The pepsin saliva test was developed to detect laryngopharyngeal reflux disease without the need for HEMII-pH [6]. To date, studies reported controversial results about the most appropriate time of saliva collection, and the related accuracy, sensitivity, specificity, and predictive values of pepsin saliva measurements. The accuracy and predictive values of the pepsin test were investigated in few studies, which reported controversial results (Table 6) [2, 6, 7, 9, 10, 14,15,16,17,18]. Overall, SE and SP ranged from 29.4 to 100% according to studies, where authors collected saliva sample in the morning, post-meals or after symptoms. Pepsin test appears to be sensitive but not specific. However, most authors included only LPR patients and the lack of control groups may significantly influence the assessment of predictive values and accuracy of pepsin test.

Our results suggested a variability of the pepsin saliva concentration throughout the day, and the lack of significant consistency with the HEMII-pH results. The morning saliva pepsin measurement appeared to be associated with the highest sensitivity and accuracy, when compared to other measurements. In 2016, Na et al. observed that the average pepsin level upon waking was higher than that measured at any other time [19]. Wang et al. corroborated these findings in a recent study where the morning saliva pepsin measurement was associated with the highest LPR detection rate [10]. The importance of the morning saliva collection was however not supported by Weitzendorfer et al. who observed higher saliva pepsin concentrations after the dinner and the lunch compared to waking concentrations [15]. In other studies, authors reported a variability between morning, post-lunch and post-dinner pepsin saliva concentrations [6, 7] without determining the most adequate time of saliva collection.

The problem of the variability of saliva pepsin concentration and the related discrepancies across studies in accuracy, sensitivity and predictive values may be tackled by the collection of multiple saliva samples. Indeed, as suggested in the present study, recent studies supported that sensitivity, specificity and predictive values may be raised when considering the highest pepsin measurement of 2 or 3 saliva sample collections within the testing day [2, 10, 15]. Considering the highest saliva pepsin measurement, sensitivity, specificity, and PPV found in the present study corroborated those of the literature (Table 6) [2, 10, 15]. Precisely, Wang et al. [10] reported that 55.7% of the true positive cases were missed by considering a single pepsin test. Similarly, Hayat et al. and Zhang et al. supported that the accuracy, sensitivity, specificity, and predictive values of pepsin saliva measurements were improved when considering the highest pepsin saliva concentration of three or four measurements, respectively [6, 7].

To date, the variability of pepsin saliva concentration throughout the day is not fully understood. Several factors may influence the gastric pepsin secretions, the esophageal motility, the relaxation of sphincters, and the related pepsin saliva concentration. First, it has been suggested that the foods and beverages consumed during the testing day may influence the pepsin saliva concentration [16, 17]. On the one hand, foods and beverages may influence the gastric secretion of pepsin, and, therefore, the pepsin concentration into the stomach content that may refluxate into the upper aerodigestive tract tissues [18, 19]. On the other hand, acid, spicy, low-protein, and high-fat foods may increase the number of transient relaxations of esophageal sphincters, leading to an increased number of pharyngeal reflux events that contain pepsin [16, 17]. Regarding the influence of diet, the differences across studies from different world regions should be interpreted according to the diet habits of populations.

Both esophageal sphincter tonicity and motility are known to be influenced by the autonomic nerve function [20, 21]. The activation of sympathetic nervous system may impair the esophageal antireflux barriers (sphincter tonicity and esophageal motility), leading to pharyngeal reflux events. In that way, patients with stress, anxiety or depressive findings at the time of the diagnosis/testing should have higher number of pharyngeal reflux events and, theoretically, higher pepsin saliva concentration compared to patients without autonomic nerve dysfunction [20, 21]. In addition to these factors, it is important to keep in mind that the saliva pepsin measurements highlight the extracellular pepsin concentrations, while recent studies suggested a potential internalization of pepsin into the Golgi apparatus of pharyngeal cells [22], which makes undetectable a part of refluxate pepsin.

To the best of our knowledge, the present report is the first study investigating accuracy, sensitivity, specificity and predictive values of pepsin saliva measurements according to the time of saliva collection. The primary limitation of the present study was the homogeneity of the study population, which mainly included patients with a positive diagnostic at the HEMII-pH and only 21 asymptomatic individuals. The lack of healthy individuals benefiting from HEMII-pH may be considered as a limitation but HEMII-pH is costly and inconvenience for asymptomatic patients. Future studies are needed to better understand the low SE and SP of pepsin test, and to investigate the presence of other gastroduodenal enzymes in the saliva of patients. Indeed, the presence of other enzymes, such as bile salts, should explain the mucosa injuries and related symptoms and findings without detected pepsin.