Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.     

Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

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.

Methods

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.

Results

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.

Conclusions

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.     

Characteristics of occult hepatitis B virus infection in the Solomon Islands

Hepatitis?B virus (HBV) infection is highly endemic in the Solomon Islands. However, little is known about the status of occult HBV infection in the Solomon Islands. This study aimed to investigate the prevalence of occult HBV infection and its clinical and virological features in the community of Solomon Islands. Blood samples were collected from a total of 564 asymptomatic individuals aged over 18 years in the Western province. The samples used in the present study consisted of 200 samples from 108 males and 92 females (mean age, 37.4 years; range, 18-71 years) that were randomly selected among the hepatitis?B surface antigen (HBsAg)-negative samples from all the participants enrolled in this study. HBV-DNA was detected by real-time?PCR in 25 (12.5%) of the 200 HBsAg-negative samples. Most of the HBV-DNA-positive individuals were infected with wild-type HBV, and only 3 strains demonstrated specific amino acid substitutions (P121X, T123N, C138S, P142S and D144E) in the α?determinant region. In conclusion, occult HBV infection was documented in 12.5% of individuals that demonstrated serologic evidence of resolved HBV infection in this study. The prevalence of occult infection was also influenced by ethnicity; it was more prevalent in Melanesians than Micronesians. In addition, occult HBV infection demonstrated a weak association with the S-variants.     

Characterization of occult hepatitis B virus infection from blood donors in China

Prevalence and characteristics of occult hepatitis B virus (HBV) infection (OBI) of genotypes B and C prevalent in China have not been extensively explored. Characterization of OBI strains obtained from Chinese blood donors was based on clinical and serological analyses, follow-up testing, and sequence analyses. Twenty-eight samples from 165,371 HBV surface antigen (HBsAg)-negative plasmas were confirmed HBsAg negative and DNA positive(HBsAg(-)/DNA(+)), of which 22 were classified as OBIs and 6 as window period infections. The OBI incidence was 1:7,517 in blood donors, whose ages ranged between 20 and 45 years (median, 28 years). OBI donors had normal alanine aminotransferase (ALT) levels and low viral loads ranging between unquantifiable amounts and 178 IU/ml (median, 14 IU/ml). Sequences from 21 basic core promoter/precore (BCP/PC) regions, five whole genomes, and two additional pre-S/S regions from OBI strains were compared to genotypes B and C HBsAg(+) reference strains. Eighty-six percent (6/7) of OBI strains were genotype C. Deletions, insertions, stop codons, and substitutions were detected in 15/21 (71%) core regulatory elements of OBI strains. Critical mutations were found in the core proteins of 5/5 OBI strains in parallel with random substitutions in pre-S/S proteins from 6/7 (86%) OBI strains. Critical mutations in core regulatory elements and core proteins might affect OBI genotype B and C strain replication. That there were few S protein substitutions suggests a minor role of the host immune defenses in OBI occurrence.     

Predictors and kinetics of occult hepatitis B virus infection in HIV-infected persons

It has been proposed that occult hepatitis B virus (HBV) infection, defined as detectable HBV‐DNA in serum with undetectable surface antigen (HBsAg^?), is associated with raised transaminases in HIV‐infected persons. The aim of this study was to determine the prevalence of occult HBV infection in two independent cohorts, and investigate its predictors, association with alanine‐aminotransferase (ALT) levels and response to antiretroviral therapy. Sera from HBsAg^? persons with core antibody (anti‐HBc⁺) were tested by real‐time PCR. Overall, 5.2% of patients were HBsAg⁺ and 39% HBsAg^?/anti‐HBc⁺. The prevalence of occult HBV infection was 48/343 (14.0%; 95% CI 10.7–18.1%), and 27/196 (13.8%) and 21/147 (14.3%) in the two cohorts. Median HBV‐DNA load was 342 (51–147,500) and 60 (25–33,850) copies/ml respectively. HBV‐DNA detection was associated with absence of surface antibody (anti‐HBs), but not with CD4 or ALT levels. Among 11 HBV‐DNA⁺ persons who started antiretroviral therapy containing lamivudine or lamivudine/tenofovir, HBV‐DNA was repeatedly undetectable over median 19 (3–43) months. However, HBV‐DNA detection was intermittent among drug‐naïve persons. Occult HBV infection is common in HBsAg^?/anti‐HBc⁺ HIV‐infected patients and predicted by undetectable anti‐HBs. The intermittent nature of HBV‐DNA detection poses a diagnostic challenge, but no association is observed with ALT levels. J. Med. Virol. 79:1464–1471, 2007. © Wiley‐Liss, Inc.     

Molecular characterization and functional analysis of occult hepatitis B virus infection in Chinese patients infected with genotype C

Occult HBV infection is defined as the persistence of HBV DNA in individuals negative for HBV surface antigen (HBsAg), and many different mechanisms have been reported in different countries. However, in China, one of the endemic areas for HBV infection, no reports have been published on occult HBV infection. The present study investigated the virological features and the mechanism of occult HBV infection in China. Full‐length HBV DNA from eight patients with occult HBV infection (S1–S8) and three HBsAg‐positive cases (SWT1–SWT3) was cloned and sequenced. Additionally, four entire linear HBV genomes from occult cases were transfected transiently into HepG2 cells. The sequencing results showed two major mutations in patients with occult HBV infection as follows: deletions in the pre‐S1 (S3, S4, and S7) and X (S1, S2, and S5) regions. Such deletions covered the S promoter and the basal core promoter (BCP), and function analysis of these variants also showed a decrease in DNA replication and antigen expression. Two patients with occult infection (S6 and S8) had no mutations capable of interfering with viral replication and gene expression in the major viral population. Thus, the deletions in the S promoter and the BCP regions that disable the regulatory elements may be the reason for the absence of HBsAg, and multiple mechanisms may be responsible for occult HBV infection. J. Med. Virol. 81:826–835, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular principles underlying hepatitis C virus diagnosis

Although the rate of new cases of HCV infection has decreased as a result of public health policies related to intravenous drug users, the overall rate of infection in the U.S. is still high. With an estimated 3.9 million people infected nationwide, combined with the observation that, for over 50% of all cases of HCV infection, a route of infection is unknown, HCV will continue to be a major cause of infectious disease related morbidity and mortality for years to come. The benefit of molecular assays resides in providing for more effective treatment of HCV-infected patients, which will certainly justify the expense.     

乙型肝炎病毒表面大蛋白检测用于筛查隐匿性HBV感染

目的 用血清学方法检测乙型肝炎病毒表面大蛋白(HBLP)来筛查隐匿性HBV感染,探讨临床隐匿性HBV感染的血清学检测策略.方法 在日常工作中从临床榆测备份管随机收集2000份用国产ELISA试剂检测HBsAg阴性结果的血清标本,双份分装-20℃冻存,单份标本实施HBLP检测,HBLP阳性标本增加另外两种共三种国产ELISA试剂双份复查HBsAg;HBLP阳性标本再经美国超灵敏MONOLISA HBsAg ULTRA试剂双份检测HBsAg,并行双份DNA定量分析、结果取均值.结果 2000份HBsAg阴性标本共检出15例HBLP阳性:HBLP阳性标本用三种国产ELISA试剂双份复查HBsAg结果一致阴性;通过美国超灵敏MONOLISA HBsAS ULTRA试剂复查HBsAg结果全部阳性;前述15份标本HBV DNA定量分析结果显示均小于500拷贝/ml,其中400～500拷贝/ml 1例,300～400拷贝/ml 3例,200～300拷贝/ml 5例,100～200拷贝/ml 4例,小于100拷贝/ml 2例.结论 用国产常规ELISA试剂检测HBsAg普遍存在漏检,漏检标本HBLP结果可能阳性,检测HBLP有利于筛查出隐匿性H BV感染,为积极寻找临床隐匿性HBV感染的检测策略提供了血清学参考.     

High prevalence of occult hepatitis B virus infection in Taiwanese intravenous drug users

The epidemiology and impact of occult HBV infection in intravenous drug users remain largely unknown. The aim of the study was to investigate the prevalence of occult HBV infection among intravenous drug users in Taiwan. Molecular assays were used to determine the level of serum HBV DNA and the genotype in 304 intravenous drug users negative for both HBsAg and anti-HCV. Of 304 intravenous drug users, 125 (41.1%) were positive for serum HBV DNA. The genotype distribution of HBV was as follows: B, 55 (44%); C, 29 (23%); and mixed B and C infections, 41 (33%). The mean and median serum HBV DNA levels in 125 intravenous drug users with occult HBV infection were 4.0 +/- 0.6 and 4.0 log(10) copies/ml, respectively. The mean serum HBV DNA level in carriers with mixed genotype B and C infections was significantly higher than those infected with HBV genotype B or genotype C alone (mean, 4.2 +/- 0.6 log(10) vs. 3.9 +/- 0.5 log(10), and 3.9 +/- 0.7 log(10) copies/ml, P = 0.01 and 0.05, respectively). The amino acid sequence determination of HBV surface gene in 20 intravenous drug users with occult HBV infection selected at random showed no mutation of amino acid at codon 145. In conclusion, the prevalence of occult HBV infection and mixed HBV genotype infections are not uncommon in intravenous drug users residing in an HBV endemic areas. In addition, intravenous drug users with occult mixed genotype B and C infections have significantly higher viral loads than those with occult infection of single HBV genotype.     

Viral deletions among healthy young Chinese adults with occult hepatitis B virus infection

To investigate the mechanism and prognosis of occult hepatitis B virus (HBV) infection (OBI) at a molecular level among healthy young adults, the presence of HBV DNA in 1176 sera samples collected from healthy young people after neonatal vaccination was assessed by nested polymerase chain reaction (PCR) using specific primers designed for the X and S regions of the HBV genome. Full-length HBV DNA from 9 patients with OBI (OB1-OB9) was cloned and sequenced. Deletions in the pre-S, basal core promoter (BCP), core (C) and polymerase (P) regions were observed. The data indicate that there is still a substantial risk of OBI in China despite neonatal vaccination. All deletions that were observed in the pre-S, BCP, C and P regions play a direct or indirect role in OBI. The presence of a deletion mutation in the pre-S1 region was considered to play a pivotal role in hepatocarcinogenesis and was found to increase the risk of hepatocellular carcinoma in the cohorts studied.     

献血人群隐匿性乙型肝炎病毒感染的流行病学和血清学研究

目的了解广州地区献血人群隐匿性乙型肝炎病毒感染（OBI）的流行病学和血清学情况。方法对广州地区199631例无偿献血者标本同时用ELISA法检测HBsAg、紫外-乳酸脱氢酶法检测ALT、核酸扩增技术（NAT）联合检测HBV/HCV/HIV及HBV单项鉴别试验,对HBsAg阴性HBV DNA阳性者进行随访,用荧光定量PCR检测病毒载量,用ELISA法检测乙肝两对半。结果 199631例标本中共检出104例HBsAg阴性HBV DNA阳性者,经随访有54例为OBI,OBI检出率为0.027%,年龄以46～55岁组检出率最高（P〈0.01）,外地身份证的献血者检出率高于广州市身份证者（P〈0.01）,OBI检出率与性别和献血次数无关（P〉0.05）。104例HBsAg阴性HBV DNA阳性的标本ALT均正常,病毒载量均〈1000IU/ml,平均值为162IU/ml。随访标本中,除6例ALT异常外其余均正常,54例OBI标本病毒载量均〈1000IU/ml,平均值为122IU/ml,乙肝两对半中抗-HBc阳性率明显高于其他项目（P〈0.01）。结论 HBsAg阴性献血者中存在OBI,有必要在献血者中开展核酸检测。     

Molecular epidemiology of chronic hepatitis B virus infection in Greece

Virological data on chronic hepatitis B virus (HBV) infection in Greece are limited. HBV genotypes, surface antigen (HBsAg) subtypes, and HBsAg “a” determinant mutations among patients infected chronically with HBV, were investigated. Serum samples from 135 HBsAg positive patients were tested. Serologic (HBsAg, anti‐HBs, HBeAg, and anti‐HBe), virologic (HBV‐DNA quantitation) and biochemical markers (serum alanine aminotransferase/ALT and aspartate aminotransferase/AST) were analyzed. HBV genotypes and HBsAg subtypes were determined by partial sequencing of the S gene. Genotyping was performed by using the National Center for Biotechnology Information online Genotyping tool and phylogenetic analysis. Nucleotide sequences were aligned pair wise with ClustalW and phylogenetic trees were constructed by the neighbor‐joining method. Sequences were also used to predict HBV HBsAg subtypes. In six patients (4%), simultaneous presence of HBsAg and anti‐HBs was determined, whereas 47 patients (35%) were HBeAg positive, 84 (62.5%) were anti‐HBe positive, and four patients (3%) were characterized by the simultaneous presence of HBeAg and anti‐HBe. Mean ALT was 238 IU/L (standard deviation = 576.84), and HBV‐DNA levels ranged from 1.02 × 10⁵ to 2.2 × 10⁷ IU/ml. Genotype D was predominant (98%), with viral groups D/ayw2 (73%) and D/ayw3 (27%). Group A/adw accounted for 1% of cases. Genotypes B and C were found exclusively in the Chinese immigrants (1%). Single or multiple point mutations were found in 35 cases (26%). Some of the most common mutations occurred at amino acid positions 129, 133, 134, 144, 145, including the “vaccine escape” mutation G145R. Mutations analysis revealed that amino acid substitutions did not affect detection by commercial immunoassays. J. Med. Virol. 83:245–252, 2011. © 2010 Wiley‐Liss, Inc.     

Role of surface promoter mutations in hepatitis B surface antigen production and secretion in occult hepatitis B virus infection

The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection.     

Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia (<10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An unusually large number of amino acid mutations was present in the immunodominant a-determinant of HBsAg (respectively 3, 6, 7, 10, and 10 mutations). Comparison of the HBV genomes in two donors to a consensus HBV genotype D sequence showed a most prominent hotspot of genetic variation in HBV nucleotides 480-570, encoding the HBsAg a-determinant. The phylogenetic comparison of separate donor HBV genes to the HBV genes of 11 reference strains (genotypes A-H) showed the donor HBV surface genes to form an outgroup, while the HBV polymerase, core and X genes closely cluster with the HBV genotype D reference strain. Maybe the HBV strains in this study represent a natural end-stage of seemingly cleared HBV infection, in which HBV maintains a low level of possibly non-infectious replication, after sacrificing its immunologically offending surface antigen, thus avoiding final clearance by the immune system.     

Molecular analysis of the hepatitis B virus presurface and surface gene in patients from eastern China with occult hepatitis B

This study was designed to detect and analyze mutations that occur within the presurface and surface (pre‐S/S) gene of HBV in patients with occult hepatitis B, and determine their relationship to that disorder. Among 254 HBsAg negative samples of blood collected in eastern China, 183 were positive for anti‐HBc alone, 61 were positive for anti‐HBe alone, and 10 samples were positive for HBeAg. Within this group, 15 samples were found to be HBV DNA positive by real‐time PCR and were designated Group I. A control group of 28 HBsAg positive samples were chosen at random from patients with chronic hepatitis B and designated Group II. The HBV pre‐S/S gene was amplified by PCR and subjected to sequencing analysis. Occult hepatitis B was found in 1.6% of the patients with anti‐HBc alone and in 3.3% of those with anti‐HBe alone. Occult hepatitis B also was found in all HBsAg negative but HBeAg positive samples. Sequencing analysis showed a significant correlation between point mutations within the “a” determinant and occult hepatitis B (P < 0.0001), and a close relationship between pre‐S deletion mutations and occult hepatitis B (P = 0.06). There were unique amino acid mutations at the G145 position other than G145R. The HBV DNA levels in patients with occult hepatitis B were significantly lower than those found in the control group. The “a” determinant mutations and pre‐S deletions may play important roles in occult hepatitis B by affecting the expression, synthesis and secretion of the S protein and by impeding viral release and replication. J. Med. Virol. 85: 979–986, 2013. © 2013 Wiley Periodicals, Inc.     

High risk of occult hepatitis B virus infection in HIV-positive patients from South Africa.

This was a retrospective, unmatched case control, laboratory-based study, investigating the impact of human immunodeficiency virus (HIV) infection on the outcome of routine laboratory detection of HBsAg and prevalence of active HBV infection in 295 samples from 167 HIV-positive and 128 HIV-negative patients. The samples were tested for HBV (HBsAg, anti-HBc, anti-HBs, HBeAg and anti-HBe) and anti-HIV 1 and 2 (Axsym assays, Abbott Laboratories), as part of routine diagnosis. A nested PCR assay, with detection limit of 800 copies/ml and employing independent sets of primers to core and surface genes, was used to investigate HBV DNA. Quantification of HBV DNA was determined with the Cobas Amplicor HBV Monitor assay (Roche Diagnostics). Of the 295 samples, the frequency of anti-HBc was almost similar; 82% for the HIV-negatives and 85% for the HIV-positives, indicating that both groups were equally exposed to HBV infection. The HIV-positives had a higher rate of anti-HBs (76.0% versus 47.7%) and a lower rate of HBsAg carriage (16.2% versus 35.2%), suggesting that HIV-positive individuals are less likely to experience chronic HBV infection. However, analysis of HBV DNA indicated that many of the anti-HBs positives (20.5% versus 8.2%) and HBsAg-negatives (22.1% versus 2.4%) had active HBV infection in the HIV-positive group. There was a statistically significant difference in the prevalence of HBV DNA in the HBsAg-negatives between the two groups (Odds ratio: 11.52; chi-square: p=0.00006). Additionally, 33.3% (5/15) of sera with "anti-HBc alone" serological pattern were HBV viremic in the HIV-positive group, compared to 0% (n=31) in the HIV-negatives. Quantification of HBV DNA from HBsAg-negative/HIV-positive patients demonstrated low level HBV viremia (below 10,000 copies/ml). In conclusion, these findings strongly support that HIV infection is a risk factor for occult HBV infections.