BRIEF COMMUNICATIONS
"e" Antigen, Dane Particles, and Serum DNA Polymerase Activity in HBsAg Carriers STEPHEN H. HINDMAN, M.D.; CLIFTON R. GRAVELLE, M.S.; BERT L. MURPHY, M.S.; DANIEL W. BRADLEY, Ph.D.; WILLIAM R. BUDGE, M.D.; and JAMES E. MAYNARD, M.D., Ph.D.; Phoenix, Arizona
Sera of 103 carriers of hepatitis B surface antigen were assayed for e-antigen and anti-e. Twenty-four were e-antigen-positivef 3 1 anti-e-positivef and 4 8 had neither detectable (e-negative). Aminotransferases were elevated in 7 5 % of the e-antigen-positive carriers compared with 2 5 % of e-negative carriers (P < 0.001) and 1 3 % of anti-e-positive carriers (P < 0.001). Serum DNA polymerase activity was significantly higher in the e-antigen-positive carriers than in carriers without e-antigen. Dane particles were shown in 10 of 12 carriers with e-antigen, compared with one of 12 e-negative carriers (P < 0.0003) and none of 12 anti-e-positive carriers (P < 0.00003). These results suggest that ongoing hepatitis B viral replication is more active in e-antigen-positive carriers than in carriers without e-antigen, a finding that may help explain the high prevalence of chronic active hepatitis described in these individuals.
T H E IMMUNOLOGIC HETEROGENICITY of hepatitis B surface
antigen (HB s Ag) is well established ( 1 , 2 ) . Although the various subtypes of HB s Ag (adw, ayw, adr) have proved to be useful epidemiologic markers, they have been of little clinical value. In 1972, Magnius and Espmark (3) described a new antigen-antibody system in chronic HB s Ag carriers using Ouchterlony double immunodiffusion, which they designated the " e " system. Although found only in patients with hepatitis B virus (HBV) infection, e-antigen is immunologically and physiochemically distinct from HB s Ag. Controversy exists as to whether it is an antigenic component of the Dane particle, the putative HBV, or a soluble host protein released in response to HBV infection. Several investigations now suggests that the presence of e-antigen in serum may have prognostic implications for patients with type B hepatitis (4-9). The detection of eantigen has been associated with chronic active hepatitis • From the Phoenix Laboratories Division, Bureau of Epidemiology, Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare; Phoenix, Arizona.
458
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and cirrhosis and appears to indicate a poor prognosis. Conversely, the presence of e-antibody (anti-e), which has been found most often in healthy HB s Ag carriers, appears to indicate a more favorable outcome. The presence of eantigen has also been implicated in the infectivity of HB s Ag carriers (9-11). We report here our attempt to further define the clinical relation of the " e " system to enzymatic evidence of active liver disease in a large group of chronic HB s Ag carriers. In addition, tests of DNA polymerase activity (an index of HBV replication) and Dane particle content of serum were made to investigate the relation of this antigen-antibody system with HBV replication. Patients and Methods
In the course of a serologic survey for HBV infection among residents of a large Midwestern state mental institution conducted between January and April 1974, 468 (33%) of the 1416 individuals surveyed had serologic evidence of past or current HBV infection (136 were HBsAg-positive and 332 had antibody to HB s Ag). One-hundred-sixteen of the 136 HBsAgpositive individuals were available for follow-up examinations that we conducted in June 1975 and, thus, represent the present study population. This population comprised 27 female patients and 89 male patients who were a mean age of 25.5 years (range, 5 to 63); 38 of the persons had Down's syndrome and the rest cerebral palsy or another form of mental retardation. Eleven of the 116 had a history of previous acute hepatitis occurring from 16 years to 1 month before their initial examination in 1974. In June 1975, none had evidence of cirrhosis or stigmata of liver failure. At that time all 116 were rebled for simultaneous but independent measurements of HBsAg and antibody against HBsAg (anti-HBs), e-antigen and anti-e, serum aspartate and alanine aminotransferase (serum glutamic oxalacetic transaminase [SGOT], serum glutamic pyruvic transaminase [SGPT]), and DNA polymerase activities. Tests of HBsAg and anti-HBs content of serum were made by solid-phase radioimmunoassay techniques using commercial reagents supplied by Abbott Laboratories; North Chicago, Illinois. Measurement of e-antigen and anti-e were done using rheophoresis plates (Abbott Laboratories) as previously described (5). Measurements of SGOT and SGPT were made by colorimetric methods using reagents provided by Sigma Chemicals (Saint Louis, Missouri) with results expressed in sigma units. The DNA polymerase activity in the serum specimens Annals of Internal Medicine 85:458-460, 1976
was measured by a standard radioisotope method (12), as described in detail elsewhere (13), with results expressed in counts per minute. The persistent HBsAg-positive individuals (103) from this population were divided into three groups according to the presence or absence of e-antigen or anti-e for comparisons of their aminotransferase and DNA polymerase levels. These same measurements were made in 13 persons who had seroconverted to anti-HBs-positive and the results used as control values for the above three groups. In addition, 12 serum specimens from each of the three groups were selected randomly and submitted under code for immune electron microscopic examination for Dane particles by a method similar to that described by Dane, Cameron, and Briggs (14). Statistical analysis was done by use of the chi square with Yates' correction, Fisher's exact test, and Cochran's modification of the ordinary /-test unless otherwise specified. Results
Of the 116 individuals in the study, 103 were still HB s Ag-positive when re tested more than 1 year later; the other 13 had become anti-HB s -positive. The mean SGOT and SGPT levels of the HB s Ag carriers (Table 1) were relatively low but were significantly higher (P < 0.01) than the mean levels of the anti-HB s -positive controls. The mean DNA polymerase activity of the carriers was also statistically higher than that of the controls (P < 0.05). Of the 103 HB s Ag carriers, the serum of 24 ( 2 3 % ) was found to be e-antigen-positive, 31 ( 3 0 % ) were anti-epositive, and 48 ( 4 7 % ) had neither detectable (e-negative). Serum of two of the 13 seroconverters contained anti-e, whereas none contained e-antigen. The distribution of e-antigen and anti-e in the 38 carriers with Down's syndrome did not differ significantly from the population as a whole; six ( 1 6 % ) were e-antigen-positive, seven ( 1 8 % ) were anti-e-positive, and 25 ( 6 6 % ) were e-negative. The mean SGOT and SGPT levels of the e-antigen-positive carriers (Table 2) were clearly elevated and were significantly higher than those of both the e-negative group ( P < 0 . 0 1 ) and the anti-e-positive group (P < 0.0025). Eighteen ( 7 5 % ) of the e-antigen-positive carriers had enzyme elevations (SGOT, > 40; SGPT, > 35) compared with 12 ( 2 5 % ) of the e-negative carriers (P < 0.001) and four ( 1 3 % ) of the anti-e-positive carriers (P < 0.001). Although the anti-e-positive carriers had the lowest mean enzyme levels of the three groups, these levels were still statistically higher than the mean enzyme levels of the controls (P < 0.025). The mean DNA polymerase activity of the e-antigenpositive carriers (Table 2) was significantly higher than Table 1. Mean Levels of Aminotransferases and DNA Polymerase in HBsAg Carriers and Anti-HBs-Positive Control Subjects*
HBV Status
HBsAg-positive Anti-HBs-positive
No.
103 13
Meanf SGOT
Meant SGPT
Mean DNA J Polymerase
± SD
+ SD
± SD
36 ± 2 8 41 ± 50 18 ± 6 13 + 6
379 ± 130 341+50
* HBV = hepatitis B virus, HBsAg = hepatitis B surface antigen, antiHBs = antibody against HBsAg, SGOT = serum glutamic oxalacetic transaminase, SGPT = serum glutamic pyruvic transaminase. t Sigma units. t Counts per minute.
Table 2. Mean Levels of Aminotransferases and DNA Polymerase in HBsAg Carriers by e Antigen-Antibody Status*
e AntigenAntibody Status
No.
Meanf SGOT
e-Antigen-positive e-Negative Anti-e-positive Total
24 48 31 103
53 31 30 36
+ SD
+ 22 +28 + 27 + 28
Meanf SGPT
Mean DNAt Polymerase
+ SD
62 38 31 41
+ ± + +
41 56 47 50
+ SD
484 356 335 379
198 79 80 130
* HBsAg = hepatitis B surface antigen, SGOT = serum glutamic oxalacetic transaminase, SGPT = serum glutamic pyruvic transaminase. t Sigma units. t Counts per minute.
that of the control subjects (P < 0.0005) and that of both the e-negative (P < 0.005) and anti-e-positive carriers (P < 0.0025). The mean DNA polymerase activity of the e-negative and anti-e-positive carriers did not differ statistically from each other or the control subjects. Fourteen ( 5 8 % ) of the e-antigen-positive carriers had DNA polymerase activities greater than 2 standard deviations above the mean of the control subjects compared with 10 ( 2 1 % ) of the e-negative carriers (P < 0.02) and none ( 0 % ) of the anti-e-positive carriers (P < 0.001). In the 24 carriers with elevated DNA polymerase activity, 18 ( 7 5 % ) had concurrent SGOT and SGPT elevations compared with similar enzyme elevations in only 16 (20%) of 79 carriers with normal DNA polymerase activity (P < 0.001). Eleven of the 36 randomly selected sera contained Dane particles, as judged by immune electron microscopy. The number of Dane particles in the positive specimens ranged from 15 to 400 Danes per grid square. The Dane particle counts showed a linear relation (R 2 = 0.779) to serum DNA polymerase activity (Figure 1). This close degree of correlation between serum DNA polymerase activity and Dane particle counts indicate the probability is quite small (P < 0.0005) that other sources of DNA polymerase (that is, host or bacterial DNA polymerase) could account for the significantly higher activities found in the e-antigenpositive carriers. Ten ( 8 3 % ) of 12 e-antigen-positive carriers had detectable Dane particles compared with one ( 8 % ) of 12 e-negative carriers (P < 0.0003) and none ( 0 % ) of 12 anti-e-positive carriers (P < 0.00003). Discussion
In this study, the e-antigen-positive carriers differed significantly from the anti-e-positive and the e-negative carriers in all variables of HBV infection tested. While the anti-e-positive and e-negative carriers did not differ statistically, the values obtained in the e-negative group were intermediate between the e-antigen-positive and anti-e-positive carriers. This result suggests that, if more sensitive tests were available, the e-negative group would be found to consist of a mixture of e-antigen and anti-e-positive persons. The significantly higher prevalence of hepatic enzyme elevations in the e-antigen-positive carriers is consistent with similar reported observations (4-9), but differs slightly in that a sizeable percentage ( 2 5 % ) had normal enzyme values. Although, the anti-e-positive carriers had the lowest prevalence of enzyme abnormalities of any of the carriers, Hindman et a\. • " e " Antigen in Hepatitis Carriers
Downloaded from https://annals.org by Tulane University user on 01/02/2019
+ + + +
459
with hepatitis B who are most likely both to develop active forms of liver disease and to be disseminators of infection. However, caution should be used in ascribing a uniformly favorable prognosis to patients with anti-e and any conclusions concerning the infectivity or lack of infectivity of anti-e-positive carriers must await more definitive studies. Finally more sensitive tests for detecting e-antigen and anti-e are needed to more definitively determine the role of the "e" system in hepatitis B infection. ACKNOWLEDGMENTS: Received 29 March 1976; revision accepted 22 July 1976. • Requests for reprints should be addressed to Stephen H. Hindman, M.D.; Phoenix Laboratories Division, Center for Disease Control; 4402 North Seventh Street; Phoenix, AZ 85014. Figure 1 . Scatter diagram of Dane particle counts by immune electron microscopy versus serum DNA polymerase activity. (Equation of best fitting regression line: Y = 0.40 X-136; correlation index of best-fitting line = 0.779; standard error of estimate = 39.7; dotted lines represent 9 5 % upper and lower confidence limits.)
References 1. L E BOUVIER GL: The heterogenicity of Australia antigen. / Infect Dis 123:671-675, 1971 2. BANCROFT WH, MUNDON FK, RUSSELL PK: Detection of addi-
tional antigenic determinants of the hepatitis B antigen. J Immunol 109:842-848, 1972 3. MAGNIUS LO, ESPMARK JA: New specificities in Australia antigen
the fact that their mean enzyme levels were significantly higher than the controls and that 13% had elevated enzymes suggests the presence of anti-e does not automatically confer a favorable prognosis. The finding that 8 3 % of the e-antigen-positive sera had detectable Dane particles by immune electron microscopy is consistent with the findings of Sheikh (15) and Nordenfelt (16) who reported the presence of Dane particles in 76% and 100%, respectively, of their e-antigen-positive carriers. Contrary to our results, Sheikh found Dane particles in five of 27 anti-e-positive carriers. The discrepancy between these observations may be due in part to the smaller sample size in our study. Nevertheless, these studies have shown a highly significant association between the presence of e-antigen in serum and the ability to detect circulating Dane particles by immune electron microscopy. The finding that the e-antigen-positive carriers were the only group that had significantly elevated mean D N A polymerase activities indicates that its presence in serum is associated with active and efficient HBV replication. The lower D N A polymerase activities found in the anti-e-positive carriers implies that this antibody is associated with less active HBV replication. These differences in the activity of HBV replication may be responsible for the significant clinical differences in the activity of liver disease shown between e-antigen-positive and anti-e-positive HB s Ag carriers. If, as previously suggested ( 1 7 ) , e-antigen is an antigenic component of the Dane particle, the association of anti-e with reduced HBV replication may be a causal one. From a clinical standpoint, screening for e-antigen and anti-e appears to be of value in identifying those patients
460
positive sera distinct from the Le Bouvier determinants. J Immunol 109:1017-1021, 1972 4. MAGNIUS LO: Characterization of a new antigen-antibody system associated with hepatitis B. Clin Exp Immunol 20:209-216, 1975 5. MAYNARD JE, BARRETT DH, MURPHY BL, et al: Relationship of
the e antigen to hepatitis B virus infection in a hyperendemic area. / Infect Dis 133:339-342, 1976 6. NEILSON JO, DIETRICHSON O, JUHL E: Incidence and meaning
of the " e " determinant among hepatitis-B-antigen positive patients with acute and chronic liver diseases. Lancet 2:913-915, 1974 7. ELEFTHERIOU N, THOMAS HC, HEATHCOTE J, et al: Incidence
and clinical significance of e antigen and antibody in acute and chronic liver disease. Lancet 2:1171-1173, 1975 8. FEINMAN SV, BERRIS B, SINCLAIR JC, et al: e antigen and anti-e
in HB s Ag carriers. Lancet 2:1173-1174, 1975 9. MAGNIUS LO, LINDHOLM A, LUNDIN P: A new antigen-antibody
system. Clinical significance in long term carriers of hepatitis B surface antigen. JAMA 231:356-359, 1974 10. OKADA K, KAMIGAMA I, INOMATA M, et al: 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 294:746-749, 1976 11. SKINH0J P, COHN J, BRADBURNE AF: Transmission of hepatitis
type B from healthy HBsAg-positive mothers. Br Med J 1:10-11, 1976 12. ROBINSON WS, ROBINSON HL: DNA polymerase in defective
Rous sarcoma virus. Virology 44:457-462, 1971 13. BRADLEY DW, MURPHY BL, SMITH JL, et al: Naturally occurring
antibody against unusually high serum DNA polymerase activity. Nature 259:594-596, 1976 14. DANE DS, CAMERON CH, BRIGGS M: Virus-like particles in serum
of patients with Australia-antigen-associated hepatitis. Lancet 1:695-698, 1970 15. SHEIKH N, WOOLF IL, GALBRAITH RM, et al: e antigen-antibody
system as indicator of liver damage in patients with hepatitis-B antigen. Br Med J 4:252-253, 1975 16. NORDENFELT E, KJELLEN L: Dane particles, DNA polymerase,
and e-antigen in two different categories of hepatitis B antigen carriers, lntervirology 5:225-232, 1975 17. NEURATH AR, TREPO C, CHEN M, et al: Identification of addi-
October 1976 • Annals of Internal Medicine • Volume 85 • Number 4
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tional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. / Gen Virol 30:277-285, 1976
"e" Antigen, Dane Particles, and Serum DNA Polymerase Activity in HBsAg Carriers STEPHEN H. HINDMAN, M.D.; CLIFTON R. GRAVELLE, M.S.; BERT L. MURPHY, M.S.; DANIEL W. BRADLEY, Ph.D.; WILLIAM R. BUDGE, M.D.; and JAMES E. MAYNARD, M.D., Ph.D.; Phoenix, Arizona
Sera of 103 carriers of hepatitis B surface antigen were assayed for e-antigen and anti-e. Twenty-four were e-antigen-positivef 3 1 anti-e-positivef and 4 8 had neither detectable (e-negative). Aminotransferases were elevated in 7 5 % of the e-antigen-positive carriers compared with 2 5 % of e-negative carriers (P < 0.001) and 1 3 % of anti-e-positive carriers (P < 0.001). Serum DNA polymerase activity was significantly higher in the e-antigen-positive carriers than in carriers without e-antigen. Dane particles were shown in 10 of 12 carriers with e-antigen, compared with one of 12 e-negative carriers (P < 0.0003) and none of 12 anti-e-positive carriers (P < 0.00003). These results suggest that ongoing hepatitis B viral replication is more active in e-antigen-positive carriers than in carriers without e-antigen, a finding that may help explain the high prevalence of chronic active hepatitis described in these individuals.
T H E IMMUNOLOGIC HETEROGENICITY of hepatitis B surface
antigen (HB s Ag) is well established ( 1 , 2 ) . Although the various subtypes of HB s Ag (adw, ayw, adr) have proved to be useful epidemiologic markers, they have been of little clinical value. In 1972, Magnius and Espmark (3) described a new antigen-antibody system in chronic HB s Ag carriers using Ouchterlony double immunodiffusion, which they designated the " e " system. Although found only in patients with hepatitis B virus (HBV) infection, e-antigen is immunologically and physiochemically distinct from HB s Ag. Controversy exists as to whether it is an antigenic component of the Dane particle, the putative HBV, or a soluble host protein released in response to HBV infection. Several investigations now suggests that the presence of e-antigen in serum may have prognostic implications for patients with type B hepatitis (4-9). The detection of eantigen has been associated with chronic active hepatitis • From the Phoenix Laboratories Division, Bureau of Epidemiology, Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare; Phoenix, Arizona.
458
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and cirrhosis and appears to indicate a poor prognosis. Conversely, the presence of e-antibody (anti-e), which has been found most often in healthy HB s Ag carriers, appears to indicate a more favorable outcome. The presence of eantigen has also been implicated in the infectivity of HB s Ag carriers (9-11). We report here our attempt to further define the clinical relation of the " e " system to enzymatic evidence of active liver disease in a large group of chronic HB s Ag carriers. In addition, tests of DNA polymerase activity (an index of HBV replication) and Dane particle content of serum were made to investigate the relation of this antigen-antibody system with HBV replication. Patients and Methods
In the course of a serologic survey for HBV infection among residents of a large Midwestern state mental institution conducted between January and April 1974, 468 (33%) of the 1416 individuals surveyed had serologic evidence of past or current HBV infection (136 were HBsAg-positive and 332 had antibody to HB s Ag). One-hundred-sixteen of the 136 HBsAgpositive individuals were available for follow-up examinations that we conducted in June 1975 and, thus, represent the present study population. This population comprised 27 female patients and 89 male patients who were a mean age of 25.5 years (range, 5 to 63); 38 of the persons had Down's syndrome and the rest cerebral palsy or another form of mental retardation. Eleven of the 116 had a history of previous acute hepatitis occurring from 16 years to 1 month before their initial examination in 1974. In June 1975, none had evidence of cirrhosis or stigmata of liver failure. At that time all 116 were rebled for simultaneous but independent measurements of HBsAg and antibody against HBsAg (anti-HBs), e-antigen and anti-e, serum aspartate and alanine aminotransferase (serum glutamic oxalacetic transaminase [SGOT], serum glutamic pyruvic transaminase [SGPT]), and DNA polymerase activities. Tests of HBsAg and anti-HBs content of serum were made by solid-phase radioimmunoassay techniques using commercial reagents supplied by Abbott Laboratories; North Chicago, Illinois. Measurement of e-antigen and anti-e were done using rheophoresis plates (Abbott Laboratories) as previously described (5). Measurements of SGOT and SGPT were made by colorimetric methods using reagents provided by Sigma Chemicals (Saint Louis, Missouri) with results expressed in sigma units. The DNA polymerase activity in the serum specimens Annals of Internal Medicine 85:458-460, 1976
was measured by a standard radioisotope method (12), as described in detail elsewhere (13), with results expressed in counts per minute. The persistent HBsAg-positive individuals (103) from this population were divided into three groups according to the presence or absence of e-antigen or anti-e for comparisons of their aminotransferase and DNA polymerase levels. These same measurements were made in 13 persons who had seroconverted to anti-HBs-positive and the results used as control values for the above three groups. In addition, 12 serum specimens from each of the three groups were selected randomly and submitted under code for immune electron microscopic examination for Dane particles by a method similar to that described by Dane, Cameron, and Briggs (14). Statistical analysis was done by use of the chi square with Yates' correction, Fisher's exact test, and Cochran's modification of the ordinary /-test unless otherwise specified. Results
Of the 116 individuals in the study, 103 were still HB s Ag-positive when re tested more than 1 year later; the other 13 had become anti-HB s -positive. The mean SGOT and SGPT levels of the HB s Ag carriers (Table 1) were relatively low but were significantly higher (P < 0.01) than the mean levels of the anti-HB s -positive controls. The mean DNA polymerase activity of the carriers was also statistically higher than that of the controls (P < 0.05). Of the 103 HB s Ag carriers, the serum of 24 ( 2 3 % ) was found to be e-antigen-positive, 31 ( 3 0 % ) were anti-epositive, and 48 ( 4 7 % ) had neither detectable (e-negative). Serum of two of the 13 seroconverters contained anti-e, whereas none contained e-antigen. The distribution of e-antigen and anti-e in the 38 carriers with Down's syndrome did not differ significantly from the population as a whole; six ( 1 6 % ) were e-antigen-positive, seven ( 1 8 % ) were anti-e-positive, and 25 ( 6 6 % ) were e-negative. The mean SGOT and SGPT levels of the e-antigen-positive carriers (Table 2) were clearly elevated and were significantly higher than those of both the e-negative group ( P < 0 . 0 1 ) and the anti-e-positive group (P < 0.0025). Eighteen ( 7 5 % ) of the e-antigen-positive carriers had enzyme elevations (SGOT, > 40; SGPT, > 35) compared with 12 ( 2 5 % ) of the e-negative carriers (P < 0.001) and four ( 1 3 % ) of the anti-e-positive carriers (P < 0.001). Although the anti-e-positive carriers had the lowest mean enzyme levels of the three groups, these levels were still statistically higher than the mean enzyme levels of the controls (P < 0.025). The mean DNA polymerase activity of the e-antigenpositive carriers (Table 2) was significantly higher than Table 1. Mean Levels of Aminotransferases and DNA Polymerase in HBsAg Carriers and Anti-HBs-Positive Control Subjects*
HBV Status
HBsAg-positive Anti-HBs-positive
No.
103 13
Meanf SGOT
Meant SGPT
Mean DNA J Polymerase
± SD
+ SD
± SD
36 ± 2 8 41 ± 50 18 ± 6 13 + 6
379 ± 130 341+50
* HBV = hepatitis B virus, HBsAg = hepatitis B surface antigen, antiHBs = antibody against HBsAg, SGOT = serum glutamic oxalacetic transaminase, SGPT = serum glutamic pyruvic transaminase. t Sigma units. t Counts per minute.
Table 2. Mean Levels of Aminotransferases and DNA Polymerase in HBsAg Carriers by e Antigen-Antibody Status*
e AntigenAntibody Status
No.
Meanf SGOT
e-Antigen-positive e-Negative Anti-e-positive Total
24 48 31 103
53 31 30 36
+ SD
+ 22 +28 + 27 + 28
Meanf SGPT
Mean DNAt Polymerase
+ SD
62 38 31 41
+ ± + +
41 56 47 50
+ SD
484 356 335 379
198 79 80 130
* HBsAg = hepatitis B surface antigen, SGOT = serum glutamic oxalacetic transaminase, SGPT = serum glutamic pyruvic transaminase. t Sigma units. t Counts per minute.
that of the control subjects (P < 0.0005) and that of both the e-negative (P < 0.005) and anti-e-positive carriers (P < 0.0025). The mean DNA polymerase activity of the e-negative and anti-e-positive carriers did not differ statistically from each other or the control subjects. Fourteen ( 5 8 % ) of the e-antigen-positive carriers had DNA polymerase activities greater than 2 standard deviations above the mean of the control subjects compared with 10 ( 2 1 % ) of the e-negative carriers (P < 0.02) and none ( 0 % ) of the anti-e-positive carriers (P < 0.001). In the 24 carriers with elevated DNA polymerase activity, 18 ( 7 5 % ) had concurrent SGOT and SGPT elevations compared with similar enzyme elevations in only 16 (20%) of 79 carriers with normal DNA polymerase activity (P < 0.001). Eleven of the 36 randomly selected sera contained Dane particles, as judged by immune electron microscopy. The number of Dane particles in the positive specimens ranged from 15 to 400 Danes per grid square. The Dane particle counts showed a linear relation (R 2 = 0.779) to serum DNA polymerase activity (Figure 1). This close degree of correlation between serum DNA polymerase activity and Dane particle counts indicate the probability is quite small (P < 0.0005) that other sources of DNA polymerase (that is, host or bacterial DNA polymerase) could account for the significantly higher activities found in the e-antigenpositive carriers. Ten ( 8 3 % ) of 12 e-antigen-positive carriers had detectable Dane particles compared with one ( 8 % ) of 12 e-negative carriers (P < 0.0003) and none ( 0 % ) of 12 anti-e-positive carriers (P < 0.00003). Discussion
In this study, the e-antigen-positive carriers differed significantly from the anti-e-positive and the e-negative carriers in all variables of HBV infection tested. While the anti-e-positive and e-negative carriers did not differ statistically, the values obtained in the e-negative group were intermediate between the e-antigen-positive and anti-e-positive carriers. This result suggests that, if more sensitive tests were available, the e-negative group would be found to consist of a mixture of e-antigen and anti-e-positive persons. The significantly higher prevalence of hepatic enzyme elevations in the e-antigen-positive carriers is consistent with similar reported observations (4-9), but differs slightly in that a sizeable percentage ( 2 5 % ) had normal enzyme values. Although, the anti-e-positive carriers had the lowest prevalence of enzyme abnormalities of any of the carriers, Hindman et a\. • " e " Antigen in Hepatitis Carriers
Downloaded from https://annals.org by Tulane University user on 01/02/2019
+ + + +
459
with hepatitis B who are most likely both to develop active forms of liver disease and to be disseminators of infection. However, caution should be used in ascribing a uniformly favorable prognosis to patients with anti-e and any conclusions concerning the infectivity or lack of infectivity of anti-e-positive carriers must await more definitive studies. Finally more sensitive tests for detecting e-antigen and anti-e are needed to more definitively determine the role of the "e" system in hepatitis B infection. ACKNOWLEDGMENTS: Received 29 March 1976; revision accepted 22 July 1976. • Requests for reprints should be addressed to Stephen H. Hindman, M.D.; Phoenix Laboratories Division, Center for Disease Control; 4402 North Seventh Street; Phoenix, AZ 85014. Figure 1 . Scatter diagram of Dane particle counts by immune electron microscopy versus serum DNA polymerase activity. (Equation of best fitting regression line: Y = 0.40 X-136; correlation index of best-fitting line = 0.779; standard error of estimate = 39.7; dotted lines represent 9 5 % upper and lower confidence limits.)
References 1. L E BOUVIER GL: The heterogenicity of Australia antigen. / Infect Dis 123:671-675, 1971 2. BANCROFT WH, MUNDON FK, RUSSELL PK: Detection of addi-
tional antigenic determinants of the hepatitis B antigen. J Immunol 109:842-848, 1972 3. MAGNIUS LO, ESPMARK JA: New specificities in Australia antigen
the fact that their mean enzyme levels were significantly higher than the controls and that 13% had elevated enzymes suggests the presence of anti-e does not automatically confer a favorable prognosis. The finding that 8 3 % of the e-antigen-positive sera had detectable Dane particles by immune electron microscopy is consistent with the findings of Sheikh (15) and Nordenfelt (16) who reported the presence of Dane particles in 76% and 100%, respectively, of their e-antigen-positive carriers. Contrary to our results, Sheikh found Dane particles in five of 27 anti-e-positive carriers. The discrepancy between these observations may be due in part to the smaller sample size in our study. Nevertheless, these studies have shown a highly significant association between the presence of e-antigen in serum and the ability to detect circulating Dane particles by immune electron microscopy. The finding that the e-antigen-positive carriers were the only group that had significantly elevated mean D N A polymerase activities indicates that its presence in serum is associated with active and efficient HBV replication. The lower D N A polymerase activities found in the anti-e-positive carriers implies that this antibody is associated with less active HBV replication. These differences in the activity of HBV replication may be responsible for the significant clinical differences in the activity of liver disease shown between e-antigen-positive and anti-e-positive HB s Ag carriers. If, as previously suggested ( 1 7 ) , e-antigen is an antigenic component of the Dane particle, the association of anti-e with reduced HBV replication may be a causal one. From a clinical standpoint, screening for e-antigen and anti-e appears to be of value in identifying those patients
460
positive sera distinct from the Le Bouvier determinants. J Immunol 109:1017-1021, 1972 4. MAGNIUS LO: Characterization of a new antigen-antibody system associated with hepatitis B. Clin Exp Immunol 20:209-216, 1975 5. MAYNARD JE, BARRETT DH, MURPHY BL, et al: Relationship of
the e antigen to hepatitis B virus infection in a hyperendemic area. / Infect Dis 133:339-342, 1976 6. NEILSON JO, DIETRICHSON O, JUHL E: Incidence and meaning
of the " e " determinant among hepatitis-B-antigen positive patients with acute and chronic liver diseases. Lancet 2:913-915, 1974 7. ELEFTHERIOU N, THOMAS HC, HEATHCOTE J, et al: Incidence
and clinical significance of e antigen and antibody in acute and chronic liver disease. Lancet 2:1171-1173, 1975 8. FEINMAN SV, BERRIS B, SINCLAIR JC, et al: e antigen and anti-e
in HB s Ag carriers. Lancet 2:1173-1174, 1975 9. MAGNIUS LO, LINDHOLM A, LUNDIN P: A new antigen-antibody
system. Clinical significance in long term carriers of hepatitis B surface antigen. JAMA 231:356-359, 1974 10. OKADA K, KAMIGAMA I, INOMATA M, et al: 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 294:746-749, 1976 11. SKINH0J P, COHN J, BRADBURNE AF: Transmission of hepatitis
type B from healthy HBsAg-positive mothers. Br Med J 1:10-11, 1976 12. ROBINSON WS, ROBINSON HL: DNA polymerase in defective
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