|Year : 2015 | Volume
| Issue : 1 | Page : 96-100
Correlation between two chemiluminescence based assays for quantification of hepatitis B surface antigen in patients with chronic hepatitis B infection
E Gupta1, P Pandey1, A Kumar2, MK Sharma2, SK Sarin2
1 Department of Virology , Institute of Liver and Biliary Sciences, Vasant Kunj, New Delhi, India
2 Department of Hepatology, Institute of Liver and Biliary Sciences, Vasant Kunj, New Delhi, India
|Date of Submission||26-Sep-2013|
|Date of Acceptance||27-Nov-2013|
|Date of Web Publication||5-Jan-2015|
Department of Virology , Institute of Liver and Biliary Sciences, Vasant Kunj, New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: Hepatitis B surface Antigen (HBsAg) is the hallmark in diagnosing hepatitis B virus (HBV) infection. In India many commercial assays are available for detection of HBsAg but very few can measure it quantitatively. The present study presents the comparative evaluation of two methods and their correlation with serum HBsAg in chronic hepatitis B (CHB) patients. Materials and Methods: Consecutive patients of CHB were included and there HBsAg levels were measured by two methods: (i) Elecsys, Roche Diagnostics, a qualitative assay and (ii) Architect, Abbott Diagnostics, a quantitative assay. The HBV DNA was measured by real-time polymerase chain reaction (qPCR). Results: Total of 136 patients were included in the study and there was a significant overall correlation between both the assays (correlation coefficient [r] = 0.83; P < 0.001). Assays correlated well with each other across all subgroups of CHB: treatment naοve (r = 0.73; P < 0.001, n = 32), on treatment (r = 0.56; P < 0.05, n = 104), hepatitis Be (HBe) antigen positive (r = 0.67; P < 0.001, n = 62) and anti-HBe positive (r = 0.61; P < 0.05, n = 74) group. On correlation with serum HBV DNA, Architect assay demonstrated good correlation (r = 0.73; P < 0.001, n = 136) as compared to the Elecsys assay (r = 0.27; P = 0.068, n = 136). Architect HBsAg QT assay (A1) also correlated well with HBV DNA in the treatment naοve group (r = 0.69; P < 0.001, n = 32). Conclusions: Our study hence proved that both the assays are comparable and a simple qualitative assay with in-house modification can be used easily for quatitation of HBsAg in clinical samples.
Keywords: Architect HBsAg, chronic hepatitis B, elecsys HBsAg, HBsAg quantitation, hepatitis B virus DNA
|How to cite this article:|
Gupta E, Pandey P, Kumar A, Sharma M K, Sarin S K. Correlation between two chemiluminescence based assays for quantification of hepatitis B surface antigen in patients with chronic hepatitis B infection. Indian J Med Microbiol 2015;33:96-100
|How to cite this URL:|
Gupta E, Pandey P, Kumar A, Sharma M K, Sarin S K. Correlation between two chemiluminescence based assays for quantification of hepatitis B surface antigen in patients with chronic hepatitis B infection. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 19];33:96-100. Available from: http://www.ijmm.org/text.asp?2015/33/1/96/148400
| ~ Introduction|| |
Hepatitis B surface Antigen (HBsAg) is the hallmark in diagnosing hepatitis B virus (HBV) infection.  In addition to its use as a qualitative marker, recently, there has been an increasing focus on quantitative HBsAg (qHBsAg) and its use in the management of patients with chronic hepatitis B virus infection (CHB). , The interest in qHBsAg started with the possible observation of its correlation with intra-hepatic HBV covalently closed circular (ccc) DNA, a true marker of HBV replication. ,, Various clinical applications of qHBsAg have been described. ,,,, It has been demonstrated that the combined use of qHBsAg and HBV DNA might help in monitoring the response to anti-viral therapy in CHB patients. In comparison with nucleos (t) ide analogues (NAs), interferon-based therapy decline in qHBsAg correlate better with prognosis. , With the aid of qHBsAg, one can anticipate an individualised approach to tailoring the treatment duration in CHB patients. ,
Quantification of HBsAg can be done by a number of methods from radioimmuno assays (RIA) to fully automated chemiluminescence immunoassays (CLIA). The CLIA is more sensitive, specific, and can detect all circulating forms of HBsAg as well as mutants.  Limited commercial assays are available for the qHBsAg. The most widely used is the Architect HBsAg quantitation (QT) assay (Abbott Diagnostics, IL, USA) but qHBsAg may also be performed using a qualitative Elecsys HBsAg assay (Roche Diagnostics GmbH, Mannheim). , Quantitative HBsAg on Elecsys is also available (Elecsys HBsAg II), and has been compared with Architect assay but this kit is not available in India. ,
Limited studies are available where performance evaluation of a qualitative assay for HBsAg and its comparison with a quantitative assay in clinical samples has been investigated. Therefore, the aim of our study was to compare qHBsAg conducted with an established commercially available assay with a simple qualitative assay and then compare them with serum HBV DNA levels in the CHB patients as per their disease status.
| ~ Materials and Methods|| |
The study was carried out from January 2012 to August 2013. Total of 136 consecutive patients of CHB attending the outpatient clinic of our Institute were included. Following patients were excluded: (a) Patients with undetectable HBV DNA levels; (b) patients with co-infection with HCV, human immunodeficiency virus (HIV) or hepatitis delta virus (HDV); (c) patients with chronic renal failure with serum creatinine >4 mg/dL; (d) patients with autoimmune liver disease; and (e) patients who did not give their consent.
Complete clinical evaluations of all the patients were reviewed using the clinical data repository of hospital information system. All study subjects' sera were tested for routine hepatitis B serological markers (HBsAg, HBeAg, anti-HBe, anti-HBc total/IgM) by commercial methods (Architect i1000SR, Abbott Diagnostics, IL, USA). All sera were subjected to HBV DNA by real-time PCR and in all of them qHBsAg was done by both, ready- to-use commercially available quantitative assay (Architect HBsAg quantitation QT assay, Abbott Diagnostics, IL, USA) (A1) and a modified qualitative assay (Elecsys HBsAg assay, Roche Diagnostics GmbH, Mannheim) (A2). All study subjects were sub-grouped as per their HBe antigen and anti-viral treatment status.
HBV DNA quantitation
HBV DNA quantitation was done on patient's plasma (500 μl) on COBAS TaqMan HBV test (Roche Diagnostics, GmbH, Mannheim, Germany). DNA was extracted using high pure extraction (Roche Diagnostics, GmbH, Mannheim, Germany) as per the manufacturer's protocol followed by real-time PCR, based on dual labelled hybridisation probe targeting the pre-core and core regions of the HBV genomes. Results were expressed as international units (IU)/ml. Lower limit of detection for the assay is 6 IU/ml and linear range 29 to 1.1 × 10 8 IU/ml.
Architect HBsAg QT assay (A1)
The qHBsAg levels were done on the fully automated Architect instrument as per the manufacturer's protocol and results were expressed as IU/ml. This is a two-step CLIA, with flexible assay protocols referred to as chemiflex. In the first step, sample and anti-HBs coated paramagnetic micro particles are combined. HBsAg present in the sample binds to the anti-HBs coated micro particles. After washing, acridinium-labelled anti-HBs conjugate is added in the second step. The resulting chemiluminescent reaction is measured as relative light units (RLUs). A direct relationship exists between the amount of HBsAg in the sample and the RLUs detected by the optical system. The concentration of HBsAg in the specimen is determined using a previously generated calibration curve. This assay allows the quantitation of HBsAg in undiluted serum/plasma samples from 0.05 to 250 IU/ml. Samples with an HBsAg level higher than 250 IU/ml require a 1:500 or greater dilution with the diluent provided by the manufacturer.
Modified Elecsys HBsAg assay (A2)
The Elecsys HBsAg assay is a two-step sandwich CLIA. The assay uses two biotinylated monoclonal HBsAg-specific capture antibodies together with a mixture of biotinylated and ruthenium-labelled polyclonal anti-HBs detection antibodies to form a sandwich complex with serum HBsAg. This complex is then bound to streptavidin-coated microparticles, with subsequent detection of ruthenium chemiluminescence. Samples were tested at a dilution of 1:400. Samples were diluted in phosphate buffered saline (PBS) and cutoff indices (c.o.i) were calculated as per the manufacturer's protocol for qualitative assay. Samples yielding a c.o.i of ≥1.0 were expressed as c.o.i ×400, while samples with a c.o.i. <1.0 were re-tested undiluted and expressed as the final c.o.i. Samples yielding values ≥1000 c.o.i were re-tested at a dilution of 1:8000 and the corresponding result was expressed as c.o.i. ×8000. The conversion factor for estimation of HBsAg units was 1 IU/mL = 18.21 c.o.i. as provided by the manufacturer based on the calibration curve generated using standards (provided by the manufacturer). Results generated were expressed as IU/mL using the above mentioned conversion factor.
Quantitative variables were expressed as median with range and qualitative variables were expressed as numbers with percentage. The correlation between HBsAg levels by both the methods and HBV DNA was done by Pearson's correlation coefficient test. Statistical analysis was done using Statistical Package for the Social Sciences software (SPSS) for Windows (Chicago, Illinois) version 17.0. All P values less than 0.05 were considered significant. Numerical data were calculated using Microsoft Excel and analysed.
| ~ Results|| |
A total of 136 eligible patients of CHB were included, out of which 97 (71.3%) were males with the median age of 46 (range: 3-76) years. Baseline characteristics of the study population are described in [Table 1].
The overall correlation between elecsys and architect assays
When both the assays, A1 and A2, were compared with each other to measure HBsAg in all the 136 patients, strong correlation was seen between the two assays (correlation coefficient [r] = 0.83; P < 0.001) [Figure 1]. The median qHBsAg by Architect assay (A1) was 1.6 × 10 3 (range: 5.7 × 10 2 -6.2 × 10 5 ) IU/ml and the median qHBsAg by Elecsys (A2) was 8.7 × 10 3 (range: 5.0 × 10 2 -3.2 × 10 5 ) IU/ml [Figure 1]. This correlation was similar, irrespective of the different subgroup of the patients [Table 2]. Correlation was better in treatment naïve and HBe antigen positive group.
|Figure 1: Correlation between A1 (architect) and A2 Elecsys qHBsAg assays|
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Comparison with serum HBV DNA levels
Measurement of serum HBV DNA is the most common monitoring tool to follow up the CHB patients, who are on the antiviral drugs, and hence qHBsAg by both the assays A1 and A2 were compared with serum HBV DNA levels in the present study. A1 showed significant correlation with serum HBV DNA (r = 0.73; P < 0.001) as compared to A2 (r = 0.27; P = 0.068) [Table 3].
|Table 3: Subgroup - wise comparison between qHBsAg and HBV DNA by both the assays|
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Comparison on the basis of antiviral status
In this study, there were 32 (23.5%) patients who were treatment naïve. The qHBsAg by A1 and A2 correlated well with each other in this group but on comparison with serum HBV DNA levels, A1 correlated more (correlation coefficient [r] = 0.69; P ≤ 0.001) as compared with A2 (correlation coefficient [r] = 0.32; P = 0.38).
However, in the remaining 104 (76.4%) patients on antiviral drugs, no correlation was seen between serum HBV DNA and qHBsAg, which was measured by both A1 and A2 assays.
Comparison on the basis HBe Ag status
In all the 136 patients, the HBeAg and anti-HBe antibody statuses were determined and the correlation of HBV DNA levels with qHBsAg was established. A total of 45.5% were HBeAg positive and rest were anti-HBe positive. In HBeAg positive patients, correlation between A1 and HBV DNA (correlation coefficient [r] = 0.54; P = 0.058) was almost similar when compared with correlation of qHBsAg levels by A2 and HBV DNA (correlation coefficient [r] = 0.36; P = 0.57).
However, no correlation was seen between HBV DNA and qHBsAg by both A1 and A2 in anti-HBe positive groups.
| ~ Discussion|| |
In this study, two chemiluminescent assays were compared with each other for the quantitation of HBsAg in clinical samples, one assay is an already established commercially available quantitative assay and the other is a commercially available qualitative assay modified for use as a quanititative assay. An overall good correlation was seen between the two assays thereby revealing that a simple qualitative assay with in-house dilution method can be a good alternative to any established quantitative assay in the measurement of HBsAg in clinical samples.
The CHB infection is a serious clinical problem because of its worldwide distribution and potential adverse sequelae. It is particularly important in the Asian-Pacific region, where the prevalence is high. The prevalence of CHB in India is in the intermediate range with an estimated 40 million subjects infected.  Long-term suppression of viral replication is critical for reducing the complications of CHB infection. Monitoring of CHB patients during antiviral treatment is important because current treatment options had limited success in achieving durable endpoints and antiviral resistance may emerge during long-term therapy. , The most common method for monitoring treatment response is serial serum HBV DNA estimation, which is expensive and technically demanding. Recently, qHBsAg has been evaluated as an alternative marker for monitoring treatment response.  Studies have shown that decline in HBsAg levels during treatment can predict the likelihood of HBsAg clearance and sustained response to treatment. , Quantitative measurement of HBsAg is therefore likely to be of increasing importance as it can be used in the individualisation of therapy. Hence, there is likely to be a growing need for reliable assays for HBsAg quantitation. In the present study, overall there was a correlation between the HBsAg levels and the serum HBV DNA, but this correlation was not persistent in all the subgroups of CHB patients, thereby indicating that it requires close monitoring of the patients as per the disease status and the phase of CHB infection. Also another important finding was that the correlation also varied depending upon the quantitative kit used.
At present there is only one commercially available kit for the quantitation of HBsAg that is Abbott Architect kit, although a new kit by Roche has recently been launched on Elecsys system but it is still unavailable in our country for commercial use. However, a qualitative kit is available on the Elecsys system for HBsAg detection. Recently, both the quantitative kits have been compared together and have shown good correlation, , but a modified qualitative kit is compared with an established kit for the first time in the present study. Finding of this study shows that both the kits can be used in clinical practice.
Despite the strong overall correlation between the assays, a few discrepancies were noted as Architect assay was better in terms of correlation with serum HBV DNA levels especially in treatment naïve group. This could be multifactorial, as one kit was qualitative, although in-house standardisation was done but absolute standardisation is needed to make it more reproducible. Other reason could be the differences in the avidity of the antibodies used in the assay. Moreover, at present, the existing data correlating HBV DNA levels with qHBsAg are very conflicting. , Hence, more studies with larger sample size should be done to evaluate this further.
Hence, in conclusion commercially available Architect kit and qualitative Elecsys kit with in-house dilution method are comparable in terms of HBsAg quantitation in clinical samples. More evidence should be collected, before we can adopt HBsAg quantitation as a monitoring tool to the follow-up patients.
| ~ Acknowledgment|| |
We acknowledge the Indian Council of Medical Research (ICMR) for the financial support.
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[Table 1], [Table 2], [Table 3]