Occult hepatitis B virus infection (OBI) is highly prevalent worldwide. In some cases, it is a consequence of infection with variant viruses mutated in the S gene and producing a surface antigen not recognized by diagnostic kits. In most cases, OBI is due to a strong inhibition of hepatitis B virus (HBV) activities exerted by host defense mechanisms. OBI may reactivate in patients undergoing immunosuppressive therapy and/or chemotherapy with the possibility of a consequent development of acute hepatitis that may lead to hepatic failure. Hematological malignancies and therapeutic schedules including rituximab are the conditions most frequently associated with OBI reactivation. However, this event may occur in a large number of additional clinical and therapeutic settings. Identification of patients prone to undergo reactivation is of great importance for promptly starting a proper antiviral therapy that may stop the HBV reactivation and prevent its clinical sequelae.
Occult hepatitis B virus infection (OBI) and its clinical implications are becoming one of the most intriguing and important topics in the field of viral hepatitis [1,2]. In particular, there is growing interest about the risk of OBI reactivation with the possible sequela of acute severe hepatitis [2,3,4,5]. This interest is largely justified for at least three main reasons: (1) epidemiological studies have clearly demonstrated that OBI is highly prevalent worldwide [although it is more widespread in the areas where hepatitis B virus (HBV) is endemic] [1,2], (2) an increasing number of cases of OBI reactivation have been observed recently as a consequence of the availability of new, potent, and efficacious immunological drugs (i.e. targeted immunomodulators or biologics) and complex chemotherapy schedules longitudinally administered through several subsequent cycles in different clinical contexts (i.e. oncology, oncohematology, and autoimmune diseases) [1,2,6,7], and (3) the availability of nucleos(t)ide analogue antivirals specifically inhibiting HBV replication, which (if properly used) represents a tool of utmost importance for preventing OBI reactivation and/or consequent acute hepatitis .
The definition of ‘occult hepatitis B virus infection' groups the clinical/virological conditions in which episomal, free HBV genomes persist for a long time in the hepatocytes of individuals testing negative for HBV surface antigen (HBsAg) . In some cases, the lack of HBsAg detection is the consequence of the infection with variant viruses carrying mutations in the HBV S gene (S-escape mutants) and producing a modified HBsAg that is not recognized by commercially available diagnostic kits [10,11]. In most cases, OBI is due to replication-competent viruses with a degree and relevance of genetic heterogeneity comparable with the HBV isolates from individuals with HBsAg-positive (namely ‘overt') infection , but subjected to a potent suppression of the replication activity and gene expression leading to the lack of HBsAg synthesis as well as to the absence (or presence in traces) of HBV DNA in the serum [1,2]. In the context of this review, it is important to stress that the replication, transcription, and protein synthesis capabilities of HBV isolates from liver of OBI individuals can be fully restored once the viruses are taken out of the host's microenvironment and transfected in hepatoma cells . In fact, the acute hepatitis that may follow OBI reactivation usually shows the typical serological profile of acute hepatitis B, with HBsAg (re)appearance and even HBeAg positivity [1,3,13]. Although the mechanisms involved in the strong inhibition of the HBV activities are far from being completely elucidated, it is clear that host factors are largely implicated. In particular, much evidence shows the central role played by immune surveillance in inhibiting HBV replication and gene expression up to the OBI phase development [14,15,16,17]. Indeed, OBI reactivation is an event usually occurring in patients immunocompromised because of a disease involving the immune system and/or because therapeutic treatments have considerably weakened the immune response [3,5,6,7]. In fact, one may depict a schematic scenario that when a subject with OBI undergoes immunosuppressive treatment, a reactivation of the viral replication and protein synthesis (including HBsAg) may occur as a consequence of the fault of the immunological control, and, once therapy is stopped and immune surveillance reconstituted, a CTL-mediated hepatocyte injury may occur leading to the development of hepatitis. Of note, aside from immunologic mechanisms, there is also evidence that - similar to other DNA viruses (i.e. Epstein-Barr virus and herpesviruses) able to develop chronic silent infections - in the case of OBI, the epigenetic modifications of histones bound to the HBV cccDNA might be a mechanism of transcriptional control and silencing of the viral activity . However, there are only few anecdotal reports and no clear evidence of the possible OBI reactivation due to drugs potentially influencing the epigenetic control of the HBV cccDNA minichromosome .
A further point that must be included in these introductory notes concerns the fact that even if the majority of OBI patients are positive for antibodies to the HBV core antigen (anti-HBc) and part of them also for the anti-HBs antibodies, about 20-25% of OBI cases are negative for all HBV serum markers [1,2]. This last subset of patients is clearly very difficult to recognize as ‘OBI carriers' since there is no sign or marker that may lead to suspect the infection and thus to perform an HBV DNA test that might allow identification of the infection. However, apart from a few anecdotal reports concerning HBV reactivation in OBI-positive/HBV-seronegative cases , OBI reactivation appears to be an event strictly occurring in anti-HBV antibody-positive subjects. In this context, it is important to stress that there are convincing data showing that the HBV-specific T cell response is much weaker in OBI seronegative individuals than in anti-HBc-positive patients, thus likely insufficient to provoke severe liver injury .
OBI Reactivation: Predisposing Conditions and Possible Predictors
Although HBV reactivation in individuals with OBI undergoing immunosuppressive therapies occurs less frequently than in cases with ‘overt' infection , it has considerable clinical relevance. In fact, considering the enormous number of potential ‘OBI carriers' (namely, anti-HBc positive individuals), it represents an everyday challenge in clinical practice. OBI reactivation may be clinically silent, but in some circumstances it may induce acute hepatitis that may have a severe and even fulminant course, or may evolve to rapidly progressive chronic hepatitis . In any case, development of acute hepatitis may determine the interruption of chemotherapy with obvious negative consequences for disease prognosis . To date, no reliable tool for predicting HBV reactivation in OBI patients is available. However, a number of clinical, therapeutic, and immunovirological profiles have been identified that - once cumulatively evaluated - may help to identify individuals at higher and those at lower risk for the occurrence of that event (table 1). The reported frequencies of OBI reactivation in the different populations according to these features are schematically summarized below.
Patients with hematological malignancies (especially non-Hodgkin lymphoma, multiple myeloma, myelomonoblastic acute leukemia, and chronic lymphocytic leukemia) have the highest risk of OBI reactivation, especially when treated with schedules including anti-CD20 monoclonal antibody (rituximab) [2,5,6,7,23,24,25]. Patients undergoing hematopoietic stem cell transplantation also show a high incidence of OBI reactivation [26,27], which is a frequent occurrence in cases of liver transplantation (but also of kidney) when the recipient is naive for HBV infection and the donor is anti-HBc positive [28,29].
Patients with rheumatologic diseases have been extensively investigated for evaluation of HBV reactivation. OBI reactivation appears to be an infrequent (but existing) event in individuals undergoing treatments including biologics (mainly anti-CD20 and anti-TNF-α drugs, see below) or with schedules containing high doses of corticosteroids [24,30,31]. Only a few cases of OBI reactivation occurring in patients with liver cancer undergoing transarterial chemoembolization and in patients with inflammatory bowel diseases under treatment with biological agents have been reported .
Finally, the real risk of OBI reactivation in patients with solid tumor undergoing chemotherapy is debatable , and there are no reliable data available about OBI reactivation in other categories of patients, such as individuals with psoriasis, in whom treatments with biological agents are applied.
Immunosuppressive Therapy and Chemotherapy
Therapeutic regimens containing rituximab [especially combinations of rituximab with cyclophosphamide, hydroxydaunorubicin, oncovin, and predniso(lo)ne] provide the highest risk of reactivation in all HBV patients, including those with OBI [5,21,23]. Apart from hematological malignancies, rituximab is also widely used in patients with autoimmune inflammatory diseases (i.e. rheumatoid arthritis, inflammatory bowel diseases, etc.). It is a monoclonal antibody directed against the CD20 antigen expressed on the B lymphocyte surface. It seems very important (and also scientifically intriguing) that a drug efficiently depleting B lymphocytes is also the most potent inductor of the restoration of HBV replication in all conditions (of course also including OBI phase) in which it appears to be suppressed (likely because of the immune control exerted by the T lymphocytes). It is thought that the central role played by B lymphocytes in the anti-HBV immune response is related to multiple aspects including, besides the production of antibodies, their activity as antigen-presenting cells and the enhancement of the cytotoxic response of CD8 T lymphocytes . Similarly, ofatumumab - another anti-CD20 monoclonal antibody used for treatment of hematological malignancies - is considered a drug with potential high propensity to induce HBV reactivation . Another biological agent utilized in the field of oncohematologic therapy and recognized as responsible for cases of OBI reactivation is the anti-CD52 monoclonal antibody alemtuzumab [5,21,23].
Anti-TNF-α drugs that are widely used in autoimmune inflammatory diseases have been reported to be associated with some cases of OBI reactivation. Of note, TNF-α is a chemokine known to suppress HBV replication .
Among the several nonbiologic drugs potentially implied in HBV reactivation (also in cases with OBI) , corticosteroids administered at high doses and for long periods must be listed. In this context, it is worth mentioning that steroids, apart from favoring the establishment of an immunosuppressive status, may directly act by stimulating HBV replication through the glucocorticoid responsive element present in the viral genome  (fig. 1).
A relevant and debated argument is whether patients positive for anti-HBc alone have a different risk of OBI reactivation compared to those positive for both anti-HBc and anti-HBs, as it was suggested that anti-HBs exerts a protective role against the rebound of HBV active replication. However, recent studies have not confirmed this different behavior of the two subsets (anti-HBs positive and anti-HBs negative) of anti-HBc-positive patients [23,36]. Indeed, a large body of evidence has demonstrated that anti-HBs/anti-HBc-positive patients undergoing immunosuppression may show a progressive decrease of the titer of anti-HBs that may finally disappear [23,26,27]. This phenomenon usually precedes the reappearance of the HBsAg in all cases with complete virological and clinical reactivation.
Similarly debated is whether the presence of minute but detectable amounts of HBV DNA in the serum before starting immunosuppressive therapy is a clear indication for the treatment with antivirals to prevent a possible massive viral reactivation. In this context, it has to be considered that patients with OBI longitudinally followed very often show phases of negativity for serum HBV DNA alternating with phases - or single time points - in which low amounts of HBV DNA are detected. This fluctuating profile has been observed in immunocompetent individuals [37,38] as well as in patients with solid tumors under chemotherapy; both conditions are usually not followed by clinically relevant viral reactivation . Of note, it has recently been reported that OBI patients with detectable traces of HBV DNA before immunosuppressive therapy have comparable risks of clinical/virological reactivation as HBV-DNA-negative subjects . In any case, it has to be stressed that HBV DNA testing before starting immune and/or chemotherapies is an undoubtedly important step for at least two main reasons: (1) detection of HBV DNA is a direct, definitive demonstration that a patient is an ‘OBI carrier' since the sole detection of anti-HBc is insufficient to prove the occult infection status , and (2) assaying HBV DNA is the only test that can identify the infrequent but existing cases of moderate/high levels of viremia (with >200 IU/ml as an arbitrary cutoff, according to the statements from an international expert meeting focused on OBI ) that may be due to infection with S-escape virus and that may require therapeutic approaches completely different compared to those adopted in cases of OBI due to suppressed viral activity (fig. 2).
Therapeutic Approaches for the Prevention or the Treatment of OBI Reactivation
The ideal treatment for OBI should rely on a drug capable of eliminating the HBV cccDNA from the hepatocyte nuclei, possibly without killing the cells. Unfortunately, no such drugs are available at present. Consequently, we have to act by trying to prevent OBI reactivation and above all the development of acute hepatitis that may follow viral reactivation, and be aware that such prevention is lifesaving in many cases.
HBsAg-Negative Patients Infected with S-Escape HBV Variants
The clinical/virological features (including serum HBV DNA levels) of these cases are substantially similar to those observed in the different conditions of the ‘overt' HBV infection; therefore, the therapeutic approaches should be similar to that adopted in the ‘typical' HBsAg-positive forms and patients undergoing chemotherapy and/or immunosuppressive therapies should be treated with entecavir or tenofovir, which are the most potent anti-HBV nucleos(t)ide analogues and (very importantly) which have the highest genetic barrier  (fig. 2).
OBI Patients with Suppressed HBV Replication and Gene Expression
This category of patients represents a difficult but frequent challenge for hepatologists. On one hand, there are too many anti-HBc-positive individuals in the world (with the highest prevalence in HBV endemic areas) to propose a preemptive therapy in all cases undergoing immunosuppressive therapy. On the other hand, the collaboration between hepatologists and the other specialists taking care of these patients (hematologists, oncologists, rheumatologists, etc.) is often not sufficiently strong enough to guarantee the very narrow biochemical and virological follow-up necessary to identify viral reactivation early enough to start antiviral therapy and prevent acute hepatitis.
Consequently, the following behavior might be suggested for anti-HBc-positive patients undergoing immunosuppressive therapy and/or chemotherapy (fig. 2): (1) all subjects suffering from the above-mentioned diseases at the highest risk of OBI reactivation should be treated with lamivudine, independently of the anti-HBs status and detectable or undetectable serum HBV DNA; (2) similarly, all individuals testing HBV DNA positive and undergoing treatment with biologics (mainly rituximab and other anti-CD20, but also anti-CD52, and anti-TNFα) and/or with a high dose of corticosteroids should be preemptively treated with lamivudine; (3) in individuals with diseases rarely associated with OBI reactivation, testing HBV DNA positive when untreated with biologics or high doses of steroids should be monitored through evaluations of HBV DNA and aminotransferase (AT) levels every 2-3 months; (4) in individuals with diseases rarely associated with OBI reactivation, testing HBV DNA negative and undergoing treatments with rituximab or other biologics should be monitored through evaluations of HBV DNA and AT levels every 2 months, and (5) all individuals with diseases rarely associated with OBI reactivation, testing HBV DNA negative and undergoing any immunosuppressive therapy or chemotherapy should be monitored by evaluations of HBV DNA and AT levels every 3 months. In individuals at points 3 and 4, if HBV DNA becomes detectable and/or there is an AT rise, the tests should be quickly repeated to verify the new profile, also including HBsAg testing (and anti-HBs titer in individuals anti-HBs positive at baseline). When the patterns of possible reactivation are confirmed, antiviral therapy with a nucleos(t)ide analogue (preferably entecavir or tenofovir) must be started.
Of note, lamivudine is efficient in preventing HBV reactivation in patients with no or very low serum HBV DNA levels , and the use of a very potent nucleos(t)ide analogue does not seem to provide significant advantages . The only warning should be the risk of the selection of lamivudine-resistant viral strains possibly archived in the HBV cccDNA pool and prone to determine a viral reactivation . This eventuality suggests continuing careful monitoring under antiviral treatment, and if the HBV DNA remains detectable (or even increases) during therapy with lamivudine, it is prudent to switch to tenofovir, which is very efficacious in suppressing lamivudine-resistant mutants
Of great importance, HBV reactivation may occur many months (up to 20 months) after stopping chemotherapy [3,5,21,40]. Consequently, a narrow follow-up for patients not treated with antivirals as well as antiviral prophylaxis for those treated must be continued for a long time (some suggest at least 18 months) after chemotherapy/immunosuppressive therapy discontinuation. In the unfortunate cases in which HBV reactivation is not prevented and acute hepatitis develops, entecavir or tenofovir should be administered as soon as possible since there are reports showing the capacity of these antivirals to avoid the fulminant course in some cases . In cases of liver transplantation (but also of kidney and bone marrow transplantation), when the donor is anti-HBc positive, antiviral prophylaxis is strongly recommended.
Although HBV reactivation in cases with OBI is much less frequent than cases with ‘overt' infection, it represents a significant challenge in clinical practice for specialists of different branches. OBI is also a challenge because the number of HBsAg-negative/anti-HBc-positive individuals (potential ‘OBI carriers') is considerably higher than HBsAg-positive individuals worldwide. Moreover, while indications for (and schedule of) antiviral treatment in HBsAg-positive subjects undergoing clinical/therapeutic conditions known to predispose to HBV reactivation (i.e. immunosuppressive therapy and/or chemotherapy) are quite well characterized, many doubts still exist about the possible therapeutic approaches in OBI patients undergoing the same conditions. This is mainly due to the fact that not all HBsAg-negative/anti-HBc-positive individuals are ‘OBI carriers', and no reliable marker (or strong indicator) of OBI reactivation has been identified so far. Nevertheless, when OBI reactivation occurs, it may determine acute hepatitis that can be fatal for the patient due to its possible fulminant course or the necessity to stop chemotherapy. Development of valid and commercially available assays allowing the certain identification of OBI in all cases in different clinical settings and particularly in conditions known to create risks for HBV reactivation has become a true necessity. In the meantime, international prospective trials involving patients at risk of OBI reactivation should be performed to better define the risk factors, the monitoring and surveillance procedures, the antiviral approaches, and their duration and endpoints.