Hepatitis C Virus and Interferon-Free Antiviral Therapeutics Revolution: Implications for Pakistan

Abstract

Hepatitis C virus (HCV) is a major health concern worldwide as a leading cause of liver-related mortalities and morbidities. Pakistan ranks second among countries with endemic HCV infection; ∼11 million cases are reported so far. HCV burden is continuously rising in Pakistan, mainly because of unsafe blood transfusions, surgical procedures, dental procedures, untrained clinicians, reuse of syringes, barbers, and ear/nose piercing tools. Lack of awareness about HCV transmission routes among the general and high-risk population is a major hurdle in disease management. HCV prevalence in the general population and healthy blood donors ranges from 3.13% to 23.83% and from 1.05% to 20.8%, respectively; whereas in the high-risk groups, HCV prevalence is up to 66%. Genotype 3 is most prevalent in Pakistan followed by genotypes 1 and 2 along with an alarming number of untypable viral genotypes in the local community. Mainly interferon-based antiviral regimens are used in Pakistan and are quite effective, because the major prevalent genotype (genotype 3) showed the best sustained virological response (SVR) with it. But a large number of individuals did not show SVR either because of infection with nonresponder genotypes or because of side effects. Due to these reasons, there was a need for interferon-free direct acting antivirals (DAAs). Recently, Sovaldi (Sofosbuvir: NS5B inhibitor) is approved on a heavy discounted rate for Pakistan; it is currently in effective use and showed good SVR. Sovaldi plus ribavirin is used alone or along with interferon to treat different viral genotypes. Sovaldi will be the future treatment regime for Pakistan, because genotype 2 and genotype 3 infected individuals achieve the best SVR with it. For the treatment of other prevalent viral genotypes, approval of some other DAAs such as Ledipasvir on discounted price is required for better disease management.

Introduction

Hepatitis C virus (HCV) is a major public health concern worldwide. It is a major causative agent for chronic hepatitis and hepatocellular carcinoma. It is a small hepatotropic virus infecting 200 million people globally (4). The World Health Organization (WHO) report states that 3% of the world population is infected with HCV, and every year, 3–4 million new cases of HCV infection occur globally (6). In Pakistan, around 11 million people are suffering from HCV, with a very high mortality and morbidity rate (59). HCV is a member of Flaviviridae with plus stranded RNA genome. Viral genome consists of 9,600 bp and is divided into 10 different genes (16). The viral RNA is translated into a single polyprotein, which is then cleaved into individual protein by the action of viral and host peptides (5). The polyprotein is co- and post-translationally processed by viral and cellular proteases to produce four structural (Core, E1, E2 and P7) and six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (9).

HCV seroprevalence in Pakistan

Pakistan ranked second in the world in terms of HCV burden, with about 11 million infections (59). Due to poor socioeconomic situation and lack of awareness among the general population of the country, viral burden is increasing. In Pakistan, the major HCV infection routes include unsafe dental and general surgery procedures, re-use of syringes, unsafe blood transfusions, unsafe barbers' tools/beauty salon equipment/ear-nose piercing guns, and so on (8). Above all, unawareness among the infected and general population about the transmission routes is further escalating the viral burden in the society. Being a cirrhotic state, the information about the prevalence of HCV in the general population is very less and is confined to urban areas only (3). A comprehensive review of previous data (2010–2016) about HCV prevalence in the general population, healthy blood donors, and high-risk groups is carried out (Table 1) (10 –15,17 –27,29 –35,37,39 –45,47,49,51 –54,57–58,60 –68,70), which showed that the prevalence of HCV in the general population ranged from 3.13% to 23.83%. In healthy blood donors, the prevalence of HCV is from 1.05% to 20.8%. Analysis of published reports on HCV prevalence in high-risk groups indicated very high seroprevalence of the virus. The investigated high-risk groups include hepatocellular carcinoma, pregnant women, multi-transfused population, prisoners, homosexual community, urological patients, surgery patients, type II diabetes patients, intravenous drug users, healthcare workers, and end-stage renal disease. The prevalence range of HCV in these high-risk groups ranged from 2.9% to 66%. The details of HCV seroprevalence in different risk groups can also be found from recent review articles (69).

Table 1.

Comprehensive Review of Hepatitis C Virus Prevalence in General Population, Healthy Blood Donors, and High-Risk Population of Pakistan (2010–2016)

	General population		Healthy blood donors		High-risk population
Year	Place/city	Sero-prevalence (%)	Place/city	Sero-prevalence (%)	Place/city	Study group	Sero-prevalence (%)
2010	Swat (58)	8.81	Multan (37)	4.90	Karachi (11)	Hepatocellular carcinoma	50
	Mansehra (13)	7	Peshawar (65)	1.57	Multan (68)	Pregnant women	7
	Thatta/Nausheroferoz	25.1			Lahore (63)	Multi-transfused population	1.5
	Karachi	23.83			Sindh (29)	Prisoners	12.8
	Karachi	3.17			Karachi (29)	Prisoners	15.2
	Multiple cities				Sindh (45)	Homosexual community	23.5
	Multan	9.6			Lahore (69)	Urological patients	13.4
	Lahore	9.4			Sukkur (49)	Surgery	13.8
	Faisalabad	8.8			Multan (37)	Type II diabetes	30.2
	Gujranwala	7.3
	Gujrat	6.8
	Sargodha	6.7
	Rawalpindi	6.7
	Sialkot	6.2
	Bahawalpur	5.0
	Islamabad	24.6
	Mansehra	10.34
	National Survey (32)	4.87
2011	Gujranwala (34)	2.32	KPK/FATA (42)	1.89	Karachi (21)	Pregnant women	5.79
	Karachi (21)	9.75	KPK/FAT (62)	2.60	KPK (14)	Multi-transfused population	15
			Karachi (24)	1.90	KPK (14)	Intravenous drug users	14.28
					KPK (14)	Dialysis patients	28
					KPK (14)	Surgery	8
					KPK (14)	Dental surgery	14.28
					Karachi (24)	Multi-transfused population	51.4
					KPK (60)	Intravenous drug users	31.5
					Peshawar (44)	Healthcare workers	4.1
					Peshawar (43)	Dialysis patients	29.2
2012	Kech (10)	5.5	Karachi (17)	2.00	Karachi (17)	Multi-transfused population	13
	Punjab (30)	3.13	Peshawar (20)	2.46	Karachi (53)	Cataract patients	11.4
	Rawalpindi (64)	17.2	Sargodha (22)	12.00	Rawalpindi (57)	Dermatological disorders	9.01
	Islamabad (64)	4
2013	Lahore (18)	4.9	Karachi (35)	2.61	Not specified (25)	Hepatocellular carcinoma	48.5
			Quetta (40)	20.8	Hyderabad (23)	Pregnant women	4.7
			Lahore (12)	15.00	Kharian (31)	Surgery	6.4
2014	Mardan (15)	11.7	Karachi (51)	1.65	Hyderabad (52)	Hepatocellular carcinoma	66
	Peshawar (33)	12.93			Islamabad (41)	Suspected for viral hepatitis	24.8
					Rawalpindi (26)	Multi-transfused population	49.5
2015	Islamabad (19)	33	Rawalpindi (54)	1.84	Islamabad (61)	Thalassemic population	55.73
					Karachi (67)	Cataract surgery	6.17
					Abbottabad (27)	Dental surgery	4.1
					Karachi (66)	End-stage renal disease	2.9
					Karachi (47)	Pregnant women	13.3
2016			Mardan (39)	1.05

HCV genotype seroprevalence in Pakistan

HCV polymerase (NS5B) is error prone and due to the error-prone nature of viral polymerase, HCV has many genotypes and subtypes. Viral genotyping is done from 5′UTR, core and NS5B region of the genome (7). Determination of the viral genotype is important for the proper management of the disease, as the antiviral therapy is genotype specific. The interferon-based and direct acting antivirals (DAAs) response is genotype dependent, and different viral genotypes showed a different sustained virological response (SVR). There are 26 reports from Pakistan regarding the HCV clade seroprevalence in Pakistan; the analysis of the available data showed that genotype 3 (GT-3) (63%) is the most common genotype in the country followed by untypable genotype (14%), GT-1 (9%), and GT-2 (8%), whereas the prevalence of GT-4 (2.5%), mixed genotype (2.25%), GT-5 (0.75%), and GT-6 (0.5%) is negligible (Fig. 1).

FIG. 1.

A comprehensive review of HCV prevalent genotypes in Pakistan. HCV, hepatitis C virus.

HCV Treatment Guidelines for Pakistan

The main purpose of chronic HCV treatment is to achieve high SVR rate and to avoid the development of liver-related complications. Previously, interferon plus ribavirin-based antiviral therapy played an important role in HCV management across the world and showed about 50–80% SVR in patients infected with HCV GT-1/GT-4 or HCV GT-2/GT-3, respectively (38). The response can vary according to the interleukin-28B (IL-28B) genotype. With the advancements of DAAs development, a combination of interferon/ribavirin and DAAs resulted in about 90% SVR in IL-28B favorable genetic makeup and also shortened the therapy duration (72). But interferon-based antivirals have many side effects and have resulted in discontinuation of therapy in a large number of individuals. Recently, the Asian Pacific Association for the Study of the Liver (APASL) published the guidelines for HCV treatments with interferon-free DAAs (55). As the most prevalent HCV genotypes are 3, 1, and 2, respectively, the prevalence of other genotypes is negligible. Keeping in mind the current scenario of Pakistan, a brief review of available literature and treatment guidelines is summarized (Table 2) (1,2,28,36,46,48,50,56,71) to help healthcare workers of the local community for the proper management of the disease. As the ratio between healthcare providers and the total population is quite unbalanced in Pakistan, they are overburdened with patient load. This summary of a treatment strategy for the local patient's pool will be of considerable help for the health department of Pakistan. The APASL guidelines defined the strategy for GT-1/GT-2/GT-3, and Sovaldi (Sofosobuvir, NS5B inhibitor) will be the future treatment drug for Pakistan. Sovaldi showed promising results against GT-3/GT-2 and also is a part of the interferon-free regimen against GT-1. However, in the near future, interferon-based regimens will also be in use because of the unavailability of other DAAs (except Sovaldi), no defined DAAs strategy against mixed infections, limited access of Sovaldi in rural areas, and no proper education of healthcare providers regarding the use of DAAs. There are also a large number of untypable viral clades circulating in the local population of Pakistan and currently, there are no defined treatment options for these patients.

Table 2.

Interferon-Free Treatment Strategy for Patients Infected with Most Prevalent Hepatitis C Virus Genotypes in Pakistan

	Treatment-naïve		Treatment-experienced: peg-interferon plus ribavirin
Genotype	Noncirrhosis	Cirrhosis	Noncirrhosis	Cirrhosis
3 (36,48,71)	Sofos (400 mg daily) +Rib for 24 weeks	Sofos (400 mg daily) +Rib for 24 weeks	Sofos (400 mg daily) +Rib for 24 weeks	Sofos (400 mg daily) +Rib for 24 weeks
2 (28,36,48,56,71)	Sofos (400 mg daily) +Rib for 12 weeks	Sofos (400 mg daily) +Rib for 12 weeks	Sofos (400 mg daily) +Rib for 12 weeks	Sofos (400 mg daily) +Rib for 12 weeks
1 (1,2,46,50)	Sofo (400 mg daily)/Ledi^a (90 mg daily) for 12 weeks	Sofo (400 mg daily)/Ledi^a (90 mg daily) for 12 weeks	Sofo (400 mg daily)/Ledi^a (90 mg daily) for 12 weeks	Sofo (400 mg daily)/Ledi^a (90 mg daily) for 12 weeks

Currently not available in Pakistan.

Ledi, Ledipasvir; Rib, Ribavirin; Sofo, Sofosbuvir.

Summary

• Pakistan is endemic for hepatitis C virus (HCV) infection, and there are ∼11 million infected individuals.

• In this study, the review of epidemiological data (2010–2016) of HCV from the general population, blood donors, and high-risk groups is summarized.

• The HCV epidemiological data of HCV genotyping from 2010 to 2016 are analyzed and comprehensively presented, which showed that genotype 3 is the major prevalent genotype followed by genotypes 1 and 2.

• The review showed that there are a significant number of diagnostically untypable HCV variants circulating in Pakistan.

• Currently, Interferon-based antiviral therapy is the main option is Pakistan, because the prevalent viral genotype is sensitive to interferon therapy and showed a good sustained virological response (SVR). But a large number of patients did not show SVR either because of side effects or because of infection with nonresponder HCV genotype.

• Recently, the approval of interferon-free antiviral; Sofosbuvir (Sovaldi: viral polymerase inhibitor: NS5B inhibitor) revolutionized the HCV management in Pakistan. This therapy is suitable for most individuals, without any side effects.

• The interferon-free direct acting antiviral (DAA)–based treatment strategy is based on existing data according to the current scenario of Pakistan.

• Finally, the review also highlights the urgent need for approval of other DAA such as Ledipasvir for better management of HCV infection in Pakistan.

Conclusion and Future Perspective

Pakistan is a developing country with a low socioeconomic status. The government's health budget is very low in comparison with population needs. In such circumstances with almost no existence of a proper health insurance system across the country, it is very difficult to treat 11 million individuals. Currently, patients are being treated with interferon based antiviral regimen, which showed quite good results, as the major prevalent HCV genotype is (GT-3) interferon sensitive. Many drawbacks of the treatment are also reported, such as side effects of therapy, relapse cases, emergence of treatment resistance, and increasing number of cirrhosis cases. Moreover, a large proportion of infected individuals were not able to be treated by this regimen. Recently, Sovaldi is approved for treatment through the Gilead global access program at the heavy discounted price of 300 USD per month. Sovaldi is used either in combination with ribavirin for 24 weeks or with Ribavirin and Peg-Interferon for 12 weeks. Initial results from Pakistan suggest that around 85% patients infected with HCV GT-3 showed SVR (55). On the basis of the earlier discussion, it can be concluded that approval of Sovaldi in Pakistan will be of great help to treat GT-2/GT-3, which are most prevalent in Pakistan. Interferon-based antiviral regimens will also be in use for the future, as they showed good SVR against favorable (GT-3) genotype. To achieve higher SVR against GT-1 and avoid side effects due to interferon-based regimens, there is an urgent need for approval of other DAAs such as Ledipasvir, on discounted price. The current APASL guidelines did not cover the mixed genotype and untypable genotype infection, so future research is needed to treat these genotypes as well as to combat the HCV infection in Pakistan.

Footnotes

Acknowledgments

The author would like to thank Mahvish Kabir for her help in English language editing and proofreading the article. There is no role of any funding agency in this study.

Author Disclosure Statement

No competing financial interests exist.

References

Afdhal

, Reddy

, Nelson

, et al. Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection. N Engl J Med, 2014; 370:1483–1493.

Afdhal

, Zeuzem

, Kwo

, et al. Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection. N Engl J Med, 2014; 370:1889–1898.

Afzal

. Are efforts up to the mark? A cirrhotic state and knowledge about HCV prevalence in general population of Pakistan. Asian Pac J Trop Med, 2016; 9:616–618.

Afzal

, Ahmed

, Zaidi

. Comparison of HCV prevalence in Pakistan and Iran; an insight into future. Hepat Mon, 2014; 14:e11466.

Afzal

, Alsaleh

, Farhat

, et al. Regulation of core expression during the hepatitis C virus life cycle. J Gen Virol, 2015; 96(Pt 2):311–321.

Afzal

, Anjum

, Zaidi

. Effect of functional interleukin-10 polymorphism on pegylated interferon-α plus ribavirin therapy response in chronic hepatitis C virus patients infected with 3a genotype in Pakistani population. Hepat Mon, 2013; 13:e10274.

Afzal

, Khan

, Ammar

, et al. Diagnostically untypable hepatitis C virus variants: it is time to resolve the problem. World J Gastroenterol, 2014; 20:17690–17692.

Afzal

, Shah

, Ahmed

. Recent HCV genotype changing pattern in the Khyber Pakhtunkhwa province of Pakistan; is it pointing out a forthcoming problem?. Braz J Infect Dis, 2016; 20:312–313.

Afzal

, Zaidi

, Dubuisson

, et al. Hepatitis C virus capsid protein and intracellular lipids interplay and its association with hepatic steatosis. Hepat Mon, 2014; 14:e17812.

10.

Ahmed

, Irving

, Anwar

, et al. Prevalence and risk factors for hepatitis C virus infection in Kech District, Balochistan, Pakistan: most infections remain unexplained. A cross-sectional study. Epidemiol Infect, 2012; 140:716–723.

11.

Ahmed

, Qureshi

, Arif

, et al. Changing trend of viral hepatitis “A twenty one year report from Pakistan Medical Research Council Research Centre, Jinnah Postgraduate Medical Centre, Karachi.”. J Pak Med Assoc, 2010; 60:86–89.

12.

Akhtar

, Khan

, Ijaz

, et al. Seroprevalence and determinants of hepatitis-C Virus infection in blood donors of Lahore, Pakistan. Pak J Zool, 2013; 45:1–7.

13.

Ali

, Ahmad

, Ali

, et al. Prevalence of active hepatitis C virus infection in district Mansehra Pakistan. Virol J, 2010; 7:334.

14.

Ali

, Siddique

, Rehman

, et al. Prevalence of HCV among the high risk groups in Khyber Pakhtunkhwa. Virol J, 2011; 8:296.

15.

Ali

, Ahmad

, Khan

, et al. Genotyping of HCV RNA reveals that 3a is the most prevalent genotype in Mardan, Pakistan. Adv Virol, 2014; 2014:606201.

16.

Anjum

, Wahid

, Afzal

, et al. Additional glycosylation within a specific hypervariable region of subtype 3a of hepatitis C virus protects against virus neutralization. J Infect Dis, 2013; 208:1888–1897.

17.

Ansari

, Shamsi

, Khan

, et al. Seropositivity of hepatitis C, hepatitis B and HIV in chronically transfused beta-thalassaemia major patients. J Coll Physicians Surg Pak, 2012; 22:610–611.

18.

Anwar

, Rahman

, Hassan

, et al. Prevalence of active hepatitis C virus infections among general public of Lahore, Pakistan. Virol J, 2013; 10:351.

19.

Asad

, Ahmed

, Zafar

, et al. Frequency and determinants of hepatitis B and C virus in general population of Farash Town, Islamabad. Pak J Med Sci, 2015; 31:1394–1398.

20.

Attaullah

, Khan

. Trend of transfusion transmitted infections frequency in blood donors: provide a road map for its prevention and control. J Transl Med, 2012; 10:20.

21.

Aziz

, Hossain

, Karim

, et al. Vertical transmission of hepatitis C virus in low to middle socio-economic pregnant population of Karachi. Hepatol Int, 2011; 5:677–680.

22.

Bhutta

, Tahir

, Ayub

, et al. Seroprevalence of anti-HCV in non-professional blood donors. Pak J Med Health Sci, 2012; 6:175–178.

23.

Bibi

, Dars

, Ashfaq

, et al. Seroprevalence and risk factors for hepatitis C virus (HCV) infection in pregnant women attending public sector tertiary care hospital in Hyderabad Sindh. Pak J Med Sci, 2013; 29:505–508.

24.

Borhany

, Shamsi

, Boota

, et al. Transfusion transmitted infections in patients with hemophilia of Karachi, Pakistan. Clin Appl Thromb Hemost, 2011; 17:651–655.

25.

Butt

, Hamid

, Wadalawala

, et al. Hepatocellular carcinoma in Native South Asian Pakistani population; trends, clinico-pathological characteristics & differences in viral marker negative & viral-hepatocellular carcinoma. BMC Res Notes, 2013; 6:137.

26.

Din

, Malik

, Ali

, et al. Prevalence of hepatitis C virus infection among thalassemia patients: a perspective from a multi-ethnic population of Pakistan. Asian Pac J Trop Med, 2014; 7S1:S127–S133.

27.

Fayyaz

, Ghous

, Ullah

, et al. Frequency of Hepatitis B and C in Patients seeking treatment at the dental section of a tertiary care hospital. J Ayub Med Coll Abbottabad, 2015; 27:395–397.

28.

Gane

, Stedman

, Hyland

, et al. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N Engl J Med, 2013; 368:34–44.

29.

Gorar

, Zulfikar

. Seropositivity of hepatitis C in prison inmates of Pakistan—a cross sectional study in prisons of Sindh. J Pak Med Assoc, 2010; 60:476–479.

30.

Hafeez-ud-din , Siddiqui

, Lahrasab

, et al. Prevalence of hepatitis B and C in healthy adult males of paramilitary personnel in Punjab. J Ayub Med Coll Abbottabad, 2012; 24:138–140.

31.

Hameed

. Frequency of hepatitis B & C in elective eye surgery. RMJ, 2013; 38:15–17.

32.

Hyder

, Ijaz

, Arshad

, et al. Age-specific frequency of screen-detected hepatitis C virus seropositivity in men from the Punjab province of Pakistan. J Med Screen, 2010; 17:214–216.

33.

Ilyas

, Ahmad

. Chemiluminescent microparticle immunoassay based detection and prevalence of HCV infection in district Peshawar Pakistan. Virol J, 2014; 11:127.

34.

Ilyas

, Iftikhar

, Rasheed

. Prevalence of hepatitis B and hepatitis C in populations of college students in Gujranwala. Biologia (Pakistan), 2011; 57:89–95.

35.

Irfan

, Uddin

, Zaheer

, et al. Trends in transfusion transmitted infections among replacement blood donors in Karachi, Pakistan. Turk J Haematol, 2013; 30:163–167.

36.

Jacobson

, Gordon

, Kowdley

, et al. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N Engl J Med, 2013; 368:1867–1877.

37.

Jadoon

, Shahzad

, Yaqoob

, et al. Seroprevalence of hepatitis C in type 2 diabetes: evidence for a positive association. Virol J, 2010; 7:304.

38.

Kanda

, Imazeki

, Yokosuka

. New antiviral therapies for chronic hepatitis C. Hepatol Int, 2010; 4:548–561.

39.

Karim

, Nasar

, Alam

, et al. Incidence of active HCV infection amongst blood donors of Mardan District, Pakistan. Asian Pac J Cancer Prev, 2016; 17:235–238.

40.

Khan

, Tareen

, Ikram

, et al. Prevalence of HCV among the young male blood donors of Quetta region of Balochistan, Pakistan. Virol J, 2013; 10:83.

41.

Khan

, Shafiq

, Mushtaq

, et al. Seropositivity and coinfection of hepatitis B and C among patients seeking hospital care in Islamabad, Pakistan. Biomed Res Int, 2014; 2014:516859.

42.

Khan

, Ali

, Ahmad

, et al. Prevalence of active HCV infection among the blood donors of Khyber Pakhtunkwa and FATA region of Pakistan and evaluation of the screening tests for anti-HCV. Virol J, 2011; 8:154.

43.

Khan

, Attaullah

, Ali

, et al. Rising burden of hepatitis C virus in hemodialysis patients. Virol J, 2011; 8:438.

44.

Khan

, Attaullah

, Ayaz

, et al. Molecular epidemiology of HCV among health care workers of Khyber Pakhtunkhwa. Virol J, 2011; 8:105.

45.

Khanani

, Somani

, Khan

, et al. Prevalence of single, double, and triple infections of HIV, HCV and HBV among the MSM community in Pakistan. J Infect, 2010; 61:507–509.

46.

Kowdley

, Gordon

, Reddy

, et al. Ledipasvir and sofosbuvir for 8 or 12 weeks for chronic HCV without cirrhosis. N Engl J Med, 2014; 370:1879–1888.

47.

Kumari

, Seetlani

, Akhter

. The emergent concern of seropositive status of hepatitis-B virus and hepatitis-C virus in the pregnant females attending a tertiary care hospital. J Ayub Med Coll Abbottabad, 2015; 27:155–157.

48.

Lawitz

, Mangia

, Wyles

, et al. Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med, 2013; 368:1878–1887.

49.

Memon

, Shaikh

, Soomro

, et al. Frequency of hepatitis B and C in patients undergoing elective surgery. J Ayub Med Coll Abbottabad, 2010; 22:167–170.

50.

Mizokami

, Yokosuka

, Takehara

, et al. Ledipasvir and sofosbuvir fixed-dose combination with and without ribavirin for 12 weeks in treatment-naive and previously treated Japanese patients with genotype 1 hepatitis C: an open-label, randomised, phase 3 trial. Lancet Infect Dis, 2015; 15:645–653.

51.

Moiz

, Moatter

, Shaikh

, et al. Estimating window period blood donations for human immunodeficiency virus type 1, hepatitis C virus, and hepatitis B virus by nucleic acid amplification testing in Southern Pakistan. Transfusion, 2014; 54:1652–1659.

52.

Munaf

, Memon

, Kumar

, et al. Comparison of viral hepatitis-associated hepatocellular carcinoma due to HBV and HCV—cohort from liver clinics in Pakistan. Asian Pac J Cancer Prev, 2014; 15:7563–7567.

53.

Naeem

, Siddiqui

, Kazi

, et al. Prevalence of hepatitis ‘B’ and hepatitis ‘C’ among preoperative cataract patients in Karachi. BMC Res Notes, 2012; 5:492.

54.

Niazi

, Bhatti

, Salamat

, et al. Impact of nucleic acid amplification test on screening of blood donors in Northern Pakistan. Transfusion, 2015; 55:1803–1811.

55.

Omata

, Kanda

, Wei

, et al. APASL consensus statements and recommendation on treatment of hepatitis C. Hepatol Int, 2016; 10:702–726.

56.

Omata

, Nishiguchi

, Ueno

, et al. Sofosbuvir plus ribavirin in Japanese patients with chronic genotype 2 HCV infection: an open-label, phase 3 trial. J Viral Hepatol, 2014; 21:762–768.

57.

Rahman

, Rizvi

, Sheikh

. Frequency of HCV infection in different dermatological disorders. J Ayub Med Coll Abbottabad, 2012; 24:58–61.

58.

Rauf

, Nadeem

, Ali

, et al. Prevalence of hepatitis B and C in internally displaced persons of war against terrorism in Swat, Pakistan. Eur J Public Health, 2010; 21:638–642.

59.

Raza

, Ahmad

, Afzal

. HCV. Interferon therapy response, direct acting antiviral therapy revolution and Pakistan: future perspectives. Asian Pac J Cancer Prev, 2015; 16:5583–5584.

60.

Rehman

, Ullah

, Ali

, et al. Active hepatitis C infection and HCV genotypes prevalent among the IDUs of Khyber Pakhtunkhwa. Virol J, 2011; 8:327.

61.

Saeed

, Waheed

, Ashraf

, et al. Estimation of hepatitis B virus, hepatitis C virus, and different clinical parameters in the thalassemic population of capital twin cities of Pakistan. Virology (Auckl), 2015; 6:11–16.

62.

Safi

, Afzal

, Waheed

, et al. Seroprevalence of hepatitis C and human immunodeficiency viruses in blood donors of northwestern Pakistan. Asian Biomed, 2011; 5:389–392.

63.

Sajid

, Khalid

, Mazari

, et al. Clinical audit of inherited bleeding disorders in a developing country. Indian J Pathol Microbiol, 2010; 53:50–53.

64.

Satti

, Mustafa

, Khan

, et al. Prevalence of hepatitis C virus in urban Ghettos of twin cities. Pak J Zool, 2012; 44:937–943.

65.

Shah

, Khattak

, Ali

, et al. Seropositivity for hepatitis B and C in voluntary blood donors. J Ayub Med Coll Abbottabad, 2010; 22:149–151.

66.

Shah

, Khan

, Alam

, et al. End stage renal disease: seroprevalence of hepatitis B and C along with associated aetiology and risk factors in children. J Trop Med, 2015; 2015:936094.

67.

Tahir

, Cheema

, Tareen

. Frequency of hepatitis-B and C in patients undergoing cataract surgery in a tertiary care Centre. Pak J Med Sci, 2015; 31:895–898.

68.

Taseer

, Ishaq

, Hussain

, et al. Frequency of anti-HCV, HBsAg and related risk factors in pregnant women at Nishtar Hospital, Multan. J Ayub Med Coll Abbottabad, 2010; 22:13–16.

69.

Umer

, Iqbal

. Hepatitis C virus prevalence and genotype distribution in Pakistan: comprehensive review of recent data. World J Gastroenterol, 2016; 22:1684–1700.

70.

Waheed

, Zafar ul

, Zaeem

, et al. Operating in a yellow nation; the frequency of hepatitis B and hepatitis C positive at a tertiary care teaching hospital. J Pak Med Assoc, 2010; 60:1058–1060.

71.

Zeuzem

, Dusheiko

, Salupere

, et al. Sofosbuvir and ribavirin in HCV genotypes 2 and 3. N Engl J Med, 2014; 370:1993–2001.

72.

Zhang

. Direct anti-HCV agents. Acta Pharm Sin B, 2016; 6:26–31.