Impact of the Rheumatoid Factor on Serological Testing Performance for Covid-19 in Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is the most common inflammatory rheumatism, affecting 0.5% to 1% of the European population. Characterized by symmetrical and distal erosive polyarthritis, its diagnosis is based on the association of the clinical assessment combined with the presence of a chronic biological inflammatory syndrome and of citrullinated cyclic antipeptide antibodies (CCP) and / or of rheumatoid factor. From a prognostic point of view, some factors allow the risk prediction of the progression to structural damage (bone erosion), such as the positivity of the rheumatoid factor or anti-CCP antibodies, and require the rapid introduction of basic immunosuppressive therapy or biotherapy. In the context of the SARS-CoV-2 pandemic, responsible for the COVID-19 disease, the interest of a serological test is essential from an epidemiological point of view in order to follow the evolution of the pandemic, and to adapt the strategy of de-confinement of the general population or of patients at risk. However, RA patients receiving biotherapy present an increased risk of infection. In this context of de-confinement, a SARS-CoV-2 serological analysis, before initiating RA patients future treatment or in order to adapt their current treatment, would be useful.

Detection of antibodies directed against SARS-CoV-2 by various detection kits is currently under evaluation. To the lack of serological test kits on the French market is added the complexity of verifying the sensitivity and specificity of these tests. On the other hand, the immune response to SARS-CoV-2 is complex and still quite unknown. The concomitant presence of several isotypes IgA, IgM and IgG is frequently observed. Thus, the isotypic switching of Ig does not seem to take place correctly in this disease case, which randers the serodiagnostics even more complicated to implement. Detection of the less specific IgMs would therefore be essential, in the case of SARS-CoV-2. Rheumatoid factor (RF) is an immunoglobulin (Ig), more often of the M type, directed against the Fc fragment of IgG. This antibody, although not directly pathogenic, is very frequently present in RA patients (70 to 80%), without being specific for this pathology since it can be observed in other autoimmune, infectious or hematological diseases as well. The presence of RF IgM, all the more in high titers, is problematic while performing many serological or immunological assays since these high levels can be responsible for false positive results.

During previous SARS epidemics (SARS-CoV-1 or MERS-CoV), false positive results of serological tests were observed. When detecting anti-SARS-CoV-1 IgG or IgM in patients who had not contracted SARS, the use of an ELISA kit, with plates coated with a cell lysate of Vero-E6 cells infected with SARS-CoV-1, had been shown to induce a low number of false positive results in healthy controls (less than 5%). However, the number of false positive results in patients with autoimmune diseases was much higher, and varied accordingly with the pathology (between 10% and 58%), the largest number of false positive results being obtained in lupus patients. This phenomenon can be explained by the use of an immune test using unpurified cell lysates, which contain antigens against which the autoantibodies present in the patient serum could react, thus distorting the outcome of the test. Other causes of increase in the false positive results rate have been described when performing SARS-CoV-1 serologies, such as the presence of active neoplasia, or because of a previous infection with another coronavirus. Indeed, other coronaviruses, such as HCoV-OC43 and HCoV-229E, display a nucleocapsid close to the one of SARS-CoV-1 : this similarity can be the source of cross-reactivity. The same phenomenon was observed with the latest coronavirus: it was therefore suggested to rather use recombinant antigens of SARS-CoV-2 in immunoassays, such as ELISA for instance Given the similarity between SARS-CoV1 and SARS-CoV2, it is likely that the various elements causing false positive results during serological tests for SARS-CoV-1, in particular the presence of auto-antibodies including RF, could also cause false positive results when performing serological tests targeting SARS-CoV-2. A recent Chinese study showed that a moderate or high rate of RF was associated with a significant rate of false positive results when searching for anti-SARS-CoV-2 IgM with a ELISA kit, which plates were however coated with recombinant antigen. The authors therefore proposed to include a urea dissociation step into the ELISA protocol, which seemed to improve the specificity of the test, without altering its sensitivity.

It thus appears essential to assess the influence of RF, for its different isotypes (IgG, IgM, IgA), on various serological tests for SARS-CoV-2 available in France, in order to better assess these tests specificity.

The investigators propose to evaluate and compare 3 different serological tests available for the detection of specific SARS-CoV-2 IgG and IgM. The rate of false positive results obtained for each type of test will be evaluated on sera originated from patients with RA who have never been in contact with SARS-CoV-2, since these sera were withdrawn before July 2019. The relation between the incidence of SARS-Cov-2 serological assays false positive results and RF antibody levels could be estimated, as well as the characterization of RF.