Periodontitis diagnostic methods, uses and kits

Abstract

An in vitro method for assessing whether a human patient has periodontitis is disclosed. The method is based on insight to determine biomarker proteins. Therefore, in a saliva sample of a patient suffering from periodontitis, the concentration of free light chain κ protein and / or free light chain λ is measured. Based on the measured concentration, a value reflecting the concentration or combined concentration for one or both of the proteins is determined. The value is compared with a threshold value reflecting the concentration or combined concentration associated with periodontitis in the same manner. The comparison allows an assessment of whether the test value indicates the presence of periodontitis in the patient. Thus, typically, a test value reflecting a concentration or combined concentration below the concentration or combined concentration reflected by the threshold indicates the absence of periodontitis in the patient, while a test value reflecting a concentration or combined concentration at or above the concentration or combined concentration reflected by the threshold indicates periodontitis in the patient.

Description

Technical Field

[0001] The present invention belongs to the field of oral care and relates to saliva-based periodontal disease diagnosis. In particular, the present invention relates to a kit, use and method for diagnosing periodontitis. Background Art

[0002] Gum inflammation, or gingivitis, is a non-destructive periodontal disease that is primarily caused by the adhesion of dental bacterial biofilm or plaque to the tooth surface. If not detected and treated, reversible gingivitis often leads to inflammation of the tissues surrounding the teeth (i.e., the periodontium), a disease known as periodontitis, which is irreversible and causes tissue destruction and alveolar bone loss, and ultimately tooth loss. As gum disease progresses, there are often clinical signs and symptoms associated with it, such as swollen gums, a change in color from pink to dark red, bleeding gums, bad breath, and gums that become more tender or tender to the touch.

[0003] Periodontitis is a chronic multifactorial inflammatory disease caused by oral microorganisms and is characterized by the progressive destruction of hard (bone) and soft (periodontal ligament) tissues, ultimately leading to tooth mobility and loss. This is to be distinguished from gingivitis, which is a reversible infection and inflammation of gum tissue. Inflammatory periodontitis is one of the most common chronic human diseases and is the main cause of tooth loss in adults. In addition to the significant negative impact of periodontitis on oral health, there is also increasing evidence that periodontitis has systemic consequences and is a risk factor for several systemic diseases, including heart disease (e.g., atherosclerosis, stroke), diabetes, pregnancy complications, rheumatoid arthritis and respiratory infections.

[0004] Therefore, early and accurate diagnosis of periodontal disease is important from both the perspective of oral and overall health.

[0005] Diagnosis of periodontal disease in common dental practice remains poor, which results in a relatively low rate of therapeutic intervention and a large number of untreated cases. Current diagnosis relies on an imprecise, subjective clinical examination of the state of oral tissues (color, swelling, degree of bleeding on probe, depth of probe pockets; and bone loss on oral x-rays) by a dental professional. These traditional methods are time consuming, and some of the techniques used (pocket depth, x-rays) reflect historical events, such as past disease activity, rather than current disease activity or susceptibility to further disease. Therefore, there is a need for a more objective, rapid, accurate, easy-to-use diagnosis, which preferably can also be performed by non-professionals. Thus, it is desirable to measure the current disease activity, and possibly the subject's susceptibility to further periodontal disease.

[0006] Saliva or oral fluid has long been advocated as a diagnostic fluid for oral and general diseases, and with the advent of miniaturized biosensors (also known as labs on a chip), there has been greater scientific and clinical interest in rapid chair-side testing for point-of-care diagnostics. In particular, for periodontal disease detection, inflammatory biomarkers associated with tissue inflammation and breakdown are readily present in saliva due to their proximity to the saliva, suggesting that saliva has great potential for use in periodontal disease detection. Indeed, this area has also generated significant interest and exciting results. For example, Ramseier et al. (JPeriodontol. 2009 Mar;80(3):436-46) identified biomarkers caused by host-type bacteria that were associated with periodontal disease. However, no definitive test has yet emerged.

[0007] Biomarkers represent biological indicators that underpin clinical manifestations and are therefore objective means for diagnosing clinical outcomes of periodontal disease. Ultimately, proven biomarkers can be used to assess the risk of future disease, identify disease in its earliest stages, determine response to initial treatment, and allow implementation of preventive strategies.

[0008] Previous limitations to the development of point-of-care tests for salivary biomarkers include the lack of technology suitable for chairside application and the inability to analyze multiple biomarkers in a personal sample. Furthermore, the selection of which biomarkers to include in such tests has not been thoroughly addressed in the literature nor has it been implemented in actual testing.

[0009] It would be desirable to provide a simpler process, and in particular a process which would only require the extraction of a small sample of saliva from the patient, and possibly by the patient himself. It would be desirable for such a sample to be input into an in vitro diagnostic device which, based on the measurement results, would allow the saliva sample to be classified so that it could return an indication of the likelihood that the patient would be classified as suffering from periodontitis. Summary of the invention

[0010] In order to better address the foregoing expectations, the present invention relates, on the one hand, to an in vitro method for assessing whether a human patient has periodontitis, the method comprising detecting the concentration of free light chain κ protein and / or free light chain λ protein in a saliva sample from the human patient; determining a test value reflecting the concentration or combined concentration determined for the one or two proteins; and comparing the test value with a threshold value associated with periodontitis that reflects the concentration or combined concentration in the same manner to assess whether the test value indicates periodontitis in the patient.

[0011] In another aspect, the present invention provides for the use of free light chain κ protein and / or free light chain λ protein in a saliva sample of a human patient as a biomarker for assessing whether the patient has periodontitis.

[0012] Optionally, the patient's age is also used as a biomarker.

[0013] In another aspect, the invention resides in a system for assessing whether a human patient has periodontitis, the system comprising:

[0014] - a detection device capable of and suitable for detecting free light chain kappa protein and / or free light chain lambda protein in a saliva sample of said human patient; and

[0015] - a processor capable and adapted to determine, based on the determined concentration of said protein, an indication that the patient has periodontitis.

[0016] The system optionally comprises a data connection to an interface, in particular a graphical user interface, which allows the display of information and preferably also the input of information, said interface being either part of the system or a remote interface.

[0017] Optionally, one or more of the aforementioned items, in particular the processor, is endowed with the capability to work properly "in the cloud", ie, not on a fixed machine, but with the aid of an Internet-based application.

[0018] In another aspect, the present invention provides a kit for detecting at least one biomarker for periodontitis in a saliva sample of a human patient, the kit comprising one or two detection reagents for detecting free light chain κ protein and / or free light chain λ protein. Typically, two detection reagents are used, each of which binds to a different biomarker. In one embodiment, the first detection reagent is capable of binding to free light chain κ, and the second detection reagent is capable of binding to free light chain λ.

[0019] In yet another aspect, the present invention provides an in vitro method for determining a change in the state of periodontitis in a human patient over a time interval from a first time point t1 to a second time point t2, the method comprising detecting the concentration of free light chain λ protein and / or free light chain κ protein in at least one saliva sample obtained from the patient at t1 and in at least one saliva sample obtained from the patient at t2, and comparing the concentrations, whereby the difference in either or both of the concentrations reflects a change in state.

[0020] In another aspect, the present invention provides a method for diagnosing whether a human patient has periodontitis, comprising detecting free light chain λ protein and / or free light chain κ protein in a saliva sample of the human patient, and assessing the presence of periodontitis in the patient based on the concentration of the one or two proteins in the saliva sample. Optionally, the method of this aspect comprises the additional step of treating periodontitis in the patient.

[0021] In yet another aspect, the present invention provides a method for detecting free light chain λ protein and / or free light chain κ protein in a human patient, comprising:

[0022] (a) obtaining a saliva sample from a human patient; and

[0023] (b) detecting the presence or absence of free light chain λ protein and / or free light chain κ protein in the sample by contacting the sample with one or more detection reagents for binding to the protein and detecting the binding between each protein and the one or more detection reagents. Typically, there is a first detection reagent that can bind to free light chain λ protein and a second detection reagent that can bind to free light chain κ protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A system for use in the methods described in the present disclosure is schematically illustrated. DETAILED DESCRIPTION

[0025] In summary, the present invention is based on the brilliant insight that even a single protein can serve as a biomarker in saliva samples of human patients for identifying the presence or absence of periodontitis. Two proteins have been identified which can be used alone or in combination to diagnose periodontitis.

[0026] These proteins are free light chain lambda proteins and / or free light chain kappa proteins. When a single biomarker protein is used, free light chain lambda proteins are preferred.

[0027] When these two proteins are detected, the sum, ratio, or difference between the two proteins may be determined in certain embodiments. The sum, ratio, and / or difference may be optionally used in combination with each other. For example, the ratio of the two chains may be combined with the sum according to one embodiment, or the ratio and the difference may be combined in another embodiment. The sum, ratio, difference, or combination thereof may optionally be further combined with the measured values ​​of free light chain λ protein and / or free light chain κ protein. For example: the κ / λ ratio may be usefully combined with the measured value of free light chain λ; the κ / λ ratio may be usefully combined with the measured value of free light chain κ; or the κ / λ ratio may be usefully combined with the measured values ​​of free light chain κ and free light chain λ.

[0028] The subject's age may optionally be included as an additional marker.

[0029] Free light chain proteins are immunoglobulin light chains. They are not associated with immunoglobulin heavy chains. Unlike typical intact immunoglobulin molecules, free light chain proteins are not covalently linked to immunoglobulin heavy chains, i.e., free light chains are not disulfide bonded to heavy chains. Typically, free light chains include about 220 amino acids. Typically, free light chain proteins include a variable region (often referred to as a light chain variable region, V L ) and constant region (often referred to as the light chain constant region, C L ). Humans produce two types of immunoglobulin light chains, named after the letters κ and λ. Each of these light chains can be further divided into multiple subgroups based on changes in the variable region, with a total of four κ subtypes (Vκ1, Vκ2, Vκ3 and Vκ4) and six λ subtypes (Vλ1, Vλ2, Vλ3, Vλ4, Vλ5 and Vλ6). Free light chain κ is usually monomeric. Free light chain λ is usually dimeric, linked by disulfide bonds (to another free light chain λ). Polymeric forms of free light chain λ and free light chain κ have been identified. These free light chains are produced by bone marrow and lymph node cells and locally in periodontal tissues by diffuse lymphocytes, and are rapidly cleared from the blood and metabolized by the kidneys. Monomeric free light chains are cleared within 2-4 hours, while dimeric free light chains are cleared within 3-6 hours.

[0030] The two proteins mentioned above are known in the art. Those skilled in the art know their structures and methods for detecting them in aqueous samples, such as saliva samples. Hereinafter, the protein biomarkers mentioned above are collectively referred to as the "biomarker group of the present invention". In one embodiment, a biomarker group of the present invention consists of the two protein biomarkers identified in the present invention, namely free light chain λ and free light chain κ. In addition to the biomarker group of the present invention, other biomarkers and / or data, such as demographic data (e.g., age, gender), may be included in the data set for determining the type of periodontitis.

[0031] When additional biomarkers are optionally included, the total number of biomarkers (ie, the biomarker panel of the invention plus the additional biomarkers) is typically 3, 4, 5, or 6.

[0032] However, an expected advantage of the present invention is that the classification of periodontitis in a patient can be determined by measuring preferably no more than two biomarkers, wherein a biomarker group consisting of both free light chain λ and free light chain κ is preferred. In particular, the determination does not involve the use of other data, which advantageously provides a simple and direct diagnostic test.

[0033] As expected, the method requires only a small amount of saliva sample to be extracted from the subject, e.g., a drop size. The size of the sample will typically range from 0.1 μl to 2 ml, e.g., 1-2 ml, whereby smaller amounts, e.g., 0.1-100 μl, can be used for in vitro device processing, and whereby extraction of larger amounts, e.g., up to 20 ml, e.g., 7.5-17 ml, is also feasible.

[0034] The sample is input into an in vitro diagnostic device, which measures the concentration of the protein or proteins involved, and the device returns a diagnosis that classifies the subject based on the likelihood of having periodontitis.

[0035] The ease of use of the present invention will make it possible to test the majority of dental patients who suffer from periodontitis or who are at high risk of developing periodontitis in a regular manner, for example as part of a regular dental check-up or even at home. This in particular allows the presence of periodontitis to be detected soon after it has developed, and therefore enables more timely oral care measures to be taken to prevent further development of periodontitis. Or, for example, for a patient who is known to be at high risk of developing periodontitis and is being tested for the first time, the method allows identification of whether periodontitis has already developed. Moreover, the method can be applied after treatment of a patient who has previously been diagnosed with periodontitis, to check whether the periodontitis has been successfully treated. In particular, the method is also suitable for self-diagnosis, whereby the steps of extracting the sample and inputting it into the device can be performed by the patient himself.

[0036] When performing the present invention to confirm the presence or absence of periodontitis, the patient may generally be known or suspected to have periodontitis. Thus, in certain embodiments, the method is used to assess whether a patient known or suspected to have periodontitis has periodontitis.

[0037] The methods of the present invention generally comprise detecting one or more of the aforementioned proteins constituting the biomarker group of the present invention, and optionally other biomarker proteins, by using one or more detection reagents.

[0038] The "saliva" tested according to the present invention can be undiluted saliva, which can be obtained by spitting or wiping, or diluted saliva, which can be obtained by gargling with a fluid. The diluted saliva can be obtained by the patient gargling or washing the mouth with sterile water (e.g., 5 ml or 10 ml) or other suitable fluid for a few seconds and then spitting into a container. Diluted saliva can sometimes be referred to as an oral rinse fluid.

[0039] "Detection" is used to refer to the measurement, quantification, scoring or determination of the concentration of a biomarker protein. Methods for evaluating biological compounds including biomarker proteins are known in the art. It is recognized that methods for detecting protein biomarkers include direct measurements and indirect measurements. Those skilled in the art will be able to select an appropriate method for determining a particular biomarker protein.

[0040] The term "concentration" in relation to protein biomarkers is given its usual meaning, i.e., the abundance of that protein in a given volume. Protein biomarkers are usually measured in units of mass per volume, most commonly mg / ml, μg / ml or ng / ml, but sometimes as low as pg / ml. Another measurement is the molar concentration (or molarity), mol / L or "M". The concentration can be determined by measuring the amount of protein in a sample of known, determined or predetermined volume.

[0041] Instead of determining the concentration, one may determine the absolute amount of a protein biomarker in a sample, or determine the mass fraction of a biomarker in a sample, such as the amount of the biomarker relative to the total amount of all other proteins in the sample.

[0042] A "detection reagent" is a reagent or composition that specifically (or selectively) binds to, interacts with, or detects a protein biomarker of interest. Such detection reagents may include, but are not limited to, antibodies, polyclonal antibodies, or monoclonal antibodies that preferentially bind to a protein biomarker.

[0043] The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact with," when referring to a detection reagent, refer to a binding reaction that is capable of determining the presence of a protein biomarker in a variety of proteins and other biological agents. Thus, under specified immunoassay conditions, a specified detection reagent (e.g., an antibody) binds to a specific protein at least twice background and does not bind substantially in significant amounts to other proteins present in the sample. Specific binding under such conditions may require that the antibody be selected for its specificity for a particular protein. Various immunoassay designs may be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays (enzyme-linked immunosorbent assays) are commonly used to select antibodies that specifically immunoreact with a protein (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically greater than 10 to 100 times the background.

[0044] "Antibody" refers to a polypeptide ligand substantially encoded by one or more immunoglobulin genes, or a fragment thereof, which specifically binds to and recognizes an antigenic epitope (e.g., an antigen). The recognized immunoglobulin genes include kappa and lambda light chain constant region genes, alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and numerous immunoglobulin variable region genes. Antibodies exist, for example, as complete immunoglobulins or as multiple well-characterized fragments produced by digestion with various peptidases. This includes, for example, Fab' and F(ab)'2 fragments. The term "antibody" when used herein also includes antibody fragments produced by alterations of complete antibodies or those synthesized de novo using recombinant DNA methods. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies or single-chain antibodies. The "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that includes one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include a heavy chain variable region. The antibody can be a bispecific antibody, for example, an antibody having a first variable region that specifically binds to a first antigen and a second variable region that specifically binds to a second different antigen. The use of at least one bispecific antibody can reduce the number of detection reagents required.

[0045] Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of people with the disease who test positive (the percentage of "true positives"). People with the disease who are not detected by the assay are "false negatives." Subjects who do not have the disease and test negative in the assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of people who test positive who do not have the disease.

[0046] The biomarker proteins of the present invention can be detected in a sample by any means. Preferred methods for biomarker detection are antibody-based assays, protein array assays, mass spectrometry (MS)-based assays, and (near) infrared spectroscopy-based assays. For example, immunoassays, including but not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitation reactions, immunodiffusion assays, fluorescent immunoassays, etc. These assays are routine and well known in the art. Only exemplary immunoassays are briefly described below (but for limiting purposes).

[0047] Immunoprecipitation protocols typically include adding the antibody of interest to the cell lysate in a lysis buffer (e.g., RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 1% aprotinin) supplemented with protein phosphatase and / or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), incubating at 4°C for a period of time (e.g., 1-4 hours), adding protein A and / or protein G agarose beads to the cell lysate, incubating at 4°C for about one hour or more, washing the beads in lysis buffer and resuspending the beads in SDS / sample buffer. The ability of an antibody to immunoprecipitate a specific antigen can be assessed by, for example, Western blot analysis. One skilled in the art will appreciate the parameters that can be modified to increase antibody binding to the antigen and reduce background (e.g., pre-clearing the cell lysate with agarose beads).

[0048] Western blot analysis generally includes preparing a protein sample, electrophoresing the protein sample in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE, depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in a blocking solution (e.g., PBS containing 3% BSA or skim milk), washing the membrane in a wash buffer (e.g., PBS-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in a blocking buffer, washing the membrane in a wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) coupled to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 125I) diluted in a blocking buffer, washing the membrane in a wash buffer, and detecting the presence of the antigen. Those skilled in the art will be able to recognize parameters that can be modified to increase the detected signal and reduce background noise.

[0049] ELISA generally involves preparing an antigen (i.e., a biomarker protein of interest or a fragment thereof), coating the wells of a 96-well microtiter plate with the antigen, adding an antibody of interest coupled to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, and detecting the presence of the antigen. In an ELISA, the antibody of interest need not be coupled to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) coupled to a detectable compound can be added to the wells. In addition, instead of coating the wells with the antigen, the antibody can be coated to the wells. In this case, after the antigen of interest is added to the coated wells, a second antibody coupled to a detectable compound can be added. Those skilled in the art will be able to appreciate the parameters that can be modified to increase the detected signal as well as other variations of the ELISA known in the art.

[0050] When multiple markers are used, a threshold is determined based on the combined concentrations of these biomarkers. When a single biomarker is used, a threshold is determined based on the concentration of that biomarker. The threshold determines whether a patient is classified as having periodontitis. The present invention reflects the insight that periodontitis can be detected with sufficient accuracy based on the measurement results of a single biomarker or a combination of two biomarkers as described above.

[0051] This insight supports another aspect of the present invention, which is to use free light chain lambda protein and / or free light chain kappa protein as one or more biomarkers in a saliva sample of a human patient to assess whether the patient suffers from periodontitis.

[0052] Such use may be carried out in a method substantially as described above and below.

[0053] When both free light chain λ and free light chain κ are detected, the method of the present invention includes determining a test value reflecting the combined concentration measured for the protein. The combined concentration value can be any value obtained by inputting the determined concentration and arithmetic operations on these values. For example, this can be a simple addition of the concentrations. It can also include multiplying each concentration by a factor reflecting the desired weight of these concentrations, and then adding the results. It can also involve multiplying the concentrations by each other, or any combination of multiplication, division, subtraction, exponentiation and addition. It may further involve seeking a certain power of the concentration. In a preferred embodiment, the sum of two biomarkers (κ+λ) is used. In other preferred embodiments, the ratio of two biomarkers (e.g., κ / λ or λ / κ) is used. In other embodiments, the difference between two biomarkers (e.g., κ-λ or λ-κ) is used.

[0054] Optionally, the test value reflects the concentration of the combined concentration determined for the protein combined with the age of the subject.

[0055] The resulting combined concentration value is compared with a threshold value which reflects in the same way the combined concentration associated with the presence of periodontitis. Said comparison allows an assessment of whether the test value indicates the presence of periodontitis in the patient whose saliva was tested.

[0056] The threshold value may be, for example, a concentration or a combined concentration value obtained in the same manner based on concentrations determined for the same protein in a reference sample associated with the presence of periodontitis, i.e., in a patient diagnosed with periodontitis. Typically, a value reflecting the same or a higher combined concentration thereby indicates the presence of periodontitis in the patient being tested. Similarly, a value reflecting a lower combined concentration in the saliva of a patient with periodontitis being tested indicates the absence of periodontitis. However, it should be understood that the threshold value may also be calculated (e.g., by using a negative multiplier) such that a test value indicative of periodontitis will be below the threshold value, while a test value indicative of the absence of periodontitis will be above the threshold value.

[0057] The threshold value may also be determined based on measuring the concentration of the present biomarker protein in a set of samples including patients with a known diagnosis of periodontitis and "non" periodontitis. From this, the measured concentration values ​​may be subjected to a statistical analysis, possibly including machine learning methods, to allow for the differentiation of patients classified as having periodontitis from patients classified as not having periodontitis with a desired sensitivity and specificity. From this, a desired threshold value may be obtained. Based on this threshold value, the same concentration measurements may be made on the samples to be tested, and the concentration values ​​may then be processed in the same manner as to obtain the threshold value, thereby determining a combined concentration value, which may be compared to the threshold value, thereby allowing for the classification of the tested samples as having periodontitis or not having periodontitis.

[0058] In an interesting embodiment, the concentration or joint concentration value is obtained in the form of a score as follows. A numerical value (protein concentration value, in units of, for example, ng / ml) is assigned to each measurement result, and these values ​​are used in linear or nonlinear combination to calculate a score between zero and one. In the case of determining a threshold based on a group of subjects as described above, a sigmoid function with the joint concentration as input is typically used to calculate a score between 0 and 1 (as further shown).

[0059] When the score exceeds a certain threshold, the method indicates that the patient has periodontitis. The threshold can be selected based on the desired sensitivity and specificity.

[0060] It will be appreciated that, according to the present invention, when a subject is "classified for periodontitis", this is performed on a subject who may be considered to be at risk of developing periodontitis or to have periodontitis. This may be known from, for example, a previously performed diagnosis of periodontitis, although that diagnosis may not distinguish the degree of periodontitis, or may be considered based on, for example, a record of the subject's oral health status.

[0061] The clinical definition accepted in the art is based on the following:

[0062] Gingival Index (GI)

[0063] The full mouth gingival index will be recorded based on the Lobene modified gingival index (MGI) graded on a scale of 0 to 4, where:

[0064] 0 = no inflammation,

[0065] 1 = Mild inflammation; slight changes in color or texture in any but not all of the marginal or papillary gingival units.

[0066] 2 = Mild inflammation; but involving the entire marginal or papillary unit,

[0067] 3 = moderate inflammation; shiny, red, edema and / or hypertrophy of the margin or papillary units,

[0068] 4 = Severe inflammation; marked erythema, edema and / or hypertrophy of marginal or papillary gingival units, spontaneous bleeding, hyperemia or ulceration.

[0069] Detection Depth (PD)

[0070] The probing depth will be recorded to the nearest millimeter using a manual UNC-15 periodontal probe. The probing depth is the distance from the tip of the probe (assumed to be at the bottom of the pocket) to the free gingival margin.

[0071] Gingival recession (REC)

[0072] Gingival recession will be recorded to the nearest millimeter using a manual UNC-15 periodontal probe. Gingival recession is the distance from the free gingival margin to the cemento-enamel junction. Gingival recession will be expressed as a positive number, while gingival overgrowth will be expressed as a negative number.

[0073] Clinical attachment loss (CAL)

[0074] Clinical attachment loss will be calculated as the sum of probing depth + recession at each site.

[0075] Detection of bleeding (BOP)

[0076] After probing, each site was assessed for probing bleeding, with the site being assigned a score of 1 if bleeding occurred within 30 seconds of probing and 0 otherwise.

[0077] The resulting subject groups (patient groups) were defined as follows, where the mild-moderate periodontitis and advanced periodontitis groups are the "periodontitis" relevant to the present invention:

[0078] - Healthy group (H): PD ≤ 3 mm in all sites (but up to four 4 mm pockets distal to the last permanent molar will be allowed), no sites have interproximal attachment loss, GI ≥ 2.0 in ≤ 10% of sites, % BOP score ≤ 10%;

[0079] - Gingivitis group (G): GI ≥ 3.0 in > 30% of sites, no sites with interproximal attachment loss, no sites with PD > 4 mm, and % BOP score > 10%;

[0080] - Moderate-mild periodontitis group (MP): interproximal PD of 5-7 mm (equivalent to approximately 2-4 mm CAL) at ≥8 teeth, %BOP score >30%;

[0081] - Advanced periodontitis group (AP): interproximal PD ≥7 mm (equivalent to approximately ≥5 mm CAL) at ≥12 teeth, %BOP score >30%.

[0082] In one embodiment, the method of the present invention utilizes Figure 1 The system schematically shown in FIG. The system may be a single device with various equipment components (units) integrated therein. The system may also have its various components or some of these components as separate devices. Figure 1 The components shown are the measuring device (A), the graphical user interface (B) and the computer processing unit (C).

[0083] As described above, the system of the present invention includes a data connection to an interface, whereby the interface itself can be part of the system or can be a remote interface. The latter refers to the possibility of using a different device (preferably a handheld device such as a smartphone or a tablet) to provide the actual interface. In these cases, the data connection will preferably involve wireless data transmission, such as via Wi-Fi or Bluetooth, or via other technologies or standards.

[0084] The measuring device (A) is configured to receive a saliva sample, for example, by placing a saliva drop on a cartridge (A1), which can be inserted into the device (A). The device can be an existing device capable of determining the concentration of at least one of free light chain lambda protein and free light chain kappa protein based on the same saliva sample.

[0085] The measuring device (A) should be able to receive a saliva sample, for example, by placing a saliva drop on a cartridge (A1), which can be inserted into the device (A). The device can be an existing device capable of determining the concentration of at least one of free light chain lambda protein and free light chain kappa protein based on the same saliva sample.

[0086] The processing unit (C) receives the numerical values of the protein concentrations from part (A). Unit (C) is provided with software (usually embedded software), which allows unit (C) to calculate a score (S) between 0 and 1. The software also includes a numerical value for a threshold (T). If the calculated value (S) exceeds (T), then unit (C) will output an indication (I) of "periodontitis" to the GUI (B), otherwise it will output "no periodontitis". Another embodiment can use a specific value of (S) to indicate the certainty of making the indication (I). This can be a probability score, whereby 0.5 is a possible threshold, for example, a score S = 0.8 will indicate the probability of periodontitis. Interesting options are:

[0087] Based on the score S, the certainty can be directly indicated, that is, S = 0.8 represents a certainty of 80% for periodontitis; or

[0088] In order to make an indication through a definition with a range of R1 - R2, such that when R1 < S < R2, the indication (I) will read "uncertain".

[0089] The specific calculation of the score can be implemented, for example, through a sigmoid function using the following formula:

[0090]

[0091] where N is the number of proteins / biomarkers used. c0, c1, etc. are coefficients (numerical values), and B1, B2, etc. are the corresponding protein concentrations.

[0092] The determination of the coefficients can be done through a training process:

[0093] - N1 subjects with periodontitis (determined by dentists according to current criteria) and N2 subjects without periodontitis (having healthy gums or gingivitis) were selected.

[0094] - A saliva sample was obtained from each subject and the protein concentration of the biomarker panel was determined as described above.

[0095] - For periodontitis, the score S is defined as 1, and for no periodontitis, the score is defined as 0.

[0096] -Apply a sigmoid function to the score and protein concentration values.

[0097] Other regression or machine learning methods (linear regression, neural networks, support vector machines) can be used, where the score S is high for periodontitis patients and low for controls without periodontitis.

[0098] With reference to the aforementioned system, in another aspect, the present invention also provides a system for assessing whether a human patient suffers from periodontitis, the system comprising:

[0099] - A detection device capable of and suitable for detecting free light chain κ protein and / or free light chain λ protein in a saliva sample of a human patient; as mentioned above, such a device is known and readily available to a person skilled in the art; typically, a container for receiving an oral sample of a subject is provided, the container being provided with the detection device.

[0100] - a processor capable and adapted to determine, from the determined concentration of said protein, an indication that the patient suffers from periodontitis.

[0101] - Optionally, the system includes a user interface (or a data connection to a remote interface) capable of presenting information, in particular a graphical user interface (GUI); a GUI is a user interface that allows a user to interact with an electronic device through graphical icons and visual indicators (such as auxiliary symbols), instead of a text-based user interface, typed command labels or text navigation (none of these interface types are excluded from the present invention); GUIs are well known and are commonly used in handheld mobile devices, such as MP3 players, portable media players, gaming devices, smartphones, and smaller home, office and industrial control devices; as mentioned above, the interface can optionally also be selected to enable the input of information, such as the subject's age, gender, BMI (body mass index).

[0102] The present invention also provides, either alone or as part of the aforementioned system, a kit for detecting at least one biomarker of periodontitis in a saliva sample of a human patient, the kit comprising one or more detection reagents for detecting free light chain κ protein and / or free light chain λ protein. Typically, the kit comprises two detection reagents, each for a different biomarker, wherein the first detection reagent is capable of binding to free light chain κ protein and the second detection reagent is capable of binding to free light chain λ protein. As discussed above with reference to the methods of the present invention, the kit may include more detection reagents, such as reagents for other proteins. In a preferred embodiment, the detection reagents available in the kit consist of detection reagents for the selected two proteins that constitute the 2-biomarker group of the present invention, as mentioned.

[0103] Preferably, the kit comprises a solid support, such as a chip, a microtiter plate, or a bead or resin containing the detection reagent. In some embodiments, the kit comprises a mass spectrometry probe, such as a ProteinChip TM .

[0104] The kit may also provide a wash solution and / or a detection reagent specific for unbound detection reagent or the biomarker (sandwich type assay).

[0105] In an interesting aspect, the identification of the biomarker panel of the present invention is applied to monitor the state of periodontitis in human patients over time. Therefore, the present invention also provides an in vitro method for determining a change in the state of periodontitis in a human patient suffering from periodontitis over a time interval from a first time point t1 to a second time point t2, the method comprising detecting the concentration of free light chain kappa protein and / or free light chain lambda protein in at least one saliva sample obtained from the patient at t1 and in at least one saliva sample obtained from the patient at t2, and comparing the concentrations, whereby preferably the difference of at least one concentration, and more preferably both concentrations, reflects a change in state. The difference can be considered as a concentration difference, so that a direct comparison can be made without first generating a number between 0 and 1 or any other classification. It will be understood that the measurements received at the two time points can also be processed in exactly the same way as when determining the state of periodontitis as above.

[0106] The present invention also provides a method for diagnosing whether a human patient suffers from periodontitis, comprising detecting free light chain κ protein and / or free light chain λ protein in a saliva sample of the human patient. The presence of periodontitis in the patient is generally assessed based on the concentration of the protein in the sample. Optionally, the method of this aspect includes another step: treating the patient's periodontitis. This optional treatment step may include administering a known therapeutic agent or dental procedure, or a combination of a therapeutic agent and a dental procedure. Known therapeutic agents include administering an antibacterial agent, such as a mouthwash, crumbs, gel or microspheres. A typical antibacterial agent used to treat periodontitis is chlorhexidine. Other therapeutic agents include antibiotics, typically oral antibiotics, and enzyme inhibitors, such as doxycycline. Known non-surgical treatment procedures include scaling and root planing (SRP). Known surgical procedures include pocket reduction, flap surgery, gingival grafting or bone grafting by surgery.

[0107] The present invention also provides a method for detecting free light chain κ protein and / or free light chain λ protein in a human patient, comprising:

[0108] (a) obtaining a saliva sample from a human patient; and

[0109] (b) detecting the presence of free light chain κ protein and / or free light chain λ protein in the sample by contacting the sample with one or more detection reagents for binding to the protein and detecting the binding between each protein and the one or more detection reagents.

[0110] The invention will be further described with reference to the following non-limiting examples.

[0111] Example

[0112] A clinical study was conducted on 153 subjects with different periodontal health conditions (determined by clinical evaluation by dental professionals according to current standards (e.g., American Academy of Periodontology standards)). Of these, 39 subjects had healthy gingiva (H), 35 subjects were diagnosed with gingivitis (G), 41 subjects were diagnosed with mild periodontitis (MP), and 38 subjects were diagnosed with advanced periodontitis (AP). The area under the ROC (receiver-operator-characteristic) curve (AUC) values ​​were obtained after repeated clinical visits at two independent clinical sites.

[0113] The performance of various biomarker combinations was evaluated by logistic regression with leave-one-out cross validation (LOOCV), resulting in the preferred biomarker combinations explained herein.

[0114] In statistics, a receiver operating characteristic curve or ROC curve is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is constructed by plotting the true positive rate (TPR) against the false positive rate (FPR) at various threshold settings. The true positive rate is also known as sensitivity, recall, or detection likelihood in machine learning. The false positive rate is also known as the false detection or false alarm likelihood and can be calculated as (1-specificity). Thus, the ROC curve is sensitivity as a function of false detection. In general, if the probability distributions for both detection and false alarm are known, the ROC curve can be generated by plotting the cumulative distribution function of the detection likelihood on the y-axis (the area under the probability distribution from -∞ to the discrimination threshold) against the cumulative distribution function of the false alarm likelihood on the x-axis. The accuracy of a test depends on how well the test separates the group being tested into those with and without the disease of interest. Accuracy is measured by the area under the ROC curve. An area of ​​1 represents a perfect test; an area of ​​0.5 represents a meaningless test. A guide to classifying the accuracy of a diagnostic test is the traditional academic scoring system:

[0115] -0.90-1=Excellent (A)

[0116] -0.80-0.90=Good (B)

[0117] -0.70-0.80 = Medium (C)

[0118] -0.60-0.70=Difference (D)

[0119] -0.50-0.60=Fail (F)

[0120] Based on the foregoing, in the results of the above clinical studies, ROC AUC values ​​above 0.75 are considered according to the present invention to represent the desired accuracy of providing a diagnostic test.

[0121] Table 1 below indicates the area under the receiver operator characteristic curve values ​​of > 0.8 obtained for detecting periodontitis using the claimed panel biomarkers.

[0122] The biomarker proteins given in the table are FLCκ, FLCλ, the ratio κ / λ, and κ+λ, and the difference κ−λ.

[0123] In this table, it can be seen that the largest result in terms of ROC AUC, 0.847, was obtained by the biomarker group consisting of the ratio κ / λ, and κ+λ, and age. A result close to this, 0.835, was obtained by FLCκ, FLCλ, the ratio κ / λ, and age. 0.835 was also obtained by FLCλ, the ratio κ / λ, and age. Scores above 0.8 were also reported for a number of other markers, including FLCλ alone.

[0124] Table 1

[0125]

[0126]

[0127] This table shows the performance of the FLC biomarker panel (optionally with age added as a marker) in classifying periodontitis and non-periodontitis by logistic regression.

[0128] After labeling κ and λ, additional predictors κ+λ, κ-λ, κ / λ were considered. No redundant groups are shown in this table. The term "redundant" here reflects the fact that groups including, for example, κ+λ and κ-λ as predictors - in logistic regression - give the same results as the corresponding groups including κ and λ as predictors.

[0129] The groups indicated in italics are not preferred due to lower relative performance. All other groups showed acceptable performance with AUC LOOCV > 0.75 (AUC LOOCV > 0.8 for most groups).

[0130] Although the present invention has been illustrated and described in detail in the drawings and in the foregoing description, such illustration and description should be considered illustrative or exemplary rather than restrictive; the present invention is not limited to the disclosed embodiments. For example, detection reagents for different biomarkers can be provided in different units. Alternatively, conveniently, the kit of the present invention can include a fixed set of detection reagents for the protein biomarkers used in all embodiments (i.e., free light chain kappa protein and / or free light chain lambda protein), and optional flexible modules including detection reagents for other biomarkers.

[0131] Other variations to the disclosed embodiments may be understood and implemented by those skilled in the art in practicing the claimed invention by studying the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain features of the invention are recited in mutually different dependent claims does not indicate that a combination of these features cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0132] In summary, the inventors have thus disclosed an in vitro method for assessing whether a human patient suffers from periodontitis. The method is based on insight to determine biomarker proteins. Therefore, in a saliva sample from a patient, the concentration of free light chain κ protein and / or free light chain λ protein is measured. Based on the measured concentration, a value reflecting the concentration or combined concentration of the protein is determined. This value is compared with a threshold value that reflects the concentration or combined concentration associated with periodontitis in the same manner. This comparison allows an assessment of whether a test value indicates the presence of periodontitis in the patient. Thus, typically, a test value reflecting a concentration or combined concentration below the concentration or combined concentration reflected by the threshold indicates that periodontitis is not present in the patient, while a test value reflecting a concentration or combined concentration reflected by the threshold or above the concentration or combined concentration indicates periodontitis in the patient.

Claims

1. A system for assessing whether a human patient has periodontitis, the system comprising: A detection device, the detection device comprising a detection reagent, the detection reagent being capable of and suitable for detecting free light chain κ protein and / or free light chain λ protein in a sample of saliva of the human patient; A processor capable and adapted to determine an indication that the human patient has periodontitis based on the concentration of the protein determined by the detection device and a concentration threshold, wherein a biomarker group including the protein has a ROC AUC value above 0.

75.

2. The system of claim 1, further comprising a container for receiving an oral fluid sample, the container comprising the detection device.

3. The system according to claim 1, further comprising: A user interface for displaying the instructions to a user; as well as A data connection between the processor and the user interface is used to transmit the indication from the processor to the user interface.

4. The system of claim 3, wherein the processor is enabled by an Internet-based application; and / or Wherein the user interface is capable of inputting information regarding the age of the human patient, and the processor is capable of and adapted to determine an indication that the human patient has periodontitis based on the determined one or more concentrations.

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