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
Page No.Title
1-3Introduction and Aim of the Work
Review of Literature
4Historical Note.1
5-20Morphology of Hepatitis B Virus.2
21-34Epidemiology of HBV.3
35-44Clinical Presentation and Sequelae.4
45-54HBV Status in Egypt.5
55-76Diagnosis of Acute and Chronic Hepatitis B.6
77-112Treatment of Chronic Hepatitis B.7
113-124Prevention of HBV.8
125-126Summary and Conclusions
127Recommendations
128-159References
Arabic Summary

III
List of Figures
Title Page
No.
Fig. (1): Schematic diagram of hepadnavirus particles. 6
Fig. (2): Electron microscopic presentation of HBV particles. 7
Fig. (3): Genome organization of HBV. 9
Fig. (4): A group of hepatitis B virions (right) and enlargements of the
two exposed cores.
10
Fig. (5): Hepatitis B Surface Proteins. 12
Fig. (6): Largest HBs proteins. 13
Fig. (7): Middle Hepatitis B proteins (MHBs). 14
Fig. (8): Schematic diagram of two loops of the "a" determinant of HBs
protein.
15
Fig. (9): Small Hepatitis B proteins (SHBs). 16
Fig. (10): HBV outer envelope contains high amounts of hepatitis B
surface proteins.
16
Fig. (11): Diagram showing Purified virions possess the HBc protein. 18
Fig. (12): HBV has evolved a unique life cycle. 20
Fig. (13): HBsAg Endemicity. 21
Fig. (14): Countries with areas with moderate to high risk of infection. 22
Fig. (15): Geographic pattern of Hepatitis B prevalence. 23
Fig. (16): Geographic distribution of HBV Genotype. 47
Fig. (17): Analysis of 1860 acute hepatitis cases. 49
Fig. (18): Evolution of HBV markers in acute infection. 61
Fig. (19): Characteristics of progression to chronic HBV infection. 62
Fig. (20): Mode of action of interferon alpha (INF-). 80
Fig. (21): Global status of countries using HepB vaccine in their
national infant immunization system.
118

IV
List of Tables
Page
No.
Title
27Table (1): Characteristics of endemic patterns of hepatitis B
virus infection.
47Table (2): Geographic Distribution of HBV Genotypes.
48Table (3): Prevalence of HCV, HBV and HBsAg in Egypt 1996.
48Table (4): Etiology of acute viral hepatitis among 200 patients.
49Table (5): The frequency of acute hepatitis B in different age
groups in year 2002 in comparison to year 1983.
64Table (6): HBeAg.
65Table (7): Interpretations of available serologic test results
for HBV.
66Table (8): Serological test findings at different stages of HBV
infection and in convalescence.
66Table (9): Hepatitis B tests.
67Table (10): Antibody to Hepatitis B Surface Antigen (HBsAg
Assay).
67Table (11): Hepatitis B Surface Antigen (Anti-HBs Assay).
68Table (12): Hepatitis B Virus Core Antigen (Anti-HBc Assay).
72Table (13): Histological Activity Index.
73Table (14): Modified HAI grading: necroinflammatory scores.
74Table (15): Glossary set by Keeffe et al., 2004 to correlate
clinical terms with laboratory results.
75Table (16): The stages of HBV in correlation with the
laboratory results.
76Table (17): Interpretation of serological markers.
79Table (18): Hepatitis B Therapies and Treatment.
81Table (19): Types and Properties of Interferon
83Table (20): Determinant of responsive factors to interferon.
104Table (21): Comparison of Three Approved Treatments of
Chronic Hepatitis B.
105Table (22): Goals of Therapy and Definitions of Response to
Therapy in Chronic Hepatitis B.
106Table (23): Available Agents for Treatment of Chronic Hepatitis
B.
112Table (24): Recommendations for Treatment of Chronic
Hepatitis B.
119Table (25): Post Exposure Prophylaxis and Post Liver
Transplant Therapies & Preventive Vaccines for HBV.
123Table (26): Results of Lamivudine Monotherapy Prior to OLT.
124Table (27): Effect of Combination Therapy with HBIG and
Lamivudine to Prevent HBV Reinfection after OLT.

V
List of Graphs
Page
No.
Title
42Graph (1): Virological response of chronic hepatitis C patients
with and without past history of HBV.
50Graph (2): Age distribution of patients with acute HBV.
86Graph (3): Lamivudine: Histologic Improvement at week 52 in
HBeAg-positive patients.
87Graph (4): Effect of Lamivudine on HBV DNA Levels.
88Graph (5): Response to Lamivudine in HBeAg-negative CHB.
90Graph (6): Treatment of Lamivudine Resistance: Prospective
study of Adefovir vs Lamivudine vs Combination Therapy.
91Graph (7): HBeAg Seroconversion with Lamivudine: Effect of
treatment Duration and Baseline ALT.
94Graph (8): HBeAg-positive patients: Adefovir vs placebo for 48
weeks.
95Graph (9): Long-term Adefovir in HBeAg-negative Patients:
Serum ALT.
96Graph (10): Long-term Adefovir in HBeAg-negative Patients:
Median Serum HBV DNA.
96Graph (11): Adefovir vs Placebo: Change from Baseline in HBV
DNA.
97Graph (12): Entecavir Treatment of Lamivudine-resistant
Hepatitis B.

VI
List of Abbreviations
Abbreviation Term
ADV Adefovir depevoxil
Anti-HBc Antibody to HBcAg
Anti-HBe Antibody to HBeAg
Anti-HBs Antibody to HBsAg
ALT Alanine aminotransferase
AST Aspartate aminotransferase
CHB Chronic hepatitis B
cccDNA covalently closed circle DNA
CTL cytotoxic T lymphocyte
ETV Entecavir
FHF Fulminant hepatic failure
GMCSF granulocyte macrophage colony stimulating factor
HBV Hepatitis B virus
HBIG Hepatitis B immune globulin
HBcAg Hepatitis B core antigen
HBeAg Hepatitis B e antigen" Envelope "
HBsAg Hepatitis B surface antigen
INF-α Interferon Alfa
LAM Lamivudine
LHBs Large HBs protein
MHBs Middle HBs protein
ORFs Open reading frames
OLT Orthotopic liver transplantation
PCR Polymerase chain reaction
Pre-S1, pre-S2 Envelope protein epitopes
SHBs Small HBs protein
ULN Upper limit of normal
WHO World health organization
WT 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.

‫االلتهاب‬ ‫وعالج‬ ‫وتشخيص‬ ‫انتشار‬ ‫في‬ ‫الحديث‬
‫ب‬ ‫الفيروسى‬ ‫الكبدى‬
‫الماجستير‬ ‫درجة‬ ‫على‬ ‫للحصول‬ ‫توطئة‬ ‫مقالة‬
‫ة‬‫ر‬‫الحا‬ ‫المناطق‬ ‫طب‬ ‫فى‬
‫مقدمة‬‫من‬
‫الرحيم‬ ‫عبد‬ ‫نعمان‬ ‫هشام‬ /‫الطبيب‬‫مصطفى‬
‫احة‬‫ر‬‫الج‬‫و‬ ‫الطب‬ ‫يوس‬‫ر‬‫بكالو‬–‫ة‬‫ر‬‫القاه‬ ‫جامعة‬
‫اف‬‫ر‬‫اش‬ ‫تحت‬ ‫وذلك‬
‫محمد‬ ‫أحمد‬ ‫ليلى‬ /‫الدكتور‬ ‫األستاذ‬
‫ة‬‫ر‬‫الحا‬ ‫المناطق‬ ‫طب‬ ‫أستاذ‬
‫الطب‬ ‫كلية‬-‫ة‬‫ر‬‫القاه‬ ‫جامعة‬
‫حسن‬ ‫لطفى‬ ‫نبيل‬ ‫أحمد‬ /‫الدكتور‬
‫ة‬‫ر‬‫الحا‬ ‫المناطق‬ ‫طب‬ ‫مدرس‬
‫الطب‬ ‫كلية‬-‫ة‬‫ر‬‫القاه‬ ‫جامعة‬
‫الطب‬ ‫كلية‬
‫ة‬‫القاهر‬ ‫جامعة‬
2004

Introduction and aim of the work
-1-
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).

Introduction and aim of the work
-2-
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.

Historical Note
-3-
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.

Review of Literature Morphology of hepatitis B virus
-4-
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).

-5-
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).

-6-
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).

-7-
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).

-8-
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).

-9-
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

-10-
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).

-11-
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.

-12-
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

-13-
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).

-14-
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).

-15-
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).

-16-
(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).

-17-
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

-18-
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).

-19-
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).

Review of Literature Epidemiology of HBV
-20-
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

-21-
anti-HBs (HBsAb) is much higher than those of chronic carriers (HBsAg-
positives) 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

-22-
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

-23-
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 viral-
host 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

-24-
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;

-25-
(vi) 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):

-26-
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).
Characteristic
Endemicity of infection
Low (%) Intermediate
(%)
High (%)
Chronic infection prevalence 0.5-2 2-7 ≥8
Past infection prevalence 5-7 10-60 70-95
Prenatal infection Rare Uncommon Common
(<10) (10-60) (>20)
Early childhood infection Rare Common Very common
(<10) (10-60) (>60)
Adolescent/adult infection Very
common
Common Uncommon
(70-90) (20-50) (10-20)

-27-
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:

-28-
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).

-29-
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

-30-
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).

-31-
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 HBV-
contaminated donor blood (Park et al., 2004).

-32-
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

-33-
blood parenterally such as from pricking or through skin abrasions.
Surgeons and dentists are particularly at risk in operating on HBsAg-
positive 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).

Review of Literature Clinical Presentation and Sequelae
-34-
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):

-35-
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).

-36-
(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 HBV-
DNA 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

-37-
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-

-38-
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 manifestations: Pleural effusion,
hepatopulmonary, portopulmonary syndrome, myocarditis,
pericarditis, and arrhythmia (Kobak et al., 2004).
(vi) Hematologic and gastrointestinal tract manifestations:
Pancreatitis, aplastic anemia, agranulocytosis and diffuse
intravascular coagulation (Haushofer et al., 2004).
(e) Post Hepatitis B Cirrhosis:

-39-
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

-40-
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).

-41-
0
20
40
60
80
At week 24 At week 48
(EVR)
At week 72
(SVR)
Virological response of chronic hepatitis C
patients with and without past history of
HBV
HBV seronegative (Group I)
HBV seropositve (Group II +
III)
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 anti-
HBe is paralleled by exacerbation of hepatitis as a result of immune-
mediated 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

-42-
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 non-
HBV 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 population-
based 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).

Review of Literature HBV status in Egypt
-43-
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.

-44-
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.

-45-
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).

-46-
Table (3): Prevalence of HCV, HBV and HBsAg in Egypt 1996
(Mohamed and Aoun, 2002):
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).
* Infection by more than one virus: 82 (41%)
** HDV in 8 patients (3 co-infection and 5 super infections)
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.
Age Adjusted Rates /100 HCV HBcAb HBsAg
Total Egypt 14.5 22.5 4.5
Rural 18.9 24.9 5.2
Urban 9.1 19.5 3.7
Males 16.4 23.9 5.0
Females 12.7 21 4.0
Etiology Total Single infection
N % N %
HAV
HEV
HBV**
HCV
68
25
63
34
34
12.5
31.5
17
34
15
29
10
17
7.5
14.5
5
Non A-E hepatitis
CMV
EBV
HGV
TTV
12
7
23
46
6
3.5
11.5
23
1
1
1
9
0.5
0.5
0.5
4.5
Undiagnosed 13 6.5

-47-
Etiology of Acute Viral Hepatitis in Egypt 1997-2000
21%
16%
13%
1%
24%
25%
HCV
HBV
All-ve
Mixed
HEV
HAV
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):
1983 2002 P
Age
groups
Total
(235) Acute HBV
Total
(200) Acute HBV
N % N %
< 5ys (36) 7 19.4 (23) 0 0
6-8ys (22) 9 40.9 (35) 0 0
9-11ys (12) 5 41.7 (22) 6 27.3 NS
12-20ys (48) 28 58.3 (26) 10 38.5 NS
21-40ys (73) 38 52.1 (73) 41 56.2 NS
41-60ys (28) 11 39.3 (19) 6 31.6 NS
>60ys (16) 4 25 (2) 0 0

-48-
Age distribution of patients with
acute hepatitis B
0
10
20
30
40
50
60
70
< 14 ys 14-30 ys 31-50 ys > 50 ys
Sedes
1
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

-49-
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:
Exposure rate =
No. of cases positive for HBcAb + No. of cases positive for
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

-50-
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 non-
schistosomal 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%)

-51-
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 non-
injecting 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:

-52-
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).

Review of Literature Diagnosis of HBV
74
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
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 anti-
HBc declines slowly, but markers of viral replication HBeAg and HBV-
DNA 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 anti-
HBc 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.

74
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
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
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
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).

74
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).

74
(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 anti-
body, 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
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
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 anti-
HBc or anti-HBs, when a patient develops anti-HBe in recovery, the
antibody will probably disappear within 12 months (Hadziyannis et al.,
2003).

Update on prevalence, diagnosis and treatment of Hepatitis B Virus

Update on prevalence, diagnosis and treatment of Hepatitis B Virus

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