rss
Gut doi:10.1136/gutjnl-2012-302344
  • Hepatology
  • Original article

Vitamin B12 supplementation improves rates of sustained viral response in patients chronically infected with hepatitis C virus

Press Release
  1. Gerardo Nardone1
  1. 1Department of Clinical and Experimental Medicine, Gastroenterology Unit, University of Naples “Federico II”, Naples, Italy
  2. 2Unit of Virology, “D. Cotugno” Hospital, Naples, Italy
  3. 3Internal Medicine Unit, University of Naples “Federico II”, Naples, Italy
  1. Correspondence to Professor Gerardo Nardone, Department of Clinical and Experimental Medicine, Gastroenterology Unit, University of Naples “Federico II”, via Pansini 5, 80131 Naples, Italy; nardone{at}unina.it
  1. Contributors AR, DC, GN: study concept and design and drafting of the manuscript. AR, DC, PC, CE, ADS: acquisition of data. AR, AB: analysis and interpretation of data; statistical analysis. PS, GN: critical revision of the manuscript for important intellectual content.

  • Revised 5 June 2012
  • Accepted 6 June 2012
  • Published Online First 17 July 2012

Abstract

Background In vitro, vitamin B12 acts as a natural inhibitor of hepatitis C virus (HCV) replication.

Objective To assess the effect of vitamin B12 on virological response in patients with chronic HCV hepatitis naïve to antiviral therapy.

Methods Ninety-four patients with chronic HCV hepatitis were randomly assigned to receive pegylated interferon α plus ribavirin (standard-of-care; SOC) or SOC plus vitamin B12 (SOC+B12). Viral response—namely, undetectable serum HCV-RNA, was evaluated 4 weeks after starting treatment (rapid viral response), 12 weeks after starting treatment (complete early viral response) and 24 or 48 weeks after starting treatment (end-of-treatment viral response) and 24 weeks after completing treatment (sustained viral response (SVR)). Genotyping for the interleukin (IL)-28B polymorphism was performed a posteriori in a subset (42/64) of HCV genotype 1 carriers.

Results Overall, rapid viral response did not differ between the two groups, whereas the rates of complete early viral response (p=0.03), end-of-treatment viral response (p=0.03) and SVR (p=0.001) were significantly higher in SOC+B12 patients than in SOC patients. In SOC+B12 patients, the SVR rate was also significantly higher in carriers of a difficult-to-treat genotype (p=0.002) and in patients with a high baseline viral load (p=0.002). Distribution of genotype IL-28B did not differ between the two groups. At multivariate analysis, only easy-to-treat HCV genotypes (OR=9.00; 95% CI 2.5 to 37.5; p=0.001) and vitamin B12 supplementation (OR=6.9; 95% CI 2.0 to 23.6; p=0.002) were independently associated with SVR.

Conclusion Vitamin B12 supplementation significantly improves SVR rates in HCV-infected patients naïve to antiviral therapy.

Significance of this study

What is already known on this subject?

  • Less than 50% of individuals infected with hepatitis C virus (HCV) genotype 1 clear the infection after treatment with the combination of pegylated interferon-α and ribavirin.

  • The non-structural 3/4A protease inhibitors boceprevir and telaprevir significantly improve the sustained viral response (SVR) rate in patients with chronic HCV genotype 1 infection naïve to treatment. However, several questions remain about the applicability of these direct-acting antiviral agents in ‘real-life’ conditions.

  • In vitro, vitamin B12 inhibits HCV translation by directly interacting with the internal ribosomal entry site of HCV-RNA.

What are the new findings?

  • In patients with chronic HCV infection naïve to antiviral therapy, vitamin B12 supplementation improves the overall rate of SVR to pegylated interferon-α and ribavirin by 34%. The effect seems to be particularly pronounced in difficult-to-treat patients—namely, those infected with HCV genotype 1 and with a high baseline viral load.

How might it impact on clinical practice in the foreseeable future?

  • The new-generation HCV antiviral drugs, such as direct-acting agents, need careful monitoring and stringent futility rules to prevent the emergence of multiresistant HCV strains and to avoid overtreatment of patients. Until eligibility criteria are well established, the standard of care+B12 combination is a safe and inexpensive alternative to improve the rate of SVR in difficult-to-treat patients. This strategy would be especially useful in those countries where, owing to limited economic means, the new-generation antiviral therapies cannot be given in routine clinical practice.

Introduction

Chronic hepatitis C virus (HCV) infection is a major worldwide health, social and economic problem. Approximately 60–80% of infected people develop chronic hepatitis and 30% of them progress to cirrhosis and to end-stage liver disease.1 The primary goal of HCV therapy is to cure the infection, which is achieved by eliminating detectable circulating HCV after cessation of treatment. The combination of pegylated interferon (peg-IFN)-α and ribavirin, which was the standard of care (SOC) for HCV chronic hepatitis up to October 2011,2–6 eradicates the infection in 40–54% of patients infected with HCV genotype 1 and in up to 80% of those infected with HCV genotypes 2 or 3. However, in about 50% of people the infection is not cleared during antiviral therapy or they relapse after treatment ends.

The recent introduction of the HCV non-structural 3/4A protease inhibitors boceprevir and telaprevir, which are direct-acting antiviral agents (DAAs), has changed the treatment options for individuals infected with HCV. The American Association for the Study of Liver Diseases recommends their use in combination with SOC therapy in patients with genotype 1 HCV infection.7 However, despite the impressive results of phase III clinical trials,8 ,9 several questions remain about the applicability of DAA therapy in ‘real-life’ conditions—namely, the emergence of drug-induced mutations, selection of candidates to avoid unnecessary treatment, adverse events and additional costs to healthcare budgets.10

HCV is a positive-sense, single-strand RNA virus that possesses an internal ribosomal entry site (IRES) at the 5′ end of its genome.11 The IRES element is a complex RNA structure containing distinct domains and can specifically interact with the ribosomal subunits and positions them directly over the initiation codon.12 The RNA structure around the initiation codon affects HCV IRES efficiency. HCV IRES-mediated initiation is part of the viral replication mechanism and, given its specificity and sensitivity to minor structural changes, it is considered one of the best targets for antiviral strategies. It has been shown in an in vitro system that vitamin B12 inhibits HCV IRES-dependent translation, probably by directly interacting with HCV IRES RNA.13 ,14

Liver is the physiological reservoir of cyanocobalamin in humans.15 Several liver diseases—such as, hepatitis, cirrhosis, hepatocellular carcinoma and metastasis, may be accompanied by relative vitamin B12 deficiency secondary to impaired liver storage consequent to the increased release during hepatic cytolysis and/or decreased clearance by the affected liver.16 Therefore, in this situation and given the natural role of vitamin B12 in the negative regulation of the HCV replication cycle,14 it is conceivable that administration of vitamin B12 might improve the rates of virological response to antiviral therapy in HCV carriers.

The aim of this open-label pilot study was to assess the effect of the addition of vitamin B12 to SOC on virological response in patients with chronic HCV infection naïve to antiviral therapy.

Patients and methods

Patients

The study group consisted of consecutive patients with chronic HCV infection, naïve to antiviral therapy, prospectively recruited among those referred to our unit between January 2006 and July 2010. Exclusion criteria were: age <18 or >70 years, previous treatment with interferon ± ribavirin, concomitant causes of liver disease, such as hepatitis B virus infection, autoimmunity and alcohol abuse (>40 g/day for men and >20 g/day for women), HIV infection, hepatocellular carcinoma, decompensated cirrhosis, severe concurrent disease or contraindications to treatment—that is, uncontrolled depression, psychosis, epilepsy, autoimmune diseases, poorly controlled hypertension, diabetes, heart failure and chronic obstructive pulmonary disease.

All patients gave written consent to participate in the study. The study protocol was approved by the ethics committee of the University of Naples Federico II.

Study protocol

Upon enrolment in the study, all patients underwent the following tests: complete blood cell count, aspartate aminotransferase, alanine aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase, total and direct bilirubin, albumin, prothrombin activity, cholinesterase and cholesterol levels. Anti-HCV antibodies were detected by a third-generation enzyme immunoassay (Ortho HCV SAVe 3.0, Raritan, New Jersey, USA). Positivity for HCV-RNA in the serum was assessed by a nested reverse transcription PCR (RT-PCR, Roche Cobas Amplicor 2.0, Roche Diagnostics, Basel, Switzerland). HCV genotype was identified with the INNO-LiPA HCV test (Innogenetics NV, Gent, Belgium).

Ultrasound-guided, fine-needle liver biopsy was performed in all cases. The threshold of adequacy for histological assessment was the presence of more than five portal tracts. Chronic hepatitis was diagnosed by an experienced liver pathologist based on the Ishak score.17 Steatosis was graded as follows: 0 (absent), 1 (30% of hepatocytes affected), 2 (30–70% of hepatocytes affected) and 3 (>70% of hepatocytes affected).

Patients enrolled in the study were randomly assigned to one of the following antiviral therapy schedules: peg-IFN plus ribavirin (SOC group) or peg-IFN plus ribavirin and vitamin B12 (SOC + B12 group). Randomisation was carried out on the basis of a computer-generated list prepared by an independent researcher not involved in the therapeutic management of the patients and unaware of the patient characteristics except for the genotype. The randomisation lists were separately generated for difficult-to-treat genotypes (genotype 1 or 4) and easy-to-treat genotypes (genotype 2 or 3) in order to balance the two treatment groups.

Peg-IFNα2b (PegIntron, Schering-Plough, New Jersey, USA) was used at a dosage of 1.5 μg/kg/week and peg-IFNα2a (Pegasys, Roche, Basel, Switzerland) at a dosage of 180 μg/week. Since no comparative studies on the efficacy of peg-IFNα had been published before starting our study, we used the two types of peg-IFNs indiscriminately. In patients infected with HCV genotype 1, ribavirin (either Rebetol, Schering-Plough, or Copegus, Roche) was administered according to body weight (1000 mg/day for patients weighing <75 kg, 1200 mg/day for patients weighing >75 kg); in the case of infection by genotype 2 or 3, a single ribavirin dose of 800 mg/day was used. The duration of treatment was 48 weeks for genotypes 1 and 24 weeks for genotypes 2 and 3. Patients in the SOC+B12 group received vitamin B12 (Dobetin, Angelini, Rome, Italy) supplementation at a dose of 5000 μg administered by intramuscular injection every 4 weeks for the duration of the antiviral therapy.

Definition of viral response

Rapid viral response (RVR) was defined as undetectable serum HCV-RNA (<50 IU/ml) 4 weeks after starting treatment. Complete early viral response (cEVR) was defined as undetectable serum HCV-RNA 12 weeks after starting treatment. The end-of-treatment viral response (ETVR) was defined as undetectable serum HCV-RNA level after completing the treatment schedule. Sustained viral response (SVR) was defined as undetectable serum HCV-RNA level at 24 weeks after stopping antiviral therapy. Virological breakthrough was defined as the reappearance of HCV-RNA during treatment. Patients who achieved an ETVR but reverted to a detectable HCV-RNA level after stopping treatment were considered ‘relapsers’. Dropout was defined as discontinuation of antiviral therapy due to adverse effects. The stopping rule consisted of treatment discontinuation in patients who either did not obtain a reduction in serum HCV-RNA concentration of at least 2 logs versus baseline at week 12 or had a detectable serum HCV-RNA level after 24 weeks of treatment. Patients who met the stopping rule criteria for treatment discontinuation were defined ‘non-responders’.

Dose modifications

The dosage of peg-IFN was reduced by half if the neutrophil count decreased to <0.75×109/l or if the platelet count decreased to <50×109/l. Peg-IFN treatment was discontinued if the neutrophil count was <0.50×109/l or the platelet count was <25×109/l. Peg-IFN dosages were reduced in 25% decrements or discontinued because of adverse events. The dosage of ribavirin was reduced in 200 mg decrements, as necessary, if the haemoglobin level decreased to <100 g/l or by ≥30 g/l, or in the event of severe cough or intolerable itching. Ribavirin treatment was discontinued if the haemoglobin level decreased to <85 g/l. Adherence to treatment was evaluated based on self-reporting by the patients during their monthly control visit and at 12-week intervals based on drug prescriptions and expressed according to the ‘80/80/80’ rule. The latter refers to the quantity of peg-IFN and ribavirin administered (percentage of the planned total dose) and the total duration of treatment (percentage of the planned duration). Patients who took at least 80% of the two drugs for at least 80% of the scheduled time were considered compliant. In addition, we evaluated the cumulative dosage of peg-IFN and ribavirin in all groups during the study.

Follow-up

Patients underwent a physical examination and complete blood count weekly for the first 4 weeks of treatment and monthly thereafter. Quantitative HCV-RNA and alanine aminotransferase levels were measured 4, 12, 48 and 72 weeks after the start of antiviral therapy. An autoimmune panel and thyroid function tests were checked every 3 months.

In order to minimise possible sources of bias of an unblinded study the record of the laboratory findings was made by medical staff not involved in treating the subjects and unaware of the study hypothesis.

Molecular biology

Genotyping for the interleukin (IL)-28B rs12979860 CT polymorphism was performed by PCR-based restriction fragment length polymorphism assay. Genomic DNA was extracted from whole blood samples using the QIAamp DNA blood mini kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. A 242 bp product was obtained with the forward primer 5′-GCTTATCGCATACGGCTAGG-3′ and the reverse primer 5′-AGGCTCAGGGTCAATCACAG-3′, which were newly designed using the NCBI Primer-Blast Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR amplification was performed in a total volume of 10 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, Tween-20 0.01%, 0.2 mM deoxyribonucleotides, 2–4 pmol of each primer, 2 mM MgCl2 and 0.5 units hot-start Taq DNA polymerase (RighTaq, Euroclone, Milan, Italy). Samples containing 10 ng of genomic DNA were subjected to 40 cycles of denaturation (at 95°C for 30 s), annealing (at 62°C for 30 s) and elongation (at 72°C for 30 s) using a TechneTC-412 thermal cycler. In a total volume of 20 μl, 10 μl of the amplicons were digested with 1 unit of the BstU-I restriction endonuclease (New England Biolabs, Hitchin, Herts, UK) at 60°C overnight. The digest fragments were 135, 82 and 25 bp for the C allele and 160 and 82 bp for the T allele variant. The fragments were resolved by electrophoresis on a 3.5% agarose gel after staining with ethidium bromide.

Statistical analysis

We set the power to 0.8, with type I error =0.05 and control to case ratio 1:1. The aim was to detect a minimal difference of 25% between SOC and SOC + B12 groups. Continuous variables were expressed as mean ± SD. When the distribution of the variables significantly deviated from normality, they were normalised by logarithmic transformation and log-transformed values were used in the analysis. Frequencies were calculated for categorical variables, with the difference between groups reported with 95% CIs. The Mann–Whitney rank sum test or the Kruskal–Wallis test with Dunn's post-test were used to compare continuous variables. Fisher's exact test was used to compare the categorical variables.

We used a univariate analysis to evaluate the independent effect of each baseline factor (sex, age, body mass index, histological staging, grading and percentage of steatosis of the baseline liver biopsy, baseline HCV-RNA levels, HCV genotype, peg-IFNα2 type, dose reduction and treatment discontinuation and vitamin B12 supplementation) on the likelihood of predicting the rate of SVR. After the conventional data had been taken into account, a logistic regression analysis was performed to identify the potential predictors of SVR by using SVR (yes/no) as dependent variable and selected variables at baseline as independent factors. To allow a comparative evaluation of the effects of the different factors on the OR to obtain the SVR, Z scores were calculated for each factor and used for the analysis. The Nagelkerke R2 was used to estimate the percentage of variance in the dependent variable explained by the independent factors. The goodness-of-fit of the final model was evaluated by the Hosmer–Lemeshow test. All statistical procedures were performed using the Statistical Package for Social Sciences (SPSS-PC V.17.0; SPSS Inc).

Statistical analysis was independently performed by two of the authors blinded to the type of treatment arm (codified as group 1 and 2) and not directly involved in the clinical management of the patients.

Results

Of the 130 patients with chronic HCV infection observed in our unit between January 2006 and July 2010, 36 were excluded from the study for the following reasons: previous treatment with interferon ± ribavirin (n=21), concomitant hepatitis B virus infection (n=3), heavy alcohol consumption (n=3), hepatocellular carcinoma (n=1), severe depression (n=2), poorly controlled diabetes (n=2), scheduled pregnancy (n=1) and refused treatment (n=3). Thus, 94 patients entered the study and were randomly assigned to receive SOC or SOC + vitamin B12 supplementation.

The baseline characteristics of the patients divided according to antiviral therapy schedule are summarised in table 1. There were no significant differences between the two treatment groups in demographic, laboratory, virological and histological parameters. Peg-IFNα2b combined with ribavirin was given in the majority of cases in both groups (68%, 95% CI 54% to 80% in the SOC group and 64%, 95% CI 49% to 76% in the SOC + B12 group, respectively). Flow of patients according to treatment arm and HCV genotype throughout the study is summarised in figure 1.

Table 1

Baseline clinical and laboratory findings of the two treatment groups

Figure 1

Flow of patients according to treatment arm and hepatitis C virus (HCV) genotype throughout the study. Grey boxes indicate the number of dropout (DO), breakthrough (BT) and non-responders (NR) at each checkpoint. *Patients who obtained a reduction in serum HCV RNA concentration of at least 2 logs versus baseline at week 12 but discontinued treatment because of laboratory abnormalities or adverse events. †One patient discontinued treatment at the 46th week. SOC, standard of care.

Virological response during antiviral therapy and at the end of follow-up is reported in table 2. Overall, RVR was obtained by 23/47=49% (95% CI 35% to 63%) patients of the SOC group and by 32/47=68% (95% CI 54% to 80%) patients of the SOC + B12 group. The difference failed to reach significance. When analysed according to HCV genotype and baseline viral load, the rates of RVR were significantly higher in the SOC + B12 group than in the SOC group only in patients with a high baseline viral load (intention-to-treat (ITT) analysis: difference 29%; 95% CI 6% to 48%; p=0.01; PP: difference 31%; 95% CI 7% to 50%; p=0.01).

Table 2

Rates of rapid, early, end-of-treatment and sustained viral response in the two treatment groups

The overall rate of cEVR was significantly higher in patients who received SOC + B12 than in those who received SOC (ITT: difference 21%; 95% CI 4% to 37%; p=0.03; PP: difference 23%; 95% CI 6% to 38%; p=0.01). The cEVR rate was significantly higher in the SOC + B12 group than in the SOC when considering HCV genotype 1 (PP: difference 30%; 95% CI 7% to 49%; p=0.01) and a high baseline viral load (ITT: difference 29%; 95% CI 8% to 48%; p=0.01; PP: difference 31%; 95% CI 10% to 49%; p=0.004).

Overall the rate of ETVR was significantly higher in SOC + B12 than in SOC patients (ITT: difference 21%; 95% CI 3% to 38%; p=0.03; PP: difference 23%; 95% CI 5% to 39%; p=0.02). It was significantly higher in SOC + B12 patients than in SOC patients when considered according to genotype (PP: difference 30%; 95% CI 6% to 49%; p=0.01) and baseline viral load (ITT: difference 32%; 95% CI 11% to 51%; p=0.005; PP: difference 34%; 95% CI 13% to 53%; p=0.002).

Overall, the rate of SVR was significantly higher in patients who received SOC + B12 than in patients who received SOC (ITT: difference 34%; 95% CI 14% to 50%; p=0.001; PP: difference 36%; 95% CI 16% to 53%; p=0.02). Interestingly, when considered according to HCV genotype and viral load, the SVR rate remained significantly higher in patients with a difficult-to-treat genotype (ITT: difference 41%; 95% CI 16% to 59%; p=0.002; PP: difference 47%; 95% CI 22% to 64%; p=0.003) and in patients with a high baseline viral load (ITT: difference 38%; 95% CI 15% to 55%; p=0.02; PP: difference 40%; 95% CI 16% to 58%; p=0.001) (table 2 and figures 2 and 3).

Figure 2

Sustained viral response (SVR) rates in patients who received standard of care (SOC) or SOC plus vitamin B12 supplementation subdivided according to HCV genotypes. Fisher's exact test. ITT, intention to treat; PP, per protocol.

Figure 3

Sustained viral response (SVR) rates in patients who received standard of care (SOC) or SOC plus vitamin B12 supplementation subdivided according to HCV viral load. Fisher's exact test. ITT, intention to treat; PP, per protocol.

The percentage of viral breakthroughs and relapsers was higher, although not significantly so, in the SOC than in the SOC + B12 group. There were 12/47=26% (95% CI 15% to 39%) non-responders in the SOC group and 4/47=9% (95% CI 3% to 20%) non-responders in the SOC + B12 group (p=0.05). At multivariate stepwise analysis, infection with HCV genotype 2 or 3 (OR=9.00; 95% CI 2.5% to 37.5%; p=0.001) and treatment with vitamin B12 (OR=6.9; 95% CI 2.0% to 23.6%; p=0.002) were the only covariates independently associated with SVR.

Adverse events, dose reduction and treatment discontinuation rates did not differ significantly between the SOC and SOC + B12 groups (table 3). Overall, 11/94=12% (95% CI 7% to 20%) patients discontinued treatment: six in the SOC group and five in the SOC + B12 group. The reasons for stopping treatment were laboratory abnormalities in two cases (severe anaemia) and adverse events in the remaining nine cases: severe depression (two), dermatitis (two), acute psychosis (one), cicatricial alopecia (one), cough and dyspnoea (one), peripheral neuritis (one) and complicated diverticulitis (one). Type and frequency of adverse events did not differ significantly between the two treatment groups. Only 19/94=20% (95% CI 13% to 29%) patients reported no adverse events.

Table 3

Frequency of adverse events and laboratory abnormalities in the two treatment groups

The dosage of peg-IFN and/or ribavirin was reduced in 24/94=26% (95% CI 18% to 35%) patients mainly because of laboratory abnormalities (anaemia in 16 cases, neutropenia in five cases and thrombocytopenia in one case). Changes in mean haemoglobin levels, neutrophil and platelet count during the antiviral therapy in patients subdivided according to treatment arm are reported in figure 4. Dose reduction was necessary because erythropoietin and granulocyte-stimulating growth factors were not licensed by the National Health System at the time the study was planned. Overall, more than 50% of patients complied with the 80/80/80 rule to a similar extent in the groups (32/47=68%; 95% CI 54% to 80% vs 27/47=57%; 95% CI 43% to 70%). The cumulative dosage of peg-IFN and ribavirin at 4, 12, 24 and 48 weeks of treatment did not differ significantly between the treatment arms nor in relation to HCV-genotype (table 4).

Figure 4

Changes in mean haemoglobin levels, neutrophil and platelet counts during the antiviral therapy in patients subdivided according to treatment arm. SOC, standard of care.

Table 4

Cumulative dosage of pegylated-interferon α and ribavirin in treatment arms by HCV-genotype

Forty-two of the 64=66% (95% CI 53% to 76%) patients with HCV genotype 1 (22 in the SOC group and 20 in the SOC + B12 group) agreed to undergo IL-28B genotyping. As shown in table 5, 11/42 (26%; 95% CI 15% to 41%) were homozygous for CC, 22/42 (52%; 95% CI 38% to 67%) were heterozygous for CT and the remaining 9/42 (21%; 95% CI 12% to 36%) were homozygous for TT, without significant differences in the distribution between the two treatment groups. The rate of SVR in patients homozygous for CC or TT did not significantly differ between the two treatment groups while it was significantly higher in patients carrying the CT genotype who received SOC + B12 (p=0.01).

Table 5

Distribution of IL-28B genotypes in relation to SVR rates in patients with HCV-genotype 1 in the two treatment groups

Discussion

Experimental evidence that vitamin B12 directly inhibits HCV replication dates back more than 10 years.14 However, thus far, no interventional study has been performed to evaluate whether vitamin B12 supplementation might be beneficial in the treatment of HCV-related chronic diseases. This open-label pilot study was designed to test the clinical efficacy of vitamin B12 supplementation in patients with chronic HCV hepatitis treated with a SOC protocol. Here we report evidence that vitamin B12 supplementation significantly improves the rate of virological response in patients naïve to antiviral therapy. Indeed, although the cumulative dosage of peg-IFN and ribavirin did not differ significantly between the two treatment arms, patients who received vitamin B12 achieved significantly higher overall rates of on-treatment viral responses (cEVR), ETVR and SVR than patients who received SOC alone. Interestingly, the most striking effect of vitamin B12 supplementation in our study occurred in patients with difficult-to-treat genotypes and a high baseline viral load. Indeed, when considering HCV genotype and viral load, the rates of SVR were respectively 41% and 38% higher in patients receiving B12 supplementation than in patients treated with SOC alone. The effect exerted by B12 in patients with HCV is consistent with the in vitro finding that vitamin B12 inhibits the HCV IRES-dependent translation in a dose-dependent manner.14 The better therapeutic outcome in our patients affected by HCV genotype 1 may reflect the fact that the plasmids used for in vitro translation inhibition assays were constructed by cloning the HCV IRES element from the Australian HCV isolate pSTDL, genotype 1.

Treatment of patients infected with HCV genotype 1 is a difficult problem. Despite intensive efforts to improve the virological response rate in these patients, the infection is cured at most in 40–50% of cases in the setting of randomised controlled trials.18–20 For each patient affected by a difficult-to-treat HCV genotype, the costs of non-SVR, including side effects, were estimated to be €12 454.00.21 In our study, vitamin B12 supplementation for the whole period of antiviral therapy, added to the overall costs of SOC therapy, amounted to only € 17.6 per patient, whereas it increased the SVR rate by 41%. In addition, vitamin B12 supplementation was safe and reduced the occurrence of severe side effects of SOC therapy without increasing the patient's treatment burden (ie, pill burden, complexity in administration and drug–drug interactions).

In a recent retrospective analysis of the medical records of 99 treatment-naïve patients with HCV-related chronic hepatitis, Rosenberg et al22 found that a high pretreatment level of B12 in serum predicted a positive ETVR. We did not measure the serum level of vitamin B12 in our patients for two reasons. First, owing to the increased release of B12 during hepatic cytolysis, the liver content of vitamin B12 does not correlate with the serum level of the vitamin,16 and second, the methodologies used to test vitamin B12 levels lack sensitivity and specificity.23

It was recently shown that a set of polymorphisms in the region of the IL-28B gene are associated with more effective clearance of the infection in patients with genotype 1 HCV chronic infection.24–26 IL-28B polymorphisms may help to explain the difference in the response rates to treatment and have become one of the best indicators for a positive outcome of treatment in this subgroup of patients. IL-28B genotyping in our patients was performed a posteriori since it was not available at the time the study was designed. Although data are not available for all patients, the distribution of IL-28B genotypes (C/C, C/T and T/T) did not differ significantly between those who received SOC and those who received SOC + B12, which indicates that the higher rate of SVR obtained in patients who received vitamin B12 supplementation was not affected by an IL-28B genotype.

We are aware that this is an open-label pilot study based on the analysis of a limited number of patients and that it lacks a placebo arm. Consequently, the results should be interpreted with due caution. However, the randomisation method used, the record of the laboratory findings made by medical staff not involved in treating the subjects and unaware of the study hypothesis, the statistical analysis independently performed by two of the authors blinded to the type of treatment arm and the objective nature of the end points used—that is, virological responses, should have, at least in part, minimised the various possible sources of bias in our study.

In conclusion, vitamin B12 supplementation significantly improves the rates of SVR in HCV-infected patients naïve to antiviral therapy, particularly those infected with genotype 1 and a high baseline viral load.

The large-scale introduction of the new-generation HCV antiviral drugs such as DAAs needs careful monitoring and stringent futility rules to prevent the emergence of multiresistant HCV strains and to avoid overtreatment of patients.10 Until eligibility criteria are well established, the SOC + B12 combination is a safe and inexpensive alternative to improve the rate of SVR in difficult-to-treat patients. This strategy would be especially useful in those countries where, owing to limited economic means, the new-generation antiviral therapies cannot be given in routine clinical practice.

Acknowledgments

We thank Jean Ann Gilder (Scientific Communication srl) for editing and revising the paper.

Footnotes

  • Competing interests None.

  • Ethics approval Ethical Committee University Federico II of Naples.

  • Provenance and peer review Not commissioned; externally peer reviewed.

References

Relevant Article