Introduction and Background - Screening for Hepatitis B ...

This systematic review update will be used by the United States Preventive Services Task Force (USPSTF) to update its recommendation from 1 , 2 on screening for hepatitis B virus (HBV) infection in nonpregnant adolescents and adults. 3 , 4 In , the USPSTF recommended screening for HBV infection in persons at high risk for infection ( B recommendation ). The USPSTF recommendation noted an HBV prevalence of two percent or greater as a reasonable threshold for deciding to screen; this includes persons born in countries and regions with a prevalence of HBV infection of two percent or greater, U.S.-born persons not vaccinated as infants whose parents were born in regions with a HBV prevalence of eight percent or greater, HIV-positive persons, persons who inject drugs, men who have sex with men, and household contacts or sexual partners of persons with HBV infection.

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Condition Background

Condition Definition

HBV is a double-stranded deoxyribonucleic acid (DNA) virus enclosed in a nucleocapsid protein (hepatitis B core antigen [HBcAg]) surrounded by an envelope protein (hepatitis B surface antigen [HBsAg]).5 Serologic markers are usually the initial tests used to determine HBV infection status ( ); subsequent tests in persons with markers indicating active infection are performed to determine the presence and level of circulating HBV DNA (viral load). Acute HBV infection (within 6 months after infection) is typically characterized by the initial appearance of HBsAg with HBV e antigen (HBeAg) and HBV DNA; immunoglobulin M (IgM) antibody to the HBV core antigen (anti-HBc) appears soon after infection, evolving to anti-HBc immunoglobulin G (IgG).6,7 Chronic infection is characterized by the persistent presence of HBsAg for longer than 6 months.6&#;8 The presence of HBeAg is usually associated with high levels of HBV DNA in serum and high infectivity.9,10 Resolution of HBV infection and disease inactivity are typically characterized by the disappearance of HBsAg and appearance of antibody to HBV surface antigen (anti-HBs). Inactive chronic HBV infection, characterized by the disappearance of HBeAg and appearance of antibody to HBeAg (anti-HBe), eventually occurs in most patients with chronic HBV infection, usually correlating with low levels of HBV DNA in serum and remission of liver inflammatory activity. Reactivation of HBV, or a flare in HBV activity in persons, can occur in persons with serological evidence of inactive or resolved (positive for anti-HBc, but negative for HBsAg) HBV infection.11

Table 1

Interpretation of Screening Tests for HBV Infection.

Prevalence and Burden of Disease/Illness

The incidence of acute symptomatic HBV infections in the United States reported to the Centers for Disease Control and Prevention (CDC)12 fell from over 20,000 cases annually in the mid-s to 2,791 cases in , with an increase to 3,409 in .12 Due to underreporting, the actual number of cases is estimated to be 6.5 times higher than the number of reported cases.12 From to , the incidence of acute HBV infection declined among all age groups.12 The highest incidence of acute HBV infections is among persons 40 to 49 years of age (2.5 cases/100,000 population in ), followed by persons 30 to 39 years of age; the rate of acute HBV infection is higher in men than women.12 Since , a rise in acute and chronic HBV infection related to drug use has been reported in several states.13&#;15

As of , the overall prevalence of chronic HBV infection in the United States is about 0.3 percent.16 In and , an estimated 847,000 people in the United States were chronically infected with HBV.12,16 Universal infant vaccination, instituted in , has reduced the incidence and prevalence of chronic HBV infection. The number of persons with serological evidence of vaccine protection from HBV rose from 57.8 million in to 68.5 million in to .16 The prevalence of HBV infection in persons 6 to 19 years of age was 0.03 percent, compared with 0.4 percent among persons 20 to 49 years of age and 0.3 percent among persons &#;50 years of age. Effects of vaccination on the overall prevalence of chronic HBV infection have been offset by immigration from places where chronic HBV is endemic, such as Asia and Africa.16 Foreign-born persons are estimated to account for approximately 95 percent of newly reported chronic HBV infections in the United States and have an estimated HBV prevalence of approximately 3.5 percent.17,18 About half of prevalent U.S. cases of chronic infection are in non-Hispanic persons of Asian descent, a group representing 5 to 6 percent of the U.S. population.19 In the National Health and Nutrition Examination Survey, the prevalence of chronic HBV infection in non-Hispanic persons of Asian descent was 3.1 percent in to , or 10 times higher than in the general population.16 The prevalence was 0.1 percent in non-Hispanic white persons, 0.6 percent in non-Hispanic black persons, and 0.06 percent in Mexican American persons. In , there were an estimated 1,727 deaths associated with HBV infection (0.46 per 100,000 persons); death rates were higher in persons age 75 years and older compared to other age groups, persons of Asian/Pacific Islander race compared to other races/ethnicities, and males compared to females.12

Etiology and Natural History

HBV is spread through percutaneous or mucous membrane exposure to blood or blood-containing body fluids (serum, semen, or saliva), including sexual contact and injection drug use; horizontal transmission of HBV also occurs among close household contacts.6,10,20 HBV infection can be transmitted from mother to infant during birth (perinatal transmission); the USPSTF addresses perinatal HBV screening in a separate review.21 The liver is the primary site of HBV replication. Acutely infected individuals may be asymptomatic or present with symptoms of acute infection, such as nausea, anorexia, fatigue, low-grade fever, and abdominal pain.5 Jaundice may also be present, and elevated liver enzymes (e.g., alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) can be seen on standard assays.

If symptoms of acute disease occur, they can take from 6 weeks to 6 months to appear.22 Acute infection generally self-resolves in 2 to 4 months, although mortality in this phase is about 1 percent. The risk of progression from acute to chronic infection varies according to age at the time of exposure. Risk of chronic infection is more than 90 percent in infants, 30 percent in children age 1 to 5 years, and less than 5 percent in those older than age 5 years.10,22 Chronic infection spontaneously resolves in 1 percent of individuals annually.8 Some chronically infected individuals are asymptomatic, although others experience a range of symptoms, including nonspecific symptoms of fatigue or other symptoms related to hepatitis, cirrhosis, or hepatocellular carcinoma.22 Extrahepatic manifestations of HBV infection include polyarteritis nodosa, membranous nephropathy, and membranoproliferative glomerulonephritis. Chronic HBV infection is characterized by several phases: 1) immune tolerant, characterized by the presence of HBeAg and very high levels of HBV DNA but normal ALT and minimal hepatic inflammation and fibrosis; 2) immune active, characterized by high levels of HBV DNA, ALT elevation, and moderate to severe hepatic inflammation; HBeAg can be present or absent (positive anti-HBe); and 3) inactive, characterized by the absence of HBeAg and presence of anti-HBe, low or undetectable levels of HBV viremia, normal ALT, and minimal hepatic inflammation.8 The immune tolerant phase has been considered a period of minimal or no disease progression, though recent studies indicate that histological activity and increased risk of hepatocellular carcinoma may occur.23 Fibrosis progression primarily occurs during the immune active phase; however, the presence and severity of fibrosis in the immune active and inactive phases is variable, as patients can transition between these phases. Although the course of chronic HBV infection varies widely, potential long-term sequelae include cirrhosis, hepatic decompensation, and hepatocellular carcinoma.24 Death from cirrhosis or hepatocellular carcinoma is thought to occur in 15 to 25 percent of those chronically infected with HBV. Increased viral load is associated with greater risk of cirrhosis, hepatocellular carcinoma, and liver-related mortality.25,26 Reactivation of HBV, or the abrupt increase in HBV activity in persons with inactive or resolved HBV, can also occur.11 Reactivation may be spontaneous, but is more commonly associated with use of immunosuppressive agents; reactivation can also occur in patients receiving direct-acting antiviral therapy for hepatitis C virus (HCV) infection.27&#;29 Clinically, the severity of reactivation ranges from mild to severe, fulminant or even fatal hepatitis. Chronically infected persons are a reservoir for person-to-person transmission of HBV infection. Presence of hepatitis D virus coinfection can impact the clinical course of HBV infection and inform treatment choices.8

Risk Factors

People born in countries with an HBV prevalence of 2 percent or greater account for 47 to 95 percent of the chronically infected population in the United States, although marked decreases in prevalence have been seen among younger persons born in these countries due to universal immunization programs.17,18,30,31 In , the prevalence of HBV infection was highest in Africa (6.1%) and in the Western Pacific region (6.2% in countries including China, the Philippines, and Vietnam), and lowest in Europe (1.6%) and the Americas (0.7%).31 Persons at higher risk for acute HBV infection in the United States include men, those age 30 to 49 years, and in recent years, non-Hispanic white persons.12 Risk factors for HBV infection include working in healthcare, having household contacts or sex partners with HBV infection (prevalence of chronic infection, 3% to 20%), HCV-positive status (1.3% to 5.8%), male sexual activity with other males (1.1% to 2.3%), injection drug use (2.7% to 11%), and HIV-positive status (6% to 15%).10,12,22,32&#;37 Settings with high proportions of persons at risk for HBV infection include sexually transmitted infection clinics, HIV testing and treatment centers, health care settings that target services toward persons who inject drugs (PWID) and men who have sex with men (MSM), correctional facilities, hemodialysis facilities, and institutions and nonresidential daycare centers for developmentally disabled persons.6

Rationale for Screening/Screening Strategies

Identification of asymptomatic persons with chronic HBV infection through screening may identify those who would benefit from earlier evaluation and management of their disease. In , an estimated 90 percent of HBsAg-positive individuals globally remained undiagnosed.38 In the United States, estimates of the proportion of persons with HBV infection unaware of their infection status range from one-third to two-thirds.22 Identification of asymptomatic chronic HBV infection could also lead to reductions in behaviors associated with more rapid progression of liver disease or interventions to decrease transmission of HBV, and identify close contacts who might also benefit from testing.38&#;40 Screening could also identify persons with evidence of HBV exposure (positive anti-HBc) who could benefit from education regarding risk of reactivation, and those who could benefit from HBV vaccination (e.g., those never exposed to HBV or those who are isolated anti-HBc positive and immunocompromised).

Interventions/Treatment

Vaccination

Screening could identify persons without prior evidence of HBV exposure (anti-HBs and anti-HBc negative), who could benefit from vaccination to protect against future infection. In persons with isolated anti-HBc positivity, vaccination is recommended in persons from low endemicity areas or those who are immunocompromised.8 In the United States, current policies are for universal vaccination of all infants at birth, catch-up vaccination of adolescents, and vaccination of high-risk adults.41 In persons not at increased risk of HBV infection, HBV serologic testing prior to vaccination is not required. HBV vaccines in the United States contain between 10 to 40 micrograms of HBsAg protein/mL for adolescents and adults, and before involved at least three intramuscular doses administered at 0, 1, and 6 months.6,10 Vaccination with the three dose vaccine results in greater than 90 percent protective antibody response after the third dose in adults and greater than 95 percent in adolescents, although protective anti-HBs titers may be attained in some persons after one or two doses.6,10 By the end of , 187 countries had introduced nationwide HBV vaccine for infants, with 105 countries targeting vaccination of all newborns.42 In , global coverage with the third infant dose of HBV vaccine reached 84 percent, and prevalence of chronic infection in children under 5 years of age dropped to 1.3 percent, compared with about 4.7 percent before vaccination programs began.31,43 In November , the U.S. Food and Drug Administration (FDA) approved a two-dose HBV vaccine44 for use in adults based on three trials showing comparable serologic outcomes to three-dose vaccines through up to 28 weeks.45 Studies of the two-dose vaccine were not designed to assess effects on risk of HBV acquisition, though vaccine-induced seroprotection is considered a surrogate of clinical protection.46

Treatment

Drugs for HBV infection are broadly categorized as interferons or nucleoside/nucleotide analogs.8,30,47,48 The interferons affect viral replication as well as immune modulation.8,9 Nucleoside/nucleotide analogues (lamivudine, adefovir, entecavir, and others) compete with binding sites on the HBV reverse transcriptase. As of October , seven antiviral drugs had been approved by the FDA for treatment of chronic HBV infection: interferon alfa-2b, pegylated interferon alfa-2a, lamivudine, adefovir, entecavir, telbivudine, tenofovir disoproxil fumarate (TDF); and the most recently approved medication (in ), tenofovir alafenamide (TAF).49,50 TAF is a prodrug of tenofovir with improved kidney and bone safety parameters compared with TDF. The American Association for the Study of Liver Diseases (AASLD) recommends pegylated interferon, entecavir, and TDF as preferred initial therapy for immune-active chronic HBV;8 TAF was recently added to the preferred list.8 Telbivudine is no longer manufactured in the United States, though it is available in other countries.

Cure rates with current antiviral therapies are low,51 and other therapies have been studied, but remain investigational.52 A number of combination therapies have also been evaluated but are not FDA approved and not recommended as first-line treatment due to unclear advantages over monotherapy in most patients, particularly in those at low risk for developing drug resistance.8 The choice of antiviral medication varies according to patient characteristics and disease activity. Factors that affect the decision to treat include the HBV DNA level, serum transaminase levels, and HBeAg status.8 Biopsy may be performed in some patients to establish the degree of liver inflammation and fibrosis, which also affect treatment, surveillance and hepatocellular carcinoma screening decision-making.8 Noninvasive alternatives to biopsy for assessing degree of hepatic fibrosis include imaging with transient elastography and various blood tests.53&#;56 The goal of treatment is to achieve sustained suppression of HBV replication and remission of liver disease in order to prevent cirrhosis, hepatic failure, and hepatocellular carcinoma.30,47 The recommended duration of treatment varies depending on the HBeAg status, presence of cirrhosis, duration of HBV DNA suppression, and choice of medication.8,22 Many patients remain on antiviral treatment indefinitely, with the exception of interferon-based therapy, which is usually recommended for a defined duration of treatment, in part due to limited tolerability and immunomodulatory effects of interferons which may result in a sustained response.8 Other treatments in patients with chronic HBV infection could include counseling or education to potentially reduce behaviors associated with accelerated progression of liver disease (such as alcohol use) or transmission, or surveillance with imaging tests to identify hepatocellular carcinoma,8 though the effectiveness of such surveillance on improving clinical outcomes is uncertain.57 Low rates of linkage and retention in treatment and use of antiviral therapy are barriers to optimal care of persons with HBV infection.58

Current Clinical Practice/Recommendations of Other Groups

Screening for HBV infection is usually performed by testing for HBsAg and anti-HBs.8 Testing for anti-HBc is not routinely recommended by AASLD8 but is recommended by the American College of Physicians (ACP)/CDC;59 it indicates prior HBV exposure status (anti-HBc does not develop after vaccination) and can help determine a patient&#;s risk for reactivation (e.g., in persons being considered for HCV therapy or immunosuppressive treatment). New rapid tests for HBsAg have recently been developed, but no rapid test has been approved by the FDA.60&#;63 The CDC recommends that FDA-approved tests be used to screen for HBsAg and a confirmatory test performed for initially reactive results.22 In persons with serologic findings suggesting chronic infection, followup includes quantitative testing for HBV viremia, presence of HBeAg, and liver transaminase levels. Current U.S. screening practices for HBV and rates of HBV testing are largely unreported. One study of over one million Americans with access to private health care found that about 20 percent were tested for HBV over a median of more than 7 years and 1.4 percent tested positive for HBV infection.64 Based on national HBV prevalence data, it was estimated that 20 to 50 percent of expected HBV infections were not identified in this cohort. Guidelines generally recommend that screening be targeted to populations and persons at increased risk for chronic HBV infection, including persons born in high-prevalence countries.8,59 However, some studies indicate that target populations are not being provided with screening and/or vaccination despite having contact with their clinician.65&#;67 HBV screening best practice advice from the ACP/CDC59 and recommendations from AASLD8 are shown in .

Table 2

HBV Screening Recommendations From the CDC and AASLD.

Both the ACP/CDC best practice advice and AASLD guideline also recommend screening of persons who engage in behaviors associated with increased risk for HBV, including men who have sex with men, persons who inject drugs, HIV-positive persons, and household contacts or sexual partners of persons with HBV infection, inmates of correctional facilities, persons with HCV infection, and persons with end-stage kidney disease.59 AASLD also recommends screening of persons with multiple sex partners or those seeking evaluation or treatment for a sexually transmitted infection, residents and staff of facilities for the developmentally disabled, and travelers to HBV endemic countries.8 Both AASLD and earlier () CDC guidelines recommend screening of U.S. born persons not vaccinated as infants whose parents were born in regions with an eight percent or greater prevalence; the CDC guideline also recommends screening needle-sharing contacts of injection drug users.8,22 The National Academies of Science, Engineering, and Medicine National Strategy cites the USPSTF recommendation on screening as an essential component of its National Strategy for Elimination of Hepatitis B and C.68

Internationally, the World Health Organization (WHO) recommends HBV testing in the general population when the prevalence is 2 percent or greater and in higher-risk populations.69 The United Kingdom&#;s National Institute for Clinical Excellence recommends HBV testing in higher-risk populations and is generally consistent with the USPSTF recommendation.70

Abstract

Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

Keywords: Hepatitis B virus, Hepatitis B surface antigen, Quantitative assay, Virology

INTRODUCTION

Hepatitis B virus (HBV) causes a wide range of clinical consequences, from acute and chronic infection to cirrhosis and hepatocellular carcinoma, and represents a global public health problem[1,2]. Historically, HBV dates to when an unknown antigen in Australia was recognized to be associated with hepatitis type B, which was later referred to as the hepatitis B surface antigen (HBsAg)[3]. Since then, HBsAg has served as a qualitative diagnostic marker for HBV infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring. A number of clinical studies have evaluated the clinical utility of HBsAg and suggested its potential roles. Yet, it needs to be underscored that a further understanding of HBsAg, not only from a clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. Therefore, in this article, we review current concepts and issues on the quantification of HBsAg titers (qHBsAg) with respect to their biologic nature, method principles, and clinically relevant topics.

STRUCTURE AND MOLECULAR VIROLOGY OF HBsAg

Components of the viral structure

HBV belongs to Hepadnaviridae and is composed of the envelope, core, DNA genome, and viral polymerase. It has a circular form of partially double-stranded DNA and is approximately nucleotides in length[4,5]. A 42-45 nm long HBV spherical form (Dane particle), which is the full virion with infectivity, can be visualized (Figure 1) under electron microscopy. It has two-layered shells. The outer shell is the envelope protein referred to as hepatitis B surface (HBs) protein, which is further divided into small, middle, and large HBs proteins (SHBs, MHBs and LHBs proteins, respectively), and the inner shell is a core protein referred to as the hepatitis B core protein in which viral polymerase and the HBV genome is enclosed. In addition to the abovementioned full virion, smaller non-infectious subviral particles are present in the serum; 17-25 nm spherical particles, mainly composed of SHBs protein, constitute the most abundant form, which is as much as 10 000-fold in excess of the full infectious virion[4,6]. Filamentous (or tubular) particles are another form, with a 20 nm diameter and variable length, and are composed of SHBs, MHBs, and the LHBs protein. The form of the HBV particles appears to be determined by the proportion of LHBs protein[7]. All three forms can be detected in serum with commercial assays and are collectively referred to as HBsAg.

Figure 1.

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Schematic model of hepatitis B surface antigen structure. Three forms of hepatitis B surface (HBs) antigen (Dane particle, filamentous particle, and spherical particle) are visualized in serum by electron microscopy. These are composed of small, middle, and large hepatitis B surface proteins. LHBs: Large HBs proteins; MHBs: Middle HBs proteins; SHBs: Small HBs proteins.

Synthesis and secretion

HBV has four distinct open reading frames (ORFs) that encode the envelope, core, polymerase, and X proteins. ORF S has three internal AUG codons encoding the SHBs, MHBs, and LHBs proteins, which correspond to the S, preS2 + S, and preS1 + preS2 + S domains, respectively (Figure 2). These proteins have a common carboxyl end but different amino ends[8].

Figure 2.

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Schematic presentation of the S/preS1/preS2 gene, RNA transcripts, and translational products. Opening reading frame S has three internal AUG codons. Transcription to produce the 2.1 kb and 2.4 kb mRNAs first occurs after translation into small hepatitis B surface proteins (SHBs), middle hepatitis B surface proteins (MHBs), and large hepatitis B surface proteins (LHBs) ensues with different promoters.

Like all other proteins, mRNA transcription is the first event to occur. Two 2.1 kb mRNAs for the M/SHBs proteins and a 2.4 kb mRNA for the LHBs protein are formed, and take a separate pathway from viral replication. Diverse transcription factors are involved and act on promoters, enhancers, and other regulatory elements, such as the glucocorticoid responsive element[9,10]. LHBs and M/SHBs expression are thought to be independently regulated with different promoters; a typical TATA box is present in the LHBs promotor (S promotor I, SPI), whereas the TATA-less promotor, which usually has multiple initiation sites, is associated with the M/SHBs promoter, thus accounting for synthesis of distinct proteins from one mRNA. In patients with active viral replication, the protein expression pattern shows a predominance of the M/SHBs protein in contrast to a predominance of the LHBs protein in inactive carriers[11]. After transcription, protein synthesis and glycosylation follows at the endoplasmic reticulum (ER) membrane resulting in a 226 amino acid SHBs protein, the MHBs protein with an additional 55 amino acids, and the LHBs protein with an additional 108-119 amino acids. Although the LHBs mRNA includes the M/SHBs sequence, it does not translate into the M/SHBs protein, and the ratio between the MHBs and SHBs protein is controlled by a complex mechanism, which is not fully understood[12]. To form a full virion, a mixture of HBs proteins in a well-balanced ratio is utilized to envelop core particles in which SHBs and LHBs protein are indispensible[13]. The virion is transported to the cell membrane through vesicles, and several conditions must be satisfied for successful secretion, because excess SHBs protein is required, whereas excess LHBs protein prevents secretion and causes dilatation of the ER with a ground-glass appearance[14-16].

Function

The primary function of the HBs protein as a virologic structure is to enclose the viral components. It also plays a major role in cell membrane attachment to initiate the infection process. Several studies have confirmed the idea that the peptide in the preS1 domain is essential in this process, showing that it specifically binds to the human liver plasma membrane and can be inhibited by a monoclonal antibody[17,18]. However, participation of the SHBs protein in attachment has also been suggested following identification of hepatocyte-bound endonexin II, which specifically binds the SHBs protein[19]. Additionally, from the host perspective, the HBs protein has the major antigenic components, including the a determinant, which is important for host-activated immunity. However, from a virologic perspective, it is postulated that excess HBs protein may divert such neutralizing antibody immune function away from the infectious virion[20].

QUANTITATIVE HBsAg ASSAYS

Methods to detect HBsAg were first described in the s using radioimmunoassays and enzyme immunoassays[21,22]. Since then, various diagnostic techniques have been developed, which are mostly confined to qualitatively diagnose HBV in clinical practice. Recently, quantitative assay of HBsAg has been developed, and two commercially available assays will be briefly introduced here.

The Architect HBsAg QT (Abbott Diagnostic, Wiesbaden, Germany) is a chemiluminescent microparticle immunoassay, which is currently the method most widely used in clinical studies[23]. The Architect HBsAg QT assay is a two-step immunoassay with flexible assay protocols, referred to as Chemiflex, for quantitatively determining human serum and plasma HBsAg concentrations. In the first step, the sample and hepatitis B surface antigen antibody (anti-HBs) coated with paramagnetic microparticles are combined. HBsAg present in the sample binds to the anti-HBs coated microparticles. After washing, acridinium-labeled anti-HBs conjugate is added. Following another wash cycle, pre-trigger and trigger solutions are added to the reaction mixture. 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 Architect Immunoassay System optics. The Architect HBsAg is a fully automated system and can detect as low as 0.2 ng/mL of HBsAg with a dynamic range of 0.05-250.0 IU/mL[24].

Elecsys HBsAg II (Roche Diagnostics, Indianapolis, IN, USA) is another method for quantitatively determining HBsAg[25]. In the first incubation step, the antigen in the sample reacts with two biotinylated monoclonal HBsAg-specific antibodies and a monoclonal/polyclonal (sheep) HBsAg-specific antibody, labeled with a ruthenium complex, to form a sandwich complex. In the second step, streptavidin-coated microparticles are added, and the complex binds to the solid phase via interaction with biotin and streptavidin. The results are reported as a cutoff index (signal sample/cutoff), and the sample is considered reactive if the index is greater than 1.0.

CLINICAL APPLICATION OF QUANTITATIVE HBsAg

Correlation with serum HBV DNA

Although measuring serum HBV DNA is the gold standard for monitoring viral load, it is relatively expensive and not yet readily available in some areas. By contrast, the technique for detecting qHBsAg is fairly easy and inexpensive, and the primary aim of initial clinical studies was to determine the relationship between qHBsAg and serum HBV DNA (Table 1). In , Deguchi et al[23] first reported the clinical significance of a high qHBsAg in patients who were hepatitis B e antigen (HBeAg) positive as opposed to those with an antibody positive to the hepatitis B e antigen (anti-HBe), and that qHBsAg correlated well with the serum HBV DNA level (r = 0.862). Although there are some contradicting results on whether qHBsAg is correlated with serum HBV DNA[26,27], it seems that they are correlated based on a number of studies[28-33]. Further studies are required to investigate the possibility of using qHBsAg as an aid, if not an alternative, for HBV DNA.

Table 1.

Recent clinical studies with quantification of hepatitis B surface antigen titers in hepatitis B virus infection

Author Antiviral therapy Correlation Prediction Clinical results Deguchi et al[23] - HBV DNA - qHBsAg is higher in HBeAg(+) Chen et al[28] - HBV DNA - qHBsAg is higher in HBeAg(+) and high HBV DNA levels, whereas qHBsAg is low in low HBV DNA level CHB Werle-Lapostolle et al[29] ADV cccDNA, HBV DNA - HBsAg and cccDNA decrease with ADV Kohmoto et al[30] LAM HBV DNA - qHBsAg is helpful for early detection of drug resistant strains Wursthorn et al[35] Peg-IFN + ADV cccDNA - Peg-IFN + ADV decreases cccDNA and HBsAg, which are well correlated Chan et al[36] Peg-IFN + LAM cccDNA Low baseline qHBsAg can predict SVR Peg-IFN + LAM decreases cccDNA and HBsAg, which are well correlated Manesis et al[31] IFN vs LAM HBV DNA Low baseline qHBsAg can predict SVR IFN induces sharper decrease in qHBsAg than LAM Wiegand et al[27] FAM ± LAM HBV DNA (not correlated) Decline of qHBsAg can predict HBsAg loss 2 log drop to below 100 IU/mL is associated with HBsAg clearance Moucari et al[32] Peg-IFN HBV DNA Early qHBsAg drop can predict SVR qHBsAg may be useful to optimize Peg-IFN therapy Brunetto et al[33] Peg-IFN ± LAM vs LAM HBV DNA On-treatment qHBsAg decline can predict sustained HBsAg loss qHBsAg < 10 IU/mL at week 48 and 1 log decline predict sustained HBsAg clearance to optimize treatment strategy Lau et al[37] Peg-IFN ± LAM - On-treatment qHBsAg can be used as an early predictor of SVR In HBeAg(+) patients, qHBsAg reduction through weeks 12, 24 and 48 were higher in patients with HBeAg seroconversion Marcellin et al[38] Peg-IFN ± LAM - qHBsAg at week 12 can predict long-term HBsAg clearance 35% of patients who had qHBsAg < IU/mL at week 12 cleared up HBsAg by 4 yr post-treatment Lu et al[39] Peg-IFN ± LAM cccDNA qHBsAg was superior to cccDNA and serum HBV DNA in predicting SVR Area under ROC curve with qHBsAg, cccDNA and HBV DNA was 0.769, 0.734, and 0.714, respectively, for predicting SVR Brunetto et al[65] Peg-IFN ± LAM - On-treatment qHBsAg can be used as an early predictor of SVR On-treatment decline in HBsAg appears to be genotype dependent. Genotype B patients showed the most rapid and pronounced decline Hou et al[66] Peg-IFN vs LAM - - Peg-IFN was superior to ADV in HBeAg seroconversion and qHBsAg decline in LAM-resistant patients Open in a new tab

Correlation with covalently closed circular DNA

An important qHBsAg issue is its association with covalently closed circular DNA (cccDNA). cccDNA is a mini-chromosome and acts as a viral template and replenishing pool for maintaining a chronic HBV infection[34]. Therefore, it is essential to understand the biology of cccDNA when considering HBV therapy. However, to examine cccDNA, an invasive procedure is required, and qHBsAg has been suggested as a surrogate marker for cccDNA. Werle-Lapostolle et al[29] reported a significant decrease in cccDNA, qHBsAg, and serum HBV DNA with adefovir (ADV) therapy, and that there was a strong correlation between cccDNA and other variables. This observation was supported by subsequent studies; Wursthorn et al[35] and Chan et al[36] also showed that cccDNA was significantly correlated with qHBsAg, suggesting that serial monitoring of qHBsAg might act as an additional marker to evaluate treatment response during antiviral therapy.

Prediction of response to antiviral therapy

After the accumulation of data confirming that qHBsAg can be utilized as a viral monitor, qHBsAg has been evaluated as a predictor of virologic response. In a study by Chan et al[36] the sensitivity, specificity, and positive and negative predictive values for sustained virologic response (SVR) in patients treated with pegylated interferon (Peg-IFN) + lamivudine (LAM) were 86%, 56%, 43%, and 92%, respectively, with baseline qHBsAg concentrations less than 10 000 IU/mL. According to the data of Manesis et al[31] achieving the complete elimination of HBsAg would probably require 10.6 years of effective LAM therapy or 5.4 years of a sustained response to interferon. Recently, the clinical usefulness of on-treatment qHBsAg in patients treated with Peg-IFN ± LAM has been suggested in both HBeAg positive and negative patients; a decline in qHBsAg of > 1 log IU/mL or specifically 0.5 and 1.0 log IU/mL at weeks 12 and 24, respectively, had high predictive value for SVR, and on-treatment HBsAg levels could be used as an early predictor of durable off-treatment response to Peg-IFN-based therapy[32,33,37]. Of note is a long-term study by Marcellin et al[38] in which 35% of patients who had qHBsAg < IU/mL at week 12 eventually cleared the HBsAg by 4 years post-treatment, which supports the clinical utility of qHBsAg. Furthermore, qHBsAg was superior to cccDNA and serum HBV DNA for predicting SVR in patients undergoing Peg-IFN-based therapy with receiver operating characteristic (ROC) curves of 0.769, 0.734, and 0.714, respectively[39].

MOLECULAR HBsAg VARIANTS

Much of our understanding of the biologic nature of the HBs protein has been gathered from various mutation and truncated protein experimental models[40,41], and it is worthwhile to address the relevance and consequences of HBsAg variants from a clinical point of view. Besides the lack of HBV proof-reading capacity[42], the development of an HBsAg mutation can be attributed to immune pressure from extensive vaccination programs, injections of hepatitis B immunoglobulin (HBIG) following liver transplantation, and the overlap with a mutation in the corresponding P gene.

Immune escape mutants

Since the introduction of an extensive vaccination program, concerns about HBsAg variants have increased after an HBV infection occurred in infants who had received an HBV vaccination and who had mounted an adequate anti-HBs response. This was presumed to be caused by immune selection pressure, because the HBsAg a determinant is the major epitope for HBV vaccination[43,44]. Changes in the amino acids within the a determinant, particularly between 137-147, disable surface antigen domain recognition by neutralizing antibodies. Of importance is the G145R mutant, because it is the most common and is replication competent with stability[45]. In a Taiwanese epidemiological study, it was reported that the prevalence of the a determinant mutation had increased from 7.8% to 28.1%, after 15 years of a universal vaccination program[46]. Fortunately, in the following years, neither the percentage increase nor any significantly adverse events with an outbreak of HBV infection actually occurred; thus, a mass vaccination program is continuing with adequate justification[47].

In addition to the extensive vaccination program, the wide use of HBIG following liver transplantation adds selection pressure to HBV. Ten of 20 patients who developed recurrent HBV infection despite hepatitis B immunoglobulin prophylaxis had amino acid substitutions involving the a determinant, which were mostly absent in pretransplantation clones[48].

Overlap and mutation in the P gene

A mutation in the P gene from prolonged oral nucleos(t)ide therapy can cause an altered sequence in the corresponding S gene due to overlap of the two genes[49], which is summarized in Table 2[50]. The nucleotide at rt204 in the P gene is associated with resistance to LAM, telbivudine (LdT), and entecavir (ETV), and the rtM204V/I mutation typically results in a sI195M, sW196S, sW196L or a terminal codon in the overlapping S gene[50]. In previous studies, LAM selected HBsAg mutants with reduced anti-HBs binding capacity, and secretion of HBsAg was prevented with a mutant strain due to the stop codon[51,52]. rt181 is another important site that confers resistance to ADV and/or LAM/LdT. Recently, Warner and Locarnini demonstrated that rtA181T caused a secretory defect and had a negative effect on secretion of the wild-type HBV virion because of a concomitant change in the envelope protein at sW172[53]. Similarly, ETV-associated rtI169T/sF161L leads to a decrease in HBsAg immunoreactivity[54].

Table 2.

Mutations in viral polymerase gene induced by oral antiviral agents and corresponding changes in hepatitis B surface antigen

Polymerase domain Mutation in polymerase Oral antiviral agents Corresponding change in HBsAg B rtI169T ETV sF161H/L rtL180M LAM, LdT No change rtA181T ADV, TFV, LAM, LdT sW rtA181T ADV, TFV, LAM, LdT sW172L rtA181V ADV, TFV, LdT sL173F rtT184A ETV No change rtT184C ETV sL175F + sL176V rtT184I ETV No change rtT184G ETV sL176V rtT184S ETV sL175F rtT184M ETV sL rtT184L ETV sL175F C rtS202C ETV No change/sS193F rtS202I ETV sV194F/S rtS202G ETV No change/sS193L rtM204V LAM sI195M rtM204I LAM, LdT sW/S/L D rtN236T ADV, TFV After end of HBsAg E rtM250I ETV After end of HBsAg rtM250V ETV After end of HBsAg Open in a new tab

The clinical significance of overlap and a common mutational substitution in the S and P gene was further extended by Kamili et al[55] who demonstrated a successful experimental infection with the rtV173L, rtL180M, and rtM204V HBV mutants that resulted in sE164D and sI195M despite high anti-HBs levels in chimpanzees[55]. Furthermore, the possibility of a vice versa phenomenon with respect to an extensive vaccination program might be postulated in that HBsAg mutants from selection pressure might harbor the corresponding P gene mutation, resulting in primary resistance to antiviral agents and therapy failure with these agents.

Detection and variants of HBsAg

As described above, an HBsAg mutation leads to diverse effects, such as decreased secretion and reduced binding capacity to anti-HBs. Of note is that not only a mutation in the a determinant but also in the S promoter or a deletion in the preS region can cause such effects[56,57]. These effects may hamper the diagnostic performance of commercial assays, and several reports have pointed to the problem of not being able to detect HBV with an a determinant mutation[58,59].

An occult HBV infection is defined as the persistence of the HBV genome in HBsAg negative individuals, and one of the explanations for occult HBV infection is a mutation in HBsAg and undetectability by available assays[60]. Both the Architect HBsAg QT and Elecsys HBsAg II seem to reliably detect HBsAg mutants with high sensitivity and specificity[24,25,61]. However, further studies are needed to validate such detection ability, because new or complex combinations of mutations can arise in this era of antiviral agents and extensive vaccination.

FUTURE PERSPECTIVES

Despite progress on qHBsAg, a number of unanswered questions still remain. Precise control mechanisms for HBsAg production in HBV are poorly understood. A discrepancy between qHBsAg and serum HBV DNA exists, although a correlation has been documented. Further research on the virologic nature of HBV could answer these two questions. Meanwhile, the role of qHBsAg in the clinical field is being actively investigated, especially as a predictor to virologic response. Of particular interest is the potential role of qHBsAg for defining the end point of oral antiviral therapy. Current American Association for the Study of Liver Diseases and European Association for the Study of the Liver guidelines with respect to an end point for therapy are unsatisfactory, because reversion to HBeAg positivity does occur after terminating therapy, and the loss of HBsAg is infrequently encountered[62,63]. In this regard, qHBsAg might be particularly helpful in patients with undetectable HBV DNA, even with a highly sensitive polymerase chain reaction assay[64]. In contrast to undetectable HBV DNA, which provides no further information for the virologic responders, HBsAg is continuously shed and detected and, based on the observations of previous studies, qHBsAg with serial monitoring in patients with undetectable HBV DNA may be utilized to determine the end point of therapy and validate the durability of antiviral agents.

CONCLUSION

HBsAg is produced and secreted through a complex mechanism that is still not fully understood. Nevertheless, quantification of serum HBsAg is currently available and there is an opportunity to make maximal use of qHBsAg to elucidate its role in clinical fields. However, a deep understanding of the virology is necessary, and it is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. Unanswered questions need to be resolved through further qHBsAg research.

Footnotes

Supported by The Grant of the Bilateral International Collaborative R&D Program from the Ministry of Knowledge Economy and the Good Health R&D Project from the Ministry for Health, Welfare and Family Affairs, South Korea (A)

Peer reviewers: Yukihiro Shimizu, MD, PhD, Kyoto Katsura Hospital, 17 Yamada-Hirao, Nishikyo, Kyoto 615-, Japan; Yogesh K Chawla, Professor, Department of Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh , India

S- Editor Wang JL L- Editor O'Neill M E- Editor Lin YP

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