Chronic Carriers of Hepatitis B Antigen (HBsAg) Histological, Biochemical, and Immunological Findings in 31 Voluntary Blood Donors J. P. V I L L E N E U V E , MD, G. RICHER, MD, PhD, J. C(3TI~, MD, R. GUt~VIN, MD, D. MARLEAU, MD, MSc, J. G. JOLY, MD, MSc, and A. VIALLET, MD
Among 289 HBsAg carriers detected by the Montreal Red Cross Blood Transfusion Service and seen by our group, 31 submitted voluntarily to liver biopsy. These 31 carriers have now been followed for 10-33 months (mean: 23) and all remained positive for HBsAg. 15 of these 31 subjects had lived in institutions during infancy or childhood and none were drug users. Histological examinations revealed 24 cases of chronic persistent hepatitis (CPH), 2 cases of chronic aggressive hepatitis, 2 with steatosis, and 3 with normal liver. On repeated determination, 16 of the 31 subjects had at least one elevated transaminase level. Transaminases levels could not be correlated with the histological diagnosis. 4 cases had positive antinuclear antibodies, all in the CPH group, a finding that could not be correlated with any clinical, biological, or histological findings. The search for other autoantibodies and the immunoglobulin determinations were totally unrewarding. Thus, it appears that chronic HBsAg carriers in Montreal voluntary blood donors often have chronic hepatitis, usually persistent, occasionally aggressive; liver biopsy still remains the most useful approach in the evaluation of these HBsAg carriers. The HBsAg-carrier state seems to be well tolerated, but further long-term studies are needed to understand the natural history of this condition.
In recent years, routine testing of blood donors for HBsAg has led to the identification of a large number of HBsAg carriers among the general population (1-3). Physicians consulted by these carriers cannot, at present, answer a number of questions related to the HBsAg-carrier state (4). Ongoing investigations will hopefully render this task easier. From the Departments of Medicine, Microbiology and Immunology, Pathology, Hematology, and from the Clinical Research Center, Hrpital Saint-Luc, and University of Montreal, Montreal, Canada. Supported in part by the Medical Research Council of Canada (MT-2061) and by a National Health Grant of Canada. Dr. J. P. Villeneuve is a recipient of a Medical Research Council Fellowship. Address for reprint requests: Dr. Andr6 Viallet, Director, Clinical Research Center, Hrpital Saint-Luc, 1058, St-Denis Street, Montreal, P. Que., Canada. 18
Of the many studies currently under way, an increasing number of reports relate to the liver histology of these carriers (5-18). However, there are wide discrepancies between these reports concerning the incidence, nature, and severity of liver abnormalities described in these HBsAg cartiers (5, 6, 9, 12). In 1971, the Canadian Red Cross Transfusion Service started routine screening for HBsAg of its voluntary blood donors by a nationwide standardized counterimmunoelectrophoretic technique (19). In Montreal, the reported incidence of HBsAg carriers is 0.51% (20), a much higher incidence than in the rest of Canada (1) and than in the U.S.A. (21). Between June, 1972, and March, 1974, 289 of these carriers volunteered to be seen by our group. The Digestive Diseases, Vol. 21, No. 1 (January 1976)
CHRONIC HBsAg CARRIERS
Fig 1. Chronic persistent hepatitis: dense mononuclear infiltrate not extending beyond the limits of the portal tract. (H & E, • 400)
characterization of the population studied and the analysis of the epidemiological factors within this population were reported elsewhere (22). The most important epidemiological data coming out of this study was that the reservoir of HBsAg carriers in Montreal was found mainly in the French-Canadian autochthonous population, half of whom lived in institutions at an early age. The present study was undertaken to assess the nature, frequency, and severity of liver disease in apparently healthy HBsAg chronic carriers in a volunteer group of these Montreal HBsAg-positive blood donors. Clinical, biochemical, and immunological parameters were included in this study. The immunological parameters were examined since it has been proposed recently that patients with chronic hepatitis could be divided into two main subDigestive Diseases, VoL 21, No. 1 (January 1976)
groups, those with positive HBsAg and those with positive autoantibodies (23-25).
MATERIALS AND METHODS Out of 289 HBsAg carriers detected by the Montreal Red Cross and seen by our group (22), 31 submitted voluntarily to percutaneous liver biopsy: the first 12 carriers were selected on the basis of a high transaminase value (over 60 units/ml) on at least one occasion; the 19 other volunteers were selected at random.
Clinical and Laboratory Study During a 48-hr period of hospitalization for liver biopsy, a complete history and physical examination were recorded. Laboratory procedures in all cases included: hemoglobin, white blood cell count, one-stage prothrombin time, platelet count, VDRL, alkaline phosphatase, total serum protein, protein electrophoresis, and se-
19
VILLENEUVE ET AL
Fig 2. Chronic persistent hepatitis: tocal accumulation of mononuclear cells in the lobular parenchyma. (H & E, x 630)
rum total bilirubin. Serum glutamic oxaloacetic (SGOT) and glutamic pyruvic transaminases (SGPT) were performed by standard laboratory procedures (26, 27): normal values in our hospital for SGOT and SGPT are 5-40 and 5-30 Karmen units, respectively. In the present study, we arbitrarily considered a value of 60 units/ml or more as being significantly elevated. Counterimmunoelectrophoresis was used for qualitative determination of HBsAg and a-foetoprotein (19). Antibody to HBsAg (anti-HBs) was searched for by passive hemagglutination (28) with commercially obtained material (Electro-Nucleonics, Bethesda, Maryland). Serum concentrations of IgG, IgA, and IgM were determined by a single radial immunodiffusion (Hyland Immunoplates). Rubella antibodies were measured by a hemagglutinationinhibition technique (29). The following antibodies were determined by the indirect immunofluorescence technique as described by our group in a previous report dealing with antimitochondrial antibody (30): antinuclear antibody (ANA) on rat liver, antimitochondrial antibody (AMA) on rat kidney, and smooth muscle antibody (SMA) and antiparietal cell antibody (PCA) on mouse
20
stomach, with a commercial preparation of fluoresceinconjugated goat antiserum to whole human immunoglobulins (Hyland). Sera were tested undiluted for PCA, at a starting dilution of 1/10 for ANA and AMA, and at 1/40 for SMA. Sera found positive by these methods were tritrated by doubling dilutions.
Histological Studies Liver biopsies were performed using the Menghini technique. All biopsies were reviewed as a whole by one pathologist. Chronic persistent hepatitis (CPH) and chronic aggressive hepatitis (CAH) were classified according to de Groote et al (31). CPH usually showed mild mononucleated infiltration of portal tract, little or no fibrosis, small foci of focal necrosis in liver parenchyma, and intact limiting plate. CAH was characterized by inflammatory infiltration by mononuclear cells, involving mainly the portal tracts, but also the parenchyma, and piecemeal necrosis. The term steatosis was used when variable degrees of fatty vacuolization of the parenchymal cells was the only demonstrable abnormality. Digestive Diseases, Vol. 21, No. 1 (January 1976)
CHRONIC HBsAg CARRIERS
Fig 3. Chronic aggressive hepatitis. Portal tract exhibiting periportal piecemeal necrosis. ( H & E, x 400)
RESULTS
Characterization of the Population Studied All the subjects were chronic carriers, and were followed by our group for 10-33 months (mean: 23); 29 were males and 2 were females. Their ages ranged from 18 to 50 years (mean: 31) at the time of their detection as HBsAg carriers. Three were born outside of Canada (France, Russia, and Haiti); all 28 others were Canadian-born of French origin. None admitted intravenous drug abuse. 15 out of 31 were orphans: 14 were placed in institutions as newborns or babies and 1 after the age of 5. Among the 16 others, 2 had a previous history of hepatitis, 3 had received blood transfusions, 2 had close contact with known cases of hepatitis, 2 had lived in the armed forces, 1 came from a family with multiple Digestive Diseases, Vol. 21, No. 1 (January 1976)
carriers, and 6 gave no relevant epidemiological history.
Histological Studies Most of the carriers (24/31) showed a histological picture compatible with a diagnosis of chronic persistent hepatitis (CPH) (Figures 1 and 2). Among the 7 other cases, 3 showed a normal histological picture, 2 had some degree of steatosis, and 2 presented chronic aggressive hepatitis (CAH) (Figure 3). The 24 carriers with CPH were subdivided in two groups: in 15 of them (group I), lesions were minimal, involving only some of the portal tracts and lobules; in the 9 other cases (group II), similar lesions were present in most of the lobules and portal tracts.
21
VILLENEUVE ET AL
Fig 4. Chronic persistent hepatitis: hepatocytes with ground-glass-appearing cytoplasm. (H & E, • 630)
The two cases of chronic aggressive hepatitis (CAH) had the typical features of piecemeal necrosis and diffuse infiltration by mononuclear cells mainly in the portal tracts. In 6 of these carriers, 2 with normal liver histology and 4 with CPH (group I), hepatocytes with "ground-glass"-appearing cytoplasm (32) could be seen (Figure 4).
Laboratory Studies As can be seen in Table 1, 8 carriers had abnormal transaminase values when first seen by us. During the follow-up period, each carrier had 311 transaminase determinations (mean: 5), and 8 more carriers presented at least one abnormal determination. The highest transaminase values (SGOT = 212, SGPT = 192) were observed in a
22
carrier with normal histology who had persistently high values on three separate determinations done over a period of 10 months. The 2 carriers with CAH had abnormal transaminase values when first seen by us: one has now been followed during 22 months, with 11 transaminase determinations, 2 of them being within normal range; in the other, followed for 28 months, 3 out of 7 transaminase determinations were within normal range. Of the 2 carriers with CAH, one was symptomatic, complaining of asthenia and anorexia, but with a normal physical examination; the other was asymptomatic but with a slight hepatomegaly. No other carrier was symptomatic, and hepatomegaly was noted in only one other carrier. The two carriers with steatosis admitted to moderate alcohol drinking; this fact was admitted to by only 8 other carriers. Digestive Diseases, Vol. 2l, No. 1 (January I976)
CHRONIC HBsAg CARRIERS TABLE 1. HISTOLOGICAL FINDINGS IN 31 HBsAG-PoSITIVE VOLUNTEER BLOOD DONORS
Hepatocytes with ' 'groundNumber glass" Histological o f cytodiagnosis carriers plasm Normal CPH I II CAH Steatosis Total
SGOT and~or SGPT > 60 U/ml Initial
Whole series
ANA positive
3
2
2
2
0
15 9 2 2 31
4 0 0 0 6
3 1 2 0 8
8 3 2 1 16
2 2 0 0 4
At the time of the biopsy, total blood count, platelet count, prothrombin time, total bilirubin, and alkaline phosphatase were normal in all carriers. Two had positive VDRL tests, one of them with a positive FTA-ABS and a past history of syphilis. During the follow-up period, 1-7 determinations (mean: 4) of the following immunological laboratory tests were performed on each carrier: all were negative for ~-foetoprotein, anti-HBs, AMA, SMA, and PCA. Rubella antibodies ranged from undetectable levels in one case to 1/80 in two cases; the titers observed were remarkably stable over the whole period of observation. Four carriers had detectable levels of ANA, and all four were in the CPH group (cf. Table 1); one was a female (CPH, group I) who had 6 determinations over a period of 2 years with titers of 1/40--I/80, without any clinical symptoms; the three others had transiently positive tests at the limit of detection of the technique (1/10). The serum concentrations of immunoglobulins were within normal limits in each group, with one of the two patients with CAH being in the upper limit of normal. However, similar values were also found in individual carriers in other groups. DISCUSSION
The prevalence of HBsAg-positive blood donors in Montreal is much higher than in the rest of Canada, and the main reservoir of HBsAg carriers comes from the autochthonous French-Canadian population (22), in contrast to similar studies done in Toronto where 66.6% of chronic carriers are of foreign origin (33). The carriers analyzed in the present study are taken from a larger epidemiological study Digestive Diseases, Vol. 21, No. 1 (January 1976)
of 289 carriers among Montreal volunteer blood donors (22). The distribution by age, sex, and ethnic origin is in good keeping with data reported for the larger study from which these carriers were taken. The most relevant epidemiological factor is that 15 of 31 had lived in institutions during infancy or childhood. This figure is also comparable with the 52% found in the epidemiological analysis of the larger study. This epidemiological factor underlines the fact that most of these carriers probably became infected during infancy and presumably have been HBsAg carriers for several years. This hypothesis, if confirmed, would tend to show, when contrasted with the remarkable lack of significant clinical, bi0chemical, and histological findings, that the HBsAgcarrier state is generally well tolerated for several years. It is interesting to note that the two carriers with chronic agressive hepatitis had not lived in an institution at an early age; thus, there is a presumption that they may have acquired the antigen later in life. On histological examination, 24 of 31 carriers showed chronic persistent hepatitis. In another Canadian study, similar results have been found by Feinman and co-workers (8), whereas in two Danish reports (10, 12) some 50% of the carriers had normal liver histology, data at variance with our own findings. In another report from Germany (9), a large study of 58 liver biopsies revealed that 33% of these carriers had normal liver histology, 8.6% had chronic persistent hepatitis, and the rest of these carriers showed various cytotoxic lesions; none of these carriers had chronic aggressive hepatitis. Other reports (6, 11, 16) indicate a higher incidence of more severe chronic liver disease such as chronic aggressive hepatitis and cirrhosis. Variations in populations studied could, in part, explain these dissimilar reports, and this has already been suggested by others (8, 34). Chronic cartiers of HBsAg in Montreal seemed to have some unusual characteristics as compared to other regions of Canada and of the United States. It is most noteworthy in the present study that none of these carriers were drug users. All were truly chronic carriers having been HBsAg positive for 10--33 months, and HBsAg having been shown to persist in all of them. This is in contrast with several reports where the populations studied were different: some of the carriers were paid blood donors (17), others were prisoners (5), known drug addicts (5, 17) or were known cases of acute viral hepatitis with a variable follow-up period (16).
23
VILLENEUVE ET AL
Although the histological interpretations (eg, minimal lesions, CPH, and "normal") may vary among the investigators, most chronic HBsAg carriers identified in routine screening among voluntary blood donors were found to have minimal liver abnormalities, while a few had chronic aggressive hepatitis. Our findings are in agreement with this data. Hepatocytes with "ground-glass" cytoplasm were detected in 6 carriers. These cells, presumably containing cytoplasmic HBsAg (32), were detected in 4 carriers with chronic persistent hepatitis, all in group I, and in two with otherwise normal liver histology. A higher proportion of carriers who underwent liver biopsy had initially elevated transaminases (8 of 31 or 25%) than in the large series from which they were selected, where only 6% showed initially elevated transaminases (22). This is explained by the bias initially introduced in the selection of carriers who underwent biopsy when this study was begun. On repeated transaminase determination performed on these 31 carriers, the number of subjects who showed at least one elevated transaminase value doubled from 8 to 16. Transaminase levels are known to fluctuate in patients with chronic persistent hepatitis (35-37) and possibly more frequent determinations could have shown elevated levels, at one time or another, in nearly all carriers. 3 cartiers were classified as having normal liver tissue on biopsy and, of these, two showed elevated transaminase levels. On the other hand, the two carriers with chronic aggressive hepatitis showed, at times, normal transaminase levels. This emphasizes the fact that one cannot rely on transaminase levels in order to determine which patients may have liver disease. Of the 31 patients, 4 had positive ANA, 3 at borderline low titers, and 1 with persisting titers of 1/40-1/80 over a 2-year period. This could not be correlated with any clinical event or with any biological evidence of aggravating liver disease. The significance of these abnormalities remains to be determined. The search for other immunological markers (immunoglobulin levels, mitochondrial antibodies, smooth muscle antibodies, parietal cell antibodies, rubella titers) were totally unrewarding. The level of rubella antibodies found in this study corresponds to that expected in the general population (29). These findings do not support the hypothesis that chronic HBsAg-associated liver disease may evolve into an autoimmune process.
24
Two carriers had chronic aggressive hepatitis One was symptomatic, the other had an enlarged liver. Both had fluctuating transaminase levels, sometimes normal and always below 200 Karmen units/ml; IgG levels were at the upper limits of normal in one of the carriers but both were negative for autoantibodies. These findings did not differentiate these two carriers from the others with or without histological abnormalities. In the present study, HBsAg carriers with chronic aggressive hepatitis could be distinguished from other carriers o.nly by liver biopsy. In conclusion, it appears that chronic HBsAg carriers among Montreal blood donors often have chronic hepatitis, usually persistent, occasionally aggressive. Extensive laboratory work-up appears to be unrewarding. Transaminase levels cannot be accurately correlated with a histological diagnosis, but they are the single parameter most likely to be abnormal. Since current biological testing cannot differentiate between patients with normal or near-normal histology and those with chronic hepatitis, persistent or aggressive, liver biopsy still remains one of the most useful approaches in the understanding of the evolution of liver disease associated with the HBsAg-carrier state. ACKNOWLEDGMENTS
We gratefully acknowledge the cooperation of many staff members from Hrpital Saint-Luc. We particularly wish to thank Mrs. Micheline Desrochers for her technical assistance and Miss C. Bessette and Mrs. F. Duchesne for typing the manuscript. REFERENCES 1. Moore BPL: Incidence of hepatitis B antigen and antibody among Canadian volunteer blood donors. Can Med Assoc J 107:396-399, 1972 2. Taswell HF: Incidence of HBAg in blood donors: An overview. Hepatitis and Blood Transfusion, GN Vyas, HA Perkins, R Schmid (eds). New York, Grune & Stratton, 1972, pp. 271-274 3. Cazal P, Robinet-Levy M, Blanc R, Lemaire JM: Grographie et drmographie de la virose Australia chez les sujets apparemment sains. Nouv Rev Fr Hematol 12:526-548, 1972 4. Chalmers TC, Alter HJ: Management of the asymptomatic Carrier of the hepatitis-associated (Australia) antigen. N Engl J Med 285:613-616, 1971 5. Bolin TD, Davis AE, Liddelow AG: Liver disease and cellmediated immunity in hepatitis-associated antigen (HAA) carriers. Gut 14:365-368, 1973 6. Eisenburg J, Meister P, Friihauf S, Greis I, Krumpoch B. Weinzierl M, Grunst J, Munte A, Rasch L: Untersuchungen zur hepatitish~tufigkeit bei klinisch gesunden Australia-antiDigestive Diseases, Vol. 21, No. 1 (January 1976)
CHRONIC HBsAg CARRIERS
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
18.
19.
20.
21.
gen-positiven blutspendern. Klin Wochenschr 51:1143-1150, 1973 Feinaru M, Levij IS: Asymptomatic hepatitis B antigen carviers: a clinicopathological study. Acta Hepato-Gastroenterol 21:185-191, 1974 Feinman SV, Cooter N, Sinclair JC, Wrobel DM, Berris B: Clinical and epidemiological significance of the HBsAg (Australia antigen): Carrier state. Gastroenterology 68:113-120, 1975 Klinge O, Kaboth U, Winckler K: Feingewebliche befunde an der leber klinisch gesunder Australia-antigen-(HB-Ag) Tr~iger. Virchows Arch A 361:35%368, 1973 lwarson S, Lindholm A, Lundin P, Hermodsson S: Hepatitis-associated antigen and antibody in Swedish blood donors. Vox Sang 22:501-509, 1972 Prince AM, Hargrove RL, Jeffries GH: The role of serum hepatitis virus in chronic liver disease. Trans Assoc Am Physicians 82:265-277, 1969 Reinicke V, Dybkjaer E, Poulsen H, Banke O, Lylloff K, Nordenfelt E: A study of Australia-antigen-p6sit~ve blood donors and their recipients, with sPecial reference to liver histology. N Engl J Med 286:867-870, 1972 Ricci G, De Bac C, Caramia F: Carriers of hepatitis B antigen: An epidemiologic and histologic study. J Infect Dis 128:125-128, 1973 Russell RI, G01dberg DM, Allan JG, MacSween RNM, Wallace J: A study of hepatic disease in Australia antigen and antibody-positive blood donors. Am J Dig Dis 19:113-121, 1974 Simon JB, Patel SK: Liver disease in asymptomatic carriers of hepatitis B antigen. Gastroenterology 66:1020-1028, 1974 Singleton JW, Fitch RA, Merrill DA, Kohler PF, Rettberg WAH: Liver disease in Australia-antigen-positive blood-donors. Lancet 2:785-787, 1971 Vittal SB, Dourdourekas D, Shobassy N, Gerber M, Telischi M, Szanto PB, Steigmann F, Clowdus BF: Asymptomatic hepatic disease in blood donors with hepatitis B antigenemia. Am J Clin Pathol 62:649-654, 1974 Woolf IL, Boyes BE, Jones DM, Whittaker JS, Tapp E, MacSween RNM, Renton PH, Stratton F, Dymock IW: Asymptomatic liver disease in hepatitis B antigen carriers. J Clin Pathol 27:348-352, 1974 Moore BPL, Meade D: Counter-immunoelectrophoresis for detection of hepatitis B antigen and antibody: A technique for large-scale use. Can J Public Health 63:453-459, 1972 Gurvin RM, Brown G, Taliano V: Donn6es statistiques concernant l'antigrne Australia pour la rrgion ouest du Qurbec. Un Med Can 102:1260-1263, 1973 Szmuness W, Prince AM, Brotman B, Hirsch RL: Hepatitis
Digestive Diseases, Vol. 21, No. 1 (January 1976)
22.
23. 24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34. 35. 36. 37.
B antigen and antibody in blood donors: an epii:lemiologic study. J Infect Dis 127:17-25, 1973 Richer G, Desrochers M, Gurvin R, Turgeon F, Viallet A: Hepatitis B antigen in Montrral blood donors: childhood institutionalization as an epidemiologic factor. Can Med Assoc J 112~4%52, 1975 Vischer TL: Australia antigen and autoantibodies in chronic hepatitis. Br Med J 2:695--698, 1970 Bulkley BH, Heizer WD, Goldfinger SE, Isselbacher KJ: Distinctions in chronic active hepatitis based on circulating hepatitis-associated antigen. Lancet 2:1323-1326, 1970 Finlayson NDC, Krohn K, Anderson KE, Jokelainen PT, Prince AM: Interrelations of hepatitis B antigen and autoantibodies in chronic idiopathic liver disease. Gastroenterology 63:646652, 1972 Karmen A, Wroblewski F, La Due JS: A note on the spectrophotometric assay of glutamic-oxaloacetic transaminase activity in human blood serum. J Clin Invest 34:131, 1955 Wroblewski F, La Due JS: Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc Soc Exp Biol Med 91:56%57i, 1956 Vyas GN, Shulman NR: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170:332-333, 1970 Richer G, Laverdirre M, Robert J, Turgeon F: Immunit6 contre la rubrole chez un groupe de femmes enceintes de la rrgion de Montrral: consid6rations sur la vaccination. Un Med Can 102:1104-1107, 1973 Richer G, Viallet A: Mitochondrial antibodies in extrahepatic biliary obstruction. Am J Dig Dis 19:740-744, 1974 de Groote J, Desmet V J, Gedigk P, Korb G, Popper H, Poulsen H, Scheuer PJ, Schmid M, Thaler H, Uehlinger E, Wepler W: A classification of chronic hepatitis, Lancet 2:626628, 1968 Hadziyannis S, Gerber MA, Vissoulis C, Popper H: Cytoplasmic hepatitis B antigen in "ground-glass" bepatocytes of carriers. Arch Pathol 96:327-330, 1973 Wrobel DM, Feinman V, Berris B, Sinclair J: Frequency of hepatitis B antigen in blood donors. Can Med Assoc J 108:570-572, 1973 Reinicke V, Dybkjaer E: Liver disease in carriers of Au antigen. Lancet 2:1032, 1971 Alpert LI, Vetancourt R: Chronic hepatitis. Arch Pathol 88:593-601, 1969 Becket MD, Scheuer PJ, Baptista A, Sherlock S: Prognosis of chronic persistent hepatitis. Lancet 1:53-56, 1970 Levine RA, Ranek L: Asymptomatic chronic hepatitis. Gastroenterology 58:371-378, 1970
25
Among 289 HBsAg carriers detected by the Montreal Red Cross Blood Transfusion Service and seen by our group, 31 submitted voluntarily to liver biopsy. These 31 carriers have now been followed for 10-33 months (mean: 23) and all remained positive for HBsAg. 15 of these 31 subjects had lived in institutions during infancy or childhood and none were drug users. Histological examinations revealed 24 cases of chronic persistent hepatitis (CPH), 2 cases of chronic aggressive hepatitis, 2 with steatosis, and 3 with normal liver. On repeated determination, 16 of the 31 subjects had at least one elevated transaminase level. Transaminases levels could not be correlated with the histological diagnosis. 4 cases had positive antinuclear antibodies, all in the CPH group, a finding that could not be correlated with any clinical, biological, or histological findings. The search for other autoantibodies and the immunoglobulin determinations were totally unrewarding. Thus, it appears that chronic HBsAg carriers in Montreal voluntary blood donors often have chronic hepatitis, usually persistent, occasionally aggressive; liver biopsy still remains the most useful approach in the evaluation of these HBsAg carriers. The HBsAg-carrier state seems to be well tolerated, but further long-term studies are needed to understand the natural history of this condition.
In recent years, routine testing of blood donors for HBsAg has led to the identification of a large number of HBsAg carriers among the general population (1-3). Physicians consulted by these carriers cannot, at present, answer a number of questions related to the HBsAg-carrier state (4). Ongoing investigations will hopefully render this task easier. From the Departments of Medicine, Microbiology and Immunology, Pathology, Hematology, and from the Clinical Research Center, Hrpital Saint-Luc, and University of Montreal, Montreal, Canada. Supported in part by the Medical Research Council of Canada (MT-2061) and by a National Health Grant of Canada. Dr. J. P. Villeneuve is a recipient of a Medical Research Council Fellowship. Address for reprint requests: Dr. Andr6 Viallet, Director, Clinical Research Center, Hrpital Saint-Luc, 1058, St-Denis Street, Montreal, P. Que., Canada. 18
Of the many studies currently under way, an increasing number of reports relate to the liver histology of these carriers (5-18). However, there are wide discrepancies between these reports concerning the incidence, nature, and severity of liver abnormalities described in these HBsAg cartiers (5, 6, 9, 12). In 1971, the Canadian Red Cross Transfusion Service started routine screening for HBsAg of its voluntary blood donors by a nationwide standardized counterimmunoelectrophoretic technique (19). In Montreal, the reported incidence of HBsAg carriers is 0.51% (20), a much higher incidence than in the rest of Canada (1) and than in the U.S.A. (21). Between June, 1972, and March, 1974, 289 of these carriers volunteered to be seen by our group. The Digestive Diseases, Vol. 21, No. 1 (January 1976)
CHRONIC HBsAg CARRIERS
Fig 1. Chronic persistent hepatitis: dense mononuclear infiltrate not extending beyond the limits of the portal tract. (H & E, • 400)
characterization of the population studied and the analysis of the epidemiological factors within this population were reported elsewhere (22). The most important epidemiological data coming out of this study was that the reservoir of HBsAg carriers in Montreal was found mainly in the French-Canadian autochthonous population, half of whom lived in institutions at an early age. The present study was undertaken to assess the nature, frequency, and severity of liver disease in apparently healthy HBsAg chronic carriers in a volunteer group of these Montreal HBsAg-positive blood donors. Clinical, biochemical, and immunological parameters were included in this study. The immunological parameters were examined since it has been proposed recently that patients with chronic hepatitis could be divided into two main subDigestive Diseases, VoL 21, No. 1 (January 1976)
groups, those with positive HBsAg and those with positive autoantibodies (23-25).
MATERIALS AND METHODS Out of 289 HBsAg carriers detected by the Montreal Red Cross and seen by our group (22), 31 submitted voluntarily to percutaneous liver biopsy: the first 12 carriers were selected on the basis of a high transaminase value (over 60 units/ml) on at least one occasion; the 19 other volunteers were selected at random.
Clinical and Laboratory Study During a 48-hr period of hospitalization for liver biopsy, a complete history and physical examination were recorded. Laboratory procedures in all cases included: hemoglobin, white blood cell count, one-stage prothrombin time, platelet count, VDRL, alkaline phosphatase, total serum protein, protein electrophoresis, and se-
19
VILLENEUVE ET AL
Fig 2. Chronic persistent hepatitis: tocal accumulation of mononuclear cells in the lobular parenchyma. (H & E, x 630)
rum total bilirubin. Serum glutamic oxaloacetic (SGOT) and glutamic pyruvic transaminases (SGPT) were performed by standard laboratory procedures (26, 27): normal values in our hospital for SGOT and SGPT are 5-40 and 5-30 Karmen units, respectively. In the present study, we arbitrarily considered a value of 60 units/ml or more as being significantly elevated. Counterimmunoelectrophoresis was used for qualitative determination of HBsAg and a-foetoprotein (19). Antibody to HBsAg (anti-HBs) was searched for by passive hemagglutination (28) with commercially obtained material (Electro-Nucleonics, Bethesda, Maryland). Serum concentrations of IgG, IgA, and IgM were determined by a single radial immunodiffusion (Hyland Immunoplates). Rubella antibodies were measured by a hemagglutinationinhibition technique (29). The following antibodies were determined by the indirect immunofluorescence technique as described by our group in a previous report dealing with antimitochondrial antibody (30): antinuclear antibody (ANA) on rat liver, antimitochondrial antibody (AMA) on rat kidney, and smooth muscle antibody (SMA) and antiparietal cell antibody (PCA) on mouse
20
stomach, with a commercial preparation of fluoresceinconjugated goat antiserum to whole human immunoglobulins (Hyland). Sera were tested undiluted for PCA, at a starting dilution of 1/10 for ANA and AMA, and at 1/40 for SMA. Sera found positive by these methods were tritrated by doubling dilutions.
Histological Studies Liver biopsies were performed using the Menghini technique. All biopsies were reviewed as a whole by one pathologist. Chronic persistent hepatitis (CPH) and chronic aggressive hepatitis (CAH) were classified according to de Groote et al (31). CPH usually showed mild mononucleated infiltration of portal tract, little or no fibrosis, small foci of focal necrosis in liver parenchyma, and intact limiting plate. CAH was characterized by inflammatory infiltration by mononuclear cells, involving mainly the portal tracts, but also the parenchyma, and piecemeal necrosis. The term steatosis was used when variable degrees of fatty vacuolization of the parenchymal cells was the only demonstrable abnormality. Digestive Diseases, Vol. 21, No. 1 (January 1976)
CHRONIC HBsAg CARRIERS
Fig 3. Chronic aggressive hepatitis. Portal tract exhibiting periportal piecemeal necrosis. ( H & E, x 400)
RESULTS
Characterization of the Population Studied All the subjects were chronic carriers, and were followed by our group for 10-33 months (mean: 23); 29 were males and 2 were females. Their ages ranged from 18 to 50 years (mean: 31) at the time of their detection as HBsAg carriers. Three were born outside of Canada (France, Russia, and Haiti); all 28 others were Canadian-born of French origin. None admitted intravenous drug abuse. 15 out of 31 were orphans: 14 were placed in institutions as newborns or babies and 1 after the age of 5. Among the 16 others, 2 had a previous history of hepatitis, 3 had received blood transfusions, 2 had close contact with known cases of hepatitis, 2 had lived in the armed forces, 1 came from a family with multiple Digestive Diseases, Vol. 21, No. 1 (January 1976)
carriers, and 6 gave no relevant epidemiological history.
Histological Studies Most of the carriers (24/31) showed a histological picture compatible with a diagnosis of chronic persistent hepatitis (CPH) (Figures 1 and 2). Among the 7 other cases, 3 showed a normal histological picture, 2 had some degree of steatosis, and 2 presented chronic aggressive hepatitis (CAH) (Figure 3). The 24 carriers with CPH were subdivided in two groups: in 15 of them (group I), lesions were minimal, involving only some of the portal tracts and lobules; in the 9 other cases (group II), similar lesions were present in most of the lobules and portal tracts.
21
VILLENEUVE ET AL
Fig 4. Chronic persistent hepatitis: hepatocytes with ground-glass-appearing cytoplasm. (H & E, • 630)
The two cases of chronic aggressive hepatitis (CAH) had the typical features of piecemeal necrosis and diffuse infiltration by mononuclear cells mainly in the portal tracts. In 6 of these carriers, 2 with normal liver histology and 4 with CPH (group I), hepatocytes with "ground-glass"-appearing cytoplasm (32) could be seen (Figure 4).
Laboratory Studies As can be seen in Table 1, 8 carriers had abnormal transaminase values when first seen by us. During the follow-up period, each carrier had 311 transaminase determinations (mean: 5), and 8 more carriers presented at least one abnormal determination. The highest transaminase values (SGOT = 212, SGPT = 192) were observed in a
22
carrier with normal histology who had persistently high values on three separate determinations done over a period of 10 months. The 2 carriers with CAH had abnormal transaminase values when first seen by us: one has now been followed during 22 months, with 11 transaminase determinations, 2 of them being within normal range; in the other, followed for 28 months, 3 out of 7 transaminase determinations were within normal range. Of the 2 carriers with CAH, one was symptomatic, complaining of asthenia and anorexia, but with a normal physical examination; the other was asymptomatic but with a slight hepatomegaly. No other carrier was symptomatic, and hepatomegaly was noted in only one other carrier. The two carriers with steatosis admitted to moderate alcohol drinking; this fact was admitted to by only 8 other carriers. Digestive Diseases, Vol. 2l, No. 1 (January I976)
CHRONIC HBsAg CARRIERS TABLE 1. HISTOLOGICAL FINDINGS IN 31 HBsAG-PoSITIVE VOLUNTEER BLOOD DONORS
Hepatocytes with ' 'groundNumber glass" Histological o f cytodiagnosis carriers plasm Normal CPH I II CAH Steatosis Total
SGOT and~or SGPT > 60 U/ml Initial
Whole series
ANA positive
3
2
2
2
0
15 9 2 2 31
4 0 0 0 6
3 1 2 0 8
8 3 2 1 16
2 2 0 0 4
At the time of the biopsy, total blood count, platelet count, prothrombin time, total bilirubin, and alkaline phosphatase were normal in all carriers. Two had positive VDRL tests, one of them with a positive FTA-ABS and a past history of syphilis. During the follow-up period, 1-7 determinations (mean: 4) of the following immunological laboratory tests were performed on each carrier: all were negative for ~-foetoprotein, anti-HBs, AMA, SMA, and PCA. Rubella antibodies ranged from undetectable levels in one case to 1/80 in two cases; the titers observed were remarkably stable over the whole period of observation. Four carriers had detectable levels of ANA, and all four were in the CPH group (cf. Table 1); one was a female (CPH, group I) who had 6 determinations over a period of 2 years with titers of 1/40--I/80, without any clinical symptoms; the three others had transiently positive tests at the limit of detection of the technique (1/10). The serum concentrations of immunoglobulins were within normal limits in each group, with one of the two patients with CAH being in the upper limit of normal. However, similar values were also found in individual carriers in other groups. DISCUSSION
The prevalence of HBsAg-positive blood donors in Montreal is much higher than in the rest of Canada, and the main reservoir of HBsAg carriers comes from the autochthonous French-Canadian population (22), in contrast to similar studies done in Toronto where 66.6% of chronic carriers are of foreign origin (33). The carriers analyzed in the present study are taken from a larger epidemiological study Digestive Diseases, Vol. 21, No. 1 (January 1976)
of 289 carriers among Montreal volunteer blood donors (22). The distribution by age, sex, and ethnic origin is in good keeping with data reported for the larger study from which these carriers were taken. The most relevant epidemiological factor is that 15 of 31 had lived in institutions during infancy or childhood. This figure is also comparable with the 52% found in the epidemiological analysis of the larger study. This epidemiological factor underlines the fact that most of these carriers probably became infected during infancy and presumably have been HBsAg carriers for several years. This hypothesis, if confirmed, would tend to show, when contrasted with the remarkable lack of significant clinical, bi0chemical, and histological findings, that the HBsAgcarrier state is generally well tolerated for several years. It is interesting to note that the two carriers with chronic agressive hepatitis had not lived in an institution at an early age; thus, there is a presumption that they may have acquired the antigen later in life. On histological examination, 24 of 31 carriers showed chronic persistent hepatitis. In another Canadian study, similar results have been found by Feinman and co-workers (8), whereas in two Danish reports (10, 12) some 50% of the carriers had normal liver histology, data at variance with our own findings. In another report from Germany (9), a large study of 58 liver biopsies revealed that 33% of these carriers had normal liver histology, 8.6% had chronic persistent hepatitis, and the rest of these carriers showed various cytotoxic lesions; none of these carriers had chronic aggressive hepatitis. Other reports (6, 11, 16) indicate a higher incidence of more severe chronic liver disease such as chronic aggressive hepatitis and cirrhosis. Variations in populations studied could, in part, explain these dissimilar reports, and this has already been suggested by others (8, 34). Chronic cartiers of HBsAg in Montreal seemed to have some unusual characteristics as compared to other regions of Canada and of the United States. It is most noteworthy in the present study that none of these carriers were drug users. All were truly chronic carriers having been HBsAg positive for 10--33 months, and HBsAg having been shown to persist in all of them. This is in contrast with several reports where the populations studied were different: some of the carriers were paid blood donors (17), others were prisoners (5), known drug addicts (5, 17) or were known cases of acute viral hepatitis with a variable follow-up period (16).
23
VILLENEUVE ET AL
Although the histological interpretations (eg, minimal lesions, CPH, and "normal") may vary among the investigators, most chronic HBsAg carriers identified in routine screening among voluntary blood donors were found to have minimal liver abnormalities, while a few had chronic aggressive hepatitis. Our findings are in agreement with this data. Hepatocytes with "ground-glass" cytoplasm were detected in 6 carriers. These cells, presumably containing cytoplasmic HBsAg (32), were detected in 4 carriers with chronic persistent hepatitis, all in group I, and in two with otherwise normal liver histology. A higher proportion of carriers who underwent liver biopsy had initially elevated transaminases (8 of 31 or 25%) than in the large series from which they were selected, where only 6% showed initially elevated transaminases (22). This is explained by the bias initially introduced in the selection of carriers who underwent biopsy when this study was begun. On repeated transaminase determination performed on these 31 carriers, the number of subjects who showed at least one elevated transaminase value doubled from 8 to 16. Transaminase levels are known to fluctuate in patients with chronic persistent hepatitis (35-37) and possibly more frequent determinations could have shown elevated levels, at one time or another, in nearly all carriers. 3 cartiers were classified as having normal liver tissue on biopsy and, of these, two showed elevated transaminase levels. On the other hand, the two carriers with chronic aggressive hepatitis showed, at times, normal transaminase levels. This emphasizes the fact that one cannot rely on transaminase levels in order to determine which patients may have liver disease. Of the 31 patients, 4 had positive ANA, 3 at borderline low titers, and 1 with persisting titers of 1/40-1/80 over a 2-year period. This could not be correlated with any clinical event or with any biological evidence of aggravating liver disease. The significance of these abnormalities remains to be determined. The search for other immunological markers (immunoglobulin levels, mitochondrial antibodies, smooth muscle antibodies, parietal cell antibodies, rubella titers) were totally unrewarding. The level of rubella antibodies found in this study corresponds to that expected in the general population (29). These findings do not support the hypothesis that chronic HBsAg-associated liver disease may evolve into an autoimmune process.
24
Two carriers had chronic aggressive hepatitis One was symptomatic, the other had an enlarged liver. Both had fluctuating transaminase levels, sometimes normal and always below 200 Karmen units/ml; IgG levels were at the upper limits of normal in one of the carriers but both were negative for autoantibodies. These findings did not differentiate these two carriers from the others with or without histological abnormalities. In the present study, HBsAg carriers with chronic aggressive hepatitis could be distinguished from other carriers o.nly by liver biopsy. In conclusion, it appears that chronic HBsAg carriers among Montreal blood donors often have chronic hepatitis, usually persistent, occasionally aggressive. Extensive laboratory work-up appears to be unrewarding. Transaminase levels cannot be accurately correlated with a histological diagnosis, but they are the single parameter most likely to be abnormal. Since current biological testing cannot differentiate between patients with normal or near-normal histology and those with chronic hepatitis, persistent or aggressive, liver biopsy still remains one of the most useful approaches in the understanding of the evolution of liver disease associated with the HBsAg-carrier state. ACKNOWLEDGMENTS
We gratefully acknowledge the cooperation of many staff members from Hrpital Saint-Luc. We particularly wish to thank Mrs. Micheline Desrochers for her technical assistance and Miss C. Bessette and Mrs. F. Duchesne for typing the manuscript. REFERENCES 1. Moore BPL: Incidence of hepatitis B antigen and antibody among Canadian volunteer blood donors. Can Med Assoc J 107:396-399, 1972 2. Taswell HF: Incidence of HBAg in blood donors: An overview. Hepatitis and Blood Transfusion, GN Vyas, HA Perkins, R Schmid (eds). New York, Grune & Stratton, 1972, pp. 271-274 3. Cazal P, Robinet-Levy M, Blanc R, Lemaire JM: Grographie et drmographie de la virose Australia chez les sujets apparemment sains. Nouv Rev Fr Hematol 12:526-548, 1972 4. Chalmers TC, Alter HJ: Management of the asymptomatic Carrier of the hepatitis-associated (Australia) antigen. N Engl J Med 285:613-616, 1971 5. Bolin TD, Davis AE, Liddelow AG: Liver disease and cellmediated immunity in hepatitis-associated antigen (HAA) carriers. Gut 14:365-368, 1973 6. Eisenburg J, Meister P, Friihauf S, Greis I, Krumpoch B. Weinzierl M, Grunst J, Munte A, Rasch L: Untersuchungen zur hepatitish~tufigkeit bei klinisch gesunden Australia-antiDigestive Diseases, Vol. 21, No. 1 (January 1976)
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