Effect of airway clearance therapies on mucociliary clearance in adults with cystic fibrosis: A randomized controlled trial.

BACKGROUND: Cystic fibrosis (CF) is an inherited disorder causing impaired mucociliary clearance within the respiratory tract, and is associated with bronchiectasis, chronic respiratory infections, and early death. Airway clearance therapies have long been a cornerstone of management of individuals with CF, although evidence supporting their use is lacking. We designed a randomized controlled trial to quantitatively compare the effects of different forms of airway clearance on mucociliary clearance.
METHODS: Three different physiotherapy methods to augment cough-clearance were studied in addition to cough-clearance alone: high-frequency chest-wall oscillating vest, oscillatory positive expiratory pressure, and whole-body vibration. We used gamma scintigraphy after inhalation of radiolabeled particles to quantify mucus clearance before, during, and after physiotherapy. As secondary endpoints, we measured concentrations of small molecules in exhaled breath that may impact mucus clearance.
RESULTS: Ten subjects were enrolled and completed study procedures. No differences were identified between any method of airway clearance, including cough clearance alone. We did identify changes in certain small molecule concentrations in exhaled breath following airway clearance.
CONCLUSIONS: Due to the limitations of this study, we do not believe the negative results suggest a change in clinical practice with regard to airway clearance. Findings pertaining to small molecules in exhaled breath may serve as future opportunities for study.

Introduction

Cystic fibrosis (CF) is a multisystem genetic disease principally characterized by lung disease with thick respiratory secretions and poor mucociliary clearance (MCC), chronic airway infections, and recurrent exacerbations [1-4]. While there are several pharmacologic strategies aimed at improving CF lung disease, airway clearance therapies (ACTs) continue to be a cornerstone of routine care [5, 6]. Many methods of ACT exist and include the use of mechanical devices that deliver pressure waves to the thoracic cavity, respiratory exercises that utilize variations in air flow and tidal breathing outside of normal respiratory patterns, and devices that provide positive expiratory pressure with or without pressure oscillations [7-10]. Exercise is also commonly used as an adjunctive ACT. However, there is little evidence to suggest superiority of one method of ACT over another, and while current guidelines in Europe, the UK, and the US all advocate for ACT, the current consensus is that the specific ACT method should be tailored to the individual patient with consideration for what is practical, acceptable to the patient, economically feasible, and subjectively effective [5]. A recently updated meta-analysis from the Cochrane Library reviewed 39 studies and did not identify clear evidence to support the favorability of any ACT technique over another based on several endpoints, including pulmonary function testing, sputum weights (wet or dry), exacerbation frequency, or participant satisfaction [11].

The mucociliary and cough clearance (MCC/CC) defect in CF has been extensively studied using gamma scintigraphy methods [12-14]. This outcome measure has been used to characterize the effect of several potential CF therapies on MCC/CC, and has shown good correlation between MCC/CC improvements and clinical benefits [15-17]. Interestingly, an early study used the MCC/CC technique to study mechanical ACTs in CF and failed to demonstrate differences in measured MCC/CC between postural drainage, positive expiratory pressure, and exercise in individuals with CF [18]. Given the improvements in the technique used for MCC/CC measurements over the last 25 years, as well as the ongoing question whether some ACTs may be superior to others, we designed a multiple crossover study of the two most commonly-used techniques in the United States as well as whole body vibration and huff-coughing alone as a control. We included whole body vibration via a vibrating platform both to evaluate the potential of this novel device as an ACT and as a control for the effects of strong mechanical stimuli not specifically targeted to the lung or developed as an ACT modality. Importantly, each of these ACTs were performed in conjunction with huff coughing, which was studied alone as a baseline comparator.

Finally, we explored the effect of ACTs on small-molecule biomarkers, including fraction of exhaled nitric oxide (FeNO) and the concentrations of other potential MCC regulators in exhaled breath condensate [19-22]. Decreased FeNO has been associated with increased disease activity in CF, and increased values have been associated with treatment [19, 20]. Metabolomic studies of airway surface biology have implicated a number of small molecules present in exhaled breath condensate as associated with regulation of airway surface liquid, most notably adenosine and other purine metabolites. Other small molecules have been associated with airway inflammation, mucus clearance activity, energy and function regulation, although their roles are poorly understood. These include some nucleosides and amino acids, small peptides, the polyamine spermine, lactate, and the mucin component sialic acid.

Our primary hypothesis was that the high-frequency chest wall oscillatory vest (HFCWO-vest) and oscillatory positive expiratory pressure (OPEP) devices would accelerate MCC/CC when compared to huff coughing alone. Our secondary hypotheses were that adenosine concentrations would increase in exhaled breath condensate as the result of mechanically-stimulated ATP release and subsequent metabolism, and that FeNO would increase in parallel with improvements in MCC.

Methods

Study design & research setting

We conducted a pilot open-label multiple crossover study to measure the effect of airway clearance therapy (ACT) on MCC in adults with CF. Subjects were recruited from and study procedures performed at the adult CF center at the University of North Carolina. The study was reviewed by the institutional review board at UNC and registered with ClinicalTrials.gov (NCT03078127), although there was a slight delay in submission to this registry which occured after subject enrollment began. This occurred due to institutional delays in assisting with registry submission. The authors confirm that all ongoing and related trials for this intervention are registered.

Subject eligibility

Subjects were eligible for enrollment if they were ≥18 years of age and had confirmed CF and a forced expiratory volume in one second (FEV1) > 30% of predicted (ppFEV1). Subjects were excluded if they were pregnant, had a recent exacerbation of lung disease requiring antibiotics within four weeks prior enrollment, or were unable to perform any of the ACT modalities.

Interventions

Subjects completed a screening visit during which eligibility criteria were confirmed and study techniques were taught and reviewed, and subjects provided written informed consent for study participation. Following this, study interventions occurred over four separate study visits, with a separate ACT modality studied at each visit. Study visits were between 3 and 21 days apart to allow for washout of ACT effects while maintaining study timeliness to prevent effects from natural disease activity. Use of hypertonic saline, dornase alfa, long-acting bronchodilators, and routine airway clearance were withheld for 12 hours prior to procedures on each study visit day. Each of three ACT modalities was studied in conjunction with prescribed huff coughing maneuvers, and a huff coughing-only study visit served as a baseline comparator. The studied ACT modalities were oscillatory positive expiratory pressure (OPEP; Aerobika®, Trudell Medical International, London, ON, Canada), high-frequency chest wall oscillation (HFCWO; The Vest®, Hill-Rom Advanced Respiratory Inc., St. Paul, MN, United States), and whole body vibration (WBV; Power Plate®, Performance Health Systems LLC, Northbrook, IL, USA). A Power Plate®, device was provided by Performance Health Systems for the execution of this study. The baseline visit was completed first, followed by three visits utilizing each of OPEP, WBV, or HFCWO, performed in a random order.

At each of the study visits, subjects would inhale radiotracer used for the primary outcome measure, and then at specified time points perform ACT procedures followed by a huff cough maneuver. Each study visit consisted of two 34-minute ACT sessions carefully timed with image acquisition for the primary outcome measure. Further details on these procedures is provided in S5 File.

Outcome measures

The primary endpoint was MCC as measured by the percent clearance of an inhaled radiolabeled marker from the whole right lung region over the 274 minutes that followed its administration. MCC was measured over 4.5 hours before, during, and after ACT sessions using a standardized methodology that involves inhalation of aerosolized Tc99m labelled sulfur colloid particles and collection of serial images over time with gamma scintigraphy, as previously described [23]. Further details on this method are described in the online supplement.

The primary outcome for this study was the average clearance (expressed as % and denoted Ave274Clr) of Tc99m activity measured within the whole right lung region of interest (ROI) from time 0 through 274 minutes in two 90-minute sessions during which ACT was performed. This was determined by averaging the percent clearance of Tc99m activity within the ROI from key images every 10 minutes, and is representative of the area under the curve of isotope clearance over time. Secondary MCC outcomes included isotope clearance from the whole lung region through 90 minutes (i.e. one ACT session), clearance from the peripheral and central ROI through 90 and 274 minutes, and isotope deposition indices (skew and central to peripheral ratio, C/P).

As part of this pilot study, we measured FeNO and collected exhaled breath condensates for measurement of small molecules before and after ACT at each study visit. We measured FeNO using a commercially-available device (NIOX MINO®, Circassia, Oxford, UK) according to the manufacturer’s instructions, and three readings were obtained and averaged. FeNO measurements were then repeated immediately after the first ACT session. Similarly, exhaled breath condensate (EBC) was collected using the R-Tube® system to non-invasively measure small-molecule concentrations in the deep lung. Subjects breathed through the R-tube device for 7 minutes to allow condensate collection, which was immediately frozen for subsequent analysis. Mass-spectrometry, using previously-described methods, was used to measure concentrations of selected small molecules implicated in CF disease activity and/or regulation of MCC as well as urea, which was used for normalization to account for variability in sample dilution [21, 22, 24, 25]. Concentrations were reported as a unitless index determined by the ratio of measured concentration of the small molecule to the measured concentration urea in the same sample. The studied compounds are listed in the results section. Additionally, all sputum expectorated during the study sessions was collected for total weight and % solid measurement.

Sample size determination

No comparable preliminary data on the effect of ACT on MCC was available to perform sample size calculation, as the only previous study used a different methodology for measuring MCC. However, our lab has collected pertinent data on the effects of inhaled 7% hypertonic saline on MCC using the same methods on 11 subjects with CF using a similar crossover study design, and calculated average mucus clearance over 180 minutes and 360 minutes respectively. From these data, we interpolated an estimated average clearance over 274 minutes (Ave274Clr), the primary endpoint for our study. At baseline, Ave274Clr was 32.4% (SD 19.0), and following hypertonic saline was 60.2% (SD 19.1%), with a mean difference of 27.7% (SD 15.1). We predicted that the observed mean change in Ave274Clr after ACT would be somewhat smaller compared to HS. We estimated a more conservative predicted effect size of 20.0%, with the same estimate of variability (SD 15.1). Using these data, a within-subject dependent effect size index, dz of 1.33 was calculated for Ave274Clr. Using G*Power (v3.1) and a paired, 2-tailed t-test to compare two means, we estimated that 7 subjects would be needed to detect a difference at 80% power with α = 0.05. Given the multiple comparisons in this study, the desire to explore relationships between MCC and secondary outcomes, and to guard against data loss due to subject drop-out, we planned to enroll 10 subjects.

Randomization

Randomization was performed by study statistician, AC, using a Latin square method to determine a block of 10 sequences, which were assigned to each subject after completion of the baseline visit. Due to the nature of the interventions, blinding was not possible. Due to the low-risk nature of this study, no interim analysis or a priori stop rules were planned,

Statistical analyses

Because the purpose and design of the study was to compare the effect of different ACT modalities when added to systematic huff coughing, a non-parametric repeated measures one-way ANOVA analysis (Friedman’s test) was used, as well as paired comparison to baseline huff-coughing alone for both the primary outcome and all secondary outcomes, adjusted for multiple comparisons using Dunn’s test. Sample size was determined based on power calculation from other studies using these methods (S4 File). For FeNO and EBC values, pre- and post- values from the different ACT methods were pooled and compared using Wilcoxon matched-pairs signed rank test. Spearman correlation analyses were performed to investigate any correlation between change in candidate biomarkers and MCC.

Results

Subjects

Ten subjects were enrolled and completed all study visits as shown in Fig 1 between January and December 2017. Subject characteristics are shown in Table 1. One subject was treated for influenza during the study period, and procedures were delayed to allow full recovery before completing the subsequent study visits. All subjects completed the study visits and tolerated study procedures well. Aside from the subject with influenza mentioned above, there were no adverse events during the study. No subject was treated with a highly effective modulator at the time of the study.

Fig 1

Subject flow diagram.

Ten subjects were recruited and completed all study procedures. Following screening, subjects performed baseline/huff-cough measurements before being randomized to a sequence in which to complete the other studied ACT methods. All recruited subjects were included in the analysis.

Table 1

Subject characteristics.

Subject	Age	Sex	FEV1	HS	Dornase
1	46	M	62%	Yes	Yes
2	56	F	69%	Yes	No
3	42	F	72%	No	No
4	35	F	47%	Yes	No
5	36	M	57%	Yes	No
6	57	F	32%	Yes	Yes
7	55	F	47%	No	Yes
8	21	F	41%	No	Yes
9	24	F	113%	No	Yes
10	32	M	89%	Yes	Yes

All subjects completed study procedures. Average age was 40.4 years and average percent predicted FEV1 was 62.9%.

Mucociliary clearance and cough clearance

Average clearance of inhaled isotope through 274 minutes for each ACT method is shown in Fig 2. No apparent differences in clearance versus time curves were observed. In the primary analysis, no differences were observed in average clearance over 274 minutes across all ACT methods (Friedman’s one-way nonparametric ANOVA, p = 0.6149), nor were any differences observed in paired comparisons between each of the three ACT methods and huff coughing alone, the baseline comparator. Similarly, no differences were observed in other MCC parameters. Because isotope deposition pattern can strongly influence subsequent clearance rates, we compared deposition skew and C/P ratio measured at each MCC study. No differences in deposition were observed between study visits that might confound interpretation of MCC rates [normalized C/P ratio (p = 0.6685); skew (p = 0.0503)]. A multiple comparisons analysis revealed a lower skew value at the OPEP visit when compared to huff-cough (1.50 vs 1.17, p = 0.0459). This difference was not believed to influence the primary outcome in a meaningful way, however.

Fig 2

Clearance of inhaled isotope as measured via gamma scintigraphy.

(A) Average clearance of inhaled isotope over time through 274 minutes. (B) Box plot of average whole lung clearance through 274 minutes. No differences were observed between ACT methods.

FeNO and EBC

The instrument used to measure FeNO was limited in that it was unavailable for 3 of the 40 study visits and has a lower limit of detection of 5 ppm. In 9 of the remaining 37 (24%) study visits, both the pre- and post-ACT FeNO values were below this threshold, and these results were not included in the analysis. One pre-ACT value was below sensitivity threshold with a post-ACT value above the threshold, and four post-ACT values were below sensitivity threshold with corresponding pre-ACT values above the threshold. In these cases, 4.5 ppm was used for the below-threshold value to allow for conservative comparisons. Changes in FeNO values are shown in Fig 3, and were decreased from baseline for each ACT type. Although there were insufficient samples within each ACT type to achieve significance, in a pooled comparison, there was a small decrease in FeNO following ACT (average decrease of 1.51 ppm, p = 0.006 using Wilcoxon signed-rank test). No correlation was identified between MCC rate and FeNO value.

Fig 3

Change in FeNO following ACT.

Median values and interquartile range are demarcated. Each ACT method showed an average decrease in FeNO following ACT, but this did not reach statistical significance until all values were pooled (p = 0.006, Wilcoxon signed-rank test).

Results of small molecule analysis from EBC are shown in Table 2. Pooling results from all ACT techniques, there appeared to be a small decrease in adenosine, nicotinamide, and phenylalanine following ACT. However, the veracity of these findings is unclear given the inherent difficulty in interpreting relatively weak signal strength when many variables are being studied. A larger and clearer effect was observed in lactate levels, which fell from a median value of 5.25 for the pre-ACT samples to 2.28 following ACT (p < 0.0001). Additionally, Spearman correlation analyses were performed for each candidate biomarker to investigate correlation between difference in concentration before and after ACT with MCC, as represented by Ave274Clr.

Table 2

Relative concentration of small molecules analyzed from exhaled breath condensate.

	Change in Concentration with ACT			Association between concentration change and MCC (Ave274Clr)
Small Molecule	Pre-ACT relative concentration, median (IQR)	Post-ACT relative concentration, median (IQR)	p-value (Wilcoxon)	Spearman correlation ρ, 95% CI	p-value
Purine Metabolism
Adenosine	0.104 (0.071–0.139)	0.0741 (0.0411–0.128)	0.041*	0.294 (-0.029–0.561)	0.066
AMP	0.173 (0.118–0.329)	0.112 (0.0629–0.270)	0.146	0.119 (-0.210–0.422)	0.466
Hypoxanthine	0.599 (0.334–1.11)	0.434 (0.204–0.876)	0.070	-0.005 (-0.325–0.315)	0.973
Nicotinamide	0.341 (0.258–0.532)	0.290 (0.200–0.437)	0.030*	-0.033 (-0.350–0.290)	0.838
Uric Acid	0.00228 (0.00108–0.00402)	0.00243 (0.00115–0.00523)	0.7446	0.038 (-0.286–0.354)	0.816
Nucleosides
Cytidine	0.0987 (0.0795–0.117)	0.0710 (0.0369–0.128)	0.183	0.201 (-0.127–0.490)	0.214
Inosine	0.142 (0.0854–0.244)	0.128 (0.0471–0.263)	0.765	0.052 (-0.273–0.366)	0.749
Amino Acids / Peptides
Isoleucine	2.41 (1.40–6.37)	1.53 (0.927–4.69)	0.128	-0.037 (-0.353–0.286)	0.820
Leucine-proline dipeptide	2.50 (1.69–3.18)	2.07 (0.934–3.00)	0.206	0.046 (-0.279–0.360)	0.780
Phenylalanine	5.50 (4.33–8.96)	4.73 (2.60–8.01)	0.041*	0.005 (-0.325–0.316)	0.976
Uridine	0.469 (0.299–0.772)	0.349 (0.223–0.827)	0.647	0.105 (-0.223–0.411)	0.521
Polyamine Synthesis Pathway
Spermine	0.0855 (0.0741–0.114)	0.0782 (0.0664–0.104)	0.438	0.173 (-0.155–0.468)	0.285
Cellular Energy Metabolites
Lactate	5.25 (0.625–7.51)	2.28 (1.09–5.67)	<0.0001***	0.313 (-0.008–0.575)	0.050
Mucin Components
Sialic Acid	0.00159 (0.000698–0.00583)	0.00176 (0.00133–0.00272)	0.196	0.017 (-0.305–0.335)	0.918

Small molecule concentration changes with ACT as well as correlation to MCC are reported. Median values and inter-quartile ranges for the relative concentrations of each candidate biomarker pre- and post-ACT are given. In order to correct for variability of dilution of samples, concentrations were normalized to urea concentration and are reported as a unitless ratio. Pre- and post-ACT samples were compared using a Wilcoxon signed rank test. Association of concentration change to Ave274Clr was investigated using Spearman correlation, ρ. These values are reported with 95% confidence interval as well as two-tailed p-value.

Six subjects expectorated sputum during at least one study visit, with complete data for all ACT types in only four subjects. Given the paucity of data, no analysis of sputum weights or percent solids was performed, but these results are available in S6 File.

Discussion

Although ACTs remain a central component of CF therapy, our study did not demonstrate any measurable short-term difference between ACT methods and huff-cough alone on MCC. Although surprising, these results corroborate an earlier gamma scintigraphy study [18]. It is also consistent with a general lack of difference between modalities on clinical outcomes such as spirometry and exacerbation frequency. In addition to studying the two most common ACT techniques used in the United States, we also studied whole-body vibration, a device developed for purposes of exercise enhancement rather than airway clearance. Some anecdotal reports indicated that this may be an effective method of ACT, and it allowed the opportunity to study the effects of mechanical stimuli similar to those in use in CF therapies (i.e. transduced oscillatory mechanical forces) but not specifically designed for that purpose. However, none of the ACT techniques we studied seemed to perform differently than any other or increase the effect of huff-cough alone.

The negative results from this study may be a result of a small sample size, as well as a study design that utilized huff-coughing as a control technique in place of a true baseline measurement. Since other ACT modalities are intended to potentiate the effect of cough-based clearance, our study design included huff-coughs to be performed with each of the other modalities. It is possible that adjunctive ACT devices do not have an effect on mucus clearance beyond huff-cough alone, however, the collective experience in CF treatment strongly advocates for the benefits of the use of adjunctive airway clearance modalities, so this conclusion seems unlikely. Alternatively, the timing between ACT treatment and MCC measurement may also have been inappropriate to detect important biological changes.

Furthermore, it may be that ACT does not contribute to mucus clearance in a manner which can be appreciated using the MCC measurement technique utilized in this study. For example, it may be that the greatest effect of ACT is to open airways obstructed by mucus plugs, an effect not detectable with this assay. Since subjects inhaled the radiotracer prior to the initiation of ACT, no isotope deposition would have occurred in airways lacking ventilation (i.e. completed obstructed). As a result, opening of these airways would not be detected.

Our secondary outcomes did reveal some unexpected findings. We included FeNO as an outcome measure because it is known to be lower in individuals with CF compared to healthy individuals, and has been associated with disease activity, and was improved in individuals treated with highly effective CFTR modulators [20, 26]. NO is known to act through guanylate cyclase to increase ciliary beat frequency, and thus may promote or reflect increased MCC [27, 28]. Indeed, this relationship was demonstrated in sheep, and whole-body vibration was implicated to accelerate MCC via increased NO release [29]. We anticipated that with stimulated MCC via ACT, FeNO would increase, but instead found a small, but opposite, effect. We speculate that this may be due to the opening of plugged airways with greater disease activity and lower levels of NO.

In vitro studies of the role of small molecules in airway biology, particularly with regard to the regulation of hydration of the airway surface liquid and the function of the mucociliary apparatus led us to hypothesize that mechanical forces applied to the airways during ACT would lead to an increase in ATP release from epithelia and appearance of purine metabolites (AMP and adenosine, in particular) in EBC following ATP metabolism [30-32]. However, we observed no such increases, and even a possible decrease in two purines. These results may suggest that ACT does not stimulate MCC through endogenous regulatory pathways, but rather may confer benefit through other mechanisms, such as mechanical disruption of mucus. The most striking result was with the marked decline of lactate in EBC, which decreased with ACT on average about 25%. While the cause of this change isn’t readily apparent from our study, this could represent improved aeration and oxygenation of plugged small airways with more aerobic metabolic activity in those diseased airways following ACT. A previous study of BALF in young children with CF showed a close positive correlation between mucin concentration and lactate in BALF, which was interpreted as mucin plugs causing localized hypoxia [33]. In addition to a rather small sample size, another limitation of the EBC analysis in our study was that FeNO measurements were taken first following ACT, and with three replicate FeNO values obtained with time required for the device to process each sample, the EBC collection did not begin until at least five minutes following completion of ACT, which may be adequate time for normalization for some of these small molecules.

We do not believe our results constitute sufficient grounds to recommend a change in clinical practice as directed by consensus guidelines in the US, UK, Europe, and Australia. However, these findings do suggest that ACT does not confer clinical benefit in the same manner as therapies which have been shown to increase MCC, such as hypertonic saline and ivacaftor for gating mutations. Future studies should include a true baseline of MCC as well as alterations in study design which may explore whether ACT has an effect on patency of airways, particularly in the era of highly effective modulators. This could be accomplished with post-ACT aerosol deposition or ventilation imaging by gamma scintigraphy or MRI. This work would be important to confirm the presumed clinical benefits of ACT and continue to justify the significant treatment burden they place on individuals with CF and their families.

CONSORT checklist.

Completed CONSORT checklist.

(DOC)

Click here for additional data file.

Mucociliary clearance data obtained from gamma scintigraphy.

Excel spreadsheeting containing MCC data used for analysis including retention and clearance in all ROIs, with deposition data (skew, C/P).

(XLSX)

Click here for additional data file.

FeNO data used in analysis.

Excel spreadsheeting containing pre- and post-ACT FeNO data for each subject and each ACT modality.

(XLSX)

Click here for additional data file.

Small molecule concentrations in exhaled breath condensate.

Excel spreadsheet containing pre- and post-ACT small molecule concentrations for each subject and each ACT modality. The raw concentrations are given, as well as those normalized to urea concentration, as was used in analysis.

(XLSX)

Click here for additional data file.

Study protocol.

Study protocol prepared a priori, as reviewed by the institutional board.

(PDF)

Click here for additional data file.

Further methods describing timing during study visits as well as additional details of the standardized methods used for MCC measurement.

(DOCX)

Click here for additional data file.

Data on sputum weights (in g) and % solids collected from subjects during study visits.

These were insufficient to be used in analysis.

(XLSX)

Click here for additional data file.

8 Jul 2021

PONE-D-21-05105

Effect of Airway Clearance Therapies on Mucociliary Clearance in Adults with Cystic Fibrosis: A randomized controlled trial

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The objective of this study is to investigate the effectiveness of airway clearance therapy on mucociliary clearance (MCC) adults with cystic fibrosis (CF). The authors considered an open-label, multiple cross-over study study. The study was approved by the Institutional Review Board at Univ. of North Carolina, and carries a legitimate NCT number. While the study objectives sound interesting, a number of shortcomings were observed, in regards to abiding by the CONSORT guidelines for conducting and reporting results of high-quality randomized clinical trials (RCTs), as well as some statistical and reporting issues:

1. Abstract:

It's better to present the Abstract in accordance to the 4 sections: Background, Methods, Results and Conclusions. Also, in the Results section, statistical significance/non-significance of findings should be expressed with p-values, and estimated precision, such as confidence intervals, or CIs (say, 95\\%). Check CONSORT checklist for Abstracts reporting of RCTs.

2. Methods:

Methods reporting appeared very messy. An orderly manner is suggested, following CONSORT guidelines, without repeating information, such as Trial Design, Participant Eligibility Criteria and settings, Interventions, Outcomes, sample size/power considerations, Interim analysis and stopping rules, Randomization (details on random number generation, allocation concealment, implementation), Blinding issues, etc. The authors are advised to create separate subsections for each of the possible topics (whichever necessary), and that way produce a very clear writeup. I see the Authors indeed made an attempt; however, they are advised to write it carefully, following nice examples in the manuscript below:

https://www.sciencedirect.com/science/article/pii/S0889540619300010

(a) For instance, the randomization technique employed (Latin square method) looks almost hidden; it should be pointed clearly. Furthermore, randomization and allocation concealment should be made very clear; the trial staff recruiting patients should not have the randomization list. Randomization should be prepared by the trial statistician, and he/she would not participate in the recruiting.

(b) I am surprised to see no statement on sample size/power in a manuscript proposing a cross-over trial. A vague statement was provided as "Sample size was determined based on..", with no specific determination of sample size wrt. some desired effect size. This is really a key here; else one has no idea why the study recruited and analyzed only 10 subjects.

(c) Friedman's one-way nonparametric ANOVA test was used, which looks OK. However, to assess correlation between changes in candidate biomarkers, the authors stated a linear regression was used. From my understanding of cross-over trials, a repeated measures analysis, via linear mixed-models is more appropriate. In that context, it is doubtful how much credible the study findings will be with only 10 subjects!

3. Results & Conclusions:

The authors ignored a thoughtful discussion of the presence/absence of a desirable 'washout' period, which is the cornerstone of a cross-over trial. The sample size enrolled is somewhat small, reflecting a specific population. I can only consider this study, at best, as a very pilot study. However, with no clear description of how they came up with 10 subjects, I am hesitant to proceed further.

For understanding and designing a nice multi-crossover study, the authors are asked to follow this article below (which also discusses the utility of using random-effects models, or the linear/nonlinear mixed-effects models):

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481185/

Reviewer #2: Thank you for the opportunity for reviewing this exciting manuscript including non-conventional outcome measures to assess the effects of different ACTs at short-term in people with cystic fibrosis. I really enjoyed reading it and learned as well. However, I would like to discuss some points with the authors.

a. The timeline of the study is a bit confusing and not easy to follow-up at first glance. Could you please try to explain it in a simpler way? You chose a complex procedure and it may be difficult for future readers to follow, especially if they are not familiar with this type of outcome measures

b. Why did you assess the effects of two ACT sessions, one followed by the other? Although two sessions per day are recommended in clinical practice, they are not back-to-back.

c. What is the rationale for assessing the whole effects of ACTs only up to 40 minutes after the end of the second session? The possibility of seeing differences over a longer period has been lost.

d. I was surprised that the autogenic drainage technique was not included in the study, as it is one of the most popular techniques in this population. Why do not include techniques that increase expiratory airflow by reducing the cross-sectional ratio of the airways when they seem to be best suited according to the mucus clearance physiology?

e. I do not agree to compare the body vibration device with exercise. The main mechanism of exercise that improves airway clearance is increased airflow. However, a body vibration device could be more similar to HFCWO device.

f. Why were the pulmonary biomarkers (FeNO and EBC) only obtained after the first ACTs session? Why not also after the second ACTs session like the MCC?

g. According to table 1, six participants use daily hypertonic saline as part of their routine treatment. Did the participants take the HS the days of the study? If so, when did they take it? How do you control this factor? Could it have influenced your results?

h. Were the participants adherent/trained to any ACTs? What were the ACTs they routinely used?

i. Could you please give us more information on the daily sputum expectoration of your participants? Alternatively, any information to understand better the level of impairment of the airway clearance in your sample?

j. I believe that huff-cough manoeuvres is a good ´comparator´ in this study. However, how many standardised cough manoeuvres performed the participants in each study visit? How many spontaneous cough manoeuvres were recorded in each study visit? Were this number similar between the different treatment arms? No data were reported in results.

k. Although no difference was observed between the treatment arms; OPEP showed a tendency to clear less Tc99m at the end of observed period (270 min). OPEP is the only ACTs analysed in this study that requires the active collaboration of the patient. It is described in the manuscript that the three ACTs were instructed to participants at the screening visit, however, were the participants able to test the OPEP device?; if participants were not sufficiently familiar with these devices, could lack of experience have affected your results?

l. Do you really think that FeNO is an appropriate outcome measure to assess the effects of ACTs in your sample considering that almost 25% of your measures did not achieve the minimum threshold? Please, could you explain this point in the discussion section? In addition, could you explain why there is an opposite direction of change in your study in this outcome measure?

m. I checked the information provided in the supplementary material and was surprised that you did not include any information on sputum quantity or % of solids in this manuscript. However, both outcomes seem to be assessed according to your initial protocol (supplementary material). Why did you not include this information in the manuscript?

n. Your data analysis is a bite confusing to me.

a. First, you suggest using non-parametrical approach in methods. I agree with this approach because of your sample size and the non-normal distribution of your outcome measures. However, it seems that you use parametric tests in the results section (e.g., page 11, line 225 it is stated that you use ANOVA; it is explained in figure 4 and in page 12, line 248 that you use t-student for paired sample; you describe your results in the figures using mean instead of median). For that reason, I recommend performing again the statistical analysis using a non-parametrical approach for all your variables and describe the findings using median and P25-P75, as you correctly did for EBC findings in table 2.

b. Why not include the effect size calculation in the results obtained from EBC? It would be useful to understand better the magnitude of the change.

c. It seems to me (based on your data and graphs) that you calculate correlations of two variables rather than linear regressions models. If this is correct, please change the information provided in the statistical analysis (page 10, line 198) and page 13, line 261) and give us information about the value of the correlation (r value) with their confidence intervals at 95%. If you finally calculate linear regressions model (I do not think it is appropriate for your sample size and the distribution of your outcome measures), you need to give us information about the odds ratio and the confidence intervals of your outcome measures.

o. Table 2 shows several molecules that have not been mentioned in the introduction or in the methods section. Could you please introduce a short paragraph to explain the role of these molecules in the airway clearance and guide to the readers?

In addition, you will find my minor comments / suggestions in the following table:

Page number Sentence / Section Issues which could be considered

Page 3, line 42 Introduction Suggest removing the word ´mechanical´ because the information is described in a general way.

Page 3, line 52 Introduction Authors referred to a SR published in 2017; however, it is not the latest version. Could the authors please check and change the information provided according to the updated version of this SR? (Cochrane Database Syst Rev. 2020 Apr 30;4(4):CD006842.

Page 3, line 42 Introduction Suggest removing the word ´mechanical´ because postural drainage and exercise are not theoretically considered mechanical ACTs.

Page 4, line 64 Introduction From my point of view, the introduction section is not the best place to describe the methodology of your study. Thus, I suggest removing the information included from line 64 to the end of the paragraph. Instead of this information, the authors may i) describe the different mechanism of action of the three techniques explored in the study; ii) or even explain why the biomarkers assessed are appropriate for evaluating the effects of ACTs because they are poorly explored in this type of studies.

Page 4, line 64 Introduction Suggest removing the word ´widely-used´ because HFCWO is not as commonly used in Europe or Asia as in the USA in this population.

Page 4, line 75 Hypothesis Please, clarify that MCC/CC was your primary endpoint and the rest of outcome measures were considered secondary endpoints.

Page 5, line 86 Methods How long was the washout period between the treatment arms? Was the washout period standardised?

Page 7, line 130 Methods Why did the authors increase the frequency each cycle? Why did the authors decide to set different pressure in each cycle? What is the reason for using this protocol?

Page 7, line 138 Methods Please, clarify that the number of coughs were registered in all clinic visits and not only in the last one.

Page 10, table 1 Results Did the participants have a chronic airway infection? Please, include additional information on possible pathogens in table 1

Page 12, fig 3 (B); fig 4 Results Please, change ´Vest´ to HFCWO

Page 14, line 276 Discussion Suggest including at short-term at the end of the sentence because you did not explore longer-term effects (24h for example).

Page 14, line 292 Discussion Suggest being caution with this sentence. In your study, you did not find any difference between ACTs plus huff-cough and huff-cough alone at very short-term and did not evaluate the most popular ACTs in this population. This does not mean that the techniques may not provide any clinical benefit in this population

Page 15, line 300 Discussion The limitation described for MCC is similar to lung clearance index to assess the effects of ACTs. Could you describe/name it?

Page 16, line 318 Discussion Suggest including your small sample size as a limitation to find a more reliable results in purines and the other small molecules.

Reviewer #3: Comments to Author:

Trimble and colleagues present a well-written paper addressing differences in airway clearance therapies on mucociliary clearance in people with CF. Although a similar study has been previously conducted with a similar sample size, the authors were able to rigorously study their research question in a well-designed study and include more current therapies and devices. Additionally, the authors were able to incorporate a novel secondary aim of evaluating small molecules as potential biomarkers of ACT clearance. Although the results of the study were negative, the question remains important and a topic for future research given widespread use of CFTR modulators and the need for individualized ACT therapies based on patient preference and cost effectiveness.

Title: No changes to suggest

Abstract: Incorporates main points concisely, no major changes to suggest

Intro:

The authors nicely provide relevant background while stating the primary objective of assessing for efficacy of ACT methods on mucociliary clearance.

P3, line 57) If word limit allows, would consider adding a sentence on how gamma scintigraphy works given the wider range of the PLOS audience (ie: technique that combines administration of radioistopes in conjunction with imaging modalities, etc).

Methods:

P5, line 85-86) Would ask authors to consider further describing timing of study visits, mentions 4 study visits but the timing of visits is not understood

Results:

P10, line 205) Would authors be able to elaborate on the number of participants/readings the FENO device malfunctioned? **It is found further down in results which would be fine to leave it placed there

Discussion:

Authors could consider commenting on small sample size of study, as it was similar to the prior study referenced in their article (ref 18). Otherwise, authors appropriately discuss limitations, notably obtaining a true baseline as the huffing group may have impacted their results. Given this study was performed prior to widespread use of CFTR modulators, it would be interesting to also include exercise as a group in future studies given that many people with CF have self-discontinued use of some ACT devices given overall improvement in clinical health.

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Reviewer #1: No

Reviewer #2: Yes: Beatriz Herrero-Cortina

Reviewer #3: No

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Submitted filename: Authors_Trimble_Comments_PlosOne21.pdf

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Submitted filename: Editor_Trimble_Comments_PlosOne21.pdf

Click here for additional data file.

21 Aug 2021

Our point-by-point responses to the reviewers is provided in the attached rebuttal letter. Our responses to the specific comments from Journal Editor Dr. Natasha McDonald in the decision letter is below:

1) We have updated the manuscript to meet the style requirements as requested.

2) We have updated the manuscript's method section to note the reason for inclusion in registry following subject enrollment (due to institutional delays with PRS system)

3) We have updated the methods section to note patient consent.

4) With regard to potential competing interests, we would like to clarify that the device we received from the commercial entity was provided to us temporarily, and was returned following completion of the study, and we therefore do not see the loan of this device as a competing interest, and we stand by our original statement with regard to competing interests. However, we would like to append the following to our original financial disclosure statement:

“This device was returned to the manufacturer following completion of the study.”

However, if the editorial board does not share this view with regard to our competing interests we provide competing interests statement to as follows:

“The authors received a PowerPlate® device from the manufacturer (Performance Health Systems LLC, Northbrook, IL, USA). This product was provided for the purposes of conducting this study without stipulation or conditions, and returned following completion of the study. No other goods, services, or any other form of compensation were provided to the authors by this entity. This does not alter our adherence to PLOS ONE policies on sharing data and materials. ”

Submitted filename: Reviewers_comments.docx

Click here for additional data file.

5 May 2022

Effect of airway clearance therapies on mucociliary clearance in adults with cystic fibrosis: a randomized controlled trial

PONE-D-21-05105R1

Dear Dr. Trimble,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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D William Cameron, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Many thanks for addressing all my comment s/ suggestions. Please continue research in this field and including other chronic respiratory diseases.

**********

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Reviewer #1: No

Reviewer #2: Yes: Beatriz Herrero Cortina

12 May 2022

PONE-D-21-05105R1

Effect of airway clearance therapies on mucociliary clearance in adults with cystic fibrosis: a randomized controlled trial

Dear Dr. Trimble:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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