The development of sensitive assays to detect small amounts of hepatitis B virus (HBV) DNA has favored the identification of occult hepatitis B infection (OBI), a virological condition characterized by a low level of HBV replication with detectable levels of HBV DNA in liver tissue but an absence of detectable surface antigen of HBV (HBsAg) in serum. The gold standard to diagnose OBI is the detection of HBV DNA in the hepatocytes by highly sensitive and specific techniques, a diagnostic procedure requiring liver tissue to be tested and the use of non-standardized non-commercially available techniques. Consequently, in everyday clinical practice, the detection of anti-hepatitis B core antibody (anti-HBc) in serum of HBsAg-negative subjects is used as a surrogate marker to identify patients with OBI. In patients with chronic hepatitis C (CHC), OBI has been identified in nearly one-third of these cases. Considerable data suggest that OBI favors the increase of liver damage and the development of hepatocellular carcinoma (HCC) in patients with CHC. The data from other studies, however, indicate no influence of OBI on the natural history of CHC, particularly regarding the risk of developing HCC.

Approximately 170 million individuals are chronically infected with hepatitis C virus (HCV) worldwide[1-4]. HCV is a small, enveloped, positive-sense, single-stranded RNA virus of the genus Hepacivirus of the Flaviviridae family. Phylogenetic analysis of HCV isolates has generated the viral classification into six major genotypes (from 1 to 6) and more than 100 subtypes[5,6]. HCV is transmitted by percutaneous exposure to infected blood through intravenous drug injection and invasive medical procedures, and by permucosal exposure through unprotected intercourse with multiple partners[7,8], particularly in human immunodeficiency virus (HIV)-positive men who have sex with men[9-12].

HCV causes acute hepatitis that is frequently asymptomatic, and in its symptomatic form, it is characterized by nausea, malaise, and jaundice. The acute HCV infection resolves spontaneously in about one-third of the cases[13,14], whereas the remaining two-thirds remain infected, circulate anti-HCV and HCV RNA, and usually show an indolent course or a slow progression to liver cirrhosis and hepatocellular carcinoma (HCC)[15]. In some cases, however, spontaneous acute exacerbations may develop, characterized by one or more peaks of the aminotransferase serum levels above the previous values[16-22], which can frequently induce a deterioration of the liver disease. In some cases the progression to liver cirrhosis and HCC is rapid[15], particularly when co-morbidities, an unfavorable genetic background, and unsafe lifestyle factors are present. Indeed, the outcome of chronic hepatitis C (CHC) is influenced by associated host factors (sex, age at infection, routes of transmission, immune response, genetic background), viral factors (HCV genotype and viral quasispecies), co-morbidities (viral co-infection, insulin-resistance, liver steatosis, immunosuppressive clinical condition) and lifestyle factors (alcohol intake)[23-30].

The development of sensitive assays to detect small amounts of hepatitis B virus (HBV) DNA has favored the identification of occult HBV infection (OBI), a virological condition characterized by a low level of HBV replication with HBV DNA detectable in the liver cells in the absence of detectable surface antigen of HBV (HBsAg) in serum. In patients with CHC, OBI has been identified in about one-third of HBsAg-negative/anti-HCV-positive subjects in the Mediterranean Basin and in more than 50% in East Asian countries[31-36]. Considerable data suggest that in patients with CHC, OBI may contribute to chronic liver damage and to the development of HCC[24,31,37-40]. Other studies, however, indicate that OBI does not influence the natural history of HCV infection, particularly as regards the risk of HCC development[41-43]. In this review article, which takes into account all the available literature data, the possible role of OBI in modifying the clinical course of CHC is evaluated and discussed.

OBI has been defined as the presence of viral DNA in the liver tissue (regardless of HBV DNA detectability in serum) of individuals testing negative for serum HBsAg[36]. The gold standard to diagnose OBI is the detection of HBV DNA in the hepatocytes by highly sensitive and specific techniques [real-time polymerase chain reaction (PCR), nested PCR, and the use of oligonucleotide primers specific for different HBV genomic regions], a diagnostic procedure requiring liver tissue to be tested and the use of non-standardized non-commercially available techniques. Consequently, in everyday clinical practice, the detection of anti-hepatitis B core antibody (anti-HBc) in serum of HBsAg-negative subjects, a sign of previous acute hepatitis B (AHB), is used as a surrogate serum marker to identify subjects with OBI[39,43-49]. This option is supported by the observation that in patients experiencing immunosuppression, OBI, as defined by the presence of HBV DNA in liver tissue, mostly occurs in HBsAg-negative/anti-HBc-positive patients[44,50,51]. The data from a previous investigation on 89 HBsAg-negative patients with CHC showed the presence of HBV DNA in plasma, peripheral blood mononuclear cells, and/or liver tissue in 60% of the anti-HBs/anti-HBc-positive patients, in 80% of the anti-HBs-negative/anti-HBc-positive patients, and in 10% of those lacking both antibodies[44].

In some cases, an underhand activity of the HBV genome is the persistence of mild hepatocellular necrosis for years after the resolution of self-limiting AHB[52,53]. The mechanism of liver damage due to OBI is still unclear, but there is some evidence that viral factors may play a role in its development and in the related liver damage. In fact, the persistent synthesis of minute undetectable amounts of the virus or other viral transcripts produced by the HBV covalently closed circular DNA (cccDNA) seems capable of maintaining the HBV-specific memory T-cell response[33,54] and the production of cytokines, such as tumor necrosis factor-α and interferon-γ[55,56]. In addition, mutations in the X region of HBV may reduce the ability of the X protein to transactivate host cellular proteins essential for viral replication, which may lead to the reduction of HBV DNA replication and the lack of HBsAg serum expression[57].

We should remember, however, that a rare escape mutation in the S region decreases the reactivity in the HBsAg detection assays[58] and is responsible for an “overt” HBsAg-negative infection that might mimic OBI.

There is biological, epidemiological, and clinical evidence demonstrating that the oncogenic potential of HBV may induce the development of HCC both in patients with cirrhosis and in those with a milder liver disease. Chronic HBV infection accounts for approximately 50% of the total cases and for virtually all childhood HCC, and prospective cohort studies showed a 5- to 100-fold increase in the risk of developing HCC among HBsAg carriers compared with uninfected subjects[75]. In spite of this, the role of OBI in the development of HCC in patients with chronic hepatitis due to etiological agents other than HBV, firstly HCV, is still a matter of debate in the scientific community. Using anti-HBc positivity or the presence of HBV DNA in plasma or liver tissue as a sign of OBI, several research groups have investigated the role of OBI in the development of HCC in HBsAg-negative patients with CHC.

The clinical impact of OBI on the natural history of CHC has been extensively investigated, but the available data are conflicting and do not allow for conclusions to be drawn on this topic. One of the main reasons for this inconsistency is the heterogeneity of the methods used to detect OBI. In fact, the detection of HBV DNA in liver tissue of HBsAg-negative subjects can be considered of high sensitivity and high specificity, and that of HBV DNA in plasma of high specificity and moderate sensitivity. In addition, the detection of anti-HBc in serum should be considered of moderate specificity and moderate sensitivity in this setting, although anti-HBc-negative subjects may show HBV DNA in the liver tissue. Furthermore, the variety of diagnostic molecular assays used to identify HBV DNA in plasma and liver tissue of HBsAg-negative subjects possess different sensitivities, bringing considerable heterogeneity in the results. Indeed, in the majority of studies, anti-HBc in serum or HBV DNA in plasma was used to detect OBI, since this method is cheaper, less invasive, and less time-consuming than the detection of HBV DNA in the liver tissue.

Other reasons for the substantial variability in the prevalence of OBI in published studies may be the differences in the extent of the spread of HBV infection in the various geographical areas, the variability in the viral characteristics, and the heterogeneity of the enrolment criteria regarding age, gender, immunological and ethnic background, and social habits of the subjects examined.

In addition, OBI itself is a virological condition of different origins; most patients having a self-limiting AHB and a minority from the pool of HBsAg chronic carriers, of whom nearly 1% per year clear serum HBsAg. Subjects with OBI of different origins may be present in different proportions in the studies published, and OBI itself may have a different outcome and a different impact on the clinical course of CHC in relation to its origin.

In light of this, we should conclude that the present knowledge on the clinical impact of OBI on the progression of liver fibrosis and on the development of HCC is still insufficient.

In order to reduce the effect of different methods with different sensitivity and specificity used to detect OBI in the published studies, we performed a comprehensive analysis of the studies in which OBI was identified by the detection of HBV DNA in the liver tissue, but the results remained conflicting. In fact, regarding the progression to cirrhosis, we have only three studies, two from the same Italian group[31,69] showing a higher rate of patients with cirrhosis in CHC with OBI than in those without, and one from another Italian group[44] showing no difference. Regarding the development of HCC, six studies were analyzed, three of which were from the same Italian research group[39,40,69], showing a higher rate of patients with HCC in the group of patients with OBI than in those without, whereas the other three studies, one from Japan[41] and two from the United States[42,43], showed no difference. The selection criteria were certainly different from one study to another, but the methods to detect HBV DNA in the liver were similar, albeit not identical. Therefore, the question whether OBI might influence the natural course of CHC remains unanswered.

A strong contribution to defining the clinical impact of OBI could come from a prospective international study considering a large number of HBsAg-negative patients with CHC selected with pre-established criteria and using as sign of OBI the detection of HBV DNA in the liver tissue performed with a highly sensitive technique in a single, high standard laboratory.

No standardized strategy, at least to our best knowledge, is at present recommended for the management of OBI in patients with CHC. In particular, because of the uncertainty surrounding the clinical impact of OBI, it is not clear whether close monitoring is an adequate measure or whether the administration of an anti-HBV nucleot(s)ide to prevent both the progression of fibrosis and the onset of HCC is necessary. In this case, the low cost of the anti-HBV nucleoside lamivudine, which is now obsolete in other HBV treatment settings because of its low genetic barrier and the consequent high risk of inducing viral resistance, might be the drug of choice to suppress the low level of HBV replication characterizing OBI.

In conclusion, some studies indicate that OBI unfavorably affects the progression of liver fibrosis and the development of HCC in patients with CHC, an observation not confirmed in other investigations. The data from prospective studies applying a careful selection of patients and a highly sensitive, standardized method to identify HBV DNA in the liver tissue may help clarify this important issue.