The Presence of Antineutrophil Cytoplasmic Antibodies and Antiphospholipid Antibodies in Patients with Severe Acute Respiratory Syndrome Coronavirus 2: A Case-Control Study among Sudanese Patients

Patients infected with COVID-19 are at an increased risk for thrombosis, suggesting a possible role of COVID-19 in the induction of coagulopathy. This study aimed to investigate the presence of prothrombotic antineutrophil cytoplasmic antibodies (ANCA) and antiphospholipid antibodies (aPLs) in the course of COVID-19 infection and to correlate these markers with severity and fatality, suggesting that COVID-19-induced autoimmune thrombosis is a possible axis in the inflammatory circuit of this infection. To investigate this, we conducted a case-control study which included patients with a positive reverse transcription-polymerase chain reaction (RT-PCR) test of COVID-19 and a control group with negative COVID-19 PCR and antibody (IgG-IgM and IgA nucleoprotein) ELISA results. An indirect immunofluorescence assay using granulocyte biochips (Aesku slides from AESKU DIAGNOSTICS, Germany) was used to detect ANCA (IgG), as well as multiplex ELISA for the detection of antiphospholipid antibodies for all patients with COVID-19 and for the control group. The results revealed the detection of antiphospholipid antibodies (IgG) in one patient out of the 45 patients in the case group. 1/45(2.2%) and 7/45(15.6%) tested positive for ANCA. Five were men and two were females, with one case revealed to be positive for both aPL and ANCA. A cytoplasmic reaction on the eosinophil granulocytes was observed in 2 cases; both were positive for ANCA. Other markers (CRP, APTT, PT, INR, ESR, and neutrophil and lymphocyte counts) were included in the study, along with demographic data. No aPL or ANCA reactions were detected for any of the control groups. These findings suggest that aPL and ANCA may be induced during the course of inflammation in COVID-19 and possibly contribute to the disease's severity and mortality.


Introduction
Coronavirus disease 2019 (COVID- 19) has spread throughout the world and caused a global health crisis with high mortality and morbidity since its emergence in December 2019 [1]. COVID-19 is caused by SARS-CoV-2, and although most patients recover, some develop severe and fatal complications related to pneumonia, acute respiratory distress syndrome (ARDS), thrombosis, cardiomyopathy, and acute renal injury (AKI) [2,3]. In addition, it was observed that many patients infected with COVID-19 had developed dysregulation of the immune system and cytokine storm syndrome, which are associated with high mortality [4].
In patients with COVID-19, the outcome is believed to be determined by the regulation versus dysregulation of the infammatory response. In the course of this infammation, elevation of cytokines, coagulation parameters like prothrombin time (PT), partial thromboplastin activation (PTT) prolongation, increased INR [5][6][7], and an increase in infammation markers such as C-reactive proteins (CRP) were observed [8]. In addition to advanced age and comorbidities, those elevated markers were also observed with mortality among COVID-19, suggesting that the fatality of COVID-19 could be in part due to thrombotic events in these individuals like deep vein thrombosis or pulmonary embolisms [9]. Including the investigation for antineutrophil cytoplasmic antibodies (ANCA) and antiphospholipid antibodies (aPLs) in patients with SARS-CoV-2 is broadening the list of possible players in the thrombosis and infammation of COVID-19 [10,11].
Antineutrophil cytoplasmic antibodies (ANCAs) are autoantibodies directed against various neutrophil antigens such as lactoferrin, cathepsin, elastase, myeloperoxidase (MPO), and proteinase 3 (PR3). Among them, in particular, ANCA in MPO and PR3 is associated with the development of vasculitis associated with ANCA (AAV). When neutrophils are activated by these antibodies; for example, they are believed to cause abnormal neutrophil activity and may damage the microvascular system [12,13]. Antiphospholipid syndrome (APS) is an autoimmune disease that is associated with the formation of autoantibodies. Tese autoantibodies react against phospholipidbinding proteins such as beta-2-glycoprotein I (β2GPI) and activate endothelial cells, platelets, and neutrophils, causing thrombosis [14,15]. Te catastrophic variant of antiphospholipid syndrome is sometimes fatal and similar to the difuse coagulopathy seen in patients with COVID-19 [16]. Te majority of studies have shown that LA is known to prolong PTT and is detected in a signifcant proportion of COVID-19 infected patients, resulting in increased fbrinogen and factor VIII levels, elevated levels of biomarkers such as CRP, and the presence of aPLs, which can afect PTT [17]. However, aPL arise temporarily in patients with critical diseases and diferent viral infections due to molecular mimicry. Te presence of these antibodies can cause thrombosis and makes it difcult to distinguish them from other types of thrombosis [18].
In this context, our current study tries to provide some insight into the possible association of ANCA and aPL with COVID-19 and their possible correlation with disease outcomes, suggesting a possible mechanism for autoimmune thrombosis that occurs in patients with COVID-19.

Study Design, Setting, and Patient Population.
Tis is a single-center prospective case-control study between May and December 2021. A total of 90 participants were recruited from Kassala Hospital, Kassala COVID-19 isolation center, Sudan. Te participants were divided into two groups: SARS-CoV-2 patients (n � 45) and controls (n � 45).

Defnitions of Case and Control.
Te cohort included patients with SARS-CoV-2 (a) who came to the COVID-19 isolation center with variable infection clinical outcomes ranging from mild asymptomatic mild to server-complicated ICU patients and (b) whose COVID-19 PCR tests were positive as a result of contact tracing. Te control group involved volunteers (a) who had no known history of acute, subacute, or chronic disease; (b) who did not take a particular medication; (c) who presented reasons other than infectious complaints; and (d) who gave consent to participate in the study in both the case and control groups.
Te demographic characteristics and medical report forms required regarding the study for both the patient group and the healthy control group were completed by qualifed physicians. Written informed consent was obtained from each participant, and the study protocol was approved by the Ministry of Health, Kassala State, Sudan. All study procedures were carried out according to the principles of the Declaration of Helsinki.

Laboratory Methods.
From patients confrmed with positive PCR for SARS-CoV-2 in the isolation center, blood samples were collected aseptically from each participant in three diferent types of vacuum blood collection tubes (K2EDTA, lithium heparin, and serum) and centrifuged at 3000 rpm for 5 min at room temperature. Samples were stored at −20°C until further testing. Te collection was carried out under strict COVID-19 isolation protocols [19].

COVID-19
Detection. Te diagnosis of COVID-19 infection was confrmed in all participants by a real-time reverse transcription-polymerase chain reaction (RT-PCR) test using qTOWER³ products (Analytik Jena, Germany). A serological test for nucleocapsid protein (NP) of SARS-CoV-2 antibodies was performed using commercial enzymelinked immunosorbent assay ELISA kits (AESKULISA ® , AESKU Diagnostics) for IgG, IgA, and IgM. Te assays were conducted according to the manufacturer's instructions.

Analytical Parameters.
All participants were evaluated for biochemical indicators such as complete blood count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), coagulation profle (PT, PTT, and INR), creatinine, urea, calcium, sodium, and potassium. (ANCA). Te serum of all participants was screened for ANCA using commercial slide biochips with ethanol-fxed granulocytes from AESKU Diagnostics. Te protocol was carried out on a fully automated indirect immunofuorescence (IIF) HELMED ® system (AESKULISA ® , AESKU Diagnostics, Wendelsheim, Germany). In brief, slides of ethanol-fxed granulocytes were incubated with diluted serum for 30 minutes at RT. After washing, the slides were treated with conjugated FITC detection antibody and incubated for 30 minutes (second incubation) and then washed with wash bufer for 5 minutes. Te mounting medium was added and then covered with a coverslip; the microscopic pattern of ANCA was determined compared to positive controls for P-ANCA and CANCA according to the AESKU pattern library.

Detection of Antiphospholipid Antibody (aPL).
Te determination of serum aPL with a 1 : 100 dilution for all participants was measured using an enzyme-linked immunosorbent assay kit (AESKULISA ® , AESKU Diagnostics, Wendelsheim, Germany), which is a multiplex test for antibodies against highly purifed human proteins (B2-glycoprotein 1, cardiolipin, phosphatidylcholine, ethanolamine, inositol, serine, and sphingomyelin). Te protocol was performed according to the manufacturer's instructions.

Statistical
Analysis. All data sets obtained from the study were analyzed using SPSS version 26 (SPSS Inc., Chicago, Illinois, USA) and Stata version 12.0 (Stata Corporation, College Station, TX). Continuous variables were presented as mean ± standard deviation and median (IQR), while categorical variables were provided as numbers and percentages. Te Mann-Whitney U test, or Fisher's exact test, was used to compare the diferences between the independent groups. Furthermore, the relationships between continuous variables were analyzed using Spearman correlation analysis; a chisquare test was used to analyze categorical variables. Te signifcance level was defned as p < 0.05 for all analyses.

Autoantibodies (ANCA/aPL) Profle among Study
Participants. Among the study participants, positivity for autoantibodies (ANCA) is observed in 7 patients. All these positive patients are cases with p � 0.002 (χ 2 � 12.27). Most of the patients are in the age group of 20-29 years (42.9%) and are male (71.4%). Among those with positive ANCA, 71.7% (n � 5) are diabetic (p � 0.024), as shown in Table 3. One patient was positive for aPL besides being positive for ANCA. Te detected patterns of ANCA were 4 (57.1%) cases of CANCA (Figure 1), one case of P-ANCA, and one case of atypical ANCA; a questionable reaction on eosinophils was observed in 2 patients, as shown in Figure 2. Te two cases that are positive for antieosinophilic cytoplasmic antibodies had a deranged coagulation panel and elevated infammatory markers, that is, ESR and CRP. SARS-CoV-2 nucleocapsid (NP) immunoglobulins (IgA, IgM, and IgG) were not signifcantly diferent between patients with positive autoantibodies and negative patients (Table 3).

Kaplan-Meier Analysis for Death Incidence.
Kaplan-Meier survival curves with logarithmic rank tests were constructed to compare the cumulative incidence of death from coagulation abnormalities, positivity for CRP and calcium ESR, levels among 45 patients with COVID-19, and the presence of diabetes among patients (see Figure 3). Along with the total of 1,178 days of follow-up, 5 patients (all among the COVID-19-infected individuals) died, making the incidence of death as 4.24 per 1,000 person-days. As in Figure 3(a), in general, having a coagulation abnormality was associated with shorter survival (50 (95% CI, 17-83.5 days) vs. 88.9 (95% CI, 70-107.5 days)) (log-rank test, p < 0.001 (17.6)). A signifcantly shorter mean survival duration was observed with low calcium levels, and no individual with normal calcium log-rank were dead (log rank test p < 0.001 (17.5)). Individuals with free DM also survived longer compared to diabetics (p � 0.002 (9.5)).

Discussion
Te incidence of autoimmune thrombosis and hemostatic changes associated with COVID-19 and other coronaviruses raises questions about possible COVID-19-related coagulopathy and thrombotic event induction [21]. ANCA and aPL have been considered as possible mechanisms that lead to proinfammatory and hypercoagulable states [22].
In our study, we screened for ANCA and aPL among a total of 90 participants, divided into 45 cases infected with COVID-19, compared to 45 healthy control individuals. Seven patients (15.6%) were found to be positive for ANCA;  [26]. Te association of these autoantibodies with the clinical outcome showed that all deaths occurred only in the cases (chi-square, p value � 0.023); of the seven positive cases, two cases (28.6%) died while 5 (71.4%) cases recovered. Fuentes Baldarrago et al., using the APACHE II scores, demonstrated that in patients with COVID-19, there was a positive correlation between worsening of the disease state and increased NETosis, suggesting that neutrophil overactivation may play a role in disease progression [27] and may lead to the generation of ANCA seen in the serum of COVID-19 patients; a hypothesis consistent with that of O'Sullivan and Holdsworth, who suggested that dysregulation of NET formation leads to the generation of ANCA [28]; furthermore, Arcanjo et al. [29] showed for the frst time that SARS-CoV-2 can activate NETosis in human neutrophils and demonstrated that this process is associated with increased levels of intracellular reactive oxygen species (ROS) [29]. Reactive oxygen species can kill pathogens directly by causing oxidative damage or indirectly, in neutrophils, by stimulating NET formation. ROS also plays a detrimental role, promoting the formation of venous thrombosis by modulating the enzymatic cascade of fbrinolysis, systems of coagulation, and the complement system. Tese fndings undoubtedly point to the critical role of neutrophils in the pathology of infection. In other severe or persistent viral infections, neutrophil-mediated alveolar damage leads to interstitial oedema, ventilation/perfusion mismatch, and respiratory failure [29].  [30].
Our results also showed a positive case of aPL, the presence of aPL in patients with COVID-19. Following the fnding of Bahramnezhad et al., which describes a case of an antiphospholipid-like condition caused by COVID-19 [31], another piece of evidence from the literature is the fnding of Moon et al. [32], who presented a hypothetical model to elucidate the pathophysiology of APS in COVID-19 and described clinicopathological similarities between CAPS and severe COVID-19 [32]. In light of this evidence and our fndings, COVID-19 may induce an antiphospholipid-like condition that plays a role in increasing the severity of disease manifestation; this hypothesis is strongly supported by the fndings of Laura et al., who examined 106 COVID-19 patients for the presence of aPL antibodies, including 30 hospitalized cases, 47 hospitalized nonneurological COVID-19 controls, and 29 COVID-19 nonhospitalized controls, and reported a high prevalence of antiphospholipid antibodies in 72 (67.9%) of all cases and 22 (73.3%) of the 30 hospitalized neurological cases [33]. An important note in our study is to mention that the positive case of aPL is also positive for the ANCA, indicating that a wide range of autoantibodies could be induced in COVID-19 infection. Our study also demonstrated the presence of a questionable eosinophil cytoplasmic reaction, possibly being an autoantibody directed against eosinophil granules. Te reaction has been observed in the serum of two patients; to our knowledge, this is the frst report of the presence of possible antieosinophil cytoplasmic antibodies. Tere is no literature available to show the exact signifcance of this fnding during the disease. However, in recent research, Gebremeskel et al. reported that MC-derived proteases and mediators are elevated in sera and lung tissues and suggested that MC activation and eosinophil activation are associated with COVID-19 infammation [34]. Based on this, we can conclude that eosinophils may be involved in the pathology of the disease. However, more research is needed to determine the exact target of the cell and the role of eosinophils in this study. Kumar et al. obtained data from 33 studies, including 16003 patients, and concluded that diabetes among patients with COVID-19 is associated with a twofold increase in the severity of COVID-19 [35], compared to nondiabetics. In our study of 7 cases who tested positive for ANCA, fve (71.7%) were diabetic (p � 0.024). One of them, in addition to being positive for ANCA, was also positive for aPL antibodies and eventually died, suggesting important roles for comorbidities in COVID-19 pathogenesis. Te most common laboratory abnormalities identifed in patients with COVID-19 include decreased albumin and lymphocyte count and elevated C-reactive protein (CRP), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), aspartate transaminase (AST), and alanine transaminase (ALT) [36,37]. Tese abnormalities are associated with worse outcomes. In our study, we compared diferent laboratory measures among controls and the two divisions of cases, i.e., recovered COVID-19 cases and dead COVID-19 cases. It is evident that most of the case groups had hemostatic abnormalities. Te groups showed signifcant diferences in total white blood cell count (p � 0.002), neutrophils, and lymphocyte count (p < 0.001). Leukocytosis, neutrophilia, and lymphopenia are more common among dead cases of COVID-19 than the other two. ESR and CRP are much higher in dead patients with COVID-19 than in the other groups (p < 0.001). Although normal renal function status was recorded between controls and recovered cases, dead cases had abnormally high records (p < 0.05). We have found signifcant diferences in INR between dead patients with COVID-19 and recovered cases (p � 0.025). Supplemented    by time-to-event analysis, the absence of coagulation abnormalities, normal calcium levels (in contrast to hypocalcemia), and the absence of diabetes mellitus showed survival benefts among cases. Tis is in line with the already known short-term prognostic indicators [35,38,39]. Tis study aimed to highlight parts of the chaotic infammatory environment in COVID-19, possibly pointing to prognostic biomarkers and therapeutic intervention pathways, and an early attempt to put a piece on the expected long-term efects which may follow the pandemic. COVID-19 is ongoing, with almost daily emerging information. Tere are several limitations to this study, in addition to the small sample size. First, the study is not suitable to reveal the complete incidence of ANCA and aPL in COVID-19. Te previous ANCA and aPL status of the subjects is unknown. Terefore, subclinical positivity for ANCA and aPL before the infection period cannot be ruled out, and the determination of aPL did not include IgM and IgA. Sample size may infuence the statistical analysis of the ANCA positive group, which prevented further statistical investigations with analyses such as multivariate regression to better demonstrate the efects of ANCA positivity on outcomes, and in the analysis, for some patients, we only had one time point. Terefore, we recommend that more studies be conducted to investigate the relevance of the observed eosinophil cytoplasmic reaction, the target antigens, and their impact on the course of the disease. ANCA and aPL are recommended to be investigated in the study of patients with COVID-19, especially in severe cases. Autoimmune screening should be of interest clinically and researchwise in the coming years.

. Conclusion
Tis prospective case-control study reveals that ANCA and aPL were detected in the COVID-19 infection group compared to the healthy control group. It suggests that COVID-19 induces the formation of ANCA and aPL in some patients and may contribute to their pathogenesis. Furthermore, the formation of ANCA in COVID-19 may indicate the activation and induction of the neutrophil extracellular trap in patients with COVID-19. Te cytoplasmic reaction of eosinophils may suggest the presence of parallel autoantibodies to ANCAs in eosinophil granules. Further studies are needed to clarify all the diferent pieces of the puzzle.