Hepatitis B, caused by Hepatitis B virus (HBV), is a major public health concern. It is a source of significant morbidity and mortality in Pakistan and around the globe. It is, along with other forms of viral hepatitis, the most common cause of chronic as well as acute liver disease, cirrhosis, and hepatocellular carcinoma. This type of the viral hepatitis Infection is usually associated with blood-borne transmission via transfusions and blood products, reuse of inadequately sterilized non-disposable needles, syringes and venous access equipment, and renal dialysis. In addition to the blood, the virus particles may be present in the saliva, semen, and stools of active cases and carriers. This makes the possibilities of transmission of the infection alarmingly very high.
VIRAL STRUCTURE
HBV is sometimes called Dane particle, named in honor of the scientist who first observed and described this virus. It is a double-stranded enveloped DNA virus belonging to the family Hepadnaviridae. The complete virion is a compact spherical structure . measuring 42 nm in diameter. The outer envelope is composed of units made from hepatitis B surface antigen (HBsAg) proteins, carbohydrates, and lipids. The envelope encloses an icosahedral 27 nm core particle. The core is made of a nucleocapsid that contains genomic DNA associated with an endogenous DNA dependent DNA polymerase surrounded by a protein capsid. This protein acts as an antigen called HBcAg.
The genomic DNA of HBV is a partially double-stranded structure comprising of a short single-stranded portion. This makes one strand short incomplete and other strand long complete. The incomplete strand is called positive or sense strand while complete stand is called negative or antisense strand. These two strands are held in a circular configuration by base pairing over approximately 250 base pairs at the 5’ termini. The 5’ termini of both the strands contain an identical stretch of 11 base pairs, which are found important in the replication process.
The DNA genome of HBV is composed of approximately 3200 nucleotide base pairs with a molecular weight of 2.3 x 106 dal. The single-stranded portion is about 600 to 2100 nucleotides long. The viral genome consists of four genes. Each gene encodes distinct protein that acts as antigen of the virus.
REPLICATION OF HBV
The viral replication primarily takes place in hepatocytes. HBV has a unique position among DNA viruses with regard to its mode of replication. It replicates through an RNA intermediate, or pregenome. The HBcAg is important in incorporating the pregenomic RNA. The replication cycle of HBV involves a reverse transcription step and pregenome has a key role here. The pregenome acts as a template for the synthesis of negative strand DNA in the presence of an enzyme called reverse transcriptase or RNA dependent DNA polymerase.
The replication of HBV begins with the transcription of negative strand DNA that leads to the production of full-length positive RNA transcripts. Many of these transcripts act as mRNAs, which are translated to synthesize various viral proteins, both functional as well as structural. The assembly of the structural proteins then takes place to develop core structures. The RNA transcript is then inserted into maturing core particle late in the process where it acts as a template for the process of reverse transcription. The product of this process is negative DNA stand. The RNA templates are then degraded by ribonucleases activity. Now the negative strand serves as template for the synthesis of positive DNA strand, although the process is not completed prior to virus maturation and release. This is why the positive strand remains as short and incomplete in the virion particle. The two strands base-pair and form a double-stranded structure. The virus is released from the host cell through budding during which it acquires the enveIope.
ANTIGENIC STRUCTURE
HBV presents a complex antigenic profile as shown in the following.
* Surface antigen or HBsAg. The envelope of the virus contains a lipoprotein that is antigenic in nature. It is referred to as surface antigen or HBsAg. This antigen has been found in the tears, breast milk, urine, and other body fluids. Development of the carrier state is indicated by the persistence of HbsAg (or HbsAg/lgM complexes) in serum after acute hepatitis. The neutralizing antibodies, directed against a common 'a’ epitope of the antigen, appears late in the serum and provides a dependable immunity. Besides a epitope, the virus possesses two sets of mutually exclusive subtype determinants, d and y, and w and r. Thus there are four major subtypes of HbsAg and adw, ayw, adr, and ayr denote the phenotypes of the virion.
* Precore antigen (HBeAg). The precore region of the HBV genome encodes a soluble protein called the precore or ‘e’ antigen (HBeAg). Its function has not been fully understood. It is stated by some researchers that HBeAg induces tolerance of the host’s immune system to itself and the core antigen, thus contributing to the persistence of chronic infection.
* Core antigen (HBeAg). The core region of the viral genome encodes a protein that is assembled to form the core. This protein on the surface of the core is called core or ‘c’ antigen (HBeAg). This antigen is also found in the infected liver homogenates and nuclei of infected hepatocytes. Its presence indicates the acute disease. The anti-HBcAg appears late during the acute infection and only persists for a month or 2. Its presence, therefore, confirms current or very recent acute infection. Its level decreases with the recovery from the acute illness.
RESISTANCE
HBV is resistant to environmental factors that inactivate most of the viruses and bacteria. it survives moist heat at 60°C for 4 hours, 100°C for 30 minutes, storage at room temperature for 6 months, and at --10 to 20°C for four and half years. The virus remains potentially infectious for months when stored at approximately 32°C. It is resistant to phenol and ether but sensitive to chlorine.
INClDENCE, TRANSMISSION, AND RISK FACTORS
HBV is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It has infected 15 million people in Pakistan and killed about 30% of them as a consequence of complications associated with hepatitis B. Every tenth person in Pakistan is the carrier of this virus. According to WHO statistics, hepatitis B-induced cirrhosis or liver cancer is the ninth most common cause of death worldwide, taking more than 1 million lives each year.
HBV is found in the blood of the infected persons and blood products. In addition to blood, the virus is also found in the body fluids, such as serum, semen, saliva, and stools of active cases and carriers. Several medical reports indicate the presence of HBsAg in tears, breast milk, and urine. The vagina is contaminated during menstruation. The distribution of HBV in blood and various body secretions establishes the mode of transmission of this virus in the community. Percutaneous and mucous membrane exposure to infected body fluids is the major source of HBV infection. The use of unsterilized syringes, sexual relations, blood transfusion, and renal dialysis are the major sources of the infection. Recent reports have confirmed that the perinatal transmission of HBV from mother to newborn is also possible. Transmission by semen donated for artificial insemination has recently been published in many research journals. The infection may be acquired by sharing of syringe among drug abusers, by heterosexuals and homosexual contacts, by the use of unsterilized hypodermic needles or instruments applied for tattooing, piercing of ears and nose, and acupuncture. Since blood contains a million infectious doses per micortiter, invisible amount of blood can transmit the infection. Medical reports are available indicating the transmission of the infection through contaminated instruments in dental clinics. BIood-sucking arthropods have not been found involved in the transmission of hepatitis B.
The most recent data from the American Center of Disease Control and Prevention (CDC) suggest that heterosexual activity is the most common risk factor for hepatitis B, followed by injection drug use, and male homosexuality making hepatitis B primarily an STD in the minds of many public health officials. In Pakistan unsafe blood transfusion practice is among important risk factors. Additional risk factors include alcohol abuse and co-existent HCV or HBV infection. The groups at high risk of acquiring HBV infection include patients and staff at hemodialysis centers, physicians, paramedical staff, dentists, medical technologists and staff working at blood banks.
The risk of developing chronic HBV infection is inversely related to age at the time of infection. Ninety percent of infants infected at birth wiil become chronically infected with HBV, as will 25-50% of children aged 1-5 and 1-5% of older children and adults. However, only a small percentage of carriers will have a history of acute infection----10% of children and 30-50% of adults.
PATHOGENESIS
After entering the host body the virus finds its way to the lymphoid tissue where it begins its replication cycle. It then travels to blood circulation and reaches the liver. In liver it penetrates the hepatocytes and initiates replication. The incubation period of the disease is 6 weeks to 6 months, average being 12-14 weeks. Because of lengthy and uneven duration of incubation period, it is very difficult to determine the origin of the infection. It is also not certain why some cases resolve and some become chronic. Few experts have correlated the chronicity of hepatitis B to certain factors including mild infection, a long incubation period, immuno-suppression, genetic predisposition, and infection early in childhood.
The development of clinical disease is directly correlated with age. Fewer than 10% of infected children and infants present with symptoms of disease, but the number increases to more than 30% in adults above the age of 30 years. The biological response to HBV infection is often complex, particularly in children. In more than 90% of adults, the infection is seIf-limiting. The serologic loss of HBsAg and presence of anti-HBsAg demonstrates resolution. However, in up to 5% of adults, as well as infants and the immunocompromised, the immune response may be incomplete, which often leads to less severe prodromal disease despite persistent viremia and continuing viral replication (positive HBeAg and HBV DNA). Patients who have evidence for persistent viral replication are more likely to develop exacerbations, cirrhosis, hepatic decompensation, death from liver failure, and HCC, while those who remain in a low replicative state have a reduced risk of more serious sequelae.
It is now a well-documented fact that HBV does not directly cause any cytopathic effects on the hepatocytes. Evidence suggests that progressive hepatic necrosis is a consequence of CMI that is initiated by HBV infection. The cytotoxic T-lymphocytes attack the virally infected hepatocytes during the acute phase of the disease and eventually kiil them.
Another important factor that plays its role in the pathogenesis of hepatitis B is the formation of immune complexes in excess HBsAg. These complexes settle in the cytoplasm and plasma membrane of hepatocytes and on or in the nuclei. Their deposition is also reported in the skin, joints, inner surfaces of blood vessels, etc. This leads to the activation of the complement system causing polyarteritis nodosa, rash, vasculitis, and arthritis.
CLINICAL MANIFESTATIONS
The clinical manifestations of hepatitis B is similar to other types of viral hepatitis. The disease commences with a prodromal phase, which has both specific and nonspecific features. It is followed by the development of jaundice.
PRODROMAL PHASE
The onset of hepatitis B is usually insidious. Acute disease is usually manifested by the gradual onset of certain nonspecific symptoms, including marked anorexia, nausea, malaise, fullness in the right upper abdominal quadrant, and low-grade fever. Persistent vomiting may occur in children but is unusual in adults unless they have fulminant disease. Smokers often complain that tobacco smoke has lost its taste. These symptoms may persist for up to a week before the onset of jaundice.
The specific clinical features of hepatitis B include polyarthritis less commonly accompanied by urticarial rash. Massive destruction of hepatocytes is accompa nied by increasing cholestasis and hence progressive darkening of the urine and loss of stool coloration are noted. The stool is usually clay-coloured. A mild diffuse epigastric or right hyochondrial pain also develops.
ICTERIC PHASE
The appearance of jaundice is associated with rapid resolution of prodromal anorexia and malaise. Abdominal pain also settles rapidly and liver frequently returns to normal size. Slight splenomegaly may occur, most frequently in children. Jaundice is most apparent in the eyes and in many cases may not be detectable in the skin or mucous membranes. Persistence of jaundice over weeks may indicate development of cholestatic syndrome. A case of hepatitis B is considered chronic if the persistence of the jaundice extends for 6 weeks or more and associated with persistently elevated serum transaminases and HBsAg. Progressive cirrhosis leads to liver failure. Moreover, patients with chronic hepatitis show a tendency of developing hepatocellular carcinoma.
LABORATORY DIAGNOSIS
There are three antigen-antibody systems associated with HBV infection that serve as the basis for current serologic tests: HBsAg and anti-HBs antibodies, HBsAg and anti-HBC antibodies, and HBeAg and anti-HBe antibodies.
DEMONSTRATION OF HBsAG
HBsAg appears in the blood 30 to 60 days after exposute to HBV, while the virus is actively replicating in hepatocytes. The antigen is produced in vast excess so that it is not only associated with new infectious virus particles, but also occurs in the serum as small noninfectious spherical or filamentous forms. It may persist for an additional several months when the infection is uncomplicated. However, in acute infection, the HBsAg usually disappears within 3 months of onset. Chronic infection may be presumed to be present when the patient maintains a positive blood test for HBsAg for six or more months. There are very few patients who convert from HBsAg positive to anti-HBs antibodies positive each year. Chronically infected patients may produce normal liver function test or positive serum alanine aminotransferase test due to various degrees of underlying liver damage.
DEMONSTRATION OF HBeAG
As mentioned earlier, HBeAg is an indicator of current viral replication and infectivity. It can be detected in blood for a short period, usually 4 to 8 weeks, while the virus is actively replicating in the liver during the acute phase of the disease. A positive HBeAg test is also associated with a positive HBV DNA test. Continued detection of HBeAg beyond first two months after the onset of infection indicates that chronic infection is likely to occur. A positive HBeAg test along with a positive HBV DNA test has been noted for many years, occasionally decades, in most chronic cases.
DEMONSTRATION OF ANTI-HBc
Immunogiobulin M (lgM) to HBcAg appears first in the serum of an active patient. As a generalization, the detection of anti-HBc is the primary indicator of acute infection. It usually appears at or just before the onset of symptoms and normally remains detectable for only 6 months. Thus, a positive test for this antibody signifies that the infection has been recent.
DEMONSTRATION OF ANTI-HBe
Anti-HBe is the second antibody to appear in the serum and is associated with the rapid clearance of HBeAg. Its titer does not persist for long under normal conditions. Its persistence for a few months or years is possible only when there is no active viral replication. Anti-HBe positive individuals show a marked improvement in ATL levels.
DEMONSTRATION OF ANTl-HBS
The antibody to HBsAg, anti-HBs, may not become detectabte for 3-6 months after acute infection. It appears in the serum after resolution of the acute infection or following vaccination and provides long-term immunity. The demonstration of anti-HBs, thus, indicates prior infection or immunity to HBV. There is, however, a range of variations in the serological profile. For example, in some patients who have perfectly recovered from hepatitis B, the anti-HBS antibody may onty be present in traces or remains below the detectable levels. In some other chronically infected patients, HBsAg and anti-HBs may be present together.
THERAPY
The antibody to HBsAg, anti-HBs, may not become detectable for 3-6 months after acute infection. It appears in the serum after resolution of the acute infection or following vaccination and provides long-term immunity. The demonstration of anti-HBs, thus, indicates prior infection or immunity to HBV. There is, however, a range of variations in the serological profile. For example, in some patients who have perfectly recovered from hepatitis B, the anti-HBs antibody may only be present in traces or remains below the detectable levels. In some other chronically infected patients, HBsAg and anti-HBs may be present together.
There is no specific antiviral therapy available for hepatitis B. Alpha interferon is the only agent known to have a lasting beneficial effect in the therapy of hepatitis B. It was first introduced in the market in 1996 in the USA. A 4 to 6 month course of therapy (5 mIU per day subcutaneously) has been shown to produce promising results in 25 to 40% of patients.
A number of new approaches to treating hepatitis B are in progress. Their objectives are to either inhibit viral replication or augment cellular immunity against the virus. Recent reports have shown the effectiveness of two new nucleoside analogues,such as famciclovir and lamivudine, against HBV. Interestingly, these nucleoside analogues were initiatly developed to treat HIV and have been found to have activity against HBV as well. Long-term trials of both the analogues are currently underway, as are studies of combination therapy with alpha interferon. Preliminary reports suggest that the regimens are well tolerated, viral load can be eliminated and often sustained, and improvement in liver histology can be maintained. It can be hoped that a combination of nucleoside analogues with or with out alfa interferon may become the regimens of choice for the therapy of hepatitis B with in next few years.
PREVENTION
GENERAL MEASURES
Hepatitis B can be prevented by adopting certain measures. These measures include the fotlowing:
* Use of sterilized syringes. 0 Use of sterilized needles and instruments for blood transfusion and surgery.
*Use of sterilized instruments for dental surgery.
*Avoidance of shaving at barbershops.
* Avoidance of sharing of towels, razors, tooth picks, and tooth brushes.
* Preference of oral medication to giving injections to patients.
* Proper screening of the blood for hepatitis B before it is used for transfusion.
* Application of safe blood bank practices.
* lmprovement in the personal and community hygiene.
* Use of safe drinking water and hygienic food.
* Availability of medical facilities to each member of the community.
* Adequate care of the patients suffering from HBV infection.
* Educating the people about the transmission and severity of HBV infection and its prevention.
* Avoidance of unsafe sexual practices.
* Immunization of the professionals, paramedics, and all other people at risk of acquiring HBV infection.
PASSIVE IMMUNIZATION
Hepatitis B antibodies are found useful for providing passive immunity if administered before or shortly after exposure to HBV. Human normal immunoglobulin (HNlG), which contains high titer of anti-HBs serum obtained from HBsAg negative donors are now available and have proved protective in many cases. Passive immunization is considered an effective, though short lived, method particularly in cases of needlestick exposure of clinical attendants of HBsAg positive patients. lt can also be used to protect sexual partners exposed to those with acute HBV infection. Doses of HBlG are given within 48 hours of exposure and at 1 month thereafter.
HBIG is recommended for all infants born to infected mothers or in endemic areas. lt helps in avoiding the chances of developing carrier state as disease contracted during infancy usually develops into carrier state.
ACTIVE IMMUNIZATION
Vaccination against HBV was introduced to control the morbidity and mortality associated with HBV. Until 1997, most of the countries had strategies aimed at immunizing only those people who were considered at risk, rather than population based immunization strategies. This attitude was changed when WHO launched a program for the control of hepatitis B and a wider immunization program was adopted.
Two types of hepatitis B vaccines are currently available. One, which is widely used in most of the countries, contains deactivated virus collected from chronic carriers. The other contains nonglycosylated particles of HBsAg expressed from recombinant DNA in yeast, This is reported as highly immunogenic and efficacious, and has remarkable safety profiles. Research work is in progress to design newer recombinant vaccines containing HBsAg and other hepatitis antigens.
All adolescents between 10 and 16 years who have not previously been vaccinated should receive 3 doses of HBV vaccine. At-risk infants, those with HBsAg positive mother or those from high prevalence communities should begin a course of 3 doses within 7 days of birth. Occupational risk groups and other at-risk adults should also be considered for HBV vaccination.
