Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Pinho, J.R.R.; Santos, C.A.; Gonzalez, C.L.M.; Bassit, L.; Barreto, C.C.; Saez-Alquezar, A.; França, A.V.C.; Carrilho, F.J.; Fonseca, L.E.P.; Chamone, D.A.F.; Silva, L. C. da

doi:10.1590/S0036-46651993000600007

Abstracts

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".

HBV; PCR; replication Markers; chronic Hepatitis

A detecção do DNA do Vírus da Hepatite B pela Reação em Cadeia da Polimerase (PCR) foi comparada com os outros marcadores sorológicos virais (AgHBs, AgHBe e anti-HBe) numa série de 49 pacientes com hepatite crônica B, incluindo 12 que apresentaram clareamento espontâneo do AgHBs. Nenhum caso AgHBs negativo foi PCR positivo, mas 33/37 (89,2%) dos casos AgHBs positivos foram PCR positivos (p < 0,0001). Entre as amostras AgHBs positivas, 9 foram AgHBc positivas e anti-HBe negativas, todas elas PCR positivas. Outros 3 pacientes foram AgHBe e anti-HBe positivos, todos também PCR positivos. Um terceiro grupo incluía 21 pacientes anti-HBe positivos e anti-HBe negativos: 19 foram PCR positivos e 2 PCR negativos. Os últimos 4 casos foram AgHBe e anti-HBe negativos, sendo 2 destes PCR positivos. A detecção de casos virêmicos anti-HBe positivos sugere que as variantes preC do VHB podem também estar presentes em nosso país. Em conclusão, a "fase de integração viral" da hepatite crônica B parece ser muito menos frequente do que se imaginava previamente, quando apenas se dispunha das técnicas de detecção de AgHBe e DNA por hibridização. O novo termo "fase de baixa replicação baixa" parece ser melhor que o anterior "fase de integração viral" .

VIROLOGY

Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Detecção do DNA do vírus da Hepatite B pela reação em cadeia da polimerase em pacientes com Hepatite Crônica B soropositivos para o anticorpo Anti-HBe

J.R.R. Pinho^I,II,III; C.A. Santos^II; C.L.M. Gonzalez^IV; L. Bassit^I; C.C. Barreto^I; A. Saez-Alquezar^I; A.V.C. França^IV; F.J. Carrilho^IV; L.E.P. Fonseca^IV; D.A.F. Chamone^I; L. C. da Silva^III

^IFundação Pró-Sangue Hemocentro de São Paulo. São Paulo, SP, Brasil

^IIVirology Branch - Instituto Adolfo Lutz. São Paulo, SP, Brasil

^IIIVirology and Hepatology Laboratories - Instituto de Medicina Tropical de São Paulo. São Paulo, SP, Brasil

^IVGastroenterology Branch - Faculdade de Medicina da Universidade de São Paulo. São Paulo, SP, Brasil

^{Address for correspondence} Address for correspondence: João Renato Rebello Pinho Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470 05403-000 São Paulo, SP, Brasil

SUMMARY

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive.

The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".

Keywords: HBV; PCR; replication Markers; chronic Hepatitis

RESUMO

A detecção do DNA do Vírus da Hepatite B pela Reação em Cadeia da Polimerase (PCR) foi comparada com os outros marcadores sorológicos virais (AgHBs, AgHBe e anti-HBe) numa série de 49 pacientes com hepatite crônica B, incluindo 12 que apresentaram clareamento espontâneo do AgHBs. Nenhum caso AgHBs negativo foi PCR positivo, mas 33/37 (89,2%) dos casos AgHBs positivos foram PCR positivos (p < 0,0001). Entre as amostras AgHBs positivas, 9 foram AgHBc positivas e anti-HBe negativas, todas elas PCR positivas. Outros 3 pacientes foram AgHBe e anti-HBe positivos, todos também PCR positivos. Um terceiro grupo incluía 21 pacientes anti-HBe positivos e anti-HBe negativos: 19 foram PCR positivos e 2 PCR negativos. Os últimos 4 casos foram AgHBe e anti-HBe negativos, sendo 2 destes PCR positivos. A detecção de casos virêmicos anti-HBe positivos sugere que as variantes preC do VHB podem também estar presentes em nosso país. Em conclusão, a "fase de integração viral" da hepatite crônica B parece ser muito menos frequente do que se imaginava previamente, quando apenas se dispunha das técnicas de detecção de AgHBe e DNA por hibridização. O novo termo "fase de baixa replicação baixa" parece ser melhor que o anterior "fase de integração viral" .

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

Recebido para publicação em 22/06/1993

Aceito para publicação em 27/08/1993.

1. BAKER, B.L.; BISCEGLIE, A.M.; KANEKO, S.; MILLER, R.; FEINSTONE, S.M.; WAGGONER, J.G. & HOOFNAGLE, J.H. - Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with virological and biochemical markers. Hepatology, 13: 632-636, 1991.
2. BONINO, F.; HOYER, B. & NELSON, J. - Hepatitis B virus DNA in the sera of HBs antigen carriers: a marker of active hepatitis B virus replication in the liver. Hepatology, 1: 386-391, 1981.
3. BRUNETTO, M.R.; STEMLER, M.; SCHODEL, F.; WILL, H.; OTTOBRELLI, A.; RIZZETTO, M.; VERME, G. & BONINO, F. - Identification of HBV variants which cannot produce precore derived HBeAg and may be responsible for severe hepatitis. Ital. J. Gastroent., 21: 151-154, 1989.
4. BRUNETTO, M.R.; STEMLER, M.; BONINO, F.; SCHODEL, F.; OLIVERI, F.; RIZZETTO, M.; VERME, G.; WILL, H. - A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B. J. Hepat., 10: 258-261, 1990.
5. CARMAN, W.F.; JACYNA, M.R.; HADZIYANNIS, S.; KARAYIANNIS, P.; McGARVEY, M.J.; MAKRIS, A. & THOMAS, H.C. - Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet, 2: 588-591, 1989.
6. CARMAN, W.F.; DOURAKIS, S.; KARAYIANNIS, P.; CROSSEY, M.; DROBNER, R. & THOMAS, H.C. - Incidence of hepatitis B viraemia, detected using the polymerase chain reaction, after successful therapy of hepatitis B virus carriers with interferon - alpha. J. Med. Virol., 34: 114-118, 1991.
7. CHEMIN, L.; BAGINSKY, I.; PETIT, M.A.; ZOULIM, F.; PICHOUD, C.; CAPEL, F.; HANTZ, O. & TREPO, C. - Correlation between HBV DNA detection by polymerase chain reaction and pre-Sl antigenemia in symptomatic asymptomatic hepatitis B virus infections. J. Med. Virol., 33: 51-57, 1991.
8. CHU, C.M.; KARAYIANNIS, P.; FOWLER, M.J.F.; MONJARDINO, J.; LIAW, Y.F. & THOMAS, H.C. - Natural history of chronic hepatitis B infection in Taiwan : studies of hepatitis B virus DNA in serum. Hepatology, 5: 431-434, 1985.
9. GARBUGLIA, A.R.; MANZIN, A.; BUDKOWSKA, A.; TALIANI, G.; CLEMENTI, M.; DELFINI, C. & CARLONI, G. - Levels of pre-S antigens and HBV DNA in sera from high and low viremic HBV carriers. J. med. Virol., 35: 273-282, 1991.
10. GERKEN, G.; PATERLINI, P.; MANNS, M.; HOUSSET, C.; TERRE, S.; DIENES, H.P.; HESS, G.; GERLICH, W.H.; BERTHELOT, P.; MEYER ZUM BUSCHENFELDE, K.H. & BRECHOT, C. - Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to pre-S and S- encoded viral surface antigens. Hepatology, 13: 158,166, 1991.
11. HOOFNAGLE, J.H. - Chronic hepatitis B. New Engl. J. Med., 323: 337-339, 1990.
12. KANEKO, S.; MILLER, R.H.; FEINSTONE, S.M.; UNOURA, M.; KOBAYASHI , K.; HATTORI, N. & PURCELL, R. - Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc. Nat. Acad. Sci. (Wash.), 86: 312-315, 1989.
13. KANEKO, S.; FEINSTONE, S.M. & MILLER, R.H. - Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J. clin. Microbiol., 27: 1930-1933, 1989.
14. KANEKO, S.; MILLER, R.H.; Di BISCEGLIE, A.M.; FEINSTONE, S.M.; HOOFNAGLE, J.H. & PURCELL, R.H. - Detection of hepatitis B virus DNA in serum by polymerase chain reaction: application for clinical diagnosis. Gastroenterology, 99: 799-804, 1990.
15. KARAYIANNIS, P.; FOWLER, M.J.F.; LOK, A.S.F.; GREENFIELD, C.; MONJARDINO, J. & THOMAS, H.C. - Detection of scrum HBV DNA by molecular hybridization: correlation with HBeAg/anti-HBe status, racial origin, liver histology and hepatocellular carcinoma. J. Hepat., 1: 99-106, 1985.
16. KORENMAN, J.; BAKER, B.; WAGGONER, J.; EVERHART, J.E.; Di BISCEGLIE, A.M. & HOOFNAGLE, J.H. - Long term remission of chronic hepatitis B after alpha-interferon therapy. Ann. intern. Med., 114: 629-634, 1991.
17. KWOK, S. & HIGUCHI, R. - Avoiding false positives with PCR. Nature, 339: 237-238, 1989.
18. LIEBERMANN, H.M.; LEBRECQUE, D.P.; KEW, M.C.; HADZIYANNIS, S.J. & SHAFRITZ, D.A. - Hepatologic detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatology, 3: 285-291, 1983.
19. PINHO, J.R.R.; FONSECA, L.E.P.; SONG, Y.; MIYAMOTO, Y.; CARRILHO, F.J.; GRANATO, C.F.H. & SILVA, L.C. - Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/anti-IIBe system, viral DNA polymerase and HBV-DNA. Rev. Inst. Med. trop. S. Paulo, 31: 328-335, 1989.
20. SAIKI, R.K.; GELFAND, D.H.; STOFFEL, S.; SCHARF, S.; HIGUCHI, R.; HORN, G.; MULLIS, K. & ERLICH, H. - Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491, 1988.
21. SILVA, L.C.; MADRUGA, C.L.A.; CARRILHO, F.J.; PINHO, J.R.R.; SAEZ-ALQUEZAR, A.; SANTOS, C.A.; BASSIT, L.; BARRETO, C.C.; ALVES, V.A.F.; GAYOTTO, L.C.C. & CHAMONE, D.A.F. - Spontaneous clearance (SC) of serum HBsAg in twenty patients with chronic hepatitis B (CHB): HBV-DNA status by Polymerase Chain Reaction (PCR) and role of viral superinfection. Gastroenterology, 104: A688, 1993.
22. SHERLOCK, S. - The natural history of hepatitis B. Postgrad. med. J., 63 (suppl. 2): 7-11, 1987.
23. SHIH, L.N.; SHEU, J.C.; WANG, J.T.; HUANG, G.T.; YANG, P.M.; LEE, H.S.; SUNG, J.L.; WANG, T.H. & CHEN, D.S. - Serum hepatitis B virus DNA in healthy HBsAg negative Chinese adults evaluated by polymerase chain reaction. J. med. Virol., 32: 257-260, 1990.
24. WANG, J.T.; WANG, T.H.; SHEU, J.C.; SHIH, L.N.; LIN, J.T. & CHEN, D.S.. - Detection of hepatitis B virus DNA by polymerase chain reaction in plasma of volunteer blood donors negative for hepatitis B surface antigen. J. infect. Dis., 163: 397-399, 1991.
25. ZOULIN, F.; MIMMS, L.; FLOREANI, M.; PICHOUD, C.; CHEMIN, I.; KAY, A.; VITVITSKI, L. & TREPO, C. - New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection. J. clin. Microbiol., 30: 1111- 1119, 1992.

Address for correspondence:

João Renato Rebello Pinho

Instituto de Medicina Tropical de São Paulo

Av. Dr. Enéas de Carvalho Aguiar, 470

05403-000 São Paulo, SP, Brasil

Publication Dates

Publication in this collection
03 July 2006
Date of issue
Dec 1993

History

Received
22 June 1993
Accepted
27 Aug 1993

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BAKER, B.L.; BISCEGLIE, A.M.; KANEKO, S.; MILLER, R.; FEINSTONE, S.M.; WAGGONER, J.G. & HOOFNAGLE, J.H. - Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with virological and biochemical markers. Hepatology, 13: 632-636, 1991.

[2] 2. BONINO, F.; HOYER, B. & NELSON, J. - Hepatitis B virus DNA in the sera of HBs antigen carriers: a marker of active hepatitis B virus replication in the liver. Hepatology, 1: 386-391, 1981.

[3] 3. BRUNETTO, M.R.; STEMLER, M.; SCHODEL, F.; WILL, H.; OTTOBRELLI, A.; RIZZETTO, M.; VERME, G. & BONINO, F. - Identification of HBV variants which cannot produce precore derived HBeAg and may be responsible for severe hepatitis. Ital. J. Gastroent., 21: 151-154, 1989.

[4] 4. BRUNETTO, M.R.; STEMLER, M.; BONINO, F.; SCHODEL, F.; OLIVERI, F.; RIZZETTO, M.; VERME, G.; WILL, H. - A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B. J. Hepat., 10: 258-261, 1990.

[5] 5. CARMAN, W.F.; JACYNA, M.R.; HADZIYANNIS, S.; KARAYIANNIS, P.; McGARVEY, M.J.; MAKRIS, A. & THOMAS, H.C. - Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet, 2: 588-591, 1989.

[6] 6. CARMAN, W.F.; DOURAKIS, S.; KARAYIANNIS, P.; CROSSEY, M.; DROBNER, R. & THOMAS, H.C. - Incidence of hepatitis B viraemia, detected using the polymerase chain reaction, after successful therapy of hepatitis B virus carriers with interferon - alpha. J. Med. Virol., 34: 114-118, 1991.

[7] 7. CHEMIN, L.; BAGINSKY, I.; PETIT, M.A.; ZOULIM, F.; PICHOUD, C.; CAPEL, F.; HANTZ, O. & TREPO, C. - Correlation between HBV DNA detection by polymerase chain reaction and pre-Sl antigenemia in symptomatic asymptomatic hepatitis B virus infections. J. Med. Virol., 33: 51-57, 1991.

[8] 8. CHU, C.M.; KARAYIANNIS, P.; FOWLER, M.J.F.; MONJARDINO, J.; LIAW, Y.F. & THOMAS, H.C. - Natural history of chronic hepatitis B infection in Taiwan : studies of hepatitis B virus DNA in serum. Hepatology, 5: 431-434, 1985.

[9] 9. GARBUGLIA, A.R.; MANZIN, A.; BUDKOWSKA, A.; TALIANI, G.; CLEMENTI, M.; DELFINI, C. & CARLONI, G. - Levels of pre-S antigens and HBV DNA in sera from high and low viremic HBV carriers. J. med. Virol., 35: 273-282, 1991.

[10] 10. GERKEN, G.; PATERLINI, P.; MANNS, M.; HOUSSET, C.; TERRE, S.; DIENES, H.P.; HESS, G.; GERLICH, W.H.; BERTHELOT, P.; MEYER ZUM BUSCHENFELDE, K.H. & BRECHOT, C. - Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to pre-S and S- encoded viral surface antigens. Hepatology, 13: 158,166, 1991.

[11] 11. HOOFNAGLE, J.H. - Chronic hepatitis B. New Engl. J. Med., 323: 337-339, 1990.

[12] 12. KANEKO, S.; MILLER, R.H.; FEINSTONE, S.M.; UNOURA, M.; KOBAYASHI , K.; HATTORI, N. & PURCELL, R. - Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc. Nat. Acad. Sci. (Wash.), 86: 312-315, 1989.

[13] 13. KANEKO, S.; FEINSTONE, S.M. & MILLER, R.H. - Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J. clin. Microbiol., 27: 1930-1933, 1989.

[14] 14. KANEKO, S.; MILLER, R.H.; Di BISCEGLIE, A.M.; FEINSTONE, S.M.; HOOFNAGLE, J.H. & PURCELL, R.H. - Detection of hepatitis B virus DNA in serum by polymerase chain reaction: application for clinical diagnosis. Gastroenterology, 99: 799-804, 1990.

[15] 15. KARAYIANNIS, P.; FOWLER, M.J.F.; LOK, A.S.F.; GREENFIELD, C.; MONJARDINO, J. & THOMAS, H.C. - Detection of scrum HBV DNA by molecular hybridization: correlation with HBeAg/anti-HBe status, racial origin, liver histology and hepatocellular carcinoma. J. Hepat., 1: 99-106, 1985.

[16] 16. KORENMAN, J.; BAKER, B.; WAGGONER, J.; EVERHART, J.E.; Di BISCEGLIE, A.M. & HOOFNAGLE, J.H. - Long term remission of chronic hepatitis B after alpha-interferon therapy. Ann. intern. Med., 114: 629-634, 1991.

[17] 17. KWOK, S. & HIGUCHI, R. - Avoiding false positives with PCR. Nature, 339: 237-238, 1989.

[18] 18. LIEBERMANN, H.M.; LEBRECQUE, D.P.; KEW, M.C.; HADZIYANNIS, S.J. & SHAFRITZ, D.A. - Hepatologic detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatology, 3: 285-291, 1983.

[19] 19. PINHO, J.R.R.; FONSECA, L.E.P.; SONG, Y.; MIYAMOTO, Y.; CARRILHO, F.J.; GRANATO, C.F.H. & SILVA, L.C. - Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/anti-IIBe system, viral DNA polymerase and HBV-DNA. Rev. Inst. Med. trop. S. Paulo, 31: 328-335, 1989.

[20] 20. SAIKI, R.K.; GELFAND, D.H.; STOFFEL, S.; SCHARF, S.; HIGUCHI, R.; HORN, G.; MULLIS, K. & ERLICH, H. - Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491, 1988.

[21] 21. SILVA, L.C.; MADRUGA, C.L.A.; CARRILHO, F.J.; PINHO, J.R.R.; SAEZ-ALQUEZAR, A.; SANTOS, C.A.; BASSIT, L.; BARRETO, C.C.; ALVES, V.A.F.; GAYOTTO, L.C.C. & CHAMONE, D.A.F. - Spontaneous clearance (SC) of serum HBsAg in twenty patients with chronic hepatitis B (CHB): HBV-DNA status by Polymerase Chain Reaction (PCR) and role of viral superinfection. Gastroenterology, 104: A688, 1993.

[22] 22. SHERLOCK, S. - The natural history of hepatitis B. Postgrad. med. J., 63 (suppl. 2): 7-11, 1987.

[23] 23. SHIH, L.N.; SHEU, J.C.; WANG, J.T.; HUANG, G.T.; YANG, P.M.; LEE, H.S.; SUNG, J.L.; WANG, T.H. & CHEN, D.S. - Serum hepatitis B virus DNA in healthy HBsAg negative Chinese adults evaluated by polymerase chain reaction. J. med. Virol., 32: 257-260, 1990.

[24] 24. WANG, J.T.; WANG, T.H.; SHEU, J.C.; SHIH, L.N.; LIN, J.T. & CHEN, D.S.. - Detection of hepatitis B virus DNA by polymerase chain reaction in plasma of volunteer blood donors negative for hepatitis B surface antigen. J. infect. Dis., 163: 397-399, 1991.

[25] 25. ZOULIN, F.; MIMMS, L.; FLOREANI, M.; PICHOUD, C.; CHEMIN, I.; KAY, A.; VITVITSKI, L. & TREPO, C. - New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection. J. clin. Microbiol., 30: 1111- 1119, 1992.