Advances in Bioscience and Biotechnology, 2013, 4, 866-871 ABB Published Online September 2013 (
Estimation of HCV viral load and liver enzymes among
different patients groups of District Gujrat, Pakistan
Asim Mushtaq1, Mujahid Azeem Tariq2, Umer Rashid1, Amber Afroz1, Nadia Zeeshan1,
Abdul R. Asif3, Muzna Zahur1,4*
1Department of Biochemistry and Molecular Biology, University of Gujrat, Gujrat, Pakistan
2Department of Pathology, Hashmat Medical College, Gujrat, Pakistan
3Department of Clinical Chemistry, University Medical Centre, Goettingen, Germany
4Department of Neurology, University Medical Centre, Goettingen, Germany
Email: *
Received 9 July 2013; revised 9 August 2013; accepted 25 August 2013
Copyright © 2013 Asim Mushtaq et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Hepatitis C has a 3% of the global disease burden
that remains endemic in many regions of the world.
According to a general statistical survey it has ap-
proximately 5.3% seroprevalence in Pakistan. HCV is
a persistent and silent disease thus making the pri-
mary diagnosis complicated. Occasionally HCV po-
sitive population could not be diagnosed by routine
HCV antibody testing therefore requires molecular
diagnosis. This study is aimed to determine the pre-
valence of HCV and to estimate the HCV viral load
by quantitative analysis among different patient groups
of District Gujrat, Pakistan. A total of 597 samples
were collected from clinically diagnosed liver patients
that were categorized into three age groups: 1) up to
25 years; 2) 26 - 50 years 3) above 50 years. All sam-
ples were subjected to real time PCR for determina-
tion and quantification of HCV RNA. Activity of liver
aminotransferases was measured. The overall preva-
lence of HCV-RNA was 73.87%. Females had slightly
higher HCV prevalence which is 74.06% while 73.45%
in males. Highest prevalence of active HCV infection
was found in age group 26 - 50. In addition, liver
function tests showed that 28.12% HCV-positive pa-
tients do not have elevated ALT level whereas 32.65%
did not show elevated AST levels. It may be assumed
that there is not a significant relationship between in-
creased viral load and liver amino transferases. The
study concluded a significantly higher rate of HCV
infection in young population. Moreover screening
with antibody and liver function tests alone does not
exclude the possibility of HCV infection.
Keywords: Hepatitis C; Viral Load; Alanine
Aminotransferase; Aspartate Aminotransferase; HCV
RNA; Gujrat
Hepatitis C virus (HCV) is a widespread infection that
causes significant morbidity and mortality all over the
world [1]. HCV infection is often asymptomatic and al-
most in 70% cases leads to chronic hepatitis. This may
results in cirrhosis, liver failure, hepatocellular carcinoma
or progressive liver disease within 20 to 30 years. Viral
load is one of the factors associated with progression of
disease in HCV infection [2]. Hepatitis C was first dis-
covered in 1989, at that time it was thought of minor
importance because of its difference from both hepatitis
A and B. At present it has become one of the major
global health issue that needs to be actively controlled
and prevented [3].
Around the globe, approximately 130 - 170 million
persons are living with chronic hepatitis C virus infection
[4]. Egypt has the largest burden of HCV in the world,
with 10% prevalence of chronic HCV infection among
the persons aged 15 - 59 years [5]. It is also a common
chronic blood-borne infection in USA, causing an esti-
mated 8000 - 10,000 death annually which will be in-
creased substantially in the next 20 years [6]. There are
almost 10 million people with HCV infection in Pakistan
Chronic HCV is usually treated with interferon (IFN)
alpha (a glycoprotein) that is considered as a backbone of
HCV treatment because of its efficient role to enhance
the immune response against virus [8]. At present, pege-
lated interferon (PEG-IFN) in combination with ribavirin
is regularly used for HCV treatment. This therapy achieves
*Corresponding author.
A. Mushtaq et al. / Advances in Bioscience and Biotechnology 4 (2013) 866-871 867
about 80% SVR for genotype 2 & 3 and 50% for geno-
type 1 [9]. Pegelated interferons are of two types, PEG-
IFN-alpha-2a and PEG-IFN-alpha-2b. They are different
in their size and configuration of the polyethylene glycol
that contains binding sites for interferon. The two for-
mulated interferons are equally good for HCV treatment
but their functioning is not compared [10]. Pre viral
loads are useful determinants of response to antiviral
therapy [11], hence initial viral load and decrease of viral
load at initial stage of antiviral therapy are useful pa-
rameters to check efficacy and to optimize antiviral
therapy [12].
There are different parameters for HCV diagnosis. In
most of the cases HCV prognosis is based on liver func-
tion tests. Abnormal level of serum amino transaminases
may lead to the prognosis of HCV infection. ALT is
most intense in liver and released into the bloodstream as
the result of liver damage that fairly indicates liver status
[13]. In fact, before the detection of HCV and the ex-
ploitation of molecular based essays for HCV RNA, the
non-A, non-B hepatitis was based on records of steadily
abnormal ALT levels [14]. On the other hand, almost
25% of patients with active HCV infection have persis-
tently normal ALT values [15]. It is considered that oc-
cult-HCV (characterized by presence of liver disease and
absence or very low levels of detectable HCV-RNA in
serum) can be indicated by abnormal liver function tests
although virus may persist for years after spontaneous
recovery or after sustained viral response [16]. In this
scenario, Viral Load is a good parameter for the presence
of hepatitis C virus and it also measures the amount of
HCV in the blood. If viral load (RNA) is found signifi-
cantly in an individual’s blood, it confirms active infec-
tion [17]. If viral load is less than 800,000 IU/mL, it is
considered as low viral load while more than 800,000
IU/mL, is called high viral load [18]. In the present study
patient data were collected from densely populated Dis-
trict Gujrat of Pakistan and tested for the presence of
HCV infection with molecular analysis. Quantification of
viral load was done to estimate the disease progression
and compared with liver enzymes activity. The data showed
high HCV prevalence in young population particularly in
females. Also a discrepancy was found between viral
load and liver function tests.
2.1. Sample Collection
A total of 597 blood samples were collected in a well
known diagnostic lab and its collection centres located in
different remote areas of Gujrat (a district of province
Punjab, Pakistan). Informed consent was added in labo-
ratory software from each patient including name, sex,
age, address and contact number. Blood samples were
collected in 5 mL chilled, sterilized disposable syringes.
Serum separation was done in 1.5 mL eppendorf tubes at
13,000 rpm for 2 min and used fresh or stored at 20˚C.
All analysis was done in accordance with the ethical
standards mentioned in the declaration of Helsinki.
2.2. RNA Extraction and Quantification
Total viral RNA was extracted from 150 μL serum sam-
ples by using Nucleospin RNA virus extraction kitac-
cording to the manufacturer’s instructions. Finally each
RNA sample was dissolved in 50 μL TE buffer.
HCV RNA was amplified by using real time amplifi-
cation and detection kit HCV Real-TM Quant (Sacace
Biotechnologies, Italy). Fluorescently labelled HCV spe-
cificprobes were used (reporter dye: cy3). FAM dye was
used to amplify HCV internal control (IC). Internal con-
trol was provided as an amplification control for each
sample being processed to detect the possible contamina-
tion risks. Two positive controls for different quantities
(quantities of standard 125 IU/mL and 12,500,000 IU/mL)
and two negative controls were carried out using cDNA
as substrate and spiked with mimic target in each run.
From the total RNA extracted, 12.5 μL was used as tem-
plate and mixed with equal volume of super mix con-
taining 300 μL of RT-PCR-mix-1, 200 μL of RT-PCR-
mix-2, 20 μL of Hot Start Taq Polymerase and 10 μL of
M-MLV Revertase. Amplification was carried out by
standard method (Stage 1: 50˚C for 1800 sec, stage 2:
95˚C for 900 sec and stage 3: 95˚C for 20 sec and 60˚C
for 40 sec for 42 repeats. PCR reaction was performed in
Smart cycler 11 (Cepheid, Germany). Results were ana-
lyzed by using smart cycler software v2.0d. Viral load
was calculated with the help of Cy3 and FamCt value
(Cy3 STD/Fam STD × 510,000 = No. of HCV IU/mL).
2.3. Assessment of ALT and AST
ALT and AST levels were measured using Ecoline re-
agents according to manufacturer’s instructions. The
threshold of 42 IU/L was used as upper normal range for
ALT and 40 IU/L for AST. Both enzyme activities were
measured at the same time when PCR for HCV quantifi-
cation was run.
2.4. Statistical Analysis
Relationship of viral load with ALT and AST in both
males and females was analysed by determining Correla-
tion Coefficient. Correlation was significant at 0.01 level
(2-tailed). Correlation coefficient was performed by us-
ing commercially available computer program SPSS
A total of 597 proposed anti HCV samples (171 detected
Copyright © 2013 SciRes. OPEN ACCESS
A. Mushtaq et al. / Advances in Bioscience and Biotechnology 4 (2013) 866-871
Copyright © 2013 SciRes.
by ELISA and 426 by ICT) were collected in Dr. Muja-
hid’s lab and FNA clinic district Gujrat and from its dif-
ferent collection centres. Out of 597 individuals 374 were
females and 223 were males. All the individuals were
categorized in three age groups (1st group: up to 25 years,
2nd group: 26 - 50 years, 3rd group: Above 50 years).
Sinceanti HCV antibodies are unable to show active
HCV infection in some cases therefore, all the samples
were subjected to RNA extraction and RT PCR to check
active infection. The PCR results detected HCV RNA in
the serum of 441 (73.87%) individuals. In males, 164
(73.45%) of 221 showed active HCV infection due to
presence of HCV RNA in their serum while in females
277 (74.06%) of 374 had active infection. Quantification
analysis further demonstrated that that population in 3rd
age group is at higher risk then the other two groups
where more than 70% of people have viral titre higher
than 800,000 IU/mL (Table 1). Distribution of active
HCV infection according to the sex and age is given in
Figures 1(a) and (b).
Liver function tests (ALT and AST) were performed
for the individuals with active infection. Among 441
confirmed patients of active HCV, 317 (71.88%) had
elevated ALT level and 297 (67.35%) had elevated AST
level (Table 2).
The correlation between viral load and ALT is more
significant than the correlation between viral load and
AST in females. Similarly, in case of males viral load
and ALT had more significant relation than viral load
and AST. Generally, males have strong relationship of
ALT and AST with viral load than females (Figures 2(a)
and (b)).
Table 1. HCV RNA viral load distribution in males and females.
Age <800,000 IU/mL >800,000 IU/mL
25 34.78% (8/23) 65.21% (15/23)
26 - 50 41.63% (92/221) 58.37% (129/221)
>50 27.27% (9/33) 72.73% (24/33)
25 36.36% (8/22) 63.64% (14/22)
26 - 50 31.58% (36/114) 68.42% (78/114) Males
>50 28.57% (8/28) 71.43% (20/28)
Table 2. Comparison of liver function tests with HCV viral load.
HCV RNA titer (IU/mL) No. of patients Serum ALT level
(mean ± SD) Serum AST level
(mean ± SD) Serum abnormal ALT
(%age) Serum abnormal ALT
104 89 47.30 ± 27.69 44.02 ± 23.44 37.08 (33/89) 35.95 (32/89)
105 96 74.22 ± 53.57 59.52 ± 36.41 76.04 (73/96) 65.62 (65.62)
106 161 87.34 ± 73.35 70.21 ± 47.91 74.53 (120/161) 68.32
107 79 113.40 ± 81.43 87.49 ± 47.57 88.61 (67/79) 75.95 (60/79)
108 15 144.80 ± 101.21 120.13 ± 67.01 93.33 (14/15) 86.66 (13/15)
109 1 128 128 ± 0 100 (1/1) 100 (1/1)
(a) (b)
Figure 1. Distribution of active HCV Infection in (a) Males; (b) Females.
A. Mushtaq et al. / Advances in Bioscience and Biotechnology 4 (2013) 866-871 869
(a) (b)
Figure 2. Relationship of viral load with ALT and AST (a) Males; (b) Females.
According to world health organization, there is about
3% world’s population infected with hepatitis C in the
world. Pakistan is one of the many developing countries
where Hepatitis C is budding as a major health issue [19,
20]. Among the major causes of this dilemma are the
absence of good preventive measures e.g. extensive reuse
of non-sterilized syringes and razors, fragile health struc-
ture, unscreened blood transfusion, general poverty and
poor education and high burden of immigrants have all
affected epidemiology of HCV in Pakistan. Punjab is
second largest province of Pakistan and has largest po-
pulation. Gujrat is a densely populated district of Punjab
located between two well-known rivers, the Jhelum and
the Chenab. It is encircled on the northwest by the Jhe-
lum River, on the northeast by Jammu and Kashmir and
southeast by Chenab River and on the west by district
Mandi Bahauddin [21].
Although Pakistan is at a high risk by HCV infection,
However, national level estimates of the prevalence of
and risk factors for hepatitis C are currently not available.
Many scientists tried to explore the rate of HCV infec-
tion and found that it varies among different population
groups and sexes like it is moderate in the general popu-
lation but very high in injecting drug users and multi-
transfused populations [22]. Similarly Ali et al. [23] stud-
ied moderate to high prevalence and risk factors of hepa-
titis C in different areas of Pakistan. A weighted average
of hepatitis C antibody was 3.0% (range 0.3% - 31.9%)
that is even higher in the high-risk subgroups. Same is
concluded by Umer et al. [24] who reviewed that the fre-
quency of hepatitis C infection in Pakistan is high (4.7%),
varying from 0.4% - 33.7%, indicating pockets of infec-
tions. In present study we found the viral loads in rela-
tion with liver aminotransferases in active HCV infected
individuals of both sexes constituting different age groups.
All the patients were anti HCV positive. The results
clearly indicated that all the anti HCV individuals had
not active HCV infection. Our results indicated that there
is a relation between liver aminotransferases and viral
load. One case study conducted in district Mansehra Pa-
kistan revealed there was 7% population having HCV
antibodies while only 3.5% population had active HCV
infection [25].
We studied a total 597 anti HCV patients of which 441
(73.87%) had active infection. The purpose of our study
was to check active HCV infection from anti HCV indi-
viduals by quantifying their viral load and to also verify
the relationship of elevated liver aminotransferases with
active HCV infection. We found that 150 of 171 anti
HCV individuals detected by ELISA had active infection
while 291 of 426 patients detected by ICT had active
infection. This clearly illustrated that ELISA is better
method for antibody detection than ICT because it gives
less false positive results. In case of males, we found that
112 (68.29) had high viral load and 52 (31.70%) had low
viral load in their serum, in females 168 (60.64%) had
high viral load and 109 (39.35%). As if viral load is less
than 800,000 IU/mL, it is considered as low viral load
while more than 800,000 IU/mL, is called high viral load
Abnormal level of serum transaminases may lead to
Copyright © 2013 SciRes. OPEN ACCESS
A. Mushtaq et al. / Advances in Bioscience and Biotechnology 4 (2013) 866-871
the prognosis of HCV infection. One study showed that
serum ALT levels, especially the AST levels were cou-
pled with liver damage [26]. Usually it is considered that
patients with chronic HCV having high HCV RNA titers
and abnormal ALT levels show active HCV reproduction
in liver and have higher risk of liver injury. Moreover,
ALT level is used for indication of histological liver
damage and a factor for starting therapy or to check anti-
viral therapy response in chronic hepatitis C [27]. We
found that 71.88% patients with active HCV infection
had elevated ALT level in their sera. As there are almost
25% of patients with active HCV infection having per-
sistently normal ALT values [18]. We found 67.35% indi-
viduals with active HCV infection having elevated AST
Patients with low viral load have 2.7 times more inci-
dence of SVR than individuals with high viral load [28].
It is also prominent that individuals with high viral load
show decreased response to antiviral therapy than indi-
viduals with low viral load and early prognosis reveals
low HCV RNA viral load. Patients with high viral load
should be treated for 24 weeks and those with low viral
load might be treated for 16 weeks if there HCV RNA
PCR is undetectable at 4th week of treatment [29,30].
Our results indicate that 36.5% (161/441) have low viral
load while 63.49% (280/441) have high viral load.
The prevalence of active HCV infection in anti HCV
individuals increases with the increase in age. Individu-
als up to 25 years had less number of anti HCV antibod-
ies and HCV RNA in their blood while with the increase
in age the chances of active HCV infection increase and
viral load also increases. Individuals with age >50 years
have highest percentage of HCV RNA titer in anti HCV
individuals. The viral load has a strong relationship with
liver enzymes (ALT and AST). With the increase in viral
load, the ALT and AST levels also increase in most cases
but in some cases of active HCV infection ALT and AST
levels remain normal.
We acknowledge support by the German Research Foundation and the
Open Access Publication Funds of the Göttingen University.
[1] Alter, H.J. and Seeff, L.B. (2000) Recovery, persistence.
and sequelae in hepatitis C virus infection: A perspective
on long-term outcome. Seminars in Liver Disease, 20, 17-
35. doi:10.1055/s-2000-9505
[2] Freeman, A.J., Dore, G.J., Law, M.G., Thorpe, M., Von
Overbeck, J., Lloyd, A.R., Marinos, G. and Kaldor, J.M.
(2001) Estimating progression to cirrhosisin chronic he-
patitis C virus infection. Hepatology, 34, 809-816.
[3] Smith, D.B., Mellor, J., Jarvis, L.M., et al. (1995) Varia-
tion of the hepatitis C virus 5’ non-coding region: Impli-
cations for secondary structure, virus detection and typing:
The International HCV Collaborative Study Group. Jour-
nal of General Virology, 76, 1749-1761.
[4] Global Burden of Disease (GBD) for Hepatitis (2004)
The Global Burden of Hepatitis C Working Group. Phar-
macology, 44, 20-29.
[5] El-Zanaty, F. and Way, A. (2009) Egypt demographic
and health survey 2008. Ministry of Health, El-Zanaty
and Associates, and Macro International, Cairo.
[6] Williams, I. (1999) Epidemiology of hepatitis C in the
United States. American Journal of Medicine, 107, 2S-9S.
[7] Waheed, Y., Shafi, T., Safi, S.Z. and Qadri, I. (2009)
Hepatitis C virus in Pakistan: A systematic review of pre-
valence, genotypes and risk factors. World Journal of
Gastroenterology, 15, 5647-5653.
[8] Caritter, R.L. and Emerson, S.S. (1997) Therapy of hepa-
titis—Meta analysis of interferon alpha 2b trials. Hepa-
tology, 26, 835-885.
[9] Fox, R.K. and Wright, T.L. (2003) Viral hepatitis: Cur-
rent diagnosis and treatment. Gastroenterology, 2, 446-
[10] Sherman, M., Shafran, S., Burak, K., Doucette, K., Wong,
W., Girgrah, N., Yoshida, E., Renner, E., Wong, P. and
Deschênes, M. (2007) Management of chronicnhepatitis
C: Consensus guidelines. Canadian Journal of Gastroen-
terology, 21, 25C-34C.
[11] Yoshioka, K., Kakumu, S. and Wakita, T. (1992) Detec-
tion of hepatitis C virus bypolymerase chain reaction and
response to interferon-a therapy: Relationship to geno-
types of hepatitis C virus. Hepatology, 16, 293-299.
[12] Ahmad, W., Ijaz, B., Javed, T.F., Jahan, S., Shahid, I.,
Khan, F.M. and Hassan, S. (2010) HCV genotype distri-
bution and possible transmission risks in Lahore, Pakistan.
World Journal of Gastroenterology, 16, 4321-4328.
[13] Lee, Y.S., Yoon, S.K., Chung, E.S., Bae, S.H., Choi, J.Y.,
Han, J.Y., Chung, K.W., Sun, H.S., Kim, B.S. and Kim,
B.K. (2001) The relationship of histologic activity to se-
rum ALT, HCV genotype and HCV RNA titers in chro-
nic hepatitis C. Journal of Korean Medical Science, 16,
[14] Dienstag, J.L. and Alter, H.J. (1986) Non-A, non-B hepa-
titis: Evolving epidemiologic and clinical perspective.
Seminars in Liver Disease, 6, 67-81.
[15] Tassopoulos, N.C. (1999) Treatment of patients with
chronic hepatitis C and normal ALT levels. Journal of
Hepatology, 31, 193-196.
[16] Carreno, V. (2006)Occult hepatitis C virus infection: A
Copyright © 2013 SciRes. OPEN ACCESS
A. Mushtaq et al. / Advances in Bioscience and Biotechnology 4 (2013) 866-871
Copyright © 2013 SciRes.
new form of hepatitis C. World Journal of Gastroen-
terology, 12, 6922-6925.
[17] Franciscus, A. (2012) Hepatitis C basics. Hepatitis C Sup-
port Project Vesion.
[18] Franciscus, A. and Highleyman, L. (2012) HCSP fact
sheet: HCV diagnostic tools (Version 4).
[19] Raza, S.A., Clifford, G.M. and Franceschi, S. (2007)
Worldwide variation in the relative importance of hepati-
tis B and hepatitis C viruses in hepato cellular carcinoma:
A systematic review. British Journal of Cancer, 96, 1127-
1134. doi:10.1038/sj.bjc.6603649
[20] Khan, S., Rai, M.A., Khan, A., Farooq, A., Kazmi, S.U.
and Ali, S.H. (2008) Prevalence of HCV and HIV infec-
tions in 2005-Earthquak areas of Pakistan. BMC Infec-
tious Diseases, 8, 147. doi:10.1186/1471-2334-8-147
[21] Anonymous (1999) Global surveillance and control of
hepatitis C. Journal of Viral Hepatitis, 6, 35-47.
[22] Waheed, Y., Shafi, T., Safi, S.Z. and Qadri, I. (2009)
Hepatitis C virus in Pakistan: A systematic review of
prevalence, genotypes and risk factors. World Journal of
Gastroenterology, 15, 5647-5653.
[23] Ali, S.A., Donahue, R.M.J., Qureshi, H. and Vermund,
S.H. (2010) Hepatitis B and hepatitis C in Pakistan preva-
lence and risk factors. International Journal of Infectious
Diseases, 13, 9-19. doi:10.1016/j.ijid.2008.06.019
[24] Umar, M., Bushra, H., Ahmad, M., Khurram, M., Usman,
S., Arif, M., Adam, T., Minhas, Z., Arif, A., Naeem, A.,
Ejaz, K., Butt, Z. and Bilal, M. (2010) Hepatitis C in
Pakistan: A review of available data. Hepatitis Monthly,
10, 205-214.
[25] Ali, A., Ahmad, H., Ali, I., Khan, S., Zaidi, G. and Idrees,
M. (2010) Prevalence of active hepatitis c virus infection
in district mansehra Pakistan. Virology Journal, 7, 334.
[26] Zechini, B., Pasquazzi, C. and Aceti, A. (2004) Correla-
tion of serum aminotransferase with HCV RNA levels
and histological findings in patients with chronic hepatitis
C: The role of serum aspartate transaminase in the eval-
uation of disease progression. European Journal of Gas-
troenterology & Hepatology, 16, 891-896.
[27] Lee, Y.S., Yoon, S.K., Chung, E.S., et al. (2001) The
Relationship of histologic activity to serum ALT, HCV
genotype and HCV RNA titers in chronic hepatitis C.
Journal of Korean Medical Science, 16, 585-591.
[28] Jensen, M.D., Morgan, T.R., Mercellin, P., Pockros, T.J.,
Reddy, K.R., Hadziyannis, S.J., Ferenci, P., et al. (2006)
Early identification of HCV genotype 1 patients respond-
ing to 24 weeks peginterferon-2a (40 kd)/ribavirin ther-
apy. Hepatology, 43, 954-960. doi:10.1002/hep.21159
[29] Bell, H., Hellum, K. and Harthug, S. (1997) Genotype,
viral load and age as independent predictors of treatment
outcome of interferon-a 2a treatment in patients with
chronic hepatitis C. Scandinavian Journal of Infectious
Diseases, 29, 17-22. doi:10.3109/00365549709008658
[30] Jimenez-Mendez, R., Uribe-Salas, F., López-Guillen, P.,
Cisneros-Garza, L. and Castañeda-Hernandez, G. (2010)
Distribution of HCV genotypes and HCV RNA viral load
in different geographical regions of Mexico. Annals of
Hepatology, 9, 33-39.
ALT: Alanine aminotransferase
AST: Aspartate aminotransferase
Ct: Cycle threshhold
HCV: Hepatitis C virus
IC: Internal control
ICT: Immunochromatographic test
M-MLV RT: Moloney murine leukemia virus reverse
PEG-IFN: Pegelated interferon