The Pigeonhole of Occult Hepatitis B

Abstract

Occult hepatitis B (OHB), or persistent hepatitis B virus (HBV) viremia in surface-antigen-HBsAg-negative patients, has been recognized as a medical concern during the last decade. The exact magnitude, pathogenesis and clinical relevance of OHB are unclear. This review organizes the published data on OHB and presents an overview of the current hypotheses on OHB's pathogenesis and clinical relevance.Many explanations have been offered for the pathogenesis of OHB, ranging from the inability of standard immunoassays to diagnose OHB to the involvement of the versatile virus-host factors. Also, special care should be taken regarding the diagnosis of OBH. It seems that both shared viral-host factors are involved in the pathogenesis of OBH. Further molecular studies on cohort patients group need to explore such association.

Headnote

Abstract- Occult Hepatitis B (OHB), or persistent hepatitis B virus (HBV) viremia in surface-antigenHBsAg-negative patients, has been recognized as a medical concern during the last decade. The exact magnitude, pathogenesis and clinical relevance of OHB are unclear. This review organizes the published data on OHB and presents an overview of the current hypotheses on OHB's pathogenesis and clinical relevance. Many explanations have been offered for the pathogenesis of OHB, ranging from the inability of standard immunoassays to diagnose OHB to the involvement of the versatile virus-host factorsAlso, special care should be taken regarding the diagnosis of OBH. It seems that both shared viral-host factors are involved in the pathogenesis of OBH. Further molecular studies on cohort patients group need to explore such association.

© 2014 Tehran University of Medical Sciences. All rights reserved.

Acta Medica Iranica, 2014;52(8):582-590.

Keywords: Occult Hepatitis B infection; HBV diagnosis; HBsAg mutations.

Introduction

Hepatitis B virus (HBV) infection is usually diagnosed when circulating hepatitis B surface antigen (HBsAg) is detected. Evidence is accumulating that HBV is present in patients who are HBsAg negative but contain HBV DNA in serum and or liver tissue. The medical literature refers to this as Occult Hepatitis B infection, or OHB.

This type of infection is detectable by polymerase chain reaction (PCR) and has been classified into seropositive and seronegative infection depending on the positivity of anti-core antibody (HBc) and hepatitis B surface antigen (anti-HBs) (1-3). Occult HBV's real place in the clinical and biological spectra of HBV infection is not well known. Long-term studies of HBV chronic and recovered infections have shown that after HBsAg is no longer detectable, HBV DNA persists for years in serum or the liver (4).

Many issues are still open regarding occult HBV infection, beginning with the diagnostic criteria, which may have implications for liver transplantation.

OHB pathogenesis

The reasons for the lack of circulating HBsAg in OHB patients are unclear. The molecular basis of HBV persistence in HBsAg-negative subjects is believed to involve a combination of viral and host-dependent factors (Figure 1). Occult HBV infection is associated with ongoing virus replication in animal models (5), and the passaged particles might be able to infect virus-naïve animals (6,7), indicating long virus persistence. A number of explanations for the persistence of HBV DNA in HBs-negative samples have been proposed, including HBV DNA in low-copy numbers (8), altered host immune response (9), genetic variations of the S gene (10), viral DNA integration in the host genome(11), infection of peripheral blood mononuclear cells (12), immune complexes in which HBsAg is hidden (13,14), and interference of other viruses such as HCV (15-18) and HIV (Figure 1) (19-22).

Rearrangement in the HBV genome is one of the exceedingly proposed mechanisms for HBV DNA persistence (Figure 1). Mutation within the surface gene of the HBV genome is one of the factors contributing to the loss of HBsAg detection by immunoassay. HBV surface gene mutants interfere with gene expression or produce an antigenically modified S protein (3). The "an" antigenic determinant is the primary determinant of HBsAg. Mutation in the "a" determinant may cause a conformational change in HBsAg, leading to undetectability of HBsAg as well as evading the host's immune response (23). The most well known mutation within the "a" determinant is a glycine-to-arginine change at codon 145, created by a G-to-A substitution at nucleotide 587 (3,23-25). Mutations at amino acid positions 120, 143, and 144 can result in poor reactivity with d/y subtyping monoclonal antibodies (26,27), and mutations in cysteine residues within the "a" determinant have been found to be essential for the antigenicity and presumably for the conformation of the protein (28).

Mutation outside the surface protein may also influence HBV replication capacity. According to earlier studies, these mutations have been reported to have a lower ''replication fit'' compared to the wild-type virus in vitro, providing a plausible explanation for the low HBV DNA levels (29,30).

Due to the absence of a crystallographic structural model, it is not possible to empirically predict the impact of a given mutation on the structure of HBsAg. Furthermore, multiple mutations and polymorphisms outside the surface protein could be found within the core, pre-S, X, and polymerase Xs, with known key regulatory functions, including enhancer regions (31,32), hyper editing APOBEC3G (31), altered methylation of HBV genome (31,32), altered RNA splicing due to deletion in the core-surface interface (33), point mutations and deletions in the Pre-S1 and Pre-S2 promoters altering the ratio between small and large surface proteins (1,32,34), defects in polymerase protein due to point mutations in TP (35), and truncated precore/core-poly start codon (1). In these cases, a weak HBV replication, leading to levels of viral particles in the serum that are insufficient to allow HBsAg detection by diagnostic assays, could be the cause of HBsAg seronegativity.

Additionally, altered host immune factors, including lower T-cell expansion (CD4) (9), aberrant expression of cytokines (36,37), DNA damage of peripheral blood lymphocytes (38), and defects in the T-cell expansion and production of cytokines (9,36), might lead to cellular demise and immune hyporesponsiveness in occult hepatitis B infected patients (Figure 1). Involvement of CTL non-catalytic response (mediated by TNF-? and IFN-gamma cytokines) in the pathogenesis of blood-borne viruses (like HBV) plays a dominant role in the clearance of virus (39). However, this type of T cell response seems unable completely to eliminate viruses including HBV, leading to cryptic infection. In this scenario, the viral genetic changes might not be the cause of occult HBV infection but could instead be characteristic of less fit viral sequences that are not cleared as quickly from the liver by the immune system (31). Thus, the low HBV replication might also be due to the kind of balancing between a minute (and undetectable) production of antigen and an inefficient host immune system.

In the aspects of mutations involvement, revealed by sequencing application in most studies, the pattern of OHB could not be unraveled by the sequencing variations alone. Specifically, the current data suggest that the types and locations of mutations do not readily distinguish occult HBV from non-occult HBV at the level of an individual sequence, even if the entire genome is analyzed. Additionally, the differences in the geographical origins of the patients could, in part, account for sequence variations. Finally, infections of HBV mutants or simultaneous infections with wild-type and mutant viruses cannot be ruled out. It is possible that a minor amount of wild-type HBV in a population pool as a quasispecies coexists with the variant but cannot be detected by the use of a sequencing analysis. Therefore, the true proportion of patients carrying HBsAg variants should be higher than the rates found in previous studies.

Nonetheless, accumulating evidence indicates that the genomic heterogeneity of the virus does not account for its occult status in most cases (18, 40-42).

OHB epidemiology

The prevalence of occult HBV infection varies significantly between countries, geographic regions and patient populations (8,40,43). The discussion below provides several possible explanations for the differences in reported prevalence rates.

First, the technical differences might explain some of the discrepancies among these studies. Because the majority of individuals with occult HBV have viral loads under 10 viral copies/mL (typically in the range of 101 to 103 copies/mL), the sensitivity and specificity of the assays are important for its complete identification (44-46). Thus, the conflicting results may be explained in part by the differences in the assay's sensitivity limits used to detect HBVDNA. For example, three studies examining OHB infection in South African AIDS patients found prevalence rates of 23%, 31%, and 88.4% (21,47,48). Also, the type and the number of HBV genomic domains examined might influence the results. In our results on the prevalence of OHB in cryptogenic cirrhosis, we found that the standard PCR-BASED based on the pre-S1 region was the most sensitive among the remaining parts of the HBV genome (42). In other studies, however, X (14,49,50), surface (49,51), poly (52), and core (14) (Karayiannis, personal communication) regions were the most sensitive parts.

Second, the nature of the specimen and the compartment explored (liver, serum, PBMC) may influence the results. Although HBVDNA detection in liver tissue is the standard for revealing OHB (53), HBV-DNA detection in serum or plasma using the correct biomolecular approach is easier to perform in large series studies because it can frequently be repeated and can identify individuals with active HBV replication. In a study by Knoll of 3,004 German donors, 552 (18.4%) had isolated anti-HBc, and among them, 44 (8%) had OHB in their serum, but in 39 liver biopsies of the same patients, 41% had OHB (54). Also, the method of extracting DNA from the specimen could have significantly affected the results.

Third, the geographical distribution of HBV infection is another potential explanation of the discrepancies among previous studies. Many studies have shown that the prevalence of occult HBV infection in HBV-endemic areas is higher than in regions with low HBV prevalence rates, and the differences in OHB prevalence might be closely related to the endemicity of HBV infection (8,55). In a Sicilian study, where the prevalence of HBV infection was quite high (15), the prevalence of OHB was reported to be 33%, whereas the distribution of 6.7% was reported in another study that used specimens from patients living in northern Italy, where the prevalence of HBV infection is lower (17).

These factors should be taken into consideration when determining the prevalence of occult HBV infection. Thus, knowledge of the prevalence of occult HBV infection is important for epidemiology and public health, especially in areas where HBV infection is prevalent.

OHB clinical relevance

OHB infection has been linked to several factors, but it is still unclear whether OHB is a result of a window period of seroconversion from acute HBV infection, reinfection, immunological complexes, sequence divergence, poor laboratory detection (due to low-level HBs antigenemia), underlying HCV/HIV co-infection, immunosuppression or other host factors (2, 48, 56-58).

In general, the research on OHB research can be divided into two major perspectives. The first view includes researchers who believe that OHB is an important problem and that immune suppression poses a significant risk for HBV reactivation in patients receiving chemotherapy who require protection with an antiviral agent (59, 60). The second set of researchers, although recognizing the condition, consider that OHB is a diagnostic problem of concomitant HBV infection with minimal or no bearing on the severity of liver disease and that OHB is more a diagnostic problem of low HBV replication and not clinically relevant for the majority of patients. The latter view underscores that OHB may simply represent the late phase of HBV infection in which specific T-cell responses keep HBV replication under control (61). Despite the observation that occult HBV genomes may be infectious, it is not clear whether cryptic infection in the absence of detectable HBsAg is associated with increased morbidity and mortality from chronic liver disease and hepatocellular carcinoma (HCC) in patients with nonHBV cirrhosis or who have had a liver transplant. Furthermore, it is not established whether such patients require long-term protection against HBV reactivation with an anti-HBV agent.

Given that occult HBV infection has been found in advanced liver diseases and in liver cancer tissue, interest is increasing as to whether occult HBV infection may contribute to acute exacerbation and development of HBV-associated diseases such as progression of liver disease, cirrhosis, and hepatocellular carcinoma. In addition, strong evidence shows that the risk of cirrhosis and HCC development is higher in patients with occult HBV (15,58,62-64). Occult HBV infection has also been reported in populations without symptomatic liver diseases, such as blood donors, individuals with normal liver tests, and general populations (65,66). In contrast, other studies have failed to demonstrate any significant association between occult HBV infection and the severity of liver disease (67,68).

Another important debate surrounds occult HBV infection transmission to others. Occult HBV has been implicated in the transmission of the HBV via transfusion of blood and blood products and transplanted solid organs (58,68-70). Other studies have also documented in the transmission and development of OHB's via transfusion of blood or blood products or organ transplantation, from mother to child, and from humans to chimpanzees (8,61,71-74). Additionally, studies have found that low levels of HBV DNA can be transmitted in humans (75), chimpanzees (76), and woodchuck animal models (77). Finally, occult HBV can be transmitted with few or no signs of ongoing viral replication (62,71,76,78).

It is unknown whether detection of very low levels of HBV DNA has any relevance in clinical practice. However, a major question about occult HBV is whether such small amounts of HBV DNA are associated with progressive liver damage. In the vast majority of patients with OHB infection, low threshold level of HBV DNA (<1,000 copies/mL) may not be sufficient to cause progressive liver disease in immunocompetent chronic HCV patients, as demonstrated in previous studies (79). In a recent survey, 49 recipients of blood transfusions traced from 10 donors with OHB in a retrospective study. Only one recipient had a sequence homology of 95% with her donor. Furthermore, after inoculation of 2 donor's serum samples to 4 chimeric mice, only one mouse contained HBV DNA (80). On the contrary, a low-level HBV infection can reactivate in the immunocompromised individual, resulting in profound liver injury and liver failure (60,81).

OHB diagnosis

Although numerous studies have examined occult HBV infection in immunocompetent patients, the precise prevalence of this clinical entity remains difficult to define. The frequency of diagnosis depends on the relative sensitivity of both HBsAg and HBV DNA assays as well as on the prevalence of HBV infection in the study population. Also, the literature has clearly demonstrated that the prevalence of occult HBV infection is dependent on the time interval from HBsAg clearance to HBV DNA assessment, with longer time intervals being associated with lower prevalence rates of occult HBV infection (15, 82).

Specimen

Considering that viremia is very low or undetectable in occult HBV infection (even when sensitive methods are used), and assuming that the viral cccDNA reservoir is located in hepatocytes, liver-tissue extracts provide the best evaluation of occult HBV prevalence in a defined set of patients (43,61). The detection of HBV DNA in the liver could simply represent integration of HBV DNA into the host genome rather than an intact virus (15). Thus, the diagnosis of occult HBV infection might be better reserved for those in whom HBV DNA is detected by two independent sets of primers in the sera or in those with cccDNA detected in the liver (72).

HBsAg Serology. HBsAg detection is the mainstay for the diagnosis of infection and the routine screening of blood donors. A wide variety of commercial assays is available for the detection of HBsAg. Clearly, the sensitivity of an assay to detect a variant is dependent on the anti-HBs used. Thus, it is not surprising that there are examples of variants that cannot be detected by all assays. Many test manufacturers have improved their HBsAg screening assays by replacing the monoclonal tracer antibody with polyclonal antibody (ies) against both wildtype and mutant HBV strains. The polyclonal capture, antibody-based assays showed a significantly better recognition of HBsAg mutants. A large number of HBsAg mutants are thought to be able to influence the performance of commercial assays (83). Although currently available HBsAg assays are superior to their predecessors, more developments are needed to improve the ongoing assessment of the ability of assays to detect a wide range of HBsAg variants. Several studies compare the sensitivity of modem assays for the detection of HBsAg variants in different panels (84-87). Regardless, if the level of antigenemia is low, as is generally the case in healthy blood donors with inactive hepatitis B, low levels of circulating HBsAg mutants may not be detected, even if the immunoassay is capable of detecting the recombinant form of the corresponding mutation (88).

In regard to OHB, the cutoff for HBsAg detection based on viral load is reported to range from 720 to 1,1431 copies/mL (180 to 2,858 IU/mL assuming 4 copies or geq/mL). Below these values, the HBsAg assay is usually nonreactive, whereas the reverse occurs at higher levels (89).

Molecular tests

In patients with OHB, serum DNA levels are frequently below the limits of detection for many of the quantitative assays. To date, there is no standardized method of viral quantification for low DNA levels in patients with occult HBV infection (Carman, personal communication) (61). Serum HBV level is usually less than 104 copies/ml in these patients (45,61). Because the majority of individuals with occult HBV infection have very low viral loads, more sensitive methods should be applied to identify occult HBV infection. A variety of testing methodologies are available to quantify HBV DNA, and it is virtually impossible to make direct comparisons due to the absence of standardization values.

Commercial molecular assays are routinely used in diagnostic laboratories, which are reported to vary significantly in sensitivity and specificity, linear range, genotype inclusivity, and sample volume; they may be insufficient for detecting very low HBV-DNA levels typical in patients with occult HBV infection. These empirical discrepancies appear to be mainly dependent on the different sensitivities and specificities of the assays used in the various studies, all of which limited testing for occult HBV to patient serum. Thus, assays of the highest sensitivity and specificity must be used for the diagnosis of OHB. As of this writing, the optimal standard for diagnosis is the analysis of HBV-DNA extracts from plasma performed by real-time, polymerase chain reaction (PCR) techniques. Quantitative real-time PCR is an effective method to detect occult HBV infection in blood centers. When the procedures are simplified, and the problem of contamination and high cost is overcome, quantitative real-time PCR should be the preferred method to detect occult HBV infection. The standard lower limit of detection (LLOD) for HBV DNA is 5 IU/mL or 30 copies/mL (89). Target amplification assays, such as the real-time PCR assay or the transcription-based mediated amplification (TMA) assay, make it possible to detect quantities of <5 IU/mL of HBV DNA and are rapidly approaching the level of a single HBV genome (89).

For research purposes and to avoid false-negative and false-positive results, PCR primers that span at least three genomic regions of the HBV genome such as the S, X, and core gene should be employed. Validation should require detection from at least two regions of the genome (53). Full-genome sequencing is the method of choice, although it is expensive and time consuming. In diagnostics, however, nested PCR would be timeconsuming and might increase the risk of PCR product carryover contamination.

OHB and HBV serologic markers

The diagnosis of OHB depends on the HBV serological marker profiles. This relationship has been studied before, and although the meaning of markers of previous HBV infection has no universal interpretation, studies have shown that the prevalence of occult HBV infection has usually been higher in subjects positive for both anti-HBs and anti-HBc than in those negative for all serologic markers (46-80% versus 20-50%) and high frequencies of HBV-DNA positivity have been observed among anti-HBc only individuals (10% to 80%) (8,15,49,67), suggesting that this antibody does not result in complete HBV elimination (90). Furthermore, most studies have suggested that detection of ''anti-HBc alone" could reflect unrecognized occult HBV infection. The term anti-HBc only has frequently been used to describe individuals infected with the human immunodeficiency virus (HIV) (56,91-94) or hepatitis C virus (HCV) (15,17,49,95-99). However, occult HBV infection has also been detected in patients who tested negative for all HBV serum markers, and prior studies have not always clearly explained their diagnostic approaches and the mechanisms of anti-HBc-negative and anti-HBs-negative serology in individuals with occult HBV infection. A possible explanation for apparent cases of "anti-HBc only" is a false negative HBsAg readings in several studies, which were reported to be a result of mutations in the "a" determinant (1,41,42).

Regardless of mutations in the surface or other HBV proteins that might influence the expression of HBsAg, such OHB infections may be detected in (a) individuals with resolving HBV infection who test positive for both anti-HBc and anti-HBs, (b) anti-HBc-only carriers who are seronegative for HBsAg in a window period of infection (15,61,63,100), and (c) false positive anti-HBc results due to the variable sensitivity and specificity of commercial assays (84-87). The possibility of false negative results for IgG anti-HBc should also be considered. Specifically, a portion of subjects with occult HBV infection who tested negative for IgG antiHBc might not have truly been negative; rather, they might have falsely tested negative due to low-antibody titer (Hollinger, personal communication.

Concluding remarks

The pathogenesis of occult hepatitis B is not yet clear, and it seems that both shared viral-host factors are involved. Further molecular studies on cohort patients group need to explore such association. HBsAg may not be an effective tool for differentiating HBV and OHB infection. Specifically, in light of this review, routinely used serological markers of HBV infection do not rule out occult and ongoing hepatitis B virus infection. This emphasizes that molecular methods should be used to detect occult HBV infection in these patients.

References

References

1. Chaudhuri V, Tayal R, Nayak B, et al. Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter. Gastroenterology 2004;127(5):1356-71.

2. Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepat 2002;9(4):243-57.

3. Kreutz C. Molecular, immunological and clinical properties of mutated hepatitis B viruses. J Cell Mol Med 2002;6(1):113-43.

4. Mosley JW, Stevens CE, Aach RD, et al. Donor screening for antibody to hepatitis B core antigen and hepatitis B virus infection in transfusion recipients. Transfusion 1995;35(1):5-12.

5. Coffin CS, Pham TN, Mulrooney PM, et al. Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection. Hepatology 2004;40(5):1053-61.

6. Michalak TI, Mulrooney PM, Coffin CS. Low doses of hepadnavirus induce infection of the lymphatic system that does not engage the liver. J Virol 2004;78(4):1730-8.

7. Lew YY, Michalak TI. In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus. J Virol 2001;75(4):1770-82.

8. Brechot C, Thiers V, Kremsdorf D, et al. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely "occult"? Hepatology 2001;34(1):194-203.

9. Zerbini A, Pilli M, Boni C, et al. The characteristics of the cell-mediated immune response identify different profiles of occult hepatitis B virus infection. Gastroenterology 2008;134(5):1470-81.

10. Carman WF. The clinical significance of surface antigen variants of hepatitis B virus. J Viral Hepat 1997;4(Suppl 1):11-20.

11. Tamori A, Nishiguchi S, Kubo S, et al. Sequencing of human-viral DNA junctions in hepatocellular carcinoma from patients with HCV and occult HBV infection. J Med Virol 2003;69(4):475-81.

12. Murakami Y, Minami M, Daimon Y, et al. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen. J Med Virol 2004;72(2):203-14.

13. Ackerman Z, Wands JR, Gazitt Y, et al. Enhancement of HBsAg detection in serum of patients with chronic liver disease following removal of circulating immune complexes. J Hepatol 1994;20(3):398-404.

14. Yotsuyanagi H, Yasuda K, Moriya K, et al. Frequent presence of HBV in the sera of HBsAg-negative, anti-HBcpositive blood donors. Transfusion 2001;41(9):1093-9.

15. 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(1):22-6.

16. De Maria N, Colantoni A, Friedlander L, et al. The impact of previous HBV infection on the course of chronic hepatitis C. Am J Gastroenterol 2000;95(12):3529-36.

17. Giannini E, Ceppa P, Botta F, et al. Previous hepatitis B virus infection is associated with worse disease stage and occult hepatitis B virus infection has low prevalence and pathogenicity in hepatitis C virus-positive patients. Liver Int 2003;23(1):12-8.

18. Rodriguez-Inigo E, Mariscal L, Bartolome J, et al. Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection. J Med Virol 2003;70(4):571-80.

19. Filippini P, Coppola N, Pisapia R, et al. Impact of occult hepatitis B virus infection in HIV patients naive for antiretroviral therapy. AIDS 2006;20(9):1253-60.

20. Lo Re V 3rd, Frank I, Gross R, et al. Prevalence, risk factors, and outcomes for occult hepatitis B virus infection among HIV-infected patients. J Acquir Immune Defic Syndr 2007;44(3):315-20.

21. Lukhwareni A, Burnett RJ, Selabe SG, et al. Increased detection of HBV DNA in HBsAg-positive and HBsAgnegative South African HIV/AIDS patients enrolling for highly active antiretroviral therapy at a Tertiary Hospital. J Med Virol 2009;81(3):406-12.

22. Mphahlele MJ, Lukhwareni A, Burnett RJ, et al. High risk of occult hepatitis B virus infection in HIV-positive patients from South Africa. J Clin Virol 2006;35(1):14-20.

23. Carman WF, Zanetti AR, Karayiannis P, et al. Vaccineinduced escape mutant of hepatitis B virus. Lancet 1990;336(8711):325-9.

24. Chen BF, Chen PJ, Jow GM, et al. High prevalence of mixed genotype infections in hepatitis B virus infected intravenous drug users. J Med Virol 2004;74(4):536-42.

25. Chen BF, Kao JH, Liu CJ, et al. Genotypic dominance and novel recombinations in HBV genotype B and C coinfected intravenous drug users. J Med Virol 2004;73(1):13-22.

26. Okamoto H, Omi S, Wang Y, et al. The loss of subtypic determinants in alleles, d/y or w/r, on hepatitis B surface antigen. Mol Immunol 1989;26(2):197-205.

27. Wallace LA, Echevarria JE, Echevarria JM, et al. Molecular characterization of envelope antigenic variants of hepatitis B virus from Spain. J Infect Dis 1994;170(5):1300-3.

28. Mangold CM, Unckell F, Werr M, et al. Secretion and antigenicity of hepatitis B virus small envelope proteins lacking cysteines in the major antigenic region. Virology 1995;211(2):535-43.

29. Pollicino T, Belloni L, Raffa G, et al. Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones. Gastroenterology 2006;130(32):823-37.

30. Sheldon J, Rodes B, Zoulim F, et al. Mutations affecting the replication capacity of the hepatitis B virus. J Viral Hepat 2006;13(7):427-34.

31. Vivekanandan P, Kannangai R, Ray SC, et al. Comprehensive genetic and epigenetic analysis of occult hepatitis B from liver tissue samples. Clin Infect Dis 2008;46(8):1227-36.

32. Fang Y, Teng X, Xu WZ, et al. Molecular characterization and functional analysis of occult hepatitis B virus infection in Chinese patients infected with genotype C. J Med Virol 2009;81(5):826-35.

33. van Hemert FJ, Zaaijer HL, Berkhout B, et al. Occult hepatitis B infection: an evolutionary scenario. Virol J 2008;5(12):146-58.

34. Cabrerizo M, Bartolome J, Caramelo C, et al. Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigennegative cases. Hepatology 2000;32(1):116-23.

35. Blum HE, Galun E, Liang TJ, et al. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication. J Virol 1991;65(4):1836-42.

36. Gujar SA, Michalak TI. Primary occult hepadnavirus infection induces virus-specific T-cell and aberrant cytokine responses in the absence of antiviral antibody reactivity in the Woodchuck model of hepatitis B virus infection. J Virol 2009;83(8):3861-76.

37. Hassanshahi G, Arababadi MK, Khoramdelazad H, et al. Assessment of CXCL12 (SDF-1alpha) polymorphisms and its serum level in posttransfusion occult HBV-infected patients in Southeastern Iran. Arch Med Res 2010;41(5):338-42.

38. Bhargava A, Khan S, Panwar H, et al. Occult hepatitis B virus infection with low viremia induces DNA damage, apoptosis and oxidative stress in peripheral blood lymphocytes. Virus Res 2010;153(1):143-50.

39. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13(1):2960.

40. Chemin I, Trepo C. Clinical impact of occult HBV infections. J Clin Virol 2005;34(Suppl 1):S15-21.

41. Pollicino T, Raffa G, Costantino L, et al. Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma. Hepatology 2007;45(2):277-85.

42. Shahmoradi S, Yahyapour Y, Mahmoodi M, et al. High prevalence of occult hepatitis B virus infection in children born to HBsAg-positive mothers despite prophylaxis with hepatitis B vaccination and HBIG. J Hepatol 2012;57(3):515-21.

43. Raimondo G, Pollicino T, Cacciola I, et al. Occult hepatitis B virus infection. J Hepatol 2007;46(1):160-70.

44. Minuk GY, Sun DF, Greenberg R, et al. Occult hepatitis B virus infection in a North American adult hemodialysis patient population. Hepatology 2004;40(5):1072-7.

45. Minuk GY, Sun DF, Uhanova J, et al. Occult hepatitis B virus infection in a North American community-based population. J Hepatol 2005;42(4):480-5.

46. Kim SM, Lee KS, Park CJ, et al. Prevalence of occult HBV infection among subjects with normal serum ALT levels in Korea. J Infect 2007;54(2):185-91.

47. Firnhaber C, Viana R, Reyneke A, et al. Occult hepatitis B virus infection in patients with isolated core antibody and HIV co-infection in an urban clinic in Johannesburg, South Africa. Int J Infect Dis 2009;13(4):488-92.

48. Mphahlele MJ, Moloto MJ. Detection of HBV DNA from serologically negative or 'silent' HBV infections--viral or host factors? S Afr Med J 2002;92(8):613-5.

49. Sagnelli E, Imparato M, Coppola N, et al. Diagnosis and clinical impact of occult hepatitis B infection in patients with biopsy proven chronic hepatitis C: a multicenter study. J Med Virol 2008;80(9):1547-53.

50. Mrani S, Chemin I, Menouar K, et al. Occult HBV infection may represent a major risk factor of non-response to antiviral therapy of chronic hepatitis C. J Med Virol 2007;79(8):1075-81.

51. Stratta P, Bruschetta E, Minisini R, et al. Prevalence and clinical relevance of occult hepatitis B virus infection in patients on the waiting list for kidney transplantation. Transplant Proc 2009;41(4):1132-7.

52. Manzini P, Girotto M, Borsotti R, et al. Italian blood donors with anti-HBc and occult hepatitis B virus infection. Haematologica 2007;92(12):1664-70.

53. Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49(4):652-7.

54. Knoll A, Hartmann A, Hamoshi H, et al. Serological pattern "anti-HBc alone": characterization of 552 individuals and clinical significance. World J Gastroenterol 2006;12(8):1255-60.

55. Fang ZL, Zhuang H, Wang XY, et al. Hepatitis B virus genotypes, phylogeny and occult infection in a region with a high incidence of hepatocellular carcinoma in China. World J Gastroenterol 2004;10(22):3264-8.

56. Grob P, Jilg W, Bornhak H, et al. Serological pattern "antiHBc alone": report on a workshop. J Med Virol 2000;62(4):450-5.

57. Wagner AA, Denis F, Weinbreck P, et al. Serological pattern 'anti-hepatitis B core alone' in HIV or hepatitis C virus-infected patients is not fully explained by hepatitis B surface antigen mutants. AIDS 2004;18(3):569-71.

58. Alavian SM, Miri SM, Hollinger FB, et al. Occult Hepatitis B (OBH) in Clinical Settings. Hepat Mon 2012;12(8):e6126.

59. Cacciola I, Pollicino T, Squadrito G, et al. Quantification of intrahepatic hepatitis B virus (HBV) DNA in patients with chronic HBV infection. Hepatology 2000;31(2):507-12.

60. Lok AS, Liang RH, Chiu EK, et al. Reactivation of hepatitis B virus replication in patients receiving cytotoxic therapy. Report of a prospective study. Gastroenterology 1991;100(1):182-8.

61. Torbenson M, Thomas DL. Occult hepatitis B. Lancet Infect Dis 2002;2(8):479-86.

62. Chemin I, Jeantet D, Kay A, et al. Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-) liver disease. Antiviral Res 2001;52(2):117-23.

63. Donato F, Gelatti U, Limina RM, et al. Southern Europe as an example of interaction between various environmental factors: a systematic review of the epidemiologic evidence. Oncogene 2006;25(27):3756-70.

64. 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(3):350-5.

65. Marusawa H, Uemoto S, Hijikata M, et al. Latent hepatitis B virus infection in healthy individuals with antibodies to hepatitis B core antigen. Hepatology 2000;31(2):488-95.

66. Shih LN, Sheu JC, Wang JT, et al. Serum hepatitis B virus DNA in healthy HBsAg-negative Chinese adults evaluated by polymerase chain reaction. J Med Virol 1990;32(4):257-60.

67. Fukuda R, Ishimura N, Niigaki M, et al. Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance. J Med Virol 1999;58(3):201-7.

68. Hui CK, Lau E, Monto A, et al. Natural history of patients with recurrent chronic hepatitis C virus and occult hepatitis B co-infection after liver transplantation. Am J Transplant 2006;6(7):1600-8.

69. Peres AA, Dias EA, Chesky M, et al. Occult hepatitis B in renal transplant patients. Transpl Infect Dis 2005;7(2):51-6.

70. Shetty K, Hussain M, Nei L, et al. Prevalence and significance of occult hepatitis B in a liver transplant population with chronic hepatitis C. Liver Transpl 2008;14(4):534-40.

71. Chazouilleres O, Mamish D, Kim M, et al. "Occult" hepatitis B virus as source of infection in liver transplant recipients. Lancet 1994;343(8890):142-6.

72. Marrero JA, Lok AS. Occult hepatitis B virus infection in patients with hepatocellular carcinoma: Innocent bystander, cofactor, or culprit? Gastroenterology 2004;126(1):347-50.

73. Raimondo G. Occult hepatitis B virus infection and liver disease: fact or fiction? J Hepatol 2001;34(3):471-3.

74. Uemoto S, Sugiyama K, Marusawa H, et al. Transmission of hepatitis B virus from hepatitis B core antibody-positive donors in living related liver transplants. Transplantation 1998;65(4):494-9.

75. Saito T, Shinzawa H, Uchida T, et al. Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B. J Med Virol 1999;58(4):325-31.

76. Thiers V, Nakajima E, Kremsdorf D, et al. Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet 1988;2(8623):1273-6.

77. Coffin CS, Michalak TI. Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis. J Clin Invest 104(2):203-12.

78. Shiota G, Oyama K, Udagawa A, et al. Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53. J Med Virol 2000;62(2):151-8.

79. Fabris P, Brown D, Tositti G, et al. Occult hepatitis B virus infection does not affect liver histology or response to therapy with interferon alpha and ribavirin in intravenous drug users with chronic hepatitis C. J Clin Virol 2004;29(3):160-6.

80. Yuen MF, Ka-Ho Wong D, Lee CK, et al. Transmissibility of Hepatitis B Virus (HBV) Infection through Blood Transfusion from Blood Donors with Occult HBV Infection. Clin Infect Dis 2011;52(5):624-32.

81. Hui CK, Yu J, Au WY, et al. Sexual transmission of hepatitis B infection despite the presence of hepatitis B virus immunity in recipients of allogeneic bone marrow transplantation. J Clin Virol 2005;32(2):173-8.

82. Koike K, Kobayashi M, Gondo M, et al. Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients. J Med Virol 1998;54(4):249-55.

83. Alavian SM, Carman WF, Jazayeri SM. HBsAg variants: Diagnostic-escape and diagnostic dilemma. J Clin Virol 2013;57(3):201-8.

84. Coleman PF, Chen YC, Mushahwar IK. Immunoassay detection of hepatitis B surface antigen mutants. J Med Virol 1999;59(1):19-24.

85. Ijaz S, Torre F, Tedder RS, et al. Novel immunoassay for the detection of hepatitis B surface 'escape' mutants and its application in liver transplant recipients. J Med Virol 2001;63(3):210-16.

86. Moerman B, Moons V, Sommer H, et al. Evaluation of sensitivity for wild type and mutant forms of hepatitis B surface antigen by four commercial HBsAg assays. Clin Lab 2004;50(3-4):159-62.

87. Zaaijer HL, Vrielink H, Koot M. Early detection of hepatitis B surface antigen and detection of HBsAg mutants: a comparison of five assays. Vox Sang 2001;81(4):219-21.

88. Jeantet D, Chemin I, Mandrand B, et al. Cloning and expression of surface antigens from occult chronic hepatitis B virus infections and their recognition by commercial detection assays. J Med Virol 2004;73(4):508-15.

89. Hollinger FB, Sood G. Occult hepatitis B virus infection: a covert operation. J Viral Hepat 2010;17(1):1-15.

90. Carman WF, Boner W, Fattovich G, et al. Hepatitis B virus core protein mutations are concentrated in B cell epitopes in progressive disease and in T helper cell epitopes during clinical remission. J Infect Dis 1997;175(5):1093-100.

91. Hofer M, Joller-Jemelka HI, Grob PJ, et al. Frequent chronic hepatitis B virus infection in HIV-infected patients positive for antibody to hepatitis B core antigen only. Swiss HIV Cohort Study. Eur J Clin Microbiol Infect Dis 1998;17(1):6-13.

92. Nunez M, Rios P, Perez-Olmeda M, et al. Lack of 'occult' hepatitis B virus infection in HIV-infected patients. AIDS 2002;16(15):2099-101.

93. Rodriguez-Torres M, Gonzalez-Garcia J, Brau N, et al. Occult hepatitis B virus infection in the setting of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection: clinically relevant or a diagnostic problem? J Med Virol 2007;79(6):694-700.

94. Shire NJ, Rouster SD, Rajicic N, et al. Occult hepatitis B in HIV-infected patients. J Acquir Immune Defic Syndr 2004;36(3):869-75.

95. Jilg W, Sieger E, Zachoval R, et al. Individuals with antibodies against hepatitis B core antigen as the only serological marker for hepatitis B infection: high percentage of carriers of hepatitis B and C virus. J Hepatol 1995;23(1):14-20.

96. Brechot C, Jaffredo F, Lagorce D, et al. Impact of HBV, HCV and GBV-C/HGV on hepatocellular carcinomas in Europe: results of a European concerted action. J Hepatol 1998;29(2):173-83.

97. Carvalho-Filho RJ, de Lucca Schiavon L, NarcisoSchiavon JL, et al. Clinical and histological impact of previous hepatitis B virus infection in patients with chronic hepatitis C. Liver Int 2009;29(1):133-40.

98. El-Sherif A, Abou-Shady M, Abou-Zeid H, et al. Antibody to hepatitis B core antigen as a screening test for occult hepatitis B virus infection in Egyptian chronic hepatitis C patients. J Gastroenterol 2009;44(4):359-64.

99. Khan MH, Farrell GC, Byth K, et al. Which patients with hepatitis C develop liver complications? Hepatology 2000;31(2):513-20.

100. Weinberger KM, Bauer T, Bohm S, et al. High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum. J Gen Virol 2000;81(Pt 5):1165-74.