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Anti-HD IgM as a marker of chronic delta infection
Journal of Hepatology. 1987;4:62-65
62
Elsevier HEP 00259
A n t i - H D IgM as a marker of chronic delta infection
Maria Buti, R. Esteban, J.I. Esteban, Helena Allende, R. Jardi and J. Guardia Departments of Internal Medicine. BiochemistO, and Pathology. Ciudad Sanitaria "Valle Hebr6n', Universidad AutOnoma, Barcelona (Spain)
(Received 22 July, 19861 (Accepted 10 October, 1986)
Summary The value of anti-HD IgM as a marker of chronic delta infection was evaluated by correlating its presence in serum with that of HD-Ag in liver cells and with the degree of inflammatory activity. Thirty-six patients with HBsAg-positive chronic hepatitis and anti-HD at high titers were studied. Overall, the liver cells of 26 patients contained HD-Ag and 27 were positive for IgM anti-HD. The correlation between both markers was excellent: 25 cases were positive for both serum anti-HD lgM and intrahepatic HD-Ag and 8 were negative for both markers. There was only 1 HD-Ag-positive patient, who was anti-HD IgM-negative. Two patients were anti-HD IgMpositive and HD-Ag-negative. Histological damage was more severe in anti-HD IgM-positive cases than in those negative for this marker (Knodell's index 13.5 vs 11.9, P < 0.01). We conclude that anti-HD lgM is a good marker of chronic active delta infection.
Introduction The diagnosis of chronic delta hepatitis is usually made in the presence of high titers of total anti-delta antibodies in serum (anti-HD) and/or that of delta antigen (HD-Ag) within the liver cells [1,2], The serological test is a sensitive indicator of exposure to the delta agent, but its specificity as a marker of active infection is uncertain since it may remain positive for a long time in patients in whom HD-Ag cannot be demonstrated in the liver, thus suggesting past or in-
active delta infection. Nevertheless, liver biopsy does not constitute the ideal procedure for the routine follow-up of patients chronically infected with the delta virus (HDV). Anti-HD of the lgM type (anti-HD IgM) which may be transiently found in some patients during acute HDV infection [4] has been proposed as a sensitive marker of chronic infection [5]. In order to evaluate the utility of anti-HD IgM as a marker of chronic active delta infection, we have investigated the presence of these antibodies and that
This work has been supported in part by a grant from the CIRIT of the Generalitat Catalunya. Correspondence address: Maria Buti, C.J. Sebastifin Bach, 4, 5, 1, Barcelona 08021, Spain, Tel. (3) 2017946. 0168-8278/87/$(13.50© 1987Elsevier Science Publishers B.V. (Biomedical Division)
ANTI-HD IgM AS MARKER OF CHRONIC DELTA INFECTION of intrahepatic H D - A g in a group of patients with anti-HD-positive chronic liver disease, and related both markers to the extent and severity of the hepatic lesion.
Patients and Methods
Thirty-six HBsAg-positive patients with histologically proven chronic liver disease and high titers of anti-HD antibodies ( > 1/1000) were studied. Twentyeight were males and 8 females. Twenty-nine were drug abusers, 1 was a multitransfused hemophiliac patient and the remaining 6 had no known risk factor for H D V infection. Thirty-two had a previous clinical episode of acute hepatitis. None of them had received antiviral or immunosuppressive therapy. Two had chronic lobular hepatitis (CLH), 4 chronic persistent hepatitis (CPH), 26 chronic active hepatitis (CAH) and 4 active cirrhosis (AC). Additional clinical data are shown in Table 1. A blood sample was drawn the same day the liver biopsy was performed. Serum HBsAg, anti-HBc, and total anti-HD antibodies were determined by RIA (Abbott Lab.) and serum H D - A g by EIA (Nochtech Lab.). Anti-HD IgM was determined by a solid phase capture RIA as described by Smedile et al. [5]; briefly, a microtiter plate was coated by goat anti-human IgM (/z-chain-specific), and overcoated with 1% BSA was incubated with 50/A of a 1:100 dilution of serum for 12 h at room temperature and then with delta antigen extracted from infected chimpanzee liver (kindly provided by J.W.-K. Shih, NIH Blood Bank, Bethesda, MD) for two more hours and finally with human IgG anti-HD labelled with 125I. TABLE 1 SUMMARY OF CLINICAL DATA OF THE 36 PATIENTS WITH CHRONIC HDV LIVER DISEASE
Age (yr) Estimated duration of liver disease (months) ALTa (normal 20 mU/ml)
Mean
Range
35
(11-68)
34 35 m U / m l
(0-108) (8-686)
a Alanine aminotransferase values at the time of liver biopsy.
63
Wells were washed 4 times with PBSfrween 20 between steps. Radioactivity was measured with a gammacounter (Beckman). Samples were considered positive when the number of cpm were 2.1 times higher than the mean negative control cpm. Severity of liver lesion was assessed using the histological indexes of Knodell et al. [6], for periportal and bridging necrosis, lobular activity, portal inflammation and fibrosis. The Student t-test was used to compare mean values of the index. Liver HD-Ag was investigated by an indirect immunoperoxidase method using a purified human IgG fraction with high anti-HD titer; the specificity and sensitivity of the procedure was verified using as controls known reference anti-sera (kindly provided by M. Rizzetto, Ospedale Molinette, Turin, and JM Sfinchez-Tapias, Hospital Clinico, Barcelona) and by blocking assay [7]. Quantification of HD-Ag positivity in liver cells was directly obtained by averaging the number of positive cells observed in 5 randomly selected fields at high magnification.
Results
Anti-HD of the IgM type was detected in 27 of the 36 cases (75%). HD-Ag was never detected in serum, but was positive in 1-90% of liver cells (mean 30%) in 26 of the 36 liver biopsy specimens (72%). There was a high degree of concordance between both findings: 25 patients were positive for both serum anti-HD IgM and liver HD-Ag and 8 additional patients were negative for both markers. In 3 cases there was a discrepancy: 1 patient was HD-Ag-positive and anti-HD IgM-negative and 2 patients were anti-HD IgM-positive and HD-Ag-negative. There were no significant differences regarding to age, duration of illness, and ALT values between patients positive or negative for liver HD-Ag and/or serum anti-HD IgM. As shown in Table 2, the correlation between the presence of IgM anti-HD in the serum and that of HD-Ag in the liver cells was excellent, so that 21 of the 26 patients with CAH and 3 of the 4 with active cirrhosis were positive for both markers.
64
M. BUTI et al.
TABLE 2 RESULTS OF LIVER HD-Ag AND SERUM ANTI-HD IgM ACCORDING TO THE TYPE OF LIVER LESION
n
HD-Ag+ only
IgM+ only
Both (+)
Both (-)
Liver lesion Chronic lobular hepatitis Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis
2 4 26 4
0 0 0 1
0 2 0 0
1 0 21 3
1 2 5 0
Total patients
36
1
2
25
8
Inflammatory liver changes were more conspicuous in the 25 patients positive for HD-Ag and antiHD IgM (Knodell's index 13.5) than in the 8 patients who were negative for both markers (index 11.9) (P > 0.01). However, when individual histological parameters were considered, no significant differences were found between these two groups.
Discussion
The results of the present study show a strong correlation (96%) between the presence of HD-Ag in liver tissue and that of anti-HD lgM in the sera of patients with chronic HBsAg-positive hepatitis and HDV superinfection. These results are similar to those obtained by Smedile et al. [5], and Farci et al. [8]. Both groups of investigators observed that antiHD IgM was positive in all patients who had HD-Ag in their liver tissue. Nonetheless, we have found 1 patient who had intrahepatic HD-Ag without serum anti-HD lgM. In 8 of our patients, as in 6 cases reported by Farci et al. [8], both markers were negative. It is possible that in such patients the HDV infection is inactive and that the finding of total antiHD simply reflected a previous exposure to the agent [9]. If this were the case, the finding of anti-HD IgM would be a more specific marker of chronic HDV infection than total anti-HD. Conversely, in some patients anti-HD IgM is found in the absence of HD-Ag in the liver; this occurred in 2 of our patients with CPH and in some of those reported by Farci et al. [8]. This might suggest that in patients with less severe forms of liver disease
the disappearance of HD-Ag from the liver precedes that of anti-HD lgM from the serum, thus indicating the absence of viral replication and, eventually, resolution of the active delta infection. It is also conceivable, however, that the immunohistochemical technique was relatively insensitive as suggested by the finding of HDV-associated RNA in some cases studied by Farci et al. [8]. The fact that 1 of the 4 patients with active cirrhosis was negative for lgM anti-HD despite the presence of HD-Ag in the liver cells might be related to the long period of time elapsed since acute HDV infection or to the concurrent HIV infection observed in this patient. Another finding of the present study was that patients who had both intrahepatic HD-Ag and serum anti-HD IgM exhibited a more severe inflammatory activity as assessed by Knodelrs indexes. This would suggest the possible value of anti-HD-IgM as a marker of active inflammatory disease and hence its usefulness in monitoring the response to potential antiviral therapy. At present, in the absence of screening techniques for detection of HDV-associated RNA in clinical laboratories, anti-HD antibodies of the IgM class seem to be the only markers which can distinguish past inactive HDV infection from ongoing chronic delta infection without the need for a liver biopsy.
Acknowledgement The authors wish to thank Ms. Sofia Garcia for her excellent laboratory assistance.
ANTI-HD IgM AS M A R K E R OF CHRONIC DELTA INFECTION
References 1 Rizzetto M, Canese MG, Arico S, et al. Immunofluorescence detection of a new antigen-antibody system (delta/anti-delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut 1977; 18: 997-1003. 2 Rizzetto M, Shi JW-K, Gerin H. The hepatitis B virus-associated delta antigen (d) isolation from liver, development of solid phase radioimmunoassay for delta and anti-delta and partial characterization of delta. J Immunol 1980; 121: 590-602. 3 Rizzetto M, Shi JW-K, Gocke D J, et al. Incidence and significance of antibodies to delta antigen in hepatitis B virus infection. Lancet 1979; ii: 986-99(I. 4 Mushahwar IK, Gerin JL, Dienstag JL, et al. Interpretation of hepatitis B virus and hepatitis delta virus serologic profiles. Pathologist 1984; 38: 648-650. 5 Smedile A, Lavarini C, Crivelli O, et al. Radioimmunoassay detection of IgM antibodies to HBV-associated delta
65
antigen: Clinical significance in delta infection, J Med Virol 1982; 9: 131-138. 6 Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981 ; 1: 431-435. 7 Buti M, Esteban R, Jard/R, et al. Evoluci6n clinico-serol6gica de la infecci6n cr6nica delta. Gastroenterol y Hepatol 1986; 90: 217-220. 8 Farci P, Gerin JL, Aragona M, et al. Diagnostic and prognostic significance of the IgM antibody to hepatitis delta virus. J Amer Med Ass 1986; 255: 1443-1446. 9 Arico S, Aragona M, Rizzetto M, et al. Clinical significance of antibody to the hepatitis delta virus in asymptomatic HBsAg carriers. Lancet 1985, ii: 356-358. 10 Rizzetto M, Verme G, Recchia S, et al. Chronic HBsAg hepatitis with intrahepatic expression of delta antigen: An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med 1983: 98: 437-441.
62
Elsevier HEP 00259
A n t i - H D IgM as a marker of chronic delta infection
Maria Buti, R. Esteban, J.I. Esteban, Helena Allende, R. Jardi and J. Guardia Departments of Internal Medicine. BiochemistO, and Pathology. Ciudad Sanitaria "Valle Hebr6n', Universidad AutOnoma, Barcelona (Spain)
(Received 22 July, 19861 (Accepted 10 October, 1986)
Summary The value of anti-HD IgM as a marker of chronic delta infection was evaluated by correlating its presence in serum with that of HD-Ag in liver cells and with the degree of inflammatory activity. Thirty-six patients with HBsAg-positive chronic hepatitis and anti-HD at high titers were studied. Overall, the liver cells of 26 patients contained HD-Ag and 27 were positive for IgM anti-HD. The correlation between both markers was excellent: 25 cases were positive for both serum anti-HD lgM and intrahepatic HD-Ag and 8 were negative for both markers. There was only 1 HD-Ag-positive patient, who was anti-HD IgM-negative. Two patients were anti-HD IgMpositive and HD-Ag-negative. Histological damage was more severe in anti-HD IgM-positive cases than in those negative for this marker (Knodell's index 13.5 vs 11.9, P < 0.01). We conclude that anti-HD lgM is a good marker of chronic active delta infection.
Introduction The diagnosis of chronic delta hepatitis is usually made in the presence of high titers of total anti-delta antibodies in serum (anti-HD) and/or that of delta antigen (HD-Ag) within the liver cells [1,2], The serological test is a sensitive indicator of exposure to the delta agent, but its specificity as a marker of active infection is uncertain since it may remain positive for a long time in patients in whom HD-Ag cannot be demonstrated in the liver, thus suggesting past or in-
active delta infection. Nevertheless, liver biopsy does not constitute the ideal procedure for the routine follow-up of patients chronically infected with the delta virus (HDV). Anti-HD of the lgM type (anti-HD IgM) which may be transiently found in some patients during acute HDV infection [4] has been proposed as a sensitive marker of chronic infection [5]. In order to evaluate the utility of anti-HD IgM as a marker of chronic active delta infection, we have investigated the presence of these antibodies and that
This work has been supported in part by a grant from the CIRIT of the Generalitat Catalunya. Correspondence address: Maria Buti, C.J. Sebastifin Bach, 4, 5, 1, Barcelona 08021, Spain, Tel. (3) 2017946. 0168-8278/87/$(13.50© 1987Elsevier Science Publishers B.V. (Biomedical Division)
ANTI-HD IgM AS MARKER OF CHRONIC DELTA INFECTION of intrahepatic H D - A g in a group of patients with anti-HD-positive chronic liver disease, and related both markers to the extent and severity of the hepatic lesion.
Patients and Methods
Thirty-six HBsAg-positive patients with histologically proven chronic liver disease and high titers of anti-HD antibodies ( > 1/1000) were studied. Twentyeight were males and 8 females. Twenty-nine were drug abusers, 1 was a multitransfused hemophiliac patient and the remaining 6 had no known risk factor for H D V infection. Thirty-two had a previous clinical episode of acute hepatitis. None of them had received antiviral or immunosuppressive therapy. Two had chronic lobular hepatitis (CLH), 4 chronic persistent hepatitis (CPH), 26 chronic active hepatitis (CAH) and 4 active cirrhosis (AC). Additional clinical data are shown in Table 1. A blood sample was drawn the same day the liver biopsy was performed. Serum HBsAg, anti-HBc, and total anti-HD antibodies were determined by RIA (Abbott Lab.) and serum H D - A g by EIA (Nochtech Lab.). Anti-HD IgM was determined by a solid phase capture RIA as described by Smedile et al. [5]; briefly, a microtiter plate was coated by goat anti-human IgM (/z-chain-specific), and overcoated with 1% BSA was incubated with 50/A of a 1:100 dilution of serum for 12 h at room temperature and then with delta antigen extracted from infected chimpanzee liver (kindly provided by J.W.-K. Shih, NIH Blood Bank, Bethesda, MD) for two more hours and finally with human IgG anti-HD labelled with 125I. TABLE 1 SUMMARY OF CLINICAL DATA OF THE 36 PATIENTS WITH CHRONIC HDV LIVER DISEASE
Age (yr) Estimated duration of liver disease (months) ALTa (normal 20 mU/ml)
Mean
Range
35
(11-68)
34 35 m U / m l
(0-108) (8-686)
a Alanine aminotransferase values at the time of liver biopsy.
63
Wells were washed 4 times with PBSfrween 20 between steps. Radioactivity was measured with a gammacounter (Beckman). Samples were considered positive when the number of cpm were 2.1 times higher than the mean negative control cpm. Severity of liver lesion was assessed using the histological indexes of Knodell et al. [6], for periportal and bridging necrosis, lobular activity, portal inflammation and fibrosis. The Student t-test was used to compare mean values of the index. Liver HD-Ag was investigated by an indirect immunoperoxidase method using a purified human IgG fraction with high anti-HD titer; the specificity and sensitivity of the procedure was verified using as controls known reference anti-sera (kindly provided by M. Rizzetto, Ospedale Molinette, Turin, and JM Sfinchez-Tapias, Hospital Clinico, Barcelona) and by blocking assay [7]. Quantification of HD-Ag positivity in liver cells was directly obtained by averaging the number of positive cells observed in 5 randomly selected fields at high magnification.
Results
Anti-HD of the IgM type was detected in 27 of the 36 cases (75%). HD-Ag was never detected in serum, but was positive in 1-90% of liver cells (mean 30%) in 26 of the 36 liver biopsy specimens (72%). There was a high degree of concordance between both findings: 25 patients were positive for both serum anti-HD IgM and liver HD-Ag and 8 additional patients were negative for both markers. In 3 cases there was a discrepancy: 1 patient was HD-Ag-positive and anti-HD IgM-negative and 2 patients were anti-HD IgM-positive and HD-Ag-negative. There were no significant differences regarding to age, duration of illness, and ALT values between patients positive or negative for liver HD-Ag and/or serum anti-HD IgM. As shown in Table 2, the correlation between the presence of IgM anti-HD in the serum and that of HD-Ag in the liver cells was excellent, so that 21 of the 26 patients with CAH and 3 of the 4 with active cirrhosis were positive for both markers.
64
M. BUTI et al.
TABLE 2 RESULTS OF LIVER HD-Ag AND SERUM ANTI-HD IgM ACCORDING TO THE TYPE OF LIVER LESION
n
HD-Ag+ only
IgM+ only
Both (+)
Both (-)
Liver lesion Chronic lobular hepatitis Chronic persistent hepatitis Chronic active hepatitis Active cirrhosis
2 4 26 4
0 0 0 1
0 2 0 0
1 0 21 3
1 2 5 0
Total patients
36
1
2
25
8
Inflammatory liver changes were more conspicuous in the 25 patients positive for HD-Ag and antiHD IgM (Knodell's index 13.5) than in the 8 patients who were negative for both markers (index 11.9) (P > 0.01). However, when individual histological parameters were considered, no significant differences were found between these two groups.
Discussion
The results of the present study show a strong correlation (96%) between the presence of HD-Ag in liver tissue and that of anti-HD lgM in the sera of patients with chronic HBsAg-positive hepatitis and HDV superinfection. These results are similar to those obtained by Smedile et al. [5], and Farci et al. [8]. Both groups of investigators observed that antiHD IgM was positive in all patients who had HD-Ag in their liver tissue. Nonetheless, we have found 1 patient who had intrahepatic HD-Ag without serum anti-HD lgM. In 8 of our patients, as in 6 cases reported by Farci et al. [8], both markers were negative. It is possible that in such patients the HDV infection is inactive and that the finding of total antiHD simply reflected a previous exposure to the agent [9]. If this were the case, the finding of anti-HD IgM would be a more specific marker of chronic HDV infection than total anti-HD. Conversely, in some patients anti-HD IgM is found in the absence of HD-Ag in the liver; this occurred in 2 of our patients with CPH and in some of those reported by Farci et al. [8]. This might suggest that in patients with less severe forms of liver disease
the disappearance of HD-Ag from the liver precedes that of anti-HD lgM from the serum, thus indicating the absence of viral replication and, eventually, resolution of the active delta infection. It is also conceivable, however, that the immunohistochemical technique was relatively insensitive as suggested by the finding of HDV-associated RNA in some cases studied by Farci et al. [8]. The fact that 1 of the 4 patients with active cirrhosis was negative for lgM anti-HD despite the presence of HD-Ag in the liver cells might be related to the long period of time elapsed since acute HDV infection or to the concurrent HIV infection observed in this patient. Another finding of the present study was that patients who had both intrahepatic HD-Ag and serum anti-HD IgM exhibited a more severe inflammatory activity as assessed by Knodelrs indexes. This would suggest the possible value of anti-HD-IgM as a marker of active inflammatory disease and hence its usefulness in monitoring the response to potential antiviral therapy. At present, in the absence of screening techniques for detection of HDV-associated RNA in clinical laboratories, anti-HD antibodies of the IgM class seem to be the only markers which can distinguish past inactive HDV infection from ongoing chronic delta infection without the need for a liver biopsy.
Acknowledgement The authors wish to thank Ms. Sofia Garcia for her excellent laboratory assistance.
ANTI-HD IgM AS M A R K E R OF CHRONIC DELTA INFECTION
References 1 Rizzetto M, Canese MG, Arico S, et al. Immunofluorescence detection of a new antigen-antibody system (delta/anti-delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut 1977; 18: 997-1003. 2 Rizzetto M, Shi JW-K, Gerin H. The hepatitis B virus-associated delta antigen (d) isolation from liver, development of solid phase radioimmunoassay for delta and anti-delta and partial characterization of delta. J Immunol 1980; 121: 590-602. 3 Rizzetto M, Shi JW-K, Gocke D J, et al. Incidence and significance of antibodies to delta antigen in hepatitis B virus infection. Lancet 1979; ii: 986-99(I. 4 Mushahwar IK, Gerin JL, Dienstag JL, et al. Interpretation of hepatitis B virus and hepatitis delta virus serologic profiles. Pathologist 1984; 38: 648-650. 5 Smedile A, Lavarini C, Crivelli O, et al. Radioimmunoassay detection of IgM antibodies to HBV-associated delta
65
antigen: Clinical significance in delta infection, J Med Virol 1982; 9: 131-138. 6 Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981 ; 1: 431-435. 7 Buti M, Esteban R, Jard/R, et al. Evoluci6n clinico-serol6gica de la infecci6n cr6nica delta. Gastroenterol y Hepatol 1986; 90: 217-220. 8 Farci P, Gerin JL, Aragona M, et al. Diagnostic and prognostic significance of the IgM antibody to hepatitis delta virus. J Amer Med Ass 1986; 255: 1443-1446. 9 Arico S, Aragona M, Rizzetto M, et al. Clinical significance of antibody to the hepatitis delta virus in asymptomatic HBsAg carriers. Lancet 1985, ii: 356-358. 10 Rizzetto M, Verme G, Recchia S, et al. Chronic HBsAg hepatitis with intrahepatic expression of delta antigen: An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med 1983: 98: 437-441.