Hepatitis B escape mutants in Scottish blood donors

Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site.     

HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.     

Surveillance for hepatitis B surface antigen mutants

Hepatitis B viral (HBV) mutants can emerge in patients as a result of selection pressure from treatment options. Some mutations that occur in the immunodominant "a" determinant of hepatitis B surface antigen (HBsAg) can present as false negative results in HBsAg immunoassays. The mutation position in HBsAg and the type of mutation impacts immunoassay performance. HBsAg mutants will continue to emerge in response to selection pressure, therefore an appropriate HBV immunoassay-testing algorithm needs to be established to ensure their detection. Mutant surveillance programs can also contribute to our understanding of the changing epidemiology of HBV infection.     

Genetic variability of the S gene of hepatitis B virus: clinical and diagnostic impact.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergence in the entire genome of >8%, HBV genomes have been classified into eight groups designated A to H. The genotypes of HBV have distinct geographical distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and natural history of infection and response to antiviral therapy, further evaluations on larger collectives of patients are necessary to give a clearer picture of the subject. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NAT) has so far been poorly investigated. Preliminary results show that new real-time NAT detect genotypes A to G with an equal sensitivity. Different mechanisms intervening at the translational or post-translational level, including conformational changes, hydrophobic changes, insertion of basic residues and reduced synthesis or secretion of HBsAg may account solely or in conjunction for escape mutations to the immune response and to detection in HBsAg immunassays. The clinical significance of S-gene mutants, needs in analogy to that of HBV genotypes, to be further investigated. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is continuously improved. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S-gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants and strategies for the (differential) screening of mutants need to be developed and evaluated.     

Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS HBsAg assay

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.     

Occult hepatitis B virus infection.

The detection of HBV DNA without HBsAg with or without the presence of HBV antibodies outside the acute phase window period defines occult HBV infection. This condition has been described in hepatocellular carcinoma (HCC), chronic hepatitis B, healthy HBV carriage and recovered infection, chronic hepatitis C and individuals without serological markers of HBV. The frequency of the diagnosis depends on the relative sensitivity of both HBsAg and HBV DNA assays. It also depends on the prevalence of HBV infection in the population. Occult HBV in blood donors has a wide range of potential origins within the natural history of the infection. It may originate from recovered infections with anti-HBs and persistent, low-level, viral replication, escape mutants undetected by the HBsAg assays or healthy chronic carriage. The last situation is mostly found with anti-HBc only. Over time, antibody markers may become undetectable leaving HBV DNA as the only marker of the infection. In all cases, the viral load is low, mostly below 10(4) IU/ml, often below 100 IU/ml. At these levels, nucleic acid testing (NAT) in pools is likely to be largely ineffective. Is occult HBV transmissible by transfusion? Carriers of anti-HBs or anti-HBc only were shown infectious in immunosuppressed organ or bone marrow transplant recipients. In immunocompetent recipients, there is no evidence that anti-HBs-containing components are infectious, even in low titre. Donations carrying anti-HBc only and HBV DNA can be infectious and this is a threat where anti-HBc is not screened. Anti-HBc screening identifies most occult HBV infection but not all. HBV NAT needs either extreme sensitivity or to be performed on individual donations to eliminate HBV DNA-containing units.     

Hepatitis B virus genotype E surface antigen detection with different immunoassays and diagnostic impact of mutations in the preS/S gene

The major neutralizing epitope, the “a” determinant of the hepatitis B virus (HBV) genotype E surface antigen (HBsAg) is most divergent from that of genotype A, which is used for preparing monoclonal antibodies used in commercially available HBV reagents. To evaluate the performance of the latest generation of HBsAg detection assays with respect to genotype E HBsAg. Three commercial assays were evaluated using sera from 200 Nigerian patients compared to the preS/S sequence of DNA positive samples. Out of 200 samples, 61 and 103 gave concordant positive and negative results between the three HBsAg assays. Of 36 samples with discordant results, 35 were confirmed negative by neutralisation. One of the three assays showed significantly high rate of false positives (29 of 35). DNA positive samples with no detectable HBsAg or reduced HBsAg detection signals (<75% of mean signal obtained with HBsAg positive samples) revealed several mutations (V14A, F46S, N48T, L49R, I49T, D51G, A53V, P54L, Q82P, F83C, L127P, A184V, T189I, S204N, V224A), mostly outside the a-determinant. Several of these mutations are found as wild type nucleotides normally in genotype A and only exceptionally in genotype E. All three assays showed comparable sensitivities for genotype E HBsAg detection (98.4–100%) but differed considerably in specificity (84–99%). Failure to detect HBsAg antigen and differences in signal intensity were mainly associated with mutations in the preS/S gene outside the “a” determinant.     

Acute hepatitis B caused by a vaccine-escape HBV strain in vaccinated subject: Sequence analysis and therapeutic strategy

HBV vaccine contains the ‘a’ determinant region, the major immune-target of antibodies (anti-HBs). Failure of immunization may be caused by vaccine-induced or spontaneous ‘a’ determinant surface gene mutants. Here, we evaluate the possible lack of protection by HBV vaccine, describing the case of an acute hepatitis B diagnosed in a 55-year-old Caucasian male unpaid blood donor, vaccinated against HBV. Sequencing data for preS–S region revealed multiple point mutations. Of all the substitutions found, Q129H, located in the “a” determinant region of HBsAg, can alter antigenicity, leading to mutants. This mutant may cause vaccine failure especially when associated with high viremia of infecting source.     

抗-HBc单项阳性患者中的隐匿性HBV感染

目的 研究抗-HBc单项强阳性患者中的隐匿性HBV感染发生率并分析发生原因.方法 收集183例血清学检测抗-HBc单项强阳性（A≤0.1）患者血清标本,采用实时定量PCR进行HBV DNA含量检测.对于DNA定量阳性的标本,PCR扩增HBV pre-S/S区基因,并进行克隆测序.结果 183例血清标本中3例HBV DNA定量结果大于103拷贝/ml,占1.6%.这3例标本中有2例得到pre-S/S区测序结果,2例标本均存在S基因＂a＂决定簇内Q129R/P点突变,且突变株与野生型共存.结论 抗-HBc单项阳性患者中存在隐匿性HBV感染,HBsAg血清免疫学方法的漏检与HBV S基因突变有关,同时与循环中HBsAg量低于检测限也有一定关系.HBV隐匿感染不仅可以造成临床诊断失误,更为严重的是献血员HBV隐匿感染造成血液的污染.     

Restoration of replication phenotype of lamivudine-resistant hepatitis B virus mutants by compensatory changes in the "fingers" subdomain of the viral polymerase selected as a consequence of mutations in the overlapping S gene

The introduction of lamivudine (LMV) for the treatment of chronic hepatitis B infection has been an important advance in the management of this disease. However, the long-term efficacy of LMV may become limited by the emergence of antiviral-resistant hepatitis B virus (HBV) mutants. The two most common LMV-resistant mutants produce changes in the viral polymerase protein (rt) of rtM204I and rtL180M/M204V (previously rtM550I and rtL526M/M550V). A number of studies have demonstrated that these HBV mutants appear to be replication impaired, both in vitro and in vivo. The detection and selection of compensatory mutations in the polymerase protein that restore the replication phenotype of these HBV mutants have been poorly described to date. The effects of mutations in the fingers subdomain of the viral polymerase protein arising as a consequence of vaccine and hepatitis B immune globulin (HBIg) selected changes in the overlapping envelope gene (S), and a determinant of the hepatitis Bs antigen (HBsAg) were analyzed in vitro. The LMV-resistant HBV mutants rtM204I and rtL180M/M204V produced substantially weaker HBV DNA replicative intermediate signals by Southern blot analysis and less total intracellular HBV DNA by real-time PCR compared to wild-type virus. The viral polymerase protein of these mutants produced little detectable radiolabeled HBV DNA in an endogenous polymerase assay. In contrast, the HBV a determinant HBIg/vaccine escape mutants sP120T, sT123N, sG145R, and sD144E/G145R (that produce rtT128N, Q130P, rtW153Q, and rtG153E respectively) yielded as much virus as wild-type HBV while the sM133L (rtY141S) mutant was replication impaired. Two of these mutants, rtT128N and rtW153Q, when introduced into a replication-competent HBV vector containing the rtL180M/M204V polymerase mutation restored the replication phenotype of this LMV-resistant mutant. These viruses produced levels of intracellular HBV DNA as determined by Southern blot and real-time PCR that were comparable to those of wild-type HBV, indicating that the changes in the fingers subdomain were able to compensate for the reduced replication of the LMV-resistant mutations. Since these viruses carry mutations in the a determinant of HBsAg that may potentially decrease the ability of anti-HBs antibody to neutralize these viruses, these HBV mutants also have the potential to behave as vaccine escape mutants.     

Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.     

Reactivation of hepatitis B virus infection with persistently negative HBsAg on three HBsAg assays in a lymphoma patient undergoing chemotherapy

In patients with occult hepatitis B virus (HBV) infection, acute exacerbation may occur when they become immunocompromised. Usually, these patients develop hepatitis B surface antigen (HBsAg) seroreversion during the flare. Here we report on a patient with occult HBV infection, who developed HBV exacerbation after chemotherapy for diffuse large B-cell lymphoma. The resurgence of HBV DNA preceded the elevation of liver enzymes for 20 weeks. Atypically, despite high viraemia, serological tests showed persistently negative HBsAg using three different sensitive HBsAg assays (i.e., Architect, Murex and AxSYM). On comparing the amino acid sequence of the index patient with the consensus sequence, five mutations were found at pre-S1, five at pre-S2 and twenty-three mutations at the S region. Six amino acid mutations were located in the ‘a’ determinant, including P120T, K122R, M133T, F134L, D144A and G145A. The mutants K122R, F134L and G145A in our patient have not been tested for their sensitivity to Architect and Murex assays by the previous investigators and might represent the escape mutants to these assays.     

Unusual naturally occurring humoral and cellular mutated epitopes of hepatitis B virus in a chronically infected argentine patient with anti-HBs antibodies

Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of the S and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate to genotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside the major hydrophilic region, while the sixth clone exhibited a genotype A "wild-type" amino acid sequence. Observed replacements included both humoral and/or cellular (major histocompatibility complex class I [MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F, P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within a given clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymous mutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for the emergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the first loop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community, since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively prevent the liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence the accuracy of the results obtained with currently used diagnostic tests.     

Hepatitis B virus markers in anti-HBc only positive individuals.

Isolated reactivity to hepatitis B virus (HBV) core antigen (anti-HBc) is observed relatively frequently in immunocompromised individuals, intravenous drug abusers (IVDA), and in the presence of HCV infection. The reason for the lack of HBsAg is not clear. The aim of the present study was to investigate which factors (genetic variability of S gene, low-level HBsAg, and immune complexes may be responsible for the failure of HBsAg detection with commercial HBsAg screening assays. Dilution series of two recombinant HBsAg escape mutants and dilutions of serum samples from chronic HBV carriers with multiple insertions in the a determinant and different HBsAg subtypes were tested with a highly sensitive assay that detects wild-type HBsAg (Elecsys HBsAg, Roche Diagnostics, Penzberg, Germany) and two assays that detect HBV wild-type and escape mutants (Murex HBsAg Version 3, Murex and Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany). Elecsys HBsAg showed in comparison to Murex HBsAg Version 3 and Enzygnost HBsAg 5.0 a reduced sensitivity for escape mutant detection. On the other hand, the best performance for HBsAg subtype detection was obtained with Elecsys HBsAg. In the second part of the study, a selected panel of isolated anti-HBc reactive (n = 104) serum samples (AxSYM Core) was submitted to testing by Elecsys HBsAg, Murex HBsAg Version 3, Enzygnost HBsAg 5.0, and HBsAg detection after immune complex dissociation (ICD) and anti-HBs determination with two different assays (AxSYM Ausab and Elecsys Anti-HBs). To assess the specificity of anti-HBc test results, all the samples were tested by a second anti-HBc assay (Elecsys Anti-HBc). Quantitative HBV DNA detection was undertaken with a commercially available HBV PCR assay (Amplicor HBV Monitor). HCV infection was present in 65.4% of anti-HBc only reactive individuals. Five AxSYM Core positive samples were negative by Elecsys Anti-HBc. Overall, 15 (14.4%) AxSYM Ausab negative samples gave positive results with Elecsys Anti-HBs (median value: 21 IU/ml). No low-level HBsAg carrier was detected among the isolated anti-HBc reactive individuals with Elecsys HBsAg. There was no evidence for the presence of immune complexes. Only one sample was repeatedly reactive by the Murex HBsAg, suggesting that the a mutant form of HBsAg was responsible for the isolated anti-HBc reactivity, however neutralisation assay was not interpretable and HBV DNA PCR was negative. Fifteen (14.4%) anti-HBc only positive individuals were HBV DNA carriers with concentrations ranging from 800 to more than >4,000,000 copies of viral DNA/ml. In conclusion, the most probable explanations for isolated anti-HBc reactivity in our study group are a possible interference of HBsAg synthesis by HCV infection (65.4%) and divergence of results of anti-HBs assays (14.4%). There is no evidence for the presence of low-level HBsAg carriers and immune complexes. HBsAg mutants cannot be excluded definitively by the test strategy used in the present evaluation.     

Infections by hepatitis B surface antigen gene mutants in Europe and North America

Hepatitis B virus (HBV) mutants have usually been studied in patients in Asia because of the wider use of HBV immunization there and the resultant emergence of viral mutants. Nevertheless, HBV surface antigen (S) gene mutants also are found in Europe and North America. In Europe and North America, HBV with mutations in the portion of the S gene coding the "a" determinant of the hepatitis B surface antigen (HBsAg) have been documented in small numbers of infants born to HBV-infected mothers following post-natal HBV vaccine and hepatitis B immune globulin (HBIG) prophylaxis and in many liver transplant recipients who develop HBV re-infection despite HBIG prophylaxis. In some cases, these mutations have included a glycine to arginine substitution at position 145 (G145R), which results in a conformational change and different reactivity to monoclonal antibody reagents than that of the wild-type virus. Mutations in the a determinant (but not G145R) also have been reported in European patients with chronic HBV infection who have not received HBV vaccine or HBIG. However, it appears that such mutations are only responsible for a small proportion of "occult" or "silent" HBV infections, which are characterized by the presence of HBV DNA in serum in the absence of detectable HBsAg. However, some of these mutant forms of HBV in cases of occult HBV may theoretically escape detection and could present a risk to blood safety.     

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

BACKGROUND: In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. RESULTS: In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. CONCLUSION: The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.     

多重PCR归化法平行检测HBV和HCV的研究

目的：建立一种多重PCR归化法并应用于对HBV、HCV平行检测。方法：利用PCR反应5′端允许添加非互补序列的原理，运用内外两对引物，经过2轮扩增，使目的产物均带上共有序列，再以共有序列为引物进行扩增，实现多重扩增。比较和筛选四种核酸提取方法。运用正交优化法，优化并确定最佳扩增条件。对28份血标本进行对比试验，并进行质量评价。结果：归化多重PCR方法对于HBV、HCV病毒合并感染患者的诊断敏感性为83.5%，诊断特异性为70.0%，诊断指数为153．3％，诊断效率为72．2％；对HBsAg阳性患者的HBV DNA的诊断敏感性为78．6％，诊断特异性为80．0％，诊断指数为158．6％，诊断效率为79．2％。对抗－HCV阳性患者的HCV RNA的诊断敏感性为75．0％，诊断特异性为90．0％，诊断指数为165．0％，诊断效率为83．3％。结论：多重PCR归化法在多基因扩增或多种病原体的同时平行检测领域具有较大应用潜力。该方法实用、准确、可靠，对HBV HCV的防治具有实际意义。     

Novel immunoassay for the detection of hepatitis B surface 'escape' mutants and its application in liver transplant recipients

Hepatitis B virus (HBV) strains with mutations in the surface gene are responsible for the failure of prophylaxis with hepatitis B immunoglobulin (HBIG) in a proportion of patients transplanted for HBsAg positive cirrhosis. So far, the emergence and evolution of these 'surface antibody escape' mutants have been studied by DNA sequencing. In this study the use of an immunoassay is described for diagnosis and characterisation of HBV recurrence after liver transplantation (OLT), based on a monoclonal antibody able to recognise both wild-type and mutant HBsAg. Pre- and post-transplant samples from 22 patients transplanted for HBsAg positive cirrhosis were studied: Group A: 12 patients who reinfected the graft despite receiving HBIG; Group B: 6 patients with no HBV recurrence with continuous HBIG; Group C: 4 patients with HBV recurrence without prophylaxis. By running the new assay in parallel with an immunoassay that is susceptible to HBsAg mutants, 4 of 12 cases were identified in Group A with HBV recurrence due to surface antibody escape mutants, whereas in 8 patients this was due to the wild-type HBV. The results from the immunoassays were confirmed in all cases by HBV DNA sequencing. The surface gene mutations in the 4 patients affected codons 144 and 145 and in one of these 4 patients HBV strains with mutations in both codons were detected before and after transplantation. The epitope recognised by the new monoclonal antibody that reacts with both wild-type and mutant HBsAg seems to remain stable in the HBIG-induced HBV mutants. This serological approach allows rapid and cost-effective screening for HBsAg escape mutants in the liver transplant setting and may be helpful in the selection of appropriate prophylaxis.     

European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty‐seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut‐off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers. J. Med. Virol. 83:95–100, 2011. © 2010 Wiley‐Liss, Inc.     

Improved Detection of Hepatitis B Virus Surface Antigen by a New Rapid Automated Assay

The performance of hepatitis B virus (HBV) surface antigen (HBsAg) screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B. In a multicenter study, a new automated rapid screening assay, Elecsys HBsAg (Roche Diagnostics), was compared to well-established tests (Auszyme Monoclonal [overnight incubation] version B and IMx HBsAg [Abbott]). Included in the evaluation were 23 seroconversion panels; sera from the acute and chronic phases of infection; dilution series of various HBsAg standards, HBV subtypes, and S gene mutants; and isolated anti-HBV core antigen-positive samples. To challenge the specificity of the new assay, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. Elecsys HBsAg showed a higher sensitivity for HBsAg subtypes ad, ay, adw2, adw4, ayw1, ayw2, ayw4, and adr detection in dilution series of different standards or sera than Auszyme Monoclonal version B and/or IMx HBsAg. Acute hepatitis B was detected in 11 to 16 of 23 seroconversion panels between 2 and 16 days earlier with Elecsys HBsAg than with the alternative assays. Elecsys HBsAg and Auszyme Monoclonal version B detected HBsAg surface mutants with equal sensitivity. The sensitivity and specificity of Elecsys HBsAg were 100%. Auszyme Monoclonal version B had a 99.9% specificity, and its sensitivity was 96.6%. IMx HBsAg showed a poorer sensitivity and specificity than the other assays. In conclusion, Elecsys HBsAg permits earlier detection of acute hepatitis B and different HBV subtypes than the alternative assays. By using highly sensitive HBsAg screening assays, low-level HBsAg carriers among isolated anti-HBV core antigen-positive individuals can be detected.