Seroprevalence of HBV infection

28 Rehman et al.
Int. J. Biosci. 2014
RESEARCH PAPER OPEN ACCESS
Seroprevalence of HBV infection and viral loads in outpatients
attending a district hospital located in Mardan, Pakistan
Habib Ur Rehman1
, Muhammad Wasim2
, Muhammad Waqar3,4*
, Asad Ullah Khan3
,
Muhammad Idrees5
,Zobia Ismail5
, Amjad Ali6
, Faisal3
, Jahangir Khan3
, Umar Javed3
,
Muhammad Saleem3
, Adnan Ahmad3
, Razi Ullah2
, Afi Ullah3
1
Department of Medicine, Mardan Complex Hospital, Mardan
2
Khyber Medical University, Peshawar Khyber Pakhtunkhaw, Pakistan
3
Department of Microbiology Hazara University Mansehra, Khyber Paktunkhwa Pakistan
4
Institute of Microbiology University of Sindh Jamshoro Sind Pakistan
5
Centre of Excellence in Molecular Biology, University of the Punjab, Lahore- 53700, Pakistan
6
Department of Biotechnology University of Malakand Khyber Paktunkhwa, Pakistan
Key words: GCMBDR, HBV, seroprevalence.
http://dx.doi.org/10.12692/ijb/4.5.28-34 Article published on March 08, 2014
Abstract
Hepatitis B is a major public health problem in Pakistan like other developing countries. HBV infection is a more contagious
disease than HCV and HIV infection. The aim of the current study is to compare seroprevalence of HBV infection in
outpatients who attended a district headquarter hospital of district Mardan, Pakistan. A total number of 270 patients were
selected for this research work. Serum of patients were tested by HBsAg and ICT strip method and those found positive were
sent to GCMBDR to confirmed for HBV DNA by Real Time PCR. Out of the total 270 screened patients, HBV DNA was
confirmed in 94(34.81%) patients using Real Time PCR of which 56(59.6%) were male while 38(40.4%) were female
outpatients. All the positive patients were divided into three categories on the bases of viral load categories such as low
(<100000 IU/ml), intermediate (100,000-10,000,000 IU/ml) and high viral load (>10,000,000 IU/ml). On the bases of these
categories 50 %( n=47) patients were found with low viral load, 33 %( n=31) with intermediate and 17 %( n=16) were found
having high viral load. Female patients were slightly more susceptible to HBV infection compared to male patients. The highest
HBV infection seroprevalence was found in patients of 21-40 age group. Majority of the sick subjects were however seen with
low viral titers.
* Corresponding Author: Muhammad Waqar  waqarkhan96@gmail.com
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print) 2222-5234 (Online)
http://www.innspub.net
Vol. 4, No. 5, p. 28-34, 2014

29 Rehman et al.
Introduction
Hepatitis is a major health problem in both developed
and under developed countries as it affects about 3.5
billion individuals globally (Wittet, 2010., Komas et
al., 2010, Awan et al., 2010, Awan et al., 2010). HBV
infection is a more contagious disease than HCV and
HIV infection. It is 10 times more contagious disease
than HCV and about 50-100 times more contagious
disease than HIV (Samuel et al., 2004). Hepatitis B
virus belongs to hepadnaviridae family and to the
genus Orthohepadna virus (Hunt, 2007). Genome of
HBV is partially circular and double-stranded circular
DNA virus (Barker et al., 1996). In 1988 six major
serotypes (A-F) of HBV were identified worldwide
(Norder et al., 1994) including genotypes G and H
(Shibayama et al., 2005) and nowadays each
genotype is further divided into multiple subtypes
with distinct properties (Schaefer et al., 2007).
Hepatitis B prevalence is high among developing
countries of Africa, the Pacific Islands and Asia while
HBV prevalence is low among the developed
countries of USA, Australia and Western Europe. In
Pakistan, HBV is also a major health problem (Khan
et al., 2010). In Japan, Eastern Europe and Russia,
about 2-8% of their populations are infected by HBV.
According to 2010 report, it is estimated that 120
million individuals are infected with HBV in China
followed by India with 40 million and Indonesia with
12 million infected individual. World Health
Organization reported that 600,000 deaths may
occur annually due to Hepatitis B virus infection
(Jakarta Post, 2011)
HBV transmission occurs as a result of exposure to
infected human blood or body fluids. It may also be
transmitted to healthy individual by sexual contact
with HBV infected persons (Fairley and Read, 2012).
It may be transmitted through contaminated blood
transfusion (Buddeberg et al., 2008) re-use of
contaminated syringes (Hughes, 2000) and from
infected mother to child during child birth (Hughes,
2000). Within family members, HBV can be
transmitted through contact with non intact skin or
mucous membrane with secretions or saliva
containing HBV (Petersen et al., 1976). Different
methods were used for detection of HBV infection.
Diagnostic assays are used to detect HBV infections
using serum or whole blood which may contain viral
antigens or antibodies produced by the host (Bonino
et al., 1987). The most frequently used method for the
detection of HBV infection is HBsAg screening.
HBsAg is the first viral antigen that may appear and
easily detectable during infection (Karayiannis et al.,
2009).
ALT is also easy method for detection of viral
hepatitis in infected individual (Dufour et al., 2000).
Nowadays, PCR test is the most specific method used
for the detection of HBV infection and also used to
measure its viral load (Zoulim et al., 2006). HBV
infection may be prevented by using safe methods i.e.
proper screening of blood before transfusion,
avoiding re-use of used syringes and needles, and by
vaccination at birth (O'Connor, 2008).
Very few number of studies are available from district
Mardan about seroprevalence of HBV
infection.Therefore, the present study was designed
to evaluate the seroprevalence of Hepatitis B virus
infection in different age groups of outpatients that
attended a district hospital in Mardan, Pakistan.
Materials and methods
Study Area
For the current study general population of District
Mardan was selected. Mardan the second largest city
of the Khyber Paktunkhwa Province of Pakistan with
a population of 1.46 million according to 1998 census
and de facto headquarters of the Yousafzai tribe
(Government of Pakistan). The study was conducted
from December 2011 to December 2012.
Sampling
All the patients included in this study were found
HBV positive by HBsAg and strip methods previously.
Whole blood samples were obtained from these 270
outpatients who attended a district hospital in
Mardan, Pakistan. Blood samples were centrifuge and
sera was separated and for further investigation
serum samples of the patients were sent to Genome

30 Rehman et al.
Center for Molecular Diagnostics & Research Lahore
(GCMBDR). All the samples were received with
proper consent of the patients at GCMBDR.
Viral DNA Isolation/Confirmation by Real Time
Polymerase Chain Reaction
Viral DNA was isolated from sera samples by using
NucleoSpin DNA Virus/Virus F kits by Machery and
Nagel. Nucleic acid isolation was done using 100 ul
serum samples according to the kit protocol. Isolated
HBV DNA was confirmed by Smart Cycler II Real-
time PCR (Cepheid, USA). Amplification was done
using real time amplification kit by Sacace
Biotechnologies Italy. Total reaction volume was 25
ul, containing 15.25 ul HBV amplification mix, 0.5 ul
Taq polymerase and 10 ul extracted DNA. For precise
check of the reaction negative and positive controls
were also included with the run. Sensitivity of the
assay is 20 IU per ml blood sample. Specificity of the
assay is about 99%.
Statistical Analysis
Statistical 9.0 software was used for analyses and
summarization of data obtained. Chi-square test was
used-value less than 0.05 was considered as
significant.
Results
Gender and Age wise Prevalence rate of HBV
infection
Out of the total 270 blood samples made of
162(60.0%) males and 108(40.0%) females, HBV
DNA was observed in 94 (34.8%) patients. Of the
total 162 male patients 56(34.5%) were found positive
for HBV DNA while of the 108 female patients,
38(35.1%) were found positive for HBV DNA by Real-
Time PCR.
Table 1. Sex wise distribution of HBV infection among outpatients studied.
Gender Total samples Total HBV positive samples Percentage P-value
Male 162 56 34.5% P=0.0081
(<0.05 Significant) through
Statistix 9 (Chi-Square)
Female 108 38 35.1%
Total 270 94 34.8%
Table 2 shows the seroprevalence of HBV infection
among the different age groups. All the outpatients
were divided into four groups i.e. 0-20, 21-40, 41-60
and above 60yrs age groups. HBV infection rate was
highest (38.9%) in 20-40 age group followed by 40-
60 age group which recorded infection rate of 36.1%,
as well as 23.1% infection rate in age group 40-60 and
low prevalence of HBV infection observed in
outpatients above 60 years of age.
Table 2. Age wise distribution of HBV infection among outpatients studied.
Age Total samples Total HBV positive samples Percentage P-value
0---20 39 9 23.1%
P<0.0001 Significant through
21---40 175 68 38.9%
41---60 36 13 36.1%
>60 20 4 20%
Total 270 94 34.8%
HBV DNA viral loads among both gender and
various age groups
On the basis of viral loads all the positive patients
were analyzed in three categories such as low
(<100000 IU/ml), intermediate (100,000-
10,000,000 IU/ml) and high viral load (>10,000,000
IU/ml). HBV viral load in both sexes are shown in
Table 3. Out of the total of 94 seropositive patients 50
%( n=47) were found to have low viral load, 33 %(
n=31) with intermediate and 17 %( n=16) were found
with high viral load. HBV viral load in various age
groups was analyzed in Table 3. All the outpatients
were divided into four groups based on their ages. Of
the 9 patients of age group 0-20, 4.2 %( n=4) were

31 Rehman et al.
found with low viral load, 1 %( n=1) with intermediate
and 4.2 %( n=4) with high viral load. In age group 21-
40, 35.1% patients were found with low, 26.5% with
intermediate and 11.7% with high viral load. 8.5%
outpatients of 41-60 age group recorded low viral
load, 3.1% with intermediate and 1% with high viral
load while 2.1% greater than 60yr old recorded
patients showed both low and high viral loads.
Table 3. Sex wise distribution of HBV viral loads among outpatients studied.
Gender
HBV Viral load(IU/ml)
<100000 100,000>10,000,000 >10,000,000 P-value
Male 27(28.7%) 23(24.4%) 06(6.3%) P=0.0382
(<0.05 Significant)
through Statistix 9
(Chi-Square)
Female 20(21.2%) 08(8.5%) 10(10.6%)
Total 47(50%) 31(33%) 16(17%)
Discussion
Mardan, a large district in Khyber Pukhtunkhwa
Pakistan, with an area of about 1632 sq.km, like other
northern areas of Pakistan lag behind in literacy rate
and health facilities, although measure towards
improvement are under way. According to a survey in
2005/06, poverty in Khyber Pukhtunkhwa is
estimated to be 29% while the literacy is 50% in the
gap in female literacy is 31 % (unic.org.pk). Therefore
it goes without saying that extensive researches on
pertinent health issues like prevalence of hepatitis B
are scarce despite the fact that due to the same lack of
advancement infectious disease like hepatitis is
continuously on the rise, this justifies the purpose of
our research. Mardan is important also because
owing to the scarcity of jobs and natural disaster like
flood and political instability a large numbers of its
natives migrates to large cities of other provinces
every year, resulting in interchanging of infectious
diseases like hepatitis B (as of 6th may 2010, 341,768
people of KPK have become internally displace people
and have been living in host communities) (WHO).
Table 4. Age distribution of HBV viral loads among outpatients studied.
Age
HBV Viral load(IU/ml) P-value
<100000 100,000>10,000,000 >10,000,000
P=0.4103(>0.05 non-
significant) through
0---20 04(4.2%) 01(1%) 4(4.2%)
21-40 33(35.1%) 25(26.5%) 11(11.7%)
41---60 08(8.5%) 03(3.1%) 01(1%)
>60 02(2.1%) 02(2.1%) 00
Total 47(50%) 31(32.9%) 16(17%)
Few Number of study is available on molecular
epidemiology of HBV infection in district Mardan.
Our research shows that gender wise, the percentage
of female affected (35.1%) is slightly more as compare
to that of males (34.5%), this contrasts with the
results from other regional studies in which more
male patients were affected than female (Khan et al.,
2011). This may be due to the fact that males migrate
to other cities and provinces in search of better jobs
and as a result acquire different infections in the
process. During their stay in these developed cities
they may get diagnosed and treated early for their
infections and may not be counted in the infected
population, but during their visits to their native land,
they may transfer these infections to their wives and
other female occupants. These females are usually
confined to their homes and city may not get
diagnosed and treated due to the poor health facilities
of the city and may get counted in the infected
population. Our study also depicts a predominance of
male patients in the low and intermediate categories.
Only in the high viral load category females showed a
significant preponderance.
As far as prevalence of HBV infection is concerned,
results obtained in this study are consistent or not

32 Rehman et al.
consistent with previous similar studies both showing
highest rate of infection in persons aged 21-40 years
(Alam et al., 2007). High rate of HBV infection in
males compare to females may be due to the fact that
they are employed outside their homes visiting barber
shops and also their involvement in blood donation
and transfusion practices, a Pakistani study shows
that in countries like Pakistan, Iran, India,
Bangladesh, Israel and Italy HBV can be transmitted
through blade sharing and barber related
instruments(Waheed et al., 2010). Blood transfusion
has been reported as potential risk factor for getting
hepatitis C and B virus infections. [29]. Poor health
facilities can leads to improper screening as well as
unsterilized dental instruments can lead to the spread
of hepatitis B. ( in a survey conducted in 2010 in
Mohmand agency KPK it was discovered that all
health sectors lacked facilities for modern screening,
dental services and detection and management of
sexually transmitted diseases(Qureshi et al., 2009).
Used syringes/needles appear to be the most
significant factor in the spread of hepatitis B and C
virus in general population of Pakistan (Ali et al.,
2009). Two major factors contributing to high re-use
of used needles/syringes leading to prostration is the
major cause of earlier while a bur dance of un
professional practitioners and lack of awareness
among the patients seems to be root cause of the
latter. As has been impisize in Mardan both these
causes are present. Un safe sexual practices also
contribute a major group of risk factors (Norman,
1997) staying frequently away from home is common
in immigrant in Mardan can lead to acquisition of
high risk behavior such as injectable drug use and
unsex paid sex.
This Study shows that out of 270 HbsAg positive
patients 94 were positive for HBV PCR. Previous
studies show that HbsAg is cleared by young carriers
earlier as compared to adults. The patients in their
early infected stage have positive HbsAg and high
viral load and as the infection grows older DNA levels
in serum decreases which is associated with the
detectable antibody that results in the clearance of
HbsAg (Norman, 1997).
According to the present study of viral loads all the
positive patients were analyzed and grouped in three
categories such as low (<100000 IU/ml),
intermediate (100,000-10,000,000 IU/ml) and high
viral load (>10,000,000 IU/ml). Total 94 positive
patients 50 %( n=47) were found with low viral load,
33 %( n=31) with intermediate and 17 %( n=16) were
found with high viral load. Data shows that more
females had high viral load (10.6%) as compared to
males (6.3%), which may be due to the fact that males
have an excess to treatment during the early stage of
disease than female.
Our results indicate that the viral load declined with
age which is supported by the previous studies
conducted in Gambia (Mendy et al., 2008). Patients
that occurred in age group 0-20, had similar
percentages for both low and high viral loads. In age
group 21-40 years 35.1% patients were found with
low, 26.5% with intermediate and 11.7% with high
viral load. Age group 41-60 year show 8.5 % patients
found with low viral load, 3.1% with intermediate and
1% with high viral load while patients with age more
than 60 year show that only 2.1% of the patients were
found with both low and high viral load and none of
the patients were found with high viral load,
supporting earlier studies and indicating the complex
pattern of HBV infection that is highly dependent
upon the age of the infection and the clearance of the
virus is greatly influenced by the host response to the
virus. According to a documented report, it has been
noticed that 95% of the adult patients develop acute
hepatitis B recover soon and remaining 5% remain
persistently infected (Norman et al., 1997).
Progression of the viral load is dependent upon the
particular genotype that the patient carries (Kumar et
al., 2011). In this particular study we did not work of
the genotype that the patient carried. Further work
has to be done.
Conclusion

33 Rehman et al.
From the present study it is concluded that female
outpatients were slightly susceptible to HBV infection
compared to the male outpatients. The highest HBV
infection rate was found in patients of 21-40 age
group and majority of the infected subjects recorded
low viral loads.
Acknowledgement
We are thankful to GCMD for supporting of this
study.Also Thanks to Mr. Murad khan and Yahya
Khan for his co-operation in this study.
Competing interest
Declare none.
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