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Hepatitis B Test

Reference Range
Hepatitis B virus (HBV) testing plays an important role in detection, classification, and
management of HBV disease.
Results of HBV serologic markers can be reported qualitatively or quantitatively as
international units (IU) or signal per cutoff (s/c) value. For example, a hepatitis B surface
antigen (HBsAg) level of less than 1 s/c is considered negative, while a level more than 5 s/c
is considered positive. Any value between 1 and 5 s/c is indeterminate and should be
repeated. For hepatitis B surface antibody (anti-HBs), a level less than 5 mIU is considered
negative, while a level more than 12 mIU is considered protective. Any value between 5 and
12 mIU is indeterminate and should be repeated.
There is no standardization between laboratories, and these cutoff values tend to vary
between manufacturers. Therefore, results are usually reported as negative or positive.
The laboratory or manufacturers insert should be referenced for quantitative measurement, if
required.
These following reference ranges are based on qualitative measurement of serologic markers
in an asymptomatic, nonimmunized population.

HBsAg: Negative

Anti-HBs: Negative; a level of greater than 10-12 mIU/mL is protective

Immunoglobulin M (IgM) hepatitis B core antibody (anti-HBc): Negative

Immunoglobulin G (IgG) anti-HBc: Negative

Hepatitis B e-antigen (HBeAg): Negative

Hepatitis B e-antibody (anti-HBe): Negative

HBV DNA: Negative

Interpretation
HBsAg usually appears 4 weeks after viral exposure but can be detected any time after the
first week. An individual positive for HBsAg is considered to be infected and is therefore

potentially infectious.[1, 2] Persistence of HBsAg is used to differentiate acute from chronic


infection. Presence of the antigen longer than 6 months after initial exposure indicates
chronic infection. However, the level of the antigen does not appear to correlate with disease
severity.[3] HBsAg can be cleared by normal immune response, and only 1% of patients with
acute HBV exposure are estimated to progress to a chronic state.[4] Detection of anti-HBs in
the serum implies either active or passive immunization that usually persists for life.
Anti-HBc is the first detectable antibody in the course of HBV disease. IgM anti-HBc
indicates acute infection and is the only serologic marker detectable during the window
period, when neither HbsAg nor anti-HBs is detectable. Once IgG anti-HBc appears in the
serum, it persists for life. Detection of IgG anti-HBc indicates previous or ongoing infection.
[5]

Individuals with positive HbeAg results have been shown to have higher rates of viral
transmission;[6, 7] therefore, the antigen is used as a marker of viral replication and infectivity.
[1]

However, HbeAg testing is indicated primarily during follow-up of chronic infection rather

than acute infection because of its variable level during the acute phase.[3]
HBV can be present in hepatocytes in two forms: as replicating virus, leading to production
of infectious particles, or as a nonreplicative form after integrating into the host DNA. Since
HBeAg is produced only during replication of the virus, it can also be used to indirectly
determine the state of HBV in the hepatocyte.[8]
Loss of HBeAg and appearance of anti-HBe in serum is called seroconversion, which is an
important clinical event in the course of HBV disease. Seroconversion can occur as early as 2
weeks in acute infection or may take years in chronic infection. Seroconversion is associated
with a lower level of HBV DNA or a low replicating state of the virus. It also implies clinical
improvement and can help predict long-term clearance of the virus.[8] However, at the time of
seroconversion, the HBV DNA level can vary.[6] Detection of HBe antibody can also be used
to monitor response to treatment and possible remission of the disease.[5]
HBV DNA (quantitative viral load) indicates viral burden and viral replication. It is used to
assess recovery from infection and candidacy for antiviral therapy and to differentiate
between inactive carrier state and chronic active hepatitis in chronic HBV infection. Proposed
cutoffs for consideration for antiviral therapy is 100,000 copies/mL or 20,000 IU/mL in

HbeAg-positive patients with chronic hepatitis and 10,000 copies/mL or 2,000 IU/mL in
HbeAg-negative patients. Note that indications for treatment are based not only on HBV
DNA, but also on serum alanine aminotransferase (ALT) levels and severity of liver disease,
which is beyond the scope of this article.[9, 10]
HBV DNA can be measured both qualitatively and quantitatively using either polymerase
chain reaction (PCR) or hybridization method. Loss of detectable HBV DNA by a solution
phase hybridization assay is an earlier indicator of response to antiviral therapy than loss of
HBeAg. PCR is the most sensitive assay in detection of HBV DNA. However, the clinical
significance of HBV DNA positivity with PCR alone in patients with past infection remains
unclear.[11] The HBV DNA cutoff to define virological response has not been determined and
varies among types of antiviral therapy.[8]
Overall, HBV testing is not based on any single marker but rather involves interpretation of
all serologic markers, HBV DNA, and liver enzymes simultaneously. These parameters can
also differentiate stages of HBV infection, as shown in Table 1.
Table 1. Interpretation of Hepatitis B Serologic Markers[5, 8] (Open Table in a new window)
HBs Anti- Anti-HBc Anti-HBc HBe AntiAg
Susceptible to infection
Immune due to natural

HBs
+

HBe
-/+

DNA
-*

infection
Immune due to

vaccination
Incubation
+
Acutely infected
-/+ Chronically infected
+
(-) is undetectable; (+) is detectable;

IgM

IgG

Ag

HBV

+
+
+
-/+**
+
-/+ -/+
(-/+) means may be detectable

*Nondetectable with non-PCR method

**May be positive in 10%-15% patients with reactivation of infection

+
+
+

Persons with chronic HBV infection can either be inactive carriers or develop chronic
hepatitis. Inactive carriers refer to HBeAg-negative individuals who have normal serum ALT
levels and low (< 2000 IU/mL) or undetectable HBV DNA, while patients with chronic
hepatitis usually have fluctuating ALT and/or HBV DNA levels. Differentiating between the
two is important in clinical management, as inactive carriers generally have a good prognosis
and antiviral treatment is not indicated. Therefore, it is recommended that ALT and HBV
DNA tests be checked every 3 months during the first year in these patients.[12]
HBsAg and anti-HBs negativity despite anti-HBc positivity should be interpreted with
caution, as there are multiple possibilities with these results.[5] These include acute infection
(if IgM is present), resolving or resolved acute infection, false-negative anti-HBs results
(especially in distantly immune patients), false-positive anti-HBc results (thus, still
susceptible), or low-level or false-negative HBsAg results in chronically infected patients.

Collection and Panels


Serologic testing for hepatitis B virus (HBV) infection relies on radioimmunoassay, enzyme
immunoassay, or immunochemiluminometric assay techniques for the detection of antigens
and antibodies in serum. This involves incubating serum in the presence of reagents within
wells coated with antigens or antibodies, which specifically react with those being tested for.
The most widely used HBsAg screening tests are enzyme-linked immunoassays (ELISAs), as
they are the most appropriate for screening large numbers of specimens on a daily basis.[1]
Hepatitis B serologic markers (HBsAg, anti-HBs, anti-HBc IgG, anti-HBc IgM, HBeAg,
anti-HBe)
Specimen: Serum or plasma
Container: Red-top tube, yellow-top tube (acid citrate dextrose tube), gel-barrier tube, plasma
preparation tube, or lavender tube (EDTA tube)
Collection method: Routine venipuncture
The specimen can be stored in a refrigerator at 2-8C when testing cannot be done promptly;
the specimen can be stable at this temperature for up to 7 days. However, some laboratory

may request to store the specimen at -20C or lower if testing cannot be done within 48
hours.[13]
HBV DNA, HBV genotype, and HBV drug resistance assays

Specimen: Serum or plasma


Container: Red-top tube, yellow-top tube (acid citrate dextrose tube), gel-barrier tube, plasma
preparation tube, or lavender tube(EDTA tube)
Collection method: Routine venipuncture
The specimen should be transfused to separate plasma/serum from cells within 6 hours and
kept frozen when testing cannot be done promptly.
The tests use PCR amplification, DNA probe hybridization, and sequencing method.
Panels

Hepatitis B serologic markers are typically part of liver function testing.


Several assays for detection of serum HBV DNA are commercially available; sensitivity
limits are given in Table 2. There is currently no standardization of HBV DNA assays among
laboratories.[8]
Table 2. Lower Detection Limits in Various Assays of HBV DNA Detection[8] (Open Table in
a new window)
Method
Hybrid capture
Branched DNA
Liquid hybridization
PCR

Detection Limit (Copies/mL)


6

3 x 10
0.7 x 106
4 x 104
102 -103

Background
Description

Hepatitis B virus (HBV) is a DNA virus in the Hepadnaviridae family. It has a lipoproteincoated surface, which is called the surface antigen (HBsAg, previously called Australia
antigen). This antigen is the first serologic marker to appear in serum after HBV infection and

is excessively produced during the life cycle of the virus. The inner particle of the virus
consists of an outer lipid envelope and an icosahedral nucleocapsid core. The nucleocapsid
encloses the viral DNA and DNA polymerase that has reverse-transcriptase activity. The outer
envelope is involved in viral binding and cellular entry.[14, 15]
There are 350-400 million people with chronic HBV infection and an estimated 500,000 to 1
million deaths from HBV disease annually worldwide.
Manifestations of acute HBV infection range from self-limited asymptomatic infection to
fulminant acute liver failure, depending on the immunologic response of each individual. The
natural course of chronic HBV infection is also variable, ranging from an inactive carrier
state to progressive chronic infection, which may evolve into cirrhosis and hepatocellular
carcinoma.[1, 16, 2]
Inactive carrier state refers to a state in which HBsAg is persistently detectable without any
evidence of disease activity, such as transaminitis or high HBV DNA levels, as opposed to
chronic active hepatitis, when serum ALT and HBV DNA levels are elevated.
HBV testing plays an important role in detection, classification and management of HBV
disease. The diagnosis of HBV infection and differentiating each clinical state of the disease
involve integrative interpretation of HBV serologic markers. These markers include the
following:

HBsAg: A coated lipoprotein particle that forms a part of the viral surface

Anti-HBs: An antibody to viral surface antigen, which provides protective


immunity to the virus

IgM anti-HBc and IgG anti-HBc: An antibody to viral genome core; the core
DNA particle is not detectable in serum but rather in hepatocytes

HBeAg: One of the viral secretory proteins associated with high titer of
HBV DNA and active liver disease

Anti-HBe: An antibody to HbeAg

HBV DNA: Quantitative measurement of HBV DNA used to assess recovery


from infection, as well as candidacy for antiviral therapy

Development of antibodies and the rise and fall of each marker differ in each clinical state.
Understanding this physiologic response is essential in interpreting these serologic markers.

The period from 6-8 months of infection, when neither HBsAg nor anti-HBs is detectable, is
called the "window period." Anti-HBc IgM may be the only positive marker during this
period.
The rise and fall of these serologic markers also help differentiate each immunologic state of
the disease, as shown in Table 3.
Table 3. Serologic Markers in Each Clinical Phase of Disease Progression[17] (Open Table in a
new window)
Phase I

Marker

Immune
Tolerance

HBsAg
HBeAg
HBeAb
ALT

Immune Clearance

Phase III

Immune
Control

Phase IV

Immune Escape

>6 months
Positive
Negative

>6 months
Positive
Seroconversion may

>6 months
Negative
Positive

>6 months
Negative
Positive

Persistently

occur
Persistently or

Persistently

Persistently or

normal

intermittently

normal

intermittently

elevated
Persistently or

< 2,000

elevated
Persistently or

IU/mL

intermittently

IU/mL

intermittently

Normal or

=20,000 IU/mL
Moderate to severe

Normal or

=20,000 IU/mL
Moderate to severe

hepatitis/cirrhosis

mild

hepatitis/ cirrhosis

HBV DNA =20,000

Liver

Phase II

histology mild
hepatitis

hepatitis

[#ReferenceRange] Indications/Applications
With recent advances in HBV treatment, including new potent antiviral agents and early
detection of hepatocellular carcinoma through effective screening methods, prompt
identification of persons with HBV infection allows the possibility of reducing morbidity and
mortally. The goal of early diagnosis is to delay or potentially reverse the progression of the

disease. Moreover, screening enables early detection of persons at risk and prevention of
ongoing HBV transmission through counseling, vaccination, and life-style modification.[5, 9]
Serologic testing for HBsAg is the primary screening test. Routine testing for chronic HBV
infection is recommended in the following populations:[1, 17, 18, 19]

Persons born in regions of high and intermediate HBV endemicity (HBsAg


prevalence, >2%)

United Statesborn persons who were not vaccinated as infants

Persons whose parents were born in regions with high HBV endemicity
(HBsAg prevalence, >8%)

Intravenous drug abusers

Men who have sex with men

Persons who need immunosuppressive therapy

Persons with elevated ALT and/or aspartate aminotransferase (AST) levels


of unknown etiology

Infants born to HBsAg-positive mothers

HIV-positive persons

Blood and organ donors

Patients on hemodialysis

Pregnant women

Close contacts of persons known to be HBsAg positive (household, needlesharing, sexual partners)

In persons with chronic HBV infection, regular monitoring of disease activity should be
performed, as viral replication and degree of liver injury can vary throughout the course of
disease. These tests include HBeAg, HBV DNA, ALT, and liver histology. Patients who are
not candidates for treatment at the time of presentation (so-called inactive carrier state)
may become candidates for treatment during follow-up if they demonstrate disease activity. It
is recommended to monitor ALT every 3-6 months and periodically measure HBV DNA for
life in these inactive carriers.[5]

HBV genotyping helps differentiate 6 genotypes of the virus (A-G). This differentiation has
epidemiologic and prognostic value. For example, patients who are infected with HBV
genotype C are more likely to develop cirrhosis and hepatocellular carcinoma, while
progression to chronic hepatitis is more common in HBV genotype A than genotype C.[20, 21]
Certain genotypes also helps predict response to therapy. For example, genotype B has been
shown to have better responses to interferon therapy than genotype C, while genotype A has
better responses than genotype D.[13, 22]
Depending on the purpose of the test, not all serologic markers need to be checked at once.
For screening purposes in asymptomatic individuals when acute infection is not suspected,
only HBsAg is adequate for the diagnosis of HBV infection. If HBsAg is positive, further
testing, including HBeAg and Ab status, HBV DNA, HBV genotyping, and ALT, should be
performed to determine disease stage and activity. If HBsAg results are negative, further
testing of anti-HBs is warranted to determine protective immunity and the need for
vaccination. When acute infection is suspected in a symptomatic patient, both HBsAg and
anti-HBc IgM should be checked initially to prevent false-negative results for HBsAg during
the window period.[23]
Considerations

When HBsAg is checked for screening purposes in a high-risk population, serologic tests to
evaluate for co-existing hepatitis C, hepatitis D, and HIV should also be performed, as coinfection with other viruses can affect the course of HBV infection, as well as efficacy of
antiviral strategies.
For treatment and monitoring, additional molecular tests, such as HBV quantification tests,
HBV genotyping, and HBV resistance assays, should be administered, as needed. Besides
diagnostic purposes, serologic markers should also be used to identify susceptible persons
and to prevent transmission.
In persons who require screening tests for chronic HBV infection, certain additional tests,
specific management, preventive measures, and monitoring should be considered and
individualized, as shown in Table 4.
Table 4. Population-Specific Testing Considerations for Routine Testing of Chronic HBV
Infection[5] (Open Table in a new window)

Population
Persons born in regions of high

Testing consideration
All persons (including immigrants, refugees,

and intermediate HBV endemicity asylum seekers, and internationally adopted


(HBsAg prevalence, >2%)

children) born in regions with high and


intermediate endemicity of HBV infection
should be tested for HBsAg, regardless of

United Statesborn persons not

vaccination status in their country of origin.


If not vaccinated as infants in the United

vaccinated as infants whose

States, these persons should be tested

parents were born in regions with regardless of maternal HBsAg status.


high HBV endemicity (>8%)
Injection-drug users

Testing for anti-HBc or anti-HBs should be


done to identify susceptible persons.

First vaccine dose should be given at the


same visit as testing for HBsAg. Susceptible
persons should complete a 3-dose hepatitis B
vaccine series to prevent infection from
ongoing exposure.

Men who have sex with men

Testing for anti-HBc or anti-HBs should be


done to identify susceptible persons.

First vaccine dose should be given at the


same visit as testing for HBsAg. Susceptible
persons should complete a 3-dose hepatitis B
vaccine series to prevent infection from
ongoing exposure.

Persons who need

Serologic testing should be used to test for all

immunosuppressive therapy,

markers of HBV infection (HBsAg, anti-HBc,

including chemotherapy,

and anti-HBs).

immunosuppression related to
organ transplantation, and
immunosuppression for
rheumatologic or gastrointestinal Because of elevated risk of fulminant
disorders

hepatitis in chronically infected persons, once


suppressive therapy is initiated and risk for
reactivation in persons with resolved
infection, persons who are HBsAg-positive
should be treated, and persons who are antiHBc positive should be monitored closely for
signs of liver disease.

Persons with elevated ALT/AST

Testing for HBsAg should be performed along

levels of unknown etiology

with laboratory testing and imaging studies in

the context of medical evaluation.


Donors of blood, plasma, organs, To prevent transmission to recipients, HBsAg,
tissues, or semen
Hemodialysis patients

anti-HBc, and HBV-DNA testing are required.


Serologic testing should be used to test for all
markers of HBV infection (HBsAg, anti-HBc,
and anti-HBs) upon admission.

To prevent transmission in dialysis settings,


hemodialysis patients should be vaccinated
against hepatitis B and revaccinated when
serum anti-HBs titer falls below 10 mIU/mL.

HBsAg-positive hemodialysis patients should


be cohorted.

Vaccine nonresponders should be tested


monthly for HBsAg.

All pregnant women

Women should be tested for HBsAg during


each pregnancy, preferably in the first
trimester.

If an HBsAg test result is not available or if the


mother was at risk for infection during
pregnancy, testing should be performed at
the time of admission for delivery.

To prevent perinatal transmission, infants of


HBsAg-positive mothers and unknown-HBsAgstatus mothers should receive vaccination
and postexposure immunoprophylaxis in
accordance with recommendations within 12
hours of delivery.

Infants born to HBsAg-positive

Testing for HBsAg and anti-HBs should be

mothers

performed 1-2 months after completion of at


least 3 doses of a licensed hepatitis B vaccine
series to assess the effectiveness of
postexposure immunoprophylaxis.

Testing should not be performed before age 9


months or within 1 month of the most recent
vaccine dose.

Household, needle-sharing, or sex Testing for anti-HBc or anti-HBs should be


contacts of persons known to be

done to identify susceptible persons.

HBsAg positive

First vaccine dose should be given at the


same visit as testing for HBsAg. Susceptible
persons should complete a 3-dose hepatitis B
vaccine series to prevent infection from
ongoing exposure.

Persons who are the sources of

Test source person for HBsAg and provide

blood or body fluids for exposures exposed person with postexposure


that might require postexposure

prophylaxis if needed.

prophylaxis (eg, needle-stick,


sexual assault)
Health-care and public safety workers with
reasonably anticipated occupational
exposures to blood or infectious body fluids
should be vaccinated against hepatitis B.

HIV-positive persons

Testing for anti-HBc or anti-HBs should be


done to identify susceptible persons.

Susceptible persons should be vaccinated

against hepatitis B to prevent transmission


from ongoing exposure.

HIV infection can accelerate progression of


HBV-related liver disease.

Antiretroviral medications used to treat HIV


infection also have anti-HBV activity. Medical
regimens for HIV management can be tailored
according to patient HBV status.

False-positive HBsAg results can occur during the postvaccination period. Transient
antigenemia of up to 4 weeks has been reported. Interpreting the result during this period
should be made with caution. If possible, testing for HBsAg within 4 weeks after HBV
vaccination should be avoided.[1, 24, 25]
The results of hepatitis B serologic tests may pose psychological, medical, and social impact
to persons infected, as well as their close contacts. Thus, the tests should always be conducted
within a context of appropriate counseling and informed consent.