International Journal of Medicine and

Pharmaceutical Sciences (IJMPS)

ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 6, Issue 2, Apr 2016, 1-6
TJPRC Pvt. Ltd.

PREVALENCE OF HEPATITIS C VIRUS GENOTYPES AND VIRAL LOAD

ISOLATED FROM IRAQI PATIENTS BY REVERSE TRANSCRIPTION-PCR
AHMED ABDULWAHID SALMA1, MOHAMMED YOUNUS NAJI AL ATBEE2,
MOHAMMED HUSSAIN WAHEED3 & KHULOOD ABDULRAZAQ KALEEL4
1
2
3

Immunologist, Al Sader Teaching Hospital, Basrah, Iraq

Lecturer, Nephrologist and Internist, Basrah Medical College, Basrah, Iraq

Laboratory Specialist, Head of Middle East Lab Molecular Lab, Basrah, Iraq
4

Laboratory Specialist, Basrah Central Public Health Lab, Basrah, Iraq

ABSTRACT
Hepatitis C virus (HCV), the major causative agent of parenterally transmitted non-A, non-B hepatitis, belongs to
the Flaviviridae family. The genome of HCV is a single-stranded RNA with positive polarity, considerable genetic
heterogeneity has been reported among isolates. HCVs genome sequence is highly variable worldwide; Six major genotypes
(1 to 6) have been identified, some of which contain several subtypes referred to as a, b, c, etc, which are approximately 80

Center in Basrah Governorate. The study is cross-sectional study and conducted in the period of May 2015 to January 2016.
Samples were collected from 131 patients attending Basrah General Hospital and Middle East Private Laboratory Center ,(
total cases are 258 , we exclude 127 patients because missing data and enrolled 131 patients included in this study)
serological test done for all patients for the detection of anti-Hepatitis C virus antibody generation 3, by Enzyme Linked
ImmunoAssay (ELISA).All ELISA positive samples were tested individually for the HCV viral load by Real time PCR ,using
patient plasma ,then RNA extraction done by QIAamp viral RNA Mini kit then amplification done by using qiagen artus
HCV RG RT PCR kit which run on Q6000 rotorgen according to manufacturer protocol the samples that contain viral
load ( 5X10^3 -8X10^8 ) were further tested for HCV genotyping by using ( Nuclear Laser Medicine HCV Rt-PCR 2.0 kit ).
This study that enrolled 131 HCV patients, 63 males and 68 females and their age range from 3 to 65 years old, mean age:
27.417 SD years with male to female ratio 0.92:1. HCV genotype that increasing frequency of genotype 1a in male in studied
patients 32 65.3% (24.4% of total cases) as compared to other genotypes which are statistically significant p= 0.047. The
distribution of HCV genotype among Iraqi patients in our study that genotype in following frequencies 1a 49 (37.4%), 1b
17(13%), 2a 2 (1.5%), 3a 7 (5.3%) , 4a 25(19.1%) and 4c 31(23.7%) . Awareness seminars regarding the risk factor of HCV
infection transmission should be regularly arranged and discovery of new vaccines by scientists is of utmost importance to
overcome this disease. The highest viral load in age between 21- 40 years old patients. So Monthly blood tests should be
necessary for the diagnosis and monitor the progression of HCV.
KEYWORDS: Basrah General Hospital and Middle East Private Laboratory Center, Progression of HCV

Received: Feb 06, 2016; Accepted: Feb 19, 2016; Published: Feb 25, 2016; Paper Id.: IJMPSAPR20161

INTRODUCTION
Hepatitis C virus (HCV), the major causative agent of parenteral transmitted non-A, non-B hepatitis , belongs to
the Flaviviridae family.1 The genome of HCV is a single-stranded RNA with positive polarity ,considerable genetic
heterogeneity has been reported among isolates .1,2 HCVs genome sequence is highly variable worldwide; Six major

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editor@tjprc.org

Original Article

subtypes have been recognized. The study was conducted in Basrah General Hospital and Middle East Private Laboratory

Ahmed Abdulwahid Salma, Mohammed Younus Naji Al Atbee,

Mohammed Hussain Waheed & Khulood Abdulrazaq Kaleel

genotypes (1 to 6) have been identified, some of which contain several subtypes referred to as a, b, c, etc , which are
approximately 80 subtypes have been recognized.4 Genotype 1 is the predominant genotype in many geographic regions,
including Europe and North America, where it accounts for 50%90% of cases. Genotypes 2 and 3 are also widely
distributed. Genotype 2 is relatively common in Europe, North America, and Japan. Genotype 3 is common in Southeast
Asia, Australia, South America, and northern Europe, in particular in intravenous drug users.5 Genotype 4 is found mainly
in Egypt, the Middle East, and central Africa, genotype 5 in southern Africa, and genotype 6 in Southeast Asia. Despite the
fact that they differ from each other by more than 30% of the RNA sequence, pathogenic differences between genotypes
have not been demonstrated, except for steatosis associated with genotype3a.5 However, because the genotype dictates the
chance of therapeutic response and duration of treatment, genotyping has become fundamental in the clinical analysis of
patients with hepatitis C.5 Genotypes 2 and 3 respond favorably, reaching 75% sustained response (SR) to 24 weeks of
treatment with PEGylated interferon and ribavirin, while genotype 1 responds poorly, at 50% SR even after 48 weeks of
treatment.5,6 In recent years, a shift in the prevalence of HCV genotypes has been reported in a number of countries around
the world . The shift in the genotype circulation may have a significant impact on clinical management of the patients as
well as on the public health.7,8
The goal of our study that to analyze the prevalence of HCV genotypes and its subtypes in Iraqi patients and its
correlation with viral loads.

CONCISE METHODS
Material: In our study we using following materials for HCV viral load and genotypes :Real time PCR (Rotorgen
Q 6000- Qiagen),Conventional PCR (Applied Biosystems 9700), Hybridization shaking water path (Memmert), Microplate
spectrophotometer reader (ELX800 biolisa reader Biokit), Deep freeze (-40 Co) ,Refrigerator (2 -8 Co),High speed
centrifuge (0.5ml tube),Centrifuge (10 -15 ml tube ),Different size micropipette (Eppendorf) 0.2,0.5,2,5,10 ml tube ,Rack
,Powder free gloves , and PCR cabinet.
Statistics: Analysis of data was carried out using the available statistical package of SPSS-20 (Statistical
Packages for Social Sciences- version 20 Statistics) for determination of statistical significance among different variables.
A descriptive statistics like mean together with analytic statistics, have been done when appropriate. A p-value of less than
0.05 was considered as significant and calculated by method of Pearson Chi square equation. The percentage in result
tables were considered for column rather than row.

METHODS
The study was conducted in Basrah General Hospital and Middle East Private Laboratory Center in Basrah
governorate. The study is cross-sectional study and conducted in the period of May 2015 to January 2016. Samples were
collected from 131 patients attending Basrah General Hospital and Middle East Private Laboratory Center, (we exclude
127 patients because missing data and enrolled 131 patients included in this study) serological test done for all patients for
the detection of anti-Hepatitis C virus antibody generation 3, by Enzyme Linked ImmunoAssay (ELISA), (Biolisa)
HCV4.0 ELISa,Biokit Spain, all ELISA positive samples were tested individually for the HCV viral load by Real time
PCR ,using patient plasma ,the RNA extraction done by QIAamp viral RNA Mini kit then amplification done using qiagen
artus HCV RG Q PCR kit which run on Q6000 Rotorgen according to manufacturer protocol the samples that contain
viral load ( 5X10^3 -8X10^8 ) were further tested for HCV genotyping by using ( Nuclear Laser Medicine HCV Rt-PCR

Impact Factor (JCC): 5.4638

NAAS Rating: 3.54

Prevalence of Hepatitis C Virus Genotypes and Viral Load

Isolated from Iraqi Patients by Reverse Transcription-PCR

2.0 kit )for amplification of sample conventional PCR used Gene Amp 9700 PCR system following the manufacturer
protocol then reveres hybridization strip assay used to determine the genotype .

RESULTS
This study that enrolled 131 HCV patients, 63 males and 68 females and their age range from 3 to 65 years old,
mean age: 27.417 SD years with male to female ratio 0.92:1, (total cases are 258, we exclude 127 patients because
missing data and enrolled 131 patients included in this study) . The table 1 show distribution of HCV genotype among
gender that increasing frequency of genotype 1a in male in studied patients 32 65.3% (24.4% of total cases) as compared to
other genotypes which are statistically significant p= 0.047. Table 2 show the distribution of HCV genotype among
patients referral from medical outpatients clinics (including gastroenterology and internal medicine clinic and dialysis and
nephrology clinic), oncology patients clinic and thalassemia clinic. The increasing frequency of medical outpatients of
genotype 4c 30 cases( 96.8%) as compare with other patients referral , which are statistically significant p= 0.0001. Table 3
show the distribution of HCV genotype among HCV viral load that increased viral loads in genotype 1a in studied patients
to more than 800 IU/L 47 cases 95.9% as compare with other genotypes. Table 4 show the distribution of HCV viral load
among age of patients that the highest viral load more than 800 IU/L in age between 21- 40 years old patients 41 cases
31.8% (31.2% of total cases).The mean viral load of HCV PCR is equal to 17289417.6565 47799026.99816 SD IU/L.
Figure 1 show distribution of HCV genotype among Iraqi patients in our study that genotype in following frequencies 1a
49 (37.4%) ,1b 17(13%) , 2a 2 (1.5%) , 3a 7 (5.3%) , 4a 25(19.1%) and 4c 31(23.7%) .
Table 1: Distribution of HCV Genotype among Gender
1a
32

HCV Genotypes
3a
4a
2
12

2a
1

1b
4c
Total
GENDR MALE Count
5
11
63
% within
65.3% 50.0% 28.6% 48.0% 29.4% 35.5% 48.1%
genotype
FEMAL Count
17
1
5
13
12
20
68
E
% within
34.7% 50.0% 71.4% 52.0% 70.6% 64.5% 51.9%
genotype
Total
Count
49
2
7
25
17
31
131
% within
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
genotype
Pearson Chi-Square = 11.2, p value =0.047, 95% Confidence Interval between 0.033-0.040
Table 2: Distribution of HCV Genotype among Patients Referral

Referral Medical
outpatient

1a
27

Count
% within
55.1%
genotype
oncology
Count
16
patient
% within
32.7%
genotype
thalassemia
Count
6
patient
% within
12.2%
genotype
Total
Count
49
% within
100.0%
genotype
Pearson Chi-Square = 39.2, p Value =0.0001
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2a
2

Genotype
3a
4a
4
18

1b
14

4c
30

Total
95

100.0% 57.1% 72.0% 82.4% 96.8% 72.5%

0

17

0.0%

0.0%

0.0%

5.9%

0.0%

13.0%

19

3.2%

14.5%

31

131

0.0%
2

42.9% 28.0% 11.8%

7

25

17

100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

editor@tjprc.org

Ahmed Abdulwahid Salma, Mohammed Younus Naji Al Atbee,

Mohammed Hussain Waheed & Khulood Abdulrazaq Kaleel

Table 3: Distribution of HCV Genotype among HCV Viral Load

Viral <800
load IU/L
>800

Genotype
HCV Viral Load 1a
2a
3a
4a
1b
4c
Total
Count
2
0
0
0
0
0
2
% within
4.1%
0.0%
0.0%
0.0%
0.0%
0.0%
1.5%
genotype
Count
47
2
7
25
17
31
129
% within
genotype
95.9% 100.0% 100.0% 100.0% 100.0% 100.0% 98.5%

Total

Count
49
2
7
25
17
31
131
% within
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
genotype
Pearson Chi-Square = 3.4, p value =0.6, 95% Confidence Interval between 0.48-0.50
Table 4: Distribution of HCV viral Load among Age of Patients
Viral Load
Age of Patients
<800 IU/L
>800 Total
AGE
<10 YR Count
0
20
20
% within LOAD
0.0%
15.5%
15.3%
10-20 Count
0
34
34
% within LOAD
0.0%
26.4%
26.0%
21-40 Count
2
41
43
% within LOAD
100.0%
31.8%
32.8%
>40YR Count
0
34
34
% within LOAD
0.0%
26.4%
26.0%
Total
Count
2
129
131
% within LOAD
100.0%
100.0%
100.0%
Pearson Chi-Square = 4.1, p value =0.2, 95% Confidence Interval between 0.25-0.26

Figure 1: Show Distribution of HCV Genotype among Iraqi Patients in our Study. HCV Genotypes in Following
Frequencies 1a 49 (37.4%), 1b 17(13%), 2a 2 (1.5%), 3a 7 (5.3%), 4a 25(19.1%) and 4c 31(23.7%)

DISCUSSIONS
The study was conducted in Basrah General Hospital and Middle East Private Laboratory Center in Basrah
Governorate. The study is cross-sectional study and conducted in the period of May 2015 to January 2016. Samples were
collected from 131 patients attending Basrah General Hospital and Middle East Private Laboratory Center, 63 males and
68 females and their age range from 3 to 65 years old, mean age: 27.417 SD years with male to female ratio 0.92:1. In our
Impact Factor (JCC): 5.4638

NAAS Rating: 3.54

Prevalence of Hepatitis C Virus Genotypes and Viral Load

Isolated from Iraqi Patients by Reverse Transcription-PCR

study HCV genotype 1a is most common and increasing frequency in Iraqi patients 32 65.3% (24.4% of total cases) as
compared to other genotypes which are statistically significant p= 0.047. HCV genotype among Iraqi patients in our study
following frequencies 1a 49 (37.4%) ,1b 17(13%) , 2a 2 (1.5%) , 3a 7 (5.3%) , 4a 25(19.1%) and 4c 31(23.7%) . This
consistent with other studies 9 ,10 that showed genotype 1a was predominant one .Patients referral from medical outpatients
clinics 95 (72.5%) ;( including gastroenterology and internal medicine clinic and dialysis and nephrology clinic) ,oncology
patients clinic 17 (13%) and thalassemia clinic 19 (14.5%) . The more frequent HCV genotype in medical outpatients is
genotype 4c 30 cases 96.8% that include mostly from hemodialysis patients, and then 1a 27 (55.1%) as compare with other
patients referral , which are statistically significant p= 0.0001. The HCV viral load that increased viral loads to more than
800 IU/L in genotype 1a 47 cases 95.9% as compare with other genotypes. The HCV viral load among age of patients that
the highest viral load more than 800 IU/L in age between 21- 40 years old patients 41 cases 31.8% (31.2% of total cases).
The mean viral load of HCV PCR is equal to 17289417.6565 47799026.99816 SD IU/L. HCV genotype among Iraqi
patients in our study that following frequencies 1a 49 (37.4%) ,1b 17(13%) , 2a 2 (1.5%) , 3a 7 (5.3%) , 4a 25(19.1%) and
4c 31(23.7%) .

CONCLUSIONS

HCV is a major global health issue affecting several individuals around the world. The prevalence of HCV
genotypes varies all over the world due to virus mutation, route of infection and population analysis. HCV
genotype 1a is most common and increasing frequency in Iraqi patients.

HCV genotype 4c is most predominant in medical and hemodialysis patients. Awareness seminars regarding the
risk factor of HCV infection transmission should be regularly arranged and discovery of new vaccines by
scientists is of utmost importance to overcome this disease.

The highest viral load in age between 21- 40 years old patients .So Monthly blood tests should be necessary for
the diagnosis and monitor the progression of HCV.

ACKNOWLEDGEMENTS
Our great thanks to all staffs in the General Basrah Hospital and Middle East Private Laboratory Center. Then,
last but not least, thanks for every one support us in this work.
Disclosures
There is no disclosure.
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editor@tjprc.org

Ahmed Abdulwahid Salma, Mohammed Younus Naji Al Atbee,

Mohammed Hussain Waheed & Khulood Abdulrazaq Kaleel
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