Differences between Obliterative and Problematic Severe in a pediatric population Teresa Bandeira, Filipa Negreiro, Marisa Salgueiro, Luísa Lobo, Pedro Aguiar, Jc Trindade

To cite this version:

Teresa Bandeira, Filipa Negreiro, Marisa Salgueiro, Luísa Lobo, Pedro Aguiar, et al.. Differences between Obliterative Bronchiolitis and Problematic Severe Asthma in a pediatric population. Pediatric , Wiley, 2011, 46 (6), pp.573. ￿10.1002/ppul.21405￿. ￿hal-00613788￿

HAL Id: hal-00613788 https://hal.archives-ouvertes.fr/hal-00613788 Submitted on 6 Aug 2011

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Differences between Obliterative Bronchiolitis and Problematic Severe Asthma in a pediatric population

For Peer Review

Journal: Pediatric Pulmonology

Manuscript ID: PPUL-10-0326.R1

Wiley - Manuscript type: Original Article

Date Submitted by the 06-Nov-2010 Author:

Complete List of Authors: Bandeira, Teresa; University Hospital Santa Maria, Pediatric Department Negreiro, Filipa; Eurotrials, Biostatistics Department Salgueiro, Marisa; University Hospital Santa Maria, Pediatric Function Laboratory Lobo, Luísa; University Hospital Santa Maria, Imaging Department Aguiar, Pedro; Eurotrials, Biostatistics Department Trindade, JC; University Hospital Santa Maria, Pediatric Department

obliterative bronchiolitis, problematic asthma, children, overlap Keywords: syndrome

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2 3 4 Title: Clinical, radiological and physiological differences 5 6 between Obliterative Bronchiolitis and Problematic Severe 7 8 Asthma in adolescents and young adults. The early origins of 9 10 11 the overlap syndrome? 12 13 Short title: Discrimination between Obliterative Bronchiolitis 14 15 and Problematic Severe Asthma in a pediatric population. 16 17 18 19 20 For Peer Review 21 Authors : Teresa Bandeira 1, Filipa Negreiro 2, Marisa Salgueiro 3, Luísa 22 23 Lobo 4, Pedro Aguiar 5, J C Trindade 6 24 25 26 Institutes and affiliations 27 28 29 1 Pediatrician. Pediatric Department, Medical School at University of 30 31 Lisbon, Hospital Santa Maria, Lisbon, Portugal 32 33 34 2 Statistician. Biostatistics Department: Eurotrials, Portugal 35 36 3 37 Cardiorespiratory Technician. Pediatric Department, Medical School at 38 University of Lisbon, Hospital Santa Maria, Lisbon, Portugal 39 4 40 Imaging Department, Medical School at University of Lisbon; Hospital 41 42 Santa Maria, Lisbon, Portugal 43 44 5 Epidemiologist. Biostatistics Department: Eurotrials, Portugal 45 46 47 6 Professor of . Pediatric Department, Medical School at 48 49 University of Lisbon, Hospital Santa Maria, Lisbon, Portugal 50 51 52 53 54 1 55 56 57 58 59 60

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2 3 Corresponding author : Dr Teresa Bandeira 4 5 Respiratory Medicine Unit, Pediatric Department 6 7 8 Hospital Santa Maria (HSM). University of Lisbon 9 10 Av. Professor Egas Moniz - 1649-035 11 12 Lisbon, Portugal 13 14 Tel: + 35 964308314 15 16 Fax: + 35 217 805 623 17 18 email: [email protected] 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 Short Title: 105 characters 31 32 33 Abstract: 245 words 34 35 Text Body: 3305 words 36 37 38 References: 43 39 40 41 Tables and Figures: 4 42 43 44 1 Online supplement 45 46 47 48 49 50 51 52 53 54 2 55 56 57 58 59 60

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2 3 4 Abstract 5 6 PURPOSE: Few reports have compared chronic obstructive lung diseases (OLD) starting 7 8 in childhood. AIMS: To describe functional, radiological and biological features of 9 10 obliterative bronchiolitis (OB) and further discriminate to problematic severe asthma 11 12 13 (PSA) or to diagnose a group with overlapping features. METHODS: data from 25 OB 14 15 patients [median (range) age: 16.3(8.6-34.7)years] from a single center were compared 16 17 with 15 PSA patients [median (range) age 14.2(8.3-24.9) years]. Lung function tests 18 19 (LFT) included , determination of lung volumes and bronchodilation. High 20 For Peer Review 21 resolution CT (HRCT) images were scored for the presence and the extent of 21 22 23 findings. Blood samples for atopic and inflammatory parameters besides skin prick 24 25 tests were performed. We used ROC curve to analyze the discriminative power of the 26 27 28 variables and cluster analysis to look for a "3rd diagnostic group". RESULTS: Patients 29 30 with OB showed a greater degree of obstructive lung defect and higher hyperinflation 31 32 (p<0.001). The most frequent HRCT features (increased lung volume, inspiratory 33 34 decreased attenuation, mosaic pattern and expiratory ) showed 35 36 significantly greater scores in OB patients. Patients with PSA have shown a higher 37 38 frequency of atopy (p<0.05). ROC curve analysis demonstrated discriminative power 39 40 for the LF variables, HRCT findings and for atopy between diagnoses. Further analysis 41 42 released 5 final variables more accurate for the identification of a third diagnostic 43 44 45 group (FVC%t, post- FEV 1 in ml, HRCT mosaic pattern, SPT and 46 47 D.Pteronyssinus specific IgE ). CONCLUSIONS: We found that OB and PSA possess 48 49 identifiable characteristic features but overlapping values may turn them 50 51 undistinguishable. 52 53 54 3 55 56 57 58 59 60

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4 5 6 Keywords: obliterative bronchiolitis, problematic asthma , children, computed 7 8 tomography, lung function, atopy, overlap syndrome 9 10

11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 4 55 56 57 58 59 60

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2 3 Introduction 4 5 6 The far most common form of bronchiolitis obliterans in children 7 8 follows a severe lower respiratorytract infection[1,2]. Other causes 9 10 11 include collagen vascular disease, toxic fume inhalation, aspiration 12 13 syndromes, and StevensJohnson syndrome[3]. The patient typically 14 15 has wheezing, , dyspnea, and persistent for weeks 16 17 or months after the initial infection. The disease may persist for years 18 19 after its onset and may worsen due to exacerbations caused by viral 20 For Peer Review 21 infections. There are no epidemiological data available, but, for 22 23 unclear reasons, OB seems to occur more frequently in the southern 24 25 hemisphere (southern Brazil, Uruguay, Argentina, Chile, New 26 27 28 Zealand, and Australia) generating a demand for hospital and clinic 29 30 services similar to that observed in patients with [2,2,3] 31 32 in other parts of the world. 33 34 35 OB is synonymous with the term “constrictive bronchiolitis” [4]. It 36 37 has been shown that there are distinctive features of lung function 38 39 (LF) and highresolution CT (HRCT) that both characterizes the 40 41 diagnosis and estimates the severity of attainment. LF results are 42 43 usually characterized by fixed obstruction, minimum response to the 44 45 administration of corticoids and normal total lung capacity [2].. HRCT 46 47 most characteristic findings are segmental or lobular areas of 48 49 hypoattenuation that are associated with narrowing of the caliber of 50 51 52 the pulmonary vessels (mosaic perfusion). These abnormalities are 53 54 5 55 56 57 58 59 60

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2 3 consistent with air trapping, and can be clearly demonstrated on CT 4 5 6 scans of the chest performed during expiration or on inspiratory or 7 8 lateral decubitus HRCT in the uncooperative pediatric patients [57]. 9 10 are also recognized and associated with OB, and tend 11 12 to be peripheral and cylindrical in nature [4]. Most features presented 13 14 by OB patients parallel those of COPD [8]. 15 16 17 In the last decade there is growing evidence for the overlap features 18 19 between obstructive lung diseases (OLD) in the adult patients [9] 20 For Peer Review 21 drawing a continuum from the variable airflow obstruction (asthma) 22 23 to the incompletely reversible airflow limitation (COPD) with the 24 25 concomitant diagnosis of asthma, chronic , or emphysema 26 27 28 being so common among OLD patients from the general population as 29 30 to account for as much as half of the OLD population who are aged 31 32 over 50 years [10]. 33 34 35 In childhood the recognition of severe asthma is not an easy task 36 37 [11]. In 2000 the ATS Proceedings classified refractory asthma 38 39 mainly based on dose of prescribed [12]. Recently 40 41 Bush et al proposes the diagnosis of problematic severe asthma 42 43 (PSA) for the poor control asthma patients that will afterwards be 44 45 divided by the subspecialist in two subgroups either as difficult into 46 47 difficulttotreat asthma or as severe therapyresistant asthma 48 49 [13,14]. 50 51 52 53 54 6 55 56 57 58 59 60

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4 5 6 The purpose of our investigation was to describe the characteristic 7 8 features and patterns distinctive for the diagnosis of OB and PSA 9 10 11 through the analysis of the results of objective assessments 12 13 consisting of conventional lung function tests, high resolution 14 15 computed tomography (HRCT) and tests for atopy and . 16 17 Our secondary goal was to determine the discriminative power of 18 19 these variables for the diagnosis of OB or PSA or for the identification 20 For Peer Review 21 of an overlap syndrome. Studying overlap syndrome may shed light 22 23 on the mechanisms of obstructive lung diseases development. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 7 55 56 57 58 59 60

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2 3 4 METHODS 5 6 7 Study design 8 9 10 A cross sectional observational study comparing two groups of 11 12 patients with the diagnosis of OB and PSA was performed. The study 13 14 was approved by the local research ethics committees and written 15 16 informed parental or own consent was obtained prior to testing. 17 18 Subjects 19 20 For Peer Review 21 OB patients were retrospectively identified in the computerized 22 23 database of outpatients attending the pediatric respiratory clinic since 24 25 1975 at H. Santa Maria (HSM), using the following key diagnoses: 26 27 28 OB, bronchiectasis, and postadenoviral infection. The identified cases 29 30 were checked to ascertain whether they met the following inclusion 31 32 criteria[13]: initial episode of a severe respiratory infection or other 33 34 relevant cause for OB in a previously healthy child followed by 35 36 persistent cough, wheezing, retractions, , abnormalities on 37 38 for months or years, a highresolution CT scan 39 40 (HRCT) showing features consistent with the diagnosis: inspiratory 41 42 decreased attenuation, expiratory air trapping and an attenuation mosaic 43 44 pattern and lung function tests demonstrating persistent irreversible 45 Comment [TB 1]: Text A 46 airflow limitation. Patients with the following diagnosis were excluded 47 48 49 based on history or tests: cystic fibrosis, bronchopulmonary 50 51 dysplasia, pulmonary tuberculosis, α1antitripsin deficiency, 52 53 54 8 55 56 57 58 59 60

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2 3 , congenital heart disease and other major 4 5 6 polimalformative syndromes. 7 8 The PSA group was selected from the Difficult Asthma Clinic at 9 10 11 Pediatric Department of HSM based on the occurrence of persistent 12 13 chronic symptoms of airways obstruction, frequent or severe 14 15 exacerbations in last year despite multiple drug treatment associated 16 17 with persistent airflow obstruction and variable obstructive pattern in 18 19 LF tests sometimes with swings in lung function where the diagnosis 20 For Peer Review 21 of asthma was plausible and an appropriate workup to exclude other 22 23 diagnosis was done [14]. No attempt for discriminating between 24 25 subgroups of PSA was done. 26 27 28 Routine OB management at our centre included 36 monthly reviews, 29 30 and the prescription of inhaled steroids and longacting beta2 31 32 agonists and ipratropium or tiotropium. When bronchiectasis were 33 34 35 present, was prescribed with advice to increase 36 37 intensity when unwell, and prescribed for exacerbations. 38 39 General health measures such as optimizing domestic/social settings 40 41 and financial support, , and exercise were strongly 42 43 promoted. Two patients were submitted to lung resection, one with 44 45 extensive unilateral MacLeod syndrome and medically uncontrollable 46 47 bronchiectasis and another after collapse of a small bronchiectatic 48 49 lung. Only one patient is on chronic home oxygenotherapy. PSA 50 51 52 patients are maintained on medium to high dose ranges inhaled 53 54 9 55 56 57 58 59 60

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2 3 steroids, longacting beta2agonists and leukotriene antagonists as 4 5 6 appropriate besides associated treatment. 7 8 9 Lung function assessments 10 11 12 Spirometry was performed using a pneumotachometer in a Jaeger 13 14 Masterscreen plethysmograph (Erich Jaeger AG, Würzburg, 15 16 Germany) by skilled pediatric pulmonary function technicians who 17 18 19 regularly work with children with obstructive lung diseases. The 20 For Peer Review 21 calibration of the plethysmograph was performed daily before each 22 23 test. FEV 1 and forced (FVC) were measured. The 24 25 reproducibility and criteria of measurements were those 26 27 recommended by the American Thoracic Society[15]. The residual 28 29 volume (RV), and total lung capacity (TLC) were determined in the 30 31 body plethysmograph [16]. Using the reference equations from 32 33 www.growinglungs.org.uk [17], spirometric values were expressed as 34 35 36 percent predicted and zscores and lung volumes in % predicted in Text D 37 Comment [TB 2]: 38 function to Zapletal or the ECCS according to age[18]. Weight and 39 40 height zscores were calculated after adjustment for age and sex. Z 41 42 score is calculated as (measured value – mean value)/Population SD. 43 44 It is a better way of expressing measurements than percent 45 46 predicted, which now should be obsolete. For example, depending on 47 48 the variance in the population, 80% predicted may be normal or 49 50 abnormal, but a Zscore of −2.5 can be seen at once always to be 51 52 53 54 10 55 56 57 58 59 60

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2 3 abnormal [19]. Postbronchodilator (BD) FEV (∆FEV ) was measured 4 1 1 5 6 15 min after the administration of 400 mcg using a 7 8 metered dose inhaler and a large volume spacer. All results were 9 10 reported from baseline tests. 11 12 13 High-resolution computed tomography (HRTC) 14 15 The HRTC studies were acquired at our institution between 2005 and 16 17 September 2009 using fourrows CT scanner. Most of the studies 18 19 (34/40) were performed between May 2008 and September 2009 20 For Peer Review 21 (online supplement). 22 23 24 25 26 27 Atopy and inflammatory markers 28 29 30 Atopy was defined as either at least one positive specific 31 32 immunoglobulin (Ig) E or one positive skin prick test to aeroallergens 33 34 (cat, dog, house dust mite, grasses and fungi)[20]. Furthermore the 35 36 presence of increased number of eosinophils (>274cel.mm 3), and /or 37 38 IgE higher that 2SD adjusted for the age was analyzed between 39 40 41 groups [20]. Inflammation pattern was considered based on a high C 42 43 reactive protein (CRP) (mg/dl), sedimentation rate (SR) mm, 44 9 45 fibrinogen (mg/dl), leukocytes (>13x10 /L) and alpha1antitrypsin 46 47 (AAT) [21]. 48 49 50 51 Statistical analysis 52 53 54 11 55 56 57 58 59 60

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2 3 4 5 Descriptive statistics[22,23], including distribution of records, age, 6 7 and gender, age at initial symptoms, hospital admission, mechanical 8 9 ventilation history and clinical characteristics were completed using 10 11 SPSS 16.0 (SPSS Inc., Chicago, IL). Results were considered 12 13 statistically significant at p < 0.05. 14 15 16 To analyze the discriminative power of the variables LFT, HRCT, atopy 17 18 and inflammation for the diagnosis of OB or PSA, Receiver Operating 19 20 For Peer Review 21 Characteristic (ROC) curves were used. For variables whose area 22 23 under the ROC curve presented a good discriminating power (> 0.70) 24 25 and shown to be statistically significant (p < 0.05) cutoffs (trim 26 27 points) were identified with sensitivity and specificity equal to or 28 29 greater than 75% (see online supplement for further details). 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 12 55 56 57 58 59 60

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2 3 4 RESULTS 5 6 Demographic and clinical characteristics of patients and comparison by group of 7 8 diagnosis are shown in Tables 1 and 2. In both groups there was a male 9 10 predominance. Patients from OB group were younger at acute episode, and 96% have 11 12 13 been admitted to hospital for lower tract infection, more frequently than PSA patients 14 15 (p < 0.05). Eight (32%) OB patients have been submitted to mechanical ventilation 16 17 (MC) at this initial episode but from the 3 PSA patients on MV, one occurred at the 18 19 neonatal period and two others were ventilated late at the course of disease because 20 For Peer Review Comment [TB 3]: Text B 21 of severe asthma attack. In 15 (60%) of the OB patients a cause was identified, 93.3 22 23 % were attributable to previous infection [8 (53.3%) to adenovirus, 2 (13.3%) to 24 25 measles, 3 (20%) to one (6.7%) varicela; in one case positive 26 27 28 for M.pneumoniae , Stevens-Johnson syndrome was diagnosed and in another (6.7%) 29 30 near-drowning in soapy water had occurred] Afterwards the majority of patients in 31 32 both groups had persistent symptoms but fine crackles were heard more frequently in 33 34 OB (60%) than in PSA patients (6.7%) (p< 0.001). . In both groups a high prevalence of 35 36 exposure to tobacco smoke was found (p=0.542). 37 38 Lung function 39 40 41 42 There was no statistical difference between the two groups in age and 43 44 height at lung function test (LFT) but patients with OB showed a 45 46 reduced zscore for weight and BMI compared with PSA patients (p< 47 48 0.05) (table 2 of the online supplement). Patients with OB showed a 49 50 significant decrease in FEV 1, FVC and FEV 1/FVC ratio. LFT of OB 51 52 53 54 13 55 56 57 58 59 60

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2 3 patients showed a range of values from 18.2 to 70.6%P for FEV (z 4 1 5 6 scores 7.3 to 2.7), from 32.8 to 91% for FVC (zscores 6.4 to 7 8 0.8), and from 46.8 to 92.6 for the FEV 1/FVC ratio (zscores 4.7 to 9 10 1.0) compared to PSA patients (p<0.05; table 3). The RV/TLC ratio 11 12 was significantly increased in patients with OB (range 27.83 to 71) (p 13 14 < 0.001), indicating higher hyperinflation compared with PSA patients 15

16 (Table 3). FEV 1 was greater than 12% in 12 (48.0%) OB patients 17 18 and 8 (53.3%) of PSA patients, but only 8 (32%) OB patients had a 19 20 200 ml improvementFor after beta2agonist Peer Review inhalation. Twelve (80%) 21 22 23 PSA patients showed an improvement after bronchodilator greater 24 25 than 200ml. The change of FEV 1 correlated significantly with baseline 26 27 FVC and FEV 1 (p<0.01) in PSA patients but not in OB patients (data 28 29 not shown). 30 31 CT findings 32 33 34 Among OB patients, the most frequent HRCT features were increased 35 36 lung volume, inspiratory decreased attenuation and mosaic pattern in 37 38 all examinations, and expiratory air trapping in all of the 24 (100%) 39 40 studies that included expiratory images. Bronchial wall thickening 41 42 43 were observed in 23 (92%) and bronchiectasis in 22 (88%). Among 44 45 PSA patients, the most frequent HRCT features were increased lung 46 47 volume in 13 out of 14 (92.9%) where this could be classified, 48 49 inspiratory decreased attenuation in 13 (86.7%) and mosaic pattern 50 51 in 11 out of 14 (78.6%). Expiratory air trapping was present in 11/14 52 53 54 14 55 56 57 58 59 60

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2 3 (78.6%) studies that included expiratory images. Bronchial wall 4 5 6 thickening was observed in 8/14 (57.1%) and bronchiectasis in 4 7 8 (26.7%). When comparing groups of diagnosis, OB patients showed 9 10 significantly higher scores for inspiratory decreased attenuation, 11 12 mosaic pattern, expiratory air trapping and bronchiectasis than PSA 13 14 (table 3 of the online supplement). 15 16 17 Atopy and inflammation 18 19 20 PSA patients had higherFor frequency Peer of at least Review 1 positive allergen skin 21 22 test (p=0.002), a greater mean number of positive skin tests per 23 24 patient (p=0.001), significantly greater median serum IgE level 25 26 (p=0.007), and mean peripheral blood eosinophil count (p=0.004) 27 28 compared with patients with OB ( table 4 of the online supplement ). 29 30 Overall 14 (93.3%) patients with PSA had at least one marker 31 32 positive versus 11 (44%) of the OB patients. 33 34 35 Inflammatory variables studied were in the normal range for both groups and there 36 37 was no significant difference between the two groups (see table E0 on online 38 39 supplement). 40 41 42 43 Variables with power to discriminate between groups of patients 44 45 46 ROC curve analysis demonstrated a good discriminative power (cut 47

48 off estimates higher than 75%) for LF variables (FVC%P, FEV 1%P, 49 50 FEV 1/FVC%P, RV%TLC e FEV 1 in ml) ( Table 5 of the online 51 52 53 54 15 55 56 57 58 59 60

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2 3 supplement), for HRCT findings ( inspiratory decreased attenuation, 4 5 6 mosaic pattern, expiratory air trapping and bronchiectasis ) ( Table 6 7 8 of the online supplement ) and for atopy (peripheral blood eosinophil 9 10 count, SPT, serum IgE level and D.Pteronyssinus specific IgE). No 11 12 inflammat ory parameter showed enough discriminative power to be 13 14 included in further analysis. Cluster analysis for the identification of a 15 16 third group diagnosis showing overlap features determined that 17 18 FVC%P, e FEV 1 in ml , mosaic pattern, SPT and D.Pteronyssinus 19 20 specific IgE were theFor best variables Peer ( online supplementReview and Table 4 ). 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 16 55 56 57 58 59 60

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2 3 4 DISCUSSION 5 6 7 To our knowledge, this is the first study to establish clinical, 8 9 radiological and physiological differences between Obliterative 10 11 Bronchiolitis (OB) and Problematic Severe Asthma (PSA) in the 12 13 transition through pediatric to young adult ages. Our main results 14 15 suggest that patients with OB start their symptoms early in life 16 17 18 usually after a severe respiratory infection. Adenovirus is the most 19 20 frequently identifiedFor etiological Peer agent [24]althoug Reviewh clinical studies 21 22 frequently miss the identification of the causal agent as it occurred at 23 Comment [TB 4]: Text C 24 this investigation [25,26]. In the second decade of life these patients 25 26 still have persistent respiratory symptoms, poor nutrition and a 27 28 significant and hyperinflation. Age and disease 29 30 severity as shown by hospital admission and the need for ventilator 31 32 support and persistent crackles distinguishes these from PSA 33 34 35 patients. Moreover although persistent symptoms and an obstructive 36 37 lung function pattern are found in PSA patients a significant less 38 39 degree of compromise is evidenced. Advances in intensive therapy 40 41 that have occurred over the last few years allowed many children to 42 43 survive who, after being affected by serious respiratory infections, 44 45 develop persistent and severe sequelae [2]. 46 47 48 Furthermore the different extension of lung compromise between OB 49 50 and PSA patients is also revealed through the HRCT findings. 51 52 53 54 17 55 56 57 58 59 60

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2 3 Technical improvements have turned HRCT in the imaging modality of 4 5 6 choice for the morphological assessment of pulmonary parenchyma, 7 8 even in children [27]. 9 10 11 We found that LFT results of obstructive ventilatory defect were 12 13 common in both OB and PSA patients. Bronchial reactivity after beta 14 15 2agonist inhalation was also commonly seen in both conditions 16 17 especially if % variation from baseline was accounted for. No 18 19 significant differences were found in FEV 1/FVC ratio between OB and 20 For Peer Review 21 PSA. Severity of LF compromise expressed by the reduction of FEV 1, 22 23 FEF 2575 and FVC must be taken in account as they tend to be more 24 25 severe in OB than in PSA otherwise being difficult to differentiate OB 26 27 28 and PSA on the basis of the LF defect alone. FVC was reduced in OB 29 30 patients and close to normal in the PSA patients and OB patients 31 32 showed higher airtrapping pattern. . Furthermore FVC and FEV 1 33 34 postBD (ml) emerged as the better LF variables capable of 35 36 identifying third group of diagnosis with overlapping features. We 37 38 have recently demonstrated that results of bronchodilation are 39 40 dependent on previous lung volumes [28] in OB patients as has been 41 42 shown by others [29,30]. Casanova et al demonstrated that a 25% 43 44 IC/TLC value had the best combined sensitivity, specificity, and 45 46 47 positive and negative predictive values for mortality in COPD patients 48 49 [29] and another study showed the same for the ratio RV/TLC[31]. 50 51 Although we have not studied the IC because there are few reference 52 53 54 18 55 56 57 58 59 60

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2 3 values determined in pediatrics [32] we found that FEV and FVC are 4 1 5 6 concomitantly decreased in OB patients determining that FEV 1/FVC 7 8 ratio is only slightly decreased and so not far different the mean 9 10 value found in PSA patients. It has been stated before that this 11 12 pattern reflects failure of the patient to inhale or exhale completely. 13 14 Another explanation, and that is our understanding, the patchy 15 16 collapse of small airways early in exhalation may have a significant 17 18 impact in the response to therapy[18,33]suggesting that the 19 20 peripheral airway involvementFor Peeris an established Review component of OB and 21 22 23 PSA patients. Early on in the evolution of the disease and in less 24 25 severe patients a simple test of gas distribution such as the 26 27 traditional VC N 2 single breath washout test would be a valuable tool 28 29 of diagnosing OB and in the future should be considered in the work 30 31 up for the diagnosis of these patients [34]. 32 33 34 Regarding highresolution CT, our findings were in agreement with 35 36 previous studies which documented that no isolated features could 37 38 accurately diagnose OB as increased lung volume, inspiratory 39 40 decreased attenuation, expiratory air trapping and mosaic pattern 41 42 were frequent both in OB and in PSA patients. However OB patients 43 44 showed higher scores for these HRCT findings and bronchiectasis 45 46 47 were by far more frequent in OB patients. In our study, mosaic 48

49 pattern provided the best power to predict the emergence of a third 50 51 group of diagnosis. The mosaic pattern of attenuation of lung tissue 52 53 54 19 55 56 57 58 59 60

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2 3 on HRCT seems to be the key finding which may offer some 4 5 6 confidence in suggesting a diagnosis of OB or overlap syndrome 7 8 rather than asthma as has been shown before [35]. Colom and Teper 9 10 demonstrated that a mosaic pattern on HRCT was strongly associated 11 12 with the diagnosis of postinfectious OB [6]. Although there are 13 14 different causes for the mosaic pattern of attenuation [36] we believe 15 16 as Jensen [35] that the mosaic pattern seen in OB results from a 17 18 combination of airtrapping due to small airways disease and 19 20 associated oligoemiaFor secondary Peer to reflex vasoconstr Reviewiction to the areas 21 22 23 of underventilated lung tissue. 24 25 We found a great proportion of patients with atopic markers in both 26 27 groups studied but variables from the atopic expression were more 28 29 frequently is PSA than in OB patients. An interaction between atopy 30 31 and induced viral wheezing is being increasingly described and a 32 33 strong association between asthma and atopy comes from large 34 35 epidemiologic studies [37,38]. 36 37 38 Although the importance of atopy as a cause of asthma in individuals 39 40 may have been overemphasized [39] and the available 41 42 epidemiological evidence suggests that the population based 43 44 proportion of asthma cases that are attributable to atopy is usually 45 46 47 less than one half, another study on difficult asthma has shown 48 49 increased proportion of atopy [40]. SPT and IgE for D.Pteronyssinus 50 51 were identified as discriminative factors between OB and PSA. This is 52 53 54 20 55 56 57 58 59 60

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2 3 in agreement with suggestion that quantitative measures of atopy 4 5 6 especially cumulative titers of IgE specific for perennial inhalant 7 8 allergens provide robust assessments of atopyassociated risk of 9 10 current asthma and its severity [38] . Atopy has also been related to 11 12 other respiratory diseases but the association between atopy and the 13 14 development of obstructive lung diseases remains to be clarified [20]. 15 16 17 The study does have a number of limitations. Patients were selected 18 19 from a specialized clinic on a third care University Hospital and so the 20 For Peer Review 21 reduced number of patients over a large time span may turn difficult 22 23 the generalization of findings. However both are rare diseases. This is 24 25 the largest group of OB patients reported in Europe [41]. PSA 26 27 28 constitutes a heterogeneous clinical diagnosis that only recently had 29 30 their distinction clarified [14] so that the present study may add 31 32 further discriminative features for severe obstructive diseases starting 33 34 in pediatric ages and suggest a different pathophysiologic defect 35 36 between them. We could have further studied peripheral airway 37 38 measurements using different diagnostic tools [34,42]but mean 39 40 values for FEF 2575 and of expiratory lung attenuation described 41 42 support the evidence of severe peripheral airway disease in both 43 44 groups of patients although more severe in the OB group. No indirect 45 46 47 acting stimulus to assess hyperreactivity was performed which could 48 49 be more precise in discriminating between diseases [9]. 50 51 52 53 54 21 55 56 57 58 59 60

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2 3 In conclusion to our knowledge we have shown for the first time that 4 5 6 a large overlap exists between the spectrum of clinical, functional, 7 8 radiological and atopic features of patients with OB and those with 9 10 PSA. Practical implications in this debate are relevant because the 11 12 management for both diseases differ as that for asthma and COPD in 13 14 adult patients. However, a growing number of researchers and 15 16 clinicians consider that is a continuum from 17 18 childhood to adulthood [9], and only prospective studies can 19 20 determine the relationshipFor between Peer adult phenotypesReview of obstructive 21 22 23 lung disease and its pediatric origins [43]. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 22 55 56 57 58 59 60

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4 5 6 Reference List 7 8 9 10 1. del Pino M, Bauer G, González Pena H et al . Growth in postviral 11 chronic lung disease. Eur J Pediatr 2006;165:845849. 12 2. Teper A, Fischer GB, Jones MH. Respiratory sequelae of viral diseases: 13 from diagnosis to treatment. Jornal de Pediatria 2002; 78(Suppl. 14 2) :S187S194. 15 16 3. Moonnumakal SP, Fan LL. Bronchiolitis obliterans in children. Curr 17 Opin Pediatr 20, 272278. 2008. 18 19 4. Chan A, Allen R. Bronchiolitis obliterans: an update. Curr Opin Pulm 20 Med 10, 133141.For 2004. Peer Review 21 22 5. CastroRodriguez JA, Daszenies C, Garcia M et al . Adenovirus 23 pneumonia in infants and factors for developing bronchiolitis 24 obliterans: A 5year followup. Pediatr Pulmonol 2006;41:947953. 25 26 6. Colom AJ, Teper AM. Clinical prediction rule to diagnose postinfectious 27 bronchiolitis obliterans in children. Pediatr Pulmonol 2009;44:1065 28 1069. 29 7. Colom AJ, Teper AM, Vollmer WM et al . Risk factors for the 30 development of bronchiolitis obliterans in children with bronchiolitis. 31 Thorax 2006;61:503506. 32 33 8. Global Initiative for Chronic . 2004. 34 http://www goldcopd com/ 2009;(Accessed Jan, 2010). 35 36 9. Gibson PG, Simpson JL. The overlap syndrome of asthma and COPD: 37 what are its features and how important is it? Thorax 2009;64:728 38 735. 39 40 10. Soriano JB, Davis KJ, Coleman B et al . The proportional Venn diagram 41 of obstructive lung disease: two approximations from the United States 42 and the United Kingdom. Chest 2003;124:474481. 43 44 11. Fleming L, Wilson N, Bush A. Difficult to Control Asthma in Children. 45 Curr Opin Allergy Clin Immunol 2007;7:190195. 46 12. Proceedings of the ATS Workshop on Refractory Asthma . Current 47 Understanding, Recommendations, and Unanswered Questions. Am J 48 Respir Crit Care Med 2000;162:23412351. 49 50 13. Bush A, Hedlin G, Carlsen KH et al . Severe childhood asthma: a 51 common international approach? Lancet 2008;372:10191021. 52 53 54 23 55 56 57 58 59 60

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2 3 14. Hedlin G, Bush A, Lordrup Carlsen K et al . Problematic severe asthma 4 in children, not one problem but many: a GA2LEN initiative. Eur Respir 5 J 2010;36:196201. 6 7 15. Miller MR, Hankinson J, Brusasco V et al . Standardisation of 8 spirometry. Eur Respir J 2005;26:319338. 9 10 16. Wanger J, Clausen JL, Coates A et al . Standardisation of the 11 measurement of lung volumes. Eur Respir J 2005;26:511522. 12 13 17. Stanojevic S, Wade A, Stocks J et al . Reference Ranges for Spirometry 14 Across All Ages: A New Approach. Am J Respir Crit Care Med 15 2008;177:253260. 16 17 18. Pellegrino R, Viegi G, Brusasco V et al . Interpretative strategies for 18 lung function tests. Eur Respir J 2005;26:948968. 19 19. Bush A. COPD: A Pediatric Disease. Journal of Chronic Obstructive 20 Pulmonary DiseaseFor 2008;5:5367. Peer Review 21 22 20. Baldacci S, Omenaas E, Oryszczyn MP. Allergy markers in respiratory 23 epidemiology. Eur Respir J 2001;17:773790. 24 25 21. Cazzola M, Macnee W, Martinez FJ et al . Outcomes for COPD 26 pharmacological trials: from lung function to biomarkers. Eur Respir J 27 2008;31:416469. 28 29 22. Aguiar P. Guia Prático de Estatística em Investigação Epidemiológica: 30 SPSS . Lisboa: Climepsi, 2007 31 32 23. Altman DG. Practical statistics for medical research . London: Chapman 33 & Hall, 1991 34 24. Colom AJ, Teper AM. Postinfectious bronchiolitis obliterans. 35 Arch.Argent.Pediatr 107, 160167. 2009. 36 37 25. Hodges IG, Milner AD, Groggins RC et al . Causes and management of 38 bronchiolitis with chronic obstructive features. Arch Dis Child 39 1982;57:495499. 40 41 26. Zhang L, Irion K, Kozakewich H et al . Clinical course of postinfectious 42 bronchiolitis obliterans. Pediatr Pulmonol 2000;29:341350. 43 44 27. Rossi UG, Owens CM. The radiology of chronic lung disease in children. 45 Arch Dis Child 2005;90:601607. 46 47 28. Bandeira T, Almodovar T, Salgueiro M et al . FEV 1 response to 48 bronchodilation in Pediatric Patients With NonTransplant Obliterative 49 Bronchiolitis and Asthma: Similarities to Adult Patients in the 50 Discriminative Value For Diagnosis. Chest Meeting Abstracts 51 2009;136:38S38c. 52 53 54 24 55 56 57 58 59 60

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2 3 29. Casanova C, Cote C, de Torres JP et al . InspiratorytoTotal Lung 4 Capacity Ratio Predicts Mortality in Patients with Chronic Obstructive 5 Pulmonary Disease. Am J Respir Crit Care Med 2005;171:591597. 6 7 30. Verbanck S, Schuermans D, Vincken W. Small airways ventilation 8 heterogeneity and hyperinflation in COPD: Response to tiotropium 9 bromide. Int J Chron Obstruct Pulmon Dis 2007;2:625634. 10 11 31. Nishimura K, Izumi T, Tsukino M et al . Dyspnea Is a Better Predictor of 12 5Year Survival Than Airway Obstruction in Patients With COPD. Chest 13 2002;121:14341440. 14 15 32. Tomalak W, Radlinski J, Pogorzelski A et al . Reference values for forced 16 inspiratory flows in children aged 715 years. Pediatr Pulmonol 17 2004;38:246249. 18 19 33. Ferguson GT. Why Does the Lung Hyperinflate? Proc Am Thorac Soc 20 2006;3:176179. For Peer Review 21 34. Robinson PD, Goldman MD, Gustafsson PM. Inert Gas Washout: 22 Theoretical Background and Clinical Utility in Respiratory Disease. 23 Respiration 2009;78:339355. 24 25 35. Jensen SP, Lynch DA, Brown KK et al . Highresolution CT Features of 26 Severe Asthma and Bronchiolitis Obliterans. Clin Radiol 2002;57:1078 27 1085. 28 29 36. Stern EJ, Swensen SJ, Hartman TE et al . CT mosaic pattern of lung 30 attenuation: distinguishing different causes. Am J Roentgenol 31 1995;165:813816. 32 33 37. PintoMendes J. Infecção na modulaçao da asma. Rev Port Pneumol 34 2008;XIV:647675. 35 36 38. Sly PD, Boner AL, Bjornsdottir US et al . Early identification of atopy in 37 the prediction of persistent asthma in children. Lancet 2008;372:1100 1106. 38 39 39. Pearce N, Pekkanen J, Beasley R. How much asthma is really 40 attributable to atopy? Thorax 1999;54:268272. 41 42 40. Bossley CJ, Saglani S, Kavanagh C et al . responsiveness 43 and clinical characteristics in childhood difficult asthma. Eur Respir J 44 2009;34:10521059. 45 46 41. Cazzato S, Poletti V, Bernardi LL et al . Airway inflammation and lung 47 function decline in childhood postinfectious bronchiolitis obliterans. 48 Pediatr Pulmonol 2008;43:381390. 49 50 42. Haruna A, Oga T, Muro S et al . Relationship between peripheral airway 51 function and patientreported outcomes in COPD: a crosssectional 52 study. BMC Pulmonary Medicine 2010;10:10. 53 54 25 55 56 57 58 59 60

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2 3 43. Bush A, MenziesGow A. Phenotypic Differences between Pediatric and 4 Adult Asthma. Proc Am Thorac Soc 2009;6:712719. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 26 55 56 57 58 59 60

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3 4 5 6 TABLES 7 8 9 10 Table 1 – Demographic characteristics and tobacco smoke exposure by group of diagnosis (OB, PA) 11 12 13 For Peer Review 14 Obliterative bronchiolitis Problematic Asthma 15 p-value 16 n=25 n=15 17 18 19 Gender: male/ female, n (%) 15/10 (60/40) 11/4 (73.3/26.7) 0.392 20 21 22 Age at study *(years), median (min ; max) 16.3 (8.6 ; 34.7) 14.2 (8.3 ; 24.9) 0.250 23 24 Time of follow-up before the study (years), 25 6.5 (2.6) 5.2 (1.74) 0.601 mean (SD) § 26 27 Gestational age (weeks), 28 39 (32 ; 41) 40 (33 ; 42) 0.397 29 median (min ; max) 30 31 Birth weight (grams), 3004.1 (911.5) a) 3274.7 (588.9) 0.316 32 mean (SD) 33 34 Duration of exclusive breastfeeding 8.25 (7.4) b) 17.38 (18.0) c) 0.100 35 (weeks), mean (SD) 36 37 Age at acute injury / initial symptoms 38 13 (3 ; 81) 36 (2 ;144) 0.031 (months), median (min ; max) 39 40 41 42 43 1 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pediatric Pulmonology Page 28 of 44

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3 4 5 6 Admission to hospital, n (%) 24 (96) 8 (53.5) 0.001 7 8 9 Mechanical ventilation, n (%) 8 (32) 3 (20) 0.408 10 11 12 13 Hospital readmission, n (%) For Peer15 (60)Review 5 (33.3) 0.108 14 15 16 Tobacco smoke exposure, n (%) 16 (64) 11 (73.3) 0.542 17 18 a) b) c) n=23; n=22; n=13 19 20 *Age at lung function tests; § only time of follow-up with the performance of LF studies is considered 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 2 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 44 Pediatric Pulmonology

1 2

3 4 5 6 Table 2 – Clinical characteristics by group of diagnosis (OB, PSA) 7 8 9 10 Obliterative bronchiolitis Problematic Asthma 11 n=25 12 % n=15 % p-value 13 For Peer Review 14 15 Past history 16 17 Eczema 3 12,0 3 20,0 0,654 18 19 20 b) 21 AOM under 2 years-old 6 25,0 9 60,0 0,029 22 23

24 b) 25 AOM after 2 years-old 3 12,5 2 13,3 >0,999 26 27 28 29 Food allergy 6 24,0 6 40,0 0,285 30

31 32 Persistent symptoms 33 34 Cough without colds or with 35 18 72,0 10 66,7 0,722 exercise 36 37

38 39 a) c) Persistent cough 9 39,1 7 53,8 0,393 40 41 42 43 3 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pediatric Pulmonology Page 30 of 44

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3 4 5

6 7 Dyspnea 20 80,0 13 86,7 0,691 8 9

10 11 Wheezing 22 88,0 15 100,0 0,279 12 13 For Peer ReviewChest sounds 14 15 16 17 11 44,0 7 46,7 0,870 18 19 20 21 Crackles 15 60,0 1 6,7 0,001 22 23 24 25 Reduced chest sounds 13 52,0 0 0,0 0,001 26 27 28 29 a) n=23, b) n=24, c) n=13 . 30 31 AOM – acute 32 33 34 35 36 37 38 39 40 41 42 43 4 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 44 Pediatric Pulmonology

1 2

3 4 5 Table 3 – Lung function results at test by group of diagnosis (OB, PSA) 6 7 8 9 Obliterative bronchiolitis Problematic Asthma 10 p-value 11 n=25 n=15 12 13 FVC% predicted For Peer Review 14 64.7(16.5) 95.2 (19.8) 15 Mean (SD) <0.001 16 63.8 (32.8 ; 91) 95.5 (46.9 ; 140) 17 Median (min; max) 18 z-score 19 FVC -3.3 (1.6) -0.5 (1.9) 20 Mean (SD) <0.001 21 -3.2 (-6.4 ; -0.8) -0.4 (-5.1 ; 3.7) 22 Median (min; max) 23 24 FEV 1% predicted 25 44.1 (14.9) 77.3 (21.5) 26 Mean (SD) <0.001 27 44.2 (18.2 ; 70.6) 81.6 (21.9 ; 112.4) 28 Median (min; max) 29

30 FEV 1 z-score 31 -4.9 (1.3) -2.0 (1.9) 32 Mean (SD) <0.001 33 -5.5 (-7.3 ; -2.7) -1.6 (-6.9 ; 1.1) 34 Median (min; max) 35 36 FEV 1/FVC% predicted 37 67.4 (13.4) 79.6 (13.0) 38 Mean (SD) 0.008 39 66.5 (46.8 ; 92.6) 81.0 (46.4 ; 101.4) 40 Median (min; max) 41 42 43 5 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pediatric Pulmonology Page 32 of 44

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3 4 5 FEV /FVC z-score 6 1 -3.3 (1.0) -2.2 (1.2) 7 Mean (SD) 0.009 8 -3.5 (-4.7 ; -1.0) -2.1 (-4.7 ; 0.2) 9 Median (min; max) 10 11 FEF 25-75 %predicted 12 18.0 (13.2) 1) 48.4 (21.2) 13 Mean (SD) For Peer Review <0.001 14 14.1 (3.3 ; 54.6) 48.0 (5.6 ; 83.3) 15 Median (min; max) 16 17 FEF 25-75 z-score 18 -5.3 (1.3) 1) -2.8 (1.5) 19 Mean (SD) <0.001 20 -5.5 (-7.3 ; -2.3) -2.6 (-6.4 ;-0.8) 21 Median (min; max) 22 TLC% predicted 23 24 108.0 (21.1) 111.9 (10.6) 25 Mean (SD) 0.506 26 106.5 (68.7 ; 152.6) 109.7 (98.5 ; 138.3) Median (min; max) 27 28 RV%TLC 29 48.6 (11.6) 32.0 (8.1) 30 Mean (SD) <0.001 31 46.4 (33.0 ; 68.6) 34.1 (18.4 ; 49.9) 32 Median (min; max) 33 34 sRtot 35 2.7 (1.9) 1.3 (0.7) 36 Mean (SD) 0.002 37 2.0 (0.8 ; 8.9) 0.9 (0.6 ; 3.0) 38 Median (min; max) 39 40 41 42 43 6 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 44 Pediatric Pulmonology

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3 4 5 ∆∆∆FEV (%) 6 1 13.6 (9.6) 15.4 (12.0) 7 Mean (SD) 0.613 8 10.9 (0.1 ; 36.7) 12.6 (0.3 ; 47.7) 9 Median (min; max) 10 11 ∆∆∆FEV 1 (ml) 12 150.4 (106.7) 334.0 (206.5) 13 Mean (SD) For Peer Review 0.005 14 150.0 (-20.0 ; 430) 260.0 (-50.0 ; 730.0) 15 Median (min; max) 16 17 1) n=24 18 19 FVC: forced vital capacity; FEV 1: forced expiratory volume in 1 second FEV 1/FVC: forced vital capacity to forced expiratory volume in 1 20 second ratio; FEF 25-75 : forced expiratory flow at 25% to 75% vital capacity; RV: residual volume; TLC total lung capacity; sRtot: 21 specic ; ∆∆∆FEV 1 difference between the pre and post bronchodilator FEV 1 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 7 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pediatric Pulmonology Page 34 of 44

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3 4 5 Table 4 – Final cluster analysis presenting the 5 variables that proved the best adequability for group of 6 diagnosis 7 8 9 10 D. pteronyssinus IgE FVC %P ∆∆∆FEV %t post-BD (ml) SPT Mosaic pattern 11 1 (class) 12 13 Cluster 1 (closer For Peer Review n 8 8 8 8 8 14 to OB) 15 16 Median (min; max) 60.6 (32.8; 82.6) 45.0 (-20.0; 110.0) 0.0 (0; 22) 0.0 (0; 6) 5.5 (3; 6) 17 18 n 4 4 4 4 4 19 Cluster 0 20 (closer to Median (min; max) 95.6 (46.9; 105.0) 530.0 (460.0; 730.0) 26.0 (7; 48) 5.0 (3; 6) 3.0 (0; 6) 21 Asthma) 22 23 24 n 23 23 23 23 23 25 26 Clus ter 3 (closer Median (min; max) 82.0 (35.3; 140.0) 220.0 (140.0; 330.0) 10.0 (0; 30) 3.0 (0; 6) 5.0 (0; 6) 27 to 3 rd group) 28

29 30 31 32 33 SPT – skin Prick tests 34 35 36 37 38 39 40 41 42 43 8 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 44 Pediatric Pulmonology

1 2 3 4 5 Online supplement : 6 Methods: 7 8 HRCT 9 10 11 12 HRCT images were acquired on inspiratory from the lung apex to the diaphragm using 13 14 1 mm slice thickness at 10 mm intervals using a bone (high spatial detail) reconstruction 15 16 17 algorithm, 50-80 mAs e 90-120 kVp (according to body volume) and 0,5 sec of rotation 18 19 time. Images were photographed at lung window settings (1600/600) and mediastinal 20 For Peer Review 21 22 window settings (300/40) before March 2009. After March 2009 images were stored in PACS 23 24 25 system. Most of the studies included inspiratory and expiratory images, typically one or two 26 27 images at selected levels through the thorax with the patient in full expiration. In some 28 29 30 cases volumetric acquisition (“combi”) was performed: 3,2/1,6 mm, 80-100 mAs e 90-120 31 32 kVp, 0,5 ms rotation time, 1,75 pitch, followed by a pre-selected multilevel acquisition (3 a 33 34 35 4) of expiratory high resolution images. 36 37 38 Each HRCT was reviewed by a -trained pediatric radiologist. A score sheet modified from 39 40 41 Jensen et al. was used to record the presence and extent of 24 HRCT findings. Each lung was 42 43 divided into upper, middle and lower zones for a total of six lung zones for each patient. 44 45 46 Each of these zones was scored separately for the presence and/or extent of the 24 HRCT 47 48 findings. Inspiratory decreased attenuation, expiratory air trapping and ground glass 49 50 51 opacities were scored according to the cross-sectional area of lung involved in each zone 0 - 52 53 no involvement; 1 – 25% of the cross-sectional area of the lung affected; 2 - 26–50% 54 55 56 affected; 3 - 51–75%; 4 - 76–100% affected, bronchiectasis were classified according to [42] 57 58 into mild with broncho-arterial (BA) ratio lower than 2, moderate, from 2 to 3 and severe if 59 60 broncho-arterial ratio was higher than 3. If a bronchiectasic collapse was found, a grade of

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1 2 3 4 5 2 or higher was considered. The remaining HRCT features shown in table 3 of the online 6 7 supplement were scored as either present (score of 1) or absent (score of 0). Score sheets 8 9 10 were than tabulated and composite scores for each HRCT feature wee than derived by 11 12 adding the scores from the six lung zones for each patient. 13 14 15 16 Statistical analysis 17 18 The demographic data and outcomes between groups were compared using two-sided χ2 19 20 For Peer Review 21 test for categorical variables and two-tailed Student’s t-tests and oneway ANOVA for 22 23 continuous variables. In small samples or in the absence of normality for continuous 24 25 26 variables, non-parametric tests (Mann-Whitney and Kruskal-Wallis) were applied. 27 28 The ROC curve analysis only identified numerical parameters associated with diagnosis and 29 30 31 cut-offs. The factors considered in the ROC analysis were: results from LFT (FVC%P; FEV 1%P; 32 33 FEV 1/FVC; RV/TLC; change in FEV 1 after bronchodilator (ml and %); results from HRCT 34 35 36 (inspiratory decreased attenuation, mosaic pattern, expiratory air trapping, bronchial wall 37 38 thickening and bronchiectasies); for atopy [(skin prick test results, eosinophils (% of total 39 40 41 cell)]; total IgE and specific IgE to D . Pteronyssinus ); inflammation (leukocytes (total number 42 43 of cells), sedimentation rate (SR), fibrinogen, C-reactive protein (CRP) and alpha-1- 44 45 46 antitrypsin (AAT) (Table 0 of the online supplement). Subsequently, in order to identify a 47 48 "3rd diagnostic group" a cluster analysis was performed (k-means clustering method, 49 50 51 SPSS®). In an initial phase two clusters were identified in order to validated this method and 52 53 followed by another cluster analysis forcing the solution of three clusters. After achieved the 54 55 56 solution of three clusters in each parameter, a cross between the final solution and 57 58 diagnosis was performed according with the following scheme (see table 1 of the online 59 60 supplement).

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1 2 3 4 5 E-Table 0: Inflammation variables for diagnosis group 6 7 OB PSA 8 9 (n=25) (n=15) p-value* 10 11 12 13 Leukocytes (cells per cubic millimeter) n 25 15 0,108 14 Missing 0 0 15 16 Mean 7,44 8,22 17 18 Median 6,58 8,02 19 20 For Peer Std.Review Deviation 2,83 1,78 21 22 Minimum 4,53 5,41 23 Maximum 14,82 11,74 24 25 26 27 Sedimentation rate (millimeters per hour) n 24 15 0,280 28 29 Missing 1 0 30 31 Mean 12,25 13,33 32 33 Median 6,50 11,00 34 Std. Deviation 14,75 10,64 35 36 Minimum 2 2 37 38 Maximum 52 35 39 40 41 42 Fibrinogen (mg/dL) n 21 13 0,710 43 44 Missing 4 2 45 Mean 321,52 314,85 46 47 Median 302,00 300,00 48 49 Std. Deviation 82,56 44,31 50 51 Minimum 227 261 52 53 Maximum 588 394 54

55 56 C-reactive-protein (mg/dL) n 25 15 0,398 57 58 Missing 0 0 59 60 Mean 0,40 0,29

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1 2 3 4 Median 0,10 0,05 5 6 Std. Deviation 0,73 0,58 7 8 Minimum 0,00 0,00 9 10 Maximum 3,00 2,20 11 12 13 14 Alpha 1 antitrypsin (mg/dL) n 24 15 0,806 15 Missing 1 0 16 17 Mean 145,83 142,80 18 19 Median 142,00 145,00 20 For Peer Review 21 Std. Deviation 22,83 27,88 22 23 Minimum 109 106 24 25 Maximum 225 203 26 27 28 29 30 31 E-Table 1: Three cluster analysis to identify the third diagnostic group (overlap syndrome) 32 33 34 OB PSA Total 35 36 Cluster 0 (nearest asthma) B A n0 37 38 39 Cluster 1 (nearest OB) D C n1 40 41 Cluster 3 (indifferent to OB and PSA / identifies the third F E n3 42 43 44 group) 45 46 47 48 49 It should be noted that: A/n0 is the positive predictive value for zero cluster for PSA. D/n1 is 50 51 the positive predictive value of a cluster for OB. After performing this analysis of 3 clusters 52 53 54 for each variable, only variables that proved simultaneously to have a good adequabilility for 55 56 identifying OB and PSA and a good appropriateness (>80%) were selected for a final cluster 57 58 59 analysis (final solution) for recognizing the third group of diagnosis (overlap) . 60

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1 2 3 4 5 For individuals correctly classified as OB or PSA in this final cluster analysis, as well as those 6 7 classified in the "3 rd diagnostic group," a comparative analysis of means and medians of the 8 9 10 variables selected for the final cluster analysis of these 3 groups were performed. With this 11 12 comparative analysis the values of central tendency that most distinguish these 3 groups 13 14 15 can be observed. Values of p<0,050 were considered to be statistically significant. 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 E-Table 2 – Anthropometric characteristics by group of diagnosis (OB, PSA) 8 9 Obliterative bronchiolitis Problematic Asthma 10 p-value 11 (n=25) (n=15) 12 Weight, z-score n 22 14 13 mean (SD) -0.54 (1.69) 0.66 (1.24) 0.028 14 median (min ; max) -0.48 (-3.41 ; 2.00) 0.89 (-1.43 ; 3.13) 15 16 Height, z-score n 22 14 17 mean (SD) -0.54 (0.92) -0.39 (0.91) 0.633 18 median (min ; max) -0.66 (-2.19 ; 1.14) -0.74 (-1.39 ; 1.50) 19 20 For Peer Review 2 21 BMI (Kg/m ) n 25 15 22 mean (SD) 20.95 (6.10) 22.41 (5.35) 0.448 23 median (min ; max) 20.55 (13.57 ; 40.14) 21.87 (15.84 ; 32.75) 24 25 BMI, z-score n 22 14 26 mean (SD) -0.34 (1.87) 0.93 (1.39) 0.036 27 median (min ; max) -0.01 (-4.26 ; 2.87) 1.70 (-1.47 ; 2.96) 28

29 30 BMI – body mass index 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 E-Table 3– Comparison of OB and PSA scores for HRCT 8 9 Obliterative Problematic 10 bronchiolitis Asthma 11 p-value 12 n=25 n=15 13 Inspiratory decreased attenuation n 25 15 14 median (min ; max) 12.0 (3.0; 23.0) 5.0 (0.0 ; 15.0) <0.001 15 16 17 Expiratory air trapping n 24 13 18 mean (SD) 12.7 (6.0) 5.2 (3.5) <0.001 19 20 For Peer Review 21 Mosaic pattern n 25 14 22 median (min ; max) 6.0 (1.0 ; 6.0) 4.0 (0.0 ; 6.0) 0.020 23 24 25 Bronchial wall thickening n 25 14 26 median (min ; max) 4.0 (0.0; 6.0) 1.5 (0.0 ; 6.0) 0.155 27 28 Bronchiectasis 29 n 25 15 30 median (min ; max) 3.0 (0.0 ; 17.0) 0.0 (0.0 ; 3.0) <0.001 31 32 33 34 35 36 37 38 39 E-Table 4 – Atopy results by group of diagnosis 40 41 42 Obliterative bronchiolitis Problematic Asthma p-value 43 n=25 n=15 44 45 Eosinophils (x10^9/L) 130 (10 ; 1570) 410 (140 ; 710) 0.004 46 median (min ; max) 47 48 Total IgE (kU/L) 33.4 (6.9 ; 3034.0) 388.0 (8.9 ; 1338.0) 0.007 49 median (min ; max) 50 51 D. pteronyssinus IgE (class) 0 (0 ; 6) 4 (0 ; 6) <0.001 52 median (min ;max) 53 54 Skin prick tests (total of mm, all tests) 55 0.00 (0 ; 22) 15.00 (0 ; 48) 0.001 56 median (min ; max) 57 58 59 60

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1 2 3 4 5 E-Table 5– Area under ROC curve (AUC) and concordance evaluation between the 2 6 clusters (cluster 1 as OB group patients and cluster 0 as PSA group patients) using 7 Kappa Cohen coefficient for Lung Function results 8 9 Kappa Cohen 10 AUC Cut-off p-value 11 coefficient 12 13 14 FVC% predicted 0.899 77.79 0.605 <0.001 15

16 FEV 1% predicted 0.899 55.00 0.641 <0.001 17 18 FEV /FVC% predicted 0.747 79.82 0.392 0.011 19 1 20 For Peer Review 21 RV%TLC 0.872 36.88 0.395 0.004 22 23 ∆∆∆FEV %t post-BD (ml) 0.800 235.00 0.333 0.012 24 1 25 26 ∆∆∆FEV 1%t post-BD (%) 0.545 ----- 0.000 >0.999 27 28 AUC – area under ROC curve 29 30 31 32 E-Table 6 – Area under ROC curve (AUC) and concordance evaluation between the 2 33 clusters (cluster 1 as OB group patients and cluster 0 as PSA group patients) using 34 35 Kappa Cohen coefficient for HR-CT findings 36 37 38 Kappa Cohen 39 AUC Cut-off p-value 40 coefficient 41 42 Inspiratory decrease 43 0,863 6,5 0,518 <0,001 attenuation 44 45 Expiratory air 46 0,856 6,5 0,480 0,001 47 trapping 48 49 Mosaic pattern 0,713 4,5 0,195 0,221 50 51 Bronchial wall 52 0,636 - 0,084 0,584 thickening 53 54 0,102 55 Bronchietasis 0,861 1,5 0,125 56 57 58 59 60

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1 2 3 4 5 6 7 E-Table 7 – Three clusters analysis. Adequability tax for LF results 8 9 Adequability for the diagnosis of Adequability for the diagnosis 10 11 OB and PSA of the 3 rd group 12 13 14 FVC% predicted 94% 87% 15

16 FEV 1% predicted 96% 66.6% 17 18 FEV /FVC% predicted 74.1 61.6 19 1 20 For Peer Review 21 RV%TLC 79.2% 37.6% 22 23 ∆FEV %t post-BD (ml) 89.5% 85.8% 24 1 25 26 ∆FEV 1%t post-BD (%) 60.7% 66.6% 27 28 29 30 31 32 33 E-Table 8– Three clusters analysis. Adequability tax for HR-CT results 34 35 36 Adequability for the diagnosis Adequability for the diagnosis of

37 rd of OB and PSA the 3 group 38 39 40 Inspiratory decreased 41 75.9% 36.4% attenuation 42 43 44 Expiratory air trapping 81.2% 76.2% 45 46 Mosaic pattern 80.8% 92.4% 47 48 49 Bronchial wall thickening 66.7% 40.0% 50 51 Bronchiectasis 58.1% 0.0% 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 E-Table 9– Three clusters analysis. Adequability tax for atopy results 8 9

10 11 Adequability for the diagnosis of Adequability for the diagnosis of

12 OB and PSA the 3 rd group 13 14 15 Skin prick tests (total of 84% 80% 16 mm, all tests) 17 18 19 Eosinophils (x10^9/L) 77.3% 77.8% 20 For Peer Review 21 Total IgE (kU/L) 68.6% 0.0% 22 23 D. pteronyssinus specific 24 88.5% 83.4% 25 IgE (class) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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