Aims: Positive serum antinuclear antibody (ANA) is present in a number of patients with chronic hepatitis C virus (HCV) infection. This study aimed to evaluate the prevalence of ANA in patients with chronic hepatitis C (CHC) and to elucidate its clinical implications in virological and histological characteristics of CHC infection.
Methods: A total of 614 CHC patients were enrolled in this prospective, hospital-based study. The serum levels of aspartate aminotransferase, alanine aminotransferase and ANA, and HCV genotype, HCV RNA level, and histological activity index scores for liver histopathology, were determined.
Results: The prevalence of positive ANA (titre >1:40) was 35.0%. Women had a significantly higher prevalence than men (41.2 vs 31.0%; p = 0.012). Patients positive for ANA were significantly older (mean (SD), 53.7 (10.5) vs 49.7 (11.3) years; p<0.001) and had higher mean (SD) alanine aminotransferase levels (186.9 (178.8) vs 155.50 (113.5) IU/l; p<0.001) and lower mean (SD) HCV RNA levels (5.2 (0.9) vs 5.4 (1.0) log IU/ml; p = 0.048) than those without ANA. Among 447 patients undergoing liver biopsy, those positive for ANA had a significantly higher mean (SD) fibrosis score (2.0 (1.3) vs 1.5 (1.1); p<0.001) and a higher frequency of F3–4 (69/187, 36.9% vs 50/260, 19.2%; p<0.001) than those negative for ANA. Multivariate logistic regression analyses showed that advanced fibrosis, lower HCV RNA levels and age were significant factors related to positive ANA.
Conclusion: ANA is associated with a more advanced liver fibrosis and lower serum HCV RNA level in patients with CHC.
- antinuclear antibody
- chronic hepatitis C
- liver fibrosis
- HCV RNA
- HCV RNA level
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Hepatitis C virus (HCV) infection frequently causes persistent infection leading to chronic liver disease, cirrhosis and hepatocellular carcinoma.1 HCV has been shown to be a lymphotropic as well as a hepatotropic virus.2 Replication of HCV in diseased extrahepatic organs and tissues may have cytopathic effects. Therefore, it may either trigger latent autoimmunity or induce de novo autoimmune disease.3 Various immunological phenomena including autoimmunity and autoimmune diseases in patients with chronic hepatitis C (CHC) infection are not uncommon.4–7 Autoantibodies such as organ-specific and non-organ-specific autoantibodies (NOSAs) are commonly found in patients with CHC infection.8 9 Antinuclear antibody (ANA), a key marker for autoimmune liver diseases and other autoimmune disorders, has been reported to occur in 4–41% of patients with CHC.8 10 11 However, the association between ANA and the clinical features of CHC remains controversial.12–14 Moreover, the impact by ANA on virological and histological characteristics of CHC has not been fully clarified.
Taiwan is a hepatitis B virus hyperendemic country, with a carrier rate of hepatitis B surface antigen (HBsAg) in the general population as high as 10–20%.15 16 The seroprevalence of HCV antibody (anti-HCV) in northern Taiwan has been reported as 1–3%.17 18 Nevertheless, our recent community-based large-scale study has shown that the prevalence of anti-HCV in southern Taiwan is 6.5%.4 Furthermore, several HCV hyperendemic townships in southern Taiwan that have an anti-HCV prevalence of more than 15% have been reported previously.19–21 Therefore, the association between ANA and the clinical features of CHC could be an important issue in southern Taiwan. However, whether there is any association between ANA and the clinical characteristics of Taiwanese patients with CHC has not been reported. We therefore conducted a prospective large-scale study to determine the prevalence of ANA in CHC patients. We also aimed to evaluate the impact of positive ANA on the clinical characteristics of CHC patients.
From December 2002 to December 2005, 614 consecutive patients (371 men, 243 women, aged 20–78 years; mean (SD) age, 51.1 (11.2) years) were enrolled in the current study at Kaohsiung Medical University Hospital, a tertiary referral centre in southern Taiwan. All patients had been positive for anti-HCV for more than 6 months and had detectable serum HCV RNA. Patients with (HBsAg), HIV infection, primary biliary cirrhosis, sclerosing cholangitis, Wilson disease, α1-antitrypsin deficiency, a current or past history of alcohol abuse (⩾20 g daily), previous liver transplantation, or evidence of hepatocellular carcinoma, were excluded from the study. All subjects were thoroughly screened for autoimmune diseases during a detailed physical examination and history taking; none of the patients had received treatment for previous or present systemic autoimmune diseases that involve multiple, diverse organs and tissues (eg, systemic lupus erythematosus, rheumatoid arthritis, Sjőgren syndrome). Liver histology was assessed by two pathologists who were blinded to the ANA status of the individual biopsies and all other clinical information. The disease activity grade and fibrosis stage were quantitatively scored according to the histological activity index (HAI).22 None of the patients had received antiviral treatment before or at the time of the study. Sera of 320 consecutive patients with positive HBsAg were analysed for ANA. The present study was approved by the ethics committee of Kaohsiung Medical University Hospital in accordance with the Declaration of Helsinki, and informed consent was obtained before the study began.
Anti-HCV was detected with a commercially available third generation ELISA kit (Abbott, North Chicago, Illinois, USA). Serum HBsAg was assayed using a commercially available kit (General Biological HBsAg radioimmunoassay; General Biological Cooperation, Hsinchu, Taiwan). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured on a multichannel autoanalyser. ANA was detected by the commercially available indirect immunofluorescence on Hep-2 cells method (Medical and Biological Laboratories, Nagoya, Japan), with an initial dilution of 1:20, following standard protocols.23 24 Positive reactions were titrated by double dilution to the end point. Serum reactivity at a dilution of at least 1:40 was considered as a positive result for all autoantibodies in the present study.
Detection of serum HCV RNA was performed using a standardised automated qualitative reverse transcription-PCR assay (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche, Branchburg, New Jersey, USA). The detection limit was 50 IU/ml. The HCV RNA levels were determined by using the branched DNA assay (Quantiplex HCV RNA 3.0; Bayer, Emeryville, California, USA), performed strictly in accordance with the manufacturer’s instructions. The quantification limit was 615 IU/ml HCV RNA. HCV genotypes 1a, 1b, 2a, 2b and 3a were determined by amplification of the core region using genotype-specific primers.25
Frequency was compared between groups using the χ2 test with Yate’s correction or Fisher’s exact test. Group means, presented as mean (SD) values, were compared using the Student t test, or nonparametric Mann–Whitney test when appropriate. Serum HCV RNA levels were expressed as means (SD) after logarithmic transformation of original values. Stepwise logistical regression was used to determine which variables were independent factors related to positive ANA. The procedures were performed using the SPSS 12.0 statistical package (SPSS, Chicago, Illinois, USA). All statistical analyses were based on two-sided hypothesis tests with a significance level of p<0.05.
The basic demographic characteristics of all 614 patients at enrolment into the study are shown in table 1. There were 306 (49.8%) patients with HCV genotype 1b infection. A total of 215 (35.0%) patients were positive for serum ANA (a titre >1:40) and the distributions of ANA titres are shown in table 1. Among 320 patients (260 men, 60 women, aged 15–71 years; mean (SD) age, 40.6 (12.5) years) with positive HBsAg, 70 (21.9%) were positive for serum ANA. The prevalence of ANA was significantly higher in patients with HCV infection than in patients with HBV infection (p<0.001). Amongst those enrolled patients, 447 patients (263 men, 184 women, aged 20–78 years; mean (SD) age, 51.4 (10.8) years) who intended to receive further antiviral treatment underwent percutaneous liver biopsy. Their mean (SD) necroinflammatory and fibrosis scores were 4.91 (2.44) and 1.73 (1.22), respectively. One hundred and nineteen (26.6%) patients with fibrosis scores of 3 or 4 were defined as having advanced fibrosis. The basic demographic characteristics between patients with and without receiving liver biopsy are shown in table 2; there were no significant differences in sex, age, AST/ALT, HCV genotype or HCV RNA levels between the two groups.
Table 3 shows the comparison of clinical characteristics by univariate analysis between patients with and without positive ANA. Female patients had a significantly higher frequency of positive ANA than males (41.2% vs 31.0%; p = 0.012). Univariate analysis also demonstrated that positive ANA status was significantly associated with older age at enrolment into the study (mean (SD) 53.7 (10.5) vs 49.7 (11.3) years; p<0.001), higher mean (SD) AST (122.3 (75.7) vs 98.7 (64.5) IU/l; p<0.001) and ALT levels (186.9 (178.8) vs 155.5 (113.5) IU/l; p = 0.010) at enrolment into the study, and lower mean (SD) HCV RNA levels (5.2 (0.9) vs 5.4 (1.0) log IU/ml; p = 0.048). Among the 447 patients underwent liver biopsy, the comparison of clinical characteristics by univariate analysis between patients with and without positive ANA are shown in table 4. ANA status was significantly associated with higher mean (SD) fibrosis scores (2.0 (1.3) vs 1.5 (1.1); p<0.001) and a higher frequency of F3–4 (69/187, 36.9% vs 50/260, 19.2%; p<0.001) on histological examination. Multivariate logistic regression analysis was conducted among 447 patients who underwent liver biopsy to clarify the independent factors associated with positive ANA. The analysis shows that severe fibrosis, lower HCV RNA levels and older age were significant independent factors associated with positive ANA (table 5).
The prevalence of positive antinuclear antibody (ANA) (titre >1:40) in patients with chronic hepatitis C (CHC) was 35.0%.
ANA seropositivity is associated with clinical characteristics including a more advanced fibrosis, lower serum hepatitis C virus RNA level, and older age, in patients with CHC.
ANA is a useful marker in predicting disease activity in patients with CHC.
In patients with HCV infection, evidence of altered immune system homeostasis is indicated by a high prevalence of NOSAs; this prevalence includes more than 50% of the infected population.12 26 Among NOSAs, anti-liver/kidney microsomal antibody type 1 has a direct influence over the enhanced severity of liver damage. However, for more frequently observed NOSAs, such as smooth-muscle antibody and ANA, there are insufficient data to provide a conclusive answer regarding their pathogenicity.27 Moreover, discrepancies in terms of prevalence between previous studies further address the difficulty in clinical elucidation.8 10 11 26 ANA is a key marker and its detection provides a simple method for autoimmune disorder screening. Therefore, it represents a sensitive and important tool for the elucidation of its impact on profiles of HCV infection. In the current study, we demonstrated that the prevalence of ANA seropositivity in Taiwanese patients with CHC was 35% in a hospital-based setting; this was much higher than the prevalence of patients with positive HBsAg (21.9%). The result was similar to a 34% prevalence of ANA seropositivity in patients CHC patients reported by Luo et al in Taiwan.14 However, because of the limitation of a hospital-based population, we did not have enough data to show the prevalence of ANA seropositivity in healthy controls; previous reports of the prevalence of ANA seropositivity in Taiwan have been 5–9.2% in healthy individuals.28 29 Noteworthy is that all the patients enrolled in the current study were clinically asymptomatic. Our results suggest that a correlation exists between chronic HCV infection and autoimmunity in Taiwanese patients with CHC. It reminds us that the autoimmune phenomenon is not uncommon and might be underestimated among patients with HCV infection in Taiwan. Further studies regarding the prevalence and characteristics of different forms of NOSAs, and the clinical manifestations, are needed to clarify this issue.
It has been shown that CHC patients with early thyroid dysfunction induced by interferon α therapy had a significantly lower pretreatment serum HCV RNA level than others.30 Meanwhile, thyroid autoantibodies, occurring either before or during combination therapy, carry a high prediction of subsequent thyroid dysfunction.5 Lower HCV RNA levels have also been shown to be correlated with CD8 cytotoxic T lymphocyte activity and CD4 cell counts in patients with CHC.31 32 Patients with intrahepatic HCV-specific cytotoxic T lymphocyte activity have been shown to have lower levels of viraemia and more active liver disease in some studies.32 33 Our recent study demonstrated that the single nucleotide polymorphism of tumour necrosis factor α promoter gene at position −308 is associated significantly with HCV RNA level.34 HCV infection is associated with presentation of multiple extrahepatic manifestations that are mainly attributed to an interplay between host immunity and HCV. There have been different results in past years concerning ANA and change of HCV viraemia; however, some previous investigations did not observe a correlation between ANA positivity and HCV RNA level in patients with CHC.12–14 Yee and coworkers observed that only the female sex was correlated with positive ANA.13 In contrast, Luo et al reported no association between ANA positivity and clinical features including age, sex, liver biochemical tests, serum HCV RNA level and HCV genotype.14 In a study by Wasmuth et al, 36 patients with CHC and NOSAs had a higher viral load compared with 42 CHC patients without autoantibodies.35 In the present study, we found that positive ANA was significantly associated with lower HCV RNA levels, and this implicated the association between immunity and HCV viral load. Whether ethnic and/or environmental factors might contribute to the discrepant results needs further studies. Subsequent changes of HCV infection confounding or interfering with host autoimmunity might be a possible explanation, and further investigation focusing on the sequential changes or interaction between ANA and HCV viraemia will be needed.
Our study demonstrated that a more advanced fibrosis stage of liver biopsies and older age in patients were associated with ANA positivity. The presence of serum ANA is associated with various factors including advancing age, genetic predisposition, environmental agents, oestrogen–androgen balance, chronic infection and neoplasm.36 Previous studies have shown that patients with ANA have more plasma cells in liver biopsies; the increased plasma cells might be a marker for B cell polyclonal activity, with a secondary clinical manifestation of increased serum immunoglobulin with or without autoantibody production13 37 indicating an association between ANA and host immune status in the liver. The immune mechanism of liver damage is still unclear and the host factors of ageing have also shown a strong association with fibrosis progression in HCV infection.38 Recently, we reported that the single nucleotide polymorphism the of interferon γ gene at position +874, and age, were independent factors associated with cirrhosis in Taiwanese patients with CHC.39 Thus, the pathogenesis and progression of liver damage might relate to host immunity and ageing. Our data showing an association between ANA and a more advanced hepatic fibrosis indicate that evaluation of ANA as a marker of liver disease activity in patients with CHC might be important and helpful in predicting a more rapid progression of fibrosis.
In conclusion, we have demonstrated that the presence of ANA seropositivity is associated with clinical characteristics including a more advanced fibrosis, lower serum HCV RNA level, and older age, in patients with CHC. ANA could be a useful marker in predicting disease activity in patients with CHC.
Competing interests: None.