Best Practice/Intervention: Aziz H. et al. (2015) Optimum predictors of therapeutic outcome in HCV patients in Pakistan. J Med Virol, 88: 100-108.

Date of Review: March 1, 2016

Reviewer(s): Christine Hu

Part A

Category: Basic Science Clinical Science Public Health/Epidemiology

Social Science Programmatic Review

Best Practice/Intervention: Focus: Hepatitis C Hepatitis C/HIV Other:

Level: Group Individual Other:

Target Population: people with HCV infection

Setting: Health care setting/Clinic Home Other:

Country of Origin: Pakistan

Language: English French Other:

Part B

YES NO N/A COMMENTS

Is the best practice/intervention a meta-analysis or primary research?

Primary research: to analyze the factors that contributes to the antiviral treatment (interferon-alpha and ribavirin) response in patients with different genotypes of hepatitis C infection.

Has the data/information been used for decision-making (e.g. program funding developments, policies, treatment guidelines, defining research priorities and funding)?

Do the methodology/results described allow the reviewer(s) to assess the generalizability of the results?

Criteria Grid Hepatitis C Research Studies, Tools, and Surveillance Systems

Are the best practices/methodology/results described applicable in developed countries?

Results are applicable to other countries as interferon alpha and ribavirin are still widely used for HCV treatment around the world.

YES NO N/A COMMENTS

Are the best practices/methodology/results described applicable in developing countries?

The research study/tool/data dictionary is easily accessed/available electronically

Full open access to the article can be found at http://onlinelibrary.wiley.com/doi/10.1002/jmv.24305/full

Is there evidence of cost effective analysis with regard to interventions, diagnosis, treatment, or surveillance methodologies? If so, what does the evidence say? Please go to Comments section

No cost effective analysis was conducted..

Are there increased costs (infrastructure, manpower, skills/training, analysis of data) to using the research study/tool/data dictionary?

How is the research study/tool funded? Please got to Comments section

No funding stated

Is the best practice/intervention dependent on external funds?

Other relevant criteria:

- Study showed 60.2% SVR to antiviral therapy - Older female patients >60 years have not shown

any response - Female younger patients <40 years have SVR

77.2% compared to male patients with 67.5% SVR - SVR rates for genotype 3 patients are significantly

higher than those other genotype patient groups - Younger age, low viral load, achieving early

inhibition of viral replication and non fatty liver are positively independently associated with SVR.

WITHIN THE SURVEILLANCE SYSTEM FOR REVIEW

Are these data regularly collected?

Study included patients who attended the Nuclear Medicine Oncology and Radiotherapy institute from 2008-2013.

Are these data regularly collected at and/or below a national level?

Are these data collected manually or electronically?

Manually

RESEARCH REPORTS

Has this research been published in a juried journal?

Journal of Medical Virology

Does the evidence utilize the existing data/surveillance information or has it generated new data and/or information?

New data/information

Journal of Medical Virology 88:100–108 (2016)

Optimum Predictors of Therapeutic Outcome inHCV Patients in Pakistan

Hafsa Aziz,* Abida Raza, and Javaid Irfan

Nuclear Medicine Oncology and Radiotherapy Institute, Islamabad, Pakistan

Hepatitis C virus (HCV) constitutes a majorpublic health issue in Pakistan. Interferon aand ribavirin is used widely in routine practicein HCV infected patients in Pakistan.Treatmentprediction is an important tool in therapymanagement. The present study aims to eval-uate trends of predictive variables of treatmentoutcome in patients with different genotypes.The analysis comprised of 921 patientsinfected with different HCV genotypes. All thepatients received IFN a-2b combined withribavirin for 24 weeks. Overall, 60.2% patientsachieved Sustained virologic response (SVR).In females sustained virologic response (SVR)was higher in age group <40 years (77.2%)than �40–50years (60%) but in male SVR wasalmost equal in both age groups. We alsofound higher SVR with low pretreatment viralload (72.4%, P< 0.0001). Sustained VirologicResponse in genotype 3a was 63.1%, 3b was55%, 1a was 36.3% and 1b was 35% 3a þ3bwas 55.0% and 1aþ3a was 42.9%. According tomultivariable logistic regression analysis age< 40 years (2.0; 95%CI, 1.49–2.84; P¼ 0.0001),low pretreatment RNA level<800,000 IU/ml(4.0; 95%CI, 2.64–6.17; P¼ 0.0001), early viro-logic response at week 12 (12.3; 95%CI,8.18–18.58; P< 0.0001) and non-fatty liver (2.5;95%CI, 3.6–6.2; P¼ 0.005) showed significancefor SVR. Nucleotide substitution in 5/UTR be-fore treatment failed to show any characteristicpattern that has correlation with sustainedresponse. Subtype 3a showed 95% presenceamong patients with age <40 years while olderpatients showed 79.9%. J. Med. Virol. 88:100–108, 2016. # 2015 Wiley Periodicals, Inc.

KEY WORDS: HCV genotypes; treatmentresponse

INTRODUCTION

Hepatitis C virus constitutes a major public healthissue and major cause of liver-related morbidity andmortality. About 130–170 million people are infectedwith HCV worldwide, which covers about 2.2–3% of

the world’s population [Alter, 2007]. In Asian-Pacificregions, the prevalence of HCV infection ranges from0.3% to 12% [Mc Caughan et al., 2007] and inPakistan 5.9% (>10 million) individuals are livingwith HCV with high morbidity and mortality [Azizet al., 2011].The combination treatment with interferon a and

ribavirin continues to be used widely in routinepractice in HCV infected patients in Pakistan be-cause PEG interferon therapy is very expensive,secondly in Pakistan infection is caused by HCV typethree which is more sensitive to interferon therapythan type one. The main treatment goal in chronichepatitis C is to suppress viral replication at maximallevel to attain sustained virologic response (SVR,defined as an undetectable serum hepatitis C virusRNA level 6 months after the end of treatment)[Strader et al., 2004]. In addition HCV infectedpatients serve as the reservoir for transmission toothers [Pyrpasopoulou et al., 2011]. However re-sponse to antiviral treatment is not uniform in allthe populations, reasons for failure of treatment arecorrelated with host, virus, and immune responsevariables [Conjeevaram et al., 2006). Additionally ithas also been found that there is correlation betweenHCV genotyping and response to therapy [Simmonds,1995]. Some studies reported genetic polymorphisminfluence antiviral response [Soler et al., 2002]. AsHCV is an RNA virus, it have conserved and hypervariable region. Most conserved region is 50UTR, itcontains replication signal as IRES (Internal riboso-mal entry site). 40S ribosomal unit bind with IRESfor initiation of translation. These changes in nucleo-tide may have dramatic effect on translation [Vanet al., 2004]. Some studies reported that nucleotidechanges in IRES region have influence on response totherapy [Th�elu et al., 2007].

Conflict of interest: None.�Correspondence to: Hafza Aziz, Nuclear Medicine Oncology

and Radiotherapy Institute Islamabad, Pakistan.E-mail: Hafsa.aziz@gmail.com

Accepted 29 May 2015

DOI 10.1002/jmv.24305Published online 16 July 2015 in Wiley Online Library(wileyonlinelibrary.com).

�C 2015 WILEY PERIODICALS, INC.

It is valuable to analyze factors that enable us topredict the response to antiviral treatment. Severalpretreatment and on treatment factors (gender, startingtreatment at younger age, low level of HCV RNA, HCVgenotype 2, and 3 and early inhibition of viral repli-cation (EVR), steatosis) have an impact on antiviraltreatment outcome [Yoshioka et al., 1992; Tsubotaet al., 1993; Suzuki et al., 1995; Backus and Boothroyd,2007; Kau et al., 2008]. However, it is not clear to whatextent these factors contribute to the response, or howthey relate to each other. Moreover we analyzedsubstitution in sub genomic 5/UTR (IIIb domain containIRES) region that may be associated with response totherapy.

MATERIALS AND METHODS

Patient Selection

A study was carried out on 921 patients with chronicHCV infection who attended the Nuclear MedicineOncology and Radiotherapy institute (NORI) frommultiple centers (Pakistan Institute of medical sciences,Islamabad specialist clinic, Federal Government Hospi-tal) from 2008–2013. Patients included in this studyhave following characteristics, Positive for anti HCVantibody with normal and raised ALT level, HCVviremia confirmed by qualitative PCR, and negative forhepatitis B surface antigen, Moreover patients enrolledin this study had a hemoglobin level >13g/dl in men or>12g/dl in women a white blood cell count >3000 cells/mm3, platelet count >100,000 cells/mm3. The study wasapproved by local ethical committee of the center andconsent form was received from all patients. Eligiblepatients received IFN 3MIU thrice a weeks for24 weeks.

Assessments of Viremia, HCV Genotypes,and ALT

Viral load was measured by using instant virusRNA kit (AJ Roboscreen Berlin, Germany) accordingto manufacturer’s protocol on real-time PCR (Rotor-Gene 3,000 Corbett Research, Sydney, Australia) atbase line, 12 week, 24 weeks, and 24 weeks aftertreatment completion for sustained virologic response(SVR defined as absence of detectable HCV RNA6 months after the completion of treatment). Thelower limit of detection of the assay is 50IU/ml. Fattyliver was detected by ultra sonography [Yajima et al.,1983] at baseline. Genotyping was performed by coreregion genotype specific primer reported by Ohnoet al. [1991] with some modification reported by Azizet al. [2013]. The primary end point of the study wasto find the factors that contribute to the sustainedvirologic response.

Sequencing

Amplified sample of selected responder and non-responder were subjected to sequencing by using ABI

automated sequencer. Chromas version 2.0 were usedto view nucleotide sequence and electro-pherograms.Ambiguities in the sequence are resolved by compar-ison of the results of both forward and reverseprimers as well as by comparison with the HCVsequences in the NCBI database. Sequence wasaligned by using CLC workbench software (www.clcbio.com). Nucleotide sequences were analyzed forsubstitutions by using software CLC Workbench.

Statistical Analysis

All the variable results were given in the form ofrate (%). Chi square method was used to evaluatevariable association with likelihood of achievingresponse to treatment. SPSS was used to carry outthe logistic regression analysis, to find associationbetween variables and SVR. P-value below 0.05 wasconsidered significant. We made a sub-analysis byadding result of 50UTR sequence analysis of patientsachieved SVR versus non-SVR.

RESULT

Study Enrolments and Disposition of Patients

Nine hundred and twenty one patients included infinal analysis that completed 24 weeks of treatmentand follow up. Five hundred twenty three werefemales (n¼ 523) and three hundred ninety eight(n¼ 398) were males. The base line characteristics ofthe patients are summarized in Table I. Surgery anddental treatment was the main mode of acquiringHCV infection.

Virologic Response

Six hundred eighty nine patients (74.8%) showedend treatment response (ETR). Two hundred thirtytwo (25.2%) patients were virologicaly non respond-ers, showing detectable HCV RNA at the end oftreatment. Of the 921 patients, 554 (60.2%) achievedsustained virologic response (SVR). Among the pa-tients (921) who completed the therapy 523 (56.8%)were females and 398 (43.2%) were males with endtreatment response rate of 75.3% and 74.1%, respec-tively (Table II). The result is an indicative of thefact that no significant difference in response to IFNa-2b plus ribavirin treatment in males and females.Over all fatty liver was observed in 21.9% of patients.SVR rates in. patient with fatty liver was lowerwhere as in patients without fatty liver was higher(66.7% vs. 36.6%, p<0.005) (Table III).The effect of different age groups on sustained

virologic response in HCV patients is shown inFigure 1. Tendency to sustained virologic response ishigher (77.2%) in females at the age of <40 years,60% in �40–50 years age group, and 27.3% in >50–60 years age group (Fig. 1). In males, sustainedvirologic response was almost equal for <40 and�40–50 years age group 66.7%. SVR 16.7% was

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Optimum Predictors of Therapeutic Outcome in HCV 101

observed in some cases of >60 year’s age group inmale patients. SVR in female >60 years of age groupwas not observed. Moreover when study populationwas categorized into four groups same findings wereobserved. In females <40 years group, the sustainedresponse rate was significantly higher (77.2%) ascompared to females with age �40 years withresponse rate of 48.8% (77.2% vs. 48.8%, P¼ 0.001)(Table III).Correlation between pretreatment viral load and

SVR was also analyzed. A wide range of virus loadswere observed among infected patients. For thepurpose of analysis patients were categorized into

two groups according to their baseline viral load(Table III). The percentage of SVR was found higherwith low pretreatment viral load (72.4% vs. 55.95%P< 0.0001). We examined SVR in HCV patientsaccording to week 12 viral decline. For this purposeall patients were evaluated at week 12. Six hundredand sixty five patients (665) showed viral decline �2log drop while two hundred and fifty six patients didnot showed 2 log drop. The SVR was observed higher73.8% (491/665) in patients who achieved earlyvirologic response (EVR) than patients who did notachieve EVR 24.6% (63/256) (Table III).

Treatment Outcome According to Genotypes

HCV genotype distribution pattern showed in all921 patients (Table I). Overall, 816 patients (88.6%)were infected with genotype 3a, 20 (2.2%) with 3aþ 3b, 44 (4.8%) with 1a, and 7 (0.8%) with both 1aþ 3a.The relation for the age groups for different subtypeswere also determined, we found subtype 3a strainsshowed higher presentation 95% among patientshaving age <40 years while older patients having age� 40 years showed 79.9%. Sub type 1a showedpresentation 10.4% among patients with age �40years where as younger patients showed only 0.7% ofsubtype 1a while studying the SVR in relation to

TABLE II. Efficacy of Treatment

SexNo of

PatientsNon responder

(%) ETR (%) SVR (%)

Female 523 129 (24.7) 394 (75.3) 316 (60.4)Male 398 103 (25.9) 295 (74.1) 238 (59.8)Total 921 232 (25.2) 689 (74.8) 554 (60.2)

End treatment response (ETR), Sustained virologic response (SVR)

TABLE I. Baseline Characteristics of HCV Patients Under-gone to IFN a-2b plus Ribavirin Treatment

Variable No. (%)

SexFemale 523 (56.8)Male 398 (43.2)

Source of infectionSurgery 120 (13)Surgery & transfusion 130 (14.8)Transfusion 80 (8.7)Dentist 306 (33.2)Unknown source 285 (30.9)

Age, Years<40 536 (58.2)�40–50 218 (23.7)>50–60 150 (16.3)�60 17 (1.8)

HCV RNA level IU/ml<100,000 51 (5.5)100,000–500,000 165 (17.9)>500,000–100,0000 182 (19.8)>100,0000 523 (56.8)

Serum ALT levelsNormal ALT 165 (17.9)Raised ALT 756 (82.1)

Genotypes1a 44 (4.8)1aþ3a 7 (0.8)1b 14 (1.5)3a 816 (88.6)3aþ3b 20 (2.2)3b 20 (2.2)

SteatosisNon-fatty liver 719 (78.0)Fatty liver 202 (21.9)

HCV, hepatitis C virus; ALT, alanine aminotransferase; raisedALT; group of patients who have serum alanine aminotransferasein male >41U/L), female >31); normal ALT; group of patients whohave serum alanine aminotransferase in male<41, female<31.

TABLE III. Variables Associated With SVR in HCV TreatedPatients

Variable

Total # oftreatedpatients

AchievedSVR

SVRRate % P-value

SexMale 398 238 59.8 NSFemale 523 316 60.4

Male age<40 Years 224 151 67.5 0.002�40 Years 174 88 50.6

Female<40 Years 312 241 77.2 0.0001�40 Years 211 103 48.8

Pre treatment viral load�8�105

IU/ml239 173 72.4 <0.0001

>8�105

IU/ml682 381 55.9

HCV genotypingGenotype 3 856 535 62.5 <0.001Othergenotypes

65 26 40.0

Response rate at week 12EVR 665 491 73.8Non-EVR 256 63 24.6 <0.0001

Pre treatment ALTRaised ALT 714 411 69.1 <0.005NormalALT

207 143 57.6

SteatosisNon-fattyliver

719 480 66.7 <0.005

Fatty liver 202 74 36.6

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102 Aziz et al.

HCV viral genotype (Table IV), response to IFNagainst patient infected with genotype 3a was 63.1%(515/816), 3b was 55% (9/20), 1a was 36.3% (16/44),and 1b was 35.0% (5/14). Rate of SVR in patientswith mixed genotype were also analyzed. SVR rate inpatients infected with 3aþ3b genotype was 55.0%and with 1aþ3a was 42.9%. Random distribution ofviral load was noticed in HCV infected patients withdifferent genotypes. A significant difference of SVRwas found between the patients with an HCV RNAlevel �800,000 IU/ml with genotype 3 and genotype 1(P< 0.0001) (Figure 2). Figure 2 also demonstratethat there is difference in response in genotype 3patient with HCV RNA �800,000 IU/ml (73.0%) thanHCV RNA <800,000 IU/ml (41.0%). High viral loadhad lower rate of SVR than with low viral load. In

addition prevalence of fatty liver was 8.1% (70/856)among HCV genotype 3 and 6.1% was observed inother group of genotype.

Biochemical Response

At the time of treatment subjects were classifiedas raised ALT group (Male �41U/L, Female �31U/L) and normal ALT group (Male <41U/L, Female<31U/L). Most of the patients 714 (77.4%) lie inthe raised ALT group with end treatment responserate of 72.7%. The proportion of the patients withSVR was slightly higher in raised ALT group thannormal ALT group that is 69.1% versuss 57.6%P< 0.005 (Table III). Serum ALT decreased tonormal level after treatment completion in 73.5%(525/713) patients. All patients were assessed after6 month of cessation of treatment for SVR andbiochemical response. During follow up it wasobserved that undetectable HCV RNA had highpredictive value for normalization of ALT level as468 of 554 (84.5%) patients with undetectable HCVRNA had normal ALT at follow up. Patients withsustained biochemical response did not show sus-tained virological response. Thus ALT normaliza-tion has low predictive value for undetectable HCVRNA.

Fig. 1. Sustained response to IFN a- 2b Plus ribavirin treatment in HCV patients based onsex and age group.

TABLE IV. Rate of Sustained Response in Patients WithDifferent HCV Genotypes

S.No

HCVgenotypes

Total treadedpatients (%) SVR (%)

NSVR(%)

1 1a 44 (4.8) 16 (36.3) 28 (63.6)2 1aþ3a 7 (0.7) 3 (42.9) 4 (57.1)3 1b 14 (1.5) 5 (35.0) 9 (64.2)4 3a 816 (88.6) 515 (63.1) 301 (36.9)5 3aþ3b 20 (2.2) 11 (55) 9 (45)6 3b 20 (2.2) 11 (55) 9 (45)

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Optimum Predictors of Therapeutic Outcome in HCV 103

Detection of Nucleotide Substitution in 5/UTRRegion

Sequence region from nucleotide 88–403 of 50UTRregion was amplified and sequences were aligned usingCLC sequence viewer to find nucleotide substitution.The sequences were graphically according to NZL1 (acD17763). The alignment of sequence (region thatconfers sub domain IIIb of 50UTR region) showed somespecific pattern from patients who have SVR and inpatients who did not have SVR (non-SVR) to antiviraltreatment (Figure 3). It was observed that non-SVRpatients have substitution at position 202 (nucleotidechange from C to T) but substitution differencebetween patients who achieved SVR and Non-SVR isnot significant.

Predictor of Sustained Virological Response toPEG IFN a-2a Univariable Logistic Regression

Analysis

By univariable logistic regression analysis wefound baseline HCV RNA level (<800,000 IU/ml OR,2.0; 95%CI, 1.50–2.85; P< 0.0001); age (<40 years;OR, 2.8; 95%CI, 2.20–3.80; P< 0.0001); HCV geno-type (Genotype 3 OR,4.0 ; 95%CI, 2.3–7.00;P< 0.0001); achieving EVR (EVR OR, 8.6; 95%CI,6.19–12.05; P< 0.0001) , Viral load (<800,000 IU/mlOR, 1.8; 95%CI, 2.1–3.2; P< 0.0001); ALT level at thetime of treatment (normal ALT OR, 1.6 ; 95%CI,1.19–2.31; P> 0.003), Steatosis (Non-Fatty liver OR,1.3;95% CI, 1.13–3.80; P< 0.005) were significantlyassociated with treatment success. Sub-group analy-sis carried to find effect of substitution at IRESbinding site on treatment response, including patientwhose 5/UTR sequence carried we found difference inresponse, though it is not significant.

Multivariable Logistic Regression Analysis

When multivariable model was built with thefactors exhibiting significance on univariable logisticregression analysis to clarify independent factor that

contributing to SVR. It showed patient age [OR (for<40 vs. �40) 2.0; 95%CI, 1.49–2.87; P¼ 0.0001], HCVRNA level [OR for (<800,000 IU/ml vs. �800,000IU/ml) 4.0; 95% CI, 2.64–6.17; P¼ 0.0001], EVR atweek 12 [OR (for EVR vs. non-EVR] 12.3; 95% CI,8.18–18.58; P< 0.0001] and steatosis [OR (for <FattyLiver vs. Non-Fatty Liver) 2.5; 95% CI, 3.6–6.2;P< 0.005] remained significance for SVR.

Tolerance and Safety Assessment

Adverse events experienced by the patients duringtreatment were recorded in Table V. Most of theHCV treated patients experience headache 53%,myalgia 45%, and fatigue 45.5%. The commonlyobserved psychiatric side effect of therapy, insomniais present in 20% of patients. Additionally it wasnoted that depression develops in 5% of patients.Diarrhea was observed in 13% cases. Ten percentpatients developed skin rash.

DISCUSSION

The current study showed SVR of 60.2% to anti-viral therapy. This SVR is higher as compare to 57%and 53% reported by others [Poynard et al., 1998;McHutchison et al., 1998]. High overall rate of HCVRNA clearance may be due to the fact that majorityof the population infected by HCV genotype 3. Thisimplies that HCV genotype 3 have better response toantiviral treatment as compared to genotype 1[Sarra-zin et al., 2001]. In a study Jacobson et al. reportedhigher rate of SVR as 78% among HCV genotype 2and 3 patients treated with Sofosbuvir with RBV[Jacobson et al., 2013]. Another study showed thatna€ıve HCV genotype 3 patients achieved SVR 95%[Zeuzem et al., 2014] after treatment with Sofosbuvirwith RBV, so the new direct antiviral agents increasethe sustained response against HCV infection.In some previous studies response to interferon in

female have not documented better than male butunable to observe such a relationship, we foundalmost equal SVR rate in male and female 59.8%versus 60.4% but the response rate was not statisti-cally significant. Furthermore multivariable logisticregression analysis shows that gender has no signifi-cant correlated to SVR. In addition results revealedthat female younger patients (<40) have SVR 77.2%as compared to male have 67.5%. In addition femaleresponse difference in younger and older female’spatients is more (28.4%) than male who have shown16.9%. difference in younger and older age. Moreover in our study we also observed no difference inresponse in male patients with age <40years and40–49 years. An important finding of the study isolder female patients >60 years have not shown anyresponse. This observation supported by Antonucciet al. [2007] who reported low chance of achievingSVR in patients older than 40 years of age. It showsthat aging impart a negative effects on the treatmentefficacy. Changing in immune system occurs as age

Fig. 2. IFN a-2b Plus ribavirin treatment outcome in relationto viral load and genotype.

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104 Aziz et al.

Fig. 3. Nucleotides alignment of 50UTR region of hepatitis C virus isolated from SVR andnon-SVR patients.

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Optimum Predictors of Therapeutic Outcome in HCV 105

increases that leads to poor responsiveness to newantigen due to decrease of na€ıve T cell, less capacityto produce interleukin, changes in cytokine profile,deficiency in T-cell receptor signal transduction andactivation [Gillis et al., 1981; Timm and Thoman,1999; Pawelec et al., 2001].The HCV viremia in a body reflects the association

with the balance between replication of virus andclearance of virus. More over HCV viral load indi-cates the therapy out come. Some studies reportedserum HCV load associated with HCV genotype[Moatter et al., 2002] but we are unable to find viralcontent association with genotype in infected HCVpatients. A clear relationship between baseline virallevels and response to IFN a-2b plus ribavirintherapy was observed. Frequency of SVR was notablydecreased as base line viral load increases in HCVpatients (72.4% vs 55.9%) (Table III) also verifiedfrom several studies [Davis et al., 1998; Jensen et al.,1999]. Another study [Martinot-Peignoux et al.,1995] demonstrated that the low baseline serum viralload (<800,000 IU/ml) is associated with significantlyhigher probability of achieving virologic responsefollowing IFN a-2b plus ribavirin therapy. The re-sults were confirmed by multivariable logistic regres-sion analysis.In this study prevalence of fatty liver was found

21.9%, this finding is in agreement with the observa-tion that HCV hijacks the lipid-producing machineryof hepatocytes for its benefit and results in steatosis[Barba et al., 1997; Shi et al., 2002]. Patients withfatty liver have markedly lower SVR (36.6%) thanthose of non-fatty liver; this observation is in con-sensus with Jian et al. who suggest that fatty liverhave adverse effect on the outcome of the treatment[Jian et al., 2006]Genotype of HCV aid physicians in tailoring anti-

viral therapy. In agreement with previous report

[Moatter et al., 2002] patients with HCV genotype 3ahave higher response (63.1%) than other genotypes.In the population with genotype other than 3, mostgenotypes observed are 1a, 1b, and 1aþ3a. Althoughnumber of these genotypes was too small in thecurrent study but other studies also reported thesegenotypes associated with poor response. From theresult it is clear that patient with genotype 3 andhave higher baseline viral load >800,000IU/ml haveresponse rate 41.0% where as patients infectedgenotype 1 have response rate 25.0% (Figure 2). Theresponse rate may be increased by using directantiviral agents. As number of studies reported thatfirst generation HCV NS3/4A protease inhibitors(boceprevir and telaprevir) increase the likelihood ofSVR to 67–75% in HCV genotype 1 patients[McHutchison et al., 2009; Kwo et al., 2010; Jacobsonet al., 2011; Poordad et al., 2011; Kanda et al., 2014].Simeprevir, second-generation hepatitis C virus pro-tease inhibitor led to SVR 77–92% [Hayashi et al.,2014]. The Food and Drug Administration in 2013approved the use of simeprevir and sofosbuvir (HCVpolymerase inhibitor) for the treatment of patientsinfected with HCV, which will improve SVR of 89%in patients infected with HCV genotype 1 [Andro-nescu et al., 2014].Random distribution of HCV RNA among infected

patients with different HCV genotypes was observed.This observation was in contrast to the study ofMoatter et al, who have reported low level of viremiain HCV genotype 3 patients [Moatter et al., 2002].But another study [Xie et al., 2005] reported viralload have no correlation with HCV genotypes. Thisdiscrepancy, in result may be caused by differentquantification method in different laboratories. Fromdata analysis we found significant association of EVRto SVR. Study showed higher SVR rate in patientswho achieved EVR as compare to patients who didnot achieve EVR (73.8% vs. 24.6% P< 0.0001)(Table III). Results also suggested that patients withearly virological response had significant higher SVR.This finding is in agreement with Sarwar et al.[2012] and Yu et al. [2007]. Patient who attains SVRbut did not attain EVR has slow response during thetreatment. 174 (26.1%) patients not showing SVRthough they showed EVR (72.2%) suggesting thatHCV RNA can persis in PBMC of infected patientswho have undetectable HCV RNA during interferontreatment [Zayed et al., 2010].The ALT value rises with development of inflam-

mation of liver. IFN a-2b plus ribavirin therapyresponse rate in patients with normal ALT was foundhigher (69.1%) to that achieved in patients withelevated ALT levels. This outcome was in consensuswith Hui et al. who revealed that the end treatmentresponse to IFN therapy in patients with normalALT levels was 59.6% and in patients with elevatedlevel was 56.6% [Hui et al., 2003]. In a study byPersico et al. [2000] 23% of their patients developedelevated ALT level, while 21% patients with normal

TABLE V. Incidences of Adverse Events During IFN a-2bPlus Ribavirin Treatment

Adverse Event No. Percentage (%)

ConstitutionalHeadache 487 53Fatigue 419 45.5Fever 276 30Myalgia 414 45

Gastro intestinalNausea 303 33Decrease in appetite 200 21.7Diarrhea 120 13

Psychiatric symptomsDepression 46 5Insomnia 184 20

Dermatological symptomsAlopasia 185 20Rash 92 10Inflammation at injection site 90 9

Respiratory disorderCough 100 10Phyringitis 105 11.4

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106 Aziz et al.

ALT developed elevated ALT level on follow up. Bothhave suggested that this raised ALT in these patientsmay be part of the natural history of patients withnormal ALT levels [Persico et al., 2000]. But in thisstudy we found only three (1.8%) patients, who hadnormal ALT during treatment but developed raisedALT levels at the end of treatment. Multivariablelogistic regression did not showed ALT as predictivefactorSequence region from nucleotide 88–403 of 50UTR

region was amplified and sequences were alignedusing CLC sequence viewer to find nucleotide sub-stitution. The sequences were graphically accordingto NZL1 (ac D17763). Alignment of sequence (regionthat confers sub domain IIIb of 50UTR region) showedthat contact point between IRES and 40S ribosomalunit (Nucleotide 128–138,145–156 and 237–250) arehighly conserved among all strains isolated frompatient showed SVR and did not showed SVR toantiviral treatment [Hazari et al., 2005]. In agree-ment with Moratorio et al. [Moratorio et al., 2007] arare nucleotide substitution is observed at G 243A,though did not disrupt stem loop III folding andsuggesting that maintaining of the structure of theinternal ribosomal entry site is prerequisite forsurvival of quasispecies in host cell (Thelu et al.,2004). In addition C–T and C–A substitution atposition 202 among patients achieved SVR and non-SVR needs further study. These findings suggest thatsequence variability found in IRES of Non-SVR doesnot affect the confirmation for binding of cellularfactors. More over consensus alignment of sequencebefore treatment did not revealed any significantcorrelation with sustained response.

CONCLUSION

SVR in patients with genotype 3 were significantlyhigher than those of patients with other genotypegroup. Subtype 3a strains showed higher presenta-tion 95% among patients having age <40years whileolder patients having age � 40years showed 79.9%.Over all, younger age, low viral load, achieving EVRand non fatty liver were independently but positivelyassociated with SVR.

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