Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 33  |  Issue : 1  |  Page : 94--100

Study of pepsin level in saliva as a noninvasive marker for diagnosis of gastroesophageal reflux disease


Hosam I Mohamed1, Siham A Khodeer2, Walaa A Shaheen3,  
1 Department of Gastroenterology and Hepatology, Menoufia University, Shebin El-Kom, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia, Egypt
3 Department of Tropical Medicine, Shebin El-Kom, Menoufia, Egypt

Correspondence Address:
Walaa A Shaheen
Shebin El-Kom, Menoufia
Egypt

Abstract

Objective This study aimed to evaluate the role of salivary pepsin level as a noninvasive marker for diagnosis of gastroesophageal reflux disease (GERD) and its endoscopic severity. Background GERD is one of the most commonly encountered gastrointestinal diseases. Confirmation of the diagnosis mostly entails the use of invasive procedures. Salivary pepsin level has been discussed previously in studies of extraesophageal symptoms of GERD. Patients and methods This study was conducted on 50 patients with clinical and endoscopic evidences of erosive reflux disease (GERD group) versus 25 patients with upper gastrointestinal symptoms not consistent with GERD and in whom endoscopic findings proved absence of GERD (non-GERD group). In addition, 20 healthy participants were selected as a control group. Patients and control were subjected to GERD Q questionnaire, upper gastrointestinal endoscopy, and quantitative measurement of salivary pepsin using ELISA kits. Results There was a highly significant increase in the mean value of salivary pepsin in GERD group (88.64 ± 46.37 ng/ml) when compared with non-GERD (38.08 ± 35.57) and control group (18.65 ± 14.71) (P = 0.0008 and 0.001, respectively), whereas there was no significant difference (P = 0.226) between non-GERD and control group. There was a highly significant increase (P = 0.0008) in mean value of salivary pepsin in patients with GERD complicated with Barrett's esophagus (152.50 ± 27.12) when compared with patients with GERD without this complication (76.48 ± 38.70). Conclusion Salivary pepsin is a simple noninvasive marker for diagnosis of GERD and its severity.



How to cite this article:
Mohamed HI, Khodeer SA, Shaheen WA. Study of pepsin level in saliva as a noninvasive marker for diagnosis of gastroesophageal reflux disease.Menoufia Med J 2020;33:94-100


How to cite this URL:
Mohamed HI, Khodeer SA, Shaheen WA. Study of pepsin level in saliva as a noninvasive marker for diagnosis of gastroesophageal reflux disease. Menoufia Med J [serial online] 2020 [cited 2020 May 25 ];33:94-100
Available from: http://www.mmj.eg.net/text.asp?2020/33/1/94/281303


Full Text



 Introduction



Gastroesophageal reflux disease (GERD) is arguably the most common disease encountered by the gastroenterologist[1].

Gastroesophageal reflux (GER) is defined by abnormal retrograde movement of gastric contents into the esophagus that causes troublesome symptoms according to Montreal consensus for definition and classification of GERD. It stipulated frequency of more than two heartburn episodes per week, which adversely affect an individual's well-being. The disease was subclassified into esophageal and extraesophageal syndromes[2].

Many tests are available for evaluating patients with suspected GERD. These tests are often unnecessary because the classical symptoms of heartburn and acid regurgitation are sufficiently specific to identify reflux disease and to begin medical treatment. However, this may not always be the case, and the clinician must choose which test to use to arrive at a diagnosis[3].

Numerous patient-related outcome questionnaires with a wide variety of characteristics have been developed for the assessment of GERD. Four well-defined parameters are noticeable in those GERD questionnaires, namely, symptoms, response to treatment, diagnosis, and burden on the quality of life of patients with GERD; however, no questionnaire is applicable to patients with alarm characteristics (i.e., dysphagia, weight loss, anemia, long-standing symptoms, and a family history of adenocarcinoma of the esophagus)[4]. These patients must be referred to upper gastrointestinal endoscopy for evaluation.

Use of upper gastrointestinal endoscopy allows for excellent view of the mucosal details and tissue sampling using biopsy and brush cytology. Endoscopy also allows for application of therapies, such as esophageal dilation and Barrett ablation[5]. Being invasive, there has been strict indications for esophagogastroduodenoscopy according to American Gastroenterological Association and American Society for Gastrointestinal Endoscopy. The sensitivity of endoscopy for GERD is 60% at best, although it has excellent specificity at 90–95%[6].

Other modalities in GERD diagnosis are used in certain situations, including pH monitoring–barium esophagram, histopathological examination, and esophageal manometry. Most of the previous tests for diagnosis of GERD are invasive tests. Some are not yielding in noncomplicated cases; hence, there was a need for a noninvasive test when dealing with patients with GERD.

Pepsin is an enzyme whose zymogen (pepsinogen) is released by the chief cells in the stomach and degrades food proteins into peptides[7]. Pepsin was considered one of the primary causes of mucosal damage during laryngopharyngeal reflux[8]. Pepsin in airways was studied as a diagnostic tool for detection of gastroesophageal reflux in patients with chronic cough, chronic lung diseases, or aspiration pneumonias. To date, there is no consensus regarding normal values of pepsin in saliva; hence, the use of pepsin as a diagnostic tool for GERD requires further validation before its use as a bedside test[9].

 Patients and Methods, and Aim of the Study



The study was approved by the Ethical Committee of Menoufia Faculty of Medicine and informed consent was obtained from each patient. This study was conducted aiming to study the role of salivary pepsin in diagnosis of GERD and correlation of pepsin level with GERD severity, on 75 patients with upper gastrointestinal symptoms who were indicated for upper gastrointestinal endoscopy and referred to endoscopy unit, Tropical Medicine Department, Menoufia University hospitals, in the period from September 2015 to April 2016. They were 33 (44%) males and 42 (56%) females. Their ages ranged between 19 and 80 years, with a mean value 47.54 ± 15.52 years. In addition, 20 healthy persons of matched age and sex were selected as controls.

Patients were classified into the following groups:

Group I (GERD group): it included 50 patients with upper gastrointestinal symptoms consistent with Montreal definition and classification of GERD and in whom endoscopic evidences of GERD were confirmed (erosive reflux disease)

Group II (non-GERD group): it included 25 patients with upper gastrointestinal symptoms not consistent with Montreal definition and classification of GERD and in whom endoscopic evidences of GERD were absent

Group III (control group): it included 20 healthy controls.

Exclusion criteria

The following were the exclusion criteria:

Previous history of esophageal or gastric surgeryAny organic esophageal diseases other than GERDPatients with anatomic facial abnormalities and oral or dental problemsAny salivary gland disease known to impair salivationPatients on antisecretory drugs (proton pump inhibitors) during the last month or drugs affecting salivary secretionLack of informed consent.

All patients and control groups were subjected to the following:

Full detailed history taking and complete clinical examinationGERD Q questionnaire[10], which has six predictors for GERD: four positive predictors [heartburn, regurgitation (the two characteristic symptoms of GERD, according to the Montreal definition), sleep disturbance because of these two reflux symptoms, and use of over the counter medication] and two negative predictors (epigastric pain and nausea). Patients were asked to reflect their symptoms over the preceding week. Scores ranging from 0 to 3 were applied for the positive predictors and from 3 to 0 (reversed order, where 3 = none) for negative predictors. The GERD Q score was calculated as the sum of these scores, giving a total score ranging from 0 to 18. A score of more than or equal to 8 was proposed as the cutoff with highest sensitivity and specificity when testing for GERD.

Upper gastrointestinal endoscopy

Olympus Evis CV 100 videoscope (Shinjuku-ku, Tokyo, Japan) was used in the endoscopy unit. Endoscopy was done after sedation using midazolam (Medathetic 5 mg/ml). Results of upper gastrointestinal endoscopy were recorded and included comment on the following: competence of cardia, inflammation, ulceration, masses, stricture of esophagus, and presence of hiatus hernia. Endoscopic classification of severity of GERD (erosive esophagitis) was based on Los Angeles classification of GERD severityMeasurement of pepsin level in saliva.

Collection

Patients were instructed to collect samples of saliva by passive drooling into a 5-ml separate specimen container (ESR tubes) containing 0.5 ml of 0.01 mol/ml citric acid, when awake and whenever experiencing symptoms such as regurgitation, heartburn, or any upper gastrointestinal symptoms (all samples from one patient were collected in one tube). The passive drooling method of saliva collection was chosen because this method does not result in stimulation of saliva secretion, which may result in wash out, dilution, or inactivation of pepsin that might have reached the mouth[11]. Sample collection lasted a day. All participants collected the samples at home and were instructed to store each sample in the refrigerator (4°C) immediately after collection and deliver the sample at the hospital the morning after the day of collection.

Storing

It was estimated that our samples were not left at room temperatures for longer than an hour. We therefore assume that storing did not affect the determination of peptic activity in our study. Samples were stored at − 80°C until they were tested for pepsin. Inappropriate storing conditions can affect the activity of gastric juice pepsin[11].

Estimation of pepsin level in saliva

Pepsin level was estimated in saliva samples using ELISA kits (Cloude Clone Corp., Houston, Texas, USA). The test principle was based on Biotin antibody sandwich technology in which pepsin was added to the wells which are coated with monoclonal antibodies labeled with biotin. This assay employed the competitive inhibition enzyme immunoassay technique. A competitive inhibition reaction was launched between biotin labeled pepsin and unlabeled pepsin (standards or samples) with the precoated antibody specific to pepsin. After incubation, the unbound conjugate is washed off. Next, avidin conjugated to horseradish peroxidase is added to each microplate well and incubated. The amount of bound horseradish peroxidase conjugate was reverse proportional to the concentration of pepsin in the sample. After addition of the substrate solution, the intensity of color developed was reverse proportional to the concentration of pepsin in the sample. The minimum detectable dose of this kit was less than 0.93 ng/ml (though nonsignificant), with a detection range of 2.47–200 ng/ml.

Statistical analysis

Data were statistically analyzed using statistical package for the social science) (IBM, Chicago, Illinois, USA) program, version 13, for Windows, and for all the analyses, a P value less than 0.05 was considered statistically significant.

 Results



GERD group patients comprised 24 (48%) males and 26 (52%) females. Their ages ranged between 19 and 62 years, with a mean value of 44.90 ± 15.10 years. Non-GERD group patients comprised nine (36%) males and 16 (64%) females. Their ages ranged between 23 and 64 years, with a mean value of 50.68 ± 14.93 years. Control group patients comprised six (30%) males and 14 (70%) females. Their ages ranged between 21 and 62 years, with a mean value of 42.45 ± 12.3 years. There was nonsignificant difference among studied groups regarding age and sex distribution.

There was a highly significant increase (P = 0.0009) of GERD Q score in GERD group (score ≥8 in all patients of this group) when compared with non-GERD group (score < 8 in all patients of this group).

Statistical analysis revealed highly significant increase in the mean value of salivary pepsin in GERD group (88.64 ± 46.37 ng/ml) when compared with non-GERD (38.08 ± 35.57 ng/ml) and control groups (18.65 ± 14.71 ng/ml) (with P = 0.0008 and 0.001, respectively), whereas there was no significant difference (P = 0.226) between non-GERD and control groups regarding mean values of salivary pepsin [Table 1].{Table 1}

Cut-off point of salivary pepsin level at highest sensitivity (81%) and specificity (81%) for diagnosis of GERD was 43.5 ng/ml. This cut-off point has a positive predictive value (PPV) of 84%, negative predictive value (NPV) of 78%, and accuracy of 81% [Table 2] and [Figure 1].{Table 2}{Figure 1}

There was a highly significant positive correlation (P = 0.001) between GERD Q score and salivary pepsin level [Figure 2].{Figure 2}

There was a significant increase in the mean value of salivary pepsin level in Los Angeles grade C (129.5 ± 37.25 ng/ml) and grade B (103.95 ± 38.69 ng/ml) when compared with grade A (69.24 ± 45.76 ng/ml) (P = 0.022 and 0.030, respectively). Mean value of salivary pepsin in Los Angeles grade C (mean) was higher than mean value of salivary pepsin in Los Angeles grade B (though nonsignificant, where P = 0.516) [Table 3].{Table 3}

We found a highly significant increase (P = 0.0008) in mean value of salivary pepsin in patients with GERD complicated with Barrett's esophagus when compared with patients without this complication.

 Discussion



The present study revealed a highly significant increase of GERD Q score in GERD group (score ≥8 in all patients of this group) when compared with non-GERD group (score < 8 in all patients of this group). The selection of patients in the present study according to GERD Q score was supported by the study by Jones et al.[10]. The authors assessed GERD Q questionnaire as a tool for diagnosis of GERD and reported that a total GERD Q score of 8 or more has the highest specificity and sensitivity and is the optimal cutoff score proposed for the diagnosis of GERD, reaching a diagnostic accuracy similar to that of a gastroenterologist. On the contrary, Moayyedi et al.[12] reported a sensitivity of heartburn and regurgitation (two positive predictors of GERD) of 30–76% for the presence of erosive esophagitis, with specificity from 62 to 96%. Moreover, Lacy et al.[13] stated that even though questionnaires to diagnose acid reflux have distinct advantages, they were not convinced that current questionnaires are effective. Therefore, use of the GERD Q in a gastroenterology/subspecialty practice may not be practical or beneficial.

In the present study, endoscopic diagnosis of GERD was performed, and its severity was based on Los Angeles classification, where 50% of patients with GERD were Los Angeles grade A, 42% Los Angeles grade B, and 8% were Los Angeles grade C. The use of this classification system in the present study agreed with Rath et al.[14] who stated that Los Angeles classification is most commonly used in USA to classify the grade of erosive esophagitis. It has been shown to have good intraobserver and interobserver agreement among experienced and inexperienced endoscopists and correlates well with the amount of esophageal acid exposure and complication of GERD.

Statistical analysis of the presenting data revealed highly significant increase in the mean value of salivary pepsin in GERD group (88.64 ± 46.37 ng/ml) when compared with non-GERD (38.08 ± 35.57 ng/ml) and control (18.65 ± 14.71 ng/ml) groups. The importance of salivary pepsin as a noninvasive marker for diagnosis of GERD was evaluated in many studies with mixed results, which might be related to different methodologies for pepsin detection in the various studies.

Potluri and colleagues used qualitative pepsin assay to compare salivary/sputum pepsin assay with 24-h esophageal pH monitoring for detection of gastric reflux into the proximal esophagus, oropharynx, and lung, and they reported that proximal esophageal reflux was documented in three of four patients with a positive salivary pepsin assay result, whereas proximal esophageal reflux was not detected in patients who had a negative pepsin assay result. They concluded that detection of pepsin in the saliva and/or sputum may provide a noninvasive method to test for the proximal reflux of gastric contents[15].

Many studies used lateral flow device (LFD) that can detect as low as 16 ng pepsin/ml saliva (positive test) for diagnosis GERD, where values below 16 ng/ml were considered negative.

Yuksel and colleagues in their study titled 'rapid salivary pepsin test: blinded assessment of test performance in GERD' reported that there was a stepwise increase in the prevalence of positive salivary pepsin among patients with GERD, being highest in those with endoscopic esophagitis (55%) followed by patients with abnormal pH findings without esophagitis (43%), and lowest in those with a symptom report of heartburn only. However, the prevalence of positive salivary pepsin was low in all tested participants: 12% in controls and up to 47% in patients with GERD with esophagitis plus pH abnormality. This may be owing to saliva samples only taken at one unspecified time point and concentrations were not determined[16].

de Bortoli and colleagues studied the use of salivary pepsin as a noninvasive diagnostic test to detect GERD and correlated it with MII-pH testing in patients with endoscopy-negative GER, and they found that salivary pepsin measured by LFD was positive in 93.7% of patients with nonerosive reflux disease (with a sensitivity of 76% and specificity of 100%) and 58.3% of hypersensitive esophagus and negative in 100% of functional heartburn group[17].

Farhath and colleagues used a pepsin enzymatic method and western blot analysis of positive samples that can detect pepsin activity at 12.5 ng/ml (positive test) in their study of pepsin in mouth swabs from preterm infants with clinical GER. The authors concluded that pepsin is detected more frequently in the mouth swabs of premature infants with clinical GER than infants without GER. However, that study used only clinical diagnosis of GERD and did not use any other confirmatory method for diagnosis. Clinical diagnosis of GER was made if there were symptoms of GER such as persistent vomiting, apnea, bradycardia, and desaturation attributed to GER or infants were on medications for GER[18].

Hayat and colleagues in their study on salivary pepsin in patients with GERD versus patients with functional heartburn and healthy controls found a higher prevalence and concentration of salivary pepsin in patients with GERD or hypersensitive esophagus when compared with functional heartburn patients. Moreover, there was a significant but weak correlation between number of postprandial reflux events and concentration of pepsin in saliva. The authors concluded that in patients with symptoms suggestive of GERD, salivary pepsin testing may complement questionnaires to assist office-based diagnosis and that this may lessen the use of unnecessary anti-reflux therapy and the need for further invasive and expensive diagnostic methods[9].

The role of salivary pepsin in diagnosis of extraesophageal reflux was supported by various studies. Ocak et al.[19] and Na et al.[20] reported that positive salivary pepsin test in a patient suspected of having laryngeopharyngeal reflux (LPR) can be a cost-effective, accurate, and alternative diagnostic method. Moreover, Kim and colleagues in their study on salivary pepsin as a pathophysiologic offender for tonsillitis found that pepsin was detected (using immunoblot analysis) in the hypertrophic tonsil and in the lymphoid follicle with excessive fibrotic appearance. In addition, pepsin staining was correlated with expression of inflammation-related factors, mainly TGF-β1 and CD68. The authors concluded that pepsin-mediated reflux caused not only direct damage to the tonsillar epithelium but also stimulated the tonsillar macrophages and tonsillar epithelial cells to secrete cytokines that attracted and activated the immune cells and may mediate the damage to the tonsil mucosa[21]. Moreover, Rosen et al.[22] in their study which aimed to correlate pepsin detection with reflux events documented with pH monitoring and upper endoscopy reported that salivary pepsin may be an important biomarker for reflux-related lung disease but cannot predict pathologic reflux in the esophagus. On the contrary, Printza et al.[23] used qualitative pepsin assay for pepsin detection in the saliva of patients who experience pharyngeal reflux and they found that pepsin was not detected with an activity assay in the saliva of patients with a clinical diagnosis of LPR and recommended that a concentration method might be more sensitive, although saliva and swallowing physiology renders the detection of pepsin in the saliva difficult.

The present study revealed that, salivary pepsin at cutoff point 43.5 ng/ml could diagnose GERD with sensitivity of 81%, specificity of 81%, accuracy of 81%, PPV of 84%, and NPV of 78%. Previous studies have reported different (higher and lower) cutoff values of salivary pepsin for diagnosis of GERD. This might be related to different protocols for salivary sampling (timing and number) as well as methodologies for pepsin detection (qualitative or quantitative).

Yuksel and colleagues used LFD in their study aimed to establish the test sensitivity, specificity, PPV, and NPV in patients with symptomatic and objective evidence of GERD compared with healthy controls. The authors reported that salivary pepsin cutoff of +1.0 equivalent to pepsin concentration of ~50 ng/ml (close to our study) has an acceptable sensitivity (87%) and specificity (87%) to diagnose GERD independent of PPI use (close to our results), and they concluded that pepsin in saliva would possibly obviate the need for expensive testing with endoscopy-negative gastroesophageal reflux disease (EGD) and/or pH monitoring.

Moreover, Hayat and colleagues used LFD in their study aimed to assess role of pepsin in saliva for the diagnosis of GERD and they reported that a cutoff point of salivary pepsin of more than 210 ng/ml (much higher than that of the present study) could diagnose GERD with sensitivity of 44%, and a specificity of 96.3%, whereas at a low concentration of less than 100 ng pepsin/ml saliva, the sensitivity and specificity were 67 and 76%, respectively. Moreover, Ocak and colleagues used immunoserologic pepsin analysis in their study for detection of salivary pepsin as a noninvasive rapid diagnostic test for LPR. The authors reported that at the detection limit of the test of 16 ng/ml, the sensitivity, specificity, PPV, and NPV for diagnosis of GER were 33, 100, 100, and 14.2%, respectively. The low sensitivity and NPV might be related to uncertainty in the sample collecting frequency (patients only collected their sputum once, when they had the worst reflux symptom).

The present study revealed significant correlation between salivary pepsin and Los Angeles classification of GERD severity. This was in agreement with results of the study conducted by Yuksel and colleagues who found that there was a stepwise increase in the prevalence of positive salivary pepsin among the patients with GERD, with values highest in those with endoscopic esophagitis followed by patients with abnormal pH findings and lowest in those with a symptom report of heartburn only and controls. The authors concluded that a positive salivary pepsin test result in a patient suspected of having GERD increases the likelihood that the patient has esophagitis and/or abnormal pH findings. On the contrary, Rosen and colleagues reported no significant relationship between pepsin positivity and an abnormal endoscopy, pH probe, or impedance.

The present study revealed that the incidence of BE in the studied of patients with GERD was 16% (eight of 50 patients), and the mean values salivary pepsin in patients with GERD complicated with BE was highly significant (P < 0.001) than those without this complication (152.50 ± 27.12 vs. 76.48 ± 38.70, respectively). When comparing these results with previous studies, lower and higher incidence rates of BE were reported. Spechler[24] reported that the prevalence of BE in patients with GERD symptoms varied between 10 and 21% depending on the indication for endoscopic referral. Peery et al.[25] reported that 10% of patients with chronic heartburn symptoms had BE, accounting for almost a half million of the visits in 2009. The higher incidence of BE in the present study may be explained by the selection of patients with GERD based on both clinical and endoscopic evidences and not on clinical diagnosis only, unlike the study of Peery and colleagues, which depended on clinical diagnosis only.

Samuels and colleagues in their study of pepsin synthesis in BE and the role of pepsin in esophageal adenocarcinoma used molecular techniques to study synthesis of pepsin by metaplastic epithelium of BE, and the authors found that pepsin protein was present in all eight Barrett's specimens and in four of eight adjacent normal specimens. Moreover, pepsinogen A mRNA was observed in two Barrett's specimens but was not observed in normal adjacent samples. Moreover, pepsinogen protein was observed in pepsinogen mRNA-positive specimens, whereas little to no pepsinogen was detected in neighboring normal tissue. This may account for the high level of pepsin in patients with GERD complicated with BE[26].

 Conclusions



From the present study, we conclude that salivary pepsin correlated positively with Los Angeles classification of GERD severity and at a cutoff point of 43.5 mg/ml, it could diagnose GERD in patients with GERD Q questionnaire score more than or equal to 8 with sensitivity of 81%, specificity of 81%, PPV of 86%, NPV of 74% and accuracy of 81%. We also concluded that, in patients with GERD complicated by BE, mean value of salivary pepsin was significantly higher when compared with patients without this complication.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Richter JE, Fredenberg FK Gastroesophageal reflux disease. In: Feldman M, Brandet LJ, eds. Sleisenger and Fordtran's gastrointestinal and liver diseases. Chapter 43. 9th ed. Philadelphia: Saunders, Elsevier; 2010. p. 772–801.
2Vakil N, van Zanten SV, Kahrilas P, Dent J, Jones R. Global Consensus Group. The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus. Am J Gastroenterol 2006; 101:1900–1920.
3Richter JE. Gastroesophageal reflux disease. In: Yamada T, ed. Gastroenterology. Chapter 32 5th ed. Hoboken, New Jersey (United States): Wiley Blackwell; 2009. p. 592-609.
4Bolier EA, Kessing BF, Smout AJ, Bredenoord AJ. Systematic review: questionnaires for assessment of gastroesophageal reflux disease. Dis Esophagus 2015; 28:105–120.
5Shaheen NJ, Weinberg DS, Denberg TD, Chou R, Qaseem A, Shekelle P. Upper endoscopy for gastroesophageal reflux disease: best practice advice from the clinical guidelines committee of the American College of Physicians. Ann Intern Med 2012; 157:808–816.
6Richter JE. Severe reflux esophagitis. Gastrointest Endosc Clin North Am 1994; 4:677.
7Florkin M. Discovery of pepsin by Theodor Schwann. Rev Méd Liège (in French) 1957; 12:139–144.
8Lillemoe KD, Johnson LF, Harmon JW. Role of the components of the gastroduodenal contents in experimental acid esophagitis. Surgery 1982; 92:276–284.
9Hayat JO, Gabieta-Somnez S, Yazaki E, Kang JY, Woodcock A, Dettmar P, et al. Pepsin in saliva for the diagnosis of gastro-oesophageal reflux disease. Gut 2014; 64:373–380.
10Jones R, Junghard O, Dent J, Vakil N, Halling K, Wernersson B, et al. Development of the GERD Q, a tool for the diagnosis and management of gastro-oesophageal reflux disease in primary care. Aliment Pharmacol Ther 2009; 30:1030–1038.
11Michishige F, Kanno K, Yoshinaga S, Hinode D, Takehisa Y, Yasuoka S, et al. Effect of saliva collection method on the concentration of protein components in saliva. J Med Investig 2006; 53:140–146.
12Moayyedi P, Talley NJ, Fennerty MB, Vakil N. Can the clinical history distinguish between organic and functional dyspepsia?. JAMA 2006; 295:1566–1576.
13Lacy BE, Chehade R, Crowell MD. Response to Vakil and Kahrilas. Am J Gastroenterol 2012; 107:323–324.
14Rath H, Timmer A, Kunkel C, Endlicher E, Grossmann J, Hellerbrand C, et al. Comparison of interobserver agreement for different scoring systems for reflux esophagitis: Impact of level of experience. Gastrointest Endosc 2004; 60:44–49.
15Potluri S, Friedenberg F, Parkman HP, Chang A, MacNeal R, Manus C, et al. Comparison of a salivary/sputum pepsin assay with 24-hour esophageal pH monitoring for detection of gastric reflux into the proximal esophagus, oropharynx and lung. Dig Dis Sci 2003;48:1813–1817.
16Yuksel E, Hong S, Strugala V, Slaughter JC, Goutte M, Garrett CG, et al. Rapid salivary pepsin test: blinded assessment of test performance in gastroesophageal reflux disease. Laryngoscope 2012; 122:1312–1366.
17De Bortoli N, Savarino E, Furnari M, Martinucci I, Zentilin P, Bertani L, et al. Use of a non-invasive pepsin diagnostic test to detect GERD: correlation with MII-pH evaluation in a series of suspected NERD patients. A pilot study. Gastroenterology 2013; 144:S–118.
18Farhath S, He Z, Saslow J, Soundar S, Vandenplas V. Detection of pepsin in mouth swab: correlation with clinical gastroesophageal reflux in preterm infants. J Matern Fetal Neonatal Med 2014; 26:819–824.
19Ocak E, Kubat G, Yorulmaz I. Immunoserologic pepsin detection in the saliva as a non-invasive rapid diagnostic test for laryngopharyngeal reflux. Balkan Med J 2015; 32:46–50.
20Na SY, Kwon OE, Lee YC, Eun YG. Optimal timing of saliva collection to detect pepsin in patients with laryngopharyngeal reflux. Laryngoscope 2016; 126:2770–2773.
21Kim J, Jeong H, Kim K, Lee Y, Jung M, Park J, et al. Extra-esophageal pepsin from stomach refluxate promoted tonsil hypertrophy. PLoS One 2016; 11:e0152336.
22Rosen R, Johnston N, Hart K, Khatwa U, Nurko S. The presence of pepsin in the lung and its relationship to pathologic gastroesophageal reflux. Neurogastroenterol Motil 2012; 24:129–e85.
23Printza A, Speletas M, Triaridis S, Wilson J. Is pepsin detected in the saliva of patients who experience pharyngeal reflux. Hippokratia 2007; 11:145–149.
24Spechler SJ. Comparison of medical and surgical therapy for complicated gastroesophageal reflux disease in veterans. The Department of Veterans Affairs Gastroesophageal Reflux Disease Study Group. N Engl J Med 1992; 326:786–792.
25Peery A, Dellon E, Lund J, Crockett S, McGowan C, Bulsiewicz W, et al. Burden of gastrointestinal disease in the United States: 2012 update. Gastroenterology 2012; 143:1179–1187. e1–3.
26Samuels T, Hoekzema C, Gould J, Goldblatt M, Frelich M, Bosler M, et al. Local synthesis of pepsin in Barrett's esophagus and the role of pepsin in esophageal adenocarcinoma. Ann Otol Rhinol Laryngol 2015; 124:893–902.