| Abstract|| |
Hepatitis B is a well-recognized global public health problem. It is estimated that nearly 2 billion people around the world have serologic evidence of past or present hepatitis B virus (HBV) infection, while 350 million people are chronically infected. This worldwide burden of hepatitis B mandates accurate and timely diagnosis of patients infected with HBV and the use of treatment strategies derived from evidence-based guidelines. HBV is a DNA virus that produces a series of viral protein products. Serologic and nucleic acid testing are critical to disease prevention and treatment objectives. Information from such testing helps determine patients' infectivity and immune status, appropriate monitoring strategies, and the efficacy of treatment, as well as providing data that contributes to a better understanding of the natural history and epidemiology of the disease. This article reviews the clinical use of serologic and nucleic acid tests as markers of disease activity.
Keywords: Diagnosis, HBV DNA, healthcare worker, hepatitis B, serology
|How to cite this article:|
Shah SM, Singh SP. Hepatitis B virus serology: Use and interpretation. Hep B Annual 2007;4:39-54
| Introduction|| |
Hepatitis B virus is a hepadnavirus with a 3200-base-pair genome that consists of partially double-stranded DNA and a lipoprotein outer envelope.  The genomic organization of the hepatitis B virus (HBV) is complex. It has 4 overlapping open reading frames with 4 major genes, designated as pre-S/S, C, P, and X. The pre-S genes (S1 and S2) code for the hepatocyte receptor-binding site, whereas the S (surface) gene codes for the hepatitis B surface antigen (HBsAg). The C (core) gene codes for hepatitis B core antigen (HBcAg), and the hepatitis B e antigen (HBeAg), whereas the P (polymerase) gene encodes the HBV DNA polymerase. The X gene encodes a protein that transactivates transcriptional promoters. Currently, routine serological diagnosis relies on the detection of three pairs of antigens and antibodies: HBsAg and anti-HBs, HBeAg and anti-HBe, and anti-HBc. HBcAg does not circulate freely in the serum.  In the 1980s, molecular hybridization assays were introduced as commercially available kits. These have now been supplanted by PCR methodology with much higher sensitivities. These assays can detect HBV infection in the absence of serological markers of infection, such as HBsAg.  Practically, however, current immunoassays for HBV antigens are so sensitive that reliance on PCR amplification of HBV DNA to establish a diagnosis of acute or chronic hepatitis B is almost never required in routine clinical practice. Additional viral markers that circulate in serum during HBV infection, such as HBV DNA polymerase, pre-S1 and pre-S2 viral envelope antigens and their respective antibodies can be detected by experimental techniques that have no routine clinical applications.
HBsAg and anti-HBs
HBsAg is the serologic hallmark of HBV infection. It is detected in the serum by radioimmunoassays (RIA) or enzyme immunoassays (EIA). It appears in serum 1-10 weeks after an acute exposure to HBV and approximately 2-6 weeks before the onset of symptoms or elevation of aminotransferases.  In patients who subsequently recover, HBsAg usually becomes undetectable after 4-6 months. In acute hepatitis B, very rarely HBsAg may be undetectable at the time of presentation, either because levels of HBsAg never reach or have already declined below the detectable threshold of the assay.  Persistence of HBsAg for more than 6 months implies chronic infection. The disappearance of HBsAg is followed by the appearance of anti-HBs. The appearance of anti-HBs marks the recovery from hepatitis B. In most patients, anti-HBs persists for life, thus conferring long-term immunity. Anti-HBs is the only protective antibody induced by most of the currently available vaccines that consist of recombinant HBsAg only. 
Although anti-HBs is produced early during the course of acute HBV infection, it is not routinely detected since HBsAg is present in excess. Thus, anti-HBs is detected only when the HBsAg levels decline in the course of acute HBV infection. Because of the sensitivity of the current assays, the previously described "window period" between the disappearance of HBsAg and the appearance of anti-HBs is rarely, if ever, observed nowadays. 
Coexistence of HBsAg and anti-HBs has been reported in about 10-25% of HBsAg positive individuals. This occurs more commonly in those with chronic hepatitis B.  In most instances, the antibodies are low level, non-neutralizing and heterotypic, i.e., directed against a different subtype of HBsAg than that present in the infected patient. The mechanism is not clear, but it may be due to the antibody formed against minor variants of the HBsAg protein. The presence of these heterotypic antibodies is not associated with any risk factors or changes in clinical course, and may occur in the setting of active liver disease and viral replication. 
HBcAg and anti-HBc
HBcAg is an intracellular antigen that is expressed in infected hepatocytes and is not detectable in serum. Its antibody, anti-HBc, can be detected throughout the course of HBV infection. During acute HBV infection, anti-HBc is predominantly immunoglobulin M (IgM) class. IgM anti-HBc is the first antibody to be detected. It usually appears within 1 month after the appearance of HBsAg, approximately 1-2 weeks before the elevation of aminotransferases.  It is the sole marker of HBV infection during the window period, that is, the time gap between the disappearance of HBsAg and the appearance of anti-HBs.  During convalescence, the titer of IgM anti-HBc declines while the titer of IgG anti-HBc rises. Thus, the detection of IgM anti-HBc is usually taken as an indication of acute HBV infection. However, in 20% of patients, IgM anti-HBc may remain detectable up to 2 years after the acute infection. Also, IgM anti-HBc can be detected in patients with chronic HBV infection during exacerbations. This may lead to misdiagnosis of acute hepatitis B infection in patients who are not previously known to have chronic HBV infection and to overestimation of the rate of progression to chronicity. 
IgG anti-HBc persists along with anti-HBs in patients who recover from acute hepatitis B and in association with HBsAg in those who progress to chronic hepatitis B. The isolated presence of IgG anti-HBc in the absence of HBsAg and anti-HBs has been reported in 0.4 to 1.7% of blood donors in low prevalence areas , and in 22% of the blood donor population in endemic areas.  Isolated detection of anti-HBc can occur in 4 settings:
- During the now rare window period of acute HBV infection, when the antibody is of IgM class;
- After many years of chronic infection when the HBsAg levels have reduced below the cut-off level of detection;
- After resolved HBV infection in the remote past as the anti-HBs levels fall below the limits of detection;
- About 50-70% of asymptomatic individuals with isolated anti-HBc have false positive test results. The evaluation of individuals with isolated anti-HBc should include repeat testing for anti-HBc (preferably with RIA rather than EIA), and for HBsAg and anti-HBs. Those who remain positive should be tested for IgM anti-HBc to rule out recent HBV infection. Individuals with evidence of chronic liver disease should be tested for HBV DNA to exclude low-level chronic HBV infection. 
HBeAg and anti-HBe
HBeAg is generally considered to a marker of HBV replication and infectivity, and is usually associated with the detection of HBV DNA.  Transmission rates of HBV infection are much higher when the source is HBeAg positive. ,
During acute infection, HBeAg appears shortly after the appearance of HBsAg. In patients who recover, HBeAg to anti-HBe seroconversion precedes HBsAg to anti-HBs seroconversion.  Anti-HBe may persist for many years after resolution of acute hepatitis B. In patients with chronic infection, HBeAg may persist for years to decades. During the HBeAg positive phase, most patients have detectable HBV DNA and active liver disease. In patients with perinatally acquired infection, there may be an immune tolerant phase with normal aminotransferase levels and minimal inflammation in the liver. , Seroconversion from HBeAg to anti-HBe is usually associated with the disappearance of HBV DNA in serum and remission of liver disease. However, a small proportion of anti-HBe positive patients continue to have active liver disease and detectable HBV DNA in serum. This may be due to low levels of wild-type HBV or the presence of pre-core mutants.
Tests for HBV DNA in serum
Qualitative (PCR-based) and quantitative molecular assays for HBV DNA in serum are commercially available.  Quantification of HBV DNA is performed with either signal or target amplification techniques. The signal amplification techniques such as the branched DNA technique are very specific; however, these assays are too insensitive to detect low levels of HBV DNA. The target amplification techniques such as the PCR-based assays are highly sensitive and are capable of detecting as few as 10 copies per ml of HBV DNA. 
Using signal amplification techniques such as the hybridization assays, HBV DNA can be detected approximately one week after the appearance of HBsAg in patients with acute hepatitis B.  Using PCR-based assays, HBV DNA can be detected much earlier, up to 2-3 weeks before the appearance of HBsAg. Recovery from acute hepatitis B is usually accompanied by the disappearance of HBV DNA. However, it may remain detectable in serum for many years if tested by PCR assays. 
Quantitative tests for HBV DNA in serum are useful to distinguish replicative from non-replicative chronic HBV infection and to monitor a patient's response to antiviral therapy. The threshold that distinguishes the replicative from the non-replicative state has been defined as an HBV DNA level of 100,000 copies per ml, although 1000-10,000 may be more accurate.  Besides, patients with high pre-treatment HBV DNA levels are less likely to respond to interferon therapy. 
Rarely, tests for HBV DNA in serum help to identify HBV as the etiology of liver disease in HBsAg-negative patients. , This is especially important in patients with fulminant hepatitis B, who may have cleared HBsAg by the time they present.  In patients with chronic liver disease, these tests may help identify HBV as the etiology for the liver disease. These patients may have sub detectable levels of HBsAg, or some may have integrated HBV DNA in the liver tissues but do not secrete HBsAg; others may be infected with HBV mutants that downregulate the production of HBsAg or produce aberrant HBsAg that cannot be detected by conventional serological assays. 
The diagnosis of acute hepatitis B is based on the detection of HBsAg and IgM anti-HBc. During the initial phase of infection, markers of HBV replication, including HBeAg and HBV DNA are present. Recovery is accompanied by the disappearance of HBV DNA, HBeAg-to-anti-HBe seroconversion, and subsequent HBsAg-to-anti-HBs seroconversion. Rarely as mentioned earlier the patients may have entered the window period at the time of presentation; IgM anti-HBc is the sole marker of acute HBV infection in these patients. This situation is more common in patients with fulminant hepatitis B, in which virus clearance tends to be more rapid. 
Past HBV infection is diagnosed by the detection of anti-HBs and IgG anti-HBc. Immunity to HBV infection after vaccination is indicated by the presence of anti-HBs only.
The diagnosis of chronic HBV infection is based on the detection of HBsAg. Additional tests for HBV replication, including detection of HBeAg and serum HBV DNA should be performed to determine whether the patient should be considered for antiviral therapy. Although PCR assays are more sensitive, the pathogenic significance of minute amounts of HBV DNA that can be detected only be PCR but not by hybridization or bDNA assays is uncertain. Thus, PCR assays should be reserved for research and not for clinical diagnosis. HBeAg negative patients who have normal aminotransferase levels do not need to be further evaluated, but those with elevated levels should be tested for HBV DNA to determine whether the liver disease is related to persistent (wild type or mutant) replication. Additional tests for hepatitis C and hepatitis D should also be performed to rule out superinfection with other hepatitis viruses.
Interpretation of HBV serologic patterns as a guide to treatment
HBV infection is characterized by several distinctive serologic and immunologic responses. The temporal profiles can serve as useful guides to monitor the course of the disease and to provide a serologic correlation with the diseases' progress. When only a single serum is available, diagnostic accuracy and efficiency are improved by reference to the complete battery of HBV markers. Analysis of various serologic profiles provides some guidance in interpreting the course of the disease and the level of infectivity. The significance and interpretation of each pattern are given below:
Pattern 1: HBV DNA+ve, HBsAg-ve, anti-HBc-ve,
This will be seen in the pre seroconversion window period and in cases of occult infection.
Pattern 2: HBV DNA+ve, HBsAg+ve, anti-HBc-ve,
This suggests early acute infection.
Pattern 3: HBV DNA+ve, HBsAg+ve, anti-HBc+ve,
This will be seen in both acute and chronic HBV infection.
Pattern 4: HBV DNA-ve, HBsAg-ve, anti-HBc+ve, anti-HBs+ve.
This suggests infection in the past with immunity.
Pattern 5: HBV DNA-ve/+ve, HBsAg-ve, anti-HBc+ve, anti-HBs-ve.
This suggests a carrier whose HBsAg levels have fallen below the level of detection in presence of a positive HBV DNA. Rarely, this pattern will be seen in the window period of an acute infection, after the disappearance of HBsAg and before the appearance of anti-HBs. This pattern can also suggest a remote HBV infection when the anti-HBs levels have dropped below detection levels; of course, the HBV DNA test will be positive in this case. About 50-70% of individuals with this pattern have a false positive reaction.
Pattern 6: HBV DNA-ve, HBsAg-ve, anti-HBc-ve, anti-HBs+ve.
This is seen in individuals who have taken and have responded to the vaccine, and are immune to HBV infection.
Pattern 7: HBV DNA-ve, HBsAg-ve, anti-HBc-ve,
This pattern excludes HBV infection.
| HBV Serology in Certain Clinical Situations|| |
In chronic renal failure (CRF)
HBV infections continue to occur in adult hemodialysis units. Occult HBV infection (HBsAg negative but HBV DNA positive) may be a contributing factor in these patients.  Further, occult HBV infection is more frequent in hepatitis C virus (HCV)-infected hemodialysis patients than otherwise normal patients with chronic HCV infection, probably because of impaired immune function in uremic patients and high risk of parenteral exposure to HBV.  The clinical significance of this finding is unknown, but HBV vaccination of hemodialysis patients and staff could be an effective way of limiting the risk of transmission of HBV infection within dialysis units.
HBV-HCV co-infection in end stage renal disease patients is related to a longer time on hemodialysis, longer duration of infection, and history of blood transfusion. Contrary to nonuremic patients, HCV co-infection is not associated with more severe forms of liver disease in ESRD patients. 
A systematic review of the published medical literature on the impact of HBsAg seropositivity on survival of renal transplant (RT) recipients showed that HBsAg was an independent and significant risk factor for death after RT . HBsAg seropositivity was an independent and significant risk factor for graft failure after RT. 
A study reported prevalence of post transplant HBV and HCV infection in 256 Indian patients with CRF and with a history of either renal transplant or hemodialysis. Seven percent had HBV infection alone, 46% were infected with HCV alone, while 37.10% were found to have co-infection of both the viruses. These findings implicate the two viruses as the major cause of post transplant hepatitis in Indian patients with CRF and indicate implementation of stringent screening procedures for these two viral infections. 
In HIV co-infection
HIV and HBV co-infection increases HIV and HBV replication, hepatitis flares, and risk of progression to chronic HBV infection, cirrhosis, and hepatocellular carcinoma. HIV and HBV co-infection decreases frequency of anti-HBe and anti-HBs seroconversion, increases risk of antiretroviral therapy-related hepatotoxicity, and reduces efficacy of HBV therapy.
HBV serology often is atypical in co-infection. Diagnosis of HBV co-infection in HIV infection is made on the basis of HBsAg-positive, anti-HBc-positive, anti-HBs-positive status. Alanine aminotransferase levels in co-infected patients often are not reliable markers of liver inflammation. HBV infection should always be treated if co-infected patients are receiving antiretroviral therapy, since immune reconstitution under antiretroviral therapy poses risk for immune-associated liver damage in these patients. 
HBV viral load and log changes in viral load
The virology labs often use "log" to report viral load. Instead of writing 100,000 copies/mL, labs may report it as 10 5 or 5 log. In mathematical jargon, a "log" equals a number multiplied by 10. If you have a viral load of 10 5 copies/mL, it is actually, 10 x 10 x 10 x 10 x 10 or 100,000. Thus a lab report that indicates HBV DNA levels greater than 100,000 copies/mL may be written HBV DNA > 5 log copies/mL or 10 5 copies/mL. Every log rise or fall is equivalent to a ten-fold increase or decrease. Besides the log value can easily be calculated using the calculator or by logging on to the website: http://www.calculator.com/calcs/calc_sci.html. HBV DNA levels are carefully monitored during treatment. During treatment, changes in viral load are often reported as logarithmic or "log changes." This simply indicates that there is a change in the value of what is being measured by a factor of 10. Thus, if the baseline viral load is 20,000 copies/ml plasma, then a 1 log increase implies a 10 fold increase to 200,000 copies/ml plasma, and a 2 log increase implies an increase in the viral load to 2,000,000 copies/ml plasma, i.e. a 100-fold increase. Similarly if the initial viral load is 20,000 copies/ml plasma, a 1 log decrease would indicate that the viral load has dropped to 2,000 copies/ml, and a 2 log decrease would indicate that the viral load has decreased to 200 copies/ml plasma. A one- or two-log decrease in viral load during therapy means an antiviral is working. On the contrary, a one- or two-log increase means an antiviral has stopped working and that viral resistance has developed.
Conversion of viral load: from one unit to another 
Although WHO has recommended that HBV DNA be expressed in terms of IU/ml, a number of different units are used to express the viral load. To convert from IU/mL to copies/mL, IU/mL value should be multiplied by 5.6. Some other equations which would help in conversion from one unit to another are as follows:
HBV-DNA 1 pg/ml = 2.83 × 10 5 copies/ml = 5.45 log 10 copies/mlHBV-DNA 10 9 genome Eq/ml = 200 million IU/mL
HBV-DNA 20000 IU/mL = 10 5 copies/ml = 0.35 pg/mL
Anti-HBs in Healthcare Workers (HCW)
In routine clinical practice, anti-HBs is rarely estimated, and its presence indicates recovery from HBV infection with seroprotection against HBV infection. Anti-HBs assays are not routinely recommended after hepatitis B vaccination. However, with regards to HCWs, the scenario is different. HCWs need to be tested for anti-HBs levels after a course of vaccination, and should ideally generate anti-HBs levels above 100 at least in the early post-vaccination period. This may decline subsequently to very low even undetectable levels. However even if undetectable, individuals in whom protective anti-HBs levels have been demonstrated once, need not be administered further doses of the vaccine again, on account of persistent immune memory; any antigenic challenge in these individuals would provoke protective anamnestic reaction.  The demonstration of post vaccination anti-HBs levels has an implication with regards to post exposure prophylaxis to be adopted, since if as per World Gastroenterology Organization Guidelines, there is no evidence of adequate anti-HBs levels in past, then the HCW needs to be administered HBIG in addition to administration of hepatitis B vaccination.  Besides, low titers of anti-HBs in HCWs call for further investigations to unravel the cause of the response after another course of hepatitis B vaccination.
HBeAg and HBV DNA in Healthcare Workers [HCW]
Initially, the public health policies to prevent transmission of HBV to patients in different countries were conventionally based on the serum HBeAg status. However, after incidents of transmission by HBeAg negative surgeons, it was felt that a more reliable marker of non-infectivity was required. It is now felt that serum HBV DNA level is a more reliable marker of non-infectivity than HBeAg status alone. In the Netherlands, current public health regulations do not allow exposure-prone procedures (EPPs) to be conducted by HCWs with HBV DNA levels above 10 5 genome equivalents per ml (geq/ml), irrespective of HBeAg status.  This HBV DNA cut-off level of 10 5 geq/ml is based on 10 7 geq/ml, below which vertical transmission is not likely to occur; a safety margin of 2 log is kept to account for natural fluctuations in viral load and variations in the assay used for quantifying HBV DNA. It is believed that the use of this HBV DNA cut-off level minimizes the risk of transmission and allows most highly educated and skilled HBV infected HCWs to continue practice. In the UK and Eire, all HBeAg positive HCW are excluded from performing EPPs, and a HBV DNA cut off level of 10 3 copies/ml is used for HBeAg negative HCWs.  In the UK, the lower cut off level of 10 3 geq/ml is based on a safety margin of 3 log as the viral load is then unlikely to rise above 10 6 copies/ml. In contrast, in the US, HCWs are excluded from performing EPPs based on the presence of HBeAg only; HBV DNA levels are not taken into consideration.  Paradoxically, it is unfortunate that in countries like India, where there is greater likelihood of such transmission mishaps, there are no regulations whatsoever.
However, there is need for caution with regards to HBV DNA levels and transmission. Recent investigations of transmissions of HBV to patients involved determining HBV DNA levels of the HCWs have shown that the transmission rate does not seem to depend on serum HBV DNA level only; the transmission rates vary greatly. A relation between HBV DNA level and transmission rate was not found in the HBeAg-positive HCWs. A possible explanation could be that transmission also depends on whether the concerned doctor performs high-risk procedures. The lowest serum HBV DNA level in a transmitting surgeon was found to be 4.0 x 10 4 geq/ml.  This creates confusion vis-à-vis which HBV DNA level should be used to allow for the conduct of exposure prone procedures by HBV infected HCWs. Public health policy should not be based on the lower levels of HBV DNA levels in these HCWs because the samples were taken at least 3 months after the actual transmission, and fluctuations in HBV DNA levels in HBeAg negative carriers occur over relatively short periods of time.  Therefore, HBV DNA levels might actually have been higher at the time of transmission.
| Conclusions|| |
Serologic assays for HBV are the mainstay diagnostic tools for HBV infection. The advent of molecular biology-based techniques has added a new dimension to the diagnosis and treatment of patients with chronic HBV infection. Serologic and nucleic acid testing are today critical to disease prevention and treatment objectives. Information from such testing not only helps determine patients' infectivity and immune status, appropriate monitoring strategies, and the efficacy of treatment, but also provides data that contributes to a better understanding of the natural history and epidemiology of the disease.
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Sunil M Shah
Consultant Gastroenterologist, Sir H. N. Hospital, Raja Ram Mohan Roy Road, Mumbai 400 004
Source of Support: None, Conflict of Interest: None