MR-proADM marker panel for early detection of sepsis

Abstract

The present invention relates to the field of diagnosis. In particular, the present invention 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 MR-proADM, determining the amount of a second biomarker in a sample of the subject, the second biomarker being selected from the group consisting of sFlt-1, GDF15 and ESM1, 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 the first biomarker being MR-proADM and the second biomarker being selected from the group consisting of sFlt-1, GDF15 and ESM1, or a detection agent specifically binding to the first biomarker and a detection agent specifically binding to the second biomarker, for assessing a subject suspected of infection. Moreover, the present invention further relates to a computer implemented method for assessing a subject suspected of having an infection, as well as a device and a kit for assessing a subject suspected of having an infection.

Description

[Technical field]

[0001] The present invention relates to the field of diagnosis. In particular, the present invention 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 MR-proADM, determining the amount of a second biomarker in a sample of the subject, the second biomarker being selected from the group consisting of sFlt-1, GDF15 and ESM1, 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 the first biomarker being MR-proADM and the second biomarker being selected from the group consisting of sFlt-1, GDF15 and ESM1, or a detection agent specifically binding to the first biomarker and a detection agent specifically binding to the second biomarker, for assessing a subject suspected of infection. Moreover, the present invention further relates to a computer-implemented method for assessing a subject suspected of having an infection, as well as devices and kits for assessing a subject suspected of having an infection. [Background technology]

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

[0003] According to the Sepsis-3 definition, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis develops rapidly, so early recognition is critical for the management of septic patients and for the initiation of correct therapeutic measures, including appropriate antibiotic treatment within the first hour of hospitalization, and 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. There is no gold standard for the diagnosis of sepsis so far in general departments and especially in emergency departments. In high-income countries, C-reactive protein (CRP), procalcitonin (PCT) and white blood cell (WBC) count are often used in emergency rooms for the detection of patients with bloodstream infections at risk for the development of sepsis, along with lactate for the detection of septic shock. In low-income countries, the diagnosis is mostly based on clinical signs and symptoms, and in some cases SIRS and SOFA criteria. However, in most current guidelines, no biomarkers are listed to diagnose sepsis (with the exception of clinical chemistry, BGE and hematological components of the SOFA score) besides lactate. 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 levels 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 2 174 143 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 for the detection or diagnosis of 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 critical or life-threatening response to a disease and / or treatment response.

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

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

[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 MR-proADM; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFlt-1, GDF15 and ESM1; (c) comparing 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 made in step (c). The present invention relates to a method comprising the steps of: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] As used below, the terms "have", "comprise" or "include" or any grammatical variants thereof are used in a non-exclusive manner. 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, and even 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 below, 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 be implemented by using alternative features, as would be understood by a person skilled in the art. Similarly, features introduced by "in one embodiment of the invention" or similar expressions are intended to be additional / alternative features, without any limitations on alternative aspects 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" means that one or more of the items followed by this term can be used according to the present invention. For example, when this term indicates that at least one sampling unit should be used, this can be understood as one sampling unit or two or more sampling units, i.e., two, three, four, five or any other number. Depending on the item to which this term refers, those skilled in the art will understand which upper limit, if any, this term can refer to.

[0016] As used herein, the term "about" means that for any number listed after the term, there exists an interval of precision within which the technical effect can be achieved. Thus, about, as referred to herein, preferably refers to the exact numerical value or a range around that exact numerical value of ±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 used to describe a chronological or chronological order.

[0018] The method of the present invention may consist of the above steps or may include additional steps, such as a step for further evaluation of the score obtained in step (d), a step for recommending a therapeutic measure, such as a treatment. Furthermore, the method of the present invention 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-mentioned method is an ex vivo method that does not require any steps to be performed on the human or animal body. Furthermore, the method may be assisted by automation. Typically, the determination of the biomarkers may be assisted by a robotic device, and the comparison and assessment may be assisted 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 at risk of suffering from sepsis, whether a subject shows worsening medical symptoms in terms of overall health status or in terms of sepsis or signs and 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 status, particularly in terms of signs and symptoms associated with sepsis and / or infection.Typically, the assessment referred to according to 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) symptoms worsening.Furthermore, when the risk of developing sepsis or the risk of a worsening health status is predicted, it will be understood that typically the prediction is performed 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, in particular at least about 48 hours after the sample is obtained. Furthermore, the risk of developing sepsis may be predicted, preferably within 24 or 48 hours after the test sample is obtained.

[0020] In one embodiment, the risk of developing sepsis within 24 hours is predicted.

[0021] In an alternative embodiment, the risk of developing sepsis within 48 hours is predicted.

[0022] A 48 hour period was analyzed in the Examples section.

[0023] 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 the condition" of the subject suspected of and / or suffering from infectious disease is well understood by those skilled in the art.This term typically relates to the worsening of the condition that may eventually lead to further medication or other intervention.

[0024] 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 of admission, if the subject is readmitted within 30 days of discharge, if the subject experiences organ dysfunction or failure as measured, for example, by SOFA score, and / or if the subject requires organ support.

[0025] One of skill in the art will appreciate when a subject's condition does not worsen. Typically, a subject's condition does not worsen if the subject does not have the results listed in the previous paragraph.

[0026] 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.

[0027] In one embodiment, predicting the risk of the subject's condition worsening is predicting the risk of the subject's antibiotic therapy being intensified.

[0028] In one embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the subject's risk of being admitted to an ICU. Thus, it is assessed whether the subject is at risk of being admitted to an ICU.

[0029] 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, it is assessed whether the subject is at risk of dying in hospital.

[0030] In yet another embodiment, the prediction of the subject's risk of worsening is a prediction of the subject's risk of death within 30 days of hospitalization. Thus, it is assessed whether the subject is at risk of dying within 30 days of admission to the 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 readmission within 30 days of discharge. Thus, it is assessed whether the subject is at risk of being readmitted within 30 days of discharge.

[0032] 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 organ failure. Organ dysfunction and organ failure can be assessed, for example, via SOFA score. Thus, the present invention further relates to the prediction of the risk of whether the subject's SOFA score increases (after the test sample is obtained). An increase in SOFA score (such as at least 1 point, at least 2 points, at least 3 points, or at least 4 points) is considered as a worsening of the condition. In contrast, if the SOFA score does not increase (but the subject does not have the highest SOFA score), the condition typically does not worsen. The prediction window may be the prediction window described above for the prediction of the risk of developing sepsis.

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

[0034] In yet another embodiment, the prediction of the risk of the subject's condition worsening is a prediction of the risk of the subject requiring organ support, such as a prediction of the risk of the subject requiring vasoactive drug therapy, hemodynamic support (e.g., fluid therapy), oxygenation (e.g., via ventilation or extracorporeal membrane oxygenation), and / or renal replacement therapy. The prediction window can 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.

[0035] 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.

[0036] As will be understood by those skilled in the art, the assessment made according to the present invention is preferably correct for 100% of the objects surveyed, but may not usually be 100% correct. This term typically requires that the statistically significant portion of the object can be accurately assessed. Whether a portion 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. Typically, the assumed confidence interval is 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.

[0037] As used herein, the term "subject" refers to an animal, preferably a mammal, more typically a human. The subject examined by the method of the present invention is a subject suspected of infection. The term "suspected of infection" as used herein means that the subject exhibits clinical parameters, signs and / or symptoms of an infectious disease. Thus, the subject according to the present invention is typically a subject suffering from an infectious disease or suspected of suffering from an infectious disease. Typically, the subject is a subject presenting to an emergency department. Advantageously, the sample is obtained at the time of consultation. Preferably, the sample is obtained at the time of consultation at the emergency department. However, the sample can also be obtained at the time of diagnosis at a primary care physician.

[0038] The term "sample" as used herein 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, or a lymphatic fluid sample. Most typically, the sample is a blood sample or a sample derived from a blood sample. Thus, the sample may be a blood, serum or plasma sample. Blood samples typically include capillary, venous or arterial blood samples. In one embodiment, the sample is an interstitial fluid sample.

[0039] 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 Sepsis-3 definition disclosed in Singer et al. (loc.cit.).

[0040] Typically, the subject to be tested is a subject suspected of suffering from an infectious disease. The term "infection" is well understood by those skilled in the art. As used herein, the term "infection" preferably refers to the invasion of the subject's body tissue by disease-causing microorganisms, their proliferation, and the reaction of the subject's tissue to the microorganisms. In one embodiment, the infectious disease is a bacterial infection. Thus, the subject is suspected of suffering from a bacterial infection.

[0041] As described elsewhere herein, the present invention allows early identification of at-risk patients. Thus, in one embodiment of the prediction described herein, the subject being tested is not suffering from sepsis at the time the sample is obtained. In a particularly preferred embodiment, the subject being tested is preferably not suffering from septic shock at the time the sample is obtained. The term "septic shock" is defined in Singer et al. (same literature). Thus, if the following criteria are met, the subject is suffering from septic shock. 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 serum lactate levels > 2 mmol / L (18 mg / dL).

[0042] It is further envisaged that the subject being tested may or may not be suffering from infection with SARS-CoV-2.

[0043] 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.

[0044] As used herein, the term "amount" refers to the absolute amount of a compound referred to herein, the relative amount or concentration of said 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 said 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 biological readout systems in response to compounds or intensity signals obtained from specifically bound ligands. It should be understood that values ​​that correlate with the above-mentioned amounts or parameters can also be obtained by all standard mathematical operations. 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.

[0045] The determination of the amount in the method of the invention can be carried out by any technique that makes it possible to detect the presence or absence or amount of the second molecule upon its release from the first molecule. Suitable techniques depend on the nature of the molecule and the characteristics of the biomarker and are discussed in more detail elsewhere in this specification.

[0046] 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 indicating 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 analytical devices such as biosensors, optical devices linked to immunoassays, biochips, 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 present invention may also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), scintillation proximity assay (SPA), nephelometry, nephelometry, latex-enhanced nephelometry or nephelometry, or solid-phase immunoassay. Further methods known in the art include 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 in the accompanying examples below.

[0047] 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 (e.g., aspartate aminotransferase). The amount of these biomarkers can also be determined by determining the activity of the enzyme in the sample.

[0048] The biomarker mid-regional pro-adrenomedullin (MRproADM) is well known in the art. It has been proposed as a marker for sepsis (Christ-Crain, M., Morgenthaler, NG, Struck, J. et al. Mid-regional pro-adrenomedullin as a prognostic marker in sepsis: an observational study. Crit Care 9, R816 (2005). https: / / doi.org / 10.1186 / cc3885). MR-proADM is a 48 amino acid long fragment derived from the proADM molecule, which is a 1:1 ratio fragment with adrenomedullin (AM). Thus, the amount of MR-proADM represents the amount and activity of adrenomedullin. AM (adrenomedullin) and PAMP (proadrenomedullin N-20 terminal peptide) are potent antihypertensive and vasodilator agents. Many actions have been reported that are most relevant to the physiological control of fluid and electrolyte homeostasis

[0049] 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 subsequently 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, etc., 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.

[0050] 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 and is expressed primarily 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 determined in a sample from a subject. The sequence of the 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, e.g., via the Uniprot database, see entry Q9NQ30 (ESM1_HUMAN). Two isoforms of ESM-1 are generated by alternative splicing, isoform 1 (having the Uniprot identifier Q9NQ30-1) and isoform 2 (having the 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).

[0051] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, ie isoform 1 has the sequence as shown under UniProt accession number Q9NQ30-1.

[0052] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, ie isoform 2 has the sequence as shown under UniProt accession number Q9NQ30-2.

[0053] In another preferred embodiment, the amount of isoform-1 and isoform-2 of the ESM-1 polypeptide, ie total ESM-1, is determined.

[0054] Aspartate aminotransferase (AST or ASAT) catalyzes the transamination of L-aspartate to α-ketoglutarate to form L-glutamate and oxaloacetate. The oxaloacetate 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 bichromatic (340 nm, 700 nm) rate technique.

[0055] 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) rate technique.

[0056] As used herein, the term "soluble Flt-1" or "sFlt-1" (abbreviation for "soluble fms-like tyrosine kinase-1") preferably refers to a polypeptide that is a soluble form of the VEGF receptor Flt1. Soluble Flt-1 was identified in the conditioned culture medium of human umbilical vein endothelial cells. The endogenous soluble Flt1 (sFlt-1) receptor is chromatographically and immunologically similar to recombinant human sFlt-1 and binds [125I]VEGF with comparable high affinity. Human sFlt-1 has been shown to form a VEGF-stabilized complex with the extracellular domain of KDR / Flk-1 in vitro. Preferably, sFlt-1 refers to human sFlt-1 as described in Kendall 1996, Biochem Biophs Res Commun 226(2):324-328 (for amino acid sequences see also, e.g., P17948, GI:125361 for human and BAA24499.1, GI:2809071 for mouse sFlt-1).

[0057] In the method according to the invention, a third biomarker may be determined. In particular, in step (b) of the method according to the invention: (i) When the amount of GDF-15 is determined as the second biomarker, the method further comprises determining the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker.

[0058] As such, 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).

[0059] The first biomarker is MR-proADM. The second biomarker is selected from sFlt-1, GDF15 and ESM1.

[0060] In one embodiment, the second biomarker is GDF-15.

[0061] In an alternative embodiment, the second biomarker is sFlt1.

[0062] In an alternative embodiment, the second biomarker is ESM1.

[0063] When GDF-15 is the second marker, the method may further comprise determining the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker. In one embodiment, MR-proADM, GDF-15 and alanine aminotransferase are determined.

[0064] In an alternative embodiment, MR-proADM, GDF-15 and aspartate aminotransferase are determined.

[0065] 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.

[0066] The term "criterion" as used herein 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, the criterion is intended to be an amount or score that allows a subject to be assigned to a group of subjects suffering from or at risk of developing a disease or condition, or not.For example, the criterion is an amount or score that allows a subject to be assigned to a group of subjects suffering from or at risk of developing sepsis (within the above-mentioned prediction window, for example within about 48 hours) or a group of subjects not at risk of developing sepsis.

[0067] The appropriate threshold amount for separating the two groups can be easily calculated by the statistical test described elsewhere herein based on the amount of biomarker from either a subject or a group of subjects known to be affected by or at risk of developing a disease or condition, or a subject or a group of subjects known to be free of or at risk of developing a disease or condition.The applicable reference amount for each subject can vary depending on various physiological parameters, such as age, sex, or subpopulation.

[0068] Typically, the reference is a reference for each biomarker derived from at least one subject known to be at risk of developing sepsis, preferably where an amount for each of the biomarkers that is essentially the same as or similar to the corresponding reference indicates a risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference indicates a subject that is not at risk of developing sepsis.

[0069] Also, typically 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 as or similar to the corresponding reference indicates a subject that is not at risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference indicates a subject that is at risk of developing sepsis.

[0070] 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.

[0071] In one embodiment, an amount of the biomarker greater than the baseline for that biomarker indicates the subject is at risk, and an amount of the biomarker lower than the baseline for that biomarker indicates the subject is not at risk (e.g., of developing sepsis as described elsewhere herein).

[0072] Reference amounts can in principle be calculated for a cohort of subjects based on a given parameter, e.g., the mean or average value of a biomarker amount, by applying standard statistical methods. Whether a test, particularly a method 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. On 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 a measure 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 accuracy of the entire 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 a suitable 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 increased risk or affected by the disease (i.e., rule out), while an optimal specificity is assumed to include / assess subjects at increased risk or affected by the disease (i.e., rule in).

[0073] 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 suspected of infection based on the amount of the biomarkers.

[0074] Thus, the amount of each 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.

[0075] Alternatively, a score can be calculated based on the amount of biomarkers, i.e., based on the amount of the first biomarker, the second biomarker, and optionally the third biomarker.The score makes it possible to assess the subject suspected of infection, for example to predict the risk of developing sepsis.Optionally, the score can be compared with a suitable reference score.

[0076] The term "comparing" as used herein includes comparing the determined amount for the biomarker referred to herein with a standard. Comparison as used herein should be understood to refer to any kind of comparison performed 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 invention, the standard is also an absolute amount, if relative amounts are determined and compared in the method of the invention, the standard is also a relative amount, etc. Alternatively, as used herein, the term "comparing" includes comparing the calculated score with an appropriate standard score. The comparison can be performed manually or computer-assisted. The values ​​of 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 an appropriate output format.

[0077] As mentioned above, it is also envisaged to calculate a score (particularly a single score) based on the amounts of the first and second biomarkers, or the first, second and third biomarkers, i.e. a single score, and to compare this score with a reference score. Preferably, the score is based on the amounts of the first and second biomarkers in a sample from the test subject, and if the amount of the third biomarker is determined, it is based on the amounts of the first, second and third biomarkers in a sample from the test subject.

[0078] The calculated score typically 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 those skilled in the art without further difficulty. 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 each biomarker as well as the structure of the decision tree can be different.

[0079] The score can be considered as a classifier parameter for assessing a subject as described herein. In particular, it allows to provide an assessment based on a single score. The reference score is preferably a value, in particular a cut-off value that allows to assess a subject suspected of infection as described herein. Preferably, the reference is a single value. Thus, it is not necessary to interpret the entire information regarding the amount of each individual biomarker. Using the scoring system described herein, advantageously, values ​​of different dimensions or units may be used for the biomarkers, because the values ​​are mathematically converted into scores. Thus, for example, values ​​for absolute concentration may be combined with peak area ratios in the score. The applied reference score may be selected based on the desired sensitivity or the desired specificity. Methods for selecting an appropriate reference score are well known in the art.

[0080] Advantageously, it has been found in the study underlying the present invention that the combination of a first biomarker with a second and preferably a third biomarker allows for reliable and early assessment of patients exhibiting signs and symptoms of infection. For example, the assessment of the subject can be performed within 5 hours after the test sample is obtained. In the study, patients presenting to the emergency department, a medical (non-surgical) emergency, were examined. For this purpose, the amounts of various biomarkers that further divided the patients into those with a high probability of sepsis and those suspected of having an infection without sepsis were determined, and the biomarkers were analyzed and mathematically combined via logistic regression analysis. The area under the receiver operating characteristic curve (AUC) was used to evaluate biomarker performance. The AUC value is the mathematical integral of the function f(x) within the interval [a][b]. The AUC was also examined for biomarker pairs and triplets. Combined, biomarker combinations were identified that showed improved AUC over the best single biomarker AUC. The results are described in the attached examples below.

[0081] Especially when these patients present, for example, in the emergency room, early assessment of the risk of developing severe complications, such as sepsis, SIRS, or a general deterioration of overall health, is crucial to initiate 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). Also included as therapeutic measures is triage to higher levels of care (e.g., intensive care unit, intermediate care unit). If there is no risk of severe complications, the patient can be discharged and managed in an outpatient setting or admitted to a lower level of care in the hospital (e.g., general ward). Thanks to the present invention, patients can be assessed at an early stage by biomarker assessment, so that life-threatening developments can be prevented. The biomarker pairs and triplets identified in the research underlying the present invention are a reliable basis for medical decisions, and assessments can be made in a time- and cost-effective manner.

[0082] As such, the method of the present 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 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.

[0083] 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 at least one broad-spectrum antibiotic, such as a cephalosporin, a beta-lactam / beta-lactamase inhibitor (e.g., piperacillin), or a carbapenem, typically as empirical broad-spectrum therapy depending on the organism and antibiotic susceptibility considered to be the likely pathogen. Fluid resuscitation administration of one or more vasoconstrictors, such as administration of norepinephrine; and Administration of one or more corticosteroids, such as hydrocortisone

[0084] The definitions given herein above apply mutatis mutandis hereinafter.

[0085] 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 MR-proADM; (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 sFlt-1, GDF15, and ESM1 (such as NTproBNP or BNP); (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 made in step (c). The present invention relates to a computer-implemented method,

[0086] The term "computer-implemented" as used herein means that the method is carried out in an automated manner on a data processing unit, typically contained in a computer or similar data processing device. The data processing unit receives values ​​for the amounts of the biomarkers. Such values ​​can be amounts, relative amounts, or any other calculated values ​​that reflect the amounts described in detail elsewhere herein. It should therefore be understood that the above-mentioned method does not require the determination of amounts for the biomarkers, but rather uses values ​​for amounts that have already been predetermined.

[0087] Typically, in step (b) of the process, (i) If a value for the amount of GDF-15 is received as the second biomarker, the method further comprises receiving a value for the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker.

[0088] 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 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 configured, when executed on a computer, to carry out a method according to one of the embodiments described herein; - 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, when the computer program is run on a computer or on a computer network, a method according to one of the embodiments described herein, - 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 having program code means in which, or capable of being stored on a storage medium, the program code means 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, and - a typically encrypted data stream signal containing the assessment provided by the method of the present invention.

[0089] The present invention relates to a device for assessing a subject suspected of infection, comprising: (a) a measuring unit for determining the amount of a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1 in a sample from a subject, the measuring unit comprising a detection system for the first biomarker and the second biomarker; (b) an evaluation unit operably 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 comprising instructions for performing a comparison of the amounts of the first and second biomarkers with the criteria, preferably as described above, 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; Including, relating to a device.

[0090] The term "device", as used herein, relates to a system comprising the above-mentioned units functionally linked to each other to allow the determination of the amount of the biomarker and its evaluation by the method of the present invention so that an assessment can be provided.

[0091] The analytical unit typically comprises at least one reaction zone having biomarker detection agents for the first and second biomarkers 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 biomarkers contained in the sample.

[0092] 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 is also connected to a detector. The connection is such that the detector can detect the binding of the biomarkers to their detection agent. The appropriate connection depends on the technique used to measure the presence or amount of the biomarker. For example, for optical detection, light transmission may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode.

[0093] The detector is adapted to detect the determination of the amount of the biomarker. The determined amount can be subsequently transmitted to an evaluation unit comprising a data processing element, such as a computer, having an implemented algorithm for determining the amount present in the sample.

[0094] The processing unit referred to in accordance with the method of the present invention typically comprises 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 over a network or in other ways to transfer data, to perform one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), cellular devices, smart or mobile devices, tablet computers, and servers, etc. In general, the data processing element includes a processor capable of executing multiple instructions (e.g., a program of software).

[0095] The evaluation unit typically includes 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 accessible by the computing device, including 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 without 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 the processor of the computing device.

[0096] According to an embodiment 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 thus operate via those control signals to transform materials remote from the computer itself. These descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey, for example, the substance of their work to others skilled in the art.

[0097] 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 that require the physical manipulation 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, in reference 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 associated with the appropriate physical quantities and are merely used herein as convenient labels applied to these quantities.

[0098] The evaluation unit may also include or have access to an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a file. According to some embodiments of the present disclosure, a computing device may perform one or more steps of the methods disclosed herein and then provide output related to the results, indicators, ratios, or other factors of the method via an output device.

[0099] Typically, the measurement unit determines a third biomarker and includes a detection system for the third biomarker, and the database includes stored references for the third biomarker, the third biomarker being: (i) When GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0100] More typically, the detection system includes at least one detection agent capable of specifically detecting each of the biomarkers.

[0101] The present invention further envisages a device for assessing a subject suspected of infection, comprising an evaluation unit, a database with stored references for a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1, and a data processor, preferably comprising instructions for performing a comparison of the amounts of the first and second biomarkers with the reference, as defined above, and for assessing the subject based on the comparison, wherein the evaluation unit is capable of receiving values ​​for the amounts of the biomarkers determined in the subject's sample.

[0102] Typically, the database includes stored references for a third biomarker, the third biomarker being (i) When GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0103] The present invention also relates in principle to the use of a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1, 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.

[0104] 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 may 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, the 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 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 usually includes at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally relevant amino acid residues of the donor antibody. Such hybrids may be prepared by several methods well known in the art. For example, the aptamer detection agent may be 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 that is the basis for the aptamer. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Specific binding of a detection agent means that it does not substantially bind, i.e. does not cross-react, with another peptide, polypeptide or substance present in the sample being analyzed. Preferably, a specifically bound biomarker should bind with an affinity at least 3 times higher than any other component of the sample, more preferably at least 10 times higher, and even more preferably at least 50 times higher. Non-specific binding may be acceptable if it can still be clearly distinguished and measured, for example according to its size on a Western blot or by its relatively higher abundance in the sample.

[0105] 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. Suitable detectable labels are any labels that can be detected by suitable detection methods. Typical 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, beta-galactosidase, luciferase, and their derivatives. 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.A suitable label may also be or include a tag, such as biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like.

[0106] Preferred agents for biomarkers such as AST, ALT, etc. are described, for example, in the Examples, see Example 1.

[0107] 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).

[0108] In one embodiment, the detection agent for ALT (ALAT) is, for example, L-alanine.

[0109] In one embodiment, the detection agent for AST (ASAT) is, for example, L-aspartate.

[0110] The determination of the biomarkers described herein may include mass spectrometry (MS) carried out 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 to be determined according to the invention, i.e. the biomarker. Preferably, mass spectrometry, as used herein, relates to GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry, e.g. MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approach using the above mentioned 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 coupled to a prior liquid chromatographic separation step. Preferably, mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS, involves two or more mass spectrometry steps, with fragmentation occurring between steps. In tandem mass spectrometry, two mass spectrometers in series are connected by a collision cell. The mass spectrometer is coupled to a chromatography device. The chromatographically separated sample is sorted and weighed in the first mass spectrometer, then fragmented by an inert gas in the collision cell, and one or more pieces are sorted and weighed in the second mass spectrometer. The fragments are fractionated and weighed in the second mass spectrometer. Identification by MS / MS is more accurate.

[0111] In one embodiment, mass spectrometry as used herein encompasses quadrupole MS. Most preferably, said quadrupole MS is carried out as follows: a) selection of the mass / charge ratio (m / z) of ions produced 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 ratio of ions produced by the fragmentation process in step b) in the additional subsequent quadrupole, steps a) to c) of the method being carried out at least once, and an analysis of the mass / charge ratio of all ions present in the mixture of substances as a result of the ionization process is carried out, the quadrupole being filled with collision gas but no accelerating voltage being applied during the analysis. Details regarding said most preferred mass spectrometry used according to the present invention can be found in WO 2003 / 073464.

[0112] More preferably, the mass spectrometry is liquid chromatography (LC) MS, such as high performance liquid chromatography (HPLC) MS, in particular HPLC-MS / MS. Liquid chromatography, as used herein, refers to all techniques that allow the separation of compounds (i.e. metabolites) in a liquid or supercritical phase.

[0113] For mass spectrometry, the analytes in the sample are ionized to generate charged molecules or molecular fragments. The mass-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.

[0114] Thus, the mass spectrometry step preferably includes an ionization step in which the biomarkers to be determined are ionized. Naturally, other compounds present in the sample / eluate are also ionized. The ionization of the biomarkers can be carried out by any method deemed appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI).

[0115] 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, if tandem MS is performed). Electrospray is a soft ionization method that results in the formation of ions without breaking chemical bonds.

[0116] More typically, a third biomarker or a detection agent that specifically binds to the third biomarker is additionally used, the third biomarker being (i) When GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0117] The present invention also relates to a kit for assessing a subject suspected of being infected, comprising a detection agent that specifically binds to a first biomarker which is MR-proADM, and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1.

[0118] As used herein, the term "kit" refers to a collection of the above-mentioned components, typically provided separately or in a single container. 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 executed on a computer or data processing device, can carry out or support 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 may be provided in a download format, such as a link to an accessible server or cloud. In addition, the kit may usually include standards for 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 required for detection of the released second molecule. In addition, the kit may partially or entirely include the device of the invention.

[0119] More typically, the kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being: (i) When GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0120] It should be understood that the definitions and explanations of terms made above apply accordingly for all embodiments described in this specification and the appended claims. The following embodiments are specific embodiments contemplated in accordance with the present invention.

[0121] 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 said first biomarker is MR-proADM; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFlt-1, GDF15 and ESM1; (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 being infected based on the amount of the biomarker; and (d) assessing the subject based on the comparisons and / or calculations made in step (c). A method comprising:

[0122] 2. In step (b), 2. The method of embodiment 1, wherein (i) if the amount of GDF-15 is determined as the second biomarker, the method further comprises determining the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker.

[0123] 3. The method of embodiment 1 or 2, wherein the subject is a subject presenting to an emergency department.

[0124] 4. The method of any one of embodiments 1 to 3, wherein the assessment is an assessment of the risk of developing sepsis and / or an assessment of the risk of the subject's condition worsening.

[0125] 5. The method of any one of embodiments 1-4, wherein the reference is a reference for each biomarker from at least one subject known to be at risk for developing sepsis, and preferably, an amount for each of the biomarkers that is essentially the same as or similar to the corresponding reference indicates that the subject is at risk for developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference indicates that the subject is not at risk for developing sepsis.

[0126] 6. The method of any one of embodiments 1-4, 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 as or similar to the corresponding reference indicates that the subject is not at risk of developing sepsis, and an amount for each of the biomarkers that differs from the corresponding reference indicates that the subject is at risk of developing sepsis.

[0127] 7. The method of any one of embodiments 1-6, wherein the subject has or is suspected of having an infectious disease.

[0128] 8. The method of any one of embodiments 1 to 7, wherein the sample is a blood sample or a sample derived therefrom.

[0129] 9. The method of any one of embodiments 1 to 8, wherein the subject is a human.

[0130] 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 MR-proADM; (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 sFlt-1, GDF15, and ESM1; (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 for suspected infection based on the amount of the biomarker; and (d) assessing the subject based on the comparisons and / or calculations made in step (c). 4. A computer-implemented method comprising:

[0131] 11. In step (b), (i) if a value for the amount of GDF-15 is received as the second biomarker, the method further comprises receiving a value for the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker; 11. The method of embodiment 10.

[0132] 12. A device for assessing a subject suspected of infection, comprising: (a) a measuring unit for determining the amount of a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1 in a sample from a subject, the measuring unit comprising a detection system for the first biomarker and the second biomarker; (b) an evaluation unit operatively coupled to the measurement unit, the evaluation unit comprising: a database with stored references for a first biomarker and a second biomarker, preferably as described in any one of embodiments 1-9; and a data processor comprising instructions for performing a comparison of the amounts of the first biomarker and the second biomarker with the references, preferably as described in any one of embodiments 1-9, 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 said subject based on the comparison, the evaluation unit being capable of automatically receiving values ​​for the amounts of the biomarkers from the measurement unit; Including, the device.

[0133] 13. The measurement unit determines a third biomarker and includes a detection system for the third biomarker, the database includes stored references for the third biomarker, and the third biomarker is: The device of embodiment 12, wherein (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0134] 14. The device of embodiment 12 or 13, wherein the detection system comprises at least one detection agent capable of specifically detecting each of the biomarkers.

[0135] 15. A device for assessing a subject suspected of infection, comprising: a database having stored criteria for a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1; and a data processor, preferably comprising instructions for performing a comparison of the amount of the first and second biomarker with the criteria as described in any one of embodiments 1 to 11, and for assessing the subject based on the comparison, the device comprising an evaluation unit capable of receiving a value for the amount of the biomarker determined in the subject's sample.

[0136] 16. The database includes stored references for a third biomarker, the third biomarker being: The device of embodiment 15, wherein (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0137] 17. Use of a first biomarker which is MR-proADM and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1, or a detection agent which specifically binds to the first biomarker and a detection agent which specifically binds to the second biomarker, for assessing a subject suspected of infection.

[0138] 18. A third biomarker or a detection agent that specifically binds to the third biomarker is additionally used, and the third biomarker is (i) The use according to embodiment 17, wherein when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0139] 19. A kit for assessing a subject suspected of being infected, comprising a detection agent that specifically binds to a first biomarker which is MR-proADM, and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1.

[0140] 20. The kit further comprises a detection agent that specifically binds to a third biomarker, the third biomarker being: 20. The kit of embodiment 19, wherein (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase.

[0141] 21. The method, device, use or kit according to any one of embodiments 1 to 20, wherein the assessment is an assessment of the risk of developing sepsis.

[0142] 22. The method, device, use or kit of any one of the preceding embodiments, wherein the risk of developing sepsis within 48 hours is predicted.

[0143] All references referenced throughout this specification are hereby incorporated in their entirety for all disclosure content not specifically mentioned above. EXAMPLES

[0144] Example 1: Biomarker Determination The Elecsys® electrochemiluminescence (ECL) technology and assay method for the determination of GDF-15 are briefly described below. The concentration of GDF-15 was determined by a cobas e 801 analyzer. The detection of GDF-15 using the cobas e 801 analyzer is based on the Elecsys® electrochemiluminescence (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 immunocomplexes. 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, in order 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 that results in the emission of photons by the ruthenium complex and TPA. The resulting electrochemiluminescent signals are recorded by photomultiplier tubes and converted to a numerical value indicative of the concentration level of each analyte.

[0145] Mid-region pro-adrenomedullin (MRproADM) was measured using the commercially available B·R·A·H·M·S MR-proADM KRYPTOR assay, a sandwich immunoassay developed for the ThermoFisher KRYPTOR platform (BRAHMS GMbH, ThermoFisher Scientific, Germany). The assay contains anti-pro-ADM sheep polyclonal antibodies conjugated with europium cryptate and anti-pro-ADM sheep polyclonal antibodies conjugated with XL665. From each plasma sample, 26 μL was used and measured undiluted on a ThermoFisher KRYPTOR analyzer (ThermoFisher Scientific, Germany).

[0146] SFLT1 or sFLT-1 (soluble fms-like tyrosine kinase-1) was measured with the commercial 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 and rutheniumylated monoclonal antibody that specifically binds to sFLT-1. 12 μL from each serum sample was used and measured undiluted on a cobas e801 analyzer (Roche Diagnostics, Germany).

[0147] 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).

[0148] ESM1 (Endothelial cell-specific molecule 1) was measured with a robust prototype ECLIA assay for ESM-1, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains 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 e 601 analyzer (Roche Diagnostics, Germany).

[0149] STREM1 or sTREM-1 (Soluble triggering receptor expressed on myeloid cells 1) was measured with the robust prototype ECLIA assay for sTREM-1, a sandwich immunoassay developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assays from Roche Diagnostics, Germany). The assay contains a biotinylated and rutheniumylated monoclonal antibody that specifically binds to sTREM-1. 50 μL was used from each serum sample and measured undiluted on a cobas e 601 analyzer (Roche Diagnostics, Germany).

[0150] ANG2 or Ang-2 (Angiopoietin-2) was measured with a robust prototype ECLIA assay for Angiopoietin-2, 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 Ang-2. 20 μL from each serum sample was used and measured undiluted on a cobas e 601 analyzer (Roche Diagnostics, Germany).

[0151] PSP (pancreatic stone protein) was measured with the commercially available Abionic Platform (Abionic, Switzerland). A nanofluidic PSP (pancreatic stone protein) immunoassay to quantify PSP from 30 μL EDTA-plasma samples. The test principle relies on the passage of the specimen, premixed for a few seconds with a solution containing fluorescently labeled detection antibodies, through a nanometer-sized channel where anti-PSP antibodies are immobilized. These antibodies capture the PSP bound to the fluorescent detection anti-PSP antibodies. The abioSCOPE reads the fluorescence emission from the PSP sensor and uses advanced signal processing to convert the signal into a concentration thanks to an embedded lot-specific calibration of the assay.

[0152] The NGAL (Neutrophil Gelatinase-Associated Lipocalin) test is a particle-enhanced turbidimetric immunoassay for the quantitative determination of NGAL. 3 μL of plasma are mixed with reaction buffer R1. After a short incubation, the reaction is started by the addition of an immunoparticle suspension (polystyrene microparticles coated with a mouse monoclonal antibody against NGAL). Assay from Roche Diagnostics (Germany). NGAL in the sample agglutinates the immunoparticles. The degree of agglutination is quantified by the amount of light scattering, measured as light absorption. The NGAL concentration in the sample is determined by interpolation on an established calibration curve. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0153] CREA (creatinine): This kinetic colorimetric assay is based on the Jaffe method. In alkaline solution, creatinine forms a yellow-orange complex with picric acid. The rate of color formation is proportional to the creatinine concentration in the sample. The assay uses "rate blanking" to minimize interference by bilirubin. Assay from Roche Diagnostics (Germany). 7.5 μL of plasma was used for the determination. Samples were measured on a cobas c 501 analyzer (Roche Diagnostics, Germany).

[0154] ALAT (Alanine Aminotransferase): Alanine aminotransferase 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 alanine aminotransferase activity and is measured using a dichroic (340 nm, 700 nm) rate technique.

[0155] ASAT (Aspartate Aminotransferase): Aspartate aminotransferase (AST) catalyzes the transamination of L-aspartate to α-ketoglutarate to form L-glutamate and oxaloacetate. The oxaloacetate 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, for example, using a dichroic (340 nm, 700 nm) rate technique.

[0156] Example 2: Analysis of Patients from the TRIAGE Study TRIAGE Study,Kantonsspital Aarau,Switzerland,Emergency Department.(Schuetz 2013,BMC emergency medicine,13(1),12).

[0157] 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 into probable sepsis cases or infected controls according to the following: ● Cases (N=64): Sepsis cases likely to have worsened / greater 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 with suspected infection but without sepsis within 48 hours of ED presentation.

[0158] Markers were mathematically combined via logistic regression and the "area under the receiver operating characteristic curve" (AUC) was used as a general measure of marker performance.

[0159] Marker pair combinations (bivariate marker combinations) with an AUC improved by at least 1 percentage point over the single markers are shown in Table 1. [Table 1]

[0160] Marker triplet combinations (trivariate marker combinations) that had an improvement in AUC over all three single markers as well as the bivariate marker pair by at least one percentage point are shown in Table 2. [Table 2]

[0161] Examples of bivariate combinations of markers that have no improvement over single markers are shown in Table 3. [Table 3]

Claims

1. 1. A method for assessing a subject suspected of infection, comprising: (a) determining the amount of a first biomarker in a sample from the subject, wherein the first biomarker is MR-proADM; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is selected from the group consisting of sFlt-1, GDF15, and ESM1; (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 comprising:

2. In step (b), 2. The method of claim 1, wherein (i) if the amount of GDF-15 is determined as the second biomarker, the method further comprises determining the amount of aspartate aminotransferase or alanine aminotransferase as a third biomarker.

3. 3. The method of claim 1 or 2, wherein the subject is a subject presenting to an emergency department.

4. 3. The method of claim 1 or 2, wherein said assessment is an assessment of the risk of developing sepsis and / or an assessment of the risk of the subject's condition worsening.

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

6. 3. The method of claim 1 or 2, wherein the subject has or is suspected of having an infectious disease.

7. 3. The method of claim 1 or 2, wherein the sample is a blood sample or a sample derived therefrom (such as blood 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 MR-proADM; (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 sFlt-1, GDF15, and ESM1; (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 the value for the amount of GDF-15 is received as the second biomarker, the method further comprises receiving a value for the amount of alanine aminotransferase or aspartate aminotransferase as a third biomarker.

9. 1. A device for assessing a subject suspected of infection, comprising: (a) a measuring unit for determining the amount of a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15, and ESM1, in a sample from the subject, the measuring unit comprising a detection system for the first biomarker and the second biomarker; (b) an evaluation unit operatively coupled to the measurement unit, the evaluation unit comprising: a database with stored reference standards for the first and second biomarkers, preferably as defined in claim 1 or 2; and a data processor comprising instructions for performing a comparison of the amounts of the first and second biomarkers, preferably as defined in claim 1 or 2, with reference standards and / or for performing a calculation of a score for assessing the subject suspected of infection based on the amounts of the biomarkers, and for assessing the subject based on said comparison; and Including, Optionally, the measurement unit determines a third biomarker and comprises a detection system for the third biomarker, the database comprises stored references for the third biomarker, and the third biomarker is (i) When GDF-15 is the second biomarker, the second biomarker is alanine aminotransferase or aspartate aminotransferase.

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

11. 1. A device for assessing a subject suspected of infection, the device comprising: a database with stored references for a first biomarker, which is MR-proADM, and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1; and a data processor, preferably as described in claim 1 or 2, comprising instructions for performing a comparison of the amounts of the first and second biomarkers with references and for assessing the subject based on said comparison, the evaluation unit being capable of receiving values ​​for the amounts of the biomarkers determined in a sample of the subject, Optionally, the database comprises stored references for a third biomarker, the third biomarker being: (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase; device.

12. Use of a first biomarker which is MR-proADM and a second biomarker selected from the group consisting of sFlt-1, GDF15 and ESM1, or a detection agent which specifically binds to the first biomarker and a detection agent which specifically binds to the second biomarker, for assessing a subject suspected of being infected.

13. A third biomarker or a detection agent that specifically binds to said third biomarker may additionally be used, said third biomarker being: (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase; 13. The use according to claim 12.

14. 1. A kit for assessing a subject suspected of having an infection, comprising a detection agent that specifically binds to a first biomarker which is MR-proADM, and a detection agent that specifically binds to a second biomarker selected from the group consisting of sFlt-1, GDF15, and ESM1, Optionally, the kit further comprises a detection agent that specifically binds to a third biomarker, wherein the third biomarker is (i) when GDF-15 is the second biomarker, it is alanine aminotransferase or aspartate aminotransferase; kit.

15. The method of claim 1 or 2, wherein the assessment is an assessment of the risk of developing sepsis.

16. The method of claim 1 or 2, wherein the risk of developing sepsis within 48 hours is predicted.

17. The device described in claim 9, wherein the assessment is an assessment of the risk of developing sepsis.

18. The device described in claim 9, which predicts the risk of developing sepsis within 48 hours.

19. The use described in claim 12 or 13, wherein the assessment is an assessment of the risk of developing sepsis.

20. The use described in claim 12 or 13, in which the risk of developing sepsis within 48 hours is predicted.

21. The kit described in claim 14, wherein the assessment is an assessment of the risk of developing sepsis.

22. The kit described in claim 14, which predicts the risk of developing sepsis within 48 hours.