Effect of vitamin D supplementation in patients with chronic hepatitis C after direct-acting antiviral treatment: a randomized, double-blind, placebo-controlled trial

Background Replacement of vitamin D (VD) among patients with chronic hepatitis C (CHC) before viral eradication has demonstrated a protective effect on serum markers associated with hepatic fibrogenesis. We therefore hypothesized that VD may facilitate further fibrosis amelioration following curative treatment with direct-acting antivirals (DAA). Methods This study was a randomized, double-blind, placebo-controlled trial conducted between February 2018 and August 2018. Patients with CHC and VD deficiency were randomized in a 1:1 ratio to either receive ergicalciferol or placebo over 6 weeks. Biochemical analysis indicators, including 25-hydroxyvitamin D (25(OH)D), fibrogenic markers [(transforming growth factor beta 1 (TGF-β1) and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1)], and fibrolytic markers [matrix metalloproteinase 9 (MMP-9) and amino terminal type III procollagen peptide (P3NP)], were assessed at baseline and at 6 weeks. Serum 25(OH)D was analyzed by a chemiluminescence immunoassay. Serum hepatic fibrogenesis markers were measured using a quantitative sandwich enzyme-linked immunosorbent assay. Results Seventy-five patients with CHC and VD deficiency were randomly assigned to VD (n = 37) and placebo (n = 38) groups. At the end of the study, the mean serum 25(OH)D level had risen to a normal level in the VD group, but was still deficient in the placebo group (41.8 ±   9.1 vs. 18.1 ±  4.6 ng/mL, p < 0.001). Upon restoration of the VD level, there were no significant mean differences in the change from baseline for TGF-β1 (−0.6 ng/mL (95% confidence interval (95% CI) [−2.8–1.7]), p = 0.63), TIMP-1 (−5.5 ng/mL (95% CI [−26.4 –15.3]), p = 0.60), MMP-9 (122.9 ng/mL (95% CI [−69.0 –314.8]), p = 0.21), and P3NP (−0.1 ng/mL (95% CI [−2.4 –2.2]), p = 0.92) between the VD and placebo groups. Conclusion Short-term VD supplementation after DAA treatment in patients with CHC does not improve serum fibrogenesis markers and may not expedite the residual liver fibrosis healing process. Future studies are warranted to evaluate the long-term effect of VD supplementation on hepatic fibrosis regression

Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Background Hepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA. Method Blood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 °C and 25 °C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to −80 °C for storage. The effect of multiple cycles of freezing and thawing of plasma at −20 °C or −80 °C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log10 copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0. Results The mean HBV pgRNA level in the initially collected plasma samples was 5.58 log10copies/mL (ranging from 3.08 to 8.04 log10 copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by −0.05 log10 copies/mL (95% confidence interval (CI) −0.095 to −0.005, p = 0.03), −0.075 log10 copies/mL (95% CI [−0.12 to −0.03], p = 0.001), −0.084 log10 copies/mL (95% CI [−0.13 to −0.039], p =  < 0.001), and −0.120 log10 copies/mL (95% CI [−0.17 to −0.076], p =  < 0.001), respectively. However, these changes were below 0.5 log10 copies/mL and thus were not clinically significant. Compared with the samples stored at 4 °C, there were no significant differences in pgRNA levels in samples stored at 25 °C for any of the storage durations (−0.01 log10 copies/mL; 95% CI [−0.708 to 0.689], p = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at −20 °C and −80 °C. Conclusion The plasma HBV pgRNA level was stable at 4 °C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable