Diagnosis of Dual c- and p-ANCA Vasculitis Following SARS-CoV-2 Infection: A Case Report

Introduction

Vasculitis refers to a group of heterogeneous pathologies that result in blood vessel inflammation. Vasculitides can affect blood vessels of all sizes and range in severity, affecting a single organ or resulting in multisystem failure [1]. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAVs) is a subtype affecting small vessels and characterized by the presence of IgG ANCA autoantibodies directed against the azurophilic granules found in the cytoplasm of neutrophils and monocytes [2]. The clinical manifestations of AAVs include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) [3]. Proteinase 3 (PR3) is the autoantigen in GPA, which is referred to as c-ANCA positive, while the autoantibodies in MPA and EGPA target myeloperoxidase (MPO) and are referred to as p-ANCA positive [4]. Emerging reports suggest that SARS-CoV-2 infection may result in autoimmune disorders, including AAVs, in susceptible individuals [5]. Cases of either c-ANCA- or p-ANCA-associated vasculitis have been documented following SARS-CoV-2 infection [6–13]. While dual ANCA positivity has been observed, no cases to date have linked its association with SARS-CoV-2 [14]. We describe the first known case of dual c-ANCA and p-ANCA vasculitis following SARS-CoV-2 infection and explore potential mechanisms underlying the pathogenesis of dual ANCA AAV in this patient.

Case Report

MAY-AUGUST 2021:

Our patient was a 70-year-old woman with a past medical history notable for atrial fibrillation, sick sinus syndrome with a pacemaker, breast cancer, gastroesophageal reflux disease, anxiety, and coronavirus disease 2019 (COVID-19) in August 2021. She received the Pfizer-BioNTech COVID-19 mRNA vaccine in May 2021.

AUGUST 2023:

In contrast to her first, largely asymptomatic infection with SARS-CoV-2, the patient was reinfected with the virus and experienced moderate fevers and a productive cough. Shortly thereafter, she developed sinusitis symptoms and continued to cough. At that time, her medications included cetirizine 10 mg per oral (PO) once per day (QD) as needed (PRN), famotidine 20 mg PO (by mouth) PRN, sertraline 25 mg PO QD, calcium carbonate 500 mg PO 3 times per day (TID) PRN, and alprazolam 0.25 mg PO PRN. She was initially evaluated by her primary care provider and was treated with a course of azithromycin 250 mg and fluticasone propionate nasal spray. However, she did not experience any improvement and received a course of amoxicillin-clavulanic acid. Over the following month, she continued to have persistent sinus pressure and developed new-onset fatigue, bilateral leg weakness, weight loss, and a lingering cough. Therefore, a CT scan of her sinuses was ordered, revealing scattered mucosal thickening of the ethmoid air cells and bilateral maxillary sinuses without air-fluid levels. Otolaryngology (ENT) was consulted, who deferred surgery and advised allergy referral.

NOVEMBER 2023:

The patient returned to her primary care physician with increasing fatigue, myalgias, and a new non-pruritic, non-painful rash on her back. Blood tests were ordered and revealed iron-deficiency anemia with a hemoglobin of 11.4 g/dL (standard range: 12–16 g/dL). She was started on ferrous sulfate 325 mg PO QD. Given her fatigue, anemia, and history of breast cancer, a PET scan was ordered, which revealed multiple prominent mediastinal and right hilar nodules in her lungs, which were concerning for granulomatous diseases and metastatic disease. A pulmonary consultation was ordered.

Several days later, she presented to the Emergency Department (ED) with progressing fatigue, unintentional 4.4 kg weight loss over the past few months, bilateral foot numbness, and anxiety. On physical examination, her heart rate was 125 beats per minute, blood pressure was 179/95 mmHg, and temperature was 37.0°C. Her cardiopulmonary and abdominal examinations were unremarkable. No skin rash was visualized. Blood tests revealed mild leukocytosis with a white blood cell count of 11.8 K/μl (standard range: 4.0–11 K/μl), normocytic anemia with a hemoglobin level of 9.8 g/dL, and an elevated lactic acid level of 2.5 mmol/L (standard range: 0.7–2.1 mmol/L). A urinalysis showed 3+ blood and 2+ protein. Creatinine was borderline elevated at 1.35 mg/dL (standard range: 0.50–1.10 mg/dL). She was treated empirically for bacteremia with ceftriaxone 1000 mg IV and normal saline 1000 mL IV and discharged home from the ED.

Five days later, she was seen in consultation by a pulmonologist for her right hilar and mediastinal lymphadenopathy. Blood work for angiotensin-converting enzyme (ACE) and alpha-1-antitrypsin levels, as well as a 24-hour urine calcium level, was ordered. A repeat CT chest without contrast revealed unchanged thoracic lymphadenopathy and no new findings.

JANUARY 2024:

The patient was evaluated at urgent care due to worsening pain, swelling, numbness, and tingling in both legs. Physical examination revealed decreased Achilles tendon reflexes bilaterally. Routine blood labs were drawn, and she was discharged home with neurology follow-up for an electromyogram later in the month to evaluate for possible neuropathy.

The patient presented to the ED 4 days later after being alerted that her urgent care labs showed a hemoglobin of 6.4 g/dL, BUN of 69 mg/dL (standard range: 8–20 mg/dL), and creatinine of 4.46 mg/dL, indicating severe anemia and acute kidney injury. She reported passing black stools, which she attributed to iron supplements. Her urinalysis showed 11–20 red blood cells (RBCs) with no RBC casts. Her blood tests from Pulmonology were reviewed at this time and were notable for an alpha-1-antitrypsin level of 760 mg/dL (standard range: 150–350 mg/dL), likely due to her underlying inflammatory process. However, her angiotensin-converting enzyme levels were within normal limits at 33 units/L (standard range: 8–53 units/L). She was transfused with leuko-reduced packed red blood cells (pRBCs) and admitted to the hospital. An iron panel was performed and was consistent with iron-deficiency anemia. Repeat labs showed worsening of BUN to 72 mg/dL and creatinine to 5.2 mg/dL. Nephrology was consulted, and further testing revealed a positive ANCA indirect immunofluorescence assay (IFA) titer (>1: 1280) (standard range: negative), elevated myeloperoxidase antibodies at 202 AU/mL (standard range: <1 AU/mL), and elevated serine proteinase 3 antibodies at 38 AU/mL (standard range: <1 AU/mL), suspicious for a diagnosis of AAV with dual p- and c-ANCA positivity. Anti-nuclear antibodies (ANA), anti-glomerular basement membrane antibodies (anti-GBM), complement component C3, and complement component C4 were all negative. Her laboratory data at that time are displayed in Table 1. She was transferred to another hospital for a renal biopsy, which showed widespread inflammation and damage to most glomeruli without immune complex deposition, confirming the diagnosis of pauci-immune necrotizing glomerulonephritis.

One day later, shortly after undergoing a bronchoscopy with hilar lymph node biopsy, she experienced acute respiratory distress. A chest X-ray revealed a dense left lower-lobe infiltrate, consistent with alveolar hemorrhage. The hilar lymph node biopsy showed granulomatous inflammation with no evidence of malignancy. Considering the biopsy result together with nasopharyngeal involvement, this dual ANCA positivity was interpreted as GPA-dominant, although her p-ANCA titers were higher. Her respiratory status normalized steadily after the initial insult.

She was transferred back to her original facility, where she underwent 1 session of therapeutic plasma exchange and was started on methylprednisolone 125 mg IV BID (twice daily), which was increased 3 days later to 250 mg IV TID (3 times daily) for a total of 3 g administered. Four days after transfer, she began 4 doses of rituximab 375 mg/m² IV weekly. She was discharged on a prednisone taper starting at 60 mg PO QD (4 times a day), the same rituximab regimen, and hemodialysis. Two days after finishing her last dosage of rituximab, she started avacopan 30 mg PO BID.

NOVEMBER 2024:

Ten months later, the patient remained stable, with prednisone 5 mg PO QD, the same dosage of avacopan, and peritoneal dialysis, awaiting a renal transplant. A timeline of the patient’s disease course is displayed in Figure 1.

In summary, this 70-year-old woman developed dual c- and p-ANCA vasculitis 2 years after initial SARS-CoV-2 infection and shortly after reinfection. Her presentation was characterized by progressive sinus and pulmonary involvement, followed by rapidly progressive renal failure.

Discussion

There are several mechanisms by which ANCA seroconversion can occur following SARS-CoV-2 infection, with neutrophil extracellular traps (NETs) playing a significant role in self-tolerance disruption and pathogenicity [15]. NETosis refers to the formation of NETs by neutrophils in response to various pathogens, antibodies, immune complexes, cytokines, and other physiological stimuli [16]. During this process, intracellular biomolecules within the neutrophil granules, such as PR3 and MPO, are expelled and/or expressed on the cell surface, where they can be recognized by circulating antibodies [16,17].

SARS-CoV-2 infection has been shown to stimulate NETosis directly through interactions with C-type lectin domain containing 5A (CLEC5A) and Toll-like receptor 2 (TLR2), which are pattern-recognition receptors on neutrophils [18]. Other viruses, such as dengue and Japanese encephalitis, have also been reported to use this same mechanism to induce NETosis, while influenza A, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV) act through different pathways [19–22]. SARS-CoV-2 infection may be unique in that NETosis induction was maintained in patients with long COVID [23]. Our patient’s prolonged respiratory symptoms may be an indication of long COVID and thus may have put her at increased risk of continuous NETosis.

Alternatively, SARS-CoV-2 infection indirectly promotes NETosis via the generation of a hyperinflammation-induced cytokine storm [24]. Some of these cytokines include TNF-α, IL-1, IL-6, and IL-18, all of which are implicated in promoting NETosis [25]. This mechanism could explain the systematic inflammatory findings in our patient, such as the leukocytosis, markedly elevated alpha-1 antitrypsin levels, and multiorgan involvement. One study found that NET formation in lung biopsy samples from patients who had died of COVID-19 was significantly associated with local IL-8 mRNA levels [26]. In addition, the generation of reactive oxygen species (ROS) by NADPH oxidase during these cytokine storms has also been shown to play a role in NETosis, specifically by inducing neutrophil nuclear membrane dissolution and histone citrullination [24]. Circulating NET levels have shown a significant association with the severity of COVID-19 [23]. A study showed that the levels of anti-NET IgG and anti-NET IgM were significantly higher in patients with severe COVID-19 compared to controls and less severe cases [27]. Increasing evidence suggests that dysregulated NETosis is positively correlated with the onset of autoimmune disease [28]. Thus, SARS-CoV-2’s accentuated ability to promote NETosis through both direct and indirect mechanisms increases the risk of self-tolerance breakdown and autoimmunity.

In addition to NETosis, there are several mechanisms by which loss of self-tolerance can be associated with this case. First, patients may have an increased risk of developing AAV due to a genetic predisposition. Several cases have demonstrated the inception of MPO AAV following SARS-CoV-2 vaccination in patients with the HLA-DRB1*09: 01 allele [29,30]. Thus, it is important to acknowledge the possibility that the Pfizer-BioNTech vaccine played a role in this patient’s new-onset vasculitis. In contrast, haplotypes such as HLA-DRB1*13: 02 have been shown to serve a protective role against multiple autoimmune diseases, including AAV [31].

Molecular mimicry can be another mechanism of ANCA-associated autoimmunity during SARS-CoV-2 infection. There is evidence of molecular mimicry between PR3 and serine proteases of Escherichia coli, Pseudomonas aeruginosa, and several other pathogens [32]. However, this mechanism has yet to be observed with SARS-CoV-2.

Finally, neoantigen formation is also possible due to the increased ROS levels generated by hyperinflammation and MPO, which could in turn modify the epitopes of PR3 and MPO, making them immunogenic. One study demonstrated that IgM isolated from MPO ANCA patients taking hydralazine had a heightened affinity for hydralazine-modified MPO [33]. However, neoantigen formation in SARS-CoV-2 has not been shown to be a mechanism of ANCA-associated pathogenesis. Nevertheless, our patient’s rapid seroconversion for c- and p-ANCA after infection supports a process of heightened autoantigen exposure, compatible with either molecular mimicry or antigen modifications.

Following the direct and indirect mechanisms of SARS-CoV-2-induced NETosis, degranulated neutrophils release many structures in their NETs, including DNA, histones, ROS, and granule and cytoplasmic proteins. Moreover, PR3 and MPO can translocate to the surface of the primed neutrophils, thus becoming available targets for circulating c- and p-ANCAs. The binding of ANCAs to their surface targets on activated neutrophils results in further degranulation and generation of NETs, leading to widespread small-vessel damage [34].

Damage from NETosis is most significant during the clinical stages of the disease process and is not commonly observed during remission of AAV, suggesting that NETs contribute to the disease’s pathological stages [35]. The primary mechanism of NET-induced cellular damage involves the direct toxicity of its components, including neutrophil elastase, MPO, and histones [36–38]. With regards to direct endothelial damage, NETs are particularly damaging to the glycocalyx layer, which is largely composed of polysaccharides anchored directly to the endothelial surface by proteoglycans and glycoproteins [39,40]. When damaged, this layer upregulates adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and selectins, increasing the recruitment and migration of neutrophils and other immune cells to the small vessels, perpetuating the disease.

Complement proteins, such as C5a, act as a significant contributor to priming neutrophils and enhancing ANCA seroconversion and disease progression [41]. Thus, medications such as avacopan, a C5a receptor inhibitor, have proven beneficial in mitigating neutrophil migration and reducing the cumulative dosage of glucocorticoids, as in our patient [42]. The resultant endothelial damage from NETosis, acting as a nidus for complement activation, can reignite the alternative complement pathway and the classical pathway via C1q, forming a pro-inflammatory loop. NETs themselves can bind and activate complement proteins C3b and C5b [43]. Furthermore, properdin, which enhances alternative pathway activation, is released from the granules of activated neutrophils and promotes further complement-mediated damage through the production of C3b [44].

Our patient’s presentation was quite severe, with alveolar hemorrhage and rapid kidney failure necessitating hemodialysis, which aligns with prior evidence that dual ANCA positivity confers a more aggressive phenotype. A retrospective study of 85 biopsy-confirmed AAV patients found that dual ANCA-positive patients had worse kidney dysfunction than patients with single ANCA positivity [14]. Similarly, Gong et al reported that dual ANCA positivity was significantly associated with increased anemia severity, higher initial serum creatinine, lower eGFR, and higher relapse rates compared to c-ANCA [45]. Our report presents a unique case of dual-AAV following SARS-CoV-2 infection. Although our patient’s phenotype led to a diagnosis of GPA-dominant vasculitis, p-ANCA was the dominant titer, which agrees with other studies [45].

Conclusions

This rare case of dual c- and p-ANCA vasculitis adds a hypothesis-generating case to the literature, suggesting an association between COVID-19 and the onset of autoimmune vasculitis. However, while the temporal relationship supports a possible link, causality cannot be established from a single case report. Other contributing factors, such as vaccination, prior medical conditions, medications, pre-existing subclinical disease, or genetic susceptibility, may have also played a role. In addition, increased surveillance after COVID-19 may increase case detection, allowing rare events to cluster by chance. Nonetheless, clinicians should maintain a high index of suspicion for AAV in patients with persistent sinus symptoms, fatigue, skin findings, nonspecific hilar lymphadenopathy, and new-onset post-infection renal dysfunction. A delayed diagnosis can lead to rapid progression of the disease, especially for patients with dual ANCA positivity, as with the development of PIGN in our patient. Future studies should focus on delineating the relationship between SARS-CoV-2-induced NETosis and ANCA seroconversion.