Liver International 2006: 26: 805–810 r 2006 The Author Journal compilation r 2006 Blackwell Munksgaard

Clinical Studies DOI: 10.1111/j.1478-3231.2006.01279.x Treatment of chronic delta with pegylated -a2b

1 2 Erhardt A, Gerlich W, Starke C, Wend U, Donner A, Sagir A, Heintges T, Andreas Erhardt , Wolfram Gerlich , 1 2 Ha¨ussinger D. Treatment of chronic hepatitis delta with pegylated interferon- Christine Starke , Ulrike Wend , a2b. 3 Andreas Donner , Abdurrahman Liver International 2006: 26: 805–810. 1 1 Sagir , Tobias Heintges and Dieter r 2006 The Author. Journal compilation r 2006 Blackwell Munksgaard Ha¨ussinger1

1Klinik fu¨r Gastroenterologie, Hepatologie und Abstract: Background/Aims: Chronic hepatitis D is difficult to treat. The present pilot study investigated the efficacy and tolerability of pegylated Infektiologie, Universita¨tsklinikum der Heinrich- Patients and Heine-Universita¨t Du¨sseldorf, Du¨sseldorf, (PEG)-interferon (IFN)-a2b in chronic hepatitis D. 2 Methods: Twelve patients with chronic hepatitis D were prospectively treated Germany, Institut fu¨r Medizinische Virologie, Justus-Liebig-Universita¨t Giessen, Giessen, with 1.5 mg/kg PEG-IFN-a2b for 48 weeks and followed for 24 weeks. 3 Germany, Institut fu¨r Pathologie, Sustained response (SR) was defined as undetectable hepatitis delta virus Universita¨tsklinikum der Heinrich-Heine-

(HDV) RNA by reverse transcriptase-polymerase chain reaction and Universita¨t Du¨sseldorf, Du¨sseldorf, Germany normalization of alanine aminotransferase (ALT) at 6 months after

treatment. Investigations included HDV RNA kinetics, determination of

virus (HBV) and HDV genotypes and histological evaluation. Key words: hepatitis – interferon – treatment – Results: An SR was achieved in two out of 12 of patients (17%). The virus

negative predictive value of a less than 3 log HDV RNA decrease at month 6 Priv.-Doz. Dr. med. Andreas Erhardt, Klinik fu¨r

was 100%. The positive predictive value of a more than 3 log HDV RNA Gastroenterologie, Hepatologie und Infektiolo- gie, Heinrich-Heine-Universita¨t Du¨sseldorf, decrease at month 6 was 67%. A marked ALT reduction at the end of treatment was observed in responders and nonresponders. Ishak histological Moorenstr. 540225, Du¨sseldorf, Germany. Tel.: 100 49 0211 8268 score was comparable at baseline and significantly improved in responders compared with nonresponders at the end of follow-up (13.5 vs. 8.0; Po0.02). Fax: 100 49 0211 8132 e-mail: [email protected] Conclusion: The present study indicates that PEG-IFN-a2b is a promising

treatment option in chronic hepatitis D. Nonresponders could be identified Received 18 January 2006, by a less than 3 log decrease of HDV RNA at 6 months of treatment. accepted 19 March 2006

Hepatitis delta virus (HDV) affects months) interferon (IFN) therapy in less than about 15 million persons worldwide (1). 10% of HDV-infected patients when sensitive Although hepatitis delta is a rare disease com- reverse transcriptase-polymerase chain reaction pared with hepatitis B or hepatitis C, its impor- (RT-PCR) testing is used as an endpoint (4, 5). tance results from a high morbidity and However, because of preexisting liver cirr- mortality. Acute hepatitis delta can lead to severe hosis and significant side effects, many patients liver disease, with the highest rate of fulminant do not tolerate high IFN doses. A biochemical courses among the hepatotropic viruses (2). response and histological improvement has been Superinfection of hepatitis B virus carriers with reported in 7–70% of patients at the end of an HDV leads to chronic hepatitis in about 90% of antiviral treatment and in 0–43% at the end - cases. Compared with chronic hepatitis C and B, of follow-up (6–10). Long-term IFN treat- progression to liver cirrhosis has been reported to ment beyond 12 months might be beneficial in be more frequent and rapid in chronic hepatitis chronic hepatitis D (11, 12). In a case report, delta (3). Furthermore, the incidence of hepato- treatment with standard IFN for 12 years was cellular carcinoma is increased in patients with able to induce a sustained virus suppression in hepatitis delta compared with patients with he- chronic HDV superinfection (13). Persistent patitis B virus (HBV) monoinfection (3). alanine aminotransferase (ALT) normalization So far, there is no effective treatment of chronic can be associated with a regression of liver hepatitis D. Sustained virus elimination can be fibrosis despite ongoing viral replication (9, 11, achieved by high-dose or prolonged (12–24 14).

805 Erhardt et al.

Lamivudine or monotherapies and Table 1. Demographic and histological data at baseline (N 5 12) lamivudine–IFN combination therapies have Age (years) 34 15 not been proven to be effective in chronic hepa- Age at diagnosis of hepatitis B 26 15 titis D (15–18). In view of the inefficiency of Age at diagnosis of hepatitis D 33 16 Gender lamivudine, it is unlikely that adefovir, tenofovir Male 9 (75%) or other nucleos(t)ide analogues might be useful Female 3 (25%) for the treatment of hepatitis D. Prenylation HBeAg status (%) inhibitors are promising HDV-specific agents HBeAg negative 100 Origin (%) but have not been studied in humans so far (19). Germany 16.7 Long-term follow-up in patients with hepatitis Mediterranean basin 16.7 delta has been performed in a limited number of Former Soviet Union 66.6 smaller studies (3, 20–23). A beneficial effect of an Risk factors of infection (%) Blood or blood products 25 antiviral therapy on the long-term clinical course Family history 25 of hepatitis D has been reported for patients who IV drugs 16.7 received a high-dose IFN treatment independent Sexual 16.7 of the sustained virological response at 6 months Unknown 16.7 Ishak scoren after the end of treatment (14). Interface hepatitis (A) 2.9 0.7 PEG-IFNs have been reported to be superior Confluent necrosis (B) 0.4 0.5 to standard IFNs with regard to sustained vir- Focal lytic necrosis, apoptosis and focal inflammation (C) 2.9 0.6 ological response in chronic hepatitis B and C Portal inflammation (D) 3.1 0.5 Total inflammation 9.3 1.3 (24, 25). The aim of the present pilot study was to Fibrosis 3.7 1.3 investigate the efficacy and safety of PEG-IFN- Total Ishak score 13 2 a2b in chronic hepatitis D. nLiver biopsy was available in 11 patients.

Patients and methods A total of 12 patients with chronic hepatitis D DNA 3.0, Bayer, Leverkusen, Germany). How- have been included in a prospective study. Pa- ever, seven patients tested positive for HBV DNA tients were treated with PEG-IFN-a2b s.c. at a by PCR. high dose of 1.5 mg/kg for 48 weeks given the high All patients were tested negative for HIV by doses of standard IFN recommended in chronic ELISA. Two patients tested positive for anti- hepatitis D. Patients were followed for another 24 HCV by ELISA but were negative for HCV weeks after termination of IFN treatment. Com- RNA (bDNA Assay 3.0, Bayer). Other liver bined ALT normalization and negativation of diseases had been excluded. HDV RNA at 24 weeks of follow-up was defined as sustained response (SR). Informed consent was HBV genotyping obtained from each patient. The study was ap- HBV genotype was determined in seven patients by proved by the local ethics committee and per- direct sequencing using primers spanning part of formed according to the ethical guidelines of the the HBV surface gene (primer sense 50- 1975 Declaration of Helsinki. tggatgtgtctgcggc-30;primerantisense50-cKttgaca- The clinical, biochemical and histological char- Dactttccaatcaatag-30). HBV DNA isolation from acteristics of patients are given in Tables 1 and 2. serum was performed with a commercial kit (High Previous IFN therapy had been performed in Pure 16 System Viral Nucleic Acid kit; Roche three patients, but IFN dose or treatment dura- Diagnostics, Mannheim, Germany). PCR amplifi- tion had been insufficient. Treatment had been cation on a LightCycler (Roche Diagnostics, Man- performed with 3 MU IFN-a thrice weekly in two nheim, Germany) was performed with 50 cycles patients and for less than 4 months in one patient. after an initial denaturing step at 95 1C, an anneal- The diagnosis of chronic hepatitis D was based ing temperature of 69–60 1C (‘touch down PCR’) on commonly accepted criteria. All patients andanelongationtemperatureof721C. Sequen- had HBsAg (Abbott Laboratories, Wiesbaden, cing was performed as described previously (26). Germany) and detectable HDV antibodies for HBV DNA could not be amplified by PCR in five more than 6 months (Ortho Diagnostics, Tokyo, patients because of the absence of viremia. Japan). HDV infection was confirmed by quali- tative HDV RNA RT-PCR in all patients at HDV RNA quantification and HDV genotyping baseline. All 12 patients with chronic hepatitis delta were HBV DNA negative in a commercial HDV RNA was isolated from 400 ml serum with HBV DNA hybridization assay (Versant HBV an automatic nucleic acid extractor and a com-

806 Treatment of chronic hepatitis delta

Table 2. Baseline data (BL), biochemical and virological response at Results the end of treatment (ET) and at follow-up (FU) Baseline characteristics BL ET FU Male gender and eastern European origin pre- ALT elevated/normal dominated among patients with hepatitis delta. N 12/0 9/3 10/2 % 100/0 75/25 83/17 Blood transfusions, a positive family history and HDV RNA positive/negative former intravenous drug abuse were the most N 12/0 10/2 10/2 common risk factors for acquisition of hepatitis % 100/0 83/17 83/17 delta (Table 1). Diagnosis of hepatitis B preceded ALT (U/l) 72 36 39 29n 52 32 AST (U/l) 39 16 32 20 39 30 that of hepatitis delta by about 7 years. Despite GGT (U/l) 37 24 40 31 36 23 the young age of the patients (34 15 years), Bilirubin (mg/dl) 0.8 0.3 0.6 0.4 0.6 0.3 incomplete cirrhosis was histologically proven in Albumin (g/dl) 4.6 0.3 4.5 0.4 4.5 0.3 three out of 11 patients (27%) at initial presenta- Platelets ( 1000/ml) 162 67 129 56w 151 72 tion. Hepatitis D genotype I or II was prevalent nALTatETcomparedwithBL:Po0.03. wPlatelets at ET compared with among the patients; there was no carrier of HDV BL: Po0.01. ALT, alanine aminotransferase; AST, aspartate aminotrans- genotype III. All patients with amplifiable HBV ferase; GGT, g glutamyl transferase; HDV, hepatitis delta virus. DNA (N 5 7) carried the HBV genotype D. mercial isolation kit (MagNA Pure Compact Nucleic Acid Isolation Kit I, Roche Diagnostics). PEG-IFN treatment Reverse transcription and PCR were performed on a LightCycler with each 10 pmol of the HDV Patients received a mean cumulative dose of sense primer (50-tccagaggaccccttca-30) and HDV 4169 1963 mg of PEG-IFN-a2b. The dose of antisense primer (50-ccgggataagcctcact-30) using a the PEG-IFN-a2b had to be reduced in four of commercial kit (LightCycler RNA amplification 12 patients because of thrombocytopenia. Overall kit for hybridization probes; Roche). Reverse there was a decrease in ALT levels during PEG- transcription was carried out at 55 1C for IFN therapy with a significant reduction in ALT 20 min. DNA amplification was performed as levels at the end of treatment (Table 2), but mean touchdown PCR with an initial annealing tem- ALT levels increased again after treatment cessa- perature of 62 1C tapering down to 48 1C using a tion. ALT decrease was most pronounced in fluorescein hybridization probe for HDV geno- sustained responders, but also nonresponders type I/II (50-gagaccgaagcgaggaggaaag-FL-30) and displayed a decrease in ALT levels from baseline a50-LC 640 nm aaagaRagcaRcggggctagc-30 hy- to the end of treatment (68 33 vs. 40 32 U/l) bridization probe. A patient’s serum that had although significance was not reached (Fig. 1). A been calibrated on the HDV plasmid pSVL 50% reduction in ALT levels at the end of LD3 was taken as the standard (27). The diag- treatment compared with baseline was observed nostic sensitivity of the HDV RNA assay was among four out of 10 nonresponders There was 150 copies/ml. For HDV genotype III, the 50- no correlation between the ALT decrease and gaagccagaacgactgggaaag-FL-30 probe was used. HDV RNA decline in the nonresponders. At the Quantitative HDV RT-PCR could not be per- end of follow-up, still two out of 10 nonrespon- formed in one patient. ders displayed a 50% decrease in ALT. A normal- ization of ALT at the end of treatment was Liver histology achieved in 27% and a negativation of HDV RNA in 17% of patients. Normalization of Baseline biopsies were available in 11 patients. ALT and negativation of HDV RNA was sus- One patient did not agree to liver biopsy. Scoring tained in 17% of the patients (sustained respon- of liver histology was performed according to the ders). A more than 3 log decrease in HDV RNA classification of Ishak et al. (28) by a blinded was identified in responders at month 6 (Fig. 2A), pathologist (A. D.). A second liver biopsy 6 but only in one out of nine nonresponders (Fig. months after the end of treatment was obtained 2B and C). The negative predictive value of a less in seven patients (two responders and five non- than 3 log HDV RNA decrease at month 6 was responders). 100%; the positive predictive value of a 3 log or more decrease of HDV RNA at month 6 was Statistics 67%. The two responders displayed lower base- Student’s t-test and w2 test were used to analyze the line HDV RNA levels (7.8 106 vs. 3.0 108 co- data. Statistical analysis was performed by using pies/ml) and slightly higher ALT levels (91 58 vs. the SPSS program (SPSS Inc., Munich, Germany). 68 33 U/l) than nonresponder patients.

807 Erhardt et al.

140 (A) 9 Nonresponders 120 8 100 7 80 6 Nonresponders 60 5 ALT [U/l] 40 4 20 Responders 3 Responders 0 2 BL ET FU

Viral load [Log copies/ml] 1 Fig. 1. Alanine aminotransferase (ALT) levels of patients at baseline (BL), end of treatment (ET) and follow-up (FU). The 10 nonresponders were grouped together ( ); the two respon- BL M1 M2 M3 M6 M12 M18 ders are shown separately (} and & ). (B) 10 9 Ishak score at baseline was not different be- 8 tween responders and nonresponders. A second 7 liver biopsy was available in seven out of 11 6 patients (64%). An improvement in the total 5 Ishak score in the second liver biopsy was seen 4 for the two responders compared with the 10 3 nonresponders. However, no relevant deteriora- 2

Viral load [Log copies/ml] 1 tion in the Isak total score and subscores from the baseline level was noticed in the nonresponder BL M1 M2 M3 M6 M12 M18 patients (Fig. 3). All patients with liver cirrhosis (C) 9 (n 5 3) were nonresponders. 8 No serious adverse events were noted in the 7 patients. Flu-like symptoms and local skin reac- 6 tion at the injection site were the most frequently 5 reported side effects (Table 3). Marked thrombo- 4 cytopenia was the most common reason for dose 3 reduction, which can be explained by the high 2 rate of patients with advanced fibrosis and cir- 1 rhosis. Viral load [Log copies/ml] BL M1 M2 M3 M6 M12 M18 Fig. 2. Hepatitis delta virus (HDV) RNA levels during the study course (BL, baseline; M1–18, months 1–18). Treatment with Discussion pegylated interferon-a2b was performed over a period of 12 months up to month 12 (M12). Follow-up of patients was 6 Chronic hepatitis D is difficult to treat. Treat- months (months 12–18). (A) The 10 nonresponders were ment regimens for hepatitis D are IFN based so grouped together ( ); the two responders are shown separately far (4, 5). The response to standard IFN is better (} and & ). (B) Individual HDV RNA courses of nonrespon- with high doses of IFN (9–10 MU thrice weekly) ders with a 50% alanine aminotransferase (ALT) reduction at the end of treatment. (C) Individual HDV RNA courses of than lower IFN doses (9). Furthermore, prolon- nonresponders without significant ALT reduction at the end of gation of treatment beyond 12 months appears to treatment. be beneficial (11–13). Sustained virological response to IFN varies between 0% and 43% in the studies reported so No predictive factors for response to IFN have far (5). However, no sustained virological re- been identified so far in chronic hepatitis D. Virus sponse was achieved in the only randomized kinetics performed in the present study suggest study that measured HDV RNA by a sensitive that patients with a less than 3 log decrease at RT-PCR method (14). One nonrandomized study month 6 are unlikely to become sustained viro- using RT-PCR measurement reported a sustained logical or biochemical responders. Virus load virological response of 10% among 21 patients determination at month 6 might be helpful for treated for 24 months (12). In the present study, a establishing a stopping rule for treatment discon- sustained virological response was achieved in tinuation. 17% of patients, which is superior to the histor- Baseline parameters were not as suited as virus ical response rates of former studies using kinetics for prediction of IFN response. There is RT-PCR determination of HDV RNA. increasing evidence that HBV genotypes play a

808 Treatment of chronic hepatitis delta

Table 3. Side effects and adverse events of pegylated interferon 14 # (PEG-IFN)-a2b 12 BL NR BL SR PEG-IFN (N 5 12) 10 FU NR * FU SR # Flulike symptoms 7 8 Local skin reaction (43cm) 5 Weight loss (5–10%) 5 6 * Fatigue 4 ISAHK Score 4 Diffuse loss of hair 4 Abdominal pain 4 2 Thrombocytopenia 4 0 Impotence 2 A B C D Total Fibrosis Total Itching 1 inflammation Ishak Fig. 3. Liver histology according to the Ishak score at baseline (BL) and follow-up (FU) in sustained responders (SR) and nonresponders (NR). nPo0.02; #Po0.02. A, interface hepatitis; major role for responsiveness to IFN in chronic B, confluent necrosis; C, focal lytic necrosis, apoptosis and focal hepatitis B (26, 29). This might also apply to inflammation; D, portal inflammation. HDV genotypes in chronic hepatitis D and to some degree to HBV genotypes, although HBV matory IFN treatment might be a treatment replication is mostly repressed in HDV infection. option. All patients with detectable genotype were car- The present study suggests that treatment of riers of HBV genotype D and HDV genotype I hepatitis D with PEG-IFN-a2b is a promising (II) in the present study. HBV genotype D is the new therapeutic option and that sustained viro- less IFN-sensitive among HBV genotypes A–D. logical response rates with PEG-IFN-a2b may be However, whether HBV genotype modulates IFN higher than those reported in former studies with response in HDV infection remains unclear. In standard IFNs. The present study furthermore general, a clear-cut discrimination of response on indicates that quantitative HDV RNA measure- the basis of baseline parameters was hampered in ment is a useful predictive parameter in hepatitis the present study by the low patient number. D. Nonresponders were characterized by a less Sustained biochemical response rates of 0–43% than 3 log decrease of HDV RNA at month 6 of have been reported in patients treated with stan- treatment. Even virological nonresponders might dard IFN (5). Interestingly, biochemical response benefit from IFN therapy because of a partial did not parallel virological response in some biochemical response. However, due to the lim- studies. A sustained biochemical response was ited number of patients in the present study larger reported in 36% of patients treated with 9 MU trials are needed to verify these conclusions. standard IFN thrice weekly despite virological nonresponse (9, 14). Long-term follow-up over a References mean of 10.8 years of these patients treated with a high dose of standard IFN (9 MU thrice weekly 1. Farci P. Delta hepatitis: an update. J Hepatol 2003; 39: for 1 year) revealed that biochemical response S212–9. 2. Hadziyannis S J. Review: hepatitis delta. J Gastroenterol even increased to 58% (seven out of 12 patients) Hepatol 1997; 12: 289–98. over time although none of the patients had 3. Fat t o v i c h G , Giustina G, Christensen E, et al. Influ- achieved a virological response at the end of ence of hepatitis delta virus infection on morbidity and treatment (14). An ALT reduction was also ob- mortality in compensated cirrhosis type B. The European served in nonresponders but was neither signifi- concerted action on viral hepatitis (Eurohep). Gut 2000; 46: 420–6. cant nor sustained. It is as yet unclear what the 4. Hussinger D, Erhardt A, Oette M. Coinfection in biochemical improvement means with respect to hepatitis. Z Gastroenterol 2004; 42: 724–30. histological progression in nonresponders. Inter- 5. Niro G A, Rosina F, Rizzetto M. Treatment of hepatitis estingly, a stabilization of histological liver score D. J Viral Hepatol 2005; 12: 2–9. was observed in the present study even in virolo- 6. Rosina F, Rizzetto M. Treatment of chronic type D (delta) hepatitis with alpha interferon. Semin Liver Dis gical nonresponders. Preliminary data in chronic 1989; 9: 264–6. hepatitis C with advanced fibrosis and cirrhosis 7. Rosina F, Pintus C, Meschievitz C, Rizzetto M. A indicate that long-term IFN treatment might randomized controlled trial of a 12-month course of re- exert antifibrotic and anti-inflammatory activity combinant human interferon-alpha in chronic delta (type even without virus clearance (30). Owing to the D) hepatitis: a multicenter Italian study. Hepatology 1991; 13: 1052–6. high rate of advanced liver fibrosis and cirrhosis 8. Madejon A, CotonatT, Bartolome J, Castillo I, Car- in hepatitis D, which was also observed in the reno V. Treatment of chronic hepatitis D virus infection present study, long-term anitfibrotic/anti-inflam- with low and high doses of interferon-alpha 2a: utility of

809 Erhardt et al.

polymerase chain reaction in monitoring antiviral response. patic expression of the delta antigen. An active and Hepatology 1994; 19: 1331–6. progressive disease unresponsive to immunosuppressive 9. Fa r c i P , Mandas A, Coiana A, et al. Treatment of chronic treatment. Ann Intern Med 1983; 98: 437–41. hepatitis D with interferon alfa-2a. N Engl J Med 1994; 330: 21. Fat t o v i c h G , Boscaro S, Noventa F, et al. Influence 88–94. of hepatitis delta virus infection on progression to cirr- 10. Gaudin J L, Tr epo C. Therapy of chronic delta hepatitis hosis in chronic hepatitis type B. J Infect Dis 1987; 155: with alpha- and beta-interferon. Prog Clin Biol Res 1993; 931–5. 382: 345–52. 22. Saracco G, Rosina F, Brunetto M R, et al. Rapidly 11. Di Marco V, Giacchino R, Ti m i ti lli A, et al. Long-term progressive hbsag-positive hepatitis in Italy. The role interferon-alpha treatment of children with chronic hepatitis of hepatitis delta virus infection. J Hepatol 1987; 5: delta: a multicentre study. J Viral Hepatol 1996; 3: 123–8. 274–81. 12. Puoti M, Rossi S, Forleo M A, et al. Treatment of 23. Verme G, Brunetto M R, Oliveri F, et al. Role of chronic hepatitis D with interferon alpha-2b in patients hepatitis delta virus infection in hepatocellular carcinoma. with human immunodeficiency virus infection. J Hepatol Dig Dis Sci 1991; 36: 1134–6. 1998; 29: 45–52. 24. Cooksley W G, Piratvisuth T, Lee S D, et al. Peginter- 13. Lau D T, Kleiner D E, Pa r k Y , Di Bisceglie A M, feron alpha-2a (40 kda): an advance in the treatment of Hoofnagle J H. Resolution of chronic delta hepatitis after hepatitis B e antigen-positive chronic hepatitis B. J Viral 12 years of interferon alfa therapy. Gastroenterology 1999; Hepatol 2003; 10: 298–305. 117: 1229–33. 25. Lindsay K L, Tr ep o C, Heintges T, et al. A randomized, 14. Fa rc i P , Roskams T, Chessa L, et al. Long-term benefit of double-blind trial comparing pegylated interferon alfa-2b to interferon alpha therapy of chronic hepatitis D: regression interferon alfa-2b as initial treatment for chronic hepatitis of advanced hepatic fibrosis. Gastroenterology 2004; 126: C. Hepatology 2001; 34: 395–403. 1740–9. 26. Erhardt A, Blondin D, Hauck K, et al. Response to 15. Garripoli A, di Marco V, Cozzolongo R, et al. Riba- interferon alfa is hepatitis B virus genotype dependent: virin treatment for chronic hepatitis D: a pilot study. Liver genotype A is more sensitive to interferon than genotype 1994; 14: 154–7. D. Gut 2005; 54: 1009–13. 16. Buti M, Esteban R, Jardi R, Rodriguez F, Guardia J. 27. Kuo M Y, Chao M, Tay lor J. Initiation of replication of Ribavirin therapy in chronic delta hepatitis. J Hepatol 1993; the human hepatitis delta virus genome from cloned DNA: 19: 318. role of delta antigen. J Virol 1989; 63: 1945–50. 17. Lau D T, Doo E, Pa r k Y , et al. Lamivudine for chronic 28. Ishak K, Baptista A, Bianchi L, et al. Histological delta hepatitis. Hepatology 1999; 30: 546–9. grading and staging of chronic hepatitis. J Hepatol 1995; 18. Wolt e rs L M, van Nu n en A B, honkoop P, et al. 22: 696–9. Lamivudine-high dose interferon combination therapy for 29. Janssen H L, van Zon n ev eld M, senturk H, et al. chronic hepatitis B patients co-infected with the hepatitis D Pegylated interferon alfa-2b alone or in combination with virus. J Viral Hepatol 2000; 7: 428–34. lamivudine for hbeag-positive chronic hepatitis B: a rando- 19. Bordier BB, Marion PL, Ohashi K, et al. A prenylation mised trial. Lancet 2005; 365: 123–9. inhibitor prevents production of infectious hepatitis delta 30. Afdhal N, Freilich B, Levine R A, Brown R S, Mon- virus particles. J Virol 2002; 76: 10465–72. sour H, O’brien C B. Colchicin versus PEG-Intron long- 20. Rizzetto M, Verme G, Recchia S, et al. Chronic hepa- term (COPILOT) trial: interim analysis of clinical outcomes titis in carriers of hepatitis B surface antigen, with intrahe- at year 2. Hepatology 2004; 40: 171A.

810