Spirometry is a conventional technique used to measure lung function in both children and adults. However, it is difficult to hold the forced expiratory flow maneuvers and to obtain an optimal measurement in early childhood even under optimal conditions. No previous studies were performed in the early years of life measuring pulmonary functions by using TBA technique in different wheezing phenotypes. This is the first study to show that airflow obstruction persisted despite appropriate treatment in children with multiple trigger wheeze.

Wheezing is the clinical sign of partial obstruction caused by turbulent flow and vibration in airways narrowed due to inflammation, mucosal edema and bronchospasm [1]. However, the pathophysiology of wheezing is unclear [2]. The prevalence of wheezing in children under five has increased in recent years [3] and currently stands at 15%-32% [4]. Although the majority of children outgrow their symptoms, infants who suffered from wheezing in early childhood are at increased risk of continued wheezing episodes (recurrent wheeze) [5-8]. In 2008, the European Respiratory Society (ERS) Task Force on preschool wheeze, proposed a classification for preschool wheeze based on symptom patterns of episodic viral wheeze (EVW) and multiple trigger wheeze (MTW) [9]. EVW is defined as wheeze triggered by viruses, and refers to children who wheeze only during respiratory infections. In this phenotype, symptoms are expected to improve significantly over time, and the risk of developing asthma and responding to asthma medications is lower [10]. MTW is defined as wheeze between respiratory infections in response to a variety of triggers such as physical exercise, laughing or crying, irritants, and allergens. In this phenotype, symptoms tend to be prolonged, response to asthma medications is lower, and these patients are considered to have "probable asthma" [9]. Additionally, MTW is more commonly associated with lung function abnormalities [11]. The EVW / MTW classification system is useful in terms of simplifying the tasc of the clinician, although transition between the phenotypes with distinct but often overlapping characteristics may occur over time [12].

However, in a study involving two different cohorts, Spycher reported that these phenotypes exhibited a tendency to remain the same, with: MTW, and to a lesser extent EVW, tending to persist regardless of wheeze severity [13]. Measuring respiratory functions from younger ages and determining risk factors for recurrent wheeze in children is important for (i) recognizing early changes linked to persistent respiratory dysfunction, (ii) understanding the pathophysiology of airways and lung parenchyma damage, and (iii) evaluating preventive measures against future lung function deterioration. Few studies to date have investigated lung function in children with recurrent wheeze and the difference between its phenotypes using different measurement methods. The results are inconsistent, some authors reported a lower lung capacity and airflow, while others observed no difference in terms of baseline pulmonary functions and bronchial hyper reactivity in wheezy children compared with healthy controls [14,15]. Spirometry is a conventional technique used to measure lung function in both children and adults. However, it is difficult to hold the forced expiratory flow maneuvers and to obtain an optimal measurement in early childhood even under optimal conditions. The development of devices capable of measuring lung volumes and airflow during tidal breathing has therefore gained increasing importance for this particular population. Tidal breath analysis (TBA) is an easily applicable standardized technique for measuring bronchial airflow and tidal vital capacity for young children [16]. This technique provides practical and replicable assessment of lung function during spontaneous quiet tidal breathing. In addition, it is non-invasive, does not require sedation, and can be applied quickly in outpatient or bedside settings [17]. No previous studies have investigated potential differences in lung function between different wheezing phenotypes in the early years of life. This study was based on the hypothesis of a difference in lung function measures between the two wheezing phenotypes of EVW and MTW in young children. The study aim was therefore to investigate lung functions in children with recurrent wheeze using TBA and to compare pre-and-post treatment lung functions between the wheezing phenotypes of EVW and MTW.

Study Patients

This retrospective cohort study was conducted at the pediatric allergy outpatient clinic of a tertiary referral hospital in Turkey between October 2019 and March 2020. Eighty children under three years of age, 44 with treatment-naive recently physician-diagnosed wheezing and 36 healthy controls were enrolled in the study. We recruited children aged under three years with recurrent wheezing, a history of hospitalization for the first wheezing episode before six months of age, and a medical history for at least the previous six months. The participants initially presented to the general pediatric outpatient clinic and were subsequently referred to the allergy clinic for further evaluation. Children with recurrent wheeze were assigned into two wheezing phenotype groups: EVW (n=19) and MTW (n=25), based on temporal wheeze patterns. The control group was recruited from age-and-sex matched healthy children admitted to the general pediatric outpatient clinic. In order to be eligible for inclusion, children in both groups were required to be non-atopic and to correctly perform lung function tests as recommended by the American Thoracic Society (ATS)/ERS task force [16].

Definitions

Recurrent wheeze was defined as ≥3 episodes of medically diagnosed wheeze [15].

EVW was defined as wheeze occuring in discrete episodes, often in association with clinical evidence of a viral cold with the child being healthy between episodes [10].

MTW was defined as wheeze triggered by infections and other causes with the child exhibiting symptoms between episodes [9].

Exclusion Criteria

Chilren with prematurity (≤37 weeks), presence of any chronic disease (bronchopulmonary dysplasia, chest wall abnormality, gastroesophageal reflux diseases, neuromuscular diseases, congenital cardiovascular diseases, immunodeficiency, asthma, adenotonsillar hypertrophy, or other respiratory diseases, etc.), or acute respiratory infections within the previous two weeks, use of any medications for respiratory tract diseases, exposure to smoking, and inability to perform lung function tests were excluded from the patient group. Children who were born prematurely (≤37 weeks), with a history of any respiratory or other chronic diseases or atopy, with acute respiratory infections in the previous two weeks or prior history of wheeze, using systemic corticosteroid and/or bronchodilators, and expose to passive smoking were excluded from the control group.

Study Design

Demographic characteristics including the patient’s age, gender, age at first wheezing episode, number and potential causes of wheezing episodes, parental atopy history (asthma, allergic rhinitis, atopic dermatitis, or food allergy), number of individuals in the household, domestic pets, and, consanguinity between parents were recorded retrospectively from the medical charts. Following initial evaluation with detailed clinical history, patients underwent physical examination, and their anthropometric characteristics (height and weight) were measured. Lung function measurements were performed on children with recurrent wheeze at baseline (first visit), and after three months of appropriate treatment (second visit), with leukotriene receptor antagonists in children with EVW or with low-dose inhaled corticosteroid in children with MTW as recommended in the relevant guideline [18]. Complete blood count findings including lymphocyte, neutrophil, eosinophil counts and, serum immunoglobulin (Ig) G, IgA, IgM and serum total IgE values were measured at baseline (first visit) and retrieved from patients’ medical records.

Tidal Breath Analysis Measurement

TBA measurement was performed in line with the recommendations of the ERS/ATS Task Force on standards for infant respiratory function testing. The sampling rate was 200 Hz as recommended for analysis of tidal breathing flow/volume loop and other sensitive parameters such as tPTEF/tE [16]. The details of TBA measurements are available as a supplementary file. All lung function measurements were performed under the supervision of the same highly- experienced nurse and physician in the pediatric allergy department lung function laboratory. In case of children with recurrent wheezing, all lung function measurements were performed during an asymptomatic period of the disease within a month from the last wheezing episode before and after treatment. The TBA equipment was connected to a pediatric respiratory function device (Jaeger / Viasys Master Screen PAED; YorbaLinda, CA, USA) by a commercially available standardized portable apparatus (Erich Jaeger GmbH, Bavaria, Marktrendwitz, Germany). The current was measured using a heated pneumotachograph providing an airflow of 0-10 L /min (Hans Rudolph Inc., KS, and USA). A transparent, latex-free, silicone-capped face mask with a low dead space was placed over the patient’s nose and mouth for breathing measurements (Rendell Baker, Soucek, Rusch UK Ltd., Bucks, and UK). The mask was tightly connected to the pneumotachograph, and the dead space was kept to a minimum. The amount of dead space was 1.66 mL in the pneumotachograph, 2.4mL in the system, and 11-14 mL in the mask, all these values being standardized. The system was checked for leakage after the transparent facemask (RendellBaker, Soucek) had been placed on the patient’s face [19,20]. Prior to each recording, the instrument was calibrated by the practitioner using a 100 mL syringe. Tidal breath measurement was initiated when the most calm, regular breathing rhythm was observed, and was stopped when movement in the body, hiccups, and impaired breathing rhythm were detected. Chloral hydrate or triclofos sodium solutions were not used for sleep induction.

A period of 2-3 minutes was allowed to elapse for adaptation to the face mask. Measurement commenced once the child’s breathing rhythm was regular. Depending on the variability of the breathing pattern, at least 60 inspiratory and expiratory breath cycles, each with a variability of less than 10%, were measured as the current-volume curve was observed on the screen. This was regarded as an epox. Respiratory patterns in which at least 20 regular respiratory cycles of each epox cycle were detected and exhibiting the best values were selected. A minimum of three epoxes was measured over no less than five minutes. The values of all epoxes were recorded, and the average value was calculated. Current volume measurement was performed using the Care Fusion pnemotact method. Volume drift was corrected by adjusting the mask properly to the face in case of a leakage. However, that infant was excluded from the study if the leak was persistent and monitoring breath patterns could not be observed, as recommended in the guideline [16]. Measured parameters included: peak tidal expiratory flow (PTEF), expiratory time (tE), the time taken to achieve peak tidal expiratory flow as a proportion of total expiratory time (tPTEF: tE), inspiratory time (tI), expiratory time (tE), tidal volume (VT), volume expired before PTEF was attained (VPTEF), total expiratory volume (VE), the volume until peak tidal expiratory flow to total expiratory volume (VPTEF: VE) and respiratory rate (RR). All parameters were calculated automatically by the TBA device computer. Environmental conditions were monitored by a thermo-hygrometer with relative humidity maintained at 30-40% and a temperature of 20-24?C.

Approval for the study was obtained from the local ethics committee (Date: 17.12.2020, protocol number: 2020/232).

Statistical Analyses

Statistical analyses were performed on SPSS 21 software (IBM Corporation, Armonk, NY, USA). Comparisons of qualitative data were performed using the chi-square test or Fisher’s exact test, while quantitative variables were compared between the study groups using either the Mann-Whitney U test or Student’s t test. Significant differences in TBA parameters between paired data before and after treatment were calculated using the paired samples -t-test or the Wilcoxon signed-rank test. A p-value <0.05 was considered statistically significant.

Ninety-one participants met the inclusion criteria and were initially enrolled in the study. However, 11 were subsequently excluded due to technically inconsistent results (six children with air leaks from the facemask and five with altered behavioral state such as crying and hiccups). The baseline demographics and the clinical features (physical examination and respiratory rate) of the patients who were unable to complete the study were not different from those of the patients included into the study. Forty-four children with recurrent wheeze (19 with EVW and 25 with MTW) and 36 healthy age-and-sex matched children were thus finally included in the study. Demographical data for children with recurrent wheeze and healthy children are presented in Table 1.

Table 1: The demographic characteristics and laboratory parameters of the participants.

Abbreviations: IQR: interquartile range, IU: international unit, mL: milliliter, µL: microliter, SD: standard deviation, ^aParametric data analysis was held by Student t test. Non-parametric data analysis was held by Mann Whitney U test. Categorical data analysis was held by Chi-square test. The significance value was accepted as p<0.05.

There was no difference between the two groups in terms of the demographic characteristics of gender or age (p>0.05). No difference was also determined between the genders in terms of any TBA parameters (p>0.05) (data not shown).

A comparison of TBA parameters between children with recurrent wheeze and healthy children is shown in Table 2. The parameters of tPTEF: tE, VPTEF: VE and VT/kg ratios were lower, while RR levels were higher in children with recurrent wheeze compared to the healthy controls (p<0.05).

Table 2: The comparison of tidal breath analysis parameters between children with recurrent wheeze and healthy children.

Abbreviations: IQR: interquartile range, SD: Standard deviation, aParametric data analysis was held by Student t test. Non-parametric data analysis was held by Mann Whitney U test. The significance value was accepted as p<0.05.

There was no difference between children with EVW and children with MTW in terms of the demographic and clinical characteristics (p>0.05). A comparison of baseline TBA parameters (measured at the first visit) between children with EVW and children with MTW is shown in Table 3. The parameters of tPTEF: tE, VPTEF: VE, tI/tE, and VT/kg ratios were lower in children with MTW compared to EVW, while minute ventilation and RR levels were higher, although the differences between the two groups were not statistically significant (p>0.05).

Table 3: Comparison of tidal breath analysis parameters between children with episodic wheeze and children with multiple trigger wheeze.

Abbreviations: IQR: interquartile range, SD: Standard deviation, ^aParametric data analysis was held by Student t test. Non-parametric data analysis was held by Mann Whitney U test. The significance value was accepted as p<0.05.

A comparison of baseline TBA parameters (measured at the first visit) between children with ETW and the healthy controls is shown in Table 4. The VPTEF: VE ratio was lower, while RR levels were higher in children with EVW compared to the healthy controls (p=0.004, and p=0.003, respectively).

A comparison of baseline TBA parameters (measured at the first visit) between children with MTW and the healthy controls is shown in Table 4.

Table 4: Comparison of tidal breath analysis parameters between children with both episodic wheeze and children with multiple trigger wheeze and healthy children.

Abbreviations: IQR: interquartile range, SD: Standard deviation, ^aParametric data analysis was held by Student t test. Non-parametric data analysis was held by Mann Whitney U test. The significance value was accepted as p<0.05.

The tPTEF: tE and VPTEF: VE ratios were lower, while RR levels were higher, in children with MTW compared to the healthy controls (p=0.043, p=0.016 and p=0.044, respectively).

A comparison of TBA parameters before and after treatment (first and second visits) in children with EVW and MTW is shown in Table 5. Minute ventilation and the VT/kg ratio increased significantly (p=0.011 and p=0.024, respectively) while tPTEF:tE, VPTEF:VE, and tI/tE ratios increased insignificantly, after treatment, in children with EVW (p>0.05). The VT/kg ratio was increased (p=0.001) while RR levels decreased (p=0.002), after treatment in children with MTW.

Table 5: Comparison of tidal breath analysis parameters between first visit and second visit both in children with episodic wheeze and children with multiple trigger wheeze.

Abbreviations: IQR: interquartile range, SD: Standard deviation, ^aParametric data analysis was held by paired sample t test. Non-parametric data analysis was held by Wilcoxon test. The significance value was accepted as p<0.05.

A comparison of post treatment TBA parameters after treatment (measured at the second visit) between children with EVW and the healthy controls is shown in Table 6. No difference in terms of TBA parameters was observed the between two groups (p>0.05), although minute ventilation was higher in children with EVW (p=0.026). A comparison of post-treatment TBA parameters between children with MTW and the healthy controls is shown in Table 6. tPTEF: tE ratio values were lower in children with MTW compared to the healthy controls (p=0.039).

Table 6: Comparison of second visit tidal breath analysis parameters between children with episodic wheeze or children with multiple trigger wheeze and healthy controls.

Abbreviations: IQR: interquartile range, SD: Standard deviation, ^aParametric data analysis was held by Student t test. Non-parametric data analysis was held by Mann Whitney U test. The significance value was accepted as p<0.05.

None of the TBA parameters was correlated with duration of the disease or recurrence of wheezing episodes (p>0.05).

The present research is one of the rare studies using TBA to investigate lung function in young children with recurrent wheeze and assessing pulmonary functions between different wheezing phenotypes EVW and MTW. The first finding of the present study was a lower tidal volume (lower VPTEF: VE and VT/kg) and airflow (lower tPTEF: tE) in children with recurrent wheeze compared to healthy children. Consistent with our findings, Dezateux reported a lower tPTEF: tE ratio in asymptomatic infants over three months of age with a prior history of wheezing, compared to healthy infants [21]. Several studies have demonstrated an association between lower lung function parameters and subsequent asthma development at older ages [5,22-25]. No difference was observed in the present study between EVW and MTW in the measurement of basal lung functions. RR was higher in both patient groups compared with the healthy children. In addition, VPTEF: VE was low in children with EVW and MTW, and tPTEF: tE in children with MTW. These results show that the baseline measurements of both groups differed from those of the healthy children, but no significant difference was determined between the two phenotypes. Interestingly, VT/kg was the main parameter improving after treatment compared to baseline values in both phenotypes EVW and MTW. However, post-treatment tPTEF: tE values remained lower than in the healthy controls in the MTW phenotype, but not in EVW. To summarize, lung function parameters reflecting airway obstruction remained low in children with MTW but returned to normal in children with EVW after three months of appropriate treatment. We suggest that persistence of bronchoconstriction reflected by a lower tPTEF: tE in children with MTW may be related to early structural or functional airway changes, basement membrane thickness, alveolar air trapping, and a gradual tendency to progression to infantile asthma. This may contribute to MTW exhibiting a more complex phenotype than that of EVW. Lack of sufficient response to an appropriate dosage and duration of inhaled corticosteroids due to unknown underlying mechanisms may therefore represent the main reason why children with MTW are more likely to develop asthma in subsequent years than those with EVW. However, this conclusion could be supported with longitudinal analysis by following up the patients into childhood.

These results are consistent with our previous report showing that airway flow and tidal volume (tPTEF, tPTEF: tE, and, VT/kg) remained significantly lower even on the 30 th day of recovery in infants hospitalized due to acute bronchiolitis compared to healthy controls [26]. Similarly to our findings, Qi et al. determined lower tPTEF: tE and VPTEF: VE ratios after clinical recovery in wheezy infants than in non-wheezing infants [27]. Martinez et al. demonstrated that infants with a lower tPTEF: tE ratio measured before 13 weeks of age, are at significantly greater risk of developing lower respiratory illnesses with wheezing by three years of age [5]. Various studies have shown that asthma-like remodeling in patients with MTW begins to develop at preschool age [28-30]. One such study observed a high basal membrane thickness compatible with eosinophilic inflammation in bronchial biopsy materials from pre-school children with severe wheezing [30]. Another study reported similar basal membrane thickness and eosinophil counts in bronchial biopsy materials between MTW and asthma in non-atopic pre-school children, but that these values were much higher than those of healthy controls [28]. The particular strengths of the present study are that lung functions were examined using non-invasive methods under observation by the same highly-experienced nurses and physician in all cases. All patients were also diagnosed by the same pediatric allergy specialist. Recently diagnosed treatment-naïve children with recurrent wheeze were included in the study in order to eliminate the potential improving effect of medications on lung function. Measurement of lung function at baseline and after treatment permitted the assessment of persistent airway obstruction and treatment response.

However, there are also some limitations to this study. Although the low number of patients is insufficient for a clear interpretation, our results are interesting since this is the only study to date on this subject. In particular, the research findings should be interpreted with caution since they cannot be used to infer causality due to the study’s retrospective design. We were also unable to determine viral agents or to follow-up our patients over an extended period.

This is the first study assessing pulmonary functions using TBA in children with different phenotypes o recurrent wheeze. Although our data are not capable of identifying whether an individual patient has EVW or MTW, TBA seems to be useful in the measurement of lung function in children under three years of age, since spirometry cannot be used in this particular population. Our findings have important implications for the interpretation of respiratory functions; (i) lung functions of children with recurrent wheeze were significantly lower than those of sex-and age-matched healthy controls, (ii) significant differences in lung function parameters were detected between the EVW and MTW groups, (iii) airflow remained low even after three months of appropriate therapy in children with MTW, but not in children with EVW, and (iv) measurements of lung function using TBA appears to be a useful, effective and safe method for a better understanding of respiratory dynamics in young children with recurrent wheeze. Due to the potential for interchange over time between the two wheezing phenotypes and the speculative prospective clinical utility of phenotyping, measurement of lung function in children with recurrent wheeze may facilitate the targeting of children who are most likely to benefit from treatment intervention, or whose symptoms are likely to be persistent. Our findings now need to be replicated in longitudinal prospective studies.

The authors are indebted to specialist nurse Nuray Ayd?n for her skills in lung function measurements.

Statement of Ethics: Approval for the study was obtained from the local ethics committee (Date: 17.12.2002, protocol number: 2020/232).

Conflict Of Interest: Elif Celik and P?nar Uysal have no conflicts of interest to declare

Funding Source: None.

All authors contributed to the study

Elif Celik: Conceptualization (Equal), Data curation (Lead), Formal analysis (Lead), Funding acquisition (Equal), Investigation (Equal), Methodology (Lead), Project administration (Lead), Resources (Equal), Software (Supporting), Validation (Supporting), Visualization (Supporting), Writing-original draft (Lead), Writing-review & editing (Equal).

Pinar Uysal: Conceptualization (Equal), Data curation (Supporting), Formal analysis (Supporting), Funding acquisition (Equal), Investigation (Equal), Methodology (Supporting), Project administration (Supporting), Resources (Equal), Software (Lead), Supervision (Lead), Validation (Lead), Visualization (Lead), Writing-original draft (Supporting), Writing-review & editing (Equal)

Abbreviations: Peak tidal expiratory flow, (PTEF); Expiratory time, (tE); The time taken to achieve peak tidal expiratory flow as a proportion of total expiratory time, (tPTEF: tE); Inspiratory time, (tI); Expiratory time, (tE); Tidal volume, (VT); Volume expired before PTEF was attained, (VPTEF); Total expiratory volume, (VE); The volume until peak tidal expiratory flow to total expiratory volume, (VPTEF:VE); Respiratory rate, (RR); kg, Kilogram.

1. Kliegman RM, Behrman RE, Jenson HB, Stanton BM. Nelson textbook of pediatrics e-book. Elsevier Health Sciences. 2007.
2. Frey U, Makkonen K, Wellman T, Beardsmore C, Silverman M. Alterations in airway wall properties in infants with a history of wheezing disorders. Am J Respir Crit Care Med. 2000; 161: 1825-1829.
3. Kuehni CE, Davis A, Brooke AM, Silverman M. Are all wheezing disorders in very young (preschool) children increasing in prevalence?. The Lancet. 2001; 357: 1821-1825.
4. Wilson N. The significance of early wheezing. Clin Exp Allergy. 1994; 24: 522-529.
5. Martinez FD, Morgan WJ, Wright AL, Holberg CJ, Taussig LM, Personnel GHMA. Diminished lung function as a predisposing factor for wheezing respiratory illness in infants. N Engl J Med. 1988; 319: 1112-1117.
6. Hancox RJ, Subbarao P, Sears MR. Relevance of birth cohorts to assessment of asthma persistence. Current allergy and asthma Reports. 2012; 12: 175-184.
7. Taussig LM, Wright AL, Holberg CJ, Halonen M, Morgan WJ, Martinez FD. Tucson children's respiratory study: 1980 to present. J Allergy Clin Immunol. 2003; 111: 661-675.
8. De Sario M, Di Domenicantonio R, Corbo G, Forastiere F, Pistelli R, Rusconi F, et al. Characteristics of early transient, persistent, and late onset wheezers at 9 to 11 years of age. J Asthma: Official J Association for the Care of Asthma. 2006; 43: 633-638.
9. Brand PL, Baraldi E, Bisgaard H, Boner AL, Castro-Rodriguez JA, Custovic A, et al. Definition, assessment and treatment of wheezing disorders in preschool children: an evidence-based approach. Eur Respir J. 2008; 32: 1096-1110.
10. Wassall HJ, Devenny AM, Daud Khan S, Ninan TK, Russell G. A comparison of virus-associated and multi-trigger wheeze in school children. J Asthma. 2005; 42: 737-744.
11. Sonnappa S, Bastardo CM, Wade A, McKenzie SA, Bush A, Aurora P, et al. Symptom-pattern phenotype and pulmonary function in preschool wheezers. J Allergy Clin Immunol. 2010; 126: 519-526.
12. Schultz A. Is it time to move on from episodic viral wheeze and multiple trigger wheeze?. Pediatr Pulmonol. 2019; 54: 1499-1500.
13. Spycher BD, Cochrane C, Granell R, Sterne JAC, Silverman M, Pedersen E, et al. Temporal stability of multi trigger and episodic viral wheeze in early childhood. Eur Respir J. 2017; 50.
14. Stick SM, Arnott J, Turner DJ, Young S, Landau LI, Lesouef PN. Bronchial responsiveness and lung function in recurrently wheezy Infants. Am Rev Respir Dis. 1991; 144: 1012-1015.
15. Borrego LM, Stocks J, Leiria-Pinto P, Peralta I, Romeira AM, Neuparth N, et al. Lung function and clinical risk factors for asthma in infants and young children with recurrent wheeze. Thorax. 2009; 64: 203-209.
16. Bates J, Schmalisch G, Filbrun D, Stocks J. Tidal breath analysis for infant pulmonary function testing. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J. 2000; 16: 1180-1192.
17. Emralino F, Steele AM. Effects of technique and analytic conditions on tidal breathing flow volume loops in term neonates. Pediatr Pulmonol. 1997; 24: 86-92.
18. Brand PL, Caudri D, Eber E, Gaillard EA, Garcia-Marcos L, Hedlin G, et al. Classification and pharmacological treatment of preschool wheezing: changes since 2008. Eur Respir J. 2014; 43: 1172-1177.
19. Frey U, Stocks J, Coates A, Sly P, Bates J. Specifications for equipment used for infant pulmonary function testing. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J. 2000; 16: 731-740.
20. Frey U, Stocks J, Sly P, Bates J. Specification for signal processing and data handling used for infant pulmonary function testing. ERS/ATS Task Force on standards for infant respiratory function testing. European Respiratory Society/American Thoracic Society. Eur Respir J. 2000; 16: 1016-1022.
21. Dezateux C, Stocks J, Dundas I, Jackson E, Fletcher M. The relationship between tPTEF: tE and specific airway conductance in infancy. Pediatr Pulmonol. 1994; 18: 299-307.
22. Young S, Arnott J, Le Souef PN, Landau LI. Flow limitation during tidal expiration in symptom-free infants and the subsequent development of asthma. J Pediatr. 1994; 124: 681-688.
23. Riikonen R, Lauhkonen E, Tormanen S, Backman K, Koponen P, Helminen M, et al. Prospective study confirms that bronchiolitis in early infancy increases the risk of reduced lung function at 10–13 years of age. Acta Paediatr. 2019; 108: 124-130.
24. Haland G, Carlsen KCL, Sandvik L, Devulapalli CS, Munthe-Kaas MC, Pettersen M, et al. Reduced lung function at birth and the risk of asthma at 10 years of age. N Engl J Med. 2006; 355: 1682-1689.
25. Martinez FD, Morgan WJ, Wright AL, Holberg C, Taussig LM, Associates GHM. Initial airway function is a risk factor for recurrent wheezing respiratory illnesses during the first three years of life. Am Rev Respir Dis. 1991; 143: 312-316.
26. Celik E, Uysal P. Pulmonary function testing with tidal breath analyze technique is useful in predicting persistant small airway damage in infants with acute bronchiolitis. Pediatr Allergy Immunol. 2020; 32: 60-66.
27. Qi Y-Y, Jiang G-L, Wang L-B, Wan C-Z, Zhang X-B, Qian L-L. Lung function in wheezing infants after acute lower respiratory tract infection and its association with respiratory outcome. Chin Med J. 2017; 130: 4-10.
28. Turato G, Barbato A, Baraldo S, Zanin ME, Bazzan E, Lokar-Oliani K, et al. Nonatopic children with multitrigger wheezing have airway pathology comparable to atopic asthma. Am J Respir Crit Care Med. 2008; 178: 476-482.
29. Cokugra? H, Akcakaya N, Camc?oglu Y, Sar?murat N, Aksoy F. Ultra structural examination of bronchial biopsy specimens from children with moderate asthma. Thorax. 2001; 56: 25-29.
30. Saglani S, Payne DN, Zhu J, Wang Z, Nicholson AG, Bush A, et al. Early detection of airway wall remodeling and eosinophilic inflammation in preschool wheezers. Am J Respir Critical Care Med. 2007; 176: 858-864.