Viral and immune factors associated with successful treatment withdrawal in HBeAg-negative chronic hepatitis B patients.

BACKGROUND & AIMS: Factors associated with a successful outcome upon nucleos(t)ide analogue (NA) treatment withdrawal in HBeAg-negative chronic hepatitis B (CHB) patients have yet to be clarified. The objective of this study was to analyse the HBV-specific T cell response, in parallel with peripheral and intrahepatic viral parameters, in patients undergoing NA discontinuation. METHODS: Twenty-seven patients without cirrhosis with HBeAg-negative CHB with complete viral suppression (>3 years) were studied prospectively. Intrahepatic HBV-DNA (iHBV-DNA), intrahepatic HBV-RNA (iHBV-RNA), and covalently closed circular DNA (cccDNA) were quantified at baseline. Additionally, serum markers (HBV-DNA, HBsAg, HBV core-related antigen [HBcrAg] and HBV-RNA) and HBV-specific T cell responses were analysed at baseline and longitudinally throughout follow-up. RESULTS: After a median follow-up of 34 months, 22/27 patients (82%) remained off-therapy, of whom 8 patients (30% of the total cohort) lost HBsAg. Baseline HBsAg significantly correlated with iHBV-DNA and iHBV-RNA, and these parameters were lower in patients who lost HBsAg. All patients had similar levels of detectable cccDNA regardless of their clinical outcome. Patients achieving functional cure had baseline HBsAg levels ≤1,000 IU/ml. Similarly, an increased frequency of functional HBV-specific CD8+ T cells at baseline was associated with sustained viral control off treatment. These HBV-specific T cell responses persisted, but did not increase, after treatment withdrawal. A similar, but not statistically significant trend, was observed for HBV-specific CD4+ T cell responses. CONCLUSIONS: Decreased cccDNA transcription and low HBsAg levels are associated with HBsAg loss upon NA discontinuation in patients with HBeAg-negative CHB. The presence of functional HBV-specific T cells at baseline are associated with a successful outcome after treatment withdrawal. LAY SUMMARY: Nucleos(t)ide analogue therapy can be discontinued in a high proportion of chronic hepatitis B patients without cirrhosis. The strength of HBV-specific immune T cell responses may contribute to successful viral control after antiviral treatment interruption. Our comprehensive study provides in-depth data on virological and immunological factors than can help guide individualised therapy in patients with chronic hepatitis B.

High prevalence of hepatitis B-antibody loss and a case report of de novo hepatitis B virus infection in a child after living-donor liver transplantation.

AIM: To assess the seroprevalence of hepatitis B virus (HBV) immunity among previously vaccinated pediatric liver transplant recipients and present a case report of de novo hepatitis B infection after liver transplantation. METHODS: This study focused on children with chronic liver diseases who received primary hepatitis B immunization and had a complete dataset of anti-HBs before and after liver transplantation between May 2001 and June 2017. Medical records were retrospectively reviewed for potential factors relating to HBV immunity loss. RESULTS: In total, 50 children were recruited. The mean time from liver transplantation to anti-HBs testing was 2.53 ± 2.11 years. The mean anti-HBs levels before and after liver transplantation were 584.41 ± 415.45 and 58.56 ± 6.40 IU/L, respectively. The rate of non-immunity (anti-HBs < 10 IU/L) in the participants was 46% (n = 26) at one year, 57% (n = 7) at two years and 82% (n = 17) at > three years following liver transplantation. The potential factors relating to HBV immunity loss after liver transplantation were identified as anti-HBs (P = 0.002), serum albumin (P = 0.04), total bilirubin (P = 0.001) and direct bilirubin (P = 0.003) before liver transplantation. A five-year-old boy with biliary cirrhosis received 4 doses of HBV vaccine with an anti-HBs titer of > 1000 IU/L and underwent liver transplantation; his anti-HBc-negative father was the donor. After liver transplantation, the boy had stenosis of the hepatic artery up to the inferior vena cava anastomosis and underwent venoplasty three times. He also received subcutaneous injections of enoxaparin for 5 mo and 20 transfusions of blood components. Three years and ten months after the liver transplantation, transaminitis was detected with positive tests for HBsAg, HBeAg, and anti-HBc (2169.61, 1706 and 8.45, respectively; cutoff value: < 1.00) and an HBV viral load of 33212320 IU/mL. CONCLUSION: The present study showed that loss of hepatitis B immunity after liver transplantation is unexpectedly common. In our case report, despite high levels of anti-HBs prior to transplantation, infection occurred at a time when, unfortunately, the child had lost immunity to hepatitis B after liver transplantation.

Detecting hepatitis B virus in surgical smoke emitted during laparoscopic surgery.

BACKGROUND: Hepatitis B virus (HBV) transmission is known to occur through direct contact with infected blood. There has been some suspicion that the virus can also be detected in aerosol form. However, this has never been directly shown. The purpose of this study was to sample and analyse surgical smoke from laparoscopic surgeries on patients with hepatitis B to determine whether HBV is present. METHODS: A total of 11 patients who underwent laparoscopic or robotic abdominal surgeries between October 2014 and February 2015 at Korea University Anam Hospital were included in this study. A high efficiency collector was used to obtain surgical smoke in the form of hydrosol. The smoke was analysed by using nested PCR. RESULTS: Robotic or laparoscopic colorectal resections were performed in 5 cases, laparoscopic gastrectomies in 3 cases and laparoscopic hepatic wedge resections in another 3 cases. Preoperatively, all of the patients had positive hepatitis B surface antigen (HBsAg). 2 patients had detectable HBsAb, and 2 were positive for hepatitis B e antigen. 3 patients were taking antihepatitis B viral medications at the time of the study. The viral load measured in the patients' blood was undetectable to 1.7×108 IU/mL. HBV was detected in surgical smoke in 10 of the 11 cases. CONCLUSIONS: HBV is detectable in surgical smoke. This study provides preliminary data in the investigation of airborne HBV infection.

Biosensors for hepatitis B virus detection.

A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor. This review focuses on the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus (HBV). The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.

Regional and ethnic aspects of viral hepatitis B among pregnant women.

OBJECTIVES: The aim of this study was to determine the prevalence of HBV infection among pregnant women in districts of Eastern Slovakia with a diverse prevalence of Roma population. METHODS: Overall 59,279 serum samples from 9 regional departments of clinical microbiology from Eastern Slovakia were collected in the period from January 2008 till December 2009 and analysed. RESULTS: The number of HBsAg positive samples overall and during pregnancy was 1.74% and 2.12%, respectively. Comparing districts with higher (> 5%) and lower (< 5%) Roma population, there was no significant difference in the prevalence of HBsAg positive samples overall (1.95% vs.1.62%). However, in the subgroup of pregnant women the prevalence of HBsAg positive samples (2.72% vs. 0.95%) differs significantly (p < 0.01). CONCLUSIONS: The prevalence of HBV infection among pregnant women in Eastern Slovakia did not rapidly exceed the estimated nationwide prevalence. However, in districts with higher Roma population the expected higher prevalence of HBV infection was confirmed. This indicates the need to pay special attention to the prevention of hepatitis B in these districts.

Prevalence of hepatitis B surface antigen in US-born and foreign-born Asian/Pacific Islander college students.

UNLABELLED: The prevalence of chronic hepatitis B (HBV) among college-age US-born Asian and Pacific Islanders (A/PI) is not well known. OBJECTIVES: To compare the prevalence of hepatitis B surface antigen (HBsAg) seropositivity in US-born to A/PI-born students at a public university. PARTICIPANTS: Undergraduate who self-identified themselves as A/PI. RESULTS: Of 145 US-born A/PI, 1.4% (confidence interval [CI] = 0.0%, 3.3%) tested positive for HBsAg compared to 3.3% (CI = 0.5%, 6.1%) of the 152 A/PI-born students. Approximately 1/3 of all students were unaware of their HBV vaccination status. CONCLUSIONS: HBsAg prevalence among A/PI undergraduates, including US-born, is considerably higher (3 to 11 times) than the mainstream US population (0.3% to 0.5%) and supports the Centers for Disease Control and Prevention (CDC) recommendations for testing all persons of A/PI ancestry, including US-born persons whose parents were born in regions with HBsAg prevalence of >or=8%. Awareness of HBV vaccination status was relatively low and vaccination did not assure that individuals were HBsAg negative.

Replication and infectivity of hepatitis B virus in HBV-related glomerulonephritis.

OBJECTIVE: To examine the presence of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in renal tissues from patients with HBV-related glomerulonephritis. METHODS: Renal tissue biopsies taken from patients with HBV-related glomerulonephritis and two control groups were prepared for immunocytochemical detection of HBsAg and HBcAg. HBV cccDNA was examined using a nested PCR. RESULTS: Of the 63 HBV-related glomerulonephritis patients studied, HBsAg was present in the renal tissues of 48 (76.2%) and HBcAg in the renal tissues of 27 (42.9%). The HBsAg and HBcAg positive rates in HBV-related glomerulonephritis patients were higher than those of the 20 patients with non-HBV-related glomerulonephritis (p<0.05). However, there was no significant difference when the HBV-related glomerulonephritis patients were compared with 12 patients with renal tuberculosis, renal atrophy, renal calculus, and renal tumor with positive serum HBV markers. In patients with HBV-related glomerulonephritis, there was no significant difference in HBsAg and HBcAg positive rates in renal tissue between patients with and without serum hepatitis B e antigen (HBeAg). By nested PCR, two of five patients with HBV-related glomerulonephritis were positive for HBV cccDNA. CONCLUSION: The location and replication of HBV in renal tissue make the kidney a potential reservoir for HBV. HBV cccDNA may be key in the search for anti-HBV drugs.

Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris.

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.

Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection.

Dendritic cells (DC) of hepatitis B virus (HBV) carriers have been reported to exhibit functional impairment. Possible explanations for this phenomenon are infection of HBV by DC or alteration of DC function by HBV. We therefore analyzed whether DC support the different steps of HBV infection and replication: uptake, deposition of the HBV genome in the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte-derived DC by adenoviral vectors, low-level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When monocyte-derived DC were subjected to wild-type HBV or a recombinant HBV expressing Renilla luciferase under a non-liver-specific promoter, intracellular HBV DNA was detected in a low percentage of cells. However, neither nuclear cccDNA was formed nor luciferase activity was detected, indicating that either uncoating or nucleocytoplasmic transport were blocked. To verify our observation in the in vivo situation, myeloid and plasmacytoid DC were isolated from blood of high viremic HBV carriers, and analyzed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DC had in vivo been exposed to more than 10(4) HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable as were viral capsids in the cytoplasm. In conclusion, circulating DC may take up HBV antigens, but neither support nucleocytoplasmic transport nor replication of HBV.

Seroprevalence of hepatitis B virus infection in individuals with clinical evidence of hepatitis in Goiânia, Goiás. Detection of viral DNA and determination of subtypes.

The presence of serological markers for hepatitis B virus (HBsAg, anti-HBc IgM and Anti-HBc total) was investigated in the serum of 1,396 individuals who had clinical suspect of hepatitis. It was observed that 50.7% of the individuals were positive and, from the total of the studied individuals, 14.5% were positive for HBsAg. From these, 8.5% were also positive for anti-HBc IgM. The analysis in relation to gender showed a higher seroprevalence index among male individuals (p < 0.0001). It was observed the occurrence of subtypes adw2 (62.7%), ayw3 (23.5%), ayw2 (9.8%) and adw4 (3.9%). The viral DNA was detected in 61 (33.9%) HBsAg positive samples and in one sample positive only for anti-HBc total. These results indicate an important incidence of the HBV infection in this population, and reinforce previous studies regarding this virus in the central west region of Brazil.

Expression of deletion mutants of the hepatitis B virus protein HBx in E. coli and characterization of their RNA binding activities.

The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.

Problems of an automated testing system for hepatitis B.

False positive hepatitis B surface and e antigen results on the Abbott Axsym analyser are causing concern. All users of this technology should be aware of the problem.

Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and p53 gene mutations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of HCC.

[Prevalence of serologic markers of hepatitis B virus in hospital personnel]. / Prevalência de marcadores sorológicos do vírus da hepatite B em trabalhadores do serviço hospitalar.

OBJECTIVE: To verify the prevalence of the anti-HBc, anti-HBs and HBsAg markers of hepatitis B virus, and to identify the risk factors determining occupational infection with this virus among hospital personnel. METHODS: Samples of serum from 210 persons both male and female who work in different occupations at a hospital university, were analysed. The technique employed was the immunoenzymatic assay using commercial kits. RESULTS: As a control group, samples of serum from 45 volunteer blood donors were utilized. It was verified that 20.5% of the hospital personnel presented a positive reaction to at least one of the markers songht, as against 6.6% of the control group. The prevalence of each marker separately was: anti-HBc 8.1%, anti-HBs 5.2%, and HBsAg 2.9% in the hospital personnel; and 4.4%, 2.2% and 0.0% in the control group. The simultaneous presence of the anti-HBc and anti-HBs markers was detected in 4.3% of the workers. In the control group, the presence of the anti-HBc and anti-HBs markers was detected, isolately, with respective prevalences of 4.4% and 2.2%. Those who presented the highest rates of positivite reaction were: laboratory technicians 24.0%, nurses 23.6%, physicians 20.8%, and cleaning personnel 18.2%. CONCLUSIONS: The findings suggest that direct contact with patients and handling of blood and other body fluids are risk factors related to occupational infection with HBV. Therefore, it is recommended that hospital personnel be vaccinated against hepatitis B.

An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli.

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.

Transmission of serum hepatitis. 1970.

There is evidence that transmission of serum hepatitis is associated with transmission of virus-like particles, approximately, 20 millimicron in diameter, containing the Australia or serum hepatitis (SH) antigen, which is currently referred to as the hepatitis associated antigen (HAA). Virus-like particles containing HAA were in the following materials, inoculation of which produced serum hepatitis: (1) a pool of human plasma, (2) serum obtained during the acute phase of hepatitis from a recipient of the plasma pool, (3) a preparation of human thrombin, and (4) serum from a proved hepatitis carrier. The HAA appeared in the serum samples of 61 individuals inoculated with these materials; serum hepatitis developed in 38 of them. Inoculation of dilutions of the plasma pool showed that serum hepatitis can be transmitted by materials containing HAA in amounts too low to be detected by current techniques.

[Concentration of hepatitis A and B virus antigens using water-soluble polymers]. / Kontsentrirovanie antigenov virusov gepatita A i B s pomoshch'iu vodorastvorimykh polimerov.

A possibility of using reversibly sedimented polymeric system for the concentration of hepatitis A and B virus antigen has been demonstrated. As polymeric systems, tetrasole-containing polyelectrolytes and interpolymeric complex polymethacryl acid-poly-1-vinylpyrrolidone were used, capable of sedimenting in acid medium and dissolving in media with the neutral pH.

[Progress in hepatitis diagnosis]. / Fortschritte in der Hepatitisdiagnostik.

Until today only five pathogens of viral hepatitis have been discovered: hepatitis virus A (HAV), B (HBV), C (HCV), D (HDV) and E (HEV). An update on the state of the art of actually established diagnostic procedures is briefly summarized. Some recent topics and problems are covered more extensively; in the case of HBV the value of HBsAG concentration as a prognostic marker, the finding 'anti-HBc only' the role of HBV mutants, the determination of circulating HBV genomic material, and modalities for HBV vaccination control; in the case of HCV the indications for study of its viral genomic material and the role of HCV subspecies are explained. Finally, first results on HEV prevalence in Switzerland are introduced.

Diagnosis of hepatitis delta virus infection.

The hepatitis delta virus (HDV) is a cause of severe liver disease in man. Several assays are available to detect both the HDV genome and its only encoded antigen, HDAg, as well as specific antibodies against HDAg (anti-HD), of both the IgG and IgM class. Different serologic patterns enable the clinician to formulate a correct diagnosis, crucial for effective treatment.

A case-control study on the association of hepatitis B virus infection and hepatocellular carcinoma in Northeast Brazil

Estudou-se a associaçäo entre carcinoma hepatocelular (CHC) e a infecçäo pelo vírus B da hepatite (VHB) em Salvador, Bahia (Brasil), utilizando-se o desenho caso-controle, pareado por idade e sexo, comparando-se a freqüência de positividade aos marcadores virais de um grupo de 40 casos de CHC com dois grupos controles: 40 indivíduos com outras neoplasias e 80 indivíduos sadios. Utilizou-se a técnica de radioimunoensaio para os testes sorológicos. Foram observadas altas proporçöes de indivíduos positivos para AgHBs (42,5 por cento) e daqueles com AgHBs ou anti HBs (65,0 por cento) entre os casos de CHC. A proporçäo de positivos para AgHBs entre os casos foi maior do que no grupo controle com outras neoplasias (7,5 por cento) e no grupo de indivíduos sadios (2,5 por cento), equivalente a estimativas de risco de 15,0 (Intervalo de Confiança (IC) a 95 por cento de 3,29, 68,30) e de 33,0 (IC a 95 por cento de 9,13, 119,28), ambos estatisticamente significantes. Näo foi observada positividade ao AgHBe AntiHBe estava presente em 41,2 por cento dos casos, sugerindo a ausência de replicaçäo viral, com integraçäo do DNA do vírus ao genoma hepático. Entre os casos de CHC houve associaçäo entre cirrose e AgHBs. Foi referida história de alcoolismo crônico mais freqüentemente por aqueles com cirrose. O estudo demonstra a relevante associaçäo entre CHC e VHB, principalmente em indivíduos jovens