| PurposesTo investigate the distribution of liver grade of necroinflammation and stage of fibrosis in chronic hepatitis B virus (HBV) infected patients, especially among patients with ALT<2×ULN.To investigate the relationship between liver grade of necroinflammation and stage of fibrosis and clinical parameters among chronic hepatitis B virus infected patients, including gender, age, serum HBsAg, HBeAg, serum HBV DNA, ALT, aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), albumin (ALB), GLO,PT-INR, white blood cell (WBC), PLT, prothrombin time international normalized ratio (PT-INR), intrahepatic HBsAg and HBcAg and so on.To find out the risk factors of moderate or severe liver fibrosis in chronic hepatitis B virus infected patients with ALT<2×ULN, we can perform liver biopsy as early as possible, thus, early-stage diagnosis and treatment can be achieved and the deterioration of HBV can be prevented or delayed.To find out the predictors of mild liver fibrosis among chronic hepatitis B virus infected patients with ALT<2×ULN, so that liver biopsy will be avoided and the dangers, painfulness and economic burden resulting from it can also be reduced. Materials and MethodsPatientsThe present study included486treatment-naive chronic HBV infected patients who were recruited to assess the status of liver histology from August2010to January2014in the Department of Infectious Disease at the First Affiliated Hospital of Anhui Medical University, China. Demographic, clinical and laboratory data were recorded, including age, gender, alcohol intake, ALT, AST, GGT, PT-INR, albumin, GLO,PLT, HBeAg status, serum HBsAg and HBV DNA levels at the time of liver biopsy or within one week and family history of HBV infection. All patients were HBsAg-positive for at least6months before enrolling in the study or verified by liver histology. Patients with concomitant liver diseases, including chronic hepatitis C or D infection, Wilson’s disease, autoimmune hepatitis, primary biliary cirrhosis, significant alcohol intake (30grams per day for male,20grams per day for female), overt cirrhosis or liver cancer, decompensated liver disease, primary liver cancer and prior antiviral treatment were excluded. All patients provided written consent prior to the liver biopsy and study entry with all clinical investigations conducted according to the principles expressed in the Declaration of Helsinki.Liver histologyAll patients received ultrasonography-guided percutaneous biopsy of the right lobe of the liver. A quick-cut and16-gauge sheathed cutting needle (BARD, purchased from American) was used for this procedure. The biopsy lengths were1.2to2.2cm with nine or more available portal areas. The biopsy specimens were fixed with10%formalin, paraffin-embedded and stained with hematoxylin and eosin for morphological evaluation and VanGieson for the assessment of fibrosis. Histologic staging of fibrosis and grading of necroinflammation were performed using the Scheuer’s classification and the guideline on prevention and treatment of chronic hepatitis B in China, the grade of hepatic inflammation was GO-4, and the stage of fibrosis was SO-4. We defined <G2as insignificant inflammation, G2-4as significant hepatic inflammation,<S2as insignificant fibrosis, and S2-4as significant hepatic fibrosis. Liver stiffness was measured by FibroScan in39cases on the day of liver biopsy.Statistical analysesStatistical analyses were performed using SPSS version16.0(SPSS Inc, Chicago, USA) software package. The Mann-Whitney U test was used to compare continuous variables with a skewed distribution; the Chi-squared test and Fisher’s exact test was used for categorical variables. Pearson’s bivariate correlation was used to demonstrate correlation between clinical parameters and histological severity. A Receiver Operating curve (ROC) analysis was performed to find a cut-off value to predict significant fibrosis or insignificant fibrosis in patients with ALT<2×ULN. Multivariate logistic regression was used to identify factors that were independently associated with significant fibrosis or inflammation. A two-sided p value of <0.05was considered statistically significant.ResultsOf all the486subjects,74.49%(362/486) were male,80.66%(392/486) had ALT<2×ULN and there were248HBeAg-positive patients and238HBeAg-negative patients. Compared with HBeAg-negative patients, HBeAg-positive patients had higher serum HBV DNA levels, serum HBsAg, PLT and AKP level, while had younger age.Significant necroinflammation and fibrosis were found in14.11%(35/248) and47.18%(117/248) of HBeAg-positive patients, respectively and11.76%(28/238) and47.90%(114/238) of HBeAg-negative patients. The proportion of expression of intrahepatic HBsAg was95.56%in HBeAg-positive group and94.54%in HBeAg-negative group. The proportion of expression of intrahepatic HBcAg in HBeAg-positive patients is higher than in HBeAg-negative patients (41.53%vs10.5%; X2=60.267,p<0.001)Many clinical parameters are related to liver fibrosis or necroinflammation. Among HBeAg-positive patients, age, PT-INR, AST and GGT positive correlated with liver fibrosis, while serum HBV DNA, serum HBsAg, HBeAg, PLT, ALB and intrahepatic HBcAg expression negative correlated with liver fibrosis; PT-INR, AST, GGT and intrahepatic HBcAg postive correlated with necroinflammation, while PLT and HBeAg negative correlated with liver necroinflammation. Higher GGT, lower PLT and HBeAg were independently associated with significant fibrosis, while higher GGT were independently associated with significant inflammation. Among HBeAg-negative patients, PT-INR, AST, AKP and GGT positive correlated with liver fibrosis, while WBC, PLT and ALB negative correlated with liver fibrosis; GLO, PT-INR and intrahepatic HBcAg postive correlated with necroinflammation, while PLT negative correlated with liver necroinflammation. Higher GGT, lower PLT and ALB were independently associated with significant fibrosis, while higher GLO and intrahepatic HBcAg were independently associated with significant inflammation.Negative correlation was found between intrahepatic HBVcccDNA levels and liver inflammation. However, the gender, HBeAg state, intrahepatic HBsAg were not significant correlated with liver histology. Among HBeAg-positive patients with ALT<2xULN, age positive correlated with liver fibrosis (r=0.188, P<0.001). The cut-off value of diagnosis of significant fibrosis was29.5, and the proportion of significant fibrosis was higher in patients with age30～40than in patients with age≤30(51.92%vs25.27%; X2=10.334, p=0.001). There was no significant difference in significant liver fibrosis or necroinflammation between patients with ALT<1xULN and1～2×ULN group, and no significant necroinflammation was found in patients with ALT<20U/L. The proportion of significant fibrosis was higher in patients with HBV DNA<51ogcopies/ml than in patients with HBV DNA>51ogcopies/ml (X2=5.009,p=0.024). Especially, the proportion of significant fibrosis reached80%among patients with age>30years and HBV DNA level<51og10copies/mL. The proportion of significant fibrosis in group of HBV DNA level (log10copies/mL)<4,4～5,5～6,6～7,7～8and>8was100%(6/6),58.82%(10/17),72.72%(24/33),45.45%(15/33),44.66%(46/103) and28.57%(16/56), respectively. HBV DNA level negative correlated with liver fibrosis among patients with ALT <2xULN (r=-0.304,p<0.001). Higher serum HBsAg, serum HBV DNA, HBeAg, PLT and ALB can predict insignificant fibrosis. Serum HBsAg and HBeAg also negative correlated with liver fibrosis among patients with ALT <2×ULN (r=-0.428, p<0.001; r=-0.440, p<0.001). The optimal level of serum HBsAg to predict insignificant fibrosis was≥>12848.87IU/mL(Yuden index:0.485; AUC=0.755, p<0.001,95%CI:0.682-0.827). The optimal level of serum HBV DNA to predict insignificant fibrosis was ≥6.65log10copies/mL (Yuden index0.373; AUC=0.728, p<0.001;95%CI:0.656-0.801)and serum HBVDNA levels≥8.44log10copies/ml was100%predictive of insignificant fibrosis. The optimal level of serum HBsAg to predict insignificant fibrosis was≥584S/CO (Yuden index0.525; AUC=0.778,p<0.001;95%CI:0.710-0.847)and serum HBsAg levels≥1600S/CO was100%predictive of insignificant fibrosis. In HBeAg-positive patients with ALT<2×ULN, the prevalence of significant fibrosis increasing with age or HBV DNA level, however, no relationship was found between liver fibrosis and age or HBV DNA level.There was no significant difference in significant fibrosis between HBV DNA<4loglOcopies/mL and HBV DNA≥41og10copies/mL group (42.48%vs50.57%; X2=1.297,p=0.255). No parameters can predict significant or insignificant fibrosis among HBeAg-positive patients with ALT<2×ULN.Among patients with ALT<2×ULN, the optimal level of liver stiffness to predict significant fibrosis was≥7.85kPa(Yuden index0.882; AUC=0.946, p<0.001;95%CI:0.862-1.031) and positive predictive value was85.71%, negative predictive value was100%. Liver stiffness levels≥11.35kPa was100% predictive of significant fibrosis.ConclusionsMany factors are correlated with liver fibrosis or necroinflammation in chronic hepatitis B infection. No significant difference was found in significant liver fibrosis or necroinflammation between HBeAg-positive patients and HBeAg-negative patients, but they had their different correlated factors with them. Among HBeAg-positive patients, higher GGT. lower PLT and HBeAg were independently associated with significant fibrosis, while higher GGT were independently associated with significant inflammation. Among HBeAg-negative patients, higher GGT, lower PLT and ALB were independently associated with significant fibrosis, while higher GLO and intrahepatic HBcAg were independently associated with significant inflammation.Among HBeAg-positive patients with ALT<2xULN, liver biopsy is recommended in patients aged30and older, especially, when they had low HBV DNA level, regardless of their ALT level. High serum HBV DNA levels, serum HBsAg and HBeAg can predict insignificant fibrosis and reduce the need for liver biopsy. Patients with serum HBsAg≥175205IU/mL or HBV DNA≥8.441og10copies/mL or HBeAg≥1600S/CO may not require immediate liver biopsy and treatment but only close follow up, regardless.No relationship was found between liver fibrosis or necroinflammation and ALT level among HBeAg-negative patients or HBeAg-positive patients with ALT<2×ULN.The ULN of ALT for chronic HBV infection recommended to lower to20U/L when they in immune-tolerance stage.The measurement of liver stiffness is fairly accurate in diagnosing significant fibrosis, and anti-virus treatment can be considered if the value over11.35kPa at two different time.HBV DNA leve≥51og10copies/mL (HBeAg-positive) or≥4log10copies/mL (HBeAg-negative) as an initiate point for anti-virus treatment or liver histology evaluation is inappropriate among chronic hepatitis B infection. |
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