Genotypes of Hepatitis C Virus and Efficacy of Direct-Acting Antiviral Drugs among Chronic Hepatitis C Patients in a Tertiary Care Hospital

Hepatitis C virus (HCV) chronic infection is a major causative factor for several chronic liver diseases, including liver cirrhosis, liver cell failure, and hepatocellular carcinoma. The HCV has seven major genotypes. Genotype 4 is the most prevalent genotype in the Middle East, including Saudi Arabia, followed by genotype 1. The HCV genotype affects the response to different HCV treatments and the progression of liver disease. Currently, combinations of direct-acting antiviral drugs (DAAs) approved for the treatment of HCV achieve high cure rates with minimal adverse effects. Because real-world data from Saudi Arabia about the efficacy of DAAs are still limited, this study was conducted to assess the effectiveness of DAAs in treating patients with chronic hepatitis C and to identify the variables related to a sustained virologic response (SVR) in a real-world setting in Saudi Arabia. This prospective cohort study included 200 Saudi patients with chronic HCV who were 18 years of age or older and had been treated with DAAs at King Abdul-Aziz Specialized Hospital in Taif, Saudi Arabia, between September 2018 and March 2021. The response to treatment was assessed by whether or not an SVR had been achieved at week 12 post treatment (SVR12). An SVR12 was reached in 97.5% of patients. SVR12 rates were comparable for patients of different ages, between men and women, and between patients with and without cirrhosis. In addition, the SVR12 rates did not differ according to the infecting HCV genotype. In this study, the presence of cirrhosis and the patient’s gender were independent predictors of who would not reach an SVR12 (known here as the non-SVR12 group) according to the results of univariate and multivariate binary logistic regression analyses based on the determinants of SVR12. In this population of patients with chronic HCV infection, all DAA regimens achieved very high SVR12 rates. The patients’ gender and the presence of cirrhosis were independent factors of a poor response.


Introduction
Chronic infection with the hepatitis C virus (HCV) is a significant risk factor for developing cirrhosis, liver cell failure, and hepatocellular cancer [1]. In addition, extra-Saudi Arabia are still limited, data collected from different ethnic groups are insufficient. This study was carried out to evaluate the efficacy of DAAs in treating chronic hepatitis C patients and to identify the factors associated with an SVR in a real-life experience from a large tertiary care hospital in Taif, Saudi Arabia. The study also aimed to report the frequency of HCV genotypes among the studied patients.

Materials and Methods
This prospective cohort, single-center study included 200 chronic HCV-infected Saudi patients (119 male and 81 female) who were 18 years of age or older and had been treated with DAAs at King Abdul-Aziz Specialized Hospital, a large tertiary care hospital in Taif, Saudi Arabia, between September 2018 and March 2021.
Inclusion criteria: This study included both treatment-naïve patients (individuals who had never been treated for HCV before) and treatment-experienced patients (those who had been treated before).

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A patient who met the following criteria was eligible for the study: 1. Positive HCV-RNA; 2.
Age ≥ 18 years. • Exclusion criteria: the following patients were excluded from the study: 1. Pregnant or breastfeeding women; 2.
Patient with systemic malignancy except after 2 years of disease-free interval; 5.
Patients with concurrent hepatitis B virus and/or HIV infection; 6.
Patients with significant illnesses such as congestive heart failure, renal failure, respiratory failure, or autoimmune diseases.
All patients were subjected to full history taking and clinical examination, abdominal ultrasonography, and routine laboratory tests, including complete blood count, international normalization ratio (INR), partial thromboplastin time (PTT), serum creatinine, serum albumin, total serum bilirubin, alanine aminotransferase (ALT), and alkaline phosphatase (ALP); gamma-glutamyl transferase (GT) and alfa fetoprotein (AFP), which were taken at the beginning of treatment (basal) and again 12 weeks after treatment ended); and HCV genotype, which was performed only at baseline. Child-Turcotte-Pugh score was calculated for each patient.
Assessment of fibrosis: The degree of fibrosis and cirrhosis was assessed using transient elastography (FibroScan): Assessment of liver stiffness using vibration controlled transient elastography is a favored and non-invasive modality. For staging of liver fibrosis, the cut-off values were F1 (>4.8) and (≤7.0 kPa), and F2 ranged between 7.0 and 9.5 kPa, while F3 equal or higher than 9.5 kPa and less than 12.0 kPa and F4 was considered if reading was ≥12.0 kPa. The XL probe was used for the examination of obese patients [31].
Both individuals who had never been treated for HCV before (treatment-naïve) and those who had been treated before (treatment-experienced) participated in the trial. Pregnant or breastfeeding women; patients with significant illnesses such as congestive heart failure, renal failure, respiratory failure, or autoimmune diseases; and patients with concurrent hepatitis B virus and/or HIV infection were excluded from this research. To assess cirrhosis and associated issues, all patients were subjected to full history taking and clinical examination in addition to a baseline abdominal ultrasound and routine laboratory investigation. The grade of liver fibrosis was assessed by using transient elastography (TE) with FibroScan.
These lab tests were done at the beginning of treatment and again 12 weeks after treatment ended: complete blood count, international normalization ratio (INR), partial thromboplastin time (PTT), serum creatinine, serum albumin, total serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (-GT). HCV genotype was performed only at baseline. Study participants were regularly monitored for 12 weeks after therapy ended. The research Ethics Committee of Taif University and King Abdul-Aziz Specialized Hospital approved this study, and each participating patient signed a written informed consent form.

Screening for HCV Infection
According to the manufacturer's instructions, patients were screened for HCV infection by MonolisaTM HCV Ag-Ab ULTRA V2 (BIO-RAD France). This assay is a qualitative enzyme immunoassay for the detection of anti-HCV antibodies and HCV capsid antigen in human serum or plasma. It uses two recombinant proteins from the non-structural region (NS3 and NS4) and a peptide from the structural region (capsid) of the HCV for the detection of the anti-HCV antibodies. A monoclonal antibody against the HCV capsid is used to detect the HCV capsid antigen.

Confirmation of HCV Infection and Quantification of HCV RNA
Identification of HCV infection was confirmed by quantitative HCV-RNA PCR using COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV Quantitative Test, version 2.0 (lower limit of detection, 15 IU/mL), according to the manufacturer's instructions. This test is used for quantifying HCV RNA genotypes 1 to 6 in human EDTA plasma or serum using the COBAS ® AmpliPrep Instrument for automated specimen processing and the COBAS ® TaqMan ® Analyzer or the COBAS ® TaqMan ® 48 Analyzer for automated amplification and detection of HCV RNA.

Determination of HCV Genotype
HCV genotype was determined by the FDA-approved Abbott RealTime HCV Genotype II assay (Abbott Molecular Inc., Des Plaines, IL, USA), according to the manufacturer's instructions. These clinical samples are processed by an RT-PCR technique that amplifies the RNA genome of HCV. In addition, at the outset of sample processing, a different RNA sequence than the HCV target sequence is injected into each specimen. For each sample, this unrelated RNA sequence is amplified alongside the target mRNA, using RT-PCR to ensure that the amplification process went well. The assay detects genotypes 1-6 and subtypes 1a and 1b using genotype-specific, fluorescent-labelled oligonucleotide probes.

The Treatment Decision
According to the AASLD-IDSA and Saudi Association for the Study of Liver Disease (SASLT) recommendations for hepatitis C, the attending physicians chose the treatment plan [31,32]. Patients were treated with one of the following treatment regimens:
The medication regimen was selected depending on genotypes, treatment history, and the presence or absence of cirrhosis. The dose of RBV for non-cirrhotic or CTP A was weight-based: 1200 mg (for patients ≥ 75 Kg) given orally daily (in two divided doses), while those >75 Kg body weight received 1000 mg orally daily (in two divided doses). For cirrhotic CTP B receiving SOF/LDV, the dose was RBV 600 mg/day, which increased by 200 mg/day every 2 weeks as tolerated. For patients with renal impairment, CrCl 30-50 mL/min 200 mg was given, alternating with 400 mg daily; and for those with CrCl < 50 mL/min and hemodialysis patients, 200 mg was given daily. For patients with baseline Hgb > 12 g, no dose adjustment was made, and RBV was discontinued if Hgb.

Assessment of Treatment Efficacy
Sustained virologic response (SVR12) was evaluated by quantitative HCV-RNA PCR at twelve weeks after the end of treatment.

Sample Size Calculation
To evaluate the difference between treatment groups (INF and SOF) and subgroups (INF; INF24, INF48, and SOF; SOF/SIM and SOF/DAC), two-way analysis of variance was performed. A total sample size of 181 was deemed sufficient to detect an effect size of 0.295 at a power of 0.95 (95%) at a partial eta squared of 0.08 at a significance level of 0.05. Taking into account a non-response rate of 10.0%, the sample size was increased to 199 individuals, i.e., 200 patients were applied. Sample size was calculated using power analysis using G*power version 3.9.1.6 for Mac OS.

Statistical Analysis
All collected data were tabulated and statistically analyzed using the following statistical tests. Descriptive data analysis was in the form of percentages, and mean and data were expressed as mean± standard deviation (SD) or number and percentages (%) as appropriate. The Pearson chi-square test or Fisher's exact test were used for group comparisons of categorical variables. Two-sample t-tests were used for all independent variables for numerical data. For categorical data, all independent factors were subjected to univariate binary logistic regression analysis, and the odds ratio with 95% confidence intervals was computed for VR evaluation. All statistical analyses were performed using the computer program SPSS software for windows version 26.0 (Statistical Package for Social Science, IBM Corp, Armonk, NY, USA). A two-tailed p-value < 0.05 was considered statistically significant.

Baseline Characteristics of the Studied Population
The study included a total of 200 chronic hepatitis C patients with complete SVR12 data. Of these patients, 119 (59.5%) were treatment-naïve, and 81 (40.5%) were treatmentexperienced. Sixty-one patients received interferon/ribavirin therapy (24 to 48 weeks), 11 patients received sofosbuvir/simeprevir therapy (12 weeks), and 9 patients received sofosbuvir/daclatasvir therapy (12 weeks). The evaluated patients were 18 to 95 years of age (mean ± SD 53.17 ± 15.71 years). Of the 200 patients studied, 119 (59.5%) were male, and 81 (40.5%) were female. Liver cirrhosis was diagnosed in 70 patients (35%), and all cirrhotic patients were compensated; 44 patients had a Child-Pugh score of B, and 24 patients had a Child-Pugh score of A. The main FibroScan score of the studied patients was 8.7 ± 4.1 kPa. Regarding associated comorbidities, most of our patients were overweight, with a mean body mass index of 28.8 ± 6.2. Approximately one-third of our patients were hypertensive, and approximately 39% had been diagnosed with diabetes; 10% had a history of coronary artery disease, and chronic kidney disease had been diagnosed in 23 patients (11.5%).

Treatment Efficacy
Overall, an SVR12 was attained in 195 patients (97.5%). As shown in Table 3, in terms of the baseline demographic and clinical characteristics, there was no statistically significant difference between the patients who achieved an SVR12 (SVR12 group) and those who did not achieve an SVR12 (non-SVR12 group) apart from the rate of cirrhosis (p = 0.032) and the distribution of HCV genotypes (p = 0.001). Similarly, the two groups did not differ in terms of the prescribed antiviral drugs (p = 0.348 and p = 0.755 for the antiviral drug combinations and concomitant RBV therapy, respectively).

Predictors of SVR12
The patient's age and gender, treatment history, presence of liver cirrhosis or chronic kidney disease, genotype, and antiviral drug regimen were assessed as potential predictors of the SVR12. Results of the univariate binary logistic regression analysis revealed a nonsignificant effect (p > 0.05) of all of the explanatory variables on the outcome. Further-

Predictors of SVR12
The patient's age and gender, treatment history, presence of liver cirrhosis or chronic kidney disease, genotype, and antiviral drug regimen were assessed as potential predictors of the SVR12. Results of the univariate binary logistic regression analysis revealed a nonsignificant effect (p > 0.05) of all of the explanatory variables on the outcome. Furthermore, the multivariate binary logistic regression analysis showed a nonsignificant effect (p > 0.05) of all of the explanatory variables on the outcome except for gender and the presence of cirrhosis (p = 0.045 and p = 0.036, respectively) ( Table 5).

Discussion
Hepatitis C virus infection is a serious challenge to global health with a significant economic impact. Chronic HCV infection is one of the main causes of liver cirrhosis, liver cell failure, and hepatocellular carcinoma. It is the most common indication for liver transplantation worldwide [33,34]. The prevalence of positive HCV antibodies in Saudi Arabia is approximately 0.7%, and the most prevalent genotype is GT4, followed by GT1 [17,35].
Prior to 2011, pegylated interferon alpha and RBV were the recommended antiviral treatments for 24 to 48 weeks. This regimen resulted in a moderate SVR and was associated with multiple side effects [36]. The introduction of the DAA-based regimens achieved high cure rates with minimal adverse effects, better tolerability, and shorter treatment duration [22,28]. Further, treatment with DAA in patients with chronic HCV infection has a positive effect on the bioelectrical brain activity, with an increase in the amplitude of evoked potentials indicating an improvement in the activity of the cerebral cortex, and this improvement was correlated with the neuroimaging parameters [29][30][31][32][33][34][35].
Long-term clinical outcomes and the health-related quality of life may increase with SVR achievement because of the decreased risk of liver disease progression [37].
In the current investigation, we monitored a total of 200 patients who were treated at a single medical facility and were found to have HCV infections. More than half of our patients had GT4 (54%), followed by GT1(32%) including subtypes a and b; this finding was consistent with the overall genotype prevalence among HCV-infected patients in Saudi Arabia [17,38]. Alarfaj et al. [39] reported in a study carried out in Riyadh, Saudi Arabia, that the most observed genotype was GT4 (63.7%), followed by GT1 (24%). According to Bawazer et al. [17], GT4 was the most prevalent genotype in Saudi Arabia, representing 65% of infections, followed by GT1 in 23% of cases. Younger age groups showed an apparent reduction in the prevalence of GT4 but had an increased rate of GT1. Moreover, several review publications and meta-analyses on the distribution of HCV genotypes reported that GT4 predominated in Middle Eastern nations, notably Egypt, Iraq, Saudi Arabia, and Syria, with rates of 86%, 60%, 56%, and 57%, respectively [40,41]. In contrast to our findings, a recent study from Bahrain revealed an increasing tendency of HCV GT1 compared with GT4 in the studied population [29].
Patients in the current study achieved high rates of SVR12 (97.5%), with therapeutic failure occurring only in five cases (2.5%). The high success rates in this study were supported by the results of Alarfaj et al. [39], who reported an SVR12 rate of 95.9% in Saudi Arabia, which is consistent with real-world data reported in Middle Eastern countries [41][42][43].
There was no statistically significant difference in SVR12 rates between age groups, genders, and individuals with or without chronic renal disease, liver cirrhosis, or genotypes. However, there was a statistically significant difference in SVR12 rates between treatmentnaïve and treatment-experienced individuals (p = 0.018). These findings were consistent with those of Yang et al. [44], who reported no statistically significant variations in SVR rates in patients with various genotypes. These results were also in agreement with the findings of Kamal et al. [45], who studied the effectiveness of DAA-based regimens in elderly Egyptian patients with chronic HCV infection and found that all of these regimens were well-tolerated, safe, and highly successful even in patients aged 75 years or beyond. They found that age did not influence the effectiveness of DAA treatment.
Regarding patients with chronic kidney disease, the results of this study were in concordance with previous studies in the literature [46,47]; they found that treatment with OBV/PTV/R and DSV with or without RBV was safe and effective in HCV-infected patients with chronic renal disease.
There was no statistically significant difference in SVR12 rates between patients treated with SOF-based regimens and those treated with other regimens regardless of whether they also used RBV. This was in line with previous research and real-world studies from all over the world, showing similar outcomes with high SVRs across a wide range of regimens, genotypes, and durations of therapy with or without the addition of RBV and in both treatment-naïve and treatment-experienced patients [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48]. Additionally, the previous use of an antiviral medication by the patient did not affect the SVR rate. Therefore, based on these findings, one can say that the proper DAA-based regimens can yield good curative outcomes.
When looking at the patient's age and gender, whether patients were treatment experienced or treatment-naïve, whether patients had chronic kidney disease or not, and the treatment regimens used and whether RBV was included in them or not, the current study was unable to show a significant difference between the SVR12 group and non-SVR group. However, there were significant differences between the SRV12 and non-SVR12 groups in terms of genotype groups, the presence of liver cirrhosis, and the Child-Pugh score of cirrhotic patients (p = 0.032, p = 0.02, and p = 0.001, respectively). Patients with cirrhosis and a Child-Pugh score of B and GT4 and GT1b had significantly lower SVR12 rates compared with patients without cirrhosis, a Child-Pugh score of A, and viral genotypes other than GT4 and GT1b, according to the results of univariate and multivariate binary logistic regression analyses based on the determinants of the SVR12 (p = 0.036). In our research, the presence of cirrhosis and a Child-Pugh score of B stood out as significant predictors of the failure to achieve an SVR12. SOF with RBV treatment resulted in an SVR in only 71.2% of patients with HCV-related cirrhosis, and more than 5% of the patients discontinued the medication owing to side effects; this was consistent with recent Egyptian research that included a large number of patients [49]. Additionally, the findings indicated a gender difference that was statistically significant (p = 0.045). In general, SVR12-associated factors were inconsistent between clinical trials and real-world investigations, making it difficult to compare the efficacies of different DAA combinations. Thus far, baseline factors (i.e., liver cirrhosis, past treatment experience, infecting HCV genotype, high viral load, increased liver enzymes, and natural polymorphisms in nonstructural HCV genes that limit drug sensitivity) have been linked to poorer SVR rates [50].
The limitation of this study was the limited number of cases (i.e., only 200 cases) compared with the prevalence rate of 1.2% in the overall population of Saudi Arabia. Furthermore, the investigation was carried out at a single center. Additionally, there was no randomization in the patient assignment to treatment [51,52]. This was also confounded by the fact that there was great heterogeneity in the regimens used. Patients younger than 18 years and those with decompensated cirrhosis were not included in the study.

Conclusions
The current study verified that DAAs were successful in treating Saudi HCV patients and achieved an SVR in 97.5% of the patients in a real-world context. The presence of cirrhosis, a Child-Pugh score of B, and viral genotypes GT4 and GT1b were significant predictors of the failure to achieve an SVR12.