Impact of IL28B (rs8099917) gene polymorphism on treatment response to Sovaldi, Daklinza, and Ribavirin in Egyptian patients with HCV infection

ABSTRACT Infection by the hepatitis C virus (HCV) is a big threat to human health all over the world. Many host factors could affect the treatment response in HCV infection. One of these factors is IL28B gene polymorphism. This research study investigated the association of IL28B (rs8099917) gene polymorphism with treatment response in patients with chronic hepatitis C virus (HCV) infection receiving a combination of Sovaldi, Daklinza, and Ribavirin in Egypt. The study included 100 patients, divided into three groups based on treatment outcomes: responders, relapsers, and non-responders. Biochemical and hematological analyses were performed, and HCV genotypes were determined using PCR. IL28B genotyping was done using real-time PCR. The results showed a high prevalence of HCV genotype 4 and a few patients with genotype 1. The G allele of IL28B was associated with a higher risk of developing relapse and non-response, while the TT genotype was significantly associated with treatment success. Viral load levels were lower in responders compared to relapsers and non-responders. The study suggests that IL28B genotype may be a valuable predictor of treatment response in chronic HCV patients treated with the specified combination therapy. Further research is needed to confirm and fully understand these findings.


Introduction
Hepatitis C is a serious illness that affects the liver and can be caused by HCV.It can range from mild to severe, and in some cases, it can lead to liver cirrhosis or cancer.The virus is typically spread through exposure to infected blood, such as unsafe injection practices or unscreened blood transfusions [1].Unfortunately, many people around the world are affected by this disease, with millions of new infections occurring each year.According to the World Health Organization (WHO), approximately 58 million people worldwide are living with chronic hepatitis C virus infection, and around 1.5 million new infections occur each year.3.2 million children and adolescents worldwide have chronic hepatitis C infection.290,000 people died from hepatitis C in 2019, primarily from cirrhosis and hepatocellular carcinoma (primary liver cancer) [2].
Antiviral medicines have been developed to cure more than 95% of people with hepatitis C infections.While vaccines exist for other forms of hepatitis such as hepatitis A and B, there is no vaccine that can prevent hepatitis C infection.Direct-acting antiviral agents (DAAs) are a class of drugs used in the treatment of HCV infections.These drugs act directly on the virus to prevent its replication and spread in the body [3].The choice of DAA regimen depends on several factors such as genotype, prior treatment history, and the presence of liver cirrhosis or other comorbidities [4].
Interleukin-28 (IL-28) is a cytokine that is produced by certain cells of the immune system, such as dendritic cells and macrophages, in response to viral infections [5].There are two isoforms of IL-28, IL-28A (also known as interferon lambda 2 or IFN-λ2) and IL-28B (also known as interferon lambda 3 or IFN-λ3) [6].In humans, IL-28 genes are located on chromosome 19 [7].IL-28 has been shown to have antiviral effects against a variety of viruses, including HCV, influenza virus, and herpes simplex virus [8].IL-28 works by binding to receptors on the surface of infected cells, triggering a signaling cascade that leads to the production of antiviral proteins and the inhibition of viral replication [9].The IL28B gene, which encodes IL-28B, has also been linked to the outcomes of HCV infection and treatment.IL28B genotypes are strongly associated with sustained virological response (SVR) in patients with chronic HCV infection treated with pegylated interferon/ ribavirin (PEG-IFN/RBV) [10,11].Thus, IL28B genotyping can be considered as a pre-treatment predictor of the outcome of Peg-IFN/RBV therapy [12].IL28B genotypes were also demonstrated at higher frequencies in infected patients with HCV who cleared the infection spontaneously [13].McCarthy et al [14]., confirmed the association of rs12979860 with treatment response in 231 Caucasians.Patients with the CC genotype had a six-fold greater rate of SVR in comparison to CT and TT genotypes.In all studies, IL28B genotypes were strongly associated with response to HCV treatment with Peg-IFN+RBV.Notably, the multivariate analysis revealed that the IL28B genotype was the strongest predictor of HCV treatment response [15].
According to our knowledge, no study has been done on the association of IL28B rs8099917 polymorphism and the treatment response of HCV genotype 4 with patients treated with Sovaldi, Daklinza, and Ribavirin.In this study, we were trying to demonstrate the role of IL28B rs8099917 gene polymorphism among treated patients of chronic HCV infection upon using the new combination of Sovaldi, Daklinza, and Ribavirin as a standard new treatment strategy in Egypt.The results showed that the TT genotype of IL28B (rs8099917) was associated with high rates of SVR and the G allele was associated with a high risk of developing relapse and non-response.

HCV patients and blood samples collection
The samples were collected as a cross-sectional study included chronic HCV patients' candidates for treatment with combined Sovaldi, Daklinza, and Ribavirin therapy.The samples were collected under a protocol approved by the Faculty of Medicine Ethical Committee Review Board, Tanta University, Tanta, Egypt (36264PR184/4/23).Patients were attending Mansoura health insurance hospital (Ibn Luqman Hospital) during the period from 2019 through 2021.The study included one hundred chronic hepatitis C patients with confirmed HCV infection, who had received the specific combination therapy of Sovaldi, Daklinza, and Ribavirin and possessed comprehensive medical records.Individuals with co-infection of other hepatitis viruses, prior HCV treatment, severe liver disease, severe immunosuppression, pregnant or breastfeeding status, and those unable to provide informed consent were excluded from this study.The hundred patients were followed up for the treatment response.Ten ml of venous blood was collected from each patient under complete aseptic conditions.Five milliliters were used for anti-HCV antibodies by ELISA and routine chemical analysis.The remaining 5 ml was distributed in small aliquots and stored at −70°C until qualitative and quantitative HCV RNA was used for HCV and IL28B genotyping.

General investigations
Liver function tests, Kidney function tests, and HCV viral load (HCV RNA) were performed during this study through clinical pathology labs of the faculty of medicine, Mansoura University.Complete blood count, leucocytic count, hemoglobin concentration, and platelet count were determined using Sysmex 800 auto-analyzer (Sysmex Europe GmbH, Germany) following the manufacturer's guidelines.Albumin, bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and uric acid were determined using kits purchased from Spinreact (Spain), and Diamond Diagnostics (Germany).The analyses have been done using Hitachi 704 auto-analyzer (Boehringer Mannheim, Germany) following the manufacturer's guidelines.

HCV viral load
HCV viral load (HCV RNA) was detected by qRT-PCR using Cobas Amplicor Auto-Analyzer (Roche Molecular Systems, USA), and Qiagen one-step RT-PCR kit (Qiagen, Germany) following the manufacturer's guidelines.The kit contains optimized components that allowed both reverse transcription and PCR amplification in a one-step reaction.The viral RNA was extracted from serum using the GeneJET Viral DNA and RNA Purification kit (Thermo Fisher Scientific, USA) based on the manufacturer's guidelines.The reaction was carried out by specific primers, 5'-AGACGTATTGAGGTCCATGC-3' (sense) and 5'-CCGCAGCGACGGTGCT GATAG-3' (antisense), designed based on core region sequences of all HCV genotypes to amplify an NS5A region.As an internal control, human GAPDH gene expression levels were measured using primers 5'-GCCATCAATGACCCCTTCATT -3' (sense) and 5'-TCTCGCTCCTGGAAGATGG -3' [16].

HCV genotyping using multiplex PCR
Patient samples containing HCV RNA were collected, and the viral RNA was isolated using QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacture protocol.Specific primers were designed to target the six known HCV genotypes (Table 1).The PCR master mix, containing Taq polymerase, PCR buffer with 3 mM MgCl2, and 400 mM of each dNTP, was prepared using Taq PCR Master mix (Qiagen, Germany).The PCR reaction was set up using the extracted HCV RNA as the template, and the specific primers for each genotype were added to the PCR master mix.The thermal cycling conditions were optimized for multiplex PCR amplification.After PCR amplification, the products were separated and visualized using agarose gel electrophoresis, with the gel stained using a DNA-specific dye (ethidium bromide), and the resulting bands observed under ultraviolet (UV) light.

IL28B genotypen
The DNA was extracted from the blood using the G-spin Total DNA Extraction mini kit (iNtRON Biotechnology, Korea).IL28B genotyping was performed by real-time PCR with ABI Prism 7000 sequence detection system (Applied Biosystems, USA) using IL28B rs8099917 real-time PCR genotyping kit (DNA-Technology, Russia).The required number of 0.2 mL PCR tubes was marked for each genotype to be tested (one tube for each sample to be tested and one extra for negative control.The mixture of PCR-buffer and Taq-AT-polymerase was prepared and added into one tube: 10 μL × (N + 1) of PCR-buffer, 0.5 μL × (N + 1) of Taq-AT-polymerase (N: number of the marked tubes including negative control).The tube was vortexed for 3-5 seconds, then spun for 1-3 seconds to collect the drops.Ten μL of PCR buffer and Taq-ATpolymerase mixture were added to each PCR tube.Five μL of DNA sample followed by one drop (20 μL) of mineral oil (purchased within the kit) were added into corresponding PCR tubes.Five μL of distilled water was used as a negative control instead of DNA samples.The tubes were spun for 1-3 seconds to collect the drops.The tubes were set to the real-time PCR instrument, then adjusted to the specified test «IL28B:rs8099917_T>G.The number and types of samples including negative controls were specified.The position of the strips was defined in the software interface according to the position they were set in the thermal block.PCR was running.
The PCR and post-PCR analysis are operated by software and held in automatic mode [17].

Statistical analysis
Data entries were performed using the Excel program (MS, Office, 2023, USA).Data were analyzed using the statistical package of social science (SPSS v. 26, USA).The quantitative data were expressed as means and standard deviations (SDs).A one-way analysis of variance (ANOVA) procedure test was used to compare the continuous variables of two different groups of patients at p less than 0.05.The qualitative data were expressed as numbers and percentages.The chi-square or Fisher exact tests were used to find the association between variables of qualitative data.SNP allele frequencies were tested from Hardy Weinberg equilibrium (HWE).Multivariate logistic regression analysis was performed using data in univariate analysis with a p-value less than 0.01.Odds ratios (ORs) were calculated and were given within a 95% confidence interval (CI).p-value less than 0.05 was considered statistically significant at CI 95%.

HCV viral load, biochemical and hematological data after the treatment course
The presented data in Table 2 showed that one hundred chronic hepatitis C patients, 81 males and 19 females with an average age of 41 years were under treatment scheduled for Sovaldi, Daklinza, and Ribavirin.After the treatment course, the patients were subjected to traditional biochemical and hematological analysis of chronic hepatitis C infection.The treatment outcomes and the viral load value (IU/ml) classified them into three main groups: responders (21 patients) with a viral load value of 8.94 IU/ml, relapsers (23 patients) with a viral load value of 6.07 × 10 4 IU/ml, and non-responders (56 patients) with a viral load value of 7.6 × 10 5 IU/ ml.Only ALT analysis showed a significantly lower value in responder groups compared to relapsers and non-responder ones.However, we could not recognize any significant differences (p < 0.05) in the leucocytic count, hemoglobin concentration, albumin concentration, bilirubin concentration, AST value, creatinine concentration, uric acid concentration, and platelet count between the three studied groups.

Distribution of IL28B (rs8099917) genotypes and alleles in the three studied groups
The distribution analysis of IL28B (rs8099917) genotypes and alleles in the three studied groups showed that the G allele was associated with a higher risk to develop relapsers and nonresponders.When testing each of the nonresponders and relapsers separately, the G allele was associated with a higher risk to develop non-response.However, no significant association was determined between IL28B genotypes, and the risk of relapse development compared to the responder.A high frequency of TT genotype was detected among responders, and it was significantly associated with SVR to the treatment.The numbers of patients with IL28B GG genotype in responders, relapsers, and non-responders were 3, 1, and 7 respectively.The numbers of patients with IL28B GT genotype in responders, relapsers, and non-responders were 7, 12, and 27, respectively.The numbers of patients with IL28B TT genotype in responders, relapsers, and non-responders were 11, 11, and 21, respectively (Figure 2).

Differences in HCV viral load according to IL28B genotypes in the three studied groups
The viral load in responders was significantly lower than those in relapsers and nonresponders.Patients.IL28B genotypes TT showed high prevalence in responder groups compared to other genotypes GG and GT.
Whereas genotypes GG and GT represented high accounts in relapsers, and nonresponders compared to responders.The viral load in patients with IL28B genotypes GT was significantly higher in non-responder than relapsers and responders.This data suggested that the G allele might be associated with the risk of treatment failure with Sovaldi, Daklinza, and Ribavirin (Figure 3).

Hardy Weinberg equilibrium of IL28B (rs8099917) genotypes
The Hardy-Weinberg equilibrium (HWE) states that allele and genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary influences.These influences include mutation, migration, and selection.Hardy-Weinberg genotype frequencies are used to test the presence of a systematic difference in allele frequencies for IL28B (rs8099917) genotypes in the three studied groups of patients.Applying HWE revealed that IL28B genotypes in responders, relapsers, and non-responders' groups were independent, and there was no evidence to accept the assumption of HWE in the selected samples (HW p < 0.05) (Table 3).

Multivariate logistic regression analysis with failure of treatment as reference category
Multivariate logistic regression analysis with treatment failures as a reference category was applied to the viral load, age, albumin concentration, bilirubin concentration, ALT value, AST value, creatinine concentration, uric acid concentration, leucocyte counts, Hb concentration, Platelets counts, and RBCs counts as covariates.The three groups were used as the main factor, and IL28B was used as a dependent factor.The analysis revealed that the IL28B GG genotype and the G allele were dependent predictors for non-response to treatment.The collected results recorded no significant differences (p < 0.05) regarding all used covariates (Table 4).

Discussion
This study involved 100 patients with chronic hepatitis C who received combination treatment with Sovaldi, Daklinza, and Ribavirin.Patients were divided into three groups based on treatment outcomes and viral load values: responders (21 patients), relapsers (23 patients), and nonresponders (56 patients).The study population consisted of 81 males and 19 females with an average age of 41 years.Analysis of traditional biochemical and hematological markers did not reveal any significant differences between the three groups.However, a significant difference was found in ALT value, with responders showing a significantly lower value compared to relapsers and non-responders.This suggests that ALT value may be a useful predictor of treatment response in patients with chronic hepatitis C. Similar results have been shown in other studies, where the AST/ALT ratio was found to be associated with liver fibrosis progression and cirrhosis [18,19].PCR analysis with specific primers to detect different HCV genotypes showed a high prevalence of HCV genotype 4, with 93 patients having this genotype.Of these, 38 were responders, 19 were relapsers, and 36 were non-responders.In contrast, only seven patients had genotype 1, with three responders, one relapser, and three non-responders.The study was unable to detect the rest of the genotypes using the specific primers.These findings suggest that genotype 4 is more prevalent than genotype 1 and the other genotypes in the study population and that there may be differences in treatment response based on genotype.This data agrees with a previous study conducted by Shemis et al.,  [20] who reported a high prevalence of HCV genotype 4 in the Egyptian population, with a percentage of 92.86%.The other genotypes found in the study were genotype 1a, genotype 1b, and genotype 3a, with prevalence percentages of 1.43%, 1.43%, and 4.28%, respectively.Analysis of the distribution of IL28B (rs8099917) genotypes and alleles in three groups of patients showed that the G allele was associated with a higher risk of developing relapse or non-response to treatment.This suggests that patients with the G allele may be less likely to respond to treatment.Multivariate logistic regression analysis found that the IL28B GG genotype and G allele were predictors of non-response to treatment, indicating that patients with these genotypes or alleles are more likely to be non-responders.Interestingly, a high frequency of the TT genotype was found among responders, and it was significantly associated with SVR to treatment.Additionally, viral load levels were correlated with treatment response, with responders having significantly lower viral load levels than relapsers and non-responders.The study also found no significant differences regarding covariates, such as age, leucocytic count, hemoglobin concentration, albumin concentration, bilirubin concentration, AST value, ALT value, creatinine concentration, uric acid concentration, and platelet count, indicating that these factors were not associated with treatment response.The IL28B genotype frequencies in three groups of patients did not conform to the Hardy-Weinberg equilibrium principle, indicating a significant difference between observed and predicted genotype frequencies.This suggests that evolutionary forces may be affecting the IL28B genotype frequencies in these patient populations, or that the population samples may not be sufficient for accurate analysis.
Previous studies have shown that IL28B polymorphisms can significantly impact virological response to HCV treatment, particularly with interferon-based therapies [10,21].Patients with certain IL28B genotypes, such as the TT genotype, have a higher likelihood of achieving SVR compared to those with other genotypes [11,22].These differences in IL28B genotype distribution may partly explain observed racial and ethnic disparities in HCV treatment outcomes.However, the exact influence of IL28B polymorphisms on treatment response is not fully understood, and further research is needed.
Our findings are also consistent with other studies that have found associations between IL28B genotype and treatment outcomes in patients with hepatitis C virus (HCV) infection.For example, Dong et al. (2015) found that patients with the CC genotype of rs12979860 were more likely to achieve an SVR to treatment than those with the CT or TT genotype [23].Another study by Chayama and Hayes (2013) found that patients infected with HCV genotype 1 were significantly more likely to achieve a sustained virological response with pegylated interferon-α plus ribavirin combination therapy if they had a common variant in the IL28B locus (rs12979860 C/C or rs8099917 T/T) [24].
Further research is needed to confirm these findings and explore the underlying mechanisms of IL28B in treatment response.The study had several limitations.First, due to the limited sample size, the study was unable to divide patients with non-G4 genotypes into subgroups to further analyze the frequencies of IL28B SNPs in different HCV genotypes.Second, it is important to note that the study did not identify which specific evolutionary forces are responsible for the deviation from HWE.Further research is needed to investigate the potential role of mutation, migration, or selection in driving the observed differences in IL28B genotype frequencies among patient groups.Third, this study did not assess the association between the IL28B genotype and histological evidence of liver inflammation in patients.Future research should explore this correlation to gain a better understanding of the role of IL28B SNPs in liver inflammation.Fourth, the study only enrolled adult patients with chronic HCV infection, and as such, the role of IL28B SNPs as a predictor of viral response in children with HCV infection remains undetermined.Further pediatric studies are needed in this field.

Conclusion
Overall, this study provides important information about the potential usefulness of ALT value as a predictor of treatment response in patients with chronic hepatitis C.However, it is important to note that further research would be needed to confirm these findings and to explore the underlying mechanisms of the observed differences in ALT value among the patient groups.The obtained results provide evidence for the potential role of the IL28B genotype as a predictor for treatment response in patients with the given condition and highlights the importance of considering genetic factors in personalized medicine approaches.The study found that the G allele of the IL28B gene (rs8099917) was associated with a higher risk of developing non-response and relapse in patients undergoing treatment.However, the TT genotype was significantly associated with SVR among responders.On the other hand, the study had several limitations, including limited sample size, and lack of assessment of histological evidence of liver inflammation.Future studies with larger sample sizes and more comprehensive assessments may be needed to better understand the role of IL28B SNPs in the treatment outcomes of patients with chronic HCV infection.

Figure 1 .
Figure 1.Distribution of HCV genotypes in the three studied groups of patients.We could not detect the rest of the genotypes.

Figure 2 .
Figure 2. Distribution of IL28B genotypes in the three studied groups of patients.

Figure 3 .
Figure 3. Variation of baseline HCV viral load in the three studied groups of patients.Asterisks represent a significant difference between the selected groups using one-way ANOVA at p < 0.01.

Table 1 .
The primers specific to the six HCV genotypes.

Table 2 .
Hepatitis C viral load, biochemical and hematological data in the three studied groups after the treatment course.

Table 3 .
Hardy Weinberg equilibrium of IL28B (rs8099917) genotypes in the three studied groups of patients.
N: number of selected patients, df: degree of freedom, HWp: Hardy Weinberg probability.

Table 4 .
Multivariate logistic regression analysis with failure of treatment as reference category.
aThe reference category is non-responder.b This parameter is set to zero because it is redundant.P: probability.