Hepat Mon.

2011;11(5):342-345 LATEST
IMPACT FACTOR

Number 34, Volume 11, Issue 5, My 2011 0.716

Official Monthly Journal of the Baqiyatallah Research Center for Gastroenterology and Liver Diseases
ISSN: Print 1735-143x, Online 1735-3408

KOWSAR
HBV DNA and ALT predict Inactive Carrier and HBsAg and HBS DNA
2-year response to LAM HBV Reactivation levels in chronic HBV

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Article Online Submission at:

Correlation between HBsAg quantitative assay results and HBV DNA levels
in chronic HBV
Azita Ganji ¹*, Abbas Esmaeilzadeh ¹, Kamran Ghafarzadegan ², Hoda Helalat ², Houshang
Rafatpanah ³, Ali Mokhtarifar ¹
1 Department of Internal Medicine , Imam Reza Hospital, Mashhad University of Medical Sciences, Mashhad, IR Iran
2 Department of Pathology, Cancer Research Center, Mashhad, IR Iran
3 Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran

A R T IC LE I NFO AB S T RAC T

Article Type: Background: Viral load has been used to diagnose and monitor patients who are being
Original Article treated for chronic hepatitis B (CHB). The Diagnosis methods are molecular-based and
expensive. Quantitation of hepatitis B surface antigen (HBsAg) by automated chemilu-
minescent micro-particle immunoassay has been proposed to be a surrogate marker.
Article history:
Quantitating HBV DNA levels molecularly is expensive; thus, a cheaper laboratory test
Received: 16 Jun 2010
as a surrogate diagnostic marker might simplify our management.
Revised: 31 Dec 2010
Objectives:We determined whether quantitative HBsAg levels correlate with HBV DNA
Accepted: 25 Jan 2011
levels in CHB.
Patients and Methods: In this cross-sectional study, all CHB patients who were referred
Keywords:
by a gastroenterologist to undergo quantitative HBV DNA assay in a qualified labora-
Chronic hepatitis B
tory in Mashhad, Iran in 2009 were enrolled, and blood samples was obtained. Pa-
Quantitative HBsAg
tients who were positive for antibodies to HCV and HDV were excluded. HBV DNA was
HBV DNA level
measured by real-time polymerase chain reaction, and serum HBsAg was quantified
byelectrochemiluminescence assay (Roche Diagnostic).
Results:Of 97 patients, 70 were male (72%) and 27 were female (28%); the mean age was
39 ± 11 years. Eighty-seven percent wasHBeAg-negative. By Mann-Whitney test,HBSAg
titer differed significantly between HBeAg-positive and -negative patients (P = 0.001),
as did HBV DNA levels (P = 0.009). By Spearman test, there was no significant correla-
tion between HBsAg and HBV DNA levels (P= 0.606 and r = 0.53).
Conclusions:HBeAg-negative patients have higher levels of HBsAg and lower levels of
HBV DNA. By electrochemiluminescence assay,HBsAg has no significant correlation
with HBV DNA levels in CHB with predominant genotype D and HBeAg negativity in
Iran.
c 2011 Kowsar M.P.Co. All rights reserved.

Implication for health policy/practice/research/medical education:

Quantitative detection of HBsAg as one of the new diagnostic methods is discussed recently in many papers. This article opens new
windows toward basic scientists, clinicians in the field of hepatology and infection diseases.

Please cite this paper as:

Ganji A, Esmaeilzadeh A, Ghafarzadegan K, Helalat H, Rafatpanah H, Mokhtarifar A. Correlation of quantitative assay of HBsAg and
HBV DNA levels in chronic HBV. Hepat Mon.2011;11(5):342-345.

* Corresponding author at: Azita Ganji, Mashhad University of Medical Sci-

Background
ences, Internal Medicine, Mashhad, IR Iran. Tel/Fax: +98-8598818.
Chronic hepatitis B is a major global problem, affecting
E-mail: Azita_ganji@yahoo.com
more than 350 million chronic Hepatitis B worldwide (1)
Copyright c 2011, BRCGL, Published by Kowsar M.P.Co All right reserved. and leading to 1 million deaths each year (2) Quantitative
HBsAg and HBV DNA levels in chronic HBV Ganji A et al. 343

levels of HBV DNA, ALT levels, and histological findings measured. Those who were positive for antibodies to HCV
are three factors to consider when determining HBV treat- and HDV were excluded.HBV DNA was measured by real-
ment. Active viral infection scan be detected by quantify- time PCR, and based on the detection limits, viral loads
ing HBV DNA, but such assays are molecular-based and that were less than 100 was considered undetectable and
expensive. Considering the distribution of HBV, particu- excluded. ALT levels were measured by ELISA. Serum HB-
larly in developing countries, a cheaper laboratory test sAg was quantified by electrochemiluminescence assay
that can be used as a surrogate marker for the molecu- (Roche Diagnostic), wherein HBsAg was expressed in IU/
lar detection of HBV DNA might make our management ml. One hundred fifteen patients with chronic hepatitis
more practical. Hepatitis B virus is a DNA virus that has B with HBeAg positive or negative were enrolled, 14 of
a circular and partially double-stranded genome, which whom were HDV-positive; and totally we excluded 4 pa-
encodes four major proteins—S, P, C, X. HBsAg is the chief tients due to missing data. SPSS 16 was used to analyze
protein of the viral envelop, and serological assays that the data. Spearman correlation coefficient was used to
detect HBsAg have guided the diagnosis of hepatitis B correlate serum levels of HBsAg and HBV DNA levels. P ≤
infection. We hypothesize that it can be a useful tool for 0.05 was considered significant.
managing patients as well.
Recently, the relationship between serum HBsAg con- Results
centrations and HBV DNA levels in hepatitis B patients Statistical Analysis
who are positive for serum HBsAg and HBeAg was exam-
ined. Serum HBsAg concentration was related to HBV Fourteen patients (12%) with CHB were HDV-positive and
DNA replication level; nevertheless, it is not feasible to excluded. Ninety-seven of 115 patients with CHB were in-
use HBsAg concentration to monitor HBV replication cluded; 70 were male (72%) and 27 were female(28%). The
levels (3). In noncirrhotic patients, HBV DNA and HBsAg mean age was 39 ± 11 years. Eighty-five CHB patients were
levels correlate negatively. HBsAg levels are low in HBeAg- HBeAg-negative (87%) and 12 (13%) were HBeAg-positive.
positive patients but higher in HBeAg-negative cases; HBV By Kolmogorov-Smimovtest, the distribution of HBsAg ti-
DNA levels are higher in HBeAg-positive patients com- ters was normal and irregular for HBV DNA levels, HBeAg
pared with HBeAg-negative cases (4). In another study, titer, AST, and ALT. Mean HBsAg level was 4021 ± 2305 IU/
however, serum HBsAg levels, using Architect HBsAg QT, ml;mean AST level was 54 ± 94 U/L and mean ALT was 62 ±
were higher in HBeAg-positive than in anti-HBe positive 84 U/L. Mean HBV DNA level was 4.69×106 ± 1.839× 107 copy/
chronic HBV carriers, correlating with the level of serum ml (Figure 1).By Mann-Whitney test, HBSAg titer differed
HBV DNA (5). Quantitative measurements of HBsAg titer significantly between HBeAg-positive and -negative pa-
constitute a simple and economical reference for HBV tients (P = 0.001). In 12 HBeAg-positive patients, less than
replication in HBV carriers as well (6). Previous studies 25% had HBsAg titer = 1088 (Q1 = 1088) and HBsAg titer in
have suggested that quantitative hepatitis B surface an- 25-50% of patients (Q2) = 2030 and in more than 75% of
tigen (HBsAg) is also a surrogate marker that can be used patients (Q3), HBsAg titer were =3143 IU/ml and in 72 pa-
to monitor patients with CHB who are being treated, and tients with HBeAg negative, HBsAg titer were in Q1= 2349,
HBsAg titer is related to HBV DNA levels (7). Q2 = 4369, and Q3 = 6108 IU/ml (Figure 2).

Objectives
Using serum HBsAg concentration as a marker of HBV
replication level in hepatitis B patients, we determined
whether quantitative HBsAg correlates with hepatitis B
virus (HBV) DNA levels in CHB in Iran.
Frequency

Patients and Methods

This descriptive analytical study (cross-sectional) was
performed to determine the correlation of serum HBSAg
level and quantitative HBV DNA level in patients with
chronic hepatitis B in the Department of Gastroenterol-
ogy and Hepatology, Imam Reza Hospital, Mashhad, Iran.
All CHB patients who were HBsAg-positive for more than
6 months and referred by a hepatologist to a qualified vi-
rology laboratory in Mashhad to undergo HBV DNA assay,
were enrolled and selected by nonrandom sampling.
After the purpose of research was explained and in-
formed consent was obtained, samples were drawn and HBS Ag titer
HBsAg, HCV Ab, HDV Ab, HBeAg, AST, and ALT levels were Figure 1. HBsAg titer in CHB (Mean = 3962.63, Std. Dev. = 2250.078, N = 82)

Hepat Mon. 2011;11(5):342-345

 344 Ganji A et al. HBsAg and HBV DNA levels in chronic HBV

cases, 87% of whom were HBeAg-negative, there was no

significant correlation between HBsAg level and HBV
DNA level—even after subdivision into HBeAg-positive
and -negative patients. Nevertheless, our analysis pro-
vides insight into the differences inHBsAg levels between
Frequency

HBeAg-positive and -negative patients, which appear to

Positive
be affected by HBeAg status. HBV DNA levels were higher

HBe Ag
in HBeAg-positive patients, but HBsAg levels were higher
in HBeAg-negative patients. In a previous study, we ob-
served patent differences in HBs levels based on HBeAg
status (14).
Studies have measured HBsAg concentration during
the treatment of HBeAg-negative patients. The lowest
rate of Sustained Virological Response (SVR) with PEG

Negative
interferon was observed in those with genotype D(15),
which experienced the smallest decline in HBsAg concen-
HBs Ag titer
tration during treatment (16) and had the lowest chance
Figure 2. Frequency of HBsAg titer in HBeAg-positive and -negative CHB of a sustained response with interferon-alpha(17). Thus,
there are differences in the importance of quantitative
HBV DNA level also differed significantly between HBsAg concentration for genotype D at baseline and dur-
HBeAg-positive and negative patients (P = 0.009). HBV ing treatment. All HBV isolates in Iranian patients are
DNA level in HBeAg-positive subjects was Q1: 8.65×10³, Q2: genotype D (8). We have an unusual subtype, "ayr," of the
9.02 ×105 and Q3: 8.38×107 and in HBeAg negatives were: virus in Iran, which is not typical for HBV genotype D (18).
Q1 (less than 25% of patients) = 515, Q2 (25-50% of patients) Based on previous studies in throughout Iran, all CHB pa-
= 3350 AND Q3 (more than 75% of patients) = 7/60 ×10 4.By tients in Iran have genotype D; thus, there was no need to
Spearman test, there was no significant correlation be- determine the genotype.
tween HBsAg and HBV DNA (P = 0.606 and r = 0.53). After In our study, we found statistically significant differenc-
the data were split, this correlation remained insignifi- es in HBsAg and HBV DNA levels between HBeAg-positive
cant inHBeAg-positive (P = 0.053 and r = -0.57) and-nega- and -negative patients, but our sample size was likely
tive patients (P = 0.605 and r = 0.057). There was no sig- too small to observe any significant correlation between
nificant link between HBsAg level and ALT level (P = 0.45). HBsAg and HBV DNA levels. Conversely, it can be due to
HBV DNA levels correlated with ALT (P = 0.05, r = 0.19). genotype D in Iran. In HBeAg-positive patients, HBsAg
correlated with serum HBV DNA, intrahepatic ccc DNA,
Discussion and total HBV DNA, but these correlations were poor in
HBeAg-negative cases (13). It appears thata cutoff value
We attempted to correlate quantitative HBsAg levels
of 1500 IU/ml for serum HBsAg during treatment can be
with HBV DNA level in an area in Iran that is predomi-
a predictor of seroconversion(19). In other studies, se-
nantly HBeAg-negative, in which nearly all patients are
rum HBsAg concentration has been used as a surrogate
infected by genotype D (8). HBsAg is a classical marker of
marker of HBV DNA in HBeAg-positive patients and those
infection with hepatitis B virus, and serological assays to
with high ALT; in their study, HBsAg titer predicted HBV
detect HBsAg have guided its diagnosis. We hypothesized
DNA levels (20). In HBeAg-negative patients, HBsAg lev-
that HBsAg can be used to manage and monitor patients
els were higher in patients with active disease than in
as well. On infection with HBV, closed circular DNA of HBV
those with inactive disease, and HBsAg remained stable
genome forms inside the nuclei of hepatocytes (9). DNA-
in HBeAg-positive patients but tended to fall gradually in
containing nucleocapsids become enveloped and are se-
HBeAg-negative patients (21). In our study, after dividing
creted into the blood (10). Hepatitis B virus ccc DNA is a
patients by active and inactive CHB (based on ALT > 50
template for viral replication, correlating robustly with
and HBV DNA > 2000), we observed was no significant
levels of total intracellular HBV DNA, serum HBV DNA,
difference between active and inactive CHB, likely due to
and HBsAg (11). HBsAg quantification indirectly reflects
the small sample size.
the number of infected hepatocytes (12).
HBsAg titer can help differentiate active from inactive
Chan, et al. observed that low pretreatment HBsAg is
carriers. Single-point combined HBsAg and HBV DNA
better than HBV DNA in predicting good responses to
quantification provide the most accurate identification
treatment (11). In another study of HBeAg-negative CHB,
of inactive carriers (22). Thus, we need more studies with
however, HBsAg correlated poorly with serum HBV DNA
larger samples in active and inactive CHB to determine
and did not correlate with intrahepatic ccc DNA or to-
whether there is any correlation in this case.
tal HBV DNA. Quantitative Immunohistochemistry for
There are 2 forms of HBsAg—one over intact virion,
hepatocyte HBsAg confirmed its relationship with viral
which includes small, medium, and large proteins in en-
replication only in HBeAg-positive patients (13). In our
velops and is related to viral infectivity, and another that

Hepat Mon. 2011;11(5):342-345

HBsAg and HBV DNA levels in chronic HBV Ganji A et al. 345

exists as subviral particles in serum and is produced in tion of quantitative assay of hepatitis B surface antigen and
great excess. These are predominantly S protein and, to HBV DNA levels in asymptomatic hepatitis B virus carriers. Eur
J Gastroenterol Hepatol. 2004;16(11):1213-8.
a lesser extent, M and L protein; these are not infectious 7. Ozaras R, Tabak F, Tahan V, Ozturk R, Akin H, Mert A, et al. Correla-
but are strongly immunogenic, stimulating antibody tion of quantitative assay of HBsAg and HBV DNA levels during
production. We propose that if we can detect 2 forms of chronic HBV treatment. Dig Dis Sci. 2008;53(11):2995-8.
HBsAg (over intact virion and subviral particle separately 8. Vaezjalali M, Alavian SM, Jazayeri SM, Nategh R, Mahmoodi M,
Hajibeigi B, et al. Genotype of Hepatitis B Virus Isolates from Ira-
and quantitate them, we can determine the relationship nian Chronic Carriers of the Virus. Hepat Mon. 2008;8(2):97-100.
between special forms of HBsAg and HBV DNA level.Based 9. Bowden S. Serological and molecular diagnosis. Semin Liver Dis.
on our results, quantitative HBsAg level cannot be used 2006;26(2):97-103.
10. Hatzakis A, Magiorkinis E, Haida C. HBV virological assessment. J
as a surrogate marker for replicative state inHBeAg-neg-
Hepatol. 2006;44(1 Suppl):S71-6.
ative patients in Iran. We recommend performing larger 11. Chan HL, Wong VW, Tse AM, Tse CH, Chim AM, Chan HY, et al. Se-
studies in Iran in different groups of CHB and patients rum hepatitis B surface antigen quantitation can reflect hepa-
who are being treated for CHB separately. HBeAg-nega- titis B virus in the liver and predict treatment response. Clin
Gastroenterol Hepatol. 2007;5(12):1462-8.
tive patients have higher levels of HBsAg and lower levels 12. Ben Slama N, Ahmed SN, Zoulim F. [HBsAg quantification: vi-
of HBV DNA. by electrochemiluminescence assay do not rological significance]. Gastroenterol Clin Biol. 2010;34(Suppl
correlate significantly with HBV DNA level in HBeAg-posi- 2):S112-8.
13. Thompson AJ, Nguyen T, Iser D, Ayres A, Jackson K, Littlejohn
tive or negative patients in Iran, necessitating additional
M, et al. Serum hepatitis B surface antigen and hepatitis B e
studies to standardize quantification assays and define antigen titers: disease phase influences correlation with viral
thresholds of HBsAg that have clinical predictive value. load and intrahepatic hepatitis B virus markers. Hepatology.
2010;51(6):1933-44.
14. Ganji A, Esmaeilzadeh A, Shovey MF, Ghaffarzadehgan K. M1904
Financial support There is NO Correlation of Serum HBsAg Level With the Degree
of Necroinflammation and Fibrosis of the Liver in Patients With
None declared. Chronic Hepatitis B. Gastroenterology. 2010;138(5):S-833.
15. Andreas E, Amei DL, Maurizia B, Florian van B, Eva W, Thomas G,
Conflict of interest et al. HBV genotypes are the strongest predictor of response to
interferon –a treatment ;multivariate evaluation in 1229 hepati-
None declared. tis b patients. Hepatol. 2008;48(suppl 1):700A.
16. Brunetto MR, Moriconi F, Bonino F, Lau GKK, Farci P, Yurday-
din C, et al. Hepatitis B virus surface antigen levels: A guide to
Acknowledgments sustained response to peginterferon alfa-2a in HBeAg-negative
chronic hepatitis B. Hepatology. 2009;49(4):1141-50.
We would like to express our appreciation to Dr. Ahmad 17. Buster EH, Hansen BE, Lau GK, Piratvisuth T, Zeuzem S, Steyerberg
Moayed and his laboratory for their contribution in col- EW, et al. Factors that predict response of patients with hepatitis
B e antigen-positive chronic hepatitis B to peginterferon-alfa.
lecting the data and to our colleagues, who helped us Gastroenterology. 2009;137(6):2002-9.
perform this study. 18. Mohebbi S, Amini-Bavil-Olyaee S, Zali N, Derakhshan F, Sabahi
F, Zali MR. An Extremely Aberrant Subtype of Hepatitis B Virus
Genotype D in Iran. Hepat Mon. 2009;9(1):73-5.
References 19. Ma H, Yang RF, Wei L. Quantitative serum HBsAg and HBeAg are
strong predictors of sustained HBeAg seroconversion to pegy-
1. Lee WM. Hepatitis B virus infection. N Engl J Med. 1997;337(24):1733-
lated interferon alfa-2b in HBeAg-positive patients. J Gastro-
45.
enterol Hepatol. 2010;25(9):1498-506.
2. Wright TL. Introduction to chronic hepatitis B infection. Am J
20. Su TH, Hsu CS, Chen CL, Liu CH, Huang YW, Tseng TC, et al. Se-
Gastroenterol. 2006;101(Suppl 1):S1-6.
rum hepatitis B surface antigen concentration correlates with
3. Lei JH, Yang X, Luo HY, Wang WL, Huang L. [Serum HBsAg con-
HBV DNA level in patients with chronic hepatitis B. Antivir Ther.
centration and HBV replication level in hepatitis B patients with
2010;15(8):1133-9.
positive serum HBsAg and HBeAg]. Zhong Nan Da Xue Xue Bao Yi
21. Chan HL, Wong VW, Wong GL, Tse CH, Chan HY, Sung JJ. A lon-
Xue Ban. 2006;31(4):548-51.
gitudinal study on the natural history of serum hepatitis B
4. Ozdil B, Cosar AM, Akkiz H, Sandikci MU, Kece C. Negative cor-
surface antigen changes in chronic hepatitis B. Hepatology.
relation between viral load and HBsAg levels in chronic HBV-
2010;52(4):1232-41.
infected patients. Arch Virol. 2009;154(9):1451-5.
22. Brunetto MR, Oliveri F, Colombatto P, Moriconi F, Ciccorossi P,
5. Deguchi M, Yamashita N, Kagita M, Asari S, Iwatani Y, Tsuchida
Coco B, et al. Hepatitis B surface antigen serum levels help to dis-
T, et al. Quantitation of hepatitis B surface antigen by an auto-
tinguish active from inactive hepatitis B virus genotype D carri-
mated chemiluminescent microparticle immunoassay. J Virol
ers. Gastroenterology. 2010;139(2):483-90.
Methods. 2004;115(2):217-22.
6. Chen CH, Lee CM, Wang JH, Tung HD, Hung CH, Lu SN. Correla-

Hepat Mon. 2011;11(5):342-345