Advertisement

Liver histology of treatment-naïve children with chronic hepatitis B virus infection in Shanghai China

  • Author Footnotes
    # These authors contributed equally to this work.
    Yao Hu
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Author Footnotes
    # These authors contributed equally to this work.
    Xia Wu
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Yingzi Ye
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Lijing Ye
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Shuzhen Han
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Xiaohong Wang
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Hui Yu
    Correspondence
    Corresponding author: Hui Yu, Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Tel: +86 21-64931184; Fax: +86 21-64931184.
    Affiliations
    Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China

    Key Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
    Search for articles by this author
  • Author Footnotes
    # These authors contributed equally to this work.
Open AccessPublished:August 22, 2022DOI:https://doi.org/10.1016/j.ijid.2022.08.017

      Highlights

      • Demographic, clinical, and histological characteristics of 278 children with chronic hepatitis B.
      • Serum alanine aminotransferase >80 U/l may strongly indicate hepatic inflammation and fibrosis.
      • Moderate fibrosis and cirrhosis can appear in children aged 3 years or younger.

      Abstract

      Objectives

      Chronic hepatitis B (CHB) is associated with high morbidity and mortality. We aimed to investigate associations between hepatic histology and clinical characteristics in treatment-naïve children with CHB in Shanghai, China.

      Methods

      The liver biopsy specimens of 278 treatment-naïve children with CHB virus infection were scored for inflammation and fibrosis, and correlations with clinical and laboratory data were determined.

      Results

      CHB clinical, virologic, and pathologic features were studied in 278 treatment-naïve children (177 [63.7%] males) in Shanghai, China. Maternal sera were positive for hepatitis B surface antigen for 277 children. At biopsy, 87.4% of patients were hepatitis B e antigen–positive. The median age at biopsy was 5.1 years (interquartile range 2.8-8.4 years). Hepatitis B virus (HBV) deoxyribonucleic acid levels were generally high (mean 7.4 log10 IU/ml), as were levels of serum alanine aminotransferase (ALT, median 105 U/l). Using the Metavir histology activity index scoring system, no, mild, moderate, and severe inflammation were seen in 2.9%, 22.3%, 73.4%, and 1.4% of patients, respectively. No fibrosis, mild fibrosis, moderate fibrosis, and cirrhosis were seen in 11.5%, 32.7%, 47.5%, and 8.3% of patients, respectively. When the serum ALT level was ≤80 (two times the upper limit of normal) and >80 U/l, the inflammation score (P <0.0001) was significantly different, and the fibrosis score was also significantly different (P <0.0001). Inflammation and fibrosis were aggravated with increasing ALT levels. Fibrosis scores were significantly higher in children aged ≤3 than aged >3 years (P <0.0001). The rates of moderate fibrosis and cirrhosis were higher in children aged ≤3 years at biopsy. No correlations were found between histologic changes and sex, HBV genotype, or HBV deoxyribonucleic acid level.

      Conclusion

      Substantial heterogeneity in inflammatory and fibrotic levels was observed in treatment-naïve children with CHB in Shanghai, China. Serum ALT levels >80 U/l may be a strong indicator of the degree of hepatic inflammation and fibrosis severity. Moderate fibrosis and cirrhosis can appear in children aged 3 years or younger.

      Graphical abstract

      Keywords

      Introduction

      Hepatitis B virus (HBV) infection is a major public health concern worldwide. Chronic HBV infection is extremely harmful and is closely associated with the development of cirrhosis, hepatocellular carcinoma (HCC), and liver failure (
      • Lozano R
      • Naghavi M
      • Foreman K
      • et al.
      Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010.
      ). Infection with HBV at a young age is known to lead frequently to the chronic hepatitis B surface antigen (HBsAg) carrier state, particularly if it occurs during the perinatal period. In China, more than half of the existing patients with chronic hepatitis B (CHB) are estimated to have been infected in the perinatal period (
      • Hou J
      • Cui F
      • Ding Y
      • et al.
      Management algorithm for interrupting mother-to-child transmission of hepatitis B virus.
      ;
      • Hou J
      • Wang G
      • Wang F
      • et al.
      Guideline of prevention and treatment for chronic hepatitis B (2015 update).
      ;
      • Wang FS
      • Fan JG
      • Zhang Z
      • et al.
      The global burden of liver disease: the major impact of China.
      ). All of these facts highlight the important role of chronic HBV infection in early childhood in the eventual development of serious chronic liver disease.
      Liver biopsy is the gold-standard method for the histologic diagnosis and management of patients with CHB. Liver biopsy can help determine the degree of hepatic inflammation and fibrosis stage and identify other coexisting liver conditions that may influence disease progression (
      • Das P
      • Ahuja A
      • Gupta SD.
      Overview of the histopathology of chronic hepatitis B infection.
      ). Several studies have revealed variable severity of disease in chronic HBV infection in children (
      • Hsu HC
      • Lin YH
      • Chang MH
      • et al.
      Pathology of chronic hepatitis B virus infection in children: with special reference to the intrahepatic expression of hepatitis B virus antigens.
      ;
      • Iorio R
      • Giannattasio A
      • Cirillo F
      • et al.
      Long-term outcome in children with chronic hepatitis B: a 24-year observation period.
      ;
      • Rodriguez-Baez N
      • Murray KF
      • Kleiner DE
      • et al.
      Hepatic histology in treatment-naïve children with chronic hepatitis B infection living in the United States and Canada.
      ;
      • Zhang HF
      • Yang XJ
      • Zhu SS
      • et al.
      Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
      ).
      Chronic HBV infection is a dynamic process that is influenced by many factors, such as host immune response, age, disease duration, viral load, and HBV genotype (
      • Das P
      • Ahuja A
      • Gupta SD.
      Overview of the histopathology of chronic hepatitis B infection.
      ;
      • Mani H
      • Kleiner DE.
      Liver biopsy findings in chronic hepatitis B.
      ). Adult patients with chronic HBV infection with elevated HBV deoxyribonucleic acid (DNA) levels and serum alanine aminotransferase (ALT) levels were revealed to be at a higher risk for developing complications (
      • Perz JF
      • Armstrong GL
      • Farrington LA
      • et al.
      The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide.
      ).
      This study was conducted to assess the correlations between histopathologic changes and the demographic, clinical, and virologic characteristics at the time of liver biopsy in treatment-naïve children with CHB in Shanghai, China.

      Patients and methods

      Inclusion and exclusion criteria

      Inclusion criteria: (i) age <18 years and HBsAg-positive, (ii) treatment-naïve, (iii) no history of blood product transfusion before biopsy, and (iv) parent willingness to provide written informed consent.
      Exclusion criteria: (i) positive test for hepatitis A, C, D, or E or for HIV, a history or evidence of chronic liver disease other than CHB or suspected HCC; (ii) a history of significant chronic pulmonary, cardiac, renal, thyroid, or immunodeficiency disease, diabetes, previous solid-organ or stem-cell transplant, or malignancy; and (iii) other definitive liver diseases, including autoimmune hepatitis, metabolic liver disease, and fatty liver disease.
      The study was approved by the ethics committee of the Children's Hospital of Fudan University, and written informed consent was obtained from every child's parent(s).

      Patients and specimens

      All patients participating in the study, who visited the hospital between January 2002 and December 2020, underwent extensive evaluation, including a detailed history and physical and laboratory examinations. Clinical and demographical information, including sex, age at the time of biopsy, transmission mode, HBV genotype, ALT level, HBV DNA level, HBsAg, and hepatitis B e antigen (HBeAg), were collected. Serum levels of HBeAg, HBsAg, anti-HBe antibodies, anti-HBs antibodies, and anti-HBc antibodies were measured by enzyme-linked immunosorbent assays. The reference range of HBsAg was 0-0.04 IU/ml, and the upper limit of detection for HBsAg was 250 IU/ml. HBV DNA levels were measured by real-time quantitative polymerase chain reaction, and the range of quantitation for HBV DNA levels was 1 × 103-1 × 108 IU/ml before 2019 and 5 × 102-1 × 108 IU/ml after 2019. HBV genotype detection was carried out by a gene sequence determination method.
      Liver biopsy specimens were collected from 286 children who were chronic HBsAg-carriers. Two of the specimens were excluded because these patients were previously treated for hepatitis B. One specimen was excluded from the study because of the patient's teratoma and chemotherapy history. One specimen was excluded owing to the presence of Wilson disease in the patient. In addition, four specimens were excluded for data integrity. Therefore, 278 liver biopsy specimens were evaluated in the final analysis.

      Histologic studies

      All specimens were stained with hematoxylin-eosin and Masson trichrome and scored for inflammation and fibrosis, using the Metavir (
      • Bedossa P
      • Poynard T.
      An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
      ) scoring systems. All biopsies were deidentified and coded and examined by a single expert histopathologist.
      To demonstrate liver HBsAg and hepatitis B core antigen (HBcAg), the deparaffinized sections were stained by the peroxidase-antiperoxidase technique (
      • Su IJ
      • Lai MY
      • Hsu HC
      • et al.
      Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection.
      ), using mouse monoclonal anti-HBs and rabbit anti-HBc (Sigma) (
      • Chang MH
      • Hsu HC
      • Lee CY
      • et al.
      Fraternal hepatocellular carcinoma in young-children in two families.
      ).

      Statistics

      For continuous data, Spearman correlation was used to determine associations between clinical characteristics and histologic data (i.e., inflammation and fibrosis scores). All P values were two-sided, with P <0.05 considered statistically significant. The Kruskal-Wallis test or Mann-Whitney test was used to analyze differences in noncontinuous data. SPSS (version 22) and GraphPad Prism (version 7) were used for all analyses and graphing.

      Results

      Patient characteristics

      Of the 278 patients whose liver biopsies and clinical characteristics were reviewed, all of them were treatment-naïve, and no obvious symptoms and signs were observed. In addition, 63.7% (177/278) of them were male (Table 1). The maternal rate of HBsAg-positive was 99.6% (277/278; from the detailed history, it was revealed that one patient had been adopted and, therefore, information regarding the child's mother's HBsAg status was not available). No patient had a history of blood product transfusion before biopsy.
      Table 1Demographics and baseline disease characteristics
      Frequency (%) or median (25th-75th percentile)
      Age at biopsy, years5.1 (2.8-8.4)
      Sex, male177 (63.7)
      HBeAg status
       Positive243 (87.4)
       Negative35 (12.6)
      ALT, U/l105 (65-175)
      HBV DNA, log10 IU/ml7.4 (6.8-8.0)
       Detectable269 (96.8)
       Undetectable9 (3.2)
      HBV genotypes, n=132
       B43 (32.6)
       C89 (67.4)
      The normal range for ALT used in this study was 0-40 U/l for both male and female individuals.
      ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.
      Up to 87.4% of the patients were HBeAg-positive, and 12.6% were HBeAg-negative at the time of biopsy. All children were HBsAg-positive (100%). Among the 132 patients for whom HBV genotype testing was performed, the HBV genotype distribution was narrow (B: 32.6%; C: 67.4%; no other genotypes).
      The median age at biopsy was 5.1 years, ranging from 8 months to 15 years and 3 months (interquartile range [IQR] 2.8-8.4 years). A total of 69 (24.8%, 69/278) patients were aged less than 3 years. At the time of the biopsy, 269 patients were HBV DNA-positive, and HBV DNA was undetectable in nine patients (<1000 IU/ml before 2019, <500 IU/ml after 2019). The mean HBV DNA level among the 269 patients in whom it was detectable was 7.4 log10 IU/ml (range 3.1-9.9 log10 IU/ml). Serum ALT ranged from 5 to 2170 U/l (median 105, IQR 65-175).

      Histologic findings

      The degree of inflammation seen in the liver biopsies ranged from none to severe inflammation, whereas the fibrosis stage ranged from none to cirrhosis (Figure 1). Using the Metavir histology activity index scoring system, no inflammation was seen in 2.9% (score 0, 8/278), mild inflammation was seen in 22.3% (score 1, 62/278), moderate inflammation was seen in 73.4% (score 2, 48.6%, 135/278; score 3, 24.8%, 69/278), and severe inflammation was seen in 1.4% (score 4, 4/278).
      Figure 1
      Figure 1Liver histology of chronic hepatitis B in children (H&E, 400x). (A) G0S0, no inflammation and fibrosis. (B) G1S1, mild inflammation and fibrosis. (C) G2S2, moderate inflammation and fibrosis, portal inflammation with extensive interface hepatitis. (D) G3S3, moderate inflammation and fibrosis, consisting of bridging fibrosis, portal lymphoid aggregate with Poulsen-type duct lesion. (E) G4S4, cirrhosis with small regenerative nodules.
      Lack of fibrosis was observed in 11.5% (score 0, 32/278), mild fibrosis was observed in 32.7% (score 1, 91/278), moderate fibrosis (including advanced fibrosis consisting of bridging fibrosis) was observed in 47.5% (score 2, 37.4%, 104/278; score 3, 10.1%, 28/278), and cirrhosis was observed in 8.3% (23/278).
      Liver HBsAg expression was examined in 278 patients. The HBsAg immunostaining positive rate was 92.1% (256/278) in biopsy specimens. HBcAg expression was examined in the liver of 276 patients. The HBcAg immunostaining positivity rate was 30.8% (85/276). Typical immune staining of HBsAg and HBcAg positive samples are shown in Figure 2, Figure 3, respectively.
      Figure 2
      Figure 2Immune staining of hepatitis B surface antigen-positive.
      Figure 3
      Figure 3Immune staining of hepatitis B core antigen-positive.

      Association between clinical characteristics and pathologic histology

      The ALT level was moderately associated with the degree of hepatic inflammation (r = 0.39, P <0.0001) and fibrosis stage (r = 0.38, P <0.0001) (Table 2, Figure 4). When the serum ALT level was less than or equal to 80 and higher than 80 U/l (two times the upper limit of normal), the degree of inflammation (P <0.0001) and fibrosis stage (P <0.0001) were significantly different (Figure 4). The percentages of moderate fibrosis and cirrhosis in children with ALT levels ≤ 80 U/l and ALT levels >80 U/l children were 39.1% (50/128) and 70.0% (105/150), respectively (P <0.0001).
      Table 2Variables for prediction of inflammation grade and fibrosis stage in multivariate logistic regression analysis.
      OutcomeVariablesβ (95% CI)SEP-value
      Inflammation gradeSex0.090 (−0.371, 0.551)0.2350.703
      Age−9.61E to 5 (−0.005, 0.005)0.0020.969
      ALT0.002 (0.001, 0.003)0.001<0.001
      HBV DNA0.181 (−0.025, 0.386)0.1050.085
      HBV genotypes
      n=132. ALT, alanine aminotransferase; HBV, hepatitis B virus.
      0.253 (−0.249, 0.756)0.2560.323
      Fibrosis stageSex0.065 (−0.385, 0.516)0.2300.776
      Age−0.005 (−0.009, 7.027E-5)0.0020.053
      ALT0.003 (0.001, 0.004)0.001<0.001
      HBV DNA0.036 (−0.164, 0.236)0.1020.721
      HBV genotypes
      n=132. ALT, alanine aminotransferase; HBV, hepatitis B virus.
      0.441 (−0.188, 1.070)0.3210.169
      a n=132.ALT, alanine aminotransferase; HBV, hepatitis B virus.
      Figure 4
      Figure 4Association between ALT and inflammation, and fibrosis.
      ALT, alanine aminotransferase.
      There was no association between inflammation degree and age at biopsy (P >0.05) (Table 2). However, for the association between fibrosis and age at biopsy, the correlation coefficient was −0.19, P = 0.0018. The fibrosis stage was significantly higher in patients aged 3 or younger than in those older than 3 years (P <0.0001) (Figure 5).
      Figure 5
      Figure 5Correlation between age at biopsy and histology.
      The percentage of children aged ≤3 years at biopsy, with ALT higher than 80 U/l was 66.7% (46/69), whereas this percentage was 49.8% (104/209) in children older than 3 years at biopsy. The percentage of patients that exhibited moderate fibrosis and cirrhosis for children aged ≤3 and >3 years with ALT levels >80 U/l were 80.4% (37/46) and 65.4% (68/104), respectively.
      No associations were found between inflammation degree and sex, HBV DNA levels, or HBV genotypes (all P values >0.05) (Table 2). No associations were found between fibrosis stage and sex, HBV DNA levels, or HBV genotypes (all P values >0.05) (Table 2).

      Discussion

      This study of 278 treatment-naïve children with CHB virus infection in China revealed heterogeneity in the severity of liver disease. Moderate inflammation was seen in a large number of children who were chronically infected with HBV in Shanghai. The mothers of most of the children were HBsAg-positive (99.6%), and none of the children had a history of blood product transfusion before biopsy. Although most of the children visited the hospital at age >1 year, we still have reason to believe that mother-to-child vertical transmission was the main route of transmission. It is well known that the major routes of HBV infection are blood contact, mother-to-child transmission (MTCT), and sexual contact (
      World Health Organization
      Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection.
      ). With improvements in detection methods and the standardization of medical procedures, MTCT has become a major transmission route (
      • Hou J
      • Wang G
      • Wang F
      • et al.
      Guideline of prevention and treatment for chronic hepatitis B (2015 update).
      ). Since China introduced the hepatitis B vaccine into its national vaccination program in 1992, the prevalence of HBsAg has dropped significantly (
      • Cui F
      • Shen L
      • Li L
      • et al.
      Prevention of chronic hepatitis B after 3 decades of escalating vaccination policy.
      ). When the use of the vaccine for CHB became government-supported in 2002, the dose provided was 5 μg/0.5 ml, which was known to prevent infection in 85-90% of children born to women who were HBsAg-positive (
      • Cui F
      • Shen L
      • Li L
      • et al.
      Prevention of chronic hepatitis B after 3 decades of escalating vaccination policy.
      ;
      • Kang W
      • Ding Z
      • Shen L
      • et al.
      Risk factors associated with immunoprophylaxis failure against mother to child transmission of hepatitis B virus and hepatitis B vaccination status in Yunnan Province, China.
      ;
      • Wang F
      • Zheng H
      • Zhang G
      • et al.
      Effectiveness of prevention of mother-to-child transmission practice in three provinces of Southern China.
      ). The transplacental transmission of HBV and intrauterine HBV infections mainly occur during the third trimester, and the risk of MTCT correlates positively with maternal HBV DNA levels (
      • Singh AE
      • Plitt SS
      • Osiowy C
      • et al.
      Factors associated with vaccine failure and vertical transmission of hepatitis B among a cohort of Canadian mothers and infants.
      ;
      • Zhang Z
      • Chen C
      • Li Z
      • et al.
      Individualized management of pregnant women with high hepatitis B virus DNA levels.
      ). In the newborns of mothers who were HBsAg- and HBeAg-positive, the incidence of MTCT was found to reach 7.1%, despite administering the hepatitis B vaccine and hepatitis B immunoglobulin at birth (
      • Pan CQ
      • Duan ZP
      • Bhamidimarri KR
      • et al.
      An algorithm for risk assessment and intervention of mother to child transmission of hepatitis B virus.
      ).
      Among these children with CHB, a higher serum ALT level was associated with a higher degree of liver inflammation and fibrosis stage, consistent with previous reports (
      • Hsu HC
      • Lin YH
      • Chang MH
      • et al.
      Pathology of chronic hepatitis B virus infection in children: with special reference to the intrahepatic expression of hepatitis B virus antigens.
      ;
      • Iorio R
      • Giannattasio A
      • Cirillo F
      • et al.
      Long-term outcome in children with chronic hepatitis B: a 24-year observation period.
      ;
      • Rodriguez-Baez N
      • Murray KF
      • Kleiner DE
      • et al.
      Hepatic histology in treatment-naïve children with chronic hepatitis B infection living in the United States and Canada.
      ;
      • Zhang HF
      • Yang XJ
      • Zhu SS
      • et al.
      Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
      ). In our study, we found that when the serum ALT level was higher than two times the upper limit of normal (80 U/l), the degree of inflammation and fibrosis stage were relatively severe. Fibrosis was present in most specimens (88.5%, 246/278) and correlated with serum ALT levels. The rates of moderate fibrosis and cirrhosis in children with ALT levels ≤80 U/l and ALT levels >80 U/l were 39.1% and 70.0%, respectively (P <0.0001). It is important to recognize that pediatricians should perform biopsies as soon as possible, even though ALT elevation might be mild, which is generally accepted as an immunotolerant phase. Therefore, providing close monitoring of pediatric patients is recommended by an increasing number of pediatricians (
      • Rodriguez-Baez N
      • Murray KF
      • Kleiner DE
      • et al.
      Hepatic histology in treatment-naïve children with chronic hepatitis B infection living in the United States and Canada.
      ;
      • Wu JF
      • Song SH
      • Lee CS
      • et al.
      Clinical predictors of liver fibrosis in patients with chronic hepatitis B virus infection from children to adults.
      ;
      • Zhang HF
      • Yang XJ
      • Zhu SS
      • et al.
      Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
      ;
      • Zhu S
      • Zhang H
      • Dong Y
      • et al.
      Antiviral therapy in hepatitis B virus-infected children with immune-tolerant characteristics: a pilot open-label randomized study.
      ).
      Cirrhosis occasionally develops during childhood (
      • Godra A
      • Perez-Atayde A
      • Jonas MM.
      Histologic features of chronic hepatitis B in children.
      ;
      • Zhang HF
      • Yang XJ
      • Zhu SS
      • et al.
      Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
      ), although, in general, it was previously believed to be rare in children. In a study of 292 children with HBV infection and elevated serum ALT levels, cirrhosis was found in 3% of the patients (
      • Bortolotti F
      • Calzia R
      • Cadrobbi P
      • et al.
      Liver cirrhosis associated with chronic hepatitis B virus infection in childhood.
      ). However, a higher hepatitis D virus (HDV) coinfection rate was reported in the children with cirrhosis than in those without cirrhosis, and this HDV coinfection was generally believed to have contributed to disease progression. In this study, 23 (8.3%) children had cirrhosis, 14 of whom were younger than 6 years. Two of the 14 patients were aged 8 months and 9 months at the time of biopsy. In addition, none of the children had HDV or hepatitis C virus coinfection. In one study of 76 HBeAg-positive children with CHB and elevated serum ALT levels, moderate-to-severe fibrosis was reported in at least 50% of them (
      • Godra A
      • Perez-Atayde A
      • Jonas MM.
      Histologic features of chronic hepatitis B in children.
      ). In our study, most of the patients had different degrees of fibrosis, and the percentage with moderate-to-severe fibrosis (cirrhosis) was up to 55.8%. All these findings suggest that chronic HBV infection in children is at risk of developing into cirrhosis, which again highlights the importance of close monitoring of pediatric patients with CHB.
      A study in adults showed an association between a higher extent of inflammation and advanced fibrosis with age over 40 years and disease duration (
      • Cadranel JF
      • Lahmek P
      • Causse X
      • Bellaiche G
      • Bettan L
      • Fontanges T
      • et al.
      Epidemiology of chronic hepatitis B infection in France: risk factors for significant fibrosis - results of a nationwide survey.
      ). In contrast, in our study, in which the median age at biopsy was 5.1 years, ranging from 8 months to 15 years and 3 months (IQR 2.8-8.4 years), with 69 (24.8%) of our patients being younger than 3 years, there was no significant correlation between the inflammation degree and the age at biopsy. However, we found that the fibrosis score was significantly higher in children aged ≤3 than those aged >3 years (P <0.0001). The percentages of moderate fibrosis and cirrhosis in children aged ≤3 years and >3 years with ALT >80 U/l were 80.4% and 65.4%, respectively (P <0.0001). One of the limitations of this study is that we did not analyze the influence of disease duration on liver pathology because of the uncertainty of the exact time of HBV infection in some children.
      In accordance with the male predominance in HCC (
      European Association for the Study of the Liver
      EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.
      ;
      Chinese Society of Infectious Diseases, Chinese Medical Association, Chinese Society of Hepatology, Chinese Medical Association
      The guidelines of prevention and treatment for chronic hepatitis B (2019 version).
      ;
      • Hou J
      • Wang G
      • Wang F
      • et al.
      Guideline of prevention and treatment for chronic hepatitis B (2015 update).
      ;
      • Terrault NA
      • Lok ASF
      • McMahon BJ
      • et al.
      Update on prevention, diagnosis, and treatment and of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
      ;
      • Vittal A
      • Ghany MG.
      WHO guidelines for prevention, care and treatment of individuals infected with HBV: a US perspective.
      ), a greater male predominance was demonstrated in children with CHB in our study (63.7%). However, no associations were found between inflammation degree or fibrosis stage and male sex, which may be because the duration of infection was not long enough. Therefore, we need to perform studies with larger sample size and a longer follow-up.
      No associations were found between inflammation degree (P >0.05) or fibrosis stage (P >0.05) and HBV DNA levels in this study, which is in accordance with the results of some previous studies (
      • Rodriguez-Baez N
      • Murray KF
      • Kleiner DE
      • et al.
      Hepatic histology in treatment-naïve children with chronic hepatitis B infection living in the United States and Canada.
      ;
      • Zhang HF
      • Yang XJ
      • Zhu SS
      • et al.
      Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
      ). No correlation was observed between genotype and the degree of inflammation or fibrosis. One reason may be that only HBV genotypes B and C were observed, and no other genotypes were observed during this study. This trend resulted in an inability to establish an association between the presence of other HBV genotypes and liver histology, which was one of the limitations. Another reason may be that the duration of infection was not long enough. It is well known that there are at least nine genotypes of HBV (i.e., A-I) (
      • Liu CJ
      • Kao JH.
      Global perspective on the natural history of chronic hepatitis B: role of hepatitis B virus genotypes A to.
      ). HBV genotypes B and C are predominant in Asia and in China (
      • Hou J
      • Cui F
      • Ding Y
      • et al.
      Management algorithm for interrupting mother-to-child transmission of hepatitis B virus.
      ;
      • Hou J
      • Wang G
      • Wang F
      • et al.
      Guideline of prevention and treatment for chronic hepatitis B (2015 update).
      ;
      • Sun YH
      • Lei XY
      • Sai YP
      • et al.
      Relationship between genotypes and clinical manifestation, pathology, and cccDNA in Chinese children with hepatitis B virus-associated glomerulonephritis.
      ;
      • Vittal A
      • Ghany MG.
      WHO guidelines for prevention, care and treatment of individuals infected with HBV: a US perspective.
      ). HBV genotype C infection is associated with a higher risk of developing chronic hepatitis, reactivation of hepatitis B, and progression to liver cirrhosis and HCC than genotype B (
      • Chu CM
      • Liaw YF.
      Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline.
      ;
      • Lin CL
      • Kao JH.
      The clinical implications of hepatitis B virus genotype: recent advances.
      ;
      • Livingston SE
      • Simonetti JP
      • Bulkow LR
      • et al.
      Clearance of hepatitis B e antigen in patients with chronic hepatitis B and genotypes A, B, C, D, and F.
      ;
      • Yu MW
      • Yeh SH
      • Chen PJ
      • et al.
      Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: a prospective study in men.
      ). Patients with HBV genotype B have a higher response rate to interferon-alpha–based therapy than those with genotype C in patients who are HBeAg-positive, and those with genotype A have a better response than those with genotype D (
      • Liu F
      • Chen L
      • Yu DM
      • et al.
      Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy.
      ;
      • Wang HY
      • Chien MH
      • Huang HP
      • et al.
      Distinct hepatitis B virus dynamics in the immunotolerant and early immunoclearance phases.
      ). A long-term, detailed clinical, virologic, immunohistologic, and pathologic study may provide more valuable insights into the natural course of HBV infection in children.
      In conclusion, the results of this study demonstrate that substantial heterogeneity in inflammation and fibrosis was observed in treatment-naïve children with CHB virus infection in Shanghai, China. Serum ALT >80 U/l may be a strong indicator of the degree of hepatic inflammation and severity of fibrosis. Moderate fibrosis and cirrhosis have already appeared in children aged ≤3 years. It is very important to closely monitor pediatric patients with CHB in clinical practice.

      Conflicts of interest

      The authors have no competing interests to declare.

      Funding

      This study was funded by the Key Development of Program of Children's Hospital of Fudan University, EK2022ZX05.

      Acknowledgments

      The authors would like to acknowledge all the staff who helped with and supported this study, especially histopathologist Prof. Xiqi Hu and his staff. The authors also acknowledge all the participating children and their families.

      Author contributions

      Hui Yu designed the study, with input from Xiaohong Wang. Yao Hu and Xia Wu analyzed and, together with Yingzi Ye, Lijing Ye, and Shuzhen Han, interpreted the data. Yao Hu drafted, and all authors critically revised the manuscript.

      Consent to participate

      Written informed consent was obtained from the parent(s)/guardian(s) of each child.

      Consent to publish

      The study was approved by the ethics committee of the hospital.

      References

        • Bedossa P
        • Poynard T.
        An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
        Hepatology. 1996; 24: 289-293
        • Bortolotti F
        • Calzia R
        • Cadrobbi P
        • et al.
        Liver cirrhosis associated with chronic hepatitis B virus infection in childhood.
        J Pediatr. 1986; 108: 224-227
        • Cadranel JF
        • Lahmek P
        • Causse X
        • Bellaiche G
        • Bettan L
        • Fontanges T
        • et al.
        Epidemiology of chronic hepatitis B infection in France: risk factors for significant fibrosis - results of a nationwide survey.
        Aliment Pharmacol Ther. 2007; 26: 565-576
        • Chang MH
        • Hsu HC
        • Lee CY
        • et al.
        Fraternal hepatocellular carcinoma in young-children in two families.
        Cancer. 1984; 53: 1807-1810
        • Chinese Society of Infectious Diseases, Chinese Medical Association, Chinese Society of Hepatology, Chinese Medical Association
        The guidelines of prevention and treatment for chronic hepatitis B (2019 version).
        Zhonghua Gan Zang Bing Za Zhi. 2019; 27: 938-961
        • Chu CM
        • Liaw YF.
        Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline.
        J Hepatol. 2005; 43: 411-417
        • Cui F
        • Shen L
        • Li L
        • et al.
        Prevention of chronic hepatitis B after 3 decades of escalating vaccination policy.
        China. Emerg Infect Dis. 2017; 23: 765-772
        • Das P
        • Ahuja A
        • Gupta SD.
        Overview of the histopathology of chronic hepatitis B infection.
        Hep B Annual. 2012; 9: 49-85
        • European Association for the Study of the Liver
        EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.
        J Hepatol. 2017; 67: 370-398
        • Godra A
        • Perez-Atayde A
        • Jonas MM.
        Histologic features of chronic hepatitis B in children.
        Hepatology. 2005; 421: 478A-479A
        • Hou J
        • Cui F
        • Ding Y
        • et al.
        Management algorithm for interrupting mother-to-child transmission of hepatitis B virus.
        Clin Gastroenterol Hepatol. 2019; 17 (e1): 1929-1936
        • Hou J
        • Wang G
        • Wang F
        • et al.
        Guideline of prevention and treatment for chronic hepatitis B (2015 update).
        J Clin Transl Hepatol. 2017; 5: 297-318
        • Hsu HC
        • Lin YH
        • Chang MH
        • et al.
        Pathology of chronic hepatitis B virus infection in children: with special reference to the intrahepatic expression of hepatitis B virus antigens.
        Hepatology. 1988; 8: 378-382
        • Iorio R
        • Giannattasio A
        • Cirillo F
        • et al.
        Long-term outcome in children with chronic hepatitis B: a 24-year observation period.
        Clin Infect Dis. 2007; 45: 943-949
        • Kang W
        • Ding Z
        • Shen L
        • et al.
        Risk factors associated with immunoprophylaxis failure against mother to child transmission of hepatitis B virus and hepatitis B vaccination status in Yunnan Province, China.
        Vaccine. 2014; 32: 3362-3366
        • Lin CL
        • Kao JH.
        The clinical implications of hepatitis B virus genotype: recent advances.
        J Gastroenterol Hepatol. 2011; 26: 123-130
        • Liu CJ
        • Kao JH.
        Global perspective on the natural history of chronic hepatitis B: role of hepatitis B virus genotypes A to.
        J. Semin Liver Dis. 2013; 33: 97-102
        • Liu F
        • Chen L
        • Yu DM
        • et al.
        Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy.
        Gut. 2011; 60: 1269-1277
        • Livingston SE
        • Simonetti JP
        • Bulkow LR
        • et al.
        Clearance of hepatitis B e antigen in patients with chronic hepatitis B and genotypes A, B, C, D, and F.
        Gastroenterology. 2007; 133: 1452-1457
        • Lozano R
        • Naghavi M
        • Foreman K
        • et al.
        Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010.
        Lancet. 2012; 380: 2095-2128
        • Mani H
        • Kleiner DE.
        Liver biopsy findings in chronic hepatitis B.
        Hepatology. 2009; 49: S61-S71
        • Pan CQ
        • Duan ZP
        • Bhamidimarri KR
        • et al.
        An algorithm for risk assessment and intervention of mother to child transmission of hepatitis B virus.
        Clin Gastroenterol Hepatol. 2012; 10: 452-459
        • Perz JF
        • Armstrong GL
        • Farrington LA
        • et al.
        The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide.
        J Hepatol. 2006; 45: 529-538
        • Rodriguez-Baez N
        • Murray KF
        • Kleiner DE
        • et al.
        Hepatic histology in treatment-naïve children with chronic hepatitis B infection living in the United States and Canada.
        J Pediatr Gastroenterol Nutr. 2020; 71: 99-105
        • Singh AE
        • Plitt SS
        • Osiowy C
        • et al.
        Factors associated with vaccine failure and vertical transmission of hepatitis B among a cohort of Canadian mothers and infants.
        J Viral Hepat. 2011; 18: 468-473
        • Su IJ
        • Lai MY
        • Hsu HC
        • et al.
        Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection.
        J Hepatol. 1986; 3: 182-189
        • Sun YH
        • Lei XY
        • Sai YP
        • et al.
        Relationship between genotypes and clinical manifestation, pathology, and cccDNA in Chinese children with hepatitis B virus-associated glomerulonephritis.
        World J Pediatr. 2016; 12: 347-352
        • Terrault NA
        • Lok ASF
        • McMahon BJ
        • et al.
        Update on prevention, diagnosis, and treatment and of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
        Hepatology. 2018; 67: 1560-1599
        • Vittal A
        • Ghany MG.
        WHO guidelines for prevention, care and treatment of individuals infected with HBV: a US perspective.
        Clin Liver Dis. 2019; 23: 417-432
        • Wang F
        • Zheng H
        • Zhang G
        • et al.
        Effectiveness of prevention of mother-to-child transmission practice in three provinces of Southern China.
        Hum Vaccin Immunother. 2015; 11: 2061-2067
        • Wang FS
        • Fan JG
        • Zhang Z
        • et al.
        The global burden of liver disease: the major impact of China.
        Hepatology. 2014; 60: 2099-2108
        • Wang HY
        • Chien MH
        • Huang HP
        • et al.
        Distinct hepatitis B virus dynamics in the immunotolerant and early immunoclearance phases.
        J Virol. 2010; 84: 3454-3463
        • World Health Organization
        Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection.
        World Health Organization, Geneva2015
        • Wu JF
        • Song SH
        • Lee CS
        • et al.
        Clinical predictors of liver fibrosis in patients with chronic hepatitis B virus infection from children to adults.
        J Infect Dis. 2018; 217: 1408-1416
        • Yu MW
        • Yeh SH
        • Chen PJ
        • et al.
        Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: a prospective study in men.
        J Natl Cancer Inst. 2005; 97: 265-272
        • Zhang HF
        • Yang XJ
        • Zhu SS
        • et al.
        Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.
        Hepatobiliary Pancreat Dis Int. 2004; 3: 395-398
        • Zhang Z
        • Chen C
        • Li Z
        • et al.
        Individualized management of pregnant women with high hepatitis B virus DNA levels.
        World J Gastroenterol. 2014; 20: 12056-12061
        • Zhu S
        • Zhang H
        • Dong Y
        • et al.
        Antiviral therapy in hepatitis B virus-infected children with immune-tolerant characteristics: a pilot open-label randomized study.
        J Hepatol. 2018; 68: 1123-1128