- Email: [email protected]
Comparative analysis of serological markers of chronic delta infection: HDV-RNA, serum HDAg and anti-HD IgM
Journal of Virolo~cal Methods
ELSEVIER
Journal of Virological
Methods 50 (1994) 59-66
Comparative analysis of serological markers of chronic delta infection: HDV-RNA, serum HDAg and anti-HD IgM Rosendo Jardi a,*, Maria Buti b, Francisco Rodriguez a, Montserrat Cotrina a, Helena Allende b, Rafael Esteban b, Jaime Guardia b a Liter Unit: Department b Liver Unit: Department
of Biochemistry, Hospital General Valle Hebrbn, Paseo Valle Hebr6n s/n, Universidad Autckoma, 08035 Barcelona, Spain of Internal Medicine, Hospital General Valle Hebrbn, Universidad Aut&oma, Barcelona, Spain Accepted
13 April 1994
Abstract To study the concordance, sensitivity and specificity of HDV-RNA determination by molecular hybridization, serum HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection by an immunoperoxidase method, a statistical analysis was applied to the results of serum sample and liver biopsy determinations in 50 patients with chronic delta hepatitis (38 positive to tissue HDAg and 12 negative).Of the 38 patients with hepatic HDAg, HDV-RNA was found in 31 (82%), serum HDAg by immunoblot in 27 (71%) and anti-HD IgM in 33 (87%). Among the 12 patients without hepatic HDAg, one was found with serum HDAg using the immunoblot technique, two (17%) had HDV-RNA, and 7 (58%) had anti-HD IgM. Serum HDAg determination by immunoblot was the most specific test, followed by HDV-RNA analysis. The least specific was the anti-HD IgM technique. The anti-HD IgM test was the most sensitive, followed by HDV-RNA and serum HDAg. The concordance with intrahepatic HDAg detection was highest for HDV-RNA determination, followed by HDAg in serum. The least degree of concordance was found with anti-HD IgM determination. These results suggest that the determination of HDV-RNA by the hybridization method can be of great value for the diagnosis and monitoring of chronic delta hepatitis. Keywords:
Chronic hepatitis D infection;
* Corresponding 0166-0934/94/$07.00
HDV-RNA;
HDAg; Anti-HD
IgM
author. Fax: + 34 3 4280443. 0 1994 Elsevier Science B.V. All rights reserved
60
R. Jardi et al. /Journal
of Virological Methods 50 (1994) 59-66
1. Introduction Hepatitis delta virus (HDV) is a defective RNA virus that replicates only in hosts simultaneously infected with hepatitis B virus (HBV) (Rizetto, 1983; Bonino et al., 1986). Chronic hepatitis delta is diagnosed by identification of the delta antigen (HDAg) in hepatic tissue and/or by determining the presence of high titers of antibodies to HDAg (anti-HD) in serum (Di Bisceglie, 1989). Anti-HD of the IgM type (anti-HD IgM) is present in serum of the majority of patients with chronic hepatitis with intrahepatic HDAg, thus anti-HD IgM has been considered a good marker of active delta infection. However, in some patients this antibody has been found in the absence of HDAg in the liver (Farci et al., 1986; Buti et al., 1987; Jardi et al., 1991; Smedile et al., 1991). Various workers have demonstrated that the detection of hepatitis delta RNA (HDV-RNA) by a molecular hybridization technique and HDAg in serum by an immunoblot method indicate HDV replication (Bermann et al., 1986; Rasshofer et al., 1988; Gupta et al., 1989; Smedile et al., 1991). Although superior in performance to conventional assays, these alternative methods are not generally available to laboratories because of the technical difficulties involved (Di Bisceglie et al., 1989). Therefore, in most cases, serological methods are still employed. The aim was to study the concordance, sensitivity and specificity of serum HDV-RNA determination by dot-blot hybridization, HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection, the current ‘gold standard’. For this purpose, a statistical analysis was carried out on serum samples and liver biopsies of 50 patients with chonic delta hepatitis examined previously (Buti et al., 1988, 1989).
2. Patients and methods 2.1. Patients Fifty HBsAg positive patients, 38 with hepatic HDAg and 12 without, with proven liver disease by histological evidence and high titers of anti-HD antibodies > l/10000 were studied. The patients with intrahepatic HDAg included 32 men and six women (mean age 22 years, range 17-30 years). All were intravenous drug addicts. The histological diagnoses were: chronic active hepatitis (CAH) in 27 cases and liver cirrhosis (LC) in 11 cases. Of the 12 patients without tissue HDAg, nine were men and three were women (mean age 26 years, range 22-50 years); seven weredrug addicts. The histological study demonstrated CAH in eight casesand LC in four cases. 2.2. Serological
analysis
HBsAg, HBeAg, anti-HBe and anti-HD were detected by commercial radioimmunoassay (Ausria II, Abbott-HBe, Antidelta; Abbott Laboratories, North Chicago, IL). Anti-HD IgM was assayed by enzyme immunoassay (Deltassay IgM, Pasteur, France)
R. Jardi et al. /Journal
of Virological Methods 50 (1994) 59-66
61
2.3. HDV-RNA assay A modified, previously described (Rasshofer et al., 1988) dot-blot hybridization technique was used for detection of HDV-RNA in serum. Briefly, 100 ~1 of serum were digested with 250 pg/ml of proteinase K for 2 h at 4S’C in a total volume of 500 ~1 containing 50 mM Tris-HCl (pH 8.01, 200 mM NaCl, 2% SDS and 10 mM EDTA. Samples were extracted once with phenol and twice with chloroform/isoamylalcohol and nucleic acids were precipitated overnight at -20°C with 1 ml of ethanol and 10 ~1 of DEAE Dextrano. Subsequently, the RNA samples were denatured at 68°C in 50% formamide, 6% formaldehyde and 1 X SSC. The samples were applied to a nitrocellulose membrane using dot-blot apparatus. The filters were dried and immobilized at -80°C under vacuum. Prehybridization was carried out as previously reported (Rasshofer et al., 1988). Hybridization was done with a cDNA of HDV-RNA representing the viral genome cloned in plasmid pSVLD3 (kindly rovided by Dr. J. Taylor, Fox r32 Chase Cancer Center, Philadelphia, USA). Radioactive P cDNA probes were labelled by random priming (Boehringer Mannheim). Filters were exposed to X-ray films in the presence of intensifying screens at -80°C. Sensitivity of the assay was 0.5 pg of plasmid pSVLD3. 2.4. HDAg in serum by Immunoblot assay HDAg was detected in serum samples by immunoblot according to a method descreibed previously (Buti et al., 1989). Briefly, 1 ml of serum was layered over 11 ml of 20% sucrose, 0.02 M Hepes, 0.01 M CaCl,, 0.01 MgCl, and 0.1% bovine serum albumin and centrifuged in a TST-41.41 rotor (Kontrom Instruments) for 5 h at 150 000 X g. The pellets were boiled in 0.05 M Tris (pH 7.41, 0.15 M NaCl and reduced with 1% 2-mercaptoethanol, run on 12% polyacrylamide gels and transferred to nitrocellulose filters. Subsequently, they were incubated with an IgG anti-HD fraction (15 pg per dl), in 0.15 M NaCl, 0.25% gelatin, 0.005 M EDTA, 0.05% Nonidet P-40 in 0.05 M Tris (pH 7.4) for 2 h. The filters were incubated for 1 h at room temperature with 1251-labelled Staphylococcus aureus protein A (Amersham L.S) and then washed, dried and autoradiographed at - 80°C. for 12 h. Samples were considered positive when two bands of approximately 24 and 27 kDa were detected. 2.5. HDAg in liver samples Liver biopsy specimens from patients were processed by standard techniques, and paraffin sections were examined for the presence of HDAg by an indirect immunoperoxidase method (Verme et al., 1986). 2.6. Statistical procedure The qualitative variables between the groups were compared using Fisher’s exact test and x2 test. To compare the sensitivity (number of patients positive for hepatic HDAg
62
R. Jardi et al. /Journal
of Virological
Methods 50 (1994)
59-66
Table 1 Comparison between the detection of HDAg by immunoblot, HDV-RNA by molecular hybridization, IgM by EIA and total anti-HD by RIA in relation to the presence or absence of hepatic HDAg Serum samples
38 Hepatic HDAg*
12 Hepatic HDAg-
HDV-RNA+ HDAg+ Anti-HD IgM+
25 (66%)
0
HDV-RNA+ HDAg+ Anti-HD IgM-
0
1 (8%)
HDV-RNA+ HDAgAnti-HD IgM+
5 (13%)
0
HDV-RNA only +
1 (3%)
1(8%1
HDAg only+
2 (5%)
1(8%)
Anti-HD IgM only +
3 (8%)
7 (58%)
Anti-HD total only +
2 (5%)
2 (17%)
anti-HD
also positive for the serological tests) and specificity (number of patients negative for hepatic HDAg also negative for serological marker) of the two methods of diagnosis, Macnemar and Fisher’s exact tests were used. The concordance study was carried out using the Kappa statistic test. A P value of less than 0.05 was considered significant.
3. Results Of the 38 patients with intrahepatic HDAg, 31 (82%) were HDV-RNA positive, 27 (71%) were serum HDAg positive by immunoblot technique and anti-HD IgM was detected in 33 cases (87%). No significant differences were found between the three types of detection. Of the 12 tissue HDAg negative chronic hepatitis cases, two (17%) had HDV-RNA and only one was positive for HDAg using the immunoblot technique, while anti-HD IgM was detected in seven cases (58%). The differences between the detection of IgM anti-HD and the other serological markers were statistically significant (P < 0.05). Table 1 shows the results obtained with determination of HDV-RNA, HDAg, anti-HD IgM and total anti-HD in serum samples of patients with chronic delta hepatitis in relation to the presence or absence of intrahepatic HDAg. It is noteworthy that in the group of patients with hepatic HDAg the three markers were positive in 66% of patients,
R. Jardi et al. /Journal
Table 2 Analysis infection
of the concordance
Intrahepatic Intrahepatic Intrahepatic
between
HDAg
of Virological
detection
HDAg/HDV-RNA HDAg/HDAg in serum HDAg/anti-HD IgM
Methods 50 (1994)
in hepatic
59-66
63
tissue and serological
Kappa
t
0.568 0.488 0.302
4.113 3.816 2.152
markers
of HDV
and that these markers were not detected together in any of the intrahepatic HDAg negative patients. The concordance analysis between the serological markers of HDV infection and HDAg detection in hepatic tissue, as well as the sensitivity, specificity and global value of these tests in relation to the presence of intrahepatic HDAg, are summarized in Table 2 and Fig. 1. The concordance with intrahepatic HDAg was highest for HDV-RNA determination (Kappa = 0.5681, followed by HDAg by immunoblot (Kappa = 0.488). The least degree of concordance was found with anti-HD IgM determination (Kappa = 0.302). The sensitivity was higher with the anti-HD IgM technique (0.868, CI = 95%, confidence range 0.719-0.956) with statistically significant differences in relation to serum HDAg determined by the immunoblot technique (P < 0.05). The HDV-RNA test was more sensitive (0.816, CI = 95%, confidence range 0.657-0.923) than serum HDAg determination (0.711, CI = 95%, confidence range 0.541-0.846); however, no statistical differences were found.
0.8 08 Q4 OS2 0 HDV-RNA
HDAg in serum
anti-HD
SENSITIVITY
0,816
0,711
SPECIFICITY
0,833
0,917
0,417
GLOBAL
0,821
0,763
0,763
VALUE
-
Fig. 1. Analysis
of sensitivity
SENSITIVITY
and specificity
i%8
SPECIFICITY
of the serological
IgM
0,868
0
GLOBAL
VALUE
tests for detection of HDV infection.
64
R. Jardi et al. /Journal
of Virological Methods 50 (1994) 59-66
The determination of serum HDAg in serum by immunoblot technique was the most specific test (0.917, CI = 95%, confidence range 0.655-0.980) followed by the determination of HDV-RNA (0.833, CI = 95%, confidence range 0.516-0.979). The least specific was the anti-HD IgM technique (0.417, CI = 95%, confidence range 0.1520.733) with statistically significant differences when compared to serum HDAg detected by the immunoblot technique (P < 0.05). The differences in specificity between determination of serum HDAg by immunoblot and HDV-RNA by hybridization in dot-blot were not statistically significant.
4. Discussion The results of this study show that there is a good degree of concordance between the detection of HDV-RNA and intrahepatic HDAg: HDV-RNA was observed in 82% of patients who presented HDAg in hepatic tissue and in only two cases 17% of HDAg hepatic negative patients. The percentage of HDV-RNA is high in the hepatic HDAg positive patients, in accordance with previous reports (Gupta et al., 1989; Smedile et al., 1991). The determination of HDV-RNA by hybridization in dot-blot has the best global relationship between sensitivity and specificity of the three methods and may be considered optimum to study serum viral replication in chronic delta hepatitis. Serum HDAg determined by the immunoblot technique was detected in 71% of the tissue HDAg positive patients and in one negative patient. This method is the most specific of the three, followed by the HDV-RNA technique. Moreover serum determinations of HDAg and HDV-RNA showed the highest degree of concordance with hepatic HDAg. However, of these two methods the analysis of serum HDV-RNA is the easiest to perform and could be applied to the evaluation of large numbers of cases with delta hepatitis. In different studies the determination of anti-HD IgM has been considered a useful and practical marker to diagnose and monitor chronic delta infection (Dimitrakakis et al., 1986; Farci et al., 1986; Buti et al., 1987). However, the correlation between serum anti-HD IgM and hepatic delta antigen is a source of controversy. Some studies have demonstrated serum anti-HD IgM in the majority of cases with active chronic HDV infection (Smedile et al., 1982) but in other works this correlation was not observed (Govindarajan et al., 1989). In our study, anti-HD IgM was detected in 87% of patients with intrahepatic HDAg and in 58% of patients without hepatic delta antigen. The concordance between anti-HD IgM and intrahepatic HDAg (Kappa = 0.302) was markedly less than was observed with serum HDV-RNA and/or serum HDAg. The sensitivity and specificity studies proved anti-HD IgM to be the most sensitive test (0.868) but the least specific (0.417, CI = 95%, confidence range 0.152-0.733). The sensitivity found for anti-HD IgM is similar to that of the most commonly used commercial kits for this marker as demonstrated in a previous study which compared two commercial kits, a radioimmunoassay (sensitivity 79%) and an enzyme immunoassay (sensitivity 71%) (Crespo et al., 1990). The low specificity of the anti-HD IgM technique may be responsible for the high percentage of anti-HD IgM detected in chronic delta hepatitis, otherwise difficult to explain, especially in patients without
R. Jardi et al. /Journal
of Virological Methods 50 (I 994) 59-66
65
HDAg in hepatic tissue. These discrepancies in the positivity of anti-HD IgM antibodies and the observation that in some patients under antiviral treatment the disappearance of hepatic HDAg preceded (up to 2 years) the loss of anti-HD IgM, demonstrates that the exclusive determination of this antibody is not reliable for deciding antiviral treatment (Lau et al., 1991). In conclusion, the results of the statistical analysis suggest that the determination of HDV-RNA by dot-blot hybridization can be an alternative method to the study of HDAg in hepatic tissue. It can be easily applied to those patients in which liver biopsy is contraindicated or in patients who need serial determinations to control the evolution of the illness or treatment. Thus, the development of a commercial test for HDV-RNA determination would provide a specific, practical tool for the diagnosis and monitoring of active delta infection.
Acknowledgements The authors wish to express their gratitude to Carme Molinos, Montserrat Gimferer and Isabel Guilera for their nursing assistance. This study was supported in part by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS-13091992).
References Bergmann, K.F. and Germ, J.L. (1986) Antigens of hepatitis delta virus in the liver and serum of humans and animal. J. Infect. Dis. 154, 702-706. Bonino, F., Heermann, K.H., Rizetto, M. and Gerlich, W.H. (1986) Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus derived envelope. J. Med. Viro1.58, 945-950. Buti, M., Esteban, J.I., Esteban, R., Allende, H., Jardi, R. and Guardia, J. (1987) Anti-HD IgM as marker of chronic delta infection. J. Hepatol. 4, 62-65. Buti, M., Esteban, R., Roggendorf, M., Femandez, J., Jardi, R., Rasshofer, R., Allende, H., Genesca, J., Esteban, J.I. and Guardia, J. (1988) Hepatitis D virus, R.N.A in acute delta infection: Serological profile and correlation with other markers of hepatitis D virus infection. Hepatology 8, 1125-1129. Buti, M., Esteban, R., Jardi, R., Rodriguez-Frias, F., Casacuberta, J., Esteban, J.L., Allende, E. and Guardia, J. (1989) Chronic delta hepatitis: Detection of hepatitis delta virus antigen in serum by immunoblot and correlation with other markers of delta viral replication. Hepatology 10, 907-910. Crespo, M., Buti, M., Jardi, R., Rodriguez-Frias, F., Allende, H., Cotrina, M., Esteban, R. and Guardia, J. (1990) Determinaci6n de anticuerpos antidelta IgM en la infection aguda y cr6nica delta: estudio comparative de dos metodos analiticos. Gastroenterologia y Hepatologia 13, 221-224. Dimitrakakis, M., Waters, M.J., Wootton, A. and Gust, I. (1986) Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus. J. Med. Virol. 20, 305-311. Di Bisceglie, A.M. and Negro, F. (1989) Diagnosis of hepatitis delta virus infection. Hepatology 6, 1014-1016. Farci, P., Germ, J.L., Aragona, M., Lindsey, I., Crivelli, O., Balastrieri, A., Smedile, A., Thomas, H.C. and Rizetto, M. (1986) Diagnostic and pronostic significance of IgM antibody to hepatitis delta virus. J. Am. Med. Assoc. 255, 1443-1446. Govindarajan, S., Gupta, S., Valinluck, B. and Redeker, A.G. (1989) Correlation of IgM anti-HDV to hepatitis delta virus, RNA in sera of chronic HDV. Hepatology 10, 34-35.
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Gupta, S., Valinluck, B. and Govindarajan, S. (1989) Detection of hepatitis delta virus in serum and liver tissue by molecular hybridization: validation of a rapid spot hybridization technique. Am. J. Clin. Pathol. 92, 218-222. Jardi, R., Buti, M., Rodriguez-Frias, F., Garcia Lafuente, A., Sjogren, M.H., Esteban, R. and Guardia, J. (19911 Clinical significance of two forms of IgM antibody to hepatitis delta virus. Hepatology 14, 25-28. Lau, J.L.N, Smith, H.M., Chaggar, K., Hansen, L.J., Portmann, B.C., Alexander, G.J.M. and Williams, R. (1991) Significance of, IgM anti-Hepatitis D virus (HDV) in chronic HDV infection. J. Med. Viral. 33, 263-276. Rasshofer, R., Buti, M., Esteban, R., Jardi, R. and Roggendorf, M. (1988) Demonstration of hepatitis D virus RNA in patients with chronic hepatitis. J. Infect. Dis. 157, 191-195. Rizetto, M. (1983) The delta agent. Hepatology 3, 729-737. Smedile, A., Lavarini, C., Crivelli, O., Raimundo, G., Fassone, M. and Rizetto, M. (1982) Radioimmunoassay detection of IgM antibodies to HBV associated delta antigen: Clinical significance in delta infection. J. Med. Virol. 9, 131-138. Smedille, A., Rosina, F., Saracco, G., Chiaberge, E., Lattore, V., Fabiano, A., Brunetto, M.R., Verme, G.. Rizetto, M. and Bonino, F. (1991) Hepatitis B replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D. Hepatology 13, 413-416. Verme, G., Amoroso, P. and Lettieri, G. (19861 A histological study of the hepatitis delta virus liver disease. Hepatology 6, 1303-1307.
ELSEVIER
Journal of Virological
Methods 50 (1994) 59-66
Comparative analysis of serological markers of chronic delta infection: HDV-RNA, serum HDAg and anti-HD IgM Rosendo Jardi a,*, Maria Buti b, Francisco Rodriguez a, Montserrat Cotrina a, Helena Allende b, Rafael Esteban b, Jaime Guardia b a Liter Unit: Department b Liver Unit: Department
of Biochemistry, Hospital General Valle Hebrbn, Paseo Valle Hebr6n s/n, Universidad Autckoma, 08035 Barcelona, Spain of Internal Medicine, Hospital General Valle Hebrbn, Universidad Aut&oma, Barcelona, Spain Accepted
13 April 1994
Abstract To study the concordance, sensitivity and specificity of HDV-RNA determination by molecular hybridization, serum HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection by an immunoperoxidase method, a statistical analysis was applied to the results of serum sample and liver biopsy determinations in 50 patients with chronic delta hepatitis (38 positive to tissue HDAg and 12 negative).Of the 38 patients with hepatic HDAg, HDV-RNA was found in 31 (82%), serum HDAg by immunoblot in 27 (71%) and anti-HD IgM in 33 (87%). Among the 12 patients without hepatic HDAg, one was found with serum HDAg using the immunoblot technique, two (17%) had HDV-RNA, and 7 (58%) had anti-HD IgM. Serum HDAg determination by immunoblot was the most specific test, followed by HDV-RNA analysis. The least specific was the anti-HD IgM technique. The anti-HD IgM test was the most sensitive, followed by HDV-RNA and serum HDAg. The concordance with intrahepatic HDAg detection was highest for HDV-RNA determination, followed by HDAg in serum. The least degree of concordance was found with anti-HD IgM determination. These results suggest that the determination of HDV-RNA by the hybridization method can be of great value for the diagnosis and monitoring of chronic delta hepatitis. Keywords:
Chronic hepatitis D infection;
* Corresponding 0166-0934/94/$07.00
HDV-RNA;
HDAg; Anti-HD
IgM
author. Fax: + 34 3 4280443. 0 1994 Elsevier Science B.V. All rights reserved
60
R. Jardi et al. /Journal
of Virological Methods 50 (1994) 59-66
1. Introduction Hepatitis delta virus (HDV) is a defective RNA virus that replicates only in hosts simultaneously infected with hepatitis B virus (HBV) (Rizetto, 1983; Bonino et al., 1986). Chronic hepatitis delta is diagnosed by identification of the delta antigen (HDAg) in hepatic tissue and/or by determining the presence of high titers of antibodies to HDAg (anti-HD) in serum (Di Bisceglie, 1989). Anti-HD of the IgM type (anti-HD IgM) is present in serum of the majority of patients with chronic hepatitis with intrahepatic HDAg, thus anti-HD IgM has been considered a good marker of active delta infection. However, in some patients this antibody has been found in the absence of HDAg in the liver (Farci et al., 1986; Buti et al., 1987; Jardi et al., 1991; Smedile et al., 1991). Various workers have demonstrated that the detection of hepatitis delta RNA (HDV-RNA) by a molecular hybridization technique and HDAg in serum by an immunoblot method indicate HDV replication (Bermann et al., 1986; Rasshofer et al., 1988; Gupta et al., 1989; Smedile et al., 1991). Although superior in performance to conventional assays, these alternative methods are not generally available to laboratories because of the technical difficulties involved (Di Bisceglie et al., 1989). Therefore, in most cases, serological methods are still employed. The aim was to study the concordance, sensitivity and specificity of serum HDV-RNA determination by dot-blot hybridization, HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection, the current ‘gold standard’. For this purpose, a statistical analysis was carried out on serum samples and liver biopsies of 50 patients with chonic delta hepatitis examined previously (Buti et al., 1988, 1989).
2. Patients and methods 2.1. Patients Fifty HBsAg positive patients, 38 with hepatic HDAg and 12 without, with proven liver disease by histological evidence and high titers of anti-HD antibodies > l/10000 were studied. The patients with intrahepatic HDAg included 32 men and six women (mean age 22 years, range 17-30 years). All were intravenous drug addicts. The histological diagnoses were: chronic active hepatitis (CAH) in 27 cases and liver cirrhosis (LC) in 11 cases. Of the 12 patients without tissue HDAg, nine were men and three were women (mean age 26 years, range 22-50 years); seven weredrug addicts. The histological study demonstrated CAH in eight casesand LC in four cases. 2.2. Serological
analysis
HBsAg, HBeAg, anti-HBe and anti-HD were detected by commercial radioimmunoassay (Ausria II, Abbott-HBe, Antidelta; Abbott Laboratories, North Chicago, IL). Anti-HD IgM was assayed by enzyme immunoassay (Deltassay IgM, Pasteur, France)
R. Jardi et al. /Journal
of Virological Methods 50 (1994) 59-66
61
2.3. HDV-RNA assay A modified, previously described (Rasshofer et al., 1988) dot-blot hybridization technique was used for detection of HDV-RNA in serum. Briefly, 100 ~1 of serum were digested with 250 pg/ml of proteinase K for 2 h at 4S’C in a total volume of 500 ~1 containing 50 mM Tris-HCl (pH 8.01, 200 mM NaCl, 2% SDS and 10 mM EDTA. Samples were extracted once with phenol and twice with chloroform/isoamylalcohol and nucleic acids were precipitated overnight at -20°C with 1 ml of ethanol and 10 ~1 of DEAE Dextrano. Subsequently, the RNA samples were denatured at 68°C in 50% formamide, 6% formaldehyde and 1 X SSC. The samples were applied to a nitrocellulose membrane using dot-blot apparatus. The filters were dried and immobilized at -80°C under vacuum. Prehybridization was carried out as previously reported (Rasshofer et al., 1988). Hybridization was done with a cDNA of HDV-RNA representing the viral genome cloned in plasmid pSVLD3 (kindly rovided by Dr. J. Taylor, Fox r32 Chase Cancer Center, Philadelphia, USA). Radioactive P cDNA probes were labelled by random priming (Boehringer Mannheim). Filters were exposed to X-ray films in the presence of intensifying screens at -80°C. Sensitivity of the assay was 0.5 pg of plasmid pSVLD3. 2.4. HDAg in serum by Immunoblot assay HDAg was detected in serum samples by immunoblot according to a method descreibed previously (Buti et al., 1989). Briefly, 1 ml of serum was layered over 11 ml of 20% sucrose, 0.02 M Hepes, 0.01 M CaCl,, 0.01 MgCl, and 0.1% bovine serum albumin and centrifuged in a TST-41.41 rotor (Kontrom Instruments) for 5 h at 150 000 X g. The pellets were boiled in 0.05 M Tris (pH 7.41, 0.15 M NaCl and reduced with 1% 2-mercaptoethanol, run on 12% polyacrylamide gels and transferred to nitrocellulose filters. Subsequently, they were incubated with an IgG anti-HD fraction (15 pg per dl), in 0.15 M NaCl, 0.25% gelatin, 0.005 M EDTA, 0.05% Nonidet P-40 in 0.05 M Tris (pH 7.4) for 2 h. The filters were incubated for 1 h at room temperature with 1251-labelled Staphylococcus aureus protein A (Amersham L.S) and then washed, dried and autoradiographed at - 80°C. for 12 h. Samples were considered positive when two bands of approximately 24 and 27 kDa were detected. 2.5. HDAg in liver samples Liver biopsy specimens from patients were processed by standard techniques, and paraffin sections were examined for the presence of HDAg by an indirect immunoperoxidase method (Verme et al., 1986). 2.6. Statistical procedure The qualitative variables between the groups were compared using Fisher’s exact test and x2 test. To compare the sensitivity (number of patients positive for hepatic HDAg
62
R. Jardi et al. /Journal
of Virological
Methods 50 (1994)
59-66
Table 1 Comparison between the detection of HDAg by immunoblot, HDV-RNA by molecular hybridization, IgM by EIA and total anti-HD by RIA in relation to the presence or absence of hepatic HDAg Serum samples
38 Hepatic HDAg*
12 Hepatic HDAg-
HDV-RNA+ HDAg+ Anti-HD IgM+
25 (66%)
0
HDV-RNA+ HDAg+ Anti-HD IgM-
0
1 (8%)
HDV-RNA+ HDAgAnti-HD IgM+
5 (13%)
0
HDV-RNA only +
1 (3%)
1(8%1
HDAg only+
2 (5%)
1(8%)
Anti-HD IgM only +
3 (8%)
7 (58%)
Anti-HD total only +
2 (5%)
2 (17%)
anti-HD
also positive for the serological tests) and specificity (number of patients negative for hepatic HDAg also negative for serological marker) of the two methods of diagnosis, Macnemar and Fisher’s exact tests were used. The concordance study was carried out using the Kappa statistic test. A P value of less than 0.05 was considered significant.
3. Results Of the 38 patients with intrahepatic HDAg, 31 (82%) were HDV-RNA positive, 27 (71%) were serum HDAg positive by immunoblot technique and anti-HD IgM was detected in 33 cases (87%). No significant differences were found between the three types of detection. Of the 12 tissue HDAg negative chronic hepatitis cases, two (17%) had HDV-RNA and only one was positive for HDAg using the immunoblot technique, while anti-HD IgM was detected in seven cases (58%). The differences between the detection of IgM anti-HD and the other serological markers were statistically significant (P < 0.05). Table 1 shows the results obtained with determination of HDV-RNA, HDAg, anti-HD IgM and total anti-HD in serum samples of patients with chronic delta hepatitis in relation to the presence or absence of intrahepatic HDAg. It is noteworthy that in the group of patients with hepatic HDAg the three markers were positive in 66% of patients,
R. Jardi et al. /Journal
Table 2 Analysis infection
of the concordance
Intrahepatic Intrahepatic Intrahepatic
between
HDAg
of Virological
detection
HDAg/HDV-RNA HDAg/HDAg in serum HDAg/anti-HD IgM
Methods 50 (1994)
in hepatic
59-66
63
tissue and serological
Kappa
t
0.568 0.488 0.302
4.113 3.816 2.152
markers
of HDV
and that these markers were not detected together in any of the intrahepatic HDAg negative patients. The concordance analysis between the serological markers of HDV infection and HDAg detection in hepatic tissue, as well as the sensitivity, specificity and global value of these tests in relation to the presence of intrahepatic HDAg, are summarized in Table 2 and Fig. 1. The concordance with intrahepatic HDAg was highest for HDV-RNA determination (Kappa = 0.5681, followed by HDAg by immunoblot (Kappa = 0.488). The least degree of concordance was found with anti-HD IgM determination (Kappa = 0.302). The sensitivity was higher with the anti-HD IgM technique (0.868, CI = 95%, confidence range 0.719-0.956) with statistically significant differences in relation to serum HDAg determined by the immunoblot technique (P < 0.05). The HDV-RNA test was more sensitive (0.816, CI = 95%, confidence range 0.657-0.923) than serum HDAg determination (0.711, CI = 95%, confidence range 0.541-0.846); however, no statistical differences were found.
0.8 08 Q4 OS2 0 HDV-RNA
HDAg in serum
anti-HD
SENSITIVITY
0,816
0,711
SPECIFICITY
0,833
0,917
0,417
GLOBAL
0,821
0,763
0,763
VALUE
-
Fig. 1. Analysis
of sensitivity
SENSITIVITY
and specificity
i%8
SPECIFICITY
of the serological
IgM
0,868
0
GLOBAL
VALUE
tests for detection of HDV infection.
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The determination of serum HDAg in serum by immunoblot technique was the most specific test (0.917, CI = 95%, confidence range 0.655-0.980) followed by the determination of HDV-RNA (0.833, CI = 95%, confidence range 0.516-0.979). The least specific was the anti-HD IgM technique (0.417, CI = 95%, confidence range 0.1520.733) with statistically significant differences when compared to serum HDAg detected by the immunoblot technique (P < 0.05). The differences in specificity between determination of serum HDAg by immunoblot and HDV-RNA by hybridization in dot-blot were not statistically significant.
4. Discussion The results of this study show that there is a good degree of concordance between the detection of HDV-RNA and intrahepatic HDAg: HDV-RNA was observed in 82% of patients who presented HDAg in hepatic tissue and in only two cases 17% of HDAg hepatic negative patients. The percentage of HDV-RNA is high in the hepatic HDAg positive patients, in accordance with previous reports (Gupta et al., 1989; Smedile et al., 1991). The determination of HDV-RNA by hybridization in dot-blot has the best global relationship between sensitivity and specificity of the three methods and may be considered optimum to study serum viral replication in chronic delta hepatitis. Serum HDAg determined by the immunoblot technique was detected in 71% of the tissue HDAg positive patients and in one negative patient. This method is the most specific of the three, followed by the HDV-RNA technique. Moreover serum determinations of HDAg and HDV-RNA showed the highest degree of concordance with hepatic HDAg. However, of these two methods the analysis of serum HDV-RNA is the easiest to perform and could be applied to the evaluation of large numbers of cases with delta hepatitis. In different studies the determination of anti-HD IgM has been considered a useful and practical marker to diagnose and monitor chronic delta infection (Dimitrakakis et al., 1986; Farci et al., 1986; Buti et al., 1987). However, the correlation between serum anti-HD IgM and hepatic delta antigen is a source of controversy. Some studies have demonstrated serum anti-HD IgM in the majority of cases with active chronic HDV infection (Smedile et al., 1982) but in other works this correlation was not observed (Govindarajan et al., 1989). In our study, anti-HD IgM was detected in 87% of patients with intrahepatic HDAg and in 58% of patients without hepatic delta antigen. The concordance between anti-HD IgM and intrahepatic HDAg (Kappa = 0.302) was markedly less than was observed with serum HDV-RNA and/or serum HDAg. The sensitivity and specificity studies proved anti-HD IgM to be the most sensitive test (0.868) but the least specific (0.417, CI = 95%, confidence range 0.152-0.733). The sensitivity found for anti-HD IgM is similar to that of the most commonly used commercial kits for this marker as demonstrated in a previous study which compared two commercial kits, a radioimmunoassay (sensitivity 79%) and an enzyme immunoassay (sensitivity 71%) (Crespo et al., 1990). The low specificity of the anti-HD IgM technique may be responsible for the high percentage of anti-HD IgM detected in chronic delta hepatitis, otherwise difficult to explain, especially in patients without
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65
HDAg in hepatic tissue. These discrepancies in the positivity of anti-HD IgM antibodies and the observation that in some patients under antiviral treatment the disappearance of hepatic HDAg preceded (up to 2 years) the loss of anti-HD IgM, demonstrates that the exclusive determination of this antibody is not reliable for deciding antiviral treatment (Lau et al., 1991). In conclusion, the results of the statistical analysis suggest that the determination of HDV-RNA by dot-blot hybridization can be an alternative method to the study of HDAg in hepatic tissue. It can be easily applied to those patients in which liver biopsy is contraindicated or in patients who need serial determinations to control the evolution of the illness or treatment. Thus, the development of a commercial test for HDV-RNA determination would provide a specific, practical tool for the diagnosis and monitoring of active delta infection.
Acknowledgements The authors wish to express their gratitude to Carme Molinos, Montserrat Gimferer and Isabel Guilera for their nursing assistance. This study was supported in part by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS-13091992).
References Bergmann, K.F. and Germ, J.L. (1986) Antigens of hepatitis delta virus in the liver and serum of humans and animal. J. Infect. Dis. 154, 702-706. Bonino, F., Heermann, K.H., Rizetto, M. and Gerlich, W.H. (1986) Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus derived envelope. J. Med. Viro1.58, 945-950. Buti, M., Esteban, J.I., Esteban, R., Allende, H., Jardi, R. and Guardia, J. (1987) Anti-HD IgM as marker of chronic delta infection. J. Hepatol. 4, 62-65. Buti, M., Esteban, R., Roggendorf, M., Femandez, J., Jardi, R., Rasshofer, R., Allende, H., Genesca, J., Esteban, J.I. and Guardia, J. (1988) Hepatitis D virus, R.N.A in acute delta infection: Serological profile and correlation with other markers of hepatitis D virus infection. Hepatology 8, 1125-1129. Buti, M., Esteban, R., Jardi, R., Rodriguez-Frias, F., Casacuberta, J., Esteban, J.L., Allende, E. and Guardia, J. (1989) Chronic delta hepatitis: Detection of hepatitis delta virus antigen in serum by immunoblot and correlation with other markers of delta viral replication. Hepatology 10, 907-910. Crespo, M., Buti, M., Jardi, R., Rodriguez-Frias, F., Allende, H., Cotrina, M., Esteban, R. and Guardia, J. (1990) Determinaci6n de anticuerpos antidelta IgM en la infection aguda y cr6nica delta: estudio comparative de dos metodos analiticos. Gastroenterologia y Hepatologia 13, 221-224. Dimitrakakis, M., Waters, M.J., Wootton, A. and Gust, I. (1986) Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus. J. Med. Virol. 20, 305-311. Di Bisceglie, A.M. and Negro, F. (1989) Diagnosis of hepatitis delta virus infection. Hepatology 6, 1014-1016. Farci, P., Germ, J.L., Aragona, M., Lindsey, I., Crivelli, O., Balastrieri, A., Smedile, A., Thomas, H.C. and Rizetto, M. (1986) Diagnostic and pronostic significance of IgM antibody to hepatitis delta virus. J. Am. Med. Assoc. 255, 1443-1446. Govindarajan, S., Gupta, S., Valinluck, B. and Redeker, A.G. (1989) Correlation of IgM anti-HDV to hepatitis delta virus, RNA in sera of chronic HDV. Hepatology 10, 34-35.
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of Virological Methods 50 (1994) 59-66
Gupta, S., Valinluck, B. and Govindarajan, S. (1989) Detection of hepatitis delta virus in serum and liver tissue by molecular hybridization: validation of a rapid spot hybridization technique. Am. J. Clin. Pathol. 92, 218-222. Jardi, R., Buti, M., Rodriguez-Frias, F., Garcia Lafuente, A., Sjogren, M.H., Esteban, R. and Guardia, J. (19911 Clinical significance of two forms of IgM antibody to hepatitis delta virus. Hepatology 14, 25-28. Lau, J.L.N, Smith, H.M., Chaggar, K., Hansen, L.J., Portmann, B.C., Alexander, G.J.M. and Williams, R. (1991) Significance of, IgM anti-Hepatitis D virus (HDV) in chronic HDV infection. J. Med. Viral. 33, 263-276. Rasshofer, R., Buti, M., Esteban, R., Jardi, R. and Roggendorf, M. (1988) Demonstration of hepatitis D virus RNA in patients with chronic hepatitis. J. Infect. Dis. 157, 191-195. Rizetto, M. (1983) The delta agent. Hepatology 3, 729-737. Smedile, A., Lavarini, C., Crivelli, O., Raimundo, G., Fassone, M. and Rizetto, M. (1982) Radioimmunoassay detection of IgM antibodies to HBV associated delta antigen: Clinical significance in delta infection. J. Med. Virol. 9, 131-138. Smedille, A., Rosina, F., Saracco, G., Chiaberge, E., Lattore, V., Fabiano, A., Brunetto, M.R., Verme, G.. Rizetto, M. and Bonino, F. (1991) Hepatitis B replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D. Hepatology 13, 413-416. Verme, G., Amoroso, P. and Lettieri, G. (19861 A histological study of the hepatitis delta virus liver disease. Hepatology 6, 1303-1307.