The underlying mechanisms for the “isolated positivity for the hepatitis B surface antigen (HBsAg)� serological profile

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http://www.springerlink.com/content/6187546p023u6608/fulltext.html

Journal Medical Microbiology and Immunology

Publisher Springer Berlin / Heidelberg

ISSN 0300-8584 (Print) 1432-1831 (Online)

Category Review

DOI 10.1007/s00430-010-0160-3

Subject Collection Biomedical and Life Sciences

SpringerLink Date Tuesday, May 11, 2010

Review

The underlying mechanisms for the “isolated positivity for the hepatitis B

surface antigen (HBsAg)†serological profile

Robério Amorim de Almeida Pondé1, 2, 3, 4

(1) Laboratório de Virologia Humana, Instituto de Patologia Tropical e Saúde

Pública, Universidade Federal de Goiás, Goiânia-Goiás, Brazil

(2) Central Goiana de Sorologia, Imuno-hematologia e Biologia Molecular,

Goiânia-Go, Brazil

(3) Hospital Materno-Infantil, Goiânia-Goiás, Brazil

(4) Rua 7A Edifício RIOL, Nº 158, 1º andar, sala 101, setor aeroporto,

Goiânia-Goiás, CEP 74-075-030, Brazil

Received: 12 February 2010 Published online: 11 May 2010

Abstract

During HBV infection, four structural antigen/antibody systems are observed:

hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs); the pre-S

antigens associated with HBsAg particles and their antibodies; the particulate

nucleocapsid antigen (HBcAg) and anti-HBc; and an antigen structurally related

to HBcAg, namely HBeAg and its antibody (anti-HBe). Through the examination of

this antigen–antibodies system, hepatitis B infection is diagnosed and the

course of the disorder may be observed. Isolated HBsAg seropositivity is a

peculiar serological pattern in HBV infection observed some times in routine

laboratory. In most cases is not clear how this profile should be interpreted

neither its significance. This pattern, however, may be associated with some

clinical and laboratorial situations of great relevance, some of which will be

addressed in this article.

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Introduction

Hepatitis B surface antigen (HBsAg) is the most important marker for laboratory

diagnosis of hepatitis B. HBsAg detection is used for the diagnosis of acute and

chronic hepatitis B virus and indicates potential infectiousness. It is one of

the first serum markers to appear during the course of HBV infection and it is

also useful as a follow-up marker, since declining concentrations are observed

in resolving hepatitis B. In patients who subsequently recover from HBV

infection, HBsAg usually becomes undetectable after 4–6 months [1]. If HBsAg

persists for more than 6 months, spontaneous clearance is very improbable and

the infected individual is considered a chronic HBV carrier [2].

However, during the acute phase of infection, following HBsAg detection, other

viral markers can be easily detectable including DNA polymerase and HBeAg. HBeAg

appears shortly after the appearance of HBsAg and disappears within several

weeks as acute hepatitis resolves. It presence in the serum correlates with

presence of viral replication in the liver. Anti-HBc IgM antibodies are

detectable at the outset of clinical disease, and as the infection evolves IgM

anti-HBc levels gradually decline, often become undetectable within 6 months and

IgG class predominates, remaining long time (sometimes life-long) at detectable

levels. As recovery and convalescence signal, antibodies to other viral proteins

appear in the blood. Anti-HBe antibodies appear after HBeAg clearance and may

persist for many years after resolution of acute HBV infection. Anti-HBs

antibodies become detectable late in convalescence, from 6 weeks to 6 months,

after HBsAg clearance, although it is produced early in the course of an acute

infection. Thus, through the examination of this antigen–antibody system,

hepatitis B infection is diagnosed and the course of the disorder may be

observed [to review see reference 3].

The aforementioned dynamic occur in the majority of cases or in other words, in

HBV infections with a typical course. Some cases, however, present with

unexpected patterns, such as, “Isolated HBsAg Seropositivityâ€. This is an

atypical serological profile and an uncommon phenomenon observed some times in

routine laboratory. This article illustrates the diverse significance of this

finding and the mechanisms could justify this peculiar pattern which is of great

importance for both clinicians and laboratorial personnel.

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Sample collected very early, before the expression of another acute phase

serological marker

Classically, 8–9 weeks following the infection, in the incubation period or

3–5 weeks before biochemical evidence of liver dysfunction and clinical

symptoms appearance, it is possible to detect HBsAg in serum, in the absence of

detectable HBeAg levels [4]. Considering that anti-HBc IgM antibodies usually

appears within 1 month of the appearance of HBsAg during acute phase, in

practice the isolated positivity for HBsAg may be really reflex of a primary

acute infection, even it is infrequent that a patient can be investigated in

such a previous moment of an infection pre-symptomatic phase [5]. This finding

demonstrates the need to properly investigate each case.

In this context, the serological profile evaluation of samples collected

subsequently will be an efficient strategy for establishing the diagnosis if

consistent with viral dynamic described earlier. However, it is important to

point out that although have been claimed that high titer anti-HBc IgM can be

accepted as an indicator for acute infection, its presence is not an absolutely

reliable marker of acute infection, since some patients with chronic hepatitis B

infection become positive for IgM antibody during flares in their disease [6].

Thus, positivity for anti-HBcIgM antibodies must be considered with caution not

to induce diagnostic mistake. On the other hand, in practice, anti-HBcIgM

negativity is important, because if this indicator is negative, the probability

of acute infection in HBsAg positive cases is nil [4].

The isolated HBsAg seropositivity, being consequence of a primary acute

infection, highlights the necessity of laboratory and blood banks especially to

adopt highly sensitive HBsAg detection methods, and have reference laboratories

with experience and sufficient technology to conclude properly the cases. The

ability to accurately detect the presence of HBsAg has a critical role in the

diagnosis of HBV infection, especially in asymptomatic people, including blood

donors [7], pregnant women [8], and patients who will be submitted to

chemotherapy treatment or drugs immunosuppressive. For this reason, the design

of tests with high analytic sensitivity has been necessary. By analyzing

seroconversion panels, it has been estimated that the new HBsAg assays with

subnanogram sensitivity (from 0.04 to 0.12 ng/ml) have shown to be more

sensitive than many licensed assays widely used (range of from 0.13 to 0.62

ng/ml) [9, 10]. These tests might potentially detect HBsAg an estimated 3–9

days earlier [9], however, they could extend further the time of persistence of

the serological profile isolated HBsAg seropositivity.

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Possible false-positive/nonspecific reactivity

The isolated reactivity to HBsAg may be, also, associated to false-positivity or

nonspecific reactivity, as may occur with all infectious disease assays [11].

The specificity relates to the ability of an antibody or a population of

antibodies to complex only one kind of molecules, and cross-reactivity is the

ability of more than one antigen competing for the same antibody-binding site as

a result of sharing of an epitope [12]. Considering that the specificity of

HBsAg assays is over 99%, false-positive results may occur because of

cross-reactivity and have been observed with heparinized samples or due to

interferences with hemoglobin or bilirubin. Besides, false-positive results may

be observed during pregnancy and in individuals with acute or chronic infections

or individuals suffering from autoimmune diseases or chronic liver diseases [13,

14].

The vast majority of the commercially available tests to HBsAg detection are

based on the sandwich enzyme immunoassay (EIA), principle which uses monoclonal

antibody and/or polyclonal anti-HBs antibody mapped to recognize the

three-dimensional structure of the ‘a’ determinant of wild type (and mutant)

HBsAg [15, 16]. These tests are highly sensitive and able to detect extremely

small quantities of HBsAg in the bloodstream, such as 0.04 ng/ml, despite this

excellent analytical sensitivity is not sufficient to detect HBV in all phases

of infection [17]. For diagnosis, manufactures determine a “cut off†value

that balances the necessary high sensitivity for detecting the antigen with the

need to avoid false-positive results. Samples with signal value above the “cut

off†are called positive. However, because the high sensitivity, is

recommended caution before establishing the diagnosis, since has been

demonstrated most of weakly positive results can be falsely positive [18]. In

addition, it is also important to consider that the current licensed HBsAg

assays have highly variable cut-off levels, which range from 0.13 to 0.62 ng/ml

(while investigational HBsAg assays are able to detect down to 0.07 ng/ml) [12],

and are different among them because of different monoclonal/polyclonal anti-HBs

antibodies (or a combination of them) bound to a solid phase and a second

labeled anti-HBs, utilized to detect the captured antigen [19]. Thus,

considering these differences, false-positive results may occur, not being

necessarily achieved by another.

In this context, it has been suggested that the result must be confirmed by an

HBsAg neutralization test, in which the percentage of HBsAg signal reduction in

the presence of antibodies against HBsAg (anti-HBs) is measured. In this test,

samples with ratio>1 (Samples/Cut Off) are incubated with anti-HBs; if HBsAg is

truly present, the anti-HBs antibody in the neutralization step blocks HBsAg,

inhibiting its binding to the reagent antibodies anti-HBs bound to a microplate

phase solid, and consequently, reducing or eliminating its signal (positive

neutralization test result) [11]. This procedure can minimize the falsely

positive results expression for this marker [20]. Additionally, nonspecific

reactivity may be reduced by overnight storage of samples at 2–8°C [21].

Despite the high HBsAg immunoassays sensitivity and specificity within the

tolerance limits for detection HBsAg, the advent of new methodologies, such as

chemiluminescent assay, has been possible to lower incidence of false-positive

results when compared to detection by immunoassay [13]. Chemiluminescent assays

are methodologies that differ from EIA tests by test principle, by using

different anti-HBs antibodies (mouse-goat-monoclonal/polyclonal) and kinetics of

the reaction (incubation time, temperature, and by different detection systems

of captured antigen [13, 22]). In addition, the procedure to measure HBsAg

involves 2 steps: constituted by initial screening followed of neutralization

assay, which serves as a confirmatory test of reactivity originally obtained

[20]. The literature has reported sensitivity and specificity after confirmation

of the results of 100% by this methodologies [13, 23], which has been able to

detect low levels of wild-type and HBsAg mutants [13, 22], eliminating

possibility of false positives results. On the other hand, considering that

enzyme-linked immunosorbent assays are licensed methodologies and widely used,

it is recommended that for the resolution of discrepant results of HBsAg or

persistent isolated HBsAg seropositivity for this methodology, neutralization

assays should be always performed to establish a conclusive serological

diagnosis. However, it is important to point out that the neutralization test

could not confirm the true reactivity in the cases of HBV surface mutants

infection, since the anti-HBs antibodies used in the step neutralization are

mapped to recognize only the wild type s-antigen three-dimensional structure.

Therefore, in these cases the neutralization test can suggest false-positive

result.

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Antigenemia after hepatitis B vaccination

Vaccines are the only effective defense mechanism against hepatitis B, and

prevention of primary infection by vaccination is an important strategy to

decrease the risk of chronic infection and its subsequent complications.

Hepatitis B vaccines have been available for almost 30 years. The

first-generation hepatitis B vaccine, an inactive plasma-derived, became

available in 1982, while the second generation of HB vaccine, a DNA recombinant

HB vaccine, was available for general use in 1986 [24]. Both of the vaccines

were proven to be safe and efficacious in preventing HBV infections.

Vaccination is essentially a simulated infection under control that stimulates

the immuno-defense mechanism of the body in order to produce protective

antibodies. Since the immunologic agent of the vaccines and serological marker

are the same [25], vaccination may create “false-positive†results in

serologic screening, originating the isolated positivity for HBsAg serological

profile.

Some studies in blood donor population have shown that a weak positive HBsAg

reaction is relatively uncommon and has been reported to occur 1–3 days after

vaccination [26]. In a study performed by Otag [27], three different vaccines

(Engerix B, Hepavax Gene and Gen Hevac were administered to 44 healthy adult

with an age range 18–60 years old, divided into three groups. All subjects

received a full dose (20 μg) of vaccine administrated via intramuscular route.

Blood samples were drawn at 1 h, 24 h and 3 days after vaccination and tested

for the presence of HBsAg by enzyme immunoassay. Each group received different

vaccine and was produced three ‘false-positive†results in blood samples

drawn at 24 h after vaccination. All subjects tested negative for HBsAg in

samples drawn at 3 days after vaccination, which indicates that antigenemia due

to vaccination is transient.

However, there has been related detectable hepatitis B surface antigenemia 5

days [28] and in until 18 days after vaccination in adults [29], extending the

period previously observed. In this context, this transient antigenemia may be

justified. A 70-Kg adult has a volume of extracellular fluid of approximately 15

l. If the adult receives a 20-μg dose of hepatitis B vaccine, then a plasma

level of 1.33 ng/ml is possible, assuming that the entire dose is soluble HBsAg.

Current HBsAg assays have sensitivities of 0.1 ng/ml, or even less. It is

therefore, not surprising that current HBsAg assays detect individual who have

been recently vaccinated against hepatitis B virus [28].

Additionally, works done with infants has indicated high incidence and long

duration for transient surface antigenemia after vaccination. Challapalli et al.

[30] described detectable levels of HBsAg in 55% of 18 newborns tested 33–56 h

after administration of engerix B. In one infant, 17 days was needed to clear

this transient antigenemia. Bernstein et al. [31] described detectable levels of

HBsAg in 12 out to 19 infants (65%) within the first 6 days after administration

of engerix B. One neonate remained seropositive at 8 days. Weintraub et al. [32]

reported detectable level HBsAg in 17% of 47 newborns tested after

administration of engerix B. Seropositive results was observed between 24 and 72

h after vaccination and all were seronegative 2 weeks after initial detection.

Finally, Koksal et al. [33] followed prospectively 39 infants vaccinated by Gen

Hevac B, and found that 69.2% of them tested positive for antigenemia at least

once. The majority of identified antigenemia cases fall into either 2–3 or

5–6 days after immunization day, whereas the longest documented duration of

antigenemia was 21 days post-vaccination [33]. The higher incidence and long

duration of transient antigenemia observed in newborn or infants might be

attributed to a relatively larger dose of vaccine in infants or level of the

development in the immuno-defense systems of the body. However, most of the

studies with adult subjects have given disparate results.

In conclusion, extreme caution should be used when interpreting HBsAg tests

performed within a limited time frame after administration of hepatitis B

vaccine. Laboratorial personnel should be aware that in all this cases the

neutralization assay will confirm the HBsAg presence in the blood, and

“false-positive†serological test results may create misdiagnosis and

needless troubles. The introduction of HBV polymerase chain reaction (PCR) in

the algorithm confirmatory may be most helpful in differentiating vaccines (who

are expected to be PCR negative) from those individuals developing acute

hepatitis B infection, who should be PCR positive.

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Immune tolerance to HBV core antigen

Isolated HBsAg seropositivity may be also explained by immune tolerance to HBV

core antigen mechanism, which consequently leads not anti-HBc antibodies

production.

The core antigen (HBcAg) nucleocapsid induces a vigorous humoral and cellular

immune response [34]. Anti-HBc antibodies become detectable in serum 3–4 weeks

after the appearance of HBsAg, and usually persist for many years, independently

of the HBV infection outcome [35]. The immune tolerance to HBcAg is known by the

incapacity of the individual to produce anti-HBc or to make it in levels not

detectable, which can be mediated by a variety of mechanisms. For example,

T-cell anergy is a tolerance mechanism in which the T cell is functionally

inactivated following an initial antigen encounter but remains alive in a

hypoactivated state [36]. T-cell anergy has been proposed as a major mechanism

for the maintenance of self-tolerance and regulation of the cellular immune

response [37]. In addition, a small number of specific peripheral T lymphocytes,

an inefficient antigen presentation or lymphokine production by the

antigen-presenting cell are among others possibilities associated with anti-HBc

production [38]. Therefore, the isolated HBsAg seropositivity and persistent

absence to anti-HBc can be associated with a selective immune system defect.

In a study, viral sequence and host immune response were investigated in an

unusual asymptomatic chronic hepatitis B virus carrier who was consistently

nonreactive for antibody to HBc and had normal ALT levels over a 5-year study

period [39]. He had not abnormalities in the number of peripheral blood T or B

cells, not HBc-specific suppressor T cells and his in vitro proliferative

response to the T-cell-dependent and T-cell-independent, were both normal, but

not to recombinant HBcAg. However, unlike other HBsAg carriers and hepatitis B

virus-immune individuals, his monocytes did not ingest HBcAg-coated beads,

suggesting that the defect could be associated with the antigen-presenting cell.

In another more recent study, the same serological profile accompanied by active

viral replication (HBsAg and HBeAg detected at high levels) with an apparent

persistent lack of anti-HBc was found in two blood donors [40]. To explore the

capacity of the two donors to produce an efficient antiviral humoral response,

several serological markers were studied and quantification of IgG was

performed. Neither donors were immunocompromised, considering that they did not

have hypogamaglobulinemia and were capable of producing antibodies to other

viruses (CMV, HAV). Additionally, nucleotide sequence analysis of the pre-C/C

region did not show mutations or deletions in encoded proteins, demonstrating

that the apparent absence of anti-HBc was not caused by a defective HBV mutant.

Although the T-cell immune response could not be explored, the T-cell immune

tolerance to HBV antigens could be the most probable explanation for this

particular immunological and clinical situation, since the persistently normal

ALT levels (which is considered an important sensitizing viral determinant in

the infected liver) had suggested the absence of liver damage, despite

serological evidence of active viral replication.

The nucleotide sequences of the precore/core and X-region of hepatitis B virus

were also studied in four subjects who were serologically negative for anti-HBc

antibodies [41]. These were persistently positive for HBsAg and were considered

to be asymptomatic HBV carriers. The nt sequences of the precore/core revealed

substitutions dispersed; however, they were not considered able to change the

epitope of the core antigen and to justify the absence of anti-HBc antibodies.

Despite X-ORF has showed nucleotide substitutions, the structure of the

X-protein and the promoter/enhancer II complex [see the following paragraphs and

reference 53–55] appeared to be conserved. These findings suggested the

absence of serum anti-HBc was not due to mutation of the HBV DNA but due to an

aberrant immune reaction of the host to HBV in accordance with Gotoh et al.

[41].

Finally, the phenomenon of the immune tolerance to HBc-antigen has been also

associated with vertical transmission of infection and consequent absence of an

antibody response to HBc in infants born to HBe-positive carrier mothers [42].

It has been postulated that HBeAg may have an immunoregulatory function in

promoting viral persistence [43]. The HBcAg and HBeAg are distinctly recognized

by antibodies but due to their extensive amino acid homology are highly

cross-reactive at the T-cell level [43]. Thus, HBe antigen, because of its small

size, may traverse the placenta and elicit HBe/HBcAg-specific T-helper cell

tolerance in utero [44], consequently may lead to fetal immunotolerance not only

to HBeAg but also to HBcAg and still to inhibit anti-HBc antibody production

when it is present in the serum [36].

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Infection by defective HBV-mutant

In all the cases of immune tolerance to HBc aforementioned, the pre-C/C regions

remained unchanged, with normal synthesis of viral antigen. However, the

individual demonstrates incapacity anti-HBc production, or do undetectable

levels. On the other hand, anti-HBc production may be affected by nucleotide

sequence deletions in the core gene, involving important epitopes to antigen

recognition by immune response cells, or in the X-region, compromising

transcriptional regulatory elements, justifying isolated seropositivity for

HBsAg. In these cases, the deficient humoral response to HBcAg may not be

accompanied by a cellular immune tolerance [45].

Infection by mutant of the core-region of hepatitis B virus

The C-gene (C-ORF) contains two regions. The core-region (183 aa) encodes for

the viral nucleocapsid (HBc-antigen/core-protein) and the precore-region (29

aa), together with the core-region, encodes for the HBe-antigen [46]. Some

functional domains within the core-protein can be highlighted, such as, the

arginine-rich C-terminus (up to aa 64) which enable the encapsidation of the

pregenomic RNA, and the remaining C-terminus (up to aa 173) which may be

important for the synthesis of plus-strand DNA and thereby for the virus

replication [47]. Furthermore, several epitopes within the core-protein differ

in immunological aspects. For example: aa sequence from 141 to 151 and aa

sequence from 84 to 101 are recognized by cytotoxic T-lymphocytes; aa sequence

from 74 to 83 and aa from 107 to 118 comprise conformational determinant

responsible for HBc antigenicity, whereas aa from 76 to 89 is linear determinant

responsible for HBe antigenicity [48]. Thus, points mutations between aa 74 and

89 and in-frame deletions involving codons 94 to 101, respectively, could reduce

HBe and HBc antigenicity and to shorten core-protein, becoming not functional

protein [48].

The nucleotide sequence of the HBV pre-C/C region has been determined after

amplification and cloning of HBV genomic fragments from serum samples of

patients HBsAg and DNA positive, but anti-HBc negative and ALT levels in the

normal limits. The result analysis of pre-C/C clones has revealed single base

deletions (T at nucleotide 79 of the pre-C and G at nt 185 of the C gene), which

generated the appearance of stop codons at nt 65–63 (pre-C) and 194–196 (C

gene), respectively, leading to the synthesis of truncated proteins.

Furthermore, in frame deletion spanning from nt 226 to nt 278 and from nt 112 to

nt 150 were also observed [49]. These fragments are important for immune

recognition because they are regions spanning nucleotides 106–158 and

226–301 which encode the main B- and T-cell epitopes of HBcAg and HBeAg [50].

Core-deletion-mutant mostly has been observed in patients with long-course HBV

infection. It has been supposed that their selection may not be favoured by

immunological features but by an enhanced expression of the polymerase [48]. The

over-expression of the polymerase is supposed to be inhibited by the two

start-codons J (nt 2163 to nt 2165) and C2 (nt2177 to nt2179), which are

situated within the C-ORF upstream of the normal polymerase-AUG (nt2307 to

nt2309) [51]. Core-deletion-mutants can lose the inhibitory J-AUG and C2-AUG,

whereas the polymerase-AUG keeps intact. Therefore, core-deleted genomes led to

a more efficient translation of the polymerase by the ribosomes which might

increase the intracellular polymerase level. This could result in an enhanced

encapsidation of the pregenomic-RNA and thus in an increased virus production

[48]. In immunocompromised patients core-deleted viruses can became preponderant

over the wild type virus. In immunocompetent patients, the appearance of

core-deletion-mutants correlates with a low level of viremia [52].

Infection by X-defective HBV mutant

The X-ORF (X-region) encodes for a 154aa protein called HBx (protein X). The

protein X is known to transactivate transcriptional regulatory elements, which

include the enhancer and core promoter elements [53]. The core promoter, located

at nt 1586 to nt 1849 [53, 54], regulates the transcription of the

core/pregenome, and enhancer II, located at nt 1687 to nt 1805 [55], is

implicated in activation of the S-promoter [53, 55]. The sequence nt 1742 to nt

1849 is called the basic core promoter. It is supposed to be sufficient for the

correct transcription initiation of the pregenome and precore mRNA [48, 53]. DR1

(direct repeated) and DR2, which are implicated in the origin of HBV DNA, are

also located in the X-region (Fig. 1).

Fig. 1 Transcription regulatory elements of hepatitis B virus. URR (upper

regulatory region) consists of a negative regulatory element

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The X-defective HBV mutant is commonly characterized by an 8-nt deletion between

nt 1770 and 1777 and a point mutation of DR2 (T-to-C) in the X-ORF [56]. This

deletion results in a C-terminally truncated X protein of 134aa (due to a loss

of 23aa and addition of 3 normal aa), which loss its transactivating activity.

The 8-nt deletion of the core promoter/enhancer II complex sequence may diminish

the function of this transcriptional regulatory element [57]. Accordingly, X-ORF

mutations may suppress the replication and expression of HBV DNA, leading to

suppression of HBcAg synthesis. Consequently, patients infected with X-defective

HBV mutant can demonstrate low serum levels of HBsAg (or be frequently negative)

and be negative for anti-HBc, despite the presence of HBV replication.

X-defective HBV-mutants have been isolated from patients with acute or chronic

hepatitis [56].

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Infection by HBV2

The existence of hepatitis B virus variants or HBV-related human hepatotropic

viruses has been suggested by some searchers to explain this atypical

serological profile of HBV infection.

The term HBV2 was introduced to describe potential variants of HBV which do not

elicit a detectable immune response to the nucleocapsid (anti-HBc) in apparently

immune-competent patients. It was reported for the first time in December 1987

by Cousaget et al. [58], when he detected a transient HBsAg reactivity among

serum samples from non-immunized Senegalese children who were negative for

antibody to the HBV core antigen and who did not seroconvert on follow-up. It

was claimed that the isolated HBsAg reactivity was specific and not due to

infection by the “classical†HBV, but to the presence of an HBV-related

virus [58].

Others serological aspects of HBV2 infection are absence of detectable levels of

serum HBeAg and anti-HBe, presence of low levels of HBV DNA with clearance rapid

of HBsAg (1–2 months), and lack of detectable anti-HBs antibody response on

recovery [59]. Although some investigators have interpreted the isolated

reactivity for HBsAg as non-specific reaction [60, 61], neutralization of the

HBsAg reactivity by human polyclonal anti-HBs [58, 62, 63], visualization of

HBsAg-like particles by electron microscopy, detection of pre-S2 epitopes by

specific monoclonal antibodies, and detection of HBV-related DNA sequences by

molecular hybridization [58], in these cases have been reported, suggesting

involvement of an HBV variant or an HBV-related agent.

HBV2 is associated with mild hepatitis, and is not commonly reported [58, 59].

However, similar cases of HBV2 infection were detected in California [64],

Taiwan [62], Spain [63] and France [65], suggesting that the putative agent can

be distributed worldwide.

Finally, it has been suggested that the transmission of the agent may be occur

by the oral route, leading to a self-limited infection with very low levels of

viral replication and absence of clinical symptoms in the majority of cases

[59]. In addition, anti-HBs resulting from vaccination do not prevent infection

with HBV2, implying that HBV2 differs from HBV in the epitopes of the envelope

[58].

Despite sequencing of HBV2 genome has not been achieved so far, it has been

suggested that this serological variant may be due to nucleotide sequence

variation in the viral core gene. Valliammai et al. [66] investigated the core

ORF sequence from patients whose infection met all the criteria for definition

as HBV2 infection. In this study, only two of fifteen patients had HBV DNA

detectable by nested or semi-nested PCR, confirming previous reports of low

levels of viremia. The amplified products from patient 1 showed deletion from

nt2021–2053 would result in loss of residues 41–51 from HBcAg. This loss

could explain the lack of anti-HBc reactivity. The amplified products from

patient 2 showed deletion of 14nt and an insertion of 1nt at nucleotide position

1970–1984, resulting in the introduction of a termination codon or a missense

mutation which would lead to loss of the core initiation codon. Both of these

mutations would abrogate the HBcAg and HBeAg synthesis. However, sequencing

studies of so-called HBV2 isolates have not been widely reported, and it is not

clear how these viruses differ from wild type HBV.

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HBcAg-anti-HBc immune-complex formation (Chronic HBV infection without anti-HBc)

The isolated HBsAg seropositivity can be also justified by immune-complex

formation of anti-HBc antibodies with the excess of HBc antigen in the

bloodstream, become anti-HBc undetectable [67]. These immune-complexes cannot be

detected by available commercial tests which are not able to detect antibodies

in the presence of excess antigen in the serum [68]. Antibodies anti-HBc

complexed could be detected only after immune-complex dissociative treatment.

Some methods are based on polyethylene glycol precipitation followed by íon

chaotropic treatment [69] or based on sucrose gradient fractionation [70].

HBcAg is not a secreted protein and exists primarily in liver and in serum

within HBV particles, not being directly accessible in the blood to the immune

system. In exceptional cases, HBc-epitopes may, however, be exposed on virion

present in the serum [71], and also during liver necrosis, when nucleocapsids

may be secreted from infected hepatocytes with high level of HBV particles,

explaining the presence of anti-HBc complexed in the bloodstream. This

phenomenon has been observed in chronic HBV carriers in phase of active

replication in a state of immunosuppression, as seen in the context of

transplantation (bone marrow, kidney, and heart), during chemotherapeutical

treatment for neoplasias [72] or an uncontrolled HIV infection, conditions could

lead to an immune system defect. HBcAg is the most immunogenic HBV component

[34] and induces the production of high titers of anti-HBc antibodies in

immunocompetent individuals who have been exposed to HBV [6]. However, the

immunosuppression conditions can potentially induce the decreased antibody

production [73, 74]. HBcAg-reactive material releases from HBV particles present

at a high level in the serum and from hepatocytes undergoing lysis consequently

could complex to low levels of anti-HBc produced in these patients and therefore

lead to no detection of antibodies.

HIV infection is the cause for depressed immunity. Chemotherapeutic and

corticosteroids drugs act as selective immunosuppressive agents either by

eliminating or inactivating cells B, compromising anti-HBc antibody production

[72]. Interestingly, in HIV-infected patient during AZT treatment, and in

individuals in chemotherapy treatment or immunosuppressive drugs use during

monitoring of treatment, low levels of anti-HBc antibodies can become detectable

[67, 72]. This suggests that partial reversibility of defect and gradual

improvement of the immune system function can occur. Therefore, in the

immunosuppression conditions, the choice of the most sensitive assay for

antibodies detection would be the most reliable diagnostic approach.

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Quantification of HBsAg and HBV DNA testing (Correlation between HBV DNA levels

and isolated HBsAg positive cases)

The concentration of HBsAg in the blood of infected individuals has been

expressed as sample to cut off ratio (S/CO) or as ng/ml, and also, UI/ml, and is

highly variable, depending of infection phase. After infection with wild-type

HBV, HBsAg may be undetectable for several weeks, even with HBsAg assays at the

detection limit <0.1 ng/ml [17], but always with a progressive increase in

concentration. In chronic infection context, HBsAg concentration may fluctuate

over time and under certain circumstances may not be detectable (in

low-replication phases; when infection resolves spontaneously or after

successful antiviral therapy) [75, 76]. However, there is limited information on

HBsAg levels and it correlation with viral load, considering the dynamic natural

course of HBV infection.

Based on the analysis of seroconversion panels, HBV viremia during primary

infection has been evaluated and categorized in DNA detection phases and HBsAg

concentrations. The called “ramp-up phase†is a period during which DNA can

be quantified and viral load and HBsAg increase progressively over time until

HBsAg detection, whereas the “before-ramp-up phase†is the period where

low-level HBV DNA is intermittently detected only by higher-sensitivity

qualitative PCR or other NAT assays. The “after-ramp-up phase†is the period

after HBsAg becomes reactive and viral load progression is either slowed or

stopped [9]. By using the most sensitive HBsAg assay (detection limit of 0.04

ng/ml), levels of 2.000 HBV genomes/ml have been calculated as average viremia

at the time of HBsAg seroconversion [77]. Using another HBsAg licensed assay

with sensitivity for detection from 0.13 to 0.62 ng/ml, the estimated viral

loads can vary from 1,757 to 11,431 gen eq/ml, at the time of HBsAg detection

[9]. After HBsAg seroconversion, the HBsAg levels and DNA increase progressively

until it reaches titers higher than 15.000 UI/ml and 2 × 107 copies per ml,

respectively, when HBeAg becomes detectable [78]. While in the early and during

acute phase of infection it has been possible to establish strong correlation

between HBsAg and HBV DNA levels, some studies have demonstrated that there is

not this correlation at the late stages of healthy HBV carriage [79] or it is

weak or missing when analyzing different phases of persistent HBV-infection

separately [80].

Despite during the window period (or in healthy carriage, infrequently) the

detection limit of sensitive HBsAg screening assay (<0.1 ng/ml) corresponds to a

concentration of HBV DNA from 300 to 400 UI/ml, Togashi et al. [81] have

recently demonstrated that HBsAg might be detected in samples without HBV DNA.

--------------------------------------------------------------------------------

Prevalence of isolated positivity for HBsAg serological profile

The prevalence of isolated positivity for HBsAg serological profile in the

hepatitis B virus infection context has not been widely established. In a

seroepidemiologic study performed in 702 children in Camerron, in 1991, the

atypical serological pattern frequency was 4.1% (29 cases) among 163 HBsAg

positive individuals [82]. In Spain has been evidenced lower frequencies ranging

from 1.3% in pregnant women to 0.05–0.6% in patients selected randomly for

study [83, 84].

However, it is important to point out that prevalence of this profile and it

significance can vary widely depending on the specific population studied, and

also, the incidence of HBV infection in the region. Taking as example the

pregnant women population in region of low HBV infection prevalence, it is

possible that the isolated reactivity for HBsAg might be less associated with

recent or chronic infection than false-reactivity, since in this population

nonspecific reactivity can occur with greater frequency [13, 14]. The same

conclusion may not be true in high prevalence regions considering the same

population of study, since the risk of exposure to the agent tend to be larger

and real in this region. In accordance with this approach, the data are limited.

Studies performed in regions with low prevalence of HBV infection have shown

that in most cases (70%) the detection HBsAg isolated can be associated with

nonspecific reactivity, since true reactivity has not been confirmed by

neutralization test or genomic detection [5]. In this geographic context, in a

few cases (3.41 per 105 persons-years), the presence of this serological profile

has been associated with recent seroconversion, estimated by the

incidence-window period model proposed by Zou et al. [85]. On the other hand, in

regions with high prevalence of HBV infection (and consequently, higher rates of

chronic infection, vertical transmission and mutation), prevalence of HBsAg

isolated profile serological tends to be increased and may correspond to almost

50% among specimens with unusual serological profile, being often related to the

altered immune response of the host or to HBV variants [86].

Seroepidemiologic studies in blood donors and in general population have been

carried out to assess the incidence of asymptomatic HBV carriers in accordance

with HBsAg detection [87, 88]. However, prevalence of HBsAg isolated has not

been reported, which would be of great value as regards its significance in the

clinical context.

In population of chronically infected individuals and in the immunosuppression

context, the frequency of this serological profile has not been evaluated

satisfactorily, despite great relevance and broad meaning.

--------------------------------------------------------------------------------

Conclusion

Hepatitis B virus surface antigen reactivity in the absence of antibodies to

core antigen is an atypical serological profile that could be associated with

several clinical situations and adverse conditions, as addressed in this review.

This profile not only can suggest acute or chronic infection but also can point

out to a possible host immune system selective defect or to immunosuppression.

On the other hand, it can be associated to HBV antigenic variant infection or,

also, to be justified by nonspecific reactivity.

Considering that diverse possibilities could explain and justify this profile,

laboratory professionals need to be aware of the performance of their HBsAg

assays applied in laboratorial routine and include the HBsAg neutralization test

to prove the true reactivity for that marker. The collection second sample is

always advisable, prior to establish the diagnosis, especially in the cases of

recent infection, before the appearance another acute phase marker, and when a

molecular test is not available to prove the reactivity for HBsAg. Samples with

discordant results, sensitivity and specificity among tests applied should be

considered, and evaluated for the presence mutant strains. Laboratories should

communicate to clinicians about status of patients with questionable HBsAg

results. Finally, parts of the clinical history including possible source of

exposure, previous immunization, immune status of the individual, and results of

any previous tests that may have been performed using another assay, include

data important to security of result and reliable serological diagnostic.

--------------------------------------------------------------------------------

References <cut>

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http://www.springerlink.com/content/6187546p023u6608/fulltext.html

Journal Medical Microbiology and Immunology

Publisher Springer Berlin / Heidelberg

ISSN 0300-8584 (Print) 1432-1831 (Online)

Category Review

DOI 10.1007/s00430-010-0160-3

Subject Collection Biomedical and Life Sciences

SpringerLink Date Tuesday, May 11, 2010

Review

The underlying mechanisms for the “isolated positivity for the hepatitis B

surface antigen (HBsAg)†serological profile

Robério Amorim de Almeida Pondé1, 2, 3, 4

(1) Laboratório de Virologia Humana, Instituto de Patologia Tropical e Saúde

Pública, Universidade Federal de Goiás, Goiânia-Goiás, Brazil

(2) Central Goiana de Sorologia, Imuno-hematologia e Biologia Molecular,

Goiânia-Go, Brazil

(3) Hospital Materno-Infantil, Goiânia-Goiás, Brazil

(4) Rua 7A Edifício RIOL, Nº 158, 1º andar, sala 101, setor aeroporto,

Goiânia-Goiás, CEP 74-075-030, Brazil

Received: 12 February 2010 Published online: 11 May 2010

Abstract

During HBV infection, four structural antigen/antibody systems are observed:

hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs); the pre-S

antigens associated with HBsAg particles and their antibodies; the particulate

nucleocapsid antigen (HBcAg) and anti-HBc; and an antigen structurally related

to HBcAg, namely HBeAg and its antibody (anti-HBe). Through the examination of

this antigen–antibodies system, hepatitis B infection is diagnosed and the

course of the disorder may be observed. Isolated HBsAg seropositivity is a

peculiar serological pattern in HBV infection observed some times in routine

laboratory. In most cases is not clear how this profile should be interpreted

neither its significance. This pattern, however, may be associated with some

clinical and laboratorial situations of great relevance, some of which will be

addressed in this article.

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Introduction

Hepatitis B surface antigen (HBsAg) is the most important marker for laboratory

diagnosis of hepatitis B. HBsAg detection is used for the diagnosis of acute and

chronic hepatitis B virus and indicates potential infectiousness. It is one of

the first serum markers to appear during the course of HBV infection and it is

also useful as a follow-up marker, since declining concentrations are observed

in resolving hepatitis B. In patients who subsequently recover from HBV

infection, HBsAg usually becomes undetectable after 4–6 months [1]. If HBsAg

persists for more than 6 months, spontaneous clearance is very improbable and

the infected individual is considered a chronic HBV carrier [2].

However, during the acute phase of infection, following HBsAg detection, other

viral markers can be easily detectable including DNA polymerase and HBeAg. HBeAg

appears shortly after the appearance of HBsAg and disappears within several

weeks as acute hepatitis resolves. It presence in the serum correlates with

presence of viral replication in the liver. Anti-HBc IgM antibodies are

detectable at the outset of clinical disease, and as the infection evolves IgM

anti-HBc levels gradually decline, often become undetectable within 6 months and

IgG class predominates, remaining long time (sometimes life-long) at detectable

levels. As recovery and convalescence signal, antibodies to other viral proteins

appear in the blood. Anti-HBe antibodies appear after HBeAg clearance and may

persist for many years after resolution of acute HBV infection. Anti-HBs

antibodies become detectable late in convalescence, from 6 weeks to 6 months,

after HBsAg clearance, although it is produced early in the course of an acute

infection. Thus, through the examination of this antigen–antibody system,

hepatitis B infection is diagnosed and the course of the disorder may be

observed [to review see reference 3].

The aforementioned dynamic occur in the majority of cases or in other words, in

HBV infections with a typical course. Some cases, however, present with

unexpected patterns, such as, “Isolated HBsAg Seropositivityâ€. This is an

atypical serological profile and an uncommon phenomenon observed some times in

routine laboratory. This article illustrates the diverse significance of this

finding and the mechanisms could justify this peculiar pattern which is of great

importance for both clinicians and laboratorial personnel.

--------------------------------------------------------------------------------

Sample collected very early, before the expression of another acute phase

serological marker

Classically, 8–9 weeks following the infection, in the incubation period or

3–5 weeks before biochemical evidence of liver dysfunction and clinical

symptoms appearance, it is possible to detect HBsAg in serum, in the absence of

detectable HBeAg levels [4]. Considering that anti-HBc IgM antibodies usually

appears within 1 month of the appearance of HBsAg during acute phase, in

practice the isolated positivity for HBsAg may be really reflex of a primary

acute infection, even it is infrequent that a patient can be investigated in

such a previous moment of an infection pre-symptomatic phase [5]. This finding

demonstrates the need to properly investigate each case.

In this context, the serological profile evaluation of samples collected

subsequently will be an efficient strategy for establishing the diagnosis if

consistent with viral dynamic described earlier. However, it is important to

point out that although have been claimed that high titer anti-HBc IgM can be

accepted as an indicator for acute infection, its presence is not an absolutely

reliable marker of acute infection, since some patients with chronic hepatitis B

infection become positive for IgM antibody during flares in their disease [6].

Thus, positivity for anti-HBcIgM antibodies must be considered with caution not

to induce diagnostic mistake. On the other hand, in practice, anti-HBcIgM

negativity is important, because if this indicator is negative, the probability

of acute infection in HBsAg positive cases is nil [4].

The isolated HBsAg seropositivity, being consequence of a primary acute

infection, highlights the necessity of laboratory and blood banks especially to

adopt highly sensitive HBsAg detection methods, and have reference laboratories

with experience and sufficient technology to conclude properly the cases. The

ability to accurately detect the presence of HBsAg has a critical role in the

diagnosis of HBV infection, especially in asymptomatic people, including blood

donors [7], pregnant women [8], and patients who will be submitted to

chemotherapy treatment or drugs immunosuppressive. For this reason, the design

of tests with high analytic sensitivity has been necessary. By analyzing

seroconversion panels, it has been estimated that the new HBsAg assays with

subnanogram sensitivity (from 0.04 to 0.12 ng/ml) have shown to be more

sensitive than many licensed assays widely used (range of from 0.13 to 0.62

ng/ml) [9, 10]. These tests might potentially detect HBsAg an estimated 3–9

days earlier [9], however, they could extend further the time of persistence of

the serological profile isolated HBsAg seropositivity.

--------------------------------------------------------------------------------

Possible false-positive/nonspecific reactivity

The isolated reactivity to HBsAg may be, also, associated to false-positivity or

nonspecific reactivity, as may occur with all infectious disease assays [11].

The specificity relates to the ability of an antibody or a population of

antibodies to complex only one kind of molecules, and cross-reactivity is the

ability of more than one antigen competing for the same antibody-binding site as

a result of sharing of an epitope [12]. Considering that the specificity of

HBsAg assays is over 99%, false-positive results may occur because of

cross-reactivity and have been observed with heparinized samples or due to

interferences with hemoglobin or bilirubin. Besides, false-positive results may

be observed during pregnancy and in individuals with acute or chronic infections

or individuals suffering from autoimmune diseases or chronic liver diseases [13,

14].

The vast majority of the commercially available tests to HBsAg detection are

based on the sandwich enzyme immunoassay (EIA), principle which uses monoclonal

antibody and/or polyclonal anti-HBs antibody mapped to recognize the

three-dimensional structure of the ‘a’ determinant of wild type (and mutant)

HBsAg [15, 16]. These tests are highly sensitive and able to detect extremely

small quantities of HBsAg in the bloodstream, such as 0.04 ng/ml, despite this

excellent analytical sensitivity is not sufficient to detect HBV in all phases

of infection [17]. For diagnosis, manufactures determine a “cut off†value

that balances the necessary high sensitivity for detecting the antigen with the

need to avoid false-positive results. Samples with signal value above the “cut

off†are called positive. However, because the high sensitivity, is

recommended caution before establishing the diagnosis, since has been

demonstrated most of weakly positive results can be falsely positive [18]. In

addition, it is also important to consider that the current licensed HBsAg

assays have highly variable cut-off levels, which range from 0.13 to 0.62 ng/ml

(while investigational HBsAg assays are able to detect down to 0.07 ng/ml) [12],

and are different among them because of different monoclonal/polyclonal anti-HBs

antibodies (or a combination of them) bound to a solid phase and a second

labeled anti-HBs, utilized to detect the captured antigen [19]. Thus,

considering these differences, false-positive results may occur, not being

necessarily achieved by another.

In this context, it has been suggested that the result must be confirmed by an

HBsAg neutralization test, in which the percentage of HBsAg signal reduction in

the presence of antibodies against HBsAg (anti-HBs) is measured. In this test,

samples with ratio>1 (Samples/Cut Off) are incubated with anti-HBs; if HBsAg is

truly present, the anti-HBs antibody in the neutralization step blocks HBsAg,

inhibiting its binding to the reagent antibodies anti-HBs bound to a microplate

phase solid, and consequently, reducing or eliminating its signal (positive

neutralization test result) [11]. This procedure can minimize the falsely

positive results expression for this marker [20]. Additionally, nonspecific

reactivity may be reduced by overnight storage of samples at 2–8°C [21].

Despite the high HBsAg immunoassays sensitivity and specificity within the

tolerance limits for detection HBsAg, the advent of new methodologies, such as

chemiluminescent assay, has been possible to lower incidence of false-positive

results when compared to detection by immunoassay [13]. Chemiluminescent assays

are methodologies that differ from EIA tests by test principle, by using

different anti-HBs antibodies (mouse-goat-monoclonal/polyclonal) and kinetics of

the reaction (incubation time, temperature, and by different detection systems

of captured antigen [13, 22]). In addition, the procedure to measure HBsAg

involves 2 steps: constituted by initial screening followed of neutralization

assay, which serves as a confirmatory test of reactivity originally obtained

[20]. The literature has reported sensitivity and specificity after confirmation

of the results of 100% by this methodologies [13, 23], which has been able to

detect low levels of wild-type and HBsAg mutants [13, 22], eliminating

possibility of false positives results. On the other hand, considering that

enzyme-linked immunosorbent assays are licensed methodologies and widely used,

it is recommended that for the resolution of discrepant results of HBsAg or

persistent isolated HBsAg seropositivity for this methodology, neutralization

assays should be always performed to establish a conclusive serological

diagnosis. However, it is important to point out that the neutralization test

could not confirm the true reactivity in the cases of HBV surface mutants

infection, since the anti-HBs antibodies used in the step neutralization are

mapped to recognize only the wild type s-antigen three-dimensional structure.

Therefore, in these cases the neutralization test can suggest false-positive

result.

--------------------------------------------------------------------------------

Antigenemia after hepatitis B vaccination

Vaccines are the only effective defense mechanism against hepatitis B, and

prevention of primary infection by vaccination is an important strategy to

decrease the risk of chronic infection and its subsequent complications.

Hepatitis B vaccines have been available for almost 30 years. The

first-generation hepatitis B vaccine, an inactive plasma-derived, became

available in 1982, while the second generation of HB vaccine, a DNA recombinant

HB vaccine, was available for general use in 1986 [24]. Both of the vaccines

were proven to be safe and efficacious in preventing HBV infections.

Vaccination is essentially a simulated infection under control that stimulates

the immuno-defense mechanism of the body in order to produce protective

antibodies. Since the immunologic agent of the vaccines and serological marker

are the same [25], vaccination may create “false-positive†results in

serologic screening, originating the isolated positivity for HBsAg serological

profile.

Some studies in blood donor population have shown that a weak positive HBsAg

reaction is relatively uncommon and has been reported to occur 1–3 days after

vaccination [26]. In a study performed by Otag [27], three different vaccines

(Engerix B, Hepavax Gene and Gen Hevac were administered to 44 healthy adult

with an age range 18–60 years old, divided into three groups. All subjects

received a full dose (20 μg) of vaccine administrated via intramuscular route.

Blood samples were drawn at 1 h, 24 h and 3 days after vaccination and tested

for the presence of HBsAg by enzyme immunoassay. Each group received different

vaccine and was produced three ‘false-positive†results in blood samples

drawn at 24 h after vaccination. All subjects tested negative for HBsAg in

samples drawn at 3 days after vaccination, which indicates that antigenemia due

to vaccination is transient.

However, there has been related detectable hepatitis B surface antigenemia 5

days [28] and in until 18 days after vaccination in adults [29], extending the

period previously observed. In this context, this transient antigenemia may be

justified. A 70-Kg adult has a volume of extracellular fluid of approximately 15

l. If the adult receives a 20-μg dose of hepatitis B vaccine, then a plasma

level of 1.33 ng/ml is possible, assuming that the entire dose is soluble HBsAg.

Current HBsAg assays have sensitivities of 0.1 ng/ml, or even less. It is

therefore, not surprising that current HBsAg assays detect individual who have

been recently vaccinated against hepatitis B virus [28].

Additionally, works done with infants has indicated high incidence and long

duration for transient surface antigenemia after vaccination. Challapalli et al.

[30] described detectable levels of HBsAg in 55% of 18 newborns tested 33–56 h

after administration of engerix B. In one infant, 17 days was needed to clear

this transient antigenemia. Bernstein et al. [31] described detectable levels of

HBsAg in 12 out to 19 infants (65%) within the first 6 days after administration

of engerix B. One neonate remained seropositive at 8 days. Weintraub et al. [32]

reported detectable level HBsAg in 17% of 47 newborns tested after

administration of engerix B. Seropositive results was observed between 24 and 72

h after vaccination and all were seronegative 2 weeks after initial detection.

Finally, Koksal et al. [33] followed prospectively 39 infants vaccinated by Gen

Hevac B, and found that 69.2% of them tested positive for antigenemia at least

once. The majority of identified antigenemia cases fall into either 2–3 or

5–6 days after immunization day, whereas the longest documented duration of

antigenemia was 21 days post-vaccination [33]. The higher incidence and long

duration of transient antigenemia observed in newborn or infants might be

attributed to a relatively larger dose of vaccine in infants or level of the

development in the immuno-defense systems of the body. However, most of the

studies with adult subjects have given disparate results.

In conclusion, extreme caution should be used when interpreting HBsAg tests

performed within a limited time frame after administration of hepatitis B

vaccine. Laboratorial personnel should be aware that in all this cases the

neutralization assay will confirm the HBsAg presence in the blood, and

“false-positive†serological test results may create misdiagnosis and

needless troubles. The introduction of HBV polymerase chain reaction (PCR) in

the algorithm confirmatory may be most helpful in differentiating vaccines (who

are expected to be PCR negative) from those individuals developing acute

hepatitis B infection, who should be PCR positive.

--------------------------------------------------------------------------------

Immune tolerance to HBV core antigen

Isolated HBsAg seropositivity may be also explained by immune tolerance to HBV

core antigen mechanism, which consequently leads not anti-HBc antibodies

production.

The core antigen (HBcAg) nucleocapsid induces a vigorous humoral and cellular

immune response [34]. Anti-HBc antibodies become detectable in serum 3–4 weeks

after the appearance of HBsAg, and usually persist for many years, independently

of the HBV infection outcome [35]. The immune tolerance to HBcAg is known by the

incapacity of the individual to produce anti-HBc or to make it in levels not

detectable, which can be mediated by a variety of mechanisms. For example,

T-cell anergy is a tolerance mechanism in which the T cell is functionally

inactivated following an initial antigen encounter but remains alive in a

hypoactivated state [36]. T-cell anergy has been proposed as a major mechanism

for the maintenance of self-tolerance and regulation of the cellular immune

response [37]. In addition, a small number of specific peripheral T lymphocytes,

an inefficient antigen presentation or lymphokine production by the

antigen-presenting cell are among others possibilities associated with anti-HBc

production [38]. Therefore, the isolated HBsAg seropositivity and persistent

absence to anti-HBc can be associated with a selective immune system defect.

In a study, viral sequence and host immune response were investigated in an

unusual asymptomatic chronic hepatitis B virus carrier who was consistently

nonreactive for antibody to HBc and had normal ALT levels over a 5-year study

period [39]. He had not abnormalities in the number of peripheral blood T or B

cells, not HBc-specific suppressor T cells and his in vitro proliferative

response to the T-cell-dependent and T-cell-independent, were both normal, but

not to recombinant HBcAg. However, unlike other HBsAg carriers and hepatitis B

virus-immune individuals, his monocytes did not ingest HBcAg-coated beads,

suggesting that the defect could be associated with the antigen-presenting cell.

In another more recent study, the same serological profile accompanied by active

viral replication (HBsAg and HBeAg detected at high levels) with an apparent

persistent lack of anti-HBc was found in two blood donors [40]. To explore the

capacity of the two donors to produce an efficient antiviral humoral response,

several serological markers were studied and quantification of IgG was

performed. Neither donors were immunocompromised, considering that they did not

have hypogamaglobulinemia and were capable of producing antibodies to other

viruses (CMV, HAV). Additionally, nucleotide sequence analysis of the pre-C/C

region did not show mutations or deletions in encoded proteins, demonstrating

that the apparent absence of anti-HBc was not caused by a defective HBV mutant.

Although the T-cell immune response could not be explored, the T-cell immune

tolerance to HBV antigens could be the most probable explanation for this

particular immunological and clinical situation, since the persistently normal

ALT levels (which is considered an important sensitizing viral determinant in

the infected liver) had suggested the absence of liver damage, despite

serological evidence of active viral replication.

The nucleotide sequences of the precore/core and X-region of hepatitis B virus

were also studied in four subjects who were serologically negative for anti-HBc

antibodies [41]. These were persistently positive for HBsAg and were considered

to be asymptomatic HBV carriers. The nt sequences of the precore/core revealed

substitutions dispersed; however, they were not considered able to change the

epitope of the core antigen and to justify the absence of anti-HBc antibodies.

Despite X-ORF has showed nucleotide substitutions, the structure of the

X-protein and the promoter/enhancer II complex [see the following paragraphs and

reference 53–55] appeared to be conserved. These findings suggested the

absence of serum anti-HBc was not due to mutation of the HBV DNA but due to an

aberrant immune reaction of the host to HBV in accordance with Gotoh et al.

[41].

Finally, the phenomenon of the immune tolerance to HBc-antigen has been also

associated with vertical transmission of infection and consequent absence of an

antibody response to HBc in infants born to HBe-positive carrier mothers [42].

It has been postulated that HBeAg may have an immunoregulatory function in

promoting viral persistence [43]. The HBcAg and HBeAg are distinctly recognized

by antibodies but due to their extensive amino acid homology are highly

cross-reactive at the T-cell level [43]. Thus, HBe antigen, because of its small

size, may traverse the placenta and elicit HBe/HBcAg-specific T-helper cell

tolerance in utero [44], consequently may lead to fetal immunotolerance not only

to HBeAg but also to HBcAg and still to inhibit anti-HBc antibody production

when it is present in the serum [36].

--------------------------------------------------------------------------------

Infection by defective HBV-mutant

In all the cases of immune tolerance to HBc aforementioned, the pre-C/C regions

remained unchanged, with normal synthesis of viral antigen. However, the

individual demonstrates incapacity anti-HBc production, or do undetectable

levels. On the other hand, anti-HBc production may be affected by nucleotide

sequence deletions in the core gene, involving important epitopes to antigen

recognition by immune response cells, or in the X-region, compromising

transcriptional regulatory elements, justifying isolated seropositivity for

HBsAg. In these cases, the deficient humoral response to HBcAg may not be

accompanied by a cellular immune tolerance [45].

Infection by mutant of the core-region of hepatitis B virus

The C-gene (C-ORF) contains two regions. The core-region (183 aa) encodes for

the viral nucleocapsid (HBc-antigen/core-protein) and the precore-region (29

aa), together with the core-region, encodes for the HBe-antigen [46]. Some

functional domains within the core-protein can be highlighted, such as, the

arginine-rich C-terminus (up to aa 64) which enable the encapsidation of the

pregenomic RNA, and the remaining C-terminus (up to aa 173) which may be

important for the synthesis of plus-strand DNA and thereby for the virus

replication [47]. Furthermore, several epitopes within the core-protein differ

in immunological aspects. For example: aa sequence from 141 to 151 and aa

sequence from 84 to 101 are recognized by cytotoxic T-lymphocytes; aa sequence

from 74 to 83 and aa from 107 to 118 comprise conformational determinant

responsible for HBc antigenicity, whereas aa from 76 to 89 is linear determinant

responsible for HBe antigenicity [48]. Thus, points mutations between aa 74 and

89 and in-frame deletions involving codons 94 to 101, respectively, could reduce

HBe and HBc antigenicity and to shorten core-protein, becoming not functional

protein [48].

The nucleotide sequence of the HBV pre-C/C region has been determined after

amplification and cloning of HBV genomic fragments from serum samples of

patients HBsAg and DNA positive, but anti-HBc negative and ALT levels in the

normal limits. The result analysis of pre-C/C clones has revealed single base

deletions (T at nucleotide 79 of the pre-C and G at nt 185 of the C gene), which

generated the appearance of stop codons at nt 65–63 (pre-C) and 194–196 (C

gene), respectively, leading to the synthesis of truncated proteins.

Furthermore, in frame deletion spanning from nt 226 to nt 278 and from nt 112 to

nt 150 were also observed [49]. These fragments are important for immune

recognition because they are regions spanning nucleotides 106–158 and

226–301 which encode the main B- and T-cell epitopes of HBcAg and HBeAg [50].

Core-deletion-mutant mostly has been observed in patients with long-course HBV

infection. It has been supposed that their selection may not be favoured by

immunological features but by an enhanced expression of the polymerase [48]. The

over-expression of the polymerase is supposed to be inhibited by the two

start-codons J (nt 2163 to nt 2165) and C2 (nt2177 to nt2179), which are

situated within the C-ORF upstream of the normal polymerase-AUG (nt2307 to

nt2309) [51]. Core-deletion-mutants can lose the inhibitory J-AUG and C2-AUG,

whereas the polymerase-AUG keeps intact. Therefore, core-deleted genomes led to

a more efficient translation of the polymerase by the ribosomes which might

increase the intracellular polymerase level. This could result in an enhanced

encapsidation of the pregenomic-RNA and thus in an increased virus production

[48]. In immunocompromised patients core-deleted viruses can became preponderant

over the wild type virus. In immunocompetent patients, the appearance of

core-deletion-mutants correlates with a low level of viremia [52].

Infection by X-defective HBV mutant

The X-ORF (X-region) encodes for a 154aa protein called HBx (protein X). The

protein X is known to transactivate transcriptional regulatory elements, which

include the enhancer and core promoter elements [53]. The core promoter, located

at nt 1586 to nt 1849 [53, 54], regulates the transcription of the

core/pregenome, and enhancer II, located at nt 1687 to nt 1805 [55], is

implicated in activation of the S-promoter [53, 55]. The sequence nt 1742 to nt

1849 is called the basic core promoter. It is supposed to be sufficient for the

correct transcription initiation of the pregenome and precore mRNA [48, 53]. DR1

(direct repeated) and DR2, which are implicated in the origin of HBV DNA, are

also located in the X-region (Fig. 1).

Fig. 1 Transcription regulatory elements of hepatitis B virus. URR (upper

regulatory region) consists of a negative regulatory element

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The X-defective HBV mutant is commonly characterized by an 8-nt deletion between

nt 1770 and 1777 and a point mutation of DR2 (T-to-C) in the X-ORF [56]. This

deletion results in a C-terminally truncated X protein of 134aa (due to a loss

of 23aa and addition of 3 normal aa), which loss its transactivating activity.

The 8-nt deletion of the core promoter/enhancer II complex sequence may diminish

the function of this transcriptional regulatory element [57]. Accordingly, X-ORF

mutations may suppress the replication and expression of HBV DNA, leading to

suppression of HBcAg synthesis. Consequently, patients infected with X-defective

HBV mutant can demonstrate low serum levels of HBsAg (or be frequently negative)

and be negative for anti-HBc, despite the presence of HBV replication.

X-defective HBV-mutants have been isolated from patients with acute or chronic

hepatitis [56].

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Infection by HBV2

The existence of hepatitis B virus variants or HBV-related human hepatotropic

viruses has been suggested by some searchers to explain this atypical

serological profile of HBV infection.

The term HBV2 was introduced to describe potential variants of HBV which do not

elicit a detectable immune response to the nucleocapsid (anti-HBc) in apparently

immune-competent patients. It was reported for the first time in December 1987

by Cousaget et al. [58], when he detected a transient HBsAg reactivity among

serum samples from non-immunized Senegalese children who were negative for

antibody to the HBV core antigen and who did not seroconvert on follow-up. It

was claimed that the isolated HBsAg reactivity was specific and not due to

infection by the “classical†HBV, but to the presence of an HBV-related

virus [58].

Others serological aspects of HBV2 infection are absence of detectable levels of

serum HBeAg and anti-HBe, presence of low levels of HBV DNA with clearance rapid

of HBsAg (1–2 months), and lack of detectable anti-HBs antibody response on

recovery [59]. Although some investigators have interpreted the isolated

reactivity for HBsAg as non-specific reaction [60, 61], neutralization of the

HBsAg reactivity by human polyclonal anti-HBs [58, 62, 63], visualization of

HBsAg-like particles by electron microscopy, detection of pre-S2 epitopes by

specific monoclonal antibodies, and detection of HBV-related DNA sequences by

molecular hybridization [58], in these cases have been reported, suggesting

involvement of an HBV variant or an HBV-related agent.

HBV2 is associated with mild hepatitis, and is not commonly reported [58, 59].

However, similar cases of HBV2 infection were detected in California [64],

Taiwan [62], Spain [63] and France [65], suggesting that the putative agent can

be distributed worldwide.

Finally, it has been suggested that the transmission of the agent may be occur

by the oral route, leading to a self-limited infection with very low levels of

viral replication and absence of clinical symptoms in the majority of cases

[59]. In addition, anti-HBs resulting from vaccination do not prevent infection

with HBV2, implying that HBV2 differs from HBV in the epitopes of the envelope

[58].

Despite sequencing of HBV2 genome has not been achieved so far, it has been

suggested that this serological variant may be due to nucleotide sequence

variation in the viral core gene. Valliammai et al. [66] investigated the core

ORF sequence from patients whose infection met all the criteria for definition

as HBV2 infection. In this study, only two of fifteen patients had HBV DNA

detectable by nested or semi-nested PCR, confirming previous reports of low

levels of viremia. The amplified products from patient 1 showed deletion from

nt2021–2053 would result in loss of residues 41–51 from HBcAg. This loss

could explain the lack of anti-HBc reactivity. The amplified products from

patient 2 showed deletion of 14nt and an insertion of 1nt at nucleotide position

1970–1984, resulting in the introduction of a termination codon or a missense

mutation which would lead to loss of the core initiation codon. Both of these

mutations would abrogate the HBcAg and HBeAg synthesis. However, sequencing

studies of so-called HBV2 isolates have not been widely reported, and it is not

clear how these viruses differ from wild type HBV.

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HBcAg-anti-HBc immune-complex formation (Chronic HBV infection without anti-HBc)

The isolated HBsAg seropositivity can be also justified by immune-complex

formation of anti-HBc antibodies with the excess of HBc antigen in the

bloodstream, become anti-HBc undetectable [67]. These immune-complexes cannot be

detected by available commercial tests which are not able to detect antibodies

in the presence of excess antigen in the serum [68]. Antibodies anti-HBc

complexed could be detected only after immune-complex dissociative treatment.

Some methods are based on polyethylene glycol precipitation followed by íon

chaotropic treatment [69] or based on sucrose gradient fractionation [70].

HBcAg is not a secreted protein and exists primarily in liver and in serum

within HBV particles, not being directly accessible in the blood to the immune

system. In exceptional cases, HBc-epitopes may, however, be exposed on virion

present in the serum [71], and also during liver necrosis, when nucleocapsids

may be secreted from infected hepatocytes with high level of HBV particles,

explaining the presence of anti-HBc complexed in the bloodstream. This

phenomenon has been observed in chronic HBV carriers in phase of active

replication in a state of immunosuppression, as seen in the context of

transplantation (bone marrow, kidney, and heart), during chemotherapeutical

treatment for neoplasias [72] or an uncontrolled HIV infection, conditions could

lead to an immune system defect. HBcAg is the most immunogenic HBV component

[34] and induces the production of high titers of anti-HBc antibodies in

immunocompetent individuals who have been exposed to HBV [6]. However, the

immunosuppression conditions can potentially induce the decreased antibody

production [73, 74]. HBcAg-reactive material releases from HBV particles present

at a high level in the serum and from hepatocytes undergoing lysis consequently

could complex to low levels of anti-HBc produced in these patients and therefore

lead to no detection of antibodies.

HIV infection is the cause for depressed immunity. Chemotherapeutic and

corticosteroids drugs act as selective immunosuppressive agents either by

eliminating or inactivating cells B, compromising anti-HBc antibody production

[72]. Interestingly, in HIV-infected patient during AZT treatment, and in

individuals in chemotherapy treatment or immunosuppressive drugs use during

monitoring of treatment, low levels of anti-HBc antibodies can become detectable

[67, 72]. This suggests that partial reversibility of defect and gradual

improvement of the immune system function can occur. Therefore, in the

immunosuppression conditions, the choice of the most sensitive assay for

antibodies detection would be the most reliable diagnostic approach.

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Quantification of HBsAg and HBV DNA testing (Correlation between HBV DNA levels

and isolated HBsAg positive cases)

The concentration of HBsAg in the blood of infected individuals has been

expressed as sample to cut off ratio (S/CO) or as ng/ml, and also, UI/ml, and is

highly variable, depending of infection phase. After infection with wild-type

HBV, HBsAg may be undetectable for several weeks, even with HBsAg assays at the

detection limit <0.1 ng/ml [17], but always with a progressive increase in

concentration. In chronic infection context, HBsAg concentration may fluctuate

over time and under certain circumstances may not be detectable (in

low-replication phases; when infection resolves spontaneously or after

successful antiviral therapy) [75, 76]. However, there is limited information on

HBsAg levels and it correlation with viral load, considering the dynamic natural

course of HBV infection.

Based on the analysis of seroconversion panels, HBV viremia during primary

infection has been evaluated and categorized in DNA detection phases and HBsAg

concentrations. The called “ramp-up phase†is a period during which DNA can

be quantified and viral load and HBsAg increase progressively over time until

HBsAg detection, whereas the “before-ramp-up phase†is the period where

low-level HBV DNA is intermittently detected only by higher-sensitivity

qualitative PCR or other NAT assays. The “after-ramp-up phase†is the period

after HBsAg becomes reactive and viral load progression is either slowed or

stopped [9]. By using the most sensitive HBsAg assay (detection limit of 0.04

ng/ml), levels of 2.000 HBV genomes/ml have been calculated as average viremia

at the time of HBsAg seroconversion [77]. Using another HBsAg licensed assay

with sensitivity for detection from 0.13 to 0.62 ng/ml, the estimated viral

loads can vary from 1,757 to 11,431 gen eq/ml, at the time of HBsAg detection

[9]. After HBsAg seroconversion, the HBsAg levels and DNA increase progressively

until it reaches titers higher than 15.000 UI/ml and 2 × 107 copies per ml,

respectively, when HBeAg becomes detectable [78]. While in the early and during

acute phase of infection it has been possible to establish strong correlation

between HBsAg and HBV DNA levels, some studies have demonstrated that there is

not this correlation at the late stages of healthy HBV carriage [79] or it is

weak or missing when analyzing different phases of persistent HBV-infection

separately [80].

Despite during the window period (or in healthy carriage, infrequently) the

detection limit of sensitive HBsAg screening assay (<0.1 ng/ml) corresponds to a

concentration of HBV DNA from 300 to 400 UI/ml, Togashi et al. [81] have

recently demonstrated that HBsAg might be detected in samples without HBV DNA.

--------------------------------------------------------------------------------

Prevalence of isolated positivity for HBsAg serological profile

The prevalence of isolated positivity for HBsAg serological profile in the

hepatitis B virus infection context has not been widely established. In a

seroepidemiologic study performed in 702 children in Camerron, in 1991, the

atypical serological pattern frequency was 4.1% (29 cases) among 163 HBsAg

positive individuals [82]. In Spain has been evidenced lower frequencies ranging

from 1.3% in pregnant women to 0.05–0.6% in patients selected randomly for

study [83, 84].

However, it is important to point out that prevalence of this profile and it

significance can vary widely depending on the specific population studied, and

also, the incidence of HBV infection in the region. Taking as example the

pregnant women population in region of low HBV infection prevalence, it is

possible that the isolated reactivity for HBsAg might be less associated with

recent or chronic infection than false-reactivity, since in this population

nonspecific reactivity can occur with greater frequency [13, 14]. The same

conclusion may not be true in high prevalence regions considering the same

population of study, since the risk of exposure to the agent tend to be larger

and real in this region. In accordance with this approach, the data are limited.

Studies performed in regions with low prevalence of HBV infection have shown

that in most cases (70%) the detection HBsAg isolated can be associated with

nonspecific reactivity, since true reactivity has not been confirmed by

neutralization test or genomic detection [5]. In this geographic context, in a

few cases (3.41 per 105 persons-years), the presence of this serological profile

has been associated with recent seroconversion, estimated by the

incidence-window period model proposed by Zou et al. [85]. On the other hand, in

regions with high prevalence of HBV infection (and consequently, higher rates of

chronic infection, vertical transmission and mutation), prevalence of HBsAg

isolated profile serological tends to be increased and may correspond to almost

50% among specimens with unusual serological profile, being often related to the

altered immune response of the host or to HBV variants [86].

Seroepidemiologic studies in blood donors and in general population have been

carried out to assess the incidence of asymptomatic HBV carriers in accordance

with HBsAg detection [87, 88]. However, prevalence of HBsAg isolated has not

been reported, which would be of great value as regards its significance in the

clinical context.

In population of chronically infected individuals and in the immunosuppression

context, the frequency of this serological profile has not been evaluated

satisfactorily, despite great relevance and broad meaning.

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Conclusion

Hepatitis B virus surface antigen reactivity in the absence of antibodies to

core antigen is an atypical serological profile that could be associated with

several clinical situations and adverse conditions, as addressed in this review.

This profile not only can suggest acute or chronic infection but also can point

out to a possible host immune system selective defect or to immunosuppression.

On the other hand, it can be associated to HBV antigenic variant infection or,

also, to be justified by nonspecific reactivity.

Considering that diverse possibilities could explain and justify this profile,

laboratory professionals need to be aware of the performance of their HBsAg

assays applied in laboratorial routine and include the HBsAg neutralization test

to prove the true reactivity for that marker. The collection second sample is

always advisable, prior to establish the diagnosis, especially in the cases of

recent infection, before the appearance another acute phase marker, and when a

molecular test is not available to prove the reactivity for HBsAg. Samples with

discordant results, sensitivity and specificity among tests applied should be

considered, and evaluated for the presence mutant strains. Laboratories should

communicate to clinicians about status of patients with questionable HBsAg

results. Finally, parts of the clinical history including possible source of

exposure, previous immunization, immune status of the individual, and results of

any previous tests that may have been performed using another assay, include

data important to security of result and reliable serological diagnostic.

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References <cut>

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