Changes of Serum Hepatitis B Virus DNA and Aminotransferase Levels during the Course of Chronic Hepatitis B Virus Infection in Children PING-ING LEE,' MEI-HWEICHANG,' CHIN-YUN LEE,' HONG-YUAN HSU,' JUEI-SAN CHEN," PEI JERCHEN3 AND DING-SHINNCHEN" 'Departments of Pediatrics, 'Clinical Pathology and "Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

*

During a follow-up period of 3.2 1.6 (1 to 8.6) yr, 1,087 serum specimens from 230 HBsAg carrier children were tested for hepatitis B virus markers.

Dividing the serum specimens into four groups according to the status of HBeAg and hepatitis B virus DNA, the frequency of abnormally elevated ALT levels in mrum was in the following order: HBeAg( + )/hepatitis B virus DNA( - ) serum (60%),HBeAg( - )/hepatitis B virus DNA(+) serum (53%). HBeAg(+)/hepatitis B virus DNA( +) serum (41%),HBeAg( - )/hepatitisB virus DNA(-) serum (11%).Analysis of the data before HBeAg clearance showed that both a high serum ALT level and a low serum hepatitis B virus DNA level correlated with an imminent clearance of HBeAg. Approximately two thirds of children with serum ALT levels higher than 100 IUD, cleared HBeAg within the following year. Clearance of HBeAg occurred within the following year in 66% (13 of 20) of cases with serum hepatitis B virus DNA level 5 1,OOO pdml, in contrast to 19% (30 of 157) of those with serum hepatitis B virus DNA level > 1,OOO pg/ml. Among 53 c h i l h n who lost HBeAg and hepatitis B virus DNA during follow-up, only nine cases did not have an identified period of abnormal serum ALT levels. For the remaining 44 children, abnormal serum ALT levels fell to normal with clearance of both HBeAg and hepatitis B virus DNA in 33 children but remained elevated in the remaining 11 cases after seroconversion. This study also demonstrated that (a) serum ALT elevations in HBsAg carrier children were usually mild in degree and infrequently exceeded 100 IUL, (b) hepatitis B virus DNA was detectable in only 1% (4 of 352) of the anti-HBe(+) sera and all showed normal ALT levels. These findings are in contrast to the adult carriers and denote some unique features of HBaAg carrier children. (HEPATOLOGY 1990;12:657-660.)

for treatment of the HBsAg carrier state through suppressing virus replication and causing resolution of hepatitic activity. It has been suggested that the treatment regimens should be administered as early as possible after onset of HBV infection to minimize the chance of virus integration into the cellular genome and to avoid the sequelae of liver damage from the hepatitic activity (2). For these reasons, it is important to delineate the natural course of chronic HBV infection in children, especially with regard to the changes of virus replication. The natural history of chronic HBV infection can be divided into three phases: (a) the early immune tolerance (highly replicative) phase, (b) the virus replicatioxdelimination (low replicative) phase and (c) the inactive v i r u s replication (nonreplicative) phase (3, 4). HBeAg and its antibody (anti-HBe) and serum HBV DNA measurements are useful in defining the state of virus replication in these phases (3-10).Few studies have defined changes of these markers in children. In this study, we report results of testing serial sera from 230 HBsAg carrier children followed over several years. Children were tested periodically for HBV markers and serum aminotransferase levels t o delineate the natural history of chronic HBV infection in children.

SUBJECTS AND METHODS

Subjects. Two hundred and thirty children, including 132 boys and 98 girls, were studied. All children were known to have HBsAg in serum for more than 6 mo and were followed at the Department of Pediatrics, National Taiwan University Hospital for at least 1yr. The ages at enrollment ranged from the newborn period to 15 yr,with a mean t S.D.of 7.9 ? 4.1 yr, and follow-upaveraged 3.2 -t- 1.6 (1to 8.6) yr. The usual 6 HBV infection is a major health problem in Taiwan mo interval between follow-up visits was shortened to 1 mo to where the HBsAg carrier rate is very high (1). New 3 mo when the serum ALT activity exceeded 100 IUL. No child therapies such as the use of a-interferon offer new hopes received antiviral or immunosuppressive therapy. Labomtory Studies. One thousand and eighty-seven serum specimenswere taken during the follow-upperiod. Serum ALT levels were assayed by autoanalyzer; the upper limit.of normal h i v e d October 31, 1989; accepted April 30, 1990. Address reprint requests to: Mei-Hwei Chang, M.D.,Department of Pedi- was set at 17 IUL, a value derived from a previous survey in normal Chinese children ( 111. HBV markers including HBsAg, atrics, National Taiwan University Hospital, Taipei. Taiwan, Republic of China. anti-HBs, HBeAg and anti-HBe were determined by RIA using 31/1/23243

657

658

LEE ET AL.

HEPATOLOGY

Total 230 cases

t I

Initial status

HBeAg(+) anti-HBe( - ) HBV DNA(*) 187 cafe6

Follow up period

HBeAgb) anti-HEe(-l HBV DNA(*) 119 ca6e.s

HBeAp(*) anli-HEe(-) HBV DNA(-) 15 cases

Y no

HBeAgbI HBeAg(-l Simultaneous enti-HBe(-l anti-HBe(-l clearance HBV DNA(-) HBV DNA(*) 23 case6 14 cases 18 ca6e6

*

I

reroconversion 134 ares

I

I

+

t

seroconversion 53 ca es

I

Final statua

HBeAg(anti-HBet.1 HBV DNA(-) 43 case6

HBeAg(- I anti-HBe(*) HBV DNA(-) 96 caoes Y

HBeAg(*) anti-HBe(-l HBV DNA(*) 134 caner

FIG.1. Changes of serum HBeAg, anti-HBe and HBV DNA in 230 HBsAg carrier children. TABLE 1. Correlation of serum HBeAg, HBV DNA and ALT levels in 1,087 serum samples from 230 children with chronic HBV infection HBeAg(-)

HBeAg(+) Serum ALT level (IU/L) 5 17 18-40 41-100 > 100 TOTAL

HBV DNA( + ) Number in sample (187 cases)

HBV DNA( - ) Number in sample (31cases)

HBV DNA( + ) Number in sample (18cases)"

HBV DNA( - ) Number in sample

379 (60%) 154 (24%) 66 (10%) 38 (6%) 637

29 (40%) 23 (32%) 11 (15%) 9 (13%) 72

14 (47%) 7 (23%) 4 (13%) 5 (17%) 30

309 (89%) 34 (10%) 4 (1%) 1(0.3%) 348

(96 casea)

"Including four cases who had reappearance of HBV DNA transiently during follow-up after seroconversion.

commercial kits from Abbott Laboratories (North Chicago, IL). Antibody to hepatitis D virus was tested by a blocking, solid-phase RIA (Abbott Laboratories, anti-delta) (12). Serum HBV DNA Assay. HBV DNA in serum was detected by the spot hybridization method as described by Scotto et al. (13) with minor modifications. Briefly, 10 pl of serum sample were treated sequentially with proteinase K (10 mg/ml) plus 10% SDS, phenol, 1N NaOH and 1 mom Tris HC1, pH 7.0 with 1.5 m o m NaCl. The sample was then applied under vacuum suction to a nitrocellulose filter (BRL No. 1051 MM, Bethesda Research Laboratories, Gaithersburg, MD) through a 96-well filtration apparatus (Manifold, Bethesda Research Laboratories). Hybridization was performed using 32P-labeledHBV DNA probe at 42" C for 16 hr. The purified HBV DNA probe, separated from the plasmid, was prepared from a head-to-tail dimer of HBV DNA (subtype adw) cloned in the EcoRI site of plasmid vector pBR322, kindly provided by Dr. W. S. Robinson (Stanford University, Stanford, CA). The specific activity of the probe was 1 to 4 x lo5 c p d n g DNA. After hybridization, the nitrocellulose paper was washed and autoradiographed at -70" C for 24 hr. The spots were assessed by a densitometer (LKB Produkter AB, Bromma, Sweden) and the amount of HBV DNA in each spot was estimated from a standard curve plotted from the reading of serially diluted, known amounts of HBV DNA. The minimum amount of HBV DNA thus detected was 0.5 pg/spot. Positive

and negative control sera were assayed simultaneously in each procedure. To avoid false-positive results from bacterial contamination, a pBR322 DNA probe was used for rehybridization after boiling nitrocellulose paper for 30 min. The assay was duplicated for each serum specimen. Definitions. Seroconversion (i.e., clearance of HBeAg and HBV DNA) was defined as the disappearance of HBeAg and HBV DNA for more than 6 mo in initially seropositive cases. Stutisticul Anulyse8. The chi-square test was used to examine the significance of differences between subgroups.

RESULTS Among the 230 carrier children, 43 cases were seronegative for HBeAg and HBV DNA at the first visit. During the follow-up period, 53 (28%) of the 187 HBeAg( + )/HBV DNA( + ) children cleared HBeAg and HBV DNA, and eight of them cleared HBsAg subsequently. The remaining 134 children continued to have HBeAg and HBV DNA in serum (Fig. 1).No consistent relationship was seen between the timing of HBeAg clearance and that of HBV DNA clearance: HBeAg disappeared at the same time as HBV DNA in 23 (43%) children, disappeared before HBV DNA in 14 (26%) children, and disappeared after HBV DNA in 16 (30%) children (Fig. 1).

TABLE 2. Correlation between serum ALT levels and probability of eubeequent HBeAg clearance during the next year in HBeAg-positive carrier children Peak ALT level

cnm)

5 17 18-40 41-100 101-200 > 200

TOTAL

659

HBV DNA AND ALT IN HBsAg CARRIER CHILDREN

Vol. 12, No. 4, 1990

Number Number

86 41 26 13 11 177

Beroconverting (%) 12 (14) 4 (10) 11 (42) 6 (46) 10 (91) 43 (24)

The 1,087 serial serum specimens from the 230 children were divided into four groups according to the status of HBeAg and HBV DNA. Frequency of abnormally elevated ALT levels was in the following order: HBeAg(+)/HBV DNA(-) serum (60%), HBeAg( - )MBV DNA( + 1 serum (53%),HBeAg( + )/HBV DNA( +) serum (41%), HBeAg( - )/HBV DNA( - ) serum (11%).The abnormal ALT values were below 100 IU/L in most sera and only seven sera exceeded 300 I U L (Table 1). Anti-HBe was positive in 352 HBeAg(-) serum samples. Four of the anti-HBe positive samples (1%) were positive for HBV DNA and all of them had normal ALT levels. In serum samples taken before clearance of HBeAg, HBV DNA levels fluctuated considerably in most cases (range = negative to 84,440 pg/ml). Subsequent followup data were available for 437 serum specimens from 177 patients to determine whether clearance of HBeAg occurred within the following year. The remaining 10 patients who cleared HBeAg during the follow-up period did not have available serum samples during the year preceding the clearance of HBeAg. A low serum HBV DNA level was found to correlate with an imminent clearance of HBeAg, occurring within the followingyear in 13 of 20 (65%) cases with low levels of HBV DNA ( 5 1,000 pg/ml) but in only 30 of 157 (19%) cases with HBV DNA levels higher than 1,000 pg/ml (x2 = 20.3, p < 0.0001). However, no consistent level of HBV DNA existed that would allow prediction with certainty of an imminent clearance of HBeAg. Indeed, among 31 cases with HBeAg( + YHBV DNA( - sera, 16 (52%) cases cleared HBeAg within the following year, whereas 15 (48%) cases did not. Elevated serum aminotransferase levels were frequently observed before clearance of HBeAg (Table 2). Approximately half of children with serum ALT levels higher than 40 I U L and two thirds of those with serum ALT levels higher than 100 IU/L cleared HBeAg within the next year. The concurrent analysis of both HBV DNA and ALT levels led to a better prediction of ensuing clearance of HBeAg (Table 3). Thus, all five children with an ALT value higher than 100 IUD, and HBV DNA value less than 1,000 pg/ml lost HBeAg within the following year. Among the 53 children who lost HBeAg and HBV DNA during follow-up, only nine cases ( 17%) did not have an

TABLE 3. Relationship between serum HBV DNA levels, serum ALT levels and the probability of subsequent HBeAg clearance in HBeAg-positive carrier children HJ3V DNA (p@’ml) and peak ALT (NL) level

HBV DNA > 1,000 ALT 5100 HBV DNA 5 1,000 ALT 1100 HBV DNA > 1,000 ALT > l o 0 HBV DNA I1,000 ALT > l o 0 TOTAL

Total

Number

number

seroconverting (%)

139

19 (14)

14

8 (57)

19

11(58)

5

5 (100)

177

2 x 4 contingency table, x 2 = 44.0, p

43 (24) i0.0001

identified period of abnormal serum ALT levels. For the remaining 44 cases, abnormal serum ALT levels were found before seroconversion and none of the values fell to normal before loss of both serum HBeAg and HBV DNA. Serum ALT levels fell to normal with clearance of both HBeAg and HBV DNA in 33 (75%)children but remained elevated in the remaining 11 (25%) cases after seroconversion. Among the latter 11 cases, 8 did not have normal serum ALT levels until an average of 4.1 t- 2.6 (1.7 to 9.8) mo of follow-upafter loss of HBeAg and HBV DNA. The three remaining children still had abnormal serum ALT levels when last seen. Antibody to hepatitis D virus was tested in serial serum samples of 185 cases, and all were negative. DISCUSSION

In children with chronic HBV infection and both HBeAg and HBV DNA in serum, the accompanyingliver disease is usually mild (3,4). In this study, only 41%of such children had abnormal ALT levels, most of which were < 100 IU/L. Thus in this phase, mild continuing liver inflammatory activity occurs. Later in the course of chronic hepatitis B, by way of as yet undefined factors that stimulate hepatitic activity or host immune responses, virus replication may be suppressed, leading to clearance of HBeAg and HBV DNA. This transitional phase is marked by the finding of high ALT and low serum HBV DNA levels with subsequent clearance of HBeAg within the year. This observation is consistent with the hypothesis that liver damage occurs because of clearance of hepatocytes supporting HBV replication rather than because HBV is directly cytopathic (4, 14, 15). Although serum HBV DNA levels tended to decline progressively before loss of HBeAg, a low HBV DNA level was not a reliable indicator for an imminent clearance of H h A g in the children studied here. Many children continued to have HBV DNA in serum during subsequent follow-up. It has been suggested that the finding of HBeAg without detectable HBV DNA in serum is a marker of imminent seroconversion from HBeAg to anti-HBe (4, 16, 17). In this study, however,

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LEE ET AL.

only 52% of children with HBeAg, but no HBV DNA in serum, cleared HBeAg during the subsequent year. Among the children studied here, elevated serum ALT levels were better markers than low serum HBV DNA levels in predicting loss of HBeAg. As shown in Table 2, more than 90% of children with ALT levels greater than 200 I U L lost HBeAg within the next year. Of course, most children had only mildly elevated serum ALT levels, in contrast to adults with chronic HBV and HBeAg, who frequently have serum ALT values above 200 I U L (14,18,19). These data are not only useful for predicting the clinical outcome for children, but also for providing important guidelines for therapy. After loss of HBeAg, both HBeAg and HBV DNA usually remained negative during follow-up. However, some patients with anti-HBe have cirrhosis or develop HCC (3,4). In this study, only 11%of children with anti-HBe and no HBV DNA had abnormal ALT levels, compared with 41% before seroconversion. Lok and Lai (20) have reported that no significant difference existed between the ALT levels in children with HBeAg and those of children with anti-HBe. The differences in results in these two studies of Asian children may be caused by sample size and duration of follow-up. In some cases, serum HBV DNA became positive transiently after loss of HBeAg. Four such occurrences were detected in this study, and none were associated with abnormal ALT values. These findings corroborate those from previous studies in Asian children with HBV (20) and differ from findings in adults in whom episodes of active virus replication after loss of HBeAg are frequently accompanied by severe abnormalities in liver function tests and liver histological findings (4, 16, 17, 21, 22). In conclusion, testing for HBeAg and HBV DNA in serum sheds light on the natural history of chronic hepatitis B in children. High levels of serum HBV DNA are usually present with HBeAg and mildly elevated serum enzymes in the early stages of disease. Progressive decline in serum HBV DNA levels and abnormal liver function tests are frequently noted in latter phases as seroconversion from HBeAg to anti-HBe occurs.After loss of HBeAg and HBV DNA, the liver disease improves and serum aminotransferases usually fall into the normal range. Further, long-term follow-up studies of these children are needed to delineate whether these early serological changes predict ultimate outcome and development of cirrhosis and HCC.

HEPATOLOGY

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