Associations between respiratory pathogens and lung function in primary ciliary dyskinesia: cross-sectional analysis from the PROVALF-PCD cohort.

Introduction: Respiratory pathogens are frequently isolated from airway samples in primary ciliary dyskinesia (PCD) patients. Few studies have investigated associations between these pathogens and lung function, with current management based on evidence from cystic fibrosis. We investigated the association between commonly isolated respiratory pathogens and lung function in PCD patients. Methods: Using a cross-sectional design, we prospectively collected clinical and concurrent microbiology data from 408 participants with probable or confirmed PCD, aged ≥5 years, from 12 countries. We used Global Lung Function Initiative 2012 references to calculate forced expiratory volume in 1 s (FEV1) z-scores. For 351 patients (86%) with complete data, we assessed the association of the four most frequently isolated pathogens with lung function by fitting multilevel linear models with country as random intercept, adjusted for age at diagnosis, age at lung function, use of antibiotic prophylaxis and body mass index z-scores. Results: Individuals with Pseudomonas aeruginosa growth in culture had significantly lower FEV1 z-scores (ß= -0.87, 95% CI -1.40- -0.34), adjusted for presence of Haemophilus influenzae, methicillin-sensitive Staphylococcus aureus and Streptococcus pneumoniae, and for covariates. When stratified by age, associations remained strong for adults but not for children. Results were similar when ciliary defects by transmission electron microscopy were included in the models and when restricting analysis to only confirmed PCD cases. Conclusions: We found that P. aeruginosa was associated with worse lung function in individuals with PCD, particularly adults. These findings suggest that it is prudent to aim for P. aeruginosa eradication in the first instance, and to treat exacerbations promptly in colonised patients.

Sulfur hexafluoride multiple breath washin and washout outcomes in infants are not interchangeable.

Objective.Sulfur hexafluoride (SF6) multiple-breath washout (MBW) assesses ventilation inhomogeneity, as an early marker of obstructive respiratory diseases. Primary outcomes are customarily washout-derived, and it is unclear whether the preceding SF6-washin can provide similar estimates. We aimed to assess comparability of primary SF6-MBW outcomes between washin and washout phases of infant SF6-MBW data measured with the WBreath (ndd Medizintechnik AG, Zurich, Switzerland) and Spiroware (Eco Medics AG, Duernten, Switzerland) MBW-setups, respectively. Approach.We assessed mean relative differences in lung clearance index (LCI) and functional residual capacity (FRC) between the washin and washout of existing SF6-MBW data from healthy infants and infants with cystic fibrosis (CF). We assessed whether these differences exceeded the mean relative within-test between-trial differences of washout-derived outcomes, which can be attributed to natural variability. We also explored non-physiological factors using a pediatric lung simulator. Main results.LCI and FRC from washin and washout were not comparable, for both setups. The mean difference (SD) in LCI between washin and washout was 2.3(10.8)% for WBreath and -9.7(8.0)% for Spiroware, while in FRC it was -4.7(7.8)% for WBreath and -2.3(9.7)% for Spiroware. These differences exceeded the within-test between-trial differences in washout-derived outcomes. Outcomes from washin and washout were also not comparable in a pediatric lung simulator. Significance.Outcomes of the washin and washout were not comparable due to an interplay of physiological and non-physiological factors, and cannot be used interchangeably.

ERS technical standard: Global Lung Function Initiative reference values for multiple breath washout indices.

BACKGROUND: Multiple breath washout is a lung function test based on tidal breathing that assesses lung volume and ventilation distribution. The aim of this analysis was to use the Global Lung Function Initiative methodology to develop all-age reference equations for the multiple breath washout indices lung clearance index (LCI) and functional residual capacity (FRC). METHODS: Multiple breath washout data from healthy individuals were collated from sites. Data were re-analysed using the latest software versions. Reference equations were derived using the lambda-mu-sigma (LMS) method using the generalised additive models of location shape and scale programme in R. The impact of equipment type, inert tracer gas, and equipment dead space volume on the derived reference ranges were investigated. RESULTS: Data from 23 sites (n=3647 test occasions) were submitted. Reference equations were derived from 1581 unique observations from participants between the ages of 2 and 81 years. Equipment type, inert tracer gas, and equipment dead space volume did not significantly affect the prediction equations for either LCI or FRC. Reference equations for LCI include age as the only predictor, whereas sex-specific reference equations for FRC included height and age. CONCLUSIONS: Global Lung Function Initiative reference equations for multiple breath washout variables provide a standard for reporting and interpretation of LCI and FRC.

Symptom trajectories in infancy for the prediction of subsequent wheeze and asthma in the BILD and PASTURE cohorts: a dynamic network analysis.

BACKGROUND: Host and environment early-life risk factors are associated with progression of wheezing symptoms over time; however, their individual contribution is relatively small. We hypothesised that the dynamic interactions of these factors with an infant's developing respiratory system are the dominant factor for subsequent wheeze and asthma. METHODS: In this dynamic network analysis we used data from term healthy infants from the Basel-Bern Infant Lung Development (BILD) cohort (435 neonates aged 0-4 weeks recruited in Switzerland between Jan 1, 1999, and Dec 31, 2012) and replicated the findings in the Protection Against Allergy Study in Rural Environments (PASTURE) cohort (498 infants aged 0-12 months recruited in Germany, Switzerland, Austria, France, and Finland between Jan 1, 2002, and Oct 31, 2006). BILD exclusion criteria for the current study were prematurity (<37 weeks), major birth defects, perinatal disease of the neonate, and incomplete follow-up period. PASTURE exclusion criteria were women younger than 18 years, a multiple pregnancy, the sibling of a child was already included in the study, the family intended to move away from the area where the study was conducted, and the family had no telephone connection. Outcome groups were subsequent wheeze, asthma, and healthy. The first outcome was defined as ever wheezed between the age of 2 years and 6 years. Week-by-week correlations of the determining factors with cumulative symptom scores (CSS) were calculated from weeks 2 to 52 (BILD) and weeks 8 to 52 (PASTURE). The complex dynamic interaction between the determining factors and the CSS was assessed via dynamic host-environment correlation network, quantified by a simple descriptor: trajectory function G(t). Wheeze outcomes at age 2-6 years were compared in 335 infants from BILD and 437 infants from PASTURE, and asthma outcomes were analysed at age 6 years in a merged cohort of 783 infants. FINDINGS: CSS was significantly different for wheeze and asthma outcomes and became increasingly important during infancy in direct comparison with all determining factors. Weekly symptoms were tracked for groups of infants, showing a non-linear increase with time. Using logistic regression classification, G(t) distinguished between the healthy group and wheeze or asthma groups (area under the curve>0·97, p<0·0001; sensitivity analysis confirmed significant CSS association with wheeze [BILD p=0·0002 and PASTURE p=0·068]) and G(t) was also able to distinguish between the farming and non-farming exposure groups (p<0·0001). INTERPRETATION: Similarly to other risk factors, CSS had weak sensitivity and specificity to identify risks at the individual level. At group level however, the dynamic host-environment correlation network properties (G(t)) showed excellent discriminative ability for identifying groups of infants with subsequent wheeze and asthma. Results from this study are consistent with the 2018 Lancet Commission on asthma, which emphasised the importance of dynamic interactions between risk factors during development and not the risk factors per se. FUNDING: The Swiss National Science Foundation, the Kühne Foundation, the EFRAIM study EU research grant, the FORALLVENT study EU research grant, and the Leibniz Prize.

An automated pipeline for computation and analysis of functional ventilation and perfusion lung MRI with matrix pencil decomposition: TrueLung.

PURPOSE: To introduce and evaluate TrueLung, an automated pipeline for computation and analysis of free-breathing and contrast-agent free pulmonary functional magnetic resonance imaging. MATERIALS AND METHODS: Two-dimensional time-resolved ultra-fast balanced steady-state free precession acquisitions were transferred to TrueLung, which included image quality checks, image registration, and computation of perfusion and ventilation maps with matrix pencil decomposition. Neural network whole-lung and lobar segmentations allowed quantification of impaired relative perfusion (RQ) and fractional ventilation (RFV). TrueLung delivered functional maps and quantitative outcomes, reported for clinicians in concise documents. We evaluated the pipeline using 1.5T data from 75 children with cystic fibrosis by assessing the feasibility of functional MR imaging, average scan time, and the robustness of the functional outcomes. Whole-lung and lobar segmentations were manually refined when necessary, and the impact on RQ and RFV was quantified. RESULTS: Functional imaging was feasible in all included CF children without any dropouts. On average, 7.9 ±â€¯1.8 (mean±SD) coronal slice positions per patient were acquired, resulting in a mean scan time of 6min 20s per patient. The whole pipeline required 20min processing time per subject. TrueLung delivered the functional maps of all the subjects for radiological assessment. Quality controlling maps and segmentations lasted 1min 12s per patient. The automated segmentations and quantification of whole-lung defects were satisfying in 88% of patients (97% of slices) and the lobar quantification in 73% (93% of slices). The segmentations refinements required 16s per patient for the whole-lung, and 2min 10s for the lobe masks. The relative differences in RFV and RQ between fully-automated and manually refined data were 0.7% (1.2%) and 2.0% (2.9%) for whole-lung quantification (median, [third quartile]), and excluding two outliers, 1.7% (3.9%) and 1.2% (3.8%) for the lobes, indicating the refinements could be potentially omitted in several patients. CONCLUSIONS: TrueLung quickly delivers functional maps and quantitative outcomes in an objective and standardized way, suitable for radiological and pneumological assessment with minimal manual input. TrueLung can be used for clinical research in cystic fibrosis and might be applied across various lung diseases.

TOLLIP and MUC5B modulate the effect of ambient NO2 on respiratory symptoms in infancy.

BACKGROUND: Current knowledge suggests that the gene region containing MUC5B and TOLLIP plays a role in airway defence and airway inflammation, and hence respiratory disease. It is also known that exposure to air pollution increases susceptibility to respiratory disease. We aimed to study whether the effect of air pollutants on the immune response and respiratory symptoms in infants may be modified by polymorphisms in MUC5B and TOLLIP genes. METHODS: 359 healthy term infants from the prospective Basel-Bern Infant Lung Development (BILD) birth cohort were included in the study. The main outcome was the score of weekly assessed respiratory symptoms in the first year of life. Using the candidate gene approach, we selected 10 single nucleotide polymorphisms (SNPs) from the MUC5B and TOLLIP regions. Nitrogen dioxide (NO2) and particulate matter ≤10 µm in aerodynamic diameter (PM10) exposure was estimated on a weekly basis. We used generalised additive mixed models adjusted for known covariates. To validate our results in vitro, cells from a lung epithelial cell line were downregulated in TOLLIP expression and exposed to diesel particulate matter (DPM) and polyinosinic-polycytidylic acid. RESULTS: Significant interaction was observed between modelled air pollution (weekly NO2 exposure) and 5 SNPs within MUC5B and TOLLIP genes regarding respiratory symptoms as outcome: E.g., infants carrying minor alleles of rs5744034, rs3793965 and rs3750920 (all TOLLIP) had an increased risk of respiratory symptoms with increasing NO2 exposure. In vitro experiments showed that cells downregulated for TOLLIP react differently to environmental pollutant exposure with DPM and viral stimulation. CONCLUSION: Our findings suggest that the effect of air pollution on respiratory symptoms in infancy may be influenced by the genotype of specific SNPs from the MUC5B and TOLLIP regions. For validation of the findings, we provided in vitro evidence for the interaction of TOLLIP with air pollution.

Optimized algorithm for speed-of-sound-based infant sulfur hexafluoride multiple-breath washout measurements.

INTRODUCTION: Major methodological issues with the existing algorithm (WBreath) used for the analysis of speed-of-sound-based infant sulfur hexafluoride (SF6) multiple-breath washout (MBW) measurements lead to implausible results and complicate the comparison between different age groups and centers. METHODS: We developed OASIS-a novel algorithm to analyze speed-of-sound-based infant SF6 MBW measurements. This algorithm uses known context of the measurements to replace the dependence of WBreath on model input parameters. We validated the functional residual capacity (FRC) measurement accuracy of this new algorithm in vitro, and investigated its use in existing infant MBW data sets from different infant cohorts from Switzerland and South Africa. RESULTS: In vitro, OASIS managed to outperform WBreath at FRC measurement accuracy, lowering mean (SD) absolute error from 5.1 (3.2) % to 2.1 (1.6) % across volumes relevant for the infant age range, in variable temperature, respiratory rate, tidal volume and ventilation inhomogeneity conditions. We showed that changes in the input parameters to WBreath had a major impact on MBW results, a methodological drawback which does not exist in the new algorithm. OASIS produced more plausible results than WBreath in longitudinal tracking of lung clearance index (LCI), provided improved measurement stability in LCI over time, and improved comparability between centers. DISCUSSION: This new algorithm represents a meaningful advance in obtaining results from a legacy system of lung function measurement by allowing a single method to analyze measurements from different age groups and centers.

Air pollution exposure during pregnancy and lung function in childhood: The LUIS study.

BACKGROUND: The adverse effects of high air pollution levels on childhood lung function are well-known. Limited evidence exists on the effects of moderate exposure levels during early life on childhood lung function. We investigated the association of exposure to moderate air pollution during pregnancy, infancy, and preschool time with lung function at school age in a Swiss population-based study. METHODS: Fine-scale spatiotemporal model estimates of particulate matter with a diameter <2.5 µm (PM2.5) and nitrogen dioxide (NO2) were linked with residential address histories. We compared air pollution exposures within different time windows (whole pregnancy, first, second, and third trimester of pregnancy, first year of life, preschool age) with forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) measured cross-sectionally using linear regression models adjusted for potential confounders. RESULTS: We included 2182 children, ages 6-17 years. Prenatal air pollution exposure was associated with reduced lung function at school age. In children aged 12 years, per 10 µg·m-3 increase in PM2.5 during pregnancy, FEV1 was 55 mL lower (95% CI -84 to -25 mL) and FVC 62 mL lower (95% CI -96 to -28 mL). Associations were age-dependent since they were stronger in younger and weaker in older children. PM2.5 exposure after birth was not associated with reduced lung function. There was no association between NO2 exposure and lung function. CONCLUSION: In utero lung development is most sensitive to air pollution exposure, since even modest PM2.5 exposure during the prenatal time was associated with reduced lung function, most prominent in younger children.

Cyclosporine A in children with ABCA3 deficiency.

BACKGROUND: Biallelic ATP-binding cassette subfamily A member 3 (ABCA3) variants can cause interstitial lung disease in children and adults, for which no proven treatments exist. Recent in vitro evidence suggested that cyclosporine A (CsA) could correct some ABCA3 variants, however for other variants this is unknown and no data in patients exist. METHODS: We retrieved the clinical data of two children aged 2 and 4 years carrying homozygous ABCA3 variants (G210C and Q1045R, respectively) and empiric CsA treatment from the Kids Lung Register database. In vitro experiments functionally characterized the two variants and explored the effects of CsA alone or combined with hydroxychloroquine (HCQ) in a human alveolar epithelial cell line (A549) derived from adenocarcinoma cells. RESULTS: Six weeks following the introduction of CsA, both children required a reduced O2 flow supply, which then remained stable on CsA. Later, when CsA was discontinued, the clinical status of the children remained unchanged. Of note, the children simultaneously received prednisolone, azithromycin, and HCQ. In vitro, both ABCA3 variants demonstrated defective lysosomal colocalization and impaired ABCA3+ vesicle size, with proteolytic cleavage impairment only in Q1045R. CsA alone corrected the trafficking impairment and ABCA3+ vesicle size of both variants with a variant-specific effect on phosphatidylcholine recycling in G210C. CsA combined with HCQ were additive for improving trafficking of ABCA3 in G210C, but not in Q1045R. CONCLUSIONS: CsA treatment might be helpful for certain patients with ABCA3 deficiency, however, currently strong clinical supporting evidence is lacking. Appropriate trials are necessary to overcome this unmet need.

Lung structural and functional impairments in young children with cystic fibrosis diagnosed following newborn screening - A nationwide observational study.

BACKGROUND: Non-invasive and sensitive clinical endpoints are needed to monitor onset and progression of early lung disease in children with cystic fibrosis (CF). We compared lung clearance index (LCI), FEV1, functional and structural lung magnetic resonance imaging (MRI) outcomes in Swiss children with CF diagnosed following newborn screening. METHODS: Lung function (LCI, FEV1) and unsedated functional and structural lung MRI was performed in 79 clinically stable children with CF (3 - 8 years) and 75 age-matched healthy controls. Clinical information was collected throughout childhood. RESULTS: LCI, ventilation and perfusion defects, and structural MRI scores were significantly higher in children with CF compared with controls, but FEV1 was not different between groups. Lung MRI outcomes correlated significantly with LCI (morphology score (r = 0.56, p < 0.001); ventilation defects (r = 0.43, p = 0.001); perfusion defects (r = 0.64, p < 0.001), but not with FEV1. Lung MRI outcomes were more sensitive to detect impairments in children with CF (abnormal ventilation and perfusion outcomes in 47 %, morphology score in 30 %) compared with lung function (abnormal LCI in 21 % and FEV1 in 4.8 %). Pulmonary exacerbations, respiratory hospitalizations, and increase in patient-reported cough was associated with higher LCI and higher structural and functional MRI outcomes. CONCLUSIONS: The LCI and lung MRI outcomes non-invasively detect even mild early lung disease in young children with CF diagnosed following newborn screening. Pulmonary exacerbations and early respiratory symptoms were risk factors for structural and functional impairment in childhood.

A computerized tool for the systematic visual quality assessment of infant multiple-breath washout measurements.

Background: Multiple-breath washout (MBW) is a sensitive method for assessing lung volumes and ventilation inhomogeneity in infants, but remains prone to artefacts (e.g., sighs). There is a lack of tools for systematic retrospective analysis of existing datasets, and unlike N2-MBW in older children, there are few specific quality control (QC) criteria for artefacts in infant SF6-MBW. Aim: We aimed to develop a computer-based tool for systematic evaluation of visual QC criteria of SF6-MBW measurements and to investigate interrater agreement and effects on MBW outcomes among three independent examiners. Methods: We developed a software package for visualization of raw Spiroware (Eco Medics AG, Switzerland) and signal processed WBreath (ndd Medizintechnik AG, Switzerland) SF6-MBW signal traces. Interrater agreement among three independent examiners (two experienced, one novice) who systematically reviewed 400 MBW trials for visual artefacts and the decision to accept/reject the washin and washout were assessed. Results: Our tool visualizes MBW signals and provides the user with (i) display options (e.g., zoom), (ii) options for a systematic QC assessment [e.g., decision to accept or reject, identification of artefacts (leak, sigh, irregular breathing pattern, breath hold), and comments], and (iii) additional information (e.g., automatic identification of sighs). Reviewer agreement was good using pre-defined QC criteria (κ 0.637-0.725). Differences in the decision to accept/reject had no substantial effect on MBW outcomes. Conclusion: Our visual quality control tool supports a systematic retrospective analysis of existing data sets. Based on predefined QC criteria, even inexperienced users can achieve comparable MBW results.

Single and multiple breath nitrogen washout compared with the methacholine test in patients with suspected asthma and normal spirometry.

BACKGROUND: Methods used to assess ventilation heterogeneity through inert gas washout have been standardised and showed high sensitivity in diagnosing many respiratory diseases. We hypothesised that nitrogen single or multiple breath washout tests, respectively nitrogen single breath washout (N2SBW) and nitrogen multiple breath washout (N2MBW), may be pathological in patients with clinical suspicion of asthma but normal spirometry. Our aim was to assess whether N2SBW and N2MBW are associated with methacholine challenge test (MCT) results in this population. We also postulated that an alteration in SIII at N2SBW could be detected before the 20% fall of forced expiratory volume in the first second (FEV1) in MCT. STUDY DESIGN AND METHODS: This prospective, observational, single-centre study included patients with suspicion of asthma with normal spirometry. Patients completed questionnaires on symptoms and health-related quality-of-life and underwent the following lung function tests: N2SBW (SIII), N2MBW (Lung clearance index (LCI), Scond, Sacin), MCT (FEV1 and sGeff) as well as N2SBW between each methacholine dose. RESULTS: 182 patients were screened and 106 were included in the study, with mean age of 41.8±14 years. The majority were never-smokers (58%) and women (61%). MCT was abnormal in 48% of participants, N2SBW was pathological in 10.6% at baseline and N2MBW abnormality ranged widely (LCI 81%, Scond 18%, Sacin 43%). The dose response rate of the MCT showed weak to moderate correlation with the subsequent N2SBW measurements during the provocation phases (ρ 0.34-0.50) but no correlation with N2MBW. CONCLUSIONS: Both MCT and N2 washout tests are frequently pathological in patients with suspicion of asthma with normal spirometry. The weak association and lack of concordance across the tests highlight that they reflect different but not interchangeable pathological pathways of the disease.

Differences in autophagy marker levels at birth in preterm vs. term infants.

BACKGROUND: Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. METHODS: In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. RESULTS: Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p = 0.025, padj = 0.050) and SIRT1 lower in preterm infants (-0.55, 95% CI -0.78;-0.31 in log2-transformed level, padj < 0.001). Furthermore, p62 decreased (padj-value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, padj < 0.001) with increasing gestational age. CONCLUSION: Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. IMPACT: To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity.

Recommendations for surveillance of pulmonary dysfunction among childhood, adolescent, and young adult cancer survivors: a report from the International Late Effects of Childhood Cancer Guideline Harmonization Group.

Childhood, adolescent, and young adult (CAYA) cancer survivors are at risk of pulmonary dysfunction. Current follow-up care guidelines are discordant. Therefore, the International Late Effects of Childhood Cancer Guideline Harmonization Group established and convened a panel of 33 experts to develop evidence-based surveillance guidelines. We critically reviewed available evidence regarding risk factors for pulmonary dysfunction, types of pulmonary function testing, and timings of surveillance, then we formulated our recommendations. We recommend that CAYA cancer survivors and healthcare providers are aware of reduced pulmonary function risks and pay vigilant attention to potential symptoms of pulmonary dysfunction, especially among survivors treated with allogeneic haematopoietic stem cell transplantation, thoracic radiotherapy, and thoracic surgery. Based on existing limited evidence and current lack of interventions, our panel recommends pulmonary function testing only for symptomatic survivors. Since scarce existing evidence informs our recommendation, we highlight the need for prospective collaborative studies to address pulmonary function knowledge gaps among CAYA cancer survivors.

Epidemiology and Clinical Characteristics of Human Metapneumovirus Infections in Hospitalized Children in Two Consecutive Postpandemic Years.

We assessed human metapneumovirus infections in children hospitalized between 2011 and 2023 and compared the strongest pre- and postpandemic seasons. After the COVID-19 pandemic, we observed offseason cases and loss of the alternating pattern of the human metapneumovirus season magnitude. Incidence rate ratio of 0- to 11-month-old versus 12- to 23-month-old children was 2.1 (95% CI: 1.0-4.8) before and 1.3 (95% CI: 0.6-2.9) after the pandemic.

Signal-correction errors in the EasyOne Pro LAB multiple-breath washout device significantly impact outcomes in children and adults.

Multiple-breath washout (MBW) is an established technique to assess functional residual capacity (FRC) and ventilation inhomogeneity in the lung. Indirect calculation of nitrogen concentration requires accurate measurement of gas concentrations. To investigate the accuracy of the CO2 concentration and molar mass (MM) values used for the indirect calculation of nitrogen concentration in a commercial MBW device [EasyOne Pro LAB (EOPL), ndd Medizintechnik AG, Switzerland] and its impact on outcomes. We used high-precision gas mixtures to evaluate CO2 and MM sensor output in vivo and in vitro. We developed updated algorithms to correct observed errors and assessed the impact on MBW outcomes and FRC measurement accuracy compared with body plethysmography. The respiratory exchange ratio (RER)-based adjustment of the measured CO2 signal used in the EOPL led to an overestimated CO2 signal (range -0.1% to 1.0%). In addition, an uncorrected dependence on humidity was identified. These combined effects resulted in an overestimation of expired nitrogen concentrations (range -0.7% to 2.6%), and consequently MBW outcomes. Corrected algorithms reduced the mean (SD) cumulative expired volume by 15.8% (9.7%), FRC by 6.6% (3.0%), and lung clearance index by 9.9% (7.6%). Differences in FRC between the EOPL and body plethysmography further increased. Inadequate signal correction causes RER- and humidity-dependent expired nitrogen concentration errors and overestimation of test outcomes. Updated algorithms reduce average signal error, however, RER values far from the population average still cause measurement errors. Despite improved signal accuracy, the updated algorithm increased the difference in FRC between the EOPL and body plethysmography.NEW & NOTEWORTHY We investigated the accuracy of the molar mass (MM) and CO2 sensors of a commercial multiple-breath washout device (ndd Medizintechnik AG, Switzerland). We identified humidity and respiratory exchange ratio-dependent errors that in most measurements resulted in an overestimation of expired nitrogen concentrations, and consequently, MBW results. Functional residual capacity and lung clearance index decreased by 6.6% and 9.9%, respectively. Despite improved signal accuracy, the difference in FRC between the EOPL and body plethysmography increased.