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Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
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.
Department of Infectious Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, ChinaKey Development of Program of Children's Hospital of Fudan University, National Children's Medical Center, Shanghai 201102, China
Demographic, clinical, and histological characteristics of 278 children with chronic hepatitis B.
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Serum alanine aminotransferase >80 U/l may strongly indicate hepatic inflammation and fibrosis.
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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.
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 (
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 (
). 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 (
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 (
). 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 (
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 (
Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection.
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, years
5.1 (2.8-8.4)
Sex, male
177 (63.7)
HBeAg status
Positive
243 (87.4)
Negative
35 (12.6)
ALT, U/l
105 (65-175)
HBV DNA, log10 IU/ml
7.4 (6.8-8.0)
Detectable
269 (96.8)
Undetectable
9 (3.2)
HBV genotypes, n=132
B
43 (32.6)
C
89 (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 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 2Immune staining of hepatitis B surface 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.
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 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 (
). 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 (
Risk factors associated with immunoprophylaxis failure against mother to child transmission of hepatitis B virus and hepatitis B vaccination status in Yunnan Province, 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 (
). 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 (
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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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.
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).
), 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 (
). 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) (
Relationship between genotypes and clinical manifestation, pathology, and cccDNA in Chinese children with hepatitis B virus-associated glomerulonephritis.
). 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 (
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.
). 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 (
). 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.
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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.
Risk factors associated with immunoprophylaxis failure against mother to child transmission of hepatitis B virus and hepatitis B vaccination status in Yunnan Province, China.
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.
Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection.
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