

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2010;8:541 545 Hepatitis B Virus DNA Level Predicts Hepatic Decompensation in Patients With Acute Exacerbation of Chronic Hepatitis B WEN JUEI JENG, I SHYAN SHEEN, and YUN FAN LIAW Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan BACKGROUND & AIMS: Acute exacerbations of chronic hepatitis B virus (HBV) infection can lead to hepatic decompensation. It is important to identify factors that predict the development of hepatic decompensation during exacerbation so that antiviral therapy can be initiated immediately. METH- ODS: Acute exacerbation, defined by an abrupt increase in alanine aminotransferase (ALT) levels to 5-fold the upper limit of normal, occurred in 110 hepatitis B e antigen (HBeAg)- seropositive non-cirrhotic patients (138 episodes). The patients were monitored every 1 to 2 weeks for serum levels of ALT, bilirubin, albumin, and prothrombin. Sex, age, HBV genotype, ALT level, HBV viral load, and the causes (spontaneous or relapse from antiviral treatment) of exacerbation were included in multivariate logistic regression analyses. The receiver operating characteristic curve was used to identify the optimal cut-off value of serum HBV DNA level to identify patients at risk for decompensation. RESULTS: Seven of the 138 episodes of acute exacerbation (5.1%) resulted in hepatic decompensation; serum HBV DNA level was the only significant risk factor (P.003). The area under the receiver operating characteristic curve was 88.6% (P.001). A serum HBV DNA cut-off value of 1.55 10 9 copies/ml predicted decompensation with a sensitivity of 85.7%, a specificity of 85.5%, a negative prediction value of 99.1%, and positive prediction value of 24.0%. CONCLUSIONS: During acute exacerbation of HBeAg-positive chronic hepatitis B, a serum HBV DNA cut-off value of 1.55 10 9 copies/ml can be used to identify patients in need of immediate antiviral therapy. View this article s video abstract at www.cghjournal.org. Keywords: Increased ALT; HBeAg Seroconversion; Lamivudine; HBV Load. Chronic hepatitis B virus (HBV) infection remains a major global health issue because of its worldwide distribution and potential adverse sequela such as hepatic decompensation and the development of cirrhosis or hepatocellular carcinoma. 1 Chronic HBV infection is a dynamic state of interaction among HBV, hepatocytes, and the host immune cells. The liver injuries are considered to be the results of an HLA class I antigen restricted cytotoxic lymphocyte-mediated immune response against HBV, with resultant apoptosis and necrosis; thus, higher alanine transaminase (ALT) level increase usually reflects more vigorous immune response against HBV and more extensive liver damage. 2 Episodic high ALT increase or hepatitis flare may occur during the natural course of chronic HBV infection, particularly in hepatitis B e antigen (HBeAg)-seropositive patients. 3 5 Given the immune-mediated pathogenesis, such flares may be associated with a decrease in viral load and eventually lead to HBeAg seroconversion with clinical remission. 2 Based on this, Asian-Pacific HBV treatment guidelines have recommended that patients with ALT level greater than 5 the upper limit of normal can be monitored for spontaneous HBeAg seroconversion if there is no concern of hepatic decompensation. 6 However, some flares may be so severe that decompensation or even hepatic failure may develop and lead to fatality. 7 9 Although factors for earlier HBeAg seroconversion have been identified, 10 little is known about the prediction of decompensation. With the advent of potent and fast-acting anti-hbv agents, timely use of these agents is able to rescue patients who are developing or who have developed hepatic decompensation. 6,11 It therefore becomes very important to identify who is going to develop decompensation so that drug therapy can be instituted in time to prevent or rescue the deterioration of liver damage. The aim of this study was to investigate factor(s) predicting the development of hepatic decompensations during acute exacerbation in HBeAg-positive chronic hepatitis B. Patients and Methods The long-term follow-up study of our patients with clinicopathologically proven chronic hepatitis B (currently 1400 patients who are HBeAg seropositive at entry) has continued. In addition, new patients with abnormal ALT levels or hepatic flares visited our outpatient clinic voluntarily or by referral from other clinics or hospitals. After initial critical evaluation, all of them were advised to be monitored regularly at an interval according to their ALT levels, as described earlier. 3 HBeAg-positive patients with noncirrhotic chronic hepatitis B who experienced acute exacerbations during follow-up evaluation were enrolled consecutively. Patients who experienced acute exacerbation during antiviral therapy, patients who already had developed decompensations, patients with increasing ALT and/or bilirubin levels who may or may not develop decompensation but had received antiviral drug that likely would alter the outcome, patients with simultaneous anti-hbe seropositivity, and those who had evidence of cirrhosis were excluded. Patients with other causes of ALT increase, such as viral superinfections (hepatitis A virus, hepatitis C virus, and hepatitis D virus), alcohol consumption, metabolic causes of liver disease, and drug-induced hepatotoxicity also were excluded. Abbreviations used in this paper: AE, acute exacerbations; ALT, alanine aminotransferase; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. 2010 by the AGA Institute 1542-3565/$36.00 doi:10.1016/j.cgh.2010.02.023

542 JENG ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6 Once acute exacerbation was recognized, the patients were monitored weekly or biweekly, including measurements of serum ALT level (normal, 36 U/L), bilirubin level (normal, 1.3 mg/dl), prothrombin time (normal, 3 s), and albumin level (normal, 3.5 g/dl). Serum HBV DNA level was measured at enrollment when the first sign of acute hepatitis flare was evident. Definitions Chronic hepatitis B (CHB) was defined as hepatitis B surface antigen seropositivity for at least 6 months, or hepatitis B surface antigen seropositive but seronegative for immunoglobulin M anti hepatitis B core when known history was shorter than 6 months and there was no evidence of cirrhosis. Acute exacerbation (AE) was defined as an abrupt increase of serum ALT level to greater than 300 U/L in patients whose original ALT level was less than 5 the upper limit of normal or a 2-fold abrupt increase of serum ALT level to greater than 5 the upper limit of normal. 2 4 AE was considered spontaneous if not related to drug therapy. AE occurring within 12 months after discontinuation of lamivudine therapy was defined as posttherapy relapse. Hepatic decompensation was defined as the presence of jaundice with a serum total bilirubin level greater than 2 mg/dl and prolongation of prothrombin time by more than 3 seconds, or development of ascites/encephalopathy. 7 Evidence of cirrhosis included well-documented histologic evidence and/or ultrasonographic findings based on a scoring system as described earlier elsewhere. 12,13 Briefly, the scoring system is based on the ultrasonographic features of liver parenchyma, liver surface, hepatic vessel, and spleen size. The diagnosis of cirrhosis was made when the score was 8 or higher. The correlation study in our unit showed that ultrasonographic findings correlated closely with histologic findings of cirrhosis (r 0.571; P.001). 13 Methods In our unit, serum HBV DNA level was measured using the hybrid Capture II assay (Digene Corp, Gaithersburg, MD; lower limit of detection, 1.4 10 5 copies/ml) before April 2007 or a standardized automated quantitative polymerase chain reaction assay (Cobas Amplicor HBV Monitor; lower limit of detection, 300 copies/ml, or the Roche COBAS TaqMan HBV Test; lower limit of detection, 69 copies/ml, or 12 IU/mL; Roche Diagnostics, Pleasanton, CA) after April 2007. HBV genotypes were determined using polymerase chain reaction restriction fragment length polymorphism of the surface gene of HBV, as described elsewhere. 14 All biochemical tests were performed with an autoanalyzer. Immunoglobulin M anti hepatitis A virus, immunoglobulin M anti hepatitis B core, hepatitis B surface antigen, HBeAg, and anti-hbe were assayed using commercially available radioimmunoassay kits (HAVABM, CORABM, Austria II; Abbott Laboratories North Chicago, IL; HBeAg/anti-HBe test; Diasorin, Saluggia, Italy). Anti hepatitis C virus was assayed by AxSYM hepatitis C virus, version 3.0 (Abbott Laboratories). Statistics In comparisons between groups, the Student t test was used for continuous variables and the chi-square test or the Fisher exact test was used for categoric variables. Stepwise logistic regression was used for the multivariate analysis. A P value of less than.05 was considered statistically significant. The optimal cut-off value of serum HBV DNA, with the best sensitivity and specificity to discriminate patients who will develop hepatic decompensation, was calculated by the receiver operating characteristic curve. A c-statistic of 0.8 to 0.9 indicates excellent diagnostic accuracy and greater than 0.7 was considered useful. The analysis was performed with SPSS version 11.5 (SPSS Inc, Chicago, IL). Results Baseline Characteristics of Patients A total of 138 episodes of acute exacerbation were documented in 110 patients. Of these 110 patients, 79 (including 5 who subsequently developed decompensation) were confirmed histologically to be noncirrhotic before their hepatitis flare. All of these 79 patients showed an ultrasound score of 6 or less at the time of liver biopsy. The demographic features are shown in Table 1. These 138 episodes occurred at a median age Table 1. Demographic Features of Patients With Acute Exacerbations in Chronic Hepatitis Type B Hepatic decompensation Characteristics All exacerbations Yes No P Number 138 7 131 Age, y a 34.7 (16 58.3) 40.8 (19.6 51.8) 34.7 (16 58.3).145 Male, n (%) 102 (81.2) 6 (87.5) 96 (73.3).676 ALT level, U/L a 419 (182 1760) 620 (306 812) 418 (182 1760).052 Total serum bilirubin level, mg/dl a 1.1 (0.6 2.0) 1.3 (1.1 1.6) 1.1 (0.6 2.0).002 Albumin level, g/dl a 4.1 (3.8 4.5) 4.1 (3.8 4.6) 4.1 (3.8 4.5).898 PT prolongation, s a 0.4 (0.0 4.3) 0.9 (0.3 1.9) 0.4 (0.0 4.3).037 Genotype b.238 B 80 (58.0) 6 (85.7) 74 (56.5) C 58 (42.0) 1 (14.3) 57 (43.5) Cause Spontaneous cause b 59 (42.7) 4 (57.1) 55 (42.0).461 Posttherapy b 79 (57.3) 3 (42.9) 76 (58.0) HBV DNA level, 10 8 copies/ml a 5.657 (0.011 16.991) 16.698 (9.349 16.983) 5.036 (0.011 16.991).001 PT, prothrombin time. a Data expressed as median (range). b Data expressed as number (%).

June 2010 HBV DNA AND HEPATIC DECOMPENSATION 543 of 34.7 years (range, 16 58.3 y), 81.2% occurred in males and 58.0% occurred in genotype B HBV infected patients. At enrollment, the median value of the ALT level was 419 U/L, total bilirubin level was 1.1 mg/dl, prothrombin time prolongation was 0.4 seconds, and the HBV DNA level was 5.657 10 8 copies/ml. (All were assayed using Digene Capture II assay.) Seven (5.1%) of the 138 episodes of acute exacerbation subsequently developed hepatic decompensation before the antiviral drug was instituted. At the decision of respective hepatologists taking care of the patients, 53 remained untreated. HBeAg seroconversion occurred in 16 (30.2%) of these 53 patients within 1 year (median, 12 wk; range, 1 46.3 wk). Factors for the Development of Decompensation The HBV DNA level at enrollment was significantly higher in patients with subsequent hepatic decompensation than the well-compensated patients (median, 16.698 10 8 copies/ml vs 5.036 10 8 copies/ml; P.001) (Table 1). The decompensation group also had significantly higher total bilirubin level (1.3 vs 1.1 mg/dl; P.002) and prolonged prothrombin time (0.9 vs 0.4; P.037) at entry, whereas ALT level was higher at marginal statistical significance (620 vs 418; P.052) (Table 1). There was no significant difference in sex, HBV genotype, age, and albumin level at entry. Multivariate logistic regression analysis was conducted including all possible factors with a P value of less than.1 (Table 1). Serum HBV DNA level was found to be the only significant risk factor for the development of hepatic decompensation (coefficient, 0.268; odds ratio, 1.31 per 10 8 copies/ml; P.003) (Table 2). Predictability of Hepatitis B Virus DNA Levels The receiver operating characteristic curve analysis revealed that the c-statistic for the prediction of hepatic decompensation by serum HBV DNA level was 0.886 (95% confidence interval, 0.811 0.961; P.001). The optimal cut-off serum HBV DNA value derived from the receiver operating characteristic curve was 1.55 10 9 copies/ml (Figure 1). Hepatic decompensation can be predicted with a sensitivity of 85.7%, specificity of 85.5%, positive predictive value of 24.0%, negative predictive value of 99.1%, positive likelihood ratio of 5.91, negative likelihood ratio of 0.17, and relative risk of 27.1 (95% confidence interval, 4.6 170.1) (Table 3). Table 2. Multivariate Analysis of the Risk Factors for the Development of Hepatic Decompensations Variable P Coefficient Odds ratio Bilirubin-t, mg/dl.062 1.567 4.79 (0.92 24.90) HBV DNA, 10 8 copies/ml.003 0.268 1.31 (1.09 1.565) Figure 1. Receiver-operating characteristic curve of HBV DNA level (10 8 copies/ml) in the prediction of decompensation in patients with HBeAg-positive chronic hepatitis B with acute exacerbation (area under the curve of all patients: c-statistic, 0.886; 95% confidence interval, 0.811 0.961; P.001). Discussion The results of the present study have shown that a serum HBV DNA level greater than 1.55 10 9 copies/ml in HBeAg-positive patients with AE may predict subsequent development of hepatic decompensation with high sensitivity, specificity, and negative predictive value. Given the mechanism of liver injuries in chronic hepatitis B patients, effective host immune response against HBV should be followed by decreasing viral load with subsequent clinical improvement. 2 In contrast, a very high HBV DNA level in cases of AE suggests that the vigorous immune attack on HBV and the resultant liver injuries will continue and therefore may lead to hepatic decompensation. Earlier case-controlled studies have identified preexisting cirrhosis, high Child Pugh score, low albumin level, high bilirubin level, prolonged prothrombin time, and high HBV DNA level as factors associated with hepatic decompensation during acute exacerbation of CHB. 9,15,16 However, except for pre-existing cirrhosis, these so-called factors are actually the presenting features of overt hepatic decompensation. Obviously, these factors have no predictive value for subsequent development of decompensation. It is conceivable that cirrhotic patients are prone to develop decompensation because patients with cirrhosis have decreased functional reserve. 17 19 Of note is Table 3. The Predictability of HBV DNA Viral Load in Patients With Hepatic Decompensation HBV DNA level, 10 8 copies/ml No Decompensation a Yes Median 5.04 16.98 Range 0.01 16.99 9.35 16.98 Cut-off value 15.5 (n 25) 19 6 15.5 (n 113) 112 1 Sensitivity, % 85.7 (50.4 97.4) Specificity, % 85.5 (83.6 86.1) PPV, % 24.0 (14.1 27.3) NPV, % 99.1 (96.9 99.8) PPV, positive predictive value; NPV, negative predictive value. a P value.001.

544 JENG ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6 that patients with evidence of cirrhosis were excluded from the present study so that the results were not confounded by this factor. Therefore, the HBV DNA level identified in this study is a genuine predictor of hepatic decompensation in chronic hepatitis B. It seems conceivable that serum HBV DNA level predictive of developing decompensation might be lower in cirrhotic patients with acute exacerbation. It was reported that lamivudine could prevent fatality in AE patients with hepatic decompensation if therapy was started before the serum bilirubin level had increased to more than 20 mg/dl. 11 A study on lamivudine therapies in patients with advanced fibrosis or cirrhosis also showed better outcome if lamivudine was started before the Child Pugh score increased by 7 or more. 18 These findings suggest that antiviral therapy should be instituted as early as possible to be able to prevent and rescue hepatic decompensation. In Asia where cost and reimbursement of antiviral therapy are critical concerns, 20 it has been recommended to monitor CHB patients with hepatitis flare for possible spontaneous HBeAg seroconversion if there is no concern of hepatic decompensation. 6 The results of the present study showed that potent and fast-acting direct antiviral agents should be given as soon as possible if an HBV DNA level greater than 1.55 10 9 copies/ml is evident in CHB patients with hepatitis flare. In contrast, the very high negative predictive value (98.7%) of this level suggests that patients with AE and an HBV DNA level less than 1.55 10 9 copies/ml have little concern of hepatic decompensation and thus can be monitored closely for spontaneous HBeAg seroconversion. This notion is supported by the observation of this study that spontaneous HBeAg seroconversion occurred in 30.2% of the patients within 1 year (median, 12 wk; range, 1 46.3 wk). It also is consistent with the findings of Kim et al 21 that patients with CHB have a higher chance of early HBeAg seroconversion if their serum HBV DNA levels are less than 10 7 copies/ml during acute exacerbation. Mutations at the basal core promoter or precore region have been considered to be prognostic factors in CHB. 22 One of the limitations of the present study was that these mutations were not studied. However, studies from Taiwan showed that both precore mutation and basal core promoter mutation played no role in the occurrence of hepatic decompensation during acute exacerbation of chronic hepatitis B. 23,24 In addition, assays for precore/basal core promoter mutation are not routinely available and thus are not practical for clinical use as a predicting marker for hepatic decompensation. The other limitation of the present study was that HBeAg-negative patients were not included. In general, serum HBV DNA level in HBeAg-negative patients is lower than that in HBeAg-positive patients 1 and acute exacerbation in HBeAg-negative CHB patients is less severe as compared with those in HBeAg-positive patients. 3 The serum HBV DNA level predictive of decompensation in HBeAg-negative patients with exacerbation requires further study. In addition, the number of episodes of hepatic decompensation may be too small to represent the true sensitivity and specificity of the test. However, it represents the true incidence of hepatic decompensation in this cohort of patients and the incidence was not lower than that reported earlier. 7 In conclusion, HBV DNA level greater than 1.55 10 9 copies/ml in patients with AE may predict subsequent occurrence of hepatic decompensation. Potent and fast-acting nucleos(t)ide analogues should be started as soon as this situation is recognized. In contrast, patients with AE and an HBV DNA level less than 1.55 10 9 copies/ml may be monitored closely to ensure that they are not developing decompensation so that we can hold antiviral therapy and wait for spontaneous HBeAg seroconversion. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Clinical Gastroenterology and Hepatology at www.cghjournal.org, and at 10.1016/ j.cgh.2010.02.023. References 1. Liaw YF, Chu CM. Hepatitis B infection. Lancet 2009;14:582 592. 2. Liaw YF. Hepatitis flares and hepatitis B e antigen seroconversion: implication in anti-hepatitis B virus therapy. J Gastroenterol Hepatol 2003;18:246 252. 3. Liaw YF, Tai DI, Chu CM, et al. Acute exacerbation in chronic type B hepatitis: comparison between HBeAg and antibody-positive patients. Hepatology 1987;7:20 33. 4. Lok AS, Lai CL. Acute exacerbations in Chinese patients with chronic hepatitis B virus (HBV) infection. Incidence, predisposing factors and etiology. J Hepatol 1990;10:29 34. 5. Nair S, Perrillo RP. Serum alanine aminotransferase flares during interferon treatment of chronic hepatitis B: is sustained clearance of HBV DNA dependent on levels of pretreatment viremia? Hepatology 2001;34:1021 1026. 6. Liaw YF, Leung N, Kao JH, et al. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update. Hepatol Int 2008;2:263 283. 7. Sheen IS, Liaw YF, Tai DI, et al. Hepatic decompensation associated with hepatitis B e antigen clearance in chronic type B hepatitis. Gasteroenterology 1985;89:732 735. 8. Chu CM, Liaw YF. Increased incidence of fulminant hepatic failure in previous unrecognized HBsAg carriers with acute hepatitis independent of etiology. Infection 2005;33:136 139. 9. Yuen MF, Sablon E, Hui CK, et al. Prognostic factors in severe exacerbation of chronic hepatitis B. Clin Infect Dis 2003;36: 979 984. 10. Liaw YF. HBeAg seroconversion as an important end point in the treatment of chronic hepatitis B. Hepatol Int 2009;3:425 433. 11. Chien RN, Lin CH, Liaw YF. The effect of lamivudine therapy in hepatic decompensation during acute exacerbation of chronic hepatitis B. J Hepatol 2003;38:322 327. 12. Lin DY, Sheen IS, Chiu CT, et al. Ultrasonographic changes of early liver cirrhosis in chronic hepatitis B: a longitudinal study. J Clin Ultrasound 1993;21:303 308. 13. Tai DI, Lin SM, Sheen IS, et al. Long-term outcome of hepatitis B e antigen-negative hepatitis B surface antigen carriers in relation to changes of alanine aminotransferase levels over time. Hepatology 2009;49:1859 1867. 14. Chien RN, Yeh CT, Tsai SL, et al. Determinants for sustained HBeAg response to lamivudine therapy. Hepatology 2003;38: 1267 1273. 15. Ikegami T, Matsuki Y, Tanaka Y, et al. Impact of determination of hepatitis B virus subgenotype and pre-core/core-promoter mutation for the prediction of acute exacerbation of asymptomatic carriers. Hepatol Res 2009;39:341 345. 16. Kusakaba A, Tanaka Y, Mochida S, et al. Case-control study for the identification of virological factors associated with fulminant hepatitis B. Hepatol Res 2009;39:648 656. 17. Liaw YF, Chen JJ, Chen TJ. Acute exacerbation in patients with liver cirrhosis: a clinicopathological study. Liver 1990;10:177 184.

June 2010 HBV DNA AND HEPATIC DECOMPENSATION 545 18. Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521 1531. 19. Chen YC, Chu CM, Yeh CT, et al. Natural course following the onset of cirrhosis in patients with chronic hepatitis B: a long-term follow-up study. Hepatol Int 2007;1:267 273. 20. Liaw YF. Antiviral therapy of chronic hepatitis B: opportunities and challenges in Asia. J Hepatol 2009;51:403 410. 21. Kim HS, Kim HJ, Shin WG, et al. Predictive factors for early HBeAg seroconversion in acute exacerbation of patients with HBeAgpositive chronic hepatitis B. Gastroenterology 2009;136:505 512. 22. Kao JH. Role of viral factors in the natural course and therapy of chronic hepatitis B. Hepatol Int 2007;1:415 430. 23. Chu CM, Yeh CT, Lee CS, et al. Precore stop mutant in HBeAgpositive patients with chronic hepatitis B: clinical characteristics and correlation with the course of HBeAg-to-Anti-HBe seroconversion. J Clin Microbiol 2002;40:16 21. 24. Tsai WL, Lo GH, Hsu PI, et al. Role of genotype and precore/basal core promoter mutations of hepatitis B virus in patients with acute exacerbation. Scand J Gastroenterol 2008;43:196 201. Reprint requests Address requests for reprints to: Yun-Fan Liaw, MD, Liver Research Unit, Chang Gung University and Memorial Hospital, 199, Tung Hwa North Road, Taipei, Taiwan 105. e-mail: liveryfl@so-net.net.tw; fax: (886) 3-3282824. Acknowledgments The authors thank the long-term grant support provided by Chang Gung Medical Research Fund (SMRPG1005) and the Prosperous Foundation, Taipei, Taiwan; and the excellent assistance of Ms Su-Chiung Chu. Conflicts of interest The authors disclose the following: Yun-Fan Liaw has been involved in clinical trials and served as a global advisory board member of Roche, Bristol-Myers Squibb, Novartis, and Gilead Sciences. The remaining authors disclose no conflicts. Funding This study was supported by grants from Chang Gung Medical Research Fund and the Prosperous Foundation, Taipei, Taiwan.