GDF15 marker panel for early detection of sepsis

Abstract

The present invention relates to a method for assessing a subject suspected of having an infection comprising the steps of: determining the amount of a first biomarker in the subject's sample, said first biomarker being GDF-15; determining the amount of a second biomarker in the subject's sample, said second biomarker being selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; comparing the amount of the biomarker with a standard for said biomarkers and / or calculating a score for assessing the subject suspected of having an infection 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, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, 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 infection. Furthermore, the present invention further relates to a computer-implemented method for assessing a subject suspected of infection, as well as a device and a kit for assessing a subject suspected of infection.

Description

[Technical field]

[0001] The present invention relates to the field of diagnosis. In particular, it relates to a method for assessing a subject suspected of infection, comprising the steps of: determining the amount of a first biomarker in a sample of the subject, the first biomarker being GDF-15; determining the amount of a second biomarker in a sample of the subject, the second biomarker being selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing the subject suspected of infection 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, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, 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 infection. Furthermore, the present invention further relates to a computer-implemented method for assessing a subject suspected of infection, as well as devices and kits for assessing a subject suspected of infection. [Background technology]

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

[0003] According to the definition of sepsis3, sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Because of its rapid onset, early recognition is important for the management of septic patients and for the initiation of correct therapeutic measures, including appropriate antibiotic therapy within the first hour of hospitalization, as well as the initiation of resuscitation with intravenous fluids and vasoactive drugs (Surviving Sepsis Campaign Guidelines 2016). Every hour of delay progressively increases morbidity and mortality.

[0004] The diagnosis of sepsis is based on clinical signs and symptoms that are non-specific and can easily be overlooked. Thus, patients are often misdiagnosed and the severity of the disease is often underestimated. So far, there is no gold standard for the diagnosis of sepsis in general, especially in the emergency department. In high-income countries, c-reactive protein (CRP), procalcitonin (PCT) and white blood cell (WBC) count are often used in emergency units for the detection of patients with bloodstream infections at risk of developing sepsis, along with lactate for the detection of septic shock. In low-income countries, the diagnosis is mainly based on clinical signs and symptoms, and occasionally SIRS and SOFA criteria. However, in the latest guidelines, other than lactate, no biomarkers are listed for diagnosing sepsis (except for clinical chemistry, BGE and hematological components of the SOFA score). PCT is only recommended to potentially ease antibiotic therapy, but with moderate evidence. The limitations of PCT in sepsis diagnosis are mainly its moderate sensitivity and specificity.

[0005] WO 2007 / 009071 discloses a method for diagnosing an inflammatory response in a test subject based on sFlt-1. The disclosed method further comprises analyzing the level of at least one of VEGF, PlGF, TNF-α, IL-6, D-dimer, P-selectin, ICAM-I, VCAM-I, Cox-2, or PAI-I.

[0006] EP 2174143 B1 discloses an in vitro method for the prognosis of patients with a primary non-infectious disease, which comprises determining the level of procalcitonin.

[0007] A number of markers have been suggested to be useful in detecting or diagnosing sepsis. These include, among others, inflammatory markers such as PCT, presepsin, GDF-15, sFLT, CRP, or interleukins, or markers specific for organ failure (see, e.g., Spanuth, 2014, Comparison of sCD14-ST(рresepsin) with еight 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] WO 2015 / 031996 describes biomarkers for the early determination of serious or life-threatening responses to disease and / or treatment responses.

[0009] However, there remains a need for biomarkers that allow reliable and early assessment of patients who exhibit signs and symptoms of infection. Summary of the Invention

[0010] Thus, the present invention provides means and methods that meet these needs.

[0011] The present invention provides a method for assessing a subject suspected of infection, comprising: (a) determining the amount of a first biomarker in a sample from a subject, wherein the first biomarker is GDF-15; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the amount of the biomarker to a standard for said biomarker and / or calculating a score for assessing the subject as suspected of being infected based on the amount of the biomarker; and (d) evaluating the subject based on the comparisons and / or calculations performed in step (c). The present invention relates to a method for assessing a subject suspected of being infected, comprising: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] It should be understood that as used in this specification and claims, "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, a reference to "an" item can mean that at least one of the item is available.

[0013] When used below, the terms "have", "comprise" or "include" or any grammatical variants thereof are used inclusively. Thus, these terms may refer both to the situation where no further features are present in the entity described in this context, in addition to the features introduced by these terms, and to the situation where one or more additional features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to the situation where no other elements are present in A, other than B (i.e., A consists solely and exclusively of B), and to the situation where, other than B, one or more further elements are present in the entity A, such as element C, elements C and D, as well as further elements. The term "comprising" has a restrictive meaning in the sense of "consisting of", also encompassing embodiments where only the mentioned items are present.

[0014] Furthermore, when used hereinafter, the terms "particularly", "more particularly", "typically" and "more typically" or similar terms are used in conjunction with additional / alternative features without limiting the possibilities of substitution. Thus, features introduced by these terms are additional / alternative features and are not intended to limit the scope of the claims in any way. The invention may also be implemented by using alternative features, as recognized by those skilled in the art. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be additional / alternative features, without any limitations on alternative embodiments of the invention, without any limitations on the scope of the invention, and without any limitations on the possibilities of combining the features so introduced with other additional / alternative or non-additional / alternative features of the invention.

[0015] Furthermore, as used herein, the term "at least one" is understood to mean that one or more of the items subsequently mentioned may be used according to the present invention. For example, if the term indicates that at least one sampling unit should be used, this may be understood as one sampling unit or more than one, i.e., two, three, four, five, or any other number of sampling units. Depending on the item to which the term refers, a person skilled in the art will understand which upper limit, if any, the term may refer to.

[0016] As used herein, the term "about" means that there is an interval of precision within which the technical effect can be achieved for any number listed after the term. Thus, when referred to herein, it preferably refers to the exact numerical value or a range around the exact numerical value ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5%.

[0017] Moreover, the terms "first," "second," "third," etc. in the specification and claims are used to distinguish between similar elements and are not necessarily intended to describe a sequential or chronological order.

[0018] The method of the invention may consist of the above steps or may include additional steps, such as a step for further evaluation of the assessment obtained in step (d), a step for recommending a therapeutic measure, such as a treatment. Furthermore, it may include steps prior to step (a), such as a step relating to the pre-treatment of the sample. However, it is preferably envisaged that the above method is an ex vivo method, which does not require any steps performed on the human or animal body. Furthermore, the method may be supported by automation. Typically, the determination of the biomarkers may be supported by a robotic device, and the comparison and assessment may be supported by a data processing device, such as a computer.

[0019] As used herein, the term "assessing" refers to assessing whether a subject suffers from sepsis, whether they are at risk of suffering from sepsis, whether they exhibit worsening medical symptoms with respect to their general health, or with respect to sepsis or symptoms associated with sepsis, or whether they exhibit symptoms associated with sepsis and / or infection.Assessing as used herein therefore includes diagnosing sepsis, predicting the risk of developing sepsis, and / or predicting any deterioration in the subject's health, particularly 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 the subject's (health) condition worsening. Furthermore, it will be understood that when the risk of developing sepsis or the risk of the health condition worsening is predicted, the prediction is typically made within a prediction window. More typically, the prediction window is preferably about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 48 hours, particularly at least about 48 hours after the sample is obtained. Furthermore, the risk of developing sepsis can be predicted preferably within 24 hours or 48 hours after the test sample is obtained.

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

[0022] In an alternative embodiment, the risk of developing sepsis within 48 hours is predicted.

[0023] A 48 hour period was analyzed in the Examples section.

[0024] In yet another embodiment, the assessment is a prediction of the risk of the subject's (health) condition worsening in the future. The term "worsening of symptoms" of a subject suspected of and / or suffering from an infectious disease is well understood by those skilled in the art. This term typically relates to a worsening of symptoms that may ultimately result in further medication or other intervention.

[0025] Preferably, the subject's condition worsens if the subject's disease severity increases, if the subject's antibiotic therapy is intensified, if the subject is admitted to an ICU or another unit for a higher level of care, if the subject requires emergency surgery, if the subject dies in the hospital, if the subject dies within 30 days after admission, if the subject is readmitted within 30 days after discharge, if the subject experiences organ dysfunction or failure, e.g., as measured by SOFA score, and / or if the subject requires organ support.

[0026] One of skill in the art will appreciate when a subject's condition does not worsen. Typically, a subject's condition does not worsen when the subject does not have the results described in the previous paragraph.

[0027] In one embodiment, the subject's condition worsens if the subject has one or more of the following outcomes: the subject is admitted to the ICU, the subject dies in the hospital, the subject dies within 30 days of admission, and / or the subject is readmitted within 30 days of discharge.

[0028] In one embodiment, predicting the risk of the subject's condition worsening is predicting the risk of the subject's antibiotic therapy being intensified.

[0029] In one embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the subject's risk of admission to an ICU. Thus, it is assessed whether the subject is at risk of admission to an ICU.

[0030] In another embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the subject's risk of dying in hospital, thus assessing whether the subject is at risk of dying in hospital.

[0031] In yet another embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the subject's risk of death within 30 days of admission to the hospital. Thus, it is assessed whether the subject is at risk of dying within 30 days of admission to the hospital.

[0032] In yet another embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the subject's risk of readmission within 30 days after discharge from the hospital. Thus, it is assessed whether the subject is at risk of being readmitted within 30 days after discharge from the hospital.

[0033] In yet another embodiment, the prediction of the risk of the subject's condition worsening is the prediction of the risk of the subject experiencing organ dysfunction or failure. Organ dysfunction and failure can be assessed, for example, through SOFA score. Thus, the present invention further relates to the prediction of the risk of the subject's SOFA score increasing or not increasing (after the test sample is obtained). An increase in SOFA score (e.g., at least one, at least two, at least three, or at least four points, etc.) is considered as a worsening of the condition. In contrast, if the SOFA score does not increase (if the subject does not have the highest SOFA score), the condition typically does not worsen. The prediction window can be the prediction window described above for the prediction of the risk of developing sepsis.

[0034] The 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 dysfunction are scored individually and summed into 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.).

[0035] In yet another embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the risk of the subject needing organ support, such as a prediction of the risk of the subject needing vasoactive therapy, hemodynamic support (e.g., fluid therapy), oxygenation (e.g., by ventilation or extracorporeal membrane oxygenation), and / or renal replacement therapy. The prediction window may be, for example, the prediction window described above for predicting the risk of developing sepsis 24 hours or 48 hours after the sample is obtained.

[0036] In one embodiment, the term "assessment" refers to the diagnosis of sepsis. Thus, a subject suspected of infection is diagnosed as suffering from sepsis or not. Preferably, assessment refers to the early detection of sepsis.

[0037] As will be understood by those skilled in the art, the assessment made according to the present invention is preferably, but usually, correct for 100% of the objects surveyed, but may not be. This term typically requires that the statistically significant part of the object can be accurately assessed. Whether the part is statistically significant can be further easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley&Sons, New York 1983. The typically assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0038] As used herein, the term "subject" refers to an animal, preferably a mammal, more typically a human. The subject investigated by the method of the present invention shall be a subject suspected of infection. As used herein, the term "suspected of infection" means that the subject shows clinical parameters, signs and / or symptoms of infection. Thus, the subject according to the present invention is typically a subject suffering from or suspected of suffering from an infectious disease. Typically, the subject is a subject who presents to an emergency department.

[0039] Advantageously, the sample is obtained during a clinic visit. Preferably, the sample is obtained during a clinic visit to an emergency department. However, the sample may also be obtained during a clinic visit to a primary care physician.

[0040] As used herein, the term "sample" refers to any sample that contains the first, second and / or third biomarkers referred to herein under physiological conditions. More typically, the sample is a body fluid sample, such as a blood sample or a sample derived therefrom, a urine sample, a saliva sample, a lymphatic sample, etc. Most typically, the sample is a blood sample or a sample derived therefrom. Thus, the sample may be a blood, serum or plasma sample. A blood sample typically includes a capillary, venous or arterial blood sample.

[0041] In one embodiment, the sample is an interstitial fluid sample.

[0042] The term "sepsis" is well known in the art. As used herein, this term refers to life-threatening organ dysfunction caused by dysregulated host response to infection. The 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 by reference in its entirety. Preferably, the term "sepsis" refers to sepsis according to the definition of sepsis-3 disclosed in Singer et al. (loc.cit.).

[0043] Typically, the subject to be tested is suspected of suffering from an infectious disease. The term "infection / infectious disease" is well understood by those skilled in the art. As used herein, the term "infection / infectious disease" preferably refers to the invasion of the subject's body tissues by disease-causing microorganisms, their proliferation, and the reaction of the subject's tissues to the microorganisms. In one embodiment, the infection is a bacterial infection. Thus, the subject is suspected of suffering from a bacterial infection.

[0044] As described elsewhere herein, the present invention allows for early identification of at-risk patients. Thus, in one embodiment of the prediction described herein, the subject being tested does not suffer from sepsis at the time the sample is obtained. In a particularly preferred embodiment, the subject being tested preferably does not suffer from septic shock at the time the sample is obtained. The term "septic shock" is defined in Singer et al. (loc.cit.). Thus, a subject suffers from septic shock if the following criteria are met: Sepsis, i.e. suspected / documented infection and a change in total SOFA score of ≥2 points as a result of infection and persistent hypotension requiring vasopressors to maintain MAP ≥ 65 mmHg despite adequate volume resuscitation, and with a serum lactate level > 2 mmol / L (18 mg / dL).

[0045] It is further envisaged that the subjects being tested may or may not be infected with SARS-CoV-2.

[0046] As used herein, the term "determining" refers to the qualitative and quantitative determination of the biomarker referred to in accordance with the present invention, i.e., the term encompasses the determination of the presence or absence, or the determination of the absolute or relative amount, of said biomarker.

[0047] As used herein, the term "amount" refers to the absolute amount of the compound referred to herein, the relative amount or concentration of the compound, as well as any value or parameter that can be correlated therewith or derived therefrom. Such values ​​or parameters include intensity signal values ​​derived from any specific physical or chemical property obtained from the compound by direct measurement, such as intensity values ​​in mass or NMR spectra. Furthermore, all values ​​or parameters obtained by indirect measurement as specified elsewhere herein are included, such as response levels determined by a biological readout system in response to a compound or intensity signal obtained from a specifically bound ligand. It should be understood that values ​​that correlate with the above-mentioned amount or parameter can also be obtained by any standard mathematical operation. When the biomarker is an enzyme, such as alanine aminotransferase (ALAT) or aspartate aminotransferase (AST or ASAT), the term "amount" can also include the activity of the enzyme.

[0048] The determination of the amount in the methods of the invention can be carried out by any technique that makes it possible to detect the presence or absence, or the amount, of said second molecule upon release from said first molecule. Suitable techniques will depend on the nature of the molecule and the characteristics of the biomarker, and are discussed in more detail elsewhere herein.

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

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

[0051] The term "growth differentiation factor-15" or "GDF-15" refers to a polypeptide that is a member of the transforming growth factor (TGF) cytokine superfamily. The terms polypeptide, peptide and protein are used interchangeably throughout this specification. GDF-15 was originally cloned as macrophage inhibitory cytokine 1 and was later identified as placental transforming growth factor-15, placental bone morphogenetic protein, nonsteroidal anti-inflammatory drug-activated gene 1, and prostate-derived factor (Bootcov loc cit; Hromas, 1997 Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26; Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998, J Biol Chem 273:13760-13767). The amino acid sequence of GDF-15 is disclosed in: WO 99 / 06445, WO 00 / 70051, WO 2005 / 113585, Bottner 1999, Gene 237:105-111, Bootcov loc.cit, Tan loc.cit., Baek 2001, Mol Pharmacol 59:901-908, Hromas loc cit, Paralkar loc cit, Morrish 1996, Placenta 17:431-441.

[0052] Insulin-like growth factor binding protein 7 (=IGFBP7) is a 30 kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y., et al., Biochem Biophys Res Comm 202 (1994) 1490-1496). Preferably, the term "IGFBP7" refers to human IGFBP7. The protein sequence is well known in the art and is accessible, for example, via GenBank (NP_001240764.1).

[0053] 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 for pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release the propeptide (108 amino acids for proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids for NT-proBNP) and the active hormone (32 amino acids for BNP). Preferably, the BNP-type peptides according to the invention are NT-proBNP, BNP and variants thereof. BNP (brain natriuretic peptide) is the active hormone and has a shorter half-life than its respective inactive counterpart NT-proBNP.

[0054] The biomarker endothelial cell specific molecule 1 (abbreviated as ESM-1) is well known in the art. The biomarker is also often referred to as endocan. ESM-1 is a secreted protein, mainly expressed in endothelial cells of human lung and kidney tissues. Public domain data suggests that it is expressed in thyroid, lung, kidney, but also cardiac tissue. See, for example, the entry for ESM-1 in the Protein Atlas database (Uhlen M.et al., Science 2015;347(6220):1260419). 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 invention, the amount of human ESM-1 polypeptide is measured in a sample from a subject. The sequence of human ESM-1 polypeptide is well known in the art (see, e.g., Lassale P. et al., J. Biol. Chem. 1996;271:20458-20464) and can be assessed, for example, via the Uniprot database (see entry Q9NQ30 (ESM1_HUMAN)). Two isoforms of ESM-1 are generated by alternative splicing, isoform 1 (having Uniprot identifier Q9NQ30-1) and isoform 2 (having Uniprot identifier Q9NQ30-2). Isoform 1 is 184 amino acids in length. In isoform 2, amino acids 101 to 150 of isoform 1 are missing. Amino acids 1 to 19 form the signal peptide (which can be cleaved).

[0055] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, ie isoform 1 has the sequence shown in UniProt accession number Q9NQ30-1.

[0056] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, ie isoform 2 has the sequence shown in UniProt Accession No. Q9NQ30-2.

[0057] In another preferred embodiment, the amount of isoform-1 and isoform-2 of the ESM-1 polypeptide, ie total ESM-1, is determined.

[0058] sTREM-1 or soluble TREM1 is a soluble form of TREM-1 (triggering receptor expressed on myeloid cells-1). Thus, 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 the innate immune response. TREM-1 is a monomeric protein of approximately 30 kD 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 short cytoplasmic domain of 5 amino acids. During infection, receptor expression is altered and sTREM-1 is released. Thus, sTREM-1 (17 kDa) is a soluble form of TREM-1 that is released from the membrane of activated phagocytes, and typically, the term "sTREM-1" encompasses all naturally occurring truncated or released forms that have at least the extracellular portion of TREM-1.

[0059] The marker "bilirubin" is well known in the art. Bilirubin is a member of the biladienes class of linear tetrapyrroles, in which the dipyrrole units are of both exo- and endo-vinyl type. It is a product of heme degradation, produced in the reticuloendothelial system by reduction of biliverdin, and transported to the liver in a complex with serum albumin. It has a role as an antioxidant. Bilirubin measurement is routine in most laboratories and can be measured by a variety of methods, such as those described in the Examples section.

[0060] The term "cardiac troponin" typically refers to human cardiac troponin T or cardiac troponin I. However, the term also includes variants of the aforementioned specific troponins, i.e. preferably variants of troponin I, more preferably variants of troponin T. Such variants have at least the same essential biological and immunological properties as the specific cardiac troponin. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to herein, for example by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize said cardiac troponin. Furthermore, the variants referred to in accordance with the present invention shall have a different amino acid sequence due to at least one amino acid substitution, deletion and / or addition, and it should be understood that the amino acid sequence of the variant is still 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% identical to the amino acid sequence of the specific troponin. The variants may be allelic variants or any other species-specific homologs, paralogs or orthologs. Furthermore, the variants referred to herein include fragments of the specific cardiac troponins or variants of the above-mentioned types, as long as these fragments have the essential immunological and biological properties referred to above. Preferably, the cardiac troponin variants have immunological properties (i.e. epitope composition) comparable to those of human troponin T or troponin I. Thus, the variants shall be recognizable by the above-mentioned means or ligands used to determine the concentration of the cardiac troponin. Thus, the variants shall be recognizable by the above-mentioned means or ligands used to determine the concentration of cardiac troponin. Such fragments may be, for example, degradation products of troponin. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation.Preferably, the biological property of troponin I and its variants is the ability to inhibit actomyosin ATPase or to inhibit angiogenesis in vivo and in vitro, which can be detected, for example, based on the assays described in Moses et al. 1999 PNAS USA 96(6):2645-2650). Preferably, the biological property of troponin T and its variants is the ability to form a complex with troponin C and I, to bind calcium ions, or to bind to tropomyosin, preferably when present as a complex of troponin C, I and T, or when present as a complex formed by troponin C, troponin I and variants of troponin T. Troponin T or troponin I can be measured by immunoassays well known in the art and commercially available, for example ELISA. Particularly preferred according to the invention is the measurement of troponin T with high sensitivity, for example using the commercially available hs-cTn assay.

[0061] CRP (C-reactive protein) is an acute phase protein that was discovered over 75 years ago to be a blood protein that binds to the C-polysaccharide of Streptococcus pneumoniae. CRP is known as a reactive inflammatory marker and is produced by distal organs (i.e., the liver) in response to or in reaction to chemokines or interleukins derived from the primary lesion site. CRP is known to consist of five single subunits that are non-covalently linked and assembled as a cyclic pentamer with a molecular weight of about 110-140 kDa. Preferably, CRP as used herein relates to human CRP. The sequence of human CRP is well known and has been disclosed, for example, by Woo et al. (J.Biol.Chem.1985.260(24),13384-13388). Levels of CRP are usually low in normal individuals, but can be elevated 100-200 fold or more due to inflammation, infection or injury (Yeh(2004)Circulation.2004;109:11-11-11-14). CRP is known to be an independent factor for predicting cardiovascular risk. CRP can be measured by immunoassays, such as ELISA, which are well known in the art and commercially available.

[0062] Procalcitonin (abbreviated as PCT) is the peptide precursor of the hormone calcitonin. Procalcitonin is therefore the inactive propeptide of calcitonin. It is composed of 116 amino acids and is produced by thyroid parafollicular cells (C cells), as well as neuroendocrine cells in the lung and intestine. 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 / s40560-019-0374-30 4). The amino acid sequence of the marker is well known in the art and is disclosed, for example, in European Patent No. 2320237 B1. PCT can be measured by immunoassays, for example ELISA, that are well known in the art and commercially available.

[0063] As used herein, the term "sFlt-1" refers to a polypeptide that is a soluble form of fms-like tyrosine kinase 1. The polypeptide is also referred to in the art as soluble VEGF receptor 1 (sVEGF R1) (see, e.g., Sunderji 2010, Am J Obstet Gynecol 202:40e1-7). It was identified in the conditioned culture medium of human umbilical vein endothelial cells. The endogenous sFlt1 receptor is chromatographically and immunologically similar to recombinant human sFlt1 and binds [125I]VEGF with comparable high affinity. Human sFlt1 has been shown to form a VEGF-stabilized complex with the extracellular domain of KDR / Flk-1 in vitro. Preferably, sFlt1 refers to human sFlt1 as described in Kendall 1996, Biochem Biophs Res Commun 226(2):324-328, see also, for amino acid sequences, e.g., Genebank Accession Nos. P17948, GI:125361 for human and BAA24499.1, GI:2809071 for mouse sFlt-1 (Genebank available from NCBI (USA) under www.ncbi.nlm.nih.gov / entrez).

[0064] Alanine aminotransferase (ALAT) catalyzes the transamination of L-alanine to α-ketoglutarate (α-KG) to form L-glutamate and pyruvate. The pyruvate formed is reduced to lactate by lactate dehydrogenase (LDH) with the concomitant 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 a dichroic (340 nm, 700 nm) kinetic technique.

[0065] Aspartate aminotransferase (AST or ASAT) catalyzes the transamination of L-aspartate to α-ketoglutarate, forming L-glutamate and oxalate. The oxalate formed is reduced to malate by malate dehydrogenase (MDH) with the concomitant 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 a dichroic (340 nm, 700 nm) kinetic technique.

[0066] Antithrombin (AT), often called antithrombin III, is a proein that inactivates several enzymes of the coagulation system, such as thrombin, matriptase-3 / TMPRSS7, and factors IXa, Xa, and XIa. It contains three disulfide bonds and a total of four possible glycosylation sites. α-antithrombin is the major form of antithrombin found in plasma and has an oligosaccharide occupying each of its four glycosylation sites. The sequence of human AT is well known in the art and can be assessed, for example, by UniProt (see accession number P01008).

[0067] In the method of the invention, a third biomarker may be determined. In particular, in step (b) of the method of the invention, (i) if the amount of sFLT1 is determined as the second biomarker, the method further comprises determining the amount of sTREM-1, antithrombin or cystatin C as a third biomarker; (ii) if the amount of cystatin C is determined as the second biomarker, the method further comprises determining the amount of bilirubin, alanine aminotransferase or aspartate aminotransferase as a third biomarker; (iii) if the amount of IGFBP-7 is determined as the second biomarker, the method further comprises determining the amount of bilirubin or procalcitonin as a third biomarker; (iv) if the amount of bilirubin is determined as the second biomarker, the method further comprises determining the amount of creatinine as a third biomarker; or (v) When the amount of sTREM-1 is determined as the second biomarker, the method further comprises determining the amount of aspartate aminotransferase as a third biomarker.

[0068] Thus, 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).

[0069] The first biomarker is GDF-15. The second biomarker is selected from sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase.

[0070] In one embodiment, the second biomarker is a cardiac troponin, such as cardiac troponin T or I, preferably troponin T.

[0071] In an alternative embodiment, the second biomarker is a BNP-type peptide, such as NT-proBNP or BNP, preferably NT-proBNP.

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

[0073] In an alternative embodiment, the second biomarker is ESM-1.

[0074] In an alternative embodiment, the second biomarker is bilirubin.

[0075] In an alternative embodiment, the second biomarker is PCT (procalcitonin).

[0076] In an alternative embodiment, the second biomarker is sFlt-1. When the amount of sFLT1 is determined as the second biomarker, the method may further comprise determining the amount of sTREM-1, antithrombin or cystatin C as a third biomarker.

[0077] In an alternative embodiment, the second biomarker is aspartate aminotransferase (ASAT).

[0078] In an alternative embodiment, the second biomarker is alanine aminotransferase (ALAT).

[0079] It should be understood that the present invention is not limited to the above markers, rather, the present invention may encompass the determination of additional markers.

[0080] As used herein, the term "criterion" refers to an amount or value that allows a subject to be assigned to either a group of subjects suffering from or at risk of developing a disease or condition, or a group of subjects not suffering from or at risk of developing said disease or condition. Such a criterion can be a threshold amount that separates these groups from each other. Thus, a criterion shall be an amount or score that allows a subject to be assigned to a group of subjects, regardless of whether they suffer from or at risk of developing a disease or condition. For example, a criterion shall be an amount or score that allows a subject to be assigned to a group of subjects that are at risk of developing sepsis or are not at risk of developing sequence (within the above-mentioned prediction window, for example within about 48 hours).

[0081] The appropriate threshold amount for separating the two groups can be calculated based on the amount of biomarker from either a subject or group of subjects known to be affected by or at risk of developing a disease or condition, or a subject or group of subjects known not to be affected by or at risk of developing a disease or condition, without further confounding by statistical tests as mentioned elsewhere herein.The reference amount applicable to an individual subject may vary according to various physiological parameters, such as age, sex, or subpopulation.

[0082] Typically, the reference is a reference for each biomarker derived from at least one subject known to be at risk of developing sepsis, and preferably, an amount for each of the biomarkers that is essentially the same as or similar to the corresponding reference is indicative of a subject at risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference is indicative of a subject not at risk of developing sepsis.

[0083] Also, typically the standards are for each biomarker derived from at least one subject known to be not at risk of developing sepsis, and preferably, an amount for each of the biomarkers that is essentially the same or similar to the corresponding standard indicates a subject that is not at risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding standard indicates a subject that is at risk of developing sepsis.

[0084] The term "at least one subject" refers to one subject or more than one subject, for example, at least 10, 50, 100, 200, or 1000 subjects.

[0085] In one embodiment, an amount of the biomarker greater than the biomarker baseline is indicative of a subject at risk (e.g., developing sepsis as described elsewhere herein), and an amount of the biomarker lower than the biomarker baseline is indicative of a subject not at risk (with the exception of antithrombin, in which case an amount of the biomarker lower than the biomarker baseline is indicative of a subject at risk, while an amount of the biomarker higher than the biomarker baseline is indicative of a subject not at risk).

[0086] Reference amounts can in principle be calculated for a cohort of subjects based on the mean or average value of a given parameter, such as the amount of a biomarker, by applying standard statistical methods. Whether a test, particularly one aimed at diagnosing an event, is accurate or not is best described by its receiver operating characteristics (ROC) (see, in particular, Zweig 1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all sensitivity / specificity pairs resulting from continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a certain prognosis or diagnosis. The ROC plot shows the overlap between the two distributions by plotting sensitivity versus 1-specificity for the entire range of thresholds suitable for making the distinction. The y-axis is the 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 a disease or condition. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative and false positive results. The x-axis is an index of specificity, calculated only from the unaffected subgroup. The true positive rate and the false positive rate are calculated completely separately by using the test results from the two different subgroups, so the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / -specificity pair that corresponds to a particular decision threshold. A test with perfect discrimination (no overlap between the two distributions of results) would 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 a test with no discrimination (the distribution of results for the two groups is identical) would be a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. If the ROC plot falls entirely below the 45° diagonal, this is 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 value can be derived from the ROC curve, which allows diagnosis or prediction of a given event with the appropriate balance of sensitivity and specificity, respectively. Therefore, the criteria used in the above-mentioned method of the present invention, i.e., the threshold value that allows distinguishing between subjects at risk and those not at risk, can usually be generated by establishing the ROC of the cohort as described above and deriving the threshold amount therefrom. Depending on the desired sensitivity and specificity of the diagnostic method, the ROC plot can lead to an appropriate threshold value. It will be understood that an optimal sensitivity is desired to exclude subjects at high risk or affected by the disease (i.e., rule out), while an optimal specificity is assumed for subjects that are assessed as at high risk or affected by the disease (i.e., rule in).

[0087] Step c) of the method of the present invention comprises comparing the amount of the biomarkers (i.e. the first biomarker, the second biomarker and optionally the third biomarker) with a standard for said biomarkers and / or calculating a score for assessing the subject as suspected of infection based on the amount of the biomarkers.

[0088] Thus, the amounts of the first biomarker, the second biomarker, and optionally the third biomarker may be compared to a standard for the first biomarker, a standard for the second biomarker, and optionally a standard for the third biomarker, respectively.

[0089] Alternatively, a score may be calculated based on the amounts of the biomarkers, i.e., the first biomarker, the second biomarker, and optionally the third biomarker. The score shall allow for the assessment of subjects suspected of infection, such as to predict the risk of developing sepsis. Optionally, the score may be compared to a suitable reference score.

[0090] As used herein, the term "comparing" includes comparing the determined amount of the biomarker referred to herein with a standard. As used herein, comparison should be understood to refer to any kind of comparison made between a value for the amount and a standard. However, it should be understood that preferably, values ​​of the same type are compared with each other, e.g., if absolute amounts are determined and compared in the method of the present invention, the standard is also an absolute amount, if relative amounts are determined and compared in the method of the present invention, the standard is also a relative amount, etc. Alternatively, as used herein, the term "comparing" includes comparing the calculated score with a suitable standard core. The comparison can be performed manually or computer-assisted. The value for the amount and the standard can, for example, be compared with each other, and the comparison can be performed automatically by a computer program implementing an algorithm for the comparison. The computer program performing the evaluation provides the desired assessment in a suitable output format.

[0091] As mentioned above, it is also envisaged to calculate a score (particularly a single score), i.e. a single score, based on the amounts of the first and second biomarkers, or the first, second or third biomarker, and to compare this score to a reference score. Preferably, the score is based on the amounts of the first and second biomarkers in the sample from the test subject, and, if the amount of the third biomarker is determined, based on the amounts of the first, second and third biomarkers in the sample from the test subject.

[0092] The calculated score combines information on the amount of at least two or three biomarkers. Moreover, in the score, the biomarkers are preferably weighted according to their contribution to the establishment of the score. Thus, the values ​​for each marker are typically weighted and the weighted values ​​are used to calculate the score. The appropriate coefficients (weights) can be determined by the skilled artisan without further ado. The score can also be calculated from a decision tree or a set (ensemble) of decision trees trained on at least two biomarkers. Based on the combination of biomarkers applied in the method of the present invention, the weights of the individual biomarkers as well as the structure of the decision tree can be different.

[0093] The score can be considered as a classification parameter for assessing a subject as described herein. In particular, it allows a person to provide an assessment based on a single score. The reference score is preferably a value, in particular a cut-off value that allows a person to assess a subject suspected of infection as described herein. Preferably, the reference is a single value. Thus, a person does not have to interpret the entire information regarding the amount of each biomarker. Using the scoring system described herein, advantageously, values ​​of different dimensions or units of the biomarkers can be used, since the values ​​are mathematically converted into scores. Thus, for example, absolute concentration values ​​can be combined into a score with peak area ratio. The applied reference score can be selected based on the desired sensitivity or the desired specificity. How to select an appropriate reference score is well known in the art.

[0094] Advantageously, in the study underlying the present invention, it was found that the combination of a first biomarker with a second biomarker, preferably a third biomarker, allows for a reliable and early assessment of patients showing signs and symptoms of infection. For example, the assessment of the subject can be performed within 5 hours after the test sample is obtained. The study investigated patients presenting to the emergency department with medical (non-surgical) emergencies. For this purpose, the patients were subdivided into those with a high probability of sepsis and those suspected of having an infection without sepsis. The amounts of the different biomarkers were determined, and the biomarkers were analyzed and mathematically combined by logistic regression analysis. The area under the receiver operating characteristic (AUC) was used to evaluate the performance of the biomarkers. The AUC value is the mathematical integral of the function f(x) in the interval [a][b]. The AUC was also investigated for biomarker pairs and triplets. Combinations of biomarkers that together show an improved AUC over the best single biomarker AUC were identified. The results are described in the attached examples below.

[0095] In particular, if these patients are presenting, for example, to an emergency unit, early assessment of the risk of developing severe complications such as sepsis, SIRS, or a general deterioration in general health dictates the initiation of therapeutic measures, including drug administration, physical or other therapeutic interventions, and / or hospitalization. These therapeutic measures may include, inter alia, for example, the rapid administration of broad-spectrum antibiotics, fluid resuscitation, vasoactive drug therapy, mechanical ventilation, other organ support (e.g., continuous hemofiltration, extracorporeal membrane oxygenation). Triage to higher levels of care (e.g., intensive care unit, intermediate care unit) is also encompassed as a therapeutic measure. If there is no risk of severe complications, the patient can be discharged home and managed in an outpatient setting or admitted to the hospital with a lower level of care (e.g., general ward). Thanks to the present invention, patients can be assessed early by biomarker determination, thus preventing life-threatening developments. The biomarker pairs and triplets identified in the studies underlying the present invention are a reliable basis for medical decisions, and the assessment can be carried out in a time- and cost-effective manner.

[0096] Thus, the method of the invention may further comprise recommending or initiating an appropriate therapeutic measure. Typically, the appropriate therapeutic measure is selected from medical guidelines or recommendations for the management of sepsis, such as the International Guidelines for the Management of Sepsis and Septic Shock (Intensive Care Med, 2017). For example, the therapeutic measure may be treatment of sepsis, or further diagnostic investigations, or other aspects of care deemed necessary by a professional.

[0097] In one embodiment, the therapeutic measures recommended or initiated if a patient is assessed as being at risk are selected from the following: • Administration of empirical broad-spectrum therapy using at least one broad-spectrum antibiotic, such as a cephalosporin, a beta-lactam / beta-lactamase inhibitor (e.g., piperacillin), or a carbapenem, typically depending on the organisms considered likely pathogens and their antibiotic susceptibility; Fluid resuscitation Administration of one or more vasoconstrictors, such as administration of norepinephrine Administration of one or more corticosteroids, such as hydrocortisone

[0098] The definitions given herein above apply mutatis mutandis below.

[0099] The present invention also provides a computer-implemented method for assessing a subject suspected of infection, comprising: (a) receiving a value for the amount of a first biomarker in a sample from a subject, wherein the first biomarker is GDF-15; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the value for the amount of the biomarker with a standard for that biomarker and / or calculating a score for assessing the subject as suspected of being infected based on the amount of the biomarker; and (d) evaluating the subject based on the comparisons and / or calculations performed in step (c). The present invention relates to a computer-implemented method for assessing a subject suspected of being infected, comprising:

[0100] As used herein, the term "computer-implemented" means that the method is performed in an automated manner on a data processing unit, typically included in a computer or similar data processing device. The data processing unit shall receive values ​​for the amounts of the biomarkers. Such values ​​may be amounts, relative amounts, or any other calculated values ​​reflecting the amounts detailed elsewhere herein. It should therefore be understood that the above-mentioned methods do not require the determination of the amounts of the biomarkers, but rather use values ​​for the amounts already pre-determined.

[0101] Typically, in the process step (b), (i) if a value for the amount of sFLT1 is received as the second biomarker, the method further comprises receiving a value for the amount of sTREM-1, antithrombin or cystatin C as a third biomarker; or (ii) if a value for the amount of cystatin C is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin, alanine aminotransferase, or aspartate aminotransferase as a third biomarker; (iii) if a value for the amount of IGFBP-7 is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin or procalcitonin as a third biomarker; (iv) if a value for the amount of bilirubin is received as the second biomarker, the method further comprises receiving a value for the amount of creatinine as a third biomarker; or (v) When a value for the amount of sTREM-1 is received as the second biomarker, the method further comprises receiving a value for the amount of aspartate aminotransferase as a third biomarker.

[0102] The present invention also in principle contemplates a computer program, a computer program product, or a computer readable storage medium tangibly embodied with said computer program, the computer program comprising instructions which, when executed on a data processing device or computer, perform the method of the present invention as described above. - a computer or a computer network comprising at least one processor, the processor being configured to execute a method according to one of the embodiments described herein, - a computer-loadable data structure adapted to carry out a method according to one of the embodiments described herein while said data structure is executed on a computer, - a computer script, the computer program being adapted to carry out the method of one of the embodiments described herein while the program is running on a computer, a computer program comprising program means for carrying out a method according to one of the embodiments described herein while said computer program is being run on a computer or on a computer network, - a computer program comprising program means according to any preceding embodiment, the program means being stored on a computer readable storage medium; a storage medium on which a data structure is stored and adapted to carry out a method according to one of the embodiments described herein after the data structure has been loaded into a primary and / or working storage device of a computer or a computer network, a computer program product comprising program code means, in which the program code means may be stored or may be stored on a storage medium for performing a method according to one of the embodiments described in this specification when the program code means are executed on a computer or a computer network, - a typically encrypted data stream signal containing data of the parameters defined elsewhere in this specification, as well as - a typically encrypted data stream signal containing the assessment provided by the method of the invention.

[0103] The present invention relates to a device for assessing a subject suspected of infection, comprising: (a) a measuring unit for measuring the amount of a first biomarker, GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase, in a sample from a subject, the measuring unit comprising a detection system for the first and second biomarkers; (b) an evaluation unit operatively linked to the measurement unit, the evaluation unit comprising a database with stored criteria for the first and second biomarkers, preferably as described above, and a data processor, preferably as described above, comprising instructions for performing a comparison of the amounts of the first and second biomarkers with the criteria and / or for performing a calculation of a score for assessing a subject suspected of infection based on the amounts of the biomarkers, and for assessing the subject based on the comparison, the evaluation unit being capable of automatically receiving values ​​for the amounts of the biomarkers from the measurement unit; The present invention relates to a device for assessing a subject suspected of infection, comprising:

[0104] As used herein, the term "device" relates to a system comprising the aforementioned units operatively linked to each other to allow the determination of the amount of a biomarker and its evaluation according to the method of the present invention, such that an assessment can be provided.

[0105] The analytical unit typically comprises at least one reaction zone having biomarker detection agents for the first and second biomarker, and preferably also for the third biomarker, in immobilized form on a solid support or carrier that is brought into contact with the sample. Furthermore, in the reaction zone it is possible to apply conditions that allow specific binding of the detection agent(s) to the biomarker contained in the sample.

[0106] The reaction zone may directly allow sample application or may be connected to a loading zone where 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 shall also be connected to a detector. The connection must be such that the detector is able to detect the binding of the biomarkers to their detection agents. The appropriate connection depends on the technique used to measure the presence or amount of the biomarkers. For example, for optical detection, light transmission may be required between the detector and the reaction zone, while for electrochemical determination, for example, a fluid connection may be required between the reaction zone and an electrode.

[0107] The detector shall be adapted to detect the determination of the amount of the biomarker. The determined amount can then be transmitted to an evaluation unit, which comprises a data processing element such as a computer with an implemented algorithm for determining the amount present in the sample.

[0108] The processing units referred to by the methods of the present invention typically comprise a central processing unit (CPU) and / or one or more graphic 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 element may be, for example, a general purpose computer or a portable computing device. It should also be understood that multiple computing devices may be used together, such as via a network or other method 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, servers, etc. In general, the data processing element comprises a processor capable of executing multiple instructions (such as a program of software).

[0109] The evaluation unit typically comprises a memory or has access to a memory. The memory is a computer-readable medium and may include, for example, a single storage device or multiple storage devices located locally with the computing device or accessible to the computing device over a network. The computer-readable medium may be any available medium that can be accessed by the computing device and includes both volatile and non-volatile media. Furthermore, the computer-readable medium may be one or both of removable and non-removable media. By way of example and not limitation, 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 cassettes, magnetic tapes, magnetic disk storage or other magnetic storage devices, or any other medium that can be accessed by a computing device and used to store a plurality of instructions that can be executed by a processor of the computing device.

[0110] According to embodiments of the present disclosure, the software may include instructions that, when executed by a processor of a computing device, may 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 may therefore operate via those control signals to transform materials remote from the computer itself. These descriptions and representations are, for example, the means used by those skilled in the art of data processing to most effectively convey the substance of their work to others skilled in the art.

[0111] The instructions may also include an algorithm, which is generally conceived to be a self-consistent sequence of steps leading to a desired result. These steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals that can be stored, transferred, transformed, combined, compared, and otherwise manipulated. It has proven convenient at times, primarily for reasons of common usage, to refer to these signals as values, characters, representations, numbers, or the like, as references to the physical items or representations that such signals are embodied or represented. It should be noted, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely used as convenient labels applied to these quantities.

[0112] The evaluation unit may also include or have access to an output device, such as, for example, a fax, a display, a printer, a file, etc. According to some embodiments of the present disclosure, a computing device may perform one or more steps of the methods disclosed herein and then provide output regarding the results, instructions, ratios, or other factors of the methods via the output device.

[0113] Typically, the measurement unit measures a third biomarker and comprises a detection system for the third biomarker, and the database includes stored references for the third biomarker, the third biomarker being: (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) When sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0114] More typically, the detection system includes at least one detection agent capable of specifically detecting each of the biomarkers.

[0115] The present invention contemplates a device for assessing a subject suspected of infection, comprising an evaluation unit comprising a database with stored criteria for a first biomarker, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase and aspartate aminotransferase, and a data processor, preferably as described above, comprising instructions for performing a comparison of the amount of the first and second biomarker with the criteria and for assessing the subject based on the comparison, wherein the evaluation unit is capable of receiving a value for the amount of the biomarker determined in a sample of the subject.

[0116] Typically, the database includes stored references for a third biomarker, the third biomarker being: (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) When sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0117] The present invention also relates in principle to the use of a first biomarker, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, 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 infection.

[0118] As used herein, the term "detection agent" typically refers to any agent that specifically binds to a biomarker, i.e., does not cross-react with other components present in the sample. Typically, detection agents that specifically bind to biomarkers referred to herein can be antibodies, antibody fragments or derivatives, aptamers, ligands of the biomarkers, receptors of the biomarkers, enzymes known to bind and / or convert the biomarkers, or small molecules known to specifically bind to the biomarkers. For example, antibodies referred to herein as detection agents include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)2 fragments capable of binding to antigens or haptens. 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 the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence typically includes at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally related amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. The aptamer detection agent may be, for example, a nucleic acid or peptide aptamer. Methods for preparing such aptamers are well known in the art. For example, random mutations can be introduced into the nucleic acid or peptide on which the aptamer is based. These derivatives can then be tested for binding according to screening procedures known in the art, for example phage display. Specific binding of the detection agent means that it should not substantially bind, i.e. cross-react, with another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound biomarker should bind with an affinity at least 3 times higher, more preferably at least 10 times higher, 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 high abundance in the sample.

[0119] The detection agent may be permanently or reversibly fused or linked to a detectable label. Suitable labels are well known to those skilled in the art. A suitable detectable label is any label that can be detected by a suitable detection method. Exemplary labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels (including "e.g. magnetic beads", paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate available as ready-made stock solutions from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). The appropriate enzyme-substrate combination may produce a colored reaction product, fluorescence or chemiluminescence, which can be measured by methods known in the art (e.g., using a light-sensitive film or a suitable camera system). For measuring enzyme reactions, the above criteria apply as well. Exemplary fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also contemplated. Exemplary radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any method known and appropriate, such as a light-sensitive film or a phosphor imager.Suitable labels may also be or include tags such as biotin, digoxigenin, His-Tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, etc.

[0120] Preferred agents for biomarkers such as AST, ALT, bilirubin and creatinine are described, for example, in the Examples, see Example 1.

[0121] When the biomarker is an enzyme such as AST or ALT, the detection agent can be a substrate for the enzyme, or any agent used for detection (see Examples).

[0122] In one embodiment, the detection agent for ALT (ALAT) is, for example, L-alanine.

[0123] In one embodiment, the detection agent for AST (ASAT) is, for example, L-aspartic acid.

[0124] The detection agent for creatinine is, for example, creatinase or any agent used for detection (see examples).

[0125] An example of a detector for albumin is bromocresol purple.

[0126] Detecting agents for bilirubin are, for example, sodium nitrite and sulfanilic acid, or any agent used for detection (see examples).

[0127] The determination of the biomarkers described herein may include mass spectrometry (MS) performed after a separation step (e.g. by LC or HPLC). Mass spectrometry as used herein encompasses all techniques that allow the determination of the molecular weight (i.e. mass) or mass variable corresponding to the compound, i.e. biomarker, determined according to the present invention. Preferably, mass spectrometry as used herein relates to GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF, or any combination approach using the aforementioned techniques. How to apply these techniques is well known to the skilled person. Moreover, suitable devices are commercially available. More preferably, mass spectrometry as used herein relates to LC-MS and / or HPLC-MS, i.e. mass spectrometry operatively linked to a prior liquid chromatographic separation step. Preferably, the mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS, involves two or more mass analysis steps, with fragmentation occurring between the steps. In tandem mass spectrometry, there are two mass spectrometers in series connected by a collision cell. The mass spectrometer is coupled to a chromatography device. The chromatographically separated sample is selected and weighed in the first mass analyzer, then fragmented by an inert gas in the collision cell, and selected and weighed in the second mass analyzer. The fragments are selected and weighed in the second mass analyzer. Identification by MS / MS is more accurate.

[0128] In one embodiment, mass analysis as used herein encompasses quadrupole MS. Most preferably, said quadrupole MS is carried out as follows: a) selection of the mass / charge quotient (m / z) of ions generated by ionization in a first analytical quadrupole of a mass spectrometer, b) fragmentation of the ions selected in step a) by applying an accelerating voltage in an additional subsequent quadrupole filled with collision gas and acting as a collision chamber, c) selection of the mass / charge quotient of ions generated by the fragmentation process of step b) in an additional subsequent quadrupole, whereby steps a) to c) of the method are carried out at least once and an analysis of the mass / charge quotient of all ions present in the mixture of substances as a result of the ionization process is carried out, whereby the quadrupole is filled with collision gas but no accelerating voltage is applied during the analysis. Details regarding said most preferred mass analysis used according to the present invention can be found in WO 2003 / 073464.

[0129] More preferably, the mass spectrometry is liquid chromatography (LC) MS, such as high performance liquid chromatography (HPLC) MS, in particular HPLC-MS / MS. As used herein, liquid chromatography refers to all techniques that allow the separation of compounds (i.e. metabolites) in a liquid or supercritical phase.

[0130] For mass spectrometry, the analytes in a sample are ionized to generate charged molecules or molecular fragments. The mass-to-charge of the ionized analytes, particularly the ionized biomarkers or fragments thereof, is then measured. Prior to ionization, the sample may be subjected to cleavage with a protease, for example trypsin. The protease cleaves the protein biomarkers into smaller fragments.

[0131] Thus, the mass analysis step preferably includes an ionization step in which the biomarkers to be determined are ionized. Of course, other compounds present in the sample / eluent are also ionized. The ionization of the biomarkers can be carried out by any method considered appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI).

[0132] In a preferred embodiment, the ionization step (for mass spectrometry) is performed by electrospray ionization (ESI). Mass spectrometry is therefore preferably ESI-MS (or ESI-MS / MS, in case of performing tandem MS). Electrospray is a soft ionization method that forms ions without breaking chemical bonds.

[0133] More typically, a third biomarker or a detection agent that specifically binds to the third biomarker is further used, the third biomarker being (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) When sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0134] 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 GDF-15, and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase and aspartate aminotransferase.

[0135] As used herein, the term "kit" refers to a collection of the above-mentioned components typically provided in separate or single containers. The container also typically contains instructions for carrying out the method of the invention. These instructions may be in the form of a manual or may be provided by a computer program code that, when implemented in a computer or data processing device, can carry out or assist in the determination of the biomarkers referred to in the method of the invention. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., compact disc), or directly on the computer or data processing device, or in a downloadable form, such as a link to an accessible server or cloud. Furthermore, the kit may typically include standards of reference amounts of biomarkers for calibration purposes, as described in detail elsewhere herein. The kit according to the invention may also include further components necessary for carrying out the method of the invention, such as solvents, buffers, washing solutions and / or reagents necessary for the detection of the released second molecule. Furthermore, it may partially or entirely include the device of the invention.

[0136] More typically, the kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) When sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0137] Therefore, it is to be understood that the above definitions and explanations of terms apply to all embodiments described in this specification and the appended claims. The following embodiments are specific embodiments contemplated in accordance with the present invention.

[0138] Embodiment 1: A method for assessing a subject suspected of infection, comprising: (a) determining the amount of a first biomarker in a sample from a subject, wherein the first biomarker is GDF-15; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the amount of the biomarker to a standard for said biomarker and / or calculating a score for assessing the subject as suspected of being infected based on the amount of the biomarker; and (d) evaluating the subject based on the comparisons and / or calculations performed in step (c). A method for assessing said subject suspected of infection, comprising:

[0139] Embodiment 2: In step (b), (i) if the amount of sFLT1 is determined as the second biomarker, the method further comprises determining the amount of sTREM-1, antithrombin or cystatin C as a third biomarker; (ii) if the amount of cystatin C is determined as the second biomarker, the method further comprises determining the amount of bilirubin, alanine aminotransferase or aspartate aminotransferase as a third biomarker; (iii) if the amount of IGFBP-7 is determined as the second biomarker, the method further comprises determining the amount of bilirubin or procalcitonin as a third biomarker; (iv) if the amount of bilirubin is determined as the second biomarker, the method further comprises determining the amount of creatinine as a third biomarker; or (v) The method of the embodiment, wherein if the amount of sTREM-1 is determined as the second biomarker, the method further comprises determining the amount of aspartate aminotransferase as a third biomarker.

[0140] Embodiment 3: The method of embodiment 1 or 2, wherein the subject is a subject presenting to an emergency department.

[0141] Embodiment 4: The method of any one of embodiments 1 to 3, wherein said assessment is an assessment of the subject's risk of developing sepsis and / or an assessment of the subject's risk of worsening of the subject's condition.

[0142] Embodiment 5: The method of any one of embodiments 1-4, wherein said references are for each biomarker derived from at least one subject known to be at risk for developing sepsis, and preferably, an amount for each of said biomarkers that is essentially the same as or similar to the corresponding reference is indicative of a subject at risk for developing sepsis, and an amount for each of said biomarkers that differs from the corresponding reference is indicative of a subject not at risk for developing sepsis.

[0143] Embodiment 6: The method of any one of embodiments 1-4, wherein said references are for each biomarker derived from at least one subject known to be not at risk of developing sepsis, and preferably, an amount for each of said biomarkers that is essentially the same or similar to the corresponding reference is indicative of a subject not at risk of developing sepsis, and an amount for each of said biomarkers that differs from the corresponding reference is indicative of a subject at risk of developing sepsis.

[0144] Embodiment 7: The method of any one of embodiments 1-6, wherein the subject has or is suspected of having an infectious disease.

[0145] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the sample is a blood sample or a sample derived therefrom.

[0146] Embodiment 9: The method of any one of embodiments 1 to 8, wherein the subject is a human.

[0147] Embodiment 10: A computer-implemented method for assessing a subject suspected of infection, comprising: (a) receiving a value for the amount of a first biomarker in a sample from a subject, wherein the first biomarker is GDF-15; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the value for the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing the subject as suspected of infection based on the amount of the biomarker; and (d) evaluating the subject based on the comparisons and / or calculations performed in step (c). A computer-implemented method for assessing a subject suspected of being infected, comprising:

[0148] Embodiment 11: In step (b), (i) if a value for the amount of sFLT1 is received as the second biomarker, the method further comprises receiving a value for the amount of sTREM-1, antithrombin or cystatin C as a third biomarker; or (ii) if a value for the amount of cystatin C is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin, alanine aminotransferase, or aspartate aminotransferase as a third biomarker; (iii) if a value for the amount of IGFBP-7 is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin or procalcitonin as a third biomarker; (iv) if a value for the amount of bilirubin is received as the second biomarker, the method further comprises receiving a value for the amount of creatinine as a third biomarker; or (v) The method of embodiment 10, wherein when a value for the amount of sTREM-1 is received as the second biomarker, the method further comprises receiving a value for the amount of aspartate aminotransferase as a third biomarker.

[0149] Embodiment 12: A device for assessing a subject suspected of infection, comprising: (a) a measuring unit for measuring the amount of a first biomarker, GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase, in a sample from a subject, the measuring unit comprising a detection system for the first and second biomarkers; (b) an evaluation unit operatively coupled to said measurement unit, said evaluation unit comprising a database with stored references for said first biomarker and said second biomarker, preferably of any one of embodiments 1-9, and a data processor comprising instructions for performing a comparison of said amounts of said first biomarker and said second biomarker with a reference and / or for performing a calculation of a score for assessing said subject suspected of infection based on said amounts of said biomarkers, and for assessing said subject based on said comparison, said evaluation unit being capable of automatically receiving values ​​for said amounts of said biomarkers from said measurement unit; A device for assessing a subject suspected of infection, comprising:

[0150] Embodiment 13: The measurement unit measures a third biomarker and comprises a detection system for a third biomarker, the database comprises stored references for a third biomarker, and the third biomarker is (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) The device of embodiment 12, wherein when sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0151] Embodiment 14: The device of embodiment 12 or 13, wherein said detection system comprises at least one detection agent capable of specifically detecting each of said biomarkers.

[0152] Embodiment 15: A device for assessing a subject suspected of infection, comprising an evaluation unit comprising a database with stored criteria for a first biomarker, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase, and a data processor, preferably according to any one of embodiments 1 to 11, comprising instructions for performing a comparison of the amounts of the first and second biomarkers with a criteria and for assessing the subject based on the comparison, wherein the evaluation unit is capable of receiving a value for the amount of the biomarkers determined in a sample of the subject.

[0153] Embodiment 16: The database comprises stored references for a third biomarker, the third biomarker being: (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) The device of embodiment 15, wherein when sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0154] Embodiment 17: The use of i) a first biomarker which is GDF-15 and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase, or ii) 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 infection.

[0155] Embodiment 18: A third biomarker or a detection agent that specifically binds to said third biomarker is additionally used, said third biomarker being: (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) The use of embodiment 17, wherein when sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0156] Embodiment 19: A kit for assessing a subject suspected of having an infection, comprising a detection agent that specifically binds to a first biomarker, which is GDF-15, and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase and aspartate aminotransferase.

[0157] Embodiment 20: The kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being: (i) if sFLT1 is the second biomarker, sTREM-1, antithrombin or cystatin C; (ii) bilirubin, alanine aminotransferase, or aspartate aminotransferase if cystatin C is the second biomarker; (iii) if IGFBP-7 is the second biomarker, bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) The kit of embodiment 19, wherein when sTREM-1 is the second biomarker, it is aspartate aminotransferase.

[0158] Embodiment 21: The method, device, use, or kit of any one of the preceding embodiments, wherein said assessment is an assessment of risk of developing sepsis.

[0159] Embodiment 21: The method, device, use or kit of any one of the preceding embodiments, wherein the risk of developing sepsis within 48 hours is predicted.

[0160] All references cited throughout this specification are hereby incorporated by reference in their entirety for the disclosure content specifically mentioned above. EXAMPLES

[0161] The examples are merely illustrative of the invention and should not be construed as limiting its scope.

[0162] Example 1: Biomarker Determination The Elecsys® Electro-ChemiLuminescence (ECL) technology and assay method for the measurement of GDF-15 are briefly described below. The concentration of GDF-15 was determined by a cobas e801 analyzer. The detection of GDF-15 by the Cobas e 801 analyzer is based on the Elecsys® Electro-ChemiLuminescence (ECL) technology. Briefly, biotin-labeled and ruthenium-labeled antibodies are combined with the respective amounts of undiluted sample and incubated in the analyzer. Subsequently, streptavidin-coated magnetic microparticles are added and incubated in the instrument to promote the binding of the biotin-labeled immune complexes. After this incubation step, the reaction mixture is transferred to the measurement cell, where the beads are magnetically captured on the surface of the electrode. Then, to separate the bound immunoassay complexes from the remaining free particles, ProCell M buffer containing tripropylamine (TPA) for the subsequent ECL reaction is introduced into the measurement cell. The induction of a voltage between the working and counter electrodes then initiates a reaction resulting in the emission of photons by the ruthenium complex and TPA. The electrochemiluminescence signal obtained by the photomultiplier tube is recorded and converted to a numerical value indicative of the concentration level of the respective analyte.

[0163] SFLT1 or sFLT-1 (soluble fms-like tyrosine kinase-1) was measured with the commercially available ECLIA assay for sFLT-1, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated monoclonal antibody and a rutheniumylated monoclonal antibody that specifically binds to sFLT-1. 12 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0164] PCT (procalcitonin) was measured with the commercially available ECLIA assay for procalcitonin, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to PCT. 18 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0165] GDF15 (growth / differentiation factor 15) was measured with the commercial ECLIA assay for GDF-15, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to GDF-15. 21 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0166] CysC2 (cystatin C) was measured with a commercially available PETIA (particle-enhanced immunoturbidimetric assay) for CysC developed for the cobas® clinical chemistry analyzer platform (Roche Diagnostics, Germany). The assay contains latex particles coated with an antibody that specifically binds to CysC. Upon mixing and incubation of the antibody reagent with the sample, the latex enhances the anti-cystatin C antibody-coated particles in the reagent and agglutinates with human cystatin C in the sample. The degree of turbidity caused by the aggregates can be determined nephelometrically at 546 nm and is proportional to the amount of cystatin C in the sample. 2 μL from each serum sample was used and measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0167] TNTHS or cTNTh (cardiac troponin T) was measured with the commercially available ECLIA assay for High Sensitivity-cTroponinT, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to cTnThs. 50 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0168] FERR (ferritin) was measured with the commercially available ECLIA assay for ferritin, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to ferritin. 10 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0169] PBNP or NTpBNP (N-terminal prohormone of brain natriuretic peptide) was measured with the commercial ECLIA assay for NTproBNP, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to NTproBNP. 15 μL was used from each serum sample and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0170] IGFBP7 (insulin-like growth factor binding protein 7) was measured with the IGFBP-7 robust prototype ECLIA assay, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to IGFBP-7. 10 μL from each serum sample was used and measured undiluted on a cobas e601 analyzer (Roche Diagnostics, Germany).

[0171] ESM1 (endothelial cell specific molecule 1) was measured with the ESM-1 robust prototype ECLIA assay, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to ESM-1. 20 μL from each serum sample was used and measured undiluted on a cobas e601 analyzer (Roche Diagnostics, Germany).

[0172] STREM1 or sTREM-1 (soluble triggering receptor expressed on myeloid cells 1) was measured with the sTREM-1 robust prototype ECLIA assay, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated monoclonal antibody and a rutheniumylated monoclonal antibody that specifically binds to TREM-1. 50 μL from each serum sample was used and measured undiluted on a cobas e601 analyzer (Roche Diagnostics, Germany).

[0173] CREP2 (creatinine): This enzymatic method is based on the conversion of creatinine to glycine, formaldehyde, and hydrogen peroxide with the aid of creatininase, creatinase, and sarcosine oxidase. When catalyzed by peroxidase, the liberated hydrogen peroxide reacts with 4-aminophenazone and HTIB a) to form a quinoneimine chromogen. The color intensity of the formed quinoneimine chromogen 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 measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0174] AT.pc (Antithrombin percentage): A kinetic colorimetric test. This test works according to the principle of the antithrombin (AT) heparin cofactor assay. Heparin and a defined amount of thrombin are added in excess to the sample. Any free antithrombin present binds to thrombin forming an inactive complex. Uninhibited thrombin liberates p-nitroaniline from the chromogenic substrate MeOCO-Gly-Pro-Arg-pNA. The remaining amount of thrombin is inversely proportional to the antithrombin content of the sample, so that the increase in absorbance at a wavelength of 415 nm can be used to calculate the antithrombin activity. Assay from Roche Diagnostics (Germany). 1 μL of plasma was analyzed. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0175] BILI (Bilirubin): Diazotized sulfanilic acid is formed by combining sodium nitrite and sulfanilic acid at 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, solubilized bilirubin, including conjugated bilirubin (monogluconides and digluconides) and delta form 2 (biliprotein-bilirubin covalently bound to albumin), is converted to diazobilirubin, a red chromophore that absorbs at 540 nm and represents total bilirubin, and is measured using a dichroic (540 nm, 700 nm) endpoint technique. Sample blank correction is used.

[0176] ALAT (Alanine Aminotransferase): Alanine aminotransferase catalyzes the transamination of L-alanine to α-ketoglutarate (α-KG) to form L-glutarate and pyruvate. The pyruvate formed is reduced to lactate by lactate dehydrogenase (LDH) with the concomitant oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance is directly proportional to the alanine aminotransferase activity and is measured using a dichroic (340 nm, 700 nm) kinetic technique.

[0177] ASAT (Aspartate Aminotransferase): Aspartate aminotransferase (AST) catalyzes the transamination of L-aspartate to α-ketoglutarate, forming L-glutamate and oxalate. The oxalate formed is reduced to malate by malate dehydrogenase (MDH) with the concomitant 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 is measured using a dichroic (340 nm, 700 nm) kinetic technique.

[0178] ALB (albumin): In the presence of a solubilizing agent, BCP binds to albumin at pH 4.9. The amount of albumin-BCP complex is directly proportional to the albumin concentration. The complex absorbs at 600 nm and is measured using a multicolor (600 nm, 540 nm, 700 nm) endpoint technique.

[0179] Example 2: Analysis of patients from the TRIAGE study TRIAGE Study,Kantonsspital Aarau,Switzerland,Emergency Department.(Schuetz 2013,BMC emergency medicine,13(1),12).

[0180] All consecutive patients seeking emergency department (ED) care for a medical emergency were included at the time of ED admission. From a total of 4000 patients, a subset of patients with suspected infection at admission was selected and classified as likely sepsis cases or infection controls according to the following: ● Cases (N=64): Sepsis cases likely to worsen / have higher severity within 48 hours of ED presentation if admitted to ICU or meet criteria from Rhee 2017, “Incidence and Trends of Sepsis in US Hospitals Using Clinical vs Claims Data, 2009-2014.” JAMA 318(13):1241-1249. ● Controls (N=207): Patients without sepsis and suspected infection within 48 hours of ED presentation.

[0181] Markers were mathematically combined by logistic regression and the "area under the receiver operating characteristic" (AUC) was used as a general measure of marker performance.

[0182] In addition to sepsis endpoints, we also assessed a "general deterioration" endpoint (i.e., whether the patient's condition worsened regardless of sepsis diagnosis) in the population of patients with suspected infection on ED admission. Patients were classified into cases and controls according to: Cases: Deterioration was defined as: increased need for care (i.e., ICU admission) or death in the hospital or death within 30 days of admission, or readmission within 30 days of discharge. Controls: Patients suspected of infection but not worsening

[0183] Marker pair combinations (bivariate marker combinations) that improved AUC over single markers by at least 1 percentage point are shown in Table 1 (for sepsis endpoints). [Table 1]

[0184] Bivariate marker pairs as well as marker triplet combinations (trivariate marker combinations) that improved the AUC by at least 1 percentage point over all three single marker combinations are shown in Table 2 (for sepsis endpoints). [Table 2]

[0185] Examples of bivariate combinations of markers that are not an improvement over single markers are shown (for sepsis endpoints) in Table 3. Table 3 demonstrates the non-obviousness of combining sepsis markers. [Table 3]

[0186] For the deterioration endpoint, combinations of marker pairs (bivariate marker combinations) that improved the AUC by at least 1 percentage point over single markers are shown in Table 4. [Table 4]

[0187] Examples of bivariate combinations of markers for exacerbation endpoints that are not improved over single markers are shown in Table 5. Table 5 demonstrates the non-obviousness of combining sepsis markers. [Table 5]

Claims

1. 1. A method for assessing a subject suspected of infection, comprising: (a) determining the amount of a first biomarker in the subject's sample, wherein the first biomarker is GDF-15; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, a cardiac troponin, e.g., cardiac troponin T or I, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing the subject as suspected of infection based on the amount of the biomarker; and (d) assessing the subject based on the comparison and / or the calculation performed in step (c). A method for assessing a subject suspected of being infected, comprising:

2. In step (b), (i) if the amount of sFLT1 is determined as the second biomarker, the method further comprises determining the amount of sTREM-1, antithrombin, or cystatin C as a third biomarker; (ii) if the amount of cystatin C is determined as the second biomarker, the method further comprises determining the amount of bilirubin, alanine aminotransferase, or aspartate aminotransferase as a third biomarker; (iii) if the amount of IGFBP-7 is determined as the second biomarker, the method further comprises determining the amount of bilirubin or procalcitonin as a third biomarker; (iv) if the amount of bilirubin is determined as the second biomarker, the method further comprises determining the amount of creatinine as a third biomarker; or (v) if the amount of sTREM-1 is determined as the second biomarker, the method further comprises determining the amount of aspartate aminotransferase as a third biomarker. The method of claim 1.

3. 10. The method of claim 1, wherein the subject is a subject presenting to an emergency department.

4. The method of claim 1 , 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. and / or wherein the reference is a reference for each biomarker derived from at least one subject known to be at risk of developing sepsis, preferably wherein an amount for each of the biomarkers is essentially the same as or similar to the corresponding reference indicative of a subject at risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference indicative of a subject not at risk of developing sepsis; 2. The method of claim 1, wherein the reference is a reference for each biomarker derived from at least one subject known to be not at risk of developing sepsis, and preferably, an amount for each of the biomarkers that is essentially the same or similar to the corresponding reference is indicative of a subject not at risk of developing sepsis, and an amount for each of the biomarkers that is different from the corresponding reference is indicative of a subject at risk of developing sepsis.

6. 10. The method of claim 1, wherein the subject has or is suspected of having an infectious disease.

7. The method of claim 1 , wherein the sample is a blood sample or a sample derived therefrom, such as serum or plasma, and / or the subject is a human.

8. 1. A computer-implemented method for assessing a subject suspected of infection, comprising: (a) receiving a value for the amount of a first biomarker in the subject's sample, wherein the first biomarker is GDF-15; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; (c) comparing the value for the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing the subject as suspected of infection based on the amount of the biomarker; and (d) assessing the subject based on the comparison and / or the calculation performed in step (c). Including, Optionally, in step (b), (i) if a value for the amount of sFLT1 is received as the second biomarker, the method further comprises receiving a value for the amount of sTREM-1, antithrombin, or cystatin C as a third biomarker; (ii) if a value for the amount of cystatin C is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin, alanine aminotransferase, or aspartate aminotransferase as a third biomarker; (iii) if a value for the amount of IGFBP-7 is received as the second biomarker, the method further comprises receiving a value for the amount of bilirubin or procalcitonin as a third biomarker; (iv) if a value for the amount of bilirubin is received as the second biomarker, the method further comprises receiving a value for the amount of creatinine as a third biomarker; or (v) if a value for the amount of sTREM-1 is received as the second biomarker, the method further comprises receiving a value for the amount of aspartate aminotransferase as a third biomarker. A computer-implemented method for assessing a subject suspected of infection.

9. 1. A device for assessing a subject suspected of infection, comprising: (a) a measuring unit for measuring the amount of a first biomarker, GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase, in a sample from said subject, said measuring unit comprising a detection system for said first biomarker and said second biomarker; (b) an evaluation unit operatively coupled to said measurement unit, said evaluation unit comprising a database with stored references for said first and second biomarkers, preferably according to claim 1, and a data processor comprising instructions for performing a comparison of said amounts of said first and second biomarkers with references and / or for performing a calculation of a score for assessing said subject suspected of infection based on said amounts of said biomarkers, and for assessing said subject based on said comparison, said evaluation unit being capable of automatically receiving values ​​for said amounts of said biomarkers from said measurement unit; A device for assessing a subject suspected of infection, comprising:

10. The measurement unit measures a third biomarker and comprises a detection system for the third biomarker, the database includes stored references for the third biomarker, and the third biomarker is (i) when sFLT1 is the second biomarker, it is sTREM-1, antithrombin or cystatin C; (ii) when cystatin C is the second biomarker, it is bilirubin, alanine aminotransferase, or aspartate aminotransferase; (iii) when IGFBP-7 is the second biomarker, it is bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) when sTREM-1 is the second biomarker, it is aspartate aminotransferase; The device of claim 9.

11. The device of claim 9 , wherein the detection system comprises at least one detection agent capable of specifically detecting each of the biomarkers.

12. A device for assessing a subject suspected of infection, comprising an evaluation unit comprising a database with stored criteria for a first biomarker, which is GDF-15, and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase and aspartate aminotransferase, and a data processor comprising instructions for carrying out a comparison of the amounts of said first and second biomarkers, preferably according to claim 1, with a criteria and for assessing said subject on the basis of said comparison, said evaluation unit being capable of receiving a value for the amount of said biomarker determined in a sample of said subject, The database includes stored references for a third biomarker, the third biomarker being: (i) when sFLT1 is the second biomarker, it is sTREM-1, antithrombin or cystatin C; (ii) when cystatin C is the second biomarker, it is bilirubin, alanine aminotransferase, or aspartate aminotransferase; (iii) when IGFBP-7 is the second biomarker, it is bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) when sTREM-1 is the second biomarker, it is aspartate aminotransferase; A device for assessing subjects suspected of infection.

13. The use of i) a first biomarker which is GDF-15 and a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptide, alanine aminotransferase, and aspartate aminotransferase, or ii) a detection agent which specifically binds to the first biomarker and a detection agent which specifically binds to the second biomarker, to assess a subject suspected of being infected.

14. A third biomarker or a detection agent that specifically binds to said third biomarker may additionally be used, said third biomarker being: (i) when sFLT1 is the second biomarker, it is sTREM-1, antithrombin or cystatin C; (ii) when cystatin C is the second biomarker, it is bilirubin, alanine aminotransferase, or aspartate aminotransferase; (iii) when IGFBP-7 is the second biomarker, it is bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) when sTREM-1 is the second biomarker, it is aspartate aminotransferase; 14. The use according to claim 13.

15. 1. A kit for assessing a subject suspected of having an infection, comprising: a detection agent that specifically binds to a first biomarker, the first biomarker being GDF-15; and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFLT1, cystatin C, IGFBP-7, bilirubin, ESM-1, sTREM-1, procalcitonin, cardiac troponin, BNP-type peptides, alanine aminotransferase, and aspartate aminotransferase; Optionally, the kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being: (i) when sFLT1 is the second biomarker, it is sTREM-1, antithrombin or cystatin C; (ii) when cystatin C is the second biomarker, it is bilirubin, alanine aminotransferase, or aspartate aminotransferase; (iii) when IGFBP-7 is the second biomarker, it is bilirubin or procalcitonin; (iv) creatinine when bilirubin is the second biomarker; or (v) when sTREM-1 is the second biomarker, it is aspartate aminotransferase; A kit for assessing subjects suspected of infection.

16. The method, device, use or kit according to any one of claims 1 to 15, wherein said assessment is an assessment of the risk of developing sepsis.

17. 16. The method, device, use or kit according to any one of claims 1 to 15, wherein the risk of developing sepsis within 48 hours is predicted.