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 manufacturer’s 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.
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
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.  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.  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, 6]
Individuals with positive HbeAg results have been shown to have higher rates of viral transmission; [7, 8] therefore, the antigen is used as a marker of viral replication and infectivity.  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. 
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. 
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.  However, at the time of seroconversion, the HBV DNA level can vary.  Detection of HBe antibody can also be used to monitor response to treatment and possible remission of the disease. 
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. [11, 12]
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.  The HBV DNA cutoff to define virological response has not been determined and varies among types of antiviral therapy. 
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.
|HBsAg||Anti-HBs||Anti-HBc IgM||Anti-HBc IgG||HBeAg||Anti-HBe||HBV DNA|
|Susceptible to infection||-||-||-||-||-||-||-|
|Immune due to natural infection||-||+||-||+||-||-/+||-*|
|Immune due to vaccination||-||+||-||-||-||-||-|
(-) is undetectable; (+) is detectable; (-/+) 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. 
HBsAg and anti-HBs negativity despite anti-HBc positivity should be interpreted with caution, as there are multiple possibilities with these results.  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. 
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 2-8°C 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 -20°C or lower if testing cannot be done within 48 hours. 
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.
Testing for hepatitis B serologic markers is typically performed as a component of liver function testing.
Several serum HBV DNA assays are available commercially. HBV DNA assays are not currently standardized among laboratories. 
The detection limits of the different methods are as follows: 
Branched DNA: 0.7 X 10 6 copies/mL
Hybrid capture: 3 X 10 6 copies/mL
PCR: 10 2 -10 3 copies/mL
Liquid hybridization: 4 x 10 4 copies/mL
Hepatitis B virus (HBV) is a DNA virus in the Hepadnaviridae family. It has a lipoprotein-coated 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. [16, 17, 18]
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, 19, 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 2.
|Marker||Phase I (Immune Tolerance)||Phase II (Immune Clearance)||Phase III (Immune Control)||Phase IV (Immune Escape)|
|HBsAg||>6 mo||>6 mo||>6 mo||>6 mo|
|HBeAb||Negative||Seroconversion may occur||Positive||Positive|
|ALT||Persistently within reference range||Intermittently or persistently elevated||Persistently within the reference range||Intermittently or persistently elevated|
|HBV DNA||=20,000 IU/mL||Intermittently or persistently or =20,000 IU/mL||< 2,000 IU/mL||Intermittently or persistently or =20,000 IU/mL|
|Liver histology findings||Normal or mild hepatitis||Moderate to severe hepatitis/cirrhosis||Normal or mild hepatitis||Moderate to severe hepatitis/cirrhosis|
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, 11]
Persons born in regions of high and intermediate HBV endemicity (HBsAg prevalence, >2%)
United States–born 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
Blood and organ donors
Patients on hemodialysis
Close contacts of persons known to be HBsAg positive (household, needle-sharing, 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. 
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. [23, 24] 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. [15, 25]
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. 
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 co-infection 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. Population-specific testing considerations for routine testing of chronic HBV infection are discussed below. 
Population-specific testing considerations for routine testing of chronic HBV infection
All persons born in areas of high and intermediate HBV endemicity (>2% HBsAg prevalence should undergo testing for HBsAg, regardless of whether they were vaccinated in their country of origin.
Persons born in the United States who went unvaccinated as infants and whose parents were born in areas of high HBV endemicity (>8% HBsAg prevalence) should undergo testing, regardless of maternal HBsAg status.
All persons who use injection drugs should undergo testing for anti-HBc or anti-HBs to identify susceptibility. In this population, the first dose of vaccine should be administered during the visit for HBsAg testing. A 3-dose hepatitis B vaccine series should be completed in all patients deemed to be susceptible in order to prevent infection due to ongoing exposure
Men who have sex with other men should also undergo anti-HBc or anti-HBs testing to determine susceptibility. In this population, the first dose of vaccine should be administered during the visit for HBsAg testing. A 3-dose hepatitis B vaccine series should be completed in all patients deemed to be susceptible in order to prevent infection due to ongoing exposure.
Persons who require immunosuppressive therapy (chemotherapy, immunosuppression for organ transplantation or gastrointestinal or rheumatic disorders) should undergo serologic testing for all markers of HBV infection (HBsAg, anti-HBc, and anti-HBs). Once immunosuppressive therapy is initiated, the risk of fulminant hepatitis in chronically infected persons and the risk for reactivation in persons with resolved infection increase, so HBsAg positivity in this population should prompt treatment, and anti-HBc positivity should prompt close monitoring for signs of liver disease.
Patients with elevated ALT/AST levels of unexplained etiology should undergo testing for HBsAg, as well as other imaging studies and laboratory testing in the context of medical evaluation.
HBsAg, anti-HBc, and HBV-DNA testing is required in donors of blood, organs, plasma, semen, or tissues to prevent transmission to recipients.
Patients undergoing hemodialysis should also undergo testing for all markers of HBV infection (HBsAg, anti-HBc, and anti-HBs) upon admission. These patients should also receive HBV vaccination and be revaccinated when serum anti-HBs levels drop below 10 mIU/mL to prevent transmission during dialysis. Such patients who are positive for HBsAG should be cohorted. Those who do not respond to vaccination should undergo monthly testing for HBsAg.
All pregnant women should undergo HBsAg testing during each pregnancy, preferably during the first trimester. If HBsAg test results are unavailable or if the mother was at risk for infection during pregnancy, she should undergo testing upon admission for delivery. Infants of mothers positive for HBsAg and of mothers with unknown HBsAg status should receive vaccination and postexposure immunoprophylaxis within 12 hours of delivery in accordance with recommendations to prevent perinatal transmission.
Infants born to mothers positive for HBsAg should undergo testing for HBsAg and anti-HBs 1-2 months after completion of at least 3 doses of a licensed hepatitis B vaccine series to evaluate the efficacy of postexposure immunoprophylaxis. Testing should occur after age 9 months or at least 1 month following the most recent vaccine administration.
Close contacts of persons with known HBsAg positivity (household contacts, persons who share needles, sex partners) should undergo testing for anti-HBc or anti-HBs to determine susceptibility. In this population, the first dose of vaccine should be administered during the visit for HBsAg testing. A 3-dose hepatitis B vaccine series should be completed in all patients deemed to be susceptible in order to prevent infection due to ongoing exposure.
In scenarios that might necessitate postexposure prophylaxis following transfer of blood or body fluids (eg, sexual assault, needle-stick), the source person of the blood or body fluid should be tested for HBsAg, and the exposed person should be provided with postexposure prophylaxis, if necessary. Health-care and public safety workers in whom occupational exposure to blood or infectious body fluids is reasonably anticipated should receive HBV vaccination.
Among persons with HIV infection, anti-HBc or anti-HBs testing should be performed to determine susceptibility. Those deemed to be susceptible should receive HBV vaccination to prevent transmission from ongoing exposure. Note that the progression of HBV-related liver disease can be accelerated by HIV infection. Antiretroviral medications used to manage HIV infection also have activity against HBV. Medical regimens for HIV management can be individualized based on 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, 27, 28]
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.