October 1977 The Journal o f P E D I A T R I C S
Hepatitis B e antigen and antibody activity in hepatitis B virus infection Groups of institutionalized subjects, volunteer blood donors with serologic evidence of asymptomatic hepatitis B virus infection, and patients with acute type B viral hepatitis were studied for the presence of hepatitis B e antigen and antibody in a sequential manner over a period of two to three years. HBeAg was detected in 9.5% o f institutionalized residents and volunteer blood donors and in 24% of patients with acute type B viral hepatitis. HBeAg positive subjects frequently had persistently elevated pyruvic glutamic transaminase levels in the serum. Anti HB e activity was observed in 26 to 32% of subjects positive for hepatitis B surface antigen. Long-term follow-up indicated that HB~tg positive subjects with anti HB e did not eventually become seronegative for HBsAg.
M. L. Tiku, M.D., G. M. Makhdoomi, M.D., K. R. Beutner, Ph.D., N. N a t h , Ph.D., and P, L. O g r a , M . D . , * B u f f a l o , N . Y .
INFECTION with hepatitis B virus may be associated infrequently with the development of a chronic carrier state for hepatitis B surface antigen in the serum, with or without clinical or biochemical evidence of active liver disease. Such carriers characteristically have persistent antibody activity against hepatitis B core antigen? detectable HB~Ag and D N A polymerase activity,2 absence of detectable antibody activity against HBsAg 3 and presence of hepatitis B e antigen in the serum? The detection of HB,Ag in HB~Ag-positive subjects has been suggested as a strong indication of infectivity in transfused blood ~ or blood products, and of the potential for fetomaternal transmission of HBV infection. 6 On the other hand, antibody to HB~Ag has been associated with the asymptomatic HB~Ag carrier state, ~ lack of infectivity in some HB~Ag containing serar and protection against fetomaternal transmission.* From the Departments of Pediatrics and Microbiology, State University of New York at Buffalo, Blood Research Laboratory, American Red Cross, and Division of Infectious Disease and Virology, Children's Hospital of Buffalo. Supported in part by research grants and contracts (A132522) (HDlO088) (A142511) (AM17050) from the National Institutes of Health. *Reprint address: Division of Infectious Disease and Virology, Children's Hospital, 219 Bryant St., Buffalo, NY 14222.
Vol. 91, No. 4, pp. 540-544
The present studies were undertaken to characterize the nature and distribution of HB~Ag and anti-HB0 in different forms Of HBV infection. An attempt was also made to study the temporal nature of HB~Ag and antiHB~ activity in the serum relative to the presence and absence of HBoAg and DNA polymerase activity in patients with HBV infection.
See related article, p..545.
Abbreviations used HBV: hepatitis B virus hepatitis B e antigen HBeAg: anti-HB~: antibody to hepatitis B e antigen HB~Ag: hepatitis B surface antigen HBcAg: antibody to hepatitis B core antigen anti-HB~: antibody to hepatitis B surface antigen SGPT: serum pyruvic glutamic transaminase DN A: deoxyribonucleic acid
MATERIALS AND METHODS Population groups (Table I). Group 1 consisted of 226 residents of an institution for mentally handicapped, including 174 subjects who were found to be HB~Ag positive, 35 subjects positive for anti-HBs but seronegative for HBsAg, and 17 subjects who were seronegative for
Volume 91 Number 4
both HB,Ag and anti-HB,. Group 2 comprised 105 volunteer blood donors who were found to be positive for HB~Ag. Group 3 included 46 subjects with acute type B viral hepatitis. The subjects in Group 1 were recruited from a population of mentally handicapped residents who are being followed in an on-going hepatitis type B epidemiologic study at this institute. These subjects ranged in age from 6 months to 60 years (mean 17.5 +_ 12.5 years). All HB~Agpositive subjects included in this group had persistence of HB~Ag in the serum for a period of six months or longer (without any clinical evidence of active liver disease or jaundice). Subjects positive for anti-HB~ and seronegative subjects included in Group 1 had no detectable HB~Ag activity in the serum for at least 6 to 12 months prior to the initiation of this study. These subjects ranged in age from 6 months to 68 years (mean 18 _+ 11 years). Group 2 subjects were recruited from the blood bank services of the regional Red Cross at Buffalo. None of the HB~Agpositive donors provided evidence of recent icteric hepatitis. These subjects ranged from 25 to 40 years in age (mean 28 _+ 7 years). Group 3 subjects were recruited from the medical services of local hospitals. Most subjects in this group were young children from 5 to 14 years in age (mean 9 _+ 5 years). Specimens. Sequential samples of serum were collected from Group 1 subjects at 4 to 6 monthly intervals. Single specimens of serum collected at the time of blood donations were available in Group 2 subjects. Single serum specimens collected at the height of jaundice or clinical disease were tested in Group 3 subjects. All samples were tested for HB~Ag, anti-HB~, HBoAg, and anti-HB,,, and selected samples were tested for serum pyruvic glutamic transaminase and deoxyribonucleic acid polymerase activity. Hepatitis testing. HB~Ag activity in the samples was determined by solid phase radioimmunoassay using Ausria II system obtained commercially? All specimens found to be positive for HB~Ag were subsequently confirmed by specific blocking procedures using human anti-HB~. The anti-HB~ activity was studied by passive hemagglutination technique using commercially available reagents. ~ The HB,Ag and anti HB, activity was determined by rheophoresis as described previously. '~ All specimens were tested with a single reference antigen or antisera provided by the CDC Hepatitis Laboratory at Phoenix, Arizona. DNA Polymerase Determination. The determination of DNA polymerase was performed in unconcentrated, aseptically collected or quick frozen ( 7 0 ~ stored serum samples by the method of Kaplan and associates, ~ as described previously.
HB~AG in H B V infection
Table I. Population groups studied for the distribution of hepatitis B e antigen (HB~Ag) and antibody (anti-HBe)
1 2 3
Asymptomatic institutionalized residents Asymptomatic volunteer blood donors Symptomatic acute type B viral hepatitis Totals
+Present; absent. SGPT levels. The activity of SGPT was determined by standard kinetic assay and the values expressed in international units at 30~ A value of over 30 IU is regarded as abnormal for all epidemiologic and clinical studies in this laboratory. RESULTS
Distribution of HB~Ag and anti HB~ in HB~Ag-positive subjects. The frequency with which HBeAg or anti-HB~ activity was detected in the three population groups is presented in Table II. Among the 174 asymptomatic HBsAg-positive institutionalized residents (Group 1) the HB~Ag was detected in 9.7% and anti-HB~ in 31%. Those subjects who were HBoAg positive were found to be younger children, with an average age of about 10 years. On the other hand, most subjects who were anti-HB, positive but negative for HBeAg appeared to be older; their average age was about 20.5 years. Of the 35 residents in Group 1 who were anti-HB~ positive but HB~Ag negative, two subjects had anti-HB~ activity; HBeAg was not detected in any of these residents. The control population of 17 HBV seronegative subjects included in Group 1 had no HB~Ag or anti-HBe activity (Table II). The frequency of HBeAg and anti-HBe in HB,Ag positive asymptomatic volunteer blood donors (Group 2) was remarkably similar to that observed in HB~Ag posi= tive residents in Group 1 (Table II). In fact, although the proportion of HBeAg and anti-HB~-positive residents in the three population groups varied somewhat, these differences did not appear to be statistically significant. Relationship to DNA polymerase activity. DNA polymerase activity was determined in 12 HBoAg positive, 20 anti-HB~ positive, and five HBeAg anti-HB~ seronegative
Tiku et al.
The Journal of Pediatrics October 1977
Table II. Distribution of hepatitis B e antigen (HBoAg) and antibody (anti-HB,) activity in
different populations with hepatitis B infection and in control subjects
No. subjects tested
174 35 17 105 46
1. Asymptomatic institutionalized residents HB~Ag+ HB~Ag--, anti-HB~+ HB~Ag-, anti-HB~2. Asymptomatic HB~Ag+ volunteer blood donors 3. Symptomatic acute type B hepatitis HB~Ag+
Table Ill. Relationship of DNA polymerase activity to the
status of hepatitis B e antigen (HB~Ag) and antibody (anti-HB~), and HB~Ag in the serum No. with DNA polymerase activity in serum
HB~Ag+ Anti-HB~+ HB~Ag-Anti
12 20 5
12 20 3
0 0 2
6 0 0
6 20 5
*Considered positive when counts per minute were 3 SD above the controls.
subjects from Group 1. Although half of the subjects positive for HB~Ag and HBeAg had DNA polymerase activity, no anti-HB, positive and HBoAg anti-HB, negative subjects who were positive for HB~Ag or anti-HB~ had any DNA polymerase activity (Table III). Temporal changes in HB~Ag status relative to HBeAg or anti-HBo activity in the serum. The HB~Ag positive and
anti-HB~ positive asymptomatic residents in Group 1 (Table II) were retested at four to six monthly intervals to detect any changes in the status of HB~Ag, anti-HBo, HB~Ag, and anti-HB~. All patients initially positive for 9HB~Ag or anti-HB~ have continued to remain so for the follow-up period of two to three years. However, certain changes in the status of HB~Ag and anti-HB~ were apparent on follow-up testing (Table IV). One subject who was initially found to be positive for HB~Ag and HB~Ag had lost the HB,Ag activity one and one-half to two years later; the remaining 16 of the 17 HBsAg a n d
HBoAg anti-HB~ negative
17 0 0 10
9.7 0 0 9.5
54 2 0 28
31.0 5.7 0 26.7
103 33 17 67
59.3 94.3 100.0 63.8
HBeAg-positive residents had no change in the preexisting status for HBsAg or anti-HB~. Of the 56 subjects who had anti-HBe activity, four initially positive for HBsAg and two residents positive for anti-HB~ lost HB~Ag and anti-HBs activity in the follow-up testing. Almost all HB~Ag-positive residents with anti HBe activity continued to remain positive for HB~Ag throughout the period of observation. The follow-up serologic changes observed in HBeAg and anti-HBe seronegative subjects were similar to those observed in anti-HB0-positive subjects. Approximately 85% of subjects had no change in the HB~Ag or anti-HB~ status, though 14.5% of HB~Ag-positive subjects lost HBsAg and about 18% of anti-HBs-positive subjects lost the anti-HBs activity in the follow-up testing. Relationship of SGPT levels to HBoAg status. SGPT levels were determined on serial samples of serum collected four to six months apart over a period of two to three years in 15 HBeAg positive, 43 anti-HBe positive, and 67 HB~Ag and anti-HBe seronegative subjects in Group 1. Most HBoAg and anti-HB~-negative or antiHB~-positive residents had normal or intermittently elevated SGPT levels. Only 15% of HB~Ag and anti-HBe seronegative subjects, and 6% of anti-HB~ positive subjects had persistentlyelevated SGPT levels (Table V), On the other hand, 46.7% of HBoAg-positive subjects had persistently elevated SGPT activity, with levels of over 50 to 100 IU observed at every testing; normal SGPT levels (below 30 IU) were observed at every testing in only 6% of such subjects. DISCUSSION The observations reported here suggest that the frequency with which HB~Ag and anti-HB~ are detected in HB~Ag-positive patients can range from 9 to 24% and 5 to 31%, respectively. Most anti-HBe-positive subjects who were initially HB~Ag positive failed to clear the antigen
Volume 91 Number 4
HB~AG in H B V infection
Table IV. Relationship of hepatitis B e antigen (HB.Ag) and antibody (anti-HB~) to the status of HB~Ag or anti-HB~ in the serum after 2 to 3 years of antibody follow-up testing
Follow-up testing (No./% subjects) Initial testing No. e status
HB~Ag+ Anti-HB, + HB~Ag-Anti-HBo--
17 56 136
No. No. HB~Ag+ Anti-HB~ + 17 54 103
0 2 33
after 2 to 3 years of follow-up, and HB~Ag-positive subjects who possessed HBeAg had persistently or intermittently elevated SGPT activity. In previously published studies, the prevalence of HBeAg in HB~Ag-positive subjects has ranged from 23 to 30% in institutionalized residents, H 6 to 14% for volunteer blood donors 1~ ~:~and approximately 10% in subjects with acute type B viral hepatitis. TM Our observations suggest a lower prevalence (9.7%) of HBeAg in institutionalized populations and higher prevalence (24.5%) in patients with acute icteric type B hepatitis. In our studies most HBsAg-positive subjects who were found to be positive for HBoAg were younger children, particularly those under ten years of age. These differences may be related to such selection factors as age, institutional setting, interval between acquisition of HBV infection, and testing for HB~Ag. This possibility is supported by the observation of generally lower prevalence of HBsAg positivity (9%) in this institutionalized population compared to a reported prevalence of 23 to 30% in similar institutions elsewhere in the United States. The prevalence of anti-HBe activity in HB~Ag-positive subjects has ranged from 15 to 23% in some studies ~2' ~ to 30 to 73% in others? ~' ~' The present data also indicate minor variability in the detection of anti-HB, in different forms of HBV infection. DNA polymerase activity was observed in only 50% of our HBoAg-positive HB~Ag-positive subjects; in other studies the degree of positive correlation between HB,Ag and DNA polymerase activity has been reported to be between 50 and 100%?" ~:~'TM It has been suggested that the presence of HBoAg in the serum may indicate active liver disease'". ~'. TM ~, ~; its detection has also been associated with infectivity of serum containing HBsAg. On the other hand, the development of anti-HB, activity has been proposed to be an indication of the clearance of HB~Ag and the restoration of normal liver function.TM TM Contrary to these reports, over 90% of anti-HBe positive subjects in the present study
Became positive HB~Ag I Anti-HB~ 0 0 0
0 0 0
1/5.9 4/7.4 15/14.5
0 2/100 6/18.2
16/94.1 50/92.6 88/85.5
Table V. Hepatitis B e antigen (HB~Ag) status and elevated levels of SGPT in HBsAg-positive subjects
No. subjects tested
No./% subjects SGPT levels elevated
Persistent* ] Intermittentt
3/6.9 82w 7/46.782w
*SGPT level over 30 IU (frequently over 100 IU) observed at every testing. tSGPT level over 30 1U observed at least on one testing. :~SGPT level below 30 IU observed at every testing. w vs H B ~ A g - anti-Hb,, : P < 0.006. HAnti-HB,+ vs HB~Ag anti-HBo: not significant. 82HB~Ag+ vs anti-HB.: P < 0.0004.
remained HB~Ag-positive during a continuous follow-up period of over two to three years. These observations do not suggest a protective role of anti-HBo against the development of a chronic HBsAg carrier state. Although immunohistologic assessment of liver damage in HB,Agpositive subjects was not possible in these patients, biochemical evidence of active liver disease was present in many HBsAg-positive subjects who also had HBeAg. We thank Mr. Bert Murphy and Dr. J. E. Maynard of the Center for Disease Control, Phoenix, Ariz., for the help rendered to us in HBeAg/anti-HB, testing. We also thank Pat Milkowski SUNYAB School of Medicine for assisting us in the statistical analysis of data. REFERENCES
1. Hoofnagle JH, Gerety RJ, Ni LY, and Barker LF: Antibody to hepatitis B core antigen: A sensitive indicator of hepatitis B virus replication, N Engl J Med 290:1336, 1974. 2. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, and Robinson WS: DNA polymerase associated with human hepatitis B antigen, J Virol 11:995, 1973.
Tiku et aL
3. Vyas GN, Roberts I, Paterson DL, and Holland PL: Nonspecific test reactions for antibodies to hepatitis B surface antigen in chronic HB~Ag carrier, Abst. No. 1155, presented at 60th Annual Meeting of Federation of American Societies for Experimental Biology, Anaheim, Calif., April 11-16, 1976. 4. Magnius LO, Lindholm A, Lundin P, and Iwarson S: A new antigen-antibody system: Clinical significance in long-term carriers of hepatitis B surface antigen, JAMA 231:356, 1975~ 5. Alter H J, Seeff LB, Kaplan PM, McAuliffe V J, Wright EC, Gerin JL, Purcell RH, Holland PV, and Zimmerman HJ: Type B hepatitis: The infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure, N Engl J Med 295:909, 1976. 6. Okada K, Kamiyama I, Inomata M, Imai M, Miyakawa Y, and Mayumi M: e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants, N Engl J Med 294:746, 1976. 7. Sheikh NE, Woolf IL, Galbraith RM, Feddleston ALW, Dymock IW, and Williams R: e antigen-antibody system as indicator of liver damage in patients with hepatitis B antigen, Br Med J 4:252, 1975. 8. Ling CM, and Overby LR: Prevalence of hepatitis B virus antigen as revealed by direct radioimmunoassay with 1~I antibody, J Immunol 109:834, 1972. 9. Vyas GN, and Shulman NR: Hemagglutination assay for antigen and antibody associated with viral hepatitis, Science 170:332, 1970. 10. Smith JL, Murphy BL, Auslander MO, Maynard JE, Schalm SS, Summerskill WHJ, and Gitnick GL: Studies of the "e" antigen in acute and chronic hepatitis, Gastroenterology 71:208, 1976.
The Journal of Pediatrics October 1977
11. Hindman SH, Gravelle CR, Murphy BL, Bradley DW, Budge WR, and Maynard JE: "e" antigen, Dane particles and serum DNA polymerase activity in HB~Ag carriers, Ann Intern Med 85:458, 1976. 12. Takahashi K, Imai M, Tsuda F, Takahashi T, Miyakawa Y, and Mayumi M: Association of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis B surface antigen, J lmmunol 117:102, 1976. 13. Imai M, Tachibana FC, Moritsugu Y, Miyakawa Y, and Mayumi M: Hepatitis B antigen-associated deoxyribonucleic acid polymerase activity and e antigen/anti-e system, Infect Immun 14:631, 1976. 14. Nielsen JO, Dietrichson O, and Juhl E: Incidence and meaning of the "e" determinant among hepatitis B antigen positive patients with acute and chronic liver diseases, Lancet 2:913, 1974. 15. Trepo CG, Magnius LO, Schaefer RA, and Prince AM: Detection of e antigen and antibody: Correlations with hepatitis B surface and hepatitis B core antigens, liver disease, and outcome in hepatitis B infections, Gastroenterology 71:804, 1976. 16. Nordenfelt E, and Andren-Sandberg M: Dane particle associated DNA polymerase and e antigen: Relation to chronic hepatitis among, carriers of hepatitis B surface antigen, J Infect Dis 134:85, 1976. 17. McAuliffe VJ, Purcell RH, and LeBouvier GL: e: A third hepatitis B antigen? N Engl J Med 294"779, 1976. 18. Feinman SV, Berris B, and Sinclair JC: e antigen and anti-e in HBsAg carriers, Lancet 2:1173, 1975. 19. Eleftheriou N, Thomas HC, Heathcote J, and Sherlock S: Incidence and clinical significance of e antigen and antibody in acute and chronic liver disease, Lancet 2:1171, 1975.