PCT marker panel for early detection of sepsis

Abstract

To provide a method for assessing individuals suspected of having an infectious disease. [Solution] The method includes the steps of: determining the amount of a first biomarker in a sample of a subject, wherein the first biomarker is PCT; determining the amount of a second biomarker in a sample of a subject, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; comparing the amount of the biomarker with a biomarker standard and / or calculating a score for rating a subject suspected of having an infection based on the amount of the biomarker; and rating the subject based on the comparison and / or calculation.

Description

Technical Field

[0001] The present invention relates to the field of diagnostics. In particular, the present invention is a method for assessing a subject suspected of having an infectious disease, comprising determining the amount of a first biomarker in a sample of the subject, wherein the first biomarker is PCT; determining the amount of a second biomarker in a sample of the subject, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; comparing the amount of the biomarker to a reference of the biomarker and / or calculating a score for assessing the subject suspected of having an infectious disease based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of a first biomarker that is PCT and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, or a detection agent that specifically binds to the first biomarker and a detection agent that specifically binds to the second biomarker, for assessing a subject suspected of having an infectious disease. Furthermore, the present invention further relates to a computer-implemented method for assessing a subject suspected of having an infectious disease, as well as a device and a kit for assessing a subject suspected of having an infectious disease.

Background Art

[0002] Infectious diseases, particularly those occurring in patients with more severe signs and symptoms, such as patients transported to an emergency room, can develop into more life-threatening medical conditions including systemic inflammatory response syndrome (SIRS) and sepsis.

[0003] According to the definition of stage 3 sepsis, sepsis is defined as a life-threatening organ dysfunction caused by a disregulated host response to infection. Because it develops rapidly, early recognition is crucial for the management of septic patients, as well as for the initiation of correct therapeutic measures, including appropriate antibiotic treatment within the first hour of hospitalization, and the initiation of resuscitation with intravenous fluids and vasoactive drugs (surviving sepsis campaign guidelines 2016). Every hour of delay gradually increases morbidity and mortality.

[0004] The diagnosis of sepsis is based on nonspecific clinical signs and symptoms and can easily be missed. Therefore, patients are frequently misdiagnosed, and the severity of the disease is often underestimated. There is currently no gold standard for diagnosing sepsis, particularly in general departments and emergency departments. In high-income countries, C-reactive protein (CRP), procalcitonin (PCT), and white blood cell (WBC) counts, along with lactate for detecting septic shock, are frequently used in emergency rooms to detect patients with bloodstream infections at risk of developing sepsis. In low-income countries, diagnosis is usually based on clinical signs and symptoms, and in some cases, SIRS and SOFA criteria. However, most current guidelines do not list biomarkers for diagnosing sepsis other than lactate (with the exception of clinical chemistry, BGE, and hematological components of the SOFA score). PCT is recommended, with moderate evidence, only to potentially reduce antibiotic treatment. The limitations of PCT in diagnosing sepsis are primarily its moderate sensitivity and specificity.

[0005] International Publication No. 2007 / 009071 discloses a method for diagnosing inflammatory responses in test subjects based on sFlt-1. The disclosed method further includes analyzing levels of at least one of the following: VEGF, PlGF5, TNF-α, IL-6, D-dimer, P-selectin, ICAM-I, VCAM-I, Cox-2, or PAI-I.

[0006] EP 2 174 143 B1 discloses an in vitro method for prognostic diagnosis of patients with a primary disease that is not infectious, which includes determining the level of procalcitonin.

[0007] Numerous markers have been suggested to be useful for the detection or diagnosis of sepsis. These include, among many others, inflammatory markers such as PCT, presepsin, GDF-15, sFLT, CRP, or interleukins, or markers specific to organ failure (see, for example, Spanuth, 2014, Comparison of sCD14-ST(presepsin) with eight biomarkers for mortality prediction in patients admitted with acute heart failure, 2014 AACC Annual Meeting Abstracts. B-331; van Engelen, 2018, Crit Care Clin 34(1):139-152).

[0008] International Publication No. 2015 / 031996 describes biomarkers for the early assessment of critical or life-threatening responses and / or treatment responses to disease.

[0009] However, there is still a need for biomarkers that enable reliable and early assessment of patients exhibiting signs and symptoms of infection. [Overview of the project]

[0010] Therefore, the present invention provides means and methods to satisfy these needs.

[0011] The present invention is a method for evaluating a subject suspected of having an infectious disease, (a) A step of determining the amount of a first biomarker in a sample of interest, wherein the first biomarker is PCT; (b) A step of determining the amount of a second biomarker in a sample of interest, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing the amount of the biomarker to a standard for the biomarker and / or the step of calculating a score for assessing a subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). This includes methods. [Modes for carrying out the invention]

[0012] As used herein and in the claims, "a" or "an" should be understood to mean one or more, depending on the context in which it is used. Yes, it exists. Therefore, for example, a reference to the "an" element (item) can mean that at least one element may be available.

[0013] When used below, the terms “have,” “comprise,” or “include,” or any grammatical variations thereof, are used in a non-exclusive manner. Therefore, these terms may refer to both situations where the entity described in this context has no further features other than those introduced by these terms, and situations where one or more further features exist. For example, the expressions “A has B,” “A comprises B,” and “A includes B” can all refer to situations where, other than B, no other elements exist in A (i.e., A consists solely and exclusively of B), as well as situations where, other than B, one or more further elements exist in entity A, such as elements C, elements C and D, or even further elements. The term “comprising” also encompasses embodiments in which only the elements mentioned exist; that is, it has a limited meaning in the sense of “consisting of.”

[0014] Furthermore, when used below, the terms “particularly,” “more particularly,” “typically,” and “more typically,” or similar terms, are used in conjunction with additional / alternative features without limiting the possibility of alternatives. Thus, features introduced by these terms are additional / alternative features and are not intended in any way to limit the scope of the claims. The present invention may be implemented by using alternative features, as will be understood by those skilled in the art. Similarly, features introduced by “in embodiments of the present invention” or similar expressions are intended to be additional / alternative features without limitation on alternative embodiments of the present invention, without limitation on the scope of the present invention, and without limitation on the possibility of combining such introduced features with other additional / alternative or non-additional / alternative features of the present invention.

[0015] Furthermore, it will be understood that, when used herein, the term "at least one" means that one or more of the elements subsequently referred to in the term may be used in accordance with the invention. For example, where the term indicates that at least one sampling unit is used, this may be understood as one sampling unit or one or more sampling units, i.e., two, three, four, five, or any other number. Depending on the element to which the term refers, if there is an upper limit to which the term may refer, those skilled in the art will understand to what extent that limit may be.

[0016] When used herein, the term "about" means that with respect to any number following the term, there is an accuracy to the extent that the technical effect can be achieved. Thus, as used herein, "about" preferably refers to a precise number or a range of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5% around the precise number.

[0017] Furthermore, terms such as “first,” “second,” and “third” in this specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order.

[0018] The method of the present invention may consist of the steps described above, or it may include additional steps, such as a step of further evaluation of the assessment obtained in step (d), or a step of therapeutic action, such as a step of recommending treatment. Furthermore, it may include a step preceding step (a), such as a step relating to sample pretreatment. However, preferably, the above method is not performed on a human or animal body. It is assumed that this is an ex vivo method that does not require any steps. Furthermore, the method may be assisted by automation. Typically, the determination of biomarkers may be supported by robotic instruments, and comparison and evaluation may be supported by data processing instruments, such as computers.

[0019] The term “assess” as used herein means to assess whether a subject has sepsis, is at risk of developing sepsis, or presents with a worsening medical condition in relation to overall health or to sepsis or signs and symptoms associated with sepsis and / or infection. Thus, assessing, as used herein, includes diagnosing sepsis, predicting the risk of developing sepsis, and / or predicting any deterioration of the subject’s health, in particular with respect to signs and symptoms associated with sepsis and / or infection.

[0020] Typically, the assessment referred to in accordance with the present invention is an assessment of the risk of developing sepsis (and thus a prediction of the risk of developing sepsis). Alternatively, the assessment is a prediction of the risk of deterioration of the subject's condition. Furthermore, if a risk of developing sepsis or deterioration of health is predicted, it will be understood that the prediction is typically made within a prediction window. More typically, the prediction window is preferably about 8, 10, 12, 16, 20, 24, 48 hours, and especially at least 48 hours after the sample was obtained. Furthermore, preferably, the risk of developing sepsis may be predicted within 24 or 48 hours after the test sample was obtained.

[0021] In one embodiment, there is a predicted risk of developing sepsis within 24 hours.

[0022] In alternative embodiments, there is a predicted risk of developing sepsis within 48 hours.

[0023] In yet another embodiment, the assessment is a prediction of the risk that the subject's (health) condition will deteriorate in the future. The term “deterioration of condition” of a subject suspected of having and / or having an infection is well understood by those skilled in the art. The term typically refers to a deterioration of condition that may ultimately lead to further drug therapy or other interventions.

[0024] Preferably, when the severity of the target disease increases, when the antibiotic treatment of the target is intensified, when the target is admitted to the ICU or another treatment room for a higher level of treatment, when the target requires emergency surgery, when the target dies in the hospital, when the target dies within 30 days of admission, when the target is readmitted within 30 days of discharge, when the target experiences organ dysfunction or failure, as measured, for example, by the SOFA score, and / or when the target requires organ support, the condition of the target is deteriorating.

[0025] One of ordinary skill in the art understands the case where the condition of the target is not deteriorating. Typically, when the target does not have the outcomes mentioned in the preceding paragraph, the condition of the target is not deteriorating.

[0026] In one embodiment, when the target has one or more of the following outcomes: when the target is admitted to the ICU, when the target dies in the hospital, when the target dies within 30 days of admission, and / or when the target is readmitted within 30 days of discharge, the condition of the target is deteriorating.

[0027] In one embodiment, the prediction of the risk that the condition of the target deteriorates is the prediction of the risk that the antibiotic treatment of the target is intensified.

[0028] In one embodiment, the prediction of the risk that the condition of the target deteriorates is the prediction of the risk that the target is admitted to the ICU. Therefore, it is evaluated whether there is a risk that the target is admitted to the ICU.

[0029] <0​​​​In yet another embodiment, the prediction of the risk of the subject's condition deteriorating is the prediction of the subject's risk of death within 30 days of hospitalization. Therefore, it is assessed whether the subject is at risk of death within 30 days of hospitalization.

[0031] In yet another embodiment, the prediction of the risk of the subject's condition worsening is the prediction of the subject's risk of readmission within 30 days of discharge. Therefore, it is assessed whether or not the subject is at risk of readmission within 30 days of discharge.

[0032] In yet another embodiment, the prediction of the risk of a subject's condition worsening is the prediction of the risk that the subject will experience organ dysfunction or failure. Organ dysfunction and failure may be assessed, for example, through a SOFA score. Thus, the present invention is further directed to predicting the risk of whether or not a subject's SOFA score will increase (after the test sample has been obtained). An increase in the SOFA score (e.g., at least 1, at least 2, at least 3, or at least 4 points) is considered a deterioration of the condition. In contrast, if the SOFA score does not increase (but the subject does not have the highest SOFA score), the condition is typically not worsening. The prediction window may be the prediction window described above for predicting the risk of developing sepsis.

[0033] Sequential organ failure assessment (SOFA) is a validated score that combines clinical assessments and laboratory measurements to quantitatively describe organ dysfunction / failure. Respiratory, coagulation, hepatic, cardiovascular, central nervous system, and renal dysfunctions are scored individually and then added together to obtain a SOFA score ranging from 0 to 24. Preferably, the SOFA score is determined as described in Vincent 1996 (Vincent et al. Intensive Care Med. 1996 Jul;22(7):707-10. doi:10.1007 / BF01709751. PMID:8844239).

[0034] In yet another embodiment, the prediction of the risk of deterioration of the subject's condition is the prediction of the risk that the subject will require organ support, for example, vasoactive drug therapy, hemodynamic support (e.g., fluid therapy), oxygen supply (e.g., ventilation or by extracorporeal membrane oxygenation), and / or kidney replacement therapy. The prediction window may be the same as the prediction window described above for predicting the risk of developing sepsis, for example, within 24 hours of obtaining the sample.

[0035] In one embodiment, the term "assessment" refers to the diagnosis of sepsis. Therefore, it is determined whether or not a subject suspected of having an infection is suffering from sepsis. Preferably, assessment refers to the early detection of sepsis.

[0036] As will be understood by those skilled in the art, the assessments performed in accordance with the present invention are preferred, but usually not 100% accurate for the subjects investigated. The term "preferred" requires that the statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be determined using various well-known statistical assessment tools, e.g., determining confidence intervals, determining p-values, etc. These can be further easily determined by those skilled in the art using methods such as the Dordent t-test and the Mann-Whitney test. Further details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typical confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. The p-values ​​are typically 0.2, 0.1, and 0.05.

[0037] The term "subject," as used herein, refers to an animal, preferably a mammal, more typically a human. The subjects examined by the method of the present invention are subjects suspected of having an infectious disease. The term "suspected of having an infectious disease," as used herein, means that the subject exhibits the clinical parameters, signs, and / or symptoms of an infectious disease. Thus, the subjects according to the present invention are typically subjects that have an infectious disease or are suspected of having an infectious disease. Typically, the subjects are subjects who visit an emergency department.

[0038] Advantageously, the sample is obtained when the patient appears. Preferably, the sample is obtained when the patient appears in the emergency department. However, the sample may also be obtained when the patient appears in the primary care physician's office.

[0039] The term “sample,” as used herein, refers to any sample containing the first, second, and / or third biomarkers referred herein under physiological conditions. More typically, a sample is a bodily fluid sample, such as a blood sample or a sample derived therefrom, a urine sample, a saliva sample, or a lymph sample. Most typically, the sample is a blood sample or a sample derived from a blood sample. Thus, a sample may be a blood, serum, or plasma sample. A blood sample typically includes a blood sample from a capillary, vein, or artery.

[0040] In one embodiment, the sample is an interstitial fluid sample.

[0041] In one embodiment, the sample is a blood, serum, or plasma sample.

[0042] The term “sepsis” is well known in the art. As used herein, the term refers to life-threatening organ failure caused by a dysregulated host response to infection. A definition of sepsis can be found, for example, in Singer et al. (Sepsis-3 The Third International Consensus Definitions for Sepsis and Septic Shock. JAMA 2016;315:801-819) (which is incorporated herein by reference in full). Preferably, the term “sepsis” refers to sepsis according to the Sepsis-3 definition disclosed in Singer et al. (loc.cit.).

[0043] Typically, the subject under test is one suspected of having an infectious disease. The term “infectious disease” (“infection”) is well understood by those skilled in the art. As used herein, the term “infectious disease” (“infection”) preferably refers to the infiltration, proliferation, and response of the subject's tissues to disease-causing microorganisms. In one embodiment, the infectious disease is a bacterial infection. Therefore, the subject is one suspected of having a bacterial infection.

[0044] As described elsewhere in this specification, the present invention enables the early identification of patients at risk. In one embodiment of the prediction described herein, the subject being tested is therefore not suffering from sepsis at the time the sample is obtained. Particularly preferred embodiment In this context, the subject being tested is preferably not suffering from septic shock at the time the sample is obtained. The term "septic shock" is defined in Singer et al. (loc.cit.). Therefore, the subject is suffering from septic shock if the following criteria are met. ● Sepsis, i.e., suspected / recorded infection, and a change in the total SOFA score of 2 or more points as a result of infection ● Sustained hypotension requiring vasoconstrictors to maintain MAP ≥ 65 mmHg despite adequate resuscitation, and serum lactate level > 2 mmol / L (18 mg / dL).

[0045] Furthermore, it is assumed that the subjects being tested may or may not have had an infection with SARS-CoV-2.

[0046] When used herein, the term “determine” refers to the qualitative and quantitative determination of a biomarker referred to in accordance with the present invention, i.e., the term includes the determination of the presence or absence of the biomarker or the determination of an absolute or relative quantity.

[0047] The term “quantity,” as used herein, refers to the absolute amount, relative amount, or concentration of a compound referred to herein, and any values ​​or parameters that correlate with or can be derived from them. Such values ​​or parameters include intensity signal values ​​derived from all specific physical or chemical properties obtained from the compound by direct measurement, e.g., intensity values ​​in mass spectra or NMR spectra. Furthermore, it includes all values ​​or parameters obtained by indirect measurement as explicitly stated elsewhere herein, e.g., the compound obtained from a specifically bound ligand or the response level determined by a biological readout system in response to the intensity signal. It should be understood that values ​​correlated with the above-mentioned quantities or parameters can also be obtained by all standard mathematical operations. If the biomarker is an enzyme, e.g., alanine aminotransferase (ALAT) or aspartate aminotransferase (AST or ASAT), the term “quantity” may also include the activity of the enzyme.

[0048] The determination of the quantity in the method of the present invention may be carried out by any technique that enables the detection of the presence or absence or quantity of the second molecule in the release from the first molecule. Preferred techniques depend on the properties of the molecules and the characteristics of the biomarkers and will be discussed in more detail elsewhere in this specification.

[0049] Typically, the amount of a biomarker referred to in accordance with the present invention can be determined by an immunoassay using a sandwich, competitive, or other assay format. The assay generates a signal indicating the presence or absence or amount of the biomarker. Further preferred methods include measuring physical or chemical properties specific to the biomarker, such as its exact molecular weight or NMR spectrum. Such methods preferably include analytical instruments such as biosensors, optical devices associated with immunoassays, biochips, mass spectrometers, NMR analyzers, surface plasmon resonance analyzers, or chromatography devices. Furthermore, methods include microplate ELISA-based methods and fully automated or robotic immunoassays (e.g., available from Roche). Preferred measurement methods according to the present invention may also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lantanide fluoroimmunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, turbidimetric analysis, latex-enhanced turbidimetry or turbidimetric analysis, or solid-phase immunoassay. Further methods known in the art, such as gel electrophoresis, 2D gel electrophoresis, etc. These methods include electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blotting. More typically, techniques specifically intended for determining the biomarkers mentioned herein are described in the accompanying examples below.

[0050] The biomarkers determined according to the present invention are well known in the art. Furthermore, methods for determining the amount of biomarkers are also known. For example, biomarkers can be measured as described in the Examples section (see Example 1). Some of the biomarkers tested are enzymes (e.g., aspartate aminotransferase). The amount of these biomarkers can also be determined by determining the activity of the enzyme in the sample.

[0051] Procalcitonin (PCT) is a peptide precursor of the hormone calcitonin. Therefore, procalcitonin is the inactive propeptide of calcitonin. It consists of 116 amino acids and is produced by parafollicular cells (C cells) of the thyroid gland, as well as neuroendocrine cells of the lungs and intestines. PCT has been widely reported as a useful biochemical marker for distinguishing sepsis from other non-infectious causes of systemic inflammation (Kondo, Y., Umemura, Y., Hayashida, K. et al. J intensive). care(2019)7:22.https: / / doi.org / 10.1186 / s40560-019-0374-4). The amino acid sequence of the marker is well known in the art and is disclosed, for example, in European Patent No. 2320237B1.

[0052] The term “cardiac troponin” typically refers to human cardiac troponin T or cardiac troponin I. The term, however, also encompasses variants of the characteristic troponins described herein, i.e., preferably cardiac troponin I, and more preferably cardiac troponin T. Such variants possess at least the same essential biological and immunological properties as the characteristic cardiac troponins. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred herein, for example, by an ELISA assay using a polyclonal or monoclonal antibody that specifically recognizes the cardiac troponin. Furthermore, variants referred to in accordance with the present invention have different amino acid sequences due to at least one amino acid substitution, deletion, and / or addition, and the amino acid sequence of the variant should still be understood to be identical to the amino acid sequence of the characteristic troponin, preferably at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99%. The variant may be an allele variant or any other species-specific homolog, paralog, or ortholog. Furthermore, variants referred to herein include fragments of characteristic cardiac troponin, or variants of the types described above, insofar as these fragments have the essential immunological and biological properties described above. Preferably, cardiac troponin variants have immunological properties (i.e., epitope composition) equivalent to human troponin T or troponin I. Therefore, the variants are recognizable by the aforementioned means or ligands used to determine the concentration of cardiac troponin. Such fragments may, for example, be degradation products of troponin. Further variants are included due to post-translational modifications such as phosphorylation or myristylation.Preferably, the biological properties of troponin I and its variants are that they can inhibit actomyosin ATPase or inhibit angiogenesis in vivo and in vitro, which can be detected, for example, based on assays described by Moses et al. 1999 PNAS USA 96(6):2645-2650. Preferably, the biological properties of troponin T and its variants are that they form complexes with troponin C and I, bind to calcium ions, or, if present, troponin C, I and T. The ability to bind to tropomyosin as a complex or as a complex formed by variants of troponin C, troponin I, and troponin T. Troponin T or troponin I can be determined by immunoassays that are well known and commercially available in the art, such as ELISA. Particularly preferred according to the present invention is the determination of troponin T with high sensitivity, for example, by using the commercially available hs-cTn assay.

[0053] Aspartate aminotransferase (AST or ASAT) catalyzes the amino group transfer from L-aspartate to α-ketoglutarate, forming L-glutamic acid and oxaloacetate. The resulting oxaloacetate is reduced to malate by malate dehydrogenase (MDH) in conjunction with the simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance over time resulting from the conversion of NADH to NAD is directly proportional to AST activity and can be measured, for example, using bichromatic rate (340, 700 nm) techniques.

[0054] Alanine aminotransferase (ALAT) catalyzes the amino group transfer of L-alanine to α-ketoglutarate (α-KG), forming L-glutamic acid and pyruvate. The resulting pyruvate is reduced to lactate by lactate dehydrogenase (LDH) in conjunction with the simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance is directly proportional to the alanine aminotransferase activity and can be measured, for example, using dichroism (340, 700 nm) rate techniques.

[0055] sTREM-1, or soluble TREM1 (or STREM1), is the soluble form of TREM-1 (Triggering Receptor Expressed on Myeloid Cells-1). Therefore, the term refers to the non-cell-bound form of TREM-1. TREM-1 is an immune receptor known to be expressed on neutrophils and monocytes / macrophages. It is a recently discovered member of the immunoglobulin superfamily involved in innate immune responses. TREM-1 is a monomeric protein of approximately 30 kDa synthesized as a 234 amino acid precursor with a 16-amino acid signal peptide, a 184-amino acid extracellular domain, a 29-amino acid transmembrane domain, and a 5-amino acid short cytoplasmic domain. During infection, receptor expression changes and sTREM-1 is released. sTREM-1 (17 kDa) is therefore the soluble form of TREM-1 that is shed from the membrane of activated phagocytic cells. Typically, the term "sTREM-1" encompasses all naturally occurring cleaved or released forms of TREM-1 that have at least an extracellular component.

[0056] The marker "bilirubin" is well known in the art. Bilirubin is a member of the class of viradienes, which are linear tetrapyrroles, and the dipyrrole units are of both exovinyl and endovinyl types. It is a product of heme degradation, produced in the reticuloendothelial system by reduction of biliverdin, and transported to the liver as a complex with serum albumin. It has an antioxidant role. Bilirubin measurement is routinely performed in most medical laboratories and can be measured by various methods (e.g., the methods described in the Examples section).

[0057] The biomarker "haptoglobin" (abbreviated as HAPT) is well known in the art. In humans, this protein is encoded by the HP gene. Haptoglobin captures free plasma hemoglobin and, in combination with it, enables heme iron recycling in the liver and prevents kidney damage. Haptoglobin also acts as an antioxidant, has antibacterial activity, and plays a role in the modulation of many aspects of the acute response. Information regarding the sequence of human haptoglobin polypeptide can be accessed via Uniprot. (See UniProtKB-P00738(HPT_HUMAN)).

[0058] As used herein, the term “BNP-type peptide” preferably includes pre-proBNP, proBNP, NT-proBNP, and BNP. More preferably, the BNP-type peptide is NT-proBNP or BNP. Most preferably, the BNP-type peptide is NT-proBNP. The pre-propeptide (134 amino acids in the case of pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release a propeptide (108 amino acids in the case of proBNP). The propeptide is further cleaved into an N-terminal propeptide (NT-propeptide, 76 amino acids in the case of NT-proBNP) and an active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptides according to the present invention are NT-proBNP, BNP, and their variants. BNP (brain natriuretic peptide) is an active hormone and has a shorter half-life than its inactive counterpart, NT-proBNP.

[0059] The biomarker heparin-binding protein (abbreviated as HBP), also known as Catationic Antimicrobial Protein of 37kDa (CAP37) or azulocidine, is a 37kDa glycoprotein synthesized in neutrophils. Furthermore, it is known to be a heparin- and neutrophil granule-derived antimicrobial, as well as a monocyte and fibroblast-specific chemotactic glycoprotein. HBP belongs to the serine protease superfamily; however, it is inactive as a protease. The amino acid sequence of human HBP can be accessed via UniProt (see UniProtKB-P20160(CAP7_HUMAN)).

[0060] The biomarker endothelial cell-specific molecule 1 (abbreviated as ESM-1) is well known in the art. Biomarkers are often also called endocans. ESM-1 is a secreted protein and is mainly expressed in endothelial cells of human lung and kidney tissue. Public domain data suggest that it is expressed not only in the thyroid, lung, and kidney, but also in cardiac tissue. See, for example, the entry for ESM-1 in the Protein Atlas database (Uhlen M. et al., Science 2015;347(6220):1260419). The expression of this gene is regulated by cytokines. ESM-1 is a proteoglycan composed of a 20 kDa mature polypeptide and a 30 kDa O-linked glycan chain (Bechard D et al., J Biol Chem 2001;276(51):48341-48349). In a preferred embodiment of the present invention, the amount of human ESM-1 polypeptide is measured in a sample from a subject. The sequence of the human ESM-1 polypeptide is well known in the art (see, for example, Lassale P. et al., J. Biol. Chem. 1996;271:20458-20464), and can be assessed via the Uniprot database, for example, see entry Q9NQ30(ESM1_HUMAN). Two isoforms of ESM-1 are produced by alternative splicing: isoform 1 (having Uniprot identifier Q9NQ30-1) and isoform 2 (having Uniprot identifier Q9NQ30-2). Isoform 1 is 184 amino acids long. Isoform 2 is missing amino acids 101 to 150 of isoform 1. Amino acids 1 to 19 form a signal peptide (which can be cleaved).

[0061] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, i.e., isoform 1 has the sequence shown under UniProt acceptance number Q9NQ30-1.

[0062] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined Therefore, isoform 2 has the sequence shown under UniProt accession number Q9NQ30-2.

[0063] In another preferred embodiment, the amounts of isoform-1 and isoform-2 of the ESM-1 polypeptide, i.e., the total ESM-1, are determined.

[0064] The marker "creatinine" is well known in the art. In muscle metabolism, creatinine is endogenously synthesized from creatine and creatine phosphate. Under conditions of normal renal function, creatinine is excreted by glomerular filtration. Creatinine determination is performed for the diagnosis and monitoring of acute and chronic kidney disease, as well as for monitoring renal dialysis. Urinary creatinine concentration can be used as a reference value for the excretion of certain analytes (albumin, α-amylase). Creatinine can be determined as described by Popper et al. (Popper H et al. Biochem Z 1937;291:354), Seelig and Wust (Seelig HP, Wust H. Arztl Labor 1969;15:34), or Bartels (Bartels H et al. Clin Chim Acta 1972;37:193). For example, sodium hydroxide and picric acid are added to the sample to initiate the formation of a creatinine-picric acid complex. In an alkaline solution, creatinine forms a yellow-orange complex with picric acid. The color intensity is directly proportional to the creatinine concentration and can be measured by photometric analysis.

[0065] In the method according to the present invention, a third biomarker may be determined. In particular, in step (b) of the method according to the present invention, (i) If the amount of cardiac troponin is determined as a second biomarker, the method further comprises determining the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If the amount of creatinine is determined as a second biomarker, the method further comprises determining the amount of aspartate aminotransferase or bilirubin as a third biomarker, or (iii) If the amount of sTREM1 is determined to be a second biomarker, the method further comprises determining the amount of aspartate aminotransferase or HBP as a third biomarker.

[0066] Therefore, the present invention relates to the determination of at least two biomarkers (i.e., the first and second biomarkers referred to herein) or at least three biomarkers (i.e., the first, second, and third biomarkers referred to herein).

[0067] The first biomarker is PCT. The second biomarker is selected from cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase.

[0068] In one embodiment, the second biomarker is cardiac troponin, for example, cardiac troponin T or I, preferably troponin T.

[0069] In an alternative embodiment, the second biomarker is a BNP-type peptide, such as NT-proBNP or BNP, preferably NT-proBNP.

[0070] In an alternative embodiment, the second biomarker is creatinine.

[0071] In an alternative embodiment, the second biomarker is ESM-1.

[0072] In an alternative embodiment, the second biomarker is haptoglobin.

[0073] In an alternative embodiment, the second biomarker is sTREM1.

[0074] In an alternative embodiment, the second biomarker is HBP (heparin-binding protein).

[0075] In an alternative embodiment, the second biomarker is aspartate aminotransferase (ASAT).

[0076] If cardiac troponin is the second marker, the method may further include determining the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker.

[0077] In one embodiment, PCT, cardiac troponin, and bilirubin are determined.

[0078] In alternative embodiments, PCT, cardiac troponin, and aspartate aminotransferase are determined.

[0079] In an alternative embodiment, PCT, cardiac troponin, and alanine aminotransferase are determined.

[0080] In an alternative embodiment, PCT, cardiac troponin, and haptoglobin are determined.

[0081] In an alternative embodiment, PCT, cardiac troponin, and ESM1 are determined.

[0082] In an alternative embodiment, PCT, cardiac troponin, and creatinine are determined.

[0083] If the amount of creatinine is determined as a second biomarker, the method may further include determining the amount of aspartate aminotransferase (ASAT) or bilirubin as a third biomarker. Thus, PCT, creatinine, and ASAT are determined. Alternatively, PCT, creatinine, and bilirubin are determined.

[0084] If the amount of sTREM1 is determined as a second biomarker, the method may further include determining the amount of aspartate aminotransferase (ASAT) or HBP as a third biomarker. Thus, PCT, sTREM1, and ASAT are determined. Alternatively, PCT, sTREM1, and HBP are determined.

[0085] It should be understood that the present invention is not limited to the markers described above. Rather, the present invention may encompass the determination of additional markers.

[0086] The term "criteria," as used herein, refers to whether or not a person is suffering from a disease or condition. This refers to a quantity or value that enables the allocation of subjects to either a group of subjects who have the disease or condition, or are at risk of developing it, or a group of subjects who do not have the disease or condition, or are not at risk of developing it. Such a criterion can be a threshold quantity that separates these groups from one another. Thus, the criterion is a quantity or score that enables the allocation of subjects to either a group of subjects who have the disease or condition, or are at risk of developing it, or a group of subjects who do not. For example, the criterion is a quantity or score that enables the allocation of subjects to either a group of subjects who are at risk of developing sepsis (within the prediction window described above, e.g., within about 48 hours), or a group of subjects who are not at risk of developing sepsis.

[0087] A suitable threshold quantity for separating the two groups can be more readily calculated by statistical tests, as referred elsewhere herein, based on the amount of biomarkers from either a subject or group of subjects known to have the disease or condition, or to be at risk of developing it, or from a subject or group of subjects known not to have the disease or condition, or not to be at risk of developing it. Applicable reference quantities for individual subjects may vary depending on various physiological parameters, such as age, sex, or subpopulation.

[0088] Typically, the criteria are criteria for each biomarker derived from at least one subject known to be at risk of developing sepsis, preferably such that the amount of each biomarker is essentially the same as or similar to the corresponding criterion indicates that the subject is at risk of developing sepsis, and such that the amount of each biomarker is different from the corresponding criterion indicates that the subject is not at risk of developing sepsis.

[0089] Typically, the criteria are also criteria for each biomarker derived from at least one subject known to be at no risk of developing sepsis, preferably such that the amount of each biomarker is essentially the same as or similar to the corresponding criterion indicates that the subject is at no risk of developing sepsis, and such that the amount of each biomarker is different from the corresponding criterion indicates that the subject is at risk of developing sepsis.

[0090] The term "at least one object" refers to one object or more than one object, for example, at least 10, 50, 100, 200, or 1000 objects.

[0091] In one embodiment, a biomarker amount greater than the baseline indicates that the subject is at risk (for example, developing sepsis). Furthermore, a biomarker amount lower than the baseline indicates that the subject is not at risk (with the exception of haptoglobin, for which a biomarker amount lower than the baseline indicates that the subject is at risk, while a biomarker amount greater than the baseline indicates that the subject is not at risk).

[0092] Reference doses can, in principle, be calculated for a cohort of subjects based on the mean or average value of a given parameter, such as the biomarker level, by applying standard statistical methods. In particular, the accuracy of tests, such as methods intended for diagnosing events, is best described by receiver operating characteristics (ROC) (see, in particular, Zweig 1993, Clin. Chem. 39:561-577). An ROC graph is a plot of all sensitivity / specificity pairs resulting from continuously varying the decision threshold across the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to accurately assign subjects to a particular prognosis or diagnosis. An ROC plot plots sensitivity versus 1-specificity over the entire range of thresholds suitable for making distinctions. This shows the overlap between the two distributions. The y-axis represents sensitivity, or true positive rate, defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. This is also called positivity in the presence of disease or symptoms. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. The x-axis is an index of specificity and is calculated only from the unaffected subgroup. Since the true positive rate and false positive rate are calculated entirely separately by using test results from two different subgroups, the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / 1-specificity pair corresponding to a specific decision threshold. A fully distinguishable test (where the two distributions of results do not overlap) will have an ROC plot that passes through the upper left corner, with a true positive rate of 1.0 or 100% (perfect sensitivity) and a false positive rate of 0 (perfect specificity). The theoretical plot for an indistinguishable test (where the distributions of results for the two groups are identical) will be a 45° diagonal from the lower left corner to the upper right corner. Most plots will fall between these two extremes. If an ROC plot falls completely below the 45° diagonal, this can be easily corrected by swapping the criteria for "positive rate" from "higher" to "lower," and vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold can be derived from the ROC curve, which allows for diagnosis or prediction of a given event with an appropriate equilibrium of sensitivity and specificity, respectively. Therefore, the criteria used in the above-described method of the present invention, namely the thresholds that enable the distinction between risk and no risk, can usually be generated by establishing the ROC of the cohort as described above and deriving threshold values ​​therefrom. Depending on the desired sensitivity and specificity for the diagnostic method, the ROC plot can derive appropriate thresholds.It is understood that optimal sensitivity is desirable for excluding subjects who are at increased risk or have the disease (i.e., rule out), while optimal specificity is assumed for subjects who are assessed as being at increased risk or have the disease (i.e., rule in).

[0093] Step c) of the method of the present invention includes comparing the amount of a biomarker (i.e., a first biomarker, a second biomarker, and optionally a third biomarker) to a standard for the biomarker and / or calculating a score for rating a subject suspected of having an infectious disease based on the amount of the biomarker.

[0094] Therefore, the amounts of the first biomarker, the second biomarker, and optionally the third biomarker may be compared to the baseline for the first biomarker, the baseline for the second biomarker, and optionally the baseline for the third biomarker.

[0095] Alternatively, the score may be calculated based on the amounts of biomarkers, i.e., based on the amounts of a first biomarker, a second biomarker, and optionally a third biomarker. The score makes it possible to assess individuals suspected of having an infection, for example, to predict the risk of developing sepsis. Optionally, the score may be compared to a preferred baseline score.

[0096] The term “compare,” as used herein, encompasses comparing a determined amount of a biomarker referred to herein with a reference. It should be understood that, as used herein, comparison refers to any type of comparison performed between a value and a reference for a quantity. However, it should be understood that, preferably, values ​​of the same type are compared with one another, for example, if an absolute quantity is determined and compared in the method of the present invention, the reference is also an absolute quantity, and if a relative quantity is determined and compared in the method of the present invention, the reference is also a relative quantity. Alternatively, the term “compare” When used herein, this includes comparing the calculated score with a suitable baseline score. The comparison can be performed manually or with computer assistance. The values ​​of the quantity and baseline can be compared with each other, for example, and the comparison can be performed automatically by a computer program that executes an algorithm for comparison. The computer program that performs the evaluation provides the desired evaluation in an appropriate output format.

[0097] As described above, it is also conceivable to calculate a score (particularly a single score) based on the amounts of the first and second biomarkers, or the first, second, or third biomarkers, i.e., a single score, and to compare this score with a baseline score. Preferably, the score is based on the amounts of the first, second, and third biomarkers in the sample from the test subject, if the amount of the third biomarker is determined based on the amounts of the first and second biomarkers in the sample from the test subject.

[0098] The calculated score combines information about the quantities of at least two or three biomarkers. Furthermore, in the score, the biomarkers are preferably weighted according to their contribution to the establishment of the rating. Thus, the values ​​for individual markers are typically weighted, and the weighted values ​​are used to calculate the score. Preferred coefficients (weights) can be determined even more readily by those skilled in the art. The score can also be calculated from a decision tree or a set of decision trees (ensemble) trained on at least two biomarkers. Depending on the combination of biomarkers applied in the method of the present invention, the structure of the decision tree may differ, in addition to the weights of the individual biomarkers.

[0099] The score can be considered a classifier parameter for rating subjects as described herein. In particular, it allows for the provision of a rating based on a single score. The reference score is preferably a value, in particular a cutoff value that allows for the rating of subjects suspected of having an infection as described herein. Preferably, the reference is a single value. Thus, it is not necessary to interpret all information regarding the amount of individual biomarkers. Using the scoring system described herein, advantageously, values ​​of different dimensions or units may be used for biomarkers, for the reason that the values ​​are mathematically converted into a score. Thus, for example, a value for absolute concentration may be combined with a peak area ratio in the score. The reference score to be applied may be selected based on the desired sensitivity or desired specificity. Methods for selecting a suitable reference score are well known in the art.

[0100] Advantageously, combinations of the first biomarker with a second, and preferably a third, biomarker have been found in the studies forming the basis of this invention to enable reliable and early assessment of patients presenting with signs and symptoms of infection. For example, assessment of a subject can be performed within 5 hours of obtaining the test sample. In the studies, patients presenting to the emergency department, which is a medical (non-surgical) emergency, were examined. For this purpose, patients were further divided into those with a high probability of having sepsis and those suspected of having an infection without sepsis. The amounts of various biomarkers were determined, and the biomarkers were analyzed and mathematically combined via logistic regression analysis. The area under the receiver operating characteristic curve (AUC) was used to evaluate biomarker performance. The AUC value is the mathematical integral of the function f(x) within the interval [a][b]. AUC was also examined for biomarker pairs and triplets. Together, biomarker combinations showing improvement in AUC compared to the best single biomarker AUC were identified. The results are described in the accompanying examples below.

[0101] In particular, if these patients appear, for example, in the emergency room, early assessment of the risk of developing severe complications, such as sepsis, SIRS, or a general deterioration of overall health, is important for drug use. It is crucial to initiate therapeutic measures, including administration, physical or other therapeutic interventions, and / or hospitalization. These therapeutic measures may include, in particular, rapid administration of broad-spectrum antibiotics, fluid resuscitation, vasoactive drug therapy, mechanical ventilation, and other organ support (e.g., continuous hemofiltration, extracorporeal membrane oxygenation). Also included as therapeutic measures is triage to a higher level of care (e.g., intensive care unit, intermediate care unit). If there is no risk of severe complications, the patient may be discharged and managed in an outpatient setting, or admitted to a lower level of care in the hospital (e.g., general ward). Thanks to the present invention, patients can be assessed at an early stage by biomarker assessment, thus preventing life-threatening onsets. The biomarker pairs and triplets identified in the research underlying the present invention provide a reliable basis for medical decisions, and assessment can be performed in a time- and cost-effective manner.

[0102] Therefore, the method of the present invention may further include recommending or initiating appropriate therapeutic measures. Typically, such appropriate therapeutic measures are selected from medical guidelines or recommendations for the management of sepsis, such as the International Guidelines for Management of Sepsis and Septic Shock (Intensive Care Med, 2017). For example, the therapeutic measures may be treatment of sepsis, or further diagnostic investigation, or other forms of care that the expert deems necessary.

[0103] In one embodiment, the treatment measures recommended or initiated when a patient is assessed as being at risk are selected from the following: ● Administration of empirical broad-spectrum therapy, typically based on the most likely pathogen and antibiotic susceptibility, such as cephalosporins, beta-lactam / beta-lactamase inhibitors (e.g., piperacillin), or at least one broad-spectrum antibiotic such as a carbapenem. ● Resuscitation with intravenous fluids ● Administration of one or more vasoconstrictors, such as norepinephrine, and ● Administration of one or more corticosteroids, such as hydrocortisone.

[0104] The definitions used herein above apply below, with any necessary modifications.

[0105] The present invention also provides a computer implementation method for evaluating a subject suspected of having an infectious disease, (a) A step of receiving a value for the amount of a first biomarker in a sample of interest, wherein the first biomarker is PCT; (b) A step of receiving a value for the amount of a second biomarker in a sample of interest, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing a value for the amount of a biomarker with a standard for the biomarker and / or the step of calculating a score for assessing a subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). This includes methods.

[0106] When used herein, the term "computer implementation" means a method that is typically contained within a computer or similar data processing device, on a data processing unit. This means that it is performed in an automated manner. The data processing unit receives a value for the amount of the biomarker. Such a value may be a quantity that reflects the amount described in detail elsewhere in this specification, a relative quantity, or any other calculated value. It should be understood that the method described above does not require the determination of the amount of the biomarker, but rather uses a value for an already predetermined quantity.

[0107] Typically, in step (b) of the method, (i) If a value for the amount of cardiac troponin is received as a second biomarker, the method further includes receiving a value for the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If a value for the amount of creatinine is received as a second biomarker, the method further includes receiving a value for the amount of aspartate aminotransferase or bilirubin as a third biomarker, or (iii) If a value for the amount of sTREM1 is received as a second biomarker, the method further includes receiving a value for the amount of aspartate aminotransferase or HBP as a third biomarker.

[0108] The present invention also, in principle, envisions a computer program, a computer program product, or a computer-readable storage medium in which the computer program is tangibly incorporated, the computer program including instructions that, when executed on a data processing device or computer, perform the methods of the present invention as described above. Specifically, this disclosure further encompasses: - A computer or computer network comprising at least one processor, the processor configured to perform a method according to one of the embodiments described herein. - A computer-loadable data structure configured to perform a method according to one of the embodiments described herein when running on a computer, - A computer script, wherein the computer program is adapted to perform one of the embodiments described herein while the program is running on a computer. - A computer program comprising programming means for performing a method according to one of the embodiments described herein when the computer program is running on a computer or on a computer network, - A computer program comprising the program means described in a prior embodiment stored on a storage medium readable by a computer, - A data structure is stored in a storage medium, and after the data structure is loaded into the main memory and / or working memory of a computer or computer network, the storage medium is adapted to perform the method according to one of the embodiments described herein. - A computer program product having program code means that can be stored or stored on a storage medium in order to perform a method according to one of the embodiments described in this specification when the program code means is executed on a computer or computer network. - A data stream signal, typically encrypted, including data about parameters defined elsewhere in this specification, and - A data stream signal, typically encrypted, including an evaluation provided by the method of the present invention.

[0109] The present invention is a device for evaluating a subject suspected of having an infectious disease, (a) The first biomarker, which is PCT, and cardiac troponin, in the sample of the subject. A measurement unit for determining the amount of a second biomarker selected from the group consisting of reatinin, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, comprising a detection system for the first biomarker and the second biomarker; and (b) An evaluation unit operably connected to a measurement unit, preferably including a database having stored references for a first biomarker and a second biomarker as defined above, and preferably including a data processor including instructions for performing a comparison of the amounts of the first biomarker and the second biomarker with references and / or for performing a score for rating subjects suspected of having an infection based on the amounts of biomarkers, and for rating said subjects based on the comparison, and capable of automatically receiving values ​​for the amounts of biomarkers from the measurement unit. This includes devices.

[0110] When used herein, the term "device" refers to a system comprising the aforementioned units functionally linked together to enable the determination and evaluation of the amount of a biomarker by the method of the present invention, so that an assessment may be provided.

[0111] The analytical unit typically includes at least one reaction zone having biomarker detectors for first and second biomarkers and, preferably, a third biomarker, in a form immobilized on a solid support or carrier that comes into contact with the sample. Furthermore, conditions can be applied in the reaction zone to enable the specific binding of the detector to the biomarkers contained in the sample.

[0112] The reaction zone may allow direct sample application, or it may be connected to a loading zone to which the sample is applied. In the latter case, the sample may be actively or passively transported to the reaction zone via a connection between the loading zone and the reaction zone. Furthermore, the reaction zone may also be connected to a detector. The connection may allow the detector to detect the binding of biomarkers to their detection agents. The preferred connection depends on the technique used to measure the presence or quantity of the biomarkers. For example, for optical detection, the propagation of light may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode.

[0113] The detector is adapted to detect the determination of the amount of a biomarker. The determined amount can then be transmitted to an evaluation unit. The evaluation unit includes a data processing element, such as a computer, which implements an algorithm for determining the amount present in the sample.

[0114] The processing units referred to in accordance with the methods of the present invention typically comprise a central processing unit (CPU) and / or one or more graphics processing units (GPUs) and / or one or more application-specific integrated circuits (ASICs) and / or one or more tensor processing units (TPUs) and / or one or more field-programmable gate arrays (FPGAs), etc. The data processing elements may be, for example, general-purpose computers or portable computing devices. It should also be understood that multiple computing devices may be used together, such as over a network or by other means of transferring data, to perform one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), cellular devices, smart or mobile devices, tablet computers, and servers. Generally, the data processing elements execute multiple instructions (e.g., software programs). Includes a processor capable of doing so.

[0115] An evaluation unit typically includes memory or has access to memory. Memory is a computer-readable medium and may include, for example, a single or multiple storage devices located locally with the computing device or accessible to the computing device via a network. The computer-readable medium may be any available medium accessible by the computing device and may include both volatile and non-volatile media. Furthermore, the computer-readable medium may be either removable or non-removable media, or both. For example, non-limitingly, the computer-readable medium may include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory, or any other memory technology; CD-ROM, digital versatile disk (DVD), or other optical disk storage; magnetic cassette, magnetic tape, magnetic disk storage, or other magnetic storage devices; or any other medium accessible by the computing device and that can be used to store multiple instructions executable by the computing device's processor.

[0116] According to embodiments of this disclosure, the software may include instructions that, when executed by the processor of a computing device, can perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to generate signals that control the operation of other machines, and thus may be actuated through those control signals to transform materials far away from the computer itself. These descriptions and expressions are means used by those skilled in the data processing technology to communicate, for example, the contents of the research to others skilled in the art in the most effective way.

[0117] Multiple instructions may also include algorithms that are generally considered to be a sequence of coherent steps leading to a desired result. These steps require the physical manipulation of physical quantities. While not always the case, these quantities typically take the form of electrical or magnetic pulses or signals that can be stored, transmitted, transformed, combined, compared, and otherwise manipulated. Primarily for reasons of general use, it may be convenient to refer to these signals as values, features, representation data, or numbers, etc., as a reference to the physical item or manifestation to which such signals are embodied or represented. However, it should be noted that all these and similar terms are used herein simply as convenient labels that are associated with and applicable to the appropriate physical quantities.

[0118] The evaluation unit may also include an output device or have access to an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a file. According to some embodiments of this disclosure, a computing device may perform one or more steps of a method disclosed herein and subsequently provide an output relating to the results of the method, an index, a ratio, or other factor via the output device.

[0119] Typically, the measurement unit includes a system for determining a third biomarker and for detecting the third biomarker, the database includes stored criteria for the third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) If sTREM1 is the second biomarker, it is either aspartate aminotransferase or HBP.

[0120] More typically, the detection system includes at least one detection agent that can specifically detect each of the biomarkers.

[0121] The present invention further envisions a device for evaluating a subject suspected of having an infectious disease, comprising an evaluation unit, the evaluation unit comprising a database having stored criteria for a first biomarker which is PCT and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, and preferably a data processor which includes instructions for performing a comparison between the amounts of the first and second biomarkers and the criteria, as defined above, and for evaluating the subject based on the comparison, wherein the evaluation unit can receive values ​​for the amounts of biomarkers determined in a sample of the subject.

[0122] Typically, the database includes stored criteria for a third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) If sTREM1 is the second biomarker, it is either aspartate aminotransferase or HBP.

[0123] The present invention also relates in principle to the use of a first biomarker, which is PCT, and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, or to the use of a detection agent that specifically binds to the first biomarker and a detection agent that specifically binds to the second biomarker, for assessing a subject suspected of having an infectious disease.

[0124] The term “detector,” as used herein, refers to any agent that specifically binds to a biomarker, i.e., an agent that does not cross-react with other components present in the sample. Typically, a biomarker-specifically binding detector as referred herein may be an antibody, an antibody fragment or derivative, an aptamer, a ligand for a biomarker, a receptor for a biomarker, an enzyme known to bind to and / or convert a biomarker, or a small molecule known to specifically bind to a biomarker. For example, antibodies referred herein as detectors include both polyclonal and monoclonal antibodies, as well as their fragments, such as Fv, Fab, and F(ab)2 fragments that can bind to an antigen or hapten. The present invention also includes single-chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence typically includes at least antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally valid amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. For example, Apta The biomarker detection agent may be a nucleic acid or a peptide aptamer. Methods for preparing such aptamers are well known in the art. For example, random mutations can be introduced into the underlying nucleic acid or peptide for the aptamer. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Specific binding of the detection agent means that it does not substantially bind to, i.e., does not cross-react with, any other peptide, polypeptide, or substance present in the sample being analyzed. Preferably, the specifically bound biomarker should bind with an affinity at least 3 times higher, more preferably at least 10 times higher, and even more preferably at least 50 times higher than any other component of the sample. Non-specific binding may be acceptable if it can still be clearly distinguished and measured, for example, according to its size on a Western blot or by its relatively higher abundance in the sample.

[0125] The detection agent may be permanently or reversibly fused or ligated to the detectable label. Suitable labels are well known to those skilled in the art. A suitable detectable label is any label detectable by a suitable detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g., magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Examples of enzymatically active labels include horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitrobluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, available as a ready-made storage solution from Roche Diagnostics), CDP-Star® (Amersham Biosciences), and ECF® (Amersham Biosciences). Appropriate enzyme-substrate combinations may produce colored reaction products, fluorescence, or chemiluminescence, which can be measured by methods known in the art (e.g., using photosensitive film or a suitable camera system). The same criteria apply to measuring enzymatic reactions. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). Furthermore, the use of quantum dots as fluorescent labels is also considered. Typical radioactive labels include 35S, 125I, 32P, and 33P. Radioactive labels can be detected by any known and appropriate method, such as a photosensitive film or a phosphor imager.Suitable labels may also include tags such as biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose-binding protein, etc., or may contain these.

[0126] Preferred detection agents for biomarkers, such as AST, ALT, and creatinine, are described, for example, in the examples (see, for example, Example 1).

[0127] If the biomarker is an enzyme, such as AST or ALT, the detection agent may be a substrate for the enzyme or any agent used for detection (see Examples).

[0128] In one embodiment, the detection agent for ALT (ALAT) is, for example, L-alanine.

[0129] In one embodiment, the detection agent for AST (ASAT) is, for example, L-aspartate.

[0130] The detection agent for creatinine is, for example, creatininase, or any agent used for detection (see Examples).

[0131] The detection agent for bilirubin is, for example, sodium nitrite and sulfanilic acid, or any agent used for detection (see Examples).

[0132] The determination of biomarkers described herein may include mass spectrometry (MS) performed after a separation step (e.g., by LC or HPLC). When used herein, mass spectrometry encompasses all techniques that enable the determination of the molecular weight (i.e., mass) or the mass variable corresponding to the biomarker, i.e., the compound to be determined according to the present invention. Preferably, when used herein, mass spectrometry relates to GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry, e.g., MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF, or any combination of approaches using the techniques described above. Methods of applying these techniques are well known to those skilled in the art. Furthermore, suitable devices are commercially available. More preferably, when used herein, mass spectrometry relates to LC-MS and / or HPLC-MS, i.e., mass spectrometry functionally coupled to a preceding liquid chromatography separation step. Preferably, mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS, involves two or more mass spectrometry steps, with fragmentation occurring between stages. In tandem mass spectrometry, two consecutive mass spectrometers are connected by a collision cell. The mass spectrometers are connected to a chromatography device. The sample separated by chromatography is sorted and weighed in the first mass spectrometer, then fragmented with an inert gas in the collision cell, and one or more fragments are sorted and weighed in the second mass spectrometer. The fragments are sorted and weighed in the second mass spectrometer. Identification by MS / MS is more accurate.

[0133] In one embodiment, mass spectrometry, as used herein, includes quadrupole MS. Most preferably, the quadrupole MS is performed as follows: a) selection of the mass / charge ratio (m / z) of ions produced by ionization at a first analytical quadrupole of the mass spectrometer; b) fragmentation of the ions selected in step a) by applying an accelerating voltage to an additional subsequent quadrupole filled with collision gas and acting as a collision chamber; c) selection of the mass / charge ratio of ions produced by the fragmentation process in step b) at an additional subsequent quadrupole, wherein steps a) to c) of the method are performed at least once, and analysis of the mass / charge ratio of all ions present in the mixture of substances as a result of the ionization process is performed, the quadrupole is filled with collision gas, but no accelerating voltage is applied during the analysis. Further details regarding the most preferred mass spectrometry used in accordance with the present invention can be found in International Publication No. 2003 / 073464.

[0134] More preferably, the mass spectrometry is liquid chromatography (LC) MS, for example, high-performance liquid chromatography (HPLC) MS, particularly HPLC-MS / MS. When used herein, liquid chromatography refers to all techniques that enable the separation of compounds (i.e., metabolites) in a liquid or supercritical phase.

[0135] For mass spectrometry, analytes in a sample are ionized to generate charged molecules or molecular fragments. The mass-charge of the ionized analytes, particularly the ionized biomarkers, or their fragments, is then measured. Prior to ionization, the sample may be subjected to cleavage with a protease, such as trypsin. The protease cleaves the protein biomarker into smaller fragments.

[0136] Therefore, the mass spectrometry step preferably includes an ionization step in which the biomarker to be determined is ionized. Naturally, other compounds present in the sample / eluate are also ionized. Ionization of the biomarker can be carried out by any method deemed appropriate, in particular by electron impulse ionization, fast atomic collision, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI).

[0137] In a preferred embodiment, the ionization step (for mass spectrometry) is carried out by electrospray ionization (ESI). Therefore, mass spectrometry is preferably ESI-MS (or ESI-MS / MS if tandem MS is performed). Electrospray is a soft ionization method that results in ion formation without breaking any chemical bonds.

[0138] More typically, a third biomarker or a detection agent that specifically binds to the third biomarker is additionally used, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) If sTREM1 is the second biomarker, it is either aspartate aminotransferase or HBP.

[0139] The present invention also relates to a kit for assessing a subject suspected of having an infection, comprising a detection agent that specifically binds to a first biomarker which is PCT, and a detection agent that specifically binds to a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase.

[0140] As used herein, the term “kit” typically refers to a collection of the components described above, provided separately or in a single container. The container also typically includes instructions for carrying out the methods of the present invention. These instructions may be in the form of a manual, or they may be provided by computer program code that can perform or support the determination of biomarkers referred to in the methods of the present invention when run on a computer or data processing device. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disk), or directly on a computer or data processing device, or it may be provided in a download format, such as a link to an accessible server or cloud. Furthermore, the kit may typically include standards for reference amounts of biomarkers for softening purposes as described in detail elsewhere herein. The kit according to the present invention may also include further components necessary for carrying out the methods of the present invention, such as solvents, buffers, washing solutions, and / or reagents required for the detection of released second molecules. Furthermore, it may include the device of the present invention in part or as a whole.

[0141] More typically, the kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being (i) If cardiac troponin is the second biomarker, bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, Is it ESM1 or creatinine, or (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) If sTREM1 is the second biomarker, it is either aspartate aminotransferase or HBP.

[0142] It should be understood that the definitions and explanations of terms used above apply appropriately to all embodiments described herein and in the appended claims. The following embodiments are specific embodiments envisioned in accordance with the present invention.

[0143] 1. A method for assessing individuals suspected of having an infectious disease, (a) A step of determining the amount of a first biomarker in a sample of interest, wherein the first biomarker is PCT; (b) A step of determining the amount of a second biomarker in a sample of interest, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing the amount of the biomarker to a standard for the biomarker and / or the step of calculating a score for assessing a subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). Methods that include...

[0144] 2. In step (b), (i) If the amount of cardiac troponin is determined as a second biomarker, the method further comprises determining the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If the amount of creatinine is determined as a second biomarker, the method further comprises determining the amount of aspartate aminotransferase or bilirubin as a third biomarker, or (iii) If the amount of sTREM1 is determined to be a second biomarker, the method further comprises determining the amount of aspartate aminotransferase or HBP as a third biomarker, according to Embodiment 1.

[0145] 3. The method of Embodiment 1 or 2, wherein the subjects are patients who visit the emergency department.

[0146] 4. Any one of Embodiments 1 to 3, wherein the assessment is an assessment of the risk of developing sepsis and / or an assessment of the risk of the subject's condition worsening.

[0147] 5. Any one of Embodiments 1 to 4, wherein the criteria are criteria for each biomarker derived from at least one subject known to be at risk of developing sepsis, and preferably, the amount of each biomarker being essentially the same as or similar to the corresponding criterion indicates that the subject is at risk of developing sepsis, and the amount of each biomarker being different from the corresponding criterion indicates that the subject is not at risk of developing sepsis.

[0148] 6. Any one of Embodiments 1 to 4, wherein the criteria are criteria for each biomarker derived from at least one subject known to be free from the risk of developing sepsis, and preferably, the amount of each biomarker being essentially the same as or similar to the corresponding criterion indicates that the subject is free from the risk of developing sepsis, and the amount of each biomarker being different from the corresponding criterion indicates that the subject is at risk of developing sepsis.

[0149] 7. One of the methods of Embodiments 1 to 6, wherein the subject is suffering from an infectious disease or is suspected to be suffering from an infectious disease.

[0150] 8. Any one of Embodiments 1 to 7, wherein the sample is a blood sample or a sample derived from a blood sample.

[0151] 9. Any one of Embodiments 1 to 8, wherein the subject is a human.

[0152] 10. A computer implementation method for assessing a subject suspected of having an infectious disease, (a) A step of receiving a value for the amount of a first biomarker in a sample of interest, wherein the first biomarker is PCT; (b) A step of receiving a value for the amount of a second biomarker in a sample of interest, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing a value for the amount of a biomarker with a standard for the biomarker and / or the step of calculating a score for assessing a subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). Methods that include...

[0153] 11. In step (b), (i) If a value for the amount of cardiac troponin is received as a second biomarker, the method further includes receiving a value for the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If a value for the amount of creatinine is received as a second biomarker, the method further includes receiving a value for the amount of aspartate aminotransferase or bilirubin as a third biomarker, or (iii) The method of Embodiment 10, further comprising receiving a value for the amount of sTREM1 as a second biomarker, wherein the method also includes receiving a value for the amount of aspartate aminotransferase or HBP as a third biomarker.

[0154] 12. A device for assessing individuals suspected of having an infectious disease, (a) A measurement unit for determining the amount of a first biomarker, which is PCT, and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, comprising a detection system for the first and second biomarkers. T; and (b) An evaluation unit operably connected to a measurement unit, preferably including a database having stored references for a first biomarker and a second biomarker, as defined in any one of embodiments 1 to 9, and a data processor including instructions for performing a comparison of the amounts of the first biomarker and the second biomarker with references and / or for performing a score for rating a subject suspected of having an infection based on the amount of the biomarkers, and for rating the subject based on the comparison, and capable of automatically receiving values ​​for the amount of the biomarkers from the measurement unit. A device that includes this.

[0155] 13. The measurement unit includes a detection system for the third biomarker, the database includes stored criteria for the third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) The device of Embodiment 12, wherein sTREM1 is aspartate aminotransferase or HBP when sTREM1 is a second biomarker.

[0156] 14. The device of Embodiment 12 or 13, wherein the detection system includes at least one detection agent capable of specifically detecting each of the biomarkers.

[0157] 15. A device for evaluating a subject suspected of having an infectious disease, comprising an evaluation unit, the evaluation unit comprising a database having stored criteria for a first biomarker which is PCT and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, and preferably a data processor which includes instructions for performing a comparison between the amounts of the first and second biomarkers and the criteria, and for evaluating the subject based on the comparison, as defined in any one of embodiments 1 to 11, wherein the evaluation unit can receive values ​​for the amounts of biomarkers determined in a sample of the subject.

[0158] 16. The database includes stored criteria for a third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) The device of Embodiment 15, wherein sTREM1 is aspartate aminotransferase or HBP when sTREM1 is a second biomarker.

[0159] 17. For assessing subjects suspected of having an infectious disease, i) the primary biomarker being PCT, as well as cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and asparagus ii) a second biomarker selected from the group consisting of ginate aminotransferases, or ii) the use of a detection agent that specifically binds to the first biomarker and a detection agent that specifically binds to the second biomarker.

[0160] 18. A third biomarker or a detection agent that specifically binds to the third biomarker is additionally used, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase, or (iii) Use of Embodiment 17, in which sTREM1 is a second biomarker, which is aspartate aminotransferase or HBP.

[0161] 19. A kit for assessing individuals suspected of having an infectious disease, comprising a detection agent that specifically binds to a first biomarker, which is PCT, and a detection agent that specifically binds to a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase.

[0162] 20. Further comprising a detection agent that specifically binds to a third biomarker, wherein the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) The kit of Embodiment 19, wherein sTREM1 is aspartate aminotransferase or HBP when sTREM1 is a second biomarker.

[0163] All references referenced throughout this specification, in addition to the disclosures specifically mentioned above, are incorporated herein in their entirety. [Examples]

[0164] Example 1: Determination of biomarkers The following is a brief description of the Elecsys® electrochemiluminescence (ECL) technique and assay method for determining cardiac troponin. Cardiac troponin concentrations were determined using a cobas e801 analyzer. Detection of cardiac troponin using the cobas e801 analyzer is based on the Elecsys® electrochemiluminescence (ECL) technique. Briefly, biotin-labeled and ruthenium-labeled antibodies are combined with their respective amounts of undiluted sample and incubated in the analyzer. Subsequently, streptavidin-coated magnetic microparticles are added to promote the binding of the biotin-labeled immunocomplex and incubated in the instrument. After this incubation step, the reaction mixture is transferred to a measurement cell, where beads are magnetically trapped on the electrode surface. Next, ProCell M buffer containing tripropylamine (TPA) for the subsequent ECL reaction is introduced into the measurement cell to separate the bound immunoassay complex from the remaining free particles. Then, the induction of a voltage between the working electrode and the counter electrode initiates a reaction resulting in the emission of photons by the ruthenium complex and TPA. The electrochemiluminescence signals obtained by the photomultiplier tube are recorded and converted into numerical values ​​indicating the concentration levels of each analyte.

[0165] PCT (procalcitonin) was measured using a commercial procalcitonin ECLIA assay, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay includes biotinylated and rutheniumized monoclonal antibodies that specifically bind to PCT. 18 μL from each serum sample was used and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0166] TNTHS or cTNThs (cardiac troponin T) was measured using a commercially available, highly sensitive ECLIA assay for cTroponin T, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay includes biotinylated and rutheniumized monoclonal antibodies that specifically bind to cTnThs. 50 μL from each serum sample was used and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0167] Ferritin (FERR) was measured using a commercial ferritin ECLIA assay, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay includes biotinylated and rutheniumized monoclonal antibodies that specifically bind to ferritin. 10 μL of each serum sample was used and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0168] PBNP or NTpBNP (the N-terminal prohormone of brain natriuretic peptide) was measured using a commercial NTproBNP ECLIA assay, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay includes biotinylated and rutheniumized monoclonal antibodies that specifically bind to NTproBNP. 15 μL from each serum sample was used and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0169] ESM1(Endothelial cell-specific molecule 1) was measured using a robust prototype ECLIA assay for ESM-1, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains biotinylated and rutheniumized monoclonal antibodies that specifically bind to ESM-1. 20 μL from each serum sample was used and measured undiluted using a cobas e601 analyzer (Roche Diagnostics, Germany).

[0170] STREM1 or sTREM-1 (Soluble triggering receptor expressed on myeloid cells 1) was measured using a robust prototype ECLIA assay for sTREM-1, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay includes biotinylated and rutheniumized monoclonal antibodies that specifically bind to sTREM-1. 50 μL was used from each serum sample, and the cobas The measurements were taken using an e601 analyzer (Roche Diagnostics, Germany) without dilution.

[0171] CREP2 (Creatinine): This enzymatic method is based on the conversion of creatinine to glycine, formaldehyde, and hydrogen peroxide with the assistance of creatininase, creatinase, and sarcosine oxidase. Hydrogen peroxide, catalyzed and released by peroxidase, reacts with 4-aminophenazone and HTIB a) to form quinone imine chromophores. The color intensity of the formed quinone imine chromophores is directly proportional to the creatinine concentration in the reaction mixture. Assay from Roche Diagnostics (Germany). 1.7 μL of plasma was analyzed. Samples were analyzed using a Cobas C 501 analyzer (Roche). Measured on Diagnostics (Germany).

[0172] KL6 (Sialic acid-added carbohydrate antigen KL-6): The sialic acid-added carbohydrate antigen KL-6 (KL-6) in the sample agglutinates with mouse KL-6 monoclonal antibody-coated latex through an antigen-antibody reaction. The KL-6 level is determined by measuring the change in absorbance caused by this agglutination. The reagent was from Sekisui Medical Co. (Japan). 2.5 μL of plasma was analyzed. The sample was measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0173] LDHI2 (Lactate Dehydrogenase): UV Assay Lactate dehydrogenase catalyzes the conversion of L-lactate to pyruvate; NAD is reduced to NADH in this process. L-lactate + NAD + LDH The initial rate of pyruvate + NADH + H + NADH formation is directly proportional to catalytic LDH activity. This is determined by measuring the increase in absorbance by photometric analysis. Assay from Roche Diagnostics (Germany). 2.2 μL of plasma was analyzed. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0174] AT.pc (Antithrombin Percentage): Kinetic colorimetric test. This test operates according to the principle of the antithrombin (AT) heparin cofactor assay. Heparin and a predetermined amount of thrombin are added in excess to the sample. All free antithrombin present binds to thrombin to form an inactive complex. Uninhibited thrombin releases p-nitroaniline from the chromogenic substrate MeOCO-Gly-Pro-Arg-pNA. The amount of residual thrombin is inversely proportional to the antithrombin content of the sample, and therefore the antithrombin activity can be calculated using the increase in absorbance at a wavelength of 415 nm. Assay from Roche Diagnostics (Germany). 1 μL of plasma was analyzed. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0175] HAPT2 (haptoglobin): Immunoturbidimetric assay for human haptoglobin, which forms a precipitate with a specific antiserum determined by turbidimetric analysis. 3.9 μL of plasma was analyzed. The sample was measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0176] HBP (Heparin-binding protein): HBP determination is based on a turbidimetric reaction between circulating HBP and a single-specific avian polyclonal antibody bound to chloromethyl microparticles under optimal pH conditions in the presence of polyethylene glycol polymer (PEG). The magnitude of the change is proportional to the amount of HBP in the test sample. Reagents from Axis-Shield Diagnostics Ltd. (Scotland). 10 μL of plasma was analyzed. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0177] BILI (bilirubin): Diazotized sulfanilic acid contains sodium nitrite and It is formed by combining sulfanilic acid at a low pH. Bilirubin (unconjugated) in the sample is solubilized by dilution in a mixture of caffeine / benzoate / acetate / EDTA. Upon addition of diazotized sulfanilic acid, the solubilized bilirubin, including conjugated bilirubin (mono and diglucolonide) and delta-2 type (biliprotein-bilirubin covalently bound to albumin), is converted to diazo-bilirubin, which is a red chromophore representing total bilirubin, measured using dichroic (540, 700 nm) endpoint techniques that absorb at 540 nm. Sample blank correction is used.

[0178] CREP2 (Creatinine): This enzymatic method is based on the conversion of creatinine to glycine, formaldehyde, and hydrogen peroxide with the assistance of creatininase, creatinase, and sarcosine oxidase. Hydrogen peroxide, catalyzed and released by peroxidase, reacts with 4-aminophenazone and HTIB a) to form quinone imine chromophores. The color intensity of the formed quinone imine chromophores is directly proportional to the creatinine concentration in the reaction mixture. Assay from Roche Diagnostics (Germany). 1.7 μL of plasma was analyzed. Samples were analyzed using a Cobas C 501 analyzer (Roche). Measured on Diagnostics (Germany).

[0179] ALAT (Alanine Aminotransferase): Alanine aminotransferase catalyzes the amino group transfer of L-alanine to α-ketoglutarate (α-KG), forming L-glutamic acid and pyruvate. The resulting pyruvate is reduced to lactate by lactate dehydrogenase (LDH) in conjunction with the simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance is directly proportional to the alanine aminotransferase activity and can be measured, for example, using dichroism (340, 700 nm) rate techniques.

[0180] ASAT (Aspartate Aminotransferase): Aspartate aminotransferase (AST) catalyzes the amino group transfer from L-aspartate to α-ketoglutarate, forming L-glutamic acid and oxaloacetate. The resulting oxaloacetate is reduced to malate by malate dehydrogenase (MDH) in conjunction with the simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance over time due to the conversion of NADH to NAD is directly proportional to AST activity and can be measured, for example, using bichromatic rate (340, 700 nm) techniques.

[0181] Example 2: Analysis of patients from the TRIAGE Study TRIAGE Study, Kantonsspital Aarau, Switzerland, Emergency Department.(Schuetz 2013,BMC emergency medicine,13(1),12).

[0182] All consecutive patients who sought emergency department (ED) treatment for medical emergencies were included in ED admissions. From a total of 4000 patients, a subset of patients suspected of having an infection at admission were selected and classified into a high-probability sepsis case group or an infection control group according to the following: ● Cases (N=64): Sepsis cases that were admitted to the ICU or met the criteria of Rhee 2017, “Incidence and Trends of Sepsis in US Hospitals Using Clinical vs Claims Data, 2009-2014.” JAMA 318(13):1241-1249, and were likely to have worsened within 48 hours of presenting in the ED / higher severity. ● Control (N=207): Patients suspected of having an infection within 48 hours of presenting with ED, but without sepsis.

[0183] We mathematically combined the markers via logistic regression and used the "Area Under the Receiver Operating Characteristic Curve" (AUC) as a general measure of marker performance.

[0184] Table 1 shows the marker pair combinations (bivariate marker combinations) that show an improvement in AUC for a single marker at least 1 percent point. [Table 1]

[0185] Table 2 shows the marker triplet combinations (trivariate marker combinations) that show an improvement in AUC for at least 1 percent of the bivariate marker pairs, as well as for all three single markers. [Table 2]

[0186] Table 3 shows examples of bivariate combinations of markers that do not show improvement compared to a single marker. [Table 3]

Claims

1. A method for assessing individuals suspected of having an infectious disease, (a) A step of determining the amount of a first biomarker in the target sample, wherein the first biomarker is PCT; (b) A step of determining the amount of a second biomarker in the sample of the subject, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing the amount of the biomarker with a standard for the biomarker and / or the step of calculating a score for evaluating the subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). Methods that include...

2. In step (b), (i) If the amount of cardiac troponin is determined as the second biomarker, the method further comprises determining the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If the amount of creatinine is determined as the second biomarker, the method further comprises determining the amount of aspartate aminotransferase or bilirubin as a third biomarker, (iii) When the amount of sTREM1 is determined as the second biomarker, the method further includes determining the amount of aspartate aminotransferase or HBP as a third biomarker. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the subject is a patient who visits the emergency department.

4. The method according to any one of claims 1 to 3, wherein the assessment is an assessment of the risk of developing sepsis and / or an assessment of the risk of the subject's condition worsening.

5. The method according to any one of claims 1 to 4, wherein the criteria are criteria for each biomarker derived from at least one subject known to be at risk of developing sepsis, and preferably, the amount of each biomarker being essentially the same as or similar to the corresponding criterion indicates that the subject is at risk of developing sepsis, and the amount of each biomarker being different from the corresponding criterion indicates that the subject is not at risk of developing sepsis.

6. The method according to any one of claims 1 to 4, wherein the criteria are criteria for each biomarker derived from at least one subject known to be free from the risk of developing sepsis, and preferably, the amount of each biomarker being essentially the same as or similar to the corresponding criterion indicates that the subject is free from the risk of developing sepsis, and the amount of each biomarker being different from the corresponding criterion indicates that the subject is at risk of developing sepsis.

7. The method according to any one of claims 1 to 6, wherein the subject is suffering from an infectious disease or is suspected to be suffering from an infectious disease.

8. The method according to any one of claims 1 to 7, wherein the sample is a blood sample or a sample derived from a blood sample.

9. The method according to any one of claims 1 to 8, wherein the subject is a human.

10. A computer implementation method for assessing individuals suspected of having an infectious disease, (a) A step of receiving a value for the amount of a first biomarker in the target sample, wherein the first biomarker is PCT; (b) Receiving a value for the amount of a second biomarker in the sample of interest, wherein the second biomarker is selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase; (c) the step of comparing a value for the amount of the biomarker with a standard for the biomarker and / or the step of calculating a score for evaluating the subject suspected of having an infectious disease based on the amount of the biomarker; and (d) A step of evaluating the subject based on the comparison and / or calculation performed in step (c). Methods that include...

11. In step (b), (i) If a value for the amount of cardiac troponin is received as the second biomarker, the method further includes receiving a value for the amount of bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine as a third biomarker, or (ii) If a value for the amount of creatinine is received as the second biomarker, the method further comprises receiving a value for the amount of aspartate aminotransferase or bilirubin as a third biomarker, (iii) When a value for the amount of sTREM1 is received as the second biomarker, the method further includes receiving a value for the amount of aspartate aminotransferase or HBP as a third biomarker. The method according to claim 10.

12. A device for assessing individuals suspected of having an infectious disease, (a) A measurement unit for determining the amount of a first biomarker, which is PCT, and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, comprising a detection system for the first biomarker and the second biomarker; and (b) An evaluation unit operably connected to the measurement unit, preferably a database having stored criteria for the first biomarker and the second biomarker as described in any one of claims 1 to 9, and preferably a system for performing comparisons of the amounts of the first biomarker and the second biomarker with criteria and / or for evaluating the subject suspected of having an infection based on the amount of the biomarkers, as described in any one of claims 1 to 9. An evaluation unit including a data processor that includes instructions for performing core calculations and for evaluating the subject based on the comparison, and which can automatically receive values ​​for the amount of the biomarker from the measurement unit. A device that includes this.

13. The measurement unit determines a third biomarker and includes a detection system for the third biomarker, the database includes stored criteria for the third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then it may be aspartate aminotransferase or bilirubin, (iii) When sTREM1 is the second biomarker, it is aspartate aminotransferase or HBP, The device according to claim 12.

14. The device according to claim 12 or 13, wherein the detection system includes at least one detection agent capable of specifically detecting each of the biomarkers.

15. A device for evaluating a subject suspected of having an infectious disease, comprising an evaluation unit, the evaluation unit comprising a database having stored criteria for a first biomarker which is PCT and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, and preferably a data processor which includes instructions for performing a comparison between the amounts of the first biomarker and the second biomarker and the criteria, and for evaluating the subject based on the comparison, as described in any one of claims 1 to 11, wherein the evaluation unit can receive values ​​for the amounts of biomarkers determined in a sample of the subject.

16. The database includes stored criteria for a third biomarker, and the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then it may be aspartate aminotransferase or bilirubin, (iii) When sTREM1 is the second biomarker, it is aspartate aminotransferase or HBP, The device according to claim 15.

17. i) the use of a first biomarker which is PCT, and a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase, for assessing a subject suspected of having an infectious disease, or i) a detection agent which specifically binds to the first biomarker and a detection agent which specifically binds to the second biomarker.

18. A third biomarker or a detection agent that specifically binds to the third biomarker is added. The third biomarker is used additively, (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase, or (iii) When sTREM1 is the second biomarker, it is aspartate aminotransferase or HBP, The use described in claim 17.

19. A kit for assessing individuals suspected of having an infectious disease, comprising a detection agent that specifically binds to a first biomarker, which is PCT, and a detection agent that specifically binds to a second biomarker selected from the group consisting of cardiac troponin, creatinine, BNP-type peptide, sTREM1, ESM-1, haptoglobin, heparin-binding protein (HBP), and aspartate aminotransferase.

20. The present invention further comprises a detection agent that specifically binds to a third biomarker, wherein the third biomarker is (i) If cardiac troponin is the second biomarker, then bilirubin, aspartate aminotransferase, alanine aminotransferase, haptoglobin, ESM1, or creatinine, (ii) If creatinine is the second biomarker, then aspartate aminotransferase or bilirubin, (iii) When sTREM1 is the second biomarker, it is aspartate aminotransferase or HBP, The kit according to claim 19.

21. The method, device, use, or kit according to any one of claims 1 to 20, wherein the assessment is an assessment of the risk of developing sepsis.

22. A method, device, use, or kit according to any one of claims 1 to 21, wherein the risk of developing sepsis within 48 hours is predicted.

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