A retrospective study of prevalence and pattern of international consensus on ANA patterns among patients with hepatitis C virus infection

Introduction

Chronic hepatitis C (CHC) is one of the significant causes of cirrhosis and hepatocellular carcinoma (HCC) worldwide. It is estimated that 74% of patients with CHC had at least one extrahepatic clinical manifestation, such as lymphoma, cryoglobulinemia, glomerulonephritis, arthritis, autoimmune thyroiditis, type 2 diabetes mellitus, and Sjögren’s syndrome (SjS) (Giuggioli et al., 2017; Cacoub & Saadoun, 2021; Negro et al., 2015; Su et al., 2014; Vergani & Mieli-Vergani, 2013). Researchers have found other autoantibodies in patients with CHC, including anti-antinuclear antibody (ANA), anti-smooth muscle antibody (ASMA), anti-mitochondrial antibody (AMA), and anti-liver/kidney microsomes type 1 (anti-LKM1). The prevalence of these autoantibodies was approximately 30% in patients with CHC (Cassani et al., 1997). ANA, detected by indirect immunofluorescence assay of HEp-2 cells (IIFA Hep-2), offers the titer of the fluorescence intensity and provides fluorescence patterns, which are helpful in diagnosing several systemic autoimmune diseases.

Two workshops are used for the standardization and classification of ANA patterns, namely, the International Autoantibody Standardization (IAS) and the International Consensus on ANA Patterns (ICAP) (Agmon-Levin et al., 2014; Damoiseaux et al., 2015). The IAS perspective promotes the standardization and improvement of autoantibodies in laboratory diagnosis. The ICAP classification presents four categories—negative, nuclear, cytoplasmic, and mitotic—and the 29 IIFA Hep-2 patterns with the clinical correlation (Chan et al., 2022; Damoiseaux et al., 2019). Each pattern is assigned a code below the descriptor, namely, anti-cell pattern 1 (AC-1) (Chan et al., 2015).

One decade ago, we used interferon (IFN)-based therapy to treat patients with hepatitis C virus (HCV) infection. Several researchers have reported that IFN-based therapies triggered the development of autoimmune diseases, extrahepatic manifestations, and autoantibodies (Covini et al., 2012; Jadali, 2013; Kajiyama et al., 2012; Marazuela et al., 1996; Novembrino et al., 2014). With the development of direct-acting antiviral agents (DAAs), the treatment of HCV infection has become feasible because DAAs showed a sustained virologic response rate >90%, less intolerance side effects, and short treatment duration compared with IFN-based therapies (Chi et al., 2021; Yen et al., 2020). Several case reports have shown a flare of autoimmune hepatitis or primary biliary cholangitis during or after DAA treatment (Choi et al., 2020; Fianchi, Ponziani & Pompili, 2020; Matsumoto et al., 2018; Montón, Escudero & Pascual, 2020; Nguyen et al., 2018). It is unknown whether the patient or the effect of DAAs triggers the autoimmunity. It is also important to know the autoimmunity status before treatment.

This study was designed to analyze the prevalence of ANA patterns in patients with CHC before DAA treatment and determine the association between different ANA patterns and patient characteristics.

Materials & Methods

Results

Discussion

In our analysis, 28.7% of the patients with CHC were ANA-positive. According to the ICAP, the most detected ANA pattern was AC-1, followed by AC-4, AC-21, AC-8, and AC-27. Of the 74 ANA-positive patients, 36 had more than one ANA pattern. ANA-positive patients showed a lower HCV RNA viral load and higher prevalence of advanced fibrosis (F3 and F4) without statistical significance. Higher positive ANA levels (>1:640) were significantly associated with a lower eGFR.

Patients with CHC are predisposed to have extrahepatic manifestations (Vergani & Mieli-Vergani, 2013). The most strongly associated disease is cryoglobulinemia (Giuggioli et al., 2017; Comarmond, Cacoub & Saadoun, 2020). Moreover, several systemic autoimmune diseases have also been reported, such as autoimmune thyroiditis (Jadali, 2013), glomerulonephritis (Negro et al., 2015), rheumatoid arthritis (Su et al., 2014), and SjS (Wang et al., 2014). The incidence of rheumatological manifestations in patients with CHC is estimated to be 38% (Priora et al., 2021). Besides, it has been noticed that several autoimmune diseases may be triggered during interferon therapy for patients with CHC (Pellicano et al., 2005; Wilson et al., 2002). Several studies have revealed that antithyroid autoantibodies and thyroid dysfunction may be triggered by IFN-alpha-based therapies for patients with CHC even without pre-existing thyroid abnormalities (Jadali, 2013; Marazuela et al., 1996). A nationwide population-based cohort study from Taiwan reported that the cumulative incidence of rheumatic diseases was lowest in HCV-uninfected cohorts (95% confidence interval (CI) [8.416%–10.734%]) compared with HCV-infected cohorts treated with (95% CI [12.417%–17.704%]) and untreated HCV-infected cohorts (95% CI [13.585%–16.479%]) (Cheng et al., 2021). This finding indicates that HCV infection was associated with some rheumatic diseases.

ANA is widely used for diagnosing several systemic autoimmune diseases, providing not only negative or positive results but also fluorescence patterns. In the past, we detected “antinuclear” antibodies and defined their patterns using immunoassay, including homogeneous, speckled, nucleolar, and nuclear rim (White & Robbins, 1987). IIFA Hep-2 allows the identification of autoantibodies targeted to antigens localized in the nucleus and cytoplasm and mitotic cells (Chan et al., 2016). The ICAP developed a classification tree for most staining patterns. There are homogeneous, speckled, dense, fine speckled, centromere, discrete nuclear dots, and nucleolar reported in nuclear patterns, and cytoplasmic patterns are fibrillar, speckled, reticular/mitochondrion-like, polar/Golgi-like, and rods and rings (Chan et al., 2022; Damoiseaux et al., 2019). These patterns were correlated with different rheumatological diseases and have been reported to play a significant role as an anti-marker of rheumatic pathology, particularly the AC-2 pattern (Pashnina et al., 2021).

There are several diseases; however, autoimmune diseases, such as hyperthyroidism, gastroparesis, and non-alcoholic fatty liver disease, were found to be associated with ANA positivity (Mitra & Ray, 2020; Parkman et al., 2022; Xun, 2021). Although the correlation remains unclear. A study from China on the relationship between ANA and various liver diseases showed that the incidence of ANA positivity in patients with hepatitis B virus (HBV) and HCV infection was 19.1% and 3.9%, respectively. The most common ANA patterns in patients with HBV infection were AC-1, AC-4, AC-21, AC-5, and AC-2. Nevertheless, the most common ANA patterns in patients with HCV were AC-21, AC-4, AC-1, and AC-5 (Wei et al., 2020). A recent study analyzed the prevalence and outcomes of autoantibodies in HCV-infected patients. The most common ANA patterns were homogeneous and speckled patterns, accounting for 43% and 36%, respectively, followed by nucleolar (7%), centromeric (7%), and rods and rings (7%) (Romano et al., 2022). Furthermore, another study revealed elevated serum markers of autoimmune hepatitis, namely, ANA, immunoglobulin G, smooth muscle actin, anti-LKM1, and soluble liver antigen, in patients with CHC (Simoes et al., 2019). However, the authors did not examine the association of these serum markers with the virological features of their patients with HCV infection or did not assess the association of these markers with different ANA patterns.

To the best of our knowledge, this is the first ANA analysis among patients with CHC with viremia according to the ICAP. Furthermore, this study provides insight onto HCV infection and autoimmunity.

In our analysis, the most detected pattern was AC-1, accounting for 25.9%, followed by AC-4, accounting for 25.2%; AC-21, accounting for 9.6%; and AC-8, accounting for 8.9%. In previous reports, the AC-1 pattern was associated with systemic lupus erythematosus, chronic autoimmune hepatitis, and juvenile idiopathic arthritis (Aringer et al., 2019; Petri et al., 2012). The AC-4 pattern is clinically indicated in systemic autoimmune rheumatic diseases, particularly SjS, lupus erythematosus, dermatomyositis, and systemic sclerosis (Aringer et al., 2019; Betteridge & McHugh, 2016; Shiboski et al., 2017; Trallero-Araguás et al., 2012). The AC-21 pattern was found in patients with primary biliary cholangitis and systemic sclerosis (Shuai et al., 2017; Zheng et al., 2017). Finally, the AC-8 pattern was found in patients with systemic sclerosis (Ceribelli et al., 2010; Mahler, Fritzler & Satoh, 2015). Our patient population has no clinical manifestations of the aforementioned rheumatological conditions. There is no prior research on ANA patterns in CHC with viremia, and we provide additional information regarding the new ANA pattern among patients with HCV infection. Thus, whether HCV per se causes the presence of such autoantibodies or triggers the subsequent development of the aforementioned rheumatological conditions requires further Investigation.

ANA positivity was mostly found in females, accounting for 63.5%. This finding is consistent with the findings of a study on the relationship between sex and autoimmune diseases, which reported that females are more likely to be ANA-positive than males (Whitacre, 2001). A study revealed that ANA-positive patients with CHC were significantly associated with higher levels of aspartate aminotransferase, alkaline phosphatase, and alpha-fetoprotein (Peng et al., 2001). Another study reported a faster rate of liver fibrosis in ANA-positive patients with CHC but without statistical significance (odds ratio = 1.8; p = 0.1452) (Yee et al., 2004). However, a recent study showed no correlation between cirrhosis, hepatic decompensation, HCC, and survival during the 10-year follow up in patients with CHC with or without autoantibody positivity (including ANA, ASMA, AMA, and anti-LKM (Gilman et al., 2018). Our data showed that the ANA(+) group had a lower HCV RNA viral load and higher prevalence of advanced fibrosis (F3 and F4). Additionally, in this study, we noticed higher ANA titers after adjusting for age and sex, which showed a significant association with a lower eGFR (p = 0.008). This study has some limitations. First, this is a retrospective study in a single institution involving a small sample. However, our study has one strength in evaluating hepatitis C viremic patients with their ANA profiles. Some patients had a positive or higher ANA titer with HCV viremia, as hepatitis C infection is associated with some autoimmune diseases. Knowing the baseline ANA profiles paves the way for further studies comparison of the evolution of ANA profile change following hepatitis C eradication. Second, there was no control group for comparison, and we didn’t have data on other autoantibodies, including ASMA, AMA, and anti-LKM1, in our cohort for further analysis. Third, we only analyzed the patients’ pretreatment data. A long-term follow-up study on patient outcomes, including fibrosis stage, hepatic decompensation, cirrhosis, HCC, and other comorbidities, is required to assess ANA pattern changes after HCV eradication.

Conclusions

We found that the overall prevalence of ANA positivity in patients with CHC was 28.7%. The most detected ANA patterns were AC-1, AC-4, AC-8, and AC-21. ANA-positive patients revealed a lower HCV RNA viral load and a higher prevalence rate of advanced fibrosis, although there was no statistical significance. Furthermore, higher ANA titer values (>1:640) were significantly associated with a lower eGFR.

Supplemental Information