Detection of hepatitis B virus in bone allografts from donors with occult hepatitis B infection

The implementation of nucleic acid testing in donor screening has improved the safety of tissue allografts. Although infectious disease transmission can be considered a rare event, the detection of occult hepatitis B infection remains challenging. The studies concerning this risk are mainly based on testing blood specimens. This work shows the correlation between results of samples obtained from donor blood and the corresponding tissue washing solution. Hepatitis B virus deoxyribonucleic acid was detected both in bone allografts from donors with serological profiles associated to active hepatitis B infection and occult hepatitis B infection. These results suggest that hepatitis B virus seems to concentrate in bone marrow even when a low viral load is present in peripheral blood. Even detection at molecular level is not enough to avoid the risk of hepatitis B virus transmission and a multiparametrical evaluation is required in tissue donor screening. The role of clinicians in recognition and reporting of allograft-associated infections is a major concern for the acquisition of experience to be applied in risk control of disease transmission.     

Comparison of hepatitis B,core, HBc,and hepatitis B antibody,anti HBs,in a presumed low risk donor population

Donors screened by medical social history interview negative for high risk behavior or communicable disease history, but subsequently exhibiting reactive serological markers, emphasize importance of duel safe guarding factors for determining donor suitability. This report examines a relationship between two immunoabsorption assay tests, hepatitis B core (HBc) antibody, a required food and drug administration (FDA) test, and hepatitis B antibody (anti HBs), non-required test. Reactive serology results, 129 cases, 3,581 donors (2008–2012) for HBc as the only initially positive serological marker were subjected to anti HBs testing in this history pre-screened donor population. Enzyme linked immunoabsorption assay kits hepatitis B, core and antibody, were used in this study. All samples were initially tested for human immunodeficiency virus, hepatitis B, and hepatitis C, utilizing nucleic acid testing and antigen antibody immunoabsorption assay. Testing was performed by a FDA-registered CLEA-certified reference laboratory. Samples were deceased donor blood samples and a limited number of pre-mortem samples, separated, stored and analyzed according to manufacturer recommendation and FDA regulations. 129 reactive HBc only samples, were subsequently tested for anti HBs. Of these 129, 94 were found to be reactive for anti HBs. This represented 72 % of samples tested for antibody, a higher percentage than anticipated for a medical history negative, low risk population.     

Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in hepatocyte death during HBV infection. We report here that the HBV core protein (HBc) is a potent inhibitor of TRAIL-induced apoptosis. Overexpressing HBc significantly decreased TRAIL-induced apoptosis of human hepatoma cells, whereas knocking-down HBc expression in hepatoma cells transfected with HBV genome enhanced it. When present in the same cell, HBc blocked the pro-apoptotic effect of the HBV X protein (HBx). The resistance of HBc-expressing cells to TRAIL-induced apoptosis was associated with a significant reduction in death receptor 5 (DR5) expression. Upon transfection, HBc significantly repressed the promoter activity of the human DR5 gene. Importantly, HBc gene transfer inhibited hepatocyte death in a mouse model of HBV-induced hepatitis; and in patients with chronic hepatitis, DR5 expression in the liver was significantly reduced. These results indicate that HBc may prevent hepatocytes from TRAIL-induced apoptosis by blocking DR5 expression, which in turn contributes to the development of chronic hepatitis and HCC. They also call into question the potential side effects of HBc-based vaccines.     

Investigation of Occult Hepatitis B Virus Infection in Anti-HBc Positive Patients from a Liver Clinic

Occult hepatitis B infection (OBI) is manifested by presence of very low levels (<200IU/mL) of Hepatitis B viral DNA (HBV DNA) in the blood and the liver while exhibiting undetectable HBV surface antigen (HBsAg). The molecular mechanisms underlying this occurrence are still not completely understood. This study investigated the prevalence of OBI in a high-risk Australian population and compared the HBV S gene sequences of our cohort with reference sequences. Serum from HBV DNA positive, HBsAg negative, and hepatitis B core antibody (anti-HBc) positive patients (study cohort) were obtained from samples tested at SEALS Serology Laboratory using the Abbott Architect, as part of screening and diagnostic testing. From a total of 228,108 samples reviewed, 1,451 patients were tested for all three OBI markers. Only 10 patients (0.69%) out of the 1,451 patients were found to fit the selection criteria for OBI. Sequence analysis of the HBV S gene from 5 suspected OBI infected patients showed increased sequence variability in the ‘a’ epitope of the major hydrophilic region compared to reference sequences. In addition, a total of eight consistent nucleotide substitutions resulting in seven amino acid changes were observed, and three patients had truncated S gene sequence. These mutations appeared to be stable and may result in alterations in HBsAg conformation. These may negatively impact the affinity of hepatitis B surface antibody (anti-HBs) and may explain the false negative results in serological HBV diagnosis. These changes may also enable the virus to persist in the liver by evading immune surveillance. Further studies on a bigger cohort are required to determine whether these amino acid variations have been acquired in the process of immune escape and serve as markers of OBI.     

Occult hepatitis B demonstrated by anti-HBc and HBV DNA in HIV-positive patients

Background:

In patients who are hepatitis B virus (HBV) DNA-positive, but HBV surface antigen (HBsAg) -negative, the infection is referred to as occult hepatitis B infection (OBI). Occult HBV infection is harmful when other liver diseases are present, and can aggravate liver damage in in patients with chronic liver diseases. In human immunodeficiency virus (HIV) infection the suppression of viral replication by the immune system might be inactivated, and classical HBV infection in OBI patients may occur. Health care professionals should be aware of OBI in HIV patients. The routine test for HBV infection in Iran is the enzyme-linked immunosorbent assay for the HBV surface antigen (ELISA HBsAg); therefore, the aim of this study was to evaluate the prevalence of OBI in Iranian HIV patients.

Methods:

This cross-sectional study was conducted in 2012 on sera from all the known and accessible HIV patients in Jahrom and Fassa, two cities in southern Iran. All samples were tested for the HBsAg, HBV core antibody (HBcAb). All the results were analyzed using SPSS.

Results:

Of the 91 patients, seven (7.7%) were HBsAg-positive and forty-five (49.5%) were HBcAb-positive. In patients with negative HBsAg (84 patients), 39 (46.4%) were HBcAb positive and 53 (63%) were positive for HBV DNA.

Conclusion:

The prevalence of HBV infection is relatively high in HIV patients, and more accurate tests than those presently in use should be used for diagnosis.Key Words: Hepatitis B, HIV infection, Occult hepatitis     

丙型病毒性肝炎母婴传播的现状、进展及未来

丙型肝炎病毒(HCV)可引起急性和慢性病毒性肝炎,可发展成肝纤维化、肝硬化,甚至肝细胞癌。HCV经典的传播途径为经血液或血液制品传播,但1992年后献血员HCV的筛检已使输血后肝炎大为减少。在发达国家,HCV传播途径正在发生改变,儿童非血液制品的丙肝日渐增多。母婴间宫内、分娩时及产后感染已成为当前及今后的重要研究课题。研究证实,HCV可经胎盘引起胎儿感染,宫内感染是HCV传播的一条重要途径。尽管人们对HCV母婴传播中所涉及的风险因素逐渐明确,但到目前为止对具体的传播机制和传播时机仍知之甚少。我们就丙型病毒性肝炎母婴传播的现状、进展及未来做简要综述。     

Occult Hepatitis B virus (HBV) infection and challenges for hepatitis elimination: A literature review

Occult hepatitis B infection (OBI) is characterized by the detection of hepatitis B virus (HBV) DNA in serum or liver but negativity for hepatitis B surface antigen. OBI, which is thought to be maintained by host, immunological, viral and/or epigenetic factors, is one of the most challenging clinical features in the study of viral hepatitis. Currently, there is no validated detection test for OBI. It is believed that OBI is widely distributed throughout the world, with a higher prevalence in populations at high-risk HBV, but the detailed worldwide prevalence patterns are unknown. We conducted a survey of recently published studies on OBI rates across all continents. High prevalence rates of OBI are observed in some specific groups, including patients with hepatitis C virus, human immunodeficiency virus co-infection or hepatocellular carcinoma. In 2016, the World Health Organization adopted strategies to eliminate viral hepatitis by 2030, but the difficulties in detecting and treating OBI currently challenge this goal. Subjects with OBI can transmit HBV, and episodes of reactivation can occur. Further studies to understanding the mechanisms that drive the development of OBI are needed and can contribute to efforts at eliminating viral hepatitis.     

Epidemiology,Risk Factors and Genotypes of HBV in HIV-Infected Patients in the Northeast Region of Colombia: High Prevalence of Occult Hepatitis B and F3 Subgenotype Dominance

Introduction

Chronic hepatitis B virus (HBV) infection is an increasing cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected individuals. HIV-positive patients are commonly co-infected with HBV due to shared routes of transmission.

Objectives

Our aim was to determine the risk factors, prevalence, genotypes, and mutations of the Surface S gene of HBV, and occult hepatitis B infection (OBI) among patients infected with HIV in a northeastern Colombian city.

Methods

A cross-sectional study was conducted with 275 HIV-positive patients attending an outpatient clinic in Bucaramanga, Colombia during 2009–2010. Blood samples were collected and screened for serological markers of HBV (anti-HBs, anti-HBc and HBsAg) through ELISA assay. Regardless of their serological profile, all samples were tested for the HBV S gene by nested-PCR and HBV genotypes were determined by phylogenetic inference. Clinical records were used to examine demographic, clinical, virological, immunological and antiretroviral therapy (ART) variables of HIV infection.

Results

Participants were on average 37±11 years old and 65.1% male. The prevalence of HIV-HBV coinfection was 12% (95%CI 8.4–16.4) of which 3.3% had active HBV infection and 8.7% OBI. The prevalence of HIV-HBV coinfection was associated with AIDS stage and ART treatment. Sequence analysis identified genotype F, subgenotype F3 in 93.8% of patients and genotype A in 6.2% of patients. A C149R mutation, which may have resulted from failure in HBsAg detection, was found in one patient with OBI.

Conclusions

The present study found a high prevalence of HIV-HBV coinfection with an incidence of OBI 2.6-fold higher compared to active HBV infection. These findings suggest including HBV DNA testing to detect OBI in addition to screening for HBV serological markers in HIV patients.     

High prevalence of occult hepatitis B virus genotype H infection among children with clinical hepatitis in west Mexico

Studies on the prevalence of infection with hepatitis B virus (HBV) among children are scarce in Latin American countries, especially in Mexico. This study was aimed to investigate the prevalence of HBV infection, occult hepatitis B infection (OBI) and HBV genotypes among children with clinical hepatitis. In total, 215 children with clinical hepatitis were evaluated for HBV infection. HBV serological markers and HBV DNA were analysed. OBI diagnosis and HBV genotyping was performed. HBV infection was found in 11.2% of children with clinical hepatitis. Among these HBV DNA positive-infected children, OBI was identified in 87.5% (n = 21/24) of the cases and 12.5% (n = 3/24) were positive for both HBV DNA and hepatitis B surface antigen. OBI was more frequent among children who had not been vaccinated against hepatitis B (p < 0.05) than in those who had been vaccinated. HBV genotype H was prevalent in 71% of the children followed by genotype G (8%) and genotype A (4%). In conclusion, OBI is common among Mexican children with clinical hepatitis and is associated with HBV genotype H. The results show the importance of the molecular diagnosis of HBV infection in Mexican paediatric patients with clinical hepatitis and emphasise the necessity of reinforcing hepatitis B vaccination in children.     

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M_r 21 000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.     

PI3K 与乙型肝炎病毒宫内感染的相关研究

慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染是全世界关注的公共卫生问题。我国是乙肝高流行区,每年约有150万乙肝病毒携带者分娩,近半数胎儿通过母婴垂直传播感染乙肝。由于婴幼儿期感染乙肝后形成的免疫耐受,往往成为慢性甚至终身携带者,逐渐发展为肝硬化、肝癌。近年来的研究发现,PI3-Akt信号通路与妊娠生及或病理过程关系密切,在感染HBV的胎盘组织中发现PI3K-Akt信号通路中相关蛋白表达异常增高,且HBx Ag干扰该通路调节凋亡功能。推断HBx Ag通过调节PI3K-Akt信号通路活性影响胎盘功能,是HBV宫内感染的一种重要分子机制。为今后阻断HBV宫内感染提供新的研究方向。     

The expression of hepatitis-C virus antigen by immunohistochemical stain and polymerase chain reaction in corneas of seropositive corneal donors

Background: Of the donor corneas rejected for transplantation, the largest group is that from donors testing seropositive for hepatitis C virus (HCV). In situations of severe shortage in supply of donor corneal tissue, we may consider the use of seropositive donors for transplantation if we can prove with high certainty the absence of HCV RNA in the donor corneal tissue. Polymerase chain reaction (PCR) is a highly sensitive and specific technique for direct detection of HCV RNA and can be used for this purpose. Nevertheless, it is not applicable for routine clinical use in most eye departments due to its unavailability and cost effectiveness.Purpose: To study the possible use of immunohistochemical method for detection of HCV antigen in corneal tissue of seropositive donors and correlate the results with those of PCR. Immunohistochemical methods have not yet been studied in donor corneal tissue.Materials and methods: Eight corneas of 4 seropositive and 8 corneas of 4 seronegative corneal donors were studied by immunohistochemical and PCR methods for the presence of HCV antigen in their corneal tissue and sera.Results: HCV RNA was not detected in the sera and corneal tissue of all seropositive and seronegative corneal donors by either PCR and immunohistochemical methods.Conclusion: Although the study is too small for conclusive results, the correlation between the immunohistochemical and PCR studies for direct detection of HCV antigen in corneal tissue of seropositive donors may raise the possibility of using the immunohistochemical method for screening of donor corneas for the detection of HCV antigen. A larger prospective study investigating the sensitivity, specificity and clinical applicability of the immunohistochemical method is warranted. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hepatitis E virus infection in two different regions of Indonesia with identification of swine HEV genotype 3

Hepatitis E is an emerging disease with a high incidence globally. Few data are available on hepatitis E virus (HEV) infection in Indonesia. To obtain molecular information on HEV infection in two regions of Indonesia with different customs and swine breeding conditions, serum samples from 137 swine farm workers, 100 blood donors and 100 swine (27 fecal samples also obtained) in Yogyakarta (Central Java) and from 12 and 64 swine farm workers, 42 and 135 local residents and 89 and 119 swine in Tulungagung (East Java) and Mengwi (Bali), respectively, from our previous study, were compared. Serological tests for anti‐HEV antibodies by ELISA, HEV‐RNA detection by RT‐PCR and phylogenetic analysis were performed. The total prevalence of anti‐HEV antibodies in humans was higher in Bali (11.6%) than in Java (5.1%; P = 0.015). No significant differences in anti‐HEV prevalence among swine farm workers and local residents in Java were found. The finding of swine HEV genotype 3 in specimens from Yogyakarta and genotype 4 from Tulungagung and Bali is somewhat different from other reports. We suggest other factors in addition to close contact with swine might play an important role in HEV transmission of non‐endemic/related custom groups. To the best of our knowledge, this is the first report on swine HEV genotype 3 in Indonesia.     

Intracellular hepatitis B virus increases hepatic cholesterol deposition in alcoholic fatty liver via hepatitis B core protein

Hepatitis B virus (HBV) infection is a prevalent infectious disease with serious outcomes like chronic and acute hepatitis, cirrhosis, and hepatocellular carcinoma. However, the metabolic alteration by HBV is rarely taken into consideration. With the high prevalence of alcohol consumption and chronic HBV infection, their overlap is assumed to be an increasing latent hazard; although the extent has not been calculated. Moreover, the impact of chronic alcohol consumption combined with HBV on cholesterol metabolism is unknown. Six-week-old male FVB/Ncrl mice were hydrodynamically injected with a pGEM-4Z-1.3HBV vector and then fed an ethanol diet for 6 weeks. Serum biomarkers and liver histology, liver cholesterol levels, and cholesterol metabolism-related molecules were measured. In vitro assays with HBx, hepatitis B surface (HBs), or hepatitis B core (HBc) protein expression in HepG2 cells costimulated with ethanol were conducted to assess the cholesterol metabolism. HBV expression synergistically increased cholesterol deposition in the setting of alcoholic fatty liver. The increase of intrahepatic cholesterol was due to metabolic alteration in cholesterol metabolism, including increased cholesterol synthesis, decreased cholesterol degradation, and impaired cholesterol uptake. Overexpression of HBV component HBc, but not HBs or HBx, selectively promoted the hepatocellular cholesterol level.     

HCV and HBV infection in 3 communities in the Neapolitan area

Using the antibody to HCV and HBc (anti-HCV, anti-HBc), we studied the prevalence of Hepatitis C and B virus in three groups: intravenous drug abusers, subjects lodging in huts and elderly clergymen. Statistical analysis was carried out by chi-square and Fisher's exact tests. Our results show statistical significance for anti-HCV seroprevalence among the three groups, while anti-HBc doesn't differ. These preliminary data seem to show that the two viruses have different ways of transmission.     

Cerumen as a potential risk for transmission of Hepatitis B virus

Hepatitis B virus (HBV) transmission via blood and other body fluids from infected individuals to healthy people has been largely demonstrated. However, in the current literature, there is little information available on the potential role of cerumen in HBV transmission. Cerumen and blood were collected from 70 patients infected with HBV and 70 volunteer healthy people were selected as the control group, and the samples were evaluated by ELISA and Real-time PCR. All the patients proved positive for HBsAg and anti HBc total. Sixty-one of the 70 cerumen samples of cases (82.1%) and 5 (7%) of controls were positive for HBV DNA with ranges from 1.53 × 102 to 2.9 × 108 and 1.3 × 102-2.6 × 105/ml, respectively. In three patients, the level of HBV DNA in cerumen was higher than that in the serums. The patients who were positive for HBeAg showed a higher rate of HBVDNA in the serum and cerumen.The results of this study showed the level of HBV DNA as a probably indicator of high risk transmission factor, which was present in the cerumen of chronic hepatitis B patients in west of Iran.     

New strategies for blood donor screening for hepatitis B virus: nucleic acid testing versus immunoassay methods

Serologic testing for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV core antigen (anti-HBc) has historically been the foundation of blood screening, while HBV nucleic acid testing (NAT) was recently developed to detect HBsAg-negative, anti-HBc-negative blood units donated during early acute infection. Comparison data on seroconversion panels using HBsAg assays of varying sensitivities and pooled- or single-sample NAT, along with viral load estimates corresponding to HBsAg assay detection limits, have provided information on the theoretical benefits of NAT relative to HBsAg. Model-derived estimates have generally been predictive of the yields of DNA-positive, HBsAg-negative window period blood units detected in a number of studies from Europe, Japan, and the US. Studies indicate that the added benefit of pooled-sample NAT is relatively small in areas of low endemicity, with greater yields in areas highly endemic for HBV. Single-sample NAT would offer more significant early window period closure and could prevent a moderate number of residual HBV transmissions not detected by HBsAg assays; however, no fully automated single-sample HBV NAT systems are currently available.Even single-sample HBV NAT may not substitute for anti-HBc screening, as indicated by studies of donors with isolated anti-HBc who have extremely low DNA levels undetectable by standard single-sample NAT and who have been associated with transfusion-transmitted HBV. Moreover, HBsAg testing may still be needed even in the setting of combined anti-HBc and NAT screening. HBsAg-positive units from donors in the chronic stage of infection may contain very low or intermittently detectable DNA levels that single-sample NAT would miss. Although such donors are usually anti-HBc reactive and would be interdicted by anti-HBc screening, some lack anti-HBc. Extensive parallel testing will be needed to determine whether single-sample NAT in combination with anti-HBc might be sufficient to detect all the infectious donors currently interdicted by HBsAg testing. In countries that do not screen for anti-HBc, HBsAg testing would be the only means of detecting donations from chronically infected individuals with low/intermittently detectable DNA, since even single-donor NAT would not identify these potentially infectious blood units. In the future, the current fully automated HBsAg assays may incorporate significant sensitivity improvements, and automated single-sample HBV NAT may become a reality. Each country will need to develop its blood screening strategy based on HBV endemicity, yields of infectious units detected by different serologic/NAT screening methods, and cost effectiveness of test methods in ensuring blood safety.     

The L60V Variation in Hepatitis B Virus Core Protein Elicits New Epitope-Specific Cytotoxic T Lymphocytes and Enhances Viral Replication

Mutations in the core protein (HBc) of hepatitis B virus (HBV) are associated with aggressive hepatitis and advanced liver diseases in chronic hepatitis B (CHB). In this study, we identified the L60V variation in HBc that generates a new HLA-A2-restricted CD8⁺ T cell epitope by screening an overlapping 9-mer peptide pool covering HBc and its variants. The nonameric epitope V60 was determined by structural and immunogenic analysis. The HBc L60V variation is correlated with hepatic necroinflammation and higher viral levels, and it may be associated with a poor prognosis in CHB patients. Immunization with the defined HBV epitope V60 peptide elicited specific cytotoxic T lymphocyte (CTL)-induced liver injury in HLA-A2⁺ HBV transgenic mice. In addition, in vitro and in vivo experiments both demonstrated that the HBc L60V variation facilitates viral capsid assembly and increases HBV replication. These data suggest that the HBc L60V variation can impact both HBV replication and HBV-specific T cell responses. Therefore, our work provides further dissection of the impact of the HBc L60V variation, which orchestrates HBV replication, viral persistence, and immunopathogenesis during chronic viral infection.