Role of Hepatitis B Surface Antigen Neutralization Test in Screening of Hepatitis B

Introduction

Hepatitis B virus (HBV) is a small, enveloped DNA virus that causes hepatitis B. Acute hepatitis B is a self-limiting disease characterized by acute inflammation and hepatocellular necrosis in association with a transient HBV infection. In contrast, chronic hepatitis B is a persistent infection marked by the evidence of hepatocellular injury, fibrosis, cirrhosis, and an increased risk of hepatocellular carcinoma.¹

The laboratory diagnosis of HBV infection primarily relies on the detection of viral markers through serological testing. These include HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc IgM and IgG. Hepatitis B surface antigen (HBsAg) serves as the principal serological marker of HBV infection. HBsAg appears in serum within 1 to 10 weeks after an acute infection with HBV. Persistence of this marker for more than six months implies chronic HBV infection.² Further evaluation of the patient for other hepatitis B viral markers, including HBV DNA and liver enzyme levels is needed to determine the stage of HBV infection and guide management.¹ Various serological assays are used to detect the presence of HBsAg in serum based on immunoassay principles. These include rapid diagnostic tests (RDTs) or laboratory-based enzyme immunoassays (EIAs), chemiluminescence immunoassays (CLIAs), and enhanced chemiluminescence immunoassays (ECLIAs).^1,3

The World Health organization (WHO) recommends a single serological assay for the detection of HBsAg in populations with an HBsAg seroprevalence of ≥0.4%, prior to further evaluation for HBV DNA and liver disease staging.3 Confirmation of HBsAg positivity using the same immunoassay with an HBsAg neutralization step, or a second, different RDT assay may be considered in populations with a low HBsAg seroprevalence (≤0.4%).3 India is classified as a region of intermediate endemicity, with an average seroprevalence of 3-4%. The reported prevalence in the general population ranges from 1.1% to 12.2%.⁴

There are not many serological tests for the detection of HBsAg with a 100% diagnostic accuracy.^4,5 Recently, qualitative HBsAg tests by ECLIA have been considered the method of choice for the screening of HBsAg. Though ECLIA is highly sensitive, certain grey areas exist where it may be difficult to interpret the result as either reactive or non-reactive. In such cases, HBsAg neutralization tests or HBV viral DNA detection can be used.^4-6

Against this background, the present study was undertaken to assess the role of the HBsAg confirmatory neutralization test in screening for hepatitis B infection and to determine the probable cut-off value for qualitative HBsAg detection using the ECLIA method.

Materials and Methods

After obtaining ethical clearance (FMIEC/CCM/253/ 2023), this ambispective study was carried out in the Microbiology Laboratory of a tertiary care center in Mangalore over a period of three years, from January 2021 to December 2023. Blood samples received for HBsAg testing from various departments of the hospital, including both outpatients and inpatients, were included in the study. Repeat blood samples from the same patient submitted for HBsAg testing were excluded.

Serum samples were subjected to qualitative ECLIA for the initial screening of HBsAg. Qualitative detection of HBsAg in human serum was performed using calibrated VITROS ECi/ECiQ Immunodiagnostic System, the VITROS 3600 Immunodiagnostic System, and the VITROS 5600 Integrated System, following the manufacturers's instructions. Quality control procedures were carried out daily in accordance with standard guidelines. Results were expressed as a normalized signal relative to the cut-off index (COI) value. A result of ≥1.00 was interpreted as a reactive sample, indicating the possible presence of HBsAg.

All samples that tested reactive by the VITROS qualitative HBsAg test (ECLIA) were retested using two RDTs: 1) TRUST Line HBsAg rapid (CIK BIOTECH, India), a lateral flow chromatographic immunoassay, and 2) HEPACARD (J. Mitra & Co. Pvt. Ltd, India), a one-step immunoassay based on the sandwich principle. Sample processing, testing, and result interpretation were performed according to the manufacturers's instructions. Results were interpreted as reactive or non-reactive based on the appearance of distinct bands/ lines at the test and control regions.

Samples that were reactive by the VITROS qualitative HBsAg test (ECLIA) with a COI value between 1 and 199 were further tested using the VITROS HBsAg confirmatory neutralization test. This test was performed for the qualitative confirmation of HBsAg in human serum and plasma (heparin) specimens, utilizing the VITROS Immunodiagnostic Products HBsAg ES assay on the VITROS ECi/ ECiQ Immunodiagnostic System, VITROS 3600 Immunodiagnostic System, and the VITROS 5600 Integrated System. The VITROS HBsAg Confirmatory Kit employs the principle of specific antibody neutralization to confirm the presence of HBsAg. One aliquot of the sample is incubated with a neutralizing reagent containing a high titer anti-HBs(the confirmatory antibody), while a second aliquot is incubated with a non-neutralizing control reagent (the sample diluent). The confirmatory antibody binds to HBsAg in the sample, thereby inhibiting its reaction in the VITROS HBsAg assay and resulting in a reduced signal compared to the non-neutralized control. Results were calculated and interpreted according to the manufacturer’s instructions. Quality controls, both positive and negative, were included with each test.

Data were recorded in an Excel sheet and analyzed using IBM SPSS Statistics version 23. Demographic variables were expressed in terms of frequencies and percentages, while continuous variables were presented as medians.

Results

A total of 77,530 venous blood samples from both outpatients and inpatients submitted for HBsAg screening were tested using the VITROS qualitative HBsAg assay (ECLIA). Of these, 636 samples were reactive, with a cut-off index (COI) >1.0, corresponding to a positivity rate of 0.82%. Among these, 536 samples (COI >200) were also reactive by RDTs. The HEPACARD rapid kit detected three cases that were not identified by the TRUST Line rapid card (Table 1).

One hundred samples with VITROS qualitative HBsAg reactive results (COI between 1-199), which were non-reactive by RDT, were subjected to the VITROS HBsAg confirmatory neutralization test. Sixty-one tested positive in the neutralization test, confirming the presence of HBs antigen in these samples, which were falsely negative by RDT. The lowest COI for which the HBsAg neutralization test came positive was 2.6. Forty-nine ECLIA reactive samples were negative by the confirmatory neutralization test and were also non-reactive by RDTs. The positive predictive value of the qualitative HBsAg ECLIA was 93.71%.

The final calculation of total confirmed cases was done by considering the HBsAg neutralization test as the confirmatory test for weakly reactive ECLIA cases. The total number of confirmed cases was 596 out of 77,530 (0.76%) over the three-year study duration, with a male predominance (male:female = 2.41:1). The most common age group affected was 40-49 years, followed by 50-69 years (Table 2).

Discussion

Hepatitis B virus infection can lead to life-threatening liver disease, and the associated illness ranges in severity from asymptomatic to symptomatic progressive disease. Since the majority of infected individuals remain asymptomatic in the initial stages and often present with late-stage disease, early and accurate detection of serum markers is crucial for the diagnosis of hepatitis B infection.⁶ The risk of developing chronic infection correlates with the age at acquisition; that is, vertical transmission from mother to neonate carries nearly a 90% risk of the neonate becoming a chronic carrier and developing progressive disease.^1,6 In our study, no neonate was found positive for HBsAg, and the most commonly affected age group was 40-49 years.

In this study, the VITROS qualitative HBsAg test (ECLIA) was used as the initial screening method to rule out all negative cases. We found a 0.82% positivity rate, which is lower than that reported in other studies by Chevaliez and Shahid et al.This lower rate could be attributed to the large number of samples submitted for HBsAg screening prior to surgery or other interventions.^7,8 Since ECLIA is a highly sensitive test, it can detect even trace amounts of HBsAg in patients at an early stage, allowing for timely initiation of treatment. However, this carries a disadvantage of occasional false-positive results, which we should be taken into consideration.⁸

RDTs serve as a good alternative to immunoassay-based methods, as they are fast, simple, and economical. Thus, they can be considered appropriate for large-scale screening and diagnosis of HBV infection in clinical settings.⁸ In the present study, cross-verification with RDTs revealed that only 84.27% of ECLIA-reactive cases were detected by these tests. RDTs failed to identify low-reactive cases, with the HEPACRD test demonstrating slightly higher sensitivity compared to the Trust Line rapid card test.

Out of 100 samples that were ECLIA qualitative HBsAg test reactive (COI 1-199), but non-reactive by RDT, 61 were positive in the HBsAg confirmatory neutralization test. These samples, with COI values ranging from 1 to 199, represented false negatives by RDT, with the lowest COI of 2.6 among the true reactive samples detected by the VITROS qualitative HBsAg test. Forty-nine samples that were non-reactive by RDT and confirmed negative by the HBsAg neutralization test had ECLIA COI values between 1 and 5.05. Based on these findings, it can be inferred that false-positive results may occur at COI values below 5. These results are consistent with the study conducted by Ghai et al., who also reported discordance between ECLIA qualitative HBsAg and RDT results between 0.9 and 32, where confirmatory testing identified four positive samples with COI values >24.7. Reliance solely on RDTs for screening may therefore lead to missed reactive cases, depending on the sensitivity and specificity of the RDT used for HBsAg detection.^8-10

According to a study by Tiwari AK et al., diagnostic accuracy can be improved by employing a sequential serological testing strategy. Such a sequential approach, previously proven for anti-HIV testing, can also be applied to HBsAg screening.⁵ Qualitative HBsAg testing by ECLIA can be used as the initial screening method to rule out non-reactive samples. As it is a highly sensitive test, cross-verification with an alternative, more economical method such as an RDT is advisable, rather than directly proceeding to the ECLIA HBsAg confirmatory neutralization test. However, for samples that are weakly reactive or yield discordant results, the ECLIA HBsAg confirmatory neutralization test should be performed before reporting the final results. The findings of our study are concordant with those of Lee et al., and Madiyal M et al., Aytac O et al., observed that using a neutralization test as a validation method for samples with HBsAg titers ≤5 COI can significantly reduce the cost of the test without requiring HBV DNA analysis.^11,12,13

This study has few limitations. The data were collected retrospectively, and rapid tests were performed only on reactive ECLIA samples. Additionally, the HBsAg neutralization test was conducted only for doubtful or weakly reactive cases.

Conclusion

Our study demonstrates that all weakly reactive samples (COI 1-5) detected by the VITROS qualitative HBsAg test (ECLIA) should be confirmed using the HBsAg neutralization test to avoid any false-positive results. A sequential testing strategy is both practical and economical for achieving accurate results. However, further studies are warranted to precisely define the grey zone of the VITROS qualitative HBsAg test.

Conflict of interest

There are no conflicts of interest to declare by any of the authors

Acknowledgements

We acknowledge the help of the faculty and technical staff of the Microbiology Department.