Abnormal laboratory results
Hepatitis B: issues in laboratory diagnosis and vaccination
David Siebert, Microbiology Registrar, Royal Children's Hospital, Melbourne; and Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, Melbourne

Summary

A laboratory diagnosis of hepatitis B (HBV) infection is dependent upon the detection of hepatitis B surface antigen in serum. The distinction between acute and chronic infection relies on the detection of other serological markers. Serum-based assays can now detect and quantify the viral DNA. These assays will have a role in therapeutic monitoring and the detection of HBV mutants.While new guidelines for vaccination have recently been published, some issues regarding revaccination and the management of people who cannot mount an adequate vaccine response are yet to be adequately resolved.

Key words: immunity, liver disease, vaccines.

Aust Prescr 1998;21:72-5

Introduction
In Australia, almost 1% of the population may be chronically infected with hepatitis B virus (HBV). This results in up to 1200 deaths annually.1 The highest carriage rates are in indigenous people (10-25%), Micronesian and South-East Asian immigrants (5-15%) and people from South-East Europe (2-5%). Despite the existence of an established maternal screening and infant vaccination program, it has been virtually impossible to identify people at risk before their exposure to the virus. Vaccination aimed at high-risk groups has therefore failed to significantly reduce the burden of chronic infection in low prevalence countries like Australia.1 For these reasons, a new program of universal vaccination has recently been recommended.2

Viral structure and products
Hepatitis B virus is a compact 42 nm sphere (Fig. 1). The outer envelope is composed of units made from hepatitis B surface antigen (HBsAg) proteins, carbohydrates and lipids. Beneath this outer layer is found the core, a 27 nm particle, made of protein subunits called hepatitis B core antigen (HBcAg).

The hepatitis B e antigen (HBeAg) is not a structural molecule; however, more than two thirds of its amino acid sequence is identical to that of the core protein. It can be secreted from hepatocytes directly into blood as a soluble protein3, which is thought to promote and maintain persistent infection.

Acute hepatitis B infection
HBV is acquired by contact with blood or body fluids containing infectious virus. Infectivity has only been clearly shown for blood, genital secretions and, on occasion, saliva. The incubation period ranges from 6-26 weeks with an average of 12 weeks (Fig. 2).

Fig. 1

A model of hepatitis B virus.

Note the 3 components of surface antigen pre-S1, pre-S2 and S.

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Fig. 2

A graphic representation of serological events in acute hepatitis B.

Major markers, hepatitis B surface antigen (HBsAg) and

anti-hepatitis B core IgM (anti-HBc IgM) are in bold.

ALT - alanine aminotransferase

Total anti-HBc = anti-HBc IgM + IgG

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Laboratory diagnosis (Table 1)
HBsAg first appears in the blood during the incubation period, while the virus is actively `replicating' in liver cells. The antigen is produced in vast excess so that it is not only associated with new infectious virus particles, but also occurs in the serum as small non-infectious spherical and filamentous forms. In acute infection, the HBsAg usually disappears within 3 months of onset. The HBeAg and HBV DNA can also be detected in blood while the virus is actively replicating in the liver (Fig. 2). Symptoms usually appear as the concentrations of bilirubin, alanine aminotransferase and each of the major viral components peak in the serum. These events coincide with the first appearance of antibodies to HBV proteins (Fig. 2).

Antibody to HBcAg (anti-HBc) rises first. As a generalisation, the detection of IgM antibody specific to the hepatitis B core is the primary indicator of acute infection. It usually appears at or just before the onset of symptoms and remains detectable for at least 6 months. The IgG component of anti-HBc usually persists for life.

Anti-HBe is the second antibody to appear and is associated with the rapid clearance of HBeAg. Later, anti-HBe declines and persists for only a few months or years if there is no active viral replication.

The antibody to HBsAg, anti-HBs, may not become detectable for 3-6 months after acute infection. It is associated with resolution of the illness. This antibody is recognised as the marker of immunity to HBV.

There is a range of variations in the serological profile and when in doubt an expert opinion can be sought. For example, in some patients who clear HBsAg and recover clinically, the anti-HBs antibody may only be present at low levels or remains below the level of detection.

Chronic hepatitis B infection
Between 1% and 10% of infected adults and older children develop chronic infection. Adults who develop chronic HBV are not usually immuno suppressed, but when they are, chronic infection is common. More than 85-95% of newborns and children infected under the age of 3 years, born to HBeAg positive mothers, become chronic carriers. This is thought to occur because of the immunological immaturity in the child and the effect of maternal HBeAg in utero. Those carriers infected in early life have an increased risk of both chronic persistent and chronic active hepatitis.

Laboratory diagnosis (Table 2)
From a laboratory perspective, chronic infection is defined as the persistence of HBsAg in the blood for a period of 6 months or more. The serological picture depends on the degree of viral activity in chronically infected hepatocytes (Fig. 3).

A recently infected chronic carrier will show evidence of ongoing viral replication in liver cells. HBeAg and HBV DNA can be detected in blood for months or years after acute infection. High levels of anti-HBc IgM antibody are no longer present.

People with long-standing HBV infection may eventually enter a phase of low level viral replication. To achieve this, chronic carriers must clear HBeAg and HBV DNA from the blood by actively producing anti-HBe antibody and clearing viral DNA from infected liver cells, mostly via cell mediated immunity.3 Several abortive attempts to clear HBeAg may occur over many years and `flares' of active hepatitis can occur (with or without symptoms) until anti-HBe is eventually made (Fig. 3). Patients who cannot clear the virus face persistent active hepatitis and are at a very high risk for the development of cirrhosis and hepatocellular carcinoma.

Table 1

Primary markers for the diagnosis of acute hepatitis B infection

Diagnosis HBV Antigens HBV DNA Anti-HBV Antibodies
HBsAg HBeAg Anti-HBc Anti-HBe Anti-HBs

IgM

Total Ig

Acute HBV

++ + + ++ + - -

Resolving

early - ± + ± ± ± ±
late - - - - + + +
Immune
vaccination - - - - - - +
past infection - - - - + - +

Table 2

Primary markers for the diagnosis of chronic hepatitis B infection

Diagnosis HBV Antigens HBV DNA Anti-HBV Antibodies
HBsAg HBeAg Anti-HBc Anti-HBe Anti-HBs

IgM

Total Ig

HBeAg positive

chronic hepatitis B

+ + ++ ++ - -
HBeAg negative chronic hepatitis B + - +/++ ++ ± -

HBe antigen negative chronic hepatitis B
Unfortunately, there are exceptions to these principles. Some people, especially those who acquire HBV in very early life, never fully clear HBV from the blood. Due to the pressure of the host's immune system, mutant forms of the HBV can be selected from the pool of infected liver cells. HBV DNA production may continue, despite the absence of HBeAg and the presence of anti-HBe in up to 30% of patients infected early in life (Fig. 3).

Testing patients for hepatitis B in practice
The most commonly used tests are enzyme immunoassays (EIA) for the detection of viral proteins and antibodies. HBV DNA can be quantified by molecular hybridisation assay and polymerase chain reaction (PCR) tests can detect minute quantities of HBV DNA in serum.

The results for HBsAg are reported as positive or negative. A positive result indicates either acute or chronic infection.

Fig. 3

A graphic representation of the clinical, histological and serological events during chronic hepatitis B in adults.

ALT - alanine aminotransferase.

The clinical presentation is age dependent with less than 30% of adults experiencing symptoms of acute hepatitis. Typically, in neonates born to HBeAg positive mothers, the infection is sub-clinical.

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Hepatitis B specific core IgM results are also reported as either positive or negative. In general, a positive result indicates recent infection.

In chronic infection, HBsAg is still present, but anti-HBc IgM has disappeared in all but a few patients with `chronic active' hepatitis. Patients who are positive for HBeAg and/or HBV DNA in the blood are highly infectious carriers. Patients with very low levels of HBV DNA in the blood, usually in the presence of anti-HBe as well, are inactive carriers with low infectivity (Table 2).

Between 10% and 20% of chronic carriers eventually clear HBsAg. Like patients recovering from acute infection, they produce anti-HBs antibody. Convalescent patients usually have IgG antibodies to both HBsAg and HBcAg. Vaccinated patients only have antibodies to HBsAg (Table 1). The titre of anti-HBs is usually expressed as IU/mL of blood. A titre of 10 IU/mL is the minimum standard for protective immunity to HBV.

Vaccination and revaccination for hepatitis B
Vaccination against HBV was introduced to control the morbidity and mortality associated with the virus. Until 1997, most Western countries, including Australia, had policies aimed at limiting the spread of the virus only in at-risk individuals, rather than population-based immunisation strategies (i.e. universal infant immunisation with a catch-up program). As part of the World Health Organization (W.H.O.) program for the control of hepatitis B, this selective policy has now been abandoned and a wider program adopted.2,4

Until HBV can be incorporated into multivalent childhood vaccines, all adolescents between 10 and 16 years who have not previously been vaccinated should receive 3 doses of standard HBV vaccine. At-risk infants, those with a HBsAg positive mother or those from high prevalence (>2%) communities (e.g. Aboriginal and Torres Strait Islanders, migrants from Africa, Oceania and other endemic areas), should start a course of 3 vaccinations within 7 days of birth. In addition to the vaccine, the children of HBsAg positive mothers should receive 100 IU hepatitis B immune globulin (HBIG) intramuscularly into the lateral thigh within 12 hours of birth.

Occupational risk groups and other at-risk adults should also be considered for HBV vaccination according to the new NHMRC guidelines (Table 3).2

Revaccination
Most people who seroconvert to the vaccine develop antibody titres >100 IU/mL within 6-8 weeks of completing vaccination. In the studies to date, the antibody response to HBsAg is measurable for 7 years or longer. The NHMRC currently recommends that at-risk individuals (e.g. persons occupationally exposed to blood and blood products) be revaccinated every 5 years, without a retest for anti-HBs titres.2 Unfortunately, the issues of failure to respond to primary vaccination, and what constitutes a protective immune titre to HBV, have not been satisfactorily resolved.

Approximately 5-15% of healthy immuno competent individuals either do not mount an antibody response to the existing recombinant vaccines (non-responders) or respond poorly (hypo-responders). Non-responders remain susceptible to HBV, but the risk for hypo-responders is unclear.5

Table 3

Individuals at increased risk of acquiring HBV infection who should be routinely vaccinated - NHMRC Guidelines 19972

Newborns of carrier mothers

Children under 10 years in high (>2%) prevalence communities

Household contacts of acute and chronic hepatitis B carriers (excluding sexual partners)

People at risk of sexual transmission

Injecting drug users

Haemodialysis patients (often poor vaccine responders)

Blood concentrate recipients

Persons with chronic liver disease/hepatitis C

Residents and staff of facilities for the intellectually disabled

Close contacts of de-institutionalised people with intellectual disabilities

Long-term prison inmates and staff of correctional facilities

Health care workers and embalmers

Others (e.g. day care personnel, police, armed forces personnel, long-stay travellers to endemic countries - see guidelines)

Vaccine non-responders
A non-responder is a person who, despite a correctly administered full course of standard HBV vaccine, does not mount an anti-HBs response when tested 8-12 weeks after the third vaccination. An anti-HBs titre of <10 IU/mL is considered negative.

One reason for failure to respond to HBV vaccine is HBV infection itself. In a patient with a relevant history, a check for HBsAg and anti-HBc can avoid a delayed diagnosis.

Pre-exposure prophylaxis in non-responders - One strategy for dealing with non-responders is to first evaluate the risk to the individual patient. If the patient has no identifiable risk factors for HBV and does not work in an environment which places the individual at high risk of HBV transmission, then revaccination is not essential. Such people should be made aware of their low risk and advised to seek passive and active vaccination with HBIG and HBV vaccine if they subsequently have a significant exposure to HBV (e.g. needle-stick injury).

Non-responders with an identifiable risk factor for HBV (Table 3) or an occupation that places them at significant risk of HBV (e.g. surgeons, dentists) should also be assessed individually. Education in universal precautions and risk reduction is essential. One further option is to attempt revaccination. A fourth or alternatively a double dose of vaccine, and then rechecking the anti-HBs titre 8-12 weeks after this vaccination will produce a response in some patients. Further doses will be unlikely to succeed.

The next generation of vaccines is likely to incorporate pre-S1 and pre-S2 components of HBsAg (Fig. 1). This has been shown to result in seroconversion after one dose in 70% of people who do not respond to the standard vaccine.5 Such vaccines are expected to become available within the next few years.

Post-exposure prophylaxis in non-responders - Any non-responder who has an identifiable or high-risk exposure to HBV (e.g. needle-stick injury) should be offered HBIG and revaccination within 72 hours of exposure. This will reduce the risk of infection and ameliorate illness. Acute and chronic infection will still be possible until newer vaccines become available. There is as yet no role for post-exposure treatment with antiviral drugs, but this may be an option in the future, after appropriate trials.

Hypo-responders
In the U.K., a healthy vaccinee who develops an anti-HBs titre of <100 IU/mL is considered to be unprotected and at risk of HBV.5 Such a classification has not been adopted in Australia. A titre of anti-HBs between 10 and 100 IU/mL takes no account of immune memory and cell-mediated immune responses to HBsAg. In addition, there are no empirical data that correlate the serum antibody titre induced by recombinant vaccine with either the degree of protection against HBV acquired by different routes of infection or the size of the infectious inoculum at the time of exposure. In theory, transfusion of a unit of HBsAg/HBV DNA positive blood into some vaccinated but poorly responsive individuals may overwhelm the `level' of protection that would normally be sufficient to prevent sexual or needle-stick transmission of HBV.

In general, policy formulation and immunisation strategies for high-risk people should be undertaken in consultation with infectious diseases physicians or a medical virologist. It may be necessary to revaccinate high-risk hypo-responders more frequently than the currently recommended 5 year interval. The practice of any one organisation will be based on the frequency, type and degree of exposure to HBV within the population under its care.

(See also Dental implications)

References
1 . Gust ID. Control of hepatitis B in Australia. The case for alternative strategies. Med J Aust 1992;156:819-21.

2
. National Health and Medical Research Council. Hepatitis B. In: The Australian Immunisation Handbook. 6th ed. Canberra: Australian Government Publishing Service, 1997:109-20.

3 . Lau JY, Wright TL. Molecular virology and pathogenesis of hepatitis B. Lancet 1993;342:1335-40.

4 . Tilzey AJ. Hepatitis B vaccine boosting: the debate continues. Lancet 1995;345:1000-1.

5 . Zuckerman JN, Sabin C, Craig FM, Williams A, Zuckerman AJ. Immune response to a new hepatitis B vaccine in healthcare workers who had not responded to standard vaccine: randomised double blind dose-response study. Br Med J 1997;314:329-33.

Self-test questions

The following statements are either true or false (click here for the answers)

1. Some patients with chronic hepatitis B do not have the hepatitis B e antigen (HBeAg).

2. After primary immunisation against hepatitis B, booster doses should not be given until the antibody titre has been checked



First published online: September 1998