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Immunologic markers, vasculitis-associated autoantibodies, and complement levels in patients with COVID-19

Background: The cause of coronavirus disease 2019 (COVID-19) is a virus which can lead to severe acute respiratory syndrome-CoV-2 (SARS-COV-2). There are evidences of involvement of immune system in pathogenesis of this disease. We investigated the presence of various vasculitis-associated autoantibodies and complement levels in patients with COVID-19. Materials and Methods: Patients with severe or critical type of COVID-19 were evaluated for symptoms, signs, and laboratory tests of vasculitis syndromes including rheumatoid factor (RF), antinuclear antibody (ANA), anti-double-stranded DNA, c and p anti-neutrophilic cytoplasmic antibody (c ANCA and P ANCA), and complement levels. Results: The study was performed in forty patients with severe or critical illness. The mean age of the participants was 48.5 ± 9.8 years. All patients had pulmonary involvement in lung computed tomography scans. Vasculitis laboratory test results included RF in two patients, ANA in three patients, and ANCA in one patient. Seventeen (42.5%) patients had hypocomplementemia in one or more complement tests. Four patients expired, of whom three had a decrease in complement level. Conclusion: Decrease in complement levels may predict a critical state of COVID-19 disease. Therefore, measuring its levels may be of great benefit in making earlier decisions to initiate disease-suppressing treatments including corticosteroids.


INTRODUCTION
Coronavirus disease-2019 (COVID-19) is caused by severe acute respiratory syndrome (SARS) coronavirus and the first cases were classified as "pneumonia of unknown etiology." The disease represents a potentially fatal disease that is of great global public health concern. By January 8, 2021, the outbreak of COVID-19 has resulted in 88,615,325 confirmed cases and 1,908,948 deaths globally. [1][2][3] Clinical presentations and laboratory manifestations of the disease include fever, cough, pulmonary and cardiac complications, headache, lymphopenia, increased lactic dehydrogenase, coagulation disorders, disease. [14][15][16] Growing consensus about the pathophysiology of SARS-CoV-2 infection has led to use some antirheumatic drugs as possible treatment options in COVID-19. [17][18][19] Among them, glucocorticoids have unique action on immune system including blocking the inflammatory cascade from the origin, global availability, low cost, quick action using the pulsed method, and its half-life compared to the long-lasting biologic agents and monoclonal antibodies. [20] Studying the relationship between vasculitis syndromes or related laboratory findings with COVID-19 may be interesting in several ways (1) could the similarity of symptoms between both diseases suggest a similar mechanism of injury? (2) Understanding the pathways of damage may be of great help in identifying the treatment, and (3) Can the treatments modify or ignite the pathophysiological processes of the disease? Designing and conducting this study may suggest ways to answer these questions, the safety pathways involved, and possibly the use of appropriate treatments. The current study also aimed at investigating the seropositivity of the tests used for the diagnosis of vasculitis syndromes in patients with severe and critical COVID-19.

Study population
We conducted a cross-sectional study in April 2020 in hospitalized patients with COVID-19 admitted in a referral hospital in Sari, North of Iran. The patients were consecutively screened by a rheumatologist for possible inclusion. Due to the lack of prior research studies on this topic, the study was performed in forty eligible patients.

Selection criteria
The diagnosis of patients with COVID-19 was based on compatible clinical symptoms with positive PCR and/or lung involvement as inclusion criteria for all patients. [21] Definition of disease severity Based on the symptoms of the disease, it was classified into these categories: asymptomatic (positive SARS-COV-2 test and no symptom), mild (fever, cough, or change in taste or smell and no dyspnea), moderate (clinical or radiologic evidence of lower respiratory tract disease and oxygen saturation ≥94%), severe (shortness of breath, respiratory rate ≥30/min, oxygen saturation ≤94% or pulmonary infiltration more than 50% within the first 24-48 h), and critical (respiratory failure, septic shock, and/or multiple organ dysfunction/failure. [22] Inclusion and exclusion criteria Patients aged 18-60 years old diagnosed with either severe or critical infection of COVID-19 were enrolled. Patients with a history of malignancies, chronic liver or kidney diseases, history of rheumatic inflammatory diseases (including systemic lupus, rheumatoid arthritis, scleroderma, and vasculitis associated with anti-neutrophil cytoplasmic antibodies [ANCA] or anti-nuclear antibodies), and patients using corticosteroids, immunosuppressive, and biological drugs were excluded.

Ethics approval
The current study was approved by the Ethics Committee of Mazandaran University of Medical Sciences (Ethic code: IR.MAZUMS.REC.1399.7427). Patients or their companions were explained about the study.

Data collection
After selecting eligible patients, their consent was obtained, and clinical and demographic information was recorded. Laboratory data including complete blood count, biochemistry, coagulation tests, urine analysis, and reports of the lung computed tomography (CT) scans were recorded. Ten milliliters of blood were drawn from patients and the sera were stored at 2-8 C.
Vasculitis syndrome tests include rheumatoid factor (RF) tests, antinuclear antibodies (ANA), antidouble-stranded DNA (anti--dsDNA), ANCA, and measurement of complement levels (C3, C4, and C50). RF was estimated by Latex method (Bionic, Iran) and ANA, anti-dsDNA antibodies, and ANCAs were performed by enzyme-linked immunosorbent assay (ELISA) (EUROIMMUN, Germany). A value greater than the upper limit of normal was considered positive. The levels of complement were also measured by ELISA (Pars Azmoon, Iran and Diametra, China) and values less than the lower limit of normal were considered low.
Leukopenia was defined as WBC <4400 cells/µL and lymphopenia was considered if absolute count was <1000 cells/µL. Anemia was defined as Hgb < 12 g/dl in women and <13.5 in men. Thrombocytopenia was considered as plt <150,000/µL. High serum creatinine level in men and women was considered as 1.13 and 0.93 mg/dl, respectively. Alanine transferase was regarded elevated if it was >55 IU/L in males and 32 IU/L in females. Hyperbilirubinemia was defined as total bilirubin >1 mg/dl and direct bilirubin >0.4 mg/dl. Creatine phosphokinase and troponin were described high if the levels were >195 U/L in men and 170 U/L in women and >100 ng/dl, respectively.

Statistical analysis
Descriptive statistics such as mean and percentage were used to describe the clinical/laboratory findings IBM SPSS ver 20, Chicago, USA. decrease in complement, 2 were RF positive and 2 were ANA positive, in one patient, both tests were positive. Among the patients expired (n = 4), three had a significant reduction in complement levels.

DISCUSSION
In the present study, we investigated immunologic and vasculitis tests and complement levels in patients with COVID-19 infection. Three patients had a positive ANA result, of whom two had hypocomplementemia and were also treated with interferon. All three patients were fully recovered and discharged from the hospital, despite severe pulmonary involvement initially. One patient was positive both for C and P ANCA, with pancytopenia but without any other ANCA-associated vasculitis manifestations. Seventeen patients showed a decrease in one or more components of the complement and of the 4 patients expired, three had a decrease in complement. The vasculitides are defined by the presence of inflammatory leukocytes in the vessel walls with damage to mural structures. They are diagnosed based on patterns of organ injury, the size of the vessels affected, histopathological and imaging features. ANA, ANCA, and serum complement levels are laboratory tests that help the physician in understanding the pathophysiology and making accurate diagnosis. [23] In this study, we did not find any classic vasculitis syndrome which might be due to small sample size, but the variety of symptoms in patients with COVID-19 can be similar and mimic the syndromes of vasculitis.
The complement system is a major component of innate immunity that acts as the effector arm of humoral immune system. The complement system is primarily perceived as a host defense system, but it may be a potentially more harmful side of innate immune pathway as an inflammatory mediator. [24] Severe sepsis is an acute condition that leads to the activation of the complement and coagulation cascades and may play central roles in multiple organ failure and severe complications. [25] Intrapulmonary activation of complement can cause acute lung injury that is complement and PMN dependent, resulting in a cytokine storm. [26] Evidence on the role of complement system as a major host mediator of SARS-CoV-induced disease suggests that complement activation regulates a systemic pro-inflammatory response to SARS-CoV infection. Inhibition of complement signaling in a mouse model is reported to be an effective treatment option following SARS-COV infection. [27] In adult patients, hypocomplementemia is an early diagnostic marker of parvovirus B19 infection. [28] Activation of the complement pathways has been reported in chronic hepatitis C virus (HCV) infection and there is an association between HCV pathogenesis and abnormal complement profiles. [29] Creating an immune complex and activating the complement system are the basic mechanisms in

RESULTS
Forty patients with severe or critical COVID-19 were entered in this study. All patients had significant changes indicating COVID-19 pneumonia in their CT scans and also clinical manifestations of the disease. The mean age of patients was 48.5 ± 9.8 years old and 27 (67.5%) were males. Demographic and basic clinical data of patients are shown in Table 1. The mean respiratory rate and saturation pressure of O 2 were 23.17 ± 5.8/min and 87.15% ± 7.46%, respectively. All patients received hydroxychloroquine and lopinavir/ritonavir combination therapy. Twenty-seven patients also received interferon beta-1 (n = 27) and 17 patients was treated with additional parenteral glucocorticoid.
The results of vasculitis tests are shown in Table 2. Of the forty patients studied, 17 (42.5%) had hypocomplementemia in one or more components (low C3 in nine patients, low C4 in five, and low CH50 in 14 patients). Six of the 15 patients with severe type of the disease (40%) and 11 of the 25 critically ill patients (44%) had low complement levels (P = 0.804). There were no significant differences between the two groups with low and normal complement levels in the percentage of lung involvement or the number of lobes involved (P > 0.05). Of the patients who had a pathophysiology of many autoimmune and vasculitis syndromes. In the present study, 40% of patients with severe disease and 44% of critically ill patients had low complement levels that could have been caused by activating and consuming complement as a result of the virus invading or reacting to it. Comparing these cases with mild cases of the disease may reveal more findings related to hypocomplementemia.
A major concern in the treatment of critically ill patients is drug-induced side effects. There is a broad spectrum of interferon-related complications i n c l u d i n g g l o m e r u l o n e p h r i t i s , s y s t e m i c l u p u s erythematosus (SLE)-like syndrome, and thrombotic microangiopathy that may be associated with hypocomplementemia. [30,31] Drug-induced lupus erythematosus is a syndrome with clinical and serological features similar to SLE that is temporally related to continuous drug exposure and resolves after discontinuation of the drug. More than 90 drugs, including interferons, are involved in causing the disease. [31] In this study, 15 of the 27 patients receiving interferon showed a deficiency of complement. Low levels of complement in these patients may be due to disease or interferon use. [32] Therefore, in addition to proper and appropriate use of the drug, care should be taken in selecting patients and monitoring its side effects. Since the onset of the COVID pandemic, several treatment regimens have been evaluated to treat this potentially dangerous disease. [33] Knowing the possible pathways in the pathogenesis of the disease may help to select appropriate treatments.
We encountered limitations in the study. This study was performed as a pilot on patients with COVID-19, and according to our knowledge, evaluation of vasculitis tests and complement levels in these patients have not yet been published. Therefore, there was no any previous study to estimate the number of samples based on them. Furthermore, patients were examined after receiving certain medications such as interferon or steroids, so subsequent studies can be performed before treatment interventions or on different days of treatment.

CONCLUSION
Based on current findings, a decrease in May predict progression to a critical COVID-19. Therefore, measuring its levels may be helpful in making earlier decisions to initiate disease suppressing treatment including corticosteroids.
This was a small pilot study and for a definite conclusion, further studies with larger sample size and inclusion of milder forms of the disease as control group are needed.

Acknowledgments
Researchers appreciate the efforts of physicians and nurses and health professionals around the world to treat and prevent COVID- 19

Financial support and sponsorship
Nil.

Conflicts of interest
There are no conflicts of interest.