We reported a pediatric patient with refractory cEGPA who experienced a life-threatening COVID-19 infection and was ultimately diagnosed with a novel de novo variant in the NFKB2 gene.

Our patient initially presented with respiratory symptoms, including asthma, recurrent respiratory tract infections, accompanied by elevated eosinophilia, and autoimmune phenomena, such as subcutaneous nodules (a manifestation of vasculitis). EGPA is classified as one of the types of AAV that affect small to medium-sized blood vessels [3]. Currently, no universally accepted diagnostic criteria for EGPA, and the classification criteria are commonly used in practice. The ACR 1990 classification criteria and Lanham criteria are frequently referenced in the literature [1, 14]. The patient exhibited: 1) a history of asthma, 2) elevated peripheral eosinophilia (eosinophilia > 10% or > 1.5 × 10^9/L), 3) pulmonary infiltrates, and 4) cutaneous and venous vasculitis. In this case, scattered eosinophils were present in the lung biopsy, while none were detected in the skin biopsy. The following reasons were considered: 1) due to the influence of specimen collection, no obvious lesion location was sampled, leading to potential false-negative results; 2) eosinophils are highly sensitive to corticosteroids, and the long-term use of steroids at the time of the skin biopsy may have contributed to their absence. Additionally, this patient meets the 2022 ACR/EULAR EGPA classification criteria, achieving a score of + 8 points (+ 3 for obstructive airway disease, + 5 for blood eosinophil count > 1 × 10^9/L) [15]. Differentiating HES from EGPA can be difficult due to their overlapping features of vasculitis and hypereosinophilia. Elevated CRP levels have been reported as a reliable diagnostic biomarker to distinguish HES from ANCA-negative EGPA with asthma, as higher CRP levels are more indicative of EGPA [16]. Our patient consistently presented with elevated CRP, supporting the diagnosis of EGPA. Data on the prevalence of pediatric cEGPA are limited. To date, there have been sporadic case reports and only four large cohort studies reporting on 33, 9, 13, and 14 patients, respectively [2, 17,18,19]. We reviewed the clinical features and treatment measures of the four cohorts, as shown in Fig. 3. Pediatric cases of EGPA exhibited more severe cardiopulmonary disease, gastrointestinal tract involvement and mortality, but had lower rates of musculoskeletal, renal and peripheral nerve manifestations compared to adults [17, 18]. Consistent with these findings, our patient exhibited involvement of the ear, nose and throat (ENT), pulmonary system, gastrointestinal tract, skin, and potential cardiac involvement, without peripheral nerve or renal disease, but lacked biopsy-confirmed findings in skin. It has been demonstrated that interstitial lung disease (ILD) in AAV is associated with poor outcomes, particularly in patients with microscopic polyangiitis (MPA) [20]. Diffuse alveolar hemorrhage (DAH) is a common and life-threatening manifestation of pulmonary involvement in MPA while the frequency of DAH is rare (4%) and presents with moderate severity in EGPA [4, 21].

The summary of four cEGPA cohort. a The clinical features of reported cohort. b The management strategies of cEGPA patients in 2009, 2013,2016 and 2018 cohort. GC: Glucocorticoids; MMF: Mycophenolate mofetil; HCQ: Hydroxychloroquine; Hydrea: Hydroxyurea; PE: Plasma exchange; IFX: Infliximab; RTX: Rituximab; OMA: Omalizumab; ADA: Adalimumab

Subsequently, a standard therapeutic regimen was initiated, involving glucocorticoids and immunosuppressants. However, during steroids tapering, she experienced recurrent respiratory distress, accompanied by fever and perianal abscess. Following interventions with IVIG and aggressive pulse steroid therapy, her symptoms were improved. Nevertheless, steroid dose reduction remained challenging. Interestingly, prolonged use of steroids and immunosuppressants may develop severe lymphopenia and increase risk of infection [22, 23]. However, recent studies have demonstrated that in patients diagnosed with both rheumatic diseases and COVID-19, DMARDs or NSAIDs do not elevate the probability of hospitalization [24]. Supporting this finding, Khalil et al. reported that the severity and mortality rates of COVID-19 were not significantly different in patients with systemic autoimmune diseases receiving immunomodulatory therapy compared to the general population [25]. In this case, the patient exhibited more severe respiratory symptoms, interstitial lung disease, and perianal abscess than patients with autoimmune diseases who were also undergoing steroid and immunosuppressive therapy during the COVID-19 pandemic in China. Therefore, we propose that her condition may not be explained by EGPA alone, and IEI should be considered as a diagnosis, despite the absence of a family history of IEI. Ultimately, genetic sequencing validated our hypothesis, indicating the importance of gene sequencing technology in refractory autoimmune diseases. Our patient meets the following criteria for IEI diagnosis: 1) Clinical manifestations: she is susceptible to a wide range of bacterial, viral, and fungal infections, including severe infections such as perianal abscesses, as well as opportunistic infections like PJP. Autoimmune phenomena are also present, such as subcutaneous nodules and rashes. 2) Laboratory tests: both humoral and cellular immunity were reduced. 3) Treatment: The standard immunosuppressants not yield effective results. 4) Genetic testing: gene panel and Sanger sequencing revealed NFKB2 variants but lacks further pathogenic functional testing. 5) Excluding secondary immunodeficiency induced by immunosuppressants. In summary, the patient is eligible for a diagnosis of IEI coexisting with EGPA.

The patient carries a novel pathogenic variant that was in intron22 of the NFKB2 gene, c.2578 + 2dup. The variant affects the C-terminal domain of the protein. This alteration may disrupt the phosphorylation sites essential for the degradation of p100 into the mature transcription factor subunit p52. Notably, most reported variants are situated near the critical phosphorylation sites S866 and S870 [26,27,28].

The non-canonical NF-κB pathway plays a key role in the generation of plasma cells, class switching, and the development of memory B cells. Patients with mutations in NFKB2 showed the immunological phenotype characterized by reduced class-switched memory B cells, impaired B cells differentiation and hypogammaglobulinemia [29, 30]. Previous reports have also shown impaired T cell proliferation and reduced NK cell cytotoxic activity, which contribute to increased susceptibility to viral infections [31,32,33,34,35]. Consistent with previous findings, serum levels of IgA and IgG in the patient were low, and both B lymphocyte and NK cell counts were reduced. However, we were unable to measure further lymphocyte subpopulations or conduct functional assays because the patient's death from respiratory failure. Mutations in NFKB2 can lead to CVID, which is often associated with autoimmune manifestations, such as vasculitis. Although a review of NFKB2 mutations did not find any cases of vasculitis [30], Mac et al. described a female patient with vasculitis-like lesions associated with an NFKB2 mutation [12]. Dysregulation of the NF-κB signaling pathway can result in excessive production of pro-inflammatory cytokines, including TNF-α and IL-6, which play a key role in the pathogenesis of vasculitis and were markedly elevated in our patient. This suggests that the NFKB2 mutation may have led to dysregulate NF-κB signaling activation, triggering a cytokine cascade and vasculitis. There is no direct evidence linking NFKB2 to IL-5. However, activated Th2 cells release eosinophil chemoattractants like IL-5, which drive the development and maturation of eosinophil, B cell differentiation, and immunoglobulin production. Increased Th17 and decreased Treg cells have been observed in patients with EGPA. NFKB2 mutations impact the non-canonical NF-κB signaling pathway, affecting inflammatory regulation and B cell growth. This suggests a potential indirect synergistic interaction between NFKB2 and IL-5, which warrants further research.

The absence of B cells and immunoglobulins increases susceptibility to infections, and an immunocompromised state may heighten the risk of sustained severe SARS-CoV-2 infection [36]. Recent studies indicate that patients with NFKB2 mutations are vulnerable to viral infections, such as Epstein-Barr Virus (EBV), CMV, herpes virus [37], and particularly SARS-CoV-2 [11]. Moreover, patients with NFKB2(p52LOF/IκBδGOF) variants produce autoantibodies that neutralize type I interferons increasing susceptibility to lethal COVID-19 pneumonia [10, 38]. Our patient showed significant characteristics of viral susceptibility, including herpes labialis, CMV infection, and life-threatening COVID-19 pneumonia. A reported case demonstrated severe SARS-CoV-2 infection associated with NFKB2 loss-of-function pathogenic variants, consistent with our patient's experience of requiring mechanical ventilation and biologics [39]. However, the outcomes differed between the two patients, possibly due to the absence of opportunistic infections such as PJP in the previously reported case. P. jirovecii and CMV exacerbated the infection during the critical phase of the illness. Additionally, our patient did not receive COVID-19 convalescent plasma (CP), which may be a crucial factor influencing prognosis.

Notably, patients with NFKB2 variants in previously reported cohorts are predisposed to adrenocortical insufficiency, autoimmune disorders, and ectodermal dysplasia. ACTH deficiency, ectodermal dysplasia, and common variable immunodeficiency associated with NFKB2 mutations are classified as deficient anterior pituitary with variable immune deficiency (DAVID) syndrome [40]. Therefore, screening for anterior and posterior pituitary deficiencies should be conducted in patients carrying NFKB2 variants [12]. In this case, this patient exhibits autoimmune susceptibility, such as subcutaneous nodules and asthma, but lacks ectodermal dysplasia and other symptoms such as alopecia, trachyonychia, and hypohidrosis. However, pituitary MRI, and assessments of ACTH and cortisol level were not performed for this patient. Consequently, the presence of ACTH deficiency, growth hormone deficiency, or thyroid-stimulating hormone deficiency remains undetermined.

Differentiating autoimmunity from infection poses a significant challenge for pediatricians, particularly in patients with prolonged use of immunosuppressants [41]. Differentiating EGPA against COVID-19 in the pandemic context presents a diagnostic challenge, due to the similarities in respiratory distress and interstitial pneumonia. But the report showed no poor outcomes in EGPA patients who had COVID-19 [42]. At present, no standardized definitions exist for remission, relapse, or refractory disease specifically for pediatric EGPA. Therefore, we apply the criteria for adults: remission of EGPA is defined as the absence of clinical signs or symptoms of active disease with or without immunosuppressive therapy. The maximal daily dose of prednisone is 5 mg. Relapse of EGPA is defined as the recurrence of clinical signs or symptoms of active disease after a period of remission. Refractory EGPA is defined as unchanged or increased signs, symptoms or other features after a period of standard induction therapy. Active disease is defined as new, persistent, or worsening clinical signs and/or symptoms associated with EGPA, independent of prior organ damage [43, 44]. The patient did not achieve remission throughout the course of the disease. Moreover, the patient displayed no signs of infection (fever, coughing, or shortness of breath) during outpatient follow-up, and no new infectious pathogens were identified. However, her disease remained active, and the glucocorticoid dose was unable to be reduced to the minimum. As a result, her condition was classified as active, refractory EGPA rather than infection-driven disease activity. Consequently, understanding the molecular and genetic mechanisms of IEI is crucial for disease screening and therapeutic strategies. Furthermore, increased vigilance is required for opportunistic infections in the context of IEI. In this case, we utilized multiple diagnostic methods to detect infection, including metagenomic sequencing of blood and BALF. mNGS is a widely applied high-throughput approach for pathogen detection, with the advantages of rapid, convenient and highly sensitive detection [45]. The detection of pediatric pneumonia was enhanced by BALF mNGS compared to conventional microbiological tests (CMTs) [46]. Our BALF mNGS analysis identified a total of seven pathogens, with P. jiroveci showing the highest sequence number, relative abundance and coverage, followed by HPyVs type 6 and CMV. Her clinical symptoms indicate that P. jiroveci and CMV are clinically significant. To the best of our knowledge, this is the first report of severe P. jiroveci infection in cEGPA. As is well known, infections are a frequent complication following immunosuppressive therapy, yet severe infections in EGPA are not common [17]. Despite routine prophylaxis with TMP-SMZ after initiating immunosuppression, she developed severe PJP. Nonetheless, it is essential to exclude background noise from the large number of pathogens detected by mNGS.

This study has certain limitations. The diagnosis of EGPA primarily relies on classification criteria, and no universally accepted diagnostic criteria currently exist for EGPA. Furthermore, due to limitations in specimens’ availability and laboratory conditions, further pathogenic functional testing of the NFKB2 variant was not conducted in this case.

In summary, given the rarity of pediatric EGPA in clinical practice, it is imperative to promptly initiate comprehensive genetic sequencing and immune function assessments in a timely manner for patients who exhibit refractory treatment outcomes. This includes challenges in reducing corticosteroid doses or the development of severe infections, such as perianal abscesses. Additionally, employing multiple methods to search for evidence of infection is essential. Further accumulation of cases is required to facilitate additional research.

We identified a novel pathogenic splicing mutation in the NFKB2 gene. This is the first report of cEGPA in a patient with an NFKB2 variant. Our findings may broaden the clinical phenotype of NFKB2 and heighten understanding of cEGPA heterogeneity. In this case, we learned that refractory cEGPA should be closely monitored for possible IEI, as life-threatening COVID-19 pneumonia was the primary cause of death in the patient with the NFKB2 variant.