Changes in thyroid-stimulating hormone and thyroxine during hepatitis C virus infection and after direct-acting antiviral therapy independent of interferon exposure: a prospective paired cohort study

Introduction

Thyroid dysfunction is a common co-morbidity across liver diseases, including viral, metabolic, and oncological etiologies (1). Hepatitis C virus (HCV) infection is of particular interest due to its high rate of extrahepatic manifestations, one being autoimmune thyroiditis (2). Thyroid disease is a well-recognized extrahepatic manifestation of HCV infection (HCVi), with prior studies reporting thyroid disorders in up to 15% of interferon-naïve HCV-positive patients. The spectrum of thyroid involvement includes hypothyroidism, hyperthyroidism, and autoimmune thyroiditis; however, many affected individuals exhibit subclinical abnormalities without overt thyroid disease (3).

Despite the development of direct-acting antiviral therapy (DAAs), 58 million individuals were infected with HCV in 2019 and it continues to be a leading cause of chronic liver disease (4). Unlike interferon treatment, which is associated with newly developed thyroid disease as a result of treatment (5), studies have revealed that DAA therapy is not associated with the development of thyroid disease (6). While the association between HCVi and autoimmune thyroid disease is documented, paired changes in thyroid hormone function before and after DAA-mediated viral clearance, independent of interferon exposure, have not been well characterized.

Prior studies evaluating thyroid function in HCV have largely been cross-sectional or conducted in the context of interferon-based therapy, limiting interpretation of virus-specific effects on thyroid physiology. Additionally, many studies have focused on overt thyroid disease or autoimmune markers rather than subtle hormonal shifts, and few have examined paired longitudinal changes before and after viral clearance (7,8). As a result, it remains unclear whether observed thyroid alterations reflect direct viral effects, systemic illness (e.g., euthyroid sick syndrome), or early autoimmune processes. This study addresses this gap by evaluating paired changes in thyroid hormone levels before and after sustained virologic response (SVR) in interferon-naïve patients treated with DAA therapy. The main goal of this hypothesis-generating study is to explore how thyroid function, quantified via thyroid-stimulating hormone (TSH) and thyroxine (T4) levels, changes during HCVi and after DAA therapy.

Methods

As previously described in detail (9), this exploratory analysis was performed using a prospective cohort of 29 adults with chronic HCVi enrolled at the National Institutes of Health Clinical Center (NCT02400216) between 29 May 2015 and 11 March 2016. Patients received DAA therapy with sofosbuvir/velpatasvir and achieved SVR. Detailed eligibility criteria, recruitment procedures, baseline clinical characteristics, and longitudinal multi-omic assessments were previously reported in the parent cohort publication (9).

Six months post SVR, 24 patients returned for reevaluation. All research was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board (No. 15-DK-0100). All subjects gave written informed consent. This study was registered at https://clinicaltrials.gov/study/NCT02400216. The registration identification number is NCT02400216.

Biochemical assays were performed on a Cobas C 501 system to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, and an Abbott Architect I2000SR Immunoassay Analyzer to measure TSH. Complete blood counts were drawn in 3 mL K2 EDTA tubes (Ref 367856, Becton, Dickinson and Company), measured on Sysmex system. T4 levels in serum were reported as relative concentration by Metabolon’s non-targeted global metabolite profiling as previously described (9).

Five patients were excluded from analysis due to levothyroxine use for hypothyroidism. TSH was measured in 19 patients during HCVi {median timepoint 3 months before treatment [interquartile range (IQR) 1.5–5]} and 18 after SVR [median timepoint 37 months post SVR (IQR 28–39.5)], with 15 patients having TSH measured at both timepoints. During infection, a percutaneous liver biopsy was performed, and patients were stratified using Ishak Fibrosis (IF) score. IF scores from 0–4 were classified as non-cirrhotic (NC) and 5–6 were classified as cirrhotic (C). Subject characteristics with measured TSH are found in Table 1. Resulting distribution of patients with TSH and T4 measurements is summarized in Table S1.

Statistical analysis

Continuous variables were summarized using medians and IQRs. Due to non-normal distribution and small sample size, non-parametric tests were used. Paired comparisons were performed using the Wilcoxon signed-rank test, and unpaired comparisons using the two-sided Mann-Whitney U test. A two-sided P value <0.05 was considered statistically significant. Analyses were conducted using GraphPad Prism version 10.2.2. Patients without paired post-SVR measurements were excluded from paired analyses.

Results

To assess how thyroid function is impacted by HCVi, TSH and T4 were measured in patients during infection and after SVR. TSH was significantly reduced during infection (median 2.01, IQR 1.06) compared to after SVR (median 2.27, IQR 1.43) (P<0.001) (Figure 1A). The C subgroup had no significant difference in TSH levels during infection compared to after SVR (P=0.13) (Figure 1B), while the NC subgroup had significantly reduced TSH during infection compared to after SVR (P=0.002) (Figure 1C). There was no significant difference in TSH levels between C and NC groups during infection (P=0.84) and after SVR (P=0.80) (Figure 1D,1E).

Figure 1 Changes in TSH and T4 levels during HCVi. TSH was significantly reduced during infection (A). In the subgroups, TSH was not significantly reduced during infection in Cs (B) but was in NCs (C). TSH also did not differ between C subgroups neither during infection (D) nor after SVR (E). T4 was significantly elevated during infection compared to after SVR (F). In the subgroups, T4 was significantly elevated in both Cs (G) and NCs (H). However, T4 did not differ between C subgroups neither during infection (I) nor after SVR (J). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. C, cirrhotic; HCVi, hepatitis C infected patients; NC, non-cirrhotic; SVR, sustained virologic response; T4, thyroxine; TSH, thyroid-stimulating hormone.

T4 was significantly elevated during infection (P<0.001) compared to after SVR (Figure 1F). Both C (P=0.01) and NC (P=0.001) (Figure 1G,1H) subgroups had significantly elevated T4 during infection compared to after SVR. There was no significant difference in the T4 levels between C and NC groups during infection (P=0.08) and after SVR (P=0.93) (Figure 1I,1J).

To further assess whether the severity of liver disease impacted thyroid function, TSH and T4 were correlated with liver disease markers. Neither TSH nor T4 correlated with any liver disease marker during infection, and the only correlations after SVR were between T4 and AST (ρ=−0.51, P=0.03) and total bilirubin (ρ=−0.63, P=0.005) (Figure 2).

Figure 2 Relationship between TSH and T4 with liver disease severity. Neither TSH nor T4 (x-axis) correlated with liver function tests (y-axis) during infection. After SVR, T4 correlated with TB and AST. Unadjusted P value: +, =0.05–0.1; *, =0.05–0.01; **, =0.01–0.001. AFP, alpha fetoprotein; ALP, alkaline phosphatase; ALT, alanine transferase; AST, aspartate transferase; DPP, dipeptidyl peptidase; GGT, gamma-glutamyl transferase; HAI, hepatic activity index; HCVi, hepatitis C infected patients; INR, international normalized ratio; SVR, sustained virologic response; T4, thyroxine; TB, total bilirubin; TSH, thyroid stimulating hormone.

Discussion

This hypothesis-generating study identified reversible alterations in TSH and T4 that were associated with active HCVi, which warrants further investigation in larger, prospective cohorts. Importantly, this analysis focuses on thyroid hormone dynamics in the contemporary DAA era. There is an abundance of literature discussing thyroid dysfunction in HCV, however the majority of research involves cohorts receiving interferon treatment, which directly mediates thyroiditis via an immune response (5,10). Analyses of thyroid functionality in treatment naïve patients with HCV has also been reported and tends to overlap with thyroid autoimmunity (11,12). There have been studies investigating the effects of DAA therapy on thyroid function suggesting clinical and subclinical thyroid dysfunction (6,13-15). Interestingly, our investigation revealed that of the patients analyzed without the presence of subclinical or clinical thyroid dysfunction as traditionally defined, there was a significant increase in TSH and decrease in T4 following DAA therapy. The findings of this study may be due to effects of either HCV or DAA therapy, and further investigation into the immunological response may provide further insight into the cause of thyroid alterations observed.

Potential mechanisms underlying these observations, which has been seen in other viral infections, include direct viral effects on thyrocytes (16), immune responses—such as cytokine and antibody enrichment, altering thyroid function like deiodinase activity (17), and modulation of the hypothalamic-pituitary-thyroid axis during chronic infection (18). These findings may also coexist with euthyroid sick syndrome, in which systemic illness alters peripheral thyroid hormone metabolism (19). The observed normalization following SVR supports a reversible process potentially driven by viral activity rather than permanent thyroid dysfunction.

The trend in TSH and T4 observed in all patients in our cohort during HCVi demonstrates the possibility that intervening early during the development of chronic HCV may aid in prevention of clinically significant thyroid dysfunction. These findings may be generalizable to broader populations of patients with Hepatitis C infection. All the findings in this study are observational and from a small sample size and would need to be further studied in a controlled experiment to validate results.

An important factor to consider in the effects of HCV on thyroid function is the state of progression of liver disease. Liver diseases including cirrhosis, hepatocellular carcinoma and cholangiocarcinoma can lead to abnormally high and/or low levels of TSH, T3, and T4 (2). However, in our cohort there was no difference between Cs (all compensated) and NCs when comparing levels of TSH or T4 nor significant correlations with TSH and T4 with liver disease markers, suggesting that thyroid alterations are independent of worsening liver disease. When subgroups were evaluated separately, only NCs were significantly different in terms of TSH. However, this was most likely compounded by small NC sample size (n=5).

The prevalence of thyroid alterations during illness, especially in conditions of high metabolic stress, is well documented in the concept of euthyroid sick syndrome, where abnormal thyroid hormone has been observed in patients with acute illness (19). This phenomenon may be indicative of a metabolic necessity for alterations in thyroid function during sickness, affecting accessibility to energy stores. Additionally, thyroid hormone has been closely associated to lymphocyte function, demonstrating a role of thyroid in immune regulation (20). In our study, the prevalence of thyroid dysfunction in treatment naïve HCV-infected patients suggests a possible viral influence on the thyroid. Recent studies have shown that HCV is capable of infecting thyrocytes in vitro (21) and in vivo (22). Additionally, antithyroid autoantibodies and autoimmune thyroiditis occur at a high prevalence in HCV infected patients (11,12), furthering evidence that HCV-associated thyroiditis may be virally induced. Given the findings of this study and the existing literature, we speculate that HCV may contribute to thyroid dysfunction, increasing the importance of early intervention and further investigation into the relationship of the thyroid to viral infection.

Conclusions

This study is limited by its small sample size and observational design, which restrict causal inference. Nonetheless, the exploratory nature of this analysis was intended to generate hypotheses regarding HCV-thyroid interactions rather than establish causality or changes to clinical practice. Thyroid autoantibodies, thyroid imaging, and serial thyroid function measurements during DAA therapy were not available, limiting the ability to distinguish autoimmune from non-autoimmune mechanisms. Thyroid hormone levels may also be influenced by physiologic and environmental factors. Additionally, the absence of a control group and the single-center nature of the cohort may limit generalizability. Despite these limitations, the paired within-patient analysis and consistent normalization of thyroid parameters following SVR support a relationship between active HCVi and reversible alterations in thyroid hormone homeostasis.

Acknowledgments

We would like to thank the patients for volunteering. We also thank the ward nursing staff, research coordinators, and the clinical team involved in the project. We also wish to thank Dr. Rebecca Brown for her insight on the project.

Footnote

Peer Review File: Available at https://aot.amegroups.com/article/view/10.21037/aot-2026-1-0012/prf

Funding: This study was funded by the intramural programs of the National Institute of Diabetes and Digestive and Kidney Diseases (No. DK054515), the National Cancer Institute, and the Clinical Center of the National Institutes of Health. The project was additionally funded by an intramural NIH Bench to Bedside award: Mechanisms of microbial translocation in hepatitis C-related liver disease 2014.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aot.amegroups.com/article/view/10.21037/aot-2026-1-0012/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All research was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board (No. 15-DK-0100). All subjects gave written informed consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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