Abstract
Oropharyngeal dysphagia frequently occurs in persons living with Alzheimer's disease and related dementias (PLWD) and results in negative health consequences. Strength-based exercises may address swallowing biomechanical impairments. Expiratory muscle strength training (EMST) is an intervention examined in other neurodegenerative populations and has demonstrated promise for improving respiratory muscle strength and airway defense physiologic capacity, potentially improving swallowing safety. We describe a case of a patient with Alzheimer's disease who participated in five consecutive weeks of EMST. We demonstrate that EMST is a feasible intervention for PLWD. Further research should be conducted to assess efficacy and benefit of EMST for PLWD.
Introduction
Oropharyngeal dysphagia, or difficulty moving food, liquid, secretions, or medications through the mouth and throat and into the esophagus, occurs in up to 86% of persons living with dementia (PLWD).1,2 These difficulties are caused by sensory and biomechanical changes to strength and/or muscle coordination, which may lead to impairments in safety (i.e., risk of material entering the airway) and/or efficiency (i.e., risk of post-swallow residue in the oral cavity or throat). Serious negative health-related outcomes are associated with dysphagia, including aspiration pneumonia, malnutrition, and dehydration. 3 The odds of pneumonia-associated mortality increases more than two-fold for PLWD compared to patients with other etiologies.1,4–6 Historically, dysphagia has been characterized as a late stage effect of disease progression; however, studies on persons living with mild Alzheimer's disease (AD) have revealed subclinical changes in neural control of swallowing and swallowing physiology that occur prior to the onset of clinically significant swallowing-related outcomes (e.g., aspiration) are observed.7,8 Other work has noted potential underreporting of dysphagia in early stages of AD, as characterized by lack of agreement between patient report of dysphagia and findings from instrumental swallowing assessments that allow for visualization of the swallow. 9
Common approaches to dysphagia management involve strategies that are reactive (implemented after dysphagia onset) and compensatory in that they alter biomechanics but do not address underlying biomechanical impairments. These include postural adjustments (e.g., chin tuck) or diet modifications (e.g., thickening liquids). These approaches, without use of rehabilitative interventions, may result in loss of muscle strength or maintenance of muscle function leading to decreases in swallowing functional reserve in persons with AD, thereby limiting potential for future rehabilitative efforts, and negatively impact quality of life. Recent studies have found that rehabilitative approaches, such as resistance-based exercise paradigms, are infrequently recommended to PLWD.10,11 To address this, a paradigm shift emphasizing proactive, patient-centered approaches to address disease progression in patients living with neurodegenerative disorders, including AD, has been proposed. 12 Proactive (in contrast to reactive) approaches include: (1) increasing swallowing physiologic reserve by using resistance-based exercise approaches; (2) interdisciplinary care to optimize the patient experience, nutritional outcomes, and swallowing safety; and (3) early discussions around advanced care planning to facilitate shared decision-making to incorporate the patient's goals of care and cultural values. Importantly, the authors note that there has been limited research elucidating the feasibility or efficacy of proactive, exercise-based approaches to target dysphagia in patients living with neurodegenerative diseases.
Expiratory muscle strength training (EMST) is an example of a strength-based intervention for dysphagia that has been studied in other neurodegenerative diseases. Prior work has assessed its feasibility and efficacy for addressing oropharyngeal dysphagia in patients living with Parkinson's disease, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis.13–19 The aims of EMST exercises are to improve respiratory muscle strength and increase subglottic expiratory generative forces to promote airway defense physiologic capacity, with potential for carryover into improved swallowing safety. EMST exercises are typically completed using pressure threshold valve devices (e.g., Philips Respironics Threshold Positive Expiratory Pressure or Aspire EMST150™ devices),20,21 which require sufficient expiratory force generation to release a spring-loaded or magnetic valve set at a specific target threshold. Results of several clinical trials have shown that EMST leads to improvements in maximum expiratory pressure,14,19,22,23 increased peak expiratory flow rate,13,14 and reduction in Penetration-Aspiration Scale scores (i.e., improved swallowing safety).22,24 While EMST has demonstrated promise for patients with other neurodegenerative diseases, no studies to date have assessed the feasibility of an EMST intervention for PLWD. Given that other studies addressing physical frailty through fall prevention interventions, 25 strength training, 26 and aerobic exercise 27 have demonstrated feasibility and positive outcomes in this population, it is likely that proactive exercise-based approaches to address dysphagia, like EMST, may also be feasible for PLWD.
This case study describes the feasibility and tolerability of an EMST approach to improve and maintain swallowing function in a PLWD, with support from care partners. This approach was selected to address unsafe swallowing (i.e., frequent aspiration) and improve peak cough flow for airway clearance. For this case, feasibility was defined as whether the patient completed all 5 weeks of a prescribed regimen and tolerability was defined as whether the patient was able to complete exercises without any reports of fatigue, discomfort, or adverse effects. We hypothesized that (1) this patient and his care partner would demonstrate good adherence to the recommended 5-week regimen, as defined by the percentage of sessions completed in his home exercise program and (2) that his expiratory pressure generation capacity (primary outcome) and voluntary peak cough flow production (secondary outcome) would increase over the duration of the exercise program. This case report was determined to be non-research by our university's Institutional Review Board and the patient signed an informed consent form agreeing to share details regarding his case history and dysphagia management.
Case description
Patient history
An 87-year-old community-dwelling Caucasian male with major neurocognitive disorder (MNCD) presented to the Speech-Language Pathology (SLP) service for dysphagia evaluation (Figure 1). His Functional Assessment Staging Tool 28 score was 4, indicating mild dementia. The patient was referred to the SLP service following comprehensive interdisciplinary assessment by a neuropsychologist and geriatrician as part of the Madison VA Virtual Geriatrics (formerly GRECC Connect) clinical program, 29 during which the patient identified occasional globus sensation (i.e., “food sticking in his throat”) and coughing when eating and drinking. Completion of a neuropsychological test battery revealed a cognitive profile suggestive of AD. This was confirmed following an MRI without contrast completed subsequent to neuropsychological testing which found “generalized cerebral volume loss with probable mild superimposed volume loss involving the hippocampi, which can be seen with AD”. Additionally, review by the staff geriatrician identified probable vascular components as the patient's medical history was significant for multiple transient ischemic attacks as well vascular risk factors including diabetes mellitus, hypertension, and dyslipidemia.
Patient interview and clinical swallow evaluation
The patient was evaluated by a SLP via telemedicine to address dysphagia concerns using a comprehensive assessment battery comprised of clinical dysphagia screening tools (e.g., 3-Ounce Water Swallow Test30,31) and patient-reported outcome measures (e.g., Eating Assessment Tool 32 ) to elicit the frequency and severity of patient-reported and family-reported swallowing symptoms. Details regarding specific measures obtained during the clinical swallow evaluation are provided in the Methods section.
Although the patient endorsed experiencing globus sensation and coughing only occasionally, his wife and children reported that these symptoms occurred every meal, necessitating interruption of meals to expectorate food. The patient's family further identified reduced oral intake; a review of his medical records indicated a 10-pound weight loss in the six months prior to the evaluation. No prior history of SLP evaluation or hospitalization due to aspiration pneumonia was noted.
At the time of clinical swallow evaluation, the patient reported consuming an unrestricted regular consistency diet with thin liquids (i.e., International Dysphagia Diet Standardization Initiative [IDDSI] 33 Level 7 for solids and Level 0 for liquids). Although the patient identified daily use of upper and lower dentures, he reported inconsistent and infrequent oral hygiene. Given symptoms described by the patient and family, a videofluoroscopic swallow study (VFSS) was recommended to characterize swallowing safety, efficiency, and presence of biomechanical impairments (see further description of this evaluation under “Methods” below).

Timeline of dysphagia care.
Methods
All measures described in this section were collected prior to initiation and following completion of EMST therapy to assess change with intervention.
Clinical swallow evaluation measures
Pulmonary function: Expiratory muscle strength. Maximum expiratory pressure (MEP) measures were obtained in accordance with guidelines from the American Thoracic Society (ATS) as part of routine clinical swallow evaluation. Five trials were completed using a handheld respiratory pressure manometer (i.e., microRPM, Vyaire Medical). The highest value of MEP (cmH2O) was recorded. This value was compared against recently published lower limits of normal for the patient's age- and sex-matched decile. 34 MEP values assisted the SLP in identifying the most suitable respiratory threshold trainer to address therapeutic targets.
Airway defense physiologic capacity: Voluntary peak cough flow. Voluntary cough flow was measured via an analog peak flow meter (i.e., Mini-Wright Standard Peak Flow Meter, Mini-Wright) as part of routine clinical swallow evaluation. The patient was seated in an upright position and was instructed to “take a deep breath and cough like something is stuck in your throat”. Five trials were completed and the highest value for peak cough flow (L/min) was recorded. This value was compared against published normative data for healthy older adults. 35
Oral intake: International Dysphagia Diet Standardization Initiative Functional Diet Scale. Recommended diet and liquid consistency levels were based on the International Dysphagia Diet Standardization Initiative (IDDSI), 33 and were subsequently scored using the IDDSI Functional Diet Scale (IDDSI-FDS). 36 The IDDSI-FDS captures the severity of oropharyngeal dysphagia as associated with the recommended degree of diet restriction. A higher score indicates a less restrictive diet, with a score of 0 indicating “nothing by mouth” (i.e., NPO or exclusive non-oral feeding) or a severe level of diet restrictedness whereas a score of 8 indicates an unrestricted diet (i.e., regular solid foods and thin liquids with no modifications).
Patient-reported outcome measure: Eating Assessment Tool. The Eating Assessment Tool (or EAT-10), 32 a 10-item questionnaire assessing dysphagia symptom severity and swallowing-related quality of life was administered to the patient as well as his spouse given presence of MNCD. Individual scores were summed out of a total of 40. On the EAT-10, a score greater than 3 is suggestive of dysphagia 32 and a score of 15 corresponds with increased aspiration risk on an objective evaluation of swallowing. 37
Objective assessment: Videofluoroscopic swallow study (VFSS)
A VFSS was conducted subsequent to the clinical swallow evaluation due to concerns regarding aspiration risk; this study served to characterize swallowing biomechanics and identify deficits for targeted intervention. This same VFSS study was repeated following completion of EMST. The VFSS was conducted on a Siemens Luminos Agile Max and was recorded at 30 frames per second and 30 pulses per second. Varibar products were used consistent with available clinic supply. 38 Bolus administration during the study was consistent with the hospital's standardized clinical protocol, and comprised of: two 5-milliliter trials of thin liquid (IDDSI 0), two 10-milliliter trials of thin liquid (IDDSI 0), two 20-milliliter trials of thin liquid (IDDSI 0), one sequential swallow trial of thin liquid (IDDSI 0), two 5-milliliter trials of mildly thick liquid (IDDSI 2), two 20-milliliter trials of mildly thick liquid (IDDSI 2), one 40-milliliter trial of mildly thick liquid (IDDSI 2), one teaspoon of barium pudding (IDDSI 4), and one solid consistency trial comprised of barium pudding with a Lorna Doone cookie (IDDSI 6). The VFSS images were interpreted through frame-by-frame review by one clinician using VaultStream EasyView™ (Olympus America) and with use of validated tools that included PAS scores 24 and the Modified Barium Swallow Impairment Profile™ (MBSImP). 39 The clinician rating VFSS images completed MBSImP™ certification and training in scoring the PAS. PAS scores were summarized by reporting the maximum score across all swallows, the mode score across all swallows, the overall percent of swallows designated as “safe” (PAS score < 3) or “unsafe” (PAS score ≥ 3), and the overall percent of swallows with “silent” aspiration (tracheal aspiration without sensory or motor response to clear aspirated material; PAS score = 8). MBSImP individual component scores as well as oral and pharyngeal sum scores were also recorded. Individual component scores range from 0 to 4, depending on the component. The highest possible oral and pharyngeal total sum scores are 22 and 29, respectively. Oral and pharyngeal total sum scores were further characterized in terms of severity according to latent class. 40
Education and goals of care discussion
Following VFSS, the patient, his wife, and children participated in an education and goal setting discussion during which aspiration and its consequences were described and options for therapy and/or compensatory approaches (e.g., postural changes, diet modification) were described. The patient and his care partners opted to participate in an EMST regimen to address aspiration and were agreeable to adopt an oral hygiene regimen to promote oral health and reduce the risk of pneumonia associated with frequent aspiration. Based on informed discussion with the patient and family, the patient continued with regular consistency solids (IDDSI 7) and thin liquids (IDDSI 0) with the goal of maintaining quality of life.
Therapy: Expiratory muscle strength training regimen
The initial EMST treatment session was conducted in clinic and consisted of education regarding the EMST regimen and practice with the therapy tool. Given the patient's short-term memory impairment, the patient's wife was present and written instructions were provided. The SLP employed a teach-back approach to promote comprehension and recall. The patient then participated in five consecutive weeks of EMST through the Vet-SHAPE (
The HEP consisted of 25 expiratory repetitions, 5 days per week for 5 weeks, resulting in 625 total repetitions over the prescribed program duration. The device facilitating exercise was a respiratory threshold trainer tool (i.e., EMST150™, Aspire Respiratory Products). 20 The patient was instructed to exercise during weekdays and rest on the weekends, similar to protocols described in the literature previously. 42 Preceding these exercises, the patient completed five voluntary cough flow trials through a digital peak flow meter (MicroLife Peak Flow Meter, MicroLife, USA) capable of measuring patient progress in cough flow generation and adherence to the exercise schedule. Trials were completed prior to completion of EMST exercises to minimize the effects of fatigue influencing peak cough flow generation.
Once weekly via VA Video Connect, 43 the patient's new maximum was determined in collaboration with the SLP. With the SLP's instruction, the patient set the device to the lowest setting (approximately 30 cmH2O) and increased the setting by turning the knob one-quarter turn each time a successful repetition was achieved (i.e., an increase in resistance of approximately 6 cmH2O each quarter turn per guidelines provided via the EMST150™ calibration directions). Opening of the valve was confirmed by the patient's wife and daughter, who also participated in each video session. Once the patient was no longer able to complete a successful repetition (i.e., he was not able to open the valve with expiratory force), the setting was lowered by one-quarter turn to the last successful setting, representing the patient's 1-repetition maximum; these instructions are consistent with other protocols utilizing telehealth for administration of EMST utilizing proxy MEP measures. 44 Two additional trials at this setting were completed to ensure consistency at this pressure. HEP targets were set to 75% of the new maximum for the following week.
Results
All outcome measures at baseline and post-treatment are summarized in Tables 1 and 2.
Modified barium swallow impairment profile individual component scores.
Pre-Treatment and post-treatment swallowing-related outcomes.
EAT-10: Eating Assessment Tool 32
IDDSI-FDS: International Dysphagia Diet Standardization Initiative – Functional Diet Scale 36
PAS: Penetration-Aspiration Scale 24
MBSImP: Modified Barium Swallow Impairment Profile 39
Patient adherence and tolerability
The patient was reported to have good tolerability to the exercise regimen with no reports of fatigue, discomfort, or adverse effects. The patient benefited from verbal cueing provided by the care partner or SLP during the first set of exercises but subsequently was able to independently complete the exercises with no additional support. Objective session data illustrated that the patient completed 24 sessions out of the total prescribed 25-session regimen (96% adherence). The patient and his family participated in 100% of follow-up SLP visits conducted over telehealth.
Pulmonary function
Maximum expiratory pressure following treatment demonstrated a 52% improvement (from 90 cmH2O to 137 cmH2O). The pre-treatment value was noted to be below the lower limits of normal for the patient's age and sex 34 ; post-treatment, this value increased above the lower limits of normal (i.e., 97.9 cmH2O). 34
Voluntary peak cough flow
Voluntary peak cough flow measures demonstrated a 25% improvement following treatment (from 280 L/min to 351 L/min). While the pre-treatment value was not, the post-treatment value was noted to be within the normal range for peak cough flow generation (i.e., 375 L/min). 35
Eating Assessment Tool
The patient's total EAT-10 score decreased by one point following treatment (from 2 to 1 out of 40 possible points). The care partner's total EAT-10 score decreased by five points following treatment (from 7 to 2 out of 40 possible points).
Videofluoroscopic swallow study
The pre-treatment VFSS revealed mild oral and pharyngeal dysphagia (see Table 2 for MBSImP sum scores) according to total sum classification. 40 MBSImP component scores for lip closure (Component 1 score = 2), tongue control (Component 2 score = 2), laryngeal vestibular closure (Component 11 score = 2), tongue base retraction (Component 15 score = 2) reflected the more substantial impairments in the swallow (see Table 1). These biomechanical impairments contributed to impaired swallow efficiency with moderate amounts of oral and pharyngeal residue (residue scale score of 2 out of 4 points). Additionally, airway invasion occurred frequently with 86% of trials considered “unsafe” (PAS ≥ 3 24 ) and 36% of trials with “silent” aspiration (PAS = 8). These events occurred only during liquid trials (i.e., thin liquids and mildly thick liquids) but were not observed during solid trials. Notably, the patient frequently aspirated greater than 25% of bolus volume during airway invasion events (characterized as a gross amount of aspiration). 45 Thickened liquids were trialed but did not improve biomechanical impairments or reduce aspiration events. Compensatory strategies, including the effortful swallow 46 and super supraglottic swallow, 47 were also trialed; these strategies were moderately successful at improving contact and coordination of oropharyngeal structures during the VFSS. However due to the complex sequence of steps and the need for step-by-step verbal instructions to complete these strategies during each swallow, the patient and his family were not receptive to utilize these as part of their home program.
The post-treatment VFSS revealed continued mild dysphagia with consistent MBSImP oral total sum scores and an improvement (decrease) in pharyngeal total sum scores (see Table 1 for MBSImP scores). Specific improvements in laryngeal elevation (Component 8) and laryngeal vestibular closure (Component 11) drove the change in pharyngeal sum score (see Table 1). Swallow efficiency as reflected by the MBSImP oral or pharyngeal residue component scores remained moderately impaired. However, the percentage of “unsafe” swallows (PAS scores ≥ 3) across all boluses decreased from 86% to 50% prior to treatment. Notably, the number of swallows with “silent” aspiration decreased from 36% of swallows pre-treatment to 7% of swallows post-treatment. Furthermore, when aspiration occurred, the amount was reduced to trace (i.e., minimal) amounts only.
Post-treatment follow-up
Post-treatment, care partner report highlighted stable weight, reduced incidents of coughing during meals, and no longer needing to get up from the table to expectorate food. Discussions regarding plans of care included maintenance of EMST exercises (5 sets of 5 exercises, 3 days per week) and oral hygiene regimen. SLP follow-up conducted three months following completion of treatment indicated no new history of pneumonia, no concerns regarding coughing or food expectoration, and continued weight stability. Finally, the patient and care partners expressed appreciation for education and discussion regarding preferences and goals of care prior to the onset of serious health consequences associated with dysphagia.
Discussion
This case report supports the feasibility, tolerability, and potential efficacy of EMST as a behavioral intervention for PLWD to serve the goal of improving swallow function, increasing expiratory muscle strength to support airway defense physiologic capacity, and maintaining quality of life. Notably, adherence to the prescribed follow-up schedule and HEP were higher than usual reports of adherence to dysphagia-related recommendations (22–50%). 48 Furthermore, this case highlights that dysphagia onset can occur in earlier stages of AD progression and should not be designated as solely a late stage sequelae. Recognizing this makes it possible to intervene earlier in the disease to optimize swallowing function for longer into progression and maintain quality of life.
Additionally, this patient consistently under-reported dysphagia at both time points with this being more pronounced prior to treatment. Family reporting of dysphagia-related symptoms was critically important in identifying a potential issue and setting the stage for further assessment and treatment. Inclusion of care partner report in a dysphagia assessment battery as well as other clinical screening measures (e.g., 3-Ounce Water Swallow Test, Test of Masticating and Swallowing Solids (TOMASS) 49 ) will be essential to determining risk for dysphagia in PLWD.
Furthermore, while dysphagia will likely occur in PLWD at some point in the disease, a proactive oropharyngeal strengthening regimen such as expiratory muscle strength training earlier in the disease process may enable patients to improve swallowing outcomes (e.g., safety), increase functional reserve for swallowing, and maintain a regular or minimally modified diet for a longer period of time. This leads to improved quality of life, optimized health through nutrition, and reduced care partner burden. For this patient and his family, changes to dietary intake were not observed using the IDDSI-FDS but this is likely due to their decision not to use dietary modifications given that they did not align with goals of care. Despite the lack of change in the IDDSI-FDS, the patient's weight stabilized following treatment which may reflect increased overall oral intake. It is also possible that the variety of foods consumed may have expanded, leading to improved nutrition. Also, improved oral hygiene combined with less frequent occurrences of airway invasion with liquids and, when aspiration did occur, more consistent responsiveness (e.g., cough, throat clear) may have allowed for maintenance of pulmonary health for this patient, even in the context of chronic dysphagia and aspiration. Furthermore, the patient and family noted improvement in swallowing-related quality of life, as characterized by changes in the EAT-10, and described fewer interruptions during mealtimes associated with coughing or needing to get up from the table to expectorate food.
Additionally, it appears from this case that the EMST intervention impacted airway closure and clearance more than pharyngeal clearance given that safety outcomes (e.g., aspiration) improved substantially but efficiency (e.g., residue) did not. PLWD with various swallowing impairments may benefit from a combination of interventions targeted to different aspects of swallowing biomechanics with these used simultaneously or sequentially, depending upon the capacity of the patient and care partner to support the patient. Future work should examine the benefit of EMST and other rehabilitative interventions on oropharyngeal dysphagia in larger cohort studies and clinical trials.
We acknowledge that there are limitations to this case study which present an opportunity for exploration in future work. Although the patient exhibited improvement across all objective measures including those that were observed on his post-treatment VFSS, we were not able to objectively assess maintenance of his functional improvement over a longer period of time. This was due to health issues experienced by his primary care partner that required prioritization. In addition, although a maintenance program was prescribed, no objective adherence data are available and detraining effects are unknown. Finally, given that this patient was in the mild stages of disease, it may be informative to observe whether EMST is preserved as an implicit skill in later stages of the disease, potentially offering an opportunity for maintenance of airway defense physiologic capacity through performance of the task at lower threshold levels. Future work should incorporate longer follow-up durations to examine maintenance of function and skill.
It should also be noted that this patient completed all follow-up visits via telehealth as he and his care partner resided in a rural area over 40 miles away from the main medical center and relied on their daughters for transportation. This necessitated the use of proxy MEP utilizing the threshold device to re-establish weekly targets. 44 This approach, which contrasts from other published EMST protocols that use a respiratory pressure meter to re-establish weekly targets in home visits conducted by an SLP, was a practical decision as it was not feasible for the patient, care partner, and other family members to travel to clinic on a once-weekly basis for re-calibration. While there are limitations in how the threshold trainer (i.e., EMST150™) quarter turn values may correspond with MEP values obtained by a respiratory pressure meter, formulas are available to support approximating the number of quarter turns on the device to corresponding expiratory pressure values. Our protocol for systematically increasing threshold trainer targets is consistent with other protocols utilizing telehealth for delivery of EMST 44 and is a critical consideration when taking into account accessibility and translation of interventions into day-to-day clinical practice.
Despite these limitations, this case study provides preliminary evidence that EMST is feasible, well-tolerated, and may be efficacious in improving oropharyngeal swallow function, with potential benefit for the growing number of individuals living with AD.
Footnotes
Acknowledgements
The manuscript was prepared at the William S. Middleton Memorial Veterans Hospital in Madison, WI (GRECC Manuscript 07-2025).
Products included or mentioned in this manuscript are not endorsed by any of the authors. This work was funded by the Department of Veterans Affairs, Veterans Health Administration, Office of Rural Health, NOMAD PROJFY-010196 and 009734. The views and content expressed in this article are solely the responsibility of the authors and do not necessarily reflect the position, policy, or official views of the NIH or Department of Veterans Affairs.
Ethical considerations
This case report was determined to be non-research by University of Wisconsin-Madison Institutional Review Board.
Consent to participate
The patient signed an informed consent form agreeing to share details regarding their case history and dysphagia management.
Author contributions
Funding
The authors acknowledge funding support as follows: JPY: Department of Veterans Affairs, Office of Rural Health; William S. Middleton Veterans Affairs Hospital, Madison, WI; University of Wisconsin-Madison ADEPT Program, supported by UW SMPH Wisconsin Partnership Program (WPP 5132) and National Institutes of Health-National Center for Advancing Translational Sciences Clinical and Translational Science Award (CTSA) 1UL1TR002373. NRP: University of Wisconsin—Madison; William S. Middleton Veteran Affairs Hospital, Madison, WI; and National Institutes of Health grant number K76AG068590.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data availability statement
Data sharing is not applicable to this article as no new datasets were generated during this study.
