Association between Salivary Substance P and Aspiration on Videofluoroscopic Swallow Study in Infants and Toddlers

Abstract

Objective:

To determine the association between salivary substance P (SP) concentration and aspiration on videofluoroscopic swallow study (VFSS) in children with oropharyngeal dysphagia.

Study Design:

We recruited children <2 years who underwent VFSS within 3 months of study enrollment. Saliva samples were collected using absorbent collection devices and SP levels measured by ELISA. We used student’s t-test to compare levels between subjects with and without aspiration. Exploratory receiver operating characteristic (ROC) analyses were used to determine optimal cut-off values and calculate sensitivity and specificity compared to gold-standard VFSS results. A subgroup analysis included only subjects with saliva collected within 1 month of VFSS.

Results:

The cohort included 50 subjects with age 8.16 ± 0.83 months at sample collection. SP concentrations were 344.7 ± 42.9 pg/ml for patients with aspiration compared to 105.7 ± 40.5 pg/ml for those without aspiration (p<0.001). On ROC analysis, area under the curve (AUC) was 0.868 (95% CI 0.721–1.0, p<0.001). A cutoff of 65 pg/ml provides 97% sensitivity (95% CI 86–99%) and 75% specificity (95% CI 43–95%) for predicting aspiration on VFSS. In a subgroup analysis with 29 subjects who had VFSS within 1 month of sample collection, AUC was 0.964 (95% CI 0.98–1.0, p<0.001). A cutoff of 63.2 pg/ml provides 100% sensitivity (95% CI 85–100%) and 83.3% specificity (95% CI 36–100%) for predicting aspiration on VFSS.

Conclusions:

Salivary substance P concentrations were found to be significantly higher in children with aspiration. Salivary substance P may represent an early candidate biomarker that could be used to predict risk of aspiration in infants and toddlers.

Introduction

Oropharyngeal dysphagia with aspiration is a major cause of severe aerodigestive symptoms in children, with increasing prevalence nationwide[1–6]. Diagnosis of aspiration is particularly difficult in children due to the high prevalence of silent aspiration in this age group[7–10]. However, appropriate diagnosis and treatment of aspiration is essential in order to prevent long-term adverse effects of chronic aspiration, including severe respiratory symptoms, bronchiectasis, and oral aversion[4; 11–13].

The current approach to diagnosis and management of oropharyngeal dysphagia in children is hindered by: 1) lack of sensitivity of observed feedings for diagnosing aspiration and 2) limitations of the gold standard videofluoroscopic swallow study (VFSS), including radiation exposure, limited timeslots available, need for patient cooperation for reliable results, and subjectivity of speech-language pathologist and radiologist interpretations of results since standardized approaches such as penetration-aspiration scale (PAS) scores or BaByVFSS Impairment Profile (VFSS-ImP) have not been universally adopted [7; 9; 14–20]. For these reasons, there is great need for a non-invasive test for aspiration, which may be used to screen, diagnose or monitor progress of pediatric swallow dysfunction[21; 22].

Several potential biomarkers of aspiration risk have been identified in adult patients, but these have not yet been evaluated in children. Substance P (SP) is a neuropeptide signaling molecule which is secreted peripherally due to activation of afferent neurons expressing TRPA1 and is involved in the pathogenesis of swallow dysfunction by 1) decreasing the activation threshold of transient receptor potential (TRP) channels, 2) enhancing the sensitivity of neurons in the pharynx, and 3) increasing cough reflex to improve airway protection[23; 24]. Pilot studies in adult populations have shown reduced SP levels in patients with neurologic causes of aspiration, including the elderly and patients with stroke and Parkinson’s disease[24–28]. In these conditions, alterations in substance P appear to be related to chronically altered signaling pathways, but less is known about dysfunctional signaling pathways in children[25; 28]. SP levels may also be helpful in predicting response to potential therapies for aspiration, such as thickened feedings or neurostimulation treatments[23; 24; 29–32].

Validation of salivary biomarkers in a pediatric population would be transformative in the care of children with oropharyngeal dysphagia by offering a non-invasive approach for predicting risk of aspiration and even potential response to thickening and novel dysphagia therapeutics[7; 33]. Despite significant overlap in pathophysiology with adults, no studies of salivary biomarkers have been performed in children with aspiration[23; 29; 34–36]. The primary aim of this pilot study was to measure salivary substance P levels in young children to determine if this biomarker could be used to predict aspiration risk.

Methods

Patients who were being evaluated for oropharyngeal dysphagia were recruited from the gastroenterology clinics and inpatient units at Boston Children’s Hospital. Subjects were included if they had had their first videofluoroscopic swallow study performed within 3 months of study enrollment and if they were an infant or toddler under 2 years of age at the time of sample collection. As this was a convenience sample of young children undergoing evaluation for oropharyngeal dysphagia, all samples were collected within a few weeks following VFSS when children were seen in clinic. Children were excluded if they had oropharyngeal/craniofacial anomalies, history of oropharyngeal/gastrointestinal procedures, or were being treated with medications that may alter SP signaling such as baclofen or ACE inhibitors due to potential for these factors affecting neuropeptide signaling. Subjects with laryngeal penetration on VFSS were also excluded for this initial validation study.

After informed consent was obtained, saliva samples were collected using infant or child-size absorbent saliva collection devices (salimetrics.com). Once more than 0.2ml of saliva was collected, each saliva sample was extracted using a 5ml syringe into a 1ml microtainer and stored at −80 degrees. Biomarker levels were run in duplicate and analyzed in blinded fashion using commercially available Enzyme-Linked Immunosorbent (ELISA) immunoassays for substance P following manufacturer’s instructions (Abcam, Waltham, MA).

Subjects’ charts were reviewed for clinical data, demographics, comorbidities and medications including proton pump inhibitors (PPI) and H2 receptor antagonists (H2RA). VFSS results were reviewed to determine the highest penetration-aspiration scale (PAS) score seen on each subjects’ most recent VFSS[19]. At our institution, all VFSS are performed and interpreted in standardized fashion with both a radiologist and a speech language pathologist.

Our primary outcome was mean salivary SP concentration in children with aspiration on VFSS compared to those without aspiration on VFSS. We used Chi-square or Fisher’s exact test and the student’s t-test to compare values between groups and linear regression to adjust for covariates. Exploratory receiver operating characteristic (ROC) analyses were used to determine optimal cut-off values based on Youden’s index and to calculate values for the sensitivity and specificity of substance P levels compared to gold standard VFSS results. We additionally performed a subgroup analysis to include only subjects who had saliva samples collected within 1 month of VFSS to further improve accuracy of the test. Positive predictive values (PPV) and negative predictive values (NPV) were calculated based on prevalence estimates. Results are presented as n (percentage) or mean ± standard error. All statistical tests were 2-sided with p<0.05 considered statistically significant. Data were analyzed using SPSS version 27.

This study was approved by the Institutional Review Board at our institution.

Results

Our cohort included 50 subjects with mean age 8.16 ± 0.84 months at the time of sample collection with samples collected between February 2024 and June 2025. Overall, 37 subjects had aspiration with PAS 8 on VFSS, 1 subject had aspiration with PAS 7 on VFSS, and 12 subjects had no aspiration with PAS 1 on VFSS. All aspiration events seen on VFSS were observed to occur during the swallow. Cohort characteristics are shown in Table 1A. There were no significant differences in cohort characteristics between subjects with and without aspiration.

Table 1A:

Full Cohort Characteristics

	Aspiration (n=38)	No Aspiration (n=12)	P-value
Age in Months at Sample Collection	8.28 ± 0.96	8.04 ± 1.8	0.92
Age in Months at VFSS	7.44 ± 0.96	7.56 ± 2.04	0.98
Months between VFSS and Sample Collection	0.82 ± 0.12	1.2 ± 0.31	0.27
Male Sex	17 (45%)	6 (50%)	0.75
Female Sex	21 (55%)	6 (50%)	0.75
History of Prematurity	12 (31%)	4 (33%)	0.91
Gestational Age (Weeks)	29.7 ± 1.3	32.8 ± 2.3	0.27
Neurologic Comorbidity	13 (34%)	2 (17%)	0.3
Cardiac Comorbidity	9 (24%)	2 (17%)	0.61
Receiving PPI	3 (8%)	0 (0%)	0.32
Receiving H2RA	5 (13%)	3 (25%)	0.38

Values reported as mean ± SE or n (%).

In the 50 patients, the SP concentration was 344.7 ± 42.9 pg/ml for those with aspiration compared to 105.7 ± 40.5 pg/ml for those without aspiration (p<0.001) as shown in Figure 1A. There were no differences in SP level when comparing by age, interval between VFSS and sample collection, prematurity, neurologic comorbidities, cardiac comorbidities, or PPI or H2RA treatment status (all p>0.14). SP ranges were noted to vary by sex in aspirating subjects, with aspirating female subjects having higher levels compared to aspirating male subjects (471.4 ± 62.7 vs 188.1 ± 25.5, p<0.001). There was no significant difference in SP level between non-aspirating female and male subjects (90.5 ± 44.7 vs 120.9 ± 71.7, p=0.73). Using a linear regression analysis with adjustment for age, comorbidities and medications, only sex and aspiration were significantly associated with SP levels (B=0.43, p=0.001 for sex and B=0.356, p=0.006 for aspiration). There were no differences in any of the baseline characteristics or comorbidities between males and females (all p>0.18). However, PAS scores were higher in females for slightly thick liquids (6.9 ± 0.6 vs 5.1 ± 0.7, p=0.06) and mildly thick liquids (6.7 ± 0.5 vs 4.5 ± 0.8, p=0.03), suggesting that this difference between males and females may represent severity of dysphagia rather than sex-based differences.

Figure 1A.

Boxplot Comparing Salivary Substance P Concentration between Children with and without Aspiration on VFSS in Full Cohort

On our exploratory ROC analysis for the whole cohort, area under the curve (AUC) was 0.868 (95% CI 0.721 – 1.0, p<0.001) as shown in Figure 2A. Using a concentration cutoff of 65 pg/ml provides 97% sensitivity (95% CI 86–99%) and 75% specificity (95% CI 43–95%) for predicting aspiration on VFSS in children under 2 years of age. Test characteristics with PPV and NPV based on prevalence estimates are shown in Table 2.

Figure 2A.

ROC Curve for Diagnosis of Aspiration in Full Cohort

Table 2:

Test Characteristics Comparing Salivary Substance P to Aspiration on VFSS

	Full Cohort (n=50)	Subgroup with VFSS within 1 Month of Saliva Sample (n=29)
Sensitivity (95% CI)	97% (86–99%)	100% (85–100%)
Specificity (95% CI)	75% (43–95%)	83% (36–100%)
General Population (Estimated Prevalence: 1%)
Positive Predictive Value (95% CI)	4% (2–10%)	6% (1–27%)
Negative Predictive Value (95% CI)	100% (99.8–100)	100% (48–100%)
Infants with Concerning Symptoms (Estimated Prevalence 25%)
Positive Predictive Value (95% CI)	57% (33–78%)	67% (25–92%)
Negative Predictive Value (95% CI)	99% (92–100%)	100% (48–100%)
High Risk Referral Population (Estimated Prevalence 69%)
Positive Predictive Value (95% CI)	90% (77–96%)	93% (69–99%)
Negative Predictive Value (95% CI)	93% (64–99%)	100% (48–100%)

On our sub-analysis comparing only subjects who had saliva samples collected within 1 month of VFSS to further improve accuracy of the test, there were 29 subjects with mean age 8.52 ± 1.08 months at the time of sample collection. In this subgroup, there were 22 subjects with aspiration with PAS 8 on VFSS, 1 subject with aspiration with PAS 7 on VFSS, and 6 subjects without aspiration with PAS 1 on VFSS. Subgroup characteristics are shown in Table 1B. There were no significant differences in characteristics between subjects with and without aspiration in the subgroup.

Table 1B:

Subject Characteristics for Subgroup of Subjects with Saliva Sample Collection within 1 month of VFSS

	Aspiration (n=23)	No Aspiration (n=6)	P-value
Age in Months at Sample Collection	8.76 ± 1.32	7.32 ± 2.52	0.61
Age in Months at VFSS	8.52 ± 1.32	7.08 ± 2.52	0.59
Months between VFSS and Sample Collection	0.27 ± 0.05	0.25 ± 0.11	0.89
Male Sex	10 (44%)	3 (50%)	0.78
Female Sex	13 (56%)	3 (50%)	0.78
History of Prematurity	6 (26%)	4 (67%)	0.14
Gestational Age (Weeks)	27.8 ± 1.6	32.8 ± 2.3	0.1
Neurologic Comorbidity	7 (30%)	1 (17%)	0.65
Cardiac Comorbidity	5 (22%)	2 (33%)	0.61
Receiving PPI	2 (9%)	0 (0%)	0.45
Receiving H2RA	3 (13%)	1 (17%)	0.82

Values reported as mean ± SE or n (%).

For this subgroup, the SP concentration was 250.9 ± 33 pg/ml for those with aspiration on VFSS compared to 57.1 ± 11.8 pg/ml for those without aspiration on VFSS (p<0.001) as shown in Figure 1B. There were no differences in SP level when comparing by age, prematurity, neurologic comorbidities, cardiac comorbidities, or PPI or H2RA treatment status (all p>0.09). SP ranges were also noted to vary by sex in aspirating subjects in this subgroup, with aspirating female infants having higher levels compared to aspirating male infants (309.5 ± 46.4 vs 174.8 ± 35.2, p=0.04). There was no significant difference in SP concentration between non-aspirating female and male subjects (62.5 ± 25.6 vs 51.7 ± 3.4, p=0.72). There were no differences in any of the baseline characteristics between males and females within the subgroup (all p>0.13). PAS scores were higher in females for mildly thick liquids (7.3 ± 0.5 vs 4 ± 1.1, p=0.007).

Figure 1B.

Boxplot Comparing Substance P Concentrations in the Subgroup of Subjects with Saliva Sample Collection within 1 month of VFSS

On our exploratory ROC analysis for the subgroup, AUC was 0.964 (95% CI 0.98–1.0, p<0.001) as shown in Figure 2B. Using a concentration cutoff of 63.2 pg/ml provides 100% sensitivity (95% CI 85–100%) and 83.3% specificity (95% CI 36–100%) for predicting aspiration on VFSS in children under 2 years of age. Test characteristics with PPV and NPV based on prevalence estimates are shown in Table 2.

Figure 2B.

ROC Curve for the Diagnosis of Aspiration in the Subgroup of Subjects with Saliva Sample Collection within 1 month of VFSS

Discussion

We present the first study to evaluate salivary substance P levels in children with oropharyngeal dysphagia. Our goal was to address two key challenges in the diagnosis and management of aspiration in young children, including the high rate of silent aspiration and the limitations of VFSS in this patient population[7–9]. Although findings should be interpreted with caution due to the modest sample size and external validation is required before clinical use, the results of our pilot study suggest that salivary SP may have high sensitivity and specificity in predicting risk of aspiration on VFSS and might hold promise as a potential biomarker of aspiration in children. Given the ease with which saliva samples can be obtained and analyzed for this purpose, this approach may provide major benefits in the clinical management of oropharyngeal dysphagia in children.

In order to evaluate the association between salivary substance P and aspiration on VFSS, we collected saliva samples from 50 infants and toddlers undergoing VFSS for evaluation of oropharyngeal dysphagia. We were able to measure SP by ELISA in all collected samples and found that in our exploratory ROC analysis that levels above 65 pg/ml provided 97% sensitivity and 75% specificity for predicting aspiration on VFSS. We chose the 3 month period between sample collection and VFSS for our primary analysis based on studies showing that improvement in pediatric swallow function tends to occur 4–6 months following diagnosis of aspiration on VFSS[37; 38]. However, it is possible that some subjects would have had a change in their extent of dysphagia within that timeframe since swallowing is dynamic, particularly in young children. We therefore also performed a more conservative subgroup analysis to improve accuracy of the test by evaluating SP levels from samples obtained within 1 month of VFSS, which likely are more closely related to swallow function at the time of sample collection. Results of our subgroup analysis showed improved sensitivity and specificity, with levels above 63.2 pg/ml providing 100% sensitivity and 83.3% specificity for predicting aspiration on VFSS. We do acknowledge, however, that these reported cutoffs are exploratory and internally derived from this pilot cohort of patients. It is also possible that swallow function could have changed over even the shorter 1 month time period.

The potential clinical utility of salivary SP may depend on the risk of aspiration in the population undergoing testing but recent reports suggest there may be increasing prevalence of dysphagia in pediatrics[1; 2]. VFSS is typically performed in infants with concerning symptoms for whom the pre-test probability of finding aspiration is higher, but the population prevalence is not known as there have been no studies of VFSS results in healthy children[39]. Therefore, in order to determine positive and negative predictive values of this test, we evaluated a range of estimated prevalence values, ranging from 1% which may be the case in the general pediatric population, to 25% which has been reported in children with concerning symptoms, and 69% which may be the prevalence in high-risk referral populations[7; 22; 39–43]. These analyses showed positive predictive values ranging from 4% to 93% and negative predictive values ranging from 93% to 100%.

SP levels were not affected by comorbidities or acid suppression medications in our cohort, but it is notable that female subjects with aspiration were found to have significantly higher SP levels compared to male subjects with aspiration. Although comorbidities did not differ between these groups, PAS scores when testing thickened liquids on VFSS were higher for female subjects, suggesting the female group may have had more severe aspiration compared to the male group. This may in part account for the higher SP values in aspirating female subjects in our cohort, but larger studies may be required to better understand this relationship.

Although salivary SP has been measured in infant studies of pain prediction and in adults at risk for aspiration, to our knowledge it has not previously been studied in children with oropharyngeal dysphagia[25; 29; 44; 45]. Prior studies in adult patients and animal models of infant aspiration have shown overlapping pathophysiology of swallow dysfunction in these groups, suggesting that disordered pharyngeal neuropeptide signaling plays a key role in aspiration in both adults and children and this is mediated by SP by way of TRP channels[23; 46–50]. However, in the present study, we found that saliva substance P levels were actually significantly higher in children with aspiration compared to children without aspiration in our cohort, which is in contrast to the findings from adult studies. Our results only show association and do not provide an explanation for this differing relationship but are supported by animal and ex vivo studies suggesting changes in intestinal neuropeptide signaling throughout development[51–55]. Additionally, studies of the salivary transcriptome show differential expression of genes that play a role in successful feeding in premature infants[56–58]. It is also important to note that the most common causes for aspiration in children differ from those in adults, with causes such as stroke and neurodegenerative conditions being most common in adults while neuro-developmental and structural airway causes are most common causes of aspiration in children; further studies of salivary SP may clarify risk of each of these potential etiologies of pediatric aspiration[37; 39; 59; 60]. It is not known whether abnormal SP is causative of aspiration in these children, if it might be a compensatory strategy to improve pharyngeal sensory perception or motor function in aspirating children, or if abnormal SP levels are instead a secondary result of some other cause of dysregulation in pharyngeal coordination. Whatever its role, our results suggest SP may be a useful biomarker of aspiration risk in children.

There are several potential limitations of this study that should be considered. Limitations may include our sample size, which may affect test characteristics, but adult aspiration studies included fewer than 30 subjects[25; 28]. Additionally, our finding of an even stronger relationship between SP levels and aspiration within the subgroup of patients with saliva collected within one month of the VFSS adds further support to the results of our primary analysis. It is also important to note that our study can only demonstrate association but future studies will be required to determine better define causal inference. PAS scores from VFSS were considered the gold-standard in diagnosing aspiration in our cohort but we acknowledge that results of a single VFSS study may not be representative of a given patient’s swallowing over time[22; 61]. We did not include otherwise healthy subjects who had not undergone VFSS in this study due to the need for a gold-standard comparison by which to evaluate swallow function, but this will be an important group for future study in order to improve predictive value of the test. All aspiration events on VFSS were found to occur during swallowing in subjects included in this study, but we note that pathophysiology and SP signaling alteration may differ in cases of oral phase/sensory delay or failure of clearance. No enrolled subjects underwent fiberoptic endoscopic evaluation of swallowing (FEES) as this diagnostic study is not commonly performed at our institution, but a comparison of SP levels with FEES results should be included in future work. We also excluded patients with only laryngeal penetration without aspiration on their VFSS. Although studies suggest that laryngeal penetration can cause similar symptoms to aspiration, there is debate about the clinical significance of laryngeal penetration depending on depth and frequency, hence the exclusion of these patients [31; 62–65]. We also focused on infants and toddlers in the present study since children under 2 years of age with feeding difficulties are at highest risk for silent aspiration[7; 8; 41]. Age was not significantly associated with SP levels in our cohort, but future studies will be required to determine SP ranges in older age groups. We also did not have the underlying cause of aspiration in this group of patients so it is not clear if the levels will vary based on the underlying etiology of swallowing dysfunction. However, regardless of the cause of aspiration, this data suggests that this test could be used to at least screen for who might need a VFSS or who might need thickening while the cause of aspiration is being evaluated. We acknowledge that reported cutoffs are exploratory and internally derived from this pilot cohort of patients and would benefit from additional validation studies in larger cohorts. Finally, it should be noted that testing of salivary SP is experimental and only limited to research studies and therefore not widely available at the present time, but our results suggest access to this testing may be beneficial for vulnerable young children at risk for aspiration.

In conclusion, results of this pilot study suggest that salivary substance P levels are significantly higher in children with aspiration on VFSS compared to children without aspiration. Salivary substance P is an early candidate biomarker requiring external validation before clinical use. However, it may hold promise as a novel biomarker that might predict risk of aspiration in infants and toddlers with high sensitivity and specificity, and may offer a useful screening tool to identify children who should undergo VFSS.

Abbreviations

H2RA

H2 receptor antagonist

PAS

Penetration-aspiration scale

PPI

Proton pump inhibitor

ROC

Receiver operating characteristic

SP

Substance P

VFSS

Videofluoroscopic swallow study

Data Availability Statement:

The data that support the findings of this study are available from the authors upon reasonable request.