Laryngopharyngeal reflux (LPR) refers to the backflow of gastric contents into the larynx, pharynx, trachea, and bronchus, resulting in several upper airway inflammatory disorders. LPR is associated with several clinical manifestations, such as laryngitis, obstructive sleep apnea, posterior glottis edema and erythema, laryngospasm, subglottic stenosis, and even otitis media [1,2]. Patients with LPR generally show no specific symptoms, making the diagnosis difficult [3].

Gastroesophageal reflux disease (GERD) is a gastric disorder and probably one of the main causes of laryngeal symptoms, especially in patients with chronic posterior laryngitis. GERD may contribute to extraesophageal syndromes via both direct (aspiration of liquid and aerosol) and indirect (vagal mediated) mechanisms (sensory neuropathic cough) [4].

Data on the prevalence of LPR have remained controversial, with certain studies reporting it to range from 18% to 80% [1,5], whereas others reporting it as low as 10% [6]. According to a study, LPR is common in 26% of patients presenting to their general practitioners (GPs) with suspected symptom profiles [7]. However, establishing an association between GERD and symptoms of laryngeal irritation is challenging. The currently used diagnostic techniques for LPR, which rely on procedures having normative standards established for the diagnosis of typical GERD (i.e., heartburn and/or regurgitation), are not appropriate for the detection of LPR.

According to the current guidelines on GERD management, all patients presenting otolaryngological and respiratory disorders should be carefully evaluated for non-GERD causes [8]. A proton pump inhibitor (PPI) test is recommended to treat extraesophageal symptoms, particularly in patients who also have typical GERD manifestations. Although a recent meta-analysis showed that a high-dose of PPIs was no longer effective in improving LPR symptoms, high dosage and long duration of PPI therapy are required for the diagnosis of atypical GERD presentation [9]. Reflux monitoring should be considered before starting PPI therapy in patients with extraesophageal manifestations, who do not have typical GERD symptoms or do not respond to PPIs. However, it is not an ideal diagnostic tool because of low sensitivity (50%–80%), low patient tolerance, major diet influence, high cost, and limited availability [10,11].

In clinical practice, primary care patients with chronic laryngeal symptoms are often referred to otolaryngologists, who frequently establish a diagnosis of posterior laryngitis by laryngoscopy and prescribe PPIs and an upper digestive endoscopy to confirm the diagnosis. However, this is a non-rationale, non-evidence-based, expensive, and ineffective approach. Therefore, a less expensive and easier to perform surrogate marker of extraesophageal reflux and LPR in the primary care is required.

A recent systematic review suggests that pepsin, a proteolytic enzyme secreted as pepsinogen from gastric mucosa and activated at acidic pH, could be a reliable marker of gastroesophageal reflux in patients with LPR. However, questions about its optimal timing, location, nature and threshold values for testing still remain unresloved [12,13]. Recently, a novel and rapid method to assess the presence of pepsin has been introduced into the market (Pep-test; RD Biomed Limited, Cottingham, UK); it is considered a non-invasive, quick, and inexpensive test for the diagnosis of LPR. Moreover. Pep-test has been favorably tested in pilot studies and in a comparative trial with multi-channel intraluminal impedance and pH monitoring [14-19].

The present study tested the reliability of Pep-test in diagnosing LPR in uninvestigated primary care attenders presenting with chronic laryngeal symptoms, and evaluated the raw pepsin intensity value in patients with LPR.

Our study compared the pepsin salivary concentration in attenders of primary care offices presenting or not with chronic laryngeal symptoms. Our data showed a high prevalence of positive Pep-test in healthy controls (higher than in LPR subjects). Therefore, our findings demonstrate that there was no difference in the prevalence of pepsin positivity between patients with suspected LPR symptoms and asymptomatic control subjects. Moreover, the concentration of pepsin did not change.

Recently, determining pepsin in saliva sample has been proposed as a non-invasive diagnostic tool for GERD and LPR [1,12,13]. Unfortunately, our findings questioned the usefulness of this non-invasive test, at least in primary care setting. In fact, we failed to find a difference in Pep-test positivity or pepsin concentration between patients with LPR symptoms and asymptomatic controls. This could be ascribed to the high presence of salivary pepsin in our control population (88%), which also included the selected group of subjects with RSI <5 and GIS=36. The high positivity of Pep-test in the control group was unexpected, especially if all previous studies reported in Table 3 are considered. There is no doubt that our control population could be affected by an increased acid reflux, despite the absence of both typical and atypical symptoms of GERD in it. We are aware that even abnormal acid reflux can be completely asymptomatic, and therefore our group of normal subjects included patients rather than controls. This is not a limitation of Pep-test itself, but it necessarily involves all methods for measuring pepsin. On the contrary, only diagnostic methods that objectively detect the burden of reflux, such as the modern 24-hour impedance-pH monitoring [23], can confirm the presence of abnormal reflux. However, these still cannot be considered as the gold standard. However, this kind of testing is not widespread and is certainly not available in the primary care setting.

It is important to emphasize that the high positivity of Pep-test in our controls is of interest for primary care physicians, because they may represent a potential population with unsuspected pathological or silent reflux, who will benefit from lifestyle changes or even medical treatment. Moreover, it would be of interest to re-test them in the long run to study their reflux burden and its consequences, especially Barrett’s esophagus and esophageal adenocarcinoma [24].

Until date, there is no validated method to assess LPR, as most procedures have shown only moderate sensitivity and specificity or are invasive and expensive. A recent systematic review suggests that pepsin could be a reliable marker of reflux in patients with LPR [13]. Indeed, the prevalence of pepsin positivity determined by Pep-test in at least one saliva sample of patients, presenting with symptoms suggestive of LPR, was 76% in our study. This result is comparable to that obtained by other studies using Pep-test for the evaluation of LPR in secondary care patients [13,25]. However, in most of the aforementioned studies (Table 3) [12,16,19,25-28], control subjects were enrolled in a secondary care setting and after a negative endoscopy and 24-hour impedance-pH test. Therefore, this may explain the discrepancy observed in our study where this invasive approach was not available. Other factors, such as the recruitment of controls on the sole basis of the absence of symptoms without any objective demonstration of their healthy status and the different timings of salivary collection, could have influenced the difference observed in our study compared with the previous ones. On the contrary, a study that included the highest number of healthy subjects reported more than one third of them as Pep-test positive [26], whereas a recent study evaluating the diagnostic utility of Pep-test, reported a high Pep-test positivity in controls with no significant difference compared to patients with LPR [25].

To the best of our knowledge, the present study is the first of its kind to use Pep-test in the primary care setting. Our results showed that primary and secondary care could produce conflicting results, most likely owing to different epidemiological distribution of such a widespread and difficult to define condition. In other words, primary care physicians tend to encounter individuals with uninvestigated symptoms, and their access to invasive diagnostic techniques is limited. On the contrary, secondary care setting has a greater opportunity than the primary care setting to perform more sophisticated tests, such as endoscopic and bioptic studies and esophageal functional tests, particularly 24-hour impedance pH monitoring. Further studies in the primary care setting, where most of these patients are seen and a high number of prescriptions occur, are required.

To avoid unnecessary, prolonged, and expensive treatments (i.e., PPIs or even anti-reflux surgery), it is clinically relevant to find a correct diagnostic approach for LPR, especially in primary care setting [27]. However, from the results of our study it appears that a physiological reflux of gastric content, especially in the post-prandial period, may occur frequently in several individuals, thus explaining the presence of pepsin in salivary samples of asymptomatic subjects. If this is true, a new hypothesis could attribute the pharyngeal and laryngeal damage observed in some asymptomatic subjects to pepsin. In any case, further studies are required to better evaluate this hypothesis.

In conclusion, performing a salivary pepsin test alone is not recommended for LPR diagnosis in primary care setting, and further invasive and accurate diagnostic tests are required to confirm the diagnosis of GERD.