Journalof Hepatology, 1992; 14: 357-360
357
@ 1992 Elsevier SciencePublishers B.V. All rights reserved. 0168-8278/92/$05.00 HEPAT 01
ectio
rlinger and J.P. Service d’Ht?patologieand INSERM V-24, H6pitai Beaujon, Ciichy, France (Received
23 April 1991)
Reactivation of chronic hepatitis e reappearance of I-I V-DNA in serum. The purpose of the study was to determine whether, before reactivation, NA would be detectable in serum: using a sensitive procedure of detection, namely polymerase chain reaction (PCR). We studied 17 patients with chronic hepatitis B who experienced an episode of reactivation, defined by the reappearance of I-IBV-DNA in serum. None of these 17 sera had HBV-DNA demonstrable by dot-blot hybridization nor liquid hybridization in sera collected before reactivation. Using PCR, I-IBVDNA was detected, before reactivation, in 13 of the 17 episodes of reactivation tested with Southern -blot and hybridization. HBV-DNA was not detectable with PCR in the serum of four patients who subsequently experienced an episode of reactivation. In conclusion, our results show low level HBV replication before reactivation in most, but not all, I-IBs-positive, HBV-DNA-negative patients. This suggests that reactivation may occur even in patients with no I-IBV-DNA demonstrable in serum with PCR prior to reactivation.
In the natural history of chronic hepatitis B virus (HBV) infection, cessation of HBV replication with seroconversion from HBeAg to anti-HBe is usually followed by a marked improvement of clinical manifestations, biochemical disorders and histologic lesions (1,2). Reappearance of HBV replication reflected by detectable serum HBV-DNA defines reactivation with abrupt increases of serum alanine aminotransferase (3-16). In anti-I-IBe-positive chronic hepatitis reactivation accounts for 62.5% of the acute exacerbations (13), with an annual incidence ranging from 7% to 35% (7,13). No predictive factor for spontaneous reactivation has been identified. Before reactivation, serum HBV-DNA is not demonstrable by direct hybridization, whose sensitivity threshold is 1 pg HBV-DNA/ml(17,18). The purpose of the study was to determine whether, before reactivation, HBV-DNA could be demonstrated in Correspondence: S. Gayno, Service d’Hepatologie
serum, with a procedure that is more sensitive than direct hybridization, namely polymerase chain reaction.
Patients
We studied 17 patients (14 men, 3 women; mean age, 43 years, range, 22-68) who had had reactivation of chronic hepatitis B in 1987, 1988 or 1989. In this study, reactivation of chronic hepatitis B was defined by the reappearance of serum HBV-DNA, associated with an increase in serum ALT, whatever the HBeAg status. Before reactivation, patients were either HBeAg-positive or -negative. During reactivation in patients who were HBeAg-negative before reactivation, HBeAg might or might not reappear. In all patients, serum HBV-DNA
and INSERM U-24, HBpital Beaujon, 92110 Clichy,
France.
S. GAYNO et al.
358 was not demonstrated before, and was demonstrated during reactivation, with both dot-blot hybridization (17) and liquid hybridization (Genostics HBV-DNA, Abbott R) (18). During reactivation all patients had elevated serum ALT which was 3-times higher than the upper limit of the normal range. During reactivation, 11 patients were HBeAg-positive: HBeAg reappeared in seven, while four were HBeAg-positive before reactivation. Six patients were HBeAg-negative and anti-HBe-positive before and during reactivation. The delay between HBeAg semconversion and reactivation, known for six of the I? antiHBe-positive patients was, on average, 16 months (range 3 months to 3 years). Reactivation was spontaneous in 13 patients, related to immunosuppressive therapy in one, and associated with HIV infection in three. Eleven patients developed clini,cal symptoms during reactivation: asthenia in ten, jaundice in four, ascites in three, encephalopathy in two, fever in two, weight loss in two, arthralgia in one. The mean duration of reactivation was 4 months (range 1 to 24 months). Two patients died during reactivation. Three of these patients had experienced several episodes of reactivation (2, 3, and 5, respectively). Liver biopsy specimens were available during reactivation from 13 patients: histological examination showed cirrhosis in five, and chronic actke hepatitis with nuclear HBcAg staining
in all 13.
Methods Study of sera collected before reactivatioti.Sera col-
lected 2-29 months before reactivation (mean, 11 months) and kept frozen C-20 “C) were available for the 17 patients. PCR was performed as previously described (19,20). Two primers from a conserved region of the S gene were used, and gave an amplified fragment of 128 base pairs. Thirty cycles of 1 min denaturation at 90 “C, 1 min annealing at 50 “C, and 1 min extension at 72 “C were performed. Amplified HBV-DNA sequences were detected with Southern blot and ethidium bromure staining (PCR-EB), and with Southern blot hybridization (PCR-SBH) with a 400 nucleotides probe which included the amplified region. The sensitivity of PCR was determined with serial dilutions of a serum with a known amount of HBV-DNA: the end-point detection was 10v4 pg/mlPrevention of contamination. To prevent contamination, the recommended precautions were used (21). As controls, the following were studied in the same sessions: (a) sera from nine patients w& drug-induced acute hepatitis, collected in the same period and kept frozen togebher with the sera studied; (b) sera from nine subjects vaccinated against HBV; (c) aliquots of reagents and primers
used for the experiment. Results were only considered valid if they were consistent in two independent experiments without contamination of the controls.
ReSUlt.9 Detectionof serum HBV-DNA before reactivation
Before reactivation, serum HBV-DNA was demonstrated by PCR-SBH in 13 of the 17 patients (76%); and by PCR-EB in eight (44%), of the 17 patients tested (Table 1). Absence of contamination
According to the recommended precautions used to prevent contamination, the absence of contamination was ascertained by the absence of positive results with control sera, i.e. sera from subjects vaccinated against HBV, and sera from p.atients with liver disease not related with HBV.
Discussion
In the present study, we defined reactivation by the reappearance of serum HBV-DNA, associated with an increase in sz=rn ALT, whatever the HBeAg status before
TABLE 1 Serum HBeAg before and during reactivation, and HBV-DNA by PCR before reactivation, in 17 patients with reactivation of chronic hepatitis B Patients
HBeAg before reactivation
HBeAg during reactivation
Delay
HBV-DNA before reactivationb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ’ 16 17
+ + + + -
+ + + + + + + + + + +
13 9 8 16 7 13 9 10 9 12 7 11 8 8 2 29 3
+ + + + + + + + + + + + + -
a Delay in months between blood collection and reactivation. b Amplified sequences of HBV-DNA detectable after Southern blot and hydridization or after agarGse gel electrophoresis and ethidiurn bromide staining.
DETECTION
OF SERUM HBV-DNA BY PCR
359
and during reactivation. Indeed, in this study, four HBeAg-positive patients experienced dramatic increases in ALT levels and the reappearance of serum HBV-DNA considered as reactivation episodes. In addition, six patients experienced reactivation episodes without the reappearance of HBeAg suggesting that pre-core HBV mutants might be implicated. In this series of 17 patients with chronic hepatitis B who experienced an episode of reactivation, serum DNA with PCR before reactivation was demon most cases (76%). These results show that low le replication was present before reactivatrorl in most of our patients. In these patients, reactivation sterns to be linked more to an increase in viral replication than to a complete reversion from absence to presence of viral replication. Serum HBV-DNA was not demonstrated with PCR before reactivation in four patients. These patients did not differ from those with demonstrable serum HBV-DNA (Table 1). There was no significant ditierence in delay from collection of sera to reactivation, HBeAg status or timing of HBeAg seroconversion. To explain this result, we suggest two hypotheses: (i) viral replication was so low
V-DNA was not detectable with ; (ii) viral replication was fluctuating and HBV replication was interrupted at the time of serum sampling. It is worth noting that the reactivation of chronic hepatitis may occur even in patients in whom H t detectable with PCR in serum. Furthermore, the presence or absence of serum HBV-DNA, demonstrated with PCR in a patient does not seem to predict the risk of reactivation or not. Our results support previous studies on the prevalence of serum HBV-DNA detection with PCR in chronic hepatitis B (22-26). However, we did not detect HBV-DNA with ?CR in one HBeAg-positive patient. We have no explanation for this surprising and conflicting result confirmed in two independent experiments, but we cannot exclude the hypothesis of PCR inhibitors in the serum sample. In conclusion, this study shows: (i) the presence of a low level HBV replication in most patients with chronic hepatitis B before reactivation; and (ii) the possibility of reactivation in patients in whom HBV-DNA is not detectable with PCR in serum.
References
11 Lai My, Chen DS, Lee SC, et al. Rerctluation of hepatitis B vicus in anti-HBe-positive chronic active type B hepatitis: molec-
1 HoofnagIe JH, Dusheiko GM, Seeff LB, Jones EA, Waggoner JG, Bales ZB. Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981; 94: 744-8. 2 Fattovich G, Rugge M, Brollo L, et al. Clinical, virologic, and histologic outcome following seroconversion from HBeAg to anti-HBe in chronic hepatitis type B. Hepatology 1986; 6: 162-72. 3 Galbraith RM, Eddleston ALWF, Williams R, Zuckerman AJ, Bagshawe KD. Eulminant hepatic failure in leukaemia and choriocarcinoma related to withdrawal of cytotoxic drug therapy. Lancet 1975; ii: 528-30. 4 Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology 1984; 86: 230-X 5 Perrilio RP, Campbell CR, Sanders GE, Regenstein FG, Bodicky CJ. Spontaneous clearance and reactivation of hepatitis B virus infection among male homosexuals with chronic type B hepatitis. Ann Intern Med 1984; 100: 43-6. 6 Davis GL, Hoofnagle JH. Reactivation of chronic type B hepatitis presenting as acute viral hepatitis. Ann Intern Med 1985; 102: 762-5. 7 Davis GL, Hoofnagle JH. Reactivation of chronic hepatitis B virus infection. Gastroenterology 1987; 92: 2028-31. 8 Koike K, Iino S, Kurai K, Mitamura K, Endo Y, Oka H, IgM anti-HBc in anti-HBe-positive chronic type B hepatitis with acute exacerbations. Hepatology 1987; 7: 573-6. 9 Liaw YF, Tai Dl, Chu CM, Pao CC, Chen TJ. Acute exacerbation in chronic type B hepatitis: comparison between HBeAG and antibody-positive patients. Hepatology 1987; 7: 20-3. 10 Tong MJ, Sampliner RE, Govindarajan S, Co RL. Spontaneous reactivation of hepatitis B in Chinese patients with HBsAg-positive chronic active hepatitis. Hepatology 1987; 7: 713-8.
ular and immunohistochemical studies. Hepato-gastroenterology 1988; 35: 17-21. 12 Vento S, Di Petri G, Luxzati R, et al. Clinical reactivation of hep atitis P in anti-HBs-positive patients with AIDS. Lancet 1989; i: 332-3. 13 Gupta S, Govindarajan S, Fong TL, Redeker AG. Spontaneous reactivation in chronic hepatitis B: patterns and natural history. J Clin Gastroenterol1998; 12: 562-8. 14 Krogsgaard K, Aldershvile J, Kryger P, et al. Reactivation of viral replication in anti-HBe-positive chronic HBsAg carriers. Liver 1990; 10: 54-8. 15 Levy P, MarceIIin P, Martinot-Peignoux M, Degott C, Nataf J, Benhamou JP. Clinical course of spontaneous reactivation of hepatitis B virus infection, in patients with chronic hepatitis B. Hepatolonv 1990; 12: 570-4. 16 Lok AS, Lai C. Acute exacerbations in Chinese patients with chronic hepatitis B virus (HBV) infection: incidence, predisposing factors and etiology. J Hepatoll990; 10: 29-34. 17 Scotto, J, Hadchouel M, Hery C, Yvart J, Tiollais P, Brechot C. Detection of hepatitis B virus DNA in serum by a simple spot hybridtiation technique: comparison with results for other viral markers. Hepatology i983; 3: 279-84. 18 Kuhns MC, McNamara AL, Cabal CM, et al. A new assay for the quantitative detection of hepatitis B viral DNA in human serum. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 258-62. 19 Larzul D, Guigue F, Sninsky JJ, Mack DH, Brechot C, Guesdon JL. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods 1988; 20: 227-37. 20 Marcellin P, Martinet-Peignoux M, Loriot MA, et al. PersistenCe of hepatitis B virus DNA demonstrated by polymerase chain reaction in serum and liver after loss of HBsAg induced by zitiviral therapy. Ann Intern Med 1990; 112: 227-8.
360
21 Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. 22 Kaneko S, Miller R, Feinstone SM, et al. Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Nat1 Acad Sci USA 1989; 86: 312-6. 23 Liang TJ, Isselbacher KJ, Wands JR. Rapid identification of low level hepatitis B-related viral genome in serum. J Clin Invest 1989; 84: 1367-71. 24 Theihnann L, Fischer M, Galle P, Nassal M. Detection of HBV-
S. GAYNO et al.
DNA in HBsAg-positive sera after amplification using the poly merase chain reaction. Liver 1989; 9: 322-8. 25 Ulrich PP, Bhat RA, Seto B, Mack D, Sninsky J, Vyas GN. Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees. J Infect Dis 1989; 160: 37-43. 26 Zeldis JB, Lee JH, Mamish D, et al. Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction. J Clin Invest 1989; 84: 1503-g.
357
@ 1992 Elsevier SciencePublishers B.V. All rights reserved. 0168-8278/92/$05.00 HEPAT 01
ectio
rlinger and J.P. Service d’Ht?patologieand INSERM V-24, H6pitai Beaujon, Ciichy, France (Received
23 April 1991)
Reactivation of chronic hepatitis e reappearance of I-I V-DNA in serum. The purpose of the study was to determine whether, before reactivation, NA would be detectable in serum: using a sensitive procedure of detection, namely polymerase chain reaction (PCR). We studied 17 patients with chronic hepatitis B who experienced an episode of reactivation, defined by the reappearance of I-IBV-DNA in serum. None of these 17 sera had HBV-DNA demonstrable by dot-blot hybridization nor liquid hybridization in sera collected before reactivation. Using PCR, I-IBVDNA was detected, before reactivation, in 13 of the 17 episodes of reactivation tested with Southern -blot and hybridization. HBV-DNA was not detectable with PCR in the serum of four patients who subsequently experienced an episode of reactivation. In conclusion, our results show low level HBV replication before reactivation in most, but not all, I-IBs-positive, HBV-DNA-negative patients. This suggests that reactivation may occur even in patients with no I-IBV-DNA demonstrable in serum with PCR prior to reactivation.
In the natural history of chronic hepatitis B virus (HBV) infection, cessation of HBV replication with seroconversion from HBeAg to anti-HBe is usually followed by a marked improvement of clinical manifestations, biochemical disorders and histologic lesions (1,2). Reappearance of HBV replication reflected by detectable serum HBV-DNA defines reactivation with abrupt increases of serum alanine aminotransferase (3-16). In anti-I-IBe-positive chronic hepatitis reactivation accounts for 62.5% of the acute exacerbations (13), with an annual incidence ranging from 7% to 35% (7,13). No predictive factor for spontaneous reactivation has been identified. Before reactivation, serum HBV-DNA is not demonstrable by direct hybridization, whose sensitivity threshold is 1 pg HBV-DNA/ml(17,18). The purpose of the study was to determine whether, before reactivation, HBV-DNA could be demonstrated in Correspondence: S. Gayno, Service d’Hepatologie
serum, with a procedure that is more sensitive than direct hybridization, namely polymerase chain reaction.
Patients
We studied 17 patients (14 men, 3 women; mean age, 43 years, range, 22-68) who had had reactivation of chronic hepatitis B in 1987, 1988 or 1989. In this study, reactivation of chronic hepatitis B was defined by the reappearance of serum HBV-DNA, associated with an increase in serum ALT, whatever the HBeAg status. Before reactivation, patients were either HBeAg-positive or -negative. During reactivation in patients who were HBeAg-negative before reactivation, HBeAg might or might not reappear. In all patients, serum HBV-DNA
and INSERM U-24, HBpital Beaujon, 92110 Clichy,
France.
S. GAYNO et al.
358 was not demonstrated before, and was demonstrated during reactivation, with both dot-blot hybridization (17) and liquid hybridization (Genostics HBV-DNA, Abbott R) (18). During reactivation all patients had elevated serum ALT which was 3-times higher than the upper limit of the normal range. During reactivation, 11 patients were HBeAg-positive: HBeAg reappeared in seven, while four were HBeAg-positive before reactivation. Six patients were HBeAg-negative and anti-HBe-positive before and during reactivation. The delay between HBeAg semconversion and reactivation, known for six of the I? antiHBe-positive patients was, on average, 16 months (range 3 months to 3 years). Reactivation was spontaneous in 13 patients, related to immunosuppressive therapy in one, and associated with HIV infection in three. Eleven patients developed clini,cal symptoms during reactivation: asthenia in ten, jaundice in four, ascites in three, encephalopathy in two, fever in two, weight loss in two, arthralgia in one. The mean duration of reactivation was 4 months (range 1 to 24 months). Two patients died during reactivation. Three of these patients had experienced several episodes of reactivation (2, 3, and 5, respectively). Liver biopsy specimens were available during reactivation from 13 patients: histological examination showed cirrhosis in five, and chronic actke hepatitis with nuclear HBcAg staining
in all 13.
Methods Study of sera collected before reactivatioti.Sera col-
lected 2-29 months before reactivation (mean, 11 months) and kept frozen C-20 “C) were available for the 17 patients. PCR was performed as previously described (19,20). Two primers from a conserved region of the S gene were used, and gave an amplified fragment of 128 base pairs. Thirty cycles of 1 min denaturation at 90 “C, 1 min annealing at 50 “C, and 1 min extension at 72 “C were performed. Amplified HBV-DNA sequences were detected with Southern blot and ethidium bromure staining (PCR-EB), and with Southern blot hybridization (PCR-SBH) with a 400 nucleotides probe which included the amplified region. The sensitivity of PCR was determined with serial dilutions of a serum with a known amount of HBV-DNA: the end-point detection was 10v4 pg/mlPrevention of contamination. To prevent contamination, the recommended precautions were used (21). As controls, the following were studied in the same sessions: (a) sera from nine patients w& drug-induced acute hepatitis, collected in the same period and kept frozen togebher with the sera studied; (b) sera from nine subjects vaccinated against HBV; (c) aliquots of reagents and primers
used for the experiment. Results were only considered valid if they were consistent in two independent experiments without contamination of the controls.
ReSUlt.9 Detectionof serum HBV-DNA before reactivation
Before reactivation, serum HBV-DNA was demonstrated by PCR-SBH in 13 of the 17 patients (76%); and by PCR-EB in eight (44%), of the 17 patients tested (Table 1). Absence of contamination
According to the recommended precautions used to prevent contamination, the absence of contamination was ascertained by the absence of positive results with control sera, i.e. sera from subjects vaccinated against HBV, and sera from p.atients with liver disease not related with HBV.
Discussion
In the present study, we defined reactivation by the reappearance of serum HBV-DNA, associated with an increase in sz=rn ALT, whatever the HBeAg status before
TABLE 1 Serum HBeAg before and during reactivation, and HBV-DNA by PCR before reactivation, in 17 patients with reactivation of chronic hepatitis B Patients
HBeAg before reactivation
HBeAg during reactivation
Delay
HBV-DNA before reactivationb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ’ 16 17
+ + + + -
+ + + + + + + + + + +
13 9 8 16 7 13 9 10 9 12 7 11 8 8 2 29 3
+ + + + + + + + + + + + + -
a Delay in months between blood collection and reactivation. b Amplified sequences of HBV-DNA detectable after Southern blot and hydridization or after agarGse gel electrophoresis and ethidiurn bromide staining.
DETECTION
OF SERUM HBV-DNA BY PCR
359
and during reactivation. Indeed, in this study, four HBeAg-positive patients experienced dramatic increases in ALT levels and the reappearance of serum HBV-DNA considered as reactivation episodes. In addition, six patients experienced reactivation episodes without the reappearance of HBeAg suggesting that pre-core HBV mutants might be implicated. In this series of 17 patients with chronic hepatitis B who experienced an episode of reactivation, serum DNA with PCR before reactivation was demon most cases (76%). These results show that low le replication was present before reactivatrorl in most of our patients. In these patients, reactivation sterns to be linked more to an increase in viral replication than to a complete reversion from absence to presence of viral replication. Serum HBV-DNA was not demonstrated with PCR before reactivation in four patients. These patients did not differ from those with demonstrable serum HBV-DNA (Table 1). There was no significant ditierence in delay from collection of sera to reactivation, HBeAg status or timing of HBeAg seroconversion. To explain this result, we suggest two hypotheses: (i) viral replication was so low
V-DNA was not detectable with ; (ii) viral replication was fluctuating and HBV replication was interrupted at the time of serum sampling. It is worth noting that the reactivation of chronic hepatitis may occur even in patients in whom H t detectable with PCR in serum. Furthermore, the presence or absence of serum HBV-DNA, demonstrated with PCR in a patient does not seem to predict the risk of reactivation or not. Our results support previous studies on the prevalence of serum HBV-DNA detection with PCR in chronic hepatitis B (22-26). However, we did not detect HBV-DNA with ?CR in one HBeAg-positive patient. We have no explanation for this surprising and conflicting result confirmed in two independent experiments, but we cannot exclude the hypothesis of PCR inhibitors in the serum sample. In conclusion, this study shows: (i) the presence of a low level HBV replication in most patients with chronic hepatitis B before reactivation; and (ii) the possibility of reactivation in patients in whom HBV-DNA is not detectable with PCR in serum.
References
11 Lai My, Chen DS, Lee SC, et al. Rerctluation of hepatitis B vicus in anti-HBe-positive chronic active type B hepatitis: molec-
1 HoofnagIe JH, Dusheiko GM, Seeff LB, Jones EA, Waggoner JG, Bales ZB. Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981; 94: 744-8. 2 Fattovich G, Rugge M, Brollo L, et al. Clinical, virologic, and histologic outcome following seroconversion from HBeAg to anti-HBe in chronic hepatitis type B. Hepatology 1986; 6: 162-72. 3 Galbraith RM, Eddleston ALWF, Williams R, Zuckerman AJ, Bagshawe KD. Eulminant hepatic failure in leukaemia and choriocarcinoma related to withdrawal of cytotoxic drug therapy. Lancet 1975; ii: 528-30. 4 Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology 1984; 86: 230-X 5 Perrilio RP, Campbell CR, Sanders GE, Regenstein FG, Bodicky CJ. Spontaneous clearance and reactivation of hepatitis B virus infection among male homosexuals with chronic type B hepatitis. Ann Intern Med 1984; 100: 43-6. 6 Davis GL, Hoofnagle JH. Reactivation of chronic type B hepatitis presenting as acute viral hepatitis. Ann Intern Med 1985; 102: 762-5. 7 Davis GL, Hoofnagle JH. Reactivation of chronic hepatitis B virus infection. Gastroenterology 1987; 92: 2028-31. 8 Koike K, Iino S, Kurai K, Mitamura K, Endo Y, Oka H, IgM anti-HBc in anti-HBe-positive chronic type B hepatitis with acute exacerbations. Hepatology 1987; 7: 573-6. 9 Liaw YF, Tai Dl, Chu CM, Pao CC, Chen TJ. Acute exacerbation in chronic type B hepatitis: comparison between HBeAG and antibody-positive patients. Hepatology 1987; 7: 20-3. 10 Tong MJ, Sampliner RE, Govindarajan S, Co RL. Spontaneous reactivation of hepatitis B in Chinese patients with HBsAg-positive chronic active hepatitis. Hepatology 1987; 7: 713-8.
ular and immunohistochemical studies. Hepato-gastroenterology 1988; 35: 17-21. 12 Vento S, Di Petri G, Luxzati R, et al. Clinical reactivation of hep atitis P in anti-HBs-positive patients with AIDS. Lancet 1989; i: 332-3. 13 Gupta S, Govindarajan S, Fong TL, Redeker AG. Spontaneous reactivation in chronic hepatitis B: patterns and natural history. J Clin Gastroenterol1998; 12: 562-8. 14 Krogsgaard K, Aldershvile J, Kryger P, et al. Reactivation of viral replication in anti-HBe-positive chronic HBsAg carriers. Liver 1990; 10: 54-8. 15 Levy P, MarceIIin P, Martinot-Peignoux M, Degott C, Nataf J, Benhamou JP. Clinical course of spontaneous reactivation of hepatitis B virus infection, in patients with chronic hepatitis B. Hepatolonv 1990; 12: 570-4. 16 Lok AS, Lai C. Acute exacerbations in Chinese patients with chronic hepatitis B virus (HBV) infection: incidence, predisposing factors and etiology. J Hepatoll990; 10: 29-34. 17 Scotto, J, Hadchouel M, Hery C, Yvart J, Tiollais P, Brechot C. Detection of hepatitis B virus DNA in serum by a simple spot hybridtiation technique: comparison with results for other viral markers. Hepatology i983; 3: 279-84. 18 Kuhns MC, McNamara AL, Cabal CM, et al. A new assay for the quantitative detection of hepatitis B viral DNA in human serum. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 258-62. 19 Larzul D, Guigue F, Sninsky JJ, Mack DH, Brechot C, Guesdon JL. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods 1988; 20: 227-37. 20 Marcellin P, Martinet-Peignoux M, Loriot MA, et al. PersistenCe of hepatitis B virus DNA demonstrated by polymerase chain reaction in serum and liver after loss of HBsAg induced by zitiviral therapy. Ann Intern Med 1990; 112: 227-8.
360
21 Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. 22 Kaneko S, Miller R, Feinstone SM, et al. Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Nat1 Acad Sci USA 1989; 86: 312-6. 23 Liang TJ, Isselbacher KJ, Wands JR. Rapid identification of low level hepatitis B-related viral genome in serum. J Clin Invest 1989; 84: 1367-71. 24 Theihnann L, Fischer M, Galle P, Nassal M. Detection of HBV-
S. GAYNO et al.
DNA in HBsAg-positive sera after amplification using the poly merase chain reaction. Liver 1989; 9: 322-8. 25 Ulrich PP, Bhat RA, Seto B, Mack D, Sninsky J, Vyas GN. Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees. J Infect Dis 1989; 160: 37-43. 26 Zeldis JB, Lee JH, Mamish D, et al. Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction. J Clin Invest 1989; 84: 1503-g.
