Hepatitis B virus serology Hepatitis B is a viral infection affecting the liver. The disease goes through different phases, which can be diagnosed by serology. Letâs start with an overview of the phases. In adults, hepatitis B virus infection begins with an acute stage. More than 95% of affected adults will subsequently recover from infection and less than 5% of cases progress to a chronic stage. Chronic hepatitis B virus infection is defined as persistent infection for at least 6 months. The term chronic infection, however, is associated with several phases. Weâll give you an initial quick overview: Often the first phase of chronic infection is the immune-active phase. In this phase, the patient has a high viral load and there is moderate to severe inflammation in the liver. In a later phase of the infection, the host may develop a partial immune response to the virus leading to low replication and remission of liver disease. However, the infection is not fully resolved, but remains quiescent for a varying period. This phase is termed as the inactive phase and patients are referred to as inactive carriers. There is another chronic phase that we would like to point out, the immune-tolerant phase. This phase is observed when infection occurs before birth through maternal transmission or early in childhood. Early exposure to hepatitis B virus leads to T cell tolerance towards the virus, resulting in minimal inflammation in the liver despite a very high viral load. The immune-tolerant phase lasts an average of 30 years and progresses into a phase with active liver disease, which, in an even later stage, can resolve to an inactive carrier state. One consequence of the long duration of immune-tolerance is that it leads to a high frequency of maternal-infant transmission in endemic countries. This list of chronic forms is not exhaustive and there are further special constellations. However, if you keep these three phases in mind, immune-active, inactive, and immune-tolerant chronic hepatitis B, that's a solid basis. Serology is also able to detect a past resolved infection. Furthermore, it can be used to detect whether a patient has been vaccinated against hepatitis B. Finally, negative hepatitis B serology indicates that there is no evidence of infection. However, a negative serology does not necessarily mean that there is no infection. Because the incubation time may be as long as 6 months, serology should be repeated if there is a well-founded suspicion of infection. An algorithm will be presented here of how these phases can be differentiated. To begin with, we will present the infectious cycle of hepatitis B virus and the antibodies and antigens involved. Hepatitis B virus consists of an envelope and a capsid, which contains the viral DNA. Three antigens are of relevance for hepatitis B serology: First: The HBs antigen is part of the envelope. âHBsâ stands for hepatitis B surface. A positive HBs antigen indicates active infection, as the antigen is present on the viral surface. Second: The HBc antigen is part of the capsid. The âcâ stands for core because the antigen is located in the core of the virus. The HBc antigen cannot be determined in serum, as it is hidden within the virus. However, infected hepatocytes express HBc on their surface, which allows the human body to develop antibodies to the HBc antigen. Anti-HBc antibodies can be detected in serum. The third antigen is the HBe antigen. The âeâ stands for envelope. HBe antigen is produced during synthesis of the HBc antigen and is then secreted from the cell. In other words, if there is viral replication, HBe antigen is secreted, which makes it a suitable marker for viral replication. The function of the secreted HBe antigen may lie in its immunosuppressive effect. It produces tolerance in T cells and thereby contributes to establishing infection. Determination of viral DNA in serum via polymerase chain reaction is a direct measurement of the viral load. It can be used in diagnostics and to determine therapeutic indications. For the purpose of this episode, we will mainly focus on the antigens and antibodies, but measurement of viral DNA has become a central element of hepatitis B analysis in recent years. The image shown provides an overview of the replication cycle of hepatitis B virus. The virus enters the hepatocyte and viral DNA is transported into the nucleus, where it is transcribed into viral RNA and replicated. RNA is transported back to the cytoplasm, where viral proteins are produced from RNA. In addition, viral DNA is produced by reverse transcriptase. Virus assembly then occurs on the cytoplasmic membrane and newly formed viruses exit the cell. Apart from the assembled virus, two further products also exit the hepatocyte: the HBe antigen and the HBs antigen. The HBe antigen is produced during protein synthesis and stems from the core protein HBc. It is secreted from the hepatocyte and as previously mentioned, one possible advantage for the virus may lie in an immunosuppressive effect. The HBs antigen is part of the virus envelope, but it also exits the cell in the form of filaments, which are basically empty virus envelopes. They are formed when the virus exits the cell and no viral DNA enters the virus envelope. Interestingly, hepatitis B virus does not cause cell destruction. Lysis of hepatocytes occurs as a result of the immune response targeted towards infected cells, which is carried out by cytotoxic T cells. In addition to the cellular defense by T cells, the body forms antibodies against the virus. The most important antibody is the anti-HBs antibody. It is able to directly bind to the viral surface and thereby neutralizes the virus. The second antibody is the anti-HBe antibody. As it does not directly bind to the virus but only to the secreted HBe antigen, it is unable to completely clear the infection. Letâs have a closer look at the interaction between antigens and antibodies over time in the next slide. In the following section, we will discuss the course of acute hepatitis B infection, which leads to subsequent recovery. After infection with hepatitis B virus, HBs antigen and HBe antigen are initially detectable in serum. During the course of the immune response, antibodies against the three viral components are formed. These antibodies can be differentiated into: anti-HBc antibody, anti-HBe antibody, and anti-HBs antibody. They have various functions and are positive in serum at different time points. We have split the antigens and antibodies into two diagrams for better clarity. The first antibody that typically increases in serum is anti-HBc. It is therefore commonly used to determine the presence of hepatitis B infection. As for all antibodies, the anti-HBc antibody can be differentiated into several classes. In hepatitis B serology, anti-HBc IgM and anti-HBc IgG are important. IgM is formed at the beginning of infection and is replaced by IgG during the course of the immune response. Usually, a serological test is used that measures the overall anti-HBc, regardless of the subtype. If anti-HBc antibodies are detectable, the subtype IgM can additionally be measured and if positive, interpreted as acute hepatitis B infection. Compared to anti-HBc, the other two antibodies, anti-HBe and anti-HBs, only appear during the later stages of infection. As a rule: anti-HBe antibodies are present in serum before anti-HBs antibodies. With the appearance of anti-HBe antibodies, HBe antigen disappears. Because anti-HBe antibodies only bind to free HBe antigen, they do not provide protection against the virus. Only the formation of anti-HBs antibodies is decisive, as they are able to eliminate the virus. This can also be seen in the slide: With the occurrence of anti-HBs antibodies, HBs antigen becomes undetectable, which indicates recovery from hepatitis B infection. In serology, the term seroconversion describes the disappearance of an antigen during the course of infection. The antigen is then replaced by the corresponding antibody, which is subsequently detectable in serum. So how does this phenomenon arise? Antigen is present in serum at the beginning of infection and is detectable. During the course of infection, the body forms antibodies. These antibodies bind to the antigen leading to neutralization. At this stage, there is no free antigen present, but also no antibodies are detectable in serum as they are bound to the antigen. Subsequently, an increasing amount of free antibody becomes available in serum, which are then detectable. As a consequence, infection may be present in the transition phase of seroconversion when both antigen and antibody are transiently undetectable in the patient and serological tests are normal. This is called a window period, which we will get back to later on. The take-home points for this slide are: anti-HBc is the first antibody to increase and is used in conjunction with HBs antigen to confirm the occurrence of infection. Anti-HBe antibodies are the next antibodies to appear and are followed by anti-HBs antibodies, which are able to resolve the infection. In this slide, we would like to take a closer look at the serological course of chronic hepatitis B infection. Chronic infection develops if the body does not form anti-HBs antibodies. The mechanism preventing the formation of individual antibodies in certain patients is not fully understood. There are several factors that may be involved. The onset of an efficient immune response is dependant on the immune status, age of infection, and comorbidities such as infection with other hepatotropic viruses. For starters, letâs discuss the following two phases of chronic infection: immune-active chronic hepatitis B and immune-tolerant chronic hepatitis B. In both cases, anti-HBs and anti-HBe antibodies are not formed. Thus, HBs and HBe antigens remain positive in the patientâs serum. In other words, there is no seroconversion. The only antibody that is increased is the anti-HBc antibody. So, what is the difference between immune-active chronic hepatitis B and immune-tolerant chronic hepatitis B. The presence of liver cell inflammation in this constellation indicates an active phase of chronic infection. On the contrary, minimal inflammation in the liver with a very high viral load indicates the immune-tolerant phase. You may be wondering how liver inflammation is measured. A reliable marker of hepatocellular injury is the enzyme alanine transaminase, in short ALT, which can be detected in blood. Besides these two phases, there is a third chronic form, which we will now have a look at on the right side of the slide. This phase is the inactive phase and, like the other two phases, there is an insufficient immune response. That is, protective anti-HBs antibodies are absent. The patient remains HBs antigen positive and although the disease is inactive, the patient is, in principle, infectious. Consistent with the other two chronic forms, anti-HBc antibodies are also detectable in the inactive carrier state. However, in contrast to the other two forms of chronic hepatitis B infection, anti-HBe antibodies are formed. Accordingly, HBe antigen becomes undetectable. This stage is associated with low levels of viremia and low-grade liver inflammation and is therefore referred to as the inactive carrier state. From a serological perspective, the hallmark of the inactive carrier state is the conversion from HBe antigen positive to HBe antigen negative. Since only anti-HBe antibodies are produced but no anti-HBs antibodies, this is also referred to as partial seroconversion. Conversion to the inactive phase may occur spontaneously or be treatment-induced. Overall, the inactive phase is associated with a more favorable prognosis compared to patients with positive HBe antigen. However, the long-term outcome varies between individuals: Exacerbation with signs of increased liver inflammation is possible, which is sometimes referred to as HBe antigen negative immune-reactivation phase. But even the recurrence of HBe antigen and the reversion back to the immune-active phase of chronic hepatitis B is possible. This may occur spontaneously or be triggered by immunosuppressive therapy. On the other hand, recovery from infection can occur by the production of anti-HBs antibodies, which clear the infection. In summary, chronic forms of hepatitis B infection are characterized by the lack of anti-HBs antibodies. However, they can be differentiated according to the formation of anti-HBe antibodies, the amount of viral replication, and the presence or absence of liver inflammation. The natural course of chronic hepatitis B virus infection is quite variable and is determined by an interplay between the host immune response and virus replication. $$ (Overview of diagnostic markers) In the following section, we will provide a summary of the various hepatitis B antigens and antibodies. If positive, HBs antigen serves as evidence of infection. It demonstrates that hepatitis B virus is present in the body and that the individual is infectious. If HBs antigen is positive for more than 6 months, it is an indication of viral persistence and implies chronic infection. Recovery from infection is possible if the patient develops antibodies against HBs antigen. This is based on the fact that HBs antigen is located on the viral surface and generally the respective antibodies can target the virus. The appearance of anti-HBs antibodies results in the disappearance of the HBs antigen and confers long-term immunity. There is a period of time between the transition from antigen to antibodies, in which both the HBs antigen and anti-HBs antibodies are below the limit of detection. This time period is referred to as a window period. As HBs antigen is negative during the window period, it is generally unsuitable as an investigative marker. This function is exerted by anti-HBc, which is, in some cases, the sole indicator of infection. Therefore, anti-HBc antibodies are a decisive marker in determining hepatitis B infection. The subgroup anti-HBc IgM is only detectable during acute infection and can be used to determine recent infection. HBe antigen and anti-HBe antibodies are important for the accurate assessment of chronic forms of infection. HBe antigen indicates the level of virus replication and is thereby a prognostic marker. It is positive in the immune-tolerant phase and in the immune-active phase and, in addition to the rate of replication, is also a marker of infectivity. Anti-HBe antibodies demonstrate the transition to infection with low-level viral replication, the inactive phase. This shift is also referred to as partial seroconversion. A note on vaccinations: Because anti-HBs antibodies mediate protection, vaccinations are performed using the HBs antigen. The antigen is recombinantly produced and used as a vaccine. So how is it possible to identify if a patient has been vaccinated? In short: Anti-HBc antibody is an indicator of infection and vaccination is performed with the HBs antigen. Thus, if a patient has anti-HBs antibodies but no anti-HBc antibodies, the individual has been vaccinated. How is hepatitis B screening performed in practice? Diagnostics can be performed in stages to avoid directly detecting all antigens and antibodies. For initial screening, determination of HBs antigen and anti-HBc is usually sufficient. Further diagnostic testing can be planned based on the results. In an environment where repeated blood collection is not possible, such as in the physicianâs office, the following may be requested: HBs antigen, anti-HBs antibody, and anti-HBc antibody. If anti-HBc antibodies are detectable, anti-HBc IgM is additionally measured. This ensures that the majority of cases are covered and that the patient doesnât have to come back to the office for subsequent blood collection. The diagnostic test request comprises the three most important parameters: HBs antigen, anti-HBs antibody, and anti-HBc antibody. These three parameters can be used to identify active infection, previous infection, and status after vaccination. In addition, anti-HBc IgM antibody can be used to confirm acute infection. The chronic forms of hepatitis B infection can be differentiated in subsequent testing for HBe antigen and anti-HBe antibody, and the presence or absence of liver cell damage. Here is an overview of the different phases and their corresponding markers, which have been discussed in this episode. It can also be found in the library, where you can go through the table in your own time. So just have a go. In the following exercise, assign the laboratory findings to the possible interpretations by dragging them to their respective fields. Please note that we have limited the quiz to the most common options. Good luck!