1

INTRODUCTION

Hepatitis is a major health problem affecting millions of people through out

the world these days. The Hepatitis was recognized centuries back but the
identity was well established only after the epidemic during the world wars1.
The term viral Hepatitis refers to a primary infection of liver by any
one of a heterogeneous group of Hepatitis viruses which currently consists of
type A, B, C, D, E and G. Type F was proposed for a putative virus, causing
transfusion associated Hepatitis distinct from type A-E. But it proved to be a
mutant of type B virus. Type F was therefore deleted from the list of
Hepatitis viruses.
Hepatitis viruses are taxonomically unrelated. Except for type B which
is a DNA virus all the other are RNA viruses. The features common to them
are their hepatotropism and ability to cause a similar icteric illness ranging in
severity from the unapparent to fulminant fatal forms.
Hepatitis may occur incidentally during many other viral infections
such as with:-
1. Yellow fever
2. Lassa fever
3. Marburg
4. Cytomegalo Virus
5. Herpes Virus
6. Varicella zoster
7. EB
8. Rubella or cowpox viruses
9. Measles

By Epidemiology and clinical criteria, two types of viral Hepatitis have been
recognized.
1. Occurring sporadically or as epidemics occurring in children and young
adults and transmitted by fecal-oral route. This is called infectious
Hepatitis later named type A Hepatitis.
2. Transmitted by inoculation originally observed in persons receiving
serum inoculation or blood transfusion. This had been given various
names such as homologous serum jaundice, serum Hepatitis and
transfusion Hepatitis. It was later called type B Hepatitis.
 2

Other types of virus causing Hepatitis are:-

• Type C virus – was identified as causing many transfusion associated

Hepatitis cases.
• A defective virus which depends on the helper function of type B was
called delta or type D Hepatitis virus.
• Another type of Hepatitis transmitted by fecal-oral route and prevalent
only in developing nations was found to be caused by Hepatitis E
virus.
• The sixth member of the group, Hepatitis G virus can also cause
Hepatitis, but its role has not yet been adequately understood.

Type B Hepatitis is the most widespread and the most important type of
viral Hepatitis. More than a third of the world’s population is estimated to be
having been identified by Hepatitis B virus (HBV). About a quarter of them
became HBV carriers. A quarter of these develop various severe diseases
like2 :-
1. Severe Liver diseases
2. Chronic Hepatitis
3. Cirrhosis
4. Primary hepatic cancer

As there is an effective vaccine against Hepatitis B virus hepatocellular

carcinoma becomes the only human cancer which is vaccine preventable. The
WHO estimates that HBV infection causes more than a million deaths a year
world wide.
 3

AIMS AND OBJECTIVES

The aims and objectives of the present study are as follows:-

• To determine the prevalence of HBsAg in Normal Healthy persons

( Routine or Normal Health check-up)

• Prevalence of HBsAg in women attending antenantal clinics.

• Prevalence of HBsAg in Hepatitis patients.

 4

REVIEW OF LITERATURE

DISCOVERY OF HBV

The discovery of HBV was serendipitous. In 1965, Blumberg, studying

human serum lipoprotein allotypes, observed in the serum of an Australian
aborigine, a new antigen which gave a clearly defined line of precipitation
with sera from two hemophiliacs who had received multiple blood
transfusions. This was named Australian antigen.
By 1968 the “Australian antigen” was found to be associated with
serum Hepatitis. It was shown to be the surface component of HBV.
Therefore the name Australian antigen was changed to ‘Hepatitis B surface
antigen (HBsAg).5
In 1970 Dane described a particle named Dane particle which is a
double walled spherical structure 42 nm in diameter. This particle is the
complete Hepatitis B virus.

HISTORY AND ETIOLOGY OF VIRAL HEPATITIS3

Viral Hepatitis is a disease of antiquity. Epidemic jaundice is mentioned in
the Talmud. Hippocrates is created with the first description of the disease in
the 4th century before Christ. Outbreaks of jaundice ravaged population
during world wars. It was an important plague in causing morbidity in
Europe. However the infectious nature of the disease was not recognized
until the end of the 19th century.
The first description of the Hepatitis B dates back to 1885 when
Lurman, a public health officer on Germany gave a detail report of an
outbreak of jaundice that developed among the workers of a local company
who had been vaccinated against small pox. The report is also noteworthy as
it points clearly towards the parental exposure as the likely mode of
transmission.
Due to importance of the Hepatitis in soldiers, transmission studies
were initiated in human volunteers during the late thirties. This was
vigorously pursued during the Second World War.
These studies provided the first insight about the infectivity, mode of
transmission and properties of the Hepatitis agents. The suggested a viral
 5

nature of the disease. It also indicated that two distinct agents lacking cross
immunity were responsible for the military outbreak.
Mc Callum introduced the classic terminology of Hepatitis A virus and
Hepatitis B virus (HBV). It was found that Hepatitis A was induced by oral
route with incubation period of about 15-30 days. The serum was responsible
for inducing the Hepatitis B virus, after several months, when given by
parental route and not by oral route.4
The discovery of Australian antigen by Blumberg and his associates
was a major breakthrough. Prince et al recognized its relation with Hepatitis
B in mid sixties was another milestone in the study of Hepatitis.
The lesson learned from the work on Hepatitis B also helped in
discovery of HAV. Feinstone et al identified HAV in immunocomplexes
from faeces to which serum of a patient convalescent from Hepatitis A had
been added. When test for the Hepatitis A and B was made available, it
became clear that two viruses did not explain all the cause of viral Hepatitis,
as many patients lacked either of the infection.
Thus the concept of a new variant of viral Hepatitis was born. It was
called non A non B Hepatitis and its diagnosis was based on exclusion
criteria. The pre serological era of non A non B Hepatitis came to an end in
the mid eighties. The breakthrough this time resulted after meticulous and
exhaustive research involving the use of sophisticated technological methods.
Thus by the end of 80’s five Hepatitis viruses were identified. They were
HAV, HBV, HCV, HDV and HEV. Recently HGV and HFV have also been
identified.
The research in this field is continuing even today. New forms of
Hepatitis viruses are being discovered now and then.
 6

VIROLOGY
CLASSIFICATION 6

Because of its unique features, HBV is assigned to family Hepadnaviridae

(hepatotrophic DNA viruses). It consists of two genera.

Orthohepadnaviruses- contains HBV as well as the woodchuck and ground

squirrel Hepatitis viruses.

Avihepadnavirus- containing the pekin duck and grey heron Hepatitis

viruses.

HBV is Hepadnavirus type 1.

The above viruses share a number of important features in relation to

the structure of their virions and associated particles, their ability to cause
both acute and chronic infections in their natural host and their association
with hepatocellular carcinoma.

STRUCTURE

Under the electron microscope, sera from type B Hepatitis patients show
three types of particles:
• The most abundant form is a ‘spherical particle’ 22 nm in diameter.
• The second type of particle is filamentous or tubular with a diameter of
22 nm and of varying length. The two particles are antigenically
identical and are surface components of HBV (HBsAg) which are
produced in great excess.
• The third particle is a double walled spherical structure 42 nm in
diameter. This particle is the complete Hepatitis B virus, known as
‘Dane particle’.
The envelope proteins expressed on the surface of the virion and the surplus
22 nm diameter spherical and filamentous particle- constitute HBsAg. It
constitute of two major polypeptide, one of which is glycosylated. The
antigen expressed on the core is called HBcAg Hepatitis B core antigen. A
 7

third antigen called Hepatitis B e antigen is a soluble non particulate

nucleocapsid protein.

Fig. 1: HEPATITIS B STRUCTURE

 8

VIRAL GENOME AND ANTIGEN

The genome has a compact structure with four overlapping genes, for the
core, surface, polymerase protein and an X protein which may act as an
activator of transcription. 7

Fig.2 HBV gene and gene products.

 9

GENES REGIONS GENES PRODUCTS

S S Major protein (S)

S + pre- S2 Middle protein (M)
S+pre-S1&S2 Large protein (L)

C C HBcAg
C+pre-C HBeAg

P DNA polymerase

X HBxAg

The S gene- It codes for the surface antigen. It consists of S region and two
pre S regions, pre S2 and pre S1. When translation begins from pre S2 region,
the M or middle protein is formed. When the entire gene from pre S1 is
translated the L or large protein results. The L protein is present only in the
virion, while the M and S proteins are found in the circulating HBsAg
particle also.

The C gene- It has two regions C and pre C. When C region alone is
translated, the core antigen is formed. HBcAg is assembled as the
nucleocapsid core particles. It is not secreted and does not circulate in blood,
but can be demonstrated in hepatocytes but immunoflorescence. When
translation begins at pre C, the resulting protein is HBeAg, a nonparticulate
antigen processing a single protein which enables it to be secreted, hence
present in circulation. The presence to HBeAg in blood provides a convenient
and readily detectable marker of HBV replication and high infectivity.

The P gene-It is the largest and codes for the DNA polymerase enzyme.

The X gene- It codes for a small nonparticulate protein (HBxAg) which has
transactivating effects on both viral and some gene. HBxAg and its antibody
are present in patients with severe chronic Hepatitis and hepatocellular
carcinoma.
 10

ANTIGEN DIVERSITY
HBsAg exhibits antigenic diversity. It contains two different antigenic
components – the common group reactive antigen ‘a’ and the two pairs of
type specific antigens d-y and w-r only member of each pair being present at
time.
It can be divided into four antigenic subtypes’ adw, adr, ayw, ayr.
These phenotypic variations reflect differences and can be used in
epidemiological studies. There are differences in the geographical
distribution of the types and also to some extent with the means of
transmission.

STABILITY
HBV is relatively heat stable virus. It remains viable at room temperature for
long periods. Heat at 600oC for 10 hours reduces infectivity by hundred to
thousand folds. It is susceptible to chemical agents. Exposure to
hydrochlorite (10,000 ppm available chlorine) or 2 % glutaraldehyde
inactivates infectivity, through HBsAg may not destroy by such treatment.

REPLICATION
Replication of viral nucleic acid starts with the hepatocyte nucleus where
viral DNA can be either free, extra chromosomal or integrated at various sites
within the host chromosome. However, integration is not essential for viral
replication.
To replicate hepadnavirus DNA, a full length RNA copy is enclosed in
core protein in the hepatocyte nucleus, this is copied to DNA by the
polymerase, the RNA is destroyed and the DNA copied, to form double
stranded as the virion matures.

CLINICAL FEATURES
There is a broad range of clinical features associated with HBV infection.
The incubation period is long, about 1-6 months. The clinical picture of
Hepatitis B is similar to that of type A, but it tends to be more severe and
 11

protracted. The onset is insidious and fever is not prominent. Exhepatic

complications like arthralgia, cirrhosis and rarely polyarteritis or
glomerunephritis may occur. These are ascribed to circulating immune
complex containing the viral surface antigen.
About 90-95 % adults with acute Hepatitis B infection recovers within
1-2 months of onset and eliminates the virus from the body within 6 months,
remaining immune thereafter. Mortality is about 0.5-2%, but may be more in
post transfusion cases. About 1% of patients, particularly those having
simultaneous delta virus infection develop fatal fulminant Hepatitis.
A proportion of cases (1-10%) remains chronically infected. They may
be asymptomatic carriers or may progress to recurrent or chronic liver disease
or cirrhosis. A few of them may develop hepatocellular carcinoma after many
decades.

PATHOGENESIS8
All types of viral Hepatitis produce similar changes at the histological level.
In the acute stage there are signs of inflammation in the portal triads. The
infiltrate is lymphocytic.
In healthy carriers the inflammatory response is mild and the affected
hepatocyte are pale staining and glassy.
In chronic Hepatitis, damage extends out from the lobular tracts giving
the piecemeal necrosis appearance. Some lobular inflammation is also seen.
As the disease progress fibrosis develops and eventually cirrhosis.
Acute disease- HBV replicates in the hepatocytes, reflected in the detection
of viral DNA and HBcAg in the nucleus and HBcAg in the cytoplasm and at
the hepatocyte membrane. Both B and T cell responses are induced by these
antigens; damage to the hepatocyte could result from both antibody-
dependent NK and cytotoxic T cell action. Expression of MHC class 1
antigens is poor in hepatocytes but can be enhanced as interferons are
produced in response to the infection. This in turn leads to increased antigen
recognition and lysis of the infected hepatocytes.
In asymptomatic carriers there may be no evidence of cell damage,
despite the presence of integrated HBV DNA and HBsAg
in the liver and plasma.
Cell-mediated responses to HBcAg are often detectable in patients with
chronic active Hepatitis. Super infection with delta virus may predispose to
progression to cirrhosis.
 12

Persistence of Hepatitis B:
• Is indicated by HBsAg present for more than 6 months.
• Occurs in 5-10% of adults, 30% of childhood and 90% of newborn
infections.
• Is more frequent in males.
• Is more likely in the immunocompromised.

Hepatocellular Carcinoma HCC9

The highest rates of HCC are found in areas where HBV is highly
endemic and where infection occurs at a very early stage. This is necessary as
there may be an interval of 30-40 years between the infection and tumor
development. Integrated viral DNA can be found in the tumor cells but the
site differs in different tumors, although the tumor is clonal in origin in each
individual. The integrated DNA is extensively rearranged and regions may be
deleted. The mechanism of carcinogenesis is not yet clear, although it is
usually associated with cirrhosis. As with other tumors, infection with the
HBV may be only one factor and others, such as genetic or chemical, may be
necessary.

MODE OF TRANSMISSION
HBV is a blood borne virus and the infection is transmitted by-
• Parenteral
• Sexual
• Perinatal modes
• Blood of carriers
• Congenital or vertical
The virus may also be present in other body fluids and secretions such
as saliva, breast milk, semen, vaginal secretions, urine, bile and feces.10
Transfusion of carrier blood, once the most widely known mode of
infection has largely been eliminated whenever donor screening is
strictly enforced. Therapeutic preparations from pooled human blood
and serum have led to Hepatitis but this risk is now minimal with
screening of donors and production techniques ensuring virus
inactivation.
 13

HBV is very highly infectious, far more than HIV. Any object or
producer that can contain very minute traces of infected blood or other
material as little as 0.00001 ml can be infectious. These include:
• Shared syringes, needles and other sharp items or endoscopes
• Personal articles such as razors, nail clippers or combs
• Practices such as acupuncture, tattooing, ritual circumcision ear
or nose piercing
Field camps for surgery or disease detection by blood testing where separate
sterile articles may not be available.
Infection by direct contact with open skin lesions such as pyoderma,
eczema, cuts and scratches is very common among young children in
developing countries.
Congenital or vertical transmission is quite common from carrier
mothers. The risk to babies is high if the mother is HBeAg positive. Infection
is acquired during birth by contact of maternal blood with the skin and
mucosa of the fetus or in the immediate post natal period. HBV infected
neonates generally do not suffer from any clinical illness but remain carriers
for life and some of them may develop HCC after many decades.
Sexual transmission of HBV occurs everywhere but is more in the
developed countries, particularly in the promiscuous homosexual. The risk of
transmission by heterosexual and homosexual contact increases with the
number of partners and the duration of such relationships. HBV infection has
occurred after artificial insemination. Semen donor screening is therefore
obligatory.
Certain groups and occupations carry a high risk of infection. These
include medical and paramedical personnel, staff of blood bank, dialysis
units, medical laboratories and mental health institutions, barbers and sex
workers. Dentists and doctors have been responsible for small outbreaks. In
non endemic countries like Britain HBV carriers are barred from invasive
medical practices. Carriers are also not permitted to be medical students.
 14

LABORATORY DIAGNOSIS11
Specific diagnosis of HBV rests on the serological demonstration of the viral
markers.

HBsAg- This is the first marker to appear in the blood after infection, being
detectable even before elimination of transaminases and onset of clinical
illness. It remains in circulation throughout the icteric or asymptomatic
course of the start of the diseases. In typical cases it may disappear within 2
months of the start of the clinical disease but may sometimes last for six
months even beyond. When it is no longer detectable antibody, anti HBs
appears and remains for a long period.

HBcAg- HBcAg is not demonstrable in circulation because it is enclosed

with in the HBsAg coat, but its antibody, anti-HBc appears in serum a week
or two after the appearance of HBsAg. It is therefore the earliest antibody
marker to be seen in blood long before anti- HBe or anti- HBs. As anti-HBc
remains life long its serves as a useful indicators of prior infection with HBV,
even after all other viral marker become an undetectable.

HBeAg- It appears in blood concurrently with HBsAg or soon afterwards.

Circulating HBeAg is an indicator of active intrahepatic viral replication and
the presence in blood of DNA polymerase, HBV DNA virions, reflecting
high infectivity.
For the diagnosis of HBV infection, detection of HBsAg on the blood
is all that ordinarily necessary. The simultaneous presence of IgM anti-HBc
indicated recent infection and the presence of IgG anti-HBc indicate remote
infection. Occasionally when the level of HBsAg is too low to be detectable,
diagnosis is made by testing for IgM anti-HBc.
HBeAg provides information about relative infectivity. Its presence
denoted high infectivity and its absence along with the presence of anti-HBe
indicates low infectivity. As it is relatively present during acute Hepatitis, its
testing is indicated only in chronic infections and carriers.
The presence of anti HBs without any other serological viral marker
indicates immunity following vaccination.
Like HBeAg, HBV DNA is also an indicator of viral replication and
infectivity. Molecular methods such as DNA:DNA hybridization and PCR at
present used for HBV DNA testing are highly sensitive and quantitative.
 15

HBV DNA level in serum reflects the degree of viral replication in the liver
and so helps to assess the progress of patients with chronic Hepatitis under
anti viral chemotherapy.

Fig. Typical course of acute Hepatitis type B.

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EPIDEMIOLOGY
Hepatitis B occurs throughout the world. There is no seasonal distribution.
The infection is usually sporadic, though occasional outbreaks have occurred
in hospitals, orphanages and institution of mentally handicapped.
The prevalence of Hepatitis carriers varies widely in different countries
in relation to their living standards.12

High Endemicity: - 10-15 % in Equatorial Africa, South East Asia, China,

parts of South America. Vertical and horizontal transmission are both
common.
Intermediate Endemicity:- 2-7% in Eastern Europe, Middle East, South Asia
and parts of South America.
Low Endemicity: - 0.1-0.5% in developed countries as North America and
Australia.
India falls in the Intermediate group with higher carrier rates in the southern
part of the country and lower rates in northern part.
Overall it is estimated that there are 200-300 millions carriers in the
world, with the preponderance of males in all populations. In area of low
Endemicty the risk of infection varies widely in different groups according to
behavior.

Table 1:Factors predisposing to increased risk of HBV infection

1. Parental drug infection.

2. Many sexual parental endemic in various parts of the world.
3. Geography: highly endemic in various parts of the world.
4. Patients in residential homes for the mentally handicapped.
5. Patients in haemodialysis, haemophilia and other units.
6. Sexual partner has one of the above risk factors.
7. Babies born to mother who have risk.
8. Health care personnel, especially those in surgery, obsterics, dental
surgery and those caring for patients in categories 4 and 5.

The relative importance of each category varies in different parts of world

and also within countries.
 17

Table 2: Acute Hepatitis B in health care workers in Englanda

Staff 1975-79 1980-84 1985-88b

Surgeons 12 25 0
Physicians 12 11 2
Laboratory staff(medical) 27 16 0
Laboratory staff(scientific) 18 37 10
Nurses 7 4 2
Mental handicap staff 31 27 10
Dentists 17 17 16
a
Rates per 100 000 per annum
b
public health laboratory service communicable diseases report
( unpublished)

Some group of patients is at increased risk of infection and of

becoming carriers. These include patients on maintenance haemodialysis
and in homes for the mentally handicapped. Finally health care personnel and
lab workers are at high risk.
The reduction in the rate of reported acute HBV infection was in
period 1985-88 probably reflects an increased awareness of the need to
adopt good working practices and the introduction of active immunization.
The only safe and effective measures for prevention is universal
active immunization .Its success has been demonstrated in some highly
endemic areas like Taiwan where the carrier rate fell from 18% in 1988 to
8% in 1993.
 18

(www)
Map. Prevalence of chronic infection with Hepatitis B virus, 2006

Now that the vaccine is manufactured in India and is available at lower cost
it should be possible to include this in national immunization schedule.
 19

PROPHYLAXIS13
General prophylaxis consists in avoiding
• Risky practices like promiscuous sex.
• Injectable drug use.
• Direct or indirect contact with blood, serum or other body fluids of
patients and carriers.
• Health education.
• Use of disposable syringes and needles.
• Screening of blood, semen and organ donors.
Above all have helped to an extent, but alone cannot eliminate the risk
altogether, particularly in the developing countries. The only certain method
appears to be universal immunization. Both passive and active immunizations
are available.

Passive Immunization – Hyperimmune Hepatitis B immunoglobulin (HBIG)

prepared from human volunteers with high titers anti-HBs, administered IM
in a dose of 300-500 i.u. soon after exposure to infection, protects against
illness and the carrier state.

Active Immunization – It is more effective. The first vaccine introduced in

1982 was prepared from pooled plasma of healthy human carries with a high
level of antigemia. The 22nm HBsAg particles separated by
ultracentrifugation were treated with proteinase; urea and formaldehyde are
used as the vaccine. This was immunogenic but became unacceptable
because its source was human plasma limited in availability and not totally
free from possible risk of unknown pathogens. It continues to be used in
some countries because it is cheaper14 .
The currently preferred vaccine is genetically engineered by cloning the
S gene of HBV I baker’s yeast. It consists of nonglycosylated HBsAg particle
alone. It is given with alum adjuvant, IM into the deltoid or in infants into the
anterolateral aspect of the thigh. Gluteal injection is not recommended as it
may result in poor immune response. Three doses given at 0, 1 and 6 months
constitute the full course. Seroconversion occurs in about 90% of the
vaccines. A special vaccine containing all antigenic components of HBsAg
(pre S1, pre S2 and S) has been developed, which gives greater
Seroconversion. Seroconversion can be checked by testing for anti-HBs
 20

which is usually detectable for about 5 years. Clinical protection is believed

from last for much longer. Booster doses are needed for those at high risk.

Combined Immunization – For immune persons exposed to HBV, combined

immunization is recommended. For babies born to carrier mother, a single
injection of 0.5 ml of HBIG given IM immediately after birth, is followed by
the full course of vaccine at a different anatomical site ,the first dose being
given within 12 hours of birth. When HBIG is not available, the vaccine
given alone has been reported to provide protection.

TREATMENT15
No specific antiviral treatment is available for acute HBV infection.
Interferon alpha, alone or in combination with other antiviral agents such as
lamivudine and famcylovir, has been beneficial in some of the chronic
Hepatitis. There is no effective treatment for the carrier state, though
spontaneous resolution takes place in some of them.

MATERIALS AND METHODS

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SPECIMEN COLLECTION AND PREPARATION 16

• The ACON HBsAg One Step Test an Hepacard strip can be performed
on the serum or plasma.

• Remove the serum or plasma from the clot of red cell as soon as
possible to avoid hemolysis. Only clear, non-hemolysed specimens
should be used.

• Testing should be performed as soon as possible after sample

collection. Do not leave the samples at room temperature for prolonged
temperature periods. Specimens can be refrigerated at 2-80 C up to 3
days. Otherwise specimens should be stored below -200 C.

• Bring specimens to room temperature prior to testing. The frozen

specimens must be completely thawed prior to testing. Specimens
should be repeatedly frozen and thawed.

• If specimens are to shipped, they should be packed in compliance with

Federal regulation covering the transportation of etiologic agents.

Screening for HbsAg

All the sera were screened for HBsAg by 2 methods:
1. The ACON HBsAg one step test.
2. Hepacard strip test.

ACON HBsAg

One Step Hepatitis B surface Antigen Test Strip

A rapid one step, to test for the qualitative detection of Hepatitis B
surface Antigen, in serum or plasma.
For professional in vitro diagnostic use

INTENDED USE
 22

ACON HBsAg One Step Test is a rapid chromatographic immunoassay for

the qualitative detection of Hepatitis B Surface Antigen in serum or plasma.

SUMMARY
Viral Hepatitis is a systematic disease primarily involving the liver. Most
cases of acute Hepatitis are caused by Hepatitis A virus, Hepatitis B virus or
Hepatitis C virus. The complex antigen found in the surface of HBV is called
HBsAg; previous designation included the Australian or Au antigen (1). The
presence of HBsAg will be detected 2-4 weeks before the ALT level becomes
abnormal and 3-5 weeks before systems or jaundice develop. HBsAg has four
principle subtypes: adw, ayw, adr, and ayr. Because of antigenic
heterogeneity of the determinant, there are 10 major serotypes of Hepatitis B
virus.
The ACON HBsAg One Step Test is a rapid test to qualitatively detect
the presence of HBsAg in serum or plasma specimens. The test utilizes a
combination of monoclonal and polyclonal antibodies to selectively detect
elevated levels of HBsAg in serum or plasma.

PRINCIPLE
The ACON HBsAg One Step Test is a qualitative, solid phase, two site
sandwich immunoassay for the detection of Hepatitis B surface Antigen
(HBsAg) in serum or plasma. The membrane is pre-coated with anti-HBsAg
antibodies on the test line region and anti-mouse antibodies on the control
region. During testing the serum or plasma samples reacts with dye conjugate
(mouse anti-HBsAg antibody-colloidal gold conjugate) which has pre-coated
in the test strip. The mixture migrated upwards on the membrane
chromatographically by capillary action to react with anti-HBsAg antibodies
on the membrane and generates a red line. Presence of this red line indicates
a positive result, while its absence indicates a negative result. Regardless of
the presence of HBsAg as the mixtures continues to migrate across the
membrane to the immobilized goat anti-mouse region, a red line at the
control region will always appear. The presence of this red line serves as
 23

verification for sufficient sample volume and proper flow as a control for the
reagents.

REAGENTS
Test strip contains mouse anti-HBsAg antibody-colloidal gold conjugate and
polyclonal anti-HBsAg antibodies coated on the membrane.

PRECAUTIONS
1. For professional in vitro diagnostic use only. Does not use after the
expiration date?

2. Do not eat, drink and smoke in the area where the specimen and kits are
handled.

3. Handle all specimens as though they contain infectious agents.

Observe established precautions against microbial hazards throughout
all procedure and follow the standard procedures for proper disposal of
specimens.

4. Wear protective clothing such as laboratory coats, disposable gloves

and eye protection when assaying samples.

STORAGE AND STABILITY

Store as packaged in the steal pouch at 4-300 C. The kit is stable within the
expiration date printed on the pouch. Do Not Freeze or use beyond the
expiration date.

PROCEDURE17
 24

Materials Provided:
• Test strip contains mouse Anti-HBsAg antibody-colloidal gold
conjugate and Polyclonal Anti-HBsAg antibodies coated on the
membrane.
• Instruction for use

Material Required But Not Provided

 Vacuum tube for serum or plasma collection
 Centrifuge
 Time

DIRECTIONS FOR USE

Allow test strip and serum or plasma samples to equilibrate to room
temperature (20 – 30°C) prior to testing.

1. Remove the ACON HBsAg test strip from the foil pouch (bring the test
to room temperature before opening the pouch). Use strip as soon as
possible but within 1 hour after removal from the pouch especially if
the room temperature is more than environment.

2. Immerse the test strip in the serum samples with arrows pointing
toward the serum or plasma.

3. Wait for red lines to appear. The test should be read in approximately
15 minutes. It is significant that the background is clear before reading
the test, and only a weak line appears in the test region (T). Do not
interpret results after 20 minutes.

INTREPRETATIONS OF RESULT
 25

POSITIVE: Two distinct red lines will appear, one in the test region (T) and
other in the control region (C).

NEGATIVE: Only a single red line appears in the control region (C). No
apparent red or pink line appears in the test region (T).

INVALID: Control line falls to appear which means improper testing

procedure or deterioration of reagents probably has occurred. In any event
repeat the test. If the problem persists, discontinue using the lot immediately
and contact your local distributor.

NOTES: The shades of red in the test line region (T) will vary depending on
the concentration of HBsAg present. However, neither the quantitative value
nor the rate of increase in HBsAg can be determined by this qualitative test.

QUALITY CONTROL
 26

A procedure control is included in the test. A red line appearing in the

Control region (C) is considered as internal positive procedure control. A
clear background in the result window is considered as internal negative
procedural control.
It is recommended that a positive HBsAg control (containing 10 ng/ml
HBsAg) and a negative HBsAg control (containing “0”ng/ml HBsAg) be
included in each day testing to verify test performance.

LIMITATIONS
• ACON HBsAg test is for in vitro diagnostic use only .This test should
be used for the detection of Hepatitis B surface Antigen in serum or
plasma sample.
• This test will only indicate the presence of Hepatitis B surface antigen
in the specimen and should not be used as the sole criteria for the
diagnosis of Hepatitis B viral infection.
• As with all diagnosis tests, all results must be considered with other
clinical information available to the physician.
• ACON HBsAg test cannot detect extremely low concentration of
HBsAg in specimen. If the test result is negative and clinical symptoms
persist, additional follow-up testing using other clinical method is
required. A negative result at any time does not preclude the possibility
of Hepatitis B

HEPACARD STRIP TEST

PRINCIPLE
Same as in ACON HBsAg one step test since both are immune
chromatographic method for test for detecting HBsAg.

TEST PROCEDURE
1. Bring the required number of Hepacard pouches and specimen to room
temperature prior to testing.
 27

2. Take out Hepacard device from the foil pouches. Zip seals the pouch
containing balance device so that they are protected from moisture.
Ensure the zip seal is perfectly locked; otherwise the devise will not
work properly.
3. Label the device with patient’s name or identification number.
4. Add 2 drops (10ul) of serum /plasma specimen into the sample well
using the dropper provided (use separate dropper/ microtip for each
Specimen)
5. Allow reaction to occur during the next 20 minutes.
6. Read reaction at 20 minutes.
7. Discard immediately after reading the result at 20 minutes ,considering
it to be potentially infectious

Fig 5

HBsAg Hepacard Test Card.

 28

OBSERVATION AND RESULT

The present study was conducted at Super Religare Laboratories Ltd.
Ludhiana (Formerly Known as SRL Ranbaxy Ltd.), for a period of four
months, from April 2010-July 2010,in which 350 sera from various groups of
patients as well as Normal Healthy Persons were tested for HBsAg.

A total of 350 blood samples were collected and tested for HBsAg using
immune chromatographic test like ACON HBsAg one step test and hepacard
strip test. Out of these 200 samples were collected from Normal Healthy
persons who were all healthy adult males and females of age group 20-45. 50
Blood sample were healthy females of the reproductive age group attending
ante-natal clinics. The remaining 100 samples were from patients with viral
Hepatitis.

Table 3:

Sr. No. Study Group No. of samples

1. Normal Healthy Persons 200

2. Women attending ante-natal clinics 50

3. Patients with viral Hepatitis 100

TOTAL 350
 29

200

150
Normal Healthy
Pesrons
100
Women attending
ante-natal clinic
50
Patient with viral
Hepatitis
0
No. %Positivity
Screened

HBsAg SEROPSITIVITY AMONG DIFFERENT GROUPS

HBsAg %
Sr.No Study group No. Screened
Positivity Positivity
Normal Healthy
1. 200 1 0.5
Persons

Women attending
2. 50 0 0
ante-natal clinics

Patients with viral

3. 100 8 8
Hepatitis

TOTAL 350 9 2.57

 30

DISCUSSION

Hepatitis B occurs throughout the world. There is no seasonal distribution.

Under development and overpopulated regions have high Endemicity, low
Endemicity in developed countries and intermediate Endemicity is noted in
Eastern Europe, Middle East, South Asia and parts of South America.
India falls in the intermediate group.

In US 3-5 % of adults have been infected with HBV. 7-10% of patient

infected with HBV become chronic carries (this statistics corresponds to a
prevalence rate of 0.3-5.0% of chronic HBV infection of adults in US).The
like hood of becoming a carrier is greater in individuals who experienced a
mild or subclinical infection.

The overall seroprevalence of HBsAg according to DOCTOR NDTV in India

is 4.2%18. In my study the prevalence is 2.57 %. This is not in accordance
with the prevalence rate in India may be because of the small sample size in
each group. Hence the study may not have been an indicator of the
prevalence rate.

Thyagarajan et al in 1996 have related India to be in intermediate prevalence

of zone ranging from 2-7%.19

In the present study ACON HBsAg One step test and Hepacard test kit are
used .one out of 200 cases was found to be positive for HBsAg among
Normal Healthy Persons showing a prevalence of 0.5%.

Prevalence rate among women attending ante-natal clinic was found to be nil.

Prevalence rate among patient with viral Hepatitis is found to be positive

showing a prevalence rate of 8 %.

HBsAg is the first marker to appear in blood after infection, being detectable
even before elevation of transaminases and onset of clinical symptoms in the
typical case it did appears with in about two months of the start of clinical
disease but may sometimes last for 6 month or even beyond.
 31

SUMMARY AND CONCLUSION

In the present study from April 2010 – July 2010 a total of 350 samples were
tested. Out of this 200 were from Normal healthy Persons, 50 samples were
from women attending ante-natal clinics and 100 were from patients with
viral Hepatitis.

• All samples were tested for HBsAg using immunochromatographic

methods
• Among women attending ante-natal clinic 50 samples were tested and
nil positivity was recorded.
• Among the healthy people i.e. normal health check-up one out of 200
samples were found positive showing a prevalence of 0.5 %.
• Among 100 patients of viral Hepatitis three were positive showing a
prevalence rate of 8 %.
• The overall prevalence rate in the entire study is 2.57 %.
 32

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