Journal of Viral Hepatitis, 2004, 11, 358–365
Occult hepatitis B virus infection in Greek patients with chronic hepatitis C and in patients with diverse nonviral hepatic diseases* S. P. Georgiadou,1 K. Zachou,1 E. Rigopoulou,2 C. Liaskos,1 P. Mina,1 F. Gerovasilis,3 E. Makri2 and G. N. Dalekos1,2 1Research Laboratory of Internal Medicine, 2Academic Liver Unit, Department of Internal Medicine, Medical School, University of Thessaly Larissa, Thessaly; and 3Gastroenterology Division at General Hospital of Volos, Magnesia, Greece Received September 2003; accepted for publication October 2003
SUMMARY. Occult hepatitis B virus (HBV) infection has been reported in patients with chronic hepatitis C who are negative for HBV surface antigen (HBsAg). However, the significance of ‘silent’ HBV in hepatitis C virus (HCV) infection is unknown. We investigated 540 subjects for the presence of occult HBV in Greek HCV patients, patients with nonviral liver diseases and healthy donors in an attempt to determine the frequency and importance of this phenomenon. One hundred and eighty-seven anti-HCV(+)/HBsAg()) patients’ sera were investigated for the presence of HBV-DNA by polymerase chain reaction. Two hundred and eighty-two selected blood donors (positive for antibodies to HBV core antigen) and 71 patients with various nonviral hepatic diseases consisted the control groups [both controls were anti-HCV())/HBsAg())]. HBV-DNA was detected in 26.2% of HCV-infected patients vs 8.5% of patients with nonviral
INTRODUCTION Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are considered to be the most common causes of liver disease worldwide. Both viruses are transmitted parenterally and they share similar risk factors for transmission. As a consequence combined HBV and HCV infection is quite frequent, especially in HBV endemic areas [1–3]. Abbreviations: c-GT, c-glutamyl-transpeptidase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, Aspartate aminotransferase; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus. *Preliminary parts of this work have been presented in abstract form in the Biennial Meeting of the IASL, Madrid, April 15–16, 2002. Roche Pharmaceuticals has partially supported this work. Correspondence: Georgios N. Dalekos, Academic Liver Unit and Research Laboratory of Internal Medicine, Medical School, University of Thessaly, Papakiriazi 22 str, 41222 Larissa, Greece. E-mail:
[email protected]
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diseases (P ¼ 0.003) and 0/282 of donors (P ¼ 0.0000). HBV-DNA was neither associated with HBV markers, nor with the clinical status of HCV and nonHCV patients. Neither epidemiological, histologic and virologic data nor the response to therapy were associated with the HBV-DNA detection. Hence one quarter of HCV-infected patients had occult HBV infection. Similar findings were not found in both control groups. Occult HBV infection in Greek patients with chronic hepatitis C does not seem to modify the progression of chronic liver disease. Further studies of longer duration are needed in order to clarify the role of ‘silent’ HBV infection in HCV-infected patients. Keywords: chronic hepatitis C, HBV-DNA, hepatitis B virus, hepatitis C virus, nonviral hepatic diseases, occult HBV infection.
Overt HBV and HCV coinfection has been reported to be associated with a more severe liver disease, increased frequency of hepatocellular carcinoma and resistance to ainterferon (a-IFN) therapy [2–7]. Several studies have also demonstrated the presence of HBV-DNA in the sera and/or liver of patients who lack serologic markers for HBV and HCV, and patients with and/or cryptogenic hepatitis, cirrhosis or hepatocellular carcinoma with an incidence ranging from 14 to 85% [8–13]. Furthermore, the reported detection rate of HBV-DNA by polymerase chain reaction (PCR) in the sera and/or liver of patients with hepatitis C who were negative for hepatitis B surface antigen (HBsAg) varies widely (0–90%) [1, 4, 14–23]. Occult HBV infection is characterized by undetectable serum HBsAg but detectable HBV-DNA in serum or liver [24]. So far, data on the prevalence and clinical significance of occult HBV infection in patients with chronic hepatitis C are missing in our country. In addition, the clinical impact of occult HBV on HCV-related disease remains controversial [4, 13–16, 18–20, 25–30]. For
Occult HBV in HCV infection these reasons, we conducted a large study to determine the prevalence and clinical significance of occult HBV infection in 187 consecutive HCV-positive/HBsAg-negative patients from Central Greece, an area with intermediate endemicity for HBV infection. As control groups we investigated 71 patients with diverse nonviral chronic liver diseases (disease control group) and 282 HBsAg-negative/ antibodies to HBV core antigen (anti-HBc)-positive healthy blood donors (healthy control group). In order to explore the possible clinical impact of occult HBV infection on HCV-related disease, the presence or absence of HBV-DNA was associated with epidemiological, clinical, laboratory, virologic, histologic data and HBV serologic markers as well as with the response to antiviral therapy.
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MATERIALS AND METHODS One hundred and eighty-seven consecutive naive patients with well-defined chronic HCV infection and 71 consecutive patients with various nonviral chronic liver diseases (disease control group) were investigated for the presence of HBVDNA using a sensitive commercially available PCR kit (HBV Monitor Cobas Amplicor, cut-off: 200 copies/mL; Roche, Mannheim, Germany). All patients were followed at the Academic Liver Unit, Department of Medicine, University of Thessaly, Medical School [mean ± standard deviation (SD), 24 ± 13.2 months]. The patients’ characteristics are shown in Table 1. Forty-four patients with HCV infection had completed antiviral treatment with a-IFN alone or in
Table 1 Characteristics of patients studied Sex (M/F) Age (mean/range, years) Disease duration (years) Chronic HCV with normal AST, ALT Chronic hepatitis C Cirrhosis Source of HCV infection Transfusion before 1990 Intra Venous drug abuse Chronic haemodialysis Multiple hospitalisations Multiple sexual partners Family member infected Needle-stick accident Unknown Extrahepatic manifestations (Yes/No) Histologic data (Yes/No) Minimal/mild inflammation Moderate/severe inflammation None or mild fibrosis Moderate or severe fibrosis Autoimmune liver diseases Alcoholic liver disease Nonalcoholic steatohepatitis Miscellaneous liver diseases
HCV patients (n ¼ 187)
Disease control group (n ¼ 71)
91/96 46.6/17–83 4.1 ± 4.3 45 109 33
36/35 48.9/15–82 3.7 ± 5.3
12
50 49 2 7 7 9 4 59 7*/180 101/86 54 47 45 56 21 18 16 16à
*Two patients had Sjogren’s syndrome (one with accompanied cryoglobulinemia), two glomerulonephritis, one cryoglobulinemia, one porphyria cutanea tarda and one Sicca syndrome. Eleven patients with autoimmune hepatitis type-1, six with primary biliary cirrhosis and four with primary sclerosing cholangitis. àThree patients with Wilson’s disease, two with Budd–Chiari’s syndrome, one with hepatic peliosis, four with cryptogenic hepatitis, six with undefined cholestatic syndrome. M, male; F, female; HCV, hepatitis C virus; AST, Aspartate aminotransferase; ALT, alanine aminotransferase. Data are given as mean ± standard deviation. 2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 358–365
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combination with ribavirin at the time of this writing. In addition, 282 selected age and sex matched blood donors (healthy control group) HBsAg-negative/anti-HBc-positive [alone or in combination with antibodies to HBV e antigen (anti-HBe) or with antibodies to HBsAg (anti-HBs)] were investigated. Every sample with detectable HBV-DNA by PCR was tested again in another experiment. Only serum samples with repeatedly detectable HBV-DNA were considered positive for HBV-DNA. All subjects consented at the time of interview to participate in the study. The human research review committee of Larissa University Hospital approved the study protocol. The diagnosis of chronic HCV infection was based on clinical, laboratory and histologic evaluation as we described previously [31–33]. Briefly, all patients included in the study met the following criteria: (a) serologic evidence of chronic infection with HCV as determined by the detection of antibodies to HCV (anti-HCV) using a third-generation enzyme immunoassay (Murex Diagnostics, Temple Hill, Dartford, UK) at least twice within 6 months before their enrolment into the study and (b) active virus replication as defined by the detection of HCV RNA using a commercially available qualitative PCR kit (HCV Monitoring Cobas Amplicor, Roche). In addition, certain epidemiologic and demographic factors conducted in the past such as operations, tattoos, injections with syringes in common use and traditional practices (for example acupuncture), sexual history, alcohol abuse, and the medical history were taken into account in HCV-infected patients. The histologic evaluation for inflammation and fibrosis in HCV-infected patients was assessed using the Knodell histologic/activity index score [34, 35]. The inflammation score was obtained by combining scores for the first three components of the Knodell index: portal, periportal and lobular inflammation (range 0–18). The Knodell fibrosis scores were 0 (no fibrosis), 1 (portal fibrosis), 2 (portal fibrosis with few septa), 3 (bridging fibrosis) and 4 (cirrhosis) [34, 35]. According to previous publications from our group [33] and for statistical reasons the patients were divided in two groups (a) according to inflammation: minimal/mild (0–8) and moderate/severe (9–18) and (b) according to fibrosis: none/ mild (0–1) and moderate/severe (2–4). Genotype was available in 107 of 187 HCV-infected patients using a hybridization assay (Inno-Lipa HCV II; Innogenetics, Gent, Belgium). Genotypes were determined according to the Simmods et al. classification [36]. Quantification of HCV-RNA levels was available in 108 patients with HCV infection by a commercially available quantitative PCR Kit (Cobas Amplicor HCV Monitor, Roche cut-off: 600 U/mL). None of the subjects studied was positive for HBsAg and antibodies to HIV (Abbott Laboratories, Wiesbaden, Germany), Venereal Disease Research Laboratory test (VDRL) and direct Coombs test. All serum samples were stored at )70 C until tested. Aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), c-glutamyl-transpeptidase (c-GT), albumin, c-globulins and platelets were determined using standard automated techniques. Serologic markers of HBV infection were also determined using standard third generation commercially available EIAs (Abbott GmbH Diagnostika, Wiesbaden-Delkenheim, Germany).
Statistical analysis All statistical calculations were performed on a Microsoft computer, using SPSS software version 10.0 (SPSS BI, Athens, Greece). Results are expressed as mean ± SD. Data were analysed by unpaired t-test, v2 (two by two with Yates’ correction) and Fisher’s exact test where applicable. Multivariate logistic analysis was used after taking into account well-defined variables (e.g HCV genotype, viral load), which may interfere in the assessment of treatment response of 44 patients who had completed antiviral treatment at the time of this writing. A two-sided P-value 0.05]. Similar findings were recorded in
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HCV patients
Disease control group
HBV-DNA HBV-DNA ()) HBV-DNA HBV-DNA (+) (n ¼ 49) (n ¼ 138) (+) (n ¼ 6) ()) (n ¼ 65) Negative markers (%) Anti-HBc (+) only (%) Anti-HBs (+) only (%) Anti-HBc (+)/ Anti-HBs(+) (%) Anti-HBs (+) (%) Anti-HBc (+) (%) Anti-HBe (+) (%)
53.1 12.2 16.3 10.2
49.3 10.9 13.8 13.8
50.0 16.7 0.0 0.0
58.5 9.2 13.8 9.2
32.7 30.6 8.2
32.6 35.5 12.3
16.7 50.0 33.3
29.2 27.7 9.2
Table 4 Prevalence (%) of HBV infection markers in patients with hepatitis C and in patients with non-viral chronic liver diseases (disease control group) according to HBV-DNA positivity
None of the patients tested HBV e antigen positive. HBV, hepatitis B virus; HCV, hepatitis C virus.
HBV-DNA Positive HCV-RNA (·105 U/mL) Genotype 1b/non-1b HCV-RNA (·105 U/mL) genotype 1b HCV-RNA (·105 U/mL) genotype non-1b
P-value
Negative
Table 5 Virologic parameters (HCVRNA levels and HCV genotype) in HBVDNA-positive and -negative patients with HCV infection
4.07 ± 4.43 (n ¼ 28) 3.53 ± 3.48 (n ¼ 80) NS 18/9 40/40 NS 4.92 ± 4.87 (n ¼ 15) 4.12 ± 3.20 (n ¼ 32) NS 3.96 ± 3.80 (n ¼ 8)
4.01 ± 3.65 (n ¼ 35) NS
HBV, hepatitis B virus; HCV, hepatitis C virus; NS, not statistically significant. the disease control group [3/6; 50% in patients with at least one HBV marker (+) vs 3/6; 50% in patients with all HBV markers ()), P > 0.05]. Furthermore, HBV-DNA positivity was not associated with virologic parameters of HCV such as, the HCV genotype and HCV-RNA levels (Table 5). In the 101 HCV-infected patients who had undergone liver biopsy, HBV-DNA positivity was not associated with liver histology (Table 6). In addition, fibrosis was not associated with the detection of HBV-DNA even if all patients with normal levels of aminotransferases and cirrhosis were included – irrespective of the presence of liver biopsy – in the none/mild and moderate/severe groups of fibrosis,
HBV-DNA Positive (n ¼ 28)
respectively (data not shown). During the follow up, three HCV-infected patients developed hepatocellular carcinoma. Only one of them had detectable HBV-DNA in serum. Concerning patients with HCV infection who completed antiviral treatment 14 of 44 (31.8%) were HBV-DNA positive. However, HBV-DNA seropositivity was not associated with the response to therapy (biochemical, virologic or histologic) either at the end of treatment or 6 months after completion of therapy (data not shown). Actually, response rates either at the end of treatment or 6 months after the completion of therapy were 73.3 and 68.4% respectively in the HCV(+)/HBV-DNA()) patients vs 58.3 and 33.3% in the
Negative (n ¼ 73)
P-value
Table 6 Histologic data in 101 HCVinfected patients according to HBV-DNA positivity
Fibrosis None or mild (%)/ 13 (46.4)/15 (53.6) 32 (43.8)/41 (56.2) NS moderate or severe (%) Activity Minimal or mild (%)/ 13 (46.4)/15 (53.6) 41 (56.2)/32 (43.8) NS moderate or severe (%) HBV, hepatitis B virus; HCV, hepatitis C virus; NS, not statistically significant. 2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 358–365
Occult HBV in HCV infection HCV(+)/HBV-DNA(+) patient group (not statistically significant difference).
DISCUSSION The present study demonstrated that almost one quarter of HCV-positive/HBsAg-negative Greek patients had detectable HBV-DNA by PCR. This prevalence of occult HBV infection among patients with chronic hepatitis C was significantly higher than that found in patients with nonviral chronic liver diseases (P ¼ 0.003) and in selected HBsAg negative Greek blood donors with markers of previous HBV infection (anti-HBc alone or in combination with anti-HBe or with anti-HBs titres
