As shown in Table 5, there was a significant association between liver inflammation and core IgM, in particular in HBeAg-positive patients. In children with increased ALT (ALTi > 1), severe liver inflammation (HAIinfl ≥9) was more frequent in patients with core IgM greater than 0.2 (P = 0.034), with an odds ratio of 5.0.
Ten HBeAg-negative children had precore mutation, one of them at the start codon and nine at nt 1896 (creating a TAG stop codon). In two children, codon 1 and 2 could not be analyzed. One HBeAg-positive child had a mixture of wild-type and precore mutant (G/A-1896). In the HBeAg-negative children, there was no difference in liver damage between carriers with precore mutants and precore wild-type strains. However, considering all children, those with precore mutations had milder liver inflammation than those with wild-type (P = 0.01), probably reflecting covariation with HBeAg status. Children with C-1858 strains (n = 17), who more rarely had precore mutations, had or tended to have more severe liver damage than those with T-1858 as measured by ALTi (P = 0.019) or HAIinfl (P = 0.13), despite similar HBV DNA levels (gm 106.7 vs. 106.6 copies/mL). Interestingly, in HBeAg-positive children (12 C-1858 and 37 T-1858), these differences were more pronounced and also were significant for HAIinfl (P = 0.03) Eight children, four of whom were HBeAg positive, had A-1764 mutation in the core promoter. The HBV DNA level tended to be lower in these 8 children with A-1764 compared with the 61 with wild-type G-1764 (gm 105.5 vs. 106.8 copies/mL;P = 0.11).
Genotypes and Origin
As expected, the observed genotypes reflected the geographic origin of the children, as shown in Table 3. The variation at nt 1858 was linked to genotypes, as reported previously (22,25), with C-1858 strains found in 79%, 0, 40%, and 0 of genotypes A, B, C and D, respectively (Table 3). The HBeAg-positive rate was higher in genotypes B and C than in genotypes A and D, probably due to an increased frequency of children with vertical transmission in the former genotypes. There was no significant difference in liver damage between genotypes.
Factors Associated With Liver Damage
Table 6 summarizes univariate analysis of parameters possibly associated with inflammation. Most associations were confined to HBeAg-positive patients. Alanine aminotransferase levels, core IgM, HBV DNA levels, and HBeAg status were highly associated with inflammation. Children with vertical transmission and adopted children (the latter group considered mostly vertically transmitted) had less liver inflammation compared with those with horizontal transmission, but this was true only in the HBeAg-positive group. Likewise, children from East Asia had less liver inflammation than those from Africa, but only in the HBeAg-positive subgroup. Accordingly, C-1858, which was found mainly in East African children infected with genotype A, was associated with more liver inflammation in the HBeAg-positive children. Mutations in the precore (1896) or in the core promoter were not associated with liver damage.
In a multiple regression analysis of the variables possibly associated with HAIinfl, only HBeAg status, ALT, and nucleotide at position 1858 in HBeAg-positive children showed statistically significant associations.
Children with chronic hepatitis B generally have no symptoms, but show a wide range of histologic changes and viremia levels. Many of the children have highly active HBeAg-positive infection, but most spontaneously seroconvert to anti-HBe before reaching adulthood, however with some geographical variation (2,4–6). Currently, there is no known parameter that can predict a severe course of infection. Moreover, our knowledge of how well noninvasive tests such as ALT and HBV DNA levels reflect histologic activity in children is insufficient.
In the current study, we found a nearly 5-log difference in HBV DNA between HBeAg-positive (median, 108.3 copies/mL) and HBeAg-negative (median, 103.4 copies/mL) patients, and no overlap between the groups. Thus, none of the HBeAg-negative children, 10 of whom were infected with precore mutant strains, had a high viremia level (all levels were less than 104.6 copies/mL). This supports previous studies, in which high viremia levels in HBeAg-negative children (detectable by hybridization assays) are rarely observed (4,26–28). This could reflect a difference from the situation in adults in which a significant proportion of HBeAg-negative carriers showed very high HBV DNA levels (and severe liver damage); such carriers almost always carry precore mutant strains (7–9,18). Seroconversion to HBe during childhood may represent a more efficient immune response, explaining the absence in this study of HBeAg-negative carriers with highly active infection. Longitudinal follow-up studies of children are required to see whether, after HBe seroconversion at a young age, low viremia levels are maintained throughout life.
None of the HBeAg-negative children showed an inflammation score greater than 7. Because all HBeAg-negative children had relatively mild inflammation and low viremia levels, no association could be detected between HBV DNA level and liver damage, as has been observed in adults (29,30). There was no association between HBV DNA level and liver damage within the HBeAg-positive children. In this group, similarly high HBV DNA levels were seen in patients with mild (i.e., with putative immune tolerance) and severe inflammation. Previous studies have shown that HBe seroconversion often is preceded by a flare of hepatitis, suggesting that viral clearance is a result of mechanisms that also may induce hepatocellular damage (27). In the current study, HBeAg-positive children with relatively low HBV DNA, who probably were likely to seroconvert, had lower ALTi and intermediate inflammation scores. Although, this study is cross-sectional, this finding suggests that an efficient immune response may parallel relatively mild inflammation of the liver, which supports the recent finding that noncytolytic mechanisms are important for controlling HBV infection (30). Previous longitudinal studies have shown that approximately 30% of children undergo HBe seroconversion in this more silent manner (31).
Core IgM, analyzed by IMx, was associated significantly with liver inflammation, as previously reported for adults (19,32). In children with elevated ALT, the proportion with severe inflammation was higher in those with core IgM index greater than 0.2 (with an odds ratio of 5).
Only 2 of 40 children with normal ALT had more pronounced inflammation (HAIinfl ≥9). Thus, the risk of missing severe inflammation if refraining from liver biopsy in this group seems to be low. In comparison, about half of the patients with increased ALT showed HAIinfl of 9 or greater. Applying a threshold of ALTi greater than 2 did not significantly increase the proportion with HAIinfl of 9 or greater, and two patients with ALT greater than this level had mild histologic changes (HAIinfl ≤3). However, combining core IgM greater than 0.2 and ALTi greater than 1 may be useful for identifying patients for liver biopsy or therapy; a HAIinfl score of 9 or greater was seen in 10 of 14 patients who met these criteria (70%).
Precore mutations have been investigated in several studies, with observed frequencies ranging from a few to 93% (11–17). Precore mutations were seen in only 1 HBeAg-positive and in 10 of the 21 HBeAg-negative children in our study, and showed no association with the degree of liver damage. The relatively low frequency probably was due partly to a large proportion of carriers infected with genotype A, which carries cytosine at nt 1858, and therefore rarely has precore mutations. Children with C-1858 strains showed more severe liver damage than those with T-1858 strains, despite equal viremia levels. The potential pathogenic importance of nt 1858 variability should be examined further because C-1858 previously has been associated with more severe liver damage in adults (18) and was shown recently to influence core promoter mutations that are linked to liver damage (33). However, the difference may reflect the impact of epidemiologic factors, as most C-1858 carriers were from Africa where perinatal infection has been reported to be rarer. An influence of epidemiologic factors on the course of infection was observed in the current study. Probable vertical transmission, which was seen in 50% of East Asian but in none of the African children, was associated with an increased rate of HBeAg positivity late in childhood, supporting the conclusion that the rate of HBe seroconversion depends on age or mode of acquisition.
The HBeAg seroconversion rates could be estimated to 0.6% per year in vertically infected/adopted children, and 4.6% in horizontally infected children, if assuming a mean duration of infection of 14 and 9 years in the respective groups (from 100% to 92% between 0 and 14 years, and from 100% to 52% between 5 and 14 years of age).
In univariate analyses ALT, core IgM, HBV DNA levels, and HBeAg were associated strongly with HAIinfl. Age, geographic origin (Africa vs. East Asia), transmission route (horizontal vs. presumed vertical), and nt 1858 (C vs. T) also showed significant associations with inflammation score, but only for the HBeAg-positive patients. In multiple regression analysis, only ALT, HBeAg status (or HBV DNA when HBeAg was not included in the analysis), and nt 1858 (C vs. T) were independently associated with HAIinfl. The reason for this could be the complex causal relationship for development of liver damage, illustrated in Figure 3, with several factors being dependent on one another. Although many of these factors were analyzed in this study, data on transmission route and age at infection were incomplete. Moreover, we do not have knowledge about HLA group and other genetic differences in the immune response, which most likely are important for the development of liver damage. The finding of more inflammation in patients with C-1858 suggests pathogenic differences between HBV variants. However, this requires confirmation because the association was seen only in the HBeAg-positive subgroup and the statistical model in this analysis was not fully satisfactory.
The AGG → TGA double mutation at nt 1762 and 1764 in the core promoter has been associated recently with more severe liver damage in adults (21,34) and with interferon response rates (35,36), but studies in children are lacking. The low frequency of the A-1764 mutation (which in general is combined with the T-1762 mutation) observed in our study indicates that analysis of the nt 1762/1764 mutation is of little use in the clinical assessment of children.
None of the 71 children in our study had cirrhosis. Other studies have shown cirrhosis in about 3% to 4% (5,37). This difference could have occurred because some of the children in these previous studies were anti-HDV positive or coinfected with hepatitis C since these studies were performed before the use of anti-HCV assays.
In summary, we found in this series of children with chronic HBV infection a nearly 5-log difference in viremia levels between HBeAg-positive and HBeAg-negative subjects, a relatively low frequency of core promoter and precore mutations, and no association of these mutations to the degree of liver damage. We also found more severe liver inflammation in children with C-1858 strains, and a significant association between geographic origin and mode of acquisition of HBV infection and HBeAg status.
The authors thank Annkatrin Gusdal for expert technical assistance.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
Hepatitis B; Children; Histology; Viremia; Mutations