Performance of hepatitis B surface antigen tests with the first WHO international hepatitis B virus genotype reference panel.

BACKGROUND: Standardization of hepatitis B surface antigen (HBsAg) tests is indispensable for consistent quality and comparability. Ideally, the assays should detect all known hepatitis B virus (HBV) genotypes equally well. OBJECTIVE: Development of an HBV genotype reference panel for HBsAg assays representing the most prevalent HBV subgenotypes to address commutability and traceability of the heat-inactivated 2nd WHO International Standard (IS) for HBsAg in relation to native HBsAg and to HBV genotypes. STUDY DESIGN: An HBV panel of 15 non-inactivated lyophilized specimens representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H was evaluated in parallel to the IS by 15 laboratories using 19 different HBsAg tests and tree unitages. The virus content of the samples was reduced by ultracentrifugation and dilution to <2×10(4) IU HBV DNA/mL. RESULTS: Twenty-two qualitative and 6 quantitative data sets were evaluated. Overall, the results demonstrated consistent detection of HBV genotypes by the majority of tests with a mean potency variability relative to the IS of 36%. Some assays showed significant genotype-dependent differences in analytical sensitivity. Some tests were more sensitive with the IS, others less. On average, one IU HBsAg corresponded to 0.88±0.20 ng HBsAg protein. CONCLUSIONS: The panel was accepted by the WHO as the "1st International Reference Panel for HBV genotypes for HBsAg-based assays". The panel is a helpful complementation to the IS to validate HBV genotype specific analytical test sensitivities.

First WHO International Reference Panel containing hepatitis B virus genotypes A-G for assays of the viral DNA.

BACKGROUND: WHO International Standards (IS) are provided for the calibration and validation of diagnostic and screening assays, e.g. for hepatitis B virus (HBV). HBV forms numerous subgenotypes and the current IS for HBV DNA reflects subgenotype A2. OBJECTIVE: A reference panel with the most prevalent subgenotypes should facilitate evaluation of genotype-specific detection efficiencies. STUDY DESIGN: 215 HBV positive plasma samples collected worldwide were characterized for HBV markers and sequenced. Fifteen subgenotype A1, A2, B2, B4, C2, D1, D3, E, F2 and G samples were selected for the panel. The lyophilized samples were tested in parallel with the IS in an international collaborative study with 16 laboratories using 13 different nucleic acid amplification techniques (NATs). RESULTS: Eight of 13 NAT had a HBV DNA detection efficiency which was independent of the genotype and consistent with the IS, while with five assays, certain deviations were noted, particularly with genotype F which was under quantitated or even missed by three assays. The panel was accepted by the WHO as the "1st WHO International Reference Panel for HBV Genotypes for HBV NAT-Based Assays". CONCLUSIONS: The evaluation of HBV DNA assays should include many different genotypes. The WHO Reference Panel is universally available for manufacturers of HBV DNA assays, diagnostic laboratories and control authorities to facilitate standardized validation of HBV genotype specific detection efficiency of both diagnostic (quantitative and qualitative) and screening NAT assays.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance.

Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti-HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV-infected patients with seroconversion to anti-HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti-HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg-encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti-HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti-HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.

Tissue donation and virus safety: more nucleic acid amplification testing is needed.

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

Core promoter mutant HBV non-responding to adefovir after viral breakthrough on lamivudine: rapid virologic response to tenofovir plus lamivudine in a cirrhotic patient.

BACKGROUND: In chronic hepatitis B patients undergoing therapy with LAM or ADV, viral breakthrough is possible due to the emergence of drug resistance. LAM resistant HBV strains are susceptible to ADV, while ADV resistant mutants remain sensitive to LAM. CASE REPORT: A male patient with HBV-related cirrhosis developed viral breakthrough (HBV DNA>1.8 x 106 IU/ml) after 4 1/2 years of treatment with LAM, and therapy was switched to ADV (10 mg/d). After three months, HBV remained highly replicative without any changes of ALT values, and ADV dose was increased (20 mg/d). Because of unchanged VL sequence analysis was performed three months later, which showed the mutation (rtS219A) and the concomitant mutation (sS210R) and 2 mutations in core promoter region (A1762T), (G1764A). During the sixth month of ADV monotherapy the patient developed liver failure. After administration of TDF plus LAM, HBV DNA became undetectable within 39 days. At day 41, the patient underwent OLT. TDF plus LAM were well tolerated, and the patient maintained undetectable HBV DNA levels, and in addition to HBIG a sustained HBsAg negative status over twenty-eight months post OLT. CONCLUSION: TDF plus LAM is a safe drug combination in case of viral breakthrough during LAM treatment and subsequent primary non-response to ADV. High VL persisting for >or= 6 months of continuous antiviral treatment may indicate drug resistance. Especially in cirrhotic patients with LAM resistance, "add on" of a nucleotide analogue is the right therapeutic strategy even before viral breakthrough gets apparent.

An international collaborative study to establish the 2nd World Health Organization International Standard for hepatitis B virus DNA nucleic acid amplification technology-based assays.

BACKGROUND AND OBJECTIVES: The aim of this study was to replace the 1st World Health Organization International Standard for hepatitis B virus DNA for nucleic acid amplification technique (NAT)-based assays (code 97/746) with a new International Standard. Two lyophilized preparations freeze dried from the same bulk were evaluated in the original collaborative study (coded 97/746 and 97/750, and termed AA and BB, respectively, in the original study). This present study re-evaluates these two preparations in terms of potency and real-time stability. MATERIALS AND METHODS: The 1(st) International Standard (97/746) and the second lyophilized preparation (97/750) were coded Samples 1 and 2, respectively, in the present study. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after long-term storage at 4 degrees C and 20 degrees C for more than 51 months. RESULTS: Data were returned from a total of nine different NAT-based assays, five in qualitative format and four in quantitative format. The results of this study confirm the results of the original collaborative study, with no significant differences being found in estimated international units (IU)/ml or polymerase chain reaction-detectable units/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (97/750). Real-time and accelerated degradation studies indicate that both samples are very stable. Storage of both preparations at 20 degrees C for more than 51 months resulted in no detectable degradation. CONCLUSIONS: On the basis of the data presented in this collaborative study, Sample 2 (code 97/750) was established as the 2nd International Standard for hepatitis B virus DNA for NAT-based assays with a potency of 10(6) IU/ml (500,000 IU/vial).

Deficiencies in the standardization and sensitivity of diagnostic tests for hepatitis B virus.

The patterns of hepatitis B virus (HBV) markers described in textbooks apply to acute and chronic infection with wild-type HBV. Deviations from these patterns occur in the very early phase, in low-level (or occult) infection and under immunosuppression. Variability may originate from the virus, the host or the test kits. In order to obtain a reliable diagnosis under these conditions, tests for all three markers of HBV infection have to be applied: HBsAg, HBV DNA and anti-HBc. All tests should be as sensitive as feasible, but even then occult infection may be missed. Reliable detection of occult or mutated HBV is particularly important in blood and organ donors and in patients before or with immunosuppression.

Outbreak of hepatitis B in a nursing home associated with capillary blood sampling.

In 2001, two residents of a nursing home in Lower Saxony, Germany, were diagnosed with acute hepatitis B virus (HBV) infection. A systematic contact investigation of 188 residents yielded 19 confirmed or probable cases of acute or recent HBV infection and three persistent asymptomatic HBsAg carriers. Sequence analysis revealed that one carrier had high viraemia (109 genomes/ml), HBV genotype A2, and the same S gene and/or X gene sequence as 16 acutely infected persons. An unmatched case-control study was conducted with the 17 cases that had sequence identity together with 26 controls. The strongest association was found for treatment by a particular general practitioner (GP) (OR > 11, P < 0.001) and blood sampling for glucose monitoring on a particular day by the GP's staff (OR 13.6, P < 0.001, adjusted OR 8.5, P = 0.017). Control measures were implemented. Serological controls after 6 and 18 months revealed that the outbreak was brought under control.

Alternative methods for validation of cell culture infection with duck hepatitis B virus.

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.

Molecular epidemiology of hepatitis B, C and D viruses in Turkish patients.

Different genotypes of the hepatitis viruses may influence the clinical outcome of the disease. The distribution of genotypes may vary according to geographical regions. The aim of this study was to evaluate hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) genotypes in Turkish patients with chronic hepatitis in a large cohort of patients. Genotyping was performed in 41, 59 and 365 patients with chronic hepatitis B, D and C, respectively, and 36 hemodialysis patients with chronic hepatitis C. Genotypes were determined by direct sequencing in hepatitis B and by polymerase chain reaction-restriction fragment length polymorphism in hepatitis C and D patients. In addition, HBV subtyping by multiplex PCR and subtype specific ELISA were performed in 83 and 71 HBsAg (+) blood donors, respectively. All hepatitis B (100%) and hepatitis D (100%) patients had genotype D and type I, respectively. HBsAg subtyping by two methods yielded that 99% of the patients were subtype ayw. S gene amino acid sequence in the 41 patients included for HBV genotyping revealed the ayw2 subtype. Genotype distribution of 365 patients with chronic C hepatitis were as follows: 306 (84%) patients genotype 1b, 43 (11%) patients genotype 1a, 10 (3%) patients genotype 2, 3 (1%) patients genotype 3, 3 (1%) patients genotype 4. Among 36 patients receiving hemodialysis, 28 (78%) patients had genotype 1b and 8 (22%) patients had genotype 1a. The study indicates that Turkish patients with chronic viral hepatitis show very little genotypic heterogeneity. Subtype ayw and the genotype D of HBV DNA, and the type I of HDV RNA represent almost 100% of related infections. The genotype 1b of HCV RNA was found to be significantly dominant in Turkish patients.

[Hepatitis B and C. Risk of transmission from infected health care workers to patients]. / Hepatitis B und CUbertragungsgefahr auf Patienten durch infiziertes medizinisches Personal.

Hepatitis B and C viruses (HBV, HCV) cause chronic infections and high viremia which often remain undetected. Health care workers have an elevated risk of acquiring HBV or HCV when performing exposure-prone procedures and transmitting these viruses to patients. The extent of viremia varies considerably in different carriers and the risk of transmission is different for the various procedures. According to reports from the last 15 years, highly viremic HBV carriers with HBeAg transmit the virus on average to 4% of their patients when performing operations with high risk of injuries. HBeAg-negative surgeons with a viremia between 10(6) and 10(7) HBV DNA molecules/ml transmit to 1.5% of the patients. The absence of reports on proven transmission caused by viremia <10(5) molecules/ml suggests a residual risk below 1:100,000 that a surgeon with lower virus load transmits to one patient within 15 years. Eight cases were reported where HCV-infected surgeons transmitted the virus to 0.15% of their patients (17/11,119) and had (as far as tested) around 10(6) HCV RNA molecules/ml or more. Current recommendations of the relevant professional associations and institutions require vaccination of medical staff against HBV with control of immunity, regular examinations of staff for HCV, and in cases of missing immunity for HBV. Infected staff with viremia must either abstain from exposure-prone procedures or have a decision from an expert committee on the acceptability of such procedures in view of the individual infection status.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in health care workers (HCWs): guidelines for prevention of transmission of HBV and HCV from HCW to patients.

The transmission of viral hepatitis from health care workers (HCW) to patients is of worldwide concern. Since the introduction of serologic testing in the 1970s there have been over 45 reports of hepatitis B virus (HBV) transmission from HCW to patients, which have resulted in more than 400 infected patients. In addition there are six published reports of transmissions of hepatitis C virus (HCV) from HCW to patients resulting in the infection of 14 patients. Additional HCV cases are known of in the US and UK, but unpublished. At present the guidelines for preventing HCW to patient transmission of viral hepatitis vary greatly between countries. It was our aim to reach a Europe-wide consensus on this issue. In order to do this, experts in blood-borne infection, from 16 countries, were questioned on their national protocols. The replies given by participating countries formed the basis of a discussion document. This paper was then discussed at a meeting with each of the participating countries in order to reach a Europe-wide consensus on the identification of infected HCWs, protection of susceptible HCWs, management and treatment options for the infected HCW. The results of that process are discussed and recommendations formed. The guidelines produced aim to reduce the risk of transmission from infected HCWs to patients. The document is designed to complement existing guidelines or form the basis for the development of new guidelines. This guidance is applicable to all HCWs who perform EPP, whether newly appointed or already in post.

Molecular approaches to validate disinfectants against human hepatitis B virus.

Disinfection is an important measure to prevent hepatitis B virus (HBV) transmission by instruments. However, virucidal testing of disinfectants against HBV is difficult, because no simple quantitative infectivity assay exists. Since molecular changes of viral epitopes and the genome may indicate virus inactivation, we measured the alteration of these constituents with 0.065% peracetic acid (PAA) for exposure times up to 1 h. Plasma of a chronic HBV carrier with 10(9) HBV genomes/ml served as viral source in the form of a 10% dilution or of a purified HB-antigen preparation. Alterations of HBV epitopes were analyzed with four monoclonal antibodies in an enzyme-linked immunosorbent assay. Changes of the HBV genomes were determined by the inability to amplify the target sequence with a quantitative real-time polymerase chain reaction, either of a short fragment (189 bp) or of the full-length (3,200 bp). The determination of the epitope and genome alteration was quantified as log10 reduction factor (RF) with the parallel line bioassay. Under a high protein load of 10% human plasma, PAA induced a HBV genome alteration of RF = 1.5 after an exposure time of 60 min. Similar RFs were seen with the four HB epitopes. Without protein load, the alteration of these epitopes amounted to a RF of more than 3.5 within 30 min. Such inhibition of PAA activity by protein load was also seen in the virucidal tests with parvovirus. Although the RF were higher in the virucidal tests, the time-dependent dose-response curves for the epitope and genome alteration and for the infectivity inactivation followed the same inactivation kinetics. The molecular alteration and disintegration epitope and genome test may therefore be suitable to measure antiviral activity of disinfectants against HBV.

An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques.

BACKGROUND AND OBJECTIVES: Twenty-two laboratories from nine countries participated in an international collaborative study to establish a World Health Organization (WHO) international standard for hepatitis B virus (HBV) DNA nucleic acid amplification techniques (NAT). MATERIALS AND METHODS: Three samples, AA, BB (both of which were lyophilized) and CC (which was a liquid preparation), were analysed using several different NAT assays. The mean HBV DNA content of each sample was determined from the study. RESULTS: Despite the range of assays (commercial and in-house) used by participants, there was good agreement among the overall mean 'equivalents'/ml obtained by the different assays, except for one laboratory (laboratory 4). The variation in estimates of log10 'equivalents'/ml was 1.75-1.25 for the three samples if results from laboratory 4 were excluded. The mean log10 'equivalents'/ml for all laboratories were 6.42 for sample AA, 6.30 for sample BB and 5.03 for sample CC (exclusion of results from laboratory 4 made little difference). The difference in titres between the two lyophilized samples (AA and BB) was not statistically significant but the titre of the frozen sample (CC) was significantly lower. Material AA (code 97/746) was accepted as the first WHO international standard for HBV DNA NAT assays and assigned a potency of 10(6) international units (IU)/ml. CONCLUSIONS: The titres (genome equivalents/ml) of three HBV preparations were determined by several laboratories using different NAT assays. This study enabled the establishment of an international standard, 97/746, for HBV DNA NAT assays.