Repeatability of Multiple Breath Washout in Pediatric Primary Ciliary Dyskinesia

I read with great interest the article by Wee et al. “Repeatability of Multiple Breath Washout in Pediatric Primary Ciliary Dyskinesia” 1, which evaluates the same-day and 28-day repeatability of Multiple Breath Washout in pediatric Primary Ciliary Dyskinesia. The authors deserve commendation for addressing a crucial methodological gap and conducting a rigorous multicenter study in a rare disease population. Their work provides valuable insights into the utility of LCI as a sensitive marker for early PCD lung disease. Nevertheless, despite the study's strengths, several methodological limitations remain, including potential variability in MBW measurements, circadian influences on lung function, and challenges related to participant cooperation, which may affect the generalizability and interpretation of results. Firstly, A major limitation of the study is the lack of standardization of pre-test airway clearance among participants, as children with primary ciliary dyskinesia typically perform airway clearance therapy at different times, intensities, and using various techniques prior to undergoing lung function testing. Without controlling these factors before multiple-breath washout (MBW) or spirometry, the study allows substantial variability in pre-test mucus burden and ventilation distribution. Such inconsistency can significantly influence lung clearance index (LCI) measurements, potentially improving or worsening repeatability depending on how effectively mucus was cleared just before testing. This may lead to misinterpretation of the intraclass correlation coefficient (ICC), with values being either overestimated or underestimated due to fluctuating mucus-related effects rather than true physiological variability. Evidence from previous work reinforces this concern; for instance, Singer et al. 2 demonstrated that LCI changes markedly during pulmonary exacerbations in relation to increased mucus load, underscoring the sensitivity of LCI to airway obstruction and mucus dynamics. This highlights that even small variations in airway clearance routines can meaningfully alter test outcomes. Therefore, standardized airway clearance protocols before lung function assessments are essential to ensure reliable and biologically meaningful LCI measurements in future PCD research. Secondly, A key limitation of the study is the absence of concurrent structural lung imaging, such as chest CT or MRI, at the time of MBW assessment. Lung Clearance Index (LCI) variability is strongly influenced by peripheral airway obstruction, mucus plugging, and heterogeneous ventilation patterns, yet without imaging performed simultaneously, the structural correlates underlying fluctuations in LCI remain unknown. This gap makes it difficult to determine whether observed variability represents true physiological change, transient airway instability, or measurement artifact. As a result, interpretation of outliers, subtle shifts across visits, or differences between patient subgroups becomes less certain, thereby limiting the study's ability to fully define repeatability across varying disease severities. The absence of imaging also restricts the opportunity to explore whether specific structural features—such as bronchiectasis extent, airway wall thickening, or segmental mucus impaction—drive disproportionate MBW variability in certain individuals. Evidence from related literature underscores the importance of structure–function linkage; for instance, Boon et al. 3 demonstrated that LCI correlates strongly with CT-detected abnormalities in patients with primary ciliary dyskinesia, highlighting how combined assessments enhance interpretation and clinical relevance. This reinforces that concurrent imaging would have strengthened the study's conclusions. Thirdly, The study did not evaluate intra-observer or inter-observer variability in multiple-breath washout (MBW) quality control, which represents an important methodological limitation. Although acceptability checks, end-tidal equilibration, and leak assessments were performed by research personnel, the absence of a reproducibility assessment means it is unclear whether different analysts applied these criteria consistently. MBW interpretation involves subjective judgment, particularly when determining the acceptability of borderline breaths or deciding when to terminate a trial. Without quantifying how reliably individual staff interpret and validate washout curves, there is a risk that variability between analysts—or even within the same analyst over time—may influence the final lung clearance index (LCI) values. This is especially relevant in small sample studies, where even minor inconsistencies in trial selection or rejection can disproportionately affect repeatability metrics and overall conclusions regarding disease severity or intervention effects. If analyst-related variability existed but went unreported, the precision of LCI outcomes may be overestimated. For instance, the study by Jensen et al. 4 demonstrates that technician-dependent differences in MBW interpretation can significantly alter LCI results, highlighting the need for standardized, reproducible quality-control procedures to ensure accurate and comparable outcomes across observers. Fourthly, In the present study, spirometry and multiple-breath washout (MBW) testing were performed at varying times of day, including both morning and afternoon sessions. It is well-established that lung function exhibits mild circadian variability, with parameters such as airway tone, airway resistance, and overall ventilatory capacity fluctuating over the course of the day. These physiological fluctuations can influence measures of lung function, including repeatability and variability metrics, independent of underlying disease status. Consequently, differences in testing time could artificially increase day-to-day variability, potentially confounding interpretation of longitudinal measurements or treatment effects. Spengler and Shea 5 demonstrated that healthy adults show significant circadian rhythms in pulmonary function, with measurable differences in spirometric indices across the day. To improve the reliability and interpretability of lung function studies, future research should standardize testing times or, at a minimum, record and account for the time of day when assessments are performed. This approach would reduce variability introduced by circadian influences and allow for more accurate comparisons across visits and participants. Lastly, A key limitation of the study is the limited inclusion of younger children (< 10 years) and those with more severe or symptomatic disease. The cohort was predominantly composed of older children with mild disease and high levels of cooperation, which allowed for successful completion of multiple-breath washout (MBW) testing. In contrast, younger or more symptomatic children often face challenges in performing MBW due to poor cooperation, fatigue, or difficulty following instructions, leading to their underrepresentation in the study. This limitation affects the study outcomes because repeatability measures, such as intraclass correlation coefficients (ICC), coefficient of variation (CV%), and coefficient of repeatability (CR), may be overestimated in this relatively “easier-to-test” group. The findings might not be generalizable to populations with more severe disease or younger age, in whom MBW variability is typically higher and test performance less reliable. Consequently, clinical interpretation of MBW repeatability based on this cohort should be approached with caution. For instance, Green et al. 6 demonstrate that younger children with primary ciliary dyskinesia exhibit greater ventilation inhomogeneity and show more variable MBW performance. Their findings highlight that age and disease severity substantially influence MBW reliability, emphasizing the need to include a broader, more diverse cohort in future studies to ensure that repeatability metrics accurately reflect all patient subgroups. Thus, Future studies should implement standardized airway clearance protocols, ensuring techniques are performed within a fixed 12–24-h window before testing to reduce variability from mucus redistribution. Incorporating same-day MRI or low-dose CT would enable structure–function correlation, enhancing interpretation of LCI variability across lung regions. Blinded cross-checking by multiple technicians, with quantification of inter-observer reliability, should be routine for MBW trial acceptance. Scheduling all baseline and repeat tests within identical time windows, such as 8–11 a.m., would minimize circadian influences. Expanding recruitment to wider age ranges and stratifying by disease severity would further improve the generalizability of findings. In conclusion, the study by Wee et al. 1 provides valuable insight into the repeatability of MBW in pediatric PCD and reinforces the potential of LCI as a clinically meaningful outcome measure. Nevertheless, several unaddressed methodological factors—including variability in airway clearance, lack of structural correlation, observer-related bias, circadian influences, and limited cohort diversity—may influence the interpretation of repeatability metrics. Addressing these elements in future studies will further strengthen the reliability and generalizability of MBW in PCD research. All authors have read and approved the final version of the manuscript. They take complete responsibility for the data's integrity and the data analysis's accuracy. All the authors meet the ICMJE authorship criteria and have made significant and equal contributions to this manuscript. All authors approved the final version and agree to be accountable for all aspects of the work, ensuring the accuracy and integrity of the data and interpretation. The authors have nothing to report. The authors have nothing to report. The authors have nothing to report. The authors declare no conflicts of interest. Artificial intelligence tools, such as ScholarAI, were utilized to assist in literature search, language refinement, and reference organization. No generative AI was used for idea creation, data interpretation, or authorship. All intellectual content, analysis, and conclusions were solely developed by the authors. The authors take full responsibility for the integrity and originality of the manuscript. The authors affirm that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned (and if relevant, registered) have been explained. Data sharing does not apply to this article as no datasets were generated during the current study; all data were sourced from published literature.