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Occult hepatitis B virus in liver tissue of individuals without hepatic disease
  Journal of Hepatology Advance publicaton March 2008
Giovanni Raimondo1, Giuseppe Navarra2, Stefania Mondello1, Lucy Costantino1, Guido Colloredo3, Eugenio Cucinotta4, Gaetano Di Vita5, Claudio Scisca4, Giovanni Squadrito1, Teresa Pollicino1
Received 19 October 2007; received in revised form 4 January 2008; accepted 7 January 2008. published online 13 February 2008. Corrected Proof

While many data are available concerning occult hepatitis B virus (HBV) infection in patients with hepatic disorders, there is little information about this cryptic infection in individuals without liver disease. The aim of this study was to investigate the prevalence of occult HBV in the general population by examining liver specimens from a large series of HBV-surface-antigen negative individuals with no clinical and biochemical evidence of liver disease.
The presence of HBV DNA was evaluated by testing, through polymerase chain reaction techniques, DNA extracts from 98 liver-disease-free individuals who underwent liver resection or needle biopsy during abdominal surgery. Sixteen of them were anti-HBV-core antigen (anti-HBc) positive and 82 were HBV serum-marker negative. All patients were negative for antibody to hepatitis C virus.
Occult HBV infection was revealed in 16 of the 98 cases (16.3%). In particular, 10/16 anti-HBc positive (62.5%) versus 6/82 (7.3%) HBV-seronegative individuals were occult carriers (p<0.0001).
This study revealed that about 1/6 of the Italian general population might be carriers of occult HBV infection, and this condition is significantly associated with the anti-HBc positive status.
Associate Editor: F. Zoulim
Through the analysis of liver DNA extracts from a large series of individuals without clinical and biochemical evidence of liver disease, we performed the first study evaluating the prevalence of occult HBV infection in the general population by the use of the gold standard method for its detection. Actually, apart from the epidemiological relevance, the information provided by this study is of importance considering that healthy occult HBV carriers might be at risk of viral reactivation and might transmit the infection. Indeed, occult HBV infection is characterized by a state of suppression of viral replication and gene expression. This status, however, may be discontinued, leading to the development of a typical hepatitis B that often has a severe - and some times even fulminant - clinical course [11], [12]. This event is usually observed in patients under condition of immunosuppression induced by therapies and/or related to diseases that involve the immune system (i.e. patients with hematological malignancies, HIV infection, hematopoietic stem cell transplantation, organ transplantation, etc.) (reviewed in [2], [3], [13], [14]). In addition, the availability of new, potent immunosuppressive drugs - like the anti-CD20, the anti-CD52 and the anti-TNF monoclonal antibodies, recently introduced in the therapeutic schedules of various clinical settings - seems to have further increased the risk of HBV reactivation in cryptically infected individuals that may present very severe clinical sequels [15], [16], [17]. In terms of risk of occult HBV transmission, while post-transfusional hepatitis B is at present a rare event in the western world [18] (although occult HBV infection of the donors is the main factor responsible for the residual cases [19], [20]), it is not infrequent in cases of OLT and the recipient may acquire a classic form of hepatitis B when the donor is an occult HBV carrier [8], [9], [10]. In fact, it has been reported that from 17% to 94% of HBsAg negative/anti-HBc positive donors may transmit HBV infection to the recipients (reviewed in [21]), while the transmission of the occult infection from individuals seronegative for all HBV markers is uncertain.
We found that 16% of individuals free from liver disease carried occult HBV infection, with a prevalence that appears to be lower compared with that observed in HCV infected patients coming from the same geographic area (about one third of these cases) [5], [6]. Moreover, when the study population was examined according to the HBV serology, it clearly emerged that the occult HBV infection strongly correlates with the anti-HBc positive status and, indeed, the vast majority of anti-HBc positive individuals (although not all of them) were occult HBV carriers. Whether the lack of HBV DNA detection in some anti-HBc cases reflects a true resolved infection with subsequent clearance of the virus, a defect of sampling at the time of liver biopsy or the existence of extra-hepatic reservoir districts of the occult viruses remains to be elucidated.
Much evidence indicates that occult HBV infection occurs quite frequently in chronic hepatitis patients seronegative for all HBV markers (more than 20% of the cases, as reviewed in [2]). Although with a lower prevalence, our study showed that the occult infection is not an infrequent finding also in HBV-seronegative individuals without liver disease since 7% of them carried occult infection. However, it has to be noted that the risk of occult HBV reactivation and/or transmission in this latter category of individuals is, for the most part, an unexplored issue.
In conclusion, in an era characterized both by the continuous emergence of new and potent immunosuppressive therapies for the treatment of various neoplastic, haematological and auto-immunitary disorders and by the increasing emigration of people from highly endemic HBV areas to more industrialized countries, occult HBV infection may become a fairly important problem of public health. While waiting for more sensitive methods for blood HBV DNA detection, anti-HBc antibody evaluation appears to be an easy way of identifying most of the occult carriers among liver-disease-free populations. This test should be strongly recommended in all patients undergoing chemotherapy and/or immunosuppressive treatments as well as all blood or organ donors, especially in view of the availability of antivirals that can specifically and properly prevent HBV reactivation and new infections.
Occult hepatitis B virus (HBV) infection is characterized by the persistence of HBV DNA in the liver of individuals negative for HBV-surface-antigen (HBsAg) [1], [2], [3]. Recent data demonstrated that occult HBV may exist in the hepatocytes as a free genome, its molecular basis being related to the long-term persistence in the hepatocyte nuclei of the viral covalently-closed-circular DNA (HBVcccDNA), a replicative intermediate that serves as template for gene transcription. Much evidence suggests that occult HBV infection may have a significant impact in several clinical contexts, since it might favour the progression of liver fibrosis and the development of hepatocellular carcinoma in patients with co-existing additional causes of liver damage, such as chronic hepatitis C virus (HCV) infection [4], [5], [6]. Moreover, occult HBV may acutely reactivate when an immunosuppressive status occurs, and it may be transmitted in the case of blood transfusion and organ transplantation, causing classic forms of hepatitis B in newly infected individuals (reviewed in [2], [3]). Mainly, carriers of occult infection may be a source of HBV transmission in the event of orthotopic liver transplantation (OLT) as an obvious consequence of the fact that the hepatocytes are the site of the virus reservoir [7], [8], [9]. At present, however, it is really difficult to predict the frequency of events such as HBV reactivation or its transmission in cases of OLT, also because of the very little information available on occult HBV prevalence in the general population. In fact, since the amount of serum HBV DNA in the occult status is very low (if not totally absent) and the viral cccDNA reservoir is located in the hepatocytes, only the analysis of liver tissue extracts can provide an exact evaluation of occult HBV prevalence in a defined set of individuals [1], [2], [3]. In fact, whereas many data are available on occult HBV infection occurring in patients with liver disease and who undergo liver biopsy for clinical/diagnostic reasons, the extent of the occult HBV tissue in individuals without hepatic injury - who obviously represent the vast category of potential liver donors - is still largely unknown.
The aim of this study was to investigate the prevalence of occult HBV infection in a large series of subjects free from liver disease through the analysis of liver DNA extracts.

In this prospective study, we examined liver tissue specimens from 98 Italian patients [39 male, 59 female, mean age 54 years, 15.7 standard deviation (SD)] consecutively admitted from 2002 to 2006 to four surgery departments located in hospitals of three different Italian cities (Messina, Bergamo and Palermo). All the patients were negative for HBsAg and anti-HCV; 16 of them were positive for the antibody to the HBV-core-antigen (anti-HBc), 5 of whom were also positive for anti-HBsAg antibody (anti-HBs). HBsAg, anti-HBs and anti-HBc were determined, respectively, by AxSYM HBsAg (V2), AxSYM CORE and AxSYM AUSAB (Abbott Diagnostics, Abbott Park, IL). All cases had normal liver biochemistry and none had a clinical history of liver disease. Moreover, the histological examination of the liver tissue fragments (obtained either through surgical resection or needle biopsy performed during surgery) showed absence of fibrosis and absence or minimal signs of inflammation in all cases, although obese subjects had steatosis of different degrees. Eight individuals underwent surgery for benign liver tumours or liver cysts, 36 for gallstones, 26 for metastases in the liver from tumours of the colon (17 cases), pancreas (3 cases), stomach (4 cases), lung (1 case) and breast (1 case); finally, 28 subjects underwent laparoscopic gastric banding for morbid obesity. Once taken, part of each liver specimen was immediately frozen for subsequent molecular analyses.
Occult HBV DNA analysis
Frozen liver specimens were tested for occult HBV DNA through the methods detailed in our previous reports [5], [6], [10]. In particular, DNA extracts from each case were examined for the presence of HBV genomes by performing four different in-house nested-PCR amplification assays to detect preS-S, precore-core, Pol and X HBV genomic regions, respectively. We considered the cases that showed positivity in at least two different viral genomic regions as HBV DNA positive. Appropriate negative and positive controls were included in each PCR experiment. In particular, as negative controls we included in each PCR experiment (a) serum and tissue DNA extracts from subjects known to be negative for HBV infection, (b) DNA-free reaction buffer, (c) water. In addition, to eliminate false negative results the beta-globin was used as a house-keeping gene. Moreover, direct sequencing of all amplified HBV sequences confirmed the specificity of the reactions. The limit of sensitivity of our nested-PCR methods was 2X10-4 copy per liver cell.
The study protocol was approved by the Ethics Committee of the Messina University Hospital and informed consent was obtained from all patients. Statistical analyses were evaluated using the Student's t-test and the χ square method.
HBV DNA sequences were detected in liver tissues from 16 out of the 98 cases examined (16.3%). In particular, among the 10 anti-HBc positive cases six were positive for all four HBV genomic regions examined (S, Core, Pol, X), two cases were positive for three genomic regions (one for Core, Pol, X; one for Pol, S and X) and two were positive for two genomic regions (one for Pol and S; one for Core and Pol); among the six HBV-seronegative individuals, three were positive for all four HBV genomic regions and three were positive for two genomic regions (two for Core, Pol; one for S and X) (Table 1). No statistical association as regards sex and age was found when occult-positive subjects were compared to occult-negative cases [male/female: 9/7 versus 30/52, p=0.1; mean age 53.4 years (18.1 SD) versus 53.7 (19.5 SD), p=0.1] (Table 2). Seven of the occult HBV carriers underwent surgery for gallstones, four for hepatic metastases, one for a benign liver tumour and four underwent gastric banding for obesity.
Occult HBV status was strongly related with the anti-HBV antibody positive status: in fact, HBV DNA was detected in 10 of the 16 (62.5%) anti-HBc positive cases versus 6 of the 82 (7.3%) HBV marker negative cases (p<0.0001). Therefore, in this set of individuals the anti-HBc test had a sensitivity of 62.5%, a specificity of 92.7%, a positive predictive value of 62.5% and a negative predictive value of 92.7% in predicting the presence of intrahepatic HBV DNA.
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