Pilot study evaluating salivary bile acids as a diagnostic biomarker of laryngopharyngeal reflux.

Bile acids in refluxate contribute to esophageal and laryngeal symptoms and are quantifiable. The aim of this study was to compare salivary bile acid concentrations across healthy controls and symptomatic patients (esophageal or laryngeal) with or without objective gastroesophageal reflux disease (GERD). This prospective study enrolled adults into three groups: esophageal symptoms (heartburn, regurgitation, chest pain); laryngeal symptoms (cough, throat clearing, sore throat, dysphonia); and controls. Symptomatic patients primarily underwent prolonged wireless reflux monitoring off acid suppression and were categorized as symptomatic no GERD (acid exposure time <4%) or esophageal/laryngeal symptoms with GERD (acid exposure time ≥4%). Controls did not undergo reflux monitoring nor upper endoscopy. Saliva samples were provided for bile acid analysis via ultraperformance liquid chromatography tandem mass spectrometry. Thirty-five participants were enrolled (mean age 47.4 years [SD 18.9], 16 [46%] male), including 10 controls and 25 symptomatic: 9 no GERD, 5 esophageal symptoms + GERD, and 11 laryngeal symptoms + GERD. Total salivary bile acids were highest in the laryngeal symptoms + GERD group (24.2 nM [SD 24.7]) compared to other groups (controls: 5.8 [6.0], P = 0.03; symptomatic no GERD: 3.1 [4.4]; P < 0.01; esophageal symptoms + GERD: 7.1 [7.1], P = 0.10). Bile acids were elevated in 45% (5/11) of the laryngeal symptoms + GERD group compared to 0% of the other three groups (P < 0.01). Salivary bile acids were higher among patients with laryngeal symptoms and objective GERD versus other groups. Salivary bile acids are a quantifiable biomarker with diagnostic potential for laryngopharyngeal reflux.

Does hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring for the diagnosis of laryngopharyngeal reflux have to be 24 h?

PURPOSE: To investigate the diagnostic value of combined multi-timepoint salivary pepsin testing (MTPSPT) and hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring (HEMII-pH) for laryngopharyngeal reflux (LPR) and whether an appropriate reduction in the duration of HEMII-pH would affect the accuracy of diagnosis of LPR. METHODS: Recruited patients were studied with both MTPSPT and HEMII-pH. The diagnosis of LPR was based on the occurrence of > 1 reflux event and/or positive results on any of the MTPSPT. The diagnostic value of combined diagnosis was studied through combining a breakdown of the 24-h HEMII-pH finding and the results of the MTPSPT. The diagnostic value was expressed in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: Based on 24-h HEMII-pH and MTPSPT, the positive rate of LPR was 83.33% and 74.69%, respectively. According to the combined diagnosis, the positive rate of LPR was 90.74%. The sensitivity and specificity of the combined diagnosis both were 89.51% and 100%, when the HEMII-pH intervals were 7 a.m.-6 p.m. and 7 a.m.-7 p.m., respectively. However, when the monitoring time was extended to 8 p.m. and bedtime, the sensitivity, specificity, PPV and NPV of the combined diagnosis both were 100%. CONCLUSIONS: The combination of MTPSPT and HEMII-pH increased the sensitivity and accuracy of diagnosis of LPR. For patients with positive MTPSPT results, the duration of HEMII-pH can be appropriately shortened to reduce patient sufferings.

Impact of bile acids on the severity of laryngo-pharyngeal reflux.

OBJECTIVES: The primary end point of this study was to evaluate the impact of bile acids on severity of laryngo-pharyngeal reflux (LPR) and the possible correlation with esophagitis and upper airway malignancies. The second end point was to evaluate if salivary bile acids and molecules other than pepsin might serve as diagnostic biomarkers of LPR. DESIGN: Observational prospective comparative study. SETTING: Otorhinolaryngology unit of a tertiary hospital. PARTICIPANTS: Sixty-two consecutive adult outpatients suspected of LPR. MAIN OUTCOME MEASURES: Bile acids, bilirubin and pepsinogen I-II were measured in saliva. Patients underwent pH metry and based on the results of bile acids were subdivided as acid, mixed and alkaline LPR. RESULTS: Significantly higher Reflux Findings Score (RFS) and Reflux Symptoms Index (RSI) were seen in patients with alkaline and mixed LPR compared to acid LPR. Salivary bile acids >1 µmol/L seem to be a reliable indicator of the severity of LPR. Compared to those without, patients with esophagitis or a history of upper airway malignancy have high concentrations of bile acids in saliva. Among the molecules studied, bile acids were the most suitable for diagnosis of LPR, with a sensitivity of 86% and a positive predictive value of 80.7%. CONCLUSIONS: Our data suggest that high concentrations of bile acids are associated with higher values of RSI and RFS in LPR as well as a higher risk of esophagitis and history of upper airway malignancies. We finally observed that bile acids provided the best biometric parameters for diagnosis of LPR among the molecules tested.

Association of pepsin and DNA damage in laryngopharyngeal reflux-related vocal fold polyps.

PURPOSE: This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR. METHODS: Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously. Hematoxylin-eosin staining was performed for pathologic analysis. Immunohistochemistry and ELISA were used to detect pepsin in tissue and saliva of VFP patients. 8-OHdG and p-H2AX expression was detected to measure oxidative DNA damage in tissue. DNA damage was investigated in human immortalized laryngeal epithelial cells exposed to pepsin. RESULTS: The pepsin concentration in saliva was significantly higher (t = 2.38, P = .024) in the pepsin positive group. There was no significant difference in pepsin expression at different sites and pathological subtypes of VFPs. The levels of 8-OHdG and p-H2AX were significantly higher in the pepsin positive group and positively correlated with the tissue expression of pepsin. The concentration of pepsin in saliva also showed a significant correlation with 8-OHdG levels. Expression of 8-OHdG and p-H2AX, and tail moment of the comet assay were elevated in human immortalized laryngeal epithelial cells following treatment with pepsin. CONCLUSION: Patients with VFPs have higher levels of oxidative DNA damage in the presence of pepsin reflux. Pepsin may induce DNA damage in laryngeal epithelial cells and participate in the pathogenesis of VFPs.

Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux.

Salivary pepsin is a promising marker for the non-invasive diagnosis of laryngopharyngeal reflux (LPR). For reliable results regarding pepsin in saliva, it is critical to standardize the collection, storage, and pre-processing methods. In this study, we optimized the saliva collection protocols, including storage conditions, i.e., solution, temperature, and time, and the pre-processing filter for pepsin. Moreover, we prepared a simple immunochromatographic strip for the rapid detection of pepsin and evaluated its sensing performance. As a result, we selected a polypropylene (PP) filter as the pre-processing filter for salivary pepsin in low resource settings, such as those where point of care testing (POCT) is conducted. This filter showed a similar efficiency to the centrifuge (standard method). Finally, we detected the pepsin using gold nanoparticles conjugated with monoclonal pepsin antibody. Under optimized conditions, the lower limit of detection for pepsin test strips was determined as 0.01 µg/mL. Furthermore, we successfully detected the salivary pepsin in real saliva samples of LPR patients, which were pre-processed by the PP filter. Therefore, we expect that our saliva collection protocol and pepsin immunochromatographic strip can be utilized as useful tools for a non-invasive diagnosis/screening of LPR in POCT.

Development of scores assessing the refluxogenic potential of diet of patients with laryngopharyngeal reflux.

OBJECTIVE: To develop clinical tools assessing the refluxogenic potential of foods and beverages (F&B) consumed by patients with laryngopharyngeal reflux (LPR). METHODS: European experts of the LPR Study group of the Young-Otolaryngologists of the International Federation of Oto-rhino-laryngological societies were invited to identify the components of Western European F&B that would be associated with the development of LPR. Based on the list generated by experts, four authors conducted a systematic review to identify the F&B involved in the development of esophageal sphincter and motility dysfunctions, both mechanisms involved in the development of gastroesophageal reflux disease and LPR. Regarding the F&B components and the characteristics identified as important in the development of reflux, experts developed three rational scores for the assessment of the refluxogenic potential of F&B, a dish, or the overall diet of the patient. RESULTS: Twenty-six European experts participated to the study and identified the following components of F&B as important in the development of LPR: pH; lipid, carbohydrate, protein composition; fiber composition of vegetables; alcohol degree; caffeine/theine composition; and high osmolality of beverage. A total of 72 relevant studies have contributed to identifying the Western European F&B that are highly susceptible to be involved in the development of reflux. The F&B characteristics were considered for developing a Refluxogenic Diet Score (REDS), allowing a categorization of F&B into five categories ranging from 1 (low refluxogenic F&B) to 5 (high refluxogenic F&B). From REDS, experts developed the Refluxogenic Score of a Dish (RESDI) and the Global Refluxogenic Diet Score (GRES), which allow the assessment of the refluxogenic potential of dish and the overall diet of the LPR patient, respectively. CONCLUSION: REDS, RESDI and GRES are proposed as objective scores for assessing the refluxogenic potential of F&B composing a dish or the overall diet of LPR patients. Future studies are needed to study the correlation between these scores and the development of LPR according to impedance-pH study.

Role of Matrix Metalloproteinases 7 in the Pathogenesis of Laryngopharyngeal Reflux: Decreased E-cadherin in Acid exposed Primary Human Pharyngeal Epithelial Cells.

Cleavage of E-cadherin and the resultant weakness in the cell-cell links in the laryngeal epithelium lining is induced by exposure to acidic contents of the refluxate. Herein, we aimed to evaluate the role of matrix metalloproteinases (MMPs) in inducing E-cadherin level changes following acid exposure to the human pharyngeal mucosal cells. E-cadherin levels were inversely correlated with the duration of acid exposure. Treatment with actinonin, a broad MMP inhibitor, inhibited this change. Immunocytochemical staining and transepithelial permeability test revealed that the cell surface staining of E-cadherin decreased and transepithelial permeability increased after acid exposure, which was significantly inhibited by the MMP inhibitor. Among the various MMPs analyzed, the mRNA for MMP-7 in the cellular component was upregulated, and the secretion and enzymatic activity of MMP-7 in the culture media increased with the acid treatment. Consequently, MMP-7 plays a significant role in the degradation of E-cadherin after exposure to a relatively weak acidic condition that would be similar to the physiologic condition that occurs in Laryngopharyngeal reflux disease patients.

Does gene expression in laryngeal subsites differ between patients with laryngopharyngeal reflux and controls?

OBJECTIVE: To identify laryngeal mRNA gene changes in patients with laryngopharyngeal reflux (LPR). METHOD: Laryngeal biopsies from non-smoking LPR patients (n=10; Reflux Symptom Index (RSI) >12 and a Reflux Finding Score (RFS) >6) and controls (n=9; RSI <12 and RFS <6) were collected from four subsites (true vocal cord, false vocal cord, medial arytenoid and posterior commissure) of the larynx. qRT-PCR analyses were conducted on 20 reflux- and inflammation-related genes, including interleukins 6 and 8, cytokeratins 8 and 14, mucin genes MUC1, MUC2, MUC3B, MUC4, MUC5B, MUC6 and MUC7 and carbonic anhydrase III. Statistical analysis (Mann-Whitney U test) compared gene expression levels between LPR and control groups at each subsite. RESULTS: Site-specific differences in squamous metaplasia and gene expression were noted in LPR patients, with the majority present in the medial arytenoid region. Significant.differences were noted in genes related to mucosal defence and inflammation, including CRNN, CD1d, TGFß-1, MUC2, MUC5B and CDH1. CONCLUSION: Whilst the posterior commissure is commonly identified as the area demonstrating the most significant macroscopic change in LPR, the histological changes and genes assessed here showed more pronounced LPR associated differences in the medial arytenoid. We identified differences in expression of mucin genes, cytokeratin-14 and molecular markers of inflammation. Whilst some of these changes may be metaplasia-related, further evaluation of the mRNA expression of these genes may provide a useful biomarker panel for diagnosis and therapeutic monitoring of LPR.

The importance of combined 24-hour multichannel intraluminal impedance-pH monitoring in the evaluation of children with suspected laryngopharyngeal reflux.

OBJECTIVE: The aim of this study was to investigate the diagnostic usefulness of combined multichannel intraluminal impedance-pH (MII-pH) monitoring in children with suspected laryngopharyngeal reflux (LPR). DESIGN, SETTING AND PARTICIPANTS: A prospective study including children in whom, due to LPR suggestive symptoms, MII-pH monitoring was performed at tertiary medical centre from February 2012 to July 2015. INTERVENTIONS: All included children underwent same diagnostic protocol which included examination by single pulmonologist and ENT specialist and underwent 24-hour MII-pH monitoring. MAIN OUTCOMES: Primary outcome was to determine MII-pH characteristics of the children in whom LPR was suspected based on symptoms and ENT examination. RESULTS: One hundred and four patients (mean age 8.9 years; range 0.4-17.9 years; male/female 57/47) participated in the study. In children with signs and symptoms suggestive of LPR, MII-pH monitoring found the median incidence of proximal gastro-oesophageal reflux (GER) of 15 (range 0-129), proximal acidic GER of 6.5 (range 0-66) and weakly acidic GER of 5 (range 0-102). There were significant positive correlations between the number of GER (proximal total, acidic and weakly acid) with Reflux Finding Score, Reflux Symptom Index and presence of eosinophils in nasal swabs. The only endoscopy ENT finding which significantly correlated with total proximal GER, acid proximal GER and weakly acidic proximal GER was arytenoid hyperaemia. CONCLUSION: Both acid and non-acid reflux seem to have a significant role in the pathogenesis of LPR.

Hypopharyngeal pepsin and Sep70 as diagnostic markers of laryngopharyngeal reflux: preliminary study.

INTRODUCTION: The management of laryngopharyngeal reflux (LPR) has been challenging. Hypopharyngeal multichannel intraluminal impedance (HMII) has shown to increase the sensitivity in diagnosing LPR. The objective of this study is to investigate the potential use of pepsin and Sep70 as diagnostic tools for detection of LPR in combination with HMII. MATERIALS AND METHODS: Tissue samples of hypopharynx, distal esophagus, and gastric cardia were collected from patients with LPR symptoms regardless of gastroesophageal reflux (GERD) diagnosis and underwent HMII to detect LPR and high esophageal reflux (HER: reflux 2 cm distal to upper esophageal sphincter) events. Patients were classified into two groups based on the presence of abnormal proximal exposure (APE), which was defined as LPR ≥1/day and/or HER ≥5/day: (1) positive-APE and (2) negative-APE. Patients with typical GERD symptoms without LPR symptoms who did not undergo HMII were used as a "control" GERD group. Protein was isolated from tissue samples and Western blot analysis of pepsin and Sep70 was performed. Pepsinogen was used as a control to differentiate pepsin from pepsinogen. Relative quantitation was performed using Image Studio Lite Software with normalization against the internal actin of each blot. RESULTS: From October 2012 to September 2013, 55 patients underwent HMII. Of 55, 20 patients underwent biopsies from hypopharynx (17 positive-APE and 3 negative-APE). Ten patients with typical GERD symptoms were identified from tissue bank as a "control" GERD group. Pepsin was detected in distal esophagus and hypopharynx in all groups without significant difference among groups. However, Sep70 in distal esophagus and hypopharynx was significantly depleted in the positive-APE group compared to the other groups (p = 0.032 and 0.002, respectively). CONCLUSION: Depletion of Sep70 with the presence of pepsin in the hypopharynx may indicate cellular injury in laryngopharynx due to constant proximal reflux. However, the normative data for these markers have to be validated.

Pepsin and bile acids in saliva in patients with laryngopharyngeal reflux - a prospective comparative study.

OBJECTIVES: Laryngopharyngeal reflux (LPR) and biliary duodenogastric reflux can cause damage to the laryngeal mucosa and voice disorders. The aim of this study was to find out whether levels of pepsin and bile acids in the saliva can serve as diagnostic markers of LPR. SETTING: A prospective comparative study. PARTICIPANTS: Twenty-eight patients with LPR proven via high-resolution manometry and combined multichannel intraluminal impedance and 24-h pH monitoring and 48 healthy controls without symptoms of LPR were included in the study. MAIN OUTCOME MEASURES: In the patients with LPR symptoms, oesophagogastroscopy with oesophageal biopsy was performed. The levels of total pepsin, active pepsin, bile acids and the pH of the saliva were determined in all participants and compared between the groups. Reflux symptom index (RSI) and reflux finding score (RFS) were also obtained and compared. The groups differed significantly in RSI (P = 0.00), RFS (P = 0.00), the levels of bile acids (P = 0.005) and total pepsin in saliva (P = 0.023). The levels of total pepsin and bile acids were about three times higher in the patients with LPR than in the healthy controls. There was a significant correlation between the RSI and RFS score and the level of total pepsin and bile acids in the saliva. Histopathological examination of the oesophageal biopsy taken 5 cm above the lower oesophageal sphincter confirmed reflux in almost 93% of patients with symptoms. CONCLUSIONS: The study results show that the levels of total pepsin and bile acids in saliva are significantly higher in patients with LPR than in the controls, thus suggesting this as a useful tool in the diagnosis of LPR and particularly biliary LPR.

Potential anti-inflammatory effect of low-level laser therapy on the experimental reflux laryngitis: a preliminary study.

The most common cause of laryngitis is the laryngopharyngeal reflux disease. The symptoms of laryngitis can be hoarseness, globus, chronic cough, voice fatigue, throat pain, and dysphagia. Low-level laser therapy (LLLT) is beneficial to reduce the pain and inflammatory response without side effects. Therefore, LLLT may be a useful tool for the treatment of laryngitis. This study proposes to analyze the effect of laser therapy in a model of reflux-induced laryngitis. The animals were randomly put into three groups: control--non-intubated; nasogastric intubation--intubated; and nasogastric intubation with laser therapy-intubated treated with 105-J/cm(2) laser irradiation. For the induction of laryngitis, the animals were anesthetized and a nasogastric tube was inserted through the nasopharynx until it reached the stomach, for 1 week. Thereafter, measurement of myeloperoxidase activity and the histopathological procedures were performed. In conclusion, we observed in this study that 105-J/cm(2) infrared laser reduced the influx of neutrophils in rats, and it improved the reparative collagenization of the laryngeal tissues.

Digestive Biomarkers of Laryngopharyngeal Reflux: A Preliminary Prospective Controlled Study.

OBJECTIVE: To investigate the digestive enzymes and biomarkers in the saliva of patients with laryngopharyngeal reflux (LPR) and asymptomatic individuals. STUDY DESIGN: Prospective controlled study. SETTING: Multicenter study. METHODS: Patients with LPR at the hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH) and asymptomatic individuals were consecutively recruited from January 2020 to April 2023 from 2 University Hospitals. The saliva of patients (off PPIs) and asymptomatic individuals was collected to measure pH, elastase, bile salts, cholesterol, gastric, and pancreatic lipases. Anxiety, symptoms, and findings were studied through perceived stress scale (PSS), reflux symptom score (RSS), and reflux sign assessment (RSA). RESULTS: Sixty-seven LPR patients and 57 asymptomatic individuals completed the evaluations. LPR patients reported higher PSS, RSS, and RSA than asymptomatic individuals. The mean saliva pH was more alkaline in LPR patients (7.23: 95% confidence interval [CI]: 7.08, 7.38) compared to controls (6.13; 95% CI: 5.95, 6.31; P = .001). The mean concentration of elastase was higher in patients (51.65 µg/mL; 95% CI: 44.47, 58.83 µg/mL) versus asymptomatic individuals (25.18 µg/mL; 95% CI: 21.64, 28.72 µg/mL; P = .001). The saliva cholesterol reported higher concentration in healthy individuals (3.43 mg/dL; 95% CI: 3.21, 3.65 mg/dL) compared to patients (1.16 mg/dL; 95% CI: 1.05, 1.27 mg/dL; P = .001). The saliva pH, and elastase concentration were significantly associated with the baseline RSS, while saliva cholesterol was negatively associated with the severity of RSS and RSA. CONCLUSION: Cholesterol, bile salts, and elastase are biomarkers of LPR and should be considered to develop future non-invasive saliva device for the detection of LPR.

[Laryngopharyngeal Reflux (LPR) in patients with persistent hoarseness]. / Refluks krtaniowo-gardlowy (LPR) u pacjentów z przewlekla chrypka

INTRODUCTION: In 2006 The Global Consensus Group in Montreal pointed out that chronic laryngitis is highly associated with gastroesophageal reflux disease (GERD). AIM OF THE STUDY: To evaluate the frequency of LPR in a selected group of patients with chronic hoarseness. We were also interested in assessment of the relationship between Reflux Symptoms Index (RSI) scores, Ryan scores from the pharyngeal pH monitoring and the morphological changes in the larynx according to Reflux Findings Score (RFS). In addition, we wanted to assess the frequency of various clinical symptoms included in the RSI questionnaire among patients with LPR. MATERIALS AND METHODS: 42 patients from an outpatient ENT clinic with chronic hoarseness and RSI ≥ 13. All subjects underwent pharyngeal pH monitoring with the Dx-pH System Restech ™ and laryngoscopy. RESULTS: Among 42 patients with chronic hoarseness, LPR was confirmed in 35 patients (83.33%). In 7 subjects pharyngeal pH monitoring was normal. Among all patients with confirmed LPR, only 5 out of 8 elements of RFS laryngoscopic changes were observed. The most frequent inflammatory changes noticed included erythema of the arytenoids and interarytenoid regions (posterior laryngitis). These findings were found in 30 out of 35 patients with LPR. Median value of RFS in patients with LPR was 4.45, which is lower than the cut off value of 7 necessary for recognition of LPR. There is statistically significant positive correlation between Ryan scores and the RFS scale results (correlation coefficient 0.91, p<0.001). CONCLUSIONS: Pharyngeal pH monitoring confirmed LPR in 83.33% selected group of patients with chronic hoarseness and RSI ≥ 13. Isolated erythema of arytenoid and interarytenoid region was the most frequent inflammatory abnormality found in the larynx. RFS values below 7 do not exclude the diagnosis of LPR. We can use RFS scales as a prognostic test of severity of LPR - due to statistically significant positive correlation between Ryan score and RFS values. The use of RSI scale revealed that the most frequent symptom among patient with LPR was throat clearing followed by hoarseness.

Pepsin detection in patients with laryngopharyngeal reflux before and after fundoplication.

BACKGROUND: Some patients with gastroesophageal reflux disease (GERD) suffer from laryngopharyngeal reflux (LPR). There is no reliable diagnostic test for LPR as there is for GERD. We hypothesized that detection of pepsin (a molecule only made in the stomach) in laryngeal epithelium or sputum should provide evidence for reflux of gastric contents to the larynx, and be diagnostic of LPR. We tested this hypothesis in a prospective study in patients with LPR symptoms undergoing antireflux surgery (ARS). METHODS: Nine patients undergoing ARS for LPR symptoms were studied pre- and postoperatively using a clinical symptom questionnaire, laryngoscopy, 24-h pH monitoring, biopsy of posterior laryngeal mucosa, and sputum collection for pepsin Western blot assay. RESULTS: The primary presenting LPR symptom was hoarseness in six, cough in two, and globus sensation in one patient. Pepsin was detected in the laryngeal mucosa in eight of nine patients preoperatively. There was correlation between biopsy and sputum (+/+ or -/-) in four of five patients, both analyzed preoperatively. Postoperatively, pH monitoring improved in all but one patient and normalized in five of eight patients. Eight of nine patients reported improvement in their primary LPR symptom (six good, two mild). Only one patient (who had negative preoperative pepsin) reported no response to treatment of the primary LPR symptom. Postoperatively, pepsin was detected in only one patient. CONCLUSIONS: Pepsin is often found on laryngeal epithelial biopsy and in sputum of patients with pH-test-proven GERD and symptoms of LPR. ARS improves symptoms and clears pepsin from the upper airway. Detection of pepsin improves diagnostic accuracy in patients with LPR.

Low-acid diet for recalcitrant laryngopharyngeal reflux: therapeutic benefits and their implications.

OBJECTIVES: Laryngopharyngeal reflux (LPR) is an expensive, high-prevalence disease with a high rate of medical treatment failure. In the past, it was mistakenly believed that pepsin was inactive above pH 4; however, human pepsin has been reported to be active up to pH 6.5. In addition, it has been shown by Western blot analysis that laryngeal biopsy samples from patients with symptomatic LPR have tissue-bound pepsin. The clinical impact of a low-acid diet on the therapeutic outcome in LPR has not been previously reported. To provide data on the therapeutic benefit of a strict, virtually acid-free diet on patients with recalcitrant, proton pump inhibitor (PPI)-resistant LPR, I performed a prospective study of 20 patients who had persistent LPR symptoms despite use of twice-daily PPIs and an H2-receptor antagonist at bedtime. METHODS: The reflux symptom index (RSI) score and the reflux finding score (RFS) were determined before and after implementation of the low-acid diet, in which all foods and beverages at less than pH 5 were eliminated for a minimum 2-week period. The subjects were individually counseled, and a printed list of acceptable foods and beverages was provided. RESULTS: There were 12 male and 8 female study subjects with a mean age of 54.3 years (range, 24 to 72 years). The symptoms in 19 of the 20 subjects (95%) improved, and 3 subjects became completely asymptomatic. The mean pre-diet RSI score was 14.9, and the mean post-diet RSI score was 8.6 (p = 0.020). The mean pre-diet RFS was 12.0, and the mean post-diet RFS was 8.3 (p < 0.001). CONCLUSIONS: A strict low-acid diet appears to have beneficial effects on the symptoms and findings of recalcitrant (PPI-resistant) LPR. Further study is needed to assess the optimal duration of dietary acid restriction and to assess the potential role of a low-acid diet as a primary treatment for LPR. This study has implications for understanding the pathogenesis, cell biology, and epidemiology of reflux disease.

Saliva Pepsin Concentration of Laryngopharyngeal Reflux Patients Is Influenced by Meals Consumed Before the Samples.

OBJECTIVES/HYPOTHESIS: To assess the impact of diet on the saliva pepsin concentration of patients with laryngopharyngeal reflux (LPR). STUDY DESIGN: Non-controlled Prospective Study. METHODS: Patients with positive LPR regarding hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH) were enrolled from three European Hospitals. Patients collected three saliva samples, respectively, in the morning (fasting), and 1 to 2 hour after lunch and dinner. Patients carefully detailed foods and beverages consumed during meals and before the pepsin samples. The 3-month treatment was based on the association of diet, proton pump inhibitors, alginate, or magaldrate regarding the HEMII-pH characteristics. Reflux Symptom Score (RSS) and Reflux Sign Assessment (RSA) were used for assessing the pre- to posttreatment clinical evolution. The Refluxogenic Diet Score and the Refluxogenic Score of a Dish (RESDI) were used to assess the refluxogenic potential of foods and beverages. The relationship between saliva pepsin concentration, HEMII-pH, RESDI, RSS, and RSA was investigated through multiple linear regression. RESULTS: Forty-two patients were included. The saliva pepsin concentration of the 24-hour period of testing was significantly associated with foods and beverages consumed during the testing period and the evening dinner (rs = 0.973, P < .001). RSS and RSA significantly improved throughout treatment. The level of saliva pepsin in the morning was a negative predictive factor of the therapeutic response regarding RSA and RSS (P < .036). CONCLUSIONS: Foods and beverages may significantly impact the saliva pepsin concentration of patients with LPR. Patients with high-level saliva pepsin in the morning had lower therapeutic response compared with those with low-level saliva pepsin. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:350-359, 2021.

Diagnostic Utility of Salivary Pepsin as Compared With 24-Hour Dual pH/Impedance Probe in Laryngopharyngeal Reflux.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is defined as the retropulsion of gastric contents into the larynx, oropharynx, and/or nasopharynx. The 24-hour combined hypopharyngeal-esophageal multichannel intraluminal impedance with dual pH probe (24h-HEMII-pH) is currently the gold standard in LPR diagnosis; however, it is invasive, user dependent, and not always tolerated. This study assesses the diagnostic utility of salivary pepsin (Peptest) at different thresholds and during symptomatic periods as compared with the 24h-HEMII-pH probe in diagnosing LPR. STUDY DESIGN: Prospective cohort study. SETTING: Private laryngology clinic in Melbourne, Australia. SUBJECTS AND METHODS: Thirty-five patients with a clinical history and endoscopic findings of LPR were recruited and simultaneously evaluated for LPR via 24h-HEMII-pH probe and salivary pepsin analysis at 5 key time points over the same 24-hour period. RESULTS: Salivary pepsin was 76.9% sensitive and had a positive predictive value (PPV) of 87.0% at a threshold of 16 ng/mL when compared with the 24h-HEMII-pH probe. If the pathologic pepsin threshold was raised to 75 ng/mL, salivary pepsin had a sensitivity of 57.7%, a specificity of 75.0%, and a PPV of 93.8%. Symptomatic testing conferred a superior specificity at 16 ng/mL (66.7%) and 75 ng/mL (100.0%) and a superior PPV at 16 ng/mL (92.3%) and 75 ng/mL (100.0%). CONCLUSION: Salivary pepsin detection is a simpler, more cost-effective, and less traumatic universal first-line alternative to 24h-HEMII-pH probe in diagnosing LPR. Superior specificities conferring greater diagnostic value may be achieved with higher thresholds and symptomatic testing. If clinical suspicion remains high following negative salivary pepsin analysis, a 24h-HEMII-pH study could provide further diagnostic information.

Usefulness of matrix metalloproteinase-7 in saliva as a diagnostic biomarker for laryngopharyngeal reflux disease.

Several diagnostic methods are currently being used to diagnose LPRD (laryngopharyngeal reflux disease), but have the disadvantage of being invasive, subjective, or expensive. Our purpose in this study was to investigate the correlation between pepsin and MMP-7 (Matrix Metalloproteinase-7) in pharyngeal secretions of subjects according to RSI (Reflux Symptom Index) score to find out the diagnostic value of MMP-7. We recruited 173 subjects aged between 19 and 85 years who completed the RSI scale. All samples were taken after waking up, and the amount of the pepsin and MMP-7 in saliva were measured by means of an enzyme activity assay. There was a significant increase of pepsin and MMP-7 activity in the study group with an RSI score of 13 or higher. The sensitivity and specificity of MMP-7 for predicting the possibility of an RSI of 13 or more was higher than that of pepsin. When MMP-7 and pepsin were combined, this sensitivity and specificity increased. An enzyme assay of MMP-7 in saliva may be a noninvasive and easy technique for diagnosing LPRD.