HEPATITIS B TESTING
PATHOLOGY OF HEPATITIS B
There are several viruses that can cause hepatitis. Each virus is known by a letter in the alphabet, such as ‘Hepatitis B’. All of the viruses cause similar problems in the body, but they are spread in different ways.
Hepatitis B can be transferred by blood, sex, or Mother to Baby transfer.
The Hepatitis B virus is present in the blood of an infected person. People can carry the virus in their blood for a long time and not have any symptoms. If infected blood enters another person’s bloodstream, that person may become infected. Infected blood can be passed from person to person by:
- Sharing needles, syringes and other injecting equipment.
- Sharing razor blades and toothbrushes.
- Piercing the skin with equipment (for example tattooing equipment, body piercing equipment and syringe needles), which has not been properly cleaned or sterilised.
- Infected blood coming into contact with the open cuts of an uninfected person.
The virus can be spread if people have sexual intercourse without a condom, especially if there is blood present.
- Mother to baby
Mothers who have long-term Hepatitis B sometimes pass the virus to their children. Most infection occurs during or shortly after birth, so if the newborn baby is given Hepatitis B immune globulin and quickly immunised, he or she can be protected from the disease.
HEPATITIS B LABORATORY INVESTIGATIONS
Specimen Choice, Collection and Transport
The specimen of choice for the diagnosis of HBV infection is blood. Serological tests for viral antigens and antibodies are typically used for diagnostic screening and can be performed on either serum or plasma. Both HBV antigens and antibody are stable at room temperature for days, at 4°C for months, and frozen at -20°C to -70°C for many years. Because modern testing involves automated enzyme immunoassays that depend on colourimetic or chemiluminescence signal measurement, care should be taken to avoid hemolysis of the sample because it may interfere with the ability of the assay to accurately detect these markers.
A number of nucleic acid-based tests, which have been the subject of recent reviews, are available to directly detect HBV-DNA in serum or plasma. Care must be taken to avoid the degradation of the viral nucleic acid in the specimen, which can result in falsely low or no measurable viral load. Serum should therefore be removed from clotted blood within 4 h of collection and stored at -20°C to -70°C , and can be subjected to up to eight short-term freeze-thaw cycles without significant loss of detectable HBV-DNA . Alternatively, the presence of EDTA in plasma is known to stabilize viral nucleic acids. EDTA blood can be stored for up to five days at 4°C without affecting the viral load . Polymerase chain reaction-based tests can use either serum or plasma, while hybridization-based assays recommend the use of serum.
The tests, called assays, for detection of hepatitis B virus infection involve serum or blood tests that detect either viral antigens (proteins produced by the virus) or antibodies produced by the host. Interpretation of these assays is complex.
The hepatitis B surface antigen (HBsAg) is most frequently used to screen for the presence of this infection. It is the first detectable viral antigen to appear during infection. However, early in an infection, this antigen may not be present and it may be undetectable later in the infection as it is being cleared by the host. The infectious virion contains an inner “core particle” enclosing viral genome. The icosahedral core particle is made of 180 or 240 copies of core protein, alternatively known as hepatitis B core antigen, or HBcAg. During this ‘window’ in which the host remains infected but is successfully clearing the virus, IgM antibodies specific to the hepatitis B core antigen (anti-HBc IgM) may be the only serological evidence of disease. Therefore, most hepatitis B diagnostic panels contain HBsAg and total anti-HBc (both IgM and IgG).
Shortly after the appearance of the HBsAg, another antigen called hepatitis B e antigen (HBeAg) will appear. Traditionally, the presence of HBeAg in a host’s serum is associated with much higher rates of viral replication and enhanced infectivity; however, variants of the hepatitis B virus do not produce the ‘e’ antigen, so this rule does not always hold true. During the natural course of an infection, the HBeAg may be cleared, and antibodies to the ‘e’ antigen (anti-HBe) will arise immediately afterwards. This conversion is usually associated with a dramatic decline in viral replication.
Ground glass hepatocytes as seen in a chronic hepatitis B liverbiopsy. H&E stain.
If the host is able to clear the infection, eventually the HBsAg will become undetectable and will be followed by IgG antibodies to the hepatitis B surface antigen and core antigen (anti-HBs and anti HBc IgG). The time between the removal of the HBsAg and the appearance of anti-HBs is called the window period. A person negative for HBsAg but positive for anti-HBs either has cleared an infection or has been vaccinated previously.
Individuals who remain HBsAg positive for at least six months are considered to be hepatitis B carriers. Carriers of the virus may have chronic hepatitis B, which would be reflected by elevated serum alanine aminotransferase (ALT) levels and inflammation of the liver, if they are in the immune clearance phase of chronic infection. Carriers who have seroconverted to HBeAg negative status, in particular those who acquired the infection as adults, have very little viral multiplication and hence may be at little risk of long-term complications or of transmitting infection to others.
PCR tests have been developed to detect and measure the amount of HBV DNA, called the viral load, in clinical specimens. These tests are used to assess a person’s infection status and to monitor treatment. Individuals with high viral loads, characteristically have ground glass hepatocytes on biopsy.