Prevalence of rheumatoid factor in asymptomatic chronic hepatitis B surface carriers in the context of endemic malaria: cross-sectional study at Cocody University Hospital, Abidjan, Côte d'Ivoire

¹Laboratory of Immunology and Haematology, University Hospital of Cocody, Abidjan, Côte d'Ivoire, ² Department of Immunology-Allergology, Medical Sciences Training and Research Unit, Félix Houphouët-Boigny University, Abidjan, Côte d'Ivoire

^&Corresponding author
Amah Patricia Victorine Goran-Kouacou, Laboratory of Immunology and Haematology, University Hospital of Cocody, Abidjan, Côte d'Ivoire

Introduction    

Rheumatoid factor (RF) is a heterogeneous family of autoantibodies directed against the crystallizable fragment (Fc) of immunoglobulin G (IgG). Primarily of the immunoglobulin M (IgM) isotype, it may also include other classes such as immunoglobulin A (IgA) and IgG [1]. These antibodies can be divided into two categories according to their affinity and function. Physiological RFs, polyreactive and of low affinity, contribute to immune regulation by facilitating the clearance of immune complexes and contributing to anti-infectious defense mechanisms [2]. Pathological RFs, monoreactive and of high affinity, are associated with various pathological conditions [2]. Initially recognized as a diagnostic marker of rheumatoid arthritis [3,4], RF is also detected in a range of diseases, including autoimmune diseases, cancers, and chronic infections [4,5]. Chronic hepatitis B, caused by the hepatitis B virus (HBV), is one such infection. It represents a major public health problem, particularly in Côte d'Ivoire, where the prevalence of hepatitis B surface antigen (HBsAg) carriage exceeds 8% [6]. This infection induces prolonged antigenic stimulation and chronic inflammation, two mechanisms known to promote the production of autoantibodies such as RF [5]. However, the relationship between HBV infection and RF production remains poorly understood. In addition, pathological RF can interfere with diagnostic tests by binding to immune complexes or through cross-reactivity with antigens, complicating serological test interpretation [7,8]. This is of particular concern in settings with limited medical resources, where diagnostic accuracy is critical to ensure appropriate patient management. Understanding the relationship between chronic HBV infection and RF production is essential to improve the interpretation of serological tests and to identify the immunopathological risks associated with this chronic viral infection. This study aimed to determine the prevalence of RF in asymptomatic chronic HBsAg carriers followed at the University Hospital of Cocody in Abidjan, Côte d'Ivoire, and to identify the clinical and immunological factors associated with RF positivity.

Methods     

Study design and setting: this was a prospective, descriptive and analytical cross-sectional study conducted over a 12-month period, from January to December 2024, at the Immunology and Haematology Laboratory of the University Hospital of Cocody, a tertiary referral center located in the commune of Cocody, Abidjan, Côte d'Ivoire. This hospital is affiliated with Félix Houphouët-Boigny University and is a center of excellence for the management of infectious, hepatic, and autoimmune diseases. The Immunology and Hematology Laboratory performs immunological, hematological, and biochemical investigations as part of diagnostic and follow-up care.

Study population: the study population consisted of asymptomatic chronic HBsAg carriers who were either being followed up or presenting for initial screening at the University Hospital of Cocody during the study period.

Inclusion criteria: i)age 15 years and older. ii) Resident in Abidjan for at least one year. iii) Confirmed chronic HBsAg carrier (persistent positivity after six months for initial screening cases). iv) Normal transaminase levels (ALT/AST). v) Negative HBeAg and positive anti-HBe antibodies. vi) Negative anti-HBc IgM antibodies. vii) No active malaria (absence of fever and negative smear).

Exclusion criteria: known autoimmune disease, ongoing antiviral or immunosuppressive treatment, acute infection at the time of enrolment, indeterminate serological results for HBsAg, HBeAg or rheumatoid factor.

Sample size: the minimum sample size was calculated assuming an expected prevalence of rheumatoid factor of 7%, as reported by Sombo et al. [9] in healthy individuals in Côte d'Ivoire. The formula used was:

Sampling method: convenience sampling was used. All patients who met the inclusion criteria and presented during the study period were recruited.

Data collection: data were collected using a structured questionnaire that included the following information: socio-demographic information (age, gender); medical history (transfusions, infections, treatments); reason for consultation (initial screening or chronic follow-up). Blood samples were taken from each patient as follows: i) Dry tube: for measurement of HBsAg, transaminases (ALT/AST), HBeAg, anti-HBe antibodies, anti-HBc IgM and rheumatoid factor. ii) Ethylenediaminetetraacetic acid (EDTA) tube: for the preparation of a Giemsa-stained thick blood smear to exclude active malaria. Patients recruited at baseline were followed up after six months to confirm their chronic HBsAg carrier status.

Laboratory analysis: virological, biochemical and immunological tests were performed using the Cobas 6000 analyser (Roche Diagnostics), according to the following principles, as summarized in Table 1. i) HBsAg, HBeAg, anti-HBe antibodies, anti-HBc IgM: measured by electrochemiluminescence immunoassay (ECLIA) on the e601 module. ii) Transaminases (ALT/AST): measured by spectrophotometric method on the c501 chemistry module. iii) Rheumatoid factor: measured by latex immunoturbidimetry on the c501 chemistry module, with a positivity threshold set at > 14 IU/mL. Internal and external quality controls were carried out to ensure the reliability of the results.

Variables studied: rheumatoid factor; age (divided into age categories); sex; reason for consultation (screening or follow-up).

Data analysis: data were entered and analysed using Epi Info software (version 3.5.1): i) Qualitative variables were expressed as frequencies and percentages. ii) Quantitative variables were expressed as mean ± standard deviation. Univariable analysis was performed using the chi-squared test, with a significance threshold of p < 0.05. iii) Multivariable logistic regression analysis was performed to identify factors independently associated with rheumatoid factor positivity. Results were expressed as adjusted odds ratios (aOR) with 95% confidence intervals and p values. Incomplete data for any variable of interest led to the exclusion of the participant from the specific analysis.

Ethical considerations: the study was conducted in accordance with the tenets of the Declaration of Helsinki. Ethical approval was obtained from the Ethics Committee of the University Hospital of Cocody, Abidjan, Côte d´Ivoire (Ref: 010/MSHP-CMU/CHU-C/DMS/RK/25). Written informed consent was obtained from all participants before enrolment. Confidentiality and anonymity of data were ensured throughout the study.

Results     

Participants and selection: a total of 150 participants were initially recruited. After applying the inclusion and exclusion criteria, 120 participants were retained for the final analysis, exceeding the minimum required size of 110 to ensure adequate statistical power (Figure 1).

Characteristics of patients: the characteristics of the study population are summarized in Table 2. Among the 120 participants, 72 (60.0%) were male and 48 (40.0%) were female, with a sex ratio of 1.5 in favor of male patients. The mean age was 36.4 ± 11.2 years (range: 16 to 65 years). Patients aged 30 years or older represented 66.6% of the study population (n= 80). Regarding the reason for consultation, 75 patients (62.5%) were recruited during initial HBsAg screening, while 45 (37.5%) were under chronic follow-up.

Prevalence of rheumatoid factor and factors associated with rheumatoid factor positivity: the rheumatoid factor was detected in 15 participants, representing a prevalence of 12.5% (95% CI: 6.54-18.46). The results of the univariable and multivariable logistic regression analyses are summarized in Table 3. Advanced age (≥30 years) was the only factor independently associated with RF positivity (aOR: 3.32; 95% CI: 1.06-10.39; p= 0.04). Sex and reason for consultation were not significantly associated with RF positivity: male sex (aOR: 1.39; 95% CI: 0.45-4.27; p= 0.768), chronic follow-up: (aOR: 1.13; 95% CI: 0.38-3.39; p= 0.799).

Discussion     

This study aimed to determine the prevalence of RF in asymptomatic chronic HBsAg carriers followed at the University Hospital of Cocody in Abidjan, Côte d´Ivoire, and to identify factors associated with RF positivity. The prevalence of RF was 12.5% (95% CI: 6.54-18.46), higher than the 7% prevalence reported in healthy individuals in Côte d´Ivoire [9]. Advanced age (≥30 years) was the only factor independently associated with RF positivity (aOR: 3.32; 95% CI: 1.06-10.39; p = 0.04). No association was found with sex or reason for consultation. Our findings support the hypothesis that chronic inflammation and prolonged antigenic exposure due to HBV infection promote RF production through multiple immunopathological mechanisms: i) Persistent antigenic stimulation: prolonged exposure to HBsAg leads to sustained activation of antigen-presenting cells (APCs), CD4+ T cells, and B cells, driven by pro-inflammatory cytokines (IL-4, IL-6, TNF-α) [10]. ii) Disruption of immune tolerance: chronic inflammation impairs regulatory T cell function, allowing autoreactive B-cell clones to expand [11]. iii) Epitope spreading and neoantigen formation: chronic HBV infection can expose cryptic self-antigens, generate neoantigens, and induce stress-related protein modifications, triggering autoantibody production [12-14]. iv) Immune complex formation: rheumatoid factor binds to IgG, forming immune complexes that activate the complement system, further amplifying chronic inflammation [15,16].

The association between advanced age and RF positivity can be explained by immunosenescence, a well-characterized process involving thymic involution, reduction in naïve T cells, dysfunction of regulatory T cells, and chronic low-grade inflammatory state (inflammaging), driven by the pro-inflammatory senescence-associated secretory phenotype (SASP) phenotype [17-19]. Cumulative antigenic exposure throughout life, particularly in chronic HBsAg carriers, may further exacerbate these immune alterations [10]. In our study, RF prevalence increased from 5% in participants under 30 years to 16% in those over 50 years, reinforcing this age-related immune dysregulation hypothesis. Interestingly, no association was found with female sex, a well-known risk factor for autoimmune diseases. Although hormonal and genetic factors contribute to increased autoimmune susceptibility in women [20,21], the chronic antigenic stimulation induced by HBV may override this sex-based predisposition in this specific population. Similarly, the absence of association with reason for consultation suggests that RF production is not necessarily influenced by the duration of HBV carriage. However, the limited number of RF-positive cases in the chronic follow-up group (6 out of 45) reduces the statistical power to fully explore this relationship. These findings have two main clinical implications: i) Rheumatoid factor screening in older HBsAg carriers may help identify patients at risk of autoimmune complications. ii) Pathological RF can interfere with serological tests, leading to diagnostic errors, particularly in resource-limited settings [7,8].

Strengths and limitations of the study: this is the first study in Côte d´Ivoire to assess RF prevalence in chronic HBsAg carriers, providing original data on autoantibody production in this context. However, some limitations must be considered: i) Cross-sectional design: this study does not allow causal inference regarding the relationship between HBV infection and RF production. ii) Recalling bias: self-reported medical history may have been affected by recall bias, despite efforts to ensure data reliability. iii) Lack of HBV viral load and inflammatory markers: the absence of virological and immunological markers prevents a more detailed exploration of HBV replication status and immune activation as potential contributors to RF production. Further longitudinal studies incorporating HBV viral load, liver enzymes levels, and inflammatory cytokines are needed to better understand the evolution of autoantibody responses in chronic HBsAg carriers. While these findings are particularly relevant in a hospital-based setting in Abidjan, their generalizability to other populations requires caution, considering potential environmental and demographic differences.

Conclusion     

This study highlights an increased prevalence of rheumatoid factor in asymptomatic chronic HBsAg carriers followed at the University Hospital of Cocody, Abidjan, Côte d´Ivoire. Advanced age was the main factor associated with RF positivity. These findings underscore the importance of immunological monitoring, particularly in older HBsAg carriers, to improve the assessment of autoimmune risks and enhance the diagnostic reliability of serological tests in this context.

Competing interests     

The authors declare no competing interest

Authors' contributions     

Amah Patricia Victorine Goran-Kouacou supervised the entire study, was responsible for the design and planning of the work, analysed the results and drafted the final version of the manuscript. Oppong Richard Yéboah contributed to the statistical analysis, interpretation of data and drafting of the methodological and results sections. Aya Ursule Aniela Assi and Yida Jocelyne Séri contributed to data collection and sample management in the laboratory, under the supervision of Adjoumanvoulé Honoré Adou. Séry Romuald Dassé provided methodological support and advice on the study design, in collaboration with Koffi N'Guessan. All the authors have read and agreed to the final manuscript.

Acknowledgments     

We extend our sincere thanks to the team of the Immunology and Haematology Laboratory of the University Hospital of Cocody for their valuable support during the analysis. We also thank the participants for their essential contribution to this study.

Tables and figures     

Table 1: summary of viral markers

Table 2: socio-demographic characteristics of patients studied

Table 3: univariable and multivariable analysis of factors associated with rheumatoid factor positivity

Figure 1: flow diagram of study participants

References