Method and kit for diagnosing laryngopharyngeal reflux disease

Abstract

The current invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol. The invention further relates to a diagnostic kit for the diagnosis of laryngopharyngeal reflux disease in a subject, said kit comprising one or more collection tubes for saliva or sputum and one or more assays for measuring the levels of one or more markers in a saliva sample obtained from said subject, wherein said one or more markers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

Description

[0001] METHOD AND KIT FOR DIAGNOSING LARYNGOPHARYNGEAL REFLUX

[0002] DISEASE

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method and kit for diagnosing laryngopharyngeal reflux disease.

[0005] BACKGROUND

[0006] Laryngopharyngeal reflux (LPR) disease is a prevalent inflammatory condition of the upper aerodigestive tract tissues related to direct and indirect effects of gastroduodenal content reflux, which induces morphological changes in the mucosa. LPR is associated with many ear, nose and throat conditions, e.g. acute or chronic otitis media, Eustachian tube dysfunction, chronic rhinosinusitis, vocal fold benign lesions, and non-functional laryngeal disorders. Both symptoms and findings of LPR are non-specific, making the clinical diagnosis difficult. The most prevalent symptoms being throat pain, dysphonia, throat clearing, cough, dysphagia and globus sensation, while the most prevalent findings include oropharyngeal wall erythema, arytenoid erythema and posterior commissure hypertrophy. The hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring (HEMII-pH) is currently the most widely used technique to diagnose LPR through the identification of acid, non-acid or weakly acid hypopharyngealesophageal reflux events (HRE). However, scientists are faced with diagnostic and therapeutic controversies and many 'gray' areas related to LPR. Symptoms are not correlated with fiberoptic signs or HEMII-pH findings, and some patients present severe LPR complaints but few HREs at the HEMII-pH.

[0007] Nowadays, it is well-established that LPR is related to transient esophageal lower and upper sphincter relaxations, and the refluxate of gastroduodenal enzymes into the laryngopharyngeal mucosa. It has been demonstrated that injury to the laryngopharyngeal mucosa is related to the presence and activity of the enzyme pepsin, which may be active in both extra- and intracellular compartments. Pepsin is an endopeptidase that breaks down proteins into smaller peptides. It is produced in the gastric chief cells of the stomach lining and is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food. The presence of pepsin in the esophagus, the throat, the mouth or the lungs is used in the diagnosis of gastroduodenal content reflux. However, the accuracy of such pepsin-based diagnostic methods is limited and a negative test cannot exclude LPR.

[0008] The aim of the invention is to provide a method which eliminates those disadvantages.

[0009] The invention thereto aims to provide an improved method and kit for diagnosing laryngopharyngeal reflux disease.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages.

[0012] To this end, the present invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject according to claim 1. More particular, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0013] Preferred embodiments of the method are shown in any of the claims 2 to 12.

[0014] In a second aspect, the present invention relates to a kit for the diagnosis of laryngopharyngeal reflux in a subject according to claim 13. More particular, the kit as described herein relates to a diagnostic kit for the diagnosis of laryngopharyngeal reflux in a subject, said kit comprising one or more collection tubes for saliva or sputum and one or more assays for measuring the levels of one or more biomarkers in a saliva or sputum sample obtained from said subject, wherein said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0015] Preferred embodiments of the kit are shown in any of the claims 14 to 15.

[0016] The current invention provides an improved method and diagnostic kit for diagnosing laryngopharyngeal reflux (LPR), having a higher specificity and / or sensitivity and / or being less expensive, less time-consuming and / or less invasive as the currently used methods and kits. Furthermore, the development of a non-invasive diagnostic method and kit for diagnosing laryngopharyngeal reflux using saliva or sputum as the specimen of choice would expand opportunities for testing in hard-to-reach populations and facilitate diagnosis in physician's offices, satellite clinics, outpatient facilities and other non-traditional testing facilities and would reduce the overall costs of LPR patient care and the healthcare system in general.

[0017] DESCRIPTION OF FIGURES

[0018] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses. Throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.

[0019] Figures 1 and 2 relate to example 1 in which suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR) were identified using a study population of 20 LPR patients and 12 control subjects.

[0020] Figure 1 shows the pancreatic elastase concentration (pg / ml) as measured in a control population and in patients suffering from LPR. Pancreatic elastase is a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0021] Figure 2 shows a Receiver Operating Curve (ROC) curve of pancreatic elastase, a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0022] Figures 3 to 6 relate to example 2 in which suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR) were identified using a study population of 45 LPR patients and 22 control subjects.

[0023] Figure 3 shows the biliary acid concentration (pmol / L) as measured in a control population and in patients suffering from LPR. Biliary acid is a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0024] Figure 4 shows a Receiver Operating Curve (ROC) curve of pancreatic elastase, a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention. Figure 5A shows the cholesterol concentration (mg / dL) as measured in a control population and in patients suffering from LPR. Cholesterol is a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention. Figure 5B shows a Receiver Operating Curve (ROC) curve of cholesterol, a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0025] Figure 6 shows a Receiver Operating Curve (ROC) curve of pH, a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0026] Figure 7 relates to example 3 in which suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR) were identified using a study population of 67 LPR patients and 35 control subjects.

[0027] Figure 7A shows the pancreatic elastase concentration (pg / ml) as measured in a control population and in patients suffering from LPR. Pancreatic elastase is a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0028] Figure 7B shows a Receiver Operating Curve (ROC) curve of pancreatic elastase, a biomarker which can be used in a method for diagnosing laryngopharyngeal reflux according to an embodiment of the invention.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention concerns an improved method and diagnostic kit for diagnosing laryngopharyngeal reflux (LPR) disease, having a higher specificity and / or sensitivity and / or being less expensive, less time-consuming and / or less invasive as the currently used methods and kits.

[0031] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0032] As used herein, the following terms have the following meanings: "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0033] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0034] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0035] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0036] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0037] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0038] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0039] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0040] "Sensitivity" and "specificity" mathematically describe the accuracy of a test which reports the presence or absence of a condition. Individuals for which the condition is satisfied are considered "positive” and those for which it is not are considered "negative".

[0041] "Sensitivity" (true positive rate) as used herein refers to the probability of a positive test, conditioned on truly being positive. "Specificity" (true negative rate) as used herein refers to the probability of a negative test, conditioned on truly being negative. In a diagnostic test, sensitivity is a measure of how well a test can identify true positives and specificity is a measure of how well a test can identify true negatives.

[0042] The positive and negative predictive values (PPV and NPV respectively) are the proportions of positive and negative results in statistics and diagnostic tests that are true positive and true negative results, respectively. The PPV and NPV describe the performance of a diagnostic test or other statistical measure. A high result can be interpreted as indicating the accuracy of such a statistic. The ideal value of the PPV, with a perfect test, is 1 (100%), and the worst possible value would be zero. With a perfect test, one which returns no false negatives, the value of the NPV is 1 (100%), and with a test which returns no true negatives the NPV value is zero. The PPV and NPV are not intrinsic to the test (as true positive rate and true negative rate are); they depend also on the prevalence. Both PPV and NPV can be derived using Bayes' theorem. In an embodiment, the method of the current invention allows to diagnose LPR with a PPV of at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%. In a preferred embodiment, said PPV is at least 50%. In an embodiment, the method of the current invention allows to diagnose LPR with a NPV of at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%. In a preferred embodiment, said NPV is at least 90

[0043] %.

[0044] Laryngopharyngeal reflux (LPR) or laryngopharyngeal reflux disease (LPRD) is a general term for the reflux of gastroduodenal contents into the laryngopharynx, oropharynx and even the nasopharynx, causing a series of symptoms and signs. It is a prevalent inflammatory condition of the upper aerodigestive tract tissues related to direct and indirect effects of gastroduodenal content reflux, which induces morphological changes in the mucosa. LPR is associated with many ear, nose and throat conditions, e.g. acute or chronic otitis media, Eustachian tube dysfunction, chronic rhinosinusitis, vocal fold benign lesions, and non-functional laryngeal disorders. Both symptoms and findings of LPR are non-specific, making the clinical diagnosis difficult. The most prevalent symptoms are throat pain, dysphonia, throat clearing, cough, dysphagia and globus sensation, while the most prevalent findings include oropharyngeal wall erythema, arytenoid erythema and posterior commissure hypertrophy.

[0045] The hypopharyngeal-esophageal multichannel intraluminal impedance-pH (HEMII- pH) monitoring is currently the most widely used method to diagnose LPR through the identification of acid, non-acid or weakly acid hypopharyngealesophageal reflux events (HRE). By using HEMII-pH monitoring, the LPR diagnosis is confirmed if there is >1 acid or non-acid hypopharyngeal reflux event. However, scientists are faced with diagnostic and therapeutic controversies and many 'gray' areas related to LPR. Symptoms are not correlated with fiberoptic signs or HEMII-pH findings, and some patients present severe LPR complaints but few HREs at the HEMII-pH. Furthermore, HEMII-pH monitoring is expensive, time-consuming, has no good tolerability in some patients and and is not available at all institutions. Moreover, the possibility that probe insertion might influence the LRP could not be completely excluded.

[0046] Reflux diagnosis typically entails expensive, invasive and time-consuming tests, such as 24 / 48 hour pH monitoring, pH Impedance, Barium Swallow, and endoscopy.

[0047] Nowadays, it is well-established that LPR is related to transient esophageal lower and upper sphincter relaxations, and the refluxate of gastroduodenal enzymes, of which pepsin is most widely known, into the laryngopharyngeal mucosa.

[0048] Measuring the level of one or more biomarkers in a saliva or sputum sample is a painless and inexpensive method to diagnose LPR.

[0049] For instance, it has been demonstrated that injury to the laryngopharyngeal mucosa was related to the presence and activity of the enzyme pepsin, which may be active in both extra- and intracellular compartments. Pepsin is an endopeptidase that breaks down proteins into smaller peptides. It is produced in the gastric chief cells of the stomach lining and is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food. The presence of pepsin in the esophagus, the throat, the mouth or the lungs is used in the diagnosis of gastroduodenal content reflux. One such diagnostic commercially available test is Peptest, a lateral flow device using monoclonal antibodies. However, sensitivity of said test is limited. When one hypopharyngeal reflux episode was used as a diagnostic threshold for LPR, sensitivity only amounted to 33% for the Peptest. (Zelenik, Karol et al. "Diagnostic Value of the PeptestTM in Detecting Laryngopharyngeal Reflux." Journal of clinical medicine vol. 10,13 2996. 5 Jul. 2021, doi: 10.3390 / jcml0132996). Sensitivity refers to a test's ability to designate an individual with disease as positive. A highly sensitive test means that there are few false negative results, and thus fewer cases of disease are missed. As sensitivity only amounted to 33% for the Peptest, a negative Peptest cannot exclude LPR.

[0050] The inventors surprisingly found that LPR patients reported significant higher saliva concentration of pancreatic elastase (p=0.001), bile acids (p=0.006) compared with healthy controls (see Example 1 below) and that the saliva pH of LPR patients was significantly higher than the pH of saliva of healthy individuals. The inventors concluded that these biomarkers could be used in a method for diagnosing laryngopharyngeal reflux (LPR) disease in a subject. In a further experiment with a bigger study population, the inventors confirmed the usefulness of pancreatic elastase and pH as good biomarkers to diagnose LPR. In addition, the inventors identified cholesterol as an additional useful biomarker to diagnose LPR and found that the concentration of bile acids can be used to determine the severity of LPR.

[0051] In a first aspect, the invention provides a method for diagnosing laryngopharyngeal reflux (LPR) disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol and wherein based on said comparison the presence or absence of said laryngopharyngeal reflux is determined. These one or more biomarkers may be used to diagnose or aid in the diagnosis of LPR.

[0052] Preferably, the saliva is collected sober (during fasting) in the morning, preferably before brushing the teeth. In a preferred embodiment, samples are obtained and kept in conditions to avoid degradation, including advantageously, storage at temperature close to 0°C or below.

[0053] As such, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject, wherein said sample is obtained in the morning from said subject when fasting, and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0054] "A subject when fasting" as used herein refers to a subject which has not digested any food in the morning on which the sample is obtained. As such, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject, wherein said sample is obtained from said subject in the morning before any food is ingested, and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0055] In an embodiment, said subject has not digested any food for at least 12 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 11 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 10 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 9 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 8 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 7 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 6 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 5 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 4 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 3 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 2 hours prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 1 hour prior to obtaining the sample from said subject. In an embodiment, said subject has not digested any food for at least 30 minutes prior to obtaining the sample from said subject.

[0056] As used herein, diagnosis or diagnostics is the process of identifying a medical condition or disease by its signs, symptoms, and from the results of various diagnostic procedures. The conclusion reached through this process is called a diagnosis. The term "diagnostic criteria" designates the combination of signs, symptoms, and test results that allows one, e.g., a physician, to ascertain the diagnosis of the respective disease. As used herein, methods for "aiding diagnosis" refers to methods that assist in making a clinical determination regarding the presence, or nature, of LPR, and may or may not be conclusive with respect to a definitive diagnosis. In an embodiment, these one or more biomarkers may be used in the prognosis of LPR, risk of LPR and / or to measure progression of LPR in confirmed LPR patients. Prognosis is a term denoting a prediction of how a patient's disease will progress, and whether there is chance of recovery. Prognosis includes methods of predicting how a patient (given their condition) may respond to treatment. Symptoms and tests may indicate favorable treatment with standard therapies. Likewise, a number of symptoms, health factors, and tests may indicate a less favorable treatment result with standard treatment (treatment prognosis) - this may indicate that an alternative treatment plan may be desired. The sample for diagnosing laryngopharyngeal reflux disease in a subject can be any type of clinically relevant sample which enables determining the pH and / or the amount of pancreatic elastase, bile acids, hormones, electrolytes, osmolality, sugar, immunoglobulin, inflammatory markers, glycoproteins, TGF-beta, lactate dehydrogenase and / or cholesterol. In an embodiment, said sample is a nasal lavage sample. In an embodiment, said sample is a blood sample. In a preferred embodiment, said sample is a saliva sample or a sputum sample.

[0057] As described above, in a preferred embodiment, said sample obtained from said subject is a saliva sample. As described above, measuring the level of one or more biomarkers in a saliva sample is a painless and inexpensive method to diagnose LPR. Furthermore, by using saliva as the specimen of choice, opportunities for testing in hard-to-reach populations are expanded and diagnosis in physician's offices, satellite clinics, outpatient facilities and other non-traditional testing facilities are facilitated, reducing the overall costs of LPR patient care and the healthcare system in general.

[0058] As such, in an embodiment, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a saliva sample obtained from said subject, wherein said saliva sample is obtained from said subject in the morning before any food is ingested, and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0059] In another embodiment, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sputum sample obtained from said subject, wherein said sputum sample is obtained from said subject in the morning before any food is ingested, and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

[0060] The invention provides methods for the diagnosis of LPR or aiding in the diagnosis of LPR, prognosis of LPR and risk of LPR in an individual by quantitatively measuring the amount of each of one or more individual biomarkers in a sample and determining whether said level is higher than a certain threshold value for said biomarker. In an embodiment, said threshold value for said biomarker is derived from logistic regression and defined by the area under the curve (AUC) from receiver operating characteristics (ROC) analysis at the highest Youden Index (as described in Examples 1, 2 and 3 below).

[0061] Preferably, said predetermined threshold value for pancreatic elastase has been obtained by measuring the salivary elastase level using the same tools on (i) at least 10 (preferably at least 15, 20, 25, 30, 40, 50, 60) patients with reflux and (ii) possibly at least 5 (preferably at least 10, 15, 20, 25, 30, 40, 50, 60) healthy persons. This allows to stratify the patients and calculate the cut-off (the predetermined threshold value). This stratification (and determination of the predetermined threshold value) is preferably also implemented for the other markers: salivary pH, cholesterol, bile acids, bilirubin, etc. In an embodiment, biomarker concentration cut-off between LPR patients and healthy controls, sensitivity (SE), specificity (SP) are evaluated with Receiver Operating Curve (ROC) and fixed at the highest Youden Index (YI) or Likelihood Ratio (LR).

[0062] In addition, the current invention identifies biomarkers that are highly sensitive and specific for LPR. In an embodiment, the method of the current invention allows for diagnosing laryngopharyngeal reflux (LPR) in a subject with a sensitivity of at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, such as 80%. In an embodiment, the method of the current invention allows for diagnosing laryngopharyngeal reflux (LPR) in a subject with a specificity of at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90 %, more preferably at least 95%, such as 100%. In an embodiment, the method of the current invention allows for diagnosing laryngopharyngeal reflux (LPR) in a subject with a combined sensitivity of at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, such as 80% and a specificity of at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90 %, more preferably at least 95%, such as 100%.

[0063] In an embodiment, the invention provides a method for determining the profile of LPR in patients, thereby sorting individuals into different classes or strata based on the characteristics of the form and nature of LPR.

[0064] In an embodiment, the invention provides a method for determining the profile of LPR in patients on the basis of the severity of the disease (e.g., mild, moderate, severe, etc.). Traditionally, the severity of LPR can for instance be determined based on the severity of symptoms and signs, which can for instance be determined based on scores obtained with the Reflux Symptoms Index questionnaire (RSI, as described in Belafsky PC, Postma GN, Koufman JA. Validity and reliability of the reflux symptom index (RSI). J Voice Off J Voice Found. 2002;16(2):274-277.) and / or the Reflux Findings Score (RFS, as described in Belafsky PC, Postma GN, Koufman JA. The validity and reliability of the reflux finding score (RFS). The Laryngoscope. 2001; 111(8): 1313-1317. doi: 10.1097 / 00005537-200108000-00001). However, studies have shown that the RSI was not specific for LPR, and its diagnostic weaknesses have been discussed repeatedly. The RSF is also nonspecific, and laryngeal signs cannot be used to determine reflux changes in many situations (e.g., in smokers, after radiation, after an upper airway infection, etc.). As such, in an embodiment, the method of the current invention allows to determine the profile of LPR in patients on the basis of the severity of the disease. In an embodiment, the method of the current invention allows to differentiate patients with severe reflux (according to the IFOS classification) from patients with mild or moderate reflux (for instance by determining the concentration of bile acids in the (saliva) sample). Based on the baseline score of quality of life of Reflux Symptom Score, the patients may be classified into: QoL-RSS thresholds acute (QoL-RSS = 6-25), recurrent (QoL-RSS = 26-38), and chronic (QoL-RSS > 38) LPR (Acute, Recurrent, and Chronic Laryngopharyngeal Reflux: The IFOSCIassification. Lechien JR, Lisan Q, Eckley CA, Hamdan AL, Eun YG, Hans S, Saussez S, Akst LM, Carroll TL. Laryngoscope. 2023 May;133(5): 1073-1080. doi: 10.1002 / lary.30322.).

[0065] Different types of reflux exist and can for instance be classified as "acid reflux", "weakly acid reflux" (also referred to as "mixed reflux"), "alkaline reflux" (also referred to as "non-acid reflux"). Traditionally, the type of reflux is often determined by a combination of 24-h multichannel intraluminal impedance combined with pH- metry (MII-pH). Using said traditional methods "Acid LPR" can be defined as the ratio of number of hypopharyngeal acid reflux episodes / number of non-acid reflux episodes being >2. "Non-acid LPR" can be defined as the ratio of number of acid reflux episodes / number of non-acid reflux episodes being <0.5. "Weakly acid LPR" can be defined as the ratio of number of hypopharyngeal acid reflux episodes / number of non-acid reflux episodes ranging from 0.51 to 2.0. "Acid hypopharyngeal acid reflux episodes" and "non-acid hypopharyngeal acid reflux episodes" are defined as episodes with pH<4.0 and pH>4.0, respectively. In an embodiment, the amount of either pancreatic elastase, bile acids or cholesterol is determined and compared to a predetermined threshold value for said specific biomarker and wherein based on said comparison the presence or absence or the severity of said laryngopharyngeal reflux is determined. As such, in an embodiment, said method comprises determining the amount of pancreatic elastase in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pancreatic elastase in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject.

[0066] As such, in an embodiment, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of pancreatic elastase in a sample obtained from said subject and comparing said amount to a predetermined threshold value for pancreatic elastase. As described above, preferably said sample is obtained in the morning from said fasting subject (a subject which has not digested any food in the morning on which the sample is obtained).

[0067] As such, in an embodiment, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of pancreatic elastase in a sample obtained from said subject, wherein said sample is obtained from said subject in the morning before any food is ingested, and comparing said amount to a predetermined threshold value for pancreatic elastase.

[0068] In another embodiment, said method comprises determining the amount of pH in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pH in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. In another embodiment, said method comprises determining the amount of bile acids in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for bile acids in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. In another embodiment, said method comprises determining the amount of cholesterol in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for cholesterol in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. In such a case, determining the amount of one biomarker in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for one biomarker is sufficient to diagnose LPR and / or determine the severity of LPR in a subject. As described above, preferably said sample is obtained in the morning from said fasting subject (a subject which has not digested any food in the morning on which the sample is obtained).

[0069] In another or further embodiment, the amount of at least two biomarkers is determined and compared to their respective threshold value in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. As such, in an embodiment, said method comprises determining the amount of pancreatic elastase in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pancreatic elastase and determining the amount of bile acids in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for bile acids.

[0070] In another embodiment, said method comprises determining the amount of pancreatic elastase in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pancreatic elastase and determining the amount of pH in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pH.

[0071] As described in example 3 below, the combination of salivary pancreatic elastase with other salivary biomarkers like pH (and cholesterol) enhances the diagnostic power.

[0072] In another embodiment, said method comprises determining the amount of pancreatic elastase in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pancreatic elastase and determining the amount of cholesterol in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for cholesterol. In another embodiment, said method comprises determining the amount of bile acids in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for bile acids and determining the amount of cholesterol in said sample obtained from said subject and comparing said amount to a predetermined threshold value for cholesterol. In such a case, determining the amount of two biomarkers and comparing said amount to a predetermined threshold value for each of said biomarkers is sufficient to diagnose LPR in a subject

[0073] In another or further embodiment, the amount of at least three biomarkers is determined and compared to their respective threshold value in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. In an embodiment, any combination of any biomarker chosen from pancreatic elastase, pH, bile acids and cholesterol can be used.

[0074] In another or further embodiment, said method comprises determining the amount of pancreatic elastase, bile acids and cholesterol in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject. As such, in an embodiment, the method comprises determining the amount of pancreatic elastase in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for pancreatic elastase, determining the amount of bile acids in said sample obtained from said subject and comparing said amount to a predetermined threshold value for bile acids and determining the amount of cholesterol in said sample obtained from said subject and comparing said amount to a predetermined threshold value specific for cholesterol. In such a case, determining the amount of three biomarkers and comparing said amount to a predetermined threshold value for each of said biomarkers is sufficient to diagnose LPR in a subject.

[0075] In another or further embodiment, said method comprises determining the amount of pancreatic elastase, pH, bile acids and cholesterol in order to diagnose or aid in diagnosing LPR and / or determine the severity of LPR in said subject.

[0076] When the material produced by the pancreas and the liver gets into the duodenum, it is mixed with duodenal fluid; altogether the duodenal fluid, liver secretion, gallbladder contents, and pancreatic secretion form a solution in the duodenum that contains bile but also many other components, such as pancreaticoduodenal enzymes. When this solution gets into the stomach and then up into the esophagus, the patient experiences duodenal gastroesophageal reflux. The liquid produced in the pancreas and the liver is concentrated in the gallbladder, goes into the duodenum, and then goes into the stomach due to duodenal gastric reflux, a physiologic condition that occurs for instance during the postprandial period. Normally, there is always a certain amount of duodenopancreatic secretion that goes into the stomach. In the stomach, this solution is mixed with gastric content, which is, most of the time, acid. This material should not normally enter the esophagus, but in patients with gastroesophageal reflux disease, the acidified gastric-duodenal contents refluxate into the esophagus. When many types of reflux substances are found in reflux fluid, the damage to the hypopharyngeal mucosa is more severe. Bile reflux is a major cause of inflammatory damage and cellular carcinoma of the laryngopharynx and is associated with laryngotracheal stenosis, tracheal fibrosis and laryngotracheal malignancy. Bile is secreted by the liver. Bile acid is the main component of bile, which maintains fat digestion and absorption, regulates inflammation, and affects the intestinal flora. In an embodiment, said one or more biomarkers comprises bile acids. Bile acids are hydroxylated steroids, synthesized in the liver from cholesterol. Duodenal emulsification of fat requires BA micelles and biliary lecithin. As such, bile acids are potent "digestive surfactants" that promote absorption of lipids (including fat-soluble vitamins), acting as emulsifiers. Bile acids represent the primary pathway for cholesterol catabolism and account for -50% of the daily turnover of cholesterol. Bile acids are minimally absorbed in the proximal small intestine, and the bile acid pool flows to the distal ileum, where the acids are reabsorbed by the entero-hepatic transport system and then returned to the liver by the portal vein. Remaining bile acids are excreted in feces. As discussed above, bile acids can enter the stomach due to duodenal gastric reflux, a physiologic condition that occurs for instance during the postprandial period. However, in patients with LPR, the acidified gastric-duodenal contents (including bile acids) refluxate into the esophagus. Bile acids can damage the laryngopharyngeal mucosa by altering the integrity of the mucosal barrier by disrupting cell function and damaging membrane structure. It has been shown that patients with more bile reflux have more severe esophageal mucosal damage, so there is a clear correlation between the amount of bile reflux and the severity of distal esophageal inflammation.

[0077] In an embodiment, said one or more biomarkers comprise bile acids.

[0078] Bile acids can be measured by any technique known from the state of the art. In an embodiment, said bile acid levels are measured using a colorimetric assay, with enzymatic methodology measured by spectrophotometry, such as the total bile acids Assay Kit (Diazyme Lab, Gregg CT Poway, CA, USA). In an embodiment, said threshold value is a value in the range between 0.02 pmol / L and 1.89 pmol / L.

[0079] In an embodiment, said one or more biomarkers comprise pancreaticoduodenal enzymes, such as pancreatic elastase. The potential role of pancreatic elastase in the development of inflammatory reaction of the upper aerodigestive tract was never studied.

[0080] In an embodiment, said one or more biomarkers comprises pancreatic elastase.

[0081] Pancreatic elastase is a form of elastase that is produced in the acinar cells of the pancreas. It is a proteolytic enzyme belonging to the class of the serine proteases or peptidases, exclusively produced in pancreas. Although the recommended name is pancreatic elastase, it can also be referred to as elastase-1, pancreatopeptidase, PE, or serine elastase. As discussed above, pancreatic elastase can enter the stomach due to duodenal gastric reflux, a physiologic condition that occurs during the postprandial period. However, in patients with LPR, the acidified gastric-duodenal contents (including pancreatic elastase) refluxate into the esophagus.

[0082] Pancreatic elastase can be measured by any technique known from the state of the art. In an embodiment, the protein level of pancreatic elastase is measured. In an embodiment, said pancreatic elastase protein level is determined by means of an immunoassay. In an embodiment, said pancreatic elastase protein level is determined by means of an Enzyme Linked Immunosorbent Assay (ELISA). In an embodiment, said threshold value is a value in the range between 10 and 60 pg / ml, preferably between 20 and 40 pg / ml, more preferably between 25 and 35 pg / ml, such as 32 pg / ml.

[0083] However, when it is necessary to diagnose LPR with a high PPV, said threshold should preferably be higher (see for instance example 2).

[0084] In a preferred embodiment, said threshold value for pancreatic elastase is a value in the range between 40 and 60 pg / ml, more preferably between 45 and 55 pg / ml, such as 45 pg / ml, 46 pg / ml, 47 pg / ml, 48 pg / ml, 49 pg / ml, 50 pg / ml, 51 pg / ml, 52 pg / ml, 53 pg / ml, 54 pg / ml, 55 pg / ml or any value in between. In an embodiment, said threshold value for pancreatic elastase is a value in the range between 45-46 pg / ml, 46-47 pg / ml, 47-48 pg / ml, 48-49 pg / ml, 49-50 pg / ml, 50-51 pg / ml, 51-52 pg / ml, 52-53 pg / ml, 53-54 pg / ml or 54-55 pg / ml.

[0085] For instance, in an embodiment, a value of more than 49 pg / ml pancreatic elastase (for instance in a saliva sample) supports the occurrence of LPR and allows to diagnose a subject with laryngopharyngeal reflux disease (as determined in example 3 below). Preferably said LPR is diagnosed with a PPV of at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% and a NPV of at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%. In an embodiment, said one or more biomarkers comprise cholesterol. As described in example 2 below, LPR patients show significantly lower cholesterol values in saliva then control patients. In an embodiment, said threshold value for cholesterol is a value in the range between 0.01 and 50 mg / dL, preferably between 0.1 and and 10 mg / dL, more preferably between 1 and 5 mg / dL, more preferably between 2 and 4 mg / dL, such as 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 mg / dL or any value in between. In a preferred embodiment, said threshold value for cholesterol is 2.1 mg / dL. For instance, in an embodiment, a value beneath 2.1 mg / dL cholesterol (for instance in a saliva sample) supports the occurrence of LPR and allows to diagnose a subject with laryngopharyngeal reflux disease.

[0086] In an embodiment, said method further comprises determining an amount of pepsin in said sample and comparing said amount of pepsin to a predetermined threshold value. As discussed above, injury to the laryngopharyngeal mucosa is related to the presence and activity of the enzyme pepsin. The abnormal secretion and activation of pepsin are crucial to the pathogenesis of LPR. Pepsin is converted from pepsinogen produced by gastric chief cells and is a major factor causing proteolysis and cell damage. Pepsin is undetectable in the laryngeal mucosa of healthy individuals. Pepsin remains active at pH 2.0-6.0. At pH 5.5 and 6.0, it has approximately 30% and 10% activity, respectively. Under neutral conditions, it remains stable, although it is inactive. The presence of pepsin in the esophagus, the throat, the mouth or the lungs can be used in the diagnosis of gastroduodenal content reflux. Pepsin can be measured by any technique known from the state of the art. In an embodiment, the protein level of pepsin is measured. In an embodiment, the protein level of pepsin is measured by means of an immunoassay. In an embodiment, said pepsin protein level is determined by means of Enzyme Linked Immunosorbent Assay (ELISA).

[0087] In an embodiment, said method further comprises determining an amount of electrolytes like Sodium, Potassium, Chloride, Calcium, Magnesium, Phosphorus or Osmolality in said sample and comparing said amount of electrolyte or Osmolality to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. They may contribute to fluid homeostasis.

[0088] In an embodiment, said method further comprises determining an amount of sugar like glucose, maltose, mannose in said sample and comparing said amount of sugar to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. They may reflect digestion. In an embodiment, said method further comprises determining an amount of immunoglobulin (including IgG, IgA, IgM) and inflammatory markers like C-reactive protein, ferritine, procalcitonin, and cytokines (including interleukin, tumor necrosis factor, growth factors) in said sample and comparing said amount of immunoglobulin and inflammatory marker to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. They may reflect the inflammatory condition.

[0089] In an embodiment, said method further comprises determining an amount of glycoproteins (including tumor markers, mucin, KL-6) in said sample and comparing said amount of glycoprotein to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. They may reflect local cellular damage.

[0090] In an embodiment, said method further comprises determining an amount of transforming growth factor beta (TGF-P) in said sample and comparing said amount of TGF-p to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. TGF-p is a marker of fibrosis.

[0091] In an embodiment, said method further comprises determining an amount of lactate dehydrogenase (LDH) in said sample and comparing said amount of LDH to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. LDH may reflect mucosal cellysis.

[0092] LPR disease may also have links to hormone imbalances. For example, low thyroid (hypothyroidism) may cause the lower esophageal sphincter valve to malfunction, allowing stomach contents to enter the esophagus. High estrogen levels can relax the lower esophageal sphincter and irritate the stomach, especially during pregnancy. Stress is another common trigger for reflux. Stress triggers the release of cortisol, the "stress hormone." Cortisol interferes with proper digestion and weakens your immune system, making individuals more prone to reflux.

[0093] In an embodiment, said method further comprises determining an amount of one or more hormones in said sample and comparing said amount of said one or more hormones to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. The amount of said one or more hormones can be determined by any suitable assay known from the state of the art. Examples of such assays are discussed below. Hormones are one of many substances made by glands in the body. Hormones circulate in the bloodstream and control the actions of certain cells or organs. Any type of suitable hormone can be used to diagnose LPR. Examples of such hormones include Thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), thyroxine (fT4), calcitonin, gastrin, Vasoactive Intestinal Peptide (VIP), Adrenocorticotropic hormone (ACTH) or cortisol. Said sample can be any type of suitable sample, but is preferably a saliva sample.

[0094] In an embodiment, said method further comprises determining an amount of ACTH in said sample and comparing said amount of ACTH to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR. Adrenocorticotropic hormone (corticotropin; ACTH) is a 39 amino acid peptide hormone produced by cells of the anterior pituitary gland and carried by the peripheral circulation to its effector organ, the adrenal cortex, where it stimulates the synthesis and secretion of glucocorticoids and, to a more modest extent, mineralocorticoids and adrenal androgens. ACTH is secreted in response to corticotropin-releasing hormone (CRH) from the hypothalamus. The actions of CRH on ACTH release are augmented by another hypothalamic hormone, arginine vasopressin. ACTH production and secretion are regulated by glucocorticoid feedback, with ACTH at the center of a homeostatic network involving the hypothalamus and pituitary. Important brain areas influencing ACTH release include the hypothalamus, amygdala, hippocampus, and prefrontal cortex. As well, ACTH release is modulated by several other stimulatory and inhibitory factors, including neurotransmitters, peptides, and immune factors. ACTH release can become dysregulated by a number of factors including stressors, psychiatric illnesses, endocrine disorders, and various diseases.

[0095] In an embodiment, said method further comprises determining an amount of cortisol in said sample and comparing said amount of cortisol to a predetermined threshold value, in order to diagnose or aid in diagnosing LPR.

[0096] Interestingly, the inventors observed that the saliva pH of LPR patients was significantly higher than the pH of saliva of healthy individuals. Moreover, the saliva pH of patients was significantly correlated with the concentration of saliva bicarbonate. Carbonic anhydrase is one of the most important defense mechanisms of laryngopharyngeal mucosa against reflux. Carbonic anhydrase is an enzyme that catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. Previous studies found that expression of carbonic anhydrase type III was significantly increased in the mucosa of posterior commissure in LPR patients. In that way, the increase of bicarbonate in saliva and the related higher pH could reflect the mucosa defense against reflux irritation. Previous studies show significantly elevated bicarbonate levels in saliva from GERD patients and suggest the possible applicability of bicarbonate as a biomarker in non- invasive diagnostics of GERD (DOSEDELOVA, Vera, Pavol DURC, J iff DOLINA, Stefan KONECNY, Frantisek FORET and Petr KUBAN. Analysis of bicarbonate, phosphate and other anions in saliva by capillary electrophoresis with capacitively coupled contactless conductivity detection in diagnostics of gastroesophageal reflux disease. Electrophoresis. HOBOKEN : WILEY-BLACKWELL, 2020, vol. 41, 1-2, p. 116-122. ISSN 0173-0835. doi: 10. 1002 / elps.201900319.).

[0097] Interestingly, the inventors found that the mean concentrations of bicarbonate did not statistically differ between LPR patients and controls. As such, bicarbonate is not a good biomarker to be used in the method of the current invention.

[0098] Furthermore, the inventors also discovered that gastric lipase does not seem to be a good biomarker to be used in the method of the current invention (see Example 2).

[0099] In an embodiment, said method further comprises measuring the pH of said sample (preferably a saliva sample) and determining whether said pH is higher than a certain threshold value. PH can be measured by any technique known from the state of the art. In an embodiment, the pH of said sample is determined by means of a pH meter or a colorimetric method like the kit DRI® pH-Detect® Test, Calibrators and Controls from ThermoScientific. For instance, in an embodiment, a pH above 7.6 (for instance in a saliva sample) supports the occurrence of LPR and allows to diagnose a subject with laryngopharyngeal reflux disease.

[0100] As described in example 3 below, the combination of salivary pancreatic elastase with other salivary biomarkers like pH (and cholesterol) enhances the diagnostic power. In a preferred embodiment, at least pancreatic elastase and pH are determined in a sample obtained from said subject and compared to a predetermined threshold value for said respective biomarker. The inventors discovered that the combination of elastase and pH is especially well suited for diagnosing laryngopharyngeal reflux disease in a subject with a sufficient positive predictive value. In an embodiment, the determination of pancreatic elastase and pH and comparison with predetermined threshold values for said respective biomarker is sufficient to diagnosing laryngopharyngeal reflux disease in a subject. As such, in a preferred embodiment, the invention relates to a method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining the amount of pancreatic elastase and the pH in a sample obtained from said subject and comparing said amount and said pH to a predetermined threshold value for said biomarker.

[0101] In an embodiment, said threshold value for pancreatic elastase is a value in the range between 30 and 40 pg / ml, more preferably between 30 and 55 pg / ml (for instance 32 pg / ml) and said threshold value for pH is between 7-8 (for instance 7.6).

[0102] However, when a high PPV is desired, said threshold value for pancreatic elastase can be increased.

[0103] In an embodiment, said threshold value for pancreatic elastase is a value in the range between 40 and 60 pg / ml, more preferably between 45 and 55 pg / ml (for instance 49 pg / ml) and said threshold value for pH is between 7-8 (for instance 7.6). For instance, in an embodiment, a value of more than 49 pg / ml pancreatic elastase and a pH above 7.6 (for instance measured in a saliva sample) supports the occurrence of LPR and allows to diagnose a subject with laryngopharyngeal reflux disease with a NPV and a PPV above at least 60%, or at least 70% or at least 80% or at least 90%. In an embodiment, positive predictive value (PPV) and negative predictive value (NPV) are calculated through contingency tables and / or ROC.

[0104] As described above, said sample is preferably a saliva sample. However, the sample can also be any other type of clinically relevant sample, such as a sputum sample, a nasal lavage sample or a blood sample. In an embodiment, said sample is for instance obtained at a first medical visit of the subject for reflux diagnosis.

[0105] As described above, said sample is preferably a saliva sample. However, the sample can also be any other type of clinically relevant sample, such as a sputum sample, a nasal lavage sample or a blood sample.

[0106] In a preferred embodiment, said saliva sample is obtained from said subject under optimal conditions. In an embodiment said sampling time (the moment during the day when the saliva sample is obtained) is optimized. Said optimal sample time can be for instance the just awake state of the subject.

[0107] In an embodiment, said sample is processed and stored under optimal conditions before measuring the level of said one or more biomarkers. In an embodiment, said sample is mixed with one or more components, such as a buffer to maintain the stability of the components and to prevent degradation of sensitive peptides in the saliva sample. In an embodiment, said saliva sample is mixed with an amount of citric acid (for instance a 2% citric acid solution). In an embodiment, said saliva sample is mixed with a Protease Inhibitor Cocktail.

[0108] In a preferred embodiment, said saliva sample is collected at room temperature (for instance in polypropylene tubes or Salivette®) and rapidly frozen to prevent degradation of sensitive peptides.

[0109] In an embodiment, said saliva sample is centrifuged to remove debris and the resulting supernatant is used for measuring the level of said one or more biomarkers. In an embodiment, said saliva sample is centrifuged at 4000 rpm for 5 minutes or at 1000 x g for 2 minutes or at 1000 x g for 10 minutes.

[0110] In an embodiment, said saliva sample is stored at a temperature of at most of 8°C. In an embodiment, said saliva sample is stored at a temperature of at most -4°C. In an embodiment, said saliva sample is stored at a temperature of at most -20°C. In an embodiment, said saliva sample is stored at a temperature of at most -80°C.

[0111] In an embodiment, said saliva sample is stored at a controlled temperature for a maximal period of time before measuring the level of said one or more biomarkers. In an embodiment, when analyzing stable biomarkers (e.g. but not limited to cholesterol, bile acids, pH, pancreatic elastase), said saliva sample is stored at a temperature of at most 8°C for a maximum period of seven days. In an embodiment, said saliva sample is stored at a temperature of at -20°C during one year or longer. Unstable biomarkers (e.g. but not limited to gastrin) need to be analyzed immediately or frozen at -20°C for storage.

[0112] In a further aspect, the invention relates to a diagnostic kit for the diagnosis of laryngopharyngeal reflux in a subject, said kit comprising one or more saliva collection tubes and one or more assays for measuring the levels of one or more biomarkers in a saliva sample obtained from said subject, wherein said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol. As described above, in a preferred embodiment both pancreatic elastase and pH are determined for diagnosing LPR with a high PPV. As such, in an embodiment, said kit comprises at least an assay for determining pancreatic elastase (for instance an ELISA assay) and an assay for determining pH (for instance a pH electrode or a colorimetric test, such as pH-Detect Test). pH-Detect Test is an end point colorimetric method which can be performed with automated clinical chemistry analyzers. The method is based on the property of acid-base indicator dyes, which produce color depending on the pH of the sample. The color change can be measured as an absorbance change spectrophotometrically. A linear two-point calibration curve can be established using the pH 3.0 and pH 11.0 calibrators.

[0113] In an embodiment, said kit further comprises a device for swabbing, such as a cotton swab. In an embodiment, said kit comprises a swab with citric acid preparation to stimulate salivation. In an embodiment, said kit comprises a swab without citric acid preparation. In an embodiment, the subject places the swab in the oral cavity and chews it for about 60 seconds to stimulate salivation, before placing the swab with the absorbed saliva in the collection tube.

[0114] Said one or more saliva collection tubes can be any suitable leak proof collection tube known from the state of the art for hygienic collection of saliva, such as a Salivette® collection tube from Sarstedt.

[0115] Said one or more assays can include any assay known from the state of the art for measuring the level of certain biomarkers, such as pancreatic elastase, pH, bile acids, cholesterol, electrolytes, osmolality, sugar, immunoglobulin, inflammatory markers, glycoproteins, TGF-beta, lactate dehydrogenase and / or hormones. Methods for pH, electrolytes, osmolality, sugar, immunoglobulin, inflammatory markers, lactate dehydrogenase and cholesterol include pH indicator paper, electrodes, colorimetry, spectrophotometry, freese point depression, enzymatic method, immunoturbidimetry, nephelometry and a colorimetric enzymatic method. Examples of such assays include: Enzyme Linked ImmunoSorbent Assay (ELISA, for instance a sandwich ELISA), homogeneous immunoassays, chemiluminescence, like ChemiLuminescence Enzyme ImmunoAssay or CLEIA, which can advantageously be achieved using the Lumipulse® platform, Electro-chemiluminescence Immunoassay (ECLIA), Chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunochromatographic strip or lateral flow immunochromatography, mass spectrometry, latex agglutination, biosensor technology using fluorescence, (magnetic) bead-based technologies (such as those based on Luminex® liquid array methodologies) and others as well as alternate newer technologies including nanotechnology, biosensors and (bead-based) lab-on-a-chip methodologies. The immunoassays can also be multiplexed or single plexed in arrays and / or beads, which can be magnetic. In an embodiment, said assay comprises an immunoassay. Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. In an embodiment, said assay for measuring the level of pancreatic elastase comprises an immunoassay. In an embodiment, said immunoassay for measuring the level of pancreatic elastase comprises an Enzyme Linked Immunosorbent Assay (ELISA). In an embodiment, said ELISA is a sandwich ELISA. In an embodiment, said assay for measuring the level of bile acids comprises a colorimetric assay, for instance a colorimetric assay measuring the rate of formation of Thio-NADH by measuring specific change of absorbance at 405 nm. In an embodiment, said amount of cholesterol in said sample is determined by means of a colorimetric enzymatic method. Colorimetric enzymatic method are known from the state of the art. The principle of this method lies in the generation of color by chemical / biochemical reaction between target analyte and reagents.

[0116] In an embodiment, levels of said one or more biomarkers comprises RIMA levels. In an embodiment, levels of said one or more biomarkers comprises protein levels.

[0117] In an embodiment, said kit further comprises an assay for measuring the protein levels of pepsin. In an embodiment, said assay for measuring the level of pepsin comprises an immunoassay. In an embodiment, said immunoassay for measuring the level of pepsin comprises an Enzyme Linked Immunosorbent Assay.

[0118] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0119] EXAMPLES AND / OR DESCRIPTION OF FIGURES

[0120] The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

[0121] Example 1. Identification of suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR) with a study population of 20 LPR patients and 12 control subjects

[0122] Aim: To investigate the presence of pancreaticoduodenal enzymes in the saliva of patients with laryngopharyngeal reflux (LPR) and identification of suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR).

[0123] Methods:

[0124] Study population and LPR diagnosis using HEMII-pH-monitoring

[0125] Patients with LPR symptoms and signs were consecutively recruited from the European Reflux Clinic consultation from January 2021 to June 2021. The LPR diagnosis was confirmed with 24-hour hypopharyngeal-esophageal multichannel intraluminal impedance pH (HEMII-pH)-monitoring. Patients with gastroesophageal reflux disease (GERD) symptoms and elderly patients (>55 years old) benefited from upper gastrointestinal endoscopy to detect the presence of esophagitis, lower esophageal sphincter insufficiency, hiatal hernia or gastritis. The following exclusion criteria were reported in the article of Lechien et al (Lechien JR, Bobin F, Dapri G, Eisendrath P, Salem C, Mouawad F, Horoi M, Thill MP, Dequanter D, Rodriguez A, Muis V, Saussez S. Hypopharyngeal-Esophageal Impedance-pH Monitoring Profiles of Laryngopharyngeal Reflux Patients. Laryngoscope. 2021; 131(2) :268-276. doi: 10.1002 / lary.28736).

[0126] Hypopharyngeal-Esophageal Multichannel Intraluminal Impedance-pH testing.

[0127] The features of HEMII-pH device and placement were reported in the article of Lechien et al (Lechien JR, Bobin F, Dapri G, Eisendrath P, Salem C, Mouawad F, Horoi M, Thill MP, Dequanter D, Rodriguez A, Muis V, Saussez S. Hypopharyngeal- Esophageal Impedance-pH Monitoring Profiles of Laryngopharyngeal Reflux Patients. Laryngoscope. 2021; 131(2) :268-276. doi: 10.1002 / lary.28736.). In summary, the device was composed of 8 impedance ring pairs and 2 pH electrodes (Versaflex Z®, Medtronic, Hauts-de-France, France). The impedance segments were placed along the esophagus zones (Z1 to Z6) and in the hypopharyngeal cavity (2 sensors). Two pH electrodes were placed 2 to 5 cm above LES and 1-2 cm above UES. The device was placed in the morning at rest. A pharyngeal reflux event was considered as an episode reaching two pharyngeal impedance sensors. The LPR diagnosis was confirmed if there was >1 acid or non-acid hypopharyngeal reflux event. Acid and non-acid reflux were defined as episodes with pH<4.0 and pH>4.0, respectively. Acid LPR was defined when the ratio of the number of hypopharyngeal acid reflux episodes / the number of nonacid reflux episodes was >2. Nonacid LPR was defined when the ratio of the number of acid reflux episodes / the number of nonacid reflux episodes <0.5. Weakly acid reflux consisted of a ratio ranging from 0.51 to 2.0. The GERD diagnosis was based on the Lyon criteria (Gyawali CP, Kahrilas PJ, Savarino E, Zerbib F, Mion F, Smout AJPM, Vaezi M, Sifrim D, Fox MR, Vela MF, Tutuian R, Tack J, Bredenoord AJ, Pandolfino J, Roman S. Modern diagnosis of GERD: the Lyon Consensus. Gut. 2018; 67(7): 1351-1362. doi: 10.1136 / gutjnl-2017-314722).

[0128] Saliva collection.

[0129] Saliva of patients was collected the morning of the HEMII-pH testing (after the 24- hour period testing), fasting and before brushing the teeth in a Salivette collection device (Sarstedt, Germany). Only patients who provided a saliva volume of 2 mL were included for analysis. Test DRI pH-Detect (Thermo Scientific, Germany), total bile acids Assay Kit (Diazyme Lab, Gregg CT Poway, CA, USA), bicarbonate CO2-L, cholesterol CHOL2 and total bilirubin BILT3 were analyzed on Cobas 8000 (Roche, Germany). Pancreatic elastase was tested with pancreatic elastase ELISA (BioServ Diagnostics, Germany).

[0130] Treatment and clinical evaluations.

[0131] Patients were treated with a 3-month low-fat, low- quick-release sugar, high-protein, alkaline and plant-based diet, and a combination of proton pump inhibitors (PPIs), alginate or magaldrate. Patients with acid and weakly acid LPR received PPIs and alginate, while those with alkaline LPR benefited from alginate or magaldrate only. Additional details about this therapeutic regimen are available in the article (Lechien JR, Bobin F, Muis V, Mouawad F, Dequanter D, Horoi M, Thill MP, Rodriguez Ruiz A, Saussez S. The efficacy of a personalised treatment depending on the characteristics of reflux at multichannel intraluminal impedance-pH monitoring in patients with acid, non-acid and mixed laryngopharyngeal reflux. Clin Otolaryngol. 2021; 46(3):602- 613. doi: 10.1111 / coa.13722.). Reflux Symptom Score (RSS)12 and Reflux Sign Assessment (RSA)13 were used to evaluate symptoms and findings throughout treatment. Findings were evaluated retrospectively in a blind manner by two laryngologists with recorded videolaryngostroboscopy and oral cavity photos of upper aerodigestive tract of patients. A control group of individuals without LPR symptoms (RSS<13) and without comorbidities was composed. The saliva of healthy individuals was collected in the morning, fasting and before brushing the teeth with the same Salivette collection device.

[0132] Statistical methods

[0133] Statistical analyses were performed using the Statistical Package for the Social Sciences for Windows (SPSS version 27.0; IBM Corp, Armonk, NY, USA and GraphPad Prism 5.0, GraphPad Software, Inc., San Diego, California, USA). According to the type of variables, the following tests were used to compare patients and controls: Mann-Whitney U test, Chi-square, and T-test. Symptom and sign changes throughout treatment were evaluated with the Wilcoxon signed-rank test. Multivariate analysis was used to study the outcome associations. Enzyme concentration cut-off between LPR patients and healthy controls was evaluated with Receiver Operating Curve (ROC) on Analyze-it (Excel, USA). A level of significance of p<0.05 was used.

[0134] Results:

[0135] Twenty patients and twelve healthy individuals completed the study. The patient group consisted of 12 acid, 4 weakly acid and 4 alkaline LPR patients. Demographic, epidemiological and clinical features are reported in Table 1 (see below). The patient group was composed of 10 males (50%) and 10 females (50%), respectively. The mean age of patients was 52.6 ± 14.3 years. The control group included 7 females and 5 males. The mean age of healthy individuals was 33.4 ± 8.2 years, which was significantly lower than the age of patients (p=0.001).

[0136] Table 1: Epidemiological and clinical features of patients.

[0137] Characteristics

[0138] Mean age (SD) 52.6 ± 14.3

[0139] Body mass index 24.1 ± 4.5

[0140] Gender (N, %)

[0141] Male 10 (50)

[0142] Female 10 (50)

[0143] Gastrointestinal endoscopy (N, %) N = 17

[0144] Normal 2 (11.8)

[0145] Esophagitis 8 (47.1)

[0146] Hiatal hernia 5 (29.4)

[0147] LES insufficiency 8 (47.1)

[0148] Gastritis 9 (52.9)

[0149] Helicobacter Pylori 1 (5.9)

[0150] Types of LPR at the HEMII-pH (N, %)

[0151] Acid LPR 12 (60)

[0152] Weakly acid LPR 4 (20)

[0153] Nonacid LPR 4 (20)

[0154] HEMII-pH feature (m ± SD)

[0155] Pharyngeal acid reflux episodes 16.3 ± 18.7 Pharyngeal nonacid reflux episodes 6.6 ± 4.8

[0156] Pharyngeal reflux episodes upright 23.0 ± 20.6

[0157] Pharyngeal reflux episodes supine 3.3 ± 4.6

[0158] Total number of pharyngeal reflux episodes 23.2 ± 22.0

[0159] GERD

[0160] Number of patients (%) 10 (50)

[0161] Percentage of time with distal pH<4 5.1 ± 10.6

[0162] DeMeester score 17.8 ± 32.5

[0163] Abbreviations: GERD=gastroesophageal reflux disease; HEMII-pH= hypopharyngeal- esophageal multichannel intraluminal impedance pH; LPR=laryngopharyngeal reflux; SD=standard deviation.

[0164] The saliva measurement data are reported in Table 2. The mean concentrations of bile acids in LPR and healthy individuals were 1.21±0.52 pmol / L and 0.17±0.48 pmol / L (p=0.006), respectively. LPR patients had a significant higher concentration of pancreatic elastase in saliva (64.89±41.33 pg / mL) compared with healthy controls (17.42±8.89 pg / mL; p=0.001) (see Figure 1). The analysis of saliva pH reported mean values of 7.76±0.64 and 7.11±0.61 for LPR and control individuals, respectively (p=0.025). The mean concentrations of bicarbonate did not statistically differ between LPR patients (5.31±3.20 mmol / L) and controls (4.55±2.44 mmol / L).

[0165] Table 2: Biomarker features.

[0166] Biomarkers LPR CT p-value

[0167] Pancreaticoduodenal enzymes

[0168] Pancreatic elastase (pg / mL) 64.89 ± 41.33 17.42 ± 8.89 0.001

[0169] Bile acids (pmol / L) 1.21 ± 0.52 0.17 ± 0.48 0.006

[0170] Saliva parameters pH 7.76 ± 0.64 7.11 ± 0.61 0.025

[0171] Bicarbonate (mmol / L) 5.31 ± 3.20 4.55 ± 2.44 NS

[0172] Abbreviations: CT=controls; LPR=laryngopharyngeal reflux; NS=Not Significant.

[0173] Otolaryngological reflux symptom score (RSS), digestive RSS and RSS total score significantly improved from pre- to 3-month posttreatment (see Table 3). Only laryngeal reflux sign assessment (RSA) reported significant improvement at 3-month posttreatment (see Table 3). Table 3: Clinical outcomes

[0174] Clinical outcomes Baseline 3 months p-value

[0175] Symptom scores

[0176] Otolaryngological RSS 50.3 ± 43.9 43.6 ± 43.9 0.038

[0177] Digestive RSS 50.8 ± 37.1 40.7 ± 39.4 0.013

[0178] Respiratory RSS 16.6 ± 25.0 9.3 ± 24.1 NS

[0179] RSS total score 117.7 ± 91.3 93.6 ± 99.9 0.008

[0180] Finding scores

[0181] Oral cavity RSA 5.4 ± 2.2 5.2 ± 1.2 NS

[0182] Pharyngeal RSA 10.5 ± 3.9 9.6 ± 2.2 NS

[0183] Laryngeal RSA 12.9 ± 5.9 9.4 ± 5.1 0.042

[0184] RSA total score 28.9 ± 7.9 24.2 ± 5.4 NS

[0185] Abbreviations: RSA=reflux sign assessment; RSS=reflux symptom score.

[0186] The multivariate analysis reported a significant association between the concentration of bile acids and the percentage of pH<4.0 in the distal esophagus (rs=0.899; p=0.006). The saliva concentration of bicarbonate was significantly correlated with the pH (rs=0.594; p<0.001) and the pancreatic elastase concentration (rs=0.355; p=0.043).

[0187] Conclusion:

[0188] LPR patients reported significant higher saliva concentration of pancreatic elastase (p=0.001) and bile acids (p=0.006) compared with controls. According to the ROC curve analysis, the cut-off of pancreatic elastase in saliva was 31.5 pg / mL, reporting sensitivity and specificity of 80% and 100% (see Figure 2). The pH of saliva was significantly higher (p=0.025) in LPR patients (7.76 ± 0.64) compared with healthy controls (7.11 ± 0.61). The multivariate analysis reported a significant association between the concentration of bile acids and the percentage of pH<4.0 in the distal esophagus (rs=0.899; p=0.006).

[0189] This is the first study to demonstrate the presence of several pancreatic enzymes in the saliva of LPR patients, highlighting the existence of a back flow of pancreaticoduodenal enzymes in the upper aerodigestive tract in LPR patients. Furthermore, this study demonstrates the potential of pancreatic elastase, pH and bile acids as biomarkers of laryngopharyngeal reflux. Example 2. Identification of suitable biomarkers for diagnosing patients with laryngopharyngeal reflux (LPR) with a study population of 45 LPR patients and 22 control subjects

[0190] A similar experiment as discussed above was performed, this time with a bigger study population consisting of 45 LPR patients and 22 control subjects. Material and methods were as discussed in example 1. Bile (biliary) acid levels, pancreatic elastase levels, cholesterol levels, gastric lipase levels and pH were determined.

[0191] 1. Bile acids:

[0192] Bile acid analysis was performed on 3 subgroups of patients with laryngopharyngeal reflux (LPR) using pH impedancemetry. The first subgroup consisted of patients with mild (acute) laryngopharyngeal reflux according to IFOS (International Federation of ORL Societies) criteria. The second subgroup included patients with moderate (acute recurrent) reflux and the third subgroup included patients with severe (chronic) reflux, the latter not easily treated with current medications. Bile acid concentrations between groups were compared using Kruskal Wallis and Mann- Whitney U tests.

[0193] LPR patients ("reflux patients", n=45) had a bile acid concentration having a mean ± standard deviation of 0.3882± 0.3672 pmol / L, whereas control subjects ("controls", n=22) had a bile acid concentration showing a mean ± standard deviation of 0.4650± 0.3640. According to the Mann-Whitney U test, both groups had no significantly different medians (p=0.1601, see Figure 3).

[0194] However, patients with severe reflux according to the IFOS classification had a significantly higher bile acid concentration than patients with mild or moderate reflux and had more episodes of pharyngeal reflux at pH impedancemetry (see Table 4 below).

[0195] Table 4: comparison different LPR patient subgroups

[0196] As such, bile acids can be considered as a biomarker of the severity of laryngopharyngeal reflux and can reflect the severity on pH impedancemetry and clinical scores (RSS for Reflux Symptom Score and RSS Quality of Life).

[0197] 2. Pancreatic elastase:

[0198] Similar to Example 1, the pancreatic elastase concentration was determined and a ROC-curve was established for the bigger study population consisting of 45 LPR patients and 22 control subjects (see Figure 4). According to the ROC curve analysis (AUC=0.850), the cut-off of pancreatic elastase in saliva was 32 pg / mL and was associated with sensitivity and specificity of 84% and 79% (see Figure 4). The inventors found that an optimized cut-off (threshold) value of 32 pg / mL can be used when a high NPV is seeked (NPV=96%, PPV=20%), while the cut-off (threshold) value can also be placed at 49 pg / mL if a high PPV is seeked (NPV=95%, PPV=100%). With a prevalence of 10% for LPR, the PPV and NPV are 20% and 96%, respectively, at the cut-off of 32 pg / mL for pancreatic elastase.

[0199] 3. Cholesterol:

[0200] Cholesterol was measured with spectrophotometry on Cobas c702 with the kit CHOL2 from Roche Diagnostics (Mannheim, Germany).

[0201] LPR patients show significantly lower cholesterol values in saliva then control patients. The mean values were 0.7±0.6 and 4. l±0.4 for LPR and control individuals, respectively (p<0.0001, see figure 5A). The cut-off (3.3 mg / dL) for differentiating LPR patients from controls was obtained with ROC analysis (see Figure 5B). Patients with LPR show cholesterol levels < 3.3 mg / dL. The PPV and NPV are both 100% at the cut-off of 3.3 mg / dL.

[0202] 4. pH

[0203] For pH a cut-off of >5.9 (NPV=99%, PPV=19%) or >7.3 (NPV=95%, PPV=55%) can be used, depending on the desired predictive value.

[0204] 5. Gastric lipase

[0205] Gastric lipase was analysed with a sandwich ELISA using a kit from Cloud-Clone Corp. However, gastric lipase was below the limit of detection for both study groups (see Table 5 below). As such, it seems that gastric lipase is not a good biomarker for diagnosing LPR. Table 5

[0206] Overall conclusion of Examples 1 and 2:

[0207] Bile acids can be considered as a biomarker for assessing the severity of laryngopharyngeal reflux disease (LPR).

[0208] Pancreatic elastase can be considered as a biomarker for LPR. A cut-off (threshold) value of 32 pg / mL elastase can be used when a high NPV is seeked (NPV=96%, PPV=20%), while the cut-off can be placed at 49 pg / mL if a high PPV is seeked (NPV=95%, PPV= 100%).

[0209] Cholesterol can be considered as a biomarker for LPR. The cut-off (threshold) value (3.3 mg / dL) for differentiating LPR patients from controls was obtained with ROC analysis. Patients with LPR show cholesterol levels < 3.3 mg / dL. The PPV and NPV are both 100% at the cut-off of 3.3 mg / dL.

[0210] PH can be considered as a biomarker for LPR. For pH a cut-off of >5.9 (NPV=99%, PPV= 19%) or >7.3 (NPV=95%, PPV=55%) can be used, depending on the desired predictive value. In the method of the current invention pH analysis should preferably be combined with another biomarker (for instance pancreatic elastase, bile acid, cholesterol or hormone levels).

[0211] Gastric lipase seems to be not a good biomarker for diagnosing LPR.

[0212] Example 3: Clinical performance study of Salivary Biomarkers in Laryngopharyngeal Reflux

[0213] A similar experiment as discussed above was performed, this time with a bigger study population consisting of 67 LPR patients and 35 control subjects.

[0214] Introduction

[0215] Laryngopharyngeal reflux (LPR) is an inflammatory condition of the upper aerodigestive tract tissues related to direct and indirect effect of gastroduodenal content reflux, which induces morphological changes in the upper aerodigestive tract. Currently, the 24-hour hypopharyngeal-esophageal multichannel intraluminal impedance pH-monitoring (HEMII-pH) is the only objective way to diagnose patients with LPR. However, HEMII-pH is costly and may be poorly tolerated by patients with laryngopharyngeal mucosa hypersensitivity. Thus, there is a need for exploring non- invasive detection methods. In this clinical performance study preliminary data are reported about the concentration of biomarkers, including a pancreaticoduodenal enzyme like elastase in the saliva of patients with LPR at the 24-hour HEMII-pH. The aim of this clinical performance study was to highlight some saliva biomarkers that may theoretically be interesting in LPR patients and, in case of detection, we aimed to propose quantitative cut-offs to distinguish with saliva of healthy controls.

[0216] Methods:

[0217] Patients and setting

[0218] Patients with LPR symptoms and signs were consecutively recruited from the European Reflux Clinic consultation from January 2021 to June 2021. The LPR diagnosis was confirmed with 24-hour HEMII-pH. Patients with gastroesophageal reflux disease (GERD) symptoms and elderly patients (>55 years old (yo)) benefited from upper gastrointestinal endoscopy to detect the presence of GERD complications, including esophagitis, esophageal stricture or Barrett metaplasia.

[0219] Hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring The features of HEMII-pH device and placement were reported in previous study. In summary, the device was placed in the morning fasting and removed after 24-hours of testing. A pharyngeal reflux event was considered as an episode reaching two pharyngeal impedance sensors. The LPR diagnosis was confirmed if there was >1 acid or nonacid hypopharyngeal reflux event. Acid and nonacid reflux were characterized as episodes with pH<4.0 and pH>4.0, respectively. Acid LPR was defined when the ratio of number of hypopharyngeal acid reflux episodes / number of nonacid reflux episodes was >2. Nonacid LPR was defined when the ratio of number of acid reflux episodes / number of nonacid reflux episodes <0.5. Weakly acid reflux consisted of a ratio ranged from 0.51 to 2.0. The GERD diagnosis was based on the Lyon criteria. The following exclusion criteria included smoker, alcohol dependence, upper respiratory tract infection within the last month, current use of anti-reflux treatment (i.e., PPIs, antihistamine, alginate and magaldrate), pregnancy, neurological or psychiatric illness, previous history of neck surgery or trauma, malignancy, history of head and neck radiotherapy and active seasonal allergies or asthma.

[0220] Saliva collection Saliva of LPR patients was collected the morning of the HEMII-pH testing (after the 24-H period testing), fasting and before brushing the teeth in a Salivette collection device (Sarstedt AG & Co., Numbrecht, Germany). The swab was put in the mouth and chewed during 1 minute. Salivette was frozen at -20°C during storage. Before analysis, Salivette was centrifuged 10 minutes at 1000g and then the cotton wool swab was removed. Only patients who provided a saliva volume of 2 mL were included for analysis.

[0221] A control group of individuals without LPR symptoms (RSS<13) and without comorbidities was composed. The saliva of healthy individuals was collected in the morning, fasting and before brushing the teeth with the same Salivette collection device.

[0222] Laboratory tests

[0223] The open channel tests DRI pH-Detect (Thermo Fisher Scientific, Dreieich, Germany) and Colorimetric Total Bile Acids Assay Kit (Diazyme Lab, Gregg CT Poway, CA, USA) were analyzed on module c502 of Cobas 8000 (Roche Diagnostics Corporation, Indianapolis, USA). The pH kit contained pH indicator dyes in an aqueous solution. The assay for bile acids measured the major forms of bile acids such as primary, secondary, and tertiary bile acids and their conjugates, including cholic acid, chenodeoxycholic acid, glycocholic acid, and taurocholic acid. The biomarkers cholesterol (CHOL2) and total bilirubin (BILT3) were analyzed on module c702 of Cobas 8000 (Roche Diagnostics Corporation, Indianapolis, USA). For cholesterol analysis, a quinone-imine derivate is measured through enzymatic colorimetry. Bilirubin is measured with colorimetric spectrophotometry through diazo-methodology. Elastase was tested with pancreatic elastase ELISA (ref. BS-86- 01 SK15 from BioServ Diagnostics GmbH, Rostock, Germany). The ELISA method for elastase detection in saliva had not been described in literature yet. The sandwich assay targeted human pancreatic elastase. It used a 96-well microtiter plate precoated with an anti-elastase capture antibody. The experimental protocol for elastase measurement followed the manufacturer's instruction according to EU registration number DE / CA81 / 2009-17: Bring kit components at room temperature, dilute rinse solution 10 times with deionized water, pipette in duplicate 50 pL of blank (rinse solution) and standards (SK15, S1-S4) in wells, followed by 50 pL of each patient sample and internal control (level 1 and 2), incubate 60 minutes at 37 degrees Celsius, invert plate to empty wells, wash three times with 200 pL of rinse solution per well, invert plate and hit onto absorbent paper to remove moisture between every wash step while holding strips firmly into frame, dilute biotinylated elastase detection antibody 1 / 201 times with rinse solution, add 50 pL of it to each well, incubate 30 minutes at 37 degrees Celsius, invert plate to empty wells, wash three times with 200 pL of rinse solution per well, invert plate and hit onto absorbent paper, add 50 pL of streptavidin conjugated horseradish peroxidase (HRP) reagent to form avidin-biotin complexes, incubate 30 minutes at 37 degrees Celsius, invert plate to empty wells, wash three times with 200 pL of rinse solution per well, invert plate and hit onto absorbent paper, add 100 pL tetramethylbenzidine substrate to react with HRP, cover plate, protect from light during incubation 20 minutes at 37 degrees Celsius, add 100 pL stop solution of sulphuric acid, read optical density within 10 minutes at wavelength 450 nm with spectrophotometer and apply blank deduction, draw a standard curve by plotting optical density on Y-axis and salivary elastase concentration in pg / mL unit on X-axis, include zero point, fit the curve to calculate concentration in unknown patient samples.

[0224] Statistical analyses

[0225] Statistical analyses were performed using the Statistical Package for the Social Sciences for Windows (SPSS version 27.0; IBM Corp, Armonk, NY, USA), Graphpad Prism 8.0.1 (GraphPad Software Inc., Boston, USA) and Analyse-it in Microsoft Excel (Analyse-it Software Ltd., Leeds, UK). A level of significance of p<0.05 and nonparametric statistics were used. Multivariate analysis was used to study the outcome associations. Biomarker concentration cut-off between LPR patients and healthy controls, sensitivity (SE), specificity (SP) were evaluated with Receiver Operating Curve (ROC) and fixed at the highest Youden Index (YI) or Likelihood Ratio (LR). Positive predictive value (PPV) and negative predictive value (NPV) were calculated through contingency tables.

[0226] Results:

[0227] Patients were 18 to 72 year-old, 64% female, mean BMI of 25, 40% presented with severe reflux, 72% had abnormal gastrointestinal endoscopy and 33% had gastroesophageal reflux disease (GERD). HEMII-pH features are displayed in table 6 below:

[0228] Table 6: HEMII-pH features

[0229] During clinical follow-up, the otolaryngological, digestive symptom scores and RSS total score significantly improved from pre- to 3-month posttreatment, as well as the laryngeal RSA .

[0230] 67 LPR patients and 35 healthy individuals completed the saliva measurements. The median concentration of elastase was 49 [interquartile range (IQR)=27-69] pg / mL in LPR patients, compared to significantly lower median of 36 [17-46] pg / mL in healthy controls (p=0.0005) (Figure 7A). The analysis of saliva pH reported median values of 7.8 [7.4-8.1] and 7.4 [7.2-7.7] for LPR and control individuals, respectively (p=0.0200). Furthermore, a significant lower median cholesterol of 0.7 [0.3-1.1] mg / dL was found in saliva of LPR versus healthy controls 4.3 [3.9-4.4] mg / dL (p<0.0001). But salivary cholesterol results often fell near the level of detection (LOD) of 0.6 mg / dL in LPR patients. Most of the detectable salivary cholesterol was found beneath the level of quantification (LOQ) of 2.2 mg / dL in LPR patients, while healthy controls reported salivary cholesterol above LOQ. For salivary elastase, both patient and control populations fell wide above LOD of 8 pg / mL and mostly above LOQ of 15 pg / mL. While salivary bile acids were found between LOD=0.14 pmol / L and LOQ=0.69 pmol / L in both populations of patients and controls.

[0231] According to the ROC curve analysis, the area under the curve (AUC) showed AUC=0.860 for salivary pH, AUC=0.980 for salivary cholesterol and AUC=0.707 for salivary elastase in diagnosing LPR patients.

[0232] The cut-off of salivary elastase was set at >49 pg / mL, reporting SE of 52% (95%CI, 42-65%) and SP of 91% (95%CI, 71-94%) (Figure 7B). Given a LPR prevalence of 10% at the Ear, Nose and Throat consultation, the PPV of salivary elastase was 40% and the NPV=95%.

[0233] The cut-off for pH was >7.6 with SE=31% (95%CI, 22-43%), SP=98% (95%CI, 87- 100%) and cholesterol <2.1 mg / dL with SE=97% (95%CI, 90-99%), SP=98% (95%CI, 87-100%). On the analytical level, the pH did not interfere with the cholesterol measurement (p=0.5979). The multivariate analysis of subpopulations reported a significant association between the concentration of bile acids and the percentage of pH<4.0 in the distal esophagus. Total bilirubin remained undetected. All laboratory methods underwent an analytical performance study for the detection of biomarkers in saliva specimen (data not shown).

[0234] Discussion:

[0235] This clinical performance study showed clinical validity for the measurement of elastase, pH, cholesterol and bile acids in the saliva of patients presenting at Ear, Nose and Throat consultation with LPR symptoms. We did not find an indication for salivary total bilirubin in LPR patients.

[0236] Bile acids and elastase are pancreaticoduodenal biomarkers, which are involved in the digestion of lipids and proteins; cholesterol metabolism is related to bile acids. Physiologically, they should be not found in gastric, esophageal, or laryngopharyngeal compartments according to the defense mechanisms against reflux of the upper digestive tract. The findings of this preliminary report are important because they support the occurrence of duodeno-gastric reflux in LPR patients, which may be associated with gastro-esophago-pharyngeal reflux and the deposit of pancreaticoduodenal content into the laryngopharyngeal mucosa. This observation raises questions about the presence of upper digestive tract dysmotility in LPR patients, e.g. pylorus sphincter disorder and duodenal dysmotility. The presence of elevated bile acids in the saliva of LPR patients has been investigated in previous clinical studies. The negative impact of refluxate bile acids on laryngopharyngeal mucosa was however demonstrated in some studies, in which authors observed that bile acids were associated with inflammatory reaction and carcinogenic potential. The potential role of elastase in the development of inflammatory reaction of the upper aerodigestive tract was never studied. Because these enzymes are active in different ranges of pH, they could have specific involvement in the development of mucosa injury according to the type of LPR (acid, weakly acid or alkaline) and the pH of saliva.

[0237] Interestingly, we observed that the saliva pH of LPR patients was significantly higher than the pH of saliva of healthy individuals. Carbonic anhydrase is one of the most important defense mechanisms of laryngopharyngeal mucosa against reflux. Carbonic anhydrase is an enzyme that catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. Previous studies found that expression of carbonic anhydrase type III was significantly increased in the mucosa of posterior commissure in LPR patients, corroborating findings of another study. In that way, the related higher pH could reflect the mucosa defense against reflux irritation.

[0238] Bilirubin has not been found in our study population, corroborating findings in literature.

[0239] The benefit of measuring salivary biomarkers is the non-invasive procedure, compared to 24-hour HEMII-pH.

[0240] The risk-benefit ratio is in favor of using salivary biomarkers in the diagnosis of LPR, because a non-invasive sampling technique is used, which enhances patient safety. The excellent NPV can rule out LPR in patients presenting at the Ear, Nose and Throat consultation when salivary elastase is lower or equal to 49 pg / mL. Moreover, a high NPV>95% has also been found with pH and cholesterol.

[0241] Conclusion:

[0242] Salivary elastase, pH, cholesterol and bile acids appear interesting biomarkers of laryngopharyngeal reflux and highlight the existence of a back flow of pancreaticoduodenal enzymes in the upper aerodigestive tract in LPR patients. Elastase above 49 pg / mL, pH above 7.6 and cholesterol beneath 2.1 mg / dL in saliva support the occurrence of LPR. The combination of salivary elastase with other salivary biomarkers like pH (and cholesterol) enhances the diagnostic power. The role of bile acids is complemental for subpopulation analysis of acid LPR in the distal esophagus.

[0243] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A method for diagnosing laryngopharyngeal reflux disease in a subject, said method comprising determining an amount of one or more biomarkers in a sample obtained from said subject and comparing said amount to a predetermined threshold value for said biomarker, said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

2. Method according to claim 1, wherein said sample is a sputum or a saliva sample.

3. Method according to any of the previous claims, wherein the amount of either pancreatic elastase, pH, bile acids or cholesterol is determined and compared to a predetermined threshold value for said biomarker.

4. Method according to any of the previous claims 1-2, wherein the amount of at least two biomarkers is determined and compared to their respective threshold value.

5. Method according to any of the previous claims 1-2, wherein said method comprises determining the amount of pancreatic elastase, bile acids and cholesterol.

6. Method according to any of the previous claims 1-2, wherein said method comprises determining the amount of pancreatic elastase, pH, bile acids and cholesterol.

7. Method according to any of the previous claims, wherein said method further comprises determining an amount of pepsin in said sample and comparing said amount of pepsin to a predetermined threshold value.

8. Method according to any of the previous claims, wherein said biomarker is pancreatic elastase and wherein said amount of pancreatic elastase is determined by means of an immunoassay.

9. Method according to any of the previous claims, wherein said biomarker is bile acids, and wherein said amount of bile acids in said sample is determined by means of a colorimetric assay.

10. Method according to claim 9, wherein said amount of bile acids allows to determine the severity of LPR.

11. Method according to any of the previous claims, wherein said biomarker is cholesterol, and wherein said amount of cholesterol in said sample is determined by means of a colorimetric enzymatic method.

12. Method according to any of the previous claims, wherein said biomarker is pH, and wherein said amount of pH in said sample is determined by means of a colorimetric method or electrodes.

13. A diagnostic kit for the diagnosis of laryngopharyngeal reflux in a subject, said kit comprising one or more collection tubes for saliva or sputum and one or more assays for determining an amount of one or more biomarkers in a saliva or sputum sample obtained from said subject, wherein said one or more biomarkers are chosen from pancreatic elastase, pH, bile acids and / or cholesterol.

14. Diagnostic kit according to claim 13, said kit further comprising an assay for measuring the amount of pepsin.

15. Diagnostic kit according to any of the previous claims 13-14, wherein one or more of said assays comprise an immunoassay or a colorimetric assay.

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