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Hepatitis B surface antigen quantification in chronic hepatitis B and its clinical utility Expert Rev. Gastroenterol. Hepatol. 8(2), 185–195 (2014)

Emilia Hadziyannis1 and Stephanos J Hadziyannis*2 1 Academic Department of Medicine, Hippokration Hospital, National and Kapodistrian University of Athens, 114 Vas. Sophias Ave, Athens 11529, Greece 2 Liver Unit and Molecular Biology Laboratory, The Evgenidion Hospital, National and Kapodistrian University of Athens, Address: 18 Papadiamantopoulou Str, Athens 11528, Greece *Author for correspondence: Tel.: +30 210 651 6807 Fax: +30 210 654 3299 [email protected]

Serum HBsAg levels have been quantified extensively in recent years with simple completely automated assays in the various phases of the natural course of chronic HBV infection, have been compared with cccDNA in the liver, with various markers of HBV replication and have been correlated with several viral, host and environmental variables. Low HBsAg levels in inactive carriers predict a spontaneous HbsAg loss. Quantification of HBsAg in serum at baseline and its decline under interferon-alfa based regimens, both in HBeAg-positive and HBeAg-negative CHB, provides important information on the prediction of sustained posttreatment outcomes and on subsequent HBsAg clearance. The value of HBsAg quantification in the monitoring of long term nucleos(t)ide analogue treatment of CHB and in the prediction of sustained response remains unclear. In this review, the most recent data regarding the overall clinical utility of HBsAg measurement in HBeAg-positive and -negative CHB and in their treatment, is critically presented. KEYWORDS: chronic hepatitis B . HBsAg . HBV . HBV DNA . inactive carriers . interferon . natural course .

nucleos(t)ide analogs



Historical flashback

Hepatitis B surface antigen (HBsAg), first discovered in the early 1960s, represents the surface protein that covers the nucleocapsid of hepatitis B virus (HBV). It is produced in excess during all phases of HBV infection and actually represents the most sensitive diagnostic seromarker of underlying HBV infection, exceeding in sensitivity even in the detection of HBV DNA in serum as assessed by the most sensitive, qualitative real-time PCR assays [1]. In the 1970s, a number of investigators evaluated serum HBsAg concentrations by determining its titers by serial dilutions, in various subsets of patients with chronic HBV infection and compared HBsAg dilution titers with the hepatitis B e antigen (HBeAg)/antiHBe status as markers of HBV replication [2,3,4]. In these studies, it was clearly shown that high HBsAg titers correlated with HBV infectivity, with vertical HBV transmission and with HBeAg positivity. In those days, the serum HBeAg/anti-HBe status had also


started to be applied in clinical practice, tested first by immunodiffusion assays and subsequently by a sensitive radioimmunoassay test [5,6]. Some of the early findings of the comparison between HBeAg/anti-HBe and HBsAg titers are summarized in (TABLE 1) [7]. In the 1980s, HBsAg was also measured by dilution titers in some early studies of standard interferon therapy of HBeAg-positive chronic hepatitis B (CHB) but subsequently, after the introduction of molecular hybridization and PCR assays, HBsAg quantification was completely abandoned being substituted by measurements of serum HBV DNA [8,9]. However, the decline in serum HBV DNA levels achieved under interferon therapy both in HBeAg-positive and -negative CHB patients, did not prove to be a reliable predictor of a sustained virologic response after stopping therapy and of subsequent HBsAg clearance either in terms of positive (PPV) or negative predictive value (NPV) [10,11]. In the 1990s, we entered the era of oral antiviral therapy with nucleos(t)ide analogs

 2014 Informa UK Ltd

ISSN 1747-4124



Hadziyannis & Hadziyannis

Table 1. Dilution titers of hepatitis B surface antigen in chronic hepatitis B virus infection in volunteer blood donors and homosexual men of New York in comparison to their HBeAg/anti-HBe status.

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Geometric mean HBsAg titer Blood donors

Homosexual men










HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus. Data taken from [7].

(NAs) in courses of finite or indefinite duration with excellent on-treatment results regarding suppression of HBV replication maintained under treatment (maintained virological response) but disappointing post-treatment results particularly in terms of sustained virological response (SVR) and of HBsAg clearance, especially in HBeAg-negative CHB [10,12]. Nevertheless, interferons came back in the treatment of CHB with their pegylated compounds (pegIFN), and large randomized controlled trials of 48 weeks’ duration documented, both in HBeAg-positive and HBeAg-negative CHB, their superiority over finite courses of NAs in terms of complete post-treatment responses [13,14]. This was particularly true for HBsAg clearance that was found to increase progressively over the years of post-treatment followup [15,16,17,18]. These data with pegIFN-a actually confirmed what had previously been well documented internationally in the treatment of HBeAg-positive CHB with standard interferons [19] as well as by pioneer studies of standard interferon therapy of HBeAg-negative CHB conducted in the Mediterranean Area [6,11,12,20,21,22,23,24]. In this framework, probable predictors of sustained response (SR) to interferon therapy and particularly of HBsAg clearance were evaluated retrospectively in a number of cohort and observational studies from various parts of the world. In such an early original study from Greece [25], HBsAg titers were evaluated retrospectively at baseline and on therapy as possible predictors of HBsAg loss in HBeAg-negative CHB patients treated by standard interferon [22] or by lamivudine [26]. This study clearly indicated that HBsAg titers decline quickly under interferon but not under lamivudine treatment and that such an ontreatment decline can be used as predictor of post-treatment loss of HBsAg. The above observations have been confirmed in several retrospective analyses of HBsAg titers in CHB patients and large evidence soon accumulated indicating that HBsAg loss induced by interferon treatment can be predicted accurately by an early on-treatment decline of HBsAg concentrations [17,27,28,29,30,31,32]. Actually in the few years that followed, there has been a flood of quantitative studies of HBsAg in the world literature covering practically all aspects of hepatitis B and mostly its predictive value in the outcome of the infection and in the response to various 186

forms of treatment. However, most of the early studies on HBsAg concentrations and their predictive value for SR and HBsAg clearance had several limitations. They represented retrospective analyses of previously treated cohorts of patients with differences in viral, host and environmental characteristics, selection biases were probable and untreated control groups were lacking. On the other hand, sensitive and reliable quantitative HBsAg assays have become commercially available [33,34] with a relatively low cost, have gained wide popularity and studies of large groups of properly documented, followed-up and treated patients from Eastern and Western countries have appeared in the world literature over the last 3–4 years. It is the purpose of the present article to review critically the hitherto reported information on HBsAg concentrations during the various phases in the natural course of chronic HBV infection and on the predictive value of HBsAg titers during treatment of CHB. Production & secretion of HBsAg from the liver

HBsAg is present in the serum of infected patients either as a component of the 42-nm infectious viral particles of HBV or as part of empty subviral particles forming filaments or spheres. These empty subviral particles are secreted from infected hepatocytes in excess (almost 100- and up to 10,000-fold) of the infectious, HBV DNA-containing virions [35]. The production of HBsAg particles is the result mainly but not exclusively of active transcription and translation of the surface genes directly from the covalently closed circular DNA (cccDNA) in HBVinfected hepatocytes [36]. A number of early [37,38] and more recent studies [17,25,28,39,40] have shown a good correlation between serum HBsAg levels and serum HBV DNA, particularly in HBeAg-positive patients. Moreover, serum HBsAg levels seemed to correlate with total intrahepatic HBV DNA as well as with cccDNA levels [41,42,43]. This correlation was of particular clinical importance and of relevance regarding antiviral treatment in CHB since serum HBV DNA does not always correlate well with the intrahepatic DNA content (either total or cccDNA) and has led to the suggestion that HBsAg could represent a marker of the total pool of infected hepatocytes that produce viral proteins (i.e., envelope proteins) and thus an indirect marker of intrahepatic cccDNA [44]. However, with more studies it has been realized that because of different mechanisms of HBsAg synthesis in the liver, the quantification of serum HBsAg cannot be used in clinical practice as a reliable surrogate marker of HBV replication [44,45,46]. Actually, there appears to be general agreement now that HBsAg is produced in the liver by the following differing pathways: .

Through translation of two RNA transcripts of the cccDNA of HBV: the pre-S1 and the preS-2/S, the former coding for the large (L) surface protein and the latter coding for the middle (M) and small (S) envelope proteins of the virus [47]. These RNA transcripts of the surface protein Expert Rev. Gastroenterol. Hepatol. 8(2), (2014)

HBsAg in hepatitis B


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Table 2. Examples of hepatitis B surface antigen cut-off values of clinical importance suggested in the literature. HBsAg cut-off value (IU/ml)

Population and/or timing

Clinical importance






Treatment Minimal inflammation

HBeAgpositive chronic hepatitis

Treatment Inactive carrier state

HBeAg-negative chronic hepatitis

Figure 1. Serum Hepatitis B surface antigen, hepatitis B virus DNA and alanine aminotransferase levels in the different phases of chronic hepatitis B virus infection. ALT: Alanine aminotransferase; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus.

During finite courses of therapy of HBeAg-positive patients, with IFN-a with or without NAs, the absolute levels of HBsAg combined with the level of its decrease have been shown to identify with relatively high probability SVR or non-response to treatment [16,31,80,81]. HBsAg levels at baseline have been found to be lower in patients with HBeAg seroconversion at the end of treatment compared with nonresponders [82,83], but the difference does not appear to be significant [84,85]. More importantly, HBsAg levels at week 12 or 24 of treatment have been lower in the responder group and a cut-off of 1500 IU/ml demonstrated a PPV of 57 and 54%, respectively, as well as high NPV, greater than 70% [81,86]. Depending on the HBV genotypes, the absence of decline at week 12 demonstrated a NPV for SVR between 82 and 97% [31]. The on-treatment decline of HBsAg titer varies according to the infecting genotype (A > B > C > D) with significant difference between A/B and C/D [87]. The highest NPV for long-term response was seen at a cut-off level of 20,000 IU/ml at week 12 or 24 of treatment [81,86]. On the basis of the above observations, two stopping rules of interferon treatment at week 12 in HBeAg-positive CHB have been formulated: no HBsAg decline and HBsAg levels >20,000 IU/ml. These proposed prediction rules have shown limited external validity and in a recent analysis of 803 HBeAg-positive patients treated in three global studies with pegIFN, a stopping rule based on absence of HBsAg decline from baseline was compared with a prediction rule that uses HBsAg levels of 20,000 IU/ml to identify patients with high and low probability of response [88].


Immune tolerance


Median HBsAg log10 IU/ml


p < 0.01


The effect of therapy on HBsAg titers has been examined in several studies most of which are retrospective. The kinetics of HBsAg under different forms of treatment continue to be thoroughly examined in order to fully understand the mechanisms of its reduction and in the cases of complete response of its subsequent clearance. Depending on the type of therapy (IFN-a or NA) and on the type of CHB (HBeAg-positive or HBeAg-negative), we have reviewed the effects of treatment on HBsAg concentrations and their predictive value on SVR and on HBsAg loss in four subgroups: IFN-a therapy in HBeAg-positive CHB, IFN-a therapy in HBeAg-negative CHB, NA therapy in HBeAg-positive CHB and NA therapy in HBeAg-negative CHB.

p < 0.01


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The effect of interferon & of NA therapy on HBsAg titers in CHB

In patients with HBsAg levels 6000 IU/ml at week 24 of treatment with pegIFN have also been shown to have high NPV (>94%), with an area under the receiver operating characteristic curve >0.8 [84,85]. Clearly, a response-guided treatment is expected to improve the cost–effectiveness of pegIFN therapy. Finally, HBsAg levels in HBeAg-positive patients with alanine aminotransferase (ALT) flares during pegIFN treatment have been associated with the type of flare (virus or host induced) [89]. Thus, subsequent to host-induced flares there has been a decline in HBsAg levels greater than 0.5–1 log10 IU/ml which, in long-term follow-up, is predictive of HBsAg loss. IFN-a therapy in HBeAg-negative CHB

IFN-a treatment, both with its standard and pegylated forms, in patients with HBeAg-negative CHB leads to a more pronounced drop in HBsAg concentrations compared with NAs [25]. In patients who achieve an SVR after stopping a 189


Hadziyannis & Hadziyannis

1. Immune tolerant HBeAg positive phase

2. Chronic hepatitis B HBeAg positive (Immune active)

3. Inactive carrier state (HBeAg negative)

cut-off levels ranging from 50 to 1000 IU/ml. Additionally, HBsAg drop >1 log10 and HBsAg

Hepatitis B surface antigen quantification in chronic hepatitis B and its clinical utility.

Serum HBsAg levels have been quantified extensively in recent years with simple completely automated assays in the various phases of the natural cours...
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