3.2 Hepatitis B virus
Hepatitis B virus (HBV) is a DNA virus, a member of the hepadnavirus (Hepadnaviridae) family. The infectious particle, the Dane particle, is 42 nm in diameter and comprises the DNA genome encapsulated in core protein, which is then covered by an envelope of surface proteins. The hepadnaviruses are characterized by the production of a vast excess of the surface proteins, commonly known as hepatitis B surface antigen (HBsAg), as spherical and filamental forms which are released into the bloodstream with the infectious Dane particles. The Dane particles can reach concentrations as high as 108/ml_1010/ml while the HBsAg particles typically reach levels of 1013/ml. The virus is transmitted parenterally, including by intimate contact, and has an incubation period of about 4_8 weeks. The prevalence of HBV varies worldwide from as low as 0.01% to 0.1% in developed countries with good public health to 20%, and even higher, in many developing countries. In the countries with a high prevalence, infection is commonly transmitted vertically from mother to infant at birth and during the early years, and horizontally during childhood through close contact. Parenteral routes commonly include: blood transfusion, notably in countries where blood is not screened; intravenous drug use; nonsterile surgical instruments, including tattooing and acupuncture instruments; and patient to staff transmission in hospital and other health care settings.
Infection may follow one of two courses: either acute infection with the subsequent clearance of the virus and development of immunity, or chronic infection with persistence of viral replication for extended periods, even through the lifetime of the individual. However, chronic infection may spontaneously resolve and the individual may then develop immunity; alternatively a stable chronic infection may reactivate with a resulting further acute episode. Although HBV infection can lead to severe disease (cirrhosis, hepatocellular carcinoma and liver failure; asymptomatic infections are very common) with most individuals infection resolves and immunity develops without any symptoms whatsoever. Figure 3.1 indicates the possible outcomes of infection with HBV.
Following infection the virus migrates to the liver and enters the hepatocytes where the viral DNA integrates with the host cell genome. The viral DNA then uses the cell's machinery to produce new viral DNA and a number of HBV proteins, including an excess of HBsAg, resulting in the sequential appearance in the bloodstream of Dane particles and uninfectious HBsAg particles; and then the other specific antigen and antibody markers of HBV infection are produced. Precisely which markers are present and for how long depends upon whether infection is acute and resolves or progresses to chronicity.
HBeAg is a non-structural protein whose specific function is not clear. It appears soon after HBsAg is first detectable and is generally considered to be a marker of viral replication and hence infectivity; while HBeAg is detectable, HBV DNA is also detectable. HBeAg, however, declines before HBsAg is cleared. Seroconversion to anti-HBe is a first indication that the infection may be acute and resolve naturally, and generally there is a short time gap between the disappearance of HBeAg and the appearance of anti-HBe.
FIGURE 3.1 Outcomes of infection with HBV
HBcAg is the protein that makes up the core of the Dane particle. It is not found in the bloodstream although it can be found free in hepatocytes. Anti-HBc, however, is a particularly useful marker of infection which appears after HBsAg and HBeAg but may persist for the lifetime of an individual. IgM anti-HBc appears first, with IgG anti-HBc gradually increasing in titre and persisting after the IgM titre has declined. IgM anti-HBc is commonly used to identify acute infections.
Anti-HBs is a protective antibody which usually appears shortly after the disappearance of HBsAg although there are some individuals in whom there is an extended delay before the appearance of anti-HBs. Titres may decline over extended periods but recovery from acute infection does confer lifetime protection.
Acute infection see Figure 3.2 The first detectable marker is HBsAg, which increases in titre quite rapidly and then starts to decline, normally being detectable for 2_12 weeks. Early on in acute infection, HBsAg may be the only marker detectable, and, in the absence of Dane particles, the individual may be initially uninfectious for a short period. HBeAg subsequently appears, but at a level below that of HBsAg, and then declines quite quickly, disappearing before HBsAg disappears. Anti-HBc (IgM + IgG) appears around the time that HBsAg levels peak, the IgM level declining gradually over a period of 2_16 weeks from first appearance, while the IgG level persists for many years, often for the lifetime of the individual. Anti-HBe appears as HBeAg disappears and may persist for many years although levels do slowly decline. Anti-HBs finally appears some time after HBsAg levels have declined and almost always marks full immunity, persisting for the lifetime of the individual. Occasional cases have been reported where anti-HBs has been detected in the presence of HBsAg.
FIGURE 3.2 Serological profile of acute HBV infection
Chronic infection see Figure 3.3 Following their appearance, HBsAg and HBeAg persist, followed subsequently by IgG anti-HBc. While HBeAg persists, an infected individual has a chronic active HBV infection. In some individuals seroconversion to anti-HBe may occur; this could be at any time, with the consequent cessation of active HBV infection. Chronic infections may resolve after a period of time with cessation of liver disease and subsequent development of full immunity.
Figure 3.3 Serological profile of chronic HBV infection
Using molecular techniques HBV DNA can be detected in serum during acute and chronic infections. In acute infections DNA can be detected in the bloodstream soon after HBsAg can be detected, but may not persist for long. Many studies have looked at the link between HBV DNA and HBeAg/anti-HBe and have found that 75% to 85% of HBeAg positive individuals are HBV DNA positive, while only 10% to 20% of anti-HBe positive individuals are HBV DNA positive. The detection of HBV DNA may have more value when performed in conjunction with anti-HBc screening to detect the HBV tail-end carriers (see 3.2.5).
3.2.4 Screening of blood donations for HBV infection
Detection of HBV infection in donated blood is best achieved by screening for HBsAg; this is universally accepted. HBsAg is the first marker to appear in the bloodstream, and persists throughout the period of infectivity. The other markers of HBV infection are of use in confirming infection (see Chapter 10) and determining the type and stage of infection, but apart from anti-HBc (see 3.2.5) have no value in routine blood screening. Detection of HBV DNA may be beneficial in assessing the individual's status, but again has limited value to blood screening: the techniques are not yet suitable for mass screening, and DNA levels can fall to below detectable levels while infectivity may remain. The sensitivity and specificity of HBsAg assays are generally good with sensitivities increasing all the time, being currently around 0.1 iu/ml.
For many years the subject of anti-HBc screening of donations has been discussed. Before specific anti-HCV screening became possible, anti-HBc screening was considered by some to be a possible way of reducing the number of cases of post-transfusion non-A, non-B hepatitis (PTNANBH), at that time seen in large numbers. The identification of the hepatitis C virus (HCV) and the advent of anti-HCV screening have now demonstrated that this particular strategy had no value in the prevention of PTNANBH.
However, others consider that the value of anti-HBc screening is to identify that small number of donors who are either resolving an acute infection or clearing a chronic infection, are apparently HBsAg negative on screening, but who may still have low level viraemia and be infectious. These people have been called "tail-end carriers". In such individuals anti-HBc IgM and/or IgG may indeed be the only detectable circulating markers of infection, thus only detectable by anti-HBc screening. This situation could explain cases of reported post-transfusion HBV apparently resulting from transfusion of donations screened as HBsAg negative; unfortunately in these situations it is often not easy to demonstrate that the patient had no other sources of infection. A major problem with anti-HBc screening is to then identify those donors who are truly anti-HBc only or who are naturally immune following infection earlier in life. Many anti-HBc screening tests have relatively poor specificity and there are also major problems confirming initial anti-HBc reactivity. Even if anti-HBs is also subsequently detected, it is generally agreed that those donors with low level anti-HBs (usually less than 100 million iu/ml) cannot be considered to be sufficiently immune to be used.
While there is no doubt that there may be a small number of individuals who may be infectious but only have detectable circulating anti-HBc, it is unclear how many blood donors would fall into this group. However, the higher the prevalence of HBV in the population, the less significant an "anti-HBc only" becomes in transfusion practice. Currently, a number of countries screen all donations for anti-HBc while others do not. At present, however, there are insufficient conclusive data available to assess the true value of anti-HBc screening.
There are currently two groups of HBV mutants identified; the pre-core mutants and the surface antigen mutants. The pre-core mutants have a normal HBsAg expression but missing or altered HBeAg expression. Currently, these mutants are not thought to present any threat to the blood supply because HBsAg expression is normal and blood screening would therefore not be compromised. However, the HBsAg mutants, originally termed vaccine escape mutants, are of concern to blood screening as HBsAg expression is altered such that some assays may fail to detect some HBsAg mutant forms. This is most likely where the assays use monoclonal antibodies, either in the solid phase or in the conjugate, as these antibodies are totally specific for the "a" determinant of HBsAg, which is the usual missing or altered protein of the HBsAg mutants. Assays using polyclonal antibodies are less likely to fail to detect HBsAg mutants as their reactivity is broader and usually encompasses other epitopes on the HBsAg molecules. Studies so far have found that most HBsAg assays from reputable international manufacturers do detect the majority of the HBsAg mutants available for study.