Diagnosis of mild or severe periodontitis

Abstract

An in vitro method for assessing whether a human patient suffering from periodontitis has mild periodontitis or severe periodontitis is disclosed. The method is based on determining a selected insight into the concentrations of three biomarker proteins. Thus, in a saliva sample of a patient suffering from periodontitis, the concentrations of pyruvate kinase (PK) and at least two of the following are measured: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9). Based on the measured concentrations, a value is determined that reflects the joint concentration for the proteins. The value is compared to a threshold value that reflects the joint concentration associated with severe periodontitis in the same way. The comparison allows assessing whether the test value is indicative of the presence of severe periodontitis or mild periodontitis of the patient. Thereby, generally, a test value that reflects a joint concentration that is lower than the joint concentration reflected by the threshold value is indicative of the patient having mild periodontitis, and a test value that reflects a joint concentration that is equal to or higher than the joint concentration reflected by the threshold value is indicative of the patient having severe periodontitis.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of oral care and relates to saliva-based diagnosis of periodontal disease. In particular, the present invention relates to a kit and method for distinguishing between mild and severe periodontitis. BACKGROUND

[0002] Gingivitis or gingival inflammation is a non-destructive periodontal disease caused mainly by the attachment of dental plaque or dental biofilm to the tooth surface, which triggers an inflammatory response in the surrounding tissues. Gingivitis is a reversible infection and inflammation of the gingival tissue and can be resolved with proper oral hygiene measures and dental professional intervention. If not detected or resolved, gingivitis often leads to inflammation of the tissues surrounding the tooth, i.e. the periodontal tissues, which is defined as periodontitis, leading to tissue destruction and loss of alveolar bone and eventually to tooth loss. During the development of gingival disease, there are often clinical signs and symptoms associated with it, such as swelling of the gums, color change from pink to deep red, gum bleeding, bad breath and gums becoming increasingly fragile or painful to touch.

[0003] Periodontitis is a chronic multifactorial inflammatory disease caused by oral microorganisms and is characterized by a gradual destruction of both hard (bone) and soft (periodontal ligament) tissues, eventually leading to tooth mobility and loss. It needs to be distinguished from gingivitis, which is a reversible infection and inflammation of the gingival tissue. Inflammatory periodontitis is one of the most prevalent chronic human diseases and is the main cause of tooth loss in adults. In addition to the substantial negative impact of periodontitis on oral health, there is also substantial evidence that periodontitis has systemic effects and that it is a risk factor for a number of systemic diseases, including heart disease (e.g. atherosclerosis, stroke), diabetes, pregnancy complications, rheumatoid arthritis and respiratory tract infections.

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

[0005] In general dental practice, the diagnosis of periodontal disease remains poor, leading to a relatively low rate of therapeutic intervention and a large number of untreated cases. Current diagnosis relies on subjective clinical examination by dental professionals of the oral tissue situation (color, swelling, degree of bleeding on probing, probing pocket depth; and bone loss from oral x-rays). 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, more objective, faster, accurate, easier to use diagnostics are desirable, which preferably can also be performed by non-specialists. Thus, it is desirable to measure 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 micro-biosensors, also known as lab-on-a-chip, point-of-care testing for rapid chair-side testing has gained greater scientific and clinical interest. In particular for periodontal disease detection, inflammatory biomarkers associated with tissue inflammation and breakdown can easily end up in saliva due to proximity, suggesting saliva has a strong potential for periodontal disease detection. Indeed, this field has therefore attracted significant interest and promising results have been presented. For example, Kido et al. (J Periodont Res 2012; 47: 488-499) identified 104 proteins in gingival crevicular fluid (GCF) samples from healthy and periodontitis sites, 64 proteins contained only in GCF from healthy sites and 63 proteins contained only in GCF from periodontitis sites. However, no definitive test has emerged.

[0007] Biomarkers represent biological indicators supporting clinical manifestations, thus they are objective measures of clinical outcomes for diagnosing periodontal disease. Ultimately, proven biomarkers can be used to assess the risk of future disease, identify disease at the earliest stage, identify response to initial treatment and allow implementation of preventive strategies.

[0008] Limitations of previous developments for point-of-care testing of saliva biomarkers include the lack of technology suitable for chair-side application and the inability to analyze multiple biomarkers in individual samples. Also, the choice of which multiple biomarkers to include in such a test has not been sufficiently addressed in the literature nor implemented in actual tests.

[0009] Furthermore, periodontitis can manifest itself in a whole range of severity from mild to severe disease. To easily assess the severity of the situation, dentists typically classify patients suffering from periodontitis in two groups - patients suffering from mild periodontitis and patients suffering from severe periodontitis. However, available methods to perform such an assessment involve labor-intensive processes that are not routinely performed by dentists for every patient and / or every visit and that cannot be performed by the consumer (self-diagnosis).

[0010] It is desirable to provide a simpler process, in particular a process that only requires obtaining (and possibly by the patient himself or herself) a small saliva sample from the patient. It is desirable to input such a sample in an in-vitro diagnostic device that will allow classifying the saliva sample based on the measurements, so that it can return an indication of the likelihood that the patient is classified as suffering from mild periodontitis or as suffering from severe periodontitis. SUMMARY

[0011] To better address the aforementioned needs, in one aspect, the present application relates to an in vitro method for assessing whether a human patient has mild periodontitis or severe periodontitis, the method comprising: in a saliva sample from the human patient, detecting the concentration of the following proteins: pyruvate kinase (PK), and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9); determining a test value reflecting the joint concentration determined for the proteins; and comparing the test value with a threshold value, the threshold value reflecting in the same way the joint concentration associated with severe periodontitis, in order to assess whether the test value indicates that the patient has mild periodontitis or severe periodontitis.

[0012] In another aspect, the present application proposes the use of proteins, pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9), as biomarkers in a saliva sample of a human patient, for assessing whether the patient has mild periodontitis or severe periodontitis.

[0013] Optionally, the age of the patient is also used as a biomarker.

[0014] In yet another aspect, the present application consists in a system for assessing whether a human patient has mild periodontitis or severe periodontitis, the system comprising:

[0015] - detection means capable and adapted to detect, in a saliva sample of a human patient, the proteins: pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9); and

[0016] - a processor capable and adapted to determine, from the determined concentrations of the proteins, an indication of a patient having mild periodontitis or severe periodontitis.

[0017] The system optionally comprises a data connection to an interface, in particular a graphical user interface, capable of presenting information, preferably also capable of inputting information such as the age of the subject and optionally other information such as the gender and / or the BMI (Body Mass Index), the interface being part of the system or a remote interface.

[0018] Optionally, one or more of the aforementioned items, in particular the processor, can be run "in the cloud", i.e. not on a fixed machine, but through means of an internet-based application.

[0019] In yet another aspect, the present application provides a kit for detecting at least three biomarkers for periodontitis in a saliva sample of a human patient, the kit comprising one or more, typically three or four, detection reagents for detecting pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9). Typically, three or more detection reagents are used, e.g. four detection reagents, each binding to a different biomarker. In one embodiment, a first detection reagent is used for detecting PK, a second detection reagent is used for detecting one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9), and a third detection reagent is used for detecting a different one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9). An optional fourth detection reagent can be used for detecting yet a different protein from the group of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9).

[0020] In yet another aspect, the present application provides an in vitro method for determining a change in periodontitis status of a human patient suffering from periodontitis over a time interval from a first time point ti to a second time point t2, the method comprising: detecting the concentration of pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9) in at least one saliva sample obtained from the patient at ti and in at least one saliva sample obtained from the patient at t2, and comparing the concentrations, whereby a difference in any one, two or all three of the concentrations reflects a change in status.

[0021] In yet another aspect, the present application provides a method of diagnosing whether a human patient has mild periodontitis or severe periodontitis, the method comprising: detecting in a saliva sample of a human patient the proteins: pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9); and based on the concentrations of the proteins in the sample, assessing the presence of mild periodontitis or severe periodontitis in the patient. Optionally, the method of this aspect comprises a further step of treating the patient for periodontitis.

[0022] In yet another aspect, the present application provides a method of detecting in a human patient having mild or severe periodontitis the proteins: pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9), the method comprising:

[0023] (a) obtaining a saliva sample from a human patient; and

[0024] (b) detecting whether pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9) are present in the sample by contacting the sample with one or more detection reagents for binding to the proteins, and detecting binding between each protein and the one or more detection reagents. Typically, there is a first detection reagent for detecting PK, a second detection reagent for detecting one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9), and a third detection reagent for detecting a different one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Schematically represents a system for use in a method as described in the present disclosure. DETAILED DESCRIPTION

[0026] In a general sense, the present invention is based on the astute insight that as few as three proteins in a saliva sample of a human patient suffering from periodontitis can serve as biomarkers for classifying said periodontitis into any one of two categories, one category being severe periodontitis, and the other category being mild or moderate (i.e. not severe, in the above as well as in the following, the term "mild periodontitis" will include moderate periodontitis, unless otherwise indicated). The latter category is hereinafter collectively referred to as mild periodontitis.

[0027] The identified protein biomarker is pyruvate kinase (PK) in combination with at least two of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8), and S100 calcium binding protein A9 (S100A9).

[0028] Optionally, the age of the subject can be included as an additional marker.

[0029] Pyruvate kinase (PK) catalyzes the last step of glycolysis. Pyruvate kinase has four tissue-specific isozymes, each with specific kinetic properties required by the different tissues.

[0030] Hemoglobin (Hb) is an iron-containing oxygen-transporting metalloprotein in the red blood cells of almost all vertebrates and in the tissues of certain invertebrates. Hemoglobin-beta (also known as beta globin, HBB, beta-globin, and hemoglobin beta subunit) is a globin protein that, together with alpha globin (HBA), makes up the most common form of hemoglobin in adults, i.e. HbA. Hb-beta typically is 146 amino acids long and has a molecular weight of 15,867 Da. Normal adult HbA is a heterotetramer consisting of two alpha chains and two beta chains. Hb-beta is typically encoded by the HBB gene on human chromosome 11. Hemoglobin delta subunit (also known as delta globin, HBD, delta-globin, and hemoglobin delta) is a globin protein that, together with alpha globin (HBA), makes up the less common form of hemoglobin in adults, i.e. HbA-2. Hb-delta typically is 147 amino acids long and has a molecular weight of 16,055 Da. Normal adult HbA-2 is a heterotetramer consisting of two alpha chains and two delta chains. Hb-delta is encoded by the HBD gene on human chromosome 11.

[0031] S100 calcium binding protein A8 (S100A8) is a calcium- and zinc-binding protein that plays a significant role in regulating inflammatory processes and immune responses. It can induce neutrophil chemotaxis and adhesion.

[0032] S100 calcium-binding protein A9 (S100A9), also known as calgranulin B, is a calcium and zinc-binding protein that plays a significant role in regulating inflammatory processes and immune responses. It can induce neutrophil chemotaxis, adhesiveness, can enhance the bactericidal activity of neutrophils by facilitating phagocytosis via activation of SYK, PI3K / AKT and ERK1 / 2, and can induce degranulation of neutrophils through a MAPK-dependent mechanism.

[0033] The above-mentioned proteins are known in the art. The skilled person knows their structure and methods for detecting them in aqueous samples, such as saliva samples. In the following, the aforementioned protein biomarkers are collectively referred to as “the biomarker panel of the invention”.

[0034] Table 1 in the example provides 12 particularly preferred combinations according to the invention.

[0035] In one embodiment, the biomarker panel of the invention comprises or consists of five protein biomarkers identified in the invention, i.e. PK, Hb-beta, Hb-delta, S100A8 and S100A9. Preferably, the biomarker panel of the invention consists of no more than four protein biomarkers identified in the invention, e.g. PK, and no more than three of the protein biomarkers Hb-beta, Hb-delta, S100A8 and S100A9. In addition to the biomarker panel of the invention, other biomarkers and / or data, such as demographic data (e.g. age, gender) can also be included in the set of data applied to determine the type of periodontitis.

[0036] An example of an additional protein biomarker is alpha-1-acid glycoprotein (A1AGP). Another exemplary additional protein biomarker is Profilin. These proteins are included in certain preferred biomarker panels in Table 1 below. Alpha-1-acid glycoprotein (A1AGP) is a plasma alpha-globulin glycoprotein that is mainly synthesized by the liver. It is also sometimes referred to as orosomucoid. It serves as a transport protein in the blood, acting as a carrier for basic and neutral charged lipophilic compounds. It is also thought to modulate the interaction between blood cells and endothelial cells. Profilin is an actin-binding protein involved in the dynamic turnover and reorganization of the actin cytoskeleton found in most cells. This is important for the spatiotemporal controlled growth of actin microfilaments, a key process for cell motility and cell shape changes. Human Profilin-1 is typically 140 amino acids long when expressed, but is usually further processed into a mature form.

[0037] Preferred extended biomarker panels are:

[0038] A1AGP + PK + S100A8 + S100A9

[0039] A1 AGP + Hb-β + Hb-δ + PK

[0040] Actin + Hb-β + Hb-δ + PK

[0041] Actin + Hb-β + PK + S100A8

[0042] When other biomarkers are optionally included, the total number of biomarkers (i.e. the biomarker panel of the present application plus the other biomarkers) is typically 4, 5 or 6.

[0043] However, a desirable advantage of the present application is that the classification of the periodontitis of a patient can be determined by preferably measuring no more than four biomarkers, and more preferably only three biomarkers, wherein the biomarker panel of (Table 1 below) is preferred. In particular, this determination does not require the use of other data, which advantageously provides a simple and straightforward diagnostic test.

[0044] As desired, the method only requires a small amount of saliva sample to be taken from the subject, e.g. the size of a water droplet. Typical ranges for the sample size are 0.1 μΐ to 2 ml, such as 1-2 ml, whereby smaller amounts (e.g. 0.1 to 100 μΐ) can be used for in vitro device handling, and whereby it is also possible to take larger samples, such as up to 20 ml, such as 7.5 to 17 ml.

[0045] The sample is input into an in vitro diagnostic device, which measures the concentration of the at least three proteins involved, and returns a diagnostic result classifying the subject as having mild periodontitis or severe periodontitis.

[0046] The ease of use of the present application will enable most dental patients having periodontitis or having a high risk of developing periodontitis to be tested on a regular basis, e.g. as part of a regular dental check-up or even at home. In particular, this allows the presence of mild periodontitis to be detected before it develops into severe periodontitis, thus enabling oral care measures to be taken more promptly to prevent the periodontitis from worsening. Alternatively, e.g. for a patient known to be at high risk of periodontitis and being tested for the first time, the method allows the identification of whether the periodontitis is mild or severe. Also, the method can be applied after treatment of a patient previously diagnosed with severe periodontitis in order to check whether the periodontitis has been improved so as to become mild. In a further scenario, an indication that a patient previously having mild periodontitis has not improved or has actually worsened after starting a treatment regime can lead to a dentist or patient deciding to change the treatment plan to help speed up the recovery process. In particular, the method is also suitable for self-diagnosis, whereby the steps of taking the sample and inputting it into the device are performed by the patient him or herself.

[0047] When the application is performed to determine whether periodontitis is mild or severe, the patient can typically be known to have periodontitis. Thus, in certain embodiments, the method is used to assess whether a human patient known to have periodontitis has mild or severe periodontitis.

[0048] The method of the application typically comprises detecting the aforementioned at least three proteins making up the biomarker panel of the application, and optionally other biomarker proteins, by use of one or more detection reagents.

[0049] The "saliva" that is tested according to the application can be undiluted saliva that can be obtained by spitting or swabbing, or diluted saliva that can be obtained by rinsing the mouth with a liquid. Diluted saliva can be obtained by the patient rinsing or swilling with sterile water (e.g. 5ml or 10ml) or other suitable liquid for a few seconds and spitting into a container. Diluted saliva can sometimes be referred to as oral rinse.

[0050] "Detecting" means measuring, quantifying, scoring or assaying the concentration of a biomarker protein. Methods of assessing biological compounds including biomarker proteins are known in the art. It will be appreciated that methods of detecting protein biomarkers include both direct and indirect measurements. The skilled person will be able to select an appropriate method of assaying a particular biomarker protein.

[0051] The term "concentration" in relation to a protein biomarker should be given its usual meaning, i.e. the abundance of the protein in a certain volume. Protein concentration is typically measured as mass per volume, most typically mg / ml or pg / ml, but sometimes as low as pg / ml. An alternative measure is molarity (or molar concentration) mol / L or "M". Concentration can be determined by detecting the amount of protein in a known, determined or predetermined volume of sample.

[0052] An alternative to determining concentration is to determine the absolute amount of protein biomarker in a sample, or to determine the mass fraction of the biomarker in the sample, e.g. the amount of biomarker relative to the total amount of all other proteins in the sample.

[0053] A "detection reagent" is a reagent or compound that specifically (or selectively) binds to, interacts with or detects a protein biomarker of interest. Such detection reagents can include but are not limited to antibodies, polyclonal antibodies or monoclonal antibodies that preferentially bind to a protein biomarker.

[0054] When referring to assay reagents, the phrase "specifically (or selectively) binds" or "specifically (or selectively) reacts with an immune response" refers to a binding reaction that identifies the presence of a heterogeneous population of proteins and protein biomarkers in other biological agents. Therefore, under specified immunoassay conditions, a specific assay reagent (e.g., an antibody) binds to a specific protein at least twice the background level and binds substantially little to other proteins present in the sample. Specific binding under these conditions may necessitate the selection of antibodies specific to a particular protein. Various immunoassay formats can be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA (enzyme-linked immunosorbent assay) is routinely used to select antibodies that specifically react with a protein (see, for example, Harlow & Lane's Antibodies, published in the 1988 Laboratory Handbook, for a description of immunoassay formats and conditions that can be used to determine specific immune responses). Typically, a specific or selective response is at least twice the background signal or noise, more typically 10 to 100 times or more than the background level.

[0055] An "antibody" refers to a polypeptide ligand essentially encoded by one or more immunoglobulin genes or fragments thereof, which specifically binds to and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the α, γ, δ, ε, and μ heavy chain constant region genes, and numerous immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as numerous well-defined fragments produced by digestion with various peptidases. This includes, for example, Fab' and F(ab)'2 fragments. As used herein, the term "antibody" also includes those antibody fragments produced by modifying intact antibodies or 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 a portion of the immunoglobulin heavy chain that includes one or more heavy chain constant regions CH1, CH2, and CH3, but excludes the heavy chain variable region. Antibodies can be bispecific antibodies, for example, 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. Using at least one bispecific antibody can reduce the number of test reagents required.

[0056] The sensitivity and specificity of diagnostic methods differ. The "sensitivity" of a diagnostic test refers to the percentage of diseased individuals who test positive (the "true positive" percentage). Diseased individuals not detected by the test are called "false negatives." Individuals without the disease who test negative are called "true negatives." The "specificity" of a diagnostic test is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of patients who test positive but do not have the disease.

[0057] The biomarker protein(s) of the present application 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 include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays, and the like. Such assays are routine and well known in the art. Exemplary immunoassays are briefly described below (but are not intended to be limiting).

[0058] An immunoprecipitation protocol generally includes lysing a cell population in a lysis buffer (such as RIPA buffer (1% NP-40 or Triton X-100, 1% deoxycholate sodium, 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), adding an antibody of interest to the cell lysate, incubating at 4°C for a period of time (e.g., 1 to 4 hours), adding protein A and / or protein G agarose beads to the cell lysate, incubating at 4°C for about an 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 particular antigen can be assessed by, for example, Western blot analysis. Those skilled in the art will appreciate parameters that can be modified to increase binding of the antibody to the antigen and decrease background (e.g., pre-clearing the cell lysate with agarose beads).

[0059] A 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 with 3% BSA or skim milk), washing the membrane in a wash buffer (e.g., PBS-Tween 20), blocking the membrane with a primary antibody (an antibody of interest) diluted in blocking buffer, washing the membrane in wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. Those skilled in the art will appreciate parameters that can be modified to increase the signal detected and decrease background noise.

[0060] ELISA typically involves preparing an antigen (i.e. a biomarker protein of interest or fragment thereof), coating the wells of a 96-well microtiter plate with the antigen, adding an antibody of interest conjugated 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 does not have to be conjugated to a detectable compound; instead, a second antibody (which recognises the antibody of interest) conjugated to a detectable compound can be added to the wells. Further, instead of coating the wells with an antigen, the wells can be coated with an antibody. In this case, after the antigen of interest is added to the coated wells, a second antibody conjugated to a detectable compound can be added. Those skilled in the art will be aware of the parameters that can be modified to increase the signal detected and other variations of ELISA known in the art.

[0061] Since multiple markers are used, a threshold is determined based on the combined concentrations of these biomarkers (and optionally age). This threshold determines whether a patient is classified as having mild periodontitis or severe periodontitis. The insight reflected in the present invention is that periodontitis can be detected as mild or severe with sufficient accuracy based on measuring a combination of the biomarkers indicated above.

[0062] This insight supports a further aspect of the present invention, the use of the proteins: pyruvate kinase (PK) and at least two of the following: haemoglobin-beta (Hb-beta), haemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9) as biomarkers in a saliva sample of a human patient suffering from periodontitis for assessing whether the patient has mild periodontitis or severe periodontitis.

[0063] This use can be implemented in the methods substantially described above and below.

[0064] The method of the present invention comprises determining a test value reflecting the combined concentrations measured for said proteins. The combined concentration value can be any value obtained by inputting the determined concentrations and performing arithmetic operations on these values. For example, this can be a simple addition of the concentrations. It can also involve 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 with each other or any combination of multiplication, division, subtraction, exponentiation and addition. It can further involve raising the concentrations to a certain power.

[0065] Optionally, the test value reflects the combined concentrations determined for said protein(s) in combination with the age of the subject.

[0066] The obtained combined concentration value is compared to a threshold value, which reflects the combined concentration in the same way that is associated with the presence of severe periodontitis. This comparison allows to assess whether the test value is indicative of the presence of severe periodontitis or mild periodontitis in the patient whose saliva was subjected to the test.

[0067] The threshold value can for example be a combined concentration value, which is obtained in the same way based on concentrations determined for the same proteins in reference samples that are associated with the presence of severe periodontitis (i.e. in patients diagnosed with severe periodontitis). Typically, values reflecting the same or higher combined concentrations are thus indicative of the presence of severe periodontitis in the patient under test. Similarly, values reflecting lower combined concentrations in the saliva of the patient under test with periodontitis are indicative of mild or moderate (i.e. non-severe) periodontitis. It is however understood that the threshold value can also be calculated (e.g. by using a negative multiplier) such that test values indicative of severe periodontitis will be lower than the threshold value, and test values indicative of mild periodontitis will be higher than the threshold value.

[0068] The threshold value can also be determined based on concentrations of the biomarker proteins now measured in a collection of samples, including patients known to be diagnosed with severe periodontitis as well as patients that are not severe (mild and / or moderate) periodontitis. The measured concentration values can thus be subjected to statistical analysis, possibly including machine learning methods, to allow to distinguish between patients classified as mild or moderate periodontitis and patients classified as having severe periodontitis with a desired sensitivity and specificity. The desired threshold value can thus be obtained. Based on this threshold value, the same concentration measurements can be performed on the sample under test, and the concentration values can then be processed in the same way as to obtain the threshold value, to determine a combined concentration value, which can be compared to the threshold value, thus allowing to classify the test sample as having mild or severe periodontitis.

[0069] In an interesting embodiment, the combined concentration value is obtained in the form of a score. Numerical values (protein concentration values, e.g. in ng / ml) are assigned to each measurement, and these values are used in a linear or non-linear combination to compute a score between 0 and 1. In the case of determining a threshold value based on a collection of subjects as mentioned above, typically a sigmoid function with the combined concentration as input is used to compute a score between 0 and 1 (as further illustrated).

[0070] When the score exceeds a certain threshold value, the method indicates that the patient has severe periodontitis. The threshold value can be chosen based on a desired sensitivity and specificity.

[0071] It is understood that, in accordance with the present application, in performing the "mild or severe periodontitis classification" on a subject, this is for a subject that can be assumed to have periodontitis. This can for example be known from a previously performed periodontitis diagnosis, although it can not be possible to distinguish its degree, or for example be assumed by a record of the subject's oral health status.

[0072] The clinically accepted definition in the art is based on the following:

[0073] Gingival index (GI)

[0074] The full mouth gingival index will be recorded based on the Löe Modified Gingival Index (MGI) on a scale from 0 to 4, wherein:

[0075] -0 = no inflammation,

[0076] -1 = mild inflammation; slight change in color and texture of any part, not entire margin or papillary gingival unit,

[0077] -2 = mild inflammation; but involving entire margin or papillary unit,

[0078] -3 = moderate inflammation; shiny, red, edematous and / or hypertrophied margin or papillary unit,

[0079] -4 = severe inflammation; margin or papillary gingival unit markedly red, edematous and / or hypertrophied, spontaneously bleeding, congested or ulcerated.

[0080] Probing depth (PD)

[0081] Probing depth will be recorded to the nearest mm using a manual UNC-15 periodontal probe. 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.

[0082] Root cementum erosion (REC)

[0083] Gingival recession will be recorded to the nearest mm 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 indicated as a positive number and gingival overgrowth will be indicated as a negative number.

[0084] Clinical attachment loss (CAL)

[0085] Clinical attachment loss will be calculated as the sum of probing depth + amount of recession for each site.

[0086] Probing bleeding on probing (BOP)

[0087] After probing, the probing bleed for each site will be assessed and a score of 1 will be assigned to the site if bleeding occurs within 30s of probing, otherwise a score of 0 will be assigned.

[0088] The resulting subject groups (patient groups) are defined as follows, whereby the mild-to-moderate periodontitis group and the severe periodontitis group are relevant for the present invention:

[0089] - Healthy group (H): PD < 3 mm in all sites (but up to four 4 mm pockets in the distal most of the last remaining molar teeth), no sites with interproximal attachment loss, GI > 2.0 in < 10% of sites, %BOP score < 10%;

[0090] - Gingivitis group (G): GI > 3.0 in > 30% of sites, no sites with interproximal attachment loss, no sites with PD > 4 mm, %BOP score > 10%;

[0091] - Mild-moderate periodontitis group (MP): interproximal PD of 5 to 7 mm, (corresponding to approximately 2 to 4 mm CAL) > 8 teeth, %BOP score > 30%;

[0092] - Advanced periodontitis group (AP): interproximal PD > 7 mm, (corresponding to approximately > 5 mm CAL) > 12 teeth, %BOP score > 30%.

[0093] In an embodiment, the method of the invention utilizes Figure 1 a schematically represented system. The system can be a single device with various device components (units) integrated therein. The system can also have its various components or some of these components as separate devices. Figure 1 The components shown are a measuring device (A), a graphical user interface (B) and a computer processing unit (C).

[0094] As mentioned above, the system of the invention comprises a data connection to an interface, whereby the interface itself can be part of the system or can be a remote interface. The latter means that a different device can be used to provide the actual interface, preferably a handheld device such as a smartphone or tablet computer. In this case, the data connection will preferably involve wireless data transfer, such as through Wi-Fi or Bluetooth or through other technologies or standards.

[0095] 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 which is capable of determining the concentration of at least the following proteins from the same saliva sample: pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9).

[0096] 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 which is able to determine the concentration of at least the following proteins from the same saliva sample: pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9).

[0097] The processing unit (C) receives the numerical values for the protein concentrations from part (A). The unit (C) is provided with software (typically embedded software) which allows it to calculate a fraction (S) between 0 and 1. The software further comprises a numerical value for a threshold (T). If the calculated value (S) exceeds (T), the unit (C) will output an indication (I) of'severe periodontitis' to the GUI (B), otherwise'mild periodontitis'. Yet another embodiment can use a specific value of (S) to indicate the certainty with which the indication (I) is made. This can be a probability fraction, whereby 0.5 is a possible threshold, and for example a fraction S = 0.8 will indicate the likelihood of severe periodontitis. Interesting options are:

[0098] Based on the fraction S, the person can make a direct indication of certainty, i.e. S = 0.8 means 80% certainty of severe periodontitis;

[0099] Based on the fraction S, the person can make a binary or a three-level indication:

[0100] -S <t->Mild periodontitis, S≥T -> Severe periodontitis;

[0101] -S <r1->Mild periodontitis, R1 < S <r2->Uncertainty,

[0102] - S > R2-> severe periodontitis;

[0103] In addition, a certainty can be added to this binary or ternary indication. This certainty will be determined by the distance of the score S to the selected threshold(s) (T, R1, R2).

[0104] The specific calculation of the score can for example be implemented by means of an S-shaped function applying the following formula:

[0105]

[0106] where N is the number of proteins / biomarkers used, co, ci, etc. are coefficients (numerical values) and Bi, B2, etc. are the respective protein concentrations.

[0107] The determination of the coefficients c i can be done by a training procedure:

[0108] - Select N1 subjects with severe periodontitis (for example as identified by a dentist using current standards) and N2 subjects with mild periodontitis.

[0109] Subjects without mild periodontitis are considered to have a score S = 0 and subjects with severe periodontitis are considered to have a score S = 1.

[0110] - Take a saliva sample from each subject and determine the protein concentrations of the biomarker combination as explained above.

[0111] - Perform a logistic regression between the protein concentrations and the scores.

[0112] Other regression or machine learning methods (linear regression, neural networks, support vector machines) can be used to train a classifier that predicts whether a subject has periodontitis or has a healthy oral situation based on the protein concentrations.

[0113] In particular, this procedure is applied (in the example) with subjects having mild periodontitis or severe periodontitis using a clinical study (identified by a clinical assessment by a dental specialist via current standards (for example, American Academy of Periodontology standards)). The performance of various biomarker combinations is evaluated by means of a Leave-1-out cross-validation resulting in the preferred biomarker combination of the present invention.

[0114] With reference to the system mentioned before, in yet another aspect, the present invention also provides a system for assessing whether a human patient has mild periodontitis or severe periodontitis, the system comprising:

[0115] - a detection device, able and adapted to detect in a saliva sample of a human patient the following proteins: pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9); as explained above, such a device is known and easily available to the skilled person; typically, a container for receiving in it an oral sample of a subject is provided, the container being provided by the detection device;

[0116] - a processor, able and adapted to determine an indication of a patient having mild periodontitis or severe periodontitis by the determined concentrations of said proteins.

[0117] Optionally, the system comprises a user interface (or a data connection to a remote interface), in particular a graphical user interface (GUI), able to present information; a GUI is a type of user interface that allows users to interact with electronic devices through graphical icons and visual indicators such as secondary notation, instead of text-based user interfaces, typed command labels or text navigation (any such interface type is not excluded in the present invention); GUIs are generally 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 controls; as said, optionally, the interface can also be chosen so as to be able to input information, such as, for example, the age, sex, BMI (Body Mass Index) of the subject.

[0118] Separately or as part of the aforementioned system, the present application also provides a kit for detecting at least three biomarkers for periodontitis in a saliva sample of a human patient, the kit comprising one or more detection reagents for detecting pyruvate kinase (PK) and at least two of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9). Thus, the kit comprises three detection reagents, each for a different biomarker, wherein a first detection reagent is for detecting PK, a second detection reagent is for detecting one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9), and a third detection reagent is for detecting a different one of hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9). As discussed above with reference to the method of the present application, the kit can comprise more detection reagents, such as in particular for inhibiting proteins or A1 AGP and / or other proteins. In preferred embodiments, the detection reagents available in the kit consist of the detection reagents for the three proteins used to select the 3-biomarker or 4-biomarker panel constituting the present application, as mentioned. In other embodiments, a separate detection reagent is provided for each biomarker protein present in the combinations exemplified in Table 1 in the example below.

[0119] Preferably, the kit comprises a solid support, such as a chip, microtiter plate or microbeads or resins comprising the detection reagents. In some embodiments, the kit comprises mass spectrometry probes, such as ProteinChip TM .

[0120] The kit can also provide a washing solution specific for the unbound detection reagents or the biomarkers (sandwich-type assay) and / or detection reagents.

[0121] In a further interesting aspect, the identification of the biomarker panel of the present application is applied to monitor the periodontitis status of a human patient over time. Thus, the present application also provides an in vitro method for determining a change in the periodontitis status of a human patient suffering from periodontitis over a time interval from a first time point ti to a second time point t2, the method comprising: detecting the concentration of the proteins: pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9) in at least one saliva sample obtained from said patient at ti and in at least one saliva sample obtained from said patient at t2, and comparing the concentrations, whereby preferably a difference in the concentration of at least two (more preferably at least three) concentrations reflects a change in status. This difference can be seen as a concentration difference, allowing for a direct comparison without first generating a number between 0 and 1 or any other classification. It is to be understood that the measurements received at the two time points can also be treated in exactly the same way as above when determining mild or severe periodontitis.

[0122] The present application also provides a method of diagnosing whether a human patient has mild or severe periodontitis, the method comprising detecting the presence of the proteins: pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9) in saliva of the patient. The presence of mild or severe periodontitis of the patient is assessed based on the concentration of the proteins in the sample. Optionally, the method of this aspect comprises a further step of treating the periodontitis of the patient. This optional treatment step can comprise administering a known therapeutic agent or dental procedure or a combination of a therapeutic agent and a dental procedure. Known therapeutic agents include administration of an agent containing an antibacterial agent, such as a mouthwash, tablet, gel or microsphere. A typical antibacterial agent for treating 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 surgical pocket reduction, flap surgery, gum grafting or bone grafting.

[0123] The present application further provides a method of detecting the proteins: pyruvate kinase (PK) and at least two of: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9) in a patient suffering from mild or severe periodontitis, the method comprising:

[0124] (a) obtaining a saliva sample from a human patient; and

[0125] (b) detecting whether the proteins are present in the saliva sample by contacting the saliva sample with first, second and third detection reagents for detecting the proteins, and detecting the binding between each protein and the detection reagent.

[0126] The application will be further illustrated with reference to the following non-limiting examples.

[0127] Example

[0128] In a clinical study with 79 subjects, of which 41 were diagnosed with mild periodontitis (including moderate periodontitis) and 38 with severe periodontitis, the area under the ROC (Receiver-Operator-Characteristic) curve (AUC) value was obtained.

[0129] In statistics, the Receiver Operating Characteristic curve or ROC curve is a plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true positive rate (TPR) against the false positive rate (FPR) for various threshold settings. The true positive rate is also known as the sensitivity, recall or detection probability in machine learning. The false positive rate is also known as the fall-out or false alarm probability and can be calculated as (1 - specificity). Thus, the ROC curve is a plot of the sensitivity versus the fall-out. Typically, if the probability distributions for detection and false alarms are known, the ROC curve can be generated by plotting, for each value of the threshold, the value of the cumulative distribution function of the detection probability (the area under the probability distribution from -∞ to the probability of the discrimination threshold) on the y-axis against the value of the cumulative distribution function of the false alarm probability on the x-axis. The accuracy of a test depends on the degree to which the test separates the population of people with or without the disease in question. The accuracy is measured by the area under the ROC curve. An area of 1 indicates a perfect test; an area of 0.5 indicates a worthless test. A guide to categorizing the accuracy of a diagnostic test is the traditional academic grading system:

[0130] - 0.90 - 1 = Excellent (A)

[0131] - 0.80 - 0.90 = Good (B)

[0132] - 0.70 - 0.80 = Fair (C)

[0133] - 0.60 - 0.70 = Poor (D)

[0134] - 0.50 - 0.60 = Fail (F)

[0135] Based on the foregoing, in the results of the aforementioned clinical study, ROC AUC values higher than 0.75 were considered to represent a desirable accuracy for providing a diagnostic test according to the present application.

[0136] Among all the biomarkers and biomarker panels determined, Table 1 below summarizes data for panels of up to four protein biomarkers representing ROC AUC values of 0.75 and above. These data indicate that 3 protein biomarkers (in the specified combinations) can provide AUC LOOCV of >0.75 to classify mild periodontitis from severe periodontitis:

[0137] Table 1

[0138]

[0139] Each of the 12 biomarker combinations in this table is highlighted as a preferred combination of the present application.

[0140] In this table, next to the marker age, 8 protein markers (not previously linked to oral health) are considered:

[0141] • A1AGP

[0142] • Hb-β

[0143] • Hb-δ

[0144] • Keratin 4

[0145] • Profilin

[0146] • Pyruvate kinase

[0147] • S100A8

[0148] • S100A9

[0149] To properly appreciate the 12 identified panels with AUC >0.75:

[0150] • Using the 8 protein markers, optionally plus age as a marker, there are 324 possible panels with up to 4 protein markers (not considering panels with only age).

[0151] • Without restricting the number of protein markers in the panel, there are 510 possible panels from these 8 markers (not considering panels with only age).

[0152] While the application has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the application is not limited to the disclosed embodiments.

[0153] For example, the detection reagents for the different biomarkers can be proposed in different units. Alternatively, and conveniently, the kit of the application can comprise a set of immobilized detection reagents for the protein biomarkers used in all the embodiments, i.e. PK and flexible modules comprising detection reagents for any one of the other biomarkers, i.e. hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9).

[0154] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed application, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain features 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.

[0155] In summary, we disclose herein an in vitro method for assessing whether a human patient suffering from periodontitis has mild periodontitis or severe periodontitis. The method is based on determining a selected insight on three biomarker proteins. Thus, in a saliva sample of a patient suffering from periodontitis, the concentration of the following proteins is measured: pyruvate kinase (PK) and at least two of the following: hemoglobin-beta (Hb-beta), hemoglobin-delta (Hb-delta), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9). Based on the measured concentrations, a value is determined that reflects the joint concentration for said proteins. This value is compared to a threshold value that reflects the joint concentration associated with severe periodontitis in the same way. This comparison allows assessing whether the test value is indicative of the presence of severe periodontitis or mild periodontitis in said patient. Thereby, generally, a test value that reflects a joint concentration lower than the joint concentration reflected by the threshold value is indicative of said patient suffering from mild periodontitis, and a test value that reflects a joint concentration equal to or higher than the joint concentration reflected by the threshold value is indicative of said patient suffering from severe periodontitis.

Claims

1. A computer processing unit configured to execute software to implement an in vitro method for assessing whether a human patient has mild or severe periodontitis, wherein the computer processing unit is configured to: - Receive the concentration of the protein, wherein the concentration of the protein is detected in a saliva sample from the human patient; - Determine a test value that reflects the combined concentration determined for the protein; - The test value is compared to a threshold to assess whether the test value indicates that the patient has mild or severe periodontitis, and the threshold reflects the combined concentrations associated with severe periodontitis in the same way. The protein described herein is composed of the following: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A9, Hb-β and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, S100A9 and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; Inhibitors of protein, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; or Inhibitor protein, Hb-β, PK and S100A8, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis.

2. The computer processing unit according to claim 1, wherein the human patient is known to have periodontitis, and / or The age of the human patient is determined, and the test value, in conjunction with the age of the human patient, reflects the combined concentration determined for the protein.

3. The computer processing unit according to any one of the preceding claims, wherein the threshold is based on the concentration of the protein determined for one or more reference samples, each sample being associated with the presence of severe periodontitis.

4. The computer processing unit according to claim 1 or 2, wherein the threshold is based on the concentration of the protein in a sample set, the sample set including samples from human patients with mild or moderate periodontitis and samples from human patients with severe periodontitis.

5. The computer processing unit according to claim 1 or 2, wherein the determined test value is arithmetically processed into a number between 0 and 1.

6. A system for assessing whether a human patient has mild or severe periodontitis, the system comprising: The detection device is capable of and adapted to detect proteins in saliva samples from the human patient. The processor is capable of and adapted to determine, based on a determined concentration of the protein, whether the patient has mild or severe periodontitis. The protein described herein is composed of the following: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A9, Hb-β and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, S100A9 and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; Inhibitors of protein, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; or Inhibitor protein, Hb-β, PK and S100A8, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis.

7. The system of claim 6 further includes a container for receiving oral fluid samples, the container including the detection device; and / or Also includes: - A user interface used to present instructions to the user; as well as - A data connection between the processor and the user interface, the data connection being used to transmit the instruction from the processor to the user interface; and / or The processor described herein is capable of functioning with the aid of internet-based applications; and / or The interface is capable of inputting information about the age of the human patient, and the processor is capable of determining, based on the determined concentration, whether the patient has mild or severe periodontitis.

8. A kit for detecting at least three biomarkers in a saliva sample of a human patient, said at least three biomarkers for assessing whether said human patient has mild or severe periodontitis, said kit comprising one or more detection reagents. The one or more detection reagents mentioned above are detection reagents used for the following: composition: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A9, Hb-β and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, S100A9 and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; Inhibitors of protein, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; or Inhibitor protein, Hb-β, PK and S100A8, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis.

9. The kit according to claim 8, wherein one or more detection reagents are contained on a solid support.

10. A computer processing unit configured to execute software to implement an in vitro method for determining changes in the periodontal status of a human patient suffering from periodontitis over a time interval from a first time point t1 to a second time point t2, said computer processing unit being configured to: The concentration of the received protein, wherein the concentration of the protein is detected in at least one saliva sample obtained from the patient at time t1 and in at least one saliva sample obtained from the patient at time t2, and The concentration at time t1 is compared with the concentration at time t2, and the difference in concentration reflects the change in state. The protein described herein is composed of the following: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to determine the state changes; PK, S100A8, and Hb-β, wherein the age of the human patient was used to determine the state changes; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to determine the state changes; PK, S100A9, Hb-β, and Hb-δ, wherein the age of the human patient was used to determine the state changes; PK, S100A8, S100A9, and Hb-β, wherein the age of the human patient was used to determine the state changes; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to determine the state changes; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient is used to determine the state change; Inhibitory proteins, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to determine the state changes; or Inhibitory proteins, Hb-β, PK, and S100A8, wherein the age of the human patient was used to determine the state changes.

11. A computer processing unit configured to execute software to implement a method for diagnosing whether a human patient has mild or severe periodontitis, said computer processing unit being configured to: The concentration of the received protein, wherein the concentration of the protein is detected in a saliva sample from the human patient; and Based on the concentration of the protein in the sample, assess whether the patient has mild or severe periodontitis. The protein described herein is composed of the following: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; PK, S100A8, and Hb-β, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; PK, S100A9, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; PK, S100A8, S100A9 and Hb-β, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; Inhibitors of protein, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis; or Inhibitor protein, Hb-β, PK and S100A8, wherein the age of the human patient was used to assess the presence of mild or severe periodontitis.

12. A system for detecting proteins in human patients with mild or severe periodontitis, the system comprising one or more detection reagents, the system being configured to: (a) Obtaining saliva samples from human patients; as well as (b) The presence of a protein in the sample is detected by contacting the sample with one or more detection reagents for binding the protein and detecting the binding between each protein and the one or more detection reagents. The one or more detection reagents mentioned above are detection reagents used for the following: composition: Pyruvate kinase PK, S100 calcium-binding protein A8, and S100 calcium-binding protein A9; PK, hemoglobin-β Hb-β, and hemoglobin-δ Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, Hb-β, and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A9, Hb-β and Hb-δ, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; PK, S100A8, S100A9 and Hb-β, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8 and S100A9; A1AGP, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; A1AGP, PK, S100A8, and S100A9, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; Inhibitors of protein, Hb-β, Hb-δ, and PK, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis; or Inhibitor protein, Hb-β, PK and S100A8, wherein the age of the human patient was used to assess whether the human patient had mild or severe periodontitis.

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