Virological significance of low-level hepatitis B virus infection in patients with hepatitis C virus associated liver disease

Cytomegalovirus (CMV) infection is an important cause of morbidity in solid organ recipients. Early markers to identify the progress of the infection and patients at high risk are required in order to apply a strategy of pre-emptive therapy. The efficacy of pre-emptive therapy relies on accurate laboratory tests to monitor CMV infection. The evaluation of CMV DNA kinetics by the polymerase chain reaction (PCR) is widely used for the management of CMV infection but markers predicting the progression of the infection and standardization of the technique are essential for the clinical interpretation of PCR results. A commercially available PCR system, the COBAS AMPLICOR Monitor (Roche Diagnostics, Brachburg, NJ), was used for the quantitation of CMV DNA in weekly blood samples (n = 504) from 47 solid organ recipients in the first 6 months after transplantation. PCR results were evaluated according to the development of clinical disease in order to find a DNA threshold and time points predicting the progression of CMV infection. Week 4 from transplantation was the earliest time point to note a significant difference between those patients who eventually developed CMV disease (n = 30) and those who remained asymptomatically infected (n = 17). At week 4, viral loads were significantly higher in patients who developed CMV disease than in asymptomatic infections (median value: 4 log(10)/10(6) leukocytes vs. 2.8, P < 0.0001). At week 4, a DNA level >/=4 log(10)/10(6) leukocytes was associated with a 45.37 odds ratio for CMV disease. Any increase >/=1 log from the first DNA detection to week 4 correlated with the clinical progression of CMV infection (odds ratio 1.74). In those patients who were treated with anti-CMV therapy, a >97% reduction of the baseline viral load was associated with a complete therapeutic success. In conclusion, CMV infection is a highly dynamic process and the quantitation of CMV DNA by PCR is a powerful marker to control successfully the infection, but a strict follow-up of the recipient and standardized PCR tests are mandatory for the best management of the infection.     

A matched case-control study of hepatitis B virus mutations in the preS and core promoter regions associated independently with hepatocellular carcinoma

This study aimed to determine hepatitis B virus (HBV) mutations associated independently with the risk of hepatocellular carcinoma (HCC), as adjusted with other mutations in the preS and core promoter regions of HBV genotypes B and C. One hundred and forty HBV-infected patients with HCC and 280 HBV-infected patients without HCC who had intact data of HBV genotyping and DNA sequencing in both regions were involved in this age-, sex-matched case-control study. Univariate and two-step stepwise multivariate regression analyses were performed to determine factors associated with the risk of HCC. Of 39 mutations evaluated, 23 in genotype C and 6 in genotype B were associated with an increased risk of HCC in the univariate analysis. Multivariate analyses established that genotype C (adjusted odds ratio [AOR] = 3.3; 95% confidence interval [CI] = 1.1-9.8), viral load (≥10(4) copies/ml) (AOR = 2.4; 95% CI = 1.0-5.8), A2962G (AOR = 18.7; 95% CI = 7.5-46.7), preS2 start codon mutation (AOR = 12.5; 95% CI = 3.4-45.5), C105T (AOR = 0.1; 95% CI = 0.0-0.2), T1753V (AOR = 3.1; 95% CI = 1.1-9.2), and A1762T/G1764A (AOR = 2.9; 95% CI = 1.1-7.3) were associated independently with HCC, adjusted for factors including mutations in both regions. By using an estimating haplotype frequencies program, it was found that a haplotypic carriage with 105C and 2962G was significantly more frequent in the patients with HCC than in those without HCC and the frequency of haplotype 2962G-preS2 start codon wildtype-105C-1762T/1764A was 47.9% in the patients with HCC and 4.3% in those without HCC. Conclusively, A2962G and T105C are novel factors associated independently with HCC. Further prospective studies are needed to confirm the role of these mutations in the development of HCC.     

Serum hepatitis C virus RNA level as a predictor of subsequent response to interferon-α therapy in Japanese patients with chronic hepatitis C

Serum hepatitis C virus (HCV) RNA level has been shown to be a good predictor of subsequent response to interferon-α (IFN) therapy in US patients in whom genotype 1a/1b are both predominant. To determine whether serum HCV RNA level is a predictor of subsequent response to IFN in Japanese patients or not, appropriately collected pre-IFN therapy serum samples from 35 Japanese patients with chronic HCV infection were studied. Serum HCV RNA level and HCV genotype were determined and correlated with the subsequent response to IFN. Response to IFN was defined by serum alanine transaminase level: complete and sustained response (n = 15), complete response followed by relapse (n = 10), and no response (n = 10). Patients with complete and sustained response had lower pre-IFN serum HCV RNA level (median: RT-PCR+, bDNA-) compared to the complete response to relapse group (median: 5.25 × 10⁶ genome equivalent/ml [eq/ml], P < 0.001) and the no response group (median: 10.63 × 10⁶ eq/ml, P < 0.001). Seven (46.7%) of the complete and sustained response patients had HCV genotype 2a and three patients had a mixture of genotypes 1b and 2a. In contrast, all 10 patients in the complete response to relapse group had genotype 1b whereas 8 of 10 patients in the non-response group had genotype 1b and 2 had genotype 2b. The patients with HCV genotype 2a had lower serum HCV RNA level than those with 1b (P = 0.002). When the HCV viremia were controlled by stratifying them into < and > 10⁶ eq/ml, patients with genotype 1 had a similar complete and sustained response rate compared to genotype 2. These data indicated that pre-IFN serum HCV RNA is also a good predictor of subsequent complete and sustained response in Japanese patients with chronic HCV infection. © 1994 Wiley-Liss, Inc.     

Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma

Chronic hepatitis B infection is a major cause of hepatocellular cancer (HCC). The pathogenesis of the carcinogenesis is not fully understood. Viral proteins such as the X protein and the truncated middle S protein have been implicated to be transactivators. In order to investigate whether any mutations within relevant parts of the hepatitis B virus (HBV) genome could be associated with the development of HCC, the genomes of 16 HBV strains from chronic HBV carriers with HCC were studied. Serum samples were subjected to PCR and the HBV DNA sequenced subsequently. Genotypes A-D were represented. The sequence analysis showed that an especially high proportion, 50% (CI 95%, 25-75%), of the patients with HCC carried HBV mutants with deletions or insertions in the N-terminal half of the pre-S2 region or had a point mutation in the start codon of pre-S2 compared with controls with chronic HBV infection, 21% (CI 95%, 3-39%). A high proportion (69%) also had mutations at position 1762 (A --> T) and/or 1764 (G --> A) in the core promoter region, but the proportion of core promoter mutations was no different from what was found in a control group of HBV carriers without HCC (68%). The pre-S2 variants, which involve deletions of immunogenic regions, may have a survival advantage as they are mostly found in long-standing HBV infection. There were no other mutations found frequently within the region coding for the X protein.     

Hepatitis B viremia is associated with increased risk of hepatocellular carcinoma in chronic carriers

The role of quantitative viral load in development of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers was evaluated using real-time PCR (TaqMan PCR), a highly sensitive method for quantitative detection of HBV DNA. Serum samples collected at study entry from HCC cases and matched controls were chosen separately from ongoing prospective cohort studies in Senegal, West Africa, and Haimen City, China. For 14 HCC cases and 28 controls from Senegal, the relative risk (RR, 95% CI) of HCC was 15.6 (2.0-124.3) for those positive by the TaqMan PCR assay. Average length of follow-up (study entry to death from HCC) among cases was 2.8 (+/-1.6) years. The paired median difference between cases and controls was 3.8 x 10(4) virions/ml, with cases higher (P = 0.09). In order to clarify the relationship with lower-titer viremia, we selected 55 cases and 55 matched controls from the Chinese cohort all negative for serum HBV DNA by conventional dot blot hybridization. In this group, the RR associated with HBV DNA positivity by TaqMan PCR was 3.1 (1.1-9.2), with an average duration of follow-up of 3.3 (+/-2.1) years. The median difference in quantitative viremia between cases and controls was 6.0 x 10(4) virions/ml, with cases higher (P < 0.0001). Increased risk appeared to be confined to subjects with viral loads >2.3 x 10(4) virions/ml. In conclusion, HBV viremia, except perhaps at extremely low levels, is associated with increased risk for HCC in prospective studies of chronic carriers in two disparate populations.     

Quantitative measurement of hepatitis B virus DNA in different areas of hepatic lobules in patients with chronic hepatitis B

Liver histology in chronic hepatitis B is marked by inflammatory infiltration involving the peripheral zones. The cause of such cellular infiltration remains unknown. The aim of the present study was to investigate the amounts of intrahepatic hepatitis B virus (HBV) DNA separately in the peripheral and central zones, using laser capture microdissection coupled with real-time quantitative polymerase chain reaction. Fourteen patients with chronic hepatitis B were included in the study. Liver biopsy samples were taken and hepatocytes were microdissected separately from peripheral and central zones. DNA was extracted from hepatocytes in each zone and evaluated the amounts of HBV-DNA. Immunohistochemical study for hepatitis B core antigen (HBcAg) was also performed. The amounts of total intrahepatic HBV-DNA in patients positive for hepatitis Be antigen (HBeAg) were greater than those in HBeAg-negative patients. There was no difference in HBV-DNA between the peripheral and central zones. Immunohistochemistry also showed that HBcAg-positive cells were distributed homogeneously in the hepatic lobules. In patients with peripherally predominant HBV-DNA, the serum alanine aminotransferase (ALT) level was lower than in patients with centrally predominant HBV-DNA. HBV-DNA was distributed homogeneously in the hepatic lobules. In patients with lower amounts of HBV-DNA in the peripheral zone, the serum ALT level tended to be higher than in other patients.     

Prevalence and impact of occult hepatitis B infection in chronic hepatitis C patients treated with pegylated interferon and ribavirin

The prevalence of occult hepatitis B, defined by absence of HBsAg and HBV DNA, ranges widely in patients with hepatitis C. This may influence the treatment of hepatitis C and the severity of liver disease. Sensitive and specific real‐time PCR techniques are available commercially and can detect more reliably low HBV DNA levels. The aim of this study was to determine the prevalence of occult hepatitis B virus infection using the COBAS Taqman assay (Roche Diagnostics, Meylan, France) in the serum and liver of HBsAg negative patients with chronic hepatitis C and to evaluate its clinical consequences on liver pathology and its impact on the response to treatment with peg‐IFNα and Ribavirin. HBV DNA detection was assessed retrospectively on 140 sera and 113 liver biopsies of HCV positive/HBsAg negative patients before treatment. A 4.4% (5/113) prevalence of occult hepatitis B was recorded in liver samples and in none of the sera. Anti‐HBc was not detected in one, three of whom were sustained virological responders to treatment, one was relapsed responder and one was non‐responder. Furthermore, in this cohort composed of 12% anti‐HBs negative/anti‐HBc positive and 20% anti‐HBs positive/anti‐HBc positive patients, anti‐HBc was not associated with pre‐therapeutic viral load, ALT serum levels, and histological activity or fibrosis. Using a commercial real‐time PCR assay, we observed a low prevalence of occult B hepatitis. This, just as anti‐HBC status, had no clinical impact in a large cohort of hepatitis C patients. It therefore does not appear useful to screen for occult hepatitis B in these patients with this test before beginning HCV treatment. J. Med. Virol. 82: 000–000, 2010. © 2010 Wiley‐Liss, Inc. J. Med. Virol. 82: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Clinical study on relationship between hepatocellular carcinoma and hepatitis C virus infection in patients with chronic liver disease]

The relationship between hepatocellular carcinoma (HCC) and hepatitis C virus (HCV) infection was investigated. Antibody to hepatitis C virus was detected in 88.8% and 87.0% of 240 patients with hepatocellular carcinoma and liver cirrhosis, respectively. A history of blood transfusion was shown in only 21.8% (21/96) of the HCV antibody positive HCC patients. Of 196 patients with chronic hepatitis type C and the HCV antibody positive liver cirrhosis, 10 developed HCC during the follow-up period of two years. A high prevalence of HCV antibody was also shown among 83 patients with alcoholic liver cirrhosis and HCC associated with alcoholic liver cirrhosis. HCV-RNA was detected in all patients with alcoholic HCC. These data support a causal association between hepatitis C virus and hepatocellular carcinoma.     

Hepatitis B virus X mutations occurring naturally associated with clinical severity of liver disease among Korean patients with chronic genotype C infection

Few reports have detailed mutation frequencies and mutation patterns in the entire X region according to clinical status. The aims of this study were to elucidate the relationships between mutation patterns and their frequencies in the X region and clinical status in a Korean cohort and determine specific X mutation types, related closely with liver disease progression. All X mutations were determined by direct sequencing in 184 patients with different clinical features. Mutation rates in the X region in patients with more severe liver disease, hepatocellular carcinoma (HCC) (3.6%) or liver cirrhosis (4%) were always significantly higher than in patients with corresponding less severe forms, chronic hepatitis (2.9%) or asymptomatic carriers (2.1%), but no significant difference in mutation rates was found in terms of HBeAg serostatus. All five mutation types (V5M/L, P38S, H94Y, I127T/N, and K130M and V131I) affecting the six codons were found to be related significantly to clinical severity. Among these, two mutation types (V5M/L and K130M and V131I) were observed more frequently in HBeAg negative patients than in HBeAg positive patients. In conclusion, the results suggest that an accumulation of mutations in the X region contributes to disease progression in chronic patients, at least Korean patients with genotype C. Specific mutation types appears to be related more to severe liver diseases such as HCC or liver cirrhosis. In particular, a novel mutation type (V5M/L) discovered firstly during the present study was found to be associated significantly with HCC.     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

High prevalence of dual or triple infection of hepatitis B, C, and delta viruses among patients with chronic liver disease in Mongolia

Mongolia is known for its high endemicity for hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infections among apparently healthy individuals. However, there are little or no data on the prevalence and genotype distribution of HBV, HCV, and HDV among patients with chronic liver disease in Mongolia. Therefore, serum samples obtained in 2004 from 207 patients (age, mean+/-standard deviation, 51.0+/-11.9 years) including those with chronic hepatitis (n=90), liver cirrhosis (n=41), and hepatocellular carcinoma (n=76) were tested for serological and molecular markers of HBV, HCV, and HDV infections. Of the 207 patients, 144 (69.6%), 106 (51.2%), and 117 (56.5%) tested positive for hepatitis B surface antigen (HBsAg) and/or HBV DNA, HCV RNA, and HDV RNA, respectively. Collectively, 172 patients (83.1%) were viremic for one or more of these viruses, including dual viremia of HBV/HDV (26.6%) or HBV/HCV (7.7%) and triple HBV/HCV/HDV viremia (30.0%). Of note, triple ongoing infection was significantly more frequent among patients with hepatocellular carcinoma than among those with chronic hepatitis (63.2% vs. 14.4%, P<0.0001). One hundred sixty patients (77.3%) had a history of blood transfusion and/or surgery. The distribution of HBV genotypes among the 116 HBV-viremic patients was: A (0.9%), B (0.9%), C (6.0%), D (88.8%), and C plus D (3.4%). All 117 HDV isolates were classified into genotype I. The 106 HCV RNA-positive samples were typed as genotype 1b (92.5%), 2a (0.9%), or 1b plus 2a (6.6%); mixed infection of two distinct HCV genotypes was found exclusively in the patients with hepatocellular carcinoma.     

Concomitant diffuse large B-cell lymphoma and hepatocellular carcinoma in chronic hepatitis C virus liver disease: a study of two cases

Chronic hepatitis C virus HCV infection progresses through liver fibrosis and cirrhosis to hepatocellular carcinoma HCC. It appears to be causally related to B-cell non-Hodgkin's lymphoma since regression after antiviral therapy has been described. Two cases are described of non-Hodgkin's lymphoma and HCC arising simultaneously in two patients. The first patient did not have cirrhosis on liver biopsy. HCV had been undetectable in plasma following successful therapy with interferon and ribavirin treatment 7 years earlier. The second patient developed an aggressive form of hepatocellular carcinoma HCC within weeks of stopping treatment with interferon and ribavirin. Therapy had induced complete viral suppression for over 40 weeks. The two cases suggest that non-Hodgkin's lymphoma and HCC can develop in the absence of detectable hepatitis C viremia and argues for continued surveillance even after sustained virological response to treatment.     

Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patients

The hepatitis A virus (HAV) is the most common etiological cause of acute hepatitis infections in humans in industrialized countries. Investigations into the viral load during HAV viremia, however, are rare. Therefore, correlation studies between viral load, biochemical, and specific serological markers have been undertaken. The group of sera comprised a series of multiple consecutive blood samples drawn from 11 patients at different times after onset of the disease. During the period up to 70 days after the onset of icterus, the individual range was at 1 x 10(3) to 3 x 10(4) HAV genome equivalents/ml. From day 75 until 120 after onset of the disease, the levels traced were at 10(3). In one case, it was possible to trace 1.25 x 10(4) genome equivalents/ml up to 180 days after onset of icterus and in two cases even up to 408 and 490 days viral load levels of 5 x 10(3) and 4 x 10(4) were detected, respectively. The same sera were used to measure IgM class antibodies to hepatitis A virus and the total anti-HAV. The results demonstrate that a direct correlation to peak levels of viral load exists with peak serum transaminase levels, but neither with peak anti-HAV IgM levels nor with total anti-HAV. Decreasing amounts of anti-HAV IgM tend to occur with decreasing amounts of HAV genome equivalents; and, vice versa, increasing amounts of total anti-HAV are accompanied by decreasing amounts of HAV genome equivalents. The longest duration of viremia was found in patients infected with HAV genotype IA.     

Substitution of amino acid 70 in the hepatitis C virus core region of genotype 1b is an important predictor of elevated alpha-fetoprotein in patients without hepatocellular carcinoma

Previous studies identified amino acid (aa) substitutions of the hepatitis C virus core region of genotype 1b (HCV-1b core region) and elevated serum alpha-fetoprotein (AFP) levels as predictors of poor virologic response to pegylated interferon (PEG-IFN) plus ribavirin (RBV), and also as risk factors for hepatocarcinogenesis. The present study evaluated the impact of aa substitutions of HCV-1b core region on AFP, as a surrogate marker of hepatocarcinogenesis, on AFP levels in 569 Japanese patients with HCV-1b but without HCC, and investigated the predictive factors of elevated AFP (> or =11 microg/L). High AFP levels were detected in 27.4% of the patients. The rate of hepatocarcinogenesis in a group of 109 patients who received IFN monotherapy and followed-up for 15 years, was significantly higher in patients with abnormal than normal AFP. Multivariate analysis of 569 patients identified fibrosis stage (F3,4), aspartate aminotransferase (> or =76 IU/L), substitution of aa 70 (glutamine or histidine), and platelet count (<15.0 x 10(4)/microl) as significant determinants of elevated AFP. In 49 patients with abnormal AFP levels and substitutions at aa 70 who were treated with PEG-IFN + RBV, the rate of normalization of AFP was significantly lower in non-virological responders (28.6%) than in transient (71.4%) and sustained (100%) virological responders. The results indicated that substitution of aa 70 of HCV-1b core region is an important predictor of elevated AFP in non-HCC patients, and that eradication of the mutant virus normalizes AFP. The results highlight the importance of eradication of mutant type virus of aa 70 for reducing the risk of hepatocarcinogenesis.     

Validation of a sensitive and specific real‐time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma however, is not well understood. A sensitive, specific, and reproducible real‐time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels, and 96 plasma samples of chronic hepatitis B patients were analyzed. Results were compared with total HBV DNA levels, individual ALT levels and the Histology Activity Index (HAI). This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR and a correlation coefficient (R) of 0.98 (P < 0.0001). cccDNA was detected in two of four international panels. Significant correlation was found between cccDNA and total HBV DNA levels in both panels (R = 0.96, and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, P < 0.0001). In 57% of these samples cccDNA was detectable. Mean level of cccDNA was 0.16% of total HBV load. Plasma cccDNA levels were higher in HBeAg positive samples than in HBeAg negative samples (4.91 log copies/ml vs. 3.88 log copies/ml, P < 0.0001). Levels of total HBV DNA and HBV genotype did not influence cccDNA detection. ALT levels and HAI‐score were not correlated with plasma cccDNA levels. These findings suggest that cccDNA levels in plasma are not the result of increased hepatocyte degeneration, but indicate that other mechanisms might be responsible. J. Med. Virol. 81:988–995, 2009. © 2009 Wiley‐Liss, Inc.     

Comparison of hepatitis B virus subgenotypes in patients with acute and chronic hepatitis B and absence of lamivudine-resistant strains in acute hepatitis B in Japan

Hepatitis B virus (HBV) has been classified into eight genotypes and can be further divided into several subgenotypes that have different geographic distributions. Because of increased human migration, the prevalence of rare subgenotypes is increasing in Japanese patients with acute hepatitis B. Lamivudine-resistant strains of HBV have begun to emerge in association with chronic hepatitis B. The aim of this study was to investigate the distribution of HBV subgenotypes and lamivudine-resistant strains in patients in Japan with acute hepatitis B. One hundred twenty-three patients with acute hepatitis B and 123 with chronic hepatitis B were studied. HBV subgenotypes and lamivudine-resistance mutations were determined by direct sequencing of the preS and polymerase region, respectively. HBV subgenotypes Aa (n=3), Ae (n=23), Ba (n=7), Bj (n=3), Cs (n=7), Ce (n=76), D (n=2), and H (n=2) were detected in patients with acute hepatitis. In patients with chronic hepatitis, HBV subgenotypes Ae (n=4), Ba (n=1), Bj (n=18), and Ce (n=100) were found. Non-common Japanese subgenotypes, that is, non-Bj and non-Ce, were detected more frequently in patients with acute hepatitis (35.8%) than in patients with chronic hepatitis (4.1%) (Odds ratio, 0.076; 95%CI, 0.029-0.200; P<0.0001). Lamivudine-resistance mutations were detected in chronic hepatitis patients with breakthrough hepatitis but not in other patients. In conclusion, the prevalence of uncommon Japanese HBV subgenotypes is expected to increase, although lamivudine-resistant strains have not yet been found in patients with acute hepatitis B.