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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
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
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
+
+
+
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.
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
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
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
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
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 whose parents were born in regions with high HBV endemicity
(HBsAg prevalence, >8%)
HIV-positive persons
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,
immunosuppressive therapy,
including chemotherapy,
and anti-HBs).
immunosuppression related to
organ transplantation, and
immunosuppression for
rheumatologic or gastrointestinal Because of elevated risk of fulminant
disorders
mothers
HBsAg positive
prophylaxis if needed.
HIV-positive persons
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.