Journal of Medical Virology 86:1828–1837 (2014)
Decreasing Hepatitis B Viral Load is Associated With a Risk of Significant Liver Fibrosis in Hepatitis B e Antigen Positive Chronic Hepatitis B Qinxiu Xie,1 Xiangyang Hu,2 Yafei Zhang,1 Xiaoping Jiang,1 Xu Li,1* and Jiabin Li1 1
Department of Infectious Disease, the First Affiliated Hospital of Anhui Medical University, Hefei, China Department of Pathology, the First Affiliated Hospital of Anhui Medical University, Hefei, China
2
Alanine aminotransferase (ALT), hepatitis B virus (HBV) DNA levels and age are used commonly to assess liver histology in chronic hepatitis B. Increasing levels of HBV DNA are associated with the increasing prevalence of significant fibrosis in HBeAg-negative patients. It is unclear whether these data can be applied to HBeAg-positive patients. In present study, liver biopsies were performed and clinical parameters were measured in 234 treatmentnaive chronic HBeAg-positive patients. The proportion of significant fibrosis in patients with ALT 1–2 ULN was similar to in patients with ALT more than 2 ULN (48.4% vs. 51.8%). Patients over 30 years of age (>30 years) had a higher prevalence of significant fibrosis than patients 30 years of age and younger (61.0% vs. 33.6%). Negative correlation between HBV DNA levels and significant fibrosis was observed in patients >30 years. The optimal level of serum HBV DNA to evaluate low risk of significant fibrosis was 6.7 log10 IU/ml. Patients with serum HBV DNA levels 8.5 log10 IU/ml all had no significant fibrosis, however, patients with HBV DNA levels 30 years. The threshold of HBV DNA levels for treatment of HBeAg-positive patients needs to be combined with age. J. Med. Virol. 86:1828– 1837, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS:
chronic hepatitis B; liver histology; HBV DNA levels; ALT; age
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION The clinical conditions associated with the hepatitis B virus (HBV) infection are variable, ranging from inactive carrier status, active chronic hepatitis B (CHB) to the development of clinical complications, including cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis B is prevalent in China, where most of the patients acquired HBV in the perinatal period or in their early stage of life [Lai and Yuen, 2007]. Histological injury and fibrosis have a good correlation with long-term risk, because repeated hepatitis flares in patients infected with hepatitis B were found to have increased necroinflammation and fibrosis on liver biopsies, resulting in increasing fibro genesis and eventual disease progression [Mani and Kleiner, 2009]. Current antiviral treatments may slow the disease progression in cirrhotic patients and reduce hepatic events and deaths in patients infected with hepatitis B with liver cirrhosis [Du et al., 2013; Wong et al., 2013]. Several guidelines for patients infected with hepatitis B recommend the use of both serum alanine aminotransferase (ALT) and HBV DNA in selecting patients for therapy, with a persistent ALT level of more than 2 upper limit of normal (ULN) [Liaw et al., 2008; Chinese guideline of prevention and treatment for chronic hepatitis B, 2011; EASL clinical practice guidelines for HBV infection, 2012]. Some patients with elevated ALT levels less than two times the ULN (2 ULN) levels in association with HBV
Grant sponsor: National Natural Science Foundation of China; Grant number: 81072342. Correspondence to: Xu Li, Department of Infectious Disease, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, Anhui 230022, China. E-mail: [email protected] Accepted 30 May 2014 DOI 10.1002/jmv.24000 Published online in Wiley Online Library (wileyonlinelibrary.com).
Liver Fibrosis and HBV DNA, ALT, Age
DNA levels >20,000 IU/ml (or 1 105 copies/ml) for HBeAg positive patients and >2,000 IU/ml (or 1 104 copies/ml) for HBeAg negative patients, required consideration of a liver biopsy to determine whether a significant histological disease was present, especially if the patient were over 40 years age. ALT is an important biochemical marker that is used in the assessment of hepatic injury, but it is limited by its poor correlation with disease severity. Some studies have shown minimal histological changes in HBeAg-positive patients with normal ALT, while other studies have shown significant necroinflammation, fibrosis, and even cirrhosis on liver biopsies [Kumar et al., 2008; Gui et al., 2010; Alam et al., 2011]. The serum HBV DNA levels reflect directly the degree of HBV replication, and are correlated strongly with long-term mortality. The risk for cirrhosis increases significantly with increasing HBV DNA levels, regardless of HBeAg status and ALT [Chen et al., 2006; Iloeje et al., 2007]. Increasing levels of HBV DNA are associated with an increasing prevalence of significant fibrosis in HBeAg-negative patients [Croagh et al., 2010]. It is unclear whether these data can be applied to HBeAg-positive patients. Previous studies in HBeAg-positive patients did not find HBV DNA to be a predictor of fibrosis [Shao et al., 2007; Park et al., 2008]. However, a previous study [Wang et al., 2008] in 28 HBeAg-positive immune-tolerant patients and reported that a lower serum HBV DNA levels, along with being over 30 years of age, was correlated independently with stage 2 fibrosis or more on liver biopsy. However, that study is limited by the small number of samples and only involved immune-tolerant stage patients. Few studies on threshold value of HBV DNA level for evaluating low risk of significant fibrosis in HBeAg-positive patients. The aims of the present study were as follows: (1) to evaluate the association between clinical parameters and significant histologic abnormalities in HBeAg-positive patients; (2) to identify high risk factors of significant fibrosis and inflammation; (3) to determine a threshold value for the HBV DNA levels which could differentiate patients who have progressive liver disease from those who are only carrying the virus. This differentiation is important for identifying the group of patients infected with HBV who are more likely to progress to advanced stages of the disease. MATERIALS AND METHODS Patients The present study included 234 treatment-naive HBeAg-positive patients infected with hepatitis B who were recruited to assess the status of liver histology from January 2010 to October 2013 in the Department of Infectious Disease at the First Affiliated Hospital of Anhui Medical University, China. Demographic, clinical and laboratory data were recorded, including age, sex, alcohol intake, ALT level, aspartate aminotransferase (AST) level, gamma-glu-
1829
tamyl transpeptidase (GGT), prothrombin time international normalized ratio (PT-INR), albumin, Platelets counts (PLT), HBeAg status, HBV DNA levels at the time of liver biopsy, and family history of HBV infection. All patients were HBsAg-positive and HBeAg-positive for at least 12 months before enrolling in the study. Patients with concomitant liver diseases, including chronic hepatitis C or D infection, Wilson’s disease, autoimmune hepatitis, primary biliary cirrhosis, significant alcohol intake (30 g per day for male, 20 g per day for female), overt cirrhosis or liver cancer, decompensated liver disease and prior antiviral treatment were excluded. Liver Biopsy All patients received ultrasonography-guided percutaneous biopsy of the right lobe of the liver. A quick-cut and 16-gauge sheathed cutting needle (BARD, purchased from American) was used for this procedure. The biopsy lengths were 1.5–2.5 cm with nine or more available portal areas. The biopsy specimens were fixed with 10% formalin, paraffin-embedded, and stained with hematoxylin and eosin for morphological evaluation and Masson’s trichrome stain for the assessment of fibrosis (Fig. 1). Histologic staging of fibrosis and grading of inflammation were performed using the Scheuer’s classification [Scheuer, 1995] and the guideline on prevention and treatment of chronic hepatitis B in China, the grade of hepatic inflammation was G0–4, and the stage of fibrosis was S0–4. G0–1 was considered as insignificant inflammation, G2–4 as significant hepatic inflammation, S0–1 as insignificant fibrosis, and S2–4 as significant hepatic fibrosis, G 2 or S 2 was considered starting points for treatment. Laboratory Assays Serum samples used for measurements were taken at the day of biopsy and stored at 80˚C. All parameters were measured using standard methodologies. Serum HBsAg, HBeAg, and antibody to the hepatitis B e antigen (anti-HBe) were measured using commercially available immunoassays (Abbott Laboratories, Chicago, IL). A serum real-time PCR technique was used for the quantitation of the serum HBV DNA levels, and the dectection range was 1,000–300,000,000 IU/ml, samples with HBV DNA levels higher than 3.0 108 IU/ml were diluted at 1:100 for retesting. All laboratory analysises were performed at the Department of Clinical Laboratory of First Affiliated Hospital of Anhui Medical University. Definition of Normal ALT The ULN of serum ALT was defined as 30 U/L for men and 19 U/L for women. All patients identified in the present study had ALT measured on at least three occasions, which were at intervals of more than J. Med. Virol. DOI 10.1002/jmv
1830
Xie et al.
Fig. 1. Histological inflammation grading and fibrosis staging were evaluated by hematoxylin-eosin and Masson’s trichrome stain method for liver biopsy specimen. A was read as having stage 2–3 fibrosis using Masson’s trichrome stain method. Original magnification is 100. B was read as having stage 2–3
fibrosis and grade 1 inflammation using HE stain method. Original magnification is 100. C was read as having grade 2 inflmmation using HE stained method. Original magnification is 100. D was read as having stage 1 fibrosis using Masson’s trichrome stain method. Original magnification is 100.
3 months apart over a period of 12 or more months before liver biopsy. The following ranges were used: 1 ULN: persistently normal ALT; 1–2 ULN: intermittent or continuously minimally elevated ALT; and >2 ULN patients with an elevated ALT >2 ULN once on the liver biopsy day or within 2 weeks prior to the liver biopsy, but having persistently normal or minimally elevated ALT (2 ULN) levels during the previous 12 months.
using SPSS version 16.0 (SPSS, Inc., Chicago, IL) software package. The Mann–Whitney U-test was used to compare continuous variables with a skewed distribution and t-test was used to compare continuous variables with a normal distribution; the Chisquared test and Fisher’s exact test was used for categorical variables. A Receiver operating curve (ROC) analysis was performed to find a cut-off value which could differentiate immune tolerant phase from immune clearance phase in HBeAg-positive patients infected with hepatitis B. Multivariate logistic regression was used to identify factors that were associated independently with significant fibrosis or inflammation. A two-sided P value of 2 ULN
P-value
(100) (14–63) (74.4) (28.3–55.8) (7–611) (11–1743) (14–660) (0.9–1.5) (26–318) (3.0–8.8) (47.4) (16.2)
122 32 97 45.1 22 38 29 1.04 154 7.29 53 12
112 29 77 43.9 34.5 80 52 1.07 162 7.42 58 26
0.144 0.060 0.078 0.05). The baseline characteristics of the study patients are depicted in Table I. Overall Liver Histology Significant inflammation and fibrosis were found in 16.2% (38/234) and 47.4% (111/234) of the total study patients, respectively. Of these, 39 (16.7%) had established cirrhosis. Significant difference were noted in age, HBV DNA, PT-INR, PLT, albumin, ALT, AST, GGT and proportion of significant inflammation
between S2 group and 2 ULN. In the above three groups, the distribution of significant fibrosis was 27.6% (8/29), 48.4% (45/93), and 51.8% (58/112), respectively, and significant inflammation was 6.9% (2/29), 10.8% (10/93), and 23.2% (26/112), respectively. The patients in group 2 had a higher proportion of significant fibrosis than did the patients in group 1 (P ¼ 0.048), but exhibited no significant difference with group 3 (P ¼ 0.628). However, the patients in group 2 had a lower proportion of significant inflammation than did the patients in group 3
TABLE II. Comparison of Clinical Characteristics Between Patients With Significant Fibrosis and Patients With Insignificant Fibrosis S2
Clinical characteristics n, % Age (years) (median, range) Male (n, %) Albumin (g/l) (median, range) GGT (U/L) (median, range) ALT (U/L) (median, range) AST (U/L) (median, range) PT-INR (median, range) PLT (109/l) (median, range) HBV DNA (log IU/ml) (median, range) Patients with significant inflammation (n, %)
111 36 85 42.3 41 58.7 43 1.1 136.2 7.1 28
(47.4) (15–63) (48.9) (28.8–55.8) (9–661) (17–1743) (14–660) (0.9–1.5) (26–265) (3.0–8.58) (25.2)
123 27 26 45.3 21 49 32 1.0 174.6 7.6 10
1 105 group (P ¼ 0.026), but it was not noted in the HBV DNA (IU/ml) 1 105 group (P ¼ 0.096). Patients were then analyzed according to different strata of age (30 and >30 years). A strong negative correlation was noted between HBV DNA levels and significant fibrosis in patients over 30 years of age (t ¼ 4.342, P < 0.001), but no correlation was found in patients 30 years of age and younger (t ¼ 0.365, P ¼ 0.716). Among patients over 30 years of age, the proportion of significant fibrosis decreased from 77.8% in the HBV DNA (IU/ml) 1 105 group to 58.0% in the HBV DNA >1 105 group (P ¼ 0.126). However, among patients 30 years of age and younger, the proportion of significant fibrosis increased from 20.0% in the HBV DNA (IU/ml) 1 105 group to 34.2% in the HBV DNA >1 105 group (P ¼ 0.662). Value of Serum HBV DNA Levels for Low Risk of Significant Fibrosis The ROC curves and AUC values of serum HBV DNA levels in evaluating low risk of significant fibrosis were performed. Serum HBV DNA levels produced a better AUC for low risk of significant fibrosis in patients with ALT 2 ULN and age over
Liver Fibrosis and HBV DNA, ALT, Age
1833
TABLE III. The Distributions of Significant Fibrosis and Inflammation in Different Level of HBV DNA According to Different Strata of ALT and Age (n ¼ 234) HBV DNA (IU/ml)
ALT (U/L) 1 ULN 1–2 ULN >2 ULN Age (years) 30 >30
>1 105 (n, %)
1 105 (n, %)
Characteristics n 4 8 10
S2 3 (75.0)a 7 (87.5)b 5 (50.0)
5 18
1 (20.0) 14 (77.8)e
G2 0 0 3 (30) 2 (40.0) 1 (5.6)
P-value
n 25 85 102
S2 5 (20.0) 38 (44.7)c 53 (52.0)
G2 2 (8.0) 10 (11.8) 23 (22.5)d
0.052,a 0.021b 0.026,c 0.026d
111 100
38 (34.2) 58 (58.0)f
13 (11.7) 22 (22.0)g
0.033,e 0.001f 0.045g
ALT, alanine aminotransferase; ULN, upper limit of normal; HBV DNA, hepatitis B virus deoxyribonucleic acid; n, number of patients; S2, significant fibrosis; G2, significant inflammation. a Comparison was between patients with serum HBV DNA (log10 IU/ml) 1 105 and >1 105 among patients with ALT 1 ULN. b Comparison was between patients with serum HBV DNA (log10 IU/ml) 1 105 and >1 105 among patients with ALT 1–2 ULN. c Comparison was between patients with ALT 1–2 ULN and 1 ULN among patients with serum HBV DNA (log10 IU/ml) >1 105. d Comparison was between patients with ALT 1–2 ULN and >2 ULN among patients with serum HBV DNA (log10 IU/ml) >1 105. e Comparison was between patients with age >30 years and 30 years among each group. f Comparison was between patients with age >30 years and 30 years among each group. g Comparison was between patients with age >30 years and 30 years among each group.
30 years (AUC 0.820, P < 0.001, 95% CI: 0.720–0.919) compared to the whole study patients with ALT 2 ULN (AUC 0.770, P < 0.001, 95% CI: 0.686–0.854). Serum HBV DNA levels did not have any evaluating value for low risk of significant inflammation (AUC 0.580 and AUC 0.520 for whole patients and patients with ALT 2 ULN, respectively). The sensitivity, specificity and predictive values of different HBV DNA levels in patients with ALT 2 ULN and age over 30 years are shown in Table IV. Based on the Youden Index, the optimal level of serum HBV DNA to evaluate low risk of significant fibrosis was 6.7 log10 IU/ml (Youden Index 0.557, sensitivity 83.9%, specificity 71.8%). Patients with serum HBV DNA levels 8.5 log10 IU/ml all had no significant fibrosis, however, patients with HBV DNA levels 4.7 log10 IU/ml all had significant fibrosis. Univariate and Multivariate Analysis for Significant Fibrosis Univariate analysis showed that in HBeAg-positive patients, age, albumin, AST, PT-INR, platelet count, GGT and HBV DNA levels were associated with significant fibrosis, and that ALT, AST, GGT, albumin, PT-INR, and platelet count (PLT) were associat-
ed with significant inflammation. Gender was not associated with either significant fibrosis or inflammation. Multivariate analysis showed that in the whole study patients, older age, lower HBV DNA levels, lower PLT and higher AST were associated independently with significant fibrosis, while higher AST and PLT were associated independently with significant inflammation. In patients with ALT 2 ULN, an older age (P ¼ 0.043), higher AST (P < 0.011) and lower HBV DNA (P ¼ 0.001) were associated independently with significant fibrosis, while no factor was associated independently with significant inflammation. The univariate and multivariate analysis of clinical parameters associated with significant inflammation or fibrosis are shown in Table V. DISCUSSION This study demonstrates that progressive liver disease in HBeAg-positive patients is driven by three main factors: which is age, a surrogate of disease duration; immune activation, as measured by ALT; and viral replication, as measured by HBV DNA levels. ALT levels are used commonly to assess liver disease, and elevated levels have been shown to be associated with active liver disease on histology while
TABLE IV. Sensitivity, Specificity and Predictive Values of Different Serum HBV DNA Levels for Predicting Insignificant Fibrosis Among Patients With ALT 2 ULN and Age >30 Years (n ¼ 70) HBV DNA (log10 IU/ml)
n
Sensitivity
Specificity
PPV
NPV
LRþ
LR
6.00 6.70 7.00 7.50 8.00 8.47
45 37 35 25 17 4
83.9% 83.9% 80.6% 58.1% 45.2% 9.7%
64.1% 71.8% 71.8% 82.1% 92.3% 100%
65.00% 70.27% 69.44% 72.00% 82.35% 100%
86.21% 84.85% 82.35% 71.11% 67.92% 59.09%
2.34 2.98 2.86 3.25 5.87 —
0.251 0.224 0.270 0.510 0.594 0.903
Insignificant fibrosis: Stage of fibrosis
Decreasing Hepatitis B Viral Load is Associated With a Risk of Significant Liver Fibrosis in Hepatitis B e Antigen Positive Chronic Hepatitis B Qinxiu Xie,1 Xiangyang Hu,2 Yafei Zhang,1 Xiaoping Jiang,1 Xu Li,1* and Jiabin Li1 1
Department of Infectious Disease, the First Affiliated Hospital of Anhui Medical University, Hefei, China Department of Pathology, the First Affiliated Hospital of Anhui Medical University, Hefei, China
2
Alanine aminotransferase (ALT), hepatitis B virus (HBV) DNA levels and age are used commonly to assess liver histology in chronic hepatitis B. Increasing levels of HBV DNA are associated with the increasing prevalence of significant fibrosis in HBeAg-negative patients. It is unclear whether these data can be applied to HBeAg-positive patients. In present study, liver biopsies were performed and clinical parameters were measured in 234 treatmentnaive chronic HBeAg-positive patients. The proportion of significant fibrosis in patients with ALT 1–2 ULN was similar to in patients with ALT more than 2 ULN (48.4% vs. 51.8%). Patients over 30 years of age (>30 years) had a higher prevalence of significant fibrosis than patients 30 years of age and younger (61.0% vs. 33.6%). Negative correlation between HBV DNA levels and significant fibrosis was observed in patients >30 years. The optimal level of serum HBV DNA to evaluate low risk of significant fibrosis was 6.7 log10 IU/ml. Patients with serum HBV DNA levels 8.5 log10 IU/ml all had no significant fibrosis, however, patients with HBV DNA levels 30 years. The threshold of HBV DNA levels for treatment of HBeAg-positive patients needs to be combined with age. J. Med. Virol. 86:1828– 1837, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS:
chronic hepatitis B; liver histology; HBV DNA levels; ALT; age
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION The clinical conditions associated with the hepatitis B virus (HBV) infection are variable, ranging from inactive carrier status, active chronic hepatitis B (CHB) to the development of clinical complications, including cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis B is prevalent in China, where most of the patients acquired HBV in the perinatal period or in their early stage of life [Lai and Yuen, 2007]. Histological injury and fibrosis have a good correlation with long-term risk, because repeated hepatitis flares in patients infected with hepatitis B were found to have increased necroinflammation and fibrosis on liver biopsies, resulting in increasing fibro genesis and eventual disease progression [Mani and Kleiner, 2009]. Current antiviral treatments may slow the disease progression in cirrhotic patients and reduce hepatic events and deaths in patients infected with hepatitis B with liver cirrhosis [Du et al., 2013; Wong et al., 2013]. Several guidelines for patients infected with hepatitis B recommend the use of both serum alanine aminotransferase (ALT) and HBV DNA in selecting patients for therapy, with a persistent ALT level of more than 2 upper limit of normal (ULN) [Liaw et al., 2008; Chinese guideline of prevention and treatment for chronic hepatitis B, 2011; EASL clinical practice guidelines for HBV infection, 2012]. Some patients with elevated ALT levels less than two times the ULN (2 ULN) levels in association with HBV
Grant sponsor: National Natural Science Foundation of China; Grant number: 81072342. Correspondence to: Xu Li, Department of Infectious Disease, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, Anhui 230022, China. E-mail: [email protected] Accepted 30 May 2014 DOI 10.1002/jmv.24000 Published online in Wiley Online Library (wileyonlinelibrary.com).
Liver Fibrosis and HBV DNA, ALT, Age
DNA levels >20,000 IU/ml (or 1 105 copies/ml) for HBeAg positive patients and >2,000 IU/ml (or 1 104 copies/ml) for HBeAg negative patients, required consideration of a liver biopsy to determine whether a significant histological disease was present, especially if the patient were over 40 years age. ALT is an important biochemical marker that is used in the assessment of hepatic injury, but it is limited by its poor correlation with disease severity. Some studies have shown minimal histological changes in HBeAg-positive patients with normal ALT, while other studies have shown significant necroinflammation, fibrosis, and even cirrhosis on liver biopsies [Kumar et al., 2008; Gui et al., 2010; Alam et al., 2011]. The serum HBV DNA levels reflect directly the degree of HBV replication, and are correlated strongly with long-term mortality. The risk for cirrhosis increases significantly with increasing HBV DNA levels, regardless of HBeAg status and ALT [Chen et al., 2006; Iloeje et al., 2007]. Increasing levels of HBV DNA are associated with an increasing prevalence of significant fibrosis in HBeAg-negative patients [Croagh et al., 2010]. It is unclear whether these data can be applied to HBeAg-positive patients. Previous studies in HBeAg-positive patients did not find HBV DNA to be a predictor of fibrosis [Shao et al., 2007; Park et al., 2008]. However, a previous study [Wang et al., 2008] in 28 HBeAg-positive immune-tolerant patients and reported that a lower serum HBV DNA levels, along with being over 30 years of age, was correlated independently with stage 2 fibrosis or more on liver biopsy. However, that study is limited by the small number of samples and only involved immune-tolerant stage patients. Few studies on threshold value of HBV DNA level for evaluating low risk of significant fibrosis in HBeAg-positive patients. The aims of the present study were as follows: (1) to evaluate the association between clinical parameters and significant histologic abnormalities in HBeAg-positive patients; (2) to identify high risk factors of significant fibrosis and inflammation; (3) to determine a threshold value for the HBV DNA levels which could differentiate patients who have progressive liver disease from those who are only carrying the virus. This differentiation is important for identifying the group of patients infected with HBV who are more likely to progress to advanced stages of the disease. MATERIALS AND METHODS Patients The present study included 234 treatment-naive HBeAg-positive patients infected with hepatitis B who were recruited to assess the status of liver histology from January 2010 to October 2013 in the Department of Infectious Disease at the First Affiliated Hospital of Anhui Medical University, China. Demographic, clinical and laboratory data were recorded, including age, sex, alcohol intake, ALT level, aspartate aminotransferase (AST) level, gamma-glu-
1829
tamyl transpeptidase (GGT), prothrombin time international normalized ratio (PT-INR), albumin, Platelets counts (PLT), HBeAg status, HBV DNA levels at the time of liver biopsy, and family history of HBV infection. All patients were HBsAg-positive and HBeAg-positive for at least 12 months before enrolling in the study. Patients with concomitant liver diseases, including chronic hepatitis C or D infection, Wilson’s disease, autoimmune hepatitis, primary biliary cirrhosis, significant alcohol intake (30 g per day for male, 20 g per day for female), overt cirrhosis or liver cancer, decompensated liver disease and prior antiviral treatment were excluded. Liver Biopsy All patients received ultrasonography-guided percutaneous biopsy of the right lobe of the liver. A quick-cut and 16-gauge sheathed cutting needle (BARD, purchased from American) was used for this procedure. The biopsy lengths were 1.5–2.5 cm with nine or more available portal areas. The biopsy specimens were fixed with 10% formalin, paraffin-embedded, and stained with hematoxylin and eosin for morphological evaluation and Masson’s trichrome stain for the assessment of fibrosis (Fig. 1). Histologic staging of fibrosis and grading of inflammation were performed using the Scheuer’s classification [Scheuer, 1995] and the guideline on prevention and treatment of chronic hepatitis B in China, the grade of hepatic inflammation was G0–4, and the stage of fibrosis was S0–4. G0–1 was considered as insignificant inflammation, G2–4 as significant hepatic inflammation, S0–1 as insignificant fibrosis, and S2–4 as significant hepatic fibrosis, G 2 or S 2 was considered starting points for treatment. Laboratory Assays Serum samples used for measurements were taken at the day of biopsy and stored at 80˚C. All parameters were measured using standard methodologies. Serum HBsAg, HBeAg, and antibody to the hepatitis B e antigen (anti-HBe) were measured using commercially available immunoassays (Abbott Laboratories, Chicago, IL). A serum real-time PCR technique was used for the quantitation of the serum HBV DNA levels, and the dectection range was 1,000–300,000,000 IU/ml, samples with HBV DNA levels higher than 3.0 108 IU/ml were diluted at 1:100 for retesting. All laboratory analysises were performed at the Department of Clinical Laboratory of First Affiliated Hospital of Anhui Medical University. Definition of Normal ALT The ULN of serum ALT was defined as 30 U/L for men and 19 U/L for women. All patients identified in the present study had ALT measured on at least three occasions, which were at intervals of more than J. Med. Virol. DOI 10.1002/jmv
1830
Xie et al.
Fig. 1. Histological inflammation grading and fibrosis staging were evaluated by hematoxylin-eosin and Masson’s trichrome stain method for liver biopsy specimen. A was read as having stage 2–3 fibrosis using Masson’s trichrome stain method. Original magnification is 100. B was read as having stage 2–3
fibrosis and grade 1 inflammation using HE stain method. Original magnification is 100. C was read as having grade 2 inflmmation using HE stained method. Original magnification is 100. D was read as having stage 1 fibrosis using Masson’s trichrome stain method. Original magnification is 100.
3 months apart over a period of 12 or more months before liver biopsy. The following ranges were used: 1 ULN: persistently normal ALT; 1–2 ULN: intermittent or continuously minimally elevated ALT; and >2 ULN patients with an elevated ALT >2 ULN once on the liver biopsy day or within 2 weeks prior to the liver biopsy, but having persistently normal or minimally elevated ALT (2 ULN) levels during the previous 12 months.
using SPSS version 16.0 (SPSS, Inc., Chicago, IL) software package. The Mann–Whitney U-test was used to compare continuous variables with a skewed distribution and t-test was used to compare continuous variables with a normal distribution; the Chisquared test and Fisher’s exact test was used for categorical variables. A Receiver operating curve (ROC) analysis was performed to find a cut-off value which could differentiate immune tolerant phase from immune clearance phase in HBeAg-positive patients infected with hepatitis B. Multivariate logistic regression was used to identify factors that were associated independently with significant fibrosis or inflammation. A two-sided P value of 2 ULN
P-value
(100) (14–63) (74.4) (28.3–55.8) (7–611) (11–1743) (14–660) (0.9–1.5) (26–318) (3.0–8.8) (47.4) (16.2)
122 32 97 45.1 22 38 29 1.04 154 7.29 53 12
112 29 77 43.9 34.5 80 52 1.07 162 7.42 58 26
0.144 0.060 0.078 0.05). The baseline characteristics of the study patients are depicted in Table I. Overall Liver Histology Significant inflammation and fibrosis were found in 16.2% (38/234) and 47.4% (111/234) of the total study patients, respectively. Of these, 39 (16.7%) had established cirrhosis. Significant difference were noted in age, HBV DNA, PT-INR, PLT, albumin, ALT, AST, GGT and proportion of significant inflammation
between S2 group and 2 ULN. In the above three groups, the distribution of significant fibrosis was 27.6% (8/29), 48.4% (45/93), and 51.8% (58/112), respectively, and significant inflammation was 6.9% (2/29), 10.8% (10/93), and 23.2% (26/112), respectively. The patients in group 2 had a higher proportion of significant fibrosis than did the patients in group 1 (P ¼ 0.048), but exhibited no significant difference with group 3 (P ¼ 0.628). However, the patients in group 2 had a lower proportion of significant inflammation than did the patients in group 3
TABLE II. Comparison of Clinical Characteristics Between Patients With Significant Fibrosis and Patients With Insignificant Fibrosis S2
Clinical characteristics n, % Age (years) (median, range) Male (n, %) Albumin (g/l) (median, range) GGT (U/L) (median, range) ALT (U/L) (median, range) AST (U/L) (median, range) PT-INR (median, range) PLT (109/l) (median, range) HBV DNA (log IU/ml) (median, range) Patients with significant inflammation (n, %)
111 36 85 42.3 41 58.7 43 1.1 136.2 7.1 28
(47.4) (15–63) (48.9) (28.8–55.8) (9–661) (17–1743) (14–660) (0.9–1.5) (26–265) (3.0–8.58) (25.2)
123 27 26 45.3 21 49 32 1.0 174.6 7.6 10
1 105 group (P ¼ 0.026), but it was not noted in the HBV DNA (IU/ml) 1 105 group (P ¼ 0.096). Patients were then analyzed according to different strata of age (30 and >30 years). A strong negative correlation was noted between HBV DNA levels and significant fibrosis in patients over 30 years of age (t ¼ 4.342, P < 0.001), but no correlation was found in patients 30 years of age and younger (t ¼ 0.365, P ¼ 0.716). Among patients over 30 years of age, the proportion of significant fibrosis decreased from 77.8% in the HBV DNA (IU/ml) 1 105 group to 58.0% in the HBV DNA >1 105 group (P ¼ 0.126). However, among patients 30 years of age and younger, the proportion of significant fibrosis increased from 20.0% in the HBV DNA (IU/ml) 1 105 group to 34.2% in the HBV DNA >1 105 group (P ¼ 0.662). Value of Serum HBV DNA Levels for Low Risk of Significant Fibrosis The ROC curves and AUC values of serum HBV DNA levels in evaluating low risk of significant fibrosis were performed. Serum HBV DNA levels produced a better AUC for low risk of significant fibrosis in patients with ALT 2 ULN and age over
Liver Fibrosis and HBV DNA, ALT, Age
1833
TABLE III. The Distributions of Significant Fibrosis and Inflammation in Different Level of HBV DNA According to Different Strata of ALT and Age (n ¼ 234) HBV DNA (IU/ml)
ALT (U/L) 1 ULN 1–2 ULN >2 ULN Age (years) 30 >30
>1 105 (n, %)
1 105 (n, %)
Characteristics n 4 8 10
S2 3 (75.0)a 7 (87.5)b 5 (50.0)
5 18
1 (20.0) 14 (77.8)e
G2 0 0 3 (30) 2 (40.0) 1 (5.6)
P-value
n 25 85 102
S2 5 (20.0) 38 (44.7)c 53 (52.0)
G2 2 (8.0) 10 (11.8) 23 (22.5)d
0.052,a 0.021b 0.026,c 0.026d
111 100
38 (34.2) 58 (58.0)f
13 (11.7) 22 (22.0)g
0.033,e 0.001f 0.045g
ALT, alanine aminotransferase; ULN, upper limit of normal; HBV DNA, hepatitis B virus deoxyribonucleic acid; n, number of patients; S2, significant fibrosis; G2, significant inflammation. a Comparison was between patients with serum HBV DNA (log10 IU/ml) 1 105 and >1 105 among patients with ALT 1 ULN. b Comparison was between patients with serum HBV DNA (log10 IU/ml) 1 105 and >1 105 among patients with ALT 1–2 ULN. c Comparison was between patients with ALT 1–2 ULN and 1 ULN among patients with serum HBV DNA (log10 IU/ml) >1 105. d Comparison was between patients with ALT 1–2 ULN and >2 ULN among patients with serum HBV DNA (log10 IU/ml) >1 105. e Comparison was between patients with age >30 years and 30 years among each group. f Comparison was between patients with age >30 years and 30 years among each group. g Comparison was between patients with age >30 years and 30 years among each group.
30 years (AUC 0.820, P < 0.001, 95% CI: 0.720–0.919) compared to the whole study patients with ALT 2 ULN (AUC 0.770, P < 0.001, 95% CI: 0.686–0.854). Serum HBV DNA levels did not have any evaluating value for low risk of significant inflammation (AUC 0.580 and AUC 0.520 for whole patients and patients with ALT 2 ULN, respectively). The sensitivity, specificity and predictive values of different HBV DNA levels in patients with ALT 2 ULN and age over 30 years are shown in Table IV. Based on the Youden Index, the optimal level of serum HBV DNA to evaluate low risk of significant fibrosis was 6.7 log10 IU/ml (Youden Index 0.557, sensitivity 83.9%, specificity 71.8%). Patients with serum HBV DNA levels 8.5 log10 IU/ml all had no significant fibrosis, however, patients with HBV DNA levels 4.7 log10 IU/ml all had significant fibrosis. Univariate and Multivariate Analysis for Significant Fibrosis Univariate analysis showed that in HBeAg-positive patients, age, albumin, AST, PT-INR, platelet count, GGT and HBV DNA levels were associated with significant fibrosis, and that ALT, AST, GGT, albumin, PT-INR, and platelet count (PLT) were associat-
ed with significant inflammation. Gender was not associated with either significant fibrosis or inflammation. Multivariate analysis showed that in the whole study patients, older age, lower HBV DNA levels, lower PLT and higher AST were associated independently with significant fibrosis, while higher AST and PLT were associated independently with significant inflammation. In patients with ALT 2 ULN, an older age (P ¼ 0.043), higher AST (P < 0.011) and lower HBV DNA (P ¼ 0.001) were associated independently with significant fibrosis, while no factor was associated independently with significant inflammation. The univariate and multivariate analysis of clinical parameters associated with significant inflammation or fibrosis are shown in Table V. DISCUSSION This study demonstrates that progressive liver disease in HBeAg-positive patients is driven by three main factors: which is age, a surrogate of disease duration; immune activation, as measured by ALT; and viral replication, as measured by HBV DNA levels. ALT levels are used commonly to assess liver disease, and elevated levels have been shown to be associated with active liver disease on histology while
TABLE IV. Sensitivity, Specificity and Predictive Values of Different Serum HBV DNA Levels for Predicting Insignificant Fibrosis Among Patients With ALT 2 ULN and Age >30 Years (n ¼ 70) HBV DNA (log10 IU/ml)
n
Sensitivity
Specificity
PPV
NPV
LRþ
LR
6.00 6.70 7.00 7.50 8.00 8.47
45 37 35 25 17 4
83.9% 83.9% 80.6% 58.1% 45.2% 9.7%
64.1% 71.8% 71.8% 82.1% 92.3% 100%
65.00% 70.27% 69.44% 72.00% 82.35% 100%
86.21% 84.85% 82.35% 71.11% 67.92% 59.09%
2.34 2.98 2.86 3.25 5.87 —
0.251 0.224 0.270 0.510 0.594 0.903
Insignificant fibrosis: Stage of fibrosis
