Evaluation of Incidence, Genotypes, and Risk Factors Associated with Newly Diagnosed Hepatitis C Cases in Khyber Pakhtunkhwa (KPK), Pakistan

Abstract

Hepatitis C is a global health concern brought on by the hepatitis C virus (HCV). The risk factors of the disease include injection drug users, contaminated medical instruments, blood transfusion, organ transplant, major surgery, dialysis, dental surgery, and acupuncture. The aim of the current study was to identify the prevalence of active HCV infection, genotypes, and their associated risk factors in newly infected patients of Khyber Pakhtunkhwa (KPK), Pakistan. A total of 230 HCV cases and 245 control subjects were enrolled. Our findings revealed that disease infection rates were higher in female patients (52.6%) than male patients (47.4%). Moreover, the disease was more prevalent in the age group 30–40 years (39.6%). The highest incidence was found in the southern region (51.30%), followed by the northern region (27.82%), and central region (18.26%). In our data set, dental surgery, major surgery, disease in family, barber community, blood received, needles, abroad travel, injection drug users, acupuncture, and organ transplantation were determined as the major associated risk factors of the disease. Odds ratio were significantly higher (10.91%) for those who used acupuncture as compared to those who did not. Similarly, odds were significantly higher (10.03%) for those who had HCV in their family, dialysis (9.377%), dental surgery (7.618%), major surgery (6.813%), barber community (5.328%), and blood received (2.252%) as compared to the control group. Also, the most frequent genotype was 3a (63%), followed by an un-typeable genotype (11.30%).

Full text

Current Trends in OMICS(CTO)
Volume 2 Issue 1, Spring 2022
ISSN(P): 2790-8283 ISSN(E): 2790-8291
Homepage: https://journals.umt.edu.pk/index.php/cto
Article QR
A publication of
Department of Life Sciences, School of Science
University of Management and Technology, Lahore, Pakistan
Title:
Evaluation of Incidence, Genotypes, and Risk Factors Associated with Newly
Diagnosed Hepatitis C Cases in Khyber Pakhtunkhwa (KPK), Pakistan
Author (s):
Ubaid Ahmad1, Najeeb Ullah Khan1, Maaz Iqbal1, Muhammad Ismail2, Adeela
Mustafa3
Affiliation (s):
1The University of Agriculture Peshawar, Pakistan
2Department of Zoology, Islamia College Peshawar
3Khyber Medical Collage
DOI:
https://doi.org/10.32350/cto.21.02
History:
Received: April 28, 2022, Revised: May 26, 2022, Accepted: June 08, 2022
Citation:
Ahmad U, Khan NU, Iqbal M, Ismail M, Mustafa A. Evaluation of incidence,
genotypes and risk factors of newly diagnosed Hepatitis C cases in Khyber
Pakhtunkhwa, Pakistan. BioSci Rev. 2022;2(1):17–34.
https://doi.org/10.32350/cto.21.02
Copyright:
© The Authors
Licensing:
This article is open access and is distributed under the terms of
Creative Commons Attribution 4.0 International License
Conflict of
Interest:
Author(s) declared no conflict of interest

Research Article
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Current Trends in OMICS
Journal of Management and
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Volume 2 Issue 1, 2022
Evaluation of Incidence, Genotypes, and Risk Factors
Associated with Newly Diagnosed Hepatitis C Cases in
Khyber Pakhtunkhwa (KPK), Pakistan
Ubaid Ahmad1, Najeeb Ullah Khan1, Maaz Iqbal1, Muhammad Ismail2, Adeela
Mustafa3
1The University of Agriculture Peshawar, Pakistan
2Department of Zoology, Islamia College, Peshawar
3Khyber Medical Colege
Abstract
Hepatitis C is a global health concern brought on by the hepatitis C virus
(HCV). The risk factors of the disease include injection drug users,
contaminated medical instruments, blood transfusion, organ transplant,
major surgery, dialysis, dental surgery, and acupuncture. The aim of the
current study was to identify the prevalence of active HCV infection,
genotypes, and their associated risk factors in newly infected patients of
Khyber Pakhtunkhwa (KPK), Pakistan. A total of 230 HCV cases and 245
control subjects were enrolled. Our findings revealed that disease
infection rates were higher in female patients (52.6%) than male patients
(47.4%). Moreover, the disease was more prevalent in the age group 30–
40 years (39.6%). The highest incidence was found in the southern region
(51.30%), followed by the northern region (27.82%), and central region
(18.26%). In our data set, dental surgery, major surgery, disease in
family, barber community, blood received, needles, abroad travel,
injection drug users, acupuncture, and organ transplantation were
determined as the major associated risk factors of the disease. Odds ratio
were significantly higher (10.91%) for those who used acupuncture as
compared to those who did not. Similarly, odds were significantly higher
(10.03%) for those who had HCV in their family, dialysis (9.377%), dental
surgery (7.618%), major surgery (6.813%), barber community (5.328%),
and blood received (2.252%) as compared to the control group. Also, the
most frequent genotype was 3a (63%), followed by an un-typeable
genotype (11.30%).
Keywords: hepatitis C virus (HCV), blood transfusion, dialysis, genotype,
surgery
*Coresponding Author: najeebkhan@aup.edu.pk ; adeelaamir@gmail.com

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Introduction
Chronic hepatitis C virus (HCV) infection is a worldwide health hazard
[1] associated with a variety of hepatic and extrahepatic illnesses,
including cancer. Hepatitis is a contagious blood viral disease caused by
HCV [2]. HCV belongs to hepaciviral genus of Flaviviridea family and
was discovered in 1989. It has a positive-sense, single-stranded RNA
genome with a size of 9.5Kb that codes for a polyprotein of 3000–3030
amino acids [3]. The infective virus leads to the inflammation of the liver,
manifested by the inflammatory cells in the infected liver tissue, which
may progress to fibrosis, followed by cirrhosis, hepatic cellular carcinoma
(HCC) (end stage of liver condition), and death [4]. HCV causes HCC by
regulating various indirect mechanisms [5]. For instance, the polyprotein
is processed by host and viral proteases which construct mature structural
and non-structural (NS) proteins [6]. HCV core protein enters the host cell
[7], where it remains present in the outside mitochondrial membrane and
endoplasmic reticulum, which enhances oxidative stress. It results in the
activation of key signaling pathways, such as the nuclear factor kappa B
and p38 mitogen-activated protein kinase pathways [8], directing the over
expression of genes involved in cytokine production and inflammation,
changes in apoptotic pathways, and tumor formation. The non-structural
proteins of HCV, NS3, and NS5A act as mediators to create oxidative
stress and inflammation.
The ideal transmission mode of HCV is either through blood or blood-
related products. It was noticed that the supply of blood was contaminated
with an unidentified agent causing post-transfusion non-A and non-B
hepatitis [9]. In developed countries, post-transfusion hepatitis C has
become relatively rare. The incidence of transfusion associated hepatitis,
traced from 1970 to 1998, decreased from 33% to the nearly eliminated
HCV transmission caused by the effectiveness of a series of donor
screening interventions [10]. In developing countries, where HCV testing
of donated blood is not feasible, receiving blood items remains a dominant
source of HCV infection. The prevalence of anti-HCV among intravenous
drug users ranged from 31% to 98% [11]. It has been reported that
injection drug use accounts for 60% and 80% of HCV infections in USA
[12] and Australia, respectively [13]. In developing countries, HCV
transmission is mainly via unsafe therapeutic injections [14] resulting in
2.34 million HCV infections every year, approximately [15]. The

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transmission of HCV through unhygienic injection equipment has been an
issue in most developing countries [16]. Other sources of HCV
transmission [17] include activities involving the potential for
percutaneous exposure to blood or blood derived body fluids, such as
tattooing [18], acupuncture [19], sharing cottons [20], and other
biologically plausible modes of transmission including body piercing,
cosmetic procedures, and commercial barbering [21].
HCV has six major genotypes and eighty subtypes with their specific
sequences [22], [23], [24]. HCV genotypes 1, 2, and 3 are found
throughout the world, while their relative prevalence differs from place to
place. HCV genotype 1 is the most common genotype, accounting for 83.4
million cases (46.2% of all HCV cases), worldwide. HCV genotype 3 is
the next most prevalent genotype with 54.3 million cases (30.1%),
worldwide. In Nepal [25], India [26], and Pakistan [27], HCV-3
(predominantly 3a) is the most common genotype [28]. The high
incidence of HCV genotype 3 is a serious concern because compared to
other genotypes, genotype 3 is associated with a higher risk of liver
consequences and overall mortality in all HCV patients [29]. In the current
study, we aimed to find the incidence of active HCV infection and
associated risk factors in newly infected individuals among the population
of Khyber Pakhtunkhwa (KPK), Pakistan.
Materials And Methods
Study Design and Sampling
In this cohort study, a total of 230 HCV cases and 245 healthy control
subjects were enrolled. All the patients and healthy blood donors were
recruited from Khyber Teaching Hospital (KTH), Peshawar. Ethical
approval from the competent authority of KTH was taken along with the
written consent of the patients. The objectives of the study were explained
to the patients before taking their data. Demographic data, such as age,
home town, locality, economic status, and time period of the disease were
collected via a standard questionnaire along with 2–3 CC blood.
Hepatitis C Virus Screening
All the samples were screened for anti-HCV using ICT (Immuno-
Chromatographic test), ELISA (Enzyme Linked Immune Sorbent Assay)
and RT (Real Time PCR).

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HCV Genotyping
The anti-HCV PCR-positive patients were further subjected to HCV
genotyping. /Complete follow up was conducted of all the pateints. The
results were obtained in the form of a document to find out their respective
genotypes. HCV genotyping was peformed using real-time hybridization
fluorescence detection method. For subtyping, HCV RNA was reverse
transcribed into cDNA by using MMLV reverse transcriptase enzyme. The
cDNA was subjected to two rounds of PCR amplification. PCR products
were electrophoresed on 2% agarose gel, stained with ethidium bromide,
and visualized on UV transilluminator.
Statistical Analysis
The odds ratio and significance level of all the possible risk factors were
assessed by using binary logistic regression view via the Statistical
Package for Social Sciences (IBM SPSS STATISTICS V20). One-way
ANOVA was carried out to determine the differences between the
different conditions (age, gender, and other parameters) of patients and
control.
Results
Demographic Characteristics of HCV Positive Cases
A total of 300 newly infected patients and 245 healthy controls were
questioned and conveniently sampled for the current study. Out of 300
HCV infected cases, 230 were found to be positive for HCV using ICT,
ELISA, and PCR, of which 121 were female and 109 were male patients
(Table 1). Based on age difference, patients were categorized into five
different age groups, namely below 30 years, 31–40 years, 41–50 years,
51–60 years, and above 60 years. Our results indicated that HCV infection
was high in age group 31–40 (35.2%), followed by age group 41–50
(23.5%) (Table 2).

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Table 1: Gender-wise Distribution of HCV Positive Cases
Count
Location
Total
Afghanistan
Southern
KP
Northern
KP
Central
KP
Gender
Female
2
14
35
70
121
Male
4
28
29
48
109
Total
6
42
64
118
230
Table 2: Age-wise Distribution of HCV Positive Cases
Age-group
No. of samples
Male
Female
Below 30
45 (19.6%)
20 (18.3)
25 (20.6)
31-40
81 (35.2%)
33 (30.2)
48 (39.6)
41-50
54 (23.5%)
25 (22.9)
29 (23.9)
51-60
38 (16.5%)
21 (19.2)
17 (14.04)
Above 60
12 (15.3%)
10 (9.17)
2 (1.6)
Total
230
109
121
Odds Ratio (OR) in Relation to HCV Risk Factors
The odds ratio and significance level of all possible risk factors were
assessed via simple and forward selection using binary logistic regression
view through Statistical Package for Social Sciences (IBM SPSS
STATISTICS V20). The package was used to analyze and find out the
percept distribution of qualitative records the various infected patients and
control group. The results revealed that the following categorical variables
(Table 3a, 3b, 3c) as significant risk factors in comparison to control. The
odds showed that the disease causing factors were statistically associated
with HCV transmission. The odds were significantly higher (10.91%) for
those patients who used acupuncture and were HCV positive as compared
to control, followed by patients with HCV in the family (10.03%), dialysis
(9.377%), dental surgery (7.628%), major surgery (6.813%), barber
community (5.328%), and blood received (2.25%).
Table 3a. Dependent Variables in Coding
Original Value
Inner Value
Yes
1
No
0

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Table 3b. Coding of All Definite Variables
Frequency
Parameter coding
(1)
Travel Abroad
Yes
42
1.000
No
432
.000
Blood Received
Yes
69
1.000
No
405
.000
I-D-Users
Yes
38
1.000
No
436
.000
Smoking
Yes
65
1.000
No
409
.000
Needles
Yes
43
1.000
No
431
.000
Barber
Yes
61
1.000
No
413
.000
O. Transplantation
Yes
20
1.000
No
454
.000
Major Surgery
Yes
104
1.000
No
370
.000
Disease in family
Yes
88
1.000
No
386
.000
Piercing
Yes
20
1.000
No
454
.000
Acupuncture
Yes
27
1.000
No
447
.000
Dialysis
Yes
52
1.000
No
422
.000
Dental Surgery
Yes
108
1.000
No
366
.000
Table 3c. Definite Variables in Calculation
Variables
B
S.E.
Wald
Df
Sig.
Exp (B)
Blood Received
.812
.377
4.624
1
.032
2.25
Barber
1.673
.490
11.658
1
.001
5.328
Major Surgery
1.919
.330
33.728
1
.000
6.813
Dental Surgery
2.030
.330
37.865
1
.000
7.618
Dialysis
2.238
.619
13.082
1
.000
9.377
Acupuncture
2.389
.883
7.329
1
.007
10.907
Disease in family
2.306
.378
37.113
1
.000
10.031
Constant
-2.151
.215
100.408
1
.000
.116

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Possible Risk Factors for HCV Transmission
A basic objective of our study was to find out the possible risk factors of
HCV transmission in the newly infected individuals. All the HCV infected
patients and also the control group were questioned as per the
questionnaire. The results revealed by applying descriptive statistics and
frequency was shown in the case study contatining the control group
also.the risk factors were significantly associated with HCV transmission
(Figure 1). Among all risk factors, dental surgery (37.39%) was found to
be the most possible route risk factor for HCV infection, followed by
major surgery (34.34%), HCV history in family (20.34%), barber
community (23.47%), blood received (23.04%), dialysis (20.86%),
needles reuse (16.08%), travel abroad (13.47%), ID-users (13.04),
acupuncture (10.86%), and organ transplantation (6.95%).
Figure 1. Possible Risk Factors for HCV Transmission in Khyber
Pakhtunkhwa
Frequency Distribution of HCV Genotypes
All HCV positive patients were assessed for HCV genotypes (Figure 2).
Out of 230 HCV samples, genotype 3a (63.04%) was found to be the most
abundant, followed by an un-typeable genotype (11.3%), 1a (9.1%), 2a

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(6.5%), 3b (4.8%), 2b (1.3%), 1b+3a (1.30%), 1b (0.86%), 1a+3a (0.86%),
and 3a+3b (0.43%).
Figure 2. Frequency Distribution of HCV Genotypes
HCV RNA was reverse transcribed into cDNA by using MMLV reverse
transcriptase enzyme. The cDNA was subjected to two rounds of PCR
amplification, which utilized genotype specific primers.
Associated Risk Factors of HCV Genotype 3a and 3b
Demographic data showed a higher percentage of association between risk
factors and HCV genotype 3a. The high percentage of barber community
(72.2%) was found to be the main associated risk factor for genotype 3a
transmission, followed by dental surgery (65.1%), blood received from
infected individuals (62.3%), major surgery (62%), HCV in the family
(59.5%), dialysis (58.3%), and acupuncture (48%) (Figure 3a). A higher
percentage association was also observed between risk factors and HCV
genotype 3b. The percenatge of infected individuals who used acupuncture
(16%) was found to be the main association factor for HCV transmission,
followed by major surgery (7.6%), dental surgery (7%), dialysis (6.2%),
HCV in the family (4.1%), and barber community (1.9%) (Figure 3b).

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a) b)
Figure 3. Associated Risk Factors of HCV Genotypes 3a and 3b. a) Association between
various risk factors and 3a genotype. b) Association between various risk factors and 3b
genotype.
Associated Risk Factors of HCV Genotypes 2a and 2b
Demographic data showed a higher percentage association between risk
factors and HCV genotype 2a (Figure 4a). The percentage of infected
individuals with HCV in the family (9.5%) was found to be the main
associated factor for HCV transmission, followed by blood received
(9.4%), dental surgery (5.8%), dialysis (4.2%), acupuncture (4%), major
surgery (3.8%), and barber community (1.9%). Demographic data also
showed a higher percentage association between risk factors and HCV
genotype 2b (Figure 4b). The percentage of infected individuals who used
acupuncture (8%) was found to be the main associated factor for HCV
transmission, followed by dialysis (2.1%), blood received from infected
individuals (1.9%), and dental surgery (1.2%).
a) b)
Figure 4: Associated Risk Factors of HCV Genotypes 2a and 2b. a) Association between
various risk factors and 2a genotype. b) Association between various risk factors and 2b
genotype.

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Associated Risk Factors of HCV Genotypes 1a and 1b
Demographic data showed a higher percentage association between risk
factors and HCV genotype 1a (Figure 5a). The percentage of patients
using dialysis (12.5%) was found to be the main associated risk factor for
HCV genotype 1a transmission, followed by blood received from infected
individuals (11.3%), individuals with HCV in the family (10.8%), major
surgery (7.6%), barber community (7.4%), dental surgery (5.8%), and
acupuncture (4%). The percentage of infected individuals who /belonged
to the same barber community (3.7%) was found to be the main associated
factor in genotype 1b transmission, followed by those who belonged to the
same dental surgery community (2.3%) (Figure 5b).
a) b)
Figure 5. Associated Risk Factors of HCV Genotypes 1a and 1b. a) Association between
various risk factors and genotype 1a. b) Association between various risk factors and
genotype 1b.
Associated Risk Factors of HCV Un-typeable and Mixed Genotypes
Demographic data showed a higher percentage association between risk
factors and un-typeable genotypes. The percentage of people using
acupuncture (20%) was found to be the main associated factor for un-
typeable genotype transmission, followed by major surgery community
(15.2%), infected individuals with HCV in the family (14.9%), dialysis
(14.6%), blood received from infected individuals (11.3%), barber
community (11.1%), and dental surgery (9.3%) (Figure 6a). A higher
percentage association was found between risk factors and HCV
genotypes 1b+3a. The percentage of infected individuals who underwent
dialysis (2.1%) was found to be main associated factor with genotypes
1b+3a transmission, followed by blood received (1.9%), barber
community (1.9%), major surgery (1.3%), and dental surgery (1.2%).

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Demographic data also showed a higher percentage association between
risk factors and HCV genotypes 3a+3b. The frequency of infected
individuals who underwent major surgery (1.3%) was found to be main
associated with genotypes 3a+3b transmission, followed by dental surgery
(1.2%), and blood received from infected individuals (1%).
a) b)
C)
Figure 6: Associated Risk Factors of HCV Un-typeable and Mixed Genotypes. a)
Association between various risk factors and un-typeable genotypes. b) Association
between various risk factors and 1b+3a genotypes. c) Association between various risk
factors and 3a+3b genotypes.
Discussion
Hepatitis is a transmissible blood viral ailment caused by the hepatitis C
virus (HCV). HCV is a worldwide health hazard related to numerous
hepatics and hepatic disorders, such as malignancies. The most essential
transmission modes of HCV are blood or blood associated products. The
known global risk factors of the disease include injection drug users,
contaminated medical instruments, blood transfusion, organ transplant,
major surgery, dialysis, household items, dental surgery, and acupuncture.

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Numerus procedures have been adopted to control the disease, including
proper screening and awareness campaigns, both in health professionals
and general public. However, theworldwide situation of the disease is still
alarming, particularly in developing countries, such as Pakistan. In this
study, we aimed to find out the exact situation of risk factors in the newly
diagnosed cases of HCV. A total of 300 HCV cases and 245 healthy
individuals were included in the current study. Out of the 300 HCV cases,
230 samples were found to be positive for active HCV using ICT, ELISA,
and RT-PCR. Our data set showed that HCV infection occurred more in
female patients as compared to male patinets. Moreover, the disease was
more prevalent in age group 31–40 years, followed by age group 41–50
years (Table 2). Recently, a similar conclusion was reached by an author
from Pakistan [30]. Our results further showed that the most probable risk
factors of HCV cases were dental surgery, followed by major surgery, and
family history. These risk factors of HCV transmission have been very
well known for the last 2–3 decades [28], [31]. It indicates that in
Pakistan, there is a lack of proper guidelines for health professionals who
consequently indulge in malpractices, even though these risk factors have
been well known to everyone including the majority of the general public.
Government officials need to ensure compliance with proper and strict
guidelines for reusing the dental and surgical equipment.
The most prevalent genotype in our data set was 3a (63.04%), followed by
the un-typeable genotype (11.3%), 1a (9.1%), 2a (6.5%), 3b (4.8 %), 2b
(1.3%), 1b+3a (1.30%), 1b (0.86%), 1a+3a (0.86%), and 3a+3b (0.43%).
Similarly, a recent study conducted by Pakistani authors from 2015 to
2018 on HCV cirrhotic patients in Peshawar hospitals [32] concluded that
the prevalence of HCV genotype 3a was 47.9%, 2a and 3b was 11% each,
1a was 6%, and 1b was 1%; while, 4.1% had mixed genotypes and 18.7%
had un-typable genotypes. Additionally, HCV genotype 3 was identified
as the most common genotype in Bangladesh, Nepal, and India [25, 33,
34]. Our sample included a substantial proportion of un-typable genotypes
in contrast to the earlier findings..
Our study identified the higher percentage association between risk
factors and HCV genotype 3b. The percentage of infected individuals who
used acupuncture (16%) was found to be the main associated factor for
HCV transmission, followed by major surgery (7.6%), dental surgery
(7%), dialysis (6.2%), HCV in the family (4.1%), and barber community

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(1.9%). Our study also showed a higher percentage association between
risk factors and HCV genotype 3a+3b. The percentage of infected
individuals who underwent major surgery (1.3%) was found to be main
associated factor with genotype 3a+3b transmission, followed by dental
surgery (1.2%), and blood received from infected individuals (1%). Blood
transfusion, dangerous intravenous injection, and surgery are all instances
of unsafe medical procedures. However, many people acquire HCV while
visiting barber shops, beauty salons, and hair salons. A large percentage of
people denied ever having been exposed to HCV. Moreover, we found no
evidence of vertical transmission. A considerable number of HCV
infections occur outside of dangerous medical procedures incosmetics, as
well as hair and barber businesses. This is presumably a result of the use
of razors, clippers, cuticle pushers, nail files, nail buffers, brushes, or any
other piercing equipment.
However, additional nationwide investigations are still required to
provide a complete picture of the distribution of HCV genotypes in
association with gender, age, risk factors, and pretreatment viral load. The
results of this study clearly showed some gaps in the epidemiology of
HCV and its associated risk factors in the Khyber Pakhtunkhwa
population.
Conclusion
In contrast to the outcomes of our previous research conducted a decade
ago, the results showed that 3a is the most common HCV genotype.
Female patients were found to have a somewhat higher prevalence of
HCV. Moreover, the majority of those infected were middle-aged (30-40
years). The findings implicate that all high and extremely high viral loads
are frequently found in all genotypes, although the most prevalent subtype
3a is associated with low and intermediate viral loads. The majority of
people in Khyber Pakhtunkhwa contract HCV because international
standards are not being followed, especially when it comes to blood
transfusion and surgery. Prior knowledge of pretreatment viral load and
HCV genotype must be considered in national planning strategies, as it
would minimize the side effects of antiviral medication, reduce the
economic bioburden, and improve response rates.
Conflict of Interests
The authors have no conflict of interests.

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Funding
The current work received no funding
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