G. Pastore, A. R. Zanetti, P. Ferroni, P. Dentico, G. Angarano, O. Schiraldi
Radioimmunoassay in the Detection of the Hepatitis B e Antigen/Antibody System in Asymptomatic Carriers of Hepatitis B Surface Antigen Correlation with Serum Dane Particle Associated DNA Polymerase Activity
Summary: A radioimmunoassay for hepatitis e antigen (HBeAg) and antibody to e (anti-HBe) was developed and sera of 7t asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), in 44 of whom liver biopsy was obtained, were tested. In addition, testing for Dane particle associated DNA polymerase activity was performed in all sera. HBeAg was detected in 14 subjects (19.7%) and anti-HBe in 46 (64.8%). The highest proportion of HBeAg positivity (40%) was found among carriers with histological evidence of chronic hepatitis, whereas anti-HBe was present in 80% of carriers with normal liver histology, in 58% of carriers with nonspecific reactive hepatitis and in 60% of carriers with chronic liver lesions. DNA polymerase activity was present in 92.8% of sera positive for HBeAg, in 13% of sera positive for antiHBe, and in 9% of sera negative for both markers. Our results demonstrate that not all HBsAg carriers reactive to HBeAg show evidence of chronic hepatitis nor, conversely, that antiHBe is invariably associated with the healthy carder state of HBsAg. Finally, circulating Dane particles, as revealed by the presence of serum specific DNA polymerase activity, may also be present in anti-HBe positive sera other than those of some HBsAg carriers lacking both HBeAg and anti-HBe.
Zusammenfassung: Ein Radioimmunassay zum Nachweis des Hepatitis-B-e-Antigen/Antik6rper-Systems bei asymptomatischen Triigern yon Hepatitis-B-Oberfliichen-Antigen. Korrelation mit der Dane-Partikel-assoziierten DNS-Polymerase-Aktivitiit. Ein Radioimmunoassay fiir das Hepatitis ,,e" Antigen (HBeAg) wurde entwickelt und Seren yon 7t asymptomatischen Tr~igern von Hepatitis-B-Oberflfichen-Antigen (HBsAg) wurden untersucht; bei 44 von ihnen wurden Leberbiopsien entnommen. Zusiitzlich wurden bei allen Seren der Test auf Dane-Partikel-assoziierte DNS-Polymerase-Aktivitiit durchgefiihrt. HBeAg wurde bei 14 Patienten (19,7%) entdeckt, anti-HBe bei 46 (64,8%). Der grOBte Anteil HBeAgpositiver Seren (40%) fand sich bei Triigern mit den histologischen Zeichen der chronischen Hepatitis, wfihrend anti-HBe bei 80% der Triiger mit normaler Leberhistologie vorhanden war, bei 58% der Tr~iger mit unspezifischer reaktiver Hepatitis und bei 60% der Triiger mit chronischem Leberschaden. Die DNS-Polymerase-Aktivitiit war bei 92,8% der HBeAg-positiven Seren vorhanden, bei 13% der anti-HBe-positiven und bei 9% der beziiglich beider Marker negativen Seren. Unsere Ergebnisse zeigen, dab nicht alle HBsAg~Triiger, die HBeAgreaktiv find, Zeichen einer chronischen Hepatitis aufweisen und dab umgekehrt auch nicht anti-HBe ausnahmslos mit dem gesunden Tr~iger-Status von HBsAg assoziiert ist. SchlieBlich kfnnen, wie dutch die Anwesenheit von spezifischer SerumDNS-Polymerase-Aktivit~it gezeigt wurde, zirkulierende Dane-Partikel auch in anderen anti-HBe-positiven Seren vorhanden sein als in denjenigen einiger HBsAg-Carder, bei denen sowohl HBeAg als auch anti-HBe fehlen.
Introduction Using a sensitive radioimmunoassay procedure, Mushahwar et al. (1) were able to demonstrate the presence of e antigen (HBeAg) or antibody to e antigen (antiHBe) in 95% of chronic carriers of hepatitis B surface antigen (HBsAg), so that the appearance of e determinant, followed by seroconversion to antibody in many patients, may be considered a normal consequence of hepatitis B virus (HBV) infection. The presence of HBeAg in HBsAg carriers has been reported to be a marker of chronicity (2, 3, 4, 5, 6, 7) and infectivity (8, 9, 10, 11), whereas anti-HBe has usually been associated with the healthy-carrier state. Furthermore, it has been established that the presence of HBeAg in serum is correlated with the circulating Dane particle and its specific D N A polymerase activity (12, 13, 14, i5), although this association is not absolute (16). In order to investigate further the significance of the HBeAg/anti-HBe system and its relationship to clinical status and liver pathology of HBeAg carriers, we used a sensitive radioimmunoassay procedure to test for HBeAg and anti-HBe in the serum of 71 asymptomatic chronic HBsAg carriers. In addition, testing for D N A polymerase activity was performed in order to define the correlation between this antigen-antibody system and circulating complete virion of HBV. Material and Methods
Patients: Sera were obtained from 7t symptom-free individuals, all of whom had been persistent HBsAg carriers for more than one year. Liver biopsy was performed in 44 subjects. In this group the following histological diagnoses were made: normal liver histology (5 cases); non-specific reactive hepatitis (24 cases) associated with steatosis in 3 cases; chronic persistent hepatitis (10 cases); moderate chronic active hepatitis (3 cases), and inactive cirrhosis (2 cases). The remaining 27 carriers were clinically and subjectively healthy persons with repeatedly normal liver function tests. Methods: HBsAg was detected by solid phase radioimmunoassay (Ausria II, Abbott Laboratories). DNA polymerase actiReceived: 13 June 1979 Dr. G. Pastore, P.. Dentico, G. Angarano, O. Schiraldi, Clinica delle Malattie Infettive, Universit~ di Bad, Policlinico, 70124 Bad, Italy; Dr. A. R. Zanetti, P. Ferroni, Istituto di Virologia, Universit~ di Milano, Via Pascal 38, 20133 Milano, Italy.
Infection 7 (1979) Nr. 6
279
G. Pastore et al.: Hepatitis B e antigen radioimmunoassay
vity was measured by the method ofKaplan et al. (17) on 25 of pellets obtained after centrifugation of 2 ml of a serum sample at 48.000 g for 6 h in a refrigerated centrifuge. The specificity of the DNA polymerase activity was confirmed by precipitation with anti-HBs of human origin (18). Detection of HBeAg and anti-HBe was carded out by solidphase radioimmunoassay employing radio-labelled specific antibody (19). IgG from an anti-HBe positive serum was prepared according to Mushahwar et al. (1) and the lz5I antibody labelling was performed by the chloramin-T method (20). For HBeAg determination, 200 ~tl of each serum to be tested was incubated overnight at room temperature with polysterene beads of 0.65 cm in diameter, coated with antibody by incubation with diluted anti-HBe positive serum. After washing four times with 5 ml water and incubating for a second time overnight at room temperature with radio-labelled antibody, the radioactivity of the beads was measured. Samples giving counts per minute of more than 2.1-fold the negative controls were considered positive. For anti-HBe determination, the beads were first coated by diluted anti-HBe containing serum, and additionally by diluted HBeAg positive serum. In this blocking test all further steps were as above. The mean positive control was assumed to give 100% binding inhibition. Samples exhibiting more than 50% inhibition were considered positive.
Table 2: Correlations between HBe-markers and detection of serum DNA polymerase activity. HBe status
No. Serum DNA polymerase activity cases positive (%) negative (%)
HBeAg Anti-HBe Neither HBeAg nor anti-HBe
14 46
13 6
(92.8) (13.0)
1 33
(7.2) (87.0)
11
1
(9.1)
10
(90.0)
°P = < 0.001
for HBeAg, anti-HBe, HBsAg, anti-HBs and anti-HBc. A ratio of 2.1 or greater was regarded as being significant. Although a strong correlation (Table 2) was found between detection of HBeAg positivity and presence of serum D N A polymerase activity (P = < 0.001), D N A polymerase was also found in 6/46 of sera positive for anti-HBe (13%) and in 1/11 of sera lacking both markers (9.1%). However, seven out of 57 sera negative for HBeAg (12.2%) showed DNA polymerase activity.
Results
Discussion
Results of HBeAg and anti-HBe testing among the various groups of HBsAg carriers are summarized in Table 1. The highest proportion of HBeAg positivity was found among carriers with histological lesions resulting from chronic hepatitis (40%), whereas HBeAg was found in four out of 27 (14.8%) healthy carriers on whom no biopsy was performed, and in four out of 24 (16.7%) carriers on whom biopsies were performed and who had non-specific reactive hepatitis. None of the five carders with normal liver histology had HBeAg, but four of them (80%) had anti-HBe. The incidence of anti-HBe in other groups of subjects tested was 70.3% among healthy carders, 58.3% among the carders with nonspecific reactive hepatitis, and 60% among the carders with chronic hepatitis. The relationship between serum D N A polymerase activity and HBe-markers is shown in Figure 1. Levels of serum D N A polymerase activity are expressed as a ratio of counts per minute (cpm) of 3H-TMP incorporation obtained related to that obtained with similarly treated sera negative
The presence of HBeAg or anti-HBe detected by radioimmunoassay procedure in sera of about 85% of chronic HBsAg carriers tested confirms previous observations (1, 21) and shows that the presence of HBeAg is a common consequence of HBV infection. Although our results show a higher prevalence of HBeAg in HBsAg carriers with histological evidence of chronic hepatitis, we also detected this antigen in about 14% of clinically healthy carriers with no sign of hepatic damage or with minimal histological lesions of the liver. The persistence of HBeAg in patients with acute hepatitis type B has been reported to be of considerable prognostic value in the development of chronic hepatitis (22, 23, 24). The finding, however, that HBeAg may be detected with a sensitive radioimmunoassay in some individuals with apparently no evidence of chronic hepatitis further emphasizes that factors other than the persistence of HBV activity are involved in the pathogenesis of chronic hepatitis. Furthermore, since anti-HBe is present in many
Table 1: Detection of HBeAg and anti-HBe by RIA in 71 asymptomatic chronic HBsAg carders according to their clinical status. No. cases
HBeAg pos.
%
Anti-HBe pos.
%
HBeAg/ anti-HBe neg.
%
No liver biopsy Liver biopsy with histology: Normal Non specific reactive hepatitis Chronic persistent hepatitis Moderate active hepatitis Inactive cirrhosis
27
4
(14.8)
19
(70.3)
4
(14.8)
5 24 10 3 2
0 4 4 1 1
(16.7) (40.0) (33.3) (50.0)
4 14 6 2 1
(80,0) (58.3) (60.0) (66.6) (50.0)
1 6 0 0 0
(20.0) (25.0) -
Total
71
14
19.7
46
64,8
11
15.5
Groups of HBsAg carders
280
Infection 7 (1979) Nr. 6
G. Pastore et al.: Hepatitis B e antigen radioimmunoassay
10
Z
0 0
0 0
Z
0 O O
0 000 0
o .... m .... v - v o
:
HBeAg pOS.
e~ q.Do
ant i - HBe pos.
I
,:.
HBeAg er~t i - H B e neg.
HBsAg carriers with histological lesions resulting from chronic hepatitis, the disappearance of HBeAg followed by sero-conversion to anti-HBe in infected patients does not invariably prevent the development of chronic hepatitis. We can also state that none of the carriers with histological lesions due to chronic hepatitis and positive for anti-HBe showed clinical or biochemical signs of liver damage, so that anti-HBe could be considered a reliable marker of chronic non-progressive and/or inactive liver disease as well as of the healthy HBsAg carrier state. Our data show a strong correlation between serum D N A polymerase activity and presence of HBeAg, but we detected this enzymatic activity in the serum in six out of 46 subjects carrying anti-HBe. Furthermore, significant levels of DNA polymerase activity were found in a carrier negative for both HBeAg and anti-HBe. These observations have been reported by other investigators (11, 13, 25, 26) and suggested that lack of HBeAg, detected with the more sensitive radioimmunoassay, not always excluded the presence of circulating Dane particles. Indeed, Dane particles were also found in combination with anti-HBe and in the absence of both indicators in approximately 30% of HBsAg carriers (3, 16, 26), so that the test of serum specific D N A polymerase activity may actually be considered a very sensitive method for the demonstration of circulating complete virion of HBV.
HB$ neg. controls
Figure 1: Relationship of DNA polymerase activity and HBe-markers in 71 asymptomatic HBsAg chronic carriers and in 25 HBsAg negative controls.
On the other hand, the infectivity of HBsAg positive blood is related to the presence of complete HBV virion and, although the number of circulating Dane particles is low in the carriers positive for anti-HBe, this does not exclude the possibility of transmitting hepatitis (27, 28, 29). Finally, more caution should be used in ascribing a uniformly favourable prognosis to patients with anti-HBe, and any conclusions concerning the infectivity of HBeAg negative or anti-HBe positive carriers must await more definitive studies. Acknowledgements
This work was supported in part by grants from Consiglio Nazionale delle Ricerche (Roma), Progetto Finalizzato Virus, Contratto no. 78.00399.84. Literature 1. Mushahwar, L K., Overby, L. R., Fr6sner, G. G., Deinhardt, F., Ling, C. M.: Prevalence of hepatitis B eantigen and its antibody as detected by radioimmunoassays. J. Med. Virol. 2 (1978) 77-87. 2. Eteftheriou, N., Thomas, H. C., Heathcote, J., Sherlock, S.: Incidence and clinical significance of e antigen and antibody in acute and chronic liver disease. Lancet II (1975) 1171-1173. 3. Sheikh, N. E. L., Woolf, L L., Galbraith, R. M., Eddleston, A. L. W. F., Dymock, L W., Williams, R.: e antigen-
Infection 7 (1979) Nr. 6
281
G. Pastore et al.: Hepatitis B e antigen radioimmunoassay antibody system as indicator of liver damage in patients with hepatitis B infection. Br. Med. J. 4 (1975) 252-253.
4. Smith, J. L., Murphy, B. L., Auslander, M. 0., Maynard, J., Schalm, S. S., Summerskill, W. H. J., Gitnick, G. L.: Studies of the "e" antigen in acute and chronic hepatitis. Gastroenterology 71 (1976) 208-209.
5. Vogten, A. J. M., Schalm, S. W., SummerskiU, W. H. J., Gitnick, G. L., Murphy, B. L., Maynard, J. E.: Behaviour of e antigen and antibody during chronic active liver disease. Relation to HB antigen-antibody system and prognosis. Lancet II (1976) 126-128. 6. Pastore, G., Dentico, P., Fera, G., Schiraldi, 0.: Prognostic value of HBeAg in chronic persistent hepatitis. Br. Med. J. 2 (1977) 1521-1522.
7. Pastore, G., Dentico, P., Fera, G., Angarano, G., Schiraldi, O.: Comportamento e significato clinico del sistema eAg/anti-e nei portatori dell' HBsAg. Boll Ist. Sieroter. Milan. 56 (1977) 333-338.
8. Magnius, L. 0., Lindholm, A., Lundin, P., lwarson, S.: A new antigen-antibody system. Clinical significance in longterm carriers of hepatitis B surface antigen. J. Am. Med. Ass. 231 (1975) 356-359. 9. Grady, G. G. et al. (Collaborative Study Group): Relation of e antigen to infectivity of HBsAg positive inoculations among medical personnel. Lancet II (1976) 492-493. 10. Okada, K., Kamiyama, L, Inomata, M., Omai, M., Miyakawa, Y., 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 (1976) 746-749. 11. Zanetti, A. R., Ferroni, P.: Presence of HBeAg, DNA polymerase activity, Dane particle in different categories of HBsAg carriers and evidence of hepatitis type B infection in their family contacts. Boll. Ist. Sieroter. Milan. 56 (1977) 79--86. 12. Nordenfelt, E., K]ellen, L.: Dane particles, DNA polymerase and e antigen in two different categories of hepatitis B antigen carriers. Intervirology 5 (1975) 225-232. 13. Hindman, S. H., Gravelle, C. R., Murphy, B. L., Bradley, D. W., Budge, W. R., Maynard, J. E.: "e" antigen, Dane particles, and serum DNA polymerase activity in HBsAg carriers. Ann Intern. Med. 85 (1976) 458--460. 14. Takahashi, K., Imai, M., Tsuda, F., Takahashi, T., Miyakama, Y., Mayumi, M.: Association of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis B surface antigen. J. Immunol. 117 (1976) 102-105. 15. Tong, M. J., Stevenson, D., Gordon, L: Correlation of e antigen, DNA polymerase activity, and Dane particles in
282
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chronic benign and chronic active type B hepatitis infections. J. Infect. Dis. 135 (1977) 980-984. 16. Trepo, C., Bird, R. G., Zuckerman, A. J.: Correlations between the detection of e antigen or antibody and electron microscopic pattern of hepatitis B surface antigen (HBsAg) associated particles in the serum of HBsAg carriers. J. Clin. Pathol. 30 (1977) 216-220. 17. Kaplan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., Robinson, W. S.: DNA polymerase associated with human hepatitis B antigen. J. Virol. 12 (1973) 995-1005. 18. Kaplan, P. M., Gerin, J. L., Alter, H. J.: Hepatitis B specific DNA polymerase activity during post-transfusion hepatitis. Nature 249 (1974) 762-764. 19. Ling, C. M., Overby, L. R.: Prevalence of hepatitis B" virus antigen as revealed by direct radioimmune assay with 12SI-antibody. J. Immunol. 109 (1972) 834-84t. 20. Hunter, W. M., Greenwood, F. C.: Preparation of iodine125 labelled human growth hormone of high specific activity. Nature (London) 194 (1962) 495--496. 21. Fields, 1-1.A., Bradley, D. W., Maynard, J. E.: RIA for the detection of HBeAg and anti-HBe. J. Immunol. 121 (1978) 930-935. 22. Woolf, L L., El Sheikh, N., Cullens, H., Eddleston, A. L. W. F., Williams, R.: HBeAg in viral hepatitis type B. Digestion 17 (1978) 317-322. 23. Villare]os, V. M., Visona, K. A., Eduarte, C. E.: e antigen and age in acute and chronic type B hepatitis. J. Med. Virol. 2 (1978) 117-125.
24. Pastore, G., Dentico, P., Angarano, G., Lapedota, E., Schiraldi, 0.: Persistence of e antigen as prognostic marker in acute hepatitis B. Infection 7 (1979) 17-20. 25. Arnold, W., Hess, G., Kaplan, M., Gerin, J. I., Mayer zum Buschenfeld, K. H.: Anti-HBc, HBeAg and DNA polymerase activity in healthy HBsAg carriers and patients with inflammatory liver diseases. Klin. Wschr. 56 (1978) 297-303. 26. Howard, C. R., Zanetti, A. R., Thai, S., Zuckerman, A. J.: Viral antigens and antibodies in hepatitis B infection. J. Clin. Pathol. 31 (1978) 681-687. 27. Schweitzer, I. L., Edwards, V. N., Brezina, M.: e antigen in HBsAg carriers mothers. N. Engl. J. Med. (letter) 293 (1975) 940. 28. Berquist, K. R., Maynard, J. E., Murphy, L.: Infectivity of serum containing HBsAg and antibody to e antigen. Lancet I (1976) 1026. 29. Shikata, T., Karasowa, T., Abe, K., Uzawa, T., Suzuki, H., Oda, T., Imai, M., Mayumi, M., Moritsugu, Y.: Hepatitis B e antigen and infectivity of hepatitis B virus. J. Infect. Dis. 136 (1977) 371-376.
Radioimmunoassay in the Detection of the Hepatitis B e Antigen/Antibody System in Asymptomatic Carriers of Hepatitis B Surface Antigen Correlation with Serum Dane Particle Associated DNA Polymerase Activity
Summary: A radioimmunoassay for hepatitis e antigen (HBeAg) and antibody to e (anti-HBe) was developed and sera of 7t asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), in 44 of whom liver biopsy was obtained, were tested. In addition, testing for Dane particle associated DNA polymerase activity was performed in all sera. HBeAg was detected in 14 subjects (19.7%) and anti-HBe in 46 (64.8%). The highest proportion of HBeAg positivity (40%) was found among carriers with histological evidence of chronic hepatitis, whereas anti-HBe was present in 80% of carriers with normal liver histology, in 58% of carriers with nonspecific reactive hepatitis and in 60% of carriers with chronic liver lesions. DNA polymerase activity was present in 92.8% of sera positive for HBeAg, in 13% of sera positive for antiHBe, and in 9% of sera negative for both markers. Our results demonstrate that not all HBsAg carriers reactive to HBeAg show evidence of chronic hepatitis nor, conversely, that antiHBe is invariably associated with the healthy carder state of HBsAg. Finally, circulating Dane particles, as revealed by the presence of serum specific DNA polymerase activity, may also be present in anti-HBe positive sera other than those of some HBsAg carriers lacking both HBeAg and anti-HBe.
Zusammenfassung: Ein Radioimmunassay zum Nachweis des Hepatitis-B-e-Antigen/Antik6rper-Systems bei asymptomatischen Triigern yon Hepatitis-B-Oberfliichen-Antigen. Korrelation mit der Dane-Partikel-assoziierten DNS-Polymerase-Aktivitiit. Ein Radioimmunoassay fiir das Hepatitis ,,e" Antigen (HBeAg) wurde entwickelt und Seren yon 7t asymptomatischen Tr~igern von Hepatitis-B-Oberflfichen-Antigen (HBsAg) wurden untersucht; bei 44 von ihnen wurden Leberbiopsien entnommen. Zusiitzlich wurden bei allen Seren der Test auf Dane-Partikel-assoziierte DNS-Polymerase-Aktivitiit durchgefiihrt. HBeAg wurde bei 14 Patienten (19,7%) entdeckt, anti-HBe bei 46 (64,8%). Der grOBte Anteil HBeAgpositiver Seren (40%) fand sich bei Triigern mit den histologischen Zeichen der chronischen Hepatitis, wfihrend anti-HBe bei 80% der Triiger mit normaler Leberhistologie vorhanden war, bei 58% der Tr~iger mit unspezifischer reaktiver Hepatitis und bei 60% der Triiger mit chronischem Leberschaden. Die DNS-Polymerase-Aktivitiit war bei 92,8% der HBeAg-positiven Seren vorhanden, bei 13% der anti-HBe-positiven und bei 9% der beziiglich beider Marker negativen Seren. Unsere Ergebnisse zeigen, dab nicht alle HBsAg~Triiger, die HBeAgreaktiv find, Zeichen einer chronischen Hepatitis aufweisen und dab umgekehrt auch nicht anti-HBe ausnahmslos mit dem gesunden Tr~iger-Status von HBsAg assoziiert ist. SchlieBlich kfnnen, wie dutch die Anwesenheit von spezifischer SerumDNS-Polymerase-Aktivit~it gezeigt wurde, zirkulierende Dane-Partikel auch in anderen anti-HBe-positiven Seren vorhanden sein als in denjenigen einiger HBsAg-Carder, bei denen sowohl HBeAg als auch anti-HBe fehlen.
Introduction Using a sensitive radioimmunoassay procedure, Mushahwar et al. (1) were able to demonstrate the presence of e antigen (HBeAg) or antibody to e antigen (antiHBe) in 95% of chronic carriers of hepatitis B surface antigen (HBsAg), so that the appearance of e determinant, followed by seroconversion to antibody in many patients, may be considered a normal consequence of hepatitis B virus (HBV) infection. The presence of HBeAg in HBsAg carriers has been reported to be a marker of chronicity (2, 3, 4, 5, 6, 7) and infectivity (8, 9, 10, 11), whereas anti-HBe has usually been associated with the healthy-carrier state. Furthermore, it has been established that the presence of HBeAg in serum is correlated with the circulating Dane particle and its specific D N A polymerase activity (12, 13, 14, i5), although this association is not absolute (16). In order to investigate further the significance of the HBeAg/anti-HBe system and its relationship to clinical status and liver pathology of HBeAg carriers, we used a sensitive radioimmunoassay procedure to test for HBeAg and anti-HBe in the serum of 71 asymptomatic chronic HBsAg carriers. In addition, testing for D N A polymerase activity was performed in order to define the correlation between this antigen-antibody system and circulating complete virion of HBV. Material and Methods
Patients: Sera were obtained from 7t symptom-free individuals, all of whom had been persistent HBsAg carriers for more than one year. Liver biopsy was performed in 44 subjects. In this group the following histological diagnoses were made: normal liver histology (5 cases); non-specific reactive hepatitis (24 cases) associated with steatosis in 3 cases; chronic persistent hepatitis (10 cases); moderate chronic active hepatitis (3 cases), and inactive cirrhosis (2 cases). The remaining 27 carriers were clinically and subjectively healthy persons with repeatedly normal liver function tests. Methods: HBsAg was detected by solid phase radioimmunoassay (Ausria II, Abbott Laboratories). DNA polymerase actiReceived: 13 June 1979 Dr. G. Pastore, P.. Dentico, G. Angarano, O. Schiraldi, Clinica delle Malattie Infettive, Universit~ di Bad, Policlinico, 70124 Bad, Italy; Dr. A. R. Zanetti, P. Ferroni, Istituto di Virologia, Universit~ di Milano, Via Pascal 38, 20133 Milano, Italy.
Infection 7 (1979) Nr. 6
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G. Pastore et al.: Hepatitis B e antigen radioimmunoassay
vity was measured by the method ofKaplan et al. (17) on 25 of pellets obtained after centrifugation of 2 ml of a serum sample at 48.000 g for 6 h in a refrigerated centrifuge. The specificity of the DNA polymerase activity was confirmed by precipitation with anti-HBs of human origin (18). Detection of HBeAg and anti-HBe was carded out by solidphase radioimmunoassay employing radio-labelled specific antibody (19). IgG from an anti-HBe positive serum was prepared according to Mushahwar et al. (1) and the lz5I antibody labelling was performed by the chloramin-T method (20). For HBeAg determination, 200 ~tl of each serum to be tested was incubated overnight at room temperature with polysterene beads of 0.65 cm in diameter, coated with antibody by incubation with diluted anti-HBe positive serum. After washing four times with 5 ml water and incubating for a second time overnight at room temperature with radio-labelled antibody, the radioactivity of the beads was measured. Samples giving counts per minute of more than 2.1-fold the negative controls were considered positive. For anti-HBe determination, the beads were first coated by diluted anti-HBe containing serum, and additionally by diluted HBeAg positive serum. In this blocking test all further steps were as above. The mean positive control was assumed to give 100% binding inhibition. Samples exhibiting more than 50% inhibition were considered positive.
Table 2: Correlations between HBe-markers and detection of serum DNA polymerase activity. HBe status
No. Serum DNA polymerase activity cases positive (%) negative (%)
HBeAg Anti-HBe Neither HBeAg nor anti-HBe
14 46
13 6
(92.8) (13.0)
1 33
(7.2) (87.0)
11
1
(9.1)
10
(90.0)
°P = < 0.001
for HBeAg, anti-HBe, HBsAg, anti-HBs and anti-HBc. A ratio of 2.1 or greater was regarded as being significant. Although a strong correlation (Table 2) was found between detection of HBeAg positivity and presence of serum D N A polymerase activity (P = < 0.001), D N A polymerase was also found in 6/46 of sera positive for anti-HBe (13%) and in 1/11 of sera lacking both markers (9.1%). However, seven out of 57 sera negative for HBeAg (12.2%) showed DNA polymerase activity.
Results
Discussion
Results of HBeAg and anti-HBe testing among the various groups of HBsAg carriers are summarized in Table 1. The highest proportion of HBeAg positivity was found among carriers with histological lesions resulting from chronic hepatitis (40%), whereas HBeAg was found in four out of 27 (14.8%) healthy carriers on whom no biopsy was performed, and in four out of 24 (16.7%) carriers on whom biopsies were performed and who had non-specific reactive hepatitis. None of the five carders with normal liver histology had HBeAg, but four of them (80%) had anti-HBe. The incidence of anti-HBe in other groups of subjects tested was 70.3% among healthy carders, 58.3% among the carders with nonspecific reactive hepatitis, and 60% among the carders with chronic hepatitis. The relationship between serum D N A polymerase activity and HBe-markers is shown in Figure 1. Levels of serum D N A polymerase activity are expressed as a ratio of counts per minute (cpm) of 3H-TMP incorporation obtained related to that obtained with similarly treated sera negative
The presence of HBeAg or anti-HBe detected by radioimmunoassay procedure in sera of about 85% of chronic HBsAg carriers tested confirms previous observations (1, 21) and shows that the presence of HBeAg is a common consequence of HBV infection. Although our results show a higher prevalence of HBeAg in HBsAg carriers with histological evidence of chronic hepatitis, we also detected this antigen in about 14% of clinically healthy carriers with no sign of hepatic damage or with minimal histological lesions of the liver. The persistence of HBeAg in patients with acute hepatitis type B has been reported to be of considerable prognostic value in the development of chronic hepatitis (22, 23, 24). The finding, however, that HBeAg may be detected with a sensitive radioimmunoassay in some individuals with apparently no evidence of chronic hepatitis further emphasizes that factors other than the persistence of HBV activity are involved in the pathogenesis of chronic hepatitis. Furthermore, since anti-HBe is present in many
Table 1: Detection of HBeAg and anti-HBe by RIA in 71 asymptomatic chronic HBsAg carders according to their clinical status. No. cases
HBeAg pos.
%
Anti-HBe pos.
%
HBeAg/ anti-HBe neg.
%
No liver biopsy Liver biopsy with histology: Normal Non specific reactive hepatitis Chronic persistent hepatitis Moderate active hepatitis Inactive cirrhosis
27
4
(14.8)
19
(70.3)
4
(14.8)
5 24 10 3 2
0 4 4 1 1
(16.7) (40.0) (33.3) (50.0)
4 14 6 2 1
(80,0) (58.3) (60.0) (66.6) (50.0)
1 6 0 0 0
(20.0) (25.0) -
Total
71
14
19.7
46
64,8
11
15.5
Groups of HBsAg carders
280
Infection 7 (1979) Nr. 6
G. Pastore et al.: Hepatitis B e antigen radioimmunoassay
10
Z
0 0
0 0
Z
0 O O
0 000 0
o .... m .... v - v o
:
HBeAg pOS.
e~ q.Do
ant i - HBe pos.
I
,:.
HBeAg er~t i - H B e neg.
HBsAg carriers with histological lesions resulting from chronic hepatitis, the disappearance of HBeAg followed by sero-conversion to anti-HBe in infected patients does not invariably prevent the development of chronic hepatitis. We can also state that none of the carriers with histological lesions due to chronic hepatitis and positive for anti-HBe showed clinical or biochemical signs of liver damage, so that anti-HBe could be considered a reliable marker of chronic non-progressive and/or inactive liver disease as well as of the healthy HBsAg carrier state. Our data show a strong correlation between serum D N A polymerase activity and presence of HBeAg, but we detected this enzymatic activity in the serum in six out of 46 subjects carrying anti-HBe. Furthermore, significant levels of DNA polymerase activity were found in a carrier negative for both HBeAg and anti-HBe. These observations have been reported by other investigators (11, 13, 25, 26) and suggested that lack of HBeAg, detected with the more sensitive radioimmunoassay, not always excluded the presence of circulating Dane particles. Indeed, Dane particles were also found in combination with anti-HBe and in the absence of both indicators in approximately 30% of HBsAg carriers (3, 16, 26), so that the test of serum specific D N A polymerase activity may actually be considered a very sensitive method for the demonstration of circulating complete virion of HBV.
HB$ neg. controls
Figure 1: Relationship of DNA polymerase activity and HBe-markers in 71 asymptomatic HBsAg chronic carriers and in 25 HBsAg negative controls.
On the other hand, the infectivity of HBsAg positive blood is related to the presence of complete HBV virion and, although the number of circulating Dane particles is low in the carriers positive for anti-HBe, this does not exclude the possibility of transmitting hepatitis (27, 28, 29). Finally, more caution should be used in ascribing a uniformly favourable prognosis to patients with anti-HBe, and any conclusions concerning the infectivity of HBeAg negative or anti-HBe positive carriers must await more definitive studies. Acknowledgements
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