Longitudinal tracking of intrabreath respiratory impedance in preschool children

Longitudinal measurements of intrabreath respiratory impedance (Zrs) in preschool‐aged children may be able to distinguish abnormal lung function trajectories in children with a history of wheezing compared to healthy ones.

Increasing height had a significant effect on all IB-OSC resistance and reactance variables when adjusted for the effect of preschool wheezing.
Conclusions: IB-OSC is feasible for tracking lung function growth in preschool-aged children and may allow abnormal lung function to be identified early in asymptomatic preschoolers with a history of persistent wheezing.

| INTRODUCTION
Longitudinal cohort studies have identified lower lung function, airway obstruction in early life, and poor lung function growth as risk factors for developing persistent asthma [1][2][3] and later chronic obstructive pulmonary disease. 4,5Spirometry and plethysmography in healthy school-aged children have shown lung function increases in an ordered manner correlating with linear growth. 6,7Epidemiological trajectory modeling techniques have identified several childhood risk factors for a low trajectory of the forced expiratory volume in 1-second (FEV 1 ), including early wheezing, asthma, atopy, early childhood lower respiratory tract infections, and tobacco smoke exposure. 8,91][12][13] First, the reliability and reproducibility of forced spirometry measurements in preschool-aged children are highly variable. 14Second, the lungs of healthy children under 6-yearold are essentially empty within 1-second during a forced expiration manoeuvre, questioning the validity of FEV 1 as an outcome variable in this age group. 15Third, small airway disease is poorly reflected by spirometry. 16Fourth, infants with low maximal flow at functional residual capacity (V' maxFRC ) using the RTC technique have been found to have average or above-average FEV 1 trajectories. 17It would be ideal to measure lung function trajectories in young children using an identical technique rather than comparing V' maxFRC and FEV 1 to confirm the hypothesis that this is secondary to catch-up lung function growth.
Respiratory oscillometry is a noninvasive lung function technique that measures the impedance of the respiratory system (Zrs) during tidal breathing by superimposing periodic variations to transrespiratory pressure and measuring the response in oscillatory gas flow. 18thin Zrs is biomechanical information about the resistance (Rrs) and reactance (Xrs) of the respiratory system where Rrs reflects airway and tissue resistive properties and Xrs reflects the elastic and inertive properties. 18Oscillometry is highly feasible in preschoolers. 19Previous oscillometry techniques-such as those using pseudorandom noise oscillations (spectral oscillometry) or recurrent impulses (impulse oscillometry)-average Zrs measurements across the entire respiratory cycle and are better suited to show between-group differences than to detect abnormal lung function in individual children. 20,21Intrabreath oscillometry (IB-OSC) is a recent variant of oscillometry using a single-frequency sinusoidal oscillation to estimate Rrs and Xrs at discrete time points during the breathing cycle.By measuring Rrs and Xrs when the gas flow (V') is zero (e.g., at moments of end-expiration and end-inspiration), the involvement of upper airway nonlinearities in Zrs can be minimized. 22The volume dependence of Rrs (ΔR) is calculated by measuring the difference between Rrs at end-expiration (ReE) and end-inspiration (ReI) and was shown by our group to detect airway obstruction in acutely wheezy children with a sensitivity of 92% and specificity of 89%. 21 aimed to assess the longitudinal changes in intrabreath Zrs in a cohort of clinically stable (i.e., asymptomatic) preschool-aged children.We hypothesized that the lung function growth trajectory in preschoolers with a history of wheezing would differ from that of healthy preschoolers who had never wheezed.We also hypothesized that the lung function growth trajectory in preschoolers with transient wheezing would be more like that of healthy preschoolers than those with persistent wheezing.

| Study subjects
This study was an extension of the Observational Research in Childhood Infectious Diseases (ORChID) community-based birth cohort study. 23 25 The methodology of the wave-tube has been described previously, 26 and the same wave-tube assembly was used for all study visits up to December 2017.A single tremoflo ® C-100, equiped with investigational software to allow measurement of IB-OSC using a 10 Hz sine wave, was used to measure lung function for all study visits from January 2018 onward.The Zrs values are comparable for identical mechanical test loads between the INCIRCLE assembly and the tremoflo ® C-100. 27single 10 Hz sinusoid was used in 16-20 s measurements to track intrabreath changes in Zrs during tidal breathing.Test occasions containing at least three technically acceptable trials with a minimum of five breathing cycles containing no evidence of signal artefact from movement, coughing, vocalization, glottic closure (e.g., swallowing), breath-holding, or leak from mouthpiece were included.All included recordings were analyzed using the software of the custom-made oscillometer.Zrs variables determined by IB-OSC included resistance at end-expiration (ReE), resistance at end-inspiration (ReI), the tidal change in resistance (ΔR=ReE-ReI), reactance at end-expiration (XeE), reactance at end-inspiration (XeI), the tidal change in reactance (ΔX=XeE-XeI), and ΔX normalized by tidal volume (ΔX/V T ).Individual breaths from each measurement trial were analysed, and variables were reported as the average of all technically acceptable measurements.

| Atopy
Atopy was defined by a positive skin prick test (≥3 mm wheal and greater than the positive histamine control) to at least one aeroallergen including cat, dog, house dust mite (Dermatophagoides pteronyssinus or Dermatophagoides farinae), mixed grasses, Bahia grass, Alternaria tenuis, and Aspergillus fumigatus performed at the final visit at 7-year of age.

| Wheeze groups
Wheeze groups were defined based on the wheezing phenotypes described in the Tucson Children's Respiratory Study. 10 Subjects were divided into four groups based on parental reports.At each visit, parents were asked, "Has your child ever had wheezing or whistling in the chest?" and "Has your child had wheezing or whistling in the chest in the past 12 months?"Based on responses, subjects were placed in one of four wheeze groups: never wheezed, transient wheeze, late-onset wheeze, or persistent wheeze (Supporting Information S1: Table 1).Only one child met the definition of late-onset wheezing and was combined into the persistent wheezing group to form three wheeze groups for analysis.

| Statistical analyses
Data are summarized as median (quartile-1, quartile-3) for continuous variables and as frequency and proportions for categorical variables.
The coefficients of variability (CV) for ReE, ReI, XeE, and XeI were calculated for each visit according to the wheeze group.Normality was checked by conducting the Shapiro-Wilks test and visual inspection of histograms.
The association between wheeze groups and differences in IB-OSC outcomes over time was investigated using linear mixed-effects models (LMMs).In all models, subjects were included as random effects to model between-subject variation.First, a null model was built for each IB-OSC outcome with no fixed effects.Then, LMMs were fitted for each IB-OSC outcome with the wheeze group and height as fixed effects.LMMs were fitted for each IB-OSC outcome with sex and height as fixed effects for the entire cohort and with inhaled corticosteroids (ICS) and wheeze group as fixed effects for only subjects with a history of wheezing.A sensitivity analysis was performed separately for subjects who never wheezed and those with transient or persistent wheezing.We compared interaction models (height-by-wheeze group, height-by-sex, and ICS-by-wheeze group) and noninteraction models for each outcome.We selected the most parsimonious model using the Akaike Information Criterion (AIC) (i.e., smallest AIC). 28Where appropriate, post hoc analysis was performed for pairwise comparison between visits and wheeze groups using Bonferroni correction.All analyses were conducted using R statistical software (R Core Team, 2022) and the lmerTest package. 29
The changes in intrabreath Zrs with growth were explored further using models containing (1) subjects as random-effect and two predictors, height and wheeze group, as fixed-effects, (2)   subjects as random-effect and height and sex as fixed-effects, and (3) subjects as random-effect and inhaled corticosteroids and wheeze group as fixed-effects are presented below.

| Association between subjects and longitudinal intrabreath Zrs
The results of the null models for each IB-OSC outcome are summarized in Supporting Information S1: Table 5.Our final models with the height and wheeze group as fixed-effects indicate that in the height and wheeze group, 45.2% of the variance in ReE (intraclass correlation coefficient [ICC] = 0.452), 30.2% of the variance in ReI, and 14.1% of the variance in ΔR is explained by between-subject differences (Supporting Information S1: Table 6).The ICC for reactance outcomes indicates that 26.8% of the variance in XeE, 14.9% of the variance in XeI, 3.7% of the variance in ΔX, and 9.6% of the variance in ΔX/V T is explained by intersubject differences.

| Association between wheezing and longitudinal intrabreath Zrs
After adjusting for height, the effect of wheezing on longitudinal intra-breath tracking of Zrs was statistically significant for ReE, XeE, ΔX, and ΔX/V T (Table 2).The subjects with transient wheezing, compared with subjects who never wheezed, had worse   3).Inhaled corticosteroids had no significant effect on the longitudinal changes of any IB-OSC variable after adjusting for the wheezing phenotype.

| Association between sex and longitudinal intrabreath Zrs
When adjusting for the effect of height, there was no statistically significant effect of sex on the longitudinal changes of intra-breath Zrs or tidal volumes for the entire cohort (Supporting Information S1: Tables 8 and 9) or for subjects who never wheezed (Supporting Information S1: Table 10).

| DISCUSSION
Increasing height between the ages of 3-7 caused a reduction in ReE and ReI values and a decrease in the magnitude (less negative) of XeE, XeI, and ΔX/V T values.With linear growth, there were small but significant changes in both ΔR (more positive) and ΔX (more negative).
T A B L E 2 Association of height and wheezing history with intrabreath impedance (n = 374 observations from 85 children).| 1889 When adjusting for the effect of height, persistent wheezers demonstrated a worse trajectory for ReE (more positive over time) and XeE, ΔX, and ΔX/V T (more negative over time) than subjects who never wheezed.The trajectory of ΔX across the preschool years was also worse (more negative) in children with transient wheezing.
Our results are consistent with the height dependence of ReE, ReI, ΔR, XeE, XeI, and ΔX in healthy African children reported by Chaya et al. 30 Rrs represents the pressure changes in-phase with gas flow (V') and reflects the sum of resistance from lung parenchyma and larger airways, where gas molecules move by bulk flow. 22In larger airways, Rrs due to laminar flow is inversely related to the radius of the airway to the fourth power (Poiseuille law). 31Our observed trend of decreasing ReE and ReI with growth was expected as airway calibre increases by 200%-300% from birth to adulthood. 32though ReE and ReI follow a similarly negative trajectory, these trajectories are not parallel and result in a small but significant increase in ΔR with preschool growth.The more gradual trajectory of ReE with height in this age group may reflect peripheral airway resistance making a greater contribution to the overall Rrs until the age of 5-year when conductance increases more rapidly. 33,34e observed trend of improving (less negative) XeE and XeI with height was also expected, as increasing lung volumes with growth would reduce the respiratory system elastance, which dominates Xrs at frequencies below the resonant frequency.Additionally, both ΔR and ΔX represent the volume dependence of Rrs and Xrs and may change in magnitude with larger tidal volumes in taller, healthy children (Supporting Information S1: Table 10).
Czӧvek and colleagues found ΔR to be highly sensitive and specific for acute airway obstruction in preschool-aged children and distinguished those with recurrent wheezing from healthy children. 21 our data, ΔR was increased in transient and persistent wheezers, but changes were not statistically significant (Table 2).This may indicate that, with only 15 persistent wheezers, the study was underpowered to detect a true difference.The measurements in the present study were also made when children were asymptomatic.ΔR in asymptomatic children with a history of wheezing may not differ significantly from healthy children due to sparing of the larger calibre airways at an early age.
The longitudinal trajectory of ΔX in asymptomatic preschool children was significantly more negative in those with a history of wheezing when the effect of growth was kept constant.Changes in Xrs reflect elastic properties of the lung and thus are influenced by the ventilation of individual lung units.Time-constant inhomogeneity, produced by changes in the mechanical properties of the smaller airways, will induce a shift in XeE to become more negative as some units are under ventilated while others are over ventilated, increasing overall lung stiffness. 18,35The abnormal (more negative) trajectory of ΔX in our cohort of children with transient and persistent wheeze may reflect an evolution of ventilation inhomogeneity over time in wheezy children that does not improve with growth in lung volumes. 21 a cross-sectional study of adults with asthma, baseline impedance data including ReE, ReI, ΔR, XeE, XeI, and ΔX distinguished those with controlled versus uncontrolled asthma. 36However, spirometry variables were unable to distinguish the difference between these two groups.In our cohort of preschool-aged children, an abnormal trajectory for ΔX may be the earliest sign of small airway dysfunction in the lung periphery. 37 report a novel outcome ΔX/V T , which normalizes the change in ΔX for tidal volume.Healthy children with no airway disease tend to have no change or a slight decrease in Xrs between end expiration and end inspiration (ΔX ≥ 0).In small airway disease, the higher elastance at end-expiration (more negative XeE values) indicates mechanical inhomogeneity of peripheral lung units, which improves with inspiration (increasing Xrs) and manifests in negative ΔX and ΔX/ V T values. 21,35Our longitudinal data support this theory by showing that the trajectory of ΔX/V T over time in preschool-aged children with persistent wheeze becomes significantly more negative than in those who have never wheezed (Table 2).This new variable may have clinical significance in other diseases, such as cystic fibrosis, where measurements of ventilation inhomogeneity are important for identifying early lung disease progression but often challenging to achieve in a clinical setting for young children. 38 the transient wheeze group, 10.4% of lung function was performed on children taking ICS.19.2% of the measurements in the persistent wheeze group were on children taking ICS.When the effect of ICS and growth was accounted for, the longitudinal trajectory of XeE and ΔX/V T were significantly worse in children with persisent wheezing than those with transient wheezing.It may be possible to use XeE and ΔX/V T to identify wheezy preschoolers at risk for developing asthma.
Our study has several strengths.This is the first longitudinal study using IB-OSC in a community-based cohort of asymptomatic preschool-aged children.IB-OSC was highly feasible with acceptable longitudinal Zrs data obtained from 78.5% of lung function naïve children aged 3-year and 98%-100% of subjects 5-year and older.This is similar to the high longitudinal feasibility of impulse oscillometry in children aged 4-6-year reported previously 39 and significantly higher than the longitudinal feasibility of impulse oscillometry (21% at 4-year, 58% at 5-year, 74% at 6-year, 79% at 7-year, 86% at 8-year) which may reflect differences in tolerance of a single sinusoid compared to an impulse waveform. 14Our feasibility is comparable to cross-sectional studies reporting the feasibility of oscillometry in preschool-aged children. 40 have demonstrated that IB-OSC can track lung function trajectories using identical methods and outcomes from age 3-year onwards instead of relying on different paediatric outcomes like maximum flow at functional residual capacity (V'maxFRC) or forced expiratory volume in 0.5 s (FEV 0.5 ) which are not useful parameters in older children and adults.
All subjects were part of a birth cohort study with nonrandom recruitment, which may contribute to selection bias, but does not change the internal validity of our study results.Our wheeze groups were based on parental annual reports of wheezing in the previous 12 months, which may be subject to responder error.There was a higher proportion of children classified with transient and persistent wheezing compared to other cohort studies which may limit the generalizability of these findings.Although we used mixed-effects models to account for subject and growth effects, our sample size was too small for more complex multivariable models.Another limitation of this longitudinal study is that the oscillometry equipment was changed from a wave-tube oscillometry assembly to the tremoflo ® C-100 during the study.Although these two devices have been compared using ventilated mechanical test loads mimicking respiratory resistance impedances, no comparison was made headto-head in children during this study.
In conclusion, this study confirms that IB-OSC is feasible for tracking longitudinal lung function in preschool-aged children.| 1891 Children who participated in ORChID were invited for follow-up in the Early Life Lung Function (ELLF) study, which involved annual visits from ages 3-7 years.Demographic characteristics, height, weight, medical history, family history, respiratory symptoms, asthma risk factors, and environmental exposures were collected at each visit.Exclusion criteria for the ORChID study applicable to the ELLF study included chronic pulmonary (except asthma) or cardiovascular disease, chronic metabolic disorder, immunodeficiency, or children taking immunosuppressive medications.The Children's Health Queensland (HREC/13/QRCH/156) and The University of Queensland (2013001291) Ethics Committees approved the study.Parents gave written, informed consent.

2. 2 |
Lung function IB-OSC was initially performed using a wave-tube oscillometry assembly designed and custom-made at the University of Szeged, Hungary, for "The International Collaboration to Improve Respiratory Health in Children" (INCIRCLE) European Respiratory Society (ERS) Clinical Research Collaboration 24 according to ERS technical standards.

WONG ET AL.
Association of inhaled corticosteroids and wheezing with intrabreath impedance (n = 217 observations from 47 children) in subjects with transient or persistent wheezing.ReE, resistance at the end-expiration; ReI, resistance at end-inspiration; XeE, reactance at end-expiration; XeI, reactance at end-inspiration; ΔR, tidal change in resistance (ΔR=ReE-ReI); ΔX, tidal change in reactance (ΔX=XeE-XeI); ΔX/VT, ΔX normalized by tidal volume.Reference category is the transient wheeze group.In the transient wheeze group, 17 occasions were performed on children taking inhaled corticosteroids.In the persistent wheeze group, 14 observations were performed on children taking inhaled corticosteroids.
a Hectopascal per second per litre.b c Hectopascal per second per litre squared.*p < .05.