Advances in Biological Research 9 (2): 69-74, 2015

ISSN 1992-0067
IDOSI Publications, 2015
DOI: 10.5829/idosi.abr.2015.9.2.9325

Distribution Pattern of Various Genotypes of HCV Circulating in

District Mardan, Khyber Pakhtunkhwa, Pakistan
Muhammad Waqas, 1Farzana Parveen, 2Muhammad Waqar, 3Amjad Ali,
Muhammad Arif Khan, 5Muhammad Wasim, 7Asad Ullah Khan, 2Zobaria Rehman,
7
Tauseef Ahmad, 6Muhammad Adnan Shereen and 2Rabia Nawaz
1

Department of Zoology, Hazara University Garden Campus Mansehra, Pakistan

Genome Center for Molecular Based Diagnostics & Research, Lahore, Pakistan
3
Department of Biotechnology, University of Malakand, Khyber Paktunkhwa, Pakistan
4
Department of Medicine D.H.Q Hospital, Mardan, Khyber Paktunkhwa, Pakistan
5
Khyber Medical Collage Peshawar, Khyber Paktunkhwa, Pakistan
6
IBMS Khyber Medical University, Peshawar, Khyber Paktunkhwa, Pakistan
7
Department of Microbiology, Hazara University Garden Campus Mansehra, Pakistan
1

Abstract: Hepatitis C is an infectious liver disease, the infection of HCV is often asymptomatic and the
chronic infection comes to be hepatocellular carcinoma, cirrhosis and end-stage liver disease. Six main
genotypes of HCV and several subtypes are recognized worldwide. HCV genotypes play important role in
distribution of frequency, clinical management and development of vaccine. Current article shows distribution
frequency of HCV genotypes present in various tehsils of district Mardan of Khyber Pakhtoonkhaw,
Pakistan.Sum of 375 patients were positive for HCV RNA by PCR and were screened using genotyping assay.
Data analysis showed that 241 out of 375 PCR positive patients were male and 134 were female. 190, 110 and
75 patients came from tehsils of Mardan, Takhat Bhai and Katlang respectively. Results showed that: Out of
the total screened patients, genotype specific PCR fragments were seen in 322 (85.8%) patient. The distribution
of typeable genotypes was as follows: 39 patients (10.4%) with genotype 1a, 11 (2.9%) patients with genotype
1b; 227 (60.5%) patients infected with genotype 3a; 24 (6.4%) patients genotype 3b; and 21(5.6%) patients
were with mixed genotypic infection. 53 (14.1%) serum samples were found un-typeable by the molecular
genotyping assay used. in Conclusion It was concluded from the current research work that different genotypes
of HCV such as 1a, 1b, 3a and 3b present in different tehsils of Mardan district and genotype 3a is most common
genotype among general population of district Mardan.
Key words: Khyber Pakhtoonkhaw

HCV

Mardan

INTRODUCTION

GCMD
the population worldwide (minor in Europe 1.03% and
maximum in Africa 5.3%) and in Pakistan 10% of the
people are infected with chronic hepatitis C virus [5, 7-9].
Hepatitis C virus has been classified as the single
member of the genus Hepacivirus of family Flaviviridae,
which have further more species the Pestivirus and the
Flavivirus [10-12]. The size of Hepatitis C virus is
approximately 55-65 nm, enveloped, with positive sense
single strand RNA virus and in human the main causative
agent of hepatitis, hepatocellular carcinoma (HCC) and
liver cirrhosis, was isolated in 1989 [2, 3, 6, 13].

Hepatitis C is an infectious liver disease, the

infection of HCV is often asymptomatic and the chronic
infection comes to be hepatocellular carcinoma, cirrhosis
and end-stage liver disease [1]. It is also expected that the
cirrhotic patients may also get other problems of cirrhosis,
hepatic cancer and liver [2-4]. All over the universe 200
million people are infected with HCV and in Pakistan there
are 17 million people infected [1, 5]. The Hepatitis C virus
was first discovered in 1989 [6]. Approximately 3.3% of

Corresponding Author: Muhammad Waqar, Institute of Microbiology, University of Sindh, Jamshoro Sindh Pakistan.
Tel: +92-321-9686002.

69

Advan. Biol. Res., 9 (2): 69-74, 2015

Complementary DNA (cDNA) Synthesis: Complementary

DNA was synthesized at 37C in 50 min using reverse
transcriptase PCR (RT-PCR) of total isolated viral RNA
(20 l) by using 100 units of Malany-murine leukima
virus reverse transcriptase
Zenzyme (M-MLVRT)
transcribed 10 l to cDNA 1 M of downstream primer
(outer antisense) according to manufacturing instructions
(Gibco BRL).

HCV can be transmitted from infected individual to

healthy individuals through various routes i.e. through
contaminated blood transfusion, sharing of razors, tooth
brushes and shaving from barber. It is also transmitted
through contaminated dental apparatus, unsafe sex and
drug abusers [14, 15, 16]. In Pakistan principal source
of HCV transmission is contaminated blood transfusion
because of lack of proper screening of blood, weak
infrastructure and untrained medical staff [16].
Due to genomic variation HCV is divided into six
main genotypes and multiple subtypes [17]. Genotypes
Distribution of HCV varies geographically. HCV subtype
1a and 1b is common in Europe and USA [18-20]. In Japan
most commonly circulated subtype is 1b [21]. In North
America, Japan and Europe subtype 2a and 2b of HCV
are distributed. Subtype 2c is mostly distributed in Italy
while Genotype 4 is present in Middle East and North
America. Genotype 5 and 6 are present in South Africa
and Hong Kong [18-22].
The present research was conducted in Mardan, a
district in the Khyber Pakhtunkhwa (KP) province of
Pakistan. The total area of the district is 1632 square
kilometers. According to 2008 survey the population of
district Mardan is 1.96 million. Mardan city is the
headquarters of the district. Few studies are available
related to the prevalence of HCV genotypes circulating
in different tehsils of Mardan. Therefore, the aims of
this study was designed for finding out different and the
most prevailing genotypes among the population of
Mardan.

HCV genotype specific PCR: For all the HCV RNA

positive individual genotyping was performed by using
HCV genotyping method described by Idrees [5]. As HCV
has 12 different genotypes, type-specific primers were
divided in two groups on the basis of differences in the
sizes of the different bands to avoid overlapping of
similar bands on agarose gel. Each first round amplified
PCR products were subjected to two second rounds
nested PCR amplifications. First with mix-A primer that are
specific for 1a, 1b, 1c, 3a, 3c and 4 genotypes and second
with mix-B primers that are specific for 2a, 2c, 3b, 5a and 6a
genotypes.
Gel Electrophoresis: Round two amplification products
were electrophoresed on a 2% agarose gel to separate
type-specific fragment. DNA ladder of 100-bp (Invitrogen,
Corp, California, USA) was used as size marker for DNA
to compare genotype specific bands.
Statistical Analysis: The data were analyzed and
summarize using statistical software Statistica version
9.0 for windows. The results for all variables were
presented in percentage (%). Fishers exact and Chi
Square tests were used to find out positive association
among the categorical variables. P<0.05 was considered as
significant.

MATERIALS AND METHODS

Study Area: The present research was conducted
in different
areas of district Mardan during
February-August,
2012.
Mardan
district
is
administratively subdivided into three tehsils that
are: Mardan, Takht Bhai and Katlang.

RESULTS
Demography of the HCV Patients: A total of 375 HCV
positive infected patients were selected from different
tehsils of district Mardan, Khyber Pakhtunkhwa, Pakistan
for specific genotyping procedure. Demography of the
diseased subjects was shown in Figure 1. Of 375 patients
64.2% (n=241) were males and 35.7% (n=134) were females
of age ranged less 10 and above 60 years. Distribution
breakup of the patients was 190, 110 and 75 from
tehsil Mardan, Takht Bhai and Katlang, respectively.
The selected samples in this setup that were HCV-RNA
positive with high viral titer were subjected for specific
genotyping assay.

Blood Sampling from HCV Positive Patients: A total of

375 HCV PCR positive patients were subjected for HCV
genotyping. HCV positive samples were collected and
data was recorded on data forms designed for study and
distributed to all collection centers in different tehsils of
district Mardan.
RNA Extraction: RNA was extracted from the serum of
HCV positive patients by using RNA isolation Kit Gentra
(Puregene Minneapolis.MN 55441, New York, USA)
according to the manufacturer's recommended protocol.
70

Advan. Biol. Res., 9 (2): 69-74, 2015

Prevalence of Specific HCV Genotypes in Different

Tehsils of District Mardan: Total 51 %( n=190) samples
were investigated from tehsil Mardan, 29%(n=110) from
tehsil Takht Bhai and 20%(n=75) samples from tehsil
Katlang. The most prevalent genotype was 3a in all
tehsils. In tehsil Mardan, 3a genotype was 64.7%(n=123),
in Takht Bhai tehsil 3a genotype was 70.9%(n=78) and in
tehsil Katlang 3a genotype was 34.6%(n=26), followed by
1a which was 9.4%(n=18) in tehsil Mardan, 6.3%(n=7) in
tehsil Takht Bhai and 18.6%(n=14) in tehsil Katlang,
3b genotype in tehsil Mardan was 6.3%(n=12), in tehsil
Takht Bhai 2.7%(n=3) and in tehsil Katlang 12%(n=9),
1b genotype in tehsil Mardan was 1(n=2)%, in tehsil
Takht Bhai 4.5%(n=5) and in tehsil Katlang 5.3%(n=4),
mixed genotypes in tehsil Mardan was 3.1%(n=6), in tehsil
Takht Bhai 5.4%(n=6) and in tehsil Katlang 12%(n=9) with
mixed genotypes and in all tehsils Mardan, Takht Bhai
and Katlang, 15.2%(n=29), 10%(n=11) and 17.3%(n=13)
patients have not shown genotype specific fragments,
hence, they were called untypable (Table I).

10%, in female 11%, 3b genotype was 6% in male and 7%

in female, 1b in male was 3% and 2% in females, 5% in
male are mixed genotype and 7% in female, the remaining
in male 17% are untypable and 10% in female.
Age Groups Wise Frequency of Hcv in District Mardan:
Now according to age groups distribution of different
HCV genotypes in district Mardan are shown in Table II.
In 20 age group totals of 1%(n=4) patients are present,
in 21-40 years age group 48.5%(n=182) patients are
investigated, in 41-60 years age group total 40.5%(152)
patients are and above 60 years age group 9.8%(n=37)
of patients are in the present study. The 3a genotype
is dominant in Mardan, according to age groups, in
20 years age group, 75 %( n=3) patients with 3a
genotype and 25%( n=1) are untypable. In 21-40 years
age group, 58.2 %( n=106) are 3a genotype, 12%( n=22)
are 1a genotype, 1.6% (n=3) are 1b genotype, 7.1 %( n=13)
are 3b genotype, 5% are mixed genotypes and 16% are
untypable. In 41-60 years age group, 58.5%(n=89) patients
have 3a genotype, 9.2%(n=14) patients have 1a genotype,
4.6 %(n=7) are 1b, 7.8%(n=13) patients have 3b genotype,
6.5%(n=10) patients have mixed genotypes and
13.1%(n=20) are untypable. In 60 years age group,
78.3%(n=29) patients have 3a genotype, 8.1%(n=3)
patients have 1a genotype and 2.7%(n=1) patients with
1b, 2.7%(n=1) with mixed genotype and 8.1%(n=3) are
untypable. In age groups wise distribution total 1a
genotype is present 10.4%, 1b is 2.9%, 3a is 60.5%, 3b is
6.6%, mixed genotypes are 5.3% and the remaining 14.1%
are untypable.

Gender Wise Frequency of HCV in District Mardan:

Figure 2 shows gender wise distribution of different HCV
genotypes in district Mardan. The 1a genotype was 10%
in both genders; 1b was 3%, 3a was 61%, 3b 6%, mixed
genotypes are 6% and the remaining 14% are untypable.
In present study 3a genotype was the predominant
genotype in both male and female in Mardan. In male,
3a genotype was 59% and in female 3a was in 63%,
followed by 1a in both male and female, in male 1a was

Table I: Prevalence of specific HCV genotypes in different tehsils of district Mardan

District

Genotype/Sub-type
------------------------------------------------------------------------------------------------------------------------------1a
1b
3a
3b
Untypable
Mixed

n= (%)

Isolated from Mardan

Isolated from Takht bhai
Isolated from Katlang

18 (9.4%)
7 (6.3%)
14(18.6%)

2 (1%)
5 (4.5%)
4 (5.3%)

123 (64.7%)
78 (70.9%)
26 (34.6%)

12 (6.3%)
3 (2.72%)
9 (12%)

29(15.2%)
11(10%)
13(17.3%)

6 (3.1%)
6 (5.4%)
9 (12%)

190 (51%)
110 (29%)
75 (20%)

Total

39 (10.4%)

11 (2.9%)

227 (60.5%)

25 (6.6%)

53 (14.1%)

20 (5.3%)

375

P= 0.0.1637 (>0.05) Non significant

Table II: Age groups wise frequency of HCV in district Mardan

Age

Genotype/Sub-type
------------------------------------------------------------------------------------------------------------------------------1a
1b
3a
3b
Untypable
Mixed

n= (%)

0------20
21----40
41----60
Above 60

0
22 (12%)
14 (9.2%)
3 (8.1%)

0
3 (1.6%)
7 (4.6%)
1 (2.7%)

3 (75%)
106(58.2%)
89 (58.5%)
29 (78.3%)

0
13 (7.1%)
12 (7.8%)
0

1 (25%)
29 (16%)
20 (13.1%)
3 (8.10%)

0 4 (1)
9 (5%)
10 (6.5%)
1 (2.7%)

182 (48.5)
152 (40.5)
37 (9.8)

Total

39 (10.4%)

11 (2.9%)

227(60.5%)

25 (6.6%)

53 (14.1%)

20 (5.3%)

375

P=0.0015 (<0.05) significant

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Advan. Biol. Res., 9 (2): 69-74, 2015

DISCUSSION

Muhammad et al. [36] that most prevalent HCV incidence

rate in Pakistan was seen in old age group. Thus these
findings suggest that untimely detection of HCV may be
due to the alertness of general population about HCV
infection in our studied population.
The important implication of current study was the
isolation of 53 (14.1%) isolates that were untypable and
no genotype specific PCR fragments were found for
these patients. These 53 serum samples with untypable
genotypes were positive for hepatitis C virus by
qualitative PCR. The rate of untypable genotypes
observed in the recent study might be due to the pedestal
that our untypable patients had already been through
standard interferon plus ribavirin therapy. Moreover
patients maybe non-responders or were relapsed
subsequently.

Commonly six HCV major genotypes and several

subtypes have been recognized globally [18]. Detection
of HCV genotype and its subtype is of great clinically
importance before recommending treatment, for the
reason that genotypes 1 and 4 show more resistance to
PEG interferon plus ribavirin treatment as compared to
genotypes 2 and 3, thus different HCV genotype needs
different period and varying anti-viral treatment dose [23].
PEG interferon plus ribavarin therapy for genotypes 1 and
4 are 48 weeks while for 2 and 3 is 24 weeks [24].
This study was conducted to analyze the HCV
genotypes circulating in various tehsils of district
Mardan. The data analysis shows that the most
predominant genotype in the studied population was 3a,
227 (60.5%) followed by 1a 39 (10.4%), 3b 24 (6.4%) 1b 11
(2.9%), 21 (5.6%) mixed genotypes and 53 (14.1%) were
untypable. The present findings were similar to previous
studies conducted in different areas of Pakistan that, 3a
is most common genotype circulating in this area
[5, 14, 25, 26]. The distribution pattern of HCV genotypes
of this study is similar to that reported from other South
Asian states such as in India [27, 28] and in Nepal [29]
where the mostly circulating genotype was 3a but
contradict from other regions of the globe such as in
Thailand [30], Japan [31], Western Europe Vietnam, USA
and where genotype 1 is the common HCV genotype [32].
In our data interpretation 21 (5.6%) individual were
infected by mixed genotypes. Factor common to these
patients was thalassemic subjects who had previously
transfused unscreened blood. These findings are
supported by other published reports indicating that
mixed genotypes were common where blood transfusion
is most prevalent particularly in thalassemia patients [33].
Furthermore individuals with mixed genotypes might
influence the antiviral treatment response and infection
progression [34]. In this study we were incapable to see a
single 4 genotype from any HCV positive patient, that is
supposed to be partially absent from Pakistan and it is the
most predominant genotype of Middle East. [35] None of
the patients were seen infected with genotype 6a and
5a; these are common in Hong Kong and South Africa
[22].
This setup showed that high incidence rate of HCV
infection (p=0.0015) was found in age group of 21-40.
These results were similar with the findings of
Inamullah et al. [26] that prevalence was seen in age
group 40 years and similar findings were observed by
Ali et al. [25] On the other hand our results deviated from

CONCLUSION
In the current study we conclude that hepatitis
C virus genotypes 1a, 1b, 3a and 3b are distributed in
different tehsil of Mardan. Genotype 3a is the
predominant genotype circulating in district Mardan.
Furthermore majority of the infected population belong
to age group 21-40 years.
Competing Interests: The authors declare that they have
no competing interests.
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