Serum Vitamin D as a Predictor to the Response of Interferon Therapy in HCV Genotype 4

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1 Med. J. Cairo Univ., Vol. 83, No. 2, September: 1-6, Serum Vitamin D as a Predictor to the Response of Interferon Therapy in HCV Genotype 4 OLFAT G. SHAKER, M.D.*; ESAM A. MOHAMED, M.Sc.**; GAMAL ESMAT, M.D.***; SAMAR A. MARZOUK, M.D.* and AMR A. ZAHRA, M.D.** The Departments of Medical Biochemistry*,** and Tropical Medicine***, Faculty of Medicine, Cairo*,*** and Fayoum** Universities, Egypt Abstract Background and Aim: Chronic infection with Hepatitis C Virus (HCV) is a huge problem both globally and at the level of the individual patient. We aimed to determine the incidence of vitamin D deficiency in patients with chronic hepatitis C. In addition, we evaluated associations of vitamin D (Vit D) serum levels with the virologic response of interferon-alfa based treatment in patients chronically infected with HCV in Egypt. Subject and Methods: Blood samples were taken from 206 patients who are suffering from chronic hepatitis C disease divided into responders (n=126) and non-responders (n=80) according to their initial response to treatment with normalization of aminotransferases (ALT and AST) levels and clearance of the virus denoted by negative HCV-RNA by PCR after 6 months of receiving treatment. Also 100 blood samples from controls were taken. The following were done: History taking, general examination, liver function tests, hepatitis markers, HCV quantitation by real time PCR and quantitation of Vit.D level by ELISA. Results: There was significant differences between responders and non responders to interferon therapy of chronic hepatitis C patients before treatment as regards the mean values of Vit D (p=0.001). Stepwise multivariate logistic regression showed that Vit D (p=0.002) was found to be significant predictor for response to interferon therapy of hepatitis C patients. Conclusion: Serum levels of Vitamin D is a significant predictor for response to interferon therapy of hepatitis C patients genotype 4. Key Words: Hepatitis C Vitamin D Interferon. Introduction HEPATITIS C virus (HCV)-related chronic liver disease is a major health problem and a key issue in antiviral research. Approximately 170 million people are infected with HCV worldwide; chronic HCV infection commonly induces immune reactive Correspondence to: Dr. Esam A. Mohamed, esamali58@yahoo.com inflammation, which results in continuous liver tissue damage and progression of liver fibrosis to cirrhosis [1]. Part of the problem relates to the fact that the current standard of care treatment for HCV, combination of pegylated interferon-alpha (PegIFNa) and Ribavirin (Rib), is expensive, associated with significant side effects, and results in only a 50% rate of sustained virological response (SVR) [2]. Predicting the likelihood of response to treatment prior to initiating therapy (or soon after starting therapy) would be very useful [3]. Infection with different HCV genotypes is a main predictor of virologic treatment response. This is evidenced by Sustained Virologic Response (SVR) rates achieved by standard treatment of only 40-50% in patients infected with genotypes 1 or 4 in contrast to SVR rates of approximately 80% in those infected with genotypes 2 or 3 [4]. In addition to this and other viral parameters, there is increasing evidence on the importance of host factors contributing to the high rate of viral persistence as well as to the outcome of antiviral therapy [5,6]. In particular, innate and adaptive immune mechanisms have been identified to be crucial for spontaneous HCV clearance as well as for the success of antiviral therapy [7]. 1, 25-dihydroxyvitamin D, the activated hormonal form of vitamin D, is an important immune modulator that has an impact on innate and adaptive immune pathways [8]. With respect to its immune regulatory function, vitamin D deficiency is associated, for example, with autoimmune diseases, lethality during septic shock, or cancer [9]. The relationship between vitamin D and liver disease is reciprocal. Liver and kidneys are required to activate vitamin D by 25-and 1-alpha-hydroxy- 1

2 2 Serum Vitamin D as a Predictor to the Response of Interferon Therapy lation, respectively. Therefore, liver diseases including chronic hepatitis C may be responsible for low serum levels of 25-hydroxyvitamin D3 (25 (OH) D3) [10]. Most of the biological activities of activated vitamin D are mediated via a nuclear Vitamin D Receptor (VDR), which serves as a ligand-activated transcription factor. Polymorphisms within the VDR gene may therefore, result in defective gene activation and consecutive impaired effector functions of vitamin D, such as calcium homeostasis, cell differentiation, or immune regulation [11]. In the present study, we aimed to detect serum level of Vitamin D in patients with chronic hepatitis C. In addition, we evaluated associations of vitamin D serum levels with the virologic response of interferon-α based treatment in patients chronically infected with HCV genotypes 4. Subjects and Methods This study was conducted on 206 patients and 100 apparent healthy individuals as controls. Patients were selected from El-Fatemeya Hospital, Cairo, as naive patients not treated before; both males and females were included. Patients treated with PEG-IFN- α 2b, at a dose of 1.5µg/kg subcutaneously every week plus ribavirin at a dose of mg/day, according to the patient's body weight for 48 weeks. Quantitative HCV RNA measurement was performed at baseline, week 4, 12, 24, 48, and during follow-up as appropriate by a quantitative reversetranscription PCR-based assay. Treatment was discontinued in patients who were HCV RNA positive at week 24. Serum was used for quantitation of 25 (OH)- vitamin D by ELISA (Immun Diagnostik, Germany). The assay utilizes of a competitive ELISA technique with a selected monoclonal antibody recognizing 25 (OH)-vitamin D. Results This study was conducted on 306 subjects with their ages ranging between years. They were classified into 2 groups: Group I: Included 206 HCV chronically infected Egyptian patients, they were 70 (34.0%) females and 136 (66.0%) males patients aged ± (mean±sd) years. Patients were treated with PEG- IFN alfa 2b 1.5µg/kg weekly and ribavirin ( mg/day) for 48 weeks. According to response to treatment patients were classified into two subgroups: 1-Respoders: Who had initial response to treatment with normalization of aminotransferases (ALT and AST) levels and clearance of the virus denoted by negative HCV-RNA by PCR after 6 months of receiving treatment and those who completed treatment for another 6 months and remained negative after completion of the treatment course. They were 48 (38.1%) females and 78 (61.9%) males patients aged ±8.800 (mean ± SD) years. 2- Non-responders: Who received treatment for 6 months and failed to clear the virus and gave positive HCV-RNA by PCR. They were 22 (27.5%) females and 58 (72.5%) males patients aged 43.25±8.628 (mean±sd) years. Group II: Included 100 healthy individuals without HCV infection. They were 62 (53.3%) males and 38 (46.7%) females aged ±8.109 (mean±sd) years. There was a highly statistically significant difference between hepatitis C patients when compared to control subjects as regards the mean values ±SD of ALT, AST, D. Bilirubin, Alkaline phosphatase (ALK), Albumin (ALB) as well as Vit D (p<0.001 each). Meanwhile, there was no statistically significant difference as regards the mean values±sd of age, sex, T. Bilirubin and alpha fetoprotein (AFP) (Table 1). The characteristics of HCV patients in this study showed a highly statistically significant difference between responders and non responders to interferon therapy before treatment as regards the mean values ± SD of AST (p=0.009), D. bil, AFP, Vit D and fibrosis (p=0.001 each). Meanwhile, there was no statistically significant difference as regards age, sex, ALT, T. bil, ALK, ALB as well as PCR (Table 2). Table (3) showed a significant differences in the prevalence of vitamin D deficiency between responders and non responders to interferon therapy of HCV patients (p=0.003). Step wise multivariate analysis showed that only ALT (p=0.040), AST (p=0.009), D.bil (p= 0.013), ALB (p=0.012), AFP (p=0.001), PC (p= 0.012) and Vit D (p=0.002) were found to be significant predictors for response, (Table 4).

3 Olfat G. Shaker, et al. 3 Table (1): Demographic and biochemical data of all subjects. Variables Age (yrs) Sex: Females% Males% Hepatitis C patients N= ± (34%) 136 (66%) Controls N= ± (46.7%) 62 (53.3%) p- value ALT (U/L) 58.56± ± * AST (U/L) 54.o3± ± * T.bil (mg/dl) 0.69± ± D.bil (mg/dl) 0.32± ± * ALK (U/L) ± ± * ALB (g/dl) 4.15± ± * AFP (ng/ml) 6.04± ± Vit D (nm/l) 69.35± ± * Table (2): Comparison between demographic and biochemical data in responders and non responders to interferon therapy before treatment. Variables Responders N=126 Non responders N=80 p- value Age (yrs) 43.18± ± Sex: - Females% - Males% within group 48 (38.1%) 22 (27.5%) (61.9%) 58 (72.5%) ALT (U/L) 57.03± ± AST (U/L) 50.37± ± * T.bil (mg/dl) 0.64± ± D.bil (mg/dl) 0.25± ± * ALK (U/L) ± ± ALB (g/dl) 4.25± ± AFP (ng/ml) 3.43± ± * Vit D (nmol/l) 78.96± ± * HCV RNA (PCR) 2.23X10 6 ±4968X X10 6 ±1979X Fibrosis: - Grades 1% - Grades 2% - Grades 3% - Grades 4% 53 (41.9%) 24 (30%) 0.001* 63 (50%) 26 (32.5%) 8 (6.5%) 30 (37.5%) 2 (1.6%) 0 (0%) Table (3): Prevalence of vitamin D deficiency in responders and non responders to interferon therapy of HCV patients. Vit D (nmol/l) Responder Response Non-responder <25: 0.003* Count % within response 2 (1.6%) 10 (12.5%) 25-50: Count % within response 27 (21.4%) 36 (45.0%) 50-75: Count % within response 40 (31.8%) 16 (20.0%) > 75: Count % within response 57 (45.2%) 18 (22.5%) p Table (4): A stepwise multivariate logistic regression showing the factors predicting response to interferon therapy of HCV patients. Variables p-value Odd ratio Odds ratio (95%CI) ALT (U/L) 0.040* ( ) AST (U/L) 0.009* ( ) D.bil (mg/dl) 0.013* 4.1X10 3 ( X10 6 ) ALB (g/dl) 0.012* ( ) AFP (ng/ml) 0.001* ( ) PC 0.012* ( ) Vit D (nmol/l) 0.002* ( ) Discussion Chronic infection with Hepatitis C Virus (HCV) is a huge problem both globally and at the level of the individual patient. Part of the problem relates to the fact that the current standard of care treatment for HCV combination pegylated interferon-alpha (PegIFNa) and Ribavirin (Rib) is expensive, associated with significant side effects, and results in only a 50% rate of sustained virological response (SVR) [2]. Egypt has a high prevalence of Hepatitis C Virus (HCV) and a high morbidity and mortality from chronic liver disease, cirrhosis, and hepatocellular carcinoma. The most prevalent genotype in Egypt is 4a, which responds less successfully to treatment [12]. Predicting the likelihood of response to treatment prior to initiating therapy (or soon after starting therapy) would be very useful [3]. There is increasing evidence on the importance of host factors contributing to the high rate of viral persistence as well as to the outcome of antiviral therapy [6]. In particular, innate and adaptive immune mechanisms have been identified to be crucial for spontaneous HCV clearance as well as for the success of antiviral therapy [7]. 1, 25-dihydroxyvitamin D, the activated hormonal form of vitamin D, is an important immune modulator that has an impact on innate and adaptive immune pathways [8]. The present study revealed that there was a highly statistically significant difference between HCV patients compared to control subjects as regards the mean values ± SD of ALT, AST, D. bilirubin, ALK, ALB and Vit D ( p<0.001 each). Meanwhile, there was no statistically significant difference as regards the mean values ± SD of age, sex, T. bilirubin and AFP. Our results are consistent with Iacob et al., [13] who reported that, there was a significant difference

4 4 Serum Vitamin D as a Predictor to the Response of Interferon Therapy between hepatitis C patients when compared to control subjects as regards the mean values ± SD of ALT, AST, D. bilirubin, ALK and ALB (p<0.001 each). Also, our results are consistent with Lange et al., [14] who reported that chronic hepatitis C virus infection is associated with vitamin D. deficiency. Also, Arteh J. et al., [10] reported that vitamin D deficiency is very common (92%) among patients with chronic liver disease, and at least one-third of them suffer from severe vitamin D. deficiency (<12ng/mL). According to our study we found that there was a highly statistically significant difference between responders and non responders to interferon therapy of chronic hepatitis C patients before treatment as regards the mean values ± SD of AST (p=0.009), D. bil, AFP, PC, Vit D, and fibrosis (p=0.001 each). Meanwhile, there was no statistically significant difference as regards the mean values ± SD of age, sex, ALT, T. bil, ALK, ALB as well as PCR. These results are consistent with previous study by Everson et al., [15] who reported that advanced hepatic fibrosis is a negative predictor of SVR to therapy. At the same time Bruno et al., [16] identified that patients who have advanced fibrosis should expect a 10% reduction in SVR compared with patients who do not have advanced fibrosis. Lange et al., [14] reported that multivariate analysis revealed that female sex (p=0.013), low serum cholesterol (p=0.023), low degrees of liver fibrosis (p=0.001), and low baseline viremia (p=0.084) are independent predictors of SVR in HCV genotype 1 patients. According to our study, we found that there was a significant difference in the prevalence of vitamin D. deficiency between responders and non responders to interferon therapy of chronic hepatitis C patients (p=0.003). Our findings are coincided with the study of Lange et al., [14] who found a significant correlation of 25-hydroxyvitamin D. serum levels and virologic response rates in HCV genotype 2 and 3 infected patients. Petta et al., [17] also reported a correlation of vitamin D. concentrations and SVR rates in HCV genotype 1 patients. Bitetto et al., [18] showed that vitamin D. supplementation improves response to antiviral treatment for recurrent hepatitis C in liver transplant recipients. Southern et al., [19] showed the beneficial effect of vitamin D. supplementation on the outcome in patients with chronic HCV genotype 2-3 infection. Nimer and Abu mouch, [20] reported that adding vitamin D. to conventional peg/rbv therapy for patients with HCV genotype 2-3 significantly improves viral response. In the present study stepwise multivariate logistic regression showed that Vit D was a good predictor of viral response (p=0.002). This finding agreed with Nimer and Abu mouch, [20] who reported that vitamin D supplementation, baseline vitamin D levels, viral load, and hepatitis C genotype are independent predictors of viral response. Also Lange et al., [14] reported that, in HCV genotype 2 and 3 patients, vitamin D. serum concentrations (p=0.009) and low baseline viremia (p=0.061) were independent predictors of SVR. We approved by stepwise multivariate logistic regression analysis the followings pre aredictors of response: ALT, AST, D. bil, ALB and AFP. Our findings were confirmed by Hosogaya et al., [21] who reported that bilirubin level is decreased with the IFN therapy as it represents the level of hepatic damage or impairment of its function, however as ALB represents the protein producing activity of the liver so it is expected to increase with the IFN therapy. Our results are consistent with Di Bisceglie et al., [22] who showed that serum AFP levels decreased significantly during therapy with pegylated interferon α -2a and ribavirin. The greater decline in AFP with SVR than relapse further indicates that elevated AFP is more likely to result from inflammation, necrosis and hepatocellular injury. However, the previous results are inconsistent with some authors [23-25], while, they are consistent with Yoneda et al., [26] who reported that AST level in the SVR group was significantly lower than that in the non-svr group ( p=.012). Also, Wang et al., [27] found that AST was a significant prognostic factors of interferon response in HCV (p=0.017). We can conclude that serum levels of Vitamin D. are significant predictors for response to interferon therapy of hepatitis C patients. Competing interests: None. Funding: This study was supported by funds from the Egyptian Science and Technology Development Fund [project number 1512 to Olfat Shaker].

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6 6 Serum Vitamin D as a Predictor to the Response of Interferon Therapy