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.

Hepatitis C virus transmission through quarantine fresh-frozen plasma.

In 1994, quarantine fresh-frozen plasma (Q-FFP) was introduced in Germany in order to reduce the risk of HIV and HCV transmission. In 1998, an acute HCV infection of a patient was reported to us. The look-back revealed that this patient had received two Q-FFP from a donor who had seroconverted for HCV in the meantime. Recipients of further plasma donations from this donor were identified. Back-up specimens of these donations were investigated in several laboratories. A total of 25 additional HCV-PCR positive plasma units had been transfused to 12 further patients. HCV infections were diagnosed in seven of these recipients, three patients had already been deceased. One of the remaining two recipients was already HCV positive prior to transfusion, in the other patient, no HCV infection was detectable. This patient had received three units of an "early" plasma donation , which was tested negative by PCR in one laboratory, but positive in the other. The subsequent, clinically infectious donation had the same discrepant PCR results. Thus, eight cases of HCV transmission were revealed and classified as "certain" with regard to causality, also due to an identical HCV genotype, i.e. 3e. Some of these infections would have been prevented by application of a different anti-HCV assay. The assay used in the respective plasmapheresis station was in-sensitive in this individual case for more than 400 days after the first PCR positive donation. This caused the release of the above mentioned infectious units. Upon re-testing the backups, three of four other anti-HCV assays revealed a positive result already 104 days after the first PCR-positive donation. The donor had increased ALAT levels (> 23 IU/L) at nine of 28 donations, two of these were higher than 2.5 times the upper normal limit, and two were higher than 68 IU/L, which is the cut-off value for male blood donors in Germany. The results of these (look-back) studies arouse several queries, i.e. differences in the diagnostic sensitivity between current anti-HCV and PCR tests, the accuracy of risk-estimates (especially when based on hemovigilance studies for Q-FFP), the value of ALAT testing, and currently practised release algorithms for Q-FFP.

Testing of individual blood donations for HCV RNA reduces the residual risk of transfusion-transmitted HCV infection.

BACKGROUND: To allow cost-effective RNA testing with NAT techniques, the national authorities of several countries have planned or already introduced tests of mixed specimens, that is, plasma pools. STUDY DESIGN AND METHODS: High-throughput extraction, amplification, and detection of HCV RNA from individual blood donations were optimized and validated. The feasibility of the method and the frequency of anti-HCV-negative, HCV RNA-positive donations were determined in a prospective study of 27,745 allogeneic and 792 autologous individual donations. RESULTS: The 50- and 95-percent detection limits of the method were determined at 44 IU per mL and 162 IU per mL, respectively (World Health Organization HCV reference material). When 201 HCV RNA-positive sera were taken as a reference, the sensitivity was 97.5 percent. The assay specificity was determined at 99.77 percent. During a 20-month period, two seronegative blood donors tested positive in HCV PCR. The viral load of these donations was 6 x 10(6) and 3 x 10(7) copies per mL, respectively. Thus, the yield of HCV RNA testing in this study was 7. 63 per 100,000 screened donations (95% CI, 1.25-22.07). In both PCR-positive donors, seroconversion was found in subsequent blood samples. CONCLUSION: This study compares the feasibility of single-donation HCV RNA screening, with the detection of a relatively high percentage of window-phase donations, to data reported from groups using HCV RNA testing of plasma pools. The relative yield of NAT of individual donations versus minipools should be directly investigated in the near future.

Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

High-throughput extraction, amplification, and detection (HEAD) of HCV-RNA in individual blood donations.

BACKGROUND: High-throughput nucleic acid amplification techniques (NATs) are required for the detection of viral genomes in individual blood donations and might be helpful in any virological laboratory. OBJECTIVE: To develop and automate a method for the detection of hepatitis C virus RNA in individual blood donations, compatible with the time schedule of routine blood bank screening an product release. STUDY DESIGN: The viral RNA was isolated with the use of target specific capture oligonucleotides and magnetic beads. This extraction method was combined with reverse transcription/amplification (RT/PCR) and fluorescence detection. We adapted our method on a pipetting robot and pipetted all steps in a single room. When the pipetting was completed, microtiter plates were heat-sealed with foils and placed into a thermocycler. Positive reactions were detected with a fluorescent dye in a second room. Aerosols were avoided with programmed slow pipetting steps and with a special device constructed for the removal of the used disposable tips. During a 7 month period, we used this method in routine testing of individual donations prior to the release of all blood components. RESULTS: The total number of 11,700 individual donations including platelet concentrates were analysed. We tested up to 192 specimens in one run within 7 h. The frequency of cross-contamination using the automated procedure was 0.1%. Five specimens have been found repeatedly reactive for HCV-RNA, four of these were anti-HCV positive, one sample from a repeat donor was negative in anti-HCV assays. A seroconversion was detectable at his next presentation, 6 months later. CONCLUSION: In this pilot study, we demonstrate that automated HCV-RT-PCR testing is practicable for individual donations in high-throughput. Additionally, the described PCR approach could easily be adapted to the detection of other viral genomes by the use of specific primers.

Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations. Eurohep Pathobiology Group.

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up to a factor of 120. The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype adw2) and genotype D (ayw2/3), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion-derived DNA reacted much less efficiently. After identification and elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.4 x 10(9) HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.0 x 10(9) HBV DNA molecules/ml in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organization reference sample.

Low prevalence of hepatitis C virus antibodies and RNA in patients with myocarditis and dilated cardiomyopathy.

We investigated the prevalence of hepatitis C virus in patients with dilated cardiomyopathy (DCM) and myocarditis in comparison to a control group of patients suffering from noninflammatory cardiac diseases such as aortic stenosis. In contrast to the results of previous studies on small numbers of patients, no significant difference in the prevalence of hepatitis C infections was observed. Our data suggest that HCV is not an important causal agent for myocarditis and DCM.

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection.

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell responses in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers. However, deficient HBs-specific T and B cell responses were observed.

Capture and RT-PCR of hepatitis C virus RNA with safety primers.

The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3'-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories.

Analysis of the precore DNA sequence and detection of precore antigen in liver specimens from patients with anti-hepatitis B e-positive chronic hepatitis.

A number of naturally occurring hepatitis B virus (HBV) mutants unable to synthesize the hepatitis B e antigen (HBeAg) have been identified in patients characterized by HBV DNA and anti-HBe in their serum. Because the analysis of the HBV-associated DNA and antigens in the liver tissue is still not complete, we investigated the precore sequence of HBV DNA and its encoded proteins in the liver tissue of 32 patients positive for HBV DNA and anti-HBe in their serum. Three different groups of patients were identified. Group I (n = 14) was characterized by viral DNA sequences with a G-A transition in the distal precore gene region, thus creating a termination codon (TAG). Liver tissue from this group was negative for HBeAg but positive for hepatitis B core antigen (HBcAg) and a peptide containing the last 10 aminoacids of the precore and the first four aminoacids of the c region. Group II (n = 6) showed variable mutations in base sequences further upstream and negative for HBeAg and the precore peptide. Group III (n = 12) contained wild-type HBV DNA sequences in the tissue and positivity for precore- and core-encoded proteins. We conclude from our study that the group of patients with HBV DNA and anti-HBe in the serum is rather heterogeneous, including HBV DNA mutations in the precore region as well as infection with the wild-type virus being negative for HBeAg in the serum. The precore peptide is produced and expressed in the tissue even when the formation of a stop codon at base 1896 prevents the complete translation of the entire HBe protein.

Immunological responsiveness of Pekin ducks to core antigen of duck hepatitis B virus.

Cellular immune responses to the HBc or HBe antigen of hepatitis B viruses contribute to the pathogenesis of hepatitis B and to the elimination of the virus. Insufficient cytotoxic immune reactions against the core antigen may be one major reason for viral persistence in the absence of severe clinical symptoms. We attempted to stop viral persistence in the animal model of congenitally infected ducks by injection of recombinant DHBc particles, together with the strong immunostimulator Freund's adjuvant. However, the duck HBc antigen (DHBcAg)-treated ducks did not develop detectable liver disease, nor was the virus persistence affected. The congenitally infected ducks did not contain antibodies against DHBcAg before injection despite continuous production of duck hepatitis B virus, and developed only a weak transient antibody response after injection of recombinant DHBcAg together with Freund's adjuvant. Noninfected ducks developed, in contrast, a strong antibody response to the injected DHBcAg. We conclude that congenitally infected ducks are immunotolerant to DHBcAg and cannot be cured by immunotherapy with exogenous recombinant DHBcAg.

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product.

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.

Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission.

A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.

Studies on the attachment and penetration of hepatitis B virus.

There are two identified liver-specific attachment sites in the preS2 domain and one in the preS1 domain. Which mechanism leads to attachment in vivo is not known. The subsequent penetration seems to require proteolysis which does not occur spontaneously in HepG2 cells, but presumably in vivo. The role of the small HBs protein for attachment remains enigmatic so far, but it must have a function because an escape mutant exists against a monoclonal antibody which binds to an epitope of the small protein. The occurrence of this escape mutant in vaccinated persons proves that the standard hepatitis B vaccine does induce neutralizing antibodies, but it also suggests very strongly that the neutralizing preS epitopes be included in future hepatitis B vaccines.

Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus.

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

Association of hepatitis C virus in human sera with beta-lipoprotein.

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03-1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19-1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-beta lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-beta lipoprotein. In 3 sera no significant precipitation could be observed.

Functions of hepatitis B surface proteins.

HBV surface proteins play a number of functional roles in cellular infection, viral synthesis and in immune responses of the host. Three coterminal proteins of differing sizes and three subdomains of the individual molecules can be recognized. In this brief review, functions of the various proteins and domains are described and their significance as potential immunogens is discussed. Although it is apparent that the surface proteins are involved in the development of persistent HBV infections, the underlying mechanisms of liver involvement remain unknown.

Absence of free core antigen in anti-HBc negative viremic hepatitis B carriers.

Using enzyme immune assay and immune electron microscopy, we have examined the sera of immune-suppressed anti-HBc negative HBV-infected patients for the presence of HBcAg. Our results suggest that free HBV core particles are absent or present only in minute amounts in the blood of chronic carriers and that at the most, only minimal amounts of core antigen are found on the surface of the virus particles.

Analysis of viral proteins in circulating immune complexes from chronic carriers of hepatitis B virus.

Sera from 54 children (mean age 5.8 years) with chronic hepatitis B virus (HBV) infection were investigated for the presence of immune complexes containing HBV proteins. Clinical diagnosis was established by histology and biochemical markers and included chronic persistent (36 cases) or chronic aggressive (seven) hepatitis, liver cirrhosis (six) and HBV-mediated membranous glomerulonephritis (five). Circulating immune complexes were precipitated with 2.5% polyethylene glycol and analysed by immune blot using monoclonal antibodies against S, pre-S2 glycopeptide, pre-S1 and HBe/c epitopes. All sera, including those from 11 healthy HBV-negative blood donors contained PEG-precipitable substances, but the amount of precipitate did not correlate with the presence or amount of HBV proteins. The great majority (36 out of 40) of HBeAg-positive patients contained HBs proteins in immune complexes, but no detectable HBe protein. The immune complexes usually contained more pre-S1 than the free HBsAg particles from the same patient. The precipitates of anti-HBe-positive patients rarely contained HBV proteins (two out of 14) and, if so, in low amounts. During follow up of six patients we found that high levels of HBs-containing immune complexes may be correlated with subsequent elimination of HBV. This elimination is possibly initiated by binding of anti-pre-S1 antibodies to HBV and HBs particles.