Liver disease is the second leading cause of death in patients infected with HIV . When compared to hepatitis C virus (HCV) monoinfection, HIV–HCV coinfection, observed in 25–30% of European and US HIV-positive patients , was found to be associated with higher rates of liver fibrosis progression  and markedly higher risks of cirrhosis and end-stage liver disease . In HIV–HCV coinfected patients, therapy-restricting contraindications, considerably lower rates of treatment initiation, and lower rates of virologic response to pegylated interferon-α-2a/2b plus ribavirin (PEGIFN + RBV) complicate chronic hepatitis C (CHC) therapy when compared to HCV monoinfection [5,6].
Low serum 25-hydroxyvitamin D [25(OH)D] levels were frequently found in both HCV monoinfected  and HIV–HCV coinfected  patients. Gal-Tanamy et al.  demonstrated in vitro that vitamin D acts directly as an antiviral agent by inhibiting HCV production in a human hepatoma cell line. Additionally, a synergistic inhibitory effect of vitamin D and interferon-α on HCV production was reported. In HCV monoinfected patients with recurrent hepatitis C after liver transplant, higher rates of virologic response were observed in those receiving vitamin D supplementation . Finally, in a randomized prospective trial including only CHC treatment-naive HCV-genotype (HCV-GT) 1 patients, higher rates of virologic response were achieved in the group receiving vitamin D supplementation .
In the two studies on 25(OH)D levels in HIV–HCV coinfected patients published to date [8,12], low 25(OH)D levels were found to be associated with advanced liver fibrosis. However, none of these studies observed an association between 25(OH)D levels and virologic response.
Thus, the aim of our study was to assess the association of low 25(OH)D levels with virologic response to PEGIFN + RBV in HIV–HCV coinfected patients while considering established risk factors for treatment failure.
Patients and methods
A total of 65 HIV–HCV coinfected patients treated with PEGIFN + RBV at the Medical University of Vienna within a prospective trial were included in this study. Inclusion requirements consisted of the availability of a liver biopsy, 25(OH)D and HCV-RNA levels, interleukin 28B (IL28B) rs12979860 single nucleotide polymorphism (SNP), CD4+ T-lymphocyte counts as well as information on virologic response. All patients had compensated liver disease, were naive to pegylated interferon-α-based CHC therapy, and free of malignancies as well as significant cardiac, pulmonary or renal disease. None of the patients was receiving vitamin D supplements.
Blood sampling and interleukin28B rs12979860 SNP testing
25(OH)D levels were retrospectively assessed using stored screening serum samples obtained within 35 days prior to CHC treatment and a commercially available enzyme immunoassay (IDS; Boldon, Tyne and Wear, UK). In order to maintain comparability with the studies on 25(OH)D in HIV–HCV coinfected individuals published to date [8,12], patients were assigned to the normal (>30 ng/ml; D-NORM), the insufficiency (10–30 ng/ml; D-INSUFF) or the deficiency (<10 ng/ml; D-DEF) group. HCV-GT and serum HCV-RNA levels were determined using commercially available assays [VERSANT HCV Genotype 2.0 Assay (LiPA) (Siemens, Vienna, Austria) and COBAS TaqMan HCV Test (Roche, Vienna, Austria)]. High HCV-RNA load was defined as HCV-RNA levels more than 6 × 105 IU/ml. Serum CD4+ T-lymphocyte counts were determined using standard flow fluorocytometry. The IL28B rs12979860 SNP was analyzed as described previously using the Step One Plus Real Time PCR System (Applied Biosystems, Carlsbad, California, USA) and a Custom TaqMan SNP Genotyping Assay .
Liver fibrosis was assessed according to the METAVIR classification . METAVIR F3 and F4 were denoted as advanced liver fibrosis.
Chronic hepatitis C treatment
All patients were treated with pegylated interferon-α-2a (180 μg) or pegylated interferon-α-2b (1.5 μg/kg body weight) once a week. HCV-GT 1/4 patients received 1000–1200 mg ribavirin, whereas HCV-GT 2/3 patients received 800 mg ribavirin daily. Treatment duration was 48 weeks, except for HCV-GT 1/4 patients without rapid virologic response (RVR), who were treated for 72 weeks. RVR, complete early virologic response (cEVR), and sustained virologic response (SVR) were defined as undetectable HCV-RNA at week 4 or 12 on treatment and 24 weeks after the end of CHC treatment, respectively.
Risk factors for treatment failure
In accordance with previous studies [15,16], HCV-GT 1/4, high HCV-RNA load, advanced liver fibrosis, and IL28B rs12979860 non-C/C SNP were considered as established risk factors for treatment failure.
Statistical analyses were performed using IBM SPSS Statistics 19 (SPSS Inc., Armok, New York, USA). Continuous variables were reported as median (range), whereas categorical variables were reported as number (proportion) of patients with the certain characteristic. One-way analysis of variance (ANOVA) with Scheffe posthoc comparisons was used for group comparisons of continuous variables when applicable. Otherwise, Kruskal–Wallis one-way ANOVA and Mann–Whitney U-test with Bonferroni correction for posthoc comparisons were applied. Group comparisons of categorical variables were performed using Fisher's Exact Test. A P value of 0.05 was considered statistically significant.
This study was conducted with the understanding and the consent of each participant and approved by the local ethics committee of the Medical University of Vienna (EKN 1099/2011).
25-hydroxyvitamin D levels
Although 37 (57%) and 15 (23%) patients presented with D-INSUFF and D-DEF, respectively, 13 (20%) patients had D-NORM (Table 1). 25(OH)D levels varied seasonally between 18.4 (71.5), 26.1 (41.2), 18.8 (34.5), and 10.95 (24.2) ng/ml in quarters 1, 2, 3, and 4, respectively (P = 0.031).
25-hydroxyvitamin D levels and risk factors for treatment failure
The prevalence of HCV-GT 1/4 and high baseline HCV-RNA load did not vary statistically significantly throughout the 25(OH)D groups (P = 0.36 and P = 0.214, respectively) (Table 1).
The proportion of patients with IL28B rs12979860non-C/C SNP in the D-INSUFF group (81%) was higher than in the D-NORM group (39%) (P = 0.007), but did not differ significantly from the D-DEF group (53%) (P = 0.081). The frequency of patients with IL28B rs12979860 non-C/C SNP observed in the D-NORM group and the D-DEF group was not statistically different (P = 0.476).
Advanced liver fibrosis was found in 63% of 25(OH)D-deficient patients, 40% of 25(OH)D-insufficient patients and 13% of patients with D-NORM. Patients with D-DEF displayed a higher prevalence of advanced liver fibrosis than patients with D-NORM (P = 0.009), whereas the difference between patients with D-INSUFF and both patients with D-DEF and D-NORM was not statistically significant (P = 0.073 and P = 0.179, respectively).
25-hydroxyvitamin D levels and virologic response
The observed RVR rates did not differ statistically significantly between the 25(OH)D groups (P = 0.406) (Table 1).
Ninety-two percent of patients with D-NORM, 68% of patients with D-INSUFF and 47% of patients with D-DEF showed cEVR. Patients with D-DEF displayed lower cEVR rates than patients with D-NORM (P = 0.016), whereas the difference between patients with D-INSUFF and both patients with D-NORM and D-DEF was not statistically significant (P = 0.078 and P = 0.213, respectively) (Table 1, Fig. 1a).
SVR was observed in 85% of patients with D-NORM, 60% of patients with D-INSUFF and 40% of patients with D-DEF. When compared to patients with D-NORM, patients with D-DEF showed lower SVR rates (P = 0.024). Although substantial, the differences in SVR rates between the D-INSUFF groups compared to both the D-NORM and the D-DEF groups were not statistically significant (P = 0.173 and P = 0.234, respectively).
25-hydroxyvitamin D levels, virologic response, and risk factors for treatment failure
The study population was separated into two subgroups: patients with 0–2 risk factors and patients with 3–4 risk factors for treatment failure (HCV-GT 1/4, advanced fibrosis, high HCV-RNA levels, and IL28B rs12979860 non-C/C SNP) (Table 1, Fig. 1b).
Among patients with 0–2 risk factors, the SVR rates in patients with and without D-DEF were comparable (86 and 76%, respectively; P = 0.67). In contrast, patients with 3–4 risk factors and D-DEF showed lower SVR rates than their counterparts without D-DEF (P = 0.012). No patient with D-DEF obtained SVR, whereas SVR was obtained in 52% of patients without D-DEF.
The fraction of patients with D-DEF that carried 3–4 risk factors was comparable to patients without D-DEF (53 and 42%, respectively; P = 0.557).
In line with previous studies, we observed a high prevalence of vitamin D insufficiency (D-INSUFF) and deficiency (D-DEF) in our study population of HIV–HCV coinfected patients [8,12].
Apart from low 25(OH)D levels in quarter 4, seasonal variation observed in this study was less pronounced when compared to studies on healthy white individuals . This finding suggests that the modulating effect of sun exposure on 25(OH)D levels in HIV–HCV coinfected patients is not as pronounced as in healthy individuals because of other interfering factors.
Although several retrospective studies, as well as randomized prospective trials, in HCV-monoinfected patients reported a statistically significant association between both 25(OH)D levels and vitamin D supplementation and virologic response [7,10,11,18,19], none of the two retrospective studies in HIV–HCV-monoinfected patients found a statistically significant relationship [8,12]. However, potential limitations of both studies have to be considered. In the study by Milazzo et al. , the analysis of factors associated with SVR was performed in a relatively small group of 51 patients, of whom 20 were not naive to interferon-based CHC treatment, which further limits their conclusions and could explain the surprisingly low rate of SVR of 24%. Another study by Terrier et al.  analyzed factors associated with SVR in 189 HIV–HCV coinfected patients, whereas the applied treatment regimens were inconsistent. Ninety-four and 95 patients received interferon–ribavirin and PEGFIFN + RBV, respectively. The corresponding SVR rates were 27 and 38%, resulting in a modest overall SVR rate of 32%. None of the two previous studies reported data on IL28B rs12979860 SNP.
In the present study, the difference between both cEVR and SVR rates among patients with D-DEF and D-NORM was statistically significant. In absolute numbers, cEVR and SVR rates were about half as high in patients with D-DEF when compared to patients with D-NORM, reflecting a clinically relevant effect. Partially conflicting results have been reported on the association of RVR with 25(OH)D levels in HCV-monoinfected patients. Although a statistically significant association was found by Bitetto et al. , no such association was reported by Lange et al. . Along with our findings, it might be concluded that the impact of 25(OH)D levels on RVR rates is less pronounced than on cEVR and SVR rates.
We demonstrated that the association between 25(OH)D levels and SVR rates is limited to difficult-to-treat patients with multiple risk factors for treatment failure. This hypothesis is supported by a study from Bitetto et al.  in HCV-monoinfected patients in which the combined influence of 25(OH)D levels and IL28B rs12979860 SNP on SVR was solely observed in patients with difficult-to-treat HCV-GT 1/4/5.
We have to acknowledge the potential limitations of our study such as the retrospective design and the potential lack of statistical power to detect differences in RVR rates.
In conclusion, our study demonstrates that low 25(OH)D levels are associated with considerably lower rates of cEVR and SVR in HIV–HCV coinfected patients. The latter finding was limited to difficult-to-treat patients with multiple risk factors for treatment failure. Thus, our findings, together with the evidence derived from the above-described prospective study in HCV-monoinfected patients , suggest that vitamin D supplementation may result in higher rates of virologic response in HIV–HCV coinfected patients. Prospective randomized trials on vitamin D supplementation in HIV–HCV coinfected patients receiving CHC therapy are highly encouraged.
Study concept and design (M.M., T.R., and M.P.R.), acquisition of data (T.R., B.A.P., A.F., F.B., M.C.A., B.S., and A.R.), measurement of 25(OH)D levels (B.O.P.), analysis and interpretation of data (M.M., T.R., M.T., and M.P.R.), drafting of the manuscript (M.M. and T.R.), critical revision of the manuscript for important intellectual content (M.M., T.R., B.A.P., A.F., B.F., M.C.A., B.O.P., A.R., M.T., and M.P.R.).
This work was supported by a grant from Roche Austria to M.P.R.
Conflicts of interest
There are no conflicts of interest.
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