We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.
Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.
Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.
Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.
Occult hepatitis B virus (HBV) infection is defined as the presence of low HBV DNA levels in the blood, liver tissue, or peripheral monocytes of hepatitis B surface antigen (HBsAg)-negative persons.
Occult HBV infection is divided into seropositive (anti-HBc (IgG) positive), and seronegative (anti-HBc (IgG) negative) cases. It has been reported that the rate of occult infection is higher in seropositive cases. Additionally, the rate of occult HBV infection is higher in areas of high HBV prevalence
In acute hepatitis B, disappearance of HBsAg indicates clearance of the virus and, thus, the absence of hepatitis. However, even after the disappearance of HBsAg and appearance of anti-HBs, low-level viremia may persist.
It remains controversial as to whether HBV occult infection has any clinical significance. However, several reports suggest the clinical relevance of occult HBV infection: its contagiousness, reactivation of occult HBV infection, linkage of occult HBV infection to chronic liver diseases and hepatocellular carcinoma (HCC), and, finally, its potential effect on the progression of liver disease.
In this study, the frequency of occult HBV infection in the liver tissue of anti-HCV positive patients was studied, and the effect of occult HBV infection on the severity of hepatitis C disease were investigated.
This study included 71 subjects with chronic liver disease who were HBsAg negative. We performed liver biopsy for the diagnosis of underlying liver disease. Of them, 18 were anti-HBs-negative and anti-HBc-positive, and 34 were anti-HBc-positive and anti-HBs-positive. The remaining 19 were anti-HBc-negative, of which 10 were anti-HBs-positive and nine were anti-HBs-negative (
Serum HBsAg and anti-HBc (IgG) was measured using the HBsAg Kit® and the Anti-HBc Kit® (Beijing North Institute of Biological Technology, Beijing, China), and anti-HBs was measured by the radioimmunoassay method using the Hepatitis B surface antigen 125I (human subtypes ad and ay) AUSAB® kit (Abbott Laboratories, Abbott Park, IL, USA).
Liver biopsies were conducted using the ACECUT® Automatic Biopsy System (TSK laboratory, Tochigi-Ken, Japan). We used an 18-gauge diameter needle and collected 5-mg specimens 15 mm in length. Biopsies were stored at below -70℃ until required. Specimens were sent for histopathological assessment by the Pathology Department. Intrahepatic HBV DNA was extracted using a QIAamp® DNA Micro Kit (Qiagen Gmbh, Germany). Extracted HBV DNA was quantified by the real-time PCR method, using a LightCycler® instrument (Roche Diagnostics, Germany) and RealArt™ HBV LC PCR reagent (Artus, Germany). According to the manufacturer, the assay detection limit was 3.5 IU/mL (18 copies/mL) and its specificity was assessed using a panel of 100 HBV-negative serum samples. Intrahepatic HBV DNA concentration was expressed as the number of viral DNA copies per 1 mg liver tissue. To verify the reliability of intrahepatic HBV DNA quantification, we measured the blood HBV DNA concentration of six HBsAg-positive cases using a Cobas Amplicor HBV Monitor™ kit (Roche Molecular Systems, Pleasanton, CA, USA) and examined its correlation with the intrahepatic concentration. This study was approved by the local Institutional Review Board and was conducted in accordance with the principles set forth in the Declaration of Helsinki.
We classified pathological findings on chronic hepatitis in patients with chronic hepatitis C into minimal, mild, moderate, and severe chronic hepatitis, according to Scheuer's scoring system,
Data are expressed as the mean ± standard deviation, or n (%) as appropriate. When comparing the baseline characteristics of patients between different groups, chi-square test and Fisher's exact test were used for categorical data, and the Student
Intrahepatic HBV DNA was detected in 23 of the 71 (32.4%) HBsAg-negative subjects. HBV DNA detection frequency was highest (9/18; 50%) in the anti-HBc (+) anti-HBs (-) group, and was detected in 10/34 (29.4%) of anti-HBc (+) anti-HBs (+) cases. Therefore, intrahepatic HBV DNA was detected in 19/52 (36.5%) of anti-HBc-positive subjects regardless of anti-HBs status (
Intrahepatic HBV DNA concentrations were 43.6±75.2, 1044.8±4224.8 and 189.1±511.8 copies/mg in the anti-HBc (+) anti-HBs (-), anti-HBc (+) anti-HBs (+) and anti-HBc (-) groups, respectively. There was no statistically significant difference between them (
Intrahepatic HBV DNA was detected in 9/32 (28.1%) of anti-HCV-positive subjects and 14/39 (35.9%) of others, suggesting that occult HBV infection was not more common in anti-HCV positive patients (
Intrahepatic HBV DNA was detected in 9 anti-HCV positive patients. We divided the 32 anti-HCV-positive individuals into 2 groups according to the presence of intrahepatic HBV DNA, and compared gender, age, serum ALT, histological activity and the frequency of primary liver cancer between the two groups. The mean fibrosis score was 3.6 in intrahepatic HBV DNA-positive and 3.2 in intrahepatic HBV DNA-negative subjects (
We compared the hepatic histological activities of the 32 cases of chronic hepatitis C according to the detection of intrahepatic HBV DNA. When they were divided into mild (1-3 point), moderate (4-6), and severe (7-9) groups, no significant difference was observed in the detection frequency of intrahepatic HBV DNA between them (
We used six HBsAg-positive subjects as positive control. In these subjects, blood HBV DNA concentrations varied from negative (below 18 copies/mL) to 1.98×107 copies/mL. Intrahepatic HBV DNA was detected in all six subjects at concentrations that varied between 105 to 109 copies/mg. Additionally, intrahepatic HBV DNA concentrations were closely correlated with those in blood (
The frequency of occult HBV infection may differ geographically, depending on HBV prevalence. Reported occult HBV infection frequencies have been as high as 70-90% of anti-HBc-positive individuals in areas of high prevalence but as low as 5-20% in areas of low prevalence.
The frequency of occult HBV infection differed according to which subjects received the test. It was reported that the frequency of occult HBV infection was as high as 30% when the subjects were patients with a chronic liver disease of unknown cause.
Additionally, it has been reported that liver cirrhosis in chronic hepatitis C patients is more common in those with occult HBV infection
On the other hand, some studies have reported no difference in serum ALT levels, histological inflammatory activity, or degree of fibrosis between patients with chronic hepatitis C accompanied by occult HBV infection and those infected with HCV alone.
Blood HBV DNA concentration in HBsAg-positive subjects was high (104 to 108 copies/mL), but in those with occult HBV infection patients it was below 102 copies/mL3. This suggests that many occult HBV infection patients would be serum PCR-negative. As such, use of liver tissue (as in the present study) may be more helpful in detecting occult HBV infection. According to one report, intrahepatic HBV DNA was detected in 37/98 (37.7%) of serum HBV DNA-negative cases by PCR.
The reason for the increasing interest in occult HBV infection is the increasing evidence of its clinical relevance. This can be divided into four categories: contagiousness of occult HBV infection, reactivation of occult HBV infection, its link to chronic liver diseases, and its link to hepatocellular carcinoma.
It is well-known that HBV infection can be excluded in anti-HBc-negative individuals, but in the present study, interestingly, intrahepatic HBV DNA was detected in some anti-HBc-negative subjects. In such cases, the possibility of false positivity may need to be considered, but according to one report,
In conclusion, this study found that intrahepatic HBV DNA was commonly detectable in HBsAg-negative subjects, and that a positive anti-HBc reaction is an important factor related to occult HBV infection. However, the frequency of occult HBV infection was not affected by chronic hepatitis C infection, and the effect of occult HBV infection on the disease severity of hepatitis C was not significant.
anti-HBc (IgG) antibody
anti-HBs antibody
hepatitis B surface antigen
hepatitis B virus
hepatocellular carcinoma
hepatitis C virus
human immunodeficiency virus
liver cirrhosis
The overall frequency of detection of intrahepatic HBV DNA was 32.4% in all of the specimens and 36.5% in anti-HBc-positive subjects. The frequency of intrahepatic HBV DNA detection did not differ significantly between the three groups. The frequency was highest (50%) in group A, followed by 29.4% in group B and 21.1% in group C. *Statistically significant when
HBV DNA levels were low in all subjects and did not differ significantly with anti-HBc or anti-HBs status. *Statistically significant when
Histologic grade or stage scores did not differ significantly with HCV status (i.e., HBV DNA-positive vs. -negative). *Statistically significant when
Correlation between intrahepatic HBV DNA and serum HBV DNA in six HBsAg-positive subjects. *Correlation coefficients; Spearman's correlation.
Clinical characteristics of the patients according to the serologic HBV status
Statistical significance test was done by Chi-square test* and Kruskal Wallis test†.
cAb, anti-HBc (IgG) antibody; sAb, anti-HBs antibody; HCV, hepatitis C virus; ALT, alanine aminotransferase.
Characteristics of the patients according to anti-HCV positivity
Statistical significance test was done by Chi-square test* and
HBV, hepatitis B virus; HCV, hepatitis C virus; ALT; alanine aminotransferase; LC, liver cirrhosis.
Characteristics of anti-HCV-positive subjects according to intrahepatic HBV DNA positivity
Statistical significance test was done by Chi-square test* and Mann-Whitney
HBV, hepatitis B virus; ALT, alanine aminotransferase.