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Long-term delta superinfection in hepatitis B surface antigen carriers and its relationship to the course of chronic hepatitis
GASTROENTEROLOGY
1983;85:235-9
Long-Term Delta Superinfection in Hepatitis B Surface Antigen Carriers and Its Relationship to the Course of Chronic Hepatitis MASSIMO COLOMBO, RITA CAMBIERI, MARIA GUIDO RONCHI, ERSILIO DEL NINNO, and ROBERTO DE FRANCHIS Clinica Medica 3 dell’Universit8 di Milano, Scuola di Specialita Ricambio, Via Pace 9 - 20122 Milano, Italy
To assess the prevalence and clinical significance of &infection in chronic hepatitis B surface antigen carriers, we examined 326 liver biopsies from 192 retrospectively selected carriers by immunofluorescence. Delta antigen was detected in 102 specimens from 50 carriers (26.2%) with peak prevalence in patients with active cirrhosis (51.5%) and generally in close association with progressive liver disease (94%). The antigen was located in the nuclei of .~‘~7&50% of the hepatocytes, without any disease-specific pattern of fluorescence. Patients with intrahepatic delta-antigen, however, had more severe liver disease than those without it. Histologic follow-up of 101 cases showed that the rates of worsening of the liver disease were similar in delta-antigen-positive and in delta-antigen-negative patients. It is concluded that delta-superinfection does play a role in worsening the histologic picture of hepatitis B surface antigen-positive chronic active hepatitis, possibly by liver injury induced acutely at the moment of infection. Since its original description (1977), the delta-agent (&agent) has been progressively recognized as a cause of acute and of chronic liver disease in carriers of the hepatitis B surface antigen (HBsAg)(l-3). Delta-infection is distributed worldwide, although it
Received September 27, 1982. Accepted February 24, 1983. Address requests for reprints to: Massimo Colombo, M.D., Clinica Medica 3, Via Pace, 9 - 20122 Milano, Italy. The authors thank Dr. Mario Rizzetto for the 6 antiserum and Dr. Benito Chinea (BBR, Milan) for the statistical analysis. 0 1983 by the American Gastroenterological Association 0016-5065/83/$3.00
GRAZIA
RUMI,
in Malattie de1 Fegato e de1
is most frequent in the Mediterranean area (2) and among drug addicts (4). Lately, evidence has been obtained that persistence of the &agent may be an important contributing factor to the deterioration of the hepatitic process, leading to chronic active hepatitis (CAH) and cirrhosis (4,s). The delta-antigen (6Ag) is almost exclusively localized within the liver cell nucleus, in association with infectious ribonucleic acid (RNA) particles (6). The recent demonstration that the antigenic reactivity of the &agent is well preserved in routine biopsy material (7) has enabled us to study &infection in chronic HBsAg carriers retrospectively. We determined the prevalence of intrahepatic 6Ag in HBsAg carriers with various degrees of liver damage. The correlation between 6Ag positivity and the severity of liver disease was calculated. In 101 patients for whom follow-up liver biopsy specimens were available, the relationship between 6Ag positivity and the course of the disease was also studied.
Material and Methods Patients
and Liver
Biopsies
Three hundred twenty-six formalin-fixed hepatic sections from 192 HBsAg carriers examined at our Institute since 1974 were examined by direct immunofluorescence (IF) for the presence of 6Ag. The epidemiologic characteristics of the patients are reported in Table 1.One hundred one patients with CAH or active cirrhosis (AC) (35 6Agpositive and 66 GAg-negative) were then followed-up for l-5 yr with serial liver biopsies [mean: 2.9 biopsies per patient). Initially, 62 patients (11 GAg-positive) were on steroid therapy (10-20 mg of methylprednisoneiday); in
236
Table
COLOMBO
GASTROENTEROLOGY
ET AL.
1. Epidemiologic Characteristics B Surface Antigen Carriers
Mean age (yr) ratio Origin Northern Italy Southern Italy Hemophiliacs Drug addicts Histologic lesions Normal liver Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis Hepatocellular carcinoma with associated cirrhosis MIF
of 192 Hepatitis 37 (8-72)
parametric data. The changes in HA1 scores in relation to therapy and S-infection were analyzed by the Wilcoxon test for paired data (11).
158134
Results
121 (63%) 71 (37%)
6
Prevalence of Delta-Antigen Surface Antigen Carriers
(3%)
4 (2%1 36 (19%)
22
(11%)
82 (42%) 33 (17%)
19 (10%)
the follow-up period, the same treatment was given to 20 additional patients with CAH or AC. The therapeutic criteria for these patients were not related to study protocol for &infection.
Methods All liver specimens were obtained with Tru-Cut (Travenol) or Menghini needles (Jamshidi), and were > 1.5 cm in length. For hepatocellular carcinoma (HCC) patients, the liver biopsy specimens were obtained at laparoscopy. In all cases, the liver disease was diagnosed following established criteria (8). In chronic hepatitis, the histologic activity was assessed blind by one of us using the numerical scoring system (HAI) of Knodell et al. (9). This system, which was originally designed to provide definitive endpoints for statistical analysis of serial changes in liver histology, combines scores for necrosis, inflammation, and fibrosis, and ranges from 0 to 22. The IF staining of 6Ag in deparaffinized sections was performed after 2 h of digestion of the specimen with 0.025% trypsin (Merck Sharp & Dohme, West Point, Pa.). After rinsing in phosphate-buffered saline, the sections were incubated with a fluorescent, high-titer antiserum to SAg (1:500), kindly provided by Dr. Rizzetto. The criteria for the specificity of this antiserum and its conjugation with fluorescein isothiocyanate were previously reported (10). The specificity controls for our IF system included a 6Agpositive liver and a SAg-negative, hepatitis B core antigen (HBcAg) positive liver. Blocking experiments of Gfluorescence were performed using another uncoupled, high-titer anti-&serum. Radioimmunoassay (RIA) for HBsAg, HBs antibody (anti-HBs), core antibody (anti-HBc), HB e antigen and antibody (HBeAg and anti-HBe) were carried out with commercial kits (Abbott, North Chicago, Ill.).
Statistical
Vol. 85, No. 2
in Hepatitis
B
Delta-antigen was detected in 102 liver biopsy specimens from 50 chronic HBsAg carriers (26.2%), with a peak prevalence in the patients with AC (51.5%) (Table 2). The majority of SAg-positive carriers were born in Northern Italy (68%) and were middle aged (Figure 1). Delta-antigen was localized within the liver cell nuclei in all but 1 patient [Figure 2). The latter was a patient with CAH in whom SAg was detected in both nucleus and cytoplasm. In the nucleus, the pattern of SAg fluorescence was always finely granular, with the nucleoli not involved, while in the cytoplasm, it was faint and scattered. Delta-antigen-positive liver cells ranged between 2% and 50% (mean: 11%) without any disease-specific pattern. In CAH and in AC, however, SAg was preferentially located in the periportal or paraseptal zones of the parenchyma-in one-third of the cases being homogenously spread all over the lobule. In HCC specimens, 6Ag was detected
only in the hepatic tissue surrounding the neoplastic foci. The prevalence of &infection appeared to correlate directly with the severity of the histologic lesions (Table 3): CAH or AC were found in 94% of the SAg-positive carriers and only in 61% of the SAgnegative patients (p < 0.001). Moreover, SAg-positive carriers with CAH or AC, or both, had higher HA1 scores than age-matched SAg-negative patients (p < 0.01) (Table 4). In these patients, however, the intrahepatic expression of SAg was unrelated to any
Fir
Analysis
The correlation between the pattern of intrahepatic &fluorescence and the HA1 score was calculated by the Spearman-Kendall analysis. The differences between HAI scores of GAg-positive patients and those of GAg-negative carriers were analyzed by the Kruskall-Wallis test for non-
l-10
11-20
2l-30
31-40
4150
51-50
w(K) AGE (Yr)
Figure
1. Age-related prevalence of delta infection in hepatitis B surface antigen carriers. Cross-hatching indicates 6Ag positive.
INTRAHEPATIC
August 1983
Table 2. Correlation Between
Intrahepatic
Delta-Antigen
and Hepatic
Lesions Serum
Number of patients
Liver histology Normal liver Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis Hepatic carcinoma
%
0
0
22
33 + 10
l/22
21122
3
13.6
a2 33 19
34 2 13 39 2 14 56 2 11
l/52 2126 1114
51152 24/26 13114
26 17 4
31.7 51.5 21.1
37 ” 148
71136
129/136
50
26.1
Positive n = 50
Discussion Immunofluorescence detection of 6Ag in routine biopsy material of retrospectively selected HBsAg carriers showed a high prevalence of 6 superinfection among these patients, and confirmed the original reports of a strong association between the &agent and progressive liver disease (1,2). This association constitutes an additional evidence of direct Gpathogenicity in HBV-related liver disease. The trend toward greater severity in &associated hepatitis may result from cumulative damage to the hepatocytes by the two agents. In chimpanzees (lo), they have distinct and possibly synergistic pathogenic mechanisms: damage by HBV is dependent on the immune response of the host (12), while that of 6 seems to result from a direct cell damage (13). However, we found no disease-specific patterns of immunohistology, nor any direct relationship between percent of GAg-positive cells and HA1 scores for hepatocellular inflammation or necrosis. The effects of Gsuperinfection on the course of HBV-related chronic hepatitis remain to be clarified. If continuing intrahepatic replication of the &agent has a damaging effect, one would expect &positive carriers to show more rapid progression of the liver lesions. Indeed, Recchia et al. (5) and Rizzetto et al. (14) showed that GAg-positive children with CAH progress to cirrhosis much more often than 6Ag-
in Status
Negative n = 142
n
%
n
%
26 17 4
52 34
a
56 16 15
39.4 11.2 10.6
47
94”
87
61.2”
x2 = 17.271913. CAH = chronic active hepatitis. HCC = hepatocellular carcinoma.
= active cirrhosis.
n
2Ol22
Table
6Ag status
a p < 0.001.
anti-HBe
2122
of Progressive Liver Disease 3. Prevalence Hepatitis B Surface Antigen Carriers, According to lntrahepatic Delta-Antigen
Total
6Ag
35 + 10
In both GAg-positive and SAg-negative patients, the histologic parameters of activity (HA1 scores) remained unchanged during follow-up, whether or not the patient was treated with steroids (Table 4). The same was true for the pattern of immunofluorescence in GAg-positive carriers. Nevertheless, in some of the patients, the histologic diagnoses of biopsy specimens obtained at follow-up did change from those of the initial biopsy (Table 5). In GAg-positive patients, the follow-up biopsy specimens did not change in 77% showed deterioration in 14% and slight improvement in 8%. In SAgnegative patients, the follow-up biopsy specimens did not change in 60% showed deterioration in 27%, and improvement in 12%. The differences in this respect between GAg-positive and GAg-negative patients were not statistically significant.
AC HCC + AC
Carriers
36
Follow-up of Carriers With Chronic Active Hepatitis or Active Cirrhosis
CAH
Antigen
237
HBV markers
HBeAg
WI
0.001).
Patients with
B Surface
Intrahepatic
of the parameters of histologic activity scored in the HA1 index. Eighteen of 35 (51%) GAg-positive carriers had past histories of acute hepatitis, as compared with 17 of 66 (25.7%) GAg-negative patients (p <
Table
in Hepatitis
Age
192
Total
DELTA AGENT IN HBsAg CARRIERS
AC
4. Follow-up Biopsies Hepatitis B Surface Liver Disease”
HBsAg positive CAH &positive &negative
Number of patients 35 66
in 101 Patients With Antigen Positive Chronic HA1 scores during (l-5 yr, mean:
follow-up 2.9 yr)
Initial
Final
13.5 2 4.5b.” 11 2 3.3”
13.6 + 4.7b 10.9 2 3.8
” Correlation between 6 infection and evolution of the histologic features of activity, scored by the Knodell index (HAI). b 6 positive vs. 6 negative: p < 0.01. c Inital vs. final: not significant. CAH = chronic active hepatitis.
238
COLOMBO
Figure
GASTROENTEROLOGY
ET AL.
2. Immunofluorescent demonstration Magnification, x250.
of delta
antigen
in paraffin
5. Histologic According
Evolution in 101 Patients with Hepatitis to Intrahepatic Delta-Antigen Status
B Surface
Intrahepatic Histologic evolution Unchanged
Deteriorated
Last follow-up
AC CAH CPH
AC CAH CPH
cirrhosis.
Positive
fluorescence
of different
Chronic
degrees.
Liver Disease Mean duration of follow-up (yr)
SAg status s(n = 66) 10 I 23
62.1%
8
s+
S-
2.9
1.8
3.8
1.7
2.5
2.5
1
3.5
2.5
2.5
13 1 ; 25.8%
CPH
CAH
li
CAH
CPH
3
CAH = chronic
nuclear
Antigen
(n fi_+3.5)
CAH
Improved AC = active
Initial diagnosis
showing
2
of deterioration were similar in SAg-positive and SAg-negative cases. This goes against the hypothesis that continuing intrahepatic replication of the 6 agent has an unfavorable effect on the course of HBVrelated chronic hepatitis. The discrepancy between our results and those of Rizzetto’s group (5,14) may derive in part from the different designs of the studies. A clue to the interpretation of the pathogenic role
negative children with CAH. In adults, combining data from four different centers, Recchia et al. and Rizzetto et al. observed a progression from CAH to cirrhosis, or from chronic persistent hepatitis (CPH) to CAH, in 50% of 66 SAg-positive patients followedup for about 3 yr. At variance with these results, only 14% of our SAg-positive patients, followed-up for a similar length of time, progressed from CAH to cirrhosis or from CPH to CAH. In addition, the rates Table
section
Vol. 85, No.
active
hepatitis.
CPH = chronic
4’ 8.6%
persistent
8
hepatitis.
12.10/r,
August
1983
of the S-agent in HBV-related chronic hepatitis might be derived from the observation that SAgpositive patients had histories of a previous episode of acute hepatitis much more frequently than SAg-negative patients (51% vs. 25.7%; p < 0.01). Possibly, the episode of acute hepatitis was the moment of superinfection with S. One might speculate, therefore, that S superinfection results in acute liver damage, worsening preexisting chronic hepatitis (15), with the further course of the disease being independent of the presence or absence of intrahepatic S-agent. It may be argued that the population in our followup study is skewed toward the patients with more severe inflammatory lesions, since it does not include follow-up biopsies from HBsAg carriers with normal liver histology. None of these latter subjects, however, had intrahepatic SAg, as also stated in the literature (16).
References Rizzetto M, Canese MG, Aricb S, et al. Immunofluorescence detection of a new antigen/antibody system (delta/anti-delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut 1977;18:997-1003. Rizzetto M, Purcell RH, Gerin JL. Epidemiology of HBVassociated delta antigen: geographical distribution and prevalence in politransfused HBsAg carriers. Lancet 198O;i:12158. Rizzetto M, Shih JWK, Gocke DJ, et al. Incidence and significance of antibodies to delta antigen in hepatitis B virus infection. Lancet 1979;ii:986-90. Raimondo G, Smedile A, Gallo L, et al. Multicentre study of prevalence of HBV-associated delta infection and liver disease in drug-addicts. Lancet 1982;i:249-51.
INTRAHEPATIC
DELTA
AGENT
IN HBsAg
CARRIERS
239
5. Recchia S, Farci P, Smedile A, et al. Chronic delta hepatitis in carriers of the HBsAg: an active and progressive disease unresponsive to treatment. 11 Fegato 1982;28(1):35-8. the 6. Rizzetto M, Hoyer B, Canese MG, et al. Delta antigen: association of delta antigen with hepatitis B surface antigen and ribonucleic acid in the serum of delta infected chimpanzees. Proc Nat1 Acad Sci USA 1980;77:6124-8. E, Gudat F, Krey G, et al. Delta-antigen in hepatitis 7. Stoecklin B: immunohistology of frozen and paraffin-embedded liver biopsies and relation to HBV-infection. Hepatology 1981; 1:238-42. S. Disease of the liver and 8. Leevy CM, Popper H, Sherlock biliary tract: standardization of nomenclature, diagnostic criteria and diagnostic methodology. Chicago: Year Book Medical Publishers Inc., 1976:9-21. RG, Ishak KG, Block WC, et al. Formulation and 9. Knodell application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;5:431-5. 10. Rizzetto M, Canese MG, Gerin JL, et al. Transmission of hepatitis B virus-associated delta antigen to chimpanzees. J Infect Dis 1980;141:590-602. 11. Steel RGD, Torrie JR. Principles and procedures of statistics. London: McGraw-Hill Book Co., 1960. 12. Bianchi L, Gudat F. Immunopathology of hepatitis B. In: Popper H, Schaffner F, eds. Progress in liver diseases. Vol VI. New York: Grune & Stratton, 1979:371-92. 13. Gerin JL, Ponzetto A, London WT, et al. Serial passage of the delta agent in chimpanzees. Fed Proc 1982;41(3):445. 14. Rizzetto M, Verme G, Recchia S, et al. Chronic HBsAg hepatitis with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med (in press). 15. Smedile A, Farci P, Verme G, et al. Influence of delta infection on severity of hepatitis B. Lancet 1982;ii:945-7. M, Crivelli 0, et al. The clinical and 16. Arico S, Rizzetto immunological significance of a new antigen/antibody system (delta/anti-delta] in chronic carriers of the HBsAg. Ital J Gastroenterol 1978;10:146-51.
1983;85:235-9
Long-Term Delta Superinfection in Hepatitis B Surface Antigen Carriers and Its Relationship to the Course of Chronic Hepatitis MASSIMO COLOMBO, RITA CAMBIERI, MARIA GUIDO RONCHI, ERSILIO DEL NINNO, and ROBERTO DE FRANCHIS Clinica Medica 3 dell’Universit8 di Milano, Scuola di Specialita Ricambio, Via Pace 9 - 20122 Milano, Italy
To assess the prevalence and clinical significance of &infection in chronic hepatitis B surface antigen carriers, we examined 326 liver biopsies from 192 retrospectively selected carriers by immunofluorescence. Delta antigen was detected in 102 specimens from 50 carriers (26.2%) with peak prevalence in patients with active cirrhosis (51.5%) and generally in close association with progressive liver disease (94%). The antigen was located in the nuclei of .~‘~7&50% of the hepatocytes, without any disease-specific pattern of fluorescence. Patients with intrahepatic delta-antigen, however, had more severe liver disease than those without it. Histologic follow-up of 101 cases showed that the rates of worsening of the liver disease were similar in delta-antigen-positive and in delta-antigen-negative patients. It is concluded that delta-superinfection does play a role in worsening the histologic picture of hepatitis B surface antigen-positive chronic active hepatitis, possibly by liver injury induced acutely at the moment of infection. Since its original description (1977), the delta-agent (&agent) has been progressively recognized as a cause of acute and of chronic liver disease in carriers of the hepatitis B surface antigen (HBsAg)(l-3). Delta-infection is distributed worldwide, although it
Received September 27, 1982. Accepted February 24, 1983. Address requests for reprints to: Massimo Colombo, M.D., Clinica Medica 3, Via Pace, 9 - 20122 Milano, Italy. The authors thank Dr. Mario Rizzetto for the 6 antiserum and Dr. Benito Chinea (BBR, Milan) for the statistical analysis. 0 1983 by the American Gastroenterological Association 0016-5065/83/$3.00
GRAZIA
RUMI,
in Malattie de1 Fegato e de1
is most frequent in the Mediterranean area (2) and among drug addicts (4). Lately, evidence has been obtained that persistence of the &agent may be an important contributing factor to the deterioration of the hepatitic process, leading to chronic active hepatitis (CAH) and cirrhosis (4,s). The delta-antigen (6Ag) is almost exclusively localized within the liver cell nucleus, in association with infectious ribonucleic acid (RNA) particles (6). The recent demonstration that the antigenic reactivity of the &agent is well preserved in routine biopsy material (7) has enabled us to study &infection in chronic HBsAg carriers retrospectively. We determined the prevalence of intrahepatic 6Ag in HBsAg carriers with various degrees of liver damage. The correlation between 6Ag positivity and the severity of liver disease was calculated. In 101 patients for whom follow-up liver biopsy specimens were available, the relationship between 6Ag positivity and the course of the disease was also studied.
Material and Methods Patients
and Liver
Biopsies
Three hundred twenty-six formalin-fixed hepatic sections from 192 HBsAg carriers examined at our Institute since 1974 were examined by direct immunofluorescence (IF) for the presence of 6Ag. The epidemiologic characteristics of the patients are reported in Table 1.One hundred one patients with CAH or active cirrhosis (AC) (35 6Agpositive and 66 GAg-negative) were then followed-up for l-5 yr with serial liver biopsies [mean: 2.9 biopsies per patient). Initially, 62 patients (11 GAg-positive) were on steroid therapy (10-20 mg of methylprednisoneiday); in
236
Table
COLOMBO
GASTROENTEROLOGY
ET AL.
1. Epidemiologic Characteristics B Surface Antigen Carriers
Mean age (yr) ratio Origin Northern Italy Southern Italy Hemophiliacs Drug addicts Histologic lesions Normal liver Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis Hepatocellular carcinoma with associated cirrhosis MIF
of 192 Hepatitis 37 (8-72)
parametric data. The changes in HA1 scores in relation to therapy and S-infection were analyzed by the Wilcoxon test for paired data (11).
158134
Results
121 (63%) 71 (37%)
6
Prevalence of Delta-Antigen Surface Antigen Carriers
(3%)
4 (2%1 36 (19%)
22
(11%)
82 (42%) 33 (17%)
19 (10%)
the follow-up period, the same treatment was given to 20 additional patients with CAH or AC. The therapeutic criteria for these patients were not related to study protocol for &infection.
Methods All liver specimens were obtained with Tru-Cut (Travenol) or Menghini needles (Jamshidi), and were > 1.5 cm in length. For hepatocellular carcinoma (HCC) patients, the liver biopsy specimens were obtained at laparoscopy. In all cases, the liver disease was diagnosed following established criteria (8). In chronic hepatitis, the histologic activity was assessed blind by one of us using the numerical scoring system (HAI) of Knodell et al. (9). This system, which was originally designed to provide definitive endpoints for statistical analysis of serial changes in liver histology, combines scores for necrosis, inflammation, and fibrosis, and ranges from 0 to 22. The IF staining of 6Ag in deparaffinized sections was performed after 2 h of digestion of the specimen with 0.025% trypsin (Merck Sharp & Dohme, West Point, Pa.). After rinsing in phosphate-buffered saline, the sections were incubated with a fluorescent, high-titer antiserum to SAg (1:500), kindly provided by Dr. Rizzetto. The criteria for the specificity of this antiserum and its conjugation with fluorescein isothiocyanate were previously reported (10). The specificity controls for our IF system included a 6Agpositive liver and a SAg-negative, hepatitis B core antigen (HBcAg) positive liver. Blocking experiments of Gfluorescence were performed using another uncoupled, high-titer anti-&serum. Radioimmunoassay (RIA) for HBsAg, HBs antibody (anti-HBs), core antibody (anti-HBc), HB e antigen and antibody (HBeAg and anti-HBe) were carried out with commercial kits (Abbott, North Chicago, Ill.).
Statistical
Vol. 85, No. 2
in Hepatitis
B
Delta-antigen was detected in 102 liver biopsy specimens from 50 chronic HBsAg carriers (26.2%), with a peak prevalence in the patients with AC (51.5%) (Table 2). The majority of SAg-positive carriers were born in Northern Italy (68%) and were middle aged (Figure 1). Delta-antigen was localized within the liver cell nuclei in all but 1 patient [Figure 2). The latter was a patient with CAH in whom SAg was detected in both nucleus and cytoplasm. In the nucleus, the pattern of SAg fluorescence was always finely granular, with the nucleoli not involved, while in the cytoplasm, it was faint and scattered. Delta-antigen-positive liver cells ranged between 2% and 50% (mean: 11%) without any disease-specific pattern. In CAH and in AC, however, SAg was preferentially located in the periportal or paraseptal zones of the parenchyma-in one-third of the cases being homogenously spread all over the lobule. In HCC specimens, 6Ag was detected
only in the hepatic tissue surrounding the neoplastic foci. The prevalence of &infection appeared to correlate directly with the severity of the histologic lesions (Table 3): CAH or AC were found in 94% of the SAg-positive carriers and only in 61% of the SAgnegative patients (p < 0.001). Moreover, SAg-positive carriers with CAH or AC, or both, had higher HA1 scores than age-matched SAg-negative patients (p < 0.01) (Table 4). In these patients, however, the intrahepatic expression of SAg was unrelated to any
Fir
Analysis
The correlation between the pattern of intrahepatic &fluorescence and the HA1 score was calculated by the Spearman-Kendall analysis. The differences between HAI scores of GAg-positive patients and those of GAg-negative carriers were analyzed by the Kruskall-Wallis test for non-
l-10
11-20
2l-30
31-40
4150
51-50
w(K) AGE (Yr)
Figure
1. Age-related prevalence of delta infection in hepatitis B surface antigen carriers. Cross-hatching indicates 6Ag positive.
INTRAHEPATIC
August 1983
Table 2. Correlation Between
Intrahepatic
Delta-Antigen
and Hepatic
Lesions Serum
Number of patients
Liver histology Normal liver Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis Hepatic carcinoma
%
0
0
22
33 + 10
l/22
21122
3
13.6
a2 33 19
34 2 13 39 2 14 56 2 11
l/52 2126 1114
51152 24/26 13114
26 17 4
31.7 51.5 21.1
37 ” 148
71136
129/136
50
26.1
Positive n = 50
Discussion Immunofluorescence detection of 6Ag in routine biopsy material of retrospectively selected HBsAg carriers showed a high prevalence of 6 superinfection among these patients, and confirmed the original reports of a strong association between the &agent and progressive liver disease (1,2). This association constitutes an additional evidence of direct Gpathogenicity in HBV-related liver disease. The trend toward greater severity in &associated hepatitis may result from cumulative damage to the hepatocytes by the two agents. In chimpanzees (lo), they have distinct and possibly synergistic pathogenic mechanisms: damage by HBV is dependent on the immune response of the host (12), while that of 6 seems to result from a direct cell damage (13). However, we found no disease-specific patterns of immunohistology, nor any direct relationship between percent of GAg-positive cells and HA1 scores for hepatocellular inflammation or necrosis. The effects of Gsuperinfection on the course of HBV-related chronic hepatitis remain to be clarified. If continuing intrahepatic replication of the &agent has a damaging effect, one would expect &positive carriers to show more rapid progression of the liver lesions. Indeed, Recchia et al. (5) and Rizzetto et al. (14) showed that GAg-positive children with CAH progress to cirrhosis much more often than 6Ag-
in Status
Negative n = 142
n
%
n
%
26 17 4
52 34
a
56 16 15
39.4 11.2 10.6
47
94”
87
61.2”
x2 = 17.271913. CAH = chronic active hepatitis. HCC = hepatocellular carcinoma.
= active cirrhosis.
n
2Ol22
Table
6Ag status
a p < 0.001.
anti-HBe
2122
of Progressive Liver Disease 3. Prevalence Hepatitis B Surface Antigen Carriers, According to lntrahepatic Delta-Antigen
Total
6Ag
35 + 10
In both GAg-positive and SAg-negative patients, the histologic parameters of activity (HA1 scores) remained unchanged during follow-up, whether or not the patient was treated with steroids (Table 4). The same was true for the pattern of immunofluorescence in GAg-positive carriers. Nevertheless, in some of the patients, the histologic diagnoses of biopsy specimens obtained at follow-up did change from those of the initial biopsy (Table 5). In GAg-positive patients, the follow-up biopsy specimens did not change in 77% showed deterioration in 14% and slight improvement in 8%. In SAgnegative patients, the follow-up biopsy specimens did not change in 60% showed deterioration in 27%, and improvement in 12%. The differences in this respect between GAg-positive and GAg-negative patients were not statistically significant.
AC HCC + AC
Carriers
36
Follow-up of Carriers With Chronic Active Hepatitis or Active Cirrhosis
CAH
Antigen
237
HBV markers
HBeAg
WI
0.001).
Patients with
B Surface
Intrahepatic
of the parameters of histologic activity scored in the HA1 index. Eighteen of 35 (51%) GAg-positive carriers had past histories of acute hepatitis, as compared with 17 of 66 (25.7%) GAg-negative patients (p <
Table
in Hepatitis
Age
192
Total
DELTA AGENT IN HBsAg CARRIERS
AC
4. Follow-up Biopsies Hepatitis B Surface Liver Disease”
HBsAg positive CAH &positive &negative
Number of patients 35 66
in 101 Patients With Antigen Positive Chronic HA1 scores during (l-5 yr, mean:
follow-up 2.9 yr)
Initial
Final
13.5 2 4.5b.” 11 2 3.3”
13.6 + 4.7b 10.9 2 3.8
” Correlation between 6 infection and evolution of the histologic features of activity, scored by the Knodell index (HAI). b 6 positive vs. 6 negative: p < 0.01. c Inital vs. final: not significant. CAH = chronic active hepatitis.
238
COLOMBO
Figure
GASTROENTEROLOGY
ET AL.
2. Immunofluorescent demonstration Magnification, x250.
of delta
antigen
in paraffin
5. Histologic According
Evolution in 101 Patients with Hepatitis to Intrahepatic Delta-Antigen Status
B Surface
Intrahepatic Histologic evolution Unchanged
Deteriorated
Last follow-up
AC CAH CPH
AC CAH CPH
cirrhosis.
Positive
fluorescence
of different
Chronic
degrees.
Liver Disease Mean duration of follow-up (yr)
SAg status s(n = 66) 10 I 23
62.1%
8
s+
S-
2.9
1.8
3.8
1.7
2.5
2.5
1
3.5
2.5
2.5
13 1 ; 25.8%
CPH
CAH
li
CAH
CPH
3
CAH = chronic
nuclear
Antigen
(n fi_+3.5)
CAH
Improved AC = active
Initial diagnosis
showing
2
of deterioration were similar in SAg-positive and SAg-negative cases. This goes against the hypothesis that continuing intrahepatic replication of the 6 agent has an unfavorable effect on the course of HBVrelated chronic hepatitis. The discrepancy between our results and those of Rizzetto’s group (5,14) may derive in part from the different designs of the studies. A clue to the interpretation of the pathogenic role
negative children with CAH. In adults, combining data from four different centers, Recchia et al. and Rizzetto et al. observed a progression from CAH to cirrhosis, or from chronic persistent hepatitis (CPH) to CAH, in 50% of 66 SAg-positive patients followedup for about 3 yr. At variance with these results, only 14% of our SAg-positive patients, followed-up for a similar length of time, progressed from CAH to cirrhosis or from CPH to CAH. In addition, the rates Table
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Vol. 85, No.
active
hepatitis.
CPH = chronic
4’ 8.6%
persistent
8
hepatitis.
12.10/r,
August
1983
of the S-agent in HBV-related chronic hepatitis might be derived from the observation that SAgpositive patients had histories of a previous episode of acute hepatitis much more frequently than SAg-negative patients (51% vs. 25.7%; p < 0.01). Possibly, the episode of acute hepatitis was the moment of superinfection with S. One might speculate, therefore, that S superinfection results in acute liver damage, worsening preexisting chronic hepatitis (15), with the further course of the disease being independent of the presence or absence of intrahepatic S-agent. It may be argued that the population in our followup study is skewed toward the patients with more severe inflammatory lesions, since it does not include follow-up biopsies from HBsAg carriers with normal liver histology. None of these latter subjects, however, had intrahepatic SAg, as also stated in the literature (16).
References Rizzetto M, Canese MG, Aricb S, et al. Immunofluorescence detection of a new antigen/antibody system (delta/anti-delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut 1977;18:997-1003. Rizzetto M, Purcell RH, Gerin JL. Epidemiology of HBVassociated delta antigen: geographical distribution and prevalence in politransfused HBsAg carriers. Lancet 198O;i:12158. Rizzetto M, Shih JWK, Gocke DJ, et al. Incidence and significance of antibodies to delta antigen in hepatitis B virus infection. Lancet 1979;ii:986-90. Raimondo G, Smedile A, Gallo L, et al. Multicentre study of prevalence of HBV-associated delta infection and liver disease in drug-addicts. Lancet 1982;i:249-51.
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5. Recchia S, Farci P, Smedile A, et al. Chronic delta hepatitis in carriers of the HBsAg: an active and progressive disease unresponsive to treatment. 11 Fegato 1982;28(1):35-8. the 6. Rizzetto M, Hoyer B, Canese MG, et al. Delta antigen: association of delta antigen with hepatitis B surface antigen and ribonucleic acid in the serum of delta infected chimpanzees. Proc Nat1 Acad Sci USA 1980;77:6124-8. E, Gudat F, Krey G, et al. Delta-antigen in hepatitis 7. Stoecklin B: immunohistology of frozen and paraffin-embedded liver biopsies and relation to HBV-infection. Hepatology 1981; 1:238-42. S. Disease of the liver and 8. Leevy CM, Popper H, Sherlock biliary tract: standardization of nomenclature, diagnostic criteria and diagnostic methodology. Chicago: Year Book Medical Publishers Inc., 1976:9-21. RG, Ishak KG, Block WC, et al. Formulation and 9. Knodell application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;5:431-5. 10. Rizzetto M, Canese MG, Gerin JL, et al. Transmission of hepatitis B virus-associated delta antigen to chimpanzees. J Infect Dis 1980;141:590-602. 11. Steel RGD, Torrie JR. Principles and procedures of statistics. London: McGraw-Hill Book Co., 1960. 12. Bianchi L, Gudat F. Immunopathology of hepatitis B. In: Popper H, Schaffner F, eds. Progress in liver diseases. Vol VI. New York: Grune & Stratton, 1979:371-92. 13. Gerin JL, Ponzetto A, London WT, et al. Serial passage of the delta agent in chimpanzees. Fed Proc 1982;41(3):445. 14. Rizzetto M, Verme G, Recchia S, et al. Chronic HBsAg hepatitis with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med (in press). 15. Smedile A, Farci P, Verme G, et al. Influence of delta infection on severity of hepatitis B. Lancet 1982;ii:945-7. M, Crivelli 0, et al. The clinical and 16. Arico S, Rizzetto immunological significance of a new antigen/antibody system (delta/anti-delta] in chronic carriers of the HBsAg. Ital J Gastroenterol 1978;10:146-51.