|Year : 2012 | Volume
| Issue : 2 | Page : 150-154
Serum hepatitis B surface antigen levels correlate with high serum HBV DNA levels in patients with chronic hepatitis B: A cross-sectional study
E Gupta1, A Kumar2, A Choudhary1, M Kumar2, SK Sarin2
1 Department of Virology, Institute of Liver and Biliary Sciences (ILBS), Vasant Kunj, New Delhi - 110 070, India
2 Department of Hepatology, Institute of Liver and Biliary Sciences (ILBS), Vasant Kunj, New Delhi - 110 070, India
|Date of Submission||23-Dec-2011|
|Date of Acceptance||09-Mar-2012|
|Date of Web Publication||28-May-2012|
Department of Virology, Institute of Liver and Biliary Sciences (ILBS), Vasant Kunj, New Delhi - 110 070
Source of Support: None, Conflict of Interest: None
Purpose : The hallmark of chronic hepatitis B (CHB) infection is the presence of hepatitis B surface antigen (HBsAg) positivity for at least 6 months. Recently, serum levels of HBsAg have been compared with serum HBV DNA as a surrogate marker to monitor CHB patients. However, data correlating these two markers are scarce. Hence, the present study was done to correlate HBV DNA with HBsAg in CHB patients. Materials and Methods: Consecutive patients of CHB were included. HBV DNA was measured by real-time polymerase chain reaction (PCR). Serum HBsAg was measured by Architect HBsAg. Results: Of the 198 patients enrolled, 166 fulfilled the inclusion criteria (mean age 43 ± 14 years, 87% males) and the median HBV DNA was 1.7 × 10 3 (range 6.0-1.1 × 10 8 ) IU/ml. Median HBsAg was 8.7 × 10 3 (range 5.0-3.2 × 10 5) IU/ml. Overall correlation between HBV DNA and HBsAg was weak but significant (Spearman ρ = 0.443, P < 0.01). Correlation in HBe antigen-positive group was better (ρ = 0.402, P < 0.01) in comparison to HBe antigen-negative group (ρ = 0.193 P = 0.05). Good correlation existed in treatment-naïve group (ρ = 0.538, P < 0.01) .Correlation was regardless of normal or raised alanine transaminase (ALT). Eighty (48%) patients had high HBV DNA (≥2000 IU/ml). Correlation in high DNA group was significant (P < 0.01). The best cut-off of HBsAg for diagnosing high DNA is 3.36 ×10 3 IU/ml. Conclusions: Serum HBsAg correlates with HBV DNA in CHB patients, especially in high serum HBV DNA, HBe antigen-positive and treatment-naïve group. HBsAg levels can be used for predicting high serum HBV DNA levels.
Keywords: Chronic hepatitis B, hepatitis B surface antigen, hepatitis B virus DNA, HBsAg quantitation
|How to cite this article:|
Gupta E, Kumar A, Choudhary A, Kumar M, Sarin S K. Serum hepatitis B surface antigen levels correlate with high serum HBV DNA levels in patients with chronic hepatitis B: A cross-sectional study. Indian J Med Microbiol 2012;30:150-4
|How to cite this URL:|
Gupta E, Kumar A, Choudhary A, Kumar M, Sarin S K. Serum hepatitis B surface antigen levels correlate with high serum HBV DNA levels in patients with chronic hepatitis B: A cross-sectional study. Indian J Med Microbiol [serial online] 2012 [cited 2019 Dec 13];30:150-4. Available from: http://www.ijmm.org/text.asp?2012/30/2/150/96664
| ~ Introduction|| |
Hepatitis B virus (HBV), a small DNA virus, is a prototype virus of the Hepadnaviridae family. HBV infection leads to a wide spectrum of liver diseases ranging from acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma.  Most adults infected with the virus recover, but 5-10% are unable to clear the virus and become chronically infected. The hallmark of chronic hepatitis B (CHB) infection is the presence of hepatitis B surface antigen (HBsAg) positivity for at least 6 months. HBsAg loss and seroconversion to anti HBs antibodies is the most desirable treatment outcome to achieve in CHB patients on treatment. However, it is attained only in 3-4% of patients. ,, Periodic serum HBV DNA is the most reliable marker to monitor CHB patients on treatment. , However, serum HBV DNA measurement has several limitations. Despite being expensive, labour-intensive HBV DNA assays lack uniformity and standardisation. Many commercial kits are available and all have different linear ranges, lower limits of detection and conversion factors. Repeated assays done preferably on the same platform are needed to monitor a patient on antivirals. Hence, there is a definite need of a monitoring tool which is economical, reliable and easy to perform. HBsAg quantitation is a recent serological marker being evaluated. Though it is not new, a fully automated version of this assay has recently been introduced.  This method is based on chemiluminescence and results are expressed as IU/ml.
Since its description was made, various studies have come up where clinical utility of HBsAg quantitation has been described, ,, but studies where theses two markers have been compared are scarce and conflicting results are available. ,,, There are no data from the Indian subcontinent where these two markers have ever been evaluated. Therefore, the present study was undertaken to correlate HBV DNA and HBsAg levels in CHB patients and also to find out the subgroup of patients where it can be more suitably used.
| ~ Materials and Methods|| |
The study was carried out from January 2010 to July 2011. All consecutive patients of CHB attending the out-patient clinic 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 D 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 consent.
Complete clinical histories 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 (Cobas e411, Roche Diagnostics, Indianapolis, IN, USA). All sera were subjected to HBV DNA by real-time polymerase chain reaction (PCR) and in all of them HBsAg quantification was done by Chemiluminiscent Immunoassay (CLIA) method (Abbott Laboratories, Chicago, IL, USA).
HBV DNA quantitation
HBV DNA quantitation was done on patient's plasma (500 μl) using COBAS TaqMan HBV test with high pure extraction (Roche Diagnostics) as per the manufacturer's protocol. This is a real-time PCR assay based on dual labeled hybridisation probe targeting the precore and core regions. Results were expressed as IU/ml. Lower limit of detection for the assay was 6 IU/ml and serum HBV DNA levels of all subjects were within the range of 6-1.1 × 10 8 IU/ml. Quantification was accurate and reproducible over a linear range of 29-1.1 × 10 8 IU/ml. Samples with HBV DNA ≥2000 IU/ml were considered as high HBV DNA. 
HBsAg levels were measured by the fully automated Architect HBsAg QT (Abbott Laboratories) assay as per the manufacturer's protocol and the results were expressed as IU/ml. This assay is calibrated against the WHO standard and allows the quantitation of HBsAg from 0.05 to 250 IU/ml. A concentration higher than 0.05 IU/ml was considered HBsAg positive. Samples with an HBsAg level higher than 250 IU/ml required a 1:500 dilution with the diluent as recommended by the manufacturer and the exact concentration of HBsAg was measured.
Quantitative variables were expressed as median with range and qualitative variables were expressed as numbers with percentage. Correlation between HBV DNA levels and HBsAg levels was determined by non-parametric two-tailed Spearman's test. Statistical analysis was done using SPSS for Windows (Chicago, IL, USA) version 17.0. All P values less than 0.05 were considered significant. Numerical data were calculated using Microsoft Excel and analysed.
| ~ Results|| |
From January 2010 through July 2011, a total of 198 eligible patients of CHB were enrolled for the study; however, 32 patients were excluded due to the following reasons: (a) Patients with undetectable HBV DNA levels (n = 21); (b) patients with co-infection with HCV, HIV or HDV (n = 1); (c) patients with chronic renal failure with serum creatinine >4 mg/dl (n = 3); (d) patients with autoimmune liver disease (n = 3) and (e) patients who did not give consent (n = 4). Hence, the remaining 166 patients were included in the study.
Overall correlation between HBsAg and HBV DNA
Out of 166 CHB patients in whom serum HBV DNA was compared with HBsAg, 87% were males with a median age of 37 (range 3-77) years. The median HBV DNA was 1.7 × 10 3 (range 6.0-1.1 × 10 8 ) IU/ml. The median HBsAg level was 8.7 × 10 3 (range 5.0-3.2 × 10 5) IU/ml [Table 1]. Overall, there was weak but significant correlation between HBV DNA and HBsAg levels (Spearman's rho ρ = 0.443, P < 0.01).
Results of the quantitative HBsAg and HBV DNA levels were also compared according to the different clinical phases of HBV infection [Table 2].
|Table 2: Correlation between HBV DNA and HBsAg level according to different phases of disease|
Click here to view
Correlation between high and low DNA groups
Patients were grouped into two depending upon the DNA levels as high and low DNA groups. 48% patients had high HBV DNA (≥2000 IU/ml). Correlation between HBsAg and HBV DNA levels in high DNA group was significant (ρ = 0.405, P < 0.01, n = 80) as compared to low DNA group (ρ = 0.142, P = 0.192, n = 86). On receiver operating characteristics curve, the area under the curve for HBsAg level for diagnosing high HBV DNA level was 0.713 (95% CI 0.636, 0.790; P < 0.001) [Figure 1]. The best cut-off of HBsAg level for predicting high DNA level is 3.36 × 10 3 IU/ml.
|Figure 1: Receiver operating characteristics curve of HBsAg quantitation for diagnosing patients with high (>2000 IU/mL) HBV DNA levels|
Click here to view
Correlation according to HBe antigen status
In all 166 patients, HBe antigen and antibody to HBe antigen were determined and correlation between HBsAg and HBV DNA was established. A total of 40% patients were HBe antigen positive and rest were HBe antigen negative. The baseline characteristics of patients according to HBe antigen status are described in [Table 3]. Quantitative HBsAg and HBV DNA were higher in HBe antigen-positive than in HBe antigen-negative patients. Correlation between HBsAg levels and HBV DNA in HBe antigen-positive group was better and significant (ρ = 0.402, P < 0.01, n = 66) as compared to HBe antigen-negative group (ρ = 0.193, P = 0.055, n = 100).
Correlation on the basis of antiviral treatment status
Out of 166 patients, 103 were treatment naïve while the rest were on some form of antiviral. On comparison of HBV DNA levels with HBsAg levels according to the treatment status of patients, good correlation was seen in treatment-naοve group (ρ = 0.538, P < 0.001, n = 103), whereas when the patients had received any form of antiviral treatment this correlation was very weak (ρ = 0.299, P < 0.05, n = 63).
Correlation according to serum ALT levels
Correlation between HBsAg and HBV DNA levels was investigated according to serum alanine aminotransferase (ALT) levels. Eighty-seven patients had normal ALT while 79 patients were found to have raised or high ALT. High ALT was defined as 2 times the upper limit of normal range (0-40 IU/ml). The correlation existed in both the groups and was regardless of normal or raised ALT levels.
| ~ Discussion|| |
The study focusses on the overall correlation between quantitative HBsAg and HBV DNA. These two markers are also correlated in different groups of CHB patients according to HBe antigen status, antiviral treatment and serum ALT levels. We found weak but significant correlation between HBsAg and HBV DNA in all the groups; this correlation was stronger in HBe antigen-positive and treatment-naïve CHB patients and in patients with high serum HBV DNA.
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. In this part of the world, majority of HBV infection is acquired perinatally or in early childhood. 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 have limited success in achieving durable endpoints and antiviral resistance may emerge during long-term therapy. Methods of monitoring treatment response include tests for serum ALT, HBV DNA, HBeAg, anti-HBe, HBsAg or anti-HBs, and liver histology. 
Recent studies have provided evidence that relationship exists between various viral antigens, such as HBeAg and HBsAg levels, and patients' response to antiviral therapy. ,,,,
One of the recent targets for quantitative serology is HBsAg. HBsAg is encoded by the envelope gene, which contains three open-reading frames: the pre-S1, pre-S2 and S domains. There is subsequent conversion to small, medium and large forms of HBsAg proteins. Newly synthesised HBsAg proteins are secreted from the hepatocyte. Similar to the HBeAg pathway, HBsAg synthesis is separate from the viral replication pathway. Existent quantitative HBsAg serology can detect all three forms of HBsAg in the circulation. Since the introduction of quantitative HBsAg, a lot of studies have come up regarding its clinical significance  Many cross-sectional studies have shown significant correlation between HBsAg levels and serum HBV DNA levels. ,, Negative or poor correlation between these two markers has also been studied by many researchers. ,
Different results from various studies may be attributed to the fact that they did not look at the correlation in different phases of HBV infection. Hence, in this study, which is the first of its kind in our geographical locale, we tried to look at the correlation between HBsAg levels and HBV DNA levels in CHB patients according to different phases of HBV infection. The phases can be divided into two, i.e. the early replicative phase characterised by the presence of HBeAg and high levels of serum HBV DNA and the non-replicative phase which is characterised by loss of HBeAg, appearance of antibodies to HBeAg in serum and marked decrease in serum HBV DNA. We found good correlation in HBe antigen-positive patients in contrast to HBe antigen-negative group. Earlier, similar results had been reported by Thompson et al.  The correlation was stronger in high serum HBV DNA group where serum HBV DNA was >2000 IU/ml and in CHB patients who were treatment naïve.
We therefore suggest that quantitative HBsAg is more related with HBV DNA levels in initial replicative phase than those in non-replicative phase of CHB. A critical question is whether quantitative HBsAg can be used instead of, or in conjunction with, HBV DNA levels to evaluate or monitor CHB patients. The study has its own limitations. Being a retrospective study, the patients were not grouped according to the type of antivirals used and they were not followed up to monitor the outcome. Better designed longitudinal studies are needed to compare the two markers.
But our findings have practical implications for the use of quantitative HBsAg as an alternative biomarker to HBV DNA. HBsAg quantitation does have potential in defining clearly a patient who requires treatment and who does not. Patients with values above the defined cut-offs would require more frequent monitoring for early detection of reactivation.
HBV DNA tests are expensive and also require a separate molecular laboratory setting with technical expertise for performance. Instead, HBsAg quantification can be carried out in a much simplified way, in a fully automated manner, in any peripheral laboratory setting. Cost per test is much cheaper for HBsAg quantitation than for HBV DNA. In resource-limited countries, cheaper methods of evaluation should be made available.
In conclusion, serum HBsAg correlates with HBV DNA in CHB patients especially in HBe antigen-positive and treatment-naïve group. HBsAg levels can reliably be used for predicting high serum HBV DNA levels.
| ~ Acknowledgments|| |
The authors thank Mr. Gaurav Singh, Mr. Sandeep Tyagi and Ms. Priyanka Pandey for their excellent technical support.
| ~ References|| |
|1.||Kao JH, Chen DS. Global control of hepatitis B virus infection. Lancet Infect Dis 2002;2:395-3. |
|2.||Gish R, Lok AS, Chang TT, de Man RA, Gadano A, Sollano J, et al. Entecavir therapy for up to 96 weeks in patients with HBeAg-positive chronic hepatitis B. Gastroenterology 2007;133:1437-44. |
|3.||Marcellin P, Heathcote EJ, Buti M, Gane E, de Man RA, Krastev Z, et al. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl J Med 2008;359:2442-55. |
|4.||Buster EH, Flink HJnone , Cakaloglu Ynone , Simon Knone , Trojan Jnone , Tabak Fnone , et al. Sustained HBeAg and HBsAg loss after long-term follow-up of HBeAg-positive patients treated with peginterferon alpha-2b.Gastroenterology 2008;135:459-67. |
|5.||Gish RG, Locarnini SA. Chronic hepatitis B: Current testing strategies. Clin Gastroenterol Hepatol 2006;4:666-76. |
|6.||Andersson KL, Chung RT. Monitoring During and After Antiviral Therapy for Hepatitis B. Hepatology 2009;49:S166-73. |
|7.||Deguchi M, Yamashita N, Kagita M, Asari S, Iwatani Y, Tsuchida T, et al. Quantitation of hepatitis B surface antigen by an automated chemiluminescent microparticle immunoassay. J Virol Methods 2004;115:217-22. |
|8.||Moucari R, Mackiewicz V, Lada O, Ripault MP, Castelnau C, Martinot-Peignoux M. Early serum HBsAg drop: A strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients. Hepatology 2009;49:1151-7. |
|9.||Wursthorn K, Jung M, Riva A, Goodman ZD, Lopez P, Bao W, et al. Kinetics of hepatitis B surface antigen decline during 3 years of telbivudine treatment in hepatitis B e antigen-positive patients. Hepatology 2010;52:1611-20. |
|10.||Chan HL, Wong VW, Tse AM, Tse C, Chim AM, Chan H, et al. Serum Hepatitis B Surface Antigen Quantitation Can Reflect Hepatitis B Virus in the Liver and Predict Treatment Response. Clin Gastroenterol Hepatol 2007;5:1462-8. |
|11.||Kuhns MC, Kleinman SH, McNamara AL, Rawal B, Glynn S, Busch MP. Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: Implications for future HBV screening policy. Transfusion 2004; 44:1332-9. |
|12.||Kohmoto M, Enomoto M, Tamori A, Habu D, Takeda T, Kawada N, et al. Quantitative detection of hepatitis B surface antigen by chemiluminiscent microparticle immunoassay during lamivudine treatment. J Med Virol 2005;75:235-9. |
|13.||Ozaras R, Tabak F, Tahan V, Ozturk R, Akin H, Mert A, et al. Correlation of quantitative assay of HBsAg and HBV DNA levels during chronic HBV treatment. Dig Dis Sci 2008;53:2995-8. |
|14.||Thakur V, Guptan RC, Kazim SN, Malhotra V, Sarin SK. Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent. J Gastroenterol Hepatol 2002;17:165-70. |
|15.||Bernard F, Raymond G, Willems B, Villeneuve JP. Quantitative assessment of serum hepatitis B e antigen, IgM hepatitis B core antibody and HBV DNA in monitoring the response to treatment in patients with chronic hepatitis B. J Viral Hepat 1997;4:265-72. |
|16.||Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW, et al. Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy. Gastroenterology 2005;128:1890-7. |
|17.||Wursthorn K, Lutgehetmann M, Dandri M, Volz T, Buggisch P, Zollner B, et al. Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B. Hepatology 2006;44:675-84. |
|18.||Liaw YF. Clinical utility of hepatitis B surface antigen quantitation in patients with chronic hepatitis B: A review. Hepatology 2011;53:2121-9. |
|19.||Sonneveld MJ, Zoutendijk R, Janssen HL. Hepatitis B surface antigen monitoring and management of chronic hepatitis B. J Viral Hepat 2011;18:449-57. |
|20.||Ozdil B, Cosar AM, Akkiz H, Sandikci MU, Kece C. Negative correlation between viral load and HBsAg levels in chronic HBV-infected patients. Arch Virol 2009;154:1451-5. |
|21.||Thompson AJ, Nguyen T, Iser D, Ayres A, Jackson K, Littlejohn M, et al. Serum hepatitis B surface antigen and hepatitis B e antigen titers: Disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers. Hepatology 2010;51:1933-44. |
[Table 1], [Table 2], [Table 3]