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Frequency and Significance of Immunoglobulin M Antibody to Hepatitis B Core Antigen in Corticosteroid-Treated Severe Chronic Active Hepatitis B
Frequency and Significance of Immunoglobulin M Antibody to Hepatitis B Core Antigen in Corticosteroid-Treated Severe Chronic Active Hepatitis B
ALBERT J . CZAJA, M.D., MARK T. S H I E L S , M.D.,* Division of Gastroenterology and Internal Medicine; HOWARD F. TASWELL, M.D., Division of Laboratory Medicine; J A M E S R. WOOD, M.D.,f Division of Gastroenterology and Internal Medicine; J Ü R G E N LUDWIG, M.D., Division of Pathology; ROBERT C. CHASE, B.S., Division of Laboratory Medicine
To a s s e s s t h e frequency a n d significance of immunoglobulin M (IgM) antibody t o h e p a t i t i s B core a n t i g e n (anti-HBc) in c o r t i c o s t e r o i d - t r e a t e d s e v e r e c h r o n i c active h e p a t i t i s B, w e t e s t e d 9 6 s e r u m s a m p l e s from 16 p a t i e n t s w h o w e r e s e r o p o s i t i v e for h e p a t i t i s B surface a n t i g e n (HBsAg) (group 1) a n d 8 H B s A g - n e g a t i v e , a n t i - H B c - p o s i t i v e p a t i e n t s (group 2) by enzyme-linked i m m u n o a s s a y . S a m p l e s o b t a i n e d in t h e p r e s e n c e a n d a b s e n c e of d i s e a s e activity before, d u r i n g , a n d after l o n g - t e r m corticosteroid t h e r a p y (mean d u r a t i o n , 42 ± 7 months) w e r e e v a l u a t e d . Seropositivity for IgM a n t i b o d y w a s demo n s t r a t e d in 12 g r o u p 1 p a t i e n t s , including 9 t e s t e d before c o r t i c o s t e r o i d t h e r a p y ; n o g r o u p 2 p a t i e n t s w e r e s e r o p o s i t i v e . Seropositivity w a s m o r e c o m m o n in s e r u m s a m p l e s o b t a i n e d d u r i n g active t h a n d u r i n g i n a c t i v e d i s e a s e (51% v e r s u s 22%; P<0.05) a n d m o r e frequent in s e r u m s a m p l e s t h a t c o n t a i n e d h e p a t i t i s B e a n t i g e n (46% v e r s u s 11%; P<0.02) a n d h e p a t i t i s B v i r u s deoxyribonucleic acid (50% v e r s u s 24%; P<0.05) t h a n in t h o s e w i t h o u t t h e s e m a r k e r s . I n some p a t i e n t s , seropositivity p e r s i s t e d o r r e c u r r e d i n t e r m i t t e n t l y d u r i n g c o r t i c o s t e r o i d t h e r a p y for up t o 57 m o n t h s . We conclude t h a t s e r o positivity for IgM a n t i b o d y c a n be d e m o n s t r a t e d frequently by e n z y m e - l i n k e d i m m u n o a s s a y in c o r t i c o s t e r o i d - t r e a t e d p a t i e n t s w i t h s e v e r e d i s e a s e . Seropositivity reflects active v i r u s r e p l i c a t i o n , a n d it is commonly a s s o c i a t e d w i t h i n f l a m m a t o r y activity. T h e d u r a t i o n of seropositivity m a y be p r o t r a c t e d d u r i n g l o n g - t e r m c o r t i c o s t e r o i d t h e r a p y .
Antibodies of t h e immunoglobulin M (IgM) class a g a i n s t t h e hepatitis B core a n t i g e n (IgM antiHBc) m a y be present in b o t h acute a n d chronic hepatitis B . 1 - 4 I n acute h e p a t i t i s B, t h e p r i m a r y IgM response p r e d o m i n a t e s early in t h e course of the infection. 5,6 L a t e r in t h e course of t h e disease, t h e i m m u n o g l o b u l i n G (IgG) a n t i b o d y becomes t h e p r e d o m i n a n t isotype a s t h e IgM
anti-HBc level wanes. 2 , 5 , 7 " 1 1 I n chronic h e p a t i t i s B, t h e frequency a n d significance of IgM antiH B c seropositivity a r e u n c e r t a i n i n a s m u c h a s several studies h a v e h a d d i s c r e p a n t findings. IgM a n t i b o d y h a s been detected frequently in p a t i e n t s with chronic active h e p a t i t i s (CAH) by r a d i o i m m u n o a s s a y 1 2 ' 1 3 b u t n o t by enzyme-linked immunoassay. 1 3 , 1 4 I n studies t h a t h a v e used t h e enzyme-linked i m m u n o a s s a y , t h e presence of IgM anti-HBc in C A H B h a s been correlated with
»Current address: Rockford Gastroenterology Associates, Rock-
l a b o r a t o r y f i n d i n g s of a c t i v e h e p a t o c e l l u l a r in-
tCurrent address: Digestive Healthcare, Minneapolis, Minnesota.
t i o n b y s o m e i n v e s t i g a t o r s 1 5 b u t n o t b y others. 1 3 ' 1 4
ford, Illinois.
flammation
Address reprint requests to Dr. A. J. Czaja, Division of Castro-
enterology, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 63:119-125,1988
a n d h e p a t i t i s B virus (HBV) replica-
D i s c o r d a n t r e s u l t s b e t w e e n s t u d i e s i n w h i c h dif-
ferent a s s a y s were used probably reflect varia119
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tions in the sensitivities of the assays, whereas differences between studies in which the same assay was used probably reflect variations in the patient populations being investigated. In this report, we assess the frequency and significance of IgM anti-HBc seropositivity by enzyme-linked immunoassay in patients with corticosteroid-treated CAH B who were selected by uniform criteria of severity of disease and were followed up systematically. In addition, we correlate the presence of IgM anti-HBc with indices of inflammatory activity and markers of virus replication, and we determine the applications and limitations of this assay in the assessment of such patients. Furthermore, we analyze the relationship among IgM anti-HBc, inflammatory activity, and serologic markers of HBV replication before, during, and after long-term corticosteroid treatment.
PATIENTS A N D METHODS Study Population.—Between 1967 and 1983,209 patients who fulfilled preestablished clinical, biochemical, and histologic criteria for severe CAH 16 were enrolled in a treatment program at our institution. Of these patients, 25 were seropositive for hepatitis B surface antigen (HBsAg). Serum and liver tissue samples had been obtained concurrently in 16 of these patients (group 1), and this material formed the basis of our report. Of the 184 patients in the treatment program who had HBsAg-negative CAH, 8 harbored antibodies to hepatitis B core and surface antigens (anti-HBc and anti-HBs) at the time of initial examination. Successive serum and liver tissue samples from these patients provided the opportunity to assess the frequency of IgM anti-HBc seropositivity in a group of patients with a different serologic profile but comparable disease severity (group 2). The clinical, biochemical, and histologic features of both groups of patients at accession are summarized in Table 1. All patients were Caucasian. Of the 24 patients, 18 (12 group 1 and 6 group 2 patients) had been assessed systematically for at least 5 years (mean, 139 ± 8 months). The other six patients were under surveillance from 0.5 to 35 months (mean, 18 ± 5 months). The mean duration of follow-up was 104 ± 14 months (range, 0.5 to 170 months) for the group
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1 patients and 118 ± 23 months (range, 20 to 200 months) for the group 2 patients. Treatment Regimens and Follow-Up.—The criteria for treatment with corticosteroids did not exclude patients with severe CAH B until after 1983. Consequently, all 16 group 1 patients had received prednisone in accordance with one of three treatment protocols. As maintenance therapy, three patients had received prednisone alone, 20 mg daily; nine had been treated with prednisone, 10 mg daily, in conjunction with azathioprine, 50 mg daily; and four had received prednisone on alternate days (mean dose, 20 mg every other day; range, 10 to 50 mg) with the dose adjusted to maintain normal serum aspartate aminotransferase values. The mean duration of corticosteroid therapy was 42 + 7 months (range, 0.5 to 91 months). As maintenance therapy for the eight group 2 patients, three had received prednisone alone, 20 mg daily; two had been treated with prednisone, 10 mg daily, and azathioprine, 50 mg daily; one had received prednisone on alternate days; and
Table 1.—Features at Time of Initial Examination of Anti-HBc-Positive Patients With (Group 1) and Without (Group 2) HBsAg* Group 1 Group 2 (N = 16) Feature (N = 8) Clinical findings Age (yr)t 49 ± 4 50 + 5 Duration of symptoms (mo)t 27 ± 9 12 ± 4 Sex (male:female) 16:0 3:5J Epidemiologie factors (no. of patients) Medical professionals 6 1 Homosexuals 1 0 Drug addicts 1 0 None 8 7 Biochemical findingsf AST (nl: <27 U/L) 507 ± 65 672 + 212 Bilirubin (nl: <1.1 mg/dl) 2.2 + 0.6 5.8 + 2.0 y-Globulin (nl: 0.7-1.6 g/dl) 2.3 + 0.2 2.6 ± 0.3 Albumin (nl: 3.1-4.3 g/dl) 3.5 ± 0.1 2.9 + 0.2§ Histologic findings (no. of patients) Periportal hepatitis 4 3 Bridging necrosis 6 0 Multilobular necrosis 1 0 Cirrhosis 5 5 *Anti-HBc = antibody to hepatitis B core antigen; AST = serum aspartate aminotransferase level; HBsAg = hepatitis B surface antigen; nl = normal laboratory value. tValues are expressed as the mean + the standard error of the mean. tSignificantly' different from group 1 (P<0.01). §Significantly different from group 1 (P<0.025).
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two had received placebo medication in accordance with our original randomized, controlled treatment trial. 16 The mean duration of corticosteroid therapy was 26 + 6 months (range, 17 to 59 months). All patients underwent assessment at regular 6-month intervals. At each examination, standard biochemical and serologic studies were performed, as described previously. 16 Serum samples were obtained at the time of entry into the study and at each follow-up visit during and after corticosteroid treatment. Specimens were frozen and stored at -20°C. Therapy was continued until previously reported criteria for remission, treatment failure, or drug intolerance had been satisfied. 16 Manifestations of relapse during or after discontinuation of corticosteroid therapy warranted reinstitution of the original treatment program. 16 Histologie interpretations were in accordance with previously published criteria. 17 Data Base.—The clinical course of each patient was assessed, and instances when the disease was active and inactive on the basis of predefined criteria were identified. Serum samples obtained at these times were analyzed for virologic markers, and the findings were correlated with the presence or absence of inflammatory activity. Results obtained before, during, and after corticosteroid therapy were compared. Disease activity was defined as histologic evidence of CAH or a serum aspartate aminotransferase level of more than 3 times the normal value. Our previous studies have demonstrated intraobserver consistency in the interpretation of the pattern and degree of histologic activity. 18,19 The reproducibility of such an interpretation by a single observer (J.L.) has been 88%. Additionally, elevation of the serum aspartate aminotransferase level to more than threefold normal during the course of severe CAH has, in our experience, been associated invariably with histologic features of CAH. 20 Ninety-six serum samples were selected from the 24 patients (mean, 4 ± 0.5 samples per patient; range, 1 to 8 samples), including 69 samples from the group 1 patients and 27 samples from the group 2 patients. Forty-nine samples had been obtained in the presence of inflammatory activity, and 47 samples had been obtained in the absence of inflammatory activity. Forty liver specimens had been obtained concurrently with
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the serum samples, including 29 from the group 1 patients and 11 from the group 2 patients. Virologic Assessments.—Each serum sample was tested for markers of HBV infection. Testing for HBsAg, anti-HBs, anti-HBc, and hepatitis B e antigen (HBeAg) and antibody to that antigen had been done in each sample by commercial radioimmunoassay or enzyme-linked immunoassay (Abbott Laboratories, North Chicago, Illinois) before the initiation of this study. Additionally, antibody to hepatitis delta virus had been sought previously in each sample by a blocking radioimmunoassay (Abbott Laboratories). These same serum samples were subsequently tested for hepatitis B virus deoxyribonucleic acid (HBV DNA) and IgM anti-HBc for the purposes of this analysis. Serum HBV DNA was sought in patients by a DNA hybridization assay that used nitrocellulose paper and a slot blotting technique similar to that described previously. 21,22 A 32P-labeled HBV probe (Bethesda Research Laboratories, Gaithersburg, Maryland) was used for the hybridization. Positive and negative serum controls, standards that contained known amounts of HBV DNA, and the serum samples to be assayed were tested in duplicate on each nitrocellulose strip. With use of this technique, a 1 pg/50 μΐ standard of HBV DNA could be detected routinely. The minimal amount of HBV DNA detected was 0.1 pg per spot after 48 hours of film exposure. IgM anti-HBc was measured by a solid-phase, commercially available, enzyme-linked immunoassay (Corzyme-M; Abbott Laboratories). Test samples were diluted at 1:1,000. Results were expressed as the absorbance value of the sample divided by a cutoff value (signal/cutoff ratio) that was calculated in accordance with the Abbott test instructions as 0.25 times the mean absorbance of the positive control plus the mean absorbance of the negative control. Test samples with absorbance values greater than the cutoff value were considered positive, and for IgM antiHBc positivity, a ratio of more than 1.0 was necessary. Statistics.—Chi-square analysis with Yates' correction was used for comparing dichotomous variables, and the unpaired t test was used to evaluate the significance of differences in means for continuous variables. The data are presented as the mean ± the standard error of the mean.
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RESULTS Frequency of IgM Anti-HBc Seropositivity.— The enzyme-linked immunoassay for IgM antiHBc was positive in 12 of the 16 group 1 patients and none of the 8 group 2 patients. The assay was positive in 9 of 14 group 1 patients who were tested at the time of initial examination. Three other group 1 patients were found to be positive during corticosteroid therapy, including two in whom the assay for IgM anti-HBc had been negative at the time of entry into the study. Of the 12 IgM anti-HBc-seropositive patients, 7 were seropositive on multiple occasions during and, in two instances, after corticosteroid therapy (mean frequency of seropositivity, 3 ± 0.4; range, 2 to 5 seropositive tests). In two patients, the assay became negative and then positive again during 36 and 37 months of corticosteroid treatment, respectively. In one of these patients, the fluctuation in seropositivity continued after withdrawal of treatment. In the five other patients, the assay remained positive in from two to four successive serum samples obtained during 6 to 57 months of corticosteroid therapy (mean, 21 ± 8 months). Relationship Between IgM Anti-HBc and Disease Activity.—IgM anti-HBc was present in 26 of 69 blood samples (38%) obtained from the 16 group 1 patients and none of 27 blood samples obtained from the 8 group 2 patients. The mean signal/cutoff ratio of samples positive for IgM anti-HBc was 1.9 + 0.2 (range, 1.1 to 5.2). IgM anti-HBc was detected in 19 of 37 samples (51%) collected from the 16 group 1 patients at times when active disease was present (Table 2). IgM anti-HBc was present more frequently in serum samples obtained when the disease was active than when it was inactive (19 of 37 versus 7 of 32; P<0.05). IgM anti-HBc seropositivity, however, did not preclude inactive disease. Of the seven samples in which IgM anti-HBc was detected in the absence of inflammatory activity (Table 2), five showed seropositivity during corticosteroid treatment. HBV DNA was present in three of the serum samples, and HBeAg was detected in all five. In the two instances in which IgM anti-HBc seropositivity accompanied inactive disease after corticosteroid treatment, HBV DNA and HBeAg were both absent. In serum samples obtained from the eight group 2 patients, IgM antibody was absent during periods of active (0 of 12) and inactive disease
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(0 of 15). HBV DNA was not detected in any of the 27 serum samples obtained from these patients. Relationship Between IgM Anti-HBc and Other HBV Markers in HBsAg-Positive CAH.—IgM anti-HBc was detected with greater frequency in HBeAg-positive than in HBeAgnegative samples (23 of 50 versus 2 of 18; P<0.02). It was also found more frequently in samples with HBV DNA than in those without HBV DNA (16 of 32 versus 8 of 34; P<0.05). IgM anti-HBc was absent in all three serum specimens that were positive for antibody to hepatitis delta virus. As shown in Table 3, the absence of IgM antiHBc in serum samples obtained from group 1 patients did not preclude the presence of HBV DNA (38%), HBeAg (63%), or the combination of HBV DNA and HBeAg (50%). IgM Anti-HBc and Corticosteroid Treatment.—Long-term corticosteroid therapy did not uniformly affect the serum levels of IgM anti-HBc (Fig. 1). Only 10 of 20 serum samples obtained in the presence of inflammatory activity during corticosteroid treatment were seropositive for IgM anti-HBc (Fig. 1). IgM anti-HBc seropositivity accompanied manifestations of inflammatory activity almost as commonly during corticosteroid therapy as at the time of initial examination (Table 2). Additionally, the degree of seropositivity in the samples assessed during corticosteroid therapy was similar to that of samples obtained initially (Fig. 1). Because corticosteroid therapy was discontinued after resolution of inflammatory activity, most serum samples obtained during this interval were associated with inactive disease (Fig. 1). In 12 of 14 such instances, IgM anti-HBc sero-
Table 2.—Frequency of IgM Anti-HBc Seropositivity in Serum Samples Obtained From HBsAg-Positive Patients During Active and Inactive Disease* Serum sampling period (no. of patients) At initial Corticosteroid therapy Finding examination During After IgM anti-HBc positive of those with active disease 9 of 14 10 of 20 Oof 3 IgM anti-HBc positive of those with inactive disease OofO 5 of 18 2 of 14 *HBsAg = hepatitis B surface antigen; IgM anti-HBc = immunoglobulin M antibody to hepatitis B core antigen.
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negativity was found. The IgM anti-HBc assay was also negative in all three instances in which inflammatory activity was present after discontinuation of corticosteroid treatment. DISCUSSION Our study indicates that IgM anti-HBc can frequently be detected by enzyme-linked immunoassay in patients with severe HBsAg-positive CAH. IgM anti-HBc seropositivity was demonstrated in 12 of 16 HBsAg-positive patients (group 1), including 9 of 14 patients who were tested at the time of initial examination and before corticosteroid therapy. Seropositivity was established in 38% of selected serum samples. IgM anti-HBc seropositivity was more common in the presence of active disease than inactive disease, and it was usually associated with the presence of HBV DNA and HBeAg in the serum. These findings suggest that active HBV replication continues to stimulate the production of a specific IgM antibody to the hepatitis B core antigen and that, in our patients, replicative activity was frequently associated with inflammatory activity. Previous studies have indicated that replicative activity does not necessarily correlate with inflammatory activity and that, in patients who may be immune tolerant of the virus, high levels of virus replication may be accompanied by minimal or no inflammatory change (for example, the chronic carrier state). 23 Because our patients were selected on the basis of the severity of inflammatory activity rather than on the degree of virus replication, our findings may not be valid in patients with lesser degrees of inflammatory activity. Importantly, HBsAg-negative, anti-HBc-positive patients (group 2) with severe disease at the time of entry into the study remained seronegative for IgM anti-
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HBc and HBV DNA throughout the period of observation. These findings indicate that IgM anti-HBc seropositivity is not a manifestation of inflammatory activity alone. IgM anti-HBc seronegativity did not preclude active virus replication. Indeed, 16 of 32 serum samples positive for HBV DNA and 27 of 50 samples positive for HBeAg were negative for IgM anti-HBc. The variability of the association between IgM anti-HBc and HBV DNA and HBeAg probably reflects differences in the sensitivities of the various assays for replicative activity and the uncertain effect of corticosteroids on each assay. A high level of virus replication may be necessary before the enzyme-linked immunoassay for IgM anti-HBc becomes positive, and fluctuations in replicative activity above and below a certain threshold may account for the absence or intermittency of IgM anti-HBc seropositivity. Similarly, IgM anti-HBc seronegativity did not preclude the presence of inflammatory activity. Five of the 14 serum specimens obtained at the time of initial examination when the liver disease was most active were negative for IgM anti-HBc, and 13 of 23 serum specimens obtained in the presence of disease activity during and after corticosteroid therapy were also negative for IgM anti-HBc (Table 2). IgM anti-HBc seronegativity in the presence of inflammatory activity may occur in chronic HBsAg-positive carriers with a superimposed non-A, non-B infection or other type of liver injury. 24 In our experience, however, seronegativity commonly occurred in patients who did not have an epidemiologic basis for non-A, non-B hepatitis or a historical basis for toxic liver injury. Additionally, IgM anti-HBc seronegativity was rarely accompanied by seropositivity for antibody to hepatitis delta virus, and it was frequently associated with HBV DNA
Table 3.—Frequency of HBV DNA and HBeAg in Serum Samples Obtained From HBsAg-Positive Patients With and Without IgM Anti-HBc* IgM anti-HBc (+) samples IgM anti-HBc (-) samples (N = 26) (N = 43) Seropositive Seronegative Serologie finding Seropositive Seronegative HBV DNA 16 8 16 26 HBeAg 23 2 27 16 HBV DNA and HBeAg 16 2 16 16 *HBeAg = hepatitis B e antigen; HBsAg = hepatitis B surface antigen; HBV DNA = hepatitis B virus deoxyribonucleic acid; IgM anti-HBc = immunoglobulin M antibody to hepatitis B core antigen.
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6.0 5.0
-
4.0
-
3.0
-
2.0
-
▲ Active disease Δ Inactive disease
Φ .Q
CO O
~°
a
1.0
Normal cutoff
<1 At presentation
Corticosteroid treatment
No corticosteroid treatment
Fig. 1. Levels of immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) in serum samples obtained from 16 patients positive for hepatitis B surface antigen in presence and absence of disease activity and corticosteroid therapy. Levels "At presentation" were determined immediately before corticosteroid therapy. A signal/cutoff ratio that exceeded 1.0 was considered a positive test result by enzymelinked immunoassay.
and HBeAg in the serum. Insensitivity of the assay for HBV replication rather than superimposed non-B infection or other type of liver injury is the most likely explanation for the discordant findings. IgM anti-HBc seropositivity was demonstrated in 7 of 32 serum samples obtained in the absence of inflammatory activity. Seropositivity in the absence of disease activity during corticosteroid therapy was usually accompanied by markers of virus replication, and this finding probably connoted immune tolerance of the virus by the host. 23 Seropositivity in the absence of disease activity after discontinuation of corticosteroid treatment was not accompanied by markers of virus replication; this finding probably connoted incomplete clearance of IgM anti-HBc after cessation of HBV replication. 2,3 Because the results of the enzyme-linked immunoassay for IgM anti-HBc were not closely associated with markers of virus replication or disease activity, the use of this test to assess these aspects of CAH B is limited. Studies in which a radioimmunoassay was used for detection of IgM anti-HBc have demonstrated a much closer association among IgM anti-HBc
seropositivity, markers of virus replication, and inflammatory activity than we have found by using the enzyme-linked immunoassay. 13,25 Long-term corticosteroid therapy is no longer used in the routine management of severe CAH B, but our findings do provide additional insight into the effects of such therapy on the markers of virus replication. IgM anti-HBc seropositivity could persist or recur intermittently during therapy (for up to 57 months, as demonstrated in our study), and in two of our patients, it developed after institution of corticosteroid therapy. These findings are supportive of other studies that have indicated that long-term corticosteroid therapy may delay clearance of the hepatitis virus. 26,27 Previous investigations have shown that the enzyme-linked immunoassay for IgM anti-HBc is a useful test for confirming the diagnosis of acute hepatitis B and distinguishing acute hepatitis B from chronic carrier states with superimposed non-A, non-B or delta virus infection.24 Our study underscores the potential difficulty of relying on this test alone to distinguish acute hepatitis B from CAH B with severe inflammatory activity. Recent studies have suggested that patients with
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acute hepatitis B have mainly 19S IgM anti-HBc and those with CAH B have predominantly 7 to 8S IgM anti-HBc. 25 Perhaps future studies will demonstrate that separation of the different types of serum IgM has diagnostic value. ACKNOWLEDGMENT We thank Linda S. Grande for assistance in the preparation of the submitted manuscript. REFERENCES 1. Brzosko WJ, Mikulska B, Cianciara J, Babiuch L: Immunoglobulin classes of antibody to hepatitis B core antigen. J Infect Dis 132:1-5, 1975 2. Cohen BJ: The IgM antibody responses to the core antigen of hepatitis B virus. J Med Virol 3:141-149, 1978 3. Tedder RS, Wilson-Croome R: IgM-antibody response to the hepatitis B core antigen in acute and chronic hepatitis B. J Hyg (Lond) 86:163-172, 1981 4. Dormeyer HH, Arnold W, Kryger P, Nielsen JO, Meyer zum Büschenfelde KH: IgM antibody to hepatitis B core antigen (anti-HB c IgM) in "healthy" HBsAg carriers: a longitudinal study of 75 cases. Klin Wochenschr 59:675678,1981 5. Lemon SM, Gates NL, Simms TE, Bancroft WH: IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 143:803-809,1981 6. Chau KH, Hargie MP, Decker RH, Mushahwar IK, Overby LR: Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 3:142-149,1983 7. Taswell HF, Czaja AJ, Nelson CA: Viral hepatitis: diagnostic test using anti-HBc (IgM). Mayo Clin Proc 60:488489,1985 8. Roggendorf M, Deinhardt F, Frösner GG, Scheid R, Bayerl B, Zachoval R: Immunoglobulin M antibodies to hepatitis B core antigen: evaluation of enzyme immunoassay for diagnosis of hepatitis B virus infection. J Clin Microbiol 13:618-626,1981 9. Kryger P, Mathiesen LR, Aldershvile J, Nielsen JO, Copenhagen Hepatitis Acuta Programme: Presence and meaning of anti-HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology 1:233-237,1981 10. Gerlich WH, Lüer W, Thomssen R, Study Group for Viral Hepatitis of the Deutsche Forschungsgemeinschaft: Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J Infect Dis 142:95-101,1980 11. Gitnick G: Immunoglobulin M hepatitis B core antibody: to titer or not to titer? to use or not to use? (editorial). Gastroenterology 84:653-655,1983 12. Feinman SV, Overby LR, Berris B, Chau K, Schable CA, Maynard JE: The significance of IgM antibodies to hepatitis B core antigen in hepatitis B carriers and hepatitis B-associated chronic liver disease. Hepatology 2:795-799,1982
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13. Sjogren M, Hoofnagle JH: Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis. Gastroenterology 89:252-258,1985 14. Govindarajan S, Ashcavai M, Chau KH, Nevalainen DE, Peters RL: Evaluation of enzyme immunoassay for antiHBc IgM in the diagnosis of acute hepatitis B virus infection. Am J Clin Pathol 82:323-325,1984 15. Nowicki MJ, Tong MJ, Nair PV, Stevenson D: Detection of anti-HBc IgM following prednisone treatment in patients with chronic active hepatitis B virus infection. Hepatology 4:1129-1133,1984 16. Soloway RD, Summerskill WHJ, Baggenstoss AH, Geall MG, Gitnick GL, Elveback LR, Schoenfield LJ: Clinical, biochemical, and histological remission of severe chronic active liver disease: a controlled study of treatments and early prognosis. Gastroenterology 63:820-833,1972 17. De Groote J, Desmet VJ, Gedigk P, Korb G, Popper H, Poulsen H, Scheuer PJ, Schmid M, Thaler H, Uehlinger E, Wepler W: A classification of chronic hepatitis. Lancet 2:626-628,1968 18. Soloway RD, Baggenstoss AH, Schoenfield LJ, Summerskill WHJ: Observer error and sampling variability tested in evaluation of hepatitis and cirrhosis by liver biopsy. Am J Dig Dis 16:1082-1086,1971 19. Czaja AJ, Davis GL, Ludwig J, Taswell HF: Complete resolution of inflammatory activity following corticosteroid treatment of HBsAg-negative chronic active hepatitis. Hepatology 4:622-627,1984 20. Czaja AJ, Wolf AM, Baggenstoss AH: Laboratory assessment of severe chronic active liver disease during and after corticosteroid therapy: correlation of serum transaminase and gamma globulin levels with histologic features. Gastroenterology 80:687-692,1981 21. Berninger M, Hammer M, Hoyer B, Gerin JL: An assay for the detection of the DNA genome of hepatitis B virus in serum. J Med Virol 9:57-68,1982 22. Wahl G: Rapid detection of DNA and RNA using Slot Blotting (Application Update #371). Keene, New Hampshire, Schleicher and Schuell, 1983 23. Chu CM, Karayiannis P, Fowler MJF, Monjardino J, Liaw Y-F, Thomas HC: Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum. Hepatology 5:431-434,1985 24. Perrillo RP, Chau KH, Overby LR, Decker RH: Antihepatitis B core immunoglobulin M in the serologic evaluation of hepatitis B virus infection and simultaneous infection with type B, delta agent, and non-A, nonB viruses. Gastroenterology 85:163-167,1983 25. Sjogren MH, Bancroft WH, Hoofnagle JH, Sosebee JL, Lemon SM: Clinical significance of low molecular weight (7-8S) immunoglobulin M antibody to hepatitis B core antigen in chronic hepatitis B virus infections. Gastroenterology 91:168-173,1986 26. Davis GL, Czaja AJ, Taswell HF, Ludwig J, Go VLW: Hepatitis B virus replication in steroid-treated severe HBsAg-positive chronic active hepatitis. Dig Dis Sei 30:97-103,1985 27. Realdi G, Alberti A, Rugge M, Bortolotti F, Rigoli AM, Tremolada F, Ruol A: Seroconversion from hepatitis B e antigen to anti-HB e in chronic hepatitis B virus infection. Gastroenterology 79:195-199,1980
ALBERT J . CZAJA, M.D., MARK T. S H I E L S , M.D.,* Division of Gastroenterology and Internal Medicine; HOWARD F. TASWELL, M.D., Division of Laboratory Medicine; J A M E S R. WOOD, M.D.,f Division of Gastroenterology and Internal Medicine; J Ü R G E N LUDWIG, M.D., Division of Pathology; ROBERT C. CHASE, B.S., Division of Laboratory Medicine
To a s s e s s t h e frequency a n d significance of immunoglobulin M (IgM) antibody t o h e p a t i t i s B core a n t i g e n (anti-HBc) in c o r t i c o s t e r o i d - t r e a t e d s e v e r e c h r o n i c active h e p a t i t i s B, w e t e s t e d 9 6 s e r u m s a m p l e s from 16 p a t i e n t s w h o w e r e s e r o p o s i t i v e for h e p a t i t i s B surface a n t i g e n (HBsAg) (group 1) a n d 8 H B s A g - n e g a t i v e , a n t i - H B c - p o s i t i v e p a t i e n t s (group 2) by enzyme-linked i m m u n o a s s a y . S a m p l e s o b t a i n e d in t h e p r e s e n c e a n d a b s e n c e of d i s e a s e activity before, d u r i n g , a n d after l o n g - t e r m corticosteroid t h e r a p y (mean d u r a t i o n , 42 ± 7 months) w e r e e v a l u a t e d . Seropositivity for IgM a n t i b o d y w a s demo n s t r a t e d in 12 g r o u p 1 p a t i e n t s , including 9 t e s t e d before c o r t i c o s t e r o i d t h e r a p y ; n o g r o u p 2 p a t i e n t s w e r e s e r o p o s i t i v e . Seropositivity w a s m o r e c o m m o n in s e r u m s a m p l e s o b t a i n e d d u r i n g active t h a n d u r i n g i n a c t i v e d i s e a s e (51% v e r s u s 22%; P<0.05) a n d m o r e frequent in s e r u m s a m p l e s t h a t c o n t a i n e d h e p a t i t i s B e a n t i g e n (46% v e r s u s 11%; P<0.02) a n d h e p a t i t i s B v i r u s deoxyribonucleic acid (50% v e r s u s 24%; P<0.05) t h a n in t h o s e w i t h o u t t h e s e m a r k e r s . I n some p a t i e n t s , seropositivity p e r s i s t e d o r r e c u r r e d i n t e r m i t t e n t l y d u r i n g c o r t i c o s t e r o i d t h e r a p y for up t o 57 m o n t h s . We conclude t h a t s e r o positivity for IgM a n t i b o d y c a n be d e m o n s t r a t e d frequently by e n z y m e - l i n k e d i m m u n o a s s a y in c o r t i c o s t e r o i d - t r e a t e d p a t i e n t s w i t h s e v e r e d i s e a s e . Seropositivity reflects active v i r u s r e p l i c a t i o n , a n d it is commonly a s s o c i a t e d w i t h i n f l a m m a t o r y activity. T h e d u r a t i o n of seropositivity m a y be p r o t r a c t e d d u r i n g l o n g - t e r m c o r t i c o s t e r o i d t h e r a p y .
Antibodies of t h e immunoglobulin M (IgM) class a g a i n s t t h e hepatitis B core a n t i g e n (IgM antiHBc) m a y be present in b o t h acute a n d chronic hepatitis B . 1 - 4 I n acute h e p a t i t i s B, t h e p r i m a r y IgM response p r e d o m i n a t e s early in t h e course of the infection. 5,6 L a t e r in t h e course of t h e disease, t h e i m m u n o g l o b u l i n G (IgG) a n t i b o d y becomes t h e p r e d o m i n a n t isotype a s t h e IgM
anti-HBc level wanes. 2 , 5 , 7 " 1 1 I n chronic h e p a t i t i s B, t h e frequency a n d significance of IgM antiH B c seropositivity a r e u n c e r t a i n i n a s m u c h a s several studies h a v e h a d d i s c r e p a n t findings. IgM a n t i b o d y h a s been detected frequently in p a t i e n t s with chronic active h e p a t i t i s (CAH) by r a d i o i m m u n o a s s a y 1 2 ' 1 3 b u t n o t by enzyme-linked immunoassay. 1 3 , 1 4 I n studies t h a t h a v e used t h e enzyme-linked i m m u n o a s s a y , t h e presence of IgM anti-HBc in C A H B h a s been correlated with
»Current address: Rockford Gastroenterology Associates, Rock-
l a b o r a t o r y f i n d i n g s of a c t i v e h e p a t o c e l l u l a r in-
tCurrent address: Digestive Healthcare, Minneapolis, Minnesota.
t i o n b y s o m e i n v e s t i g a t o r s 1 5 b u t n o t b y others. 1 3 ' 1 4
ford, Illinois.
flammation
Address reprint requests to Dr. A. J. Czaja, Division of Castro-
enterology, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 63:119-125,1988
a n d h e p a t i t i s B virus (HBV) replica-
D i s c o r d a n t r e s u l t s b e t w e e n s t u d i e s i n w h i c h dif-
ferent a s s a y s were used probably reflect varia119
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tions in the sensitivities of the assays, whereas differences between studies in which the same assay was used probably reflect variations in the patient populations being investigated. In this report, we assess the frequency and significance of IgM anti-HBc seropositivity by enzyme-linked immunoassay in patients with corticosteroid-treated CAH B who were selected by uniform criteria of severity of disease and were followed up systematically. In addition, we correlate the presence of IgM anti-HBc with indices of inflammatory activity and markers of virus replication, and we determine the applications and limitations of this assay in the assessment of such patients. Furthermore, we analyze the relationship among IgM anti-HBc, inflammatory activity, and serologic markers of HBV replication before, during, and after long-term corticosteroid treatment.
PATIENTS A N D METHODS Study Population.—Between 1967 and 1983,209 patients who fulfilled preestablished clinical, biochemical, and histologic criteria for severe CAH 16 were enrolled in a treatment program at our institution. Of these patients, 25 were seropositive for hepatitis B surface antigen (HBsAg). Serum and liver tissue samples had been obtained concurrently in 16 of these patients (group 1), and this material formed the basis of our report. Of the 184 patients in the treatment program who had HBsAg-negative CAH, 8 harbored antibodies to hepatitis B core and surface antigens (anti-HBc and anti-HBs) at the time of initial examination. Successive serum and liver tissue samples from these patients provided the opportunity to assess the frequency of IgM anti-HBc seropositivity in a group of patients with a different serologic profile but comparable disease severity (group 2). The clinical, biochemical, and histologic features of both groups of patients at accession are summarized in Table 1. All patients were Caucasian. Of the 24 patients, 18 (12 group 1 and 6 group 2 patients) had been assessed systematically for at least 5 years (mean, 139 ± 8 months). The other six patients were under surveillance from 0.5 to 35 months (mean, 18 ± 5 months). The mean duration of follow-up was 104 ± 14 months (range, 0.5 to 170 months) for the group
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1 patients and 118 ± 23 months (range, 20 to 200 months) for the group 2 patients. Treatment Regimens and Follow-Up.—The criteria for treatment with corticosteroids did not exclude patients with severe CAH B until after 1983. Consequently, all 16 group 1 patients had received prednisone in accordance with one of three treatment protocols. As maintenance therapy, three patients had received prednisone alone, 20 mg daily; nine had been treated with prednisone, 10 mg daily, in conjunction with azathioprine, 50 mg daily; and four had received prednisone on alternate days (mean dose, 20 mg every other day; range, 10 to 50 mg) with the dose adjusted to maintain normal serum aspartate aminotransferase values. The mean duration of corticosteroid therapy was 42 + 7 months (range, 0.5 to 91 months). As maintenance therapy for the eight group 2 patients, three had received prednisone alone, 20 mg daily; two had been treated with prednisone, 10 mg daily, and azathioprine, 50 mg daily; one had received prednisone on alternate days; and
Table 1.—Features at Time of Initial Examination of Anti-HBc-Positive Patients With (Group 1) and Without (Group 2) HBsAg* Group 1 Group 2 (N = 16) Feature (N = 8) Clinical findings Age (yr)t 49 ± 4 50 + 5 Duration of symptoms (mo)t 27 ± 9 12 ± 4 Sex (male:female) 16:0 3:5J Epidemiologie factors (no. of patients) Medical professionals 6 1 Homosexuals 1 0 Drug addicts 1 0 None 8 7 Biochemical findingsf AST (nl: <27 U/L) 507 ± 65 672 + 212 Bilirubin (nl: <1.1 mg/dl) 2.2 + 0.6 5.8 + 2.0 y-Globulin (nl: 0.7-1.6 g/dl) 2.3 + 0.2 2.6 ± 0.3 Albumin (nl: 3.1-4.3 g/dl) 3.5 ± 0.1 2.9 + 0.2§ Histologic findings (no. of patients) Periportal hepatitis 4 3 Bridging necrosis 6 0 Multilobular necrosis 1 0 Cirrhosis 5 5 *Anti-HBc = antibody to hepatitis B core antigen; AST = serum aspartate aminotransferase level; HBsAg = hepatitis B surface antigen; nl = normal laboratory value. tValues are expressed as the mean + the standard error of the mean. tSignificantly' different from group 1 (P<0.01). §Significantly different from group 1 (P<0.025).
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two had received placebo medication in accordance with our original randomized, controlled treatment trial. 16 The mean duration of corticosteroid therapy was 26 + 6 months (range, 17 to 59 months). All patients underwent assessment at regular 6-month intervals. At each examination, standard biochemical and serologic studies were performed, as described previously. 16 Serum samples were obtained at the time of entry into the study and at each follow-up visit during and after corticosteroid treatment. Specimens were frozen and stored at -20°C. Therapy was continued until previously reported criteria for remission, treatment failure, or drug intolerance had been satisfied. 16 Manifestations of relapse during or after discontinuation of corticosteroid therapy warranted reinstitution of the original treatment program. 16 Histologie interpretations were in accordance with previously published criteria. 17 Data Base.—The clinical course of each patient was assessed, and instances when the disease was active and inactive on the basis of predefined criteria were identified. Serum samples obtained at these times were analyzed for virologic markers, and the findings were correlated with the presence or absence of inflammatory activity. Results obtained before, during, and after corticosteroid therapy were compared. Disease activity was defined as histologic evidence of CAH or a serum aspartate aminotransferase level of more than 3 times the normal value. Our previous studies have demonstrated intraobserver consistency in the interpretation of the pattern and degree of histologic activity. 18,19 The reproducibility of such an interpretation by a single observer (J.L.) has been 88%. Additionally, elevation of the serum aspartate aminotransferase level to more than threefold normal during the course of severe CAH has, in our experience, been associated invariably with histologic features of CAH. 20 Ninety-six serum samples were selected from the 24 patients (mean, 4 ± 0.5 samples per patient; range, 1 to 8 samples), including 69 samples from the group 1 patients and 27 samples from the group 2 patients. Forty-nine samples had been obtained in the presence of inflammatory activity, and 47 samples had been obtained in the absence of inflammatory activity. Forty liver specimens had been obtained concurrently with
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the serum samples, including 29 from the group 1 patients and 11 from the group 2 patients. Virologic Assessments.—Each serum sample was tested for markers of HBV infection. Testing for HBsAg, anti-HBs, anti-HBc, and hepatitis B e antigen (HBeAg) and antibody to that antigen had been done in each sample by commercial radioimmunoassay or enzyme-linked immunoassay (Abbott Laboratories, North Chicago, Illinois) before the initiation of this study. Additionally, antibody to hepatitis delta virus had been sought previously in each sample by a blocking radioimmunoassay (Abbott Laboratories). These same serum samples were subsequently tested for hepatitis B virus deoxyribonucleic acid (HBV DNA) and IgM anti-HBc for the purposes of this analysis. Serum HBV DNA was sought in patients by a DNA hybridization assay that used nitrocellulose paper and a slot blotting technique similar to that described previously. 21,22 A 32P-labeled HBV probe (Bethesda Research Laboratories, Gaithersburg, Maryland) was used for the hybridization. Positive and negative serum controls, standards that contained known amounts of HBV DNA, and the serum samples to be assayed were tested in duplicate on each nitrocellulose strip. With use of this technique, a 1 pg/50 μΐ standard of HBV DNA could be detected routinely. The minimal amount of HBV DNA detected was 0.1 pg per spot after 48 hours of film exposure. IgM anti-HBc was measured by a solid-phase, commercially available, enzyme-linked immunoassay (Corzyme-M; Abbott Laboratories). Test samples were diluted at 1:1,000. Results were expressed as the absorbance value of the sample divided by a cutoff value (signal/cutoff ratio) that was calculated in accordance with the Abbott test instructions as 0.25 times the mean absorbance of the positive control plus the mean absorbance of the negative control. Test samples with absorbance values greater than the cutoff value were considered positive, and for IgM antiHBc positivity, a ratio of more than 1.0 was necessary. Statistics.—Chi-square analysis with Yates' correction was used for comparing dichotomous variables, and the unpaired t test was used to evaluate the significance of differences in means for continuous variables. The data are presented as the mean ± the standard error of the mean.
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RESULTS Frequency of IgM Anti-HBc Seropositivity.— The enzyme-linked immunoassay for IgM antiHBc was positive in 12 of the 16 group 1 patients and none of the 8 group 2 patients. The assay was positive in 9 of 14 group 1 patients who were tested at the time of initial examination. Three other group 1 patients were found to be positive during corticosteroid therapy, including two in whom the assay for IgM anti-HBc had been negative at the time of entry into the study. Of the 12 IgM anti-HBc-seropositive patients, 7 were seropositive on multiple occasions during and, in two instances, after corticosteroid therapy (mean frequency of seropositivity, 3 ± 0.4; range, 2 to 5 seropositive tests). In two patients, the assay became negative and then positive again during 36 and 37 months of corticosteroid treatment, respectively. In one of these patients, the fluctuation in seropositivity continued after withdrawal of treatment. In the five other patients, the assay remained positive in from two to four successive serum samples obtained during 6 to 57 months of corticosteroid therapy (mean, 21 ± 8 months). Relationship Between IgM Anti-HBc and Disease Activity.—IgM anti-HBc was present in 26 of 69 blood samples (38%) obtained from the 16 group 1 patients and none of 27 blood samples obtained from the 8 group 2 patients. The mean signal/cutoff ratio of samples positive for IgM anti-HBc was 1.9 + 0.2 (range, 1.1 to 5.2). IgM anti-HBc was detected in 19 of 37 samples (51%) collected from the 16 group 1 patients at times when active disease was present (Table 2). IgM anti-HBc was present more frequently in serum samples obtained when the disease was active than when it was inactive (19 of 37 versus 7 of 32; P<0.05). IgM anti-HBc seropositivity, however, did not preclude inactive disease. Of the seven samples in which IgM anti-HBc was detected in the absence of inflammatory activity (Table 2), five showed seropositivity during corticosteroid treatment. HBV DNA was present in three of the serum samples, and HBeAg was detected in all five. In the two instances in which IgM anti-HBc seropositivity accompanied inactive disease after corticosteroid treatment, HBV DNA and HBeAg were both absent. In serum samples obtained from the eight group 2 patients, IgM antibody was absent during periods of active (0 of 12) and inactive disease
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(0 of 15). HBV DNA was not detected in any of the 27 serum samples obtained from these patients. Relationship Between IgM Anti-HBc and Other HBV Markers in HBsAg-Positive CAH.—IgM anti-HBc was detected with greater frequency in HBeAg-positive than in HBeAgnegative samples (23 of 50 versus 2 of 18; P<0.02). It was also found more frequently in samples with HBV DNA than in those without HBV DNA (16 of 32 versus 8 of 34; P<0.05). IgM anti-HBc was absent in all three serum specimens that were positive for antibody to hepatitis delta virus. As shown in Table 3, the absence of IgM antiHBc in serum samples obtained from group 1 patients did not preclude the presence of HBV DNA (38%), HBeAg (63%), or the combination of HBV DNA and HBeAg (50%). IgM Anti-HBc and Corticosteroid Treatment.—Long-term corticosteroid therapy did not uniformly affect the serum levels of IgM anti-HBc (Fig. 1). Only 10 of 20 serum samples obtained in the presence of inflammatory activity during corticosteroid treatment were seropositive for IgM anti-HBc (Fig. 1). IgM anti-HBc seropositivity accompanied manifestations of inflammatory activity almost as commonly during corticosteroid therapy as at the time of initial examination (Table 2). Additionally, the degree of seropositivity in the samples assessed during corticosteroid therapy was similar to that of samples obtained initially (Fig. 1). Because corticosteroid therapy was discontinued after resolution of inflammatory activity, most serum samples obtained during this interval were associated with inactive disease (Fig. 1). In 12 of 14 such instances, IgM anti-HBc sero-
Table 2.—Frequency of IgM Anti-HBc Seropositivity in Serum Samples Obtained From HBsAg-Positive Patients During Active and Inactive Disease* Serum sampling period (no. of patients) At initial Corticosteroid therapy Finding examination During After IgM anti-HBc positive of those with active disease 9 of 14 10 of 20 Oof 3 IgM anti-HBc positive of those with inactive disease OofO 5 of 18 2 of 14 *HBsAg = hepatitis B surface antigen; IgM anti-HBc = immunoglobulin M antibody to hepatitis B core antigen.
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negativity was found. The IgM anti-HBc assay was also negative in all three instances in which inflammatory activity was present after discontinuation of corticosteroid treatment. DISCUSSION Our study indicates that IgM anti-HBc can frequently be detected by enzyme-linked immunoassay in patients with severe HBsAg-positive CAH. IgM anti-HBc seropositivity was demonstrated in 12 of 16 HBsAg-positive patients (group 1), including 9 of 14 patients who were tested at the time of initial examination and before corticosteroid therapy. Seropositivity was established in 38% of selected serum samples. IgM anti-HBc seropositivity was more common in the presence of active disease than inactive disease, and it was usually associated with the presence of HBV DNA and HBeAg in the serum. These findings suggest that active HBV replication continues to stimulate the production of a specific IgM antibody to the hepatitis B core antigen and that, in our patients, replicative activity was frequently associated with inflammatory activity. Previous studies have indicated that replicative activity does not necessarily correlate with inflammatory activity and that, in patients who may be immune tolerant of the virus, high levels of virus replication may be accompanied by minimal or no inflammatory change (for example, the chronic carrier state). 23 Because our patients were selected on the basis of the severity of inflammatory activity rather than on the degree of virus replication, our findings may not be valid in patients with lesser degrees of inflammatory activity. Importantly, HBsAg-negative, anti-HBc-positive patients (group 2) with severe disease at the time of entry into the study remained seronegative for IgM anti-
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HBc and HBV DNA throughout the period of observation. These findings indicate that IgM anti-HBc seropositivity is not a manifestation of inflammatory activity alone. IgM anti-HBc seronegativity did not preclude active virus replication. Indeed, 16 of 32 serum samples positive for HBV DNA and 27 of 50 samples positive for HBeAg were negative for IgM anti-HBc. The variability of the association between IgM anti-HBc and HBV DNA and HBeAg probably reflects differences in the sensitivities of the various assays for replicative activity and the uncertain effect of corticosteroids on each assay. A high level of virus replication may be necessary before the enzyme-linked immunoassay for IgM anti-HBc becomes positive, and fluctuations in replicative activity above and below a certain threshold may account for the absence or intermittency of IgM anti-HBc seropositivity. Similarly, IgM anti-HBc seronegativity did not preclude the presence of inflammatory activity. Five of the 14 serum specimens obtained at the time of initial examination when the liver disease was most active were negative for IgM anti-HBc, and 13 of 23 serum specimens obtained in the presence of disease activity during and after corticosteroid therapy were also negative for IgM anti-HBc (Table 2). IgM anti-HBc seronegativity in the presence of inflammatory activity may occur in chronic HBsAg-positive carriers with a superimposed non-A, non-B infection or other type of liver injury. 24 In our experience, however, seronegativity commonly occurred in patients who did not have an epidemiologic basis for non-A, non-B hepatitis or a historical basis for toxic liver injury. Additionally, IgM anti-HBc seronegativity was rarely accompanied by seropositivity for antibody to hepatitis delta virus, and it was frequently associated with HBV DNA
Table 3.—Frequency of HBV DNA and HBeAg in Serum Samples Obtained From HBsAg-Positive Patients With and Without IgM Anti-HBc* IgM anti-HBc (+) samples IgM anti-HBc (-) samples (N = 26) (N = 43) Seropositive Seronegative Serologie finding Seropositive Seronegative HBV DNA 16 8 16 26 HBeAg 23 2 27 16 HBV DNA and HBeAg 16 2 16 16 *HBeAg = hepatitis B e antigen; HBsAg = hepatitis B surface antigen; HBV DNA = hepatitis B virus deoxyribonucleic acid; IgM anti-HBc = immunoglobulin M antibody to hepatitis B core antigen.
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6.0 5.0
-
4.0
-
3.0
-
2.0
-
▲ Active disease Δ Inactive disease
Φ .Q
CO O
~°
a
1.0
Normal cutoff
<1 At presentation
Corticosteroid treatment
No corticosteroid treatment
Fig. 1. Levels of immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) in serum samples obtained from 16 patients positive for hepatitis B surface antigen in presence and absence of disease activity and corticosteroid therapy. Levels "At presentation" were determined immediately before corticosteroid therapy. A signal/cutoff ratio that exceeded 1.0 was considered a positive test result by enzymelinked immunoassay.
and HBeAg in the serum. Insensitivity of the assay for HBV replication rather than superimposed non-B infection or other type of liver injury is the most likely explanation for the discordant findings. IgM anti-HBc seropositivity was demonstrated in 7 of 32 serum samples obtained in the absence of inflammatory activity. Seropositivity in the absence of disease activity during corticosteroid therapy was usually accompanied by markers of virus replication, and this finding probably connoted immune tolerance of the virus by the host. 23 Seropositivity in the absence of disease activity after discontinuation of corticosteroid treatment was not accompanied by markers of virus replication; this finding probably connoted incomplete clearance of IgM anti-HBc after cessation of HBV replication. 2,3 Because the results of the enzyme-linked immunoassay for IgM anti-HBc were not closely associated with markers of virus replication or disease activity, the use of this test to assess these aspects of CAH B is limited. Studies in which a radioimmunoassay was used for detection of IgM anti-HBc have demonstrated a much closer association among IgM anti-HBc
seropositivity, markers of virus replication, and inflammatory activity than we have found by using the enzyme-linked immunoassay. 13,25 Long-term corticosteroid therapy is no longer used in the routine management of severe CAH B, but our findings do provide additional insight into the effects of such therapy on the markers of virus replication. IgM anti-HBc seropositivity could persist or recur intermittently during therapy (for up to 57 months, as demonstrated in our study), and in two of our patients, it developed after institution of corticosteroid therapy. These findings are supportive of other studies that have indicated that long-term corticosteroid therapy may delay clearance of the hepatitis virus. 26,27 Previous investigations have shown that the enzyme-linked immunoassay for IgM anti-HBc is a useful test for confirming the diagnosis of acute hepatitis B and distinguishing acute hepatitis B from chronic carrier states with superimposed non-A, non-B or delta virus infection.24 Our study underscores the potential difficulty of relying on this test alone to distinguish acute hepatitis B from CAH B with severe inflammatory activity. Recent studies have suggested that patients with
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acute hepatitis B have mainly 19S IgM anti-HBc and those with CAH B have predominantly 7 to 8S IgM anti-HBc. 25 Perhaps future studies will demonstrate that separation of the different types of serum IgM has diagnostic value. ACKNOWLEDGMENT We thank Linda S. Grande for assistance in the preparation of the submitted manuscript. REFERENCES 1. Brzosko WJ, Mikulska B, Cianciara J, Babiuch L: Immunoglobulin classes of antibody to hepatitis B core antigen. J Infect Dis 132:1-5, 1975 2. Cohen BJ: The IgM antibody responses to the core antigen of hepatitis B virus. J Med Virol 3:141-149, 1978 3. Tedder RS, Wilson-Croome R: IgM-antibody response to the hepatitis B core antigen in acute and chronic hepatitis B. J Hyg (Lond) 86:163-172, 1981 4. Dormeyer HH, Arnold W, Kryger P, Nielsen JO, Meyer zum Büschenfelde KH: IgM antibody to hepatitis B core antigen (anti-HB c IgM) in "healthy" HBsAg carriers: a longitudinal study of 75 cases. Klin Wochenschr 59:675678,1981 5. Lemon SM, Gates NL, Simms TE, Bancroft WH: IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 143:803-809,1981 6. Chau KH, Hargie MP, Decker RH, Mushahwar IK, Overby LR: Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 3:142-149,1983 7. Taswell HF, Czaja AJ, Nelson CA: Viral hepatitis: diagnostic test using anti-HBc (IgM). Mayo Clin Proc 60:488489,1985 8. Roggendorf M, Deinhardt F, Frösner GG, Scheid R, Bayerl B, Zachoval R: Immunoglobulin M antibodies to hepatitis B core antigen: evaluation of enzyme immunoassay for diagnosis of hepatitis B virus infection. J Clin Microbiol 13:618-626,1981 9. Kryger P, Mathiesen LR, Aldershvile J, Nielsen JO, Copenhagen Hepatitis Acuta Programme: Presence and meaning of anti-HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology 1:233-237,1981 10. Gerlich WH, Lüer W, Thomssen R, Study Group for Viral Hepatitis of the Deutsche Forschungsgemeinschaft: Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J Infect Dis 142:95-101,1980 11. Gitnick G: Immunoglobulin M hepatitis B core antibody: to titer or not to titer? to use or not to use? (editorial). Gastroenterology 84:653-655,1983 12. Feinman SV, Overby LR, Berris B, Chau K, Schable CA, Maynard JE: The significance of IgM antibodies to hepatitis B core antigen in hepatitis B carriers and hepatitis B-associated chronic liver disease. Hepatology 2:795-799,1982
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