The Diagnosing Challenge of a Positive ANCA Vasculitis in the Paediatric Age

ANCA-positive systemic vasculitides, rare in paediatric age, present multiorganic involvement. A female teenager presented with a history of subglottic stenosis diagnosed at the age of 12. From the investigation carried out, we highlight hematoproteinuria and negative ANCAs. At 15 years old, she was admitted for gastrointestinal symptoms and respiratory distress. She presented poor peripheral perfusion, pulmonary haemorrhage, respiratory failure, and severe renal insufficiency. She was started mechanical ventilation and emergency haemodialysis. The immunological study revealed ANCA MPO positive. A presumptive diagnosis of ANCA-positive vasculitis was made, and she was started corticotherapy, cyclophosphamide, and plasmapheresis. A renal biopsy, performed later, showed crescentic glomerulonephritis with chronicity signs. Positive ANCA vasculitis may progress slowly or suddenly. The diagnosis was confirmed by a biopsy; however, we can make a presumptive diagnosis based on clinical findings and in a positive ANCA test in order to start an early treatment and decrease the associated morbimortality.


Introduction
Vasculitides are disorders characterized by in ammation of the blood vessel that may occur as a primary process or secondary to an underlying disease. Apart from common vasculitides such as Henoch-Schonlein purpura and Kawasaki disease, most of the primary vasculitic syndromes are rare in childhood [1][2][3][4]. e antineutrophil cytoplasmic antibody-(ANCA-) associated vasculitides (AAV) are a group of chronic, multiorgan, and often relapsing diseases [3,5]. e three classic AAV are granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA).
EGPA is an eosinophilic-rich necrotizing vasculitis, exceedingly rare in childhood, characterized by chronic rhinosinusitis, asthma, and peripheral blood eosinophilia [4,6]. In EGPA, ANCA is found in a minority of children [4].
MPA is a necrotizing nongranulomatous vasculitis that a ects small size blood vessels [3,4,9]. is disease a ects children signi cantly younger than those that GPA a ects [7]. Comparatively to GPA, almost all children have constitutional symptoms (85%) in MPA; however, pulmonary manifestations are less frequent (44%) and less severe [7].
Renal involvement is also frequent (75%) and tends to be more severe than that in paediatric GPA [7]. In MPA, contrary to GPA, the upper respiratory tract is spared [4,7]. MPA is commonly associated with elevated titers of ANCA directed at myeloperoxidase (MPO) that lead to perinuclear staining of neutrophils (p-ANCA) [4,7,8].
Although GPA is primarily associated with PR3-ANCA and MPA with MPO-ANCA, about 25% of the patients with GPA or MPA have the alternative ANCA [10]. e priorities in the management of a child with AAV are the prompt recognition and early treatment, as these diseases can be severe and life-threatening if not appropriately managed [11]. e diagnosis of AAV is strongly suggested by a positive ANCA test; however, 5-10% of the patients do not develop ANCA, so a negative result does not exclude a diagnosis of AAV [8,11]. e biopsy of an a ected organ remains the most de nitive method to establish a diagnosis [8,11]. Treatment comprises remission induction, with initial immunosuppressive therapy and maintenance immunosuppressive therapy for a variable period to prevent relapse [4,5,8,9,11].
Despite treatment, AAV still carries considerable disease-related morbidity and mortality mainly due to progressive renal failure or aggressive respiratory involvement [5,6,9].

Case Presentation
A fteen-year-old female was transferred to the intensive paediatric care unit due to pulmonary haemorrhage, requiring mechanical ventilation, and renal insu ciency.
She has no relevant family history and has neonatal antecedents of extreme prematurity and bronchopulmonary dysplasia.
At eleven years of age, she started having episodes of respiratory distress, wheezing, coughing, and stridor that lead to multiple hospitalizations despite therapy with β2 long-acting agonists, antileukotrienes, and inhaled corticosteroids. In the exacerbations, she presented weak response to salbutamol and improvement with adrenaline and methylprednisolone. In this context, an etiological investigation was carried out at the hospital in her residential area: negative allergy screening; normal immunoglobulin and complement levels; normal alpha-1 antitrypsin level; negative sweat test; negative Ziehl-Neelsen staining and Mycobacterium tuberculosis culture in gastric juice; and normal upper gastrointestinal endoscopy, esophagealgastroduodenal transit, and thoracic computed tomography.
At twelve years old, she developed progressive dysphonia showing no improvement with oral and inhaled corticosteroids. She was observed in otolaryngology consultation at a central hospital: the chest radiograph showed the steeple sign ( Figure 1) and the broncho broscopy revealed a pleated and hardened supraglottic mucosa and subglottic stenosis. Firstly, she underwent laser surgery, and two months later, an emergency tracheotomy for respiratory insu ciency. After the tracheostomy, she needed reinterventions due to the worsening of the subglottic stenosis. From the investigation carried out in this hospital, we highlight normal angiotensin-converting enzyme level, negative antinuclear antibodies (ANA), negative ANCA (by a combination of both indirect immuno uorescence technique (IFT) and enzyme-linked immunosorbent assays (ELISAs) for PR3 and MPO), the presence of hematoproteinuria (proteins 5 mg/dl and erythrocytes > 25/ eld), mild changes in renal function (creatinine 0.86 mg/dl and glomerular ltration rate 65 ml/min/1.73 m 2 ), and laryngeal biopsy with squamous epithelial hyperplasia and polymorphic in ltrate, without granulomas or malignancy signs.
Four months later, at the age of fteen, she was admitted to the hospital in her residential area with a history of abdominal pain, vomiting, diarrhoea, di culty in breathing and coughing for the past two days, and one-month history of aphonia. Examination ndings revealed apyrexia, pallor, poor peripheral perfusion, and di culty in breathing with global retraction and bilateral crepitations at pulmonary auscultation. She also presented hypertension (144/99 mmHg), generalized mild oedema, oliguria, and bleeding through the tracheostomy hole. Initial investigations revealed mixed acidosis (pH 7.01; pCO 2 62.3 mmHg; HCO 3 15.5 mmol/L; and lactate 1.1 mmol/L), severe anaemia with normal leukocyte and platelet accounts (haemoglobin 5.3 g/dl; leukocytes 12.700/uL; and platelets 318.000/uL), severe renal impairment (urea 339 mg/dl and creatinine 18 mg/dl), and hyperkalaemia (7.18 mmol/L). e remaining ionogram, liver function, coagulation screen, and C-reactive protein were within normal limits. e chest radiograph disclosed di used opacity with ill-de ned edges (like cotton wool) in the lower half of both lung elds, and ultrasound revealed moderate left pleural e usion. Bronchoscopy revealed bronchial serohematic content. e patient's condition deteriorated rapidly, and she was transferred to the intensive paediatric care unit, in our hospital, for respiratory and renal support.
In the intensive paediatric care unit, while reviewing the patient's electronic clinical process, we detected previous analyses that had not been valued. Given the clinical picture of renal failure with hematoproteinuria associated with pulmonary haemorrhage with positive MPO-ANCA, a presumptive diagnosis of vasculitis anti-MPO ANCA positive was made, and she immediately started treatment with daily methylprednisolone (650 mg/day) and support measures (red blood cells concentrate, antihypertensives, sodium bicarbonate, calcium gluconate, salbutamol, and insulin) and haemodialysis. On the fourth day of treatment, methylprednisolone was changed to oral prednisolone (1 mg/kg/day) and she started oral cyclophosphamide (2 mg/kg/day) and plasma exchange for seven days. She also received prophylactic cotrimoxazole.
From the laboratorial investigation carried out in the intensive paediatric care unit, we highlight immunoglobulins and C4 within normal values, C3 slightly decreased (77.1 mg/dl), negative anti-glomerular basement membrane antibodies, negative ANA, and positive ANCA MPO (IFT: ANCA 1/640, p-ANCA pattern; ELISA: anti-MPO 55.8 UQ, anti-PR3 14.6 UQ-normal range < 20 UQ). oracic computed tomography showed areas with a bilateral ground glass appearance and hilar adenomegalies. Echocardiogram revealed a small pericardial e usion without further changes. e patient was ventilated for a total of 13 days and remained clinically stable with progressive hypertension control and with no new episodes of pulmonary haemorrhage. She maintained oligoanuric renal insu ciency with the need of dialysis treatment. A renal biopsy was performed, and the histologic examinations revealed crescentic glomerulonephritis with a strong brosis degree. A pauciimmune pattern was observed in immuno uorescence microscopy.
On the thirty-third day of hospitalization, cyclophosphamide treatment was suspended due to leukopenia and induction therapy was started with rituximab (750 mg/m 2 /dose, two doses with fteen-day breaks). She was discharged after 36 days of hospitalization with oral prednisolone that was slowly tapered.
Currently, half a year later, she maintains haemodialysis therapy; however, she has improved from the tracheal stenosis allowing her better vocal quality. ere were no further complications. e ANCA became negative with reduction of the anti-MPO concentration to <2.3 UQ (normal range < 20 UQ).

Discussion
AAV are rare in the paediatric age and can progress slowly over months or explosively over days. In this case report, the long period between the onset of symptoms and the diagnosis clearly illustrates the di culty faced in recognizing AAV.
e respiratory symptoms, present since she was eleven years old, constituted an early manifestation of the disease. Nevertheless, the diagnosis of tracheal stenosis was only made six months later, after the development of dysphonia. After this diagnosis, an etiological investigation was carried out at the otolaryngology centre and they found hematoproteinuria and mild renal insu ciency that was not properly valued since the immunological study was normal. Later, the investigation was repeated, and she presented a worsening of hematoproteinuria and renal function with positive ANCA. Nonetheless, these results were not valued before the life-threatening episode observed in the admission.
In the intensive care unit, a presumptive diagnosis of MPO-ANCA-positive vasculitis was made, allowing for the start of immediate treatment. Later, a renal biopsy was performed, and it was compatible with the previous presumptive diagnosis; however, unfortunately a high chronicity degree was also observed, and the patient was considered with end-stage renal disease.
Classi cation criteria of vasculitides were developed in order to standardize clinical de nitions of di erent vasculitides but not to diagnose disease in individuals [12]. e goal was to de ne homogeneous populations to facilitate clinical research studies. Once childhood vasculitides are rare, experience of individual physicians in diagnosing and caring for children with chronic vasculitis is also limited, as was recognized by Ozen in an survey in which the median number of patients with an AAV diagnosed by any rheumatologist in a single year was <1 [13]. us, clinicians with limited experience to these particular conditions may use classi cation criteria to assist in the diagnosis of individual patients [13,14]. e rst classi cation criteria, and most widely used, were developed by the American College of Rheumatology (ACR); however, these criteria are based upon data from adult patients [15]. Once some individual criteria are more frequent or characteristic of childhood presentation (like subglottic stenoses in GPA) [16], the European League Against Rheumatism (EULAR) and the Paediatric Rheumatology European Society (PRES) proposed in 2005 a system of classi cation for vasculitides in children, which was validated in 2008 (EULAR/PRINTO/PRES criteria) [17,18]. Unfortunately, due to the few cases of the less frequent forms of AAV in their population, classi cation criteria were not developed for MPA. European Medicines Agency (EMA) to attempt to solve these limitations developed an algorithm which includes a list of surrogate markers for GPA, re ecting those features most typical of GPA not occurring in MPA, like subglottic stenosis [19]. ereby, this algorithm in a sequential manner applies di erent criteria (including ACR or EULAR/PRINTO/PRES criteria for GPA), de nitions, and surrogate markers, from most speci c to least speci c, in a stepwise approach allowing the di erentiation between CSS, GPA, and MPA [19].
Applying EULAR/PRINTO/PRES criteria, we can a rm that our patient has GPA (she ful lls four out of six GPA criteria-upper respiratory tract involvement, laryngotracheobronchial obstruction, lung involvement, ANCA positivity, and renal involvement-and only three criteria are required for the diagnosis) [17]. Although GPA is typically associated with PR3-ANCA, MPO-ANCA can be found in about 25% of these patients [10]. us, the presence of MPO-ANCA does not rule out GPA diagnosis as shown by the EULAR/PRINTO/PRES criteria that only included in their criteria ANCA positivity, which could be for MPO or PR3 [17]. e goal of AAV therapy is the induction of complete remission which is de ned as the absence of active disease.

Case Reports in Pediatrics
Initial immunosuppressive therapy in AAV typically consists of glucocorticoids combined with either cyclophosphamide or rituximab [4]. In our case, and in addition to this therapy, plasmapheresis was also performed, as this child had pulmonary haemorrhage and severe renal insu ciency with the need for dialysis [4,20,21]. In this case report, and despite the child maintaining end-stage renal disease after induction immunosuppressive therapy, she presented tracheal and pulmonary disease remission and ANCA titers declined to become negative. ere are controversies about the role of ANCA titers in determining e cacy of treatment and in the prediction of the development of a relapse. e absence of ANCA is usually evidence against a are, but the opposite is not true [4].
ere is little knowledge about the optimal treatment of AAV patients who developed severe renal insu ciency. ese patients have less probability of responding to therapy compared with preserved renal function patients and additionally present an increased risk for immunotherapy adverse e ects. A recent study with patients requiring dialysis at the time of diagnosis showed that, in the absence of active extrarenal vasculitis, continued immunosuppressive therapy beyond four months is very unlikely to bene t patients who remain dependent on dialysis [22]. us, in this case, due to the need of dialysis therapy since the admission and the strong chronicity degree observed in kidney biopsy, we decided not to administer maintenance immunosuppressive therapy.
e authors intend to point out the di culty in diagnosing AAV and distinguishing between these diseases, as they are rare in paediatric age, their clinical features and serologies often overlap, and they have a varied clinical course, which is sometimes indolent. In this case, the diagnosis was even more di cult since the serologies were initially negative, and the child was monitored in di erent centres which may have hampered the clinical data integration. Although the classi cation criteria for AAV were not elaborated for the purpose of individual diagnosis, given the rarity and di culty of diagnosing these diseases, they could be used by physicians for diagnosis when their experience with patients with AAV is limited [13,14].