Xin Shu,Hixi Sun,Xion Yng,Yifn Ji,Peiyn Xu,Hong Co,*,K Zhng,*

aDepartment of Infectious Diseases,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,China

bDepartment of Infectious Diseases,Huadu District People's Hospital of Guangzhou,Guangzhou,China

ABSTRACT

Background and aims:Effective hepatic blood flow(EHBF)decreases with liver disease progression,and identifying liver pathology is critical for patients with liver disease.This study was designed to elucidate the correlation between EHBF and liver pathology and explore the potential of EHBF for predicting the degree of liver pathology.

Methods:In this study,207 patients with hepatitis B virus(HBV)who underwent liver biopsy and indocyanine green(ICG)clearance tests were enrolled.EHBF was measured using the ICG clearance test,and liver tissue was histologically analyzed to determine the pathological stage according to the Scheuer scoring system.Demographic data,biochemical indexes,and FibroScan data were collected for statistical analysis.

Results:EHBF levels decreased as the liver histological stages of inflammation and fibrosis increased(P＜0.01).EHBF was significantly negatively associated with the levels of alanine aminotransferase,aspartate aminotransferase,gamma-glutamyl transpeptidase,alkaline phosphatase,aspartate aminotransferase-to-platelet ratio index, fibrosis index based on the four factors,and liver stiffness measurement(P＜0.05).The EHBF levels of patients without liver inflammation(G0)were significantly higher than those of patients with liver inflammation(G1-4)(P＜0.001).The area under the receiver operating characteristic curve(AUROC)value for discriminating patients without liver inflammation was 0.827,and the optimal cutoff value was 0.936 L/min.The EHBF levels of patients with severe liver inflammation(G4)were significantly lower than those of patients with G0-3 liver inflammation(P＜0.001).The AUROC value for discriminating patients with severe liver inflammation was 0.792,and the optimal cutoff value was 0.552 L/min.The EHBF levels of patients without liver fibrosis(S0)were significantly higher than those of patients with liver fibrosis(S1-4)(P＜0.001).The AUROC value for discriminating patients without liver fibrosis was 0.633,and the optimal cutoff value was 1.173 L/min.The EHBF levels of patients with liver cirrhosis(S4)were significantly lower than those of patients with S0-3 liver fibrosis(P＜0.001).The AUROC value for discriminating patients with liver cirrhosis(S4)was 0.630,and the optimal cutoff value was 0.562 L/min.

Conclusions:EHBF levels and liver pathology are significantly correlated.EHBF could effectively reflect liver inflammation and fibrosis in patients infected with HBV,especially for patients without liver inflammation or liver fibrosis.

Keywords:

Hepatitis B virus(HBV)

Liver inflammation

Liver fibrosis

Effective hepatic blood flow(EHBF)

Liver disease

1.Introduction

Liver disease remains an important public health problem.One recent report of the Lancet Standing Commission on Liver Disease in the UK has asserted that“liver disease is on a trajectory to become the biggest cause of death in England and Wales”.1The continuing increase in the burden of liver disease is worsening in deprived areas.2Therefore,a great deal of work remains to be performed toward its prevention.

Liver inflammation and fibrosis are crucial components of the pathological changes occurring in liver disease and have been considered primary risk factors for developing liver cirrhosis and hepatocellular carcinoma(HCC).Liver biopsy(LB)is the gold standard for identifying the grade of liver inflammation and fibrosis.3However,its widespread use was restricted because of sampling errors,observer discrepancies,and severe complication risks.4Frequently,biochemical parameters,such as serum transaminases,including alanine aminotransferase(ALT)and aspartate aminotransferase(AST),are the most commonly used for evaluating the severity of liver inflammation in liver diseases.5,6Transient elastography has been widely recommended by the guidelines of chronic hepatitis B virus(HBV)management for assessing hepatic fibrosis staging.7However,several studies have reported that patients with normal or mildly elevated ALT levels are not necessarily free from liver injury or liver-associated mortality.8Furthermore,our previous study has found differences between clinical and pathological diagnoses,and the levels of ALT failed to be consistent with the stages of liver pathological diagnosis.9Some confounders may affect the diagnostic accuracy of a transient elastography device in staging liver fibrosis.Therefore,novel biomarkers that can accurately assess and monitor the severity of liver inflammation and fibrosis remain urgently needed.

The indocyanine green(ICG)retention test is a quantitative function test reflecting liver functional reserve and blood flow.ICG correlates with the presence,degree,and complications of portal hypertension,reflecting modifications of liver blood flow.10Effective hepatic blood flow(EHBF)is measured using the ICG clearance test;the formula to calculate EHBF was deduced as follows:EHBF=R/(ICGa-ICGhv),where R is the liver ICG clearance rate,ICGa is the ICG concentration in the hepatic artery and portal vein blood,and ICGhv is the ICG concentration in the hepatic vein blood.ICGa has the same concentration as ICG in peripheral venous blood because other organs do not clear ICG.The general approach is to assume that the liver clearance rate is 100%(because the liver has a high first-pass effect on ICG),and ICGhv is identified as 0 accordingly.The role of vascular alterations,subsequent impaired liver blood flow,and hepatic hypoxia in the pathophysiology of liver disease has been convincingly demonstrated in several animal models.11Persistent inflammation is a strong stimulus for pathological angiogenesis and vascular remodeling,which contributes to progression from liver fibrosis to cirrhosis;imbalance between the blood flow in the hepatic artery and that in the portal vein increases hepatic artery in flow,which is beneficial to the formation of cirrhotic nodules.12Reduced hepatic blood flow is a significant finding in patients with severe hepatic ischemic reperfusion injury.13There was a significant correlation between liver function and hepatic blood flow,which was measured using xenonenhanced computed tomography(Xe-CT)in alcoholic liver cirrhosis.14Likewise,in chronic hepatitis C(CHC)or non-alcoholic fatty liver disease(NAFLD),there were significant correlations between hepatic tissue blood flow measured using Xe-CT and liver function;and hepatic tissue blood flow significantly decreased with fibrosis progression.15Therefore,theoretically,EHBF level should change with the stage of liver inflammation and fibrosis and thus can be used for evaluating the severity of liver inflammation and fibrosis.Unfortunately,most studies have involved patients without HBV infection and were conducted at the blood levels.So far,no relevant literature on EHBF has been reported at the pathological level,especially in patients infected with HBV.

This study was designed to elucidate the change in EHBF level with the grade of liver inflammation and fibrosis and to explore EHBF as a biomarker for the severity of liver pathologies for patients with HBV infection.

2.Patients and methods

2.1.Ethical approval

This study was conducted according to the principles of the Declaration of Helsinki for ethical research.The study protocol was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.Written informed consent was obtained from the participants,and all data were identified.

2.2.Patients

Patients with liver disease infected with HBV who underwent ICG clearance tests and LB at the Department of Infectious Diseases of the Third Affiliated Hospital of Sun Yat-sen University between January 2017 and December 2019 were included in this study.The inclusion criteria consisted of patients with documented liver disease who were 18-65 years of age and underwent an ICG clearance test and LB.The exclusion criteria were as follows:patients infected with other viruses or human immunodeficiency virus;those with HCC,cardiovascular disease,diabetes,and kidney disease;and those who are pregnant,among others.

2.3.LB

All patients underwent percutaneous LB performed using ultrasound localization.Liver samples were obtained using 16-or 18-gage needles and were fixed in formalin and embedded in paraffin.Hematoxylin and eosin and reticular fiber staining or Masson's trichome staining was performed on each section.Liver samples with less than three portal tracts were excluded from this study.The LB specimens were graded for inflammation(i.e.,G0,G1,G2,G3,and G4)and fibrosis(S0,S1,S2,S3,and S4)according to a modified Scheuer scoring system by two experienced clinical pathologists.The stages of liver inflammation and fibrosis were classified as previously described.16The grades for liver inflammation(G)were defined as follows:G0=none;G1=mild inflammation; G2 = mild to moderate inflammation G3=moderate inflammation;and G4=severe inflammation.Meanwhile,the stages of liver fibrosis(S)were defined as follows:S0=no fibrosis;S1=minimal fibrosis;S2=moderate fibrosis;S3=severe fibrosis;and S4=cirrhosis.

2.4.Biochemistry assay

Blood samples were obtained within 1 day before LB.Serum biochemical tests were conducted on a 7600-020 ISE automatic analyzer(Hitachi,Tokyo,Japan),including serum ALT,AST,gammaglutamyl transpeptidase(GGT),alkaline phosphatase(ALP),and total bilirubin.A blood routine examination was performed using whole blood by a routine blood test instrument,including platelet(PLT)and white blood cell counts.The AST-to-PLT ratio index(APRI)and fibrosis index based on the four factors(FIB-4)were calculated according to the following formula:APRI=100×(AST/upper limit of normal AST)/PLT;FIB-4=(age×AST)/(PLT×square root of ALT).17

2.5.Liver stiffness measurement(LSM)

LSM was performed using transient elastography(Fibro Scan 502,Echosens,Paris,France)with the 3.5-MHz standard probe by the same operator.17The specific operation method was followed as previously described.

2.6.ICG test

ICG clearance tests were performed under hemodynamically stable conditions without high-rate fluid infusion or transfusion.A total of 4 h or more of fasting was needed before the ICG clearance tests were performed.According to the patients’body weight(0.5 mg/kg,maximum 25 mg),ICG(Liaoning,China)dissolved in 5 mL saline was injected into the periphery within 10 s.The ICG disappearance rate was measured using pulse spectrophotometry(DDG-3300K,Nihon Kohden,Tokyo,Japan).The ICG retention rate at 15 min(R15%)and EHBF were automatically calculated.18The operation process was performed as previously described.

2.7.Statistical analysis

Statistical Package for the Social Sciences(SPSS,version 21.0,Armonk,NY,USA)was used for all statistical analyses.Continuous variables were expressed as mean±standard deviation or median(range)depending on the result of normality testing and analyzed using a t test or the Wilcoxon test,as appropriate.The Shapiro-Wilk test was used as the normality test.Student's t test and two-way analysis of variance were used for parametric data,and the Kruskal-Wallis test and Mann-Whitney U-test were used for nonparametric data.Correlation coefficients were evaluated using Pearson's or Spearman's correlation.The performances of the serum models in predicting liver histological scores were assessed using receiver operating characteristic(ROC)curve analyses and area under the ROC curves(AUROCs).All P-value was two-tailed,and P-value＜0.05 was considered statistically significant.

3.Results

3.1.Patient characteristics

In this study,207 patients with HBV were enrolled.Table 1 shows the age,gender,EHBF level,and baseline clinical characteristics of these patients.

3.2.Distribution of EHBF according to liver histological severity

Statistical analysis using the Kruskal-Wallis test revealed significant differences between liver inflammation grades and the levels of EHBF,ALT,AST,GGT,and ALP(P＜0.05)(Table 2).The results showed that EHBF levels decreased with increasing histological inflammation grades(Table 2).However,the differences of EHBF levels between G0 and G1,G2,G3,and G4 were statistically significant(P＜0.001)(Fig.1A).Similarly,we found that the EHBF levels were significantly higher in G0,G1,G2,and G3 patients than those in G4 patients(P＜0.001)(Fig.1A).No differences were observed among G1,G2,and G3 patients(P＞0.05).Furthermore,patients with mild and moderate inflammation(G1,G2,and G3)showed significantly lower EHBF levels than those without inflammation(G0)(Fig.1B).Patients with severe inflammation(G4)showed significantly lower EHBF levels than those with mild and moderate inflammation(G1,G2,and G3)and those without inflammation(G0)(Fig.1B).

In terms of liver fibrosis stages,significant differences were observed among the levels of EHBF,APRI,FIB-4,and LSM(P＜0.01)(Table 3).The results revealed that EHBF levels decreased with increasing liver histological fibrosis stages(Table 3).However,the differences between S0 and S1,S2,S3,and S4 were statistically significant(P＜0.01)(Fig.2A).However,the EHBF levels were not significantly different among S1,S2,S3,and S4 patients(P＞0.05)(Fig.2A).Furthermore,patients with minimal to severe fibrosis(S1,S2,and S3)showed significantly lower EHBF level than those without fibrosis(S0)(Fig.2B).Patients with cirrhosis(S4)showed significantly lower EHBF level than those without fibrosis(S0)(Fig.2B).No difference was observed between cirrhosis(S4)and minimal to severe fibrosis(S1,S2,and S3)(Fig.2B).

Table 1Baseline characteristics of patients with hepatitis B virus(N=207).

3.3.Correlations between EHBF and biochemical parameters indicating liver inflammation

ALT and AST are biochemical parameters widely used to indicate inflammatory fluctuations in liver diseases.Correlation analysis indicated that EHBF was significantly negatively associated with ALT and AST levels(Table 4).Furthermore,GGT and ALP were negatively correlated with EHBF levels(Table 4).ALT,AST,GGT,and ALP levels were significantly positively associated with each other(Table 4).

3.4.Correlations between EHBF and parameters indicating liver fibrosis

EHBF was significantly negatively associated with APRI,FIB-4,and LSM(P＜0.01)(Table 5).Furthermore,APRI,FIB-4,and LSM were significantly positively associated with each other(P＜0.01)(Table 5).

3.5.Differentiating histologically liver inflammation by EHBF

To investigate whether EHBF can predict the absence or presence of inflammation in patients with liver disease,we first compared EHBF levels between patients without liver inflammation(G0)and patients with liver inflammation(G1-4).EHBF levels were significantly higher in patients without liver inflammation than in patients with liver inflammation(P＜0.001)(Fig.3A).To evaluate the performance of EHBF in predicting whether patients have liver inflammation,ROC curves were employed.The AUROC value for discriminating liver disease patients without liver inflammation was 0.827(95% confidence interval(CI),0.762-0.892),and the optimal cutoff value was 0.936 L/min(Fig.3B).

Table 2Comparison of EHBF,ALT,AST,GGT,ALP,and TBIL levels in different liver inflammation grades.

Fig.1.Distribution of EHBF according to liver histological inflammation grades.(A)Comparison of the EHBF levels according to liver inflammation grades.(B)Comparison of the EHBF levels among patients without inflammation(G0),mild and moderate inflammation(G1/G2/G3),and severe inflammation(G4).***P＜0.001.Liver inflammation grades were defined as follows:G0=none;G1=mild inflammation;G2=mild to moderate inflammation;G3=moderate inflammation;and G4=severe inflammation.Abbreviation:EHBF,effective hepatic blood flow.

Table 3Comparison of EHBF,APRI,FIB-4,and LSM values in different liver fibrosis stages.

Fig.2.Distribution of EHBF according to liver histological fibrosis stages.(A)Comparison of the EHBF levels according to the liver histological fibrosis stages.(B)Comparison of the EHBF levels among patients with no fibrosis(S0),minimal to severe fibrosis(S1/S2/S3),and cirrhosis(S4).**P＜0.01,***P＜0.001.NS,not significant.Liver fibrosis stages were defined as follows:S0=no fibrosis;S1=minimal fibrosis;S2=moderate fibrosis;S3=severe fibrosis;and S4=cirrhosis.Abbreviation:EHBF,effective hepatic blood flow.

To investigate whether EHBF can differentiate severe liver inflammation (G4)from absence,mild,and moderate liver inflammation(G0-3),EHBF levels were compared.EHBF levels were significantly lower in patients with severe liver inflammation(G4)than in patients with G0-3 liver inflammation(P＜0.001)(Fig.3C).Furthermore,ROC curves were employed to evaluate the performance of EHBF in predicting severe liver inflammation.The AUROC value for discriminating patients with severe inflammation was 0.792(95%CI,0.693-0.892),and the optimal cutoff value was 0.552 L/min(Fig.3D).

Table 4Associations among EHBF,ALT,AST,GGT,and ALP.

Table 5Associations of EHBF,APRI,FIB-4,and LSM.

Fig.3.Differential diagnostic performance of the EHBF in discriminating liver histological inflammation grades.(A)Comparison of the EHBF-value between patients without inflammation(G0)and patients with inflammation(G1/G2/G3/G4).(B)Receiver operating characteristic(ROC)curve for predicting the absence of inflammation(G0).(C)Comparison of the EHBF-value between patients with severe inflammation(G4)and patients with no,mild,and moderate inflammation(G0/G1/G2/G3).(D)ROC curve for predicting severe inflammation(G4).***P＜0.001.Liver inflammation grades were defined as follows:G0=none;G1=mild inflammation;G2=mild to moderate inflammation;G3=moderate inflammation;and G4=severe inflammation.Abbreviations:AUROC,area under the receiver operating characteristic curve;EHBF,effective hepatic blood flow.

3.6.Histologically differentiating liver fibrosis using EHBF

To explore whether EHBF can be a biomarker for differentiating the stages of liver fibrosis in patients with HBV infection,we first compared EHBF levels between patients without liver fibrosis(S0)and those with liver fibrosis(S1-4).As shown in Fig.4A,the level of EHBF was significantly higher in patients without liver fibrosis(S0)than in patients with liver fibrosis(S1-4)(P＜0.001).To evaluate the performance of EHBF in predicting liver fibrosis,ROC curves were employed.The AUROC value for discriminating patients without liver fibrosis was 0.633(95%CI,0.539-0.727),and the optimal cutoff value was 1.173 L/min(Fig.4B).

To investigate whether EHBF can differentiate liver cirrhosis(S4)from absence,minimal,and severe liver fibrosis(S0-3),EHBF levels were compared.The level of EHBF was significantly lower in S4 patients than in S0-3 patients(P＜0.001)(Fig.4C).Furthermore,ROC curves were employed to evaluate the performance of EHBF in predicting liver cirrhosis.The AUROC value for discriminating patients with liver cirrhosis was 0.630(95%CI,0.530-0.729),and the optimal cutoff value was 0.562 L/min(Fig.4D).

Fig.4.Differential diagnostic performance of EHBF in discriminating liver fibrosis stages.(A)Comparison of the EHBF-value between patients without fibrosis(S0)and patients with liver fibrosis(S1/S2/S3/S4).(B)Receiver operating characteristic(ROC)curve for predicting the absence of fibrosis(S0).(C)Comparison of the EHBF-value between patients with cirrhosis(S4)and patients with no or minimal to severe fibrosis(S0/S1/S2/S3).(D)ROC curve for predicting liver cirrhosis(S4).***P＜0.001.Liver fibrosis stages were defined as follows:S0=no fibrosis;S1=minimal fibrosis;S2=moderate fibrosis;S3=severe fibrosis;and S4=cirrhosis.Abbreviations:AUROC,area under the receiver operating characteristic curve;EHBF,effective hepatic blood flow.

4.Discussion

Liver inflammation is the primary cause of fibrosis,cirrhosis,and HCC in patients with liver disease.19Various disadvantages limit the wide application of LB.Therefore,noninvasive markers to predict significant liver inflammation and fibrosis in patients with liver diseases are needed.Recently,many studies have focused on this topic.Wang et al.20have found that the GGT-to-PLT ratio has a better diagnostic value than conventional parameters in predicting liver inflammation in chronic hepatitis B(CHB)patients with normal or mildly elevated ALT levels.20The serum hepatitis B core antibody was a strong indicator for assessing the hepatic inflammatory degree and was used for antiviral treatment decisions in CHB patients with normal ALT levels.21Serum Golgi protein 73(GP73)combined with AST and GGT reflected moderate to severe liver inflammation in patients with CHB.22Furthermore,a logistic regression model established based on biochemical parameters could be used to effectively assess liver necroinflammation in patients with CHB who have normal or minimally elevated ALT levels.23A new noninvasive algorithm incorporating age,ALT,and GGT was developed for determining significant liver histological changes in CHB patients with normal or mildly elevated ALT levels.24Serum liver fibrosis markers,including hyaluronic acid,collagen type IV,N-terminal propertied of type III procollagen,and laminin,may be strong indicators for discriminating significant liver inflammation,especially in CHB patients with normal or near-normal ALT levels.16Nevertheless,almost all of these indicators are based on serum biochemical parameters.Thus,detection bias may affect the specificity and sensitivity of these indicators.

Many studies have shown that hepatic blood flow gradually decreases as liver disease progresses in all patients with chronic liver disease.14Van Beers et al.,25reported that hepatic perfusion parameters measured using CT were significantly altered and correlated with the severity of chronic liver disease.In this study,EHBF measured using the ICG clearance test was used to evaluate the severity of liver inflammation.We found that EHBF levels decreased as the liver histological inflammation grades increased and were significantly negatively associated with ALT,AST,ALP,and GGT levels.These results support the proposal that EHBF is an indicator of liver inflammation.Moreover,ICG-R15 correlated with the presence,degree,and complications of portal hypertension,reflecting modifications of liver blood flow in patients with initial cirrhosis and well-preserved liver function.10Vascular alterations and subsequent impaired hepatic blood flow in early liver inflammation contributed to the progression of the disease by inducing increased intrahepatic vascular resistance and consequently a relative hypoxic environment in the liver disease tissue.11Kelly et al.13have reported that reduced hepatic blood flow is a significant finding in patients with severe hepatic ischemia-reperfusion injury and that hepatic blood flow plays an important role in hepatic ischemia-reperfusion injury.The preservation of hepatic blood flow improves survival and prevents hepatic inflammation following hemorrhagic shock.26MC-2 peptide may be an intermediary substance for liver inflammation and hepatic blood flow.A study has found that MC-2 decreased the levels of ALT and enhanced EHBF.27In short,sinusoidal narrowing caused by liver inflammation and swollen hepatocytes may damage the blood flow to the liver,which increases the intrahepatic vascular resistance.Increased intrahepatic vascular resistance early in the development of liver disease has been convincingly demonstrated in several animal models of NAFLD,whereas increased portal pressure,a consequence of increased intrahepatic vascular resistance,has been proven in patients with NAFLD.11Therefore,EHBF measured by the ICG clearance test could directly reflect the degree of liver damage.

In this study, EHBF was independently associated with the absence of liver inflammation and achieved the best diagnostic efficiency(AUROC=0.827)for the presence of liver inflammation.Until recently,there have been few studies exploring indicators to discriminate the presence or absence of liver inflammation.Several studies have showed that patients with normal or mildly elevated ALT levels still suffer from liver injury.8The level of ALT is affected by patients’demographics and other factors.28A recent study has found that using the ALT level to reflect liver necroinflammation is difficult in hepatitis B e antigen-negative patients with CHB who have minimally elevated ALT levels.23In this study,EHBF measured using the ICG clearance test could directly reflect the degree of liver damage and achieved the best diagnostic efficiency in the differentiation of patients with HBV infection without liver inflammation.Furthermore,EHBF was independently associated with severe liver inflammation(G4)and achieved the best diagnostic efficiency(AUROC=0.792)in the differentiation of HBV-infected patients with severe liver inflammation.Clinically,ALT and AST are the most commonly used markers for evaluating the severity of liver inflammation in liver diseases.However,the levels of ALT and AST are affected by drug treatment and testing bias.Because of poor liver function,LB is difficult to promote in patients with severe liver inflammation.A recent study found that EHBF was significantly lower in patients with acute-on-chronic liver failure (ACLF) and was related to the severity of HBV-ACLF.18Therefore,EHBF is a potential marker that can reflect the degree of liver damage. Of course, a larger cohort is needed to validate this finding,and a cutoff value is also needed.

Recently,APRI,FIB-4,and LSM have been widely investigated as noninvasive methods for quantifying liver fibrosis in HBV-infected patients with liver diseases.Regrettably,the values of APRI,FIB-4 and LSM are substantially limited by active liver inflammation and are significantly correlated with serum AST and ALT levels.29,30In this study,EHBF levels decreased as the liver histological fibrosis stages increased and were significantly negatively associated with APRI,FIB-4,and LSM values.However,no differences were found between the groups with liver fibrosis.Similarly,we also performed the same analysis on the diagnostic efficiency of EHBF for the presence of liver fibrosis and liver cirrhosis.Finally,although statistically significant differences were found,the diagnostic effect appeared to be relatively weak.This is not overall consistent with the findings of Shigefuku et al.15This discrepancy might be because the etiopathology of the liver disease in our patients has been considered.Certainly,the number of cases could also be the cause.In summary,EHBF levels decreased as the liver histological fibrosis stages increased but cannot be beneficial to make an effective diagnosis for liver fibrosis.

Several limitations of this study should be noted.First,the number of patients included in this study was relatively small,which is a limitation that should not be neglected.Classification and diagnostic biases are unavoidable.A longitudinal cohort study with a larger sample would be desirable to confirm the findings of this study.Second,this study was retrospective and used data obtained from a single center.Third,the results of this study are based only on cross-sectional study without any longitudinal research.Considering the individual differences,a series of cohort studies are needed to validate the findings.

5.Conclusions

In conclusion,there is a significant correlation between the level of EHBF and liver pathology.EHBF could effectively reflect liver inflammation and fibrosis in patients infected with HBV,especially for those without liver inflammation or liver fibrosis.

Authors’contributions

X.Shu and H.Sun contributed equally to this work.X.Shu,H.Sun,H.Cao,and K.Zhang contributed to the design,data analysis and interpretation,manuscript writing,and manuscript revision.Y.Jia,X.Yang,and P.Xu contributed to the sample collection and processing.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Science and Technology Program of Guangzhou,China(No.202002030044).