Adult patients with chronic HCV infection (defined as the presence of HCV RNA in serum for > 6 mo) referred to the Tehran Blood Transfusion Hepatitis Clinic between March 2011 and August 2013 were enrolled in the study. Patients with no concomitant hepatic illness, negative results for human immunodeficiency virus antibody and hepatitis B surface antigen, and no history of prior antiviral therapy for HCV infection were included in the study. A majority of the patients underwent percutaneous liver biopsy, for which specimens were evaluated according to the modified Knodell score grading and staging system by Ishak et al[13].

Patients received weekly subcutaneous injections of Peg-IFN-alpha-2a (180 μg/mL Pegaferon^®; Pooyesh Darou, Tehran, Iran) and daily oral ribavirin (Ribabiovir^®; Bakhtar Bioshimi, Kermanshah, Iran): 800 mg for HCV genotype-3 and 1000-1200 mg according to the patient’s weight (< or ≥ 75 kg) for HCV genotype-1.

Treatment duration was 24 wk for those infected with HCV genotype-3, or 48 wk for infection with HCV genotype-1, but could be shortened or extended depending on the patient’s response and compliance.

The patients were closely monitored throughout the treatment course, with monthly complete blood cell count and liver enzyme tests, and HCV RNA level assessment before treatment, at weeks 4, 12, 24, at the end of treatment, and 24 wk after treatment completion. Informed consent was obtained from all participating patients. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committee of the Iranian Blood Transfusion Organization.

Quantitative assessment of HCV RNA was performed using the COBAS TaqMan HCV Test v2.0 (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions and a detection limit of 10 IU/mL. HCV genotyping was performed using HCV genotype-specific primers[14].

The IFNL SNPs were assessed using the PCR-restriction fragment length polymorphism method[15]. Briefly, genomic DNA was extracted from patients’ peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The PCR was performed using Accupower PCR PreMix (Bioneer Corp., Daejeon, South Korea) with the following conditions: 94 °C for 5 min, 35 cycles of 94 °C for 20 s, 66 °C for 20 s, and 72 °C for 20 s, followed by 72 °C for 5 min. Primers used for the reaction included: rs12979860, 5’-GCGGAAGGAGCAGTTGCGCT-3’ and 5’-GGGGCTTTGCTGGGGGAGTG-3’; or rs8099917, 5’-CCCACTTCTGGAACAAATCGTCCC-3’ and 5’-TCTCCTCCCCAAGTCAGGCAACC-3’. The PCR amplicons were then digested for ≥ 1 h with 10 U of restriction endonuclease: BstUI for rs12979860 or BsrDI for rs8099917 (Fermentas of Thermo Fisher Scientific, Waltham, MA, United States). The digested PCR products were separated on 3% agarose gels revealing the following sized fragments: rs12979860, 196 and 45 bp for the CC genotype, 241, 196, and 45 bp for the CT genotype, or 241 bp for the TT genotype; rs8099917, 552 bp for the TT genotype, 552, 322 and 230 bp for the GT genotype, and 322 and 230 bp for the GG genotype.

Therapeutic responses were categorized as: rapid virologic response (RVR; HCV RNA undetectable after 4 wk of treatment), early virologic response (EVR; HCV RNA undetectable or ≥ 2 log decreased at 12^th wk of treatment), or sustained virologic response (SVR; HCV RNA undetectable at 6 mo after the end of treatment). Achievement of SVR was considered as treatment success, and thus used to describe patients as responders or non-responders. HCV breakthrough was used to describe cases where HCV RNA was detected during treatment after a previous period where it was undetectable, and resistant infections were cases where HCV RNA was detectable at all times. If HCV RNA was undetectable at the end of therapy, but then detected 6 mo later, this was considered a relapse[16].

Statistical analyses were performed using SPSS v16.0 software (SPSS Inc., Chicago, IL, United States). The association of each nominal variable with SVR achievement was evaluated with cross tabulation and χ² testing. Independent sample Student’s t tests were used for continuous variables with a normal distribution, otherwise the Mann-Whitney U test was used. All baseline variables that had a P < 0.2 in univariate tests were entered into a logistic regression model, and P < 0.05 was considered statistically significant. The statistical methods used in this study were reviewed by Maryam Sharafkhah, MS, of the Biostatistics in Digestive Diseases Research Institute.

A total of 152 patients (141 men and 11 women; mean age 41.9 ± 10.0 years) with chronic HCV infection were included in this study. The mean HCV RNA level was 3920258 ± 6856760 IU/mL (interquartile range: 4420288 IU/mL). A larger proportion of patients (93/152; 61.2%) were infected with HCV genotype-1 (1a: n = 86; 1b: n = 7), and the remaining patients were infected with HCV genotype-3a (n = 56), a mixed genotype-1a/3a (n = 2), or mixed genotype-1a/4 (n = 1).

The frequencies of IFNL rs12979860 CC, CT, and TT genotypes were 37.5%, 47.3%, and 13.2%, respectively, and were 60.2%, 37.2%, and 2.7% for rs8099917 TT, GT, and GG genotypes, respectively. There were no significant differences in rs12979860 and rs8099917 subtype frequencies with respect to HCV genotype.

Liver biopsies were performed in 99/152 (65.1%) patients, indicating stage 0 (n = 8), stage 1 (n = 23), stage 2 (n = 26), stage 3 (n = 27), or stage 4 (n = 4) fibrosis, or cirrhosis (n = 11).

Treatment duration was reduced to < 48 wk for 24/94 (25.5%) patients with HCV genotype-1 infection (15 with shortened treatment course, two as a result of non-compliance, and seven following treatment failure), and to < 24 wk in one non-compliant HCV genotype-3 subject. Treatment was prolonged up to 72 wk in 14/94 (14.9%) patients with HCV genotype-1 infection because of cirrhosis or the continued presence of HCV RNA at 12 wk. Similarly, 10 patients with HCV genotype-3 infection required an extended 48-wk therapy course. Treatment course was withdrawn in 1 subject due to attempted suicide. The 11 patients with cirrhosis did not show signs of ascites or thrombocytopenia, and treatment was therefore administered.

The treatment outcomes for all patients and according to HCV genotypes are shown in Table 1. Although the rates of EVR and SVR did not differ between the HCV genotype-1 and genotype-3 groups, the rate of RVR was significantly higher in patients with HCV genotype-3 than in patients with HCV genotype-1 (P < 0.01). Furthermore, 44/62 (71.0%) patients who did not demonstrate RVR and 7/14 (50.0%) patients who did not show EVR eventually achieved SVR. Therefore, overall, RVR and EVR were significantly related to treatment success (P < 0.01).

The age, baseline HCV RNA level, liver fibrosis stage, serum alanine and aspartate transaminase levels, and body mass index were compared among treatment responders and non-responders within the HCV genotype groups (Table 2). Patients with mixed HCV genotypes were excluded from analyses due to the small sample size. The baseline characteristics did not differ between HCV genotype groups, although responders were significantly younger (P < 0.01), and had lower HCV RNA levels (P = 0.04) than non-responders. These differences were not significant within the HCV genotype subgroups. In contrast, body mass index did not differ within the entire cohort with regard to treatment response, however, responders with HCV genotype-1 infection had a significantly higher body mass index than non-responders (P = 0.03).

Table 3 shows the prevalence of rs12979860 and rs8099917 genotypes with respect to treatment response. Among subjects with rs12979860 CC genotype, 91.2% (52/57) responded successfully to treatment, whereas 82.1% (78/95) of patients with a non-CC genotype responded. Treatment success occurred in 86.7% (39/45) of patients with the rs8099917 TT genotype, and in 89.7% (61/68) of those with a non-TT genotype. Univariate analyses revealed that there were no differences in the prevalence of the IFNL genotypes between responders and non-responders for either polymorphism. However, among patients with HCV genotype-1 infection, the rs12979860 CC genotype was significantly related to treatment success (94.1% vs 75.0%; P = 0.02). This was not observed in the HCV genotype-3 group.

Patients with the rs12979860 CC genotype had significantly higher HCV RNA levels compared to those with a non-CC genotype (60.9 × 10⁵± 81.8 × 10⁵ IU/mL vs 26.4 × 10⁵± 45.7 × 10⁵ IU/mL, P = 0.01). Further analyses also revealed that RNA levels of HCV genotype-1 were significantly higher in patients with rs12979860 CC vs other genotypes (71.6 × 10⁵± 88.1 × 10⁵ IU/mL vs 28 × 10⁵± 40.2 × 10⁵ IU/mL, P < 0.01). However, this was not the case for patients in the HCV genotype-3 group. In addition rs12979860 CC and rs8099917 TT genotypes were significantly related to achievement of RVR (both P < 0.05), but not EVR. RVR was also significantly associated with rs12979860 CC and rs8099917 TT genotypes in patients infected with HCV genotype-1 (P = 0.01).

Logistic regression analysis of treatment success was conducted according to age, categorical HCV RNA level, stage of liver fibrosis, rs12979860 CC genotype, and aspartate transaminase level (variables identified by P < 0.2 in univariate analyses). The results showed that only rs12979860 CC genotype was a predictor of SVR achievement (P = 0.03). Table 4 shows the results of multivariate analysis.

Chronic hepatitis C virus (HCV) infection is a major public health problem that can result in cirrhosis and lead to hepatocellular carcinoma or even death. For the past decade, Pegylated interferon (Peg-IFN) has been the primary therapeutic intervention for chronic HCV infection, although its efficacy is influenced by many factors, including single nucleotide polymorphisms in or near the IL28B gene.

Many studies have reported wide ranging efficacies for Peg-IFN and ribavirin in the treatment of chronic HCV infection. Although patient age, viral load, and ethnicity can substantially affect treatment success, the role of host genetic factors, such as single nucleotide polymorphisms in relevant genes, are becoming more apparent. Thus, further understanding of the influence of these factors will help improve patient outcome.

The high response rate to Peg-IFN and ribavirin is an important finding of this study, and may support the decision to accept treatment by many patients with chronic HCV infection, particularly those in developing countries. The high response rate of Iranian patients to Peg-IFN and ribavirin in this study highlights that the conventional dual therapy can still be considered the first-line treatment for these patients.

The results of this study show that the rs12979860 CC genotype is a strong predictor of treatment response. Thus, patients with this rs12979860 genotype can achieve greater benefit from Peg-IFN and ribavirin treatment.

Interferon-λ is a cytokine that is involved in host defense against viral infections, such as hepatitis C. Single nucleotide polymorphisms are genetic variations within the DNA that are common within a population.

The authors carried out a cross-sectional multivariate analysis to describe positive predictive factors for response to HCV treatment with Peg-IFN alpha-2a plus ribavirin in a naïve Iranian cohort. Special attention is given to IL28B polymorphisms as a target to predict sustained viral response. They observed a similar finding to previous reports showing a relationship between the IFNL rs12979860 CC genotype and a higher sustained virologic response probability. The patient cohort demonstrated a high sustained virologic response rate overall, which could be related to ethnic factors that render the Iranian population more sensitive to Peg-IFN plus ribavirin treatment.