Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment.

Variability during spirometry can persist despite control of technical and personal factors. We postulate spirometry induces gastro-oesophageal reflux (GOR), which may cause variability and affect results of spirometry. Fifty-eight (58) subjects undergoing GOR investigation with oesophageal manometry and 24hr pH monitoring were recruited. Oesophageal dysmotility and GOR were assessed as part of clinical care. Subjects performed 2 sets of spirometry separated by a 10-minute rest period. The assessment of GOR during spirometry procedure (defined by a lower oesophageal pH<4) started from the first set of spirometry and concluded when the second set of spirometry was completed. We calculated variability (%) of FEV1, FVC and PEFR within each set as well as changes over 10-minutes. Twenty-six subjects (45%) recorded GOR during assessment. Of these, 23 subjects recorded GOR during the 10-minute rest period. Four subjects had GOR recorded only during spirometry tests. We did not find variability of spirometry parameters between the groups with and without GOR during spirometry procedure. However, in subjects with GOR, we found small but significant reductions of PEFR (0.5L/s, 8%, p<0.001) and FEV1 (84 mL, 3%, p = 0.048) in the second set of spirometry compared to the first spirometry set. This pilot study demonstrates that GOR can occur during and following spirometry. Presence of GOR during spirometry in this patient population caused small decreases in PEFR and FEV1 when it is repeated 10-minutes later however not increase variability in a single series of measurements.

Introduction

Spirometry is the most widely used and accessible lung function test in respiratory medicine. Adequate repeatability is required to ensure high confidence in lung function interpretation. Thus, spirometry guidelines identify technical sources of variation during testing [1]. However, when the patient is employing an appropriate technique to perform the test there may be legitimate biological sources of variation in response to spirometry in persons with established lung disease. For example, patients with active asthma may have bronchoconstriction due to the forced manoeuvre [2]. Other sources of pathophysiological variation have yet to be identified. In addition to individual test variation, some patients may show decreases in spirometry parameters 10-minutes following a bronchodilator assessment that is possibly independent of the effects of a standard dose of beta2-agonist. One possible pathophysiological event could be acid reflux induced by spirometry which could cause reflex bronchoconstriction [3] or upper airway acidification [4]. It may be possible upper airway acidification to reflux may impact upper airway function and negatively impact spirometry performance [5]. Lavorini et al., [6] observed cough-like expiratory efforts (“deflation cough”) during spirometry manoeuvres, which the researchers correlated to gastro-oesophageal reflux (GOR). The frequency of deflation cough during spirometry was reduced after antacid administration.

There is a strong association between GOR disease and pulmonary diseases, including chronic obstructive pulmonary disease [7] and asthma [8], but the underlying mechanisms are unclear. It is suggested that hyperinflation, vigorous cough, or bronchospasm may increase intra-abdominal pressure and decrease the diaphragmatic contribution to sphincter tone, thereby promoting reflux of gastric contents [9-13]. Therefore, vigorous breathing manoeuvres performed as part of spirometry, from inspiration to total lung capacity (TLC) followed by forced expiration, may challenge the integrity of the gastro-oesophageal junction.

We hypothesise a standard spirometry manoeuvre may induce GOR events in reflux susceptible patients. We performed a pilot study to understand if GOR events associated with spirometry manoeuvres in persons with reflux symptoms referred for high-resolution oesophageal manometry and ambulatory 24-hour oesophageal pH monitoring. Secondary aims were to investigate if GOR occurring with spirometry affects spirometry results and variability.

Materials and methods

Recruitment

From July 2016 –October 2017, patients attended the GI Motility Clinic, Camden Hospital, NSW Australia, for suspected GOR evaluation. All patients underwent routine oesophagus function assessment (high-resolution oesophageal manometry) and 24 hr pH monitoring study, described below. Potential participants that met the inclusion criteria (adults with no history of major oesophageal motility disorders e.g. achalasia, oesophageal spasm) were approached and informed of the study. The 58 participants that provided informed written consent took part in a spirometry procedure, described in detail below. All procedures were in accordance with the Helsinki Declaration, and the study was approved by the local institutional review board: South Western Sydney Local Health District Human Ethics committee (Ref: HREC/15/LPOOL/462). The participants in this study are representative of a population that experiences frequent GOR symptoms and/or are diagnosed with GOR.

High-resolution oesophageal manometry and 24-hour oesophageal pH monitoring

Patients referred to the Gastrointestinal Motility Clinic, Camden Hospital with reflux symptoms assessed by high-resolution oesophageal manometry (ManoScan ESO System, Medtronic, Minneapolis, MN, USA) and ambulatory 24-hour pH monitoring (Digitrapper pH-Z testing system, Medtronic, Minneapolis, MN, USA) as per standard protocols [14, 15].

We assessed oesophageal motility physiology in accordance to Chicago Classification Version 3.0 criteria [16]. Following manometry, the patients were intubated through the nose with a pH catheter adjusted to position the pH sensor 5 cm above the lower oesophageal sphincter. Patients went about their usual activities over a 24-hour period with the pH probe in-situ and GOR during this period was quantified by the DeMeester score. GORD was diagnosed based on the 24-hour DeMeester score of ≥14.72 [17] from the acquired recordings, (AccuView Software, Version 5.2 Medtronic, Minneapolis, MN, USA).

Lower oesophageal pH monitoring during the assessment period of spirometry

A gastroenterologist (JZ) trained in spirometry enrolled patients into the study at the end of their 24-hour pH monitoring with the pH catheter still intubated. Subjects performed two sets of spirometry manoeuvres separated by a 10-minute rest period which mimicked the protocol for a pre and post bronchodilator spirometry test where two separate sets of spirometry are required. The assessment period for GOR started with the first set of spirometry and ended when the second set of spirometry was complete. The inspiratory and expiratory components of spirometry attempts were marked using the software, including the rest period on the pH recording device. The pH catheter was removed upon the completion of the second set of spirometry.

GOR is defined when lower oesophagus pH falls below 4 [17]. Two study investigators (JZ, VH) independently reviewed the oesophageal pH from the assessment period to determine the presence of GOR. They also assessed whether GOR occurred during the inspiratory phase or the forced expiratory phase of spirometry. Differences in their assessment by consensus discussion.

Acid reflux was quantified by the number of reflux episodes during each set of spirometry and during the rest period using AccuView software, as well as the number of reflux events and time-in-reflux during spirometry sets and rest period. A reflux event is denoted by the period between the pH below 4 and the pH returning to >4. Time-in-reflux is the average time of reflux events over the assessment period.

Spirometry

Spirometry was conducted (QRS SpiroCard & Office Medic Software Version 4.4 Vectracor, Totowa, NJ, USA) via a laptop computer in accordance with the American Thoracic Society/European Respiratory Society spirometry standard [18] to accommodate for the presence of the pH catheter within the nose. We made a minor modification to not use a nose clip due to discomfort from the intubated pH catheter. Instead participants were asked to pinch their nose closed. Prior to conducting the first set of spirometry, the study investigator checked that the patient was not experiencing reflux immediately prior to the manoeuvre. Subjects were instructed to inhale to total lung capacity before performing a forced expiratory manoeuvre and expiring to residual volume, for a minimum of three attempts and no more than five. We recorded the forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1) and peak expiratory flow rate (PEFR).

Subjects then had a 10-minute rest period, before performing a second set of spirometry (Fig 1).

Fig 1

Example of pH-monitoring during spirometry study.

Acid reflux events (pH <4.0) and duration are recorded by device software. Spirometry attempts are signified by grey diamond markers (top) which represent the beginning of a forced exhalation manoeuvre. Ten minute rest period is indicated with blue bar. Participants are in a seated position during the entire period of testing.

Data analysis

Statistical analysis was performed (SPSS 24.0 for Windows, SPSS Inc, Chicago, IL, USA). Data distribution was determined with Shapiro-Wilk normality test; mean and standard deviation calculated for normally distributed data, while median and interquartile range calculated for non-normally distributed data. Data groups were compared using the Student’s t-test or Wilcoxon rank-sum test as appropriate for the distribution. Chi-square test was used to compare nominal variables between groups. Percentage of time-in-reflux was calculated as the total duration of all reflux events/duration of particular time period multiplied by 100. Variability within a set of spirometry was calculated for FEV1, FVC and PEFR using the formula: Variability = (max value–min value)/max value x 100 (expressed as a % value). Data is expressed as mean±SD unless otherwise indicated.

Results

Subject demographic and clinical investigation results

We enrolled 58 subjects (mean age 52yrs; range 21–79; 24 (41%) males, 34 (59%) females), with reflux symptoms, consecutively at the end of their 24-hour pH monitoring and all subjects completed the required 2 sets of spirometry. High-resolution oesophageal manometry assessment identified, 48 subjects (83%) with abnormalities predisposing to reflux (hiatus hernia and/or reduced lower oesophageal sphincter resting tone)while 10 (17%) had normal oesophageal function. Based on a cut-off DeMeester Score of 14.72, 39 (71%) subjects had clinically significant reflux on the preceding 24-hour oesophageal pH monitoring. The median DeMeester Score in these patients with significant reflux was 45.3 (IQR: 21.8 to 57.4) [19].

GOR during spirometry assessment

Twenty-six (45%) subjects recorded GOR during spirometry assessment, and were compared with the 32 subjects that did not record GOR in Table 1. A greater proportion of subjects with GOR during spirometry assessment had reflux pathophysiology (89% vs 78%). Subjects with GOR during spirometry were also more likely to be diagnosed with GORD (77% vs 59%; P = 0.27) and receive a higher DeMeester Score (mean 39.8±36.3 vs mean 26.4±24.8; P = 0.06) on the preceding 24-hour oesophageal pH monitoring, however these differences only approached statistically significance.

Table 1

Subject demographic data and clinical information from 24hr GORD assessment (n = 58).

	Spirometry assessment		Statistics
	GOR present (n = 26)	GOR absent (n = 32)	p-values
Gender
Male	11 (42%)	13 (41%)	0.90 (χ2)
Female	15 (58%)	19 (59%)
Age (mean ± SD) years	49 ± 16	55 ± 14	0.15 (t-test)
24 hr pH study
Diagnosed GORD	20 (77%)	19 (59%)	0.27 (χ2)
No GORD diagnosis	6 (23%)	13 (41%)
DeMeester (median ± IQR)a	27 ± 35.9	21 ± 28.5	0.06 (Wilcoxon)
Oesophageal manometry
Hiatus hernia present	23 (88%)	22 (69%)	0.07 (χ2)
Average resting LOS pressure (median ± IQR) mmHg	14 ± 14.7	20 ± 17.3	0.06 (Wilcoxon)
Reflux pathophysiologyb	23 (89%)	25 (78%)	0.01 (χ2)
Normal physiology	3 (11%)	7 (22%)

aGastro-oesophageal reflux disease (GORD) diagnosis based on DeMeester score ≥14.72 on 24-hour oesophageal pH monitoring; LOS: lower oesophageal sphincter

bReflux pathophysiology refers to the presence of a hiatus hernia or low resting LOS pressure <13 mmHg); ns: non-significant (p >0.05).

Of the 26 subjects who recorded GOR during spirometry assessment, GOR was most frequently observed during the 10-minute rest period (23/26, 88%; Table 2). A smaller proportion of subjects (17/26, 65%) had GOR during spirometry manoeuvres. A minority of subjects, had GOR exclusively during the first (1/25, 4%) and second sets (3/26, 12%) of spirometry.

Table 2

Analysis of GOR present during spirometry assessment (n = 26).

Time period	GOR present (%)a	Reflux events (median ± IQR)	Time in reflux (median ± IQR) %
1st set spirometry	6 (23%)	2.5 ± 3.5	21.3 ± 39.7
10-min rest	23 (88%)	2.0 ± 3.0	3.6 ± 18.1
2nd set spirometry	11 (42%)	1.0 ± 2.0	5.8 ± 25.1

aTotal in this column exceeds 26 as some subjects have GOR in 2 or more time periods.

bReflux index: no. of GOR events divided by number of spirometry attempts.

The 17 subjects recorded GOR during spirometry manoeuvre had a combined total of 46 GOR events (distinct inverse peaks where pH<4) during testing, of which, 32 (70%) coincided with the inspiratory and expiratory phases of spirometry. Examples of GOR events during inspiration and expiration manoeuvres are shown in Fig 2A and 2B.

Fig 2

A) Examples of GOR occurring throughout assessment period. Black arrowheads indicate GOR events during forced expiratory manoeuvres while white arrowheads indicate GOR events during inspiration to Total Lung Capacity. B) blue arrows indicate subtle GOR events.

We found the highest mean number of GOR events of 3.2±1.9 in the 1st set of spirometry, compared to 2.9±2.9 and 2.3±2.4 during the rest period and second set of spirometry, however these differences were not significantly different (P = 0.24).

Spirometry results and variability

The mean lung function values in this population were FVC 3.39L±1.0 and FEV1 2.72L±0.9. Comparisons of spirometry variables between 1st and 2nd sets are presented in Table 3. Subjects with observed GOR during spirometry assessment had a significantly lower mean PEFR in the second spirometry compared to the first spirometry (0.5±0.6L/s lower, P<0.001). Although, the mean FEV1 was also reduced in the second set of spirometry (84mL±0.2 lower, P = 0.048) the difference in volume would not appear to be of clinical significance. Mean FVC did not change between the two sets of spirometry.

Table 3

Analysis of spirometry results (mean ± SD).

GOR present during spirometry assessment (n = 25)a
	1st set	2nd set	Difference	Paired T-test
FVC (L)	3.41 ± 0.97	3.36 ± 0.94	0.048 ± 0.20	0.25
FEV1 (L)	2.77 ± 0.87	2.68 ± 0.85	0.084 ± 0.20	0.048b
PEFR (L/s)	6.64 ± 2.18	6.14 ± 2.16	0.50 ± 0.60	<0.001b
No GOR present during spirometry assessment (n = 32)
	1st set	2nd set	Difference	Paired T-test
FVC (L)	3.35 ± 0.99	3.44 ± 1.11	-0.092 ± 0.27	0.06
FEV1 (L)	2.68 ± 0.90	2.76 ± 0.99	-0.082 ± 0.23	0.06
PEFR (L/s)	6.61 ± 2.20	6.47 ± 2.27	0.14 ± 0.72	0.30

aOne spirometry study was omitted due to technical fault

bstatistical significance p value <0.05

In the group of participants without GOR during spirometry assessment, there was no significant difference in mean FEV1, FVC and PEFR between the two sets of spirometry.

The variability of FVC, FEV1, and PEFR within each sets of spirometry is comparable between GOR present and GOR absent groups (Table 4). There is a slight reduction in variability within the second set compared to the first set for all FVC, FEV1 and PEFR values but the differences were not statistically significant (P<0.05).

Table 4

Comparison of parameter variability (median% ± IQR) within a set of spirometry.

	Spirometry assessment
	GOR present (n = 26)		GOR absent (n = 32)
Variability	1st set	2nd set	1st set	2nd set
FVC	4.3 ± 7.4	4.0 ± 5.4	4.2 ± 4.9	4.2 ± 3.6
FEV1	5.4 ± 7.4	5.4 ± 9.6	5.9 ± 5.1	4.5 ± 8.7
PEFR	13.0 ± 14.1	11.4 ± 18.1	13.3 ± 15.2	8.6 ± 13.1

aVariability within a set of spirometry was calculated for FEV1, FVC and PEFR using the formula: Variability = (max value–min value)/max value x 100 (expressed as a % value)

Discussion

This pilot study demonstrates that GOR events occur during or following spirometry in almost half of patients presenting for assessment of GORD using 24-hour oesophageal pH monitoring. These findings support our primary hypothesis that spirometry can induce GOR.

Our study recruited a clinical population symptomatic of GOR undergoing high-resolution oesophageal manometry and 24-hour oesophageal pH monitoring. This was appropriate for a pilot study of this nature as we wanted to see if we were able to detect the physiological changes to support the primary hypothesis.

We found that GOR is associated with both inspiratory (inhaling to TLC from functional residual capacity during tidal breathing) and expiratory phases of spirometry. This has been observed in a similar study where the occurrence of deflation cough during maximal expiratory spirometry manoeuvres were associated with clinical features of GOR [6]. A potential mechanism for this, is the reduced intra-thoracic pressure during inspiration acts as a suctioning force that could draw gastric contents into the oesophagus. The diaphragmatic contraction during inspiration would result in increased lower oesophageal sphincter tone and would normally prevent GOR. However, in subjects with a hiatus hernia, this diaphragmatic squeeze acting on the hiatus hernia may propel contents within the hernia back into the oesophagus. Considering our initial findings, we propose that during forced expiration, the increase in intra-abdominal pressure may exceed intra-thoracic pressure, thus this pressure differential could cause movement of gastric contents into the oesophagus. Further, GOR may also be facilitated by diaphragmatic contraction being reduced during expiration. The reduced diaphragmatic tone in turn may reduce overall gastro-oesophageal junction tone, enabling retro-grade movement of gastric contents. Thus, the observed strong association between GOR and forced expiration could be due to the combination of increased pressure gradients and reduced gastro-oesophageal tone during expiration manoeuvre.

A novel finding in this study was that the majority of GOR occurred during the 10-minute rest period. We postulate that the mechanical stresses associated with spirometry could temporarily weaken gastro-oesophageal junction tone leading to increased reflux susceptibility for a period of time after the first set of spirometry manoeuvres. This disruption of the junction integrity is likely to be transient as a low mean percentage time in reflux was recorded during the rest period and fewer GOR events were recorded in the second set of spirometry.

In the group of patients with reflux during spirometry assessment, we found a slightly lower PEFR in the second set of spirometry. Although a lower PEFR is commonly due to poorer patient effort, a possible explanation is reduction of large airway calibre, for instance from bronchoconstriction. Acid-induced bronchoconstriction caused by neurogenic inflammation as a result of tachykinin (substance P/neurokinin A) release upon acid stimulation has been demonstrated [20, 21]. The proposed mechanisms of this involve proton-activation of central vagal reflex and subsequent oesophageal-airway local axon reflex or microaspirate (reflux) stimulation of capsaicin-sensitive sensory neurons [22]. The possibility of reflux-induced bronchoconstriction having an effect on spirometry is supported further by our observation of the small but statistically significant reduction of FEV1. However these changes were small and their clinical significance is debatable. Changes in PEF and FEV1 could also be due to changes in upper airway function during a forced manoeuvre [23]. There was no change in the variability of FEV1, FVC or PEFR within the second spirometry set, and therefore we did not have evidence to suggest that poorer spirometry effort was the reason for the lower PEFR and FEV1. The small changes in spirometry parameters may be attributed to participants in this study cohort having normal lung function and no history for respiratory diseases. Future study within a population with respiratory disease may lead to greater variability in lung function in the presence of acid reflux during spirometry.

Our findings are consistent with previous studies that observed indirect induction of reflex bronchoconstriction by gastric content that reaches the upper airways [10, 24] via tracheal irritant receptors believed to be situated in the upper airway epithelium [10]. A number of animal studies have demonstrated the activation of these receptors is associated with a vagally mediated reflex bronchoconstriction [25]. Exposures to acidic aerosols have also been shown to stimulate reflex bronchoconstriction [26, 27], most likely by stimulation of irritant receptors in the tracheobronchial tree.

Acute upper airway acidification has also been shown to cause changes in upper airway calibre representing upper airway dysfunction, for example variability vocal cord abduction or increased adduction [3, 26, 27]. A limitation of our pH measurement was limited to the mid-distal oesophagus, and we could not measure acid reflux in the upper oesophagus, pharynx or larynx. Therefore we cannot confirm a possible cause for upper airway dysfunction if this were responsible, as knowing this would be able to reveal if these changes could be due to upper airway reflux. Further studies should investigate this mechanism in more detail by identify changes in upper airway pH with spirometry in both susceptible subjects who demonstrate decreases in post bronchodilator spirometry and including the effects in a well characterised healthy population.

Conclusion

This study demonstrates that GOR events do occur during spirometry in a group of patients who are predisposed to GOR or have significant GOR disease. Further, a high prevalence of GOR occurred during a 10-minute rest period between two sets of spirometry. The observation of reductions in some spirometry parameters 10-minutes after an initial set of spirometry manoeuvres suggests reductions in airway calibre may be associated with spirometry-induced GOR or the presence of GORD. Further research investigating this relationship may determine if these reductions in spirometry parameters are reflective of the predisposition to GOR events and increases in airway acidity.

Study data.

Summary of participant characteristics, oeosphagogastric findings, 24hr pH study results, and spirometry evaluation.

(TXT)

Click here for additional data file.

27 Nov 2019

PONE-D-19-25405

Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment

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Reviewer #1: This study demonstrates GOR may result in very mild physiologic changes but not clinically significant changes in the patients studied. The authors imply specific changes despite not proven by statistics.

Reviewer #2: The present work was aimed to show the occurrence of gastro-oesophageal reflux (GOR) during or soon after spirometry (i.e.: forced expiratory maneuver following full inspiration to total lung capacity) and to assess the potential consequences of in-expiratory effort-related GOR in terms of subsequent respiratory functional results and their variability.

The rational is that in individuals who potentially may have gastro-oesophageal reflux disease (GORD), as suggested by peculiar symptoms or anatomic-functional predisposing conditions, the increased positive pressure gradient occurring during in-expiratory efforts between abdomen and thorax may induce distal or even proximal acid reflux causing reflex bronchoconstriction or upper airway stimulation with subsequent increase in airflow resistance.

In this cohort of subjects with high probability of having GORD, the Authors found that GOR was present during or following spirometry in almost half of the subjects (26/58). In contrast with subjects without GOR during or following spirometry, those with GOR showed in the subsequent set of spirometry a significant reduction of some functional parameters, suggesting the occurrence of the GOR-related airflow obstruction.

In addition, the results show that in 39 subjects over 58 suspected of GORD who were diagnosed as having GORD by standard criteria based on 24-hours oesophageal pH monitoring, about 50% (20/39) exhibited spirometry-induced GOR and the resting 50% (19/39) not. Conversely, in 19 subjects who did not have GORD, 6/19 (about one/third) exhibited spirometry-induced GOR and 13/19 not. Thus, repeated spirometry seems not useful to increase the diagnosis of GORD.

General comments

The presence of GOR during spirometry is not a novelty and in fact deflation-related peak expiratory flows occurring near residual volume at the end of maximal expiratory maneuvers has been suggested as a marker of GOR (see Lavorini et al. Chest 2011;140:690).

The new findings of this work are the more frequent occurrence of GOR throughout 10 minutes after spirometry than during spirometry and the mild reduction of some functional indices (such as PEF and FEV1) in the subsequent set of spirometry.

Surprisingly GOR was recorded also during slow inspiration toward TLC before the maximal forced expiration.

The methods to assess GOR are up to date.

The numbers in the tables 1 and 2 are difficult to follow and sometimes do not fit with the text. Please check it.

The statistics are adequate.

Minor comments

I do not think these findings may explain the paradoxical increase of airflow obstruction after a bronchodilator test performed to assess the reversibility of baseline airway obstruction and I will skip this sentence from the discussion.

Some English mistakes here and there need to be amended.

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7 Dec 2019

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Manuscript ID: [PONE – D-19-25405] – [EMID:d5e0088e3ed7c23b]

Title: Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment

Authors: Jerry Zhou, Ming Teo, Vincent Ho, John D. Brannan

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4. Fig 1 and 2 uploaded and converted in PACE digital diagnostic tool

In Response to Reviewer #1

1. This study demonstrated the presence of acid reflux during and immediately following spirometry in individuals with reflux symptoms. This cohort of individuals who have GOR symptoms was selected to maximise the chance of reflux during spirometry. The presence of reflux was also noted to have a significance effect on FEV1 and PEFR (Table 2), although as the reviewer correctly states the differences are small and may not be of clinical significance. This may be due to the test group selection and may translate to larger variations in a test group with lung function disorders. The manuscript has been amended to address the small changes in FEV1 and PEFR, and added a section discussing the choice of test group.

In Response to Reviewer #2

1. The study by Lavorini et al., has been acknowledged and the amended manuscript compares its results to this study.

2. The novelty of reflux immediately after spirometry has been emphasised in the amended manuscript

3. Tables 1 & 2 have been amended, data that were not utilised in the text and discussion are removed to improve readability. Data removed from Table 1: Hiatus hernia size, Presence of low resting pressure, minor motility disorder combined to reflux pathophysiology. Data removed from Table 2: GOR only in this period, Mean reflux index. Values in text have also been updated to match table values.

4. “Paradoxical increase of airflow obstruction after a bronchodilator test performed to assess the reversibility of baseline airway obstruction” sentence is removed in amended manuscript.

5. English mistakes amended.

7 Jan 2020

PONE-D-19-25405R1

Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment

PLOS ONE

Dear Dr. Jerry Zhou,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authos should revise the manuscript following reviewers' suggestions.

In addition, I found old papers cited in the reference list concerning the mechanisms of association between GOR and bronchospasm/asthma and, in particular, I would add some sentences about the role of neurogenic inflammation as a main mechanism of bronchoconstriction due to airway acidification (protons-induced bronchoconstriction). I suggest to update the reference list.

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Reviewers' comments:

Reviewer 1:

This study demonstrates GOR may result in very mild physiologic changes but not clinically significant changes in the patients studied. The authors imply specific changes despite not proven by statistics.

Reviewer 2:

The present work was aimed to show the occurrence of gastro-oesophageal reflux (GOR) during or soon after spirometry (i.e.: forced expiratory maneuver following full inspiration to total lung capacity) and to assess the potential consequences of in-expiratory effort-related GOR in terms of subsequent respiratory functional results and their variability.

The rational is that in individuals who potentially may have gastro-oesophageal reflux disease (GORD), as suggested by peculiar symptoms or anatomic-functional predisposing conditions, the  increased positive pressure gradient occurring during in-expiratory efforts between abdomen and thorax may induce distal or even proximal acid reflux causing reflex bronchoconstriction or upper airway stimulation with subsequent increase in airflow resistance.

In this cohort of subjects with high probability of having GORD, the Authors found that GOR was present during or following spirometry in almost half of the subjects (26/58). In contrast with subjects without GOR during or following spirometry, those with GOR showed in the subsequent set of spirometry a significant reduction of some functional parameters, suggesting the occurrence of the GOR-related airflow obstruction.

In addition, the results show that in 39 subjects over 58 suspected of GORD who were diagnosed as having GORD by standard criteria based on 24-hours oesophageal pH monitoring, about 50% (20/39) exhibited spirometry-induced GOR and the resting  50% (19/39) not. Conversely, in 19 subjects who did not have GORD, 6/19 (about one/third) exhibited spirometry-induced GOR and 13/19 not. Thus, repeated spirometry seems not useful to increase the diagnosis of GORD.

General comments

The presence of GOR during spirometry is not a novelty and in fact deflation-related peak expiratory flows occurring near residual volume at the end of maximal expiratory maneuvers has been suggested as a marker of GOR (see Lavorini et al. Chest 2011;140:690).

The new findings of this work are the more frequent occurrence of GOR throughout 10 minutes after spirometry than during spirometry and the mild reduction of some functional indices (such as PEF and FEV1) in the subsequent set of spirometry.

Surprisingly GOR was recorded also during slow inspiration toward TLC before the maximal forced expiration.

The methods to assess GOR are up to date.

The numbers in the tables 1 and 2 are difficult to follow and sometimes do not fit with the text. Please check it.

The statistics are adequate.

Minor comments

I do not think these findings may explain the paradoxical increase of airflow obstruction after a bronchodilator test performed to assess the reversibility of baseline airway obstruction and I will skip this sentence from the discussion.

Some English mistakes here and there need to be amended.

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Jan 2020

In Response to Journal Requirements:

1. The manuscript has been formatted in accordance with PLOS ONE style

2. Manuscript amended to address how consent was provided: “Participants were informed of the study and written consent was provided from those participating in the study.”

3. Additional section added to Methods: Recruitment; detailing a) recruitment date range b) inclusion criteria c) representation of larger population e) recruitment process

4. Fig 1 and 2 uploaded and converted in PACE digital diagnostic tool

5. Support file attached (study data) along with update in manuscript

6. The role of neurogenic inflammation as a mechanism for reflux-induced bronchoconstriction is a salient point and supports our findings that GOR affects spirometry tests. Amendment has been made to highlight its significance in the Discussion (Line 265-269) along with the proposed mechanisms of neurogenic inflammation in bronchoconstriction as a result of vagal reflex and microaspirate stimulation.

In Response to Reviewer #1

1. This study demonstrated the presence of acid reflux during and immediately following spirometry in individuals with reflux symptoms. This cohort of individuals who have GOR symptoms was selected to maximise the chance of reflux during spirometry. The presence of reflux was also noted to have a significance effect on FEV1 and PEFR (Table 2), although as the reviewer correctly states the differences are small and may not be of clinical significance. This may be due to the test group selection and may translate to larger variations in a test group with lung function disorders. The manuscript has been amended to address the small changes in FEV1 and PEFR, and added a section discussing the choice of test group.

In Response to Reviewer #2

1. The study by Lavorini et al., has been acknowledged and the amended manuscript compares its results to this study.

2. The novelty of reflux immediately after spirometry has been emphasised in the amended manuscript

3. Tables 1 & 2 have been amended, data that were not utilised in the text and discussion are removed to improve readability. Data removed from Table 1: Hiatus hernia size, Presence of low resting pressure, minor motility disorder combined to reflux pathophysiology. Data removed from Table 2: GOR only in this period, Mean reflux index. Values in text have also been updated to match table values.

4. “Paradoxical increase of airflow obstruction after a bronchodilator test performed to assess the reversibility of baseline airway obstruction” sentence is removed in amended manuscript.

5. English mistakes amended.

4 Feb 2020

Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment

PONE-D-19-25405R2

Dear Dr. Jerry Zhou,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Fabio Luigi Massimo Ricciardolo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Author responses are appropriate and ms acceptable for publication. The ms will serve as a guide to pulmonologists when interpreting pulmonary function studies in patients with GERD

Reviewer #2: General comments

The amended version of this work is definitively improved, more easily readable and better written than the original paper.

I do not have more questions or criticisms to rise.

The main practical advice following the results of this study is that in patients with baseline obstructive ventilatory defect, knowing to have GORD or predisposing conditions to GOR, the spirometric response to a subsequent test of bronchial responsiveness using short-acting bronchodilators (if required) should be assessed after an interval of time longer than 10 minutes (20 or better 30 min) to avoid spurious results.

Perhaps this suggestion could be added at the end of discussion section if Authors agree.

**********

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Reviewer #1: Yes: Arthur F Gelb MD

Reviewer #2: No

7 Feb 2020

PONE-D-19-25405R2

Prevalence and effects of gastro-oesophageal reflux during spirometry in subjects undergoing reflux assessment

Dear Dr. Zhou:

I am 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.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

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on behalf of

Professor Fabio Luigi Massimo Ricciardolo

Academic Editor

PLOS ONE