Update on prevalence, diagnosis and treatment of hepatitis b virus

UPDATE ON PREVALENCE, DIAGNOSIS AND TREATMENT OF HEPATITIS B VIRUS ESSAY Submitted for the partial Fulfillment of the Master degree in Tropical Medicine Presented by

Dr. Hesham Noaman Abdel Raheem Mustafa (M.B., B.Ch.)

Under Supervision of

Dr. Laila Ahmad Mohammad Professor of Tropical Medicine Faculty of Medicine Cairo University

Dr. Ahmad Nabil Lotfy Hassan Lecturer of Tropical Medicine Faculty of Medicine Cairo University

Faculty of Medicine Cairo University 2005

I

ACKNOWLEDGEMENT I would like to start by thanking "God" for his help during this work as a part of his generous help through out my life.

I am deeply indebted to Professor. Dr. LAILA AHMAD MOHAMMAD (Professor of Tropical Medicine, Faculty of Medicine, Cairo University) for suggesting, planning and supervising this work and for her continuous guidance and encouragement .

I would like to express my deepest thanks and gratitude to DR. AHMAD NABIL LOTFY HASSAN (Lecturer of Tropical Medicine, Faculty of Medicine, Cairo University) for his wise guidance, criticism and valuable suggestions throughout the present study.

Last, but not least, to all patients who are suffering, hoping that this work will give them hope for a better future.

Hesham Noaman Abdel Raheem (M.B., B.Ch.) December 2005

II

Table of Contents Title Introduction and Aim of the Work

Page No. 1-3

Review of Literature 1

Historical Note.

4

2

Morphology of Hepatitis B Virus.

5-20

3

Epidemiology of HBV.

21-34

4

Clinical Presentation and Sequelae.

35-44

5

HBV Status in Egypt.

45-54

6

Diagnosis of Acute and Chronic Hepatitis B.

55-76

7

Treatment of Chronic Hepatitis B.

77-112

8

Prevention of HBV.

113-124

Summary and Conclusions

125-126

Recommendations References Arabic Summary

127 128-159

III

List of Figures Title Fig. (1): Schematic diagram of hepadnavirus particles.

Fig. (2): Electron microscopic presentation of HBV particles.

Fig. (3): Genome organization of HBV. Fig. (4): A group of hepatitis B virions (right) and enlargements of the two exposed cores.

Fig. (5): Hepatitis B Surface Proteins.

Fig. (6): Largest HBs proteins.

Fig. (7): Middle Hepatitis B proteins (MHBs).

Fig. (8): Schematic diagram of two loops of the "a" determinant of HBs protein.

Fig. (9): Small Hepatitis B proteins (SHBs).

Fig. (10): HBV outer envelope contains high amounts of hepatitis B surface proteins.

Fig. (11): Diagram showing Purified virions possess the HBc protein.

Fig. (12): HBV has evolved a unique life cycle.

Fig. (13): HBsAg Endemicity.

Fig. (14): Countries with areas with moderate to high risk of infection.

Fig. (15): Geographic pattern of Hepatitis B prevalence.

Fig. (16): Geographic distribution of HBV Genotype.

Fig. (17): Analysis of 1860 acute hepatitis cases.

Fig. (18): Evolution of HBV markers in acute infection.

Fig. (19): Characteristics of progression to chronic HBV infection.

Fig. (20): Mode of action of interferon alpha (INF-).

Fig. (21): Global status of countries using HepB vaccine in their national infant immunization system.

Page No. 6 7 9 10 12 13 14 15 16 16 18 20 21 22 23 47 49 61 62 80 118

IV

List of Tables Title Table (1): Characteristics of endemic patterns of hepatitis B virus infection.

Table (2): Geographic Distribution of HBV Genotypes.

Table (3): Prevalence of HCV, HBV and HBsAg in Egypt 1996.

Table (4): Etiology of acute viral hepatitis among 200 patients.

Table (5): The frequency of acute hepatitis B in different age groups in year 2002 in comparison to year 1983.

Table (6): HBeAg. Table (7): Interpretations of available serologic test results for HBV. Table (8): Serological test findings at different stages of HBV infection and in convalescence.

Table (9): Hepatitis B tests.

Table (10): Antibody to Hepatitis B Surface Antigen (HBsAg Assay).

Table (11): Hepatitis B Surface Antigen (Anti-HBs Assay).

Table (12): Hepatitis B Virus Core Antigen (Anti-HBc Assay).

Table (13): Histological Activity Index.

Table (14): Modified HAI grading: necroinflammatory scores.

Table (15): Glossary set by Keeffe et al., 2004 to correlate clinical terms with laboratory results.

Table (16): The stages of HBV in correlation with the laboratory results.

Table (17): Interpretation of serological markers.

Page No. 27 47 48 48 49 64 65 66 66 67 67 68 72 73 74 75 76

Table (18): Hepatitis B Therapies and Treatment.

Table (19): Types and Properties of Interferon Table (20): Determinant of responsive factors to interferon.

79 81 83

Table (21): Comparison of Three Approved Treatments of Chronic Hepatitis B. Table (22): Goals of Therapy and Definitions of Response to Therapy in Chronic Hepatitis B. Table (23): Available Agents for Treatment of Chronic Hepatitis B. Table (24): Recommendations for Treatment of Chronic Hepatitis B. Table (25): Post Exposure Prophylaxis and Post Liver Transplant Therapies & Preventive Vaccines for HBV. Table (26): Results of Lamivudine Monotherapy Prior to OLT. Table (27): Effect of Combination Therapy with HBIG and Lamivudine to Prevent HBV Reinfection after OLT.

104 105 106 112 119 123 124

V

List of Graphs Title Graph (1): Virological response of chronic hepatitis C patients with and without past history of HBV. Graph (2): Age distribution of patients with acute HBV. Graph (3): Lamivudine: Histologic Improvement at week 52 in HBeAg-positive patients.

Graph (4): Effect of Lamivudine on HBV DNA Levels.

Graph (5): Response to Lamivudine in HBeAg-negative CHB. Graph (6): Treatment of Lamivudine Resistance: Prospective study of Adefovir vs Lamivudine vs Combination Therapy.

Graph (7): HBeAg Seroconversion with Lamivudine: Effect of treatment Duration and Baseline ALT. Graph (8): HBeAg-positive patients: Adefovir vs placebo for 48 weeks.

Graph (9): Long-term Adefovir in HBeAg-negative Patients: Serum ALT. Graph (10): Long-term Adefovir in HBeAg-negative Patients: Median Serum HBV DNA. Graph (11): Adefovir vs Placebo: Change from Baseline in HBV DNA. Graph (12): Entecavir Treatment of Lamivudine-resistant Hepatitis B.

Page No. 42 50 86 87 88 90 91 94 95 96 96 97

VI

List of Abbreviations Abbreviation ADV Anti-HBc Anti-HBe Anti-HBs ALT AST CHB cccDNA CTL ETV FHF GMCSF HBV HBIG HBcAg HBeAg HBsAg INF-α LAM LHBs MHBs ORFs OLT PCR Pre-S1, pre-S2 SHBs ULN WHO WT

Term Adefovir depevoxil Antibody to HBcAg Antibody to HBeAg Antibody to HBsAg Alanine aminotransferase Aspartate aminotransferase Chronic hepatitis B covalently closed circle DNA cytotoxic T lymphocyte Entecavir Fulminant hepatic failure granulocyte macrophage colony stimulating factor Hepatitis B virus Hepatitis B immune globulin Hepatitis B core antigen Hepatitis B e antigen" Envelope " Hepatitis B surface antigen Interferon Alfa Lamivudine Large HBs protein Middle HBs protein Open reading frames Orthotopic liver transplantation Polymerase chain reaction Envelope protein epitopes Small HBs protein Upper limit of normal World health organization Wild Type

UPDATE ON PREVALENCE, DIAGNOSIS AND TREATMENT OF HEPATITIS B VIRUS ESSAY Submitted for the partial Fulfillment of the Master degree in Tropical Medicine Presented by

Dr. Hesham Noaman Abdelraheem Mustafa (M.B., B.Ch.)

Under Supervision of

Dr. Laila Ahmad Mohammad Professor of Tropical Medicine Head of Tropical Medicine Department, Faculty of Medicine Cairo University

Dr. Ahmad Nabil Lotfy Hassan Lecturer of Tropical Medicine Tropical Medicine Department, Faculty of Medicine Cairo University

Faculty of Medicine Cairo University 2004

INTRODUCTION AND AIM OF THE WORK Hepatitis B virus (HBV) infection is a serious global health problem, with 2 billion people infected worldwide, and 350 million suffering from chronic HBV infection (Lavanchy, 2004).

Being the 10th leading cause of death worldwide, HBV infections result in 0.5 to 1.2 million deaths per year caused by chronic hepatitis, cirrhosis, and hepatocellular carcinoma; the last accounts for 320 000 deaths per year In Western countries, the disease is relatively rare and acquired primarily in adulthood, whereas in Asia and most of Africa, chronic HBV infection is common and usually acquired perinatally or in childhood (Alter, 2003; Lavanchy, 2004).

Hepatitis B is a major health problem in Egypt (Attia, 1998). As for acute HBV infection, the prevalence of HBV in Egypt is not yet adequately estimated after the use of hepatitis B vaccine (Zakaria et al., 2000).

However, Orfi (2002) stated that the prevalence of acute HBV infection was 12% in children 4-14 years old and 50.9% in adults > 14 years old. The most common age group infected by HBV ranged from 21-30 years (42.4%) whereas the least infected age group was from 4-8 years (3%). Sherif et al. (1985) reported that HBV was more prevalent in Upper Egypt (11.7%) than in Lower Egypt (8.8%).

Initial testing for hepatitis B should include hepatitis B surface antigen and hepatitis B core antibody, (which is frequently reported as total core antibody and subsequently fractionated into its IgM and IgG components) (Russo, 2004). The presence of hepatitis B core IgM indicates acute infection or, in some cases, reactivation; the presence of hepatitis B core IgG indicates prior infection (Russo, 2004).

The primary aim of antiviral therapy is durable suppression of serum HBV DNA to the lowest level possible. The

threshold level of HBV DNA for determination of candidacy for therapy is >10

copies/ml for patients with (HBeAg) positive chronic hepatitis B

(Keeffe et al, 2004). A lower serum HBV DNA threshold is appropriate for patients with HBeAg-negative chronic hepatitis B and those with decompensated cirrhosis, and the scientists recommends thresholds of 10

copies/ml and 10³ copies/ml respectively (Keeffe et.al., 2004).

Effective vaccines for hepatitis B virus have been available

since 1982; infant and childhood vaccination programs introduced in the 1990s have resulted in a marked decrease in new infections. Risk factors for progression to chronic infection include age at the time of infection and impaired immunity (Lin and Kirchner, 2004). From 15 to 30 percent of patients with acute hepatitis B infection progress to chronic infection. Medical therapies for chronic hepatitis B include interferon alfa-2b, lamivudine, and the nucleotide analog adefovir dipivoxil (Lin and Kirchner, 2004).

Chronic infection with hepatitis B and its sequelae remains a major

global

health

concern.

Despite

recommendations

and

implementation of vaccination programs, the health and economic burdens are still significant (Tran and Martin, 2004). People in endemic areas and immigrants from these areas need to be adequately screened and treated. HBeAg-negative chronic hepatitis is increasingly recognized with additional challenges in management. Programs implementing primary prophylaxis strategies such as vaccination of high-risk adult and adolescent groups should continue (Tran and Martin, 2004).

The aim of the present work is to reveal of the prevalence of HBV worldwide and in Egypt and to discuss updated clinical issues in the diagnosis and modern modalities of treatment.

REFERENCES: 1. Alter M.J. (2003): Epidemiology of hepatitis B in Europe and worldwide.J Hepatol; 39 Suppl 1:S64-9.

2. Attia M.A. (1998): prevalence of hepatitis B and C in Egypt and Africa.Antiver ther; 3(supl 3):1-9.

3. Keeffe E. B., Dieterich D.T.,Han S.H., Jacobson I.M. et. al.

(2004): A Treatment Algorithm for the Management of Chronic Hepatitis B Virus Infection in the United States. Clinical Gastroenterology and Hepatology; 2: 87-106.

4. Lavanchy D. (2004): Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat ; 11(2):97-107.

5. Lin, K.W. and Kirchner JT. (2004): Hepatitis B. Am Fam Physician; 69(8):1863.

6. Orfi M. (2002): The role of hepatitis B virus as a cause of acute viral hepatitis after the wide use of hepatitis B vaccine in Egypt.

M.Sc. Thesis: Tropical Medicine, Cairo University: 63-71.

7. Russo M. W. (2004): Care and Management of Chronic Hepatitis B: An Overview for Patients and Family Caregivers Hepatitis B. Emergency Medicine.

8. Sherif M.M., Abou-Atia M.A., Pazzagalia G. et.al. (1985): Hepatitis B Virus infection in Upper and Lower Egypt. J. Med. Virol; 15(2); 129-35.

9. Tran, T.T. and Martin, P. (2004): Hepatitis B: epidemiology and natural history. Clin Liver Dis.; 8(2):255-66.

10.Zakaria S.; Zakaria M. and Fouad R. (2000): Sero-prevalence of viral hepatitis markers in a rural and semi-rurual Egyptian district.Antiviral therapy; 5(suppl 1):12.

‫الحديث في انتشار وتشخيص وعالج االلتهاب‬ ‫الكبدى الفيروسى ب‬

‫مقالة توطئة للحصول على درجة الماجستير‬ ‫فى طب المناطق الحارة‬ ‫مقدمة من‬

‫الطبيب‪ /‬هشام نعمان عبد الرحيم مصطفى‬ ‫بكالوريوس الطب والجراحة – جامعة القاهرة‬ ‫وذلك تحت اشراف‬

‫األستاذ الدكتور‪ /‬ليلى أحمد محمد‬ ‫أستاذ طب المناطق الحارة‬

‫كلية الطب‪ -‬جامعة القاهرة‬

‫الدكتور‪ /‬أحمد نبيل لطفى حسن‬ ‫مدرس طب المناطق الحارة‬ ‫كلية الطب‪ -‬جامعة القاهرة‬ ‫كلية الطب‬

‫جامعة القاهرة‬ ‫‪2004‬‬

Introduction and aim of the work

INTRODUCTION AND AIM OF THE WORK Hepatitis B virus (HBV) infection is a serious global health problem, with 2 billion people infected worldwide, and 350 million suffering from chronic HBV infection (Alter, 2003).

Being the 10th leading cause of death worldwide, HBV infections result in 0.5 to 1.2 million deaths per year caused by chronic hepatitis, cirrhosis, and hepatocellular carcinoma; the last accounts for 320 000 deaths per year In Western countries, the disease is relatively rare and acquired primarily in adulthood, whereas in Asia and most of Africa, chronic HBV infection is common and usually acquired perinatally or in childhood (Lavanchy, 2004).

Hepatitis B is a major health problem in Egypt (Abdelhamed et al., 2002). As for acute HBV infection, the prevalence of HBV in Egypt is not yet adequately estimated after the use of hepatitis B vaccine (Zakaria et al., 2000).

Sherlock and Doodley, 2002 stated that chronic hepatitis B is often a silent disease. The patient may be virtually symptom-free with only biochemical evidence of continued activity and may simply complain of fatigue and being generally unwell. The diagnosis is made after a routine medical check. Lok and McMahon, 2001 reported that 15-40% of HBV carriers would develop serious sequelae during their lifetime.

Initial testing for hepatitis B should include hepatitis B surface antigen and hepatitis B core antibody, (which is frequently reported as total core antibody and subsequently fractionated into its IgM and IgG components) (Russo, 2004).The presence of hepatitis B core IgM indicates acute infection or, in some cases, reactivation; the presence of hepatitis B core IgG indicates prior infection (Russo, 2004).

-1-

Introduction and aim of the work

The primary aim of antiviral therapy is durable suppression of serum HBV DNA to the lowest level possible. The threshold level of HBV DNA for determination of candidacy for therapy is >10 copies/ml for patients with (HBeAg) positive chronic hepatitis B (Keeffe et al, 2004). A lower serum HBV DNA threshold is appropriate for patients with HBeAg-negative chronic hepatitis B and those with decompensated cirrhosis, and the scientists recommends thresholds of 10

copies/ml and 10³ copies/ml respectively (Keeffe et.al., 2004).

Effective vaccines for hepatitis B virus have been available since

1982; infant and childhood vaccination programs introduced in the 1990s have resulted in a marked decrease in new infections. Risk factors for progression to chronic infection include age at the time of infection and impaired immunity (Lin and Kirchner, 2004). From 15 to 30 percent of patients with acute hepatitis B infection progress to chronic infection.

Medical therapies for chronic hepatitis B include interferon alfa-2b, lamivudine, and the nucleotide analog adefovir dipivoxil (Lin and Kirchner, 2004).

Chronic infection with hepatitis B and its sequelae remains a major global health concern. Despite recommendations and implementation of vaccination programs, the health and economic burdens are still significant (Tran and Martin, 2004). People in endemic areas and immigrants from these areas need to be adequately screened and treated.

HBeAg-negative chronic hepatitis is increasingly recognized with additional challenges in management. Programs implementing primary prophylaxis strategies such as vaccination of high-risk adult and adolescent groups should continue (Tran and Martin, 2004).

The aim of the present work is to reveal the prevalence of HBV worldwide and in Egypt and to discuss updated clinical issues in the diagnosis and modern modalities of treatment.

-2-

Historical Note

HISTORICAL NOTE The HBV was discovered in 1966 (Blumberg et al., 1967). HBV, the causative agent of B-type hepatitis in humans, is a Hepatotrophic DNA-containing virus that replicates via reverse transcription (Shen et al., 2004). HBV is the only known DNA virus that has hepatocytes specificity (Lu et al., 2004a).

Hepatitis B virus (HBV) was the first human hepatitis virus from which the proteins and genome were identified and characterized. Before discovery of the hepatitis viruses, two types of hepatitis transmission were differentiated based on epidemiological observations. Type A was considered to be predominantly transmitted by the fecal-oral route, whereas type B was transmitted parenterally (Seo et al., 2004).

In 1966, Blumberg, in a research for polymorphic serum proteins, discovered a previously unknown antigen in the blood of an Australian Aborigine (Australia antigen). Four years later, it was recognized that the appearance of this antigen was related to type B hepatitis (Mao et al., 2004). Using immune electron microscopy, Dane eventually discovered virus-like particles that carried this antigen on their surface, in the serum of hepatitis B patients, and these particles were considered the hepatitis B virus (Lee et al., 2004).

In 1973, the viral nature of the particles discovered by Dane was confirmed by the detection of an endogenous DNA polymerase activity within their core (Schiefke et al., 2004).

This enzyme allowed Shen et al. (2004) to detect and characterize the HBV genome as a small, circular, partially double-stranded DNA molecule.

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Review of Literature

Morphology of hepatitis B virus

CHAPTER I MORPHOLOGY OF HEPATITIS B VIRUS The virion of hepatitis B (Dane particle) consists of surface and core. The core is formed in hepatocyte nucleus and the surface particles are made in the cytoplasm. The core contains a DNA polymerase. The structure is double-stranded and circular. It is approximately 3200 nucleotides in length and has a single-stranded gap of 600-2100 nucleotides (Sherlock and Dooley 2002). The DNA-polymerase reaction appears to repair the gap. The core contains a core antigen and another antigen called “e” is a protein subunit of the core (Shindo et al., 2004).

Hepatitis B virus is spherical with a diameter of 42nm. Using negative staining of virions adsorbed to the electron microscopic grids, a double-shelled structure of the virions becomes apparent. The outer protein shell (or envelop) is formed by the HBs proteins (Kumar and Agrawal, 2004). Surface structure details such as knobs or spikes as observed on many other enveloped viruses are found on HBV (Sugauchi et al., 2004).

The inner protein shell is referred to as the core particle or capsid, having a diameter of 34nm in cryoelectron microscopy (Hanazaki, 2004). It is composed of HBc protein and encloses the viral DNA, which is often positively stained (Tsitsilonis et al., 2004).

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Morphology of hepatitis B virus

Fig. (1) Schematic diagram of hepadnavirus particles. Individual subunits containing SHBs protein only, HBs protein plus pre-S2 (MHBs), and HBs protein plus pre-S1 and pre-S2 (LHBs) is shown in intact virus, among filamensts and spheres. The virus particles contain an internal nucleocapsid (HBc) and viral genome (Lai et al., 2003a).

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Morphology of hepatitis B virus

Fig. (2): Electron microscopic presentation of HBV particles. The round 42 nm particles represent infectious virions (Dane particle). The small empty spheres and the filaments are non-infectious. The preparation was enriched in virus particles (Guptan et al., 2002).

HBV Genome: The complete virus comprises a lipoprotein coat, the HBsAg, enveloping a nucleocapsid core that contains a small, circular DNA molecule. Four open reading frames (ORFs) have been identified on the HBV DNA genome. They encode seven proteins, including a HBV DNA polymerase molecule with reverse transcriptase activity. The replication of the virus resembles that of retroviruses and occurs predominantly but not exclusively in hepatocytes. Virus variants involving genomic mutations have been identified (Michael et al., 2002).

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Review of Literature

Morphology of hepatitis B virus

There are four partially overlapping ORFs encoding the envelope (pre-S/S), core (precore/core), polymerase (P) and X proteins. The pre S/S ORF encodes the large (L), middle (M) and small (S) surface glycoproteins (Peksen et al., 2004). The precore/core ORF is translated into a precore polypeptide, which is modified into a soluble protein HBeAg and the nucleocapsid protein HBcAg. The p protein functions as a reverse transcriptase as well as a DNA polymerase. The X protein is a potent transactivator and may play a role in hepatocarcinogenesis (Hahne et al., 2004).

Expression of the viral gene products is regulated by four promoters directing the synthesis of a set of viral transcripts, which are heterogeneous at their 5’ ends but coterminal at their 3’ ends (Benhamou, 2004).

HBV envelope is composed of three polypeptides of different size major, middle and large envelope protein (Kirschberg et al., 2004). The HBsAg contains several antigenic determinants termed ad, y, w and r while some 10 subtypes combinations are recognized; the most common are adw, adr, ayr. These have uneven distribution worldwide and epidemiologically have proved useful in identifying the source of hepatitis B outbreaks (Huo et al., 2004).

The HBeAg minus strain is associated with more severe disease (acute and chronic) and a high rate of progression to cirrhosis (Lin and Kirchner, 2004).

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Review of Literature

Morphology of hepatitis B virus

Fig. (3): Genome organization of HBV. The outer lines represent the different classes of transcripts; the bold inner circles the DNA genome as present in the virion. The four major ORFs (preC/C, preS1/preS2/S, P and X) are indicated in the center (Kawaguchi et al., 2003).

The Virion DNA: Using electron microscopy, the DNA of HBV was shown to be circular; partially double-stranded (Marcellin, 2002).

In vivo, HBV particles are obviously secreted from the infected cell before the double strand is completed. Thus, the incomplete plus strand has a defined 5’ end but a variable 3’ end (Perrillo, 2002).

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Review of Literature

Morphology of hepatitis B virus

The complete minus strand has defined 5’ and 3’ ends, with a terminal redundancy of 9 bases, in the region in which the genome is triple-stranded (Robek et al., 2002).

Fig. (4): A group of hepatitis B virions (right) and enlargements of the two exposed cores (Tong and Tu, 2004).

The viral polymerase is covalently bound at tyrosine 63 via a phosphodiester bridges to the 5’ end of the minus strand (Humphries and Dixon, 2003). This linkage caused extraction of virion-derived DNA to the phenol phase during classical DNA extraction (Hadziyannis et al., 2003). The 5’ end of the plus-DNA strand is formed by an 18-base-long oligoribnucleotide, which is capped in the same manner as a messenger RNA-mRNA (Lai et al., 2003b). The genome contains two directly repeated sequences of 10 or 11 bases, DR1 and DR2 (Tong and Tu, 2004).

From the nucleotide sequence of a double-stranded version of the DNA genome, it is possible to get six different sequences of amino acid encoding triplets of nucleotides (codons). If a sequence of potential codons does not encode a protein, one of the three possible stop codons randomly interrupts it, whereas protein-encoding sequences are usually

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Review of Literature

Morphology of hepatitis B virus

free of stop codons for a distance of at least 50 codons (Carretero and Herraiz, 2004).

Protein composition of HBV particles: (1) Surface proteins (HBs proteins): Virions and large structures such as the HBs proteins are built from protein subunits that are held together by noncovalent interactions, and, in the case of HBV proteins, by disulfide bonds between cysteines of different protein molecules (Honkoop and de Man, 2003).

In HBs proteins, six protein bands are visible ranging from 24000 to 42000 apparent molecular weight. All of these proteins can also be stained specifically by immune blotting with an antibody against the smallest component, P24, which demonstrates that at least one epitope of the smallest protein is present in the larger proteins. Four of the proteins contain an oligosccharide linked to one or two of their asparagines residues (N-linked glycan). These glucoproteins migrate as somewhat larger molecules in electrophoresis than the nonglycosylated forms (Sakai et al., 2003).

Enzymatic removal of the glycan shows that HBs proteins contain only three different HBs polypeptides: (i)

The largest (LHBs) is converted by partial glycosylation in vivo from p39 to GP42.

(ii)

A middle-sized protein (MHBs) that is either single or double glycosylated at GP33 or GP36 and.

(iii)

A small protein (SHBs) that may be glycosylated at GP27 or nonglycosylated at P24 (Trifan and Stanciu, 2003).

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Review of Literature

Morphology of hepatitis B virus

Fig. (5): Hepatitis B Surface Proteins (Trifan and Stanciu, 2003).

(I) Largest Hepatitis B proteins (LHBs): It contains two domains; Pre-S (composed of the pre-S1 and pre-S2 sequence) and S. In the mature virions, the pre-S domains are accessible for antibodies; receptors and proteases (Saruc et al., 2002). The S domain and parts of the pre-S1 sequence are hidden by the pre-S2 sequence of LHBs. During biosynthesis, the entire pre-S domain of HBs seems to stay initially at the cytosol; thus, the asparagine 4 of the pre-S2 sequence is not glycosylated (Liu and Schinazi, 2002).

The amino end of the pre-S domain carries the sequence methionine-glycine, which, together with other less well-defined neighboring amino acids, serves as a signal for the replacement of the methionine by the C14 fatty acid, myristic acid (Zheng et al., 2002).

Myristylation is not essential for virion formation, but is essential for virion infectivity of HBV (Zhou et al., 2002).

During virion maturation, the pre-S domains reconfigure in about 50% of the LHBs molecules and translocate to the surface of the particle.

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Review of Literature

Morphology of hepatitis B virus

The other 50% remain on the cytosolic side. The significance of this dual topology of LHBs is obvious; most studies on the attachment of HBV to the target cells showed the necessity of the pre-S domain for binding to the cellular receptors, implying that externally localized pre-S is essential for the viral life cycle. The pre-S domain is one of the most variable regions of the HBV genome (Duseja et al., 2002).

Fig. (6): largest HBs proteins (Duseja et al., 2002).

(II) Middle Hepatitis B proteins (MHBs): This minor component of the HBV virion is composed of the S domain and of the 55 amino acid long pre-S2 domain (Wen et al., 2002).

The pre-S2 domain is hydrophilic and does not contain cysteins. It is very sensitive to proteases and can be removed selectively from HBs particles without destroying the S domain. Thus, MHBs is virtually absent in HBV vaccines that contain protease-treated HBs particles from carrier plasma (Shang et al., 2002). The pre-S2 domain is located at the surface and partially covers the S domain of MHBs. The central pan of the pre-S2 - 12 -

Review of Literature

Morphology of hepatitis B virus

domain (amino acid 7-20), which also forms the major epitope, binds a modified form of serum albumin (Li et al., 2003). HBV carriers with more than 10mg HBsAg/ml have usually free albumin binding sites on their particles, whereas at lower HbsAg concentrations all binding sites are occupied (Tang et al., 2003).

Fig. (7): Middle Hepatitis B proteins (MHBs) (Shang et al., 2002).

(III) Small Hepatitis B proteins (SHBs): The amino acid sequence of SHBs at the amino and carboxy ends has been determined biochemically. The internal sequence could be only partially analyzed by this approach (Cho et al., 2002). However, together with the protein sequence predicted by the nucleotide sequence, it became clear that the sequence of SHBs begins at the third conserved AUG of ORFS (Open reading frames), and that it ends at the stop codon of ORFS (Chen et al., 2002a). SHBs are rich in hydrophobic amino acids. It has many tryptophans, but few tyrosines, and thus, unlike most proteins possesses an ultraviolet absorption spectrum similar to tryptophan.

Furthermore, it contains a very high number of 14 cysteines, which are cross-linked with each other (Chang et al., 2002).

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Morphology of hepatitis B virus

At asparagine 146, there is a signal for addition of an N-linked glycan, which is present in approximately half of the molecules (Jung et al., 2002). This glycan has two complex antennas with terminal sialic acids (Suzuki et al., 2002).

SHBs occur in stable subtypes that were originally defined by antibodies. Antigen reactivities that were present on all known HBs isolates were considered as determinant “a”. The best-known subtypical determinants are d or y and w or r (Thakur et al., 2002a).

Fig. (8): Schematic diagram of two loops of the "a" determinant of HBs protein. The major epitope is located in the second loop between amino acids 139 and 147 (Hwang et al., 2003).

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Review of Literature

Morphology of hepatitis B virus

Fig. (9): Small Hepatitis B proteins (SHBs) (Hayashi and Furusyo, 2004).

Furthermore, a subdivision of w into w1 and w4 is possible (Wang et al., 2002a). Subtyping has been done by DNA sequencing of the SHBs gene (De Gottardi and Negro, 2004). Because all SHBs subtypes are able to induce cross-protection after immunization, the significance of serological or other subtyping is mainly of epidemiological and phylogenetic interest (Hayashi and Furusyo, 2004).

Fig. (10): HBV outer envelope contains high amounts of hepatitis B surface proteins. The envelope surrounds the inner nucelocapsid which is comprised of 180 hepatitis B core proteins arranged in an icosahedral arrangement with T=3 and T=4 symmetry. The nucleocapsid also contains at least one hepatitis B polymerase protein as well as the HBV genome (Lacarnini, 2004).

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Review of Literature

Morphology of hepatitis B virus

(2) Core proteins: (I) HBc Protein: This protein contains many hydrophilic and charged amino acids. It does not contain lipid or glycan, but if expressed in eukaryotic cells, it becomes phosphorylated (Carreno et al., 2004). It is synthesized in the cytosol of the infected cells. As an essential step in the viral life cycle, it packages its own mRNA and the viral polymerase after formation of the RNA-polymerase complex and assembles into core particles. A protein kinase is also packaged. These particles are then enveloped by patches of the ER membrane, which contains the three HBc proteins (Carretero and Herraiz, 2004).

Once core particles are assembled and have left the reducing environment of the cytosol, their structure is stabilized by disulfide bonds, but the conserved cysteines are not required for assembly or envelopment (Kondili et al., 2004).

The significance of the HBc protein for the viral life cycle is evident. After assembly of core particles and encapsidation of the viral genome, the particles allow envelopment by the HBs proteins and formation of the virus. Furthermore, some regions within the carboxy terminal part of the HBc subunits seem to be essential for genome maturation (Kobak et al., 2004).

Besides being enveloped, the core particle may play an important role in delivering the mature viral genome into the nucleus of the infected cell, leading to restoring and amplification of HBV DNA in the nucleus of the persistently infected hepatocytes (Ding et al., 2004).

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Morphology of hepatitis B virus

Fig. (11): a diagram showing Purified virions possess the HBc protein, which aggregates to form the core particle (Ding et al., 2004).

(II) HBe Protein: All hepadnaviruses have evolved to the ability to express a secretory form of their HBc proteins. They achieve this by the 5’ terminal part of the ORF C called the pre-C sequence. The pre-C sequence encodes a hydrophobic -helix, which is a secretion signal and allows for translocation of the HBe protein into the lumen of the Endoplasmic Reticulum (ER) (Wan et al., 2004). Part of the HBe protein is transported to the plasma membrane (Tanaka et al., 2004).

Another part of the HBe protein does not reach the ER lumen and is not cleaved at all. Furthermore, uncleaved HBe precursor protein accumulates as phosphoprotein (Zhou and Wu, 2004).

HBe protein is not essential for the viral life cycle. Variants without functional pre-C sequence and HBe protein arise often during acute or chronic HBV infection, especially during interferon therapy.

High levels of secreted HBe protein are found in low symptomatic highly - 17 -

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Morphology of hepatitis B virus

viraemic virus carriers. Elimination of HBeAg is usually accompanied by a flare-up of immune pathogenesis and a decrease of viraemia (Kim and Sherker, 2004).

These observations suggest that HBe protein may somehow suppress the immune elimination of HBV producing hepatocytes. It appears that a functional pre-C sequence inhibits partially HBV replication (Lai and Terrault, 2004).

The life cycle of the virus: As with all other viruses, the life cycle of HBV and its relatives in the animal kingdom can be divided into several steps: Attachment of the virus to the host cells, Penetration into the cell, Release of the viral genome, Expression of viral gene products, Replication of the viral genome, finally, Assembly of virions, and Release of the virus (Zhu et al., 2004).

HBV have a unique pathway, allowing the entry of newly synthesized viral DNA from the cytosol into the nucleus (Zhang, 2004).

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Morphology of hepatitis B virus

Fig. (12): HBV has evolved a unique life cycle that results in the production of enormous viral loads during active replication without actually killing the infected cell directly (Lacarnini, 2004).

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Epidemiology of HBV

CHAPTER II EPIDEMIOLOGY OF HBV The world health organization (WHO) estimated that 2 billion people have been infected by HBV worldwide of these more than 300 millions are chronically infected carriers of whom 25% are at risk of serious illness and eventually death from cirrhosis or hepatocellular carcinoma (WHO, 2004). Three quarters of the world population lives in areas where there are significant levels of infection (Tsai, 2004).

Fig. (13): HBsAg Endemicity (CDC, 2003).

It is estimated that worldwide, more than 50 million new infections with HBV occur yearly, and as many as 1 million deaths annually can be attributed to the effects of this infection. Studies of the prevalence of chronic infections (chronic carriers) have yielded estimates of 250 to 350 million individuals, or about 5% of the earth’s population. Prevalence of - 20 -

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Epidemiology of HBV

anti-HBs (HBsAb) is much higher than those of chronic carriers (HBsAgpositives) in all populations (Deng et al., 2004).

The annual incidence rate of acute hepatitis B is estimated to be approximately 7.4 per 100.000 in Western Europe and approximately 3.7 per 100.000 in the U.S.A. (Hahne et al., 2004).

Fig. (14): Countries with areas with moderate to high risk of infection (WHO, 2003).

The prevalence of HBV infection varies markedly throughout regions of the world (Huo et al., 2004). Hepatitis B is highly endemic in developing regions with large population such as South East Asia, China, Sub-Saharan Africa and the Amazon Basin, where at least 8% of the population are HBV chronic carrier (Wang et al., 2004b). In these areas, 70–95% of the population shows past or present serological evidence of HBV infection. Most infections occur during infancy or childhood. Since - 21 -

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Epidemiology of HBV

most infections in children are asymptomatic, there is little evidence of acute disease related to HBV, but the rates of chronic liver disease and liver cancer in adults are high (Yang et al., 2004).

Fig. (15): Geographic pattern of Hepatitis B prevalence (WHO, 2004).

Asia and Africa have previously been classified as areas of high endemicity for HBV, but in some countries, highly effective vaccination programmes have shifted this pattern towards intermediate or low endemicity. Thus, China is now the only country in Asia where HBV endemicity is high (Cooksley, 2004).

Hepatitis B is moderately endemic in parts of Eastern and Southern Europe, the Middle East, Japan, India, Korea, the Philippines, Taiwan and Thailand and part of South America. Between 10–60% of the population have evidence of infection, and 2-7% is chronic carriers. Acute disease related to HBV is common in these areas because many infections occur - 22 -

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in adolescents and adults; however, the high rates of chronic infection are maintained mostly by infections occurring in infants and children (Suskind and Rosenthal, 2004). In these areas, mixed patterns of transmission exist, including infant, early childhood and adult transmission (Lu et al., 2004b).

The endemicity of HBV is low in most developed areas, such as North America, Northern and Western Europe and Australia (Song et al., 2004). In these regions, HBV infects 5–7% of the population, and only 0.5–2% of the population is chronic carriers (Flisiak et al., 2004). In these areas, most HBV infections occur in adolescents and young adults in relatively well-defined high-risk groups, including injection drug user, homosexual males, health care workers and patients who require regular blood transfusion or hemodialysis (Wang et al., 2004b).

Most countries in Africa have high HBV endemicity, with the exceptions of Tunisia and Morocco, which have intermediate endemicity (Lada et al., 2004). Zambia has borderline intermediate/high endemicity.

In the Middle East, Bahrain, Iran, Israel and Kuwait are areas of low endemicity, Egypt, Saudi Arabia, Cyprus, Iraq and the United Arab Emirates have intermediate endemicity, and Jordan, Oman, Palestine and Yemen have high endemicity (Zhu et al., 2004).

The outcome of HBV infection is the result of complicated viralhost interactions. As in other infections with non cytopathic viruses, the immune response is thought to play a crucial role in disease pathogenesis but there is increasing evidence that a variety of viral mechanisms, some depending on the function of virally encoded proteins, have a profound impact on the infected hepatocytes, the liver microenvironment and host anti-viral responses (Brunetto and Bonino, 2004).

Park et al. (2004) found that chronic hepatitis B is often a silent disease. The patient may be virtually symptom-free with only - 23 -

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biochemical evidence of continued activity and may simply complain of fatigue and being generally unwell. The diagnosis is made after a routine medical check. Lin et al. (2004b) reported that 15-40% of HBV carriers would develop serious sequelae during their lifetime.

The detection and quantification of hepatitis B virus genomes in molecular biology-based assays appear to be the most reliable methods for monitoring HBV infection and assessing responses to antiviral treatment (Akarca et al., 2004).

The endemicity of HBV infection varies greatly worldwide and is influenced primarily by the predominant age at which infection occurs.

Endemicity of infection is considered high in those parts of the world where at least 8% of the population is HBsAg-positive. In these areas, 70% to 90% of the population generally has serological evidence of previous HBV infection. Almost all infections occur either during the perinatal period or early in childhood, which accounts for the high rates of chronic HBV infection in these populations (Tong and Tu, 2004).

Occult HBV infection: Characterized by the presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg). Serum HBV level is usually less than 104 copies /ml in these patients. Diagnosis of occult HBV infection requires sensitive HBV-DNA using the polymerase chain reaction (PCR) assay. Several possibilities have been hypothesized as the mechanisms of occult HBV infection. These include: (i)

Mutations of HBV-DNA sequence;

(ii)

Integration of HBV-DNA into host’s chromosomes;

(iii)

Infection of peripheral blood mononuclear cells by HBV;

(iv)

Formation of HBV- containing immune complex;

(v)

Altered host immune response; - 24 -

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(vi)

Epidemiology of HBV

Interference of HBV by other viruses. The precise prevalence of occult HBV infection remains to be defined (Wright, 2004).

Loss of HBsAg is associated with marked improvement in clinical

and serum biochemical features of chronic HBV; yet mild degrees of hepatitis and low levels of viral DNA may persist in the liver (Perrillo, 2004). Occult HBV infection if associated with HCV, increases the severity of HCV, unfavorable response to IFN therapy and increase risk for HCC (Allain, 2004).

El-zayadi (2005) described occult HBV infection, as it is the detection of very low HBV-DNA in individuals who lost all serologic markers of HBV. May be sero-positive for Anti-HBc +ve, Anti-HBs +ve or sero-negative for them. It is associated with very low levels of HBV rather than HBV mutants. No data on the use of antiviral treatment in patients with occult HBV infection. Its prevalence in high endemic areas 70-90% and in low endemic areas 5-20% but in Intermediate endemic areas unknown.

No data are available about the amount of hepatitis B virus genomes in liver of patients with chronic HBV infection. Intrahepatic HBV DNA was assessed in patients with serological and clinical evidence of HBV infection with either active or suppressed viral replication (Lin and Kirchner 2004). Cases with suppressed HBV activity, despite the very low levels of viraemia, maintain a relatively high amount of intrahepatic viral genomes. This virus reservoir is likely involved in HBV reactivation, which is usually observed after stopping lamivudine treatment (Lacarnini, 2004).

Table (1): Characteristics of endemic patterns of hepatitis B virus infection (Alter, 2003):

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Characteristic Chronic infection prevalence Past infection prevalence Prenatal infection Early childhood infection Adolescent/adult infection

Epidemiology of HBV Endemicity of infection Low (%) Intermediate (%) 0.5-2 2-7 5-7 10-60 Rare Uncommon (<10) (10-60) Rare Common (<10) (10-60) Very Common common (70-90) (20-50)

High (%) ≥8 70-95 Common (>20) Very common (>60) Uncommon (10-20)

There are significant differences in geographic distribution of HBV genotypes and subtypes (Guillevin et al., 2004). The global prevalence of chronic HBV infection varies widely, from high ( 8%) to intermediate (2-7%) and low (< 2%). The predominant routes of transmission vary according to the endemicity of the HBV infection. In areas of high endemicity, prenatal transmission is the main route, whereas in areas of low endemicity, sexual contact amongst high-risk adults is predominant (Yegane et al., 2004).

Hepatitis B Virus major health problem: Viral hepatitis with various forms of acute and chronic liver disease is with potential and ultimately fatal sequelae, causing a public health problem worldwide (Cui et al., 2002).

Hepatitis B is the most important of several hepatitis viruses of man because of the number of cases of the disease and the frequent occurrence of persistent infection that may lead to cirrhosis and cancer of the liver (Lee et al., 2002). HBV infection is the most common cause of chronic liver disease worldwide (Kao et al., 2002a).

Roughly, one third of the world population has been infected with HBV (Liu et al., 2003).

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Epidemiology of HBV

Although hepatitis B is an ancient disease, most of our knowledge of its epidemiology, prevention, pathogenesis, natural history and treatment were made in the last 30 years (Tibbs and Smith, 2003).

Fulminat HBV infection is an important cause of acute liver failure and is responsible for approximately 100 to 200 deaths per year in the United States (Pramoolsinsup, 2002).

1 million persons die each year from HBV-related chronic liver disease (Akbar et al., 2004). Worldwide HBV infection is one of the ten leading causes of death (Candotti et al., 2004).

Personal risk factors: Genetic factors influence disease outcome. Persistent HBV segregated within families in a manner suggestive of autosomal recessive trait and the degree of concordance for HBsAg status is significantly higher in monozygotic twins than in dizygoyic twins. Also, the allele HLA-DRB1* 1302 was associated with spontaneous elimination of infection (Zhou et al., 2002).

The prevalence of HBV is closely correlated with age ranging from 2% in those under 12 to 59% in subjects over 39 (Wang et al., 2002a).

However, those aged 15-24 are at the highest risk (Yao et al., 2004).

Lai (2004) found that infection with HBV is found to be inversely related to the educational level and is directly related to the frequency of the receipt of parenteral injection for medical purposes. They stated that risk factors for HBsAg positivity were male sex, age < or = 50 years, and a family history of HCC. Risk groups:

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Epidemiology of HBV

HBV infection is also more prevalent in certain groups in developed countries, such as immigrants from endemic areas and persons with multiple sex partners (Dandri et al., 2002).  Infants under three years of age in areas of hyperendemicity.

The risk is not only of vertical transmission from mother to infant and intrafamilial contact at home, but also through other points of close contact, for example, at nurseries (Poynard, 2002).  Health care workers and the staff and inmates of prisons and residential institutions are recognized as high-risk groups for hepatitis B because of their exposure to blood and body fluids (Sherlock and Dooley, 2002).  Drug abusers are very important risk group for HBV and the risk of HBV infection increased significantly with years of drug abuse and not associated with age and sex (Agarwal et al., 2003).  Homosexuals are one of the risk groups and infection among homosexuals is related to duration of homosexual activity, number of sexual contacts and anal contact (Stranksy, 2004).  Patients with renal failure, cancer and organ transplant are also groups at high risk (Aliyu et al., 2004).  Post transfusion populations are at risk in countries where donors are not screened (Hm et al., 2004).  The prevalence among blood donors in the United States was found to be less than 0.1% (Peksen et al., 2004). While it is as high as 15% in some countries such as Taiwan, Singapore and Hong Kong (Carretero and Herraiz 2004).

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Epidemiology of HBV

Transmission of HBV Percutanous exposure to blood, sexual transmission and peri-natal transmission account for the majority of cases of HBV infections in humans (Wang et al., 2002c).

Infection by faeces; urine, tears breast milk, bile or pancreatic juice has never been demonstrated even though HBsAg or HBV particles been detected in such fluids (Shang et al., 2002).

1- Peri-natal transmission: Transmission from mother to neonate may occur through contact with maternal blood and other infectious fluids during labour, colostrums and rarely through breast milk or placental transmission (Thakur et al., 2002b). Between 90-100% of the neonates infected by this route will themselves go on to become carriers and in turn infect their offspring’s (Saudy et al., 2003). Almost all (HBeAg) positive mothers transmit HBV to their infants who usually become chronic HBsAg/HBeAg carriers. This probably because of a tolerogenic effect of HBeAg, this crosses the placenta inducing immunologic tolerance in utero (Saab et al., 2003).

In high-carriage rate areas, HBV infection is acquired by passage from the mother to the baby. The infection is usually not via the umbilical vein, but from the mother at the time of the birth and during close contact afterward. The risk of transmission increases as term approaches and is greater in acute than in chronic carriers (Villamil, 2003). The mother is HBsAg positive, and also, but not always, hepatitis B "e" antigen (HBeAg) positive.

Antigenaemia develops in the baby within 2 months of birth and tends to persist (Tamori et al., 2003). There is an inverse relationship between the risk of chronicity and the age of infection, the risks being - 29 -

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Epidemiology of HBV

80% to 90% for infections before the age of 1 year and 20% to 50% for infections in early childhood (Tang et al., 2003).

A curious, but yet not fully explained observation is that peri-natal transmission is much more common in Asia than in Africa (Song et al., 2004). This finding may be because HBsAg positive women in Asia are much more likely to be HBeAg positive and to have higher levels of circulating HBV DNA than women in Africa. However, even when mothers in Africa are HBeAg positive, their babies do not become HBsAg positive until 6 moths to one year of age, whereas in Asia, exposed babies tend to become HBsAg positive by 3 months after birth (Zhang, 2004).

2- Sexual contact: HBV-DNA has been detected in seminal fluid, vaginal secretions and saliva suggesting that these fluids are likely to be infectious. Studies in patients attending clinics for sexually transmitted diseases have demonstrated a link between promiscuous sexual activity and the risk of hepatitis B infection and in terms of population risk, sexual transmission represents the most important route of transmission in the developed world (Ferraro et al., 2003). Hepatitis B was previously considered a sexually transmitted disease predominantly related to homosexual activity. In recent years, however, changes in sexual practice among the homosexual community, prompted by health concerns over human immune deficiency virus, have slowed the spread of HBV among this population (Giannini et al., 2003).

This decrease of homosexual transmission has high lighted the relative importance of heterosexual transmission and led to the increased recognition of HBV infection through heterosexual activity (Trifan and Stanciu, 2003).

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Epidemiology of HBV

In low prevalence areas, such as the United States, most infections (80% to 85%) occur in adults who are exposed through sexual contact to a chronically infected person (Papatheodoridis and Hadziyannis, 2004).

The Centers for Disease Control and Prevention CDC (2002) had a study of acute hepatitis B in sentinel countries (1992-1993) (such as Asian-Pacific Islanders, Alaskan natives, and Asian-Americans). The study showed that heterosexual contact with multiple partners or sexual contact with a person with hepatitis B in the 6 months preceding the acute case accounted for 41% of cases. History of homosexual contact was elicited from another 9% of participants. Infection is frequent in homosexuals and is related to duration of homosexual activity, number of sexual partners and anal contact (Marcellin et al., 2003). Thus, sexual contact is the most frequent route of acquisition of hepatitis B in the United States and probably in other developed countries (Lee et al., 2004).

3- Blood and blood products: Post transfusion hepatitis B continues to be the most common cause of HBV infection; screening of the donor units for HBsAg by ELISA does not exclude all blood units infectious for HBV. Additional measures to ensure safety of blood supply should be sought (Mark, 2003).

Blood transfusion continues to cause hepatitis B in countries where donor blood is not screened for HBsAg. Transmission is more likely with blood from paid donors than from volunteer blood (Lin and Kirchner, 2004). In the U.S.A and other developed countries, transfusion acquired hepatitis B is rare because of the testing and elimination of HBVcontaminated donor blood (Park et al., 2004).

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4- Parentral drug abuse: Parental drug abusers develop hepatitis from using shared, unsterile equipments. The mortality may be very high in this group Multiple attacks are seen and chronicity is frequent (Papatbeodoridis et al., 2002). Liver biopsy may show, in addition to acute or chronic hepatitis, foreign material, such as a chalk, injected with the elicited drug (Rivero et al., 2002).

5- Opportunities for parentral infection: Opportunities for parenteral infection include the use of unsterile instruments for dental treatment, ear piercing, subcutaneous injections, acupuncture and tattooing. Parenteral drug abusers develop hepatitis from using shared, unsterile equipment (Sherlock and Dooley, 2002).

6- Transmission in high endemic areas: High endemic areas such as Africa, Greece and the Far East, the transmission is in the childhood and probably horizontal through kissing, shared utensils such as toothbrushes and razors and injections (Vanlandschoot et al., 2002). Blood sucking arthropods such as mosquitoes or bed bugs may be important vectors, particularly in the tropics although insecticide spraying of dwellings had no effect on HBV infection (Wang et al., 2002d).

7- Exposure of unknown origin: Despite these, apparently obvious routes of transmission, in many cases the route of transmission cannot be identified (Suskind and Rosenthal, 2004).

8- Health Care Workers: Hospital staff and health care workers in contact with patients, and especially patient’s blood, usually have a higher carrier rate than the general population. This applies particularly to staff on renal dialysis or oncology units. The patient’s attendant is infected through contact with - 32 -

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Epidemiology of HBV

blood parenterally such as from pricking or through skin abrasions.

Surgeons and dentists are particularly at risk in operating on HBsAgpositive patients with a positive HBeAg. Spread from a surgeon to patients is rare and usually involves major operation. When standard cleaning procedures are used, there is no evidence that HBV infection is spread by endoscopes (Sherlock and Dooley, 2002).

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Clinical Presentation and Sequelae

CHAPTER III CLINICAL PRESENTATION AND SEQUELAE (a) Acute HBV Infection: The incubation period ranges from 2 to 20 weeks (average 8-12 weeks). The onset is usually insidious beginning with non-specific prodromal constitutional and gastrointestinal symptoms including; malaise, anorexia, nausea and vomiting, and flu-like symptoms of pharyngitis, cough, coryza, photophobia, headache and myalgias.

Prodromal symptoms abate or disappear with onset of jaundice, although anorexia, malaise and weakness may persist. These events can be related to circulating immune complexes (Livezey et al., 2002).

The usual clinical attack diagnosed in the adult tends to be more severe than for hepatitis A or C, however, the overall picture is similar.

The self-limited, benign icteric disease usually lasts less than 16 weeks, jaundice rarely exceeds 4 weeks. Occasionally, a prolonged benign course is marked by increased serum transaminase value for more than 16 weeks, relapses are rare. Cholestatic hepatitis with prolonged deep jaundice and pruritus is unusual (Craxi and Cooksley, 2003). Physical examination reveals mild tender hepatomegaly in over 70% of cases.

Mild splenomegaly and posterior cervical lymphadenopathy is found in 15-20% of cases (D. Valla, 2003).

Nevertheless, the clinical course of acute HBV infection may be anicteric. The high carriage rate of serum markers in those who give no history of acute HBV infection suggests that subclinical episodes must be extremely frequent. The non-icteric cases are more liable to become chronic than the icteric ones (Akarca et al., 2004).

(b) Fulminant Hepatic Failure (FHF): - 34 -

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Clinical Presentation and Sequelae

Based on the centers for Disease Control and Prevention Viral Hepatitis Surveillance program and sentinel countries study data, the total number of annual deaths due to fulminant viral hepatitis in the United States is approximately 2000 of which approximately half are due to hepatitis B (Desmet, 2003).

In fulminant hepatitis B (FHF), the surface antigen may be in low titer or undetectable (De Villa et al., 2003). Some cases may be negative for HBsAg because of infection by mutant strains of the virus that cause infection and disease, but do not produce HBsAg or HBeAg. It has been suggested that acute infection with the precore mutant strain of HBV that is unable to synthesize HBeAg is more frequently associated with a fulminant presentation than is the acute wild type HBV infection (Tamori et al., 2003).

The diagnosis may be made by only finding serum IgM anti-HBc (Ferraro et al., 2003) or by the detection of HBV-DNA by PCR in serum or liver of patients infected with mutant forms of the virus (Alter, 2003).

The precore mutant has been associated with fulminant disease in Israel (Barcena et al., 2003) and Japan (Friedman, 2004), but not in France (Lok et al., 2004) or in North America (Benhamou, 2004).

FHF is characterized by rapidly evolving hepatic dysfunction, coagulopathy is early fetor hepaticus confusion and drowsiness are ominous signs. Flapping tremors may only be transient or absent, but rigidity is usual. Cerebral oedema (usually without papilloedema) is the most common identifiable cause of death. There is marked elevation of serum bilirubin and transaminases, but the latter may decline towards normal despite disease progression. Hypoglycaemia, hyponatraemia, hypokalaemia and leucocytosis are common laboratory findings. The development of multi-organ failure worsens the prognosis. Case fatality is 80% without liver tranplantation (Brunetto and Bonino, 2004).

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Clinical Presentation and Sequelae

(c) Chronic Hepatitis B: Chronic HBV hepatitis is found predominantly in males. Males are six times more likely to become carriers than females. Approximately 10% of patients contracting HBV as adults and 90% of those infected as neonates will not clear HBsAg from the serum within 6 months (Goldstein et al., 2002).

Chronicity may follow an unresolved acute attack. The attack is usually mild. The patient with an explosive onset and deep jaundice usually recovers completely. Similarly, survivors of fluminant viral hepatitis seldom, if ever, develop progressive disease. After an attack, serum transaminase levels fluctuate with intermittent jaundice (Buti et al., 2002).

The patient may be virtually symptom free with only biochemical evidence of continued activity, and may simply complain of fatigue and being generally unwell. Diagnosis may even be made in a symptom-free patient after a routine medical check or at the time of blood donation (Candotti et al., 2004).

Chronic hepatitis B is often a silent disease. Symptoms do not correlate with the severity of liver damage. Apparently, stable patients with chronic HBV disease may have a clinical relapse. This is marked by fatigue and rise in serum transaminase values. Relapse may be related to seroconversion from an HBeAg positive state to an HBeAg and HBVDNA negative one. Serconversion may be spontaneous in 10% to 15% of patients per annum (Yoshida et al., 2004).

HBV-DNA can remain positive even when anti-HbeAb has developed (Carreno et al., 2004). Spontaneous reactivation from HBeAg negative to HBeAg and HBV-DNA positive state has also been

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described. The clinical picture ranges from absence of manifestations to fulminant hepatic failure (Lai et al., 2003a).

(d) Inactive carrier: Seroconversion from HBeAg to HBe antibody (anti-HBe) is usually accompanied by marked decrease in serum HBV DNA level (< 5 log 10 copies/ml) and normalization of ALT. Patients who have undergone seroconversion with suppression of HBV replication and normalization of ALT are considered to be in an inactive carrier state. It is now recognized that HBV replication persists during this phase. Using sensitive PCR assays, HBV DNA can be detected in the sera of most inactive carriers, but levels rarely exceed 4 log 10 copies/ml (Stephen, 2004).

(e) Extra-hepatic manifestations: Some extra-hepatic conditions are associated with circulating immune complexes containing HBsAg. The accompanying chronic liver disease is usually mild and slowly progressive (Haushofer et al., 2002).

Extra-hepatic manifestations include: (i)

Joint and neurologic manifestations: Guillain-Barré syndrome, encephalitis,

aseptic

meningitis,

mononeuritis

multiplex,

arthralgias and arthritis (serum sickness) (Papatheodoridis and Hadziyannis, 2004). (ii)

Glomerulonephritis: Membranous GN (MGN), mainly in children with prevalence rate 11-56.2%. The mechanism is that HBV antigens (i.e., HBsAg, HBeAg) act as triggering factors eliciting immunoglobulins and thus forming immune complexes, which are dense irregular deposits in the glomerular capillary basement membranes. INF-a therapy has been successful in treating HBV-

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related GN. A regimen of 5 million units of IFN-a subcutaneously daily for 4 months (Song et al., 2004). (iii)

Polyarteritis nodosa: High prevalence (36-69%) of HBsAg in patients with polyarteritis nodosa (PAN). Circulating immune complexes containing HBsAg, immunoglobulins (IgG and IgM), and complement have been demonstrated in the walls of the affected vessels. The clinical manifestations of the disease include cardiovascular (e.g. hypertension, pericarditis, heart failure), renal (e.g. hematuria, proteinuria, renal insufficiency), gastrointestinal (e.g. abdominal pain, mesenteric vasculitis), musculoskeletal (e.g. arthralgias, arthritis), neurological (e.g. mononeuritis), and dermatological (e.g. rashes) involvement. Small and medium-sized arteries and arterioles are affected. Adenine arabinoside, an antiviral drug and IFN-a, an immunomodulator and antiviral protein, have been used in conjunction with plasmapheresis and short course of corticosteroids (Lacarnini, 2004).

(iv)

Skin manifestations: Hives and fleeting maculopapular rash.

Women are more affected. Papular acrodermatitis, recognized as Gianotti-Crosti syndrome, has been associated with hepatitis B in children (Kumar and Agrawal, 2004).

(v)

Cardiopulmonary hepatopulmonary,

manifestations: portopulmonary

Pleural syndrome,

effusion, myocarditis,

pericarditis, and arrhythmia (Kobak et al., 2004). (vi)

Hematologic

and

Pancreatitis,

aplastic

gastrointestinal anemia,

tract

manifestations:

agranulocytosis

intravascular coagulation (Haushofer et al., 2004).

(e) Post Hepatitis B Cirrhosis:

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Clinical Presentation and Sequelae

Many patients with chronic HBV infection evolving over many years present with established liver cirrhosis, with jaundice, ascites or portal hypertension. Encephalopathy is unusual at presentation (Honkoop and de Man, 2003). In many cases, cirrhosis is clinically silent.

Development of hepatic cirrhosis in a patient with chronic HBV infection could be suspected if the patient has mild pyrexia, vascular spiders, palmer erythema, epistaxis or lower limb oedema (Guillevin et al., 2004).

Firm hepatomegaly is common, but progressive hepatocyte destruction and fibrosis gradually reduce liver size as the disease progresses. Jaundice is usually mild when it first appears (Friedman, 2004). With disease progression, easy bruising becomes more common, endocrine changes are noticed, more readily in men, signs of portal hypertension including splenomegaly, collateral vessel formation, ascites, and upper gastrointestinal tract hemorrhage develop. Evidence of hepatic encephalopathy also becomes increasingly common with advancing disease (Ding et al., 2004).

(f) The relation with HDV: The delta agent is a very small RNA particle coated with HBsAg. It is capable of infection only when activated by the presence of HBV. The interaction between the two viruses is very complex (Cui et al., 2002).

Hepatitis B and delta infection may be simultaneous (co-infection). Delta infection is strongly associated with intravenous drug abuse, but can affect all risk groups for HBV infection (Dandri et al., 2002).

Delta infection is world wide, but is particularly prevalent in Southern Europe, the Balkans, the Middle East, South India and parts of Africa (Kato et al., 2002). With co-infection, the acute delta hepatitis is usually self-limited, as the delta cannot outlive the transient HBV - 39 -

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Clinical Presentation and Sequelae

antigenaemia, the clinical picture is usually indistinguishable from hepatitis resulting from HBV alone. However, a biphasic rise in ALT may be noticed, the second rise being due to the acute effects of delta (D. Valla, 2003).

With super infection, the acute attack may be severe and even fulminant, or may be marked only be rise in serum transaminases. Delta infection should always be considered in any HBV carrier, usually clinically stable, who has a relapse (Fabrega et al., 2003). Co-infection is more likely to be followed by recovery, with the patient becoming immune to delta. Superinfection is followed by a complete recovery in only 10% of patients and there is a high chronicity rate. Both modes of infection can have a fulminant course (Giannini et al., 2003).

g) HCV and previous HBV infection: Prevalence of anti-HBc is high among HCV positive individuals 50-55% (Hu, 2002). Previous HBV infection among anti-HCV patients is associated with worse disease stage (Hadziyannis et al., 2004). Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections account for most of the cases of liver disease worldwide (Hahne et al., 2004).

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Clinical Presentation and Sequelae

Virological response of chronic hepatitis C patients with and without past history of HBV 80 60

HBV seronegative (Group I)

40

HBV seropositve (Group II + III)

20 0 At week 24

At week 48 (EVR)

At week 72 (SVR)

Graph (1): (Esmat, 2005).

Chronic Hepatitis B and Hepatocellualr Carcinoma The history of HBV is influenced by the age at which the infection is acquired, integrity of the host's immunity, and exposure to environmental cofactors. Chronic infection with HBV is more frequent in men

than

in

women,

in

neonates

than

in

adults,

and

in

immunocompromised patients than in immunocompetent ones (Wen et al., 2002). The risk of chronicity decline 60% during the second year of life to 10% by 5 years of age (Wolters et al., 2002).

In the immunocompetent patient, persistence of HBeAg-producing strains is associated with hepatic inflammation. Seroconversion to antiHBe is paralleled by exacerbation of hepatitis as a result of immunemediated liver cell necrosis and progressive clearance of infected hepatocytes and serum HBV DNA "healthy carriers" (Leung, 2002). The condition of healthy carriers is clinically a long-term benign situation. A prospective cohort study of 92 Italian healthy carriers showed that the - 41 -

Review of Literature

Clinical Presentation and Sequelae

prognosis for these subjects was excellent, with a low risk of developing cirrhosis or HCC over 10 years (Ohshiro et al., 2002).

Conversely, HBeAg-seropsitive patients with replicating HBV display various degrees of liver damage, from benign forms of chronic lobular hepatitis to more severe forms of active cirrhosis and HCC (Rodriguez et al., 2003). Persistent HBV replication is instrumental in the progression of the disease to cirrhosis and HCC. Virus heterogenecity is another important factor in the natural history of HBV infection (Stephen, 2004).

Hepatocellular carcinoma (HCC) is a frequent sequela of chronic HBV infection. In endemic areas, the risk of developing HCC among individuals chronically infected with HBV is up to 100 times that of nonHBV carriers (Tanaka et al., 2004). The risk to carriers, however, varies substantially from region to region because of factors not clearly understood (Tsai, 2004).

The classic work of Beasley, 1988 in adult Taiwanese civil service workers reported an incidence rate of 495 per 100.000 person-years in HBsAg-positive subjects McMahon et al., 1990 studying a populationbased cohort of Alaskan natives, have found an HCC incidence of 387 per 100.000 person-years in HBV carrier males of all ages. Wright (2004) have reported HCC incidence rates for a cohort of 1.069 HBV carriers in Toronto with an average follow up length of 26 months. He found HCC incidence rates of 657 per 100.000 person-years in males and 122 per 100.000 person-years in females (Zhang, 2004).

In a multicenter European study of 349 patients with compensated cirrhosis, secondary to chronic HBV infection, HCC developed in 9% during a mean follow up period of 6 years. The yearly incidence of cirrhosis among chronic HBV patients is 2.4% to 7%, with approximately 1.5% of cirrhotic developing HCC every year (Yang et al., 2004).

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Review of Literature

HBV status in Egypt

CHAPTER IV HBV STATUS IN EGYPT Hepatitis B is and will for some time be a major health problem in Egypt (Marzaban, 2003). It is recommended to consolidate the Egyptian program of infant hepatitis B vaccination, and to extend it to older children and high-risk adult groups (Sallam, 2000). The prevalence of HBsAg carriers in Egypt varies widely with age, sex, community (urban or rural), schistosomiasis and/or chronic liver disease, exposure to certain risk factor (Marzaban, 2003).

Egypt was reported by Andre (2000) to be an area of high prevalence for HBV; however, Poynard (2002) reported it to be an intermediate area. It was also reported that the carrier rate to be 8% among primary school children (Esmat, 2005).

In the early 90s, carrier rate of 3.25% and 3.6% were reported in Alexandria and Cairo by Marzaban (2003); and Hasseb (2000) found that, among school children 5-15 years from a rural village in Dakahlia, the exposure rate for HBV infection was 22% with frequency of HBsAg of 4% by counter immune electrophoresis, 16% by reversed passive haemagglutination and 18% by other tests. The frequency of HBsAb was 4.5%.

Labib et al., 2002 stated that the maximum HBsAg percentage 13.37% was found in the age group 2-5 years, decreasing afterwards to reach 12.85% in the age group 5-12 years.

In the mild 80s, Abdelaziz (2004) reported higher prevalence as follows; 8.8% in Lower Egypt and 11.7% in Upper Egypt with more prevalence in young adults and in males than females in both communities.

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HBV status in Egypt

The overall seroprevalence of HBV when HBsAg and/or HBcAb were assessed was found to increase progressively with age peaking in the 40-67 years old group at a rate of 66%, which is an extraordinarily high seroprevalence rate (Abdelaziz et al., 2000). Abdelhamed et al.

(2002) found it to be 20.7% collectively in all age groups in 1996 and 19.6% in 1997 (Orfi, 2002). There is the impression that HB carriage rate is decreased from 10% (Marzaban, 2003) to 3.2% (Abdelaziz, 2004).

As for acute HBV infection, the prevalence of HBV in Egypt is not yet adequately estimated after the use of hepatitis B vaccine (Abdelaziz, 2004). However, Orfi (2002) stated that the prevalence of acute HBV infection was 12% in children 4-14 years old and 50.9% in adults > 14 years old. The most common age group infected by HBV ranged from 21-30 years (42.4%) whereas the least infected age group was from 4-8 years (3%). The most common risk factor for infection with acute HBV was accidental puncture in (56.1%), followed by dental procedures in (48.5%) and surgical intervention in (24.2%).

Saudy et al. (2003) reported that HBV genotype D is the most prevalent in Egypt. Abdelhamed et al. (2004) found genotype D in more than 75% of Egyptian patients with positive HBV DNA.

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HBV status in Egypt

Fig. (16): Geographic distribution of HBV Genotype (Hayashi and Furusyo, 2004).

Table (2): Geographic Distribution of HBV Genotypes (Hou et al., 2005): Genotypes Distributions A (Aa, Ae) White Caucasians in Europe, Black Americans in US (Ae), Black Africans, South Africa (Aa), Asia (Aa), India B (Ba, Bj) Southern China (Ba), Taiwan (Ba), Vietnam (Ba), Asians in the USA, Japan (Bj) C China (Mainland and Taiwan), Japan, Thailand, Asians in the USA D White Caucasians (Southern Europe), Arabs (North Africa and the Middle East), India E West Africa F Central and South America G United States, France H Central America

Kamel et al., 1994 described the HBV situation in Egypt as hyperendemic in egypt, with seroprevelence rates ranging from 24% in the general population to 66% in persons 40-67 years of age (Darwish et al., 1996).

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HBV status in Egypt

Table (3): Prevalence of HCV, HBV and HBsAg in Egypt 1996 (Mohamed and Aoun, 2002): Age Adjusted Rates /100 Total Egypt Rural Urban Males Females

HCV 14.5 18.9 9.1 16.4 12.7

HBcAb 22.5 24.9 19.5 23.9 21

HBsAg 4.5 5.2 3.7 5.0 4.0

Table (4): Etiology of acute viral hepatitis among 200 *patients presented with acute symptomatic hepatitis to Embaba Fever Hospital between December 2001 and September 2002 (Zakaria et al., 2004).

Etiology

Total N 68 25 63 34

% 34 12.5 31.5 17

Single infection N % 34 17 15 7.5 29 14.5 10 5

HAV HEV HBV** HCV Non A-E hepatitis CMV 12 6 1 EBV 7 3.5 1 HGV 23 11.5 1 TTV 46 23 9 Undiagnosed 13 6.5 * Infection by more than one virus: 82 (41%) ** HDV in 8 patients (3 co-infection and 5 super infections)

0.5 0.5 0.5 4.5

Abdelhamed et al. (2002) explained this discrepancy about prevalence rates, by the fact that all the previous studies were performed on hospitalized patients with moderate to severe illness and not on the large number of mild and asymptomatic infection that must have occurred in the community.

- 46 -

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HBV status in Egypt

Etiology of Acute Viral Hepatitis in Egypt 1997-2000

21%

25%

16%

HCV HBV All-ve Mixed HEV HAV

24% 1%

13%

Fig. (17): Analysis of 1860 acute hepatitis cases (Mohamed and Aoun, 2002).

Table (5): The frequency of acute hepatitis B in different age groups in year 2002 in comparison to year 1983 (Zakaria et al., 2004): Age groups

1983 Total (235)

< 5ys 6-8ys 9-11ys 12-20ys 21-40ys 41-60ys >60ys

(36) (22) (12) (48) (73) (28) (16)

Acute HBV N % 7 19.4 9 40.9 5 41.7 28 58.3 38 52.1 11 39.3 4 25

2002 Total (200) (23) (35) (22) (26) (73) (19) (2)

- 47 -

P Acute HBV N % 0 0 0 0 6 27.3 10 38.5 41 56.2 6 31.6 0 0

NS NS NS NS

Review of Literature

HBV status in Egypt

Age distribution of patients with acute hepatitis B 70 60 50 40 Sedes 1

30 20 10 0 < 14 ys

14-30 ys

31-50 ys

> 50 ys

Graph (2): Age distribution of patients with acute hepatitis B (Zakaria et al., 2004).

Although HBV vaccination was effective in decreasing the infection in those below 10 years (Esmat, 2005). HBV is still responsible about a high percentage of acute viral Hepatitis disease in Egypt (Abdelaziz, 2004).

HBV Status in Egypt in Various groups: (1) Among the general population: Based on HBsAg positivity alone, Esmat (2005) reported 6.7% carriage rate in two rural villages in the Nile Delta and El-Zayadi (2005) reported 8% carriage rate among primary school children. Studies which included the overall seroprevalence of HBV (HBsAg and/or anti-HBc), gave higher percentage rates of 20.7% (Saudy et al., 2003) and 19.6% (Hasseb, 2000) among different regions of Egypt.

Hasseb (2000) determined the prevalence of HBV in Egypt by studying 3186 normal Egyptian born and living in 4 Egyptian governorates (Kaliobia, Menoufia, Gharbia and Aswan). They represented both sexes, both urban and rural populations and include all - 48 -

Review of Literature

HBV status in Egypt

age groups (except neonates and infants in their first year of life), thus permitting a mass evaluation of HBV in Egypt. Serum from each participant was tested for HBsAg, HBcAb and HBsAb. They found that the prevalence of HBsAg in the whole population was 6.35% and HBcAb was 38.68% of those studied (Marzaban, 2003).

Sallam (2000) measured the exposure rate of hepatitis B in Egypt.

Exposure rate depends on both HBsAg and HBcAb where: No. of cases positive for HBcAb + No. of cases positive for Exposure rate =

HBsAg alone Total number of cases studied

He reported that the exposure rate to hepatitis B virus was found to be 40.3%. This percent was similar to El-Zayadi (2005), who showed that the exposure rate for HBV in whole population studies was found to be (40.37%).

Heikal (2005) reported that HBV was more prevalent in Upper Egypt (11.7%) than in Lower Egypt (8%). Sallam (2000) showed that the highest prevalence of HBV in Egypt was in Aswan.

The high prevalence of HBsAg together with high exposure rate to HBV found in Gaffar studies and El-Sahly studies are sufficient to transfer Egypt from an area of intermedicity for HBV as categorized by the WHO to an area of high endemicity of this virus (Abdelhamed et al., 2002).

(2) Among patients with acute hepatitis: Marzaban (2003) studied the etiology of acute hepatitis in Egyptian children of both urban and rural areas. Two groups of patients were studied. Group one consisted of 77 infants and children with acute hepatitis admitted to pediatric department of Abbasia fever hospital (urban area), group (2) consisted of 21 infants and children with acute hepatitis admitted to Banha fever hospital (rural area). The ages of the - 49 -

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HBV status in Egypt

children in both studies ranged between 2 months and 17 years. In urban group HBV was responsible for (41.8%) of the cases, HDV (whether a coinfection or as superinfection) was responsible for (26%) of the cases (Abdelaziz, 2004).

(3) Among Chronic Liver Disease patients: Abdelaziz (2004) found that 43% of patients with nonschistosomal chronic liver disease and 36% of patients with hepatocellular carcinoma were HBsAg carriers in their series.

Another study by Aoki et al. (2002) was done on 135 adult patients living in Alexandria governorate, mostly in rural areas of the Nile Delta, and who had established chronic liver disease showed a carrier rate of HBsAg of 16%, and 64% of anti-HBcAb. Similarly, Attia et al. (1998) found a 21% carrier rate of HBsAg and 54% of anti-HBcAb among cirrhotic patients in their own study.

(4) Schistosomal infection: Schistosomiasis contributes to significantly increased HBV infection (Abdelaziz, 2004).

A study was applied on patients with acute viral hepatitis who were followed up for 12 months. Patients with concomitant schistosomiasis had higher mean values for liver function test results and a greater proportion had abnormal liver function test results during hospitalization and follow-up than those with acute viral hepatitis only. Concomitant schistosomiasis increased the prevalence and prolonged splenomegaly and morbidity due to acute viral hepatitis. Both male sex and concomitant schistosomiasis prolonged the HBsAg carrier state. Acute viral hepatitis frequently

converts

uncomplicated

intestinal

schistosomiasis

to

hepatosplenic schistosomiasis (Marzaban, 2003).

High prevalence of chronic HBs antigenaemia (58%) has been demonstrated in children with schistosomal hepatic fibrosis but only (2%) - 50 -

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HBV status in Egypt

in normal children, this denotes that children with SHF represent a dangerous reservoir for hepatitis B infection to the community (Abdelhamed et al., 2002). It is said that, patients suffering from heaptosplenic schistosomiasis experience 28% higher HBsAg carrier rate.

An important observation is the diminished anti HBs rate in such patients.

This may be due to an immunological defect, resulting in an unsatisfactory antibody response and chronic hepatitis B antigenemia (Abdelhamed et al., 2002).

Schistosomiasis does not only increase the severity of HBV infection but also elevates to risk of HCC over that associated with the HBV infection alone (Badawi and Michael, 1999).

However, a study by Marzaban (2003) revealed that the primary residence in the Nile delta and Valley areas where shistosomiasis is highly endemic was a statistically significant risk factor for HCV, but not HBV infection.

(5) Drug abusers: Labib et al. (2002) studied the prevalence of HBV among Egyptian drug abusers and it was 57.75 and 15.4% in injecting and noninjecting drug abusers respectively. The former group showed significantly more common signs of liver disease as hepatomegaly, elevated enzymes, cirrhosis and history of jaundice. These manifestations correlated positively with the duration of addiction thus all injecting drug abuses cases > 10 years duration were infected by HBV but this was less obvious in non-injecting ones.

(6) Children to carrier mothers & peri-natal infection: 17% of newborn infants to HBsAg positive mothers were HBsAg positive and none of the mother was HBeAg positive (Abdelaziz, 2004).

(7) Among Blood Donors: - 51 -

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HBV status in Egypt

The carrier rate among blood donors was 3.9% for villagers (Annual report Agouza center, 1982) whereas Abdelaziz (2004) and Marzaban (2003) in Alexandria and Cairo reported a carrier rate of 3.25% and 3.6% respectively. A more recent study El-Zayadi (2005) on 90 blood donors showed 4.4% HBsAg positivity.

(8) Among the immunocompromised: Several workers reported an increased susceptibility to hepatitis B virus in immuncompromised patients. A study done by Sallam (2000) on 137 patients with an immuncomprimising illness (Leprosy, Bronchial asthma and diabetes mellitus) along with 25 healthy individuals serving as controls indicated that HBsAg carrier rate was 4% for the control healthy group, 7% for bronchial asthma, 10% for diabetes and 24% for leprosy.

(9) Dental field: The exposure rate of HBV among dentists working or studying at the Faculty of Oral and Dental Medicine, Cairo University was found to be 27.1% with a carrier rate of 7.1% (Abdelhamed et al., 2002).

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Diagnosis of HBV

CHAPTER V DIAGNOSIS OF ACUTE AND CHRONIC HEPATITIS B (1) Acute Hepatitis: Newly infected subjects have an incubation period averaging 8-12 weeks after exposure and before clinical symptoms, and the length of time depends on size of inoculum and host factors (Akahane et al., 2002). HBsAg appearance accompanies the prodromal phase, the arthralgia and skin rash that sometimes appear are thought to be related to formation of HBsAg anti-HBs complexes. All of this occurs before ALT elevations and other manifestations of liver involvement (Akcam et al., 2002).

HBsAg concentrations peak at or shortly after an increase in serum ALT. The duration of HBsAg positivity can be highly variable and usually has little relationship to recovery, but the ALT and HBsAg decline and disappear together. HBsAg is cleared from serum early in 10% of patients by the time they present to physicians (Aoki et al., 2002).

Such a serological event can cause diagnosis problems, but in such cases, the detection of IgM anti-HBc can help to confirm the diagnosis. The presence of a strong IgM anti-HBc is indicative of acute phase infection (Arase et al., 2002).

HBeAg and HBV-DNA is detected in sera of patients before symptoms develop and at about the same time that HBsAg is detected.

Both are considered markers of viral replication. The disappearance of these markers and the seroconversion to anti-HBe precede clearance of HBsAg, and such events predict recovery. The best serological indicator of recovery is the appearance of anti-HBs, but in many cases, it is not 74

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Diagnosis of HBV

detected before recovery is already clinically evident (Beckebaum et al., 2002).

2. Chronic outcomes of HBV infection: In most adult cases of acute hepatitis B serum HBsAg disappears within 12 to 16 weeks after exposure, but in about 10% of patients antigenaemia will be detected for more than 6 months. A 6 months persistence of HBsAg by convention defines the carrier state because these patients have a reduced likelihood of recovery. Most remain chronically infected and experience several possible outcomes; every year about 1% of adult-onset carriers will spontaneously lose HBsAg and seroconvert to anti-HBs. In contrast, 90% of babies who have been infected perinatally of within the first 5 years of life become carriers and have little chance of spontaneous recovery during their lifetime (D. Valla, 2003).

After the acute phase, a typical marker pattern is evident; IgM antiHBc declines slowly, but markers of viral replication HBeAg and HBVDNA

remain

detectable, with

anti-HBe

and

anti-HBs

usually

undetectable. Elevated ALT values indicate ongoing active hepatitis.

Some carriers will have persistently active hepatitis, and some will progress to cirrhosis and possibly hepatocellular carcinoma (Craxi and Cooksley, 2003).

At intervals after the acute phase that are yet unpredictable, many patients become asymptomatic carriers. That is, while HBsAg and antiHBc persist, ALT levels return to near normal and seroconversion from HBeAg to anti-HBe occurs. HBV-DNA declines, but patients remain infectious. This transition from active to asymptomatic chronic infection can occur directly after the acute phase, or it may happen years later, often after a flare of symptoms and a brief increase in ALT levels.

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Diagnosis of HBV

Occasionally, asymptomatic carriers experience a return to active hepatitis with reappearance of HBeAg and HBV-DNA. A few chronic carries may have serum levels of HBsAg below detectable limits.

Although asymptomatic carriers appear to be in an inactive state of hepatitis, they remain at significantly high risk of cirrhosis and hepatocellular carcinoma (Cooksley, 2004).

Serology and molecular testing: Because

the

clinical

symptoms

of

HBV

infection

are

indistinguishable from other forms of viral hepatitis, definitive diagnosis is dependent on serologic testing for HBV infection. Varieties of tests are available to make the diagnosis of HBV infection (Berger and Preiser, 2002). Acute HBV infection is characterized by the presence of HBsAg in serum and the development of IgM class antibody (IgM anti-HBc) (Fabrega et al., 2003).

Both serologic and molecular assays are useful in the diagnosis of viral hepatitis. They may detect early infections before other sings of disease appear, differentiate acute from chronic infections, and detect persistence of viraemia or verify development of immunity (Bienzle et al., 2003).

(I) HBsAg: The discovery of the Australia antigen and its association with hepatitis was a major advance in the laboratory diagnosis of viral hepatitis (Bonacini et al., 2002). The Australia antigen (HBsAg) could be detected in most of the patients with acute and chronic disease by simple assay procedures

as

agar-gel

diffusion

(AGD)

or

counter

immunoelectrophoresis (Goldstein et al., 2002). Following exposure to HBV, HBsAg can be detected in serum for several weeks before increased serum amino tansferase levels are observed. HBsAg persists 74

Review of Literature

Diagnosis of HBV

during the prodromal phase and is not usually cleared from the serum until convalescence (Chan et al., 2002).

Hepatitis B surface antigen (HBsAg) subtyping method based on a commercial enzyme immunoassay (EIA) for detection of HBsAg in which the procedure was modified to include the use of monoclonal antibodies with restricted anti-HBs specificities. This method, which was able to classify HBsAg as: ayw1, ayw2, ayw3, ayw3* (intermediate between ayw3 and ayw4), ayw4, ayr, adw2, adw4 and adr. This reliable procedure, derived from commercially available reagents, can be easily used in several applications such as large epidemiologic studies and as a substitute for nucleotide sequencing genotyping which is not adapted for large-scale screening and not applicable on samples from nonviremic hepatitis B virus (HBV) carriers (Cerenzia et al., 2002).

It was subsequently found that most of the remaining patients with hepatitis B also had serum HBsAg, but that tests with significantly greater sensitivity were necessary to detect those (Chan et al., 2002). Ferraro et al. (2003) develop a modified "sandwich" radio immunoassay (RIA) to detect HBsAg this diagnostic method has a dilution sensitivity more than 100 times that of AGD and can detect less than 0.5ng HBsAg/ml serum (Craxi and Cooksley, 2003). In the last decade, enzyme sandwich immunoassays (EIAs) have largely replaced RIAs. Recent modified EIAs that use micro-particles, computerized instrumentation produce very rapid and completely automated micro-particle enzyme immunoassay (MEIAs) for HBsAg (Conjeevaram and Lok, 2003).

HBsAg is often used as the serological marker to screen for HBV infection in the investigation of liver cirrhosis (Chang, 2003). HBsAg is the most important serological marker of acute and chronic hepatitis B infection. Therefore, sensitivity of the currently used detection system for 74

Review of Literature

Diagnosis of HBV

HBsAg is critical to blood screening, diagnosis of HBV infection and therapy monitoring of HBV infected individuals (Giannini et al., 2003).

The performance of HBsAg screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B (De Villa et al., 2003). HBV-associated HCC expresses HBsAg on its cell surface, and this may serve as a tumor-associated antigen (Desmet, 2003).

The most commonly used definition of the carrier state is presence of HBsAg in serum for at least 6 months. It is important to recognize that occasionally it may take a few more months for some individuals to clear HBsAg, but HBsAg should be undetectable 1 year after acute HBV infection (Friedman, 2004).

Chronic carriers of HBV usually show HBsAg in their sera.

However, in some individuals this antigen can't be detected by routine serological assays lack of HBsAg might be mutations in the part of the molecule recognized by specific antibodies. At least some of the chronic low-level carriers where HBsAg is not detected could be infected by diagnostic escape mutants and/or by variants with impaired replication (Brown, 2005).

(II) HBsAb: Antibodies are formed against several antigenic sites HBsAg and are all generally designated as HBsAb. Some of these are unique to specific viral strains, but all wild strains of HBV contain a common immunological determinant commonly referred to as "a". HBsAb/a is the most prominent antibody in convalescent sera or in vaccines (Cui et al., 2002).

The group specific antibodies directed against the HBsAg prevent viral infection or reinfection and reduce the virus load in body fluids (Dandri et al., 2002). Clearance of HBsAg from the sera was observed 74

Review of Literature

Diagnosis of HBV

within 6 months after disease onset, and the corresponding antibody appeared within 12 months (Duseja et al., 2002).

HBsAb or their immune complexes were found in 83% of acute hepatitis B and in 37% of chronic ones. Their detection in a single serum sample should not be considered as an evidence of elimination of the infection (Hu, 2002). Circulating HBsAb secreting B cells were significantly higher in early acute hepatitis B or early after HBs vaccination than in chronic hepatitis B and decreased in the follow-up as a result of compartmentalization to lymphoid tissues (Haushofer et al., 2004).

HBV replication progressively disappears in most of the patient after seroconversion of HBsAg to HBsAb (Haushofer et al., 2002). In patients who recover from acute hepatitis B, seroconversion to anti-HBs occurs shortly after disappearance of HBsAg. There may be subjects who have an extended period between loss of antigenaemia and appearance of anti-HBs, and this period is referred to as the "core window", a time when antibodies to hepatitis B core proteins are the only serological indicators of HBV infection (Friedman, 2004). This core window may last for a few days to several months. In an HBV infection, serum anti-HBs indicate lifelong immunity to re-infection by hepatitis B. In vaccine recipients, the immune response is not usually as strong as the immune response to infection, and vaccine-induced anti-body does not persist as long, but declines predictably after the final inoculations (Flisiak et al., 2004). Nonetheless, the efficacy of these vaccines proves that humoral anti-HBs responses to these surface antigens are protective against HBV infection (Haushofer et al., 2004).

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Diagnosis of HBV

Fig. (18): Evolution of HBV markers in acute infection (Guillevin et al., 2004).

Fig. (19): Characteristics of progression to chronic HBV infection (Guillevin et al., 2004).

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Diagnosis of HBV

(III) HBcAg: Hepatitis B core antigen (HBcAg) is not directly detectable in serum because of the HBsAg envelope that surrounds it. Furthermore, any exposed HBcAg reacts with circulating antibody, thus blocking its detection. However, HBcAg can be identified in liver biopsies by immunofluorescent techniques, and the histochemical detection of this antigen is occasionally used as a marker of viral replication (Gheit et al., 2002). HBcAg does not circulate in serum (Hübscher and Potmann, 2002).

(IV) HBcAb: Detection of IgM HBcAb is a useful marker for HBV acute infection (Goldstein et al., 2002). IgA HBcAb is a sensitive marker for HBV replication, and its absence may exclude HBV replication (Kao et al., 2002b).

Anti-HBc (antibody to HBcAg), is perhaps the most serological prominent marker of HBV exposure. HBcAg is very immunogenic and consequently high anti-HBc titers early in an infection suggest a period of active viral replication. The antibody is detectable in serum shortly after the appearance of HBcAg, but usually before elevations in ALT, and it persists in serum throughout disease and recovery (Kao and Clsen, 2002). The earliest anti-HBc in acute disease is predominantly. IgM antibody, with lower activities of IgG and IgA anti-HBc also present concurrently. There is no evidence; however, that serum anti-HBc offers any immune protection (Mao et al., 2004).

(V) HBeAg / Anti-HBe: HBeAg in serum can be detected by a sandwich immunoassay format similar to that for HBsAg. The antigen is captured by antibody affixed to a solid phase and is then detected with a second labeled antibody (Gotsman et al., 2002). The HBeAg positive chronic hepatitis 74

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Diagnosis of HBV

patients displayed significantly higher transaminase levels than those negative for HBeAg (Guptan et al., 2002).

Before the introduction of DNA testing, HBeAg was used as a marker for active HBV replication and infectivity and as a criterion for treatment (Brunetto and Bonino, 2004). Its role as a marker of active viral replication is associated with the increased risk of HCC. The risk of HCC was increased by a factor of 10 among the men who were HBsAg positive only and by a factor of 60 among those who were HBsAg and HBeAg positive. The striking increase in cases of HCC in men who were HBeAg+ve might suggest an oncogenic role of HBeAg (Guillevin et al., 2004).

Table (6): HBeAg (Liang and Ghany, 2002).

Analysis of 505 cases history of patients among men with viral hepatitis demonstrates that the frequency of HBsAg detection by Enzyme Immune Assay (EIA) in saliva of patients in acute period was found to correlate with the frequency of its detection in serum. In early 74

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Diagnosis of HBV

convalescence the frequency of detection of that antigen in serum (59.5% of patients) was significantly higher than in saliva (23.8%).The frequencies of HBeAg detection by EIA in saliva samples was significantly higher than that in serum samples in both acute phase (84.3% and 28.1% of patients, respectively) and in early convalescence (56.2% and 3.1% of patients, respectively). HBeAg became undetectable in blood whereas HBs-antigenemia was still pronounced, and a month after the beginning of the disease it was not found in serum specimens. In saliva, HBeAg was detected in 95.8% of patients observed directly after admission. A month after the beginning of the disease it was detected in saliva of 66.7% of patients and, by the end of observation period, in 12.5% of patients recovered from viral hepatitis. HBV DNA revealed by PCR in saliva and serum of HBV-infected patients was detected in acute period not only in serum (84.6% of cases) but also in saliva (46.2% of cases) (Hakozaki et al., 2002).

Persistence of HBeAg has been associated with progression to chronicity; persistence of HBeAg for more than 12 weeks indicates chronicity, whereas early seroconversion to anti-HBe signals recovery (Hayashi and Furusyo, 2004). A competitive assay procedure is used to detect anti-HBe, using the same kit reagent provided for HBeAg. Serum concentrations of HBeAg and anti-HBe are typically low and, unlike antiHBc or anti-HBs, when a patient develops anti-HBe in recovery, the antibody will probably disappear within 12 months (Hadziyannis et al., 2003).

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Diagnosis of HBV

Table (7) Interpretations of available serologic test results for HBV (Lacarnini, 2004): HBsAg IGM ANTIHBC + -

TOTAL ANTIHBC -

ANTIHBS

INTERPRETATION

-

+

+

+

-

-

+

+

-or +

+

-

+

-

-

-

-

+

-

-

+

+

Early HBV infection before anti-HBc response.

Early HBV infection. Because IgM antiHBc is positive, the onset is within 6 months. IgG antibody usually appears shortly after IgM; therefore, both are usually posi-tive when IgM is positive.

Recent acute HBV infection (within 4-6 months) with resolution; i.e., HBsAg has already disappeared. Anti-HBs usually appear within a few weeks or months of HBsAg disappearance.

HBV infection, onset at least 6 months earlier because IgM anti-HBc has disappeared. Prob-able chronic HBV infection.

Response to hepatitis B vaccine. No evidence of infection.

Past HBV infection, recovered.

Table (8): Serological test findings at different stages of HBV infection and in convalescence (Honkoop and de Man, 2003).

Stage of infection

HBsAg AntiHBs Late incubation period + Acute hepatitis B or + persistent carrier state HBsAg –ve acute hepatitis Recovery with loss of anti-HBs Healthy HBsAg carrier ++ + Chronic hepatitis B, + persistent carrier state HBV infection in recent ++ past, convalescence HBV infection in distant + or past, recovery Recent HBV vaccination, ++ repeated exposure to antigen without infection,

74

ANTI-HBC IGG IGM

HBeAg

AntiHbe

+

+ or +

+

-

-

+

-

-

+

-

-

-

++ + ++ + ++

+ or -

-

+

+ or + or -

+

-

-

+

+ or -

-

-

-

-

-

-

-

Review of Literature

Diagnosis of HBV

or recovery from infection with loss of detectable antiHBc

Table (9): Hepatitis B tests (Perillo, 2004).

Hepatitis B Hepatitis B (HBV) antibodies and/or antigens are detected. Additional (HBV) tests may be needed to determine whether you have an acute or chronic HBV infection.  Hepatitis B surface antigen (HBsAg) indicates an active infection. If levels remain high, this indicates a chronic carrier. You can infect others with HBV.  Hepatitis B surface antibody (HBsAb) indicates end of active infection and protection against HBV for life. It can also indicate protection from receiving the HBV vaccine.  Hepatitis B core antibody (HBcAb) indicates that you have been infected with HBV. It does not distinguish between a past or present infection.  Hepatitis B e-antigen (HBeAg) indicates an active contagious state.  Hepatitis B e-antibody (HBeAb) indicates the end of an HBV infection. You are less contagious but can still infect others.  HBV DNA testing detects genetic material (DNA) from the hepatitis B virus.

FDA- Approved Tests for Hepatitis B Table (10): Antibody to Hepatitis B Surface Antigen (HBsAg Assay) (Lok et al., 2004).

Tradename(s) Format Sample

Approval Date Serum / Donor Abbott Laboratories 4/1/1985 Plasma Screen & Abbott Park, IL Conf. Kit US License 0043 Serum / Donor Bio-Rad 12/28/1999 Plasma / Screen & Laboratories Blood Cadaveric Conf. Kit Virus Division Serum Redmond, WA US License 1109 Serum / Donor Ortho-Clinical 7/23/1971 Plasma Screen & Diagnostics, Inc. Conf. Kit Raritan, NJ US License 1236

Auszyme Monoclonal

EIA

Genetic Systems HBsAg EIA 2.0

EIA

Ortho Antibody to HBsAg ELISA Test System 2 AUK-3

EIA

RIA

Serum / Plasma

ETI-MAK-2

EIA

Serum / Plasma

Use

Manufacturer

Donor Screen & Conf. Kit Donor Screen & Conf. Kit

DiaSorin s.r.l.

Saluggia, Italy US License 1257 DiaSorin s.r.l.

74

5/9/1986

4/18/1995

Review of Literature

Diagnosis of HBV

Table (11): Hepatitis B Surface Antigen (Anti-HBs Assay) (Lok et al., 2004).

Tradename(s) Format Sample Use

Manufacturer

Ausab

RIA

Serum / Anti-HBs Plasma

Ausab EIA

EIA

AB-AUK-3

RIA

Serum / Anti-HBs Plasma Serum / Anti-HBs Plasma

ETI-AB-AUK

EIA

Serum / Anti-HBs Plasma

Approval Date Abbott Laboratories 2/5/1975 Abbott Park, IL US License 0043 Abbott Laboratories 11/18/1982 DiaSorin s.r.l.

Saluggia, Italy US License 1257 DiaSorin s.r.l.

6/27/1989

4/18/1995

Table (12): Hepatitis B Virus Core Antigen (Anti-HBc Assay) (Lok et al., 2004).

Trade name(s) Corzyme

Format

Sample Use

Manufacturer

EIA

Serum / Donor Screen Plasma

Ortho HBc EIA ELISA Test System

Serum / Donor Screen Plasma

ABCOREK

RIA

Serum / Donor Screen Plasma

ETI-ABCOREK

EIA

Serum / Donor Screen Plasma

Approval Date Abbott Laboratories 3/19/1991 Abbott Park, IL US License 0043 Ortho-Clinical 4/18/1991 Diagnostics, Inc. Raritan, NJ US License 1236 DiaSorin s.r.l. 3/19/1991 Saluggia, Italy US License 1257 DiaSorin s.r.l.

3/19/1991

(VI) HBV DNA: HBV DNA is regarded to be the most sensitive marker of viral replication and infectivity, which was previously related to the presence of HBsAg in serum and HBcAg in liver cells. In liver tissue, different molecular patterns can be recognized as free viral DNA and integrated sequences. Introduction of the PCR allows the detection of very small amounts of viral DNA and has markedly improved diagnostic sensitivity.

The study of HBV DNA has become a valuable part of the routine

74

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diagnosis in chronic hepatitis B facilitating more precise statements about the course and prognosis of the disease (Chu and Lok, 2002).

Immunoaffinity capture of intact HBV with biotinylated pre-S1 antibodies coupled to streptavidin-coated magnetic beads is apparently an optimal method of sample preparation for amplification of HBV DNA in patients in the pre-HBsAg window period, and for detecting low-level viraemia persistent in several individuals who were former chronic HBV carriers who had seroconverted i.e. developed anti-HBs (Kao and Clsen, 2002).

The principle of the polymerase chain reaction (PCR) is the amplification of DNA using a heat stable polymerase. It has been used to detect extremely small quantities (i.e. organisms) of HBV DNA in serum (Berger and Preiser, 2002). This principle of target amplification techniques is to synthesize a large number of copies of the viral genome (amplicons) in a cyclic enzymatic reaction then, they can be detected by various methods, and the amount of viral genomes in the clinical sample can be quantified. The PCR method uses several temperatures and one enzyme, a thermostable DNA polymerase. The amplicons are double – stranded DNA. PCR can be applied to HBV DNA directly after extraction of nucleic acids or lysis of the viral envelope. Each complete PCR cycle doubles the number of DNA copies; after n cycles, 2 n copies of each number of DNA molecules present at the beginning of the reaction are therapeutically synthesized. In fact, the reaction is saturable and reaches a plateau generally after 35 to 45 cycles (Pawlotsky, 2002).

Real-time PCR techniques have been developed. The principle is to detect amplicon synthesis and to deduce the amount of viral genomes in the starting clinical sample during rather than at the end of the PCR reaction. These methods are theoretically more sensitive than classical 74

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Diagnosis of HBV

target amplification techniques and are not prone to carryover contamination (Kakimi et al., 2002b).

Viral genome sequence analysis is aimed at identifying signature sequences and/or amino acid substitutions specific positions. Signature sequences are used to classify viral strains into phylogenetic groups, called genotypes. This method can be applied to the detection of known clinically relevant motifs or mutations (Kakimi et al., 2002a).

PCR-ISH is a technique that combines the sensitivity of PCR with the localizing ability of in situ hybridization (ISH). PCR-ISH is a sensitive technique for localizing HBV in tissue sections and the low level of HBV replication persists in HCC cells (Kajiya et al., 2002).

HBV DNA can be detected on average 21 days before the appearance of HBsAg, even when HBsAg is assayed with the most sensitive test (Jiang et al., 2002). HBV DNA continued to be detectable 1-3 weeks after the production of the HBsAg (Jung and Pape, 2002).

PCR is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed paraffin embedded tissues.

Freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore formalin-fixed paraffin embedded tissues may be used for the detection of viral DNA sequences by PCR (lsikawa et al., 2002).

PCR assay was developed for the direct detection of HBV in paraffin embedded liver tissue and applied this assay to determine whether HBV DNA exists in livers. The limit of detection of HBV DNA by these methods was estimated to be one genomic copy of HBV DNA per cell (Liaw et al., 2002).

Detection of HBV DNA is a reliable evidence of the presence of the viral agent and its replication. With the PCR, it became possible to 74

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Diagnosis of HBV

extend the sensitivity by amplification of viral sequences. A study was done to test whether viral sequences could be found in liver tissue specimens negative for HBV DNA by conventional hybridization techniques and confirmed PCR to be more sensitive method to detect HBV DNA in the liver compared with the conventional hybridization technique (Irshad et al., 2002).

PCR is a highly sensitive technique for the detection of HBV-DNA in serum, liver tissue and peripheral mononuclear blood cells. In chronic HBV, it is particularly useful for identification of the infectious subjects who are HBsAg positive and anti HBeAg antibody positive and for the follow up of HBV infections in liver transplantation programs (Sherlock and Dooley, 2002). Hwang et al. (2003) stated that HBV DNA in serum detected by PCR is a good marker of the level of viraemia. It can be correlated with serum transaminase levels and parallels the presence of HBsAg in serum. It can be found in serum and liver after the loss of HBsAg. PCR was used to detect HBV DNA in the sera and livers of patients with chronic HBV after treatment-induced or spontaneous loss of serum HBsAg. HBV DNA may be detectable by PCR in liver tissue years after the disappearance of HBsAg even in the absence of detectable HBV DNA in serum (Humphries and Dixon, 2003).

Serum HBV DNA level is strongly correlated with liver HBV DNA levels in chronic HBV with cirrhosis. Liver free HBV DNA can still be detected in about half of the cirrhotic patients with undetectable serum HBV DNA. Serum HBeAg is not a good predictor of serum or liver HBV DNA levels in cirrhotic patients (Hou et al., 2005).

(VII) Liver biopsy: Histologic examination of liver biopsy material is still the best way of assessing the severity of chronic hepatitis B and establishing the 74

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Diagnosis of HBV

prognosis (Pawlotsky, 2002). The purpose of a liver biopsy is to assess the liver damage and to rule out other causes of liver disease. The histologic diagnosis of chronic hepatitis should include the etiology, grade of necroinflammatory activity and stage/extent of fibrosis (Liu et al., 2003).

Histological Activity Index by Knodell (Kao et al., 2002a): The Knodell system was the first formal numerical scoring system to be proposed. It involves assessment of four main histological features, as summarized in Table (13). Scores for the individual categories can be added to produce an overall "histological activity index' ranging from 022. The knodell system has been widely used in many studies throughout the world.

Table (13): Histological Activity Index by Knodell (Kao et al., 2002a): PERIPORTAL ± BRIDGING NECROSIS. 0 = NONE. 1 = MILD PIECEMEAL NECROSIS. 3 = MODERATE PIECEMEAL NECROSIS (<50% OF CIRCUMFERENCE OF MOST TRACTS).

4 = MARKED PIECEMEAL NECROSIS ({50% OF CIRCUMFERENCE OF MOST TRACTS).

5 = MODERATE PIECEMEAL NECROSIS AND BRIDGING NECROSIS. 6 = MARKED PIECEMEAL NECROSIS AND BRIDGING NECROSIS. 10 = MULTILOBULAR NECROSIS. INTRALOBULAR DEGENERATION AND FOCAL NECROSIS 0 = NONE 1 = MILD (ACIDOPHILIC BODIES, BALLOONING DEGENERATION AND/OR SCATTERED FOCI OF NECROSIS IN < 1/3 OF LOBULES OR NODULES. 3 = MODERATE (INVOLVEMENT OF 1/2-2/3 LOBULES OR NODULES) 4 = MARKED (INVOLVEMENT OF > 2/3 OF LOBULES OR NODULES).

PORTAL INFLAMMATION 0 = NONE. 1 = MILD (FEW INFLAMMATORY CELLS IN < 1/3 OF TRACTS).

3 = MODERATE (INCREASED INFLAMMATORY CELLS IN 1/3-2/3 OF TRACTS).

4 = MARKED (NUMEROUS INFLAMMATORY CELL IF > 2/3 OF TRACTS).

FIBROSIS 0 = NO FIBROSIS 1 = FIBROSIS PORTAL EXPANSION. 3 = BRIDGING FIBROSIS (PORTAL-PORTAL OR PORTAL-CENTRAL).

4= CIRRHOSIS.

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Histological Grading and Staging of Chronic Hepatitis (Kawaguchi et al., 2003): This scheme represents an extension of the original knodell system, with a number of minor modifications. Firstly, a continuous scale is used for scoring of each of the features assessed. Secondly, necroinflammatory activity and fibrosis are considered as separate categories. Thirdly, confluent necrosis is separated from periportal hepatitis and is included as a separate category of necoinflamamtory activity.

Table (14): Modified Hai Grading: Necroinflammatory Scores (Kawaguchi et al., 2003): SCORE A. Periportal or periseptal interface hepatitis (Piecemeal necrosis) Absent Mild (focal, few portal areas) Mild/moderate (focal most portal areas) Moderate (continuous around < 50% of tracts or septa).

Severe (continuous around > 505 of tracts of septa) B. Confluent necrosis Absent Focal confluent necrosis Zone 3 necrosis in some areas Zone 3 necrosis in most areas Zone 3 necrosis + occasional portal-central (P-C) bridging.

Zone 3 necrosis + multiple P-C bridging.

Panacinar or multiacinar necrosis.

C. Focal (spotty) Iytic necosis, apoptosis and focal inflamamtion Absent One focus or less per 10 x objective.

Two to four foci per 10 x objective.

Five to ten foci per 10 x objective.

More than ten foci per 10 x objective.

D. Portal inflammation None Mild, some or all protal areas.

Moderate, some or all protl areas.

Moderate/marked, all protal areas Marked, all portal areas.

Maximum possible score

0 1 2 3 4 0 1 2 3 4 5 6 0 1 2 3 4 0 1 2 3 4 18

Table (15) a glossary set by Keeffe et al., 2004 to correlate clinical terms with laboratory results as follows: 74

Review of Literature

Diagnosis of HBV

Definitions: - Chronic hepatitis B: Chronic necroinflammatory disease of the liver caused by persistent infection with HBV. Chronic hepatitis B can be subdivided into HBeAg-positive and HBeAg-negative chronic hepatitis B. - Inactive HBsAg carriers state: Persistent HBV infection of the liver without significant, ongoing necroinflamamtory disease.

- Resolved hepatitis B: Previous HBV infection without further virolgoic, biochemical, or histologic evidence of active virus infection or disease.

- Acute exacerbation or flare of hepatitis B: Intermittent elevations of aminotransferase activity to more than 10 times the upper limit of normal and more than twice the baseline value.

- Reactivation of hepatitis B: Reappearance of active necroinflammatory disease of the liver in a person known to have the inactive HBsAg carrier state or resolved hepatitis B. - HBeAg clearance: Loss of HBeAg in a person who was previously HBeAg positive.

- HBeAg seroconversion: Loss of HBeAg and detection of HBeAb in a person who was previously HBeAg positive and HBeAb negative, associated with decrease in serum HBV DNA to < 10 copies/mL. - HBeAg reversion : Reappearance of HBeAg in a person who was previously HBeAg negative and HBeAb positive.

Diagnostic criteria : a) Chronic hepatitis B: 1. HBsAg positive > 6 months.

2. Serum HBV DNA > 10 copies/ml.

3. Persistent or intermittent elevation in ALT/AST levels.

4. Liver biopsy showing chronic hepatitis (necroinflammatory score ≥ 4) b) Inactive HBsAg carrier state : 1. HBsAg positive > 6 months.

2. HBeAg negative, HBeAb positive.

3. Serum HBV DNA < 10 copies/ml.

4 Persistently normal ALT/ AST levels.

5. Liver biopsy confirms absence of significant hepatitis (necroinflammatory score <4). c) Resolved hepatitis : 1. Previous known history of acute or chronic hepatitis B or the presence of HBcAb ± HBsAb. 2. HBsAg negative.

3. Undetectable serum HBV DNA. 4. Normal ALT level.

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Table (16): The stages of HBV in correlation with the laboratory results (Kim and Sherker 2004): DISEASE MARKER HBsAb HBsAb HBV DNA HBeAg HBeAb HBcAb AST/ALT

REPLICATIVE PHASE

INTEGARATIVE PHASE

Stage 1 + +(strongly) + + Normal

Stage 3 + + Normal

Stage 2 + + + + elevated

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Stage 4 + + + Normal

Diagnosis of HBV

Review of Literature

Diagnosis of HBV

Table (17): Interpretation of serological markers according to symptoms, transaminases and histological features (Poynard, 2002).

Acute

HBSAG HBSAB HBEAG HBEAB HBCAB HBCAB HBVIGG IGM DNA + + +/+ + +

SYMPTOMS ALT HAI AND FIBROSIS +/+/+ +

Chronic carrier wild type

+

-

+

-

+

-

+

+/-

+/-

+

Chronic carrier pre-core + mutant

-

-

+

+

-/+

+

+/-

+/-

+

'Healthy carrier"

+

-

+

-

+

-

-

-

-

-

"Immune tolerance"

+

-

+

-

+

-

++

-

-

-

Recovery/Immunity

-

+

-

+

+

-

-

-

-

-

from -

+

-

-

-

-

-

-

-

-

-

+/-

-

-

+/-

-

+/-

+/-

+/-

+/-

Review of Literature

Immunity vaccination Occult infection

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Treatment of Chronic Hepatitis B

CHAPTER VI TREATMENT OF CHRONIC HEPATITIS B Effective treatment of chronic hepatitis B is defined as sustained clearance of circulating HBeAg and HBV-DNA with production of HBeAb and improvement in liver disease as determined by normalization of serum ALT level and reduction of necro-inflammation on liver biopsies. The most widely available agents for the treatment of chronic hepatitis B are interferon and Lamivudine (Pramoolsinsup, 2002).

Definitive treatment: The kinetics of HBV clearance during anti-viral therapy is slow requiring long-term therapy to control viral replication. Emergence of resistant mutants is accelerated by high HBV replication and hepatocyte turnover, which are common features in patients with chronic HBV infection. These mutants have a decreased priming and elongation activity, and remain sensitive to novel nucleoside analogues (Kiss et al., 2002).

Decision to treat: it must be taken individually, based on precisely weighted parameters. Elevated ALT activity, a liver biopsy showing chronic hepatitis with or without cirrhosis and the presence of significant levels of HBV DNA are strong arguments about initiating antiviral therapy. The precise HBV DNA cutoff that discriminates between low and high pretreatment replication needs to be determined, using standardized quantification units (De Gottardi and Negro, 2004).

Treatment monitoring: HBV DNA quantification with repeated ALT determinations and HBeAg/HBeAb assessment in HBeAg positive patients are critical in monitoring treatment (Kirschberg et al., 2004).

Aims of treatment: are to achieve sustained suppression of HBV - 75 -

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replication and remission of liver disease. The end points used to assess treatment response include normalization in serum ALT level, undetectable serum HBV, loss of HBeAg with or without detection of HBeAb and improvement in liver histology (Kobak et al., 2004).

Inconsistencies

in

the

definition

of

response,

lack

of

standardization of HBV DNA assays, and heterogeneity in patient populations make it difficult to compare response rates in clinical trials of treatment of chronic hepatitis B. Responses to antiviral therapy of chronic hepatitis B are categorized as biochemical, virologic or histologic and as on-therapy or sustained off therapy (Kondili et al., 2004). ALT level and HAI, rather than low HBV DNA level were found to be important predictors for response (Kumar and Agrawal, 2004).

Established treatment: treatments as studied by Kuo et al. (2004) including the developed therapeutic modalities are based either on antiviral drugs or focus on attempts to augment anti viral immune response.

Their results have been largely disappointing. The treatment of chronic hepatitis B is based on IFN-α administration at a dose of 5-10 million units 3 times a week subcutaneously for 16-32 weeks or lamivudine at a dose of 100 mg /day orally for 48 weeks or longer. Which of these two drugs should be chosen for first line treatment for chronic hepatitis B is controversial. Patients with a low HBV DNA are more likely than the others who are not to have a sustained response to IFN- i.e. HBe seroconversion (Lacarnini, 2004).

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Table (18): Hepatitis B Therapies and Treatment (Lok et al., 2004).

Drug Name Intron A (interferon alfa-2b) Hepsera (adefovir dipivoxil) Epivir-HBV (lamivudine; 3TC)

Drug Class

Manufacturer

interferon

Schering-Plough

Baraclude (entecavir)

nucleoside analogue

Bristol-Myers Squibb

interferon

Roche Labs

Pegasys (peginterferon alfa-2a) Emtricitabine (FTC) Telbivudine (LdT) Clevudine (L-FMAU) Elvucitabine (ACH 126, 443) Valtorcitabine Racivir BAM 205 HepX-B

nucleotide analogue nucleoside analogue

Gilead Sciences GlaxoSmithKline

nucleoside analogue nucleoside analogue nucleoside analogue nucleoside analogue nucleoside analogue nucleoside analogue small molecule monoclonal antibody

Gilead Sciences

FDA Status FDAapproved FDAapproved FDAapproved about to got FDAapproval FDAapproved Phase III NDA filed

Idenix

Phase III

Gilead Sciences

Phase II

Achillon Pharmaceuticals

Phase II

Idenix

PhaseII

Pharmasset

Phase II

Novelos

Phase II/III

XTLBiopharm

Phase II Phase II Phase II in combo with Epivir-HBV AND Orphan drug for liver cancer in US Phase II Phase II Phase I Phase I Phase I Phase I

HE 2000

immune stimulant

Hollis-Eden

Zadaxin (thymosin-alpha)

immune stimulant

SciClone

Theradigm EHT 899 MCC 478 MIV 210 Hepavir B HBV DNA vaccine

immune stimulant viral protein nucleoside analogue nucleoside analogue nucleoside analogue immune stimulant

Epimmune Enzo Biochem Eli Lily Medivir Ribapharm PowderJect

Prednisone priming: The rationale for administering a tapering course of steroids prior to antiviral therapy (prednisone priming) is that recovery of immune function following steroid withdrawal may be beneficial particularly if - 77 -

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Treatment of Chronic Hepatitis B

this is timed with the initiation of IFN- therapy (Lada et al., 2004).

Although a small subset of patients may benefit from prednisone priming, there is a risk of fatal exacerbations in patients with underlying cirrhosis. Therefore, prednisone priming is not recommended as a primary treatment of chronic hepatitis B (Lai, 2004).

(1) Immunomodulating agents and chronic hepatitis B: Interferon therapy: IFN- has direct effect on the immune system, including enhanced expression of HLA-class 1 molecules and stimulation of CD8+ cytotoxic T-cell activity, and treatment-induced alanine transaminase (ALT) flares are a surrogate marker of these properties (Lai and Terrault, 2004).

Interferon is cytokine with Immunomodulatory; antiproliferative and antiviral properties. Interferon alpha (INF-) was licensed in 1992 and it is the first approved treatment for chronic hepatitis B in most countries (Waked, 2005).

Fig. (20): Mode of action of interferon alpha (INF-) (Cooksley, 2004).

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Treatment of Chronic Hepatitis B

Natural interferon is a protein produced by the body's cells in response to viral infections. There are three types: IFN-alfa (also known as leukocyte interferon), IFN-beta (also known as fibroblast interferon) and IFN-gamma (also known as immune interferon). IFN-alpha and IFN-beta are also referred to as Type I interferon and IFN-gamma as Type II. There are approximately 20 subtypes of IFN-alpha but only one IFN-beta and IFN-gamma (Cooksley, 2004).

Commercially produced (genetically engineered) interferon alfa mimics the activity of naturally-occurring interferon alfa produced by the body, although the mechanism of action of the drug is not well understood (Lin and Kirchner, 2004).

Table (19): Types and Properties of Interferon (Lin and Kirchner, 2004).

Property Previous designations Genes pH2 stability Inducers

Principal source

Alpha Leukocyte IFN Type I >20 Stable Viruses (RNA>DNA) dsRNA Leukocytes, Epithelium

Interferon Beta Fibroblast IFN Type I 1 Stable Viruses(RNA>DNA) dsRNA

Gamma Immune IFN Type II 1 Labile Antigens, Mitogens

Fibroblasts

Lymphocytes

Intron a (interferon alfa-2b) is one commercial form of interferon approved by the US Food and Drug Administration for the treatment of Hepatitis B (Leung, 2002).

Multiple studies of interferon therapy in chronic HB have demonstrated beneficial effects. A 4-month course of interferon alfa-2b treatment results in virological response in 30-40% of patients with significant reduction of serum HBV-DNA expression, normalization of ALT level and loss of HBeAg. Seroconversion from HBeAg to Anti-HBe - 79 -

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Treatment of Chronic Hepatitis B

occurs in 15-20% of interferon treated patients (Lai et al., 2003b).

INF-α therapy has been accepted as an effective therapy with a good response rate for patients with chronic hepatitis B who were HBeAg-positive, had high serum ALT levels and low HBV-DNA levels (Lee et al., 2004).

Selection of patients and indications for interferon therapy: a) Patients with compensated chronic hepatitis with active viral replication that is elevated serum ALT levels, detectable HBeAg and/or HBV DNA and histology of active hepatitis (Chan et al., 2002b). b) Patients who are HBeAg-negative but HBV DNA positive and with elevated ALT levels, usually respond temporary to interferon therapy with decreases in serum levels of both HBV-DNA and ALT. These patients are commonly seen in Mediterranean region and Asia, and have a low sustained response and common relapse but requiring longer course of interferon treatment (Leung, 2002). c) Patients in the immune-tolerant phase of chronic hepatitis B infection, with circulating HBeAg and HBV-DNA but have normal or near normal ALT values, have very low response rates to interferon therapy, they are commonly seen in Asia among perinatally infected children, the response rate is less than 10% so they are not treated in most of the authorities (Li et al., 2003). d) Patients with decompensated cirrhosis may benefit from interferon therapy by inhibiting viral replication but it may produce significant adverse events such as serious infection and hepatic failure (Lin et al., 2004b).

Dosage regimen: The recommended regimen of interferon alfa-2b 5 MU or 10 MU - 80 -

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Treatment of Chronic Hepatitis B

three times per week for 4-months course subcutaneously. In case of HBeAg +ve for 16 weeks while in case of HBeAg –ve for 12 months (Perrillo et al., 2002).

Long term twice weekly INF- therapy could be a worldwide strategy for HBeAg positive patients with serum HBV-DNA level of less than 200 Meq/ml and patients with transient acute exacerbation of ALT during or after INF- therapy could often respond well after exacerbation of ALT (Arase et al., 2002).

The FDA-approved dose of standard interferon alfa for treatment of hepatitis. FDA-approved treatment for hepatitis B is five million units daily for 16 weeks (Liaw, 2002).

Patients with lower ALT, higher HBV-DNA, and immunosuppressed patients have a poorer response to IFN- and can be dangerous in cirrhosis (De Gottardi and Negro, 2004).

Table (20): Determinant of responsive factors to interferon (Lin and Kirchner, 2004).      

Favorable outcome HBeAg positive Low serum HBV-DNA level High serum ALT Active liver histology Female-western adult Acquired or recent HBV infection

     

Unfavorable outcome HBeAg negative High serum HBV-DNA level Normal serum ALT Inactive liver histology Male Asian Long standing HBV infection

  

Non-cirrhotic Anti-HIV negative HDV negative

  

Cirrhotic patients Anti-HIV positive HDV positive

Side effects of interferon: Common side effects include: Flu like symptoms, including headaches, Extreme weariness (fatigue), Muscle aches, Fever, Hair loss, Depression and other mood - 81 -

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disorders, Decreased number of platelets and Decreased white blood cell count (Pramoolsinsaip, 2002).

Rare side effects include: Thyroid problems, Confusion, Excessive amounts of protein in the urine, indicating poor kidney function and Heart problems (Perillo, 2004).

Peters et al. (2004) found that Pegylated Interferon-Alfa has primary role in treatment of Chronic hepatitis B whether HBeAg-positive or HBeAg-negative. Its concept is the production of a molecule which maintains prolonged biologic effect, thus reducing the opportunity for the virus to “escape.” There are 2 Pegylated interferons — alfa-2a (40 KD) and alfa-2b (12 KD).

Cooksley (2004) added that pegylated interferon-Alfa was more effective than conventional interferon. Its dose is 1.5 mcg/kg body weight interferon alfa-2b for the first 24 weeks and 1 mcg/kg for the second 24 weeks. At follow-up 6 months after the end of therapy.

(2) Antiviral nucleoside analogues: Nucleoside analogues suppress HBV-DNA replication mainly by inhibiting the viral polymerase. The lower side effect profile of the nucleoside analogues has made them preferred agents for first-line therapy, although concerns about resistance developing during monotherapy has tempered enthusiasm somewhat (Pfeifer et al., 2002).

Nucleoside analogues have been introduced in the management of chronic HBV infection. They mainly act by inhibition of HBV polymerase activity resulting in decrease of viral replication. They are administered orally and most of them have excellent tolerance and safety profile (Kidd et al., 2002).

Lamivudine is the only nucleoside analogue licensed for chronic - 82 -

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hepatitis B. It has potent activity against HBV and 12 month course achieves clearance of HBeAg in 20-30% of HBeAg positive patients and both biochemical and virological remission in more than 65-70% of HBeAg-negative chronic hepatitis B patients (Lagget and Rizzetto, 2002).

Prolonged effective antiviral therapy is required for eradication of chronic HBV infection, but long-term treatment with nucleoside analogues has been found to be associated with progressively increasing rates of viral resistance because of emergence of resistant HBV mutant strains (Lalekha et al., 2002).

Virological breakthroughs usually develop after the first 6 months of lamivudine monotherapy and their rate ranges between 15% and 30% at 12 months and exceeds 50% after 3 years of therapy. Resistant HBV mutant strains harbor point mutations in the HBV polymerase gene and predominantly in the well-conserved tyrosine methionine aspartate aspartate (YMDD) motif. Although resistant HBV strains may have impaired replication capacity compared with the wild HBV, their clinical significance has not been completely clarified yet. No significant biochemical breakthroughs with or without deterioration of liver function may develop in others. There is no proven effective therapy for the resistant HBV mutant strains, although adefovir and entecavir seem to be interesting candidates (Le Pogam and Shih, 2002). (a) Lamivudine Lamivudine is a cytosine analogue with potent inhibitory effect in the RNA-dependant DNA polymerase of HBV, HIV and it is approved for the treatment of chronic hepatitis B (Liaw, 2002).

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Treatment of Chronic Hepatitis B

Graph (3): (Schiff et al., 2003b).

Lamivudine may also reverse the T-cell hypo responsiveness to hepatitis B viral antigens observed in patients who have chronic hepatitis B virus infection (Rodriguez et al., 2003).

Graph (4): (Roque et al., 2002).

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Treatment of Chronic Hepatitis B

Efficacy and tolerability: Worldwide studies of lamivudine in patients with HBeAg-positive chronic hepatitis B have demonstrated good efficacy based on improved hepatic histology, HBeAg seroconversion, suppression of serum HBVDNA and normalization of ALT levels (Roque et al., 2002).

Lamivudine monotherapy is also effective in suppressing HBV replication and ameliorating disease in patients who are HBeAg negative (Sakai et al., 2002).

Graph (5): (Saab et al., 2003).

Studies in Asian and Western patients with elevated ALT activity and markers of HBV replication (HBeAg and HBV-DNApositive) have responses to lamivudine with HBeAg seroconversion 1618%, HBeAg loss in 30-33%, sustained normalization of ALT in 41-49% and histology improvement and reduction in histologic activity index of more than 2 points in 50-60% of treated patients (Sakai et al., 2002).

Reductions of HBV-DNA serum levels were detected after 2 weeks of lamivudine therapy in 97% of patients and eventually became undetectable in all patients (Spijkerman et al., 2002). However, after - 85 -

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Treatment of Chronic Hepatitis B

cessation therapy, serum HBV-DNA in these patients always reappeared, this may be attributed to the persistence of HBV covalently closed circular DNA in the nuclei of hepatocytes (Squadrito et al., 2002).

This competitor of cytidine is 80% bioavailable and devoid of side effects at the oral dose of 100mg daily; tolerance continues for therapies up to 3 years. It induces a rapid decline of ALT accompanied by improvement of histology; however, there is a delayed accumulating seroconversion to HBeAb and the switch to HBsAb is rare (Sobao et al., 2002).

Over the long term, its activity is abolished by the emergence of specific viral mutations YMDD mutants that rekindle the disease. The indication to lamivudine therapy in HBeAg negative chronic hepatitis B is currently under investigation. Lamivudine is highly efficacious in preventing HBV reinfection in liver transplants (Sakugawa et al., 2002).

It is well tolerated after 1 year of treatment, HBeAg seroconversion rate increased with higher pretherapy ALT levels, suggesting that patients with stronger endogenous antiviral defenses to kill hepatocytes harboring viral DNA. In addition, it is beneficial in HBeAg negative chronic hepatitis B, and patients with decompensated cirrhosis and HBV replication. However, genotypic resistant YMDD mutations start to emerge after 9-10 months of lamivudine therapy, and their incidence increases more quickly than the HBeAg seroconversion rate durating prolonged therapy (Tedder et al., 2002).

Thus, the benefits of long-term lamivudine must be balanced against concern about YMDD mutations and the durability of treatment response. There are encouraging preliminary results for adefovir dipivoxial, entecavir, emtricitabine, clevudine and other nucleoside analogues in the early stages of appraisal; entecavir and adefovir dipivoxil appear effective in patients with YMDD mutants (Tamori et - 86 -

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Treatment of Chronic Hepatitis B

al., 2003).

Treatment of patients with lamivudine results in loss of detectable levels of HBV DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated (Abdelhamed et al., 2002).

Samuel (2004) concluded the following results; 1) Lamivudine resistant mutants are likely to be cross resistant to other L-cytidine analogues; 2) antiviral therapy using a single reverse transcriptase (RT) inhibitor is likely to fail to eradicate viral DNA and 3) new nucleoside analogues with unique mode of action (inhibition of priming or elongation of RT, or DNA polymerase activity) and activity against lamivudine resistant strains are emerging. Combination of these new anti HBV agents with DNA based immunization may prove useful to eradicate viral infection.

Graph (6): (Peters et al., 2004).

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Treatment of Chronic Hepatitis B

Long-term efficacy of Lamivudine: Studies in Asia have shown that the frequency of HBeAg seroconversion increases with longer duration of treatment from 22% at 1 year to 27% at 2 years and to 40% at 3 years (Schiff et al., 2003a). The role of indefinite maintenance treatment with lamivudine after HBe seroconversion is unclear during a maintenance therapy; lamivudine resistant mutants increased from 14-40% of treated patients and in other study 66% of patients developed YMDD mutation after 4 years of lamivudine therapy (Schiff et al., 2003b).

Graph (7): (Schiefke et al., 2004).

Predictors of Response to Lamivudine Therapy: The pretreatment factors that predict the response of patients with HBeAg-positive chronic hepatitis to lamivudine are the high baseline ALT and high baseline HAI; they are very similar to those for IFN except that baseline HBV DNA may not be as important a predictor for lamivudine efficacy (Perrillo et al., 2002).

Sekiya et al. (2002) clearly showed that baseline ALT is highly - 88 -

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Treatment of Chronic Hepatitis B

predictive of lamivudine efficacy but high ALT is also predictive of spontaneous HBeAg seroconversion.

Lamivudine Resistance: The likelihood of developing drug resistance increases with duration of therapy. After 1 year of lamivudine therapy, 14 to 32% of individuals have genotypic resistant mutations. This figure increases with each year of extended Lamivudine therapy (Selimo et al., 2002).

Lamivudine resistance is more likely to develop in individuals with high baseline HBV DNA and/or high baseline ALT (Seo et al., 2004), high HAI score, and high body mass index (31) (Wright, 2004).

Consequences of Drug Resistance: It remains possible for individuals who have developed drug resistance to seroconvert and to have an improvement in liver histology (Wong et al., 2002). The presence of lamivudine-resistant mutations may not cause major adverse effects during short-term follow-up, especially in immunocompetent

patients

with

mild

disease.

However,

in

immunocompromised patients, (particularly those co-infected with HIV) and in immunocompetent patients with cirrhosis; hepatic decompensation and even death have been described (Shiao et al., 2002).

Thus, consequences of drug resistance may be:  Exacerbation of hepatitis.  Decreased rate of seroconversion.  Rapid re-infection of liver graft.  Rapid disease progression (after liver transplantation).  Severe hepatitis HBV/HIV co-infection.  Transmission of drug resistance (Tanaka et al., 2004).

Possible Actions to Take When Lamivudine Resistance Develops: - 89 -

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1) Continue lamivudine monotherapy if serum ALT levels remain normal despite reactivation of HBV DNA (Yang et al., 2002).

2) Stop Lamivudine. If Lamivudine treatment is stopped in those who have developed drug resistance, viral replication returns to wild type.

The cessation of treatment in this situation has been discouraged by the relevant pharmaceutical company, but there have been two reports which suggest that cessation of therapy in individuals whose ALT and HBV DNA have become elevated once again is safe (Sinturk et al., 2002).

3) Add another drug effective against the drug-resistant mutant. The alternative to stopping lamivudine if ALT and HBV DNA levels become elevated once more is to add an antiviral agent effective against

lamivudine-resistant

mutations

(Yano

et al.,

2002),

particularly if the patient has cirrhosis, it is probably safest to take this action. None is currently licensed but several are known to be effective, and adefovir dipivoxil {approved by the Food and Drug Administration (FDA)} has already been shown in vivo to be very effective after liver transplantation (Zhang et al., 2002). Yano et al.

(2003) suggest from preliminary results of an ongoing study that adefovir dipivoxil alone may be as effective as adefovir dipivoxil and lamivudine in this setting. (b) Other Nucleoside Analogues: A number of other agents are either approved by the Food and Drug Administration (FDA) like (adefovir dipivoxil, entecavir) or in the process of phase III evaluation (clevudine, emtricitabine) (Yao et al., 2002).

Adefovir Dipivoxil Adefovir dipivoxil is a nucleotide analogue of adenosine - 90 -

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Treatment of Chronic Hepatitis B

monophosphate that inhibits both HBV reverse transcriptase and DNA polymerase activity. Adefovir has been shown to be effective in suppressing not only wild-type HBV but also lamivudine-resistant HBV mutants (Sakai et al., 2002). It does not lead to the emergence of resistant virus after up to 60 weeks of therapy but it is unclear whether resistance mutations will emerge after longer therapy or not (Xu et al., 2002).

Graph (8): (Brabin et al., 2002).

It received approval by the FDA in September 2002. A 1-year randomized, controlled study involving 515 individuals with HBeAgpositive hepatitis indicated that the rate of HBeAg seroconversion in individuals receiving a 10-mg dose (12%) was significantly greater than that observed in the placebo group (6%), P <0.05 (Pasquetto et al., 2002).

The number of individuals whose serum ALT fell within the normal range was also higher in those randomly assigned to treatment (10 or 30 mg), 48% and 55%, respectively (compared with 16% in the control group). Serum HBV DNA levels fell to less than 400 copies/ml in 21% of those who received 10 mg/day and in 39% of those who received 30 mg/day and this effect was not observed in any of those who received placebo (Chang et al., 2003).

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Treatment of Chronic Hepatitis B

Graph (9): (Dahmen et al., 2004).

At the 10 mg/day dose, a significant improvement in both the necro-inflammatory and the fibrosis scores on liver biopsy was observed after 1 year of therapy The major advantages of adefovir dipivoxil appear to be the apparent lack of development of drug resistant mutations (Chu et al., 2002) and its antiviral efficacy in the presence of lamivudineresistant mutations (Danalioglu et al., 2004).

Graph (10): (Dando et al., 2003).

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Treatment of Chronic Hepatitis B

Graph (11): (Fattovich, 2003).

Entecavir: On March 29, 2005, FDA approved Baraclude (entecavir) for the treatment of chronic hepatitis B virus infection in adults with evidence of active viral replication, and either evidence of persistent elevations in serum aminotransferases (ALT or AST), or histologically active disease (Gish et al., 2005a).

Entecavir is a guanosine nucleoside analogue, which is a potent selective inhibitor of hepatitis B virus, and is undergoing phase III development worldwide (Gish et al., 2005b). In the woodchuck model, it is highly active, and importantly has shown reduction in CCC DNA (covalently closed circular DNA) levels and reduction in development of HCC with prolonged survival (Brown, 2005).

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Treatment of Chronic Hepatitis B

Graph (12): (He et al., 2002).

Famciclovir: It is the oral pro-drug of penciclovir, an acyclic deoxyguanosine analogue. Penciclovir triphosphate competes with dGTP for incorporation into the nascent HBV DNA chains and for the priming of reverse transcription. Given the low virological response rates, potential risk of drug resistance and the need for thrice daily administration, the role of famciclovir in the management of chronic HBC is limited (Kao, 2002).

Emtricitabine/ coviracil: It is a cytosine nucleoside analogue with potent anti viral activity against HBV. Because of the structural similarity with lamivudine, longterm therapy with emtricitabine may select the same resistant mutants (Kim et al., 2004).

Others: - 94 -

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Treatment of Chronic Hepatitis B

Three simple related nucleosides -L-2 deoxycytidine, -Lthymidine and -L 2 deoxyadenosine; have been discovered to be potent, specific and selective inhibitors of the replication of HBV. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. These compounds have the potential for use in combination therapy with goal of achieving superior viral suppression and diminishing the onset of resistance (Lohr et al., 2002).

BAY 41-4109 is a member of a class of heteroaiyl-pyimidines that was identified as potent inhibitors of human HBV replication and antiHBV drug candidate (Manesis et al., 2002). Pharmacokinetic studies in mice have shown rapid absorption, a bioavailability of 30% and dose proportional plasma concentrations (Manesis et al., 2002).

Oligodeoxynucleotide TGO20 can be transfected into cells by packing with liposme and can be combined with HBV DNA. TGO20 can effectively inhibit replication of HBV (Moola et al., 2002).

Novel 2-amino-6 arylthio-9-2 {(phosphonomethoxy) ethyl} purine bis (2, 2, 2-trifluoroethyl) esters (PMEA) showed considerably high anti- HBV activity, and exhibited low cytotoxicity (Gotsman et al., 2002). It is a lipophilic prodrug of 9-(2-phosphonylmethOxyethyl) adenine (PMEA), designated PMEALO, and incorporated it into reconstituted lactosylated high-density lipoprotein (LacNeoHDL) was internalized by the asialogycoprotein receptor on parenchymal liver cells and converted into its active diphosphorylated metabolite. Asialofetuin, an established ligand for the asialoglycoprotein receptor, inhibited the association by > 75%, which confirms the role of the asialoglycoprotein receptor. Association of the prodrug-loaded particles to HepG2 cells was coupled to degradation. LacNeoHDL-associated PMEALO effectively - 95 -

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Treatment of Chronic Hepatitis B

inhibited HBV DNA synthesis (Lo et al., 2003).

Effective antiviral agents are thought to inhibit HBV DNA synthesis irreversibly by chain determination because RT lack an exonucleolytic activity that can remove incorporated nucleotides. The HBV-RT pyrophosphorolytic activity may be a determinant of antiviral drug efficacy and could serve as a target for antiviral drug therapy. The strong inhibitory effect of cytoplasmic pyrophosphate concentration on viral DNA synthesis may also partly account for the apparent slow rate of HBV genome replication (Myers et al., 2003).

(3) Combination therapy: A number of subsequent investigator-initiated trials of combination IFN and lamivudine therapy also suggest that this combination is more efficacious than either therapy given alone. In the study reported by Hakozaki et al. (2002), 150 patients were randomly assigned to interferon alfa-2b 9 MU 3 times a week and lamivudine 100 mg daily for 24 weeks or to lamivudine 100 mg daily as monotherapy for 52 weeks.

Follow-up was conducted for a further 48 weeks. The rate of HBeAg seroconversion was 35% with combination therapy and 19% with monotherapy. A repeat liver biopsy was performed. At the end of treatment, improvement in inflammation (≥2 points fall in the HAI score from baseline) was seen in 46% of those assigned to combination and in 27% of those assigned to lamivudine monotherapy. An improvement in fibrosis was reported in 42% with the combination and in 24% of those with monotherapy (Ogawa et al., 2002).

The combination of IFN and lamivudine is associated with an enhanced rate of virologic response when compared with either agent alone. From a theoretical perspective, the combination of lFN with one or more nucleoside analogues may be the most effective way to treat - 96 -

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Treatment of Chronic Hepatitis B

HBV infection in many clinical situations (Fattovich, 2003).

The combination of large dosage IFN- 2b and lamivudine therapy in children was compared at the end of therapy and 6 monthsafter therapy, normalization of ALT and the clearances of HbeAg and HbsAg in both groups were directly proportional to the duration of treatment. However, the higher complete response rate at 12 months of combination therapy was not statistically different from that at 6 months (Hanazaki, 2004). However, Hadziyannis et al. (2004) in their study found that the response rate of IFN- and lamivudine combination therapy in children with chronic hepatitis B was similar to that of IFN- monotherapy.

Combination IFN- 2b and thymosin-1 treatment may provide a safe and effective therapeutic approach for the difficult to treat HbeAb positive chronic hepatitis B patients (Haushofer et al., 2004).

Combination of IFN- plus GMCSF (granulocyte macrophage colony stimulating factor) was effective in non-responders (Hayashi and

Furusyo, 2004).

Combination or sequential therapy of nucleoside analogues may help to better achieve the goals of treatment for chronic hepatitis B in the new century (Hm et al., 2004).

(4) Therapeutic vaccine: HBcAg based therapeutic vaccine was found to induce T cell proliferative responses in chronically infected hepatitis B patients to the T helper epitope. The induction of a T helper 0/T helper 2 CD4+ T cell response rather than T helper 1 response. Thus, its combination with IFN- or IL-12, which might reverse the CD4+ cell response, should be considered. It is likely that different types of combination therapy may have to be tailor made for chronic HBV infection with different - 97 -

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virological and immunological profiles and different degrees of liver damage (Gheit et al., 2002).

PreS2/S vaccination of chronic HBV carriers led to a reduction in HBV replication or clearance of virus in 30% of treated patients (Gotsman et al., 2002). Combination of IFN-α with Pre S2 containing vaccine (Genhevac-B) is effective for the vaccine failures and may increase sustained response compared to IFN-α alone (Hou et al., 2005).

(5) Gene therapy: A hammerhead ribosome, RzC was designed to target the sequence encoding the tail region of the HBV core protein suggesting the possibility of hammerhead ribosome mediated gene therapy for the treatment of HBV infections (Hübscher and Potmann, 2002). As an alternative to ribozymes, the use of DNA containing, phosphorothioatemodified minimized hammerhead ribozymes (minizymes) to inhibit HBsAg expression and viral replication. Minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection (Humphries and Dixon, 2003).

(6) Immunotherapy: Administration of monoclonal antibodies against HBsAg to HBVTrimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA positive mice in a dose dependent manner. They were more effective than a polyclonal antibody preparation in both inhibitions of HBV liver infection and reduction of viral load (Hunter, 2002).

CTL (cytotoxic T lymphocyte) reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration (Rapicetta et al., 2002). IL-12 plays a central role in mounting an effective cellular immune response directed towards - 98 -

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Treatment of Chronic Hepatitis B

elimination of intracellular pathogens. Subcutaneously administered recombinant human IL-12 treated for 12 and 10 consecutive weeks was generally well tolerated, but was associated with temporary decrease in neutrophils and lymphocyte counts, and with elevation in serum transaminases and bilirubin. Serum IL-12 levels do not appear to be advantageous in comparison to other currently available treatment (Sakugawa et al., 2002).

(7) Other modalities under trial: Peptide aptamers are molecules, which can block viral replication by interfering with capsid formation. Specifically bound to be HBV core protein under intracellular conditions. One of them, named CL-I, efficiently inhibited viral capsid formation and consequently, HBV replication and virion production (Tsamandas et al., 2002).

Hepatocyte nuclear factor (HNF 3) inhibits nuclear hormone receptor-mediated viral replication. Inhibition of HBV replication by Hepatocyte nuclear factor (HNF-) is associated with the preferential reduction in the level of the pregenornic RNA compared with that of precore RNA. HBeAg encoded by the precore RNA mediates part of the inhibition of viral replication by Hepatocyte nuclear factor (HNF 3- ) (Torbenson and Thomas, 2002).

The amino terminal transcriptional activation domain of Hepatocyte nuclear factor (HNF 3- ) is essential for the inhibition of HBV replication. The activation of transcription by (HNF3) from HBV promoters downstream from the nucleocapsid promoter appears to contribute indirectly to the reduction in the steady state level of 3.5-Kb HBV RNA, possibly by interfering with the elongation rate of these transcripts.

Therefore,

transcriptional

interference

mediated

by

Hepatocyte nuclear factor (HNF-) may also regulate HBV RNA - 99 -

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Treatment of Chronic Hepatitis B

synthesis and viral replication (Tang and Mclachlan, 2002).

Higher doses of bifendate (Bethanechol chloride) taken for a long term has remarkable anti- HBV efficacy in treatment of chronic hepatitis B (Saruc et al., 2002).

All patients receiving indomethacin 75 mg daily for 6 months exhibited a total HBeAb immunoglobulin response. The prostaglandin pathway is involved in the pathogenesis of the immune response against HBV, and that the suppression of viral replication is achieved as indicated by the disappearance HBeAg and HBV DNA in healthy chronic HBV carriers (Tibbs and Smith, 2003).

Capsid-targeted viral inactivation is a conceptually powerful approach that exploits virion structural proteins to target a degradative enzyme specifically into viral particles. C proximal fusion to the HBV capsid protein of the Ca 2+ dependent nuclease SN from staphylococcus aureus yields a chimeric protein, core SN. The antiviral effect depends on both an enzymatically active SN and on the core domain. Core SN does not block assembly of RNA containing nucleocapsids but interferes with proper synthesis of viral DNA inside the capsid or leads to rapid DNA degradation. An intracellular nuclease activation that owing to the characteristics of HBV morphogenesis, is nonetheless highly virus specific (Saudy et al., 2003).

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Table (21): Comparison of Three Approved Treatments of Chronic Hepatitis B (Shen et al., 2004).

Indications

IFN-

HBeAg+, normal ALT

Not indicated

HBeAg+ chronic Indicated hepatitis HBeAg- chronic hepatitis Indicated Duration of Treatment HBeAg+ chronic 4-6 months hepatitis HBeAg- chronic hepatitis 1 year Route Subcutaneous Side Effects Drug Resistance

Lamivudine Not indicated

Not indicated

Indicated

Indicated

Indicated

Indicated

≥1 year

≥1 year

>1 year Oral

>1 year Oral Potential nephrotoxicity None, year 1 3%, year 2 Intermediate

Many

Negligible

-

0%, year 1 70%, year 5 Low

Cost* High * Based on treatment duration of 1 year.

Adefovir

Kawaguchi et al. (2003) reported that even after resolution of hepatitis B and recovery, there is persistence of very small amounts of HBV DNA. Some of organs or tissues, for instance, the renal tubular epithelium and the choroids plexus, are not readily accessible to T cells due to a closed basal membrane and could represent immunoprivileged sites.

Table (22): Goals of Therapy and Definitions of Response to Therapy in Chronic Hepatitis B (Papatheodoridis and Hadziyannis, 2004): Goals of therapy

Ideal: HBV eradication (clearance HBsAg) Realistic: Sustained suppression of HBV HBeAg-positive CHB: sustained seroconversion of HBeAg to anti-HBe HBeAg-negative CHB: sustained biochemical and virological response

Types of response Biochemical: normalization of serum ALT/AST activity Virological: HBeAg-positive CHB: loss of HBeAg (and development of anti-HBe) and decrease in serum HBV-DNA to low levels* HBeAg-negative CHB: undetectable serum HBV-DNA by PCR assays (or perhaps decrease in serum HBV-DNA from initially

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high to low levels*) Complete: Both biochemical and virological response as well as loss of serum HBsAg Histological: Decrease in necroinflammatory activity by >/= 2 points without worsening in fibrosis (compared with pre-treatment histological findings) Time of assessment of response

On-therapy response: response during therapy Initial response: response achieved at any time within the first 12 months of therapy Maintained on-therapy response: response that persists throughout therapy End-of-therapy response: response at the end of a defined course of therapy Off-therapy or sustained response: maintenance of response for >/= 12 months after discontinuation of therapy

High and low serum HBV-DNA levels are considered as >/= and < 105 copies/ml.

Table (23): Available Agents for Treatment of Chronic Hepatitis B (Papatheodoridis and Hadziyannis, 2004).

Interferon-alpha

Lamivudine

Adefovir dipivoxil

Anti-viral activity

+

++

++

Immunomodulatory activity

++

-

-

Frequent and potentially severe

Minimal

Minimal

High

Low

High

None

25%, 40%, >50% at 1, 2 and 3 years

Minimal (< 2% at 2 years)

17-32% (controls: 611%)

24% (controls: 11%)

Side-effects Cost Efficacy of courses of finite duration*

In HBeAg-positive CHB HBeAg loss

HBeAg seroconversion

33% (controls: 12%) 18% (more than controls)

16-17% 12% (controls: 4(controls: 6%) 6%)

In HBeAg-negative CHB End-of-therapy response

46-54%

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70-75%

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Treatment of Chronic Hepatitis B

Sustained off-therapy response

22-30%

< 10-15%

Unknown

Efficacy of long-term therapy In HBeAg-positive CHB HBeAg seroconversion at 24 months

Unknown

27%

21%

HBeAg seroconversion at 36 months

Unknown

33%

Unknown

HBeAg seroconversion at 48 months

Unknown

47%

Unknown

In HBeAg-negative CHB On-therapy 24-month response

46%

55-65%

71-73%

On-therapy 36-month response

Unknown

40-50%

Unknown

On-therapy 48-month response

Unknown

35-40%

Unknown

* Duration of interferon therapy: 4-6 months in HBeAg-positive CHB and 12-24 months in HBeAg-negative CHB. Duration of lamivudine and adefovir dipivoxil therapy: 12 months in all cases.

Anti-HBc is more prevalent in chronic hepatitis C patients than previously reported in the general population. The seropositivity of antiHBc is often associated with severe liver fibrosis. Pegylated interferon based therapy was more effective than standard interferon in HCV patients with previous exposure to HBV (Hwang et al., 2003).

HBV specific antibodies and T cells, in turn, may prevent viral spread and reinfection and therefore control remaining virus. Activation or transmission has been reported during immunosuppression and after transplantation of organs from recovered (anti-HBs positive) donors into immunosuppressed recipients (Keeffe et al., 2004). Post exposures to HBV affect the severity & the response to the treatment in patients with chronic HCV (Esmat, 2005).

Treatment strategies for HBV Antiviral therapies are able to inhibit replication of-hepatitis B with varying efficiency. Introduction of treatment activates the host’s immune - 103 -

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response to the virus either directly (IFN) or indirectly, secondary to a fall in viral load (lamivudine) (Vanlandschoot et al., 2002).

IFN is contraindicated in the treatment of occult HBV, poorly tolerated and ineffective. The more suitable is Lamivudine 100 mg/day or Adefovir 10 mg/day (Allain, 2004).

The likelihood of success can be predicted by: 1) Baseline levels of ALT: Patients who are HBeAg positive with normal or nearly normal ALT levels will not respond to either IFN or lamivudine. Such individuals are generally in the immune tolerant phase of their disease; less often, they may be just at the turning point of spontaneous seroconversion. (The level of serum HBV DNA will determine which situation is more likely, HBV DNA levels being very high in the former and relatively low in the latter) (Wang et al., 2002d).

The higher the baseline ALT, the more likely the individual is to seroconvert upon introduction of antiviral therapy (and the more likely they are to seroconvert spontaneously); Thus, prior to initiating treatment the patient should be monitored for at least 3 months to assess whether or not the process of spontaneous seroconversion is about to occur. In one study, 37% of individuals with HBcAg-positive chronic hepatitis observed for 3 months prior to recruitment to a therapeutic trial were noted to seroconvert spontaneously (Weber et al., 2002).

2) High baseline HBV DNA Generally (but not always) predicts poor response to IFN (Wolters et al., 2002), this is not necessarily the case for lamivudine, although it is predictive of the development of drug resistance. Rate of fall of HBV DNA after introduction of therapy may be helpful in predicting subsequent seroconversion (Xu et al., 2002).

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Antiviral therapy can prevent or reduce fibrosis progression. Thus, even when baseline ALT is not high, if the liver biopsy results indicate severe disease, treatment is appropriate (Yang et al., 2002).

The following strategies are recommended for patients with (I) HBeAg-positive moderate or severe chronic hepatitis without cirrhosis: A 4-6 months course of interferon alpha (5 MU daily or 9-10 MU thrice weekly, or 6 MU/m2 thrice weekly for children may be used as an initial therapy. If interferon is contraindicated, ineffective or poorly tolerated, lamivudine should be given at a dose 100 mg daily for at least 1 year. Adefovir should be given in a dose 10 mg daily for at least 1 year.

Treatment with lamivudine or Adefovir should be continued for 4-6 months after virological response is achieved. If a virological response is not achieved after 1 year, decision to continue treatment should weigh likelihood of a sustained response against the risk of development of drug resistance particularly with lamivudine (D. Valla et al., 2003).

(II) For patients with HBeAg-negative moderate or severe chronic hepatitis

without

cirrhosis,

the

following

strategies

are

recommended: A 12-24 month course of interferon alpha, 5-6 MIU thrice weekly may be considered as an initial therapy. If interferon is contraindicated, ineffective or poorly tolerated, lamivudine or Adefovir should be considered. Because HBeAg is already undetectable, the end point of treatment is not established. Sustained suppression of HBV replication is associated with histological improvement and therefore appears a realistic goal of treatment. The optimal duration of treatment is not known. Most patients will require more than one year of therapy, decision to continue treatment should weigh the likelihood of a sustained response against the - 105 -

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Treatment of Chronic Hepatitis B

risk of development of drug resistance or drug toxicity (Liu et al., 2003).

Hepatitis relapses on stopping Lamivudine therapy, whether HBeAg positive or negative, reintroduction of the drug as a maintenance therapy if the patient has not developed drug resistance (Haushofer et al., 2004).

(III) If a breakthrough on lamivudine therapy (for HBeAg positive or negative chronic hepatitis B) is thought to be due to emergence of lamivudine-resistant mutants, treatment options include: a. Continue lamivudine if serum HBV DNA and aminotransferases levels are lower than they were pretreatment. b. Discontinue lamivudine in patients without underlying cirrhosis and who are not immunosuppressed and c. Change to or add adefovir if available (Giannini et al., 2003).

Patients with cirrhosis, but without clinical or laboratory signs of decompensation can be managed like non-cirrhotic patients. Particular care should be paid to these as flares due to antiviral response, antiviral resistance or after cessation of treatment can lead to severe decompensation.

(IV) Decompensated cirrhotic patients should be evaluated for liver transplantation. If they show active viral replication, they should receive antiviral therapy. Several options are available: a. Start lamivudine early, in the hope that a successful virological response may delay or obviate the need for liver transplantation.

Adefovir can be added or substitute lamivudine when lamivudine resistance develops. b. Start lamivudine only when transplant is imminent (within the next 6 months). c. Use adefovir as first line therapy with close monitoring for the renal function (Aliyu et al., 2004).

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(V) Post transplant patients with recurrent hepatitis B who have not previously received lamivudine should be treated with lamivudine or adefovir. Breakthrough during lamivudine therapy should be treated with adefovir and careful monitoring of the renal function is required (Hm et al., 2004).

(VI) In HBV infected patients requiring immunosuppressive therapy Lamivudine is generally preferable to interferon as antiviral therapy.

Treatment can be started 2-4 weeks before immunosuppression or at the first sign of an exacerbation of the hepatitis. (Yao et al. 2004).

Table (24): Recommendations for treatment of Chronic Hepatitis B (Lok et al., 2004): HBeAg +

HBV DNA* +

+

+

ALT

Treatment Strategy

≤2 × ULN >2 × ULN

Low efficacy with current treatment. Observe; consider treatment when ALT becomes elevated IFN- , LAM, or ADV may be used as initial therapy

-

+

>2 × ULN

-

-

≤2 ×

End point of treatment - seroconversion from HBeAg to anti-HBe Duration of therapy IFN- : 16 weeks Lamivudine: minimum 1 year, continue for 3-6 months after HBeAg seroconversion Adefovir: minimum 1 year IFN nonresponders/contraindications to IFN- › LAM or ADV LAM resistance › ADV IFN , LAM or ADV may be used as initial therapy, IFN- or ADV is preferred because of the need for long-term therapy End point of treatment - sustained normalization of ALT and undetectable HBV DNA by PCR assay Duration of therapy IFN- : 1 year Lamivudine: >1 year Adefovir: >1 year IFN nonresponders/contraindications to IFN › LAM or ADV LAM resistance › ADV No treatment required

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ULN Cirrhosis Compensated: LAM or ADV Decompensated: LAM (or ADV); coordinate treatment with transplant center. Refer for liver transplant. IFN- contraindicated ± Cirrhosis Compensated: Observe Decompensated: Refer for liver transplant 5 N.B: DNA > 10 copies/ml; this value is arbitrarily chosen. ±

+

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Review of Literature

Prevention of HBV

CHAPTER VII PREVENTION OF HBV Risk personnel: Persons who should be screened for HBV infection: persons born in hyperendemic areas, men who have sex with men, injecting drug users, dialysis patients, HIV-infected individuals, pregnant women, family members, household members, and sexual contacts of HBV infected persons (Chang et al., 2002).

(A) Passive Immunization: (1) Hepatitis B immunoglobulin (HBIG): HBIG is administered to prevent hepatitis B infection after exposure of HBV-contaminated body fluids and in infants born to HBsAg-positive mothers (Yang et al., 2004).

Polyclonal HBIG has been shown to reduce HBV recurrence after liver transplantation and to decrease the possibility of graft loss by rejection (Wang et al., 2004b).

(2) Passive immunization in adults: Hepatitis B immunoglobulin is prepared from pooled plasma with a high titer of hepatitis B surface antibody and may confer temporary passive immunity under certain defined conditions. The major indication for the administration of hepatitis B immunoglobulin is a single acute exposure to hepatitis B virus, such as occurs when blood containing surface antigen is inoculated or splashed onto mucous membranes and the conjunctiva. The optimal dose has not been established, but doses in the range of 250 to 500 IU have been used effectively. It should be administered as early as possible after exposure and preferably within 48 hours, usually 3 ml (containing 200 IU of anti-HBs per milliliter) in - 109 -

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Prevention of HBV

adults. It should not be administered more than 7 days after exposure. It is generally recommended that two doses of hepatitis B immunoglobulin should be given 30 days apart (Tsai, 2004).

(3) Passive immunization in babies: Results with the use of hepatitis B immunoglobulin for prophylaxis in babies at risk of infection with hepatitis B virus are encouraging if the immunoglobulin is given as soon as possible after birth or within 12 hours of birth, and the chance of the baby developing the persistent carrier state is reduced by about 70%. More recent studies using combined passive and active immunization indicate an efficacy approaching 90%. The dose of hepatitis B immunoglobulin recommended in the newborn is 1 to 2 ml (200 IU of anti-HBs per milliliter) (Vierling, 2005).

(B) Active immunization: Vaccination: HB vaccine (HBsAg vaccine) was started soon after its identification in 1970. The first plasma-derived vaccine was licensed in the United States in 1981 (Heptavax B®, Merck Sharp and Dohme, USA). It was derived from the plasma of patient with chronic HBV infections. Despite its excellent efficacy and safety profile in millions of individuals worldwide, acceptance of the vaccine was low because of illegitimate fears of transmission of live HBV and other blood borne pathogens (Tong and Tu, 2004). Clinical trials with the yeast-derived recombinant hepatitis B vaccine started in 1984 and in 1986 EngerixTMB (SmithKline Beecham Biologicals, Rixensart, Belgium) became the first human vaccine manufactured using recombinant DNA technology to be marketed. A second similar vaccine, Recombivax HB, (Merck Sharp and Dohme, USA) was licensed in the U.S. in 1989. These second- 110 -

Review of Literature

Prevention of HBV

generation recombinant hepatitis B vaccines rendered plasma derived vaccines obsolete and they progressively disappeared in the course of the 1990s. Meanwhile hundreds of millions of vaccines doses have been administered. The different vaccines turned out to be safe and immunogenic (Wang et al., 2004a).

The International strategy to eliminate hepatitis B virus (HBV) transmission is based on: 1) Screening all pregnant women for hepatitis B surface antigen and post-exposure vaccination of infants of infected mothers.

2) Vaccinating all infants as part of the childhood vaccination schedule.

3) Vaccinating children and adolescents not vaccinated previously.

4) Vaccinating adolescents and adults in groups at increased risk for infection (Yano et al., 2003).

In areas of high endemicity, where mothers may not always be screened for hepatitis B infection, routine vaccination of infants at birth with a course of hepatitis B vaccine alone should be highly protective even for very high-risk infants of HBeAg-positive mothers (Lacarnini, 2004).

Needle stick injuries and non-reporting of needle stick injuries were highly prevalent in nursing students. More intensive education programs should be directed at students to increase their awareness of and compliance with universal precautions before commencing their practical work experience. Students need to practice prompt post-exposure evaluation so that the need for early intervention can be assessed. Any public health and infection control strategy should include a universal catch-up

HBV

vaccination

program

among

students

before

commencement of internship (Toniutto et al., 2004).

The WHO recommended that by the end of the 20 th century hepatitis B vaccine be incorporated into routine infant and childhood - 111 -

Review of Literature

Prevention of HBV

immunization programmes for all countries. The efficacy of universal immunization has been shown in different countries with striking reductions of the prevalence of HBV carriage in children. Most important, hepatitis B vaccination can protect children against HCC and fulminant hepatitis (Yegane et al., 2004).

Infection with HBV has become a vaccine preventable disease. In order for recombinant HBV vaccines to eradicate HBV, a universal vaccination of neonates and or children needs to be implemented however; there are major obstacles on the road to global hepatitis B vaccination i.e. poverty and scarcity of human resources in those parts of the world who are most badly in need of these vaccines. Despite their proven immunogenicity, hepatitis B vaccines are unable to induce an adequate immune response in 5-10% of healthy adults. The nonresponsiveness of these subjects is a selective phenomenon and not the expression of a general immune deficiency (Akbar et al., 2004).

Immunization against hepatitis B is required for groups that are at an increased risk of acquiring this infection. These groups include individuals requiring repeated transfusions of blood or blood products, prolonged in-patient treatment, patients who require frequent tissue penetration or need repeated access to the circulation, patients with natural or acquired immune deficiency, and patients with malignant disease (Candotti et al., 2004). Viral hepatitis is an occupational hazard among health care personal. High rates of infection with hepatitis B occur in narcotic drug addicts and drug abusers, homosexuals and prostitutes.

Individuals working in high endemic areas are also at increased risk of infection. Young infants, children and susceptible persons living in certain tropical and subtropical areas where present socioeconomic conditions are poor and the prevalence of hepatitis B is high should also be immunized (Lai, 2004).

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Prevention of HBV

Many countries, including the United States, introduced universal immunization for infants in 1992. More than 85 countries have introduced hepatitis B vaccine into their national immunization programs (Yao et al., 2004).

Available vaccines are highly effective in inducing long-term seroprotection and are considered of equivalent immunogenicity for most purposes. Immunologic memory maintains seroprotection in the absence of measurable HBsAb. Innovations have included the 2-dose, 10mg Recombivax HB regimen for those 11-15 years old and Twinrix, a combination hepatitis A and B vaccine. The immunocompromised are at increased risk for hypo or non-response to HBV vaccine and loss of immunologic memory. These patients should have post vaccination testing for HBsAb titers as well as periodic testing thereafter. If their titer decreases to less than 10 MIU/, they should receive a booster dose (Hm et al., 2004).

Types of vaccines: 1- Human plasma derived vaccines: The first active protective vaccines were introduced in 1981 (Schiff et al., 2003a).

2- Recombinant DNA vaccine: The first human vaccine produced with molecular biologic techniques (Kakimi et al., 2002b).

3- Hybrid virus vaccine: Potential live vaccines using recombinant vaccine viruses to HB, rabies and herpes simplex (Chen et al., 2002b).  Immune response to HB vaccines in special groups: Vaccination of special groups who are at increased risk of infection shows lower immune response to the vaccines. For example, only 60% of - 113 -

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Prevention of HBV

haemodialysis patients develop anti HBs after vaccination (Keeffe et al., 2004) and mentally retarded patients who have significantly lower response rates (Perillo, 2004).

Hepatitis B vaccines are effective in producing protection against HBV infection in hemodialysis patients but antibody response is variable (Yoshida et al., 2004). Non-responders have higher morbidity and mortality. Thus can be considered as a risk factor in the Haemodialysis populations (Zhang, 2004).

Fig. (21): Global status of countries using HepB vaccine in their national infant immunization system (WHO, 2003).

I.D. administration of recombinant HB vaccine in repeated small injections is absolutely effective in Haemodialysis patients (gave higher antibody titers in at least 50% of the follow-up measurements) (Zhou and Wu, 2004).

Zhu et al. (2004) investigated the immune response of liver transplantation to HB vaccines they stated that the total response rate to - 114 -

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Prevention of HBV

HB vaccine in liver transplant patients, is much lower than in general population and there is a rapid decline of antibody titers probably due to immune suppression. The role of booster in these patients should be clarified (Wright, 2004).

Table (25): Post Exposure Prophylaxis and Post Liver Transplant Therapies & Preventive Vaccines for HBV (Lok et al., 2004): Post Exposure Prophylaxis and Post Liver Transplant Therapies Brand Name

Activity

Manufacturer

FDA Status

HBV immune Bayer (US) Globulin HBV immune Nabi Nabi-HB Globulin HBV immune Cangene (Canada) VariZIG Globulin Preventive Vaccines for HBV

FDAapproved FDAapproved BLA filed with FDA

Brand Name

Activity

Manufacturer

FDA Status

Recombivax HB

Hepatitis B

Merck & Co.

BayHepB

FDAapproved Hepatitis B GlaxoSmithKline Energix-B FDAapproved Hepatitis A GlaxoSmithKline Twinrix FDAand B approved Hepatitis B GlaxoSmithKline Pediarix* FDAand other approved Pediarix: a new combination vaccine that protects infants against diphtheria, tetanus, pertussis (whooping cough), polio, and disease due to the hepatitis B virus.

Prophylaxis against hepatitis B recurrence following liver transplantation: Innovations in the management of HBV infection before and after orthotopic liver transplantation (OLT) have revolutionized outcomes for hepatitis B virus (HBV)-infected recipients. In the absence of preventative therapies, OLT for patients with acute or chronic replicating HBV infections resulted in universal reinfection of the allograft, progressive graft failure and excessive mortality, even with retransplantation (Weber et al., 2002). Thus, HBV-related liver disease - 115 -

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Prevention of HBV

was initially regarded as a contraindication to OLT (Tan et al., 2002) and was excluded as an indication by Medicare in the U.S.A. (Tai et al., 2002).

Over the past 15 years, sequential application of therapeutic strategies to prevent recurrent HBV infection after OLT and to inhibit HBV replication before and after OLT has steadily improved outcomes (Shang

et

al.,

2002).

Indefinite

administration

of

passive

immunoprophylaxis with hepatitis B immune globulin (HBIG, a source of high titer, polyvalent anti-HBs antibodies) resulted in a significant reduction in recurrent hepatitis B, especially among recipients without active HBV replication at the time of OLT (Sobao et al., 2002).

Pre-OLT inhibition of HBV replication using lamivudine (LAM) rendered patients with active replication eligible for transplantation, prevented recurrence post-OLT (unless LAM-resistant escape mutations developed) and improved the outcome of patients who became reinfected (Squadrito et al., 2002). The combination of HBIG and lamivudine enhanced prevention of HBV reinfection (Lalekha et al., 2002) and the advent of adefovir dipoxil (ADV) provided a safe and efficacious therapeutic option for patients with LAM-resistant infection (Lai and Terrault, 2004). As a result of this progress in prevention and treatment of HBV reinfection, acute or chronic hepatitis B is now universally accepted as an excellent indication for OLT (Squadrito et al., 2002).

Transplantation of livers from donors with isolated anti-HBcpositivity (i.e. negative for HBsAg and anti-HBs) has resulted in additional challenges, since isolated anti-HBc-positive livers are capable of transferring HBV infection to 50-78% of HBV-naïve recipients in the absence of preventative therapy (Thakur et al., 2002).

In addition, HBV-naïve recipients who have not been vaccinated to - 116 -

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Prevention of HBV

prevent hepatitis B prior to OLT have an ongoing risk of acquiring parenterally transmitted HBV infections that require prompt diagnosis and treatment (Torbenson and Thomas, 2002). Mandatory hepatitis B vaccination for all HBV-naive patients with diseases that could require OLT could significantly reduce this risk (Tsamandas et al., 2002).

Two mechanisms have been implicated in allograft reinfection: 1) rapid reinfection by HBV in the circulation of the recipient and/or 2) reinfection from HBV replicating in extrahepatic sites (Vanlandschoot et al., 2002). Since the former mechanism predominates in recipients with high HBV viral loads at the time of OLT, patients with active replication detected by insensitive molecular hybridization assays were considered as inferior candidates (Wen et al., 2002).

LAM has been extensively studied in OLT candidates, is well tolerated in decompensated cirrhosis and results in undetectable HBV DNA using molecular hybridization in 63-100% of patients within 2-3 months (Ohshiro et al., 2002). LAM is effective for both wild type (WT) and precore mutant (HBeAg-negative) strains (Gotsman et al., 2002). Uninterrupted therapy is required prior to OLT, since premature cessation results in recurrent HBV replication (Wolters et al., 2002).

Unfortunately, prolonged therapy, which is necessary for clinical benefit, increases the risk of developing LAM-resistant mutations as a result of amino acid substitutions in the YMDD motif encoded by the HBV RNA-dependent DNA polymerase gene (Wong et al., 2002).

The incidence of such mutations was 15-20% per year in the studies summarized in the Table (26), and development of mutations can worsen liver failure (Brabin et al., 2002). Results of OLT in patients with YMDD mutations before transplantation have been reported for only a few patients and were conflicting (Pramoolsinsup, 2002).

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Prevention of HBV

Combination HBIG and LAM prevented recurrence in some (Buti et al., 2002), but not all recipients (Chan et al., 2002). Since ADV and tenofovir have excellent efficacy against LAM-resistant HBV mutants (Perrillo, 2002), they should be used to treat patients with LAM resistance prior to OLT (Dando and Plosker, 2003).

Adefovir Monotherapy is an excellent therapy for pre-OLT patients with LAM-resistant infections (Schiff et al., 2003b).

Entecavir Monotherapy is a potent antiviral agent for both WT (Wild type) and LAM-resistant forms of HBV (Honkoop and de Man, 2003) that reduces HBV DNA levels to a greater degree than LAM (Barcena et al., 2003).

ETV (Entecavir) is currently in phase III

clinical trials in non-transplant patients (Toniutto et al., 2004).

The combination of LAM therapy pre- and post-OLT with HBIG post-OLT has become the standard of care for most liver transplant programs (Samuel, 2004).

Table (26): Results of Lamivudine Monotherapy Prior to OLT (*Mean or Median. NR; not reported) (Dahmen et al., 2004).

Authors

N=

Grellier et al., Markowitz et al., Villeneuve et al., Lo et al., Perrillo et al., Yao et al., Seehofer et al., Rosenau et al., Marzano et al., Fontana et al., Andreone et al., Fontana et al.,

17 10 35 31 30 23 17 19 33 162 25 154

Duration of Therapy*(months) >1 2.7 19 3.2 29 13 7.2 12 16 16 4.5 5.7

- 118 -

Negative HBV DNA (%) 100 100 100 63 74 100 88 NR 73 67 92 >80

Resistant Mutants (%) NR NR 25 NR 22 10 18 10.5 3 11 8 27

Review of Literature

Prevention of HBV

Adoptive Transfer of HBV Immunity from Donor Livers Effective adoptive transfer of humoral immunity to hepatitis B was first demonstrated in recipients of bone marrow transplants from immune donors (Chang, 2003). Transplantation of livers from HBV vaccinated woodchucks into HBV-infected recipients also has been shown to reduce or delay severity of reinfection, presumably due to the effects of HBVspecific memory cells among passenger leukocytes (Dahmen et al., 2004). An initially low recurrence rate in a Chinese cohort receiving LAM monotherapy was hypothesized to be due to transplantation of passenger leukocytes in HBV-immune donors that produced anti-HBs in the recipients (Papatheodoridis and Hadziyannis, 2004). A recent report ascribed the successful elimination of a de novo HBV infection post-OLT with development of anti-HBs to the fact that the liver donor had been immunized against hepatitis B (Toniutto et al., 2004).

Table (27): Effect of Combination Therapy with HBIG and Lamivudine to Prevent HBV Reinfection after OLT. (IV, intravenous; IM, intramuscular, NR, not reported) (Toniutto et al., 2004).

Authors

N=

HBV DNANegative at OLT (%)

HBIGRoute of Administration

HBV Reinfection (%)

14

Pre-OLT Duration* of LAM(mos) 3

Markowitz et al., Yao et al., Yoshida et al., Angus, et al.

Marzano et al., McCaughan et al., Rosenau et al., Roche et al., Han et al., Seehofer et al.,

93

IV

0

10 7 37 33

8.6 NR 3.2 4.6

80 100 NR 100

IV then IM IM IM IV

10 0 3 4

9

0

NR

IM

0

21 15 59 17

4.6 4.6 NR 10.6

77 73 NR 71

IV IV IV IV

10 7 0 18

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Review of Literature

Prevention of HBV

Vaccination after Transplantation The prospect of generating active immunity against HBsAg epitopes remains an intriguing strategy that could obviate the need for passive immunoprophylaxis (Ohshiro et al., 2002).

Because of the shortage of deceased donor organs, livers from isolated anti-HBc-positive donors (negative for HBsAg and anti-HBs) are being routinely transplanted into HBV-naïve recipients (Moola et al., 2002). In the absence of preventative measures, HBV infection occurred in 50% to 78% of such recipients in Spain (Arase et al., 2002) and the U.S.A. (Chang et al., 2003), respectively. However, livers from antiHBc-positive donors can be successfully used without reinfection if recipients receive prophylactic therapy with a combination of HBIG and LAM (Fabrega et al., 2003) or LAM monotherapy (Saab et al., 2003).

Recent reports also indicate that anti-HBc positivity in the donor is not a contraindication for live donor liver transplantation when effective prophylaxis is given (Hwang et al., 2003).

- 120 -

Summary

SUMMARY & CONCLUSION HBV is a Hepatotropic DNA-containing virus, discovered in 1966

by Blumberg. The virion of hepatitis B (Dane particle) consists of surface and core with a diameter of 42 nm (Kumar and Agrawal, 2004).

The protein composition of HBV particles; either surface protein (HBs proteins) composed of LHBs (largest Hepatitis B proteins), MHBs (middle Hepatitis B proteins), SHBs (small Hepatitis B proteins) or core proteins; composed of HBc protein and HBe protein.

The world health organization (WHO, 2004) estimated that 2 billion people have been infected by HBV worldwide; of these more than 300 millions are chronically infected carriers of whom 25% are at risk of serious illness and eventually death from cirrhosis or hepatocellular carcinoma. The prevalence of HBV infection varies markedly throughout regions of the world; highly endemic in South East Asia, moderately endemic in Eastern and Southern Europe and low endemic areas as in North America (Tsai, 2004).

Concerning transmission of HBV; there is peri-natal transmission, sexual contact, blood and blood products, parentral drug abuse, opportunities for parentral infection, transmission in high endemic areas, exposure of unknown origin is still present.

As regards clinical presentation and sequelae; HBV can present as acute infection, fulminant hepatic failure (FHF), chronic hepatitis, extrahepatic manifestations, post hepatitis B cirrhosis or combinations with HDV or HCV. Occult HBV infection is characterized by the presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg).

Concerning the diagnosis of acute and chronic hepatitis B; the advances in molecular biology techniques led to the development of hybridization and polymerase chain reaction (PCR) assays for direct - 121 -

Summary

determination of HBV DNA. The diagnosis of HBV infection can also be made by the detection of HBsAg or HBcAg in liver tissues by immunohistochemical staining and of HBV DNA by Southern hybridization, in-situ hybridization, or PCR. Treatment of chronic hepatitis B include Interferon therapy, nucleoside analogues such as Lamivudine, Adefovir Dipivoxil, Entecavir, Famciclovir, Emtricitabine/ coviracil, Combination therapy, Therapeutic vaccine, Gene therapy and Immunotherapy.

Prophylaxis against viral B infection is highly recommended using vaccination alone or combined with hepatitis B immunoglobulin for infants and individuals at risk of exposure.

- 122 -

Recommendations

RECOMMENDATIONS  Hepatitis B vaccination is the best protection as it provides protection against hepatitis B for 15 years and possibly much longer. It is recommended that all infants, health care workers and persons at risk of exposure e.g. sexual partners of chronically infected persons; should be vaccinated.  Hepatitis B Immune globulin is recommended for accidentally exposed persons, ideally within 24 hours of exposure and no later than 7 days. A repeated dose is necessary 28 - 30 days later.  Newborns of HBV infected mother should receive HBIG plus the hepatitis B vaccine within l2 hours of birth and two additional doses of vaccine at one and six to twelve months of age.  Strict governmental instructions for hygiene and sterilization should be followed particularly in risky procedures e.g. surgical intervention, dental procedures, endoscopies and blood or body fluids sampling.  Strict observation of blood donors, the presence of normal ALT, AST are not sufficient. Evaluation for occult HBV infection by determination of HBV DNA in serum or tissues should be considered in the context of the prevalence of HBV infection in this geographical area and the type of population.  In order to prevent HBV reinfection after liver transplantation, it is recommended to combine Lamivudine therapy pre- and posttransplantation with HBIG post-transplantation. This regimen has become the standard of care for most liver transplant programs.

- 123 -

References

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Hepatol Vol. 38, No. 4, Suppl. 1 Oct.43-89.  Sakugawa, H., Nakazone, H., Nakayosld, T. et al. (2002): Preponderance of hepatitis B virus genotype B contributes to a better prognosis of a chronic HBV infection in Okinawa, Japan. J Med Virol; 67(4): 484-9.  Sallam, T. (2000): Prevalence of HCV, HBV and HIV infections in chronic drug abusers. M.Sc. thesis, Tropical Medicine, Cairo University; 39-42.  Samuel, D. (2004): Management of hepatitis B in liver transplantation patients. Semin Liver Dis. 24 (Suppl 1):55-62.

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Weber, B., Schlemmer, K.H., Hartmann, E. et al. (2002): Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res; 54(2): 69-78.

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- 143 -

‫الملخص العربى‬ ‫تتناول هذة الرسالة كل ماهو جديد فى طريقة العدوى والظوواهر اكليينكككوة‪ ،‬وطور‬ ‫التشووصكو والعوومر لمووره اكلتكووا‬

‫اللروودى ال يروسووى‬

‫الووذ ت و التشوواف عووا‬

‫هووذا ال يوورو‬

‫‪ 1966‬رواسطة ريومريرر‪ ،‬ويتلون من سطح ونواة قطرها ‪ 42‬نانومككرون‬ ‫أم ووا الس ووطح فك ووو يتل ووون م وون مجموع ووة م وون الرروتين وواب ارروتين وواب اكلتك ووا‬

‫اللر وودى‬

‫اللريو ورة‬

‫والمتوسووطة واليوو)يرةم أمووا ةالنسووةة لينوواة فكو تحتووو عيووى رووروتين ‪ HBc‬وبووروتين ‪ HBe‬وكوول‬ ‫ه و ووذة المكون و وواب تس و ووتصد ف و ووى التش و ووصكو وتيعو و و‬

‫دو ار هام و ووا ف و ووى الظ و وواهرة اكليينككك و ووة ليم و ووره‬

‫واكستجاةة ليعمر وانتشار العدوى‬ ‫وطةق ووا لم ووا تو و نشو ورة ف ووى المق وواكب العيمك ووة المتصيي ووة وج وود أن ع وودد األش ووصاو‬ ‫الميووارين ةاكلتكووا‬

‫اللروودى ال يروسووى‬

‫كقوودر ةح ووالى ‪ 2‬رييووون شووصو عيووى مسووتوى العووال ‪،‬‬

‫وذلو طةقووا لمووا تو نشورة فووى تقريوور منظمووة اليووحة العالمكووة ا‪2004‬م موون هوواكم الميووارين يوجوود‬ ‫ح ووال ‪ 300‬مييووون شووصو موون حوواميى المووره‪ ،‬موونك ‪ %25‬معرضووين لميوواةة ةمضوواع اب‬ ‫المره التى قد تيل الى فشل كردى وسرطان أولوى ةاللرود وقود وجود أن هنوا ت واوب كريور فوى‬ ‫درجووة اسووتكطان اكلتكووا‬

‫اللروودى‬

‫فووى كافووة أنحووام العووال حيو‬

‫توجوود منوواط عالكووة اكسووتكطان‬

‫مثل جنو شر اسكا ومناط منص ضة اكستكطان مثل أمريكا الشمالكة‬ ‫أمووا فكمووا يصووو طوور نقوول العوودوى ةووال يرو‬

‫اللروودى‬

‫فكووى متعووددة وأهمكووا عوون‬

‫طريو نقوول الوود ومشووتقات ‪ ،‬اكتيووال الجنسو ‪ ،‬موون األ حاميووة المووره إلووى الجنووين‪ ،‬وعوون طريو‬ ‫استصدا األدواب والحقن المستعمية‪ ،‬كما توجد طر عدوى أصرى تعترر الى اكن مجكولة‬ ‫أما ةالنسوةة ليم ارحول اكليينكككوة والمضواع اب الصايوة ة يورو‬ ‫اكلتكووا‬

‫اللروودى الحوواد‪ ،‬اكلتكووا‬

‫اللروود الحوواد النش و ‪ ،‬اكلتكووا‬ ‫‪-1-‬‬

‫فكوى تتنوو مون‬

‫اللروود الموونمن‪ ،‬ث و ةعوود ذل و‬

‫مرحيووة التييووك اللروودى والمضوواع اب مثوول ال شوول اللروودى واألو ار اللردكووة وممووا ينيوود حوودة وسوورعة‬ ‫التدهور اكليينككى فى هاكم المرضى هو اكياةة ةألثر من فيرو‬ ‫ال يرو‬

‫جان‬

‫مثل فيرو‬

‫‪ D‬أو ‪ C‬الى‬

‫اللردى‬ ‫أمووا فكمووا يصووتو ةطوور التشووصكو‪ ،‬فقوود أدى التطووور المووذهل فووى تقنكووة الكندسووة‬

‫الوراثكووة واكحكووام الجنيئكووة الووى ظكووور ت وواعمب مثوول التكجووين و رووى سووى ار والتووى سوواعدب عيووى‬ ‫اللشك عن ال دى ان اكة الصاو ةال يرو‬

‫وتعترر هذة الطر من أه الطر التى‬

‫اللردى‬

‫تسووتصد فووى التشووصكو وفووى ال حووو ماقروول العوومر ومتاةعووة نتووائ العوومر‪ ،‬وكووذل تعتروور الطوور‬ ‫الوحي وودة الت ووى ت ووادى ال ووى التش ووا‬

‫الع وودوى ة ووال يرو‬

‫المس ووتتر وال ووذى كي ووت التش ووافة‬

‫اللر وودى‬

‫ةالطر التشصكيكة التقييدكة‪ ،‬وهذا النو من أه العوامل التى تادى الى انتشار ال يورو‬

‫اللرودى‬

‫وصاية عن طري نقل الد ومشتقاتة فى رنو الد والتى تعتمد عادة عيى فحوو المستضواد‬ ‫السطحى لي يرو‬ ‫وقوود تناولووب الرسووالة أكضووا أه و الطوور التووى تسووتصد فووى عوومر اكلتكووا‬ ‫ال يروسى‬

‫اللروودى‬

‫وه متعوددة ومت اوتوة فوى اكسوتجاةة وتشومل العومر ةواكنترفيرون‪ ،‬أو العقواقير مثول‬

‫كمك يوودين‪ ،‬أدك ووفير ديرك وكسويل‪ ،‬إنتككوافير‪ ،‬فامسوككيوفير‪ ،‬إمتريسويتارين ‪/‬كوفي ارسويل‪ ،‬أو العومر‬ ‫العمجو أو العومر‬ ‫رواسطة الجمع رين اثنين أو ألثر من األدوكة الساةقة ةاإلضافة إلى التطعك‬ ‫ّ‬ ‫ةالجينواب أو العوومر المنواع‬

‫وتناولووب المقالوة أكضووا الحوودي‬

‫فوى عوومر موره اكلتكووا‬

‫اللروودى‬

‫قرل وةعد نراعة اللرد وأوضحب أهمكة العمر لكاكم المرضى‬ ‫وتووت الوقاكووة مون اكلتكووا‬

‫اللروود ال يروسو‬

‫إمووا ةووالتطعك السووير رواسووطة نقوول‬

‫األجسا المناعكة المضادة ليةوال)ين أو لطط وال الرضوع أو ةوالتطعك اكيجوار رواسوطة رروتينواب‬ ‫ال يرو‬

‫المشتقة من الةمنما الةشرية أو المصيقة رواسطة الكندسة الوراثكة‬

‫‪-2-‬‬

‫الحديث في انتشار وتشخيص وعالج االلتهاب الكبدى‬ ‫الفيروسى ب‬ ‫مقالة توطئة للحصول على درجة الماجستير‬ ‫فى طب المناطق الحارة‬ ‫مقدمة من‬

‫الطبيب‪ /‬هشام نعمان عبد الرحيم مصطفى‬ ‫بكالوريوس الطب والجراحة – جامعة القاهرة‬ ‫وذلك تحت اشراف‬

‫األستاذ الدكتور‪ /‬ليلى أحمد محمد‬ ‫أستاذ طب المناطق الحارة‬ ‫كلية الطب‪-‬جامعة القاهرة‬

‫الدكتور‪ /‬أحمد نبيل لطفي حسن‬ ‫مدرس طب المناطق الحارة‬ ‫كلية الطب‪-‬جامعة القاهرة‬

‫كلية الطب‬ ‫جامعة القاهرة‬ ‫‪2005‬‬