INTRODUCTION

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The envelope protein of hepatitis B virus (HBV), hepatitis B surface antigen (HBsAg), is a glycosylated lipoprotein usually shed in large amounts in the serum of infected individuals, where it is found as spherical particles with diameters of 22 nm or filaments of similar diameter (39). The a determinant of HBsAg, a predicted double-loop structure projecting from the surface of the HBV particle (36), is the major neutralizing epitope. Antibodies to the a determinant confer protection in adults against all the common subtypes of HBV. Subtype determinants d or y and w or r are also located within the predicted loop regions. Nine serotypes have been described (10). These have been related to six genomic groups, A through F, based on sequencing of the S gene of isolates from different geographical regions (26, 27).

HBsAg is one of the first serum markers to appear during the course of HBV infection and can be detected 2 to 8 weeks before biochemical evidence of liver dysfunction and the onset of jaundice. HBsAg is cleared within a few months in self-limiting illness. If HBsAg persists for more than 6 months, spontaneous clearance is very improbable and the infected individual is considered a chronic HBV carrier.

Among the many commercially licensed HBsAg assays available, enzyme-linked immunosorbent assays are currently the most frequently used. These assays use either monoclonal or polyclonal anti-HBs bound to a solid phase and a second labelled anti-HBs to detect the captured antigen. Despite the high-level performance of screening assays, transfusion-associated HBV infection is still reported (17, 21). There are three possible explanations of false-negative results in commercial assays. In chronic HBV carriers, the HBsAg level may be below the detection limit, i.e., a high proportion of individuals with antibodies against HBV core antigen (anti-HBc) as the only serological marker of infection are low-level chronic carriers of the virus (14, 20). Another explanation is that virus variants yield sequences that are not recognized by the antibodies employed in the assays. In different geographic locations, vaccine-escape mutants are emerging under the selective pressure of active immunization, and there is a danger that they will become dominant strains as vaccination becomes universal (6, 19). Breakthrough infections due to point mutations of the a determinant have been identified in Europe, Africa, and Asia (8, 13, 28, 40). Vaccine-escape mutants within the a determinant of the S gene are not recognized as effectively by conventional diagnostic tests as the wild-type particle (9, 18). A third possible explanation is that there are variants in other parts of the genome that downregulate the production of HBsAg (7).

To reduce the residual risk of transfusion-associated hepatitis B, the sensitivity of HBsAg screening assays is continuously improved. Elecsys HBsAg (Roche Diagnostics, Penzberg, Germany) is a new, fully automated, rapid assay which permits the qualitative detection of HBsAg directly from the patient blood collection tube in a total incubation time of 18 min. In a multicenter study, it was compared with two alternative well-established serological assays.

	MATERIALS AND METHODS

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Elecsys HBsAg. Elecsys HBsAg is a two-step sandwich assay for the qualitative detection of HBsAg in human serum or plasma, with a total incubation time of 18 min. All the reaction steps of Elecsys HBsAg are performed automatically by the Elecsys 2010 system. The assay can be performed directly from the primary patient blood collection tube, and random access is possible.

Elecsys HBsAg confirmatory test. Repeatedly HBsAg-reactive samples are preincubated during 30 min at room temperature or overnight at 2 to 8°C in a ratio depending on the index value with anti-HBs-positive plasma in order to confirm the presence of HBsAg by specific antibody neutralization. In parallel, the same sample volume is incubated with control reagent (anti-HBs-negative serum). If HBsAg is present in the specimen, it will form an immune complex with the blocking anti-HBs antibodies, thereby reducing the number of binding sites available for the biotinylated and ruthenylated antibodies of Elecsys HBsAg. A specimen is considered positive if the reduction in absorbance of the neutralized specimen is 50% or greater compared to that of the nonneutralized control.

Comparative assays. Elecsys HBsAg was compared to two antigen detection assays, Auszyme Monoclonal and IMx HBsAg (Abbott, Delkenheim, Germany). For the Auszyme Monoclonal assay, overnight incubation was performed (version B) in order to achieve a high level of sensitivity, and the assay will be referred to hereafter as Auszyme Monoclonal B.

HBV DNA PCR. HBV DNA PCR was performed as described by Berger et al. (1). Briefly, DNA was extracted from serum samples (25 µl) by using the QIAmp blood kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. For DNA amplification, a nested PCR protocol was performed. For the first PCR, primers HBV1 (5'-TCg TgT TAC Agg Cgg ggT TT) and HBV2 (5'-CgA ACC ACT gAA CAA Atg gC) flanking a 513-bp fragment of the S genome region were used. With the inner primers HBV3 (5'-gCC AAA ATT CgC AgT CCC CA) and HBV4 (5'-AgA TgA ggC ATA gCA gCA gg), a 129-bp sequence was amplified. The reaction mixture (50 µl) consisted of 5 µl of extracted DNA, 5 µl of 10× PCR buffer (Boehringer Mannheim, Mannheim, Germany), 5 µl of deoxynucleoside triphosphate (dNTP) mix (2 mM), 1 µl of each primer, and 0.4 µl of Taq polymerase (5 U/µl) in 32.6 µl of H₂O. Amplification was performed with a Gene Amp 9600 device (Perkin-Elmer, Norwalk, Conn.) and involved 30 cycles in which primers were denatured at 95°C (30 sec), annealed at 55°C (30 sec), and extended at 72°C (45 sec). Amplificates were visualized by 4% agarose gel electrophoresis and ethidium bromide staining.

Specimens. The intra- and interassay precision was assessed by testing replicates of calibrators, controls, and positive and negative serum samples (Tables 1 and 2). The technical performance of Elecsys HBsAg was assessed by testing HBsAg-positive and HBsAg-negative samples spiked with hemoglobin, bilirubin, triglyceride, biotin, or rheumatoid factor and sera with increasing concentrations of IgG, IgA, and IgM.

Statistical analysis. The performance of Elecsys HBsAg was compared to those of Auszyme Monoclonal B and IMx HBsAg for the seroconversion panels. The mean number of days by which HBsAg was detected with Elecsys HBsAg in comparison to Auszyme Monoclonal B and IMx HBsAg was determined for the 23 seroconversion panels tested. The statistical significance of the results for each test was determined by using the Wilcoxon test for matched pairs (5). Anti-HBc, anti-HBc-IgM, anti-HBs, anti-HBe, and HBeAg determinations were performed for the resolution of discrepant results between HBsAg assays. Discordant samples were considered for the calculation of sensitivity and specificity only if results from at least three comparative assays and/or a neutralization assay were available. A sample was considered a true positive if it was repeatedly reactive in at least two assays and confirmed by neutralization assay (if enough sample material was available) and if it was anti-HBc positive and anti-HBs negative. For seroconversion panels and sensitivity panels, repeated reactivity in a single assay was considered as a true positive result. Conversely, a test result was interpreted as a true negative if it was negative with Elecsys HBsAg and in at least one comparative assay and confirmatory testing (if enough sample material was available) and if it showed one of the following serological constellations: anti-HBc and anti-HBs negative (HBV negative), anti-HBc and anti-HBs positive (resolved infection), or anti-HBc negative and anti-HBs positive (HBV vaccination).

	RESULTS

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Technical performance. The intra-assay precision was validated by testing 20 replicates of two calibrators, positive and negative controls, and one HBsAg-negative and two HBsAg-positive serum samples (Table 1). The intra-assay coefficients of variation (CV) ranged from 1.2 to 6.9%. The interassay reproducibility of Elecsys HBsAg was evaluated by testing in nine different runs the positive and negative calibrators, one positive and one negative control, one borderline-negative, one low-positive, and two negative serum samples (Table 2). The interassay CV ranged between 3.1 and 11.4%. No interference was observed with HBsAg-positive and -negative serum samples spiked with hemoglobin (up to 1.4 g/dl), triglycerides (up to 2,884 mg/dl), bilirubin (up to 30.9 mg/dl), and rheumatoid factor (up to 3,500 IU/ml) and samples with high concentrations of IgG (up to 6,900 mg/dl), IgM (up to 3,400 mg/dl), and IgA (up to 3,600 mg/dl). With biotin concentrations higher than 60 ng/ml, a weakly HBsAg-positive sample (0.06 IU/ml) was determined to be a false negative.

Sensitivity. Overall, Elecsys HBsAg showed a higher sensitivity than Auszyme Monoclonal B and IMx HBsAg for detecting HBsAg subtypes ad and ay in dilution series of HBsAg PEI and WHO standards, BBI HBsAg sensitivity panel PHA 805, and the Abbott sensitivity panel (Table 4). The calculation of the detection limit by interpolation yielded 0.014 PEIU/ml (0.033 IU/ml) and 0.017 PEIU/ml (0.055 IU/ml) for PEI standards ad and ay, respectively. No difference in sensitivity between Elecsys HBsAg and Auszyme Monoclonal B was observed for HBsAg-positive specimens 4/ay and 10/ad and BBI HBsAg low-titer panels 102 and 103 (Table 4). IMx HBsAg showed a higher detection limit in HBsAg low-titer panel 102 than in the alternative assays (Table 4). Two specimens of low-titer panel 103 tested negative with IMx HBsAg but were positive with Elecsys HBsAg and Auszyme Monoclonal B. 

HBsAg subtypes and mutants. Elecsys HBsAg showed a two- to eightfold-higher sensitivity than Auszyme Monoclonal B for subtypes ayw1, ayw2, ayw4, adw2, adw4, adrq⁺, and adrq of the CNTS panel (Table 6). Of the seven subtyped sera of various geographical origins, Elecsys HBsAg detected subtypes adw2 and ayw4 and the mixed subtype adr/ayr with a sensitivity better than that of Auszyme Monoclonal B (Table 6). No difference in sensitivity was observed between both tests for HBsAg surface mutants (Table 6). The insertion mutant was detected only at a low dilution of 1/5 with both assays.

Specificity. One sample of 490 preselected HBsAg-negative specimens from South African blood donors was repeatedly reactive and confirmed by the Elecsys confirmatory assay (Table 7). IMx HBsAg gave a negative result, while Auszyme Monoclonal B showed a very weak reactivity. The serological profile (anti-HBc positive, anti-HBc-IgM negative, anti-HBe positive, HBeAg negative, and anti-HBs negative) was compatible with an HBsAg-positive HBV carrier (see Table 9). Further, five samples were repeatedly weakly reactive with IMx HBsAg but negative in the neutralization assay and Auszyme Monoclonal B. All five serum samples showed the serological profile of resolved HBV infection (anti-HBs positive, anti-HBc positive, anti-HBe positive, and anti-HBc-IgM and HBeAg negative) (see Table 9).

	DISCUSSION

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The results of the present study show that Elecsys has a sensitivity better than those of well-established HBsAg screening assays. Furthermore, Elecsys HBsAg combines a high sensitivity (100%) and specificity (100%), whereas usually these criteria are mutually exclusive. By testing samples from a total of 4,120 unselected blood donors in different blood banks (supplied by A. Mühlbacher and U. Michl, Blutzentrale der Landeskrankenanstalten Salzburg, Salzburg, Austria; R. Camacho, Hospital Egas Moniz, Laboratorio di Imunohemoterapia, Lisbon, Portugal; and J. H. Hernandez, Banco de Sangre de ICS, Hospital Valle de Hebron, Barcelona, Spain), a specificity of 100% was obtained (data not shown).

The benefit of high sensitivity was particularly evident in seroconversion panels (Table 5). Elecsys HBsAg detected acute hepatitis B significantly earlier than the comparative assays. Elecsys was also more sensitive than the Monolisa Ag HBs second generation assay (Sanofi Pasteur, Marnes-la-Coquette, France). Among 15 commercially available HBsAg tests, including Auszyme Monoclonal B and IMx HBsAg, Elecsys HBsAg achieved the highest sensitivity in BBI seroconversion panels (29). Comparing the results obtained from 21 BBI seroconversion panels as reported by Palmer et al. (29) with our results, Elecsys HBsAg detected acute HBV infection earlier than the Monolisa Ag HBs second generation assay in 8 of 21 seroconversion panels. Elecsys HBsAg also showed a better performance in BBI seroconversion panels than an alternative new automated microparticle chemiluminescence immunoassay (16).

A high rate (2.4%) of false-positive results was obtained with IMx HBsAg for specimens from blood donors, hospitalized patients, pregnant women, and IVDAs (Tables 3, 7, and 9). In contrast, Auszyme Monoclonal B showed 99.9% specificity. Previous reports (31, 35) showed a false-positivity rate of 0.2 to 0.3% with Auszyme Monoclonal B. Nonspecific reactivity may be reduced by overnight storage of samples at 2 to 8°C (31).

For the resolution of discrepant results of HBsAg screening assays, it is important to consider additional HBV markers. HBsAg in combination with anti-HBc determination shows a positive and negative predictive value of 100%. However, during acute hepatitis B, anti-HBc may not be detected in the first days to weeks in up to 8% of the cases (24). Another explanation for isolated HBsAg-positive results may be the absence of anti-HBc antibody response as a consequence of iatrogenic or acquired immunodeficiency (25). Occasionally, anti-HBc reactivity is absent in patients with selective immunodeficiency or immunocompromised HBsAg carriers (11, 23, 24, 30).

Depending on the prevalence of HBV infection and the patient group investigated, 1 to 32% of positive anti-HBc results are isolated positive findings (15). Isolated anti-HBc reactivity is frequently observed in IVDAs, HIV-infected individuals (32, 33), HBV and HCV coinfected patients (20), and pregnant women (14). Several reports have shown that 10 to 40% of individuals positive solely for anti-HBc are chronic carriers (2, 3, 14, 20, 34, 41) and that they can transmit HBV via blood transfusion or from mother to child (38). Anti-HBc-positive organ donors are a potential source of transmission of HBV to their recipients (12). The infectivity of these individuals can be confirmed by the detection of HBV DNA by hybridization (22) or PCR (14, 32, 37, 38, 41). A collection of isolated anti-HBc-positive individuals was included in our study in order to test the hypothesis that a highly sensitive HBsAg assay may detect low-level HBsAg in HBV carriers. With Elecsys HBsAg, 2.7% of isolated anti-HBc-reactive samples that were negative with IMx HBsAg tested positive (Table 1); only one of these samples was PCR positive. Of 2,000 antenatal clinical attendees in Papua New Guinea, 5% of HBsAg-positive subjects were negative by a widely used monoclonal assay but positive by PCR. The monoclonal assay had a sensitivity of 0.5 to 1 ng/ml. These samples were reactive in another assay with a sensitivity of 0.1 ng/ml. Over 50% of these discordant samples had rare or unique variants of the major hydrophilic region of HBsAg (7). These data also demonstrate that the sensitivity of current screening HBsAg assays should be increased, arguing for implementation of detection of HBV DNA in blood donors. In the clinical diagnostic laboratory, potentially infectious low-level HBV carriers may also be detected by using highly sensitive anti-HBc-IgM assays (4). Another reason for the possible failure of commercial assays in detecting surface antigen in isolated anti-HBc reactive HBV carriers is that the a determinant is more variable and shows a larger variability of the whole protein than HBsAg-positive controls (43). Polyclonal-antibody-based assays do not guarantee full sensitivity (42). Modification of commercial assays is necessary to increase the sensitivity of detection of S gene variants. By using monoclonal antibodies directed against different S gene mutants in a prototype assay, it was possible to achieve a higher sensitivity in dilution series of HBsAg variants than with serological assays (unpublished data).

Elecsys HBsAg and Auszyme Monoclonal B showed a high sensitivity for three surface antigen mutants with amino acid substitutions in positions 133 (ML), 103 (GT), and 126 (IN). However, one mutant with an amino acid insertion behind position 113 (T) was detected only at a low dilution of 1/5 (Table 6). These data illustrate that the sensitivity of commercial assays is variable for the different surface mutants. It is conceivable that false-negative results may be obtained with serological assays, especially for low-level HBV mutant carriers.

Elecsys HBsAg detected subtypes adw2 and ayw1 (genotype A), adw4 (genotype F), ayw2 (genotype D), ayw4 (genotype E), and adr (genotype C) with a sensitivity better than that of Auszyme Monoclonal B. According to these data, Elecsys HBsAg seems to be more accurate for HBsAg screening in different geographical locations.

In conclusion, Elecsys HBsAg is highly sensitive and specific and represents a major improvement over the alternative assays for the detection of HBsAg in blood donors and in routine laboratory diagnostics. Future developments of serological assays should include monoclonal antibodies that recognize epidemiologically relevant surface antigen mutants and further optimization of sensitivity. An alternative would be the screening of blood donors with nucleic acid amplification techniques. However, technology for nucleic acid testing in blood screening laboratories has not yet been developed. The limitations of these procedures are mainly linked to the difficulties related to the automation of sample processing and to the possibilities of cross-contamination of samples due to the high sensitivity of amplification methods.