JCM OCCULT HBV INFECTION. Occult HBV Infection. Original Article

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1 Occult HBV Infection OCCULT HBV INFECTION D. Jelev, A. Ivanova, Z. Krastev St.Ivan Rilski University Hospital, Clinic of Gastroenterology, MU - Sofia Key words: occult HBV infection; OBI; HBsAg; anti-hbc total; HBV DNA Address for correspondence: D. Jelev St.Ivan Rilski University Hospital, Clinic of Gastroenterology, 15 Acad. Ivan Geshov Blvd, 1431 Sofia, Bulgaria, tel: , fax: , dejelev@yahoo.com Abstract: We presented 6 cases of occult HBV infection in patients with known other aetiology of liver disease. All patients were HBsAg (-), anti-hbc total (+) and with detectable serum HBV DNA. Four of them were with ongoing HCV infection. Serum HBV DNA and HCV RNA were determined by real-time PCR assay (Applied Biosystems, USA). Serum level of HBV DNA was > cp/ml in all subjects. Two out of 6 had HBV DNA level even > cp/ml. A severe reactivation of HBV infection with reappearance of HBsAg and elevated ALT up to 18 times above the upper limit of normal was observed in one out of 6 cases. The reactivation occurred during immunosuppressive therapy, despite the maintenance treatment with lamivudine. In this patient HBV DNA level > cp/ml was found both in serum and saliva. Original Article Introduction This is the first report regarding detectable HBV DNA in the saliva of patient with occult HBV infection. The results from HBV DNA testing suggested that a viraemia over cp/ml is possible in subjects with occult HBV infection. In some of presented cases HBV DNA level was even > cp/ml, which predetermines a high infectious risk. Such a viraemia, in combination with elevated aminotransferases, is an indication for antiviral therapy. HBsAg is a marker for hepatitis B virus (HBV) infection, as it is positive both in acute and chronic infection. It has been reported that HBV infection can persist even in the absence of circulating HBsAg 1-6. This Occult hepatitis B Infection (OBI) was most frequently discovered in subjects with anti-hbc total (+) status, but was found also in some of anti-hbc total (-) as well 3,7. Currently, OBI is defined with HBsAg (-) status and detectable HBV DNA in the serum and/or in the liver 3,7. It is considered that the serum level of HBV DNA in OBI is very low < cp/ml (<200 UI/ml) 3,7. Despite the progress of bio-molecular technologies, numerous aspects of OBI remain unclear and some of the reported results are controversial. There are no data from large clinical trials on this matter. The publications concern mostly isolated cases or small groups of patients. The following important questions related to OBI need a more detailed elucidation: the role of OBI in the HBV transmission after organ and blood donation and carcinogenesis, as well as the risk of reactivation of HBV infection and development of active liver disease, especially in patients receiving immunosuppressive therapy. Materials and Methods Patients We present 6 cases (3 males and 3 females) with OBI in patients with known other aetiology of liver disease: 2 patients were with complicated course of HCV infection 1 patient - with HCV infection after polychemotherapy given for treatment of acute myelogenous leukemia 1 patient - with HCV related liver cirrhosis and 22

2 J Clin Med. 2009; 2(2):22-27 hepatocellular carcinoma (HCC) 1 patient - with an advanced primary biliary cirrhosis (PBC) 1 patient - after liver transplantation. All subjects were HBsAg (-) and anti-hbc total (+) and 5 of 6 cases were anti-hbs (-). Only 1 out of 6 was anti-hbs (+) with the anti-hbs titer of 143 IU/l. HBsAg, anti-hbc total and anti-hbs antibodies were tested by using commercially available standard ELISA kits. The serum levels of HBV DNA and HCV RNA were determined by real-time PCR assay (Applied Biosystems, USA). The characteristics of studied patients are presented in table 1. Results All patients with OBI had serum levels of HBV DNA > 200 IU/ml (>1 000 cp/ml), while 2 out of 6 had viraemia above 2000 IU/ml (> cp/ml) figure 1. Serum HBV DNA levels are shown in figure 1. The corresponding HCV RNA serum levels of subjects co-infected with HCV are also shown for comparison. The presented cases in this study are too heterogeneous, unified mainly by the presence of OBI. For that reason they will be reviewed separately. Patient 1 RIT is a 43-year-old female. In 1991 after blood transfusion during delivery an acute hepatitis B was found. The patient subsequently lost HBsAg. In

3 Occult HBV Infection abnormal aminotransferases due to HCV infection was found. In 1997 a liver biopsy was performed. Histological examination revealed chronic hepatitis with a low level of necro-inflammatory activity and initial fibrosis F1. Interferon-alpha (IFN) therapy was initiated, but later it was stopped due to the lack of treatment response and side effect - depressive syndrome. In 2008 an OBI was found: HBsAg (-), anti- HBs < 10 IU/l, anti-hbc total (+), HBV DNA cp/ ml and HCV RNA IU/ml. Patient 2 KID is a 41-year-old female. In 2002 on occasion of blood donation, HCV infection was found with proven HBsAg (-) status. Probably the patient had been infected with HCV through a blood transfusion performed in She also had a sister with hepatitis B infection. In 2005 the patient was with slightly elevated ALT levels ( times above the ULN), as well as with serum HCV RNA of IU/ml and HCV genotype 1b. In November 2005, chronic hepatitis was histologically confirmed: METAVIR A2; F2. A combination therapy with IFN and ribavirin was administered for 18 months. The subject was with good end of treatment virological and biochemical response, but relapsed six months post therapy. This patient was also non-responder to further antiviral treatment with amantadine. In June 2008 she was retested for Patient 5 VSD is a 42-year-old female with primary biliary cirrhosis, which was diagnosed in She was HBsAg and anti-hcv negative. In the last three years hepatitis B serology due to the complicated course a progressive decrease in the liver function has been of HCV infection. HBsAg-negative serologic status was confirmed, but positive anti-hbc total as well as anti- HBs antibodies (anti-hbs titer IU/l) were found. OBI was confirmed further by serum level of HBV DNA of cp/ml and HCV RNA level of IU/ml. Patient 3 HHS is a 29-year-old male who was diagnosed with an acute myelogenous leukemia in September A remission was achieved through 3 induction and 7 reinduction courses of polychemotherapy, followed by a consolidation therapy. In October 2007, because of elevated aminotransferases, a HCV infection was discovered. In February 2008, an active virus replication was found with HCV RNA level of IU/ml and HCV genotype 1. In March 2008 the patient was with highly elevated ALT (15 times above the ULN). The remission of leukemia was confirmed several times through control bone marrow examination. The virological investigation in March 2008 revealed: anti-hav IgM (-), HBsAg (-); anti-hbc total (+); anti- HBs (-) serological status with serum HBV DNA of 9231 cp/ml. Combined IFN and ribavirine therapy was initiated which induced a rapid normalization of the aminotransferases on the 1 st month. Serum HCV RNA and HBV DNA decreased to the undetectable levels on the 3 rd treatment month. The combined therapy is still in progress. Patient 4 BTP - a 51-year-old male with HCV (+) liver cirrhosis Child B, which was diagnosed in 2003 because of esophageal varices haemorrhage. In 2007, an abdominal ultrasound examination revealed a lesion in the liver with characteristics of hepatocellular carcinoma (HCC). The control ultrasonography in 2008 indicated the progression of liver lesion, which was confirmed by spiral CT scan. The level of alphafetoprotein was fourfold elevated. The virological testing revealed: HBsAg (-); anti-hbc total (+) and anti-hbs (-) serology and serum levels of HBV DNA and HCV RNA cp/ml and IU/ml respectively. observed with increasing bilirubin levels above 400 µmol/l, prolonged prothrombine time and low serum albumin level. In 2008, the patient progressed to decompensated cirrhosis Child B with 2 nd degree esophageal varices. The patient was included in the waiting list for liver transplantation and OBI was found during the further laboratory examinations: HBsAg (-), anti-hbc total (+) and anti-hbs (-), serum HBV DNA level of 5929 cp/ml. Patient 6 MGC is a 24-year-old female. In 2003 an urgent liver transplantation was performed due to acute liver failure, caused by a fulminant hepatitis B. Since the transplantation, the patient received immunosuppressive therapy and lamivudine treatment. The serological tests after the transplantation showed: HBsAg (-), anti-hbs (-) and HBeAg (-) with undetectable serum HBV DNA. In February 2008 an 24

4 J Clin Med. 2009; 2(2):22-27 OBI was found with low serum HBV DNA level of 470 cp/ml. She remained HBsAg (-), HBeAg (-), anti-hbe (-), anti-hbc total (+) and anti-hbs (-), but 1 month later the viraemia increased to 4773 cp/ml and similar HBV DNA level of 4260 cp/ml was detected in saliva. In October 2008 ALT flare occurred with elevated ALT levels up to 18 times above the ULN. The virological testing revealed reappearance of HBsAg and anti-hbc IgM antibodies simultaneously with the increasing level of serum HBV DNA to 5286 cp/ml. Hepatitis A, hepatitis D, hepatitic C, EBV and CMV infections were excluded. The other serology results presented: HBeAg (-); anti-hbe (+); anti-hbs (-). Discussion Serum HBV DNA levels observed in the discussed 6 patients suggest that a viraemia over cp/ml is possible in OBI. In some of the presented cases HBV DNA was found to be even > cp/ml which predetermines a high infectious risk. Such a viraemia, in combination with elevated aminotransferases, is an indication for active antiviral therapy 7. The presence of high viraemia is likely to be due to the suppressed immune system of the reviewed patients. Two of them were treated with immunosuppressive drugs, while other two were with decompensated anti-hbc total (+) methadone maintained patients in liver cirrhosis (combined with HCC in one of the New York Сity 23. cases). In addition, 4 out of 6 were with chronic HCV infection, which also causes immunosuppression and thus compromises the development of an effective immune response after a standard vaccination against hepatitis B 8,9. The combination of OBI and hepatitis C has been reported by other author as well 10,11. In some studies OBI in patients with HCV infection has been associated with more severe liver damage and more rapid progression to liver cirrhosis The presence of OBI is a risk factor for the development of HCC, especially among subjects with HCV infection or alcohol induced liver disease 1, In one of the discussed patients with OBI and HCV coinfection we also found Child B cirrhosis, complicated with HCC. OBI in patients with chronic HCV infection is associated with poor therapeutic response to IFN and ribavirine combination therapy 10-12,17. We did not achieve sustained treatment response in two of our patients treated with IFN, even though one of them received a prolonged 18-month combination therapy. In patient 3, we observed a good early biochemical and virologic response to the IFN and ribavirine combination. It resulted in serum HBV DNA loss, but since the treatment hasn t finished yet, the sustained therapeutic effect still remains unknown. Another important clinical aspect related to OBI is the risk of transmitting HBV infection through donation of blood and organs In 2008 two cases of clinically manifested acute hepatitis B after transfusion of erythrocyte concentrate and fresh frozen plasma to two separate recipients from a single donor were published. The serological status of donor was HBsAg (-), anti-hbc total (+), anti-hbs (+) with low serum HBV DNA level of 180 IU/ml (~950 cp/ml) 21. A complete identity between the HBV DNA of the donor and recipients was demonstrated. This is a concerning fact as one of our recent studies has shown that 30% of the blood donors in Sofia are anti-hbc total (+). However, not all of anti-hbc total (+) carriers have OBI. Minuk et al 22 have defined that 18% of anti-hbc total (+) subjects are with detectable levels of serum HBV DNA. Serum HBV DNA has been detected in 26% of There is also an increasing number of reports regarding HBV DNA in the saliva of patients with chronic HBV infection 24,25. HBV DNA has been detected in saliva even in subjects with very low viraemia 26. In patient 6 we found similar HBV DNA levels both in serum and saliva in parallel probes collected in the same day. The female patient discussed was with the lowest level of viraemia among the reviewed 6 cases with OBI. It is worth knowing that this is the first report regarding detectable HBV DNA in the saliva of patient with OBI. The reactivation of OBI occurs mostly after powerful immunosuppressive treatment or in the presence of diseases that might cause such an effect over the immune system 3, It has been reported that reactivation of OBI may result in a severe fulminate hepatitis 30,31. These data suggest that testing for anti- 25

5 Occult HBV Infection HBc total (and HBV DNA if appropriate) is needed in all patients scheduled for immunosuppressive therapy. Patients with OBI should be monitored and if the HBV DNA levels would increase, they should be scheduled for treatment with nucleoside analogues 7. In our study we observed a reactivation of HBV infection in a patient with liver transplantation. A reappearance of HBsAg and anti-hbc IgM antibodies in serum was found with a rapid increase of ALT up to 18 times above the ULN. The reactivation occurred 5 years after the liver transplantation, despite the antiviral treatment with lamivudine. Most likely this was due to lamivudine resistance which usually occurs in about 70% of the patients on the 5 th year after treatment initiation 7. References: 1. Brechot C, Thiers V, Kremsdorf D et al. Persistent hepatitis B virus infection in subject without hepatitis B surface antigen: clinically significant or purely occult? Hepatology 2001; 34: Conjeevaram H, Lok A. Occult hepatitis B virus infection: Ahidden menace? Hepatology 2001; 34: Raimondo G, Allain J, Brunetto R et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008; 49: Raimondo G, Balasano C, Craxi A et al. Occult hepatitis B virus infection. Digest and Liver Disease 2000; In conclusion, OBI is a result of the persistence 5. Raimondo G, Pollicino T, Cacciola I et al.occult hepatitis B virus infection. J Hepatol 2007; 46: through years and decades of the covalently closed circular HBV DNA (ccchbv DNA) in the nucleuses of 6. Raimondo G. Occult HBV infection and liver disease: fact the infected hepatocytes 3,32. This phenomenon only or fiction? J Hepatol 2001; 34: confirms the thesis that viral eradication remains an unrealistic treatment goal at the present level of 7. European Association for the Study of the Liver. EASL knowledge. We can only achieve an inactive carrier Clinical Practice Guidelines: Management of chronic hepatitis B. J state of HBV infection 33. However, in certain cases Hepatol 50 (2009), in press - doi: /j.jhep the reactivation of HBV infection can cause severe, even fulminant hepatitis. It can develop even after 8. Leroy V, Bourliere M, Durand M et al. The antibody OBI transmission through blood transfusion or organ response to hepatitis B virus vaccination is negatively influenced by the hepatitis C virus viral load in patients with chronic hepatitis C: a transplantation. Coexisting OBI with liver disease of case-control study. Eur J Gastroenterol Hepatol 2002; 14: other aetiology may lead to enhanced progression to cirrhosis. The prolonged OBI is an important risk factor 9. Wiedmann M, Liebert U, Oesen U et al. Decreased for the development of HCC, as its oncogenic role is immunogenicity of recombinant hepatitis B vaccine in chronic probably related to the ability of the virus DNA to hepatitis C. Hepatology 2000; 31: integrate with the host genome. 10. Cacciola I, Pollicino T, Squadrito G et al. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 1999; 341: Fucuda R, Ishimura N, Niigaki M et al. Serologically silent hepatitis B virus coinfection in patients with hepatitis C virusassociated chronic liver disease: clinical and virological significance. J Med Virol 1999; 58: De Maria N, Colantoni A, Freidlander L et al. The impact of previous HBV infection on the course of chronic hepatitis C.Am J Gastroent 2000; 95: Sagnelli E, Coppola N, Scolastico C et al. HCV genotype and silent HBV coinfection: two main risk factors for a more severe liver disease. J Med Virol 2001; 64:

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