Gastroenterology
Volume 130, Issue 3 , Pages 678-686, March 2006

Predicting Cirrhosis Risk Based on the Level of Circulating Hepatitis B Viral Load

  • Uchenna H. Iloeje

      Affiliations

    • Global Epidemiology and Outcomes Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, (Wallingford, Connecticut)
    • ,
  • Hwai–I. Yang

      Affiliations

    • Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
    • ,
  • Jun Su

      Affiliations

    • Global Epidemiology and Outcomes Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, (Wallingford, Connecticut)
    • ,
  • Chin–Lan Jen

      Affiliations

    • Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
    • ,
  • San–Lin You

      Affiliations

    • Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
    • ,
  • Chien–Jen Chen

      Affiliations

    • Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan
    • Genomics Research Center, Academia Sinica, Taipei, Taiwan
    • Corresponding Author InformationAddress requests for reprints to: Professor Chien–Jen Chen, Genomics Research Center, Academia Sinica, 128 Academia Rd, Section 2, Nankang, Tapei 115, Taiwan; fax: (886) 2-23938683.
    • ,
  • The Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-In HBV (the REVEAL-HBV) Study Group

Received 27 April 2005; accepted 9 November 2005. published online 23 November 2005.

Article Outline

Background & Aims: Cirrhosis develops as a result of hepatic inflammation and subsequent fibrosis in chronic hepatitis B infection. We report on the relationship between hepatitis B viremia and progression to cirrhosis in chronic hepatitis B infection. Methods: This was a population-based prospective cohort study of 3582 untreated hepatitis B–infected patients established in Taiwan from 1991 to 1992. Serum samples were tested for HBV DNA on cohort entry serum samples and the diagnosis of cirrhosis was by ultrasound. Results: During a mean follow-up time of 11 years, the 3582 patients contributed 40,038 person-years of follow-up evaluation and 365 patients were newly diagnosed with cirrhosis. The cumulative incidence of cirrhosis increased with the HBV-DNA level and ranged from 4.5% to 36.2% for patients with a hepatitis B viral load of less than 300 copies/mL and 106 copies/mL or more, respectively (P < .001). In a Cox proportional hazards model adjusting for hepatitis B e-antigen status and serum alanine transaminase level among other variables, hepatitis B viral load was the strongest predictor of progression to cirrhosis relative risk [95% confidence interval] was 2.5 [1.6–3.8]; 5.6 [3.7–8.5]; and 6.5 [4.1–10.2] for HBV-DNA levels ≥104 − <105; ≥105 − <106; ≥106 copies/mL, respectively. Conclusions: These data show that progression to cirrhosis in hepatitis B–infected persons is correlated strongly with the level of circulating virus. The risk for cirrhosis increases significantly with increasing HBV-DNA levels and is independent of hepatitis B e-antigen status and serum alanine transaminase level.

 

Infection with hepatitis B virus (HBV) is particularly prevalent in the Asia-Pacific region and sub-Saharan Africa.1, 2 Chronic HBV infection can lead to chronic and progressive liver disease. Globally, this is the major risk factor for the development of hepatocellular carcinoma,3 cirrhosis, and decompensated liver disease.4

The reported incidence rates of cirrhosis in chronic HBV infection have come mostly from tertiary care centers, and range from 2% to 7% annually.5, 6, 7, 8, 9 In contrast, in a study of 1506 asymptomatic HBV carriers, the cirrhosis incidence rate was 0.7% annually.10 Reported risk factors for chronic HBV-related cirrhosis include the presence of hepatitis B e-antigen (HBeAg), advanced age, increased alanine transaminase level, co-infection with the hepatitis delta virus, and diabetes mellitus.6, 7, 11, 12 Patients with cirrhosis are estimated to progress to decompensated cirrhosis at a rate of 3% annually.13 In cirrhotic patients, the presence of HBeAg (reflecting active viral replication) increases the risk for liver decompensation.14 The 5-year survival rate is 84% among compensated cirrhosis patients and 14%–28% in decompensated cirrhosis patients.13, 15

Effective suppression of viral replication in chronic hepatitis B patients has been shown to slow disease progression and improve patient outcomes.6, 8, 14, 15, 16, 17, 18 Patients who lose HBeAg and seroconvert to anti-HBe antibody–positive status are known to have a more durable suppression of viral replication.19 Loss of viral control in patients who develop HBV strains resistant to the antiviral agent lamivudine leads to a more rapid disease progression when compared with patients not infected with the resistant virus.16, 20 These data confirm that ongoing viral replication leads to progression of chronic hepatitis B disease in treated patients.

Data on the incidence and predictors of cirrhosis are lacking in individuals with chronic HBV infection from nonclinical settings. Highly sensitive polymerase chain reaction–based methods are available for measuring serum HBV-DNA levels, and make it possible to examine carefully the relationship between HBV-DNA level and disease progression in this population. We examined the incidence of cirrhosis in a population of untreated chronic HBV-infected persons, and determined the relative importance of the HBV-DNA level as a predictor of progression to cirrhosis in this population.

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Materials and Methods 

Enrollment of Study Cohort 

From 1991 to 1992, all 89,293 residents between the ages of 30 and 65 years (47,079 men and 42,214 women) living in 7 townships in Taiwan were invited to participate in a prospective cohort study. The initial invitation was by mailed letters, and follow-up telephone calls subsequently were made to those not responding to the initial invitation. Of those invited to participate in the study, a total of 11,973 men (25%) and 11,847 women (28%) provided informed consent, enrolled in the study, and were free of hepatocellular carcinoma at screening. Of the 23,820 enrolled, 4155 (2445 men and 1710 women) were hepatitis B surface antigen (HBsAg) positive at enrollment, and were followed up every 6–12 months with ultrasound and medical examinations. The data cut-off date for these analyses was June 30, 2004. Serum samples that were frozen at enrollment were tested for HBV-DNA level in 3851 of 4155 (93%) patients with available samples. All study patients provided informed consent, and assistance was provided by study personnel to ensure appropriate understanding of the informed consent by all patients. Results of all abnormal tests were provided to patients, and those with ultrasound evidence of cirrhosis, fatty liver, or a focal lesion, and laboratory evidence of an increased α-fetoprotein or alanine transaminase level were instructed specifically in a letter to see their physicians for follow-up evaluation. The study protocol was approved by the Institutional Review Board of the College of Public Health at National Taiwan University.

Data Collection and Laboratory Testing 

Patients were interviewed in person by trained public health nurses at recruitment by using a structured questionnaire. Information on sociodemographic characteristics, dietary habits, cigarette smoking, alcohol consumption, betel quid chewing, personal medical and surgical history, and family history of cancers and other major diseases was collected. Complete gynecologic information also was collected from female patients. Information on cigarette smoking and alcohol consumption was collected in the questionnaire as a binary variable (yes or no), and for those who smoked cigarettes or consumed alcohol, additional questions were asked to gather information on the quantity of cigarettes smoked and alcohol consumed, and the frequency of cigarette smoking and alcohol consumption. A 10-mL blood sample was collected from each participant at the baseline and follow-up examinations. The serum samples were fractionated on the day of collection and stored at −70°C until tested. Laboratory testing was performed using commercial kits: HBsAg and HBeAg by radioimmunoassay (Abbott Laboratories, North Chicago, IL), antibodies against hepatitis C virus (HCV) by enzyme immunoassay using second-generation test kits (Abbott Laboratories), alanine transaminase by serum chemistry autoanalyzer (model 736; Hitachi Co., Tokyo, Japan) using commercial reagents (bioMe’rieux, Mercy-I’Etoile, France), and serum HBV DNA by polymerase chain reaction (COBAS Amplicor; Roche Diagnostics, Indianapolis, IN).

Sample Selection and Ascertainment of Cirrhosis 

Of the 3851 patients in the cohort, 198 were removed (195 had evidence of HCV co-infection and 3 did not have anti-HCV testing). Seventy-one other patients were excluded from the analyses (2 men died within 6 months of cohort entry from nonhepatic causes and 69 were diagnosed with cirrhosis within 6 months of enrollment), leaving a final sample of 3582 patients; these 3582 patients comprise the cohort under study.

Cirrhosis was diagnosed by high-resolution real-time ultrasound based on a quantitative scoring system derived from the appearance of the liver surface, liver parenchymal texture, intrahepatic blood vessel size, and splenic size.10, 21 Every participant had a screening ultrasound at baseline; ultrasounds also were performed every 6–12 months during follow-up evaluation. At the time of the ultrasound examinations, the HBV-DNA level was unknown and hence the tests were performed and results were interpreted blinded to the HBV-DNA level. All ultrasounds were performed and interpreted according to a standardized protocol by a team of investigators coordinated by one of the authors (S.–L.Y.). All cases were confirmed by retrieval of sonographic records. In total, 365 newly diagnosed cirrhosis cases were included in these analyses. To confirm the complete ascertainment of cirrhosis cases, we conducted data linkage to the National Health Insurance profiles in Taiwan.

Statistical Analysis 

The person-years of follow-up evaluation for each participant were calculated from the date of enrollment to the date of the diagnosis of cirrhosis, the date of death, or the last date of linked data available from National Health Insurance profiles, whichever came first. Censored patients included those who died before the occurrence of cirrhosis during follow-up evaluation, or those still living but free of cirrhosis by the end of follow-up evaluation. Incidence rates of cirrhosis were determined for different categories of serum HBV-DNA level. Cumulative incidence of cirrhosis was derived for 5 categories of HBV-DNA level (<300; 300–9.9 × 103; 1.0–9.9 × 104; 1.0–9.9 × 105; and ≥1.0 × 106 copies/mL), using the Nelson–Aalen method, a nonparametric method for estimating the cumulative hazard.22, 23, 24 The statistical software Stata (StataCorp LP, College Station, TX) was used to obtain the corresponding estimates and figures.

We examined the significance of the relationship between serum HBV-DNA level and cirrhosis risk within different strata of other risk factors. Cox proportional hazards models were used to estimate the relative risk for cirrhosis. Variables that were adjusted for included HBeAg status, serum alanine transaminase level, sex, age, anti-HCV antibody status, cigarette smoking, and alcohol drinking. All covariates were treated as binary variables except age, which was treated as a continuous variable. Statistical significance was set at an α value of 0.05 using 2-tailed tests. The significance of the relationship between increasing serum HBV-DNA level and cirrhosis risk, adjusted for other risk factors, was examined using a test for trend.

Sensitivity analyses were conducted, restricting the analyses to the subset of cirrhosis patients who either had cirrhosis confirmed on 2 or more separate ultrasound examinations or in whom other evidence of cirrhosis (thrombocytopenia <150,000 cells/mm3, elongated prothrombin time >3 seconds, liver or splenic enlargement, and portal vein >12 mm on ultrasound) was documented.

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Results 

The flow of patients in this cohort is shown in Figure 1, and the frequency of monitoring during the study follow-up period is described in Table 1. The 3582 patients under study were followed up for a mean of 11 years and contributed 40,038 person-years of observation and by June 30, 2004; a total of 1053 (29.4%) participants dropped out of follow-up evaluation. There were 365 (10%) patients newly diagnosed with cirrhosis. Of the cirrhosis patients, 261 (72%) cases were confirmed by 2 or more separate ultrasound examinations, 4 (1%) had 1 ultrasound documenting cirrhosis and clinical evidence of cirrhosis was documented, and 100 (27%) were diagnosed on the basis of 1 ultrasound examination.

Table 1. Frequency of Ultrasound, Health Examinations, and Blood Samples During Follow-Up Period
Number of testsUltrasound examinations (%)Blood sampling and health examinations (%)
≤52265(63.2)1753(48.9)
6–10999(27.9)1195(33.4)
>10318(8.9)634(17.7)

N = 3582.

As shown in Table 2, the cohort under study is very similar to the base population but the cirrhosis patients were more likely to be older, male, drink alcohol, be HBeAg positive, have higher HBV-DNA and serum ALT levels, and more likely to have died during follow-up evaluation.

Table 2. Demographic Characteristics of Different Subsets of the Study Population
Base study population (N = 3851)Without HCV co-infected patients (N = 3653)Cohort under study (N = 3582)Cirrhosis cases under study (N = 365)
Median age45454549
Males, N (%)2367(61.5)2260(61.9)2196(61.3)287(78.6)
Alcohol drinkers, N (%)467(12.1)451(12.4)431(12.0)57(15.6)
HBeAg positive, N (%)581(15.1)565(15.5)545(15.2)135(37.0)
Median HBV DNA, copies/mL103103103105
ALT ≥ 45 IU, N (%)250(6.5)218(6.0)203(5.7)49(13.4)
Number of patients who died during follow-up period, N (%)374(9.7)346(9.5)310(8.7)76(20.8)

The distribution of patients by HBV-DNA level at enrollment shows that 869 had an undetectable HBV-DNA level (Table 3). The 1563 patients (44%) with a level of 104 copies/mL or more accounted for 274 (75%) of the cirrhosis cases. After adjusting for age, sex, smoking, and alcohol use, an enrollment HBV-DNA level of 106 copies/mL or more was associated with the highest incidence of cirrhosis (relative risk, 9.8; 95% confidence interval, 6.7–14.4; P < .001). Increasing HBV-DNA level was associated with increasing risk for cirrhosis incidence (test of trend, P < .001). The cumulative incidence of cirrhosis was 4.5%, 5.9%, 9.8%, 23.5%, and 36.2% in patients with serum HBV-DNA levels of less than 300, 300–9.9 × 103, 1.0–9.9 × 104, 1.0–9.9 × 105, and 106 copies/mL or more (Figure 2).

Table 3. Incidence of Cirrhosis by HBV-DNA Level
Serum HBV-DNA level, copies/mLSample sizePerson-years of follow-up evaluationCirrhosis casesIncidence (per 100,000 person-years)Adjusted relative riska (95% confidence interval)
Undetectableb86910,048.834338.8Reference
300–9.9×103115013,259.057429.91.4(0.9–2.2)
1.0–9.9×1046287105.555774.02.5(1.6–3.8)c
1.0–9.9×1053333460.0651878.65.9(3.9–9.0)c
=1.0×1066026164.31542498.39.8(6.7–14.4)c

NOTE. N = 3582.

a Adjusted for age, sex, cigarette smoking, and alcohol consumption.

b <300 copies/mL.

c P < .001; test of trend P < .001.

The relationship between increasing HBV-DNA level and cirrhosis risk also was examined in stratified analyses as shown in Table 4. Within each stratum, the relative risk for cirrhosis adjusted for the other variables in Table 4 increased with the HBV-DNA level. The adjusted relative risk for cirrhosis was highest in those with the highest HBV-DNA level (≥106 copies/mL). Across all strata, the association between increasing viral load and increased risk for cirrhosis was highly significant at a P value of less than .001. In addition, men and older patients were at a higher risk for cirrhosis within each HBV-DNA category.

Table 4. Association Between HBV-DNA Level and Cirrhosis Risk Stratified by Several Variables
VariablesAdjusted relative riska (95% confidence interval) for HBV DNA in copies/mL
<300300–9.9 × 1031.0–9.9 × 1041.0–9.9 × 105≥106
Sex
FemaleReference1.2(0.5–2.9)0.9(0.3–2.7)7.4(3.0–18.3)10.3(4.8–22.0)
Male2.2(1.0–4.9)3.3(1.6–7.1)6.7(3.1–14.2)12.7(6.0–26.9)21.3(10.3–44.1)
Age, y
≤50Reference1.0(0.5–1.7)1.6(0.9–2.8)3.4(2.0–5.9)5.0(3.1–8.0)
>500.8(0.4–1.6)1.7(1.0–3.0)3.3(1.9–5.8)8.2(4.8–14.1)16.0(9.9–26.0)
Cigarette smoking
NoReference1.3(0.7–2.2)2.3(1.3–4.0)5.5(3.1–9.7)10.8(6.6–17.6)
Yes0.9(0.5–1.9)1.6(0.9–2.9)2.7(1.5–5.0)6.0(3.3–10.6)7.7(4.5–13.3)
Alcohol consumption
NoReference1.3(0.8–2.1)2.7(1.7–4.3)6.1(3.9–9.7)10.6(7.0–16.0)
Yes1.1(0.5–2.8)2.6(1.3–5.0)1.7(0.7–4.4)5.6(2.8–11.4)7.9(4.5–13.9)

NOTE. N = 3582.

a Adjusted for age, sex, cigarette smoking, and alcohol consumption except for the stratifying variable.

Table 5 shows the multivariate-adjusted relative risks for cirrhosis by serum HBV-DNA level for various subsets of the cohort under study. After adjusting for age, sex, cigarette smoking, alcohol consumption, HBeAg status, and alanine transaminase level, the multivariate-adjusted relative risk (95% confidence interval) increased with higher HBV-DNA levels. Those with HBV-DNA levels of 106 copies/mL or more had the greatest risk; the adjusted relative risk was 6.5 (95% confidence interval, 4.1–10.2; P < .001). This was unchanged in the different subset analyses performed. The other independent predictors for cirrhosis risk were sex, age, HBeAg status, and increased serum alanine transaminase level.

Table 5. Multiple Cox Proportional Hazard Regression Analyses of Risk Factors Associated With Cirrhosis Among Those Chronically Infected With HBV and Negative for Anti-HCV Antibodies
VariablesMultivariate-adjusted relative risk (95% confidence interval)
All patients (N = 3582)HBeAg-negative patients (N = 3037)HBeAg-negative patients with a normal ALT level (N = 2923)
Sex
Female1.0(reference)1.0(reference)1.0(reference)
Male2.5(1.9–3.3)a2.7(1.9–3.9)a2.5(1.7–3.7)a
Age
1-year increment1.05(1.04–1.06)a1.03(1.02–1.05)a1.03(1.02–1.05)a
Cigarette smoking
No1.0(reference)1.0(reference)1.0(reference)
Yes1.0(0.8–1.2)1.0(0.8–1.4)1(0.8–1.4)
Alcohol drinking
No1.0(reference)1.0(reference)1.0(reference)
Yes0.9(0.7–1.3)1.0(0.7–1.5)1.1(0.8–1.6)
HBeAg
Negative1.0(reference)Not includedNot included
Positive1.7(1.2–2.3)b
ALT (IU)
<451.0(reference)1.0(reference)Not included
≥451.5(1.1–2.1)c1.6(1.0–2.6)
HBV DNA, copies/mLd
Undetectable (<300)1.0(reference)1.0(reference)1.0(reference)
300–9.9 × 1031.4(0.9–2.2)1.4(0.9–2.1)1.4(0.9–2.1)
1.0–9.9 × 1042.5(1.6–3.8)a2.4(1.5–3.7)a2.5(1.6–3.9)a
1.0–9.9 × 1055.6(3.7–8.5)a5.4(3.5–8.3)a5.6(3.6–8.7)a
≥1.0 × 1066.5(4.1–10.2)a6.7(4.1–11.0)a6.6(3.9–11.2)a

NOTE. N = 3582.

a P < .001.

b P < .01.

c P < .05.

d P < .001.

In Table 6, we restricted the analyses further by removing all patients diagnosed with cirrhosis on the basis of 1 ultrasound examination (N = 100), leaving an analysis cohort of 3482 patients. In these analyses, the risk for cirrhosis associated with increasing HBV-DNA level was even greater. The patients with an HBV-DNA level of 300 or more to less than 104 copies/mL were now at a significantly higher risk for cirrhosis than those below the limit of detection (P < .05). The greatest risk was found in patients with HBV-DNA levels of 106 copies/mL or more; the adjusted relative risk was 10.6 (95% confidence interval, 5.7–19.6; P < .001). The other independent predictors were age, sex, HBeAg status, and increased serum alanine transaminase level. The association between increased cirrhosis risk and increasing HBV-DNA level also remained strong in the subset analyses.

Table 6. Multiple Cox Proportional Hazard Regression Analyses of Risk Factors Associated With Cirrhosis Among Those Chronically Infected With HBV and Negative for Anti-HCV Antibodies
VariablesMultivariate-adjusted relative risk (95% confidence interval)
All patients (N = 3482)HBeAg-negative patients (N = 2960)HBeAg-negative patients with a normal ALT level (N = 2850)
Sex
Female1.0(reference)1.0(reference)1.0(reference)
Male2.6(1.9–3.7)a2.8(1.8–4.5)a2.6(1.6–4.1)a
Age
1-year increment1.06(1.05–1.08)a1.05(1.03–1.07)a1.05(1.03–1.07)a
Cigarette smoking
No1.0(reference)1.0(reference)1.0(reference)
Yes1.0(0.8–1.3)1.1(0.7–1.5)1.1(0.8–1.6)
Alcohol drinking
No1.0(reference)1.0(reference)1.0(reference)
Yes0.8(0.6–1.2)0.9(0.5–1.4)0.9(0.6–1.5)
HBeAg
Negative1.0(reference)Not includedNot included
Positive2.0(1.3–2.9)a
ALT(IU)
<451.0(reference)1.0(reference)Not included
≥451.6(1.2–2.3)†1.7(1.0–3.0)
HBV DNA, copies/mLa
Undetectable(<300)1.0(reference)1.0(reference)1.0(reference)
300–9.9 × 1032.0(1.1–3.6)b1.9(1.0–3.5)b2.1(1.1–4.0)b
1.0–9.9 × 1043.6(2.0–6.6)a3.4(1.8–6.2)a3.7(2.0–7.1)a
1.0–9.9 × 1059.7(5.4–17.3)a9.1(5.0–16.4)a10.4(5.6–19.6)a
≥1.0 × 10610.6(5.7–19.6)a11.6(6.1–22.1)a12.3(6.1–25.1)a

NOTE. The 100 cases of cirrhosis diagnosed on the basis of 1 ultrasound test are excluded. N = 3482.

a P < .001.

b P < .05.

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Discussion 

This 11-year population-based prospective cohort study offers a unique opportunity to evaluate the natural history of chronic HBV infection in the absence of interferon-alfa or nucleoside analogue therapies. Although treatment options for chronic hepatitis B became available during the period of this study, the Taiwanese Bureau of National Health Insurance did not start reimbursing chronic hepatitis B treatment until 2003. Therefore, these patients received no specific chronic hepatitis B treatment for most of the follow-up period. This is consistent with the finding that only .4% of patients in a recently published report from a cohort of Taiwanese HBV-infected men had received any antiviral therapy.25 To ensure that participants were receiving standard-of-care treatment, all patients with abnormal α-fetoprotein levels, alanine transaminase levels, or ultrasound tests were informed in a letter of the results and instructed to see their physician for follow-up evaluation. Patients were free to discontinue participation at any time for any reason. In addition, all patients with suspected hepatocellular carcinoma were referred by the investigators to a medical center for further evaluation and treatment. Also, on the availability of reimbursement for HBV therapy in October of 2003, we instituted formal referral to the National Taiwan University Hospital for evaluation for patients meeting the established treatment guidelines. Results from this cohort contributed to the reimbursement decision made by the Bureau of National Health Insurance in Taiwan for HBV therapies.

In this natural history study of chronic HBV-infected persons, we used a sensitive polymerase chain reaction assay to determine HBV-DNA level. Other comparable natural history studies, such as the study of another cohort of Taiwanese patients and the Alaskan natives study, did not test for HBV-DNA level.10, 26, 27

These results show a very strong association between serum HBV-DNA level and risk for cirrhosis in this population. As shown in Table 3 and Figure 2, the incidence of cirrhosis is consistently higher with higher viral load. As shown in Table 4, there is a highly significant association between serum viral load and risk for cirrhosis within different strata of several risk factors, suggesting an independent effect of HBV DNA.

For all patients, serum HBV-DNA level was established on serum samples collected before the identification of cirrhosis, and the ultrasound examinations were performed blinded to the HBV-DNA level. By removing all patients diagnosed with cirrhosis within 6 months of enrollment we ensured that the risk category (HBV-DNA level) was established before the diagnosis of cirrhosis for all patients, thereby improving our ability to evaluate a causal relationship.

In the multivariate Cox proportional hazards regression models, serum HBV-DNA level was the strongest predictor of progression to cirrhosis. Alcohol consumption was not associated with risk for cirrhosis in this study. A similar finding was reported in the previously referenced study of 1506 Taiwanese men, and may be owing to the relatively low quantity of alcohol consumed by Taiwanese people.10

In this cohort we do not know the exact duration of infection in the patients before enrollment, but it is presumed that the patients were infected during birth or in childhood, as is the case in most Taiwanese HBV-infected persons. The exact mechanism by which patients with high levels of circulating virus are at higher risk for progressing to cirrhosis cannot be addressed by the data presented in this current epidemiology study. Animal models, however, show that chronic immune reaction to HBV is both necessary and sufficient to induce chronic liver damage.28 This chronic immune reaction is associated with chronic hepatic inflammation and is triggered by immune recognition of HBsAg-producing hepatocytes. In human beings infected early in life by HBV, there is a long immune tolerance phase with high levels of viremia and little evidence of liver damage.29 This is followed by an immune clearance phase, characterized by recurrent episodes of liver inflammation and episodic increases in serum alanine transaminase levels. These episodes of liver inflammation are associated initially with high levels of serum HBV DNA that subsequently decrease, increased HBeAg titer, and increased HBeAg/anti-HBe antibody immune complexes.30, 31 During the phase of immune clearance in chronic hepatitis B patients, hepatocytes harboring viruses are targeted, leading to liver cell death, regeneration, fibrosis, and, eventually, cirrhosis. In Asians, the frequency and severity of acute liver injury during immune clearance has been associated with the development of cirrhosis, whereas HBeAg to anti-HBe–antibody seroconversion, the desired outcome of immune clearance, is not always associated with a cessation of disease progression to cirrhosis.9, 32 In the study of Alaskan natives, multiple episodes of seroreversion from HBeAg-negative to HBeAg-positive status was shown to increase the risk significantly for hepatocellular carcinoma.26 These studies suggest that high levels of circulating virus may be associated with the severity of liver injury during the immune clearance phase, which in turn may lead to the development of cirrhosis in these patients. We hypothesize, therefore, that high levels of circulating viral DNA precede and provide the trigger for the immune responses that lead to liver injury in human beings.30, 33 Accumulation of liver injury over time then manifests as end-organ damage. Consistent with this hypothesis is the finding that in Asian patients, cirrhosis and hepatocellular carcinoma develop many years after HBeAg to anti-HBe antibody seroconversion,34 and an estimated 65% of liver complications occur after HBeAg seroconversion has occurred.29

In this study, the diagnosis of cirrhosis was performed blinded to the HBV-DNA level, and was based on an ultrasound scoring system that was validated against laparoscopic biopsy examination–confirmed cirrhosis cases as reported by Lin et al.21 This allowed for all patients in the cohort to be screened in a similar manner, minimizing the potential for ascertainment bias. More recent evidence of the value of this ultrasound scoring system for diagnosing cirrhosis was reported in a study by Hung et al35 in 2003, which provides additional validation of this ultrasound protocol in patients infected with HBV and HCV. Lin et al21 reported a sensitivity of 82.8% and a specificity of 80% for an ultrasound score of 7 or more in their study of 29 chronic hepatitis B patients. Hung et al35 reported on 210 patients (67 with HBV and 143 with HCV infection) and showed that the ultrasound scores were correlated significantly with the fibrosis scores, and in HBV-infected patients the sensitivity and specificity for a score of 7 or more on ultrasound was 77.8% and 92.5%, respectively. Other prospective studies appear to confirm the findings of these 2 studies.36, 37, 38 Nishiura et al38 studied 103 patients with chronic liver disease (91 from viral hepatitis B or C) who also had an ultrasound within 15 days of their liver biopsy examination. In this study published in 2005, there was a strong correlation between the ultrasound score and fibrosis on liver biopsy examination (Spearman’s rank correlation coefficient, 0.9524). The reported sensitivity for an ultrasound score of 6.5 or more in this study was 100%. Gaiani et al37 reported on 212 patients with compensated liver disease (160 with evidence of HBV or HCV infection) in 1997. By using a quantitative ultrasound scoring system derived from the appearance of the liver surface, liver size and morphology, echogenicity, portal vein diameter and flow, and splenic size, they reported a sensitivity of 81.8% and a specificity of 84.7% with a diagnostic accuracy of 84% for ultrasound-diagnosed cirrhosis. Others, however, have shown poor correlation between ultrasound and histologic findings of liver disease.39, 40 Neither one of these studies used a quantitative scoring system similar to that used in our study. In the study by Sanford et al,39 11 of the 125 patients (8.8%) had chronic hepatitis (cause was not specified further), of whom 7 had cirrhosis, by contrast, 54 of 125 (43%) had alcoholic liver disease, of whom 26 had cirrhosis. In the study by Celle et al,40 87 patients had cirrhosis (34.5% and 52.9% of viral and alcoholic causes, respectively) and the positive predictive value of ultrasonography for the diagnosis of cirrhosis was 95.2% with a low negative predictive value (42.5%). On reviewing these studies, there are noteworthy inferences that can be made: (1) the use of a systematic ultrasound scoring system based on the structural appearance of the liver surface, liver parenchyma, intrahepatic blood vessels, and the spleen, as was performed in our study, appears to improve the diagnostic accuracy of ultrasonography; (2) studies in patients with viral hepatitis B or C are associated with better correlation with biopsy examination findings when compared with studies of more heterogenous patient populations; and (3) ultrasonography is more likely to result in underdiagnosis rather then overdiagnosis of cirrhosis; this means that the bias is toward the null rather than an overestimation of risk.

Although liver biopsy examination is viewed as the gold standard for diagnosing cirrhosis, its use in a population-based epidemiology study of this type would be very challenging clinically, and prohibitive from a cost perspective. It also may lead to underascertainment of cirrhosis. In the study of Alaskan natives, only biopsy examination–confirmed cirrhosis cases were reported, with an incidence rate of 107 per 100,000 person-years for male patients.27 This was lower than the rate for hepatocellular carcinoma (386/100,000 person-years), suggesting an underascertainment of cirrhosis cases. Notably the hepatocellular carcinoma cases either were biopsy examination confirmed or based on a combination of radiographic evidence of a liver mass and an α-fetoprotein level of more than 400 ng/mL. By using ultrasound, it is possible that we may have introduced some misclassification. This is expected to be a nondifferential bias because all patients were HBsAg-positive, followed up in a similar manner for the diagnosis of cirrhosis and the predictive variable, and the HBV-DNA level was not known at the time of the ultrasound. Any misclassification therefore will have the effect of biasing the results toward the null. The sensitivity analyses (Table 6) appears to confirm this. By restricting the cirrhosis cases only to those confirmed on at least 2 ultrasound examinations or with clinical signs, the strength of the association between baseline HBV-DNA level and subsequent cirrhosis risk was greater. The calculated incidence of cirrhosis in our study was 0.9% per annum, lower than the reported 2%–7% rate from clinical populations with presumed or documented chronic hepatitis B,5, 6, 9 and similar to the 0.7% reported in a similar study.10 Therefore, it is unlikely that we have overestimated the incidence of cirrhosis in this study.

The current hepatitis B treatment guidelines generally recommend that an HBV-DNA level of 105 copies/mL or more be considered the threshold for active disease and therefore for treatment interventions.1, 41 The current recommendations are based in part on data using relatively insensitive HBV-DNA assays to estimate disease progression risk. In our study the use of a more sensitive assay made it possible to define the risk for cirrhosis at different HBV-DNA levels and the data show that lower HBV-DNA levels are associated significantly with progression of HBV infection to cirrhosis. We found that HBV-DNA level not only predicts the risk for cirrhosis independent of serum alanine transaminase level or HBeAg status but was the strongest risk predictor. In the HBeAg-negative patients with normal serum alanine transaminase levels at cohort entry, we found a significant increase in the risk for cirrhosis as the HBV-DNA level increased.

We have not accounted for the impact of HBV genotype on cirrhosis risk in this cohort because complete information on the HBV genotype of the patients in this study currently is not available. However, in Taiwan the majority of HBV infections are caused by genotype B or C infection42, 43 and we believe that these 2 genotypes are responsible for the majority of infections in our study cohort.

From this large sample size prospective cohort study, cohort entry serum HBV-DNA level strongly predicted progression to cirrhosis in the future. Our findings suggest that the level of circulating serum HBV-DNA level is the strongest predictor of future cirrhosis risk, and a level of 104 copies/mL or more is associated with a significant risk for progression to cirrhosis independent of HBeAg status and serum alanine transaminase level.

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 Supported by a grant from the Department of Health, Executive Yuan, Taipei, Taiwan, and by a grant from Bristol-Myers Squibb Company.

PII: S0016-5085(05)02276-6

doi:10.1053/j.gastro.2005.11.016

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