High prevalence of occult hepatitis B in Baltimore injection drug users



Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in a serum or liver in the absence of hepatitis B surface antigen (HBsAg). The prevalence and clinical correlates of occult hepatitis B remain incompletely defined. A cross-sectional study was performed to determine the prevalence of occult hepatitis B in a high-risk cohort composed of 188 injection drug users in Baltimore, Maryland. All individuals had chronic hepatitis C viral infections confirmed by RNA detection and liver biopsy. Serologic assays for HBsAg and core antibody (HBcAb) were performed. Serum HBV DNA was detected using the COBAS HBV AMPLICOR monitor assay (lower limit of detection, 200 HBV copies per milliliter) and a semi-nested polymerase chain reaction (PCR) assay (lower limit of detection, 15 HBV copies per milliliter). Although almost all individuals (96%) were anti-HBC positive, only 8 of 188 (4%) were HBsAg positive. Occult hepatitis B was not identified using the COBAS assay, but was found in 81 of 180 (45%) of individuals using semi-nested PCR. Of the 8 HBsAg positive individuals, HBV DNA was found in 1/8 using the COBAS assay and 6/8 using the nested PCR assay. Overall, liver disease was mild, with a median serum alanine aminotransferase (ALT) of 38 IU/L, median activity grade of 3/18, and median fibrosis stage of 1/6. No association was found between the serum AST (aspartate aminotransferase), activity grade, or stage of liver disease and the presence of occult hepatitis B. Serum ALT levels were slightly higher in patients without occult hepatitis B (46 vs. 35 IU/L), and the median years since first injection drug use was somewhat longer in those without occult hepatitis B (24 vs. 20 years). In conclusion, although further research is needed to assess its clinical significance, there is a high prevalence of occult HBV infection in this cohort of HCV-infected injection drug users. (HEPATOLOGY 2004;39:51–57.)

Worldwide, hepatitis B virus (HBV) infection is the primary cause of cirrhosis and hepatocellular cancer (HCC) and is 1 of the 10 leading causes of death.1 After acute HBV infection, most adults appear to recover, because viral proteins or DNA are typically not detectable in their blood2–4 and they generally are not considered to be at risk for disease. In other individuals, HBV infections persist, and at least three distinct clinical states of viral persistence have been defined based on serologic findings in adults who acquire chronic HBV infection: (1) replicative phase, (2) nonreplicating or low-replicating phase, and (3) the more recently defined occult hepatitis B. In addition, individuals infected in infancy have an initial immune tolerance phase.

Chronic hepatitis B is defined by the repeated detection of hepatitis B surface antigen (HBsAg) for 6 or more months after acute infection.2–4 Chronic hepatitis B is associated with higher serum levels of HBV DNA, high levels of hepatitis B circulating e antigen (unless the virus has mutations that prevent e protein expression), a high risk of viral transmission, elevated liver enzymes, liver inflammation, and the highest risk of cirrhosis and HCC.3, 5 In contrast, in the silent carrier state, HBsAg remains detectable in sera, but there are lower levels of serum HBV DNA, hepatitis B e antibody (HBeAb) is present, hepatitis B circulating e antigen is not detected, and liver enzymes are repeatedly normal (or minimally elevated); such individuals are at lower risk for progression to cirrhosis or HCC.6, 7 The final viral persistence state, occult hepatitis B, more recently has been identified using sensitive polymerase chain reaction (PCR) assays that detect low levels of HBV DNA in the sera and/or liver of persons who are HBsAg negative.5 Occult hepatitis B infection has been documented in a number of patient subgroups, including those with hepatitis C viral (HCV) infection, human immunodeficiency virus (HIV) infection, hepatocellular carcinoma, and cryptogenic advanced liver fibrosis.5 It is not clear whether occult hepatitis B is common in urban settings in the United States and whether it is associated with advanced liver disease. Therefore, we assessed the prevalence and clinical significance of occult hepatitis B in a community-based group of HCV-infected injection drug users with available liver biopsies who were at high risk for all forms of chronic HBV infection.


HBV, hepatitis B virus; HCC, hepatocellular cancer; HBsAg, hepatitis B surface antigen; HBeAB, hepatitis B e antibody; PCR, polymerase chain reaction; HCV, hepatitis C viral; HIV, human immunodeficiency virus; ALT, alanine aminotransferase; HBcAb, hepatitis B core antigen; PCR, polymerase chain reaction; AST, aspartate aminotransferase.

Patients and Methods

Persons Studied.

Between 1988 and 1989, a cohort was recruited in Baltimore, Maryland, of persons who had injected illicit drugs in the preceding 10 years, were more than 17 years of age, and were free of manifestations of acquired immunodeficiency syndrome.8 Within this cohort, a subset of 1667 individuals was identified as the HCV subcohort because they had antibodies to HCV and at least one follow-up visit.9 Between 1996 and 1998, 210 individuals from the HCV subcohort were enrolled in a study through which liver biopsies were performed to ascertain the magnitude of clinically inapparent liver disease (Fig. 1).10 The 210 persons in the biopsy subgroup were similar to those in the overall HCV subcohort who were not biopsied in terms of age, gender, race, and drug use. However, the biopsied group had slightly higher serum alanine aminotransferase (ALT) levels (median, 37 IU/L vs. 32 IU/L) and a lower frequency of HIV infection (28% vs 49%), reflecting entry criteria that excluded persons with advanced HIV (CD4 count less than 200 cells/mm3) and enriched sampling for persons with persistently elevated ALT values.10 Overall, 18.8% of the entire cohort and 27.4% of the biopsy subgroup had ALT levels of 60 IU/L (1.5 times the upper limit of normal) or more. Seventy-six percent of those not in the biopsy subgroup were hepatitis B core antibody (HBcAb) positive. The clinical and histologic findings in this biopsy subgroup have been reported previously in detail.10 Twenty-two of the 210 individuals in the biopsy substudy eventually cleared their HCV infections, whereas 188 did not. In this study, we retrospectively examined the sera of the 188 individuals positive for HCV RNA for the presence of occult hepatitis B. Because of the scarcity of serum, those who cleared HCV RNA were not further studied. HCV RNA viral load was measured by Cobas Amplicor Monitor Assay (Roche Molecular Diagnostics, Branchburgh, NJ). HCV genotype information was not available in most cases and was not studied further.

Figure 1.

The study group of 188 HCV-infected individuals was derived from a larger cohort of HCV-infected injection drug users who underwent liver biopsy.

All participants in the cohort were offered hepatitis A virus and HBV vaccinations if they were not immune. Participants provided consent for all aspects of this investigation using protocols approved by the Institutional Review Board of the Johns Hopkins Schools of Public Health and Medicine.

Serologic Analyses.

Antibodies to hepatitis B core antigen (anti-HBC; total immunoglobulin M and immunoglobulin G) were detected using the Abbott Corzyme immunoassay (Abbott Laboratories, Abbott Park, IL), whereas HBsAg was detected using the Abbott Auszyme immunoassay (Abbott Laboratories). Both assays were used as per the manufacturer's recommendations without modifications. Overall, the median time interval from acquisition of the tested sera to liver biopsy was 2.2 months, with 95% of serum samples collected within 6 months of the liver biopsy. We did not investigate for hepatitis B surface antibodies because HBV vaccination status at the time of the biopsy was unknown in many individuals.

HBV DNA Detection.

Serum HBV DNA was assessed first using the Roche COBAS HBV AMPLICOR monitor assay (COBAS-MA; Roche Molecular Diagnostics, Branchburgh, NJ) and second using a semi-nested polymerase chain reaction (PCR) assay. Briefly, the COBAS system is based on a single round of PCR using biotin-labeled primers that targets a conserved region of the core-precore region. As the primers are incorporated into the amplicon, the amount of fluorescence increases proportionately to the amount of product. During sample preparation, a known amount of a noninfectious 2,766-bp linearized plasmid is added to each sample and is referred to as the quantitation standard. This plasmid has primer binding regions that are identical to the conserved region of the core-precore region, but has a unique probe capture site that allows separation from hepatitis B genome amplicons. The ratio of the quantitation standard to that of the hepatitis B amplicon is then used to calculate the number of hepatitis B copies per milliliter. The lower limit of detection is 200 HBV genome copies per milliliter (roughly equivalent to 33 IU), although in some samples, HBV DNA can be detected, but not quantified, when levels of less than 200 genome copies per milliliter are present.

For the semi-nested PCR assay, genomic DNA was extracted from 200 μL of serum or plasma using the QIAmp DNA Blood Mini Kit (Qiagen, Valenica, CA) and resuspended in 100 μL of elution buffer or water. Ten micrograms of salmon sperm DNA per sample was used as a carrier in the extraction process, which significantly enhanced detection of DNA at low copy numbers in our laboratory. Thirty-three microliters of the extracted DNA was then used for first-round PCR in a 50-μL reaction using the S1 and S1R primers and cycling conditions described by Hou et al.11 to amplify a 689-bp conserved region of the HBV surface gene. This was followed by a second round of PCR using S1 and P29 primers with 5 μl of sample from the first round PCR as template, yielding a final amplicon of 672 bp.

To evaluate the sensitivity of the seminested assay, a commercial source of human plasma with complete HBV particles (Accurun 325, series 600; Boston Biomedica, West Bridgewater, MA) was diluted serially (Base matrix diluent; Boston Biomedica, West Bridgewater, MA), extracted as described above, and amplified. The assay routinely detected down to 1 copy per PCR reaction, equivalent to 15 HBV genome copies per milliliter. This was then repeated with two patient samples from individuals not in this study who were HBsAg positive and had HBV DNA levels quantitated using the COBAS HBV MONITOR assay. With these two patient samples, the seminested assay detected to 3 copies per reaction, equivalent to 45 HBV genome copies per milliliter. Thus, the seminested assay in our laboratory detected as low as 15–45 HBV genome copies per milliliter (roughly equivalent to 2.5–7.5 IU).

To prevent sample degradation as a result of frequent freeze and thaws, the samples were pre-aliquoted. To avoid contamination, sample preparation, PCR setup, and PCR amplification steps took place in separate areas. Eight-strip PCR tubes with individual caps also were used to avoid contamination (Eppendorf, Westbury, NY). Typically, one water control was included per eight samples, and these were negative in all cases. Amplicons were detected by electrophoresis in 1% agarose gels with ethidium bromide and, when possible, were sequenced directly and compared with others as an added precaution against contamination.

Liver Biopsies.

Percutaneous liver biopsy specimens were obtained by a hepatologist using a 16-gauge needle. The specimens were then fixed in formalin and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin and Masson's trichrome. All of the tested serum and plasma samples were obtained within 6 months of the liver biopsies.

Biopsy specimens were graded and staged using the Modified Hepatic Activity Index (also known as “Ishak”).12 In the Modified Hepatic Activity Index system, inflammation is scored on an ordinal scale of 0 through 4 for portal inflammation, interface activity, and lobular inflammation and a scale of 0 through 6 for confluent necrosis. The individual components are then summed for a maximum score of 18. In our patient population, confluent necrosis is essentially nonexistent, so the effective maximum score is 12. The fibrosis is staged on an ordinal scale of 0 to 6, with categories for no fibrosis (0), portal fibrosis (1, 2), bridging fibrosis (3, 4), and incomplete and established cirrhosis.5, 6 All biopsies were staged and graded by a single liver pathologist (M.T.) who was blinded to all serologic and clinical information.

Statistical Analysis.

Medians were compared using the Wilcoxon rank-sum test. Proportions were compared with chi-square tests. SYSTAT version 10 (SPSS 2000, Richmond, VA) was used for data analysis.


Of the 188 study participants, 180 (96%) were anti-HBC positive, whereas HBsAg was detected in just 8 of 188 (4%). Using the COBAS assay, HBV DNA was detected in a single HBsAg-reactive individual who had an HBV DNA level of less than 200 copies per milliliter. HBV DNA was not detected in individuals nonreactive for HBsAg with the COBAS assay. In contrast, HBV DNA was found with seminested PCR in 6 of 8 individuals (75%) reactive for HBsAg and 81 of 180 (45%) individuals who were HBsAg nonreactive (Fig. 2). Direct sequencing of the 689-bp amplicon was accomplished in 37 cases (47%) of occult hepatitis B and in three cases of HBsAg-positive hepatitis B. Based on sequence analysis,13 all cases were genotype A. Alignment of the protein sequences of this region shows many changes, but no strong clustering in the major hydrophilic region of the hepatitis B S gene (Fig. 3). Base pair changes were noted in both those with and without HBsAg. Although the number of sequenced cases with HBsAg were small, there was no apparent difference in the number or location of base pair changes.

Figure 2.

A Venn diagram showing the relationship between HBsAg reactivity and HBV DNA positivity by COBAS MONITOR assay and seminested PCR.

Figure 3.

HBV surface protein sequence changes in 22 cases. The consensus sequence and numbering is from Stuyver et al.13 As per Pumpens et al.,38 the locations of five antigenic regions are shown by solid bars and the locations of two antigenic loops in the a determinant are indicated by boxes.

The 81 persons with occult hepatitis B were similar to the 99 HBV DNA-negative individuals in terms of age, race, and gender (Table 1). The overall study group showed mild elevations in serum transaminase levels, with a median serum ALT of 38 IU/L and aspartate aminotransferase (AST) of 38 IU/L. The mild biochemical abnormalities were accompanied by mild liver disease on biopsy: median activity grade of 3/12, and median fibrosis stage of 1/6. Statistical analysis revealed no differences between those with and without occult hepatitis B in terms of serum AST, total activity grade, individual grade components (interface activity, portal inflammation, and lobular inflammation), fibrosis stage, and HIV seropositivity (all P > .05). However, compared with those with occult hepatitis B, the median serum ALT level (Fig. 4) was higher in patients without occult hepatitis B (46 vs. 35 IU/L; P = .015) and the median number of years since first injection drug use, a reasonable surrogate of the duration of HBV infection, was longer (24 vs. 20; P = .014). The HCV viral load did not differ significantly between those with and without occult HBV, with means of 6.8 and 6.7 million copies/mL, respectively (P = .11, Kruskal-Wallis).

Table 1. Demographic Information, Serologic Findings, and Liver Biopsy Findings
 Total Group (N = 188)Occult HBV Negative (n = 99)Occult HBV Positive (n = 81)HBsAg Positive (n = 8)
  • *

    Stage 0 or 1.

  • Stages 3–6.

Years of age (median)42434144
 M150 (80%)86 (86%)58 (72%)6 (75%)
 F38 (20%)13 (13%)23 (28%)2 (25%)
 Black179 (95%)94 (95%)78 (96%)7 (88%)
 Nonblack9 (5%)5 (5%)3 (4%)1 (12%)
HIV seropositive57 (30%)30 (30%)24 (29%)2 (38%)
HBVcAb seropositive181 (96%)96 (96%)78 (96%)7 (88%)
No. years since first injection drug use (median)22241923
ALT/AST in IU/L (medians)38/3846/3834/3738.5/58
MHAI grade (median)3334
MHAI stage (median)1110.5
No. with no or mild fibrosis*123 (65%)61 (61%)55 (68%)7 (88%)
No. with advanced fibrosis20 (11%)11 (11%)8 (10%)1 (12%)
Figure 4.

Median ALT levels were slightly higher in those without occult HBV.


In this cohort of HCV-infected injection drug users, we found a very high prevalence of HBV exposure as evidenced by positivity for HBcAb. Interestingly, this high rate of exposure was coupled with a very low prevalence of typical HBsAg-positive infections (4%) and, in contrast, a very high prevalence of occult hepatitis B infections (45%). In both the occult hepatitis B infections and the HBsAg-positive infections, HBV DNA levels were low. Thus, in this population of injection drug users with chronic HCV infections, chronic HBV infections are often HBsAg negative and are characterized by very low DNA levels.

Some interesting parallels exist between our findings and those reported by McMahon et al.14 in Alaskan Natives, another high-risk group for chronic HBV infection that has predominately horizontal viral transmission during childhood and early youth. In a population-based study of 52,000 individuals, they found a prevalence of 3.1% HBsAg positivity. They further reported a prevalence of occult hepatitis B of 52% in a subcohort of 33 individuals tested a median of 5 years after HBsAg clearance.15

Worldwide, the prevalence of occult hepatitis B seems to be influenced strongly by risk factors for typical chronic HBV infection.5, 16 Occult hepatitis B infection has been identified in individuals with HCC, those with chronic HCV infections who are also positive for HBcAb, and those with cryptogenic advanced liver fibrosis.5, 16 Given their high risk of chronic hepatitis B infection, it is not surprising that injection drug users also are at high risk for occult hepatitis B.

All of the sequenced cases in this study were genotype A. Genotype A also was reported in 5 of 13 injection drug users with typical HBsAg-positive chronic hepatitis B from Seattle, Washington.17 In contrast to Hou et al.,11 we did not find an increased frequency of amino acid changes in the major hydrophilic region of the S gene, a difference that may reflect the differences between HBV infection in this cohort and those from other regions of the world. This observation underscores the possibility that additional findings in this study may not be applicable broadly to other groups with occult hepatitis B infection.

Regarding the clinical significance of occult hepatitis B infection, no association was found in this study between the presence of occult hepatitis B and serum AST level, or grade and stage of liver disease on biopsy. There was a statistically significant difference in serum ALT levels, although it is of questionable clinical relevance, given the small magnitude of the difference, and was, in fact, lower in those with occult hepatitis B. Others have reported similar findings, with no observable differences in the amount of liver inflammation and fibrosis between those with and without occult hepatitis B,18 whereas some have found the opposite and have shown occult hepatitis B to be associated with greater inflammation19, 20 and fibrosis on liver biopsies21 and with advanced cryptogenic fibrosis.22 To date, most of the information on the clinical significance of occult hepatitis B has come from cross-sectional studies. Longitudinal studies likely will be important in clarifying the clinical significance of occult hepatitis B. In particular, paired biopsy studies may well be necessary to understand the association between occult hepatitis B and liver fibrosis progression.

One limitation to our results is the overall mild liver disease seen on liver biopsy, which may have obscured any relationship between occult hepatitis B and inflammation or fibrosis. The mild liver disease seen in this study is partly a reflection of the enrollment criteria for the liver biopsy cohort, which specifically enriched for those with no or mild ALT elevations and excluded those with concomitant serious medical conditions that may have increased the risk for fibrosis, such as those who were inebriated and those with advanced HIV-related immunosuppression. Our study is also limited by the small number of cases with HBsAg-positive chronic hepatitis B, because we were not able to compare the degree of liver disease between those with HBsAg-positive hepatitis B and those with occult hepatitis B.

In the literature, most individuals with occult hepatitis B are anti-HBC and/or anti-HBS reactive, although up to 20% of individuals with occult hepatitis B can be negative for both anti-HBC and anti-HBS.5 In our study, 96% of individuals were HBcAb positive. An individual's HBV antibody status likely reflects the time course of the original infection as well as episodes of re-exposure. In fact, in a study by Komori et al.23 of 15 patients with chronic HBV infection followed up for a median of 4.4 years after HBsAg clearance, serum HBV DNA was no longer detectable in 13 of 15 individuals, but HBV DNA was found in the liver of all tested patients. Most individuals retained their HBcAb positivity, although titers declined over time. Thus, the serologic findings associated with occult hepatitis B in our study may reflect the persistence of HBcAb following a more typical chronic HBV infection, perhaps with enhanced persistence or even new subclinical infections resulting from episodes of re-exposure.

In this study, occult hepatitis B required nested PCR for detection, and positive cases seemed to have low copy numbers as indicated by negative COBAS-MA testing, results similar to that reported by others.24 Although the reasons for this are not clear, the actual HBV genome copy numbers are not necessarily less than 200 (lower limit of detection of COBAS-MA), because other factors may have played a role, such as the increased coefficient of variation of the COBAS assay at very low HBV DNA copy numbers.25, 26 In addition, there are other potential factors that have not been investigated. In this regard, the lower limit of detection for the COBAS-MA assay has typically been studied using dilutions of plasmids containing wild-type HBV genome.25, 26 In occult HBV, however, frequently there are large numbers of base pair changes that may alter both the primer and hybridization sites of the COBAS-MA assay. In addition, low levels of PCR inhibitors may have little notable effect with abundant HBV copy numbers, but may reduce the detection of occult HBV in some cases. The nearly doubling of PCR cycles in the nested assays then may partially offset low-level inhibitors. PCR sensitivity also depends on the amount and quality of starting serum and DNA extraction methods. Optimizing both of these components likely enhanced our ability to detect low levels of HBV DNA.

We did not investigate mechanisms that may explain the low HBV viral replication rates in this population. However, HCV viral replication has been shown to inhibit HBV replication in a number of experimental and epidemiologic studies, providing one possible explanation for the low HBV replicative rates in this population. For example, HCV core proteins suppress HBV replication by two- to fourfold in cell culture,27 and HCV coinfection reduces HBV core and surface antigen expression in the liver.28 None of the patients in this study had undergone antiviral therapy for either HCV or HBV before the study visits, excluding this as a confounding factor. Others have identified viral mutations that may impair viral replication, particularly mutations in the X gene29 and core promoter region.30 In addition, the host immune response seems to play a role, because immunosuppression from cytotoxic therapy31, 32 and solid organ transplantation33–35 can lead to reactivation of occult hepatitis B, with re-emergence of surface antigen expression and hepatitis. In this regard, we did not find a difference in the prevalence of occult hepatitis B between those seronegative and seropositive for HIV, although the selection criteria used for the biopsy subgroup may have biased the results by excluding those individuals with advanced HIV disease. Finally, reports of detectable HBV DNA years after recovery from acute hepatitis B infection36, 37 suggest the possibility that hepatitis B may persist after infection without causing disease similarly to other DNA viruses, such as cytomegalovirus and Epstein-Barr virus.

In conclusion, in this urban community-based cohort of injection drug users, there is a high prevalence of persistent HBV infection that is characterized by low levels of viral replication, with most infections falling into the category of occult hepatitis B. This high prevalence of occult hepatitis B likely reflects the nearly universal exposure to hepatitis B in this population. No association was found between occult hepatitis B and serum AST or histologic markers of disease severity, although the overall mild liver disease in this cohort may have limited this study's ability to detect such an association.