Introduction

Hepatitis C virus (HCV) is a leading cause of liver-related mortalities and morbidities and is a major health concern worldwide. With the emergence of approximately 3-4 million new cases of HCV infection every year, it is estimated that 2%-3% (130-170 million) of the world popula­tion are chronically infected 1. HCV transmission occurs principally via the exposure to contaminated blood or blood products. The most important mode of HCV transmission is needle sharing among intravenous (IV) drug users. Blood transfusion, tattooing, and sexual contacts are possible ways of spreading HCV infection 2. Although the infection is preliminary acute, about 75%-85% of infected patients fail to clear the virus within 6 months and gradually progress to chronic infection 3. Among chronically infected cases, 10%-20% of patients develop liver cirrhosis over a period of 20-30 years, and of those with cirrhosis, 1%-5% will develop hepatocellular carcinoma (HCC) annually. As a result, on a global scale, chronic HCV infection is responsible for approximately 250 000 to 350 000 deaths annually, which are mostly related to the decompensation of cirrhosis, end-stage liver disease, and HCC 4,5,6.

Diagnostic tests for HCV infection use serological assays to detect specific antibodies to HCV (anti-HCV) and molecular techniques to detect HCV-RNA in blood serum or plasma. A positive HCV antibody immunoassay test reveals an exposure to HCV; however, as patients who have spontaneously cleared the disease may produce false-positive anti-HCV results, it should be followed with a qualitative or quantitative polymerase chain reaction (PCR) test to detect the presence of HCV RNA in the blood 7,8.

Based on sequence homology analysis, at least seven major HCV genotypes and numerous distinct subtypes have been determined. Due to the genetic heterogeneity of HCV, evolution or existence of different HCV quasispecies in each patient, and the low efficient virus propagation, the development of HCV vaccine remains challenging.

The proximate goal of HCV treatment is viral eradication, which is achieved by eliminating detectable circulating HCV 6 months after treatment cessation. Between 2001 and 2011, standard treatment for chronic HCV infection was based on the use of PegIFN α-2a or 2b in combination with ribavirin, known as SOC. This treatment regimen yields an SVR in 40– 80% of cases depending on different viral and host parameters 9,10. The recently developed DAAs are consisting of protease inhibitors, NS5A inhibitors, nucleotide analog NS5B polymerase inhibitor and the non-nucleotide polymerase inhibitor 7,11,12,13. These new treatments improve SVR rates significantly in difficult cases such as genotype 1 HCV infection 13. Despite these advantages, in most of low- and middle income countries including Iran, access to these drugs is limited, mostly due to their high cost and unavailability 14. Population based epidemiological studies have estimated that the prevalence of HCV in the Iranian general population is less than 0.5%, which is relatively low 15. Among the infected patients with genotype 1a, 3a and 1b genotypes are known to be the most common genotypes 16,17,18. Available studies have showed that several host factors including host genotypes (race, age, gender, IL28B), degrees of hepatic fibrosis prior to treatment response and virological genotypes and RNA levels can be used to predict SVR rates.

HCV is an enveloped RNA virus belonging to the family Flaviviridae 19. The virus possesses an internal ribosomal entry site (IRES) at the 5′ untranslated region (UTR). The IRES is essential for the translation of viral polyprotein precursors. It is a complex RNA structure responsible for the correct assembling of ribosomal subunits at the viral start codons, which act through a mechanism that bypasses the canonical eukaryotic mRNA translation initiation. The unique structure and high sequence conservation of the 5′ UTR rendering IRES RNA represents a unique structure for drug targets.

Cobalamins, the class of compounds to which vitamin B12 belongs, are mostly stored in the human liver 20. Recent studies have reported that various liver diseases such as acute hepatitis, cirrhosis, HCC, and metastatic liver diseases are associated with major alterations in plasma vitamin B12 concentrations 21,22,23. Moreover, the results from an available study have reported that vitamin B12 plays a natural role in the negative regulation of the HCV replication cycle 24. Based on these evidences, the possible role of vitamin B12 supplementation in improving the rates of viral response to antiviral therapy in HCV carriers has been investigated. Recently, vitamin B12 supplementation in HCV carriers showed a significant improvement in response to treatment with PegIFN and ribavirin at all time-points during therapy 25. However, this study was conducted on a limited number of patients and no clinical study has investigated the implication of adding B12 to SOC in the setting of HCV treatments. The aim of this randomized clinical trial was to assess the effect of vitamin B12 addition to SOC on the viral response to antiviral therapy in patients with naïve chronic HCV infection.

Methods

Subjects

Patients with chronic HCV infection and naïve to antiviral therapy were enrolled among those referred to the hepatology clinic of Rasoul-e-Akram university Hospital between 2014 and 2016. Patients either aged <18 or >65 years, with the history of treatment course with interferon and/or ribavirin, having conditions with consequences for liver disease, such as alcoholic liver disease, HBV or HIV infection, autoimmune hepatitis, HCC, decompensated cirrhosis, as well as severe synchronous disease or contraindications to treatment (depression, psychosis, epilepsy, uncontrolled hypertension, cytopenia (haemoglobin<12 g ⁄dL, leukocyte count <3000 ⁄mm, platelet count <100 000 ⁄mm), or having severe cardiac and pulmonary disease were excluded from the study. Written consent to participate in the study was obtained from all patients. The study protocol was approved by the ethic committee of the Iran University of Medical Sciences (ethic code: Approval date; 2015-04-15, 1394/01/26-Ethics committee No; 25592) and registered by the Iranian Registry Clinical Trial (IRCT Code: IRCT Id IRCT2015041720178N3).

Results

Of the 90 chronically infected patients that referred to our hepatology clinic, 16 were excluded from the study for the following reasons: having the history of treatment with interferon ± ribavirin (n=6), concomitant hepatitis B virus infection (n=2), heavy alcohol consumption (n=1), HCC (n=1), severe depression (n=3), poorly controlled diabetes (n=1), and refused treatment (n=2). Thus, 74 eligible patients were randomly assigned to receive one of these treatment regimens: PegIFN in combination with ribavirin (SOC (or PegIFN in combination with ribavirin plus vitamin B12 injections (SOC + Vit B12).

Patient’s mean ages were respectively 44±12 and 41±10 years in SOC + Vit B12 and SOC grops. Majority of our patients were male in both groups. Dominant genotype of HCV was genotype 3 in both groups (A=19/36 and B=20/38). The treatment groups did not show significant differences regarding to demographic, laboratory, virological and fibroscan parameters (Table 1). The distributions of IL28B genotypes were not significantly different between the two treatment groups (Table 2).

Viral response during antiviral therapy, at the end of treatment and during 24 weeks follow-up was reported in Table 3. Using ITT analysis, our results showed that the rates of cEVR (88% 68%, p=0.033), ETVR (83 % . 68%, p=0.001) and SVR (80% . 68%, p=0.0001) were significantly higher in group SOC + Vit B12 compared to group SOC. However, the rate of RVR in group SOC + Vit B12 and group SOC was not significantly different (78% . 77%, p=0.097). The relapse, drop out and non-responder rates were significantly lower in group SOC + Vit B12 compared to group SOC. Viral breakthrough rate was not significantly different between two treatment groups (Table 3). Adverse events (especially in Fatigue, Headache, Fever, Flu-like, Nausea, Generalized Hair Loss, and Weight loss) were significantly lower in group SOC + Vit B12 compared to group SOC (55%. 63% P=0.0001). The results of laboratory abnormality showed that, anemia was significantly lower in group treated with SOC + Vit B12 compared to group treated with SOC alone. Besides, necessitation of dose adjustment was significantly higher in group using SOC compared to group using SOC + Vit B12 (25% 8%, P=0.0001). The dosage of PegIFN and/or ribavirin was respectively reduced in 9/38 (24%) and 3/36 (8%) patients in SOC and SOC + Vit B12 groups, mainly because of laboratory abnormalities (anaemia in SOC = 6 and A=2 cases, neutropenia in group SOC + Vit B12 =1 and SOC =1 cases and thrombocytopenia in group SOC = 1 case). Group SOC also showed significantly higher rate of treatment discontinuation compared to group SOC + Vit B12 (6/38 patients (16%) . 1/36 patients (2%), P = 0.0001). Treatment discontinuation was caused by severe anemia (1 case), severe headache (2 cases), severe flu-like symptom (1 case), major depression and weight loss (1 case), thyroid function test abnormality (1 case) in group SOC and irritability and insomnia (1 case) in group SOC + Vit B12 (Table 4). When baseline parameters of HCV genotype (1 or 3), viral load, fibrosis score and IL28B genotype (CC, TT and CT) were considered, the results of logistic regression analysis demonstrated that SVR rate was significantly higher in group SOC + Vit B12 patients who had higher viral count (>600,000 IU/ml) and IL28B CC genotype, and insignificantly higher in patients with HCV genotype 1 (Table 5).

Regarding multivariate analysis, adding vitamin B12 to treatment and genotype IL28B allele CC were considered as a prognostic factor to achieve SVR in this study (Table 6,Table 7).

Overall, the compliance with the 80/80/80 rule was > 50% and group SOC + Vit B12 was significantly more adherent to treatment regimen than group SOC (30/36 (83%) in group SOC + Vit B12 vs 26/38 (68%) in group SOC). The flow of patients according to treatment arm and HCV genotype throughout the study is summarized in figure 1 (Figure 1).

Discussion

For decades, PegIFN in combination with ribavirin was recommended as SOC for chronic hepatitis C virus infection 28. The rate of SVR in patients receiving this treatment regimen is indicated to be 40– 80%, depending on various baseline parameters, such as HCV genotype, IL28B polymorphisms, gender and HCV RNA levels 9. The introduction of DAAs has remarkably improved SVR rates in shorter duration of treatment, and with better tolerability in comparison to SOC. However, treatment with PegIFN in combination to RBV is still used in countries where DAAs are not available or are expensive. As only a fraction of patients with chronic hepatitis C infection would have access to such expensive therapies, optimizing efficacy of PegIFN and RBV combination therapy has become a research of interest over the past few years. For instance, available reports have showed that the addition of Sofosbuvir and vitamin D to the combination therapy with PegIFN plus Ribavirin can increase the rate of SVR among HCV-infected patients 29,30,31.

Direct inhibitory effects of vitamin B12 on HCV IRES-dependent translation have been indicated by experimental evidences almost two decades ago 24. Since then, several studies have reported the correlation between vitamin B12 and virological response of chronic hepatitis C infection receiving SOC 25,32,33. Considering these evidences, the aim of the current study was to evaluate the possible effects of adding vitamin B12 by injection (at a dose of 5000 microgram) to PegIFN and RBV combination therapy on the virological response in treatment-naive patients with chronic HCV.

Here we provide evidence that addition of vitamin B12 to SOC significantly improves the rate of virological response in chronically HCV infected patients. Our findings showed that patients who received vitamin B12 during therapy achieved significantly higher rates of cEVR, ETVR and SVR compared to patients receiving PegIFN and RBV alone. Moreover, relapse, drop out and non-responder rates were significantly lower in this group. Patients who received PegIFN and RBV along with vitamin B12 experienced a significantly lower frequency of treatment side effects compared to those who received PegIFN and RBV only. Besides, necessitation of dose adjustment and treatment discontinuation was lower in patients who received vitamin B12 together with PegIFN and RBV. Our findings showed that SVR rate was significantly higher in patients receiving PegIFN and RBV plus vitamin B12, who had high viral count >600,000 IU/ml and IL28B CC genotype. The association of rs12979860 (C/T) polymorphism with both spontaneous and treatment-induced HCV clearance has been indicated in different studies 34,35. Regarding multivariate analysis, our findings showed that adding vitamin B12 to treatment and genotype IL28B allele CC were prognostic factors to achieve SVR. The results of IL-28B genotyping in our patients did not show any significant differences in the distribution of IL-28B genotypes (C/C, C/T and T/T) between two treatment groups, indicating that the higher rate of SVR observed in SOC + B12 patients was not affected by IL-28B genotype.

Our findings were in agreement with the open-label pilot study by Rocco 25, which reported that the addition of vitamin B12 to SOC significantly improved the rate of virological response in patients with chronic HCV infection. Accordingly, Rosenberg and Hagen 32, in their work showed that high levels of vitamin B12 in serum is favorable for achieving an ETVR in CHC patients treated with SOC. Contrary to these results, Mechie 21 reported that patients with lower baseline levels of serum vitamin B12 (cut-off value of 570 ng/L) achieved higher rate of SVR compared to patients with vitamin B12 above the cut-off value. However, as the liver is the main physiological reservoir of human, and in hepatic diseases, the release of vitamin B12 is accrued due to hepatic necrosis. Thus, in these situations, the liver content of vitamin B12 was not necessarily correlated with the serum levels of vitamin B12.

Conclusions

In conclusion, our findings showed that the addition of vitamin B12 to SOC significantly improved the rates of treatment response in HCV-infected patients. We are aware that the main limitations of our study were the small number of patients and lack of a placebo arm. Moreover, patients were not blinded to taking vitamin B12 and their baseline serum levels of vitamin B12 were not measured. Thus, the results should be interpreted cautiously. However, our findings along with previous research indicate that the addition of vitamin B12 improves the rate of virological response in chronic hepatitis C patients. As this treatment regimen is safe and inexpensive, it represents a promising option for improving the effectiveness of the HCV treatment with SOC, particularly in resource-limited settings.

Abbreviations

cEVR : Complete Early Viral Response

DDA : Direct Acting Antivirals

ETVR : End-of-Treatment Viral Response

HCV : Hepatitis C virus

IL28B : Interleukin 28B

IRES : Internal Ribosomal Entry Site

PegIFN : Pegylated Interferon

PCR : Polymerase Chain Reaction

RVR : Rapid Early Viral Response

SOC : Standard of Care

SVR : Sustain Early Viral Response

UTR : Untranslated Region

Competing Interests

The authors declared no competing interests.

Authors' Contributions

Marjan Mokhtare, Ahmad Darvishi Zeidabadi, Mansour Bahardoust: developed the theory, carried out the experiment, performed the measurements, contributed to sample preparation.

Saeid Safari, Mitra Barati, Shahram, Agah, Mahsa Motavaf verified the analytical methods, contributed to the interpretation of the results, wrote the article.

All authors read and approved the final manuscript.