RU2775090C2 - Proadrenomedullin as marker indicating unfavorable event - Google Patents

Abstract

FIELD: medical diagnostics.

SUBSTANCE: method for the prediction of a lethal outcome in a subject suffering from sepsis is disclosed, including the determination of a proadrenomedullin (hereinafter – proADM) level in a sample of this subject, where the proADM level lower than 0.88 nmol/l indicates the survival of the subject during approximately 28 days, while the subject has an assessment on a dynamic organ failure assessment scale (SOFA scale) ≤ 6.

EFFECT: invention provides for a fast and reliable method for the prediction of a lethal outcome in a subject.

15 cl, 4 dwg, 9 tbl, 2 ex

Description

The present invention relates to the diagnosis, prediction, risk assessment and/or risk stratification of an adverse event, in particular death, in a subject. The invention relates to a method comprising determining the level of proadrenomedullin (proADM) in a sample of a subject, where the level of proADM indicates an adverse event in this subject, and/or determining the level of at least one histone, in particular histone H2B, H4, H2A and/or H3 in a subject's sample, wherein the level of at least one histone is indicative of an adverse event in that subject. The invention further relates to kits for carrying out the methods according to the invention.

State of the art

The intensive care unit (ICU) in a hospital is usually the one with the most severe cases and the highest mortality (Kaneko-Wada Fde, Dominguez-Cherit et al. 2015). It is a very expensive component in any healthcare system, mainly because of the long ICU stay, modern and expensive technology, and the overall complexity of intensive care (Halpern and Pastores 2010). Despite great efforts in intensive care, mortality in the ICU ranges from 6.4% to 40%, depending on the analyzed patient population and their severity (Mayr, Dunser et al. 2006). The leading causes of poor outcome and death in the ICU are associated with organ failure such as multiple organ failure syndrome (MOS) and sepsis (Vincent 2008; Ferreira and Sakr 2011). Mortality in a subgroup of patients with sepsis in Europe is even higher, ranging from 27% to 54% (Vincent 2008).

To identify patients with high risk factors with adverse or fatal events/outcomes (i), to assist clinicians in making early decisions after admission to the ICU (ii), to optimize clinical management and resources (iii) and finally to reduce mortality in ICU (iv), knowledge about determinants such as short and long term outcomes is important (Mayr, Dunser et al. 2006). So far, severity rating scales have been mainly used to assess risk and severity, and to predict outcome in critically ill patients. The most commonly used scoring scales in the ICU are the Acute Physiological and Chronic Health Assessment (APACHE), the Simplified Acute Physiological Assessment Scale (SAPS), and the Dynamic Organ Failure Assessment (SOFA) scale, based on 17, 14, or 6 physiological parameters or specific parameters of organ dysfunction, respectively (Bouch and Thompson 2008). Despite the ability to predict patient outcome, such scales also have various disadvantages. For example, it is necessary to define and evaluate each individual parameter of the scales. Thus, scores are time consuming to determine (≥1 day), which is particularly undesirable in ICUs where rapid tests are highly valued. In addition, such scales depend on the subjectivity of each evaluating clinician, need to be reviewed frequently, and rely on several individually determined parameters.

Accordingly, there is a need for simple, rapid and objective criteria for diagnosing, predicting, assessing risk and/or stratifying the risk of an adverse event in a subject, in particular a critically ill subject.

Therefore, the technical object underlying the invention is to provide means and methods that provide a fast and reliable method for predicting an adverse event, in particular a fatal event, for example, death/death, in a subject.

The technical problem is solved by providing the embodiments presented below in this application and described in the attached claims.

Description of the invention

The invention relates to a method for diagnosing, predicting, assessing the risk and/or stratifying the risk of an adverse event in a subject, including determining the level of proadrenomedullin (proADM) in a sample of a given subject, where this level of proADM or a given fragment indicates a given adverse event in a given subject.

In addition, the invention relates to a method for diagnosing, predicting, assessing risk and/or stratifying the risk of an adverse event in a subject, including determining the level of at least one histone or its fragment in a sample of a given subject, where this level of at least one histone or a given fragment indicates a given adverse event in a given subject.

In addition, the invention relates to a method for diagnosing, predicting, assessing the risk and / or stratifying the risk of an adverse event in a subject, including determining the level of at least one histone and the level of proadrenomedullin (proADM) and / or their fragment (s) in a sample of this subject, where a given level of at least one histone and a given level of proADM and/or fragment(s) thereof are indicative of a given adverse event in a given subject. In the context of this application, the term "proADM" also covers fragments such as CP-proADM and mature ADM, unless otherwise indicated.

The present invention solves the above technical problem. As set forth herein below and in the accompanying examples, it was unexpectedly found in a clinical study that the levels of at least one histone, in particular histone H2B, H4, H2A and H3, and/or the level of proADM, in particular CP-proADM , demonstrate(s) a strong statistical relationship with an adverse event, such as death, in subjects; see Illustrative Example 1. Accordingly, the present application shows that a given at least one histone protein and/or a given proADM, eg CP-proADM, can be used as a marker to predict an adverse event. In particular, the examples confirm that such markers can be used to predict survival or non-survival, ie death, of a subject.

In the accompanying examples, it has surprisingly been demonstrated that a subject's proADM (particularly CP-proADM) level is less than or equal to approximately a reference (control) level, e.g., approximately 0.9 nmol/L, indicating that an adverse event will not occur in within approximately 28 days. A reference level of approximately 0.9 nmol/L proADM provides 100% sensitivity in detecting non-survivors, meaning a level below this threshold reliably identifies patients who survive a 28-day period. A reference level of approximately 1.8 nmol/L proADM also reliably predicts an adverse event, such as death, in the accompanying examples. Accordingly, the level of proADM indicates whether the subject will survive or not, for example, within about 28 days.

It has also been demonstrated that proADM (particularly CP-proADM) reliably discriminates against non-survivors or survivors if the subjects were stratified according to the severity of the disease and/or condition, such as organ failure. The predictive method works in general for subjects having any number of organ failures, ie any SOFA score. Detection of the level of proADM (in particular, CP-proADM) allows the identification of non-survivors in all severity groups, that is, for any score on the SOFA scale. This is particularly important for less severely ill patients (SOFA score < 6) as this group represents either the earliest clinical manifestation of sepsis and/or a less severe form of this disease in the clinical setting (eg, ICU or NDE) . Thus, proADM (particularly CP-proADM) is a reliable marker for monitoring the course of sepsis at an early stage, since it provides a rapid prognostic value and helps guide diagnostic interventions and make treatment decisions.

The accompanying examples show that subjects can be classified into less severely ill subjects (eg, SOFA score ≤ 6), moderately ill subjects (eg, SOFA score 7 to 12), or severely ill subjects (eg, with a SOFA score ≥ 13). Accordingly, subjects can be classified based on the SOFA score. The predictive value of a proADM (particularly SR-proADM) can be improved by combining it with determining a subject's SOFA score. However, the predictive value of this marker is such that a SOFA score is not necessary; in other words, in some aspects of the method according to the invention, the level of proADM (preferably CP-proADM) of the subject is determined, but the score on the SOFA scale is not determined. In the attached examples, it was shown that the level of proADM (in particular, CP-proADM) demonstrates the best efficacy among the studied markers in patients with low severity of the disease (less severely ill). Such subjects may have a SOFA score of ≤ 6. In particular, a proADM (particularly CP-proADM) level in less severely ill subjects below the reference level of approximately 1.8 nmol/L has been shown to indicate that the adverse event does not occur, in particular death, for approximately 28 days; see example 2, table 9. In addition, proADM also has advantages in predicting an adverse event compared to other markers such as lactate in the case of moderately severely ill subjects. Such subjects may have a SOFA score of 7 to 12. In particular, a proADM level in moderately severely ill subjects below the reference level of approximately 3.2 nmol/L has been shown to indicate that the adverse event does not occur, in particular, death, within approximately 28 days; see example 2, table 9. Finally, the level of proADM also reliably predicts an adverse event in critically ill subjects. In particular, it has been demonstrated that a proADM level in critically ill subjects below the reference level of 5.6 nmol/l indicates that the adverse event does not occur, in particular death, for approximately 28 days; see example 2, table 9.

Accordingly, the level of proADM (particularly CP-proADM) predicts a subject's risk and thus assists the clinician in deciding which type of therapy would be most appropriate. Thus, the level of proADM improves the treatment of the patient in the clinic. Therefore, the level of proADM can also be used to monitor the course of the disease in the intensive care unit, in particular, to identify subjects who do not need further intensive care, for example, treatment in the ICU or emergency department (EDS). In particular, a proADM reference value of less than about 0.9 nmol/L (preferably 0.88 nmol/L) will "rule out" death within 28 days of admission to the ICU or NDE. Thus, this cut-off threshold is suitable for guiding early clinical decisions when the clinical manifestations of overt organ failure are not yet evident. Therefore, the method according to the invention can be used for this purpose. Identification of subjects who do not need (further) intensive care (e.g. ICU or DCO) but can, for example, be transferred to a general ward reduces costs for the carrier and frees up ICU or DCO beds.

In a specific aspect, the method of the invention is used to predict mortality or stratify subjects who have not yet shown clinical signs of organ failure (e.g., patients corresponding to subjects with a low SOFA score such as ≤ 6, preferably ≤ 4, more preferably ≤ 3).

In addition, the accompanying examples show that the level of proADM (in particular, SR-proADM) can also be used to modify the score of a given subject on the SOFA scale. For example, the SOFA can be adapted, eg increased, based on the subject's proADM level, where the modified SOFA score is indicative of the adverse event.

In particular, a subject's SOFA score may be increased by one if the subject's proADM level is greater than about 1.8 nmol/L. This modified SOFA score further improves prediction; see example 2.

Instead of or in addition to the SOFA score, an accelerated SOFA score (qSOFA) may also be determined.

In the accompanying examples, it was also surprisingly shown that the level of at least one histone, in particular, H2B, H4, H2A and H3, is strongly associated with an adverse event, such as death, in a subject. In particular, the accompanying examples show that histone levels are statistically associated with the onset of an adverse event, such as death, within about 28 days, within about 7 days, or within about 3 days. In the accompanying examples, it was found that an increase in the level of the marker indicates that the subject will experience an adverse event, such as death; see attached example 1, for example tables 1-6. Moreover, histone levels have been shown to be particularly strongly correlated with short-term adverse events, such as occurring within 7 days or 3 days. In addition, the level of at least one histone in samples of subjects suffering from a respiratory disease was found to correlate with the onset of an adverse event, such as death, within 7 days; see, for example, Table 4. In addition, the level of at least one histone in samples from subjects suffering from a urinary tract infection was found to correlate with the onset of an adverse event, such as death, within 28 days; see, for example, Table 5. In addition, the level of at least one histone in a sample of a subject suffering from malignant neoplasms was found to correlate with the onset of an adverse event, such as death, within 28 days; see, for example, table 6.

In addition, in the accompanying examples, it was also unexpectedly shown that the level of proADM, for example, the level of the CP-proADM fragment, is closely correlated with an adverse event, for example, death, in a subject. In particular, the accompanying examples show that the level of proADM is statistically associated with the onset of an adverse event, such as death, within about 28 days, within about 7 days, or within about 3 days; see, for example, tables 2 and 3. In particular, in the attached examples, it is shown that the level of proADM indicates an adverse event, such as death, occurring within 28 days or within 7 days; see, for example, tables 2 and 3.

In addition, the accompanying examples show that prediction is further improved if a combination of marker levels is determined. In particular, determining the level of at least one histone in addition to the level of proADM further improved the prediction of an adverse event, such as death; see tables 1 to 3. The accompanying examples show that determining the level of an additional marker and/or parameter in addition to the level of at least one histone or proADM further improves the prediction of an adverse event, such as death; see Explanatory Tables 1 to 3. For example, the accompanying examples show that determining a score on a clinical scale also improves prediction based on proADM or histone levels. In addition, determining the level of a biomarker, such as aldolase B, improves prediction based on the level of proADM or histones. Accordingly, the invention also relates to a method including determining the level of an additional marker and/or parameter, ie using a panel of markers.

The present invention has, inter alia, the following advantages over conventional methods: The methods and kits of the invention are fast, objective, easy to use, and accurate in predicting an adverse event: The methods and kits of the invention are markers that are easily measurable in everyday practice in hospitals, since histone and proADM levels can be determined in conventionally obtained blood samples or other biological fluids obtained from the subject. In addition, the determination of histone or proADM levels is very fast. Therefore, the methods and kits of the invention are useful for rapid evaluation as well as diagnosis and prediction of an adverse event, such as death. Accordingly, rapid determination is also suitable for rapidly making a treatment decision to avoid or reduce the risk of an adverse event occurring. In addition, due to the simple result of measuring the biomarker, which is one specific value, when using this method or kits according to the invention, there is no estimation error based on subjective perception by medical personnel. The reproducibility is thus improved compared to the subjective assessment of physiological parameters, which are used, for example, in the evaluation of the SOFA scale. The histone or proADM level can also be combined with other marker(s) and/or parameter(s), such as clinical scores such as the SOFA score, to further improve prediction and tailor the assay to specific sensitivity and specificity. to assess the general condition of critically ill patients.

Accordingly, the methods and kits provided herein are advantageous for diagnosing, predicting, assessing risk, and/or stratifying the risk of an impending adverse event, such as death/death, in a subject.

As described above in this application and in the accompanying examples, it was unexpectedly found that the level of histones and the level of proADM correlate with an adverse event, such as death, in subjects. Accordingly, the invention relates to methods and kits for diagnosing, predicting, assessing risk and/or stratifying the risk of an adverse event, in particular death, in a subject.

In addition, the invention relates to methods and kits for monitoring, guiding therapy and/or monitoring therapy of subjects, where the level of at least one histone and/or proADM indicates an adverse event, in particular, death. The definitions above and below in this application also apply to such aspects.

In particular, the invention relates to a method for diagnosing, predicting, assessing risk and/or stratifying the risk of an adverse event, in particular a fatal outcome, in a subject, including

(i) determining the level of at least one histone in a sample of the subject, where the level of at least one histone is indicative of a given adverse event, in particular death, in the subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in a sample of the subject, where the level of proADM is indicative of a given adverse event, particularly death, in the subject.

As used herein, the term "determining the level of at least one histone" or the like refers to determining the level of a histone or fragment thereof in a sample of a subject, or determining the level of more than one histone or fragments thereof in a sample of a subject. In particular, "determining the level of at least one histone" may refer to determining the level of a histone in a sample of a subject, where preferably the histone is selected from the group consisting of histone H2B, H4, H2A and H3. In particular, the level of histone H2B is determined. In addition, "determining the level of at least one histone" may refer to determining the level of a histone in a sample of a subject, where, in particular, determine the level of histone H4. In addition, "determining the level of at least one histone" may refer to the determination of the level of two histones in a sample of the subject, where preferably determine the levels of histones H2B and H4. In addition, "determining the level of at least one histone" may refer to determining the level of three histones in a sample of the subject, where preferably determine the levels of histones H2B, H4 and H2A. In addition, "determining the level of at least one histone" may refer to determining the level of four histones in a sample of the subject, where preferably determine the levels of histones H2B, H4, H2A and H3.

Accordingly, in the context of the present invention, “determining the level of at least one histone” may refer to determining the level of H2B histone, the level of H4 histone, the level of H2A histone, and/or the level of H3 histone.

In particular, the term "determining the level of at least one histone" or the like may refer to determining the level of a histone in a sample of a subject. Accordingly, the invention also relates to a method for diagnosing, predicting, assessing risk, risk stratifying and/or monitoring an adverse event in a subject, including determining the level of a histone or a given fragment in a sample of a given subject, where a given level of histone or a given fragment is indicative of a given adverse event. , in particular fatal.

In the context of this application, the term "histone" or "histone protein" or "histones" or "histone proteins" refers to the canonical histone(s) such as H1, H2A, H2B, H3 or H4, as well as to histone variant(s) such as H3.3, H2A.Z, etc., or fragment(s) thereof. The histones form an octameric particle around which the DNA wraps, creating the chromatin structure (Luger, Nature. 1997 Sep 18; 389(6648):251-60). For example, the histone proteins H2A, H2B, H3, and H4 (two of each) form an octamer around which 165 base pairs of DNA wrap to form the basic subunit of chromatin, the nucleosome. Histones are also found outside the nucleus in many pathophysiological processes (WO 2009/061918). The presence of extracellular histones in the blood of patients suffering from diseases of various etiologies characterized by inflammatory processes has been described. Release of histones from activated immune cells may be mediated by extracellular traps. Activated neutrophils, as the main mechanism for controlling and eliminating infection, can release extracellular fibers, the so-called neutrophil extracellular traps (NET) (Brinkmann V., et al. Science 2004; 303(5663): p. 1532-5). Other mechanisms by which histones can be released into the patient's bloodstream include cell apoptosis, necrosis, pyroptosis, or cell necroptosis.

In particular, at least one histone is selected from the group consisting of H2B, H4, H2A and H3. Accordingly, the histone level to be determined in the methods and kits of the invention is in particular the level of the histone(s) H2B, H4, H2A and/or H3. Histone sequences are known to the person skilled in the art. Examples of histone sequences are shown in SEQ ID NOs: 1-4. An example of the amino acid sequence of histone H4 is given in SEQ ID NO: 1. An example of the amino acid sequence of histone H2A is given in SEQ ID NO: 2. An example of the amino acid sequence of histone H3 is given in SEQ ID NO: 3. An example of the amino acid sequence of histone H2B is given in SEQ ID NO: 4. In particular, at least one histone is selected from the group consisting of H2B, H4, H2A and H3. More specifically, at least one histone is selected from the group consisting of H2B, H4 and H2A. More specifically, at least one histone is H2B and H4. More specifically, at least one histone is H2B or H4.

"Determining the level of at least one histone" or the like is meant to refer to determining the level of at least one histone or a fragment of at least one histone in a sample. In particular, the level of histone H2B, H4, H2A and/or H4 is determined in the sample. Accordingly, at least one histone identified in the sample may be a free histone, or at least one histone identified in the sample may occur and may be part of a macromolecular complex, such as an octamer, nucleosome, and/or NET. Therefore, the level of at least one histone in the sample may include the level of free histone protein and/or histone protein that is part of the macromolecular complex.

In private aspects of the invention, the level of histone or its fragment that is not part of a macromolecular complex, such as a nucleosome, an octamer, or a neutrophil extracellular trap (NET) can be determined in a sample. Such(s) histone(s) in this application is called "free(s) histone(s)". Accordingly, the level of at least one histone may in particular be the level of at least one free histone.

The level of such free histones can be determined by detecting amino acid sequences or epitopes within the histone structure that are not available within a stoichiometric macromolecular complex such as a mononucleosome or an octamer. In such structures, certain histone regions are closed and thus sterically inaccessible, as shown for neutrophil extracellular traps ("NETs") (Brinkmann V., et al. Science 303(5663): p. 1532-5, 2004). In addition, in the octamer or nucleosome, histone regions are also involved in intramolecular interactions, for example, between individual histones. Accordingly, the region/peptide/epitope of the histone to be determined in the context of the invention makes it possible to identify whether the histone is free or is part of a macromolecular complex. For example, in an immunoassay based method, the antibodies used may not detect histones, such as H4, when they are part of the octameric core of the nucleosomes, because the epitopes are then structurally inaccessible. The following are examples of histone regions/peptides/epitopes that can be used to determine free histone. For example, regions/peptides/epitopes of the N-terminal or C-terminal histone tail can be used to detect histones, whether they are within a macromolecular complex or are free histones according to the present invention.

It is contemplated that the definition of histones may include post-translationally modified histone proteins. Accordingly, post-translational modifications may include deacetylation or acetylation, phosphorylation, methylation, ubiquitylation, and citrullination of amino acids.

"Stoichiometric" in this context refers to intact complexes, for example, mononucleosome or octamer. "Free histone proteins" may also include histones not associated with chromatin. For example, "free histone proteins" may also include single histone proteins or non-octameric histone complexes. Free histones can (eg, temporarily) bind to individual histones, eg, histones can form homo- or heterodimers. Free histones can also form homo- or heterotetramers. The homo- or heterotetramer may be composed of four histone molecules, eg H2A, H2B, H3 and/or H4. A typical heterotetramer is formed by two heterodimers, where each heterodimer consists of H3 and H4. The present application also contemplates that the heterotetramer can be formed by H2A and H2B. The present application also provides that the heterotetramer may be formed by one heterodimer consisting of H3 and H4 and one heterodimer consisting of H2A and H2B. Thus, free histones are referred to herein as, and may be, monomeric, heterodimeric, or tetrameric histone proteins that are not part of a (“stoichiometric”) macromolecular complex consisting of a histone octamer bound to a nucleic acid, e.g., a nucleosome. In addition, free histones can also be associated with nucleic acids, and while these free histones are not part of a ("stoichiometric") macromolecular complex, such as an intact nucleosome. Preferably free(s) histone(s) essentially does not contain(at) nucleic acids.

A fragment of at least one histone may be of any length, such as at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as the fragment is unambiguous in determining the level of a particular histone. A fragment of at least one histone refers to an independent fragment of histones, for example, histones H2B, H4, H2A and H3. Below in this application are various examples of fragments of histones suitable for determining the level of histone in the sample of the subject. This application also contemplates that the level of histones can be determined by determining the fragment spanning the N-terminal or C-terminal histone tail. In addition, the histone or fragment to be defined in the context of the present invention can also be modified, for example, by post-translational modification. Examples of post-translational modifications may be acetylation, citrullination, deacetylation, methylation, demethylation, deimination, isomerization, phosphorylation and ubiquitination.

In the context of this application, the term "proadrenomedullin" or "proADM" refers to proadrenomedullin or a fragment thereof, in particular SR-proADM. "ProADM level definition" or the like is meant to refer to the definition of a proADM or a fragment thereof. The fragment may be of any length, such as at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as the fragment is unambiguous in determining the level of proADM. In particular preferred aspects of the invention, "determination of the level of proADM" refers to the determination of the level of average regional (median) proadrenomedullin (CP-proADM). CP-proADM is a fragment of proADM. The peptide adrenomedullin (ADM) was discovered as a 52 amino acid hypotensive peptide that was isolated from human pheochromocytoma (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a 185 amino acid precursor peptide ("preproadrenomedullin" or "pre-proADM"). An example of the amino acid sequence of ADM is given in SEQ ID NO: 5. ADM contains positions 95-146 of the pre-proADM amino acid sequence and is a splicing product thereof. "Proadrenomedullin" ("proADM") refers to a pre-proADM that does not contain a signal sequence (amino acids 1 to 21), that is, pre-proADM amino acid residues 22 to 285. "Mid-regional proadrenomedullin" ("CP-proADM") refers to amino acids 42-95 of pre-proADM. An example of the amino acid sequence of CP-proADM is given in SEQ ID NO: 6. This application also provides that the peptide pre-proADM or CP-proADM and its fragment can be used for the methods described in this application. For example, a peptide or fragment thereof may comprise pre-proADM amino acids 22-41 (PAMP peptide) or pre-proADM amino acids 95-146 (mature adrenomedullin). The C-terminal fragment of proADM (amino acids 153 to 185 pre-proADM) is called adrenotensin. ProADM peptide fragments or CP-proADM fragments may contain, for example, at least about 5, 10, 20, 30 or more amino acids. Accordingly, the proADM fragment may, for example, be selected from the group consisting of CP-proADM, PAMP, adrenotensin and mature adrenomedullin, preferably in this application the fragment is CP-proADM.

The present application also contemplates that polypeptides having sequence identity with proADM or with at least one histone can be defined. For example, the methods and kits of the invention can detect polypeptides that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to SEQ ID NO: 5 or 6, or respectively SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, where higher sequence identity values are preferred . According to the present invention, the terms "sequence identity", "homology" or "percent homology", or "identical", or "percent identity", or "percent identity" in the context of two or more amino acid sequences refers to two or more sequences or subsequences, that are the same, or have a certain percentage of amino acids that are the same, when compared and aligned for maximum match within the comparison window (preferably over the entire length) or in a specific region, when measured using a sequence comparison algorithm known in the art, or by manual alignment and visual inspection. Sequences identical by, for example, 70% to 90% or more (preferably 95% or more) can be considered substantially identical. This definition also applies to the complement of the sequence under study. Preferably, the described identity occurs over a region that is at least about 10 to about 15 amino acids in length, more preferably over a region that is at least about 20 to about 35 amino acids in length, most preferably over its entire length. Those skilled in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on the computer program CLUSTALW (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB ( Brutlag Comp. App. Biosci. 6 (1990), 237-245), as is known in the art.

In the context of this application, the term "level" of a marker refers to the amount of a marker molecule in a sample. In other words, the concentration of the marker is determined in the sample. For example, in a subject sample, the concentration of proADM or a fragment thereof, preferably CP-proADM, and/or the concentration of histone(s) H2B, H4, H3 and/or H2A or fragment(s) is determined.

As used herein, the term "level of at least one histone" refers to the amount of at least one histone molecule, e.g., the amount of H2B, H4, H2A and/or H3, or a fragment thereof, in a sample obtained from a subject. In other words, the concentration of at least one histone protein or fragment thereof is determined in the sample.

In the context of the present application, the term "proadrenomedullin marker (proADM) level" or "proadrenomedullin marker (proADM) marker level or fragment thereof" refers to the amount of the proadrenomedullin marker molecule or fragments thereof in a sample obtained from a subject. In other words, the concentration of the marker is determined in the sample. Therefore, the term "regional mean proadrenomedullin (SR-proADM) marker level" refers to the amount of the regional mean proadrenomedullin (SR-proADM) marker molecule in a sample obtained from a subject. As described above, the present application also contemplates that a fragment of proadrenomedullin (proADM), preferably CP-proADM, can be detected and quantified. CP-proADM fragments can also be detected and quantified. Suitable methods for determining the level of proADM or a fragment thereof (preferably CP-proADM) or determining the level of at least one histone or fragment thereof are described below in this application.

In the context of this application, "adverse event" means a health condition/status of a subject that is or would be life threatening. Therefore, an adverse event refers to a critical deterioration in a subject's health/status compared to an earlier health condition of the same subject, e.g. diagnosed and confirmed, e.g. approximately 28 days, 14 days, 7 days, 3 days, 1 day, 12 hours, 5 hours, 1 hour or less before deterioration; or compared to the health status of subjects suffering from similar disease(s) or medical condition(s), i.e. the progression of the disease(s) or medical disorder(s) is or becomes threatening the life of the subject under study. An adverse event may also refer to a critical deterioration in the health/status of a subject compared to the health of healthy subjects. As used herein, a critical deterioration in health/status, a life-threatening condition/status of a subject, or life-threatening progression may mean that the subject is at an increased risk of death, i.e., the subject is likely to die. Critical deterioration, critical health condition/status, or life-threatening health condition/status can also be assessed/diagnosed using clinical scores, such as the SOFA score. For example, a score on the SOFA scale above 14 indicates a very serious health condition, indicating a critical state of health of the subject. A SOFA score of 0 to 6 indicates less severe health, and a SOFA score of 7 to 14 indicates severe health. For example, the subject's health may be critically degraded due to organ dysfunction, multiple organ dysfunction, disease(s), or medical disorder(s) such as an infection. Accordingly, the term "adverse event" may refer to dysfunction of an organ, dysfunction of multiple organs, a disease or medical condition such as an infection that is or will be life threatening. For example, dysfunction of an organ or dysfunction of multiple organs can lead to such a critical deterioration, and thus the term "adverse event" can also refer to dysfunction of an organ or dysfunction of multiple organs, in particular organ failure or multiple organ failure. For example, a disease or medical disorder may lead to such a critical deterioration, and thus the term "adverse event" may also refer to the disease(s) or medical disorder(s) where the disease(s) or medical( f) disorder(s) may refer to a disease(s) or medical disorder(s) that are life threatening. The subject may also be suffering from a disease(s) and/or medical disorder(s), and an adverse event in such a situation is a condition in which the progression of the disease(s) or medical disorder(s) becomes(are) life-threatening(s), that is, an adverse event may also refer to a life-threatening progression of a disease or disorder. The disease or disorder may be selected from the group consisting of respiratory disease, urinary tract infection, infectious and non-infectious inflammatory response, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, organ(s) failure (s), cardiovascular disease, hematological disease, disseminated blood clotting, diabetes mellitus, malignancy, liver disease, kidney disease, immunosuppression, viral infection, fungal infection, bacterial infection, gram-negative bacterial infection, gram-positive bacterial infection, abdominal infection and suppression of immunity. The disease may also be an infection(s). In particular aspects, the subject is suffering from a viral infection, a fungal infection, a bacterial infection, a Gram-negative bacterial infection, and/or a Gram-positive bacterial infection. Respiratory disease can also refer to a respiratory infection (such as community-acquired pneumonia (CAP) or lower respiratory tract infection (LRTI)). In a separate particular aspect, the subject suffers from a fungal infection.

In the context of the present application, the term "subject with depressed immunity" refers to a subject with a depressed immune system compared to at least one healthy subject. Such an immunocompromised subject may be suffering from human immunodeficiency virus (HIV) or may be a subject undergoing radiation therapy and/or receiving immunosuppressive drugs.

Preferably, in the present application, the subjects are suffering from severe sepsis or septic shock, preferably based on the American College of Thoracic Medicine/Society for Critical Care Consensus Conference definition of SEPSIS-2.

In particular aspects of the invention, the adverse event is a fatal outcome. In other words, in particular aspects of the invention, the adverse event is the death of the subject. Accordingly, the term "adverse event" specifically means "fatal" or "death". In the context of this document, the term "lethal" means that the subject dies, that is, means a fatal outcome. Accordingly, the methods and kits of the invention make it possible to determine/predict whether a subject will die and/or is at increased risk of death.

In the context of this application, the general term "organ dysfunction" may also mean that there may be dysfunction of more than one organ, that is, it can also refer to dysfunction of organs, unless otherwise indicated. The term "organ dysfunction" or "organ dysfunction" refers to a condition in a subject in which an organ or more than one organ fails to perform its/its normal function(s) compared to an unaffected organ, such as for example, the organ(s) of at least one healthy subject. For example, the organ(s) may have reduced activity, or the organ(s) may be abnormally active in a subject with organ dysfunction compared to the organ(s) of at least one healthy subject. Preferably, the organ(s) with dysfunction of the organ(s) may have decreased or increased activity of at least about 10%, 20%, 30%, 50%, 70%, 90%, 100%, or 200% according to compared with unaffected(s) organ(s), for example, at least one healthy subject. In particular, dysfunction of the organ(s) may lead to organ(s) failure(s). Accordingly, "impaired function of the organ(s)" can preferably also refer to organ(s) insufficiency(s). "Organ failure(s)" refers to dysfunction of an organ(s) to the extent that normal homeostasis can no longer be maintained, eg without external clinical intervention. "Organ failure(s)" may refer to dysfunction of an organ(s) to the extent that normal homeostasis of the organ(s) can no longer be maintained, eg without external clinical intervention. "Organ failure(s)" can also refer to dysfunction of an organ(s) to the extent that the subject's normal homeostasis can no longer be maintained, for example, without external clinical intervention. In particular aspects of the invention, an organ dysfunction is an organ failure or failure of at least one organ. The general term "organ failure" can also mean that there is a failure of more than one organ, that is, it can also refer to organ failure, unless otherwise indicated. As used herein, organ dysfunction may also be referred to as multi-organ dysfunction. As used herein, organ failures may also be referred to as multiple organ failure. Examples of organ dysfunction or organ failure are circulatory shock, hematological failure, liver failure, neurological failure, renal failure, respiratory failure, and metabolic acidosis. Accordingly, in the context of the invention, dysfunction of an organ or dysfunction of at least one organ may preferably be selected from the group consisting of circulatory shock, hematological failure, liver failure, neurological failure, renal failure, respiratory failure, and metabolic acidosis. In this application, it is understood that the subject may also have a dysfunction or deficiency of more than one organ, for example, a combination of dysfunctions or deficiencies of two, three, four organs selected from the group consisting of circulatory shock, hematological insufficiency, liver failure, neurological insufficiency , renal failure, respiratory failure and metabolic acidosis.

In particular aspects of the invention, the level of at least one histone and/or the level of proADM, in particular CP-proADM, indicates(s) the onset of this adverse event within approximately 28 days. In the context of this application, the term "onset of an adverse event during" means that the subject is likely to experience/have/suffer from, experience/have/suffer from, or experience/have/suffer from an adverse event during that particular time period.

In the context of this application, the term "non-occurrence of an adverse event" means that the event does not occur, for example, within approximately 28 days. For example, the death of the subject does not occur within this time range. Accordingly, this indicates the survival of the subject, for example, for about 28 days.

The level of at least one histone and / or the level of proADM, in particular CP-proADM, also indicates the occurrence of this adverse event within about 28 days, about 25 days, about 20 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. The level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the onset of this adverse event within 14 days. In particular, the level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the onset of this adverse event within 7 days. In particular, the level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the occurrence of this adverse event within 3 days.

In particularly preferred aspects of the invention, the level of at least one histone and/or the level of proADM, in particular CP-proADM, indicates(s) the onset of a lethal outcome of the subject within about 28 days. The level of at least one histone and/or the level of proADM, in particular CP-proADM, can also indicate the onset of death within about 28 days, about 25 days, about 20 days, about 15 days, about 14 days, about 13 days , about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. The level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the onset of death within 14 days. In particular, the level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the onset of death within 7 days. In particular, the level of at least one histone and/or the level of proADM, in particular CP-proADM, may also indicate the onset of death within 3 days.

In preferred aspects of the invention, a given CP-proADM level of a given subject is indicative of the onset of an adverse event, particularly death, in that subject within about 28 days, or within about 7 days.

In particular aspects of the invention, the level of at least one histone indicates the onset of an adverse event, in particular death, within about 7, or preferably within about 3 days. In particular, the H2B level indicates the onset of an adverse event even within about 7, or preferably within about 3 days.

In particular aspects of the invention, the level of at least one histone indicates the onset of an adverse event, in particular death, within about 7, or preferably within about 3 days. In particular, the H4 level indicates the onset of an adverse event even within about 7, or preferably within about 3 days.

In particular aspects of the invention, the level of at least one histone and the level of proADM indicate the onset of an adverse event, in particular death, in a given subject within approximately 28 days.

The term "indicates a given adverse event" means that the subject has or is likely to experience/have/suffer from a given adverse event, such as death. Therefore, the level of at least one histone and/or the level of a subject's proADM is(are) indicative of an adverse event, eg, death, in the subject.

The method according to the invention also relates to a method in which the level of at least one histone in a sample of the subject is determined, where this level of at least one histone is compared with a reference level of at least one histone, and where this level of at least one histone is indicative of this adverse event.

Preferably, the invention also relates to a method in which the level of proADM, in particular CP-proADM, is determined in a sample of a subject, where this level of proADM, in particular CP-proADM, is compared with a reference level of proADM, and where this level proADM, in particular CP-proADM, indicates this adverse event.

The method also relates to a method in which the level of at least one histone is determined and the level of proadrenomedullin (proADM), in particular CP-proADM, is determined in a sample of the subject, where this level of at least one histone is compared with a reference level of at least one histone, and where a given proADM level is compared to a reference proADM level, and where a given adverse event in a given subject is identified based on the comparison step.

As used herein, the term "compared to a reference level of at least one histone" or grammatical variations thereof means that the level of at least one histone of a subject is compared to a reference level of that at least one histone. Thus, the subject's histone level is compared to a corresponding reference level of the same histone. For example, the H2B histone level determined in a subject's sample is compared to a reference H2B histone level. The same principle applies similarly to other histones. The reference level of at least one histone is, in particular, the level of H2B histone, the level of H4 histone, the level of H2A histone and/or the level of H3 histone.

In the context of this application, the term "compared to a reference proADM level" or its grammatical variants means that the proADM level of a subject is compared to a reference proADM level. When determining the level of fragment(s) of at least one histone and/or proADM, the reference level may also be the level of the corresponding fragment(s).

In the context of the present application, "reference level" may reflect the normal level of the corresponding marker, indicating the absence of an adverse event in the preferred aspects of the invention. Accordingly, such a reference level reflects the normal level of the corresponding marker, not indicative of a life-threatening condition or, in particular, death/lethal outcome of the subject. Accordingly, the reference level may be the level of at least one histone and/or the proADM level of at least one reference subject, where the reference subject(s) does/have no adverse events for approximately 28 days, preferably within within about 14 days, more preferably within about 7 days, or particularly preferably within about 3 days.

The reference level may be the level of at least one histone and/or the level of proADM of a group of healthy subjects (eg, a cohort). Accordingly, the reference level is preferably the level of at least one histone and/or the level of proADM, in particular CP-proADM, of healthy subjects. A healthy subject is one without a diagnosed (and confirmed) disease and/or disorder. Healthy subjects may preferably have normally functioning organs, i.e. no dysfunction and/or organ failure(s), and/or no diagnosed disease(s) or medical disorder(s), such as those described above. Accordingly, the reference(s) level(s) may(may) represent the level of at least one histone and/or the level of proADM, determined in samples of healthy subjects. Reference subjects or healthy subjects in the present application is preferably defined as a group of subjects or a group of healthy subjects, for example, a cohort of subjects. Healthy reference subjects do not have organ dysfunction or organ failure. Accordingly, the reference level may be the level of at least one histone and/or the level of proADM in subjects without organ dysfunction or organ failure. Accordingly, the reference level may be a level indicating the absence of organ dysfunction or organ failure.

In preferred embodiments, the reference level of proADM (preferably herein CP-proADM) is from about 0.7 nmol/L to about 2.0 nmol/L. In more preferred embodiments, the reference level of proADM is from about 0.8 nmol/L to about 1.9 nmol/L. In more preferred embodiments, the reference level of proADM is from about 0.9 nmol/L to about 1.8 nmol/L. In more preferred embodiments, the reference level of proADM is from about 0.88 nmol/L to about 1.79 nmol/L. In more preferred embodiments, the reference level of proADM is about 0.9 nmol/L or about 1.8 nmol/L. In the most preferred embodiments, the reference level of proADM is about 0.88 nmol/L or about 1.79 nmol/L. The reference level used depends on the desired predictive specificity and sensitivity, i.e. a lower threshold such as approximately 0.9 nmol/L (preferably 0.88 nmol/L) proADM (preferably CP-proADM) reliably identifies subjects not experiencing adverse events (in particular, death, that is, it specifically identifies survivors within 28 days).

In particular embodiments, a subject's proADM level (i) less than or equal to a reference proADM level (e.g., from about 0.9 nmol/L to 1.8 nmol/L) indicates that the adverse event is not occurring, specifically , fatal, within 28 days; or (ii) greater than the reference level of proADM (for example, from about 0.9 nmol/l to 1.8 nmol/l), indicates that an adverse event, in particular death, occurs or will occur within 28 days.

The sensitivity and specificity of the proposed methods depend not only on the analytical quality of the test. Sensitivity and specificity also depend on the definition of what constitutes an abnormal (eg, death) or normal result. The distribution of the levels of at least one histone and/or proADM, preferably the level of SR-proADM, for subjects with and without an adverse event may overlap. Under such conditions, the test does not always distinguish between normal and dysfunctional states with 100% accuracy. The person skilled in the art will appreciate that the state of the subject as such, or at least one additional marker and/or parameter of the subject, can aid in the interpretation of the data, and that this additional information allows for a more reliable prediction in areas of overlap. The condition as such may refer to the general health of the subject. The subject's general health may influence the decision to select a reference level. The attached examples show which reference levels have proven to be the most appropriate for a particular condition. Based on this information, the reference level can be further adapted.

For example, the optimal reference level may depend on the general health of the subject. General health status can be determined based on the SOFA score. For example, a proADM reference level of approximately 1.8 nmol/L may be used in less severely ill subjects, such as subjects with a SOFA score ≤ 6. A proADM reference level of approximately 3.2 nmol/L may be used in moderately severely ill subjects, e.g., subjects with a SOFA score of 7 to 12. A proADM reference level of approximately 5.5 nmol/L may be used in severely ill subjects, e.g., subjects with a SOFA score ≥ 13.

In preferred embodiments, the proADM reference level may be approximately 1.8 nmol/L if the subject's SOFA score is ≤ 6, the proADM reference level may be approximately 3.2 nmol/L if the subject's SOFA score is 7 to 12; or the proADM reference level may be approximately 5.6 nmol/L if the subject's SOFA score is ≥ 13. In most preferred aspects, the proADM reference level may be approximately 1.8 nmol/L if the subject's SOFA score is ≤ 6 , or the proADM reference level may be approximately 3.2 nmol/L if the subject's SOFA score is 7 to 12.

Accordingly, the optimal reference level can be selected by determining the level(s) of at least one additional marker and/or parameter (eg, sex, group, and age). Levels of at least one additional marker and/or parameter can also be compared to reference levels, where similar or identical values/levels of a given at least one additional marker and/or subject parameter are compared to corresponding levels of a given at least one additional marker and/or parameter data reference levels indicates that the risk of the subject to experience/have/suffer from an adverse event is reduced, and/or where higher or lower levels/values of this at least one additional marker and/or parameter compared to corresponding levels of this at least one additional marker and/or parameter of these reference levels indicates that the risk of experiencing/having/suffering from an adverse event is increased.

In addition, ROC tests can be used to select the optimal reference level that provides the most appropriate prediction, as described below and in the accompanying examples.

The disease/disorder from which the subject suffers can also influence the optimal reference level. For example, the accompanying examples show that subjects suffering from a fungal infection exhibit the highest levels of proADM. Accordingly, if the subject is suffering from a fungal infection, the reference level of proADM may be 5.6 nmol/L.

In addition, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffer(s) from a disease and/or medical disorder, and where the progression of the disease or disorder is not life threatening. In other words, the reference level may also be the level of at least one histone and/or the level of proADM of at least one reference subject, where the given reference subject(s) is/are suffering from a disease and/or medical condition. disorder, and where the adverse event, such as death, does not occur within 28 days, 7 days, or 3 days.

In addition, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and/or medical condition. disorders and infections (such as sepsis or septic disorders), and where the adverse event, in particular death, does not occur within 28 days, 7 days, or 3 days. In other words, the adverse event in the reference subject does not occur within 28 days, 7 days, or 3 days. In particular, the reference subject suffers from the same disease or medical condition/disorder as the study subject.

In addition, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and/or medical condition. disorder and does not suffer from infection, and where the adverse event, in particular death, does not occur within 28 days, 7 days or 3 days.

In addition, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and/or medical condition. disorders, including SIRS, where the subject(s) do not suffer from infection, and where the adverse event, in particular death, does not occur within 28 days, 7 days, or 3 days.

In the context of the present application, at least one reference subject refers to more than one reference subject. In particular, at least one reference subject is a group or cohort of reference subjects. As described below in this application, means and methods for determining the levels of markers are described, for example, in the cited sources.

The reference level in the context of the present application is usually a predetermined level, that is, it has been predetermined as a reference level for later use at the point of care, for example, ICU or NDE.

In preferred aspects of the invention, the adverse event is a fatal outcome. In these aspects, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where the reference subject(s) does not die(s) for about 28 days, for about 14 days, for about 7 days, or for about 3 days. In addition, in these aspects, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and /or a medical disorder, and where the reference(s) subject(s) does not die(s) for about 28 days, for about 14 days, for about 7 days, or for about 3 days. In addition, in these aspects, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and /or a medical disorder and infection, and where the reference subject(s) does not die(s) for about 28 days, for about 14 days, for about 7 days, or for about 3 days. In addition, in these aspects, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and /or a medical disorder and not from an infection, and where the reference subject(s) does not die(s) for about 28 days, for about 14 days, for about 7 days, or for about 3 days. In addition, in these aspects, the reference level can also be the level of at least one histone and/or the level of proADM of at least one reference subject, where this reference subject(s) suffers from a disease and /or a medical disorder, including SIRS, where the subject(s) do not suffer from an infection, and where the reference subject(s) do not die(s) for approximately 28 days, for approximately 14 days, for about 7 days, or for about 3 days.

As described herein, an increased level of at least one histone (or an increase in the level of at least one histone) and/or an increased level of proADM (or an increase in the level of proADM), in particular CP-proADM compared to a reference level, indicates an unfavorable event, in particular death. In particular, the adverse event occurs within 28 days, 7 days or 3 days as described above. Accordingly, a given level of at least one histone and/or a given level of proADM indicative of a given adverse event may be an elevated level compared to a reference level.

Accordingly, the method of the invention comprises a method comprising determining the level of at least one histone in a sample of a given subject, wherein an elevated level of the given at least one histone of the given subject compared to a reference level of at least one histone is indicative of a given adverse event, in particular , fatal, in this subject.

In addition, the invention includes a method comprising determining the level of proADM, in particular CP-proADM, in a sample of a given subject, where an elevated level of a given proADM, in particular CP-proADM, in a given subject compared to a reference level of a given proADM, in particular , CP-proADM, indicates a given adverse event, in particular a fatal outcome, in a given subject.

In addition, the invention includes a method comprising determining the level of proADM, in particular CP-proADM, in a sample of a given subject, where an elevated level of proADM, in particular CP-proADM, compared with a reference level of proADM, in particular CP-proADM, indicates a given adverse event in a given subject, where preferably the given adverse event occurs within about 28 days; or where a reduced level or equivalent level of proADM, in particular CP-proADM, compared with a reference level of proADM, in particular CP-proADM, indicates that the adverse event in this subject does not occur, preferably within about 28 days .

In addition, the invention includes a method comprising determining the level of at least one histone in a sample of a given subject and determining the level of proADM, in particular CP-proADM, in a sample of a given subject, where an increased level of this at least one histone in a given subject and an increased the level of a given proADM, in particular CP-proADM, in a given subject, compared with a reference level of a given at least one histone and a reference level of a given proADM, in particular CP-proADM, indicate this adverse event, in particular death, in this subject.

The invention also relates to a method comprising

(i) determining the level of at least one histone in a sample of the subject,

where a given level of at least one histone is compared with a reference level of at least one histone,

and where an elevated level of a given at least one histone in a given subject compared to a given reference level of at least one histone is indicative of a given adverse event in the given subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in the subject's sample,

where a given proADM level is compared to a reference proADM level

and where an elevated level of a given proADM in a given subject compared to a given reference level of proADM is indicative of a given adverse event in that subject.

In other words, the invention also relates to a method comprising

(i) determining the level of at least one histone in a sample of the subject,

where a given level of at least one histone is compared with a reference level of at least one histone,

and where the increase in the level of at least one histone compared to the reference level of at least one histone indicates this adverse event in this subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in the subject's sample,

where a given proADM level is compared with a reference proADM level,

and where an increase in the level of proADM compared to the reference level of proADM indicates this adverse event in this subject.

The invention may also relate to a method comprising

(i) determining the level of at least one histone in a sample of the subject,

where a given level of at least one histone is compared with a reference level of at least one histone of the same subject, obtained from a previous analysis,

and where an increase in the level of at least one histone compared to a given reference level of at least one histone indicates a given adverse event in a given subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in the subject's sample,

where this level of proADM is compared with the reference level of proADM of the same subject, obtained from a previous analysis,

and where an increase in the level of proADM compared to a given reference level of proADM indicates a given adverse event in a given subject.

In the context of this application, the term "derived from a previous analysis" refers to a previous determination of the level of a marker in a sample of the same subject, for example, 28 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before the next analysis, and where in such aspects this previously determined marker level is considered as a reference level.

In the context of the present application, the term "elevation of the level of (given) marker" means that the level of the marker is increased, that is, refers to an increased level of the marker. Accordingly, the term "increase in the level of (given) marker" in this application is used interchangeably with the term "increased level of (given) marker". Elevated marker level or elevated marker level of a subject means that the marker level is at least about 15%, preferably at least about 20%, more preferably at least about 25%, or even more preferably at least about 30%. above the reference level of the marker.

In the context of the invention, the term "an increase in the level of at least one histone compared to a reference level" or the like is used interchangeably in this application with the term "an increased level of at least one histone in a given subject compared to a given reference level" or the term "increased the level of at least one histone compared to this reference level", or the like. Such terms mean that the level of at least one histone, e.g., H2B level, H4 level, H2A level, and/or H3 level, in a subject is at least about 15%, preferably at least about 20%, more preferably at least at least about 25%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least at least about 70% more preferably at least about 80%, more preferably at least about 90%, most preferably at least about 100% higher than the reference level of at least one histone. In other words, in the most preferred aspects, the elevated level of a given at least one histone (or an increase in a given level) can be approximately twice as high as a given reference level of at least one histone. Increasing the level by about a factor of two further increases the risk of an adverse event, such as death. For example, a hazard ratio of 1.45, as defined by H4 as indicated in Table 1, means that the risk of an adverse event is further increased by 45%. Therefore, such values are also indicative of a close correlation and optimal markers for diagnosis.

In the context of this application, the term "increased level of proADM compared to a given reference level" or the like is used interchangeably with the term "increased level of proADM in a given subject compared to a given reference level" or the term "increased level of proADM compared to a given reference level", or the like. Such terms mean that the level of proADM, in particular the level of CP-proADM, in the subject is at least about 10%, preferably at least about 15%, more preferably at least about 20%, more preferably at least about 25%, or even more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least at least about 70%, more preferably at least about 80%, more preferably at least about 90%, most preferably at least about 100% higher than the proADM reference level, in particular CP-proADM. In other words, in the most preferred aspects, the elevated level of a given proADM may be approximately twice as high as a given reference level of proADM.

In the context of this application, the term "decrease in the level of proADM compared to the reference level" or the like is used interchangeably with the term "reduced level of proADM in this subject compared to the given reference level" or the term "reduced level of proADM compared to the given reference level", or the like. Such terms mean that the level of proADM, in particular the level of CP-proADM, in the subject is at least about 10%, preferably at least about 15%, more preferably at least about 20%, more preferably at least about 25%, or even more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least at least about 70%, more preferably at least about 80%, more preferably at least about 90%, most preferably at least about 100% lower than the proADM reference level, in particular CP-proADM. In other words, in the most preferred aspects, the reduced level of a given proADM may be approximately two times lower than a given reference level of proADM.

The term "equal" also includes a level that may be similar to a reference level. Accordingly, the term "equal" refers to a subject's proADM level of +/- 10%, preferably +/- 5%, more preferably +/- 2%, or most preferably the same or the same as a reference proADM level.

In the context of this application, the term "reduction or equal level of proADM compared to the reference level" means that the level of proADM, in particular the level of SR-proADM, in the subject is +/- 10%, +/-5%, +/- 2%, or is the same or the same as the reference level, or at least about 10%, preferably at least about 20%, more preferably at least about 25%, or even more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% most preferably at least about 100% lower than the proADM reference level, in particular ti, SR-proADM.

In this application, it is understood that reference levels and determined levels of markers (i.e., levels determined for an individual subject at the point of care, for example, ICU or NDE) may vary depending on the analysis/method by which levels are determined. For example, the reference level and detectable marker level determined by mass spectrometric methods may differ from the corresponding levels determined using immunoassays. The accompanying examples demonstrate that marker levels can be determined by several methods, such as immunoassay methods and mass spectrometric methods. Reference levels can be optimized by statistical methods as shown in the accompanying examples, such as Cox regression analysis. Risk ratios can also be calculated. For example, and as shown in the accompanying examples, hazard ratios greater than 0 (HR>0) indicate a worse prognosis, i.e. an adverse event is likely to occur, and hazard ratios less than 0 indicate a good prognosis, i.e. an adverse event is not likely to occur. will come. Accordingly, one skilled in the art will know how to determine reference levels. For example, levels (including reference levels) can be determined using an immunoassay, for example, by determining the level of at least one histone and/or the level of proADM, eg, CP-proADM, in samples of subjects (or reference subjects).

In certain aspects of the invention, the reference level of at least one histone may be about 100 ng/ml, more preferably about 90 ng/ml, more preferably about 80 ng/ml, more preferably about 70 ng/ml, more preferably about 60 ng/ml , more preferably about 50 ng/ml, more preferably about 45 ng/ml, more preferably about 40 ng/ml, or most preferably about 35 ng/ml. In certain aspects of the invention, the reference level of at least one histone may be about 10 ng/mL, more preferably about 15 ng/mL, more preferably about 20 ng/mL, more preferably about 25 ng/mL, more preferably about 30 ng/mL. or most preferably about 35 ng/ml.

In certain aspects of the invention, the reference level of at least one histone may be from about 10 ng/ml to about 100 ng/ml, from about 10 ng/ml to about 90 ng/ml, more preferably from about 10 ng/ml to about 60 ng/ml, more preferably from about 10 ng/ml to about 40 ng/ml, more preferably from about 15 ng/ml to about 40 ng/ml, or most preferably from about 20 ng/ml to about 40 ng/ml.

In certain aspects of the invention, the proADM reference level may be about 4 nmol/L, more preferably about 5 nmol/L, more preferably about 7 nmol/L, more preferably about 8 nmol/L, more preferably about 9 nmol/L, or more preferably about 6 nmol/l.

Moreover, levels (including reference levels) can be determined using mass spectrometric methods, such as methods that determine the relative amount or determine the absolute amount of the protein or fragment of interest.

Determining the relative amount of "SRM" can be done by:

1. Determination of increased or decreased presence of a target protein by comparing the area of the characteristic SRM peak (selected reactions monitoring) of a given target fragmented peptide detected in a sample with the area of the same characteristic SRM peak of the target fragmented peptide at least in the second, third, fourth or more biological samples.

2. Determination of increased or decreased presence of a target protein by comparing the characteristic SRM peak area of a given target peptide detected in a sample with the areas of characteristic SRM peaks obtained from fragmented peptides of other proteins in other samples obtained from different and separate biological sources, where comparison the area of the characteristic SRM peak of two samples in relation to the fragmented peptide is normalized, for example, to the amount of protein analyzed in each sample.

3. Determination of increased or decreased presence of a target protein by comparing the area of the characteristic SRM peak for a given target peptide with the areas of the characteristic SRM peaks of other fragmented peptides derived from different proteins in the same biological sample, in order to normalize the changing levels of histone proteins to the levels of other proteins , which do not change their expression levels under different cellular conditions.

4. These assays can be applied to both unmodified fragmented peptides and modified fragmented peptides of target proteins, where modifications include, but are not limited to, phosphorylation and/or glycosylation, acetylation, methylation (mono-, di-, tri-), citrullination , ubiquitinylation, and where the relative levels of modified peptides are determined in the same manner as the determination of the relative amounts of unmodified peptides.

Determining the absolute amount of a given peptide can be done by:

1. Comparison of the SRM/MRM characteristic peak area for a given fragmented peptide from target proteins in a single biological sample with the SRM/MRM characteristic peak area of the fragmented peptide internal standard added to the protein lysate from the biological sample. The internal standard can be a labeled synthetic version of the fragmented peptide from the target protein under study, or a labeled recombinant protein. This standard is added to the sample in known amounts before (required for a recombinant protein) or after digestion, and the SRM/MRM characteristic peak area can be determined for both the fragmented peptide internal standard and the native fragmented peptide in the biological sample separately, followed by comparison. areas of both peaks. This method can be applied to unmodified fragmented peptides and modified fragmented peptides, where modifications include, but are not limited to, phosphorylation and/or glycosylation, acetylation, methylation (e.g., mono-, di-, or trimethylation), citrullination, ubiquitinylation, and where the absolute levels modified peptides can be determined in the same way as the absolute levels of unmodified peptides.

2. Peptides can also be quantified using external calibration curves. The normal curve approach uses a constant amount of heavy peptide as an internal standard and a varying amount of light synthetic peptide added to the sample. To construct standard curves taking into account the influence of the matrix, it is necessary to use a representative matrix similar to the matrix of the samples under study. In addition, the inverse curve method (internal standard method) circumvents the problem of endogenous analyte in the matrix, where a constant amount of light peptide is added to the endogenous analyte to obtain an internal standard, and different amounts of heavy peptide are added to obtain a set of standard concentrations. In the test samples for comparison with normal or reverse curves, add an amount of standard peptide equal to the amount of internal standard added to the matrix used to create the calibration curve.

Accordingly, the person skilled in the art knows how to determine the levels of markers and, in particular, the optimal reference levels, which is also illustrated in the attached examples.

The methods and kits of the present invention may also include determining at least one additional marker and/or parameter in addition to at least one histone and/or proADM.

In the context of this application, a parameter is a characteristic, feature, or measurable that can help identify a particular system. The parameter is an important element of health and physiology assessments, such as the risk of a disease/disorder/clinical condition, preferably an adverse event. In addition, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. An exemplary parameter may be selected from the group consisting of Acute Physiological Conditions and Chronic Health Scale (APACHE I-IV scale), Simplified Acute Physiological Conditions Scale (SAPS I-III scale), Dynamic Organ Failure Scale score ( SOFA scores), Accelerated SOFA scores (qSOFA), Simplified Acute Physiological Conditions II scores (SAPSII), Probability of Death Model (MPM I-III), Multiple Organ Failure Scale (MODS), scores Effectiveness of Care (TISS), Nine Equivalents of Nursing Care Scale (NEMS), World Federation of Neurosurgical Societies (WFNS) and Glasgow Coma Scale (GCS), CURB-65 Pneumonia Severity Scale, Pneumonia Severity Index ( PSI), age, gender, family history, ethnicity, body weight, body mass index (BMI), cystoscopy report, white blood cell count, scan computed tomography, blood pressure, heart rate, antihypertensive medication, fluid intake, wheezing, body temperature, presence of diabetes, and current smoking habit.

In the context of this application, terms such as "marker", "mimic", "prognostic marker", "indicator" or "biomarker", or "biological marker" are used interchangeably and refer to measurable and quantifiable biological markers (for example, the concentration of a certain protein or enzyme or fragment thereof, the concentration of a certain hormone or fragment thereof, or the presence of biological substances or a fragment thereof), which serve as indicators for assessments of health and physiology, such as the risk of a disease/disorder/clinical condition, preferably an adverse event. A marker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers can be measured in a sample (such as a blood, plasma, urine, or tissue test). The subject's at least one additional marker may be selected from the group consisting of procalcitonin, calcitonin, endothelin-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP), neutrophil gelatinase-associated lipocalin ( NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic stone protein (PSP), myeloid cell-expressed trigger receptor 1 (TREM1), interleukin-6 (IL-6), interleukin- 1, interleukin-24 (IL-24), other interleukins, presepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), alpha-1 antitrypsin, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP9), chromogranin A, S100A protein, S100B protein and tumor necrosis factor-α (TNFα), or a fragment thereof.

In particular aspects of the invention, at least one additional marker and/or parameter of a given subject may be selected from the group consisting of the level of aldolase B in a given sample, the level of copeptin in a given sample, the level of proendothelin-1 in a given sample, the level of lactate in a given sample , the level of procalcitonin (PCT) in the given sample, the subject's Dynamic Organ Failure Assessment (SOFA) score, the Simplified Acute Physiological Conditions (SAPSII) score, the Acute Physiological and Chronic Health II (APACHE II) scores ) of a given subject and the level of soluble fms-like tyrosine kinase-1 (sFlt-1) in a given sample.

In certain aspects of the invention, at least one additional marker and/or parameter of a given subject may be selected from the group consisting of procalcitonin, calcitonin, endothelin-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP) , neutrophil gelatinase-associated lipocalin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic stone protein (PSP), trigger receptor expressed on myeloid cells 1 (TREM1), interleukin-6 (IL-6), interleukin-1, interleukin-24 (IL-24), other interleukins, presepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), alpha-1 antitrypsin, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP9), chromogranin A, S100A protein, S100B protein, tumor necrosis factor-α (TNFα), age, sex, family history, ethnicity, body weight, body mass index (BMI) ), c histoscopic report, white blood cell count, computed tomography scan, blood pressure, heart rate, antihypertensive treatment, fluid intake, wheezing, body temperature, presence of diabetes mellitus, current smoking habit, scores on the Acute Physiological Conditions and Chronic Health APACHE I-IV), Simplified Acute Physiological Conditions Scale (SAPS I-III scale), Dynamic Organ Failure Assessment (SOFA) scores, Probability of Death Model (MPM I-III), Multiple Organ Failure Scale scores (MODS), Treatment Effectiveness Scale (TISS), Nine Nursing Equivalents Scale (NEMS), World Federation of Neurosurgical Societies (WFNS) scores, and Glasgow Coma Scale (GCS) scores.

In the context of this application, the term "aldolase B" refers to fructose bisphosphate aldolase B or liver type aldolase, which is one of the three isoenzymes (A, B and C) of the class I fructose 1,6-bisphosphate aldolase enzyme. The level of aldolase B in sample of the subject can be determined by mass spectrometric methods.

"Copeptin" is also called "CT-proAVP" or "C-terminal vasopressin". Vasopressin is a powerful vasoconstrictor. The level of CT-proAVP can be measured in the plasma or serum of a subject using immunoassays, as described below.

In the context of this application, the term "lactate" refers to the maximum concentration of lactate measured in the blood. Typically, the lactate concentration is measured daily or even more frequently. The concentration of lactate in the blood can be determined by analyzing lactate oxidase by spectrophotometric methods.

In the context of the present application, the term "procalcitonin" or "PKT" refers to a peptide covering amino acid residues 1-116, 2-116, 3-116 or fragments thereof. Thus, procalcitonin fragments are at least 12 amino acids in length, preferably more than 50 amino acids, more preferably more than 110 amino acids. PCT may contain post-translational modifications such as glycosylation, lipidation or derivatization. Procalcitonin is the precursor of calcitonin and catacalcin. Thus, under normal conditions, circulating PCT levels are very low (less than about 0.05 ng/mL). The level of PCT in a subject's sample can be determined using immunoassays, as described below.

In the context of this application, the term "dynamic organ failure score" or "SOFA score" is one of the rating scales used to track the condition of a patient during a stay in the intensive care unit (ICU) or emergency department (EDD). The SOFA scale is a scoring system that determines the degree of dysfunction or organ failure in a patient. The scale is based on six different scores, one each for the respiratory, cardiovascular, hepatic, coagulation, renal, and neurological systems. Both the average and the highest SOFA scores are predictive of outcome. An increase in SOFA score during the first 24-48 hours of ICU stay or NDE predicts a mortality rate of at least 50% to 95%. Scores less than 9 predict a fatality rate of 33%, while scores above 14 may be close to 95% or higher.

In the context of this application, "SAPS II" or "Simplified Acute Physiology Score II" refers to a classification system for the severity of a disease or disorder (see Le Gall JR et al., A new Simplified Acute Physiology Score (SAPS II) based on a European /North American multicenter study JAMA 1993;270(24):2957-63). The SAPS II scale consists of 12 physiological parameters and 3 disease-related parameters. The score is calculated on the basis of 12 routine physiological measurements, information about the previous state of health and some information obtained upon admission to the ICU or NCO. The SAPS II score can be determined at any time, preferably on day 2. The "worst" measurement is defined as the measure that corresponds to the highest score. The score on the SAPS II scale ranges from 0 to 163 points. The classification system includes the following parameters: age, heart rate, systolic blood pressure, temperature, Glasgow Coma Scale score, mechanical ventilation or CPAP (constant positive pressure ventilation), PaO2, FiO2, urine output, blood urea nitrogen, sodium, potassium, bicarbonate, bilirubin, leukocytes, chronic diseases and type of hospitalization. There is a sigmoidal relationship between death and overall SAPS II score. The lethal outcome of the subject is 10% with a SAPSII score of 29, 25% with a SAPSII score of 40, 50% with a SAPSII score of 52, 75% with a SAPSII score of 64 points, and 90% with a SAPSII score of 77 points (Le Gall loc. cit.).

In the context of this document, the term "APACHE II" or "acute physiological conditions and chronic health status scale II" is a system for assessing the severity of the disease (Knaus et al., 1985). It can be used up to 24 hours after a patient is admitted to the intensive care unit (ICU) or emergency department (ED) and can be determined based on 12 different physiological parameters.

In the context of this document, the term "accelerated SOFA score" or "qSOFA score" is a scoring system that indicates impaired organ function or risk of death in a patient. The rating scale is based on three criteria: 1) a change in mental status, 2) a decrease in systolic blood pressure to a level of less than 100 mm Hg. Art., 3) the frequency of respiratory movements is more than 22 breaths per minute. Patients with two or more of these conditions are at greater risk of organ failure or death.

In the context of this application, the term "soluble fms-like tyrosine kinase-1" or "sFlt-1" is a tyrosine kinase protein that deactivates proteins that cause blood vessel growth. Soluble Flt-1 (sFlt-1) is a splice variant of VEGF receptor 1 (Flt-1) that is produced by various tissues. The level of sFLT1 in a subject's sample can be determined by mass spectrometry and by immunoassays.

In the context of the present application, the "CURB-65" scale (Lim et al., 2003) refers to the following subject risk factors:

New onset confusional state (defined as a score on the Abbreviated Intelligence Test (AMTS) (Hodkinson, 1972) of 8 or less),

Blood urea nitrogen more than 7 mmol/l (19 mg/dl),

Respiratory rate of 30 breaths per minute or more

Blood pressure less than 90 mm Hg. Art., systolic or diastolic blood pressure equal to 60 mm Hg. Art. or less

Age 65 years and older.

Each risk factor is assigned one point, with a maximum score of 5.

As used herein, "pneumonia severity index" (PSI) refers to a clinical predictive rule for calculating the likelihood of morbidity and mortality among patients with community-acquired pneumonia (Fine et al., 1997).

Methods or kits according to the invention may include determining the level of at least one histone and/or the level of proADM and the level of an additional marker and/or parameter, as described above.

In preferred aspects, these methods or kits of the invention may include determining a given proADM level and a given SOFA score. In addition, these methods or kits of the invention may include determining a given proADM level, a given SOFA score, and a given lactate level in a given subject.

The methods and kits used according to the present invention may also include the determination of at least one additional parameter, such as a SAPS II score, a SOFA score, and/or an APACHE II score. For example, the method may include determining the level of at least one histone and/or the level of proADM in a sample of the subject and scoring the subject's SAPS II scale. Preferably, the method comprises determining a given level of at least one histone in a given sample of a given subject and a given SAPSII score of the subject. The level of at least one histone and the score on the SAPSII scale may indicate the onset of an adverse event, in particular death, in this subject within 28 days.

Preferably, the method comprises determining the level of proADM in a given sample of a given subject and a given SAPSII score of the subject. The proADM level and SAPSII score may indicate the onset of an adverse event, in particular death, in a given subject within 28 days or within 7 days.

In addition, the method may include determining the level of at least one histone and/or the level of proADM in a sample of the subject and assessing the subject on the SOFA scale.

In addition, the method may include determining the level of at least one histone and/or the level of proADM in the sample of the subject and the level of PCT in the sample of the subject. In addition, the method may include determining the level of at least one histone and/or the level of proADM in a sample of the subject and the level of aldolase B in the sample of the subject.

In the context of this application, the term "subject" (or "patient") may refer to a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, in particular mammals, and other organisms. Thus, the methods proposed in this application are applicable to both humans and animals. Accordingly, the subject may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, cattle, horse, camel, or primate. Preferably the subject is a mammal. More preferably, the subject is a human.

The method proposed in this application can be applied to any subject, which is a healthy subject or a subject suffering from any(their) disease(s) or disorder(s). In preferred aspects, the subject is suffering from a disease, disorder, or medical condition. The subject to be investigated may be a critically ill patient, preferably where the subject has been admitted to an intensive care unit. In the context of this application, the term "critically ill" means that the subject or patient is in a life-threatening situation.

The subject or reference subject(s) may be suffering from a disease or medical condition/disorder, where the disease or medical condition/disorder may be selected from the group consisting of respiratory disease, urinary tract infection, inflammatory response associated with infection and non-infectious etiology, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, organ failure(s), cardiovascular disease, diabetes mellitus, malignancy(s), liver disease, disease kidney, immunosuppression, viral infection, fungal infection, bacterial infection, gram-negative bacterial infection, gram-positive bacterial infection and immune suppression. In the present application, the method according to the invention, based on the detection of the level of proADM (in particular, the level of SR-proADM), is particularly effective in predicting an adverse event in subjects suffering from a fungal infection. It is also particularly effective in subjects suffering from respiratory tract infections, in particular lower respiratory tract infections.

The subject may also be suffering from an infection following a bone and/or tissue defect, such as a broken bone.

In addition, the subject may also be selected for surgical intervention. This means that the subject will undergo surgery. This surgical intervention may be related to the disease and/or disorder that is the cause or symptom on admission to the hospital.

In particular, the subject suffers from respiratory disease, urinary tract infection and/or malignant(s) neoplasm(s).

"Systemic inflammation" in the context of the invention preferably refers to a condition characterized by the release of pro-inflammatory cytokines and activation of the innate immune system, which may be caused by biological factors, chemical factors or genetic factors. Severe "systemic inflammation" can lead to organ failure and death.

"SIRS" in the context of the invention is a systemic inflammatory response syndrome without signs of infection. It includes, but is not limited to, more than one of the following clinical manifestations: (1) body temperature above 38°C or below 36°C; (2) heart rate greater than 90 beats per minute; (3) tachypnea, manifested by a respiratory rate of more than 20 breaths per minute, or hyperventilation, as indicated by PaCO ₂ less than 32 mm Hg. Art.; and (4) a change in white blood cell count, such as a count greater than 12,000/mm ³ , a count less than 4,000/mm ³ , or the presence of greater than 10% immature neutrophils (Bone et al., CHEST 101(6): 1644-55, 1992).

"Sepsis" in the context of the invention refers to the systemic response to infection. Alternatively, sepsis can be considered as a combination of SIRS with a confirmed infectious process or infection. Sepsis can be characterized as a clinical syndrome defined by the presence of both infection and a systemic inflammatory response (Levy MM et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med. 2003 Apr;31(4) :1250-6). The term "sepsis" in the context of this application includes, but is not limited to, sepsis, severe sepsis, septic shock. Severe sepsis in this context means sepsis associated with dysfunction of an organ, pathology associated with hypoperfusion, or hypotension caused by sepsis. Pathologies associated with hypoperfusion include lactic acidosis, oliguria, and acute mental status change. Sepsis-induced hypotension is defined as systolic blood pressure less than about 90 mmHg. Art. or its decrease by approximately 40 mm Hg. Art. or greater than baseline in the absence of other causes of hypotension (eg, cardiogenic shock). Septic shock is defined as severe sepsis with sepsis-induced hypotension persisting despite appropriate fluid therapy, along with the presence of hypoperfusion-related pathologies or organ dysfunction (Bone et al., CHEST 101(6): 1644-55, 1992).

The term "sepsis" also includes severe sepsis or septic shock based on the definition of SEPSIS-2 (Bone et al., 2009). The term "sepsis" also includes subjects that fall under the definition of SEPSIS-3 (Singer et al., 2016).

The term "sepsis" in the context of this application refers to all possible stages of development of sepsis.

In the context of the present application, the term "infection" in the scope of the invention means a pathological process caused by the penetration into normal sterile tissue or fluid of pathogenic or potentially pathogenic microorganisms, and refers to infection(s) by bacteria, viruses, fungi and/or parasites. Accordingly, the infection may be a bacterial infection, a viral infection, and/or a fungal infection. The infection may be a local or systemic infection. In addition, a subject suffering from an infection may be suffering from more than one source of infection at the same time. For example, a subject suffering from an infection may suffer from a bacterial infection and a viral infection; from viral infection and fungal infection; from bacterial and fungal infection, and from bacterial infection, fungal infection and viral infection.

In particular, the subject is suffering from sepsis. In addition, the subject may preferably be suffering from a respiratory disease, preferably a lower respiratory tract infection. In the context of the present application, a disease of the respiratory system includes pathological conditions that affect organs and tissues, allowing gas exchange in higher organisms, and also includes conditions of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, as well as nerves and muscles, involved in respiration.

In particular, in the methods and kits of the invention, the level of at least one histone in a given sample of a given subject suffering from a given respiratory disease indicates the onset of an adverse event, in particular death within 7 days.

In particular, in the methods and kits of the invention, a subject is suffering from a urinary tract infection, wherein a given level of at least one histone in a given sample of a given subject suffering from a given urinary tract infection is indicative of an adverse event, in particular death within 28 days.

In particular, in the methods and kits of the invention, a subject is suffering from a cancer, wherein a given level of at least one histone in a given sample of a given subject suffering from a given cancer is indicative of the occurrence of an adverse event, such as death within 7 days. .

In the context of this application, the term "sample" means a biological sample obtained from a subject. In the context of the present application, "sample" may, for example, refer to a biological fluid or tissue sample obtained for the purpose of diagnosing, predicting, or evaluating a subject of interest, such as a patient. Preferably, in the present application, the sample is a biological fluid sample such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In particular, the sample is blood, blood plasma, blood serum or urine. Samples may be processed (pre-treated) eg by fractionation or purification, eg separation of whole blood into serum or plasma components. Such pretreatments may also include, but are not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation, or dialysis. Pre-treatments may also include the addition of chemicals or biochemicals to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, complexing agents. Preferably the sample is a blood sample, more preferably a serum sample or a plasma sample.

"Plasma" in the context of the present invention is a practically cell-free blood supernatant containing an anticoagulant obtained after centrifugation. Examples of anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparinates or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (eg, citrated, EDTA or heparinized blood), for example, for at least 15 minutes at 2000-3000 g.

"Serum" in the context of the present invention is the liquid fraction of whole blood, which is collected after the blood is allowed to clot. When coagulated blood (clotted blood) is centrifuged, serum can be obtained as a supernatant.

In the context of this application, "urine" is a body fluid excreted by the kidneys in a process called urination (or urination) and excreted through the urethra.

As described above, the level of at least one histone and/or proADM is determined in a sample of a subject. The person skilled in the art is aware of methods/assays that can be used to determine the level of biomarkers in a sample.

As described above, the level of at least one histone is determined, in particular, the level (s) of histones H2B, H4, H2A and/or H3 is determined. In particular, a histone or a fragment of a histone can be determined. Examples of such fragments are given below in this application.

Such a histone or fragment thereof may also be a free histone. For example, methods, kits and antibodies according to the present invention may, in particular, provide for the detection of peptides or epitopes of free histones. Such amino acid regions are also referred to herein as histone cores or parts. The following describes peptides or epitopes that can also be used to detect free histones using the methods proposed in this application.

In particular, at least one histone may be an H4 histone, wherein at least one peptide with a sequence spanning amino acid residues 22 to 102 of histone H4 according to SEQ ID NO: 1 is determined. In particular, at least one histone represents histone H4, at least a peptide with a sequence selected from the group consisting of amino acid sequences covering residues 60 to 67 of SEQ ID NO: 1, residues 46 to 56 of SEQ ID NO: 1, residues 67 to 78 SEQ ID NO: 1, residues 22 to 30 of SEQ ID NO: 1, residues 67 to 78 of SEQ ID NO: 1, residues 92 to 102 of SEQ ID NO: 1, residues 22 to 34 of SEQ ID NO: 1, residues 46 to 102 of SEQ ID NO: 1, residues 46 to 55 of SEQ ID NO: 1, residues 80 to 91 of SEQ ID NO: 1, residues 24 to 35 of SEQ ID NO: 1 and residues 68 to 77 SEQ ID NO: 1. For example, two peptides can be identified.

In particular, at least one histone is histone H4, and peptides are defined with amino acid sequences spanning residues 46 to 56 of SEQ ID NO: 1 and residues 67 to 78 of SEQ ID NO: 1.

In addition, at least one histone is an H2A histone, wherein at least a peptide with a sequence spanning amino acid residues 20 to 118 of histone H2A according to SEQ ID NO: 2 is determined. In particular, at least one histone is is histone H2A, wherein at least a peptide with a sequence selected from the group consisting of amino acid sequences spanning residues 21 to 53 of SEQ ID NO: 2, residues 21 to 29 of SEQ ID NO: 2, residues 30 to 53 SEQ ID NO: 2, residues 120 to 129 SEQ ID NO: 2, residues 21 to 29 SEQ ID NO: 2, residues 82 to 88 SEQ ID NO: 2, residues 89 to 95 SEQ ID NO: 2 and residues 100 to 118 of SEQ ID NO: 2.

In addition, at least one histone is an H3 histone, and at least one peptide with a sequence spanning amino acid residues 27 to 62 of histone H3 according to SEQ ID NO: 3 is determined. In addition, at least one histone is is histone H3, wherein at least a peptide with a sequence spanning amino acid residues 27 to 37 of SEQ ID NO: 3 and/or spanning amino acid residues 52 to 62 of SEQ ID NO: 3 is determined.

In addition, at least one histone is histone H2B, and at least a peptide with a sequence spanning amino acid residues 41 to 69 of histone H2B according to SEQ ID NO: 4 is determined.

In addition, at least one histone is histone H2A, wherein at least a peptide or fragment thereof is selected from the group consisting of SEQ ID NOs: 7, 8, 9, and 10.

In addition, at least one histone is histone H4, wherein at least a peptide or fragment thereof is selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15 and 16.

In the present application it is understood that one, two, three, four or more peptides can be defined.

The level of markers, for example, at least one histone or fragment thereof and/or proADM or fragment thereof, can be determined using any assay that reliably determines the concentration of the marker. In particular, mass spectrometry (MS) and/or immunoassays can be used, as shown in the accompanying examples. In the context of the present application, an immunoassay is a biochemical test that measures the presence or concentration of a macromolecule/polypeptide in solution using an antibody or binding fragment of an antibody or immunoglobulin.

Alternatively, instead of antibodies, other capture molecules or molecular scaffolds that specifically and/or selectively recognize histone sequences, histone epitopes, and histone structural conformations may be included within the scope of the present invention. In the context of this application, the term "capture molecules" or "molecular scaffolds" includes molecules that can be used to bind molecules of interest or interest, i.e., analytes (eg, histone(s) and/or proADM), from a sample. Thus, capture molecules must have an appropriate shape, both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, to specifically bind target or molecules of interest. Thus, binding may, for example, be mediated by interactions based on ionic, van der Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bonds, or a combination of two or more of the above interactions, or covalent interactions between capture molecules or molecular scaffolds and target or molecules of interest. In the context of the present invention, capture molecules or molecular scaffolds may, for example, be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, a peptide, and a glycoprotein. Capture molecules or molecular scaffolds include, for example, aptamers, DARpin (engineered ankyrin repeat proteins), affimers, and the like.

In the context of the present application, the term "antibody" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin (Ig) molecules, that is, molecules containing an antigen-binding site that specifically binds an antigen (which enters into an immune reaction with it). According to the invention, the antibodies can be both monoclonal and polyclonal antibodies. In particular, antibodies are used that specifically bind to at least one histone and/or specifically bind to proADM. An antibody is considered specific if its affinity for a molecule of interest, e.g., at least one histone and/or proADM, or fragment thereof, is at least 50-fold higher, preferably 100-fold higher, most preferably at least 1000 times higher than with respect to other molecules contained in the sample containing the molecule of interest. It is well known in the art how to design and select antibodies with a given specificity. In the context of the invention, monoclonal antibodies are preferred. An antibody or binding fragment thereof specifically binds to markers or fragments thereof as defined herein. In particular, the antibody, or binding fragment thereof, binds to the peptides of at least one histone protein as defined herein. Thus, the peptides defined herein may also be epitopes to which antibodies specifically bind. In addition, the methods and kits of the invention employ an antibody or binding fragment thereof that specifically binds to proADM, in particular CP-proADM. Examples of immunoassays can be luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescently labeled bead-based matrices, magnetic beads, protein microarray-based assays, accelerated methods of analysis, analysis using rare earth cryptates. In addition, assays suitable for point-of-care testing and accelerated test methods such as, for example, immunochromatographic test strips can be used.

In certain aspects of the invention, the method is a method for diagnosing, predicting, assessing risk and/or stratifying the risk of an adverse event in a subject, including

(i) obtaining a sample from the subject;

(ii) detecting the level of at least one histone and/or the level of proadrenomedullin (proADM) in the subject's sample by contacting the sample with the antibody(s) or its (their) antigen-binding fragment(s) or derivative(s) specific for an epitope of a given at least one histone and/or a given proADM, and detection of binding between the antibody(s) or its (their) antigen-binding fragment(s) or derivative(s) and the given at least one histone and /or proADM data;

(iii) where a given level of at least one histone and/or a given level of proadrenomedullin (proADM) indicate(s) a given adverse event in a given subject.

In certain aspects of the invention, the method is an immunoassay comprising the following steps:

a) bringing the sample into contact with

(i) a first antibody or antigen-binding fragment or derivative thereof specific for a first histone epitope or proADM, and

(ii) a second antibody or antigen-binding fragment or derivative thereof specific for a second histone epitope or proADM; and

b) detecting the binding of the first and second antibodies or their antigen-binding fragments or derivatives to a histone or proADM.

Preferably, one of the antibodies may be labeled and the other antibody may be associated with the solid phase or may be selectively associated with the solid phase. In a particularly preferred aspect of the assay, one of the antibodies is labeled while the other is either bound to the solid phase or may be selectively bound to the solid phase. The first antibody and the second antibody may be present in a dispersed form in a liquid reaction mixture, where the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, is associated with the first antibody, and the second labeling component of this labeling system is associated with the second antibody so that upon binding of both antibodies to the at least one histone detection data and/or proADM data, a measurable signal is generated allowing the resulting sandwich complexes to be detected in the measurement solution. The labeling system may contain a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

In a preferred embodiment, the method is carried out as a heterogeneous sandwich immunoassay in which one of the antibodies is immobilized on a randomly selected solid phase, e.g. on the walls of coated tubes (e.g., polystyrene tubes; coated tubes; CT) or microtiter plates, e.g. consisting of polystyrene, or on particles, such as, for example, magnetic particles, while the other antibody has a group that resembles a detectable label or allows selective attachment to the label, and which serves to detect the formed sandwich structures. It is also possible to temporarily delay the immobilization or to carry it out later using suitable solid phases.

The method of the present invention can also be carried out as a homogeneous method, where the sandwich complexes formed by the antibody/antibodies and a marker, for example, a detectable histone or proADM or a fragment thereof, remain suspended in the liquid phase. In this case, it is preferable that in the case of using two antibodies, both antibodies are labeled with parts of the detection system, which results in signal generation or signal triggering if both antibodies are combined in one sandwich. Such techniques should be implemented, in particular, as methods for detecting fluorescence enhancement or fluorescence quenching. A particularly preferred aspect relates to the use of detection reagents used in pairs, such as, for example, those described in US Pat. In this way, measurements are possible in which only reaction products containing both labeling components in one immune complex are detected directly in the reaction mixture. For example, such technologies are offered under the trademarks TRACE ^® (Time Resolved Amplified Cryptate Emission) or KRYPTOR ^® , which implement the ideas of the patent applications cited above. Therefore, in particular preferred aspects, a diagnostic device is used to implement the method proposed in this application. For example, the level of histone or proADM or their fragment, and/or the level of any other marker from the method proposed in this application is determined. In particular preferred aspects, the diagnostic device is a KRYPTOR ^® .

In addition, the immunoassay methods of the present invention may preferably employ a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an epitope(s) of at least one histone and/or proADM. For example, below and above in this application, peptides are described that may be suitable for determining the level of proADM and/or at least one histone.

For example, the immunoassay methods of the present invention may preferably use a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H4 histone epitope(s), wherein the first epitope and/or the second epitope is the H4 histone epitopes present in the sequence spanning amino acid residues 22 to 102 of SEQ ID NO: 1.

In addition, the immunoassay methods of the present invention may employ a first antibody, an antigen-binding fragment thereof, or a derivative specific for an H4 histone epitope present in the sequence spanning amino acid residues 46 to 56 of SEQ ID NO: 1, and a second antibody, an antigen-binding fragment thereof. or a derivative specific for an H4 histone epitope present in the sequence spanning amino acid residues 67 to 78 of SEQ ID NO: 1.

For example, the immunoassay methods of the present invention may preferably use a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H2B histone epitope(s), wherein the first epitope and/or the second epitope are H2B histone epitopes present in the sequence spanning amino acid residues 41 to 69 of SEQ ID NO: 4.

In addition, the immunoassay methods of the present invention may preferably use a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H2A histone epitope(s), wherein the first epitope and/or or the second epitope is the H2A histone epitopes present in the sequence spanning amino acid residues 20 to 118 of SEQ ID NO: 2.

In addition, the immunoassay methods of the present invention may use a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H2A histone epitope(s), wherein the first epitope and/or the second epitope are H2A histone epitopes present in the sequence spanning amino acid residues 21 to 53, 20 to 118, or 120 to 129 of SEQ ID NO:2.

In addition, the immunoassay methods of the present invention may employ a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H3 histone epitope(s), wherein the first epitope and/or the second epitope is the H3 histone epitopes present in the sequence spanning amino acid residues 27 to 62 of SEQ ID NO: 3.

More preferably, the immunoassay methods of the present invention may employ a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H4 histone epitope(s), wherein the epitope(s) is selected. (s) from the group consisting of the amino acid sequence comprising residues 22 to 30 of SEQ ID NO:1, residues 46 to 56 of SEQ ID NO:1, residues 67 to 78 of SEQ ID NO:1, residues 92 to 102 SEQ ID NO:1, residues 22 to 34 of SEQ ID NO: 1 and residues 46 to 102 of SEQ ID NO: 1.

In addition, the immunoassay methods of the present invention may employ a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H2A histone epitope(s), wherein the epitope(s) is selected. (s) from the group consisting of the amino acid sequence comprising residues 21 to 53 of SEQ ID NO: 2, residues 21 to 29 of SEQ ID NO: 2, residues 30 to 53 of SEQ ID NO: 2, and residues 120 to 129 SEQ ID NO: 2.

In addition, the immunoassay methods of the present invention may use a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for the H2B histone epitope(s) spanning (covering) residues with 41 to 69 SEQ ID NO: 4.

In addition, the immunoassay methods of the present invention may employ a first antibody and/or a second antibody, or antigen-binding fragment(s) or derivative(s) thereof, specific for an H3 histone epitope(s) spanning (spanning) residues with 27 to 37 of SEQ ID NO: 3 and/or enclosing (covering) residues 52 to 62 of SEQ ID NO: 3.

In addition, the immunoassay methods of the present invention may use a first antibody and/or a second antibody, or an antigen-binding fragment or derivative thereof, specific for an H2A histone epitope present in the sequence spanning amino acid residues 21 to 53 and/or 120 to 129. the sequence of histone H2A shown by SEQ ID NO: 2.

In addition, the immunoassay methods of the present invention may employ a first antibody, an antigen-binding fragment thereof, or a derivative specific for an H4 histone epitope present in the sequence spanning amino acid residues 22 to 102 of SEQ ID NO: 1, and a second antibody, an antigen-binding fragment thereof. or a derivative specific for the free histone epitope H2A, H2B or preferably H3.

In addition, the immunoassay methods of the present invention may employ a first antibody, an antigen-binding fragment thereof, or a derivative specific for an H2B histone epitope present in the sequence spanning amino acid residues 41 to 69 of SEQ ID NO: 4, and a second antibody, an antigen-binding fragment thereof. or a derivative specific for the free histone epitope H2A, H4 or H3.

In addition, the immunoassay methods of the present invention may employ a first antibody, an antigen-binding fragment thereof, or a derivative specific for an H2B histone epitope present in the sequence spanning amino acid residues 20 to 118 of SEQ ID NO: 2, and a second antibody, an antigen-binding fragment thereof. or a derivative specific for the free histone epitope H2B, H4 or H3.

In addition, the immunoassay methods of the present invention may use a first antibody, an antigen-binding fragment thereof, or a derivative specific for an H2B histone epitope present in the sequence spanning amino acid residues 27 to 62 of SEQ ID NO: 3, and a second antibody, an antigen-binding fragment thereof. or a derivative specific for the epitope of the free histone H2B, H4 or H2A.

In addition, the immunoassay methods of the present invention may use a first antibody, an antigen-binding fragment thereof, or a derivative thereof specific for an H2A histone epitope present in the sequence spanning amino acid residues 21 to 53, 120 to 129, or 20 to 118 of SEQ ID NO : 2, and a second antibody, an antigen-binding fragment or derivative thereof, specific for the free histone epitope H3, H4, or preferably H2B.

The invention also relates to an antibody, or an antigen-binding fragment or derivative thereof, specific for an epitope of a histone protein or fragment thereof, as described in detail above. Examples of antibodies useful for detecting histones or proADM, preferably CP-proADM, are shown in the accompanying examples. Thus, the present invention relates to an antibody(s), its (their) antigen-binding fragment(s) or derivative(s), specific(s) for the histone epitope H2B, H4, H2A, H3 and/or proADM, preferably SR-proADM. Examples of epitopes or peptides that antibodies specifically bind to are given above and below in this application.

The level of the marker, for example, at least one histone and/or proADM, can also be determined using mass spectrometric (MS) analysis, as described in the attached examples. Such a method may include detecting the presence, amount, or concentration of one or more modified or unmodified fragmented peptides, e.g., proADM and/or histone, in a given biological sample or protein cleavage product (e.g., trypsin cleavage) from that sample, and optionally separating sample by chromatographic methods and subjecting the prepared and optionally separated sample to MS analysis. For example, MS analysis can use mass spectrometry with selected reaction monitoring (SRM), multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM), in particular to determine the amounts of at least one histone peptide. In this application, the term "mass spectrometry" or "MS" refers to an analytical technique that allows the identification of compounds by their mass. To improve the resolution and mass determination capabilities of mass spectrometry, samples can be processed prior to MS analysis. Accordingly, the invention relates to MS detection methods that can be combined with immunoenrichment technologies, sample preparation methods and/or chromatographic methods, preferably liquid chromatography (LC), more preferably high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography ( UHPLC). Sample preparation methods include lysis, fractionation, sample digestion to peptides, depletion, enrichment, dialysis, desalting, alkylation, and/or peptide reduction. However, these steps are optional. Selective detection of analyte ions can be performed using tandem mass spectrometry (MS/MS). Tandem mass spectrometry is characterized by a mass selection step (in the context of this application, the term “mass selection step” means the isolation of ions having a certain m/z or a narrow range of m/z) followed by fragmentation of the selected ions and analysis of the mass of the (fragmented) ions of the resulting product.

One skilled in the art will know how to quantify the level of a marker in a sample using mass spectrometric techniques. For example, the determination of the relative amount of "rSRM" or the determination of the absolute amount, as described above, can be used.

In the context of the present application, "diagnosis" in the context of the present invention refers to the recognition and (early) detection (detection) of an adverse event, in particular death, in a subject, and may also include differential diagnosis. The term "diagnosis" may also cover the assessment of the severity of an adverse event. For example, an assessment of how critical the condition is and how likely an adverse event is to occur. In addition, the diagnosis means that the time when the subject will experience an adverse event can be predicted, for example, within at least about 28, 7, or 3 days.

"Prognosis" refers to predicting a subject's outcome or a particular risk of experiencing/having/suffering from an adverse event, in particular death. It may also include an assessment of the likelihood of recovery or the likelihood of a poor outcome for a given subject.

The invention also relates to methods and kits for monitoring, guiding therapy and/or monitoring therapy of subjects, where the method includes

(i) determining the level of at least one histone in a sample of the subject, where the level of at least one histone is indicative of a given adverse event in the subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in a sample of the subject, where the level of proADM is indicative of a given adverse event in the subject.

The methods and kits of the invention can also be used for monitoring. "Monitoring" refers to tracking an already diagnosed disease or medical condition, such as analyzing the progression of a disease or medical condition, or the effect of a particular treatment on progression, and predicting an adverse outcome, such as a life-threatening condition or even death.

The term "control therapy" in the context of the present invention refers to the monitoring and/or adjustment of therapeutic treatment of the subject. Adjustment of therapeutic treatment may also include a decision as to whether further treatment of the subject will be carried out as it was done before, or whether the treatment will be adapted. For example, an adjustment in therapeutic treatment may be whether the subject will be in the ICU or NDE, or whether he will be discharged.

In this application, the terms "risk assessment" and "risk stratification" refer to the grouping of subjects into different risk groups in accordance with their future prognosis. Risk assessment also refers to stratification for the application of preventive and/or therapeutic measures.

In the context of this application, the term "direction of therapy" refers to the use of certain therapies or medical interventions based on the value of one or more biomarkers and/or clinical parameters and/or scores on clinical scales.

The methods of the present invention may be partially computerized. For example, the step of comparing a detected marker level, eg proADM level and/or histone level, with a reference level can be carried out in a computer system. In a computer system, a certain level of marker(s) may be combined with levels of another marker and/or subject parameters to calculate a score used for diagnosis, prediction, risk assessment and/or risk stratification. For example, the determined values may be entered (either manually by a healthcare professional or automatically from the device(s) in which the level(s) of the respective marker(s) has been determined) into the computer system. The computer system may be located directly at the point of care (for example, the ICU or NCO) or in a remote location connected via a computer network (for example, via the Internet). Typically, a computer system will store values (e.g., marker level or parameters such as age, blood pressure, body weight, gender, etc.) on a computer-readable medium and calculate a score based on predetermined and/or pre-stored reference levels. or reference values. The resulting score will be displayed and/or printed out to the user (typically a healthcare professional such as a doctor). Alternatively or additionally, the associated prognosis, diagnosis, score, or stratification will be displayed and/or printed to the user (typically a healthcare professional such as a physician).

Therefore, the present invention in an additional aspect relates to a system for diagnosing, predicting, risk assessment and/or risk stratification of an adverse event in a subject, including

(i) a sample analyzer for determining the level of proADM and/or histone in the subject's sample (and, optionally, levels of additional markers); and

(ii) a computer subsystem programmed to calculate a diagnosis, prediction, risk assessment, and/or risk stratification of an adverse event in a subject, usually based on a comparison of the determined level(s) with the reference level(s).

The computer subsystem can combine the detectable(s) level(s) of the marker with additional(s) level(s) and/or parameter(s), as described in this application. The output of the device may be an assessment or immediate diagnosis, prognosis, risk assessment, and/or risk stratification of an adverse event in a subject.

In a related aspect, the invention relates to a computer program product embodied in a computer-readable medium that, when executed on a computer, performs the steps of determining a diagnosis, predicting, assessing risk, and/or stratifying the risk of an adverse event in a subject based on a determined level of proADM and/or histone in subject sample. Typically, this stage is based on a comparison of the determined(s) level(s) with the reference(s) level(s), as described in this application.

The invention also relates to kits, the use of kits and methods in which such kits are used. The invention relates to kits for implementing the methods presented above and below in this application. The definitions presented in this application, for example, those presented in relation to the methods, are also applicable to the kits according to the invention. In particular, the invention relates to kits for diagnosis, prognosis, risk assessment and/or risk stratification, where this kit contains

(i) detection reagents for determining a given level of at least one histone in a given sample of a given subject, and

reference data comprising a given reference level of at least one histone, wherein an elevated level of the given at least one histone in a given sample of a given subject compared to a given reference level of at least one histone is indicative of an adverse event in the given subject; and/or

(ii) detection reagents for determining a given level of proADM in a given sample of a given subject, and

reference data including this reference level of proADM, and

where an elevated level of a given proADM in a given sample of a given subject compared to a given reference level of proADM is indicative of an adverse event in that subject.

In addition, the invention preferably relates to a kit and its use for carrying out the method according to the invention, where this kit contains detection reagents for determining a given level of proADM in a given sample of a given subject and reference data, including a given reference level of proADM, and where the increase in the level of proADM in a given sample of a given subject compared to a given reference level of proADM indicates an adverse event in that subject.

In addition, the invention preferably relates to a kit and its use for carrying out the method according to the invention, where this kit contains detection reagents for determining a given level of proADM in a given sample of a given subject and reference data, including a given reference level of proADM, and where the decrease in the level of proADM or a similar level of proADM in a given sample of a given subject compared to a given reference level of proADM indicates that an adverse event in that subject will not occur within approximately 28 days.

The invention also relates to the kit and its use in the diagnosis, prognosis, risk assessment and/or risk stratification of an adverse event in a subject,

(i) where determine the level of at least one histone in the sample of the subject,

where the level of at least one histone is compared with a reference level of at least one histone, and

where an adverse event in a given subject is identified based on a comparison of the level of at least one histone determined in the sample, and the reference level of at least one histone; and/or

(ii) where determine the level of proADM in the sample of the subject,

where the level of proADM is compared with the reference level of proADM, and

where the adverse event in the subject is identified based on a comparison of the proADM level determined in the sample and the reference proADM level.

The invention also relates to the kit and its use in the diagnosis, prognosis, risk assessment and/or risk stratification of an adverse event in a subject,

(i) where the kit contains detection reagents for determining the level of at least one histone in the subject's sample, and

where the level of at least one histone indicates an adverse event in this subject; and/or

(ii) where the kit contains detection reagents for determining the level of proADM in the subject's sample, and

where a given level of a given proADM indicates a given adverse event in a given subject.

In the context of the present application, "reference data" includes reference level(s) of at least one histone and/or proADM, in particular CP-proADM. Levels of at least one histone and/or proADM in a subject's sample can be compared to reference levels contained in the reference data in the kit. An elevated level of detectable marker(s) indicates an adverse event, in particular death. A reduced level or an equal level of the detectable marker(s) indicates that an adverse event, in particular death, will not occur. Reference levels are described above in this application. The reference data may also include a reference sample against which the level of at least one histone and/or the level of proADM is compared. Reference data may also include guidance on the use of kits according to the invention.

In the context of this application, the term "detection reagent" or the like are reagents suitable for determining the marker(s) described herein, for example, at least one histone and/or proADM. Examples of such detection reagents are, for example, ligands, such as antibodies or fragments thereof, that specifically bind to the peptide or epitopes of the marker(s) described herein. Such ligands can be used in immunoassays as described above. Additional reagents used in immunoassays to determine the level of the marker(s) may also be included in the kit and are considered in this application as detection reagents. Detection reagents can also refer to reagents used for the detection of markers or their fragments by MS methods. Thus, such a detection reagent can also be reagents, such as enzymes, chemicals, buffers, etc., used to prepare a sample for MS analysis. The mass spectrometer can also be considered as a detection reagent. The detection reagents according to the invention can also be a calibration solution(s), for example, which(s) can be used to determine and compare the level of the marker(s).

These definitions and explanations are similarly applicable to the following objects. The present invention also relates to the following objects:

1. A method for diagnosing, predicting, assessing risk and / or stratifying the risk of an adverse event in a subject, including

(i) determining the level of at least one histone in a sample of the subject, where the level of at least one histone is indicative of a given adverse event in the subject; and/or

(ii) determining the level of proadrenomedullin (proADM) in a sample of the subject, where the level of proADM is indicative of a given adverse event in the subject.

2. The method according to claim 1, where the adverse event occurs within 28 days, or where the adverse event does not occur within 28 days.

3. The method according to paragraph 1 or 2, where

(i1) a given level of at least one histone is compared with a reference level of at least one histone; and/or

(ii1) where this proADM level is compared with a reference proADM level; and

(iii) where a given adverse event in a given subject is identified based on the comparison in step (i1) and/or (ii1), respectively.

4. The method according to any of the objects 1-3, where

(i) an increase in the level of at least one histone compared to the reference level of at least one histone indicates this adverse event in this subject; and/or

(ii) an increase in the level of proADM compared to the reference level of proADM indicates this adverse event in this subject, where preferably this adverse event occurs within about 28 days, or

a decrease or equal level of a given proADM in a given subject compared to a given reference level of a given proADM indicates that no adverse event has occurred, preferably within about 28 days.

5. A method according to any one of 1-4, wherein the adverse event is selected from the group consisting of death, organ dysfunction, multiple organ dysfunction, and a disease or medical disorder such as an infection.

6. The method according to any one of 1-5, wherein the adverse event is a fatality.

7. The method according to any one of items 3-6, wherein the reference level of proADM is from about 0.9 nmol/L to about 1.8 nmol/L.

8. The method according to any of the objects 1-7, where

(i) an elevated level of the at least one histone is approximately twice the given reference level of the at least one histone; and/or

(ii) the elevated level of the given proADM is approximately twice the given reference level of the proADM.

9. The method according to any one of items 3-8, wherein the reference level of at least one histone and/or the reference level of proADM is(are) the level of at least one histone and/or the level of proADM of at least one reference subject.

10. The method according to any one of items 3-9, where the reference(s) subject(s) is(are) a healthy(s) subject(s), not having(their) adverse event.

11. The method according to any one of items 3-10, wherein the given reference level of at least one histone and/or reference level of proADM is the level of at least one histone and/or proADM obtained from a previous analysis of this subject.

12. The method according to any one of items 1-11, wherein the proADM is mid-regional proadrenomedullin (SR-proADM).

13. The method according to any one of items 1-12, wherein a given proADM level in a given subject is indicative of a given adverse event, preferably death, in that subject within 28 days.

14. The method according to any one of items 1-13, wherein a given proADM level in a given subject is indicative of a given adverse event, preferably death, in that subject within 7 days.

15. A method according to any one of items 1-14, wherein the at least one histone is an H2B histone, an H2A histone, an H3 histone, and/or an H4 histone.

16. The method according to any one of items 1-15, wherein the at least one histone is an H2B histone.

17. The method according to any one of items 1-16, wherein a given level of at least one histone in a given subject is indicative of a given adverse event, preferably death, in that subject within 7 days or within 3 days.

18. The method according to any one of items 1-17, further comprising determining at least one marker and/or parameter of a given subject selected from the group consisting of a lactate level in a given sample, a level of at least one histone, an aldolase B level in a given sample, copeptin level in this sample, lactate level in this sample, procalcitonin (PCT) level in this sample, scores on the Dynamic Organ Failure Assessment (SOFA) scale of the subject, scores on the Simplified Acute Physiological Conditions Scale (SAPSII score), scores on the scale of acute physiological conditions and chronic health conditions II (APACHE II) of this subject and the level of soluble fms-like tyrosine kinase-1 (sFlt-1) in this sample.

19. The method according to any one of items 1-18, further comprising determining a score on the Dynamic Organ Failure Assessment (SOFA) score of the subject.

20. The method according to any one of items 3-19, where

the reference level of proADM is approximately 1.8 nmol/L if the subject's SOFA score is ≤ 6;

the reference level of proADM is approximately 3.2 nmol/L if the subject's SOFA score is between 7 and 12; or

the proADM reference level is approximately 5.5 nmol/L if the subject's SOFA score is ≥ 13.

21. The method according to any one of objects 1-20, where

a decrease or equal level of proADM in this subject compared to the reference level of proADM of approximately 1.8 nmol/l, and the subject's SOFA score ≤ 6 indicate that this adverse event does not occur;

a decrease or equal level of proADM in the subject compared to the reference level of proADM of approximately 3.2 nmol/l, and the subject's SOFA score of 7 to 12 indicate that the adverse event is not occurring; or

a decrease or equal level of proADM in this subject compared to the reference level of proADM of approximately 5.6 nmol/l, and the subject's SOFA score ≥ 13 indicates that this adverse event does not occur; and

where preferably the adverse event does not occur within approximately 28 days.

22. The method according to any one of objects 1-20, where

an increase in the subject's proADM level compared to the reference proADM level of approximately 1.8 nmol/L and the subject's SOFA score ≤ 6 indicate an adverse event in the subject;

an increase in the subject's proADM level compared to the reference proADM level of approximately 3.2 nmol/L and the subject's SOFA score of 7 to 12 indicate an adverse event in the subject; or

an increase in the subject's proADM level compared to the reference proADM level of approximately 5.6 nmol/L and the subject's SOFA score ≥ 13 indicate an adverse event in the subject,

where preferably this adverse event occurs within about 28 days.

23. The method of any one of aspects 19-23, wherein the subject's SOFA score is increased based on the subject's proADM level, and wherein the modified SOFA score is indicative of a given adverse event.

24. The method of claim 23, wherein the subject's SOFA score is incremented by one if the subject's proADM level is greater than about 1.8 nmol/L.

25. A method according to any one of items 1-24, comprising determining a given level of at least one histone and a given level of proADM in a given sample of a given subject.

26. The method of claim 25, wherein the given level of at least one histone and the given level of proADM are indicative of the occurrence of a given adverse event, preferably death, in a given subject within 28 days.

27. A method according to any one of items 1-26, comprising determining a given level of at least one histone in a given sample of a given subject and a given SAPSII score of the given subject.

28. The method of claim 27, wherein a given level of at least one histone and a given SAPSII score indicate the occurrence of a given adverse event, preferably death, in a given subject within 28 days.

29. A method according to any one of items 1-28, comprising determining a given level of proADM in a given sample of a given subject and a given SAPSII score of the given subject.

30. The method of claim 29, wherein the given proADM level and the given SAPSII score indicate the onset of a given adverse event, preferably death, in a given subject within 28 days or within 7 days.

31. A method according to any one of items 1-30, comprising determining a given proADM level, a given SOFA score, and a given lactate level in a given subject.

32. The method according to any one of items 1-31, wherein the subject is suffering from a disease or medical condition.

33. The method according to any one of items 1-32, wherein the subject is a critically ill patient, preferably wherein the subject is admitted to an intensive care unit.

34. The method according to any one of items 1-33, wherein said subject suffers from a disease or medical condition selected from the group consisting of respiratory disease, urinary tract infection, inflammatory response associated with infectious and non-infectious etiology, systemic inflammatory response syndrome ( SIRS), sepsis, severe sepsis, septic shock, organ failure(s), cardiovascular disease, diabetes mellitus, malignancy, liver disease, kidney disease, immunosuppression, viral infection, fungal infection, bacterial infection, Gram-negative bacterial infection, Gram-positive bacterial infection and immunosuppression.

35. The method according to any one of items 1-34, wherein the subject is suffering from sepsis.

36. The method according to any one of items 1-35, wherein the subject is an immunocompromised subject, such as a subject suffering from human immunodeficiency virus (HIV), a subject undergoing radiation therapy, and/or a subject receiving immunosuppressive drugs.

37. The method according to any one of items 1-36, wherein the subject is suffering from a respiratory disease, preferably a lower respiratory tract infection.

38. The method of claim 37, wherein a given level of at least one histone in a given sample of a given subject suffering from a given respiratory disease is indicative of death within 7 days.

39. The method according to any one of items 1-38, wherein the subject is suffering from a urinary tract infection.

40. The method of claim 39, wherein a given level of at least one histone in a given sample of a given subject suffering from a given urinary tract infection is indicative of death within 28 days.

41. The method of any one of items 1-40, wherein the subject is suffering from a cancer.

42. The method of claim 41 wherein a given level of at least one histone in a given sample of a given subject suffering from a given cancer is indicative of death within 7 days.

43. The method according to any one of items 1-42, where the level of the marker or parameter is determined within approximately 12 hours after the admission of this subject.

44. The method according to any one of items 1-43, where the sample is a biological fluid, blood, blood plasma, blood serum or urine.

45. A method according to any one of items 1-44, wherein a given level of at least one histone and/or proADM is determined using a method selected from the group consisting of mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA ), chemiluminescent and fluorescent immunoassays, enzyme-linked immunosorbent assays (EIAs), enzyme-linked immunosorbent assays (ELISAs), fluorescent labeled bead-based arrays, magnetic-beaded arrays, protein microarray assays, accelerated assays and assays using rare earth cryptates elements.

46. The method according to claim 45, where the method is an immunoassay, and where the analysis is carried out in a homogeneous phase or in a heterogeneous phase.

47. The method according to paragraph 45 or 46, where the method is an immunoassay comprising the following steps:

a) bringing the sample into contact with

(i) a first antibody or antigen-binding fragment or derivative thereof specific for a first histone epitope or proADM, and

(ii) a second antibody or antigen-binding fragment or derivative thereof specific for a second histone epitope or proADM; and

b) detecting the binding of the first and second antibodies or their antigen-binding fragments or derivatives with this first histone or with proADM.

48. The method of claim 47, wherein one of the first or second antibodies is labeled and the other antibody is or can be selectively bound to the solid phase.

49. The method according to claim 47 or 48, where the first antibody and the second antibody are present in dispersed form in the liquid reaction mixture, and where the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, is associated with the first antibody, and the second labeling component of the labeling system is coupled to the second antibody such that upon binding of both antibodies to either the at least one detectable histone or the proADM, a measurable signal is generated allowing the resulting sandwich complexes to be detected in the measurement solution.

50. The method according to claim 49, wherein the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, particularly of the cyanine type.

51. The method of claim 45, wherein the MS method of analysis is Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), or Parallel Reaction Monitoring (PRM).

52. The method according to any one of items 1-51, wherein the at least one histone is histone H4, and where at least a peptide with a sequence spanning amino acid residues 22 to 102 of histone H4 according to SEQ ID NO: 1 is determined. .

53. The method according to any one of items 1-52, wherein the at least one histone is histone H4, and wherein at least a peptide with a sequence selected from the group consisting of amino acid sequences spanning residues 47 to 59 of SEQ ID is determined. NO: 1, residues 68 to 79 SEQ ID NO: 1, residues 60 to 67 SEQ ID NO: 1, residues 22 to 30 SEQ ID NO: 1, residues 67 to 78 SEQ ID NO: 1, residues with 92 to 102 SEQ ID NO: 1, residues 22 to 34 SEQ ID NO: 1, residues 46 to 102 SEQ ID NO: 1, residues 46 to 55 SEQ ID NO: 1, residues 80 to 91 SEQ ID NO : 1, residues 24 to 35 of SEQ ID NO: 1 and residues 68 to 77 of SEQ ID NO: 1.

54. The method according to any one of items 1-53, wherein the at least one histone is histone H2A, and where at least a peptide with a sequence spanning amino acid residues 20 to 118 of histone H2A according to SEQ ID NO: 2 is determined. .

53. The method according to any one of items 1-54, wherein the at least one histone is histone H2A, and wherein at least a peptide with a sequence selected from the group consisting of amino acid sequences spanning residues 21 to 53 of SEQ ID is determined. NO: 2, residues 21 to 29 SEQ ID NO: 2, residues 30 to 53 SEQ ID NO: 2, residues 120 to 129 SEQ ID NO: 2, residues 21 to 29 SEQ ID NO: 2, residues with 82 to 88 of SEQ ID NO: 2, residues 89 to 95 of SEQ ID NO: 2, and residues 100 to 118 of SEQ ID NO: 2.

56. The method according to any one of items 1-55, wherein the at least one histone is histone H3, and where at least a peptide is determined with a sequence spanning amino acid residues 27 to 62 of histone H3 in accordance with SEQ ID NO: 3 .

57. The method according to any one of items 1-56, wherein the at least one histone is histone H3, and wherein at least a peptide with a sequence spanning amino acid residues 27 to 37 of SEQ ID NO: 3 and/or spanning amino acid residues 52 to 62 of SEQ ID NO: 3.

58. The method according to any one of items 1-57, wherein the at least one histone is an H2B histone, and wherein at least a peptide with a sequence spanning amino acid residues 41 to 69 of histone H2B according to SEQ ID NO: 4 is determined. .

59. The method according to any one of items 1-59, wherein the at least one histone is an H2A histone, and wherein at least a peptide or fragment thereof selected from the group consisting of SEQ ID NOs: 7, 8, 9 and ten.

60. The method according to any one of items 1-59, wherein the at least one histone is histone H4, and wherein at least a peptide or fragment thereof is determined, selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15 and 16.

61. Set for implementing the method according to any of objects 1-60, containing

(i) detection reagents for determining a given level of at least one histone in a given sample of a given subject, and

reference data comprising a given reference level of at least one histone, where an increase in the level of at least one histone in a given sample of a given subject compared to a given reference level of at least one histone indicates an adverse event in the given subject; and/or

(ii) detection reagents for determining a given level of proADM in a given sample of a given subject, and

reference data including this reference level of proADM, and

where an increase in the level of proADM in a given sample of a given subject compared to a given reference level of proADM indicates an adverse event in this subject.

62. A kit for carrying out the method according to any one of items 1-29, comprising detection reagents for determining a given level of proADM in a given sample of a given subject and reference data, including a given reference level of proADM, and where a decrease in the level of proADM or a close level of proADM in a given sample of this subject compared to a given reference level of proADM indicates that an adverse event will not occur in this subject, preferably within about 28 days.

63. Use of a kit according to claim 61 or 62 in a method according to any one of items 1-60.

64. The use of the kit according to paragraph 63 for the diagnosis, prognosis, risk assessment and / or risk stratification of an adverse event in a subject,

(i) where define a given level of at least one histone in a given sample of a given subject,

where a given level of a given at least one histone is compared with a given reference level of at least one histone, and

where a given adverse event in a given subject is identified based on a comparison of a given level of at least one histone determined in a given sample and a given reference level of at least one histone; and/or

(ii) where a given level of proADM is determined in a given sample of a given subject,

where the given proADM level is compared with the given reference proADM level, and

where a given adverse event in a given subject is identified based on a comparison of a given level of proADM determined in a given sample and a given reference level of proADM.

65. The use of a kit for diagnosing, predicting, assessing the risk and / or stratifying the risk of an adverse event in a subject,

(i) where this kit contains detection reagents for determining the level of at least one histone in the sample of the subject, and

where a given level of at least one histone indicates a given adverse event in a given subject; and/or

(ii) where this kit contains detection reagents for determining the level of proADM in the sample of the subject, and

where a given level of a given proADM indicates a given adverse event in a given subject.

The present invention in preferred embodiments also relates to the following objects:

1. A method for diagnosing, predicting, assessing risk and / or stratifying the risk of an adverse event in a subject, including

determining the level of proadrenomedullin (proADM) in the subject's sample, where the given proADM level is indicative of a given adverse event in the subject.

2. The method according to paragraph 1, where

this level of proADM is compared with a reference level of proADM; and

where a given adverse event in a given subject is identified based on the comparison.

3. The method according to paragraph 1 or 2, where

where a decrease in the level, or a lower level, or an equal level of a given proADM in a given subject compared to a given reference level of a given proADM indicates that this adverse event does not occur, preferably within about 28 days; or

where an increase in the level of proADM or a higher level of proADM compared to the reference level of proADM indicates this adverse event in this subject, where preferably this adverse event occurs within about 28 days.

4. The method according to any one of items 1-3, where the reference level of proADM is from about 0.7 nmol/l to about 2.0 nmol/l, preferably from about 0.8 nmol/l to about 1.9 nmol/l .

5. A method according to any one of items 1-4, wherein the adverse event is a fatality.

6. The method of claim 5, wherein a proADM level greater than about 0.9 nmol/l, preferably greater than about 1.9 nmol/l, more preferably greater than about 2.0 nmol/l, indicates non-survival of the subject for about 28 days.

7. The method of claim 1 or 2 wherein the proADM level is below about 1.0 nmol/L, preferably below about 0.9 nmol/L, most preferably below about 0.88 nmol/L is indicative of survival of the subject for about 28 days.

8. The method according to any one of items 1-7, which further determines the score on the scale of the dynamic assessment of organ failure (SOFA scale) of the subject.

9. The method according to any one of items 2-5, where

the reference level of proADM is approximately 1.8 nmol/l for subjects with symptoms consistent with a SOFA score ≤ 6;

the reference level of proADM is approximately 3.2 nmol/l for subjects with symptoms consistent with a SOFA score of 7 to 12; or

the reference level of proADM is approximately 5.5 nmol/L for subjects with symptoms consistent with a SOFA score ≥ 13.

10. The method according to any of the objects 1-5 or 9, where

a decrease or equal level of proADM in this subject compared to the reference level of proADM of approximately 1.8 nmol/l, and the subject's SOFA score ≤ 6 indicate that this adverse event does not occur;

a decrease or equal level of a given proADM in that subject compared to a reference proADM level of approximately 3.2 nmol/L and a SOFA score of the subject of 7 to 12 indicates that the adverse event is not occurring; or

a decrease or equal level of this proADM in this subject compared to the reference level of proADM of approximately 5.6 nmol/l, and the subject's SOFA score ≥ 13 indicates that this adverse event does not occur; and

where preferably the adverse event does not occur within approximately 28 days.

11. The method according to any of the objects 1-5 or 9, where

an increase in the proADM level in this subject compared to the reference level of proADM of approximately 1.8 nmol/l, and the subject's SOFA score ≤ 6 indicate this adverse event in this subject;

an increase in the level of proADM in this subject compared to the reference level of proADM of approximately 3.2 nmol/l, and a score on the SOFA scale of this subject from 7 to 12 indicate this adverse event in this subject; or

an increase in the subject's proADM level compared to the reference proADM level of approximately 5.6 nmol/L, and the subject's SOFA score ≥ 13 indicate this adverse event in this subject,

where preferably this adverse event occurs within about 28 days.

12. The method of any one of 8-11, wherein the subject's SOFA score is incremented based on the subject's proADM level, and wherein the modified SOFA score is indicative of a given adverse event.

13. The method of claim 12, wherein the subject's SOFA score is increased by one if the subject's proADM level is greater than about 1.8 nmol/L.

14. The method according to any one of items 3-5 and 9-13, wherein the elevated level of a given proADM is approximately twice the given reference level of proADM.

15. The method according to any one of items 1-14, wherein the proADM is mid-regional proadrenomedullin (SR-proADM) or mature ADM.

16. The method according to any one of items 1-15, wherein a given proADM level in a given subject is indicative of a given adverse event, preferably death, in that subject within 28 days.

17. The method according to any one of objects 1-16, further comprising determining at least one marker and/or parameter of a given subject selected from the group consisting of a level of at least one histone, a level of procalcitonin (PCT) in a given sample, assessing the Simplified Acute Physiological Conditions Scale (SAPSII score), the subject's Acute Physiological Conditions and Chronic Health II (APACHE II) score, the subject's Pneumonia Severity Index (PSI) score, and the subject's CURB-65 score.

18. The method of claim 17, wherein the level of the marker or parameter is determined within approximately 12 hours of admission of the subject.

19. The method according to any one of items 1-18, wherein the proADM level is determined within about 12 hours of the subject's admission.

20. A method according to any one of items 1-19, comprising determining a given proADM level and scoring the subject's SOFA score.

21. A method according to any one of items 1-20, wherein the subject is suffering from a disease or medical condition.

22. The method according to any one of items 1-21, wherein the subject is a critically ill patient, preferably where the subject is admitted to an intensive care unit or emergency room, preferably where the subject is admitted to an intensive care unit.

23. The method according to any one of items 1-22, where the subject suffers from a disease or medical condition selected from the group consisting of respiratory disease, urinary tract infection, inflammatory response associated with infectious and non-infectious etiology, systemic inflammatory response syndrome ( SIRS), sepsis, severe sepsis, septic shock, organ failure(s), cardiovascular disease, diabetes mellitus, malignancy, liver disease, kidney disease, immunosuppression, viral infection, fungal infection, bacterial infection, Gram-negative bacterial infection, Gram-positive bacterial infection and immunosuppression.

24. The method according to any one of items 1-23, wherein the subject is suffering from sepsis.

25. The method according to any one of items 1-24, wherein the subject is an immunocompromised subject, such as a subject suffering from human immunodeficiency virus (HIV), a subject undergoing radiation therapy, and/or a subject receiving immunosuppressive drugs.

26. A method according to any one of items 1-25, wherein the sample is a biological fluid, blood, blood plasma, serum, or urine.

27. The method according to any one of items 1-26, where a given level of proADM is determined using a method selected from the group consisting of mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay analysis (EIA), enzyme-linked immunosorbent assay (ELISA), bead-based arrays with luminescent labels, magnetic bead-based arrays, protein microarray assays, accelerated assays and rare earth cryptate assays.

28. The method according to claim 27, wherein the method is an immunoassay and wherein the assay is carried out in a homogeneous phase or in a heterogeneous phase.

29. The method according to claim 28, where the method is an immunoassay comprising the following steps:

a) bringing the sample into contact with

(i) a first antibody or antigen-binding fragment or derivative thereof specific for the first proADM epitope, and

(ii) a second antibody or antigen-binding fragment or derivative thereof specific for a second proADM epitope; and

b) detecting the binding of the first and second antibodies or their antigen-binding fragments or derivatives with proADM.

30. The method of claim 29 wherein one of the first or second antibodies is labeled and the other antibody is or can be selectively bound to the solid phase.

31. The method according to claim 29 or 30, wherein the first antibody and the second antibody are present in dispersed form in the liquid reaction mixture, and where the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, is associated with the first antibody, and the second labeling component of the labeling system is coupled to the second antibody such that upon binding of both antibodies to the detectable proADM, a measurable signal is generated allowing the resulting sandwich complexes to be detected in the measurement solution.

32. The method according to claim 31, wherein the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, particularly of the cyanine type.

33. A kit for carrying out the method according to any one of objects 1-32, containing detection reagents for determining a given level of proADM in a given sample of a given subject and reference data, including a given reference level of proADM, and where the increase in the level of proADM in a given sample of a given subject is compared with a given proADM reference level indicates an adverse event in that subject.

34. A kit for carrying out the method according to any one of items 1-32, comprising detection reagents for determining a given level of proADM in a given sample of a given subject and reference data, including a given reference level of proADM, and where a decrease in the level of proADM or a close level of proADM in a given sample of this subject compared to this reference level of proADM indicates that an adverse event in this subject will not occur within approximately 28 days.

35. Use of a kit according to claim 33 or 34 in a method according to any one of items 1-29.

36. The use of a kit according to paragraph 33 or 34 for the diagnosis, prognosis, risk assessment and / or risk stratification of an adverse event in a subject,

where the given level of proADM is determined in the given sample of the given subject,

where the given proADM level is compared with the given reference proADM level, and

where a given adverse event in a given subject is identified based on a comparison of a given level of proADM determined in a given sample and a given reference level of proADM.

37. The use of a kit for diagnosing, predicting, assessing risk and / or stratifying the risk of an adverse event in a subject,

where this kit contains detection reagents for determining the level of proADM in the sample of the subject, and

where a given level of a given proADM indicates a given adverse event in a given subject.

38. The method according to any one of items 2-32, where this comparison is carried out in a computer system.

39. The method of any one of entities 1-32, wherein the determined proADM value is used in computing a score indicative of a given diagnosis, prognosis, risk assessment, and/or risk stratification of an adverse event in a subject.

40. The method of claim 40, wherein additionally at least one additional marker and/or subject parameter is used to calculate the score.

41. The method according to claim 40, wherein at least one additional marker and/or parameter of the subject is selected from the group consisting of at least one histone level, the level of procalcitonin (PCT) in the given sample, a score on a simplified scale of acute physiological conditions (SAPSII score), the subject's Acute Physiological Conditions and Chronic Health II (APACHE II) scores, the subject's Pneumonia Severity Index (PSI) scores, and the subject's CURB-65 scores.

In the context of this application, the terms "comprising" and "comprising" or their grammatical variants should be understood as denoting at least these features, integers, steps or components, but not excluding the addition of one or more additional features, integers, steps, components or their groups. This term includes the terms "consisting of" and "consisting essentially of", which are intended to mean only the mentioned features, integers, steps or components, to the exclusion of any additional features.

Thus, the terms "comprising"/"comprising"/"having" mean that any additional component (or, likewise, features, integers, steps, and the like) may be present.

The term "consisting of" means that no additional components (or, likewise, features, integers, steps, and the like) are present.

The term "consisting essentially of" or its grammatical variations when used in this application should be understood as denoting the mentioned features, integers, steps or components, but not excluding the addition of one or more additional features, integers, steps, components or groups thereof, but only if additional features, integers, steps, components or groups thereof do not significantly change the main and new characteristics of the claimed composition, device or method.

Thus, the term "consisting essentially of" means that such specific additional components (or, similarly, features, integers, steps, and the like) may be present, namely those that do not significantly affect the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of" (which may be used interchangeably herein with the term "comprising essentially") allows for the presence of other components in a composition, device, or method in addition to the required components (or, similarly, features, integers, stages and the like), provided that the presence of other components does not significantly affect the essential characteristics of the device or method.

The term “method” refers to techniques, means, techniques, and procedures for performing a given task, including, but not limited to, those techniques, tools, techniques, and techniques that are either known or can be easily derived from known techniques, tools, techniques. and methodologies by practitioners in chemistry, biology and biophysics.

The term "approximately" preferably refers to ±10% of a given numerical value, more preferably to ±5% of a given numerical value, and in particular to an exact given numerical value.

In the context of the present application, the term "approximately" refers to ±10% of a given numerical value, and in particular to ±5% of a given numerical value. Each time the term "approximately" is used, specific reference is also made to an exact numerical value. When the term "approximately" is used in relation to a parameter that is quantified in whole numbers, such as the number of nucleotides in a given nucleic acid, numbers corresponding to ±10% or ±5% of that numerical value should be rounded to the nearest whole number. For example, the expression "about 25 amino acids" refers to the range of 23 to 28 amino acids, in particular the range of 24 to 26 amino acids, and preferably refers to the specific value of 25 amino acids.

The sensitivity and specificity of a diagnostic and/or prognostic test depends not only on the analytical "quality" of the test, but also on the definition of what constitutes an abnormal result. In practice, receiver operating characteristic curves (ROC curves) are typically calculated by plotting the value of a variable against its relative frequency in "normal" (that is, apparently healthy people with no prenatal disorders or conditions) and "sick" populations, eg, subjects experiencing/having/suffering from an adverse event, eg, death. For any particular marker (such as CP-proADM), the distribution of marker levels for subjects with or without the disease/condition is likely to overlap. Under such conditions, the test does not always distinguish normal from disease with 100% accuracy, and the area of overlap may indicate where the test fails to distinguish normal from disease. A threshold is selected below which a test is considered abnormal and above which a test is considered normal, or below or above which tests indicate a particular condition, such as an abnormal event. The area under the ROC curve is a measure of the likelihood that the observed measurement will correctly identify the condition. ROC curves can be used even if the test results do not necessarily give an exact number. As long as the results can be ranked, the ROC curve can be created. For example, test results of "diseased" (or adverse event) samples may be ranked according to grade (eg, 1=low, 2=normal, and 3=high). This ranking can be related to results in a "normal" population and an ROC curve can be generated. These methods are well known in the art; see, for example, Hanley et al. 1982. Radiology 143: 29-36. Preferably the threshold is chosen to provide an area under the ROC curve of greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0. 9. The term "approximately" in this context refers to +/- 5% of a given measurement.

The horizontal axis of the ROC curve is (1-specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the degree of true positive results. Thus, for a particular selected cutoff value (1-specificity) can be determined and an appropriate sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that the measured marker level will correctly identify an adverse event, in particular a fatal outcome. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an odds ratio, or an hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disorder or condition (poor outcome), i.e., a "sick group". In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "patient" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "patient" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the study group; and a value less than 1 indicates that a negative result is more likely in the control group.

In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "sick" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.

In the case of a hazard ratio, a value of 1 indicates that the relative risk of an end state (eg, death) in the "sick" and "control" groups is equal; a value greater than 1 indicates that the risk is higher in the sick group; and a value less than 1 indicates that the risk is higher in the control group.

One skilled in the art will recognize that associating a diagnostic or prognostic indicator with a diagnosis or predicted risk of a future clinical outcome is a statistical analysis. For example, a marker level less than X may signal that a patient is more likely to suffer from an adverse event/outcome than patients with a level greater than or equal to X, as determined by the level of statistical significance. In addition, the change in marker concentration from baseline may reflect the patient's prognosis, and the degree of marker level change may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and/or p-value; see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals according to the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5% , 99.9% and 99.99%, and the preferred p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 and 0.0001.

Unless otherwise indicated, established genetic engineering techniques were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001), incorporated herein by reference in its entirety.

The present invention is further described with reference to the following non-limiting figures.

Fig. 1 : Kaplan-Meier analysis to predict death at 28 days

Fig. 2 : AUROC analysis to identify non-survivors at 28 days (whole cohort)

Fig. 3 : AUROC analysis to identify non-survivors at 28 days, depending on biomarker levels in the three severity groups.

Fig. 4 : AUROC analysis to identify non-survivors at 28 days based on SOFA score.

The present invention is further described with reference to the following non-limiting examples.

Example 1

METHODS:

Study population

From June 1, 2013 to June 14, 2014, two hundred and thirty-seven critically ill patients were consecutively admitted to the intensive care unit of the University Hospital of Dijon, Burgundy region (“Centre hospitalier universitaire (CHU) de Dijon Bourgogne”). ). Patients under 18 years of age were excluded from the study. The study was approved by the local institute review board. Written informed consent was obtained from patients or their immediate family prior to enrollment in the study. All patients had a wide range of medical conditions, including cardiovascular disease, diabetes mellitus, malignancy, respiratory disease, liver disease, kidney disease, and immunosuppression, and were followed up until discharge or death in hospital. Based on a retrospective review of medical records, diagnostic imaging studies, and microbiological findings, two independent clinicians classified patients on the day of admission as non-septic (with or without systemic inflammatory response syndrome (SIRS)), severe sepsis, or septic shock at the according to international standard criteria (Bone, Balk et al. 1992). A blood sample was taken on the day of admission, that is, within the first 24 hours. Baseline questionnaire and clinical data were recorded, including medical history, physical examination findings, routine blood tests (eg, blood cultures), non-laboratory diagnostic tests (eg, SIRS criteria, organ failure criteria), therapeutic interventions (eg, mechanical ventilation (ALV) , vasopressor agents, and renal replacement therapy (RRT)), as well as outcome measures (eg, length of stay, overall mortality). At admission, a Dynamic Organ Failure Assessment (SOFA) score based on six organ parameters and a Simplified Acute Physiological Conditions Scale (SAPS II) score based on 17 predominantly physiological parameters were calculated (Le Gall, Lemeshow et al. 1993; Vincent , Moreno et al. 1996).

Biomarkers

Serum lactate levels were measured by colorimetric analysis using the e501 modular analyzer from Roche Diagnostics, Meilan, France. The reference limit for lactate was 0.5-2.2 mmol/L.

CP-proADM (mid-regional proadrenomedullin), copeptin, and PCT (procalcitonin) levels were determined in plasma samples using ultrasensitive assays such as the KRYPTOR random access analyzer (Thermo Scientific B⋅R⋅A⋅H⋅M⋅S). The levels of histone H2A, H2B, H3 and H4, as well as the level of aldolase B were determined in plasma samples, for example, using selected reaction monitoring or multiple reaction monitoring (SRM/MRM) assays, as described below. Specific peptides derived from markers were measured using LC-MS/MS technology (mass spectrometer (MS) TSQ Quantiva; ThermoFisher Scientific). It was found that the identified peptide sequences and their fragmented ions, the so-called transitions, for each peptide are suitable mimic standards for monitoring the levels of marker proteins in a blood sample. Optimization was performed on synthetic peptides that can be highly labeled with isotopes. The best peptides were selected in terms of signal-to-noise ratio. For each peptide, the optimal retention time and at least 4 best transitions were set.

Example of MS quantification and selection of peptides and junctions

5 μl of each clinical plasma preparation was added to 20 μl of 8 M urea/2.5% n-propanol/300 mM Tris/10 mM DTT, pH 8.5, and incubated at 37° C. for one hour. 500 mM iodoacetic acid prepared in 1 M ammonium bicarbonate was added to each sample well and incubated in the dark at room temperature for one hour. 113 μl 50 mM Tris/5 mM CaCl2, pH 8.0 was added to each well. Trypsin (Thermo Fisher Scientific) was rehydrated with 150 μl of 25 mM acetic acid and added at a ratio of 1:10 (total protein:protease) and incubated at 37° C. for 20 hours. Finally, the digestion was stopped by adding another 2 μl of formic acid. Glucagon (1 ng/µl) and standard heavy peptides were then added prior to administration.

SRM analyzes were developed on a TSQ Quantiva triple quadrupole mass spectrometer in combination with an Ultimate 3000 HPLC (Thermo Fisher Scientific). Separation by reverse phase chromatography was carried out in a 20-minute linear concentration gradient from 5 to 40% B, with a total analysis time of 40 minutes (solvent A: aqueous 0.2% FA (formic acid), solvent B: ACN 0.2 %FA). The flow rate during the linear concentration gradient was set to 240 µl/min. The total volume of the injected sample was 160 μl for all samples and points on the curve. An Accucore aQ 150 mm x 2.1 mm column (ThermoFisher Scientific) was run at 50°C.

Optimization was performed on heavy labeled synthetic peptides containing labeled 13C and 15N arginine or lysine (ThermoFisher Scientific or New England Peptide). Individual instrument parameters, such as collision energy, tube lens, and measurement time, were automatically checked for each transition. After several runs, an optimized list of peptides and transitions (i.e., signal with the highest intensity and least overlap with other transitions) and corresponding retention times was obtained with a selection of at least four fragment transitions per selected peptide.

Peptides were identified by co-elution of light and heavy labeled transitions in chromatographic separation. Pinpoint (Thermo Fisher Scientific) and Skyline (MacCoss Lab) software was used for time alignment, relative transition quantification, and target protein quantification.

Relative and absolute marker quantitations were performed using exemplary methods as described below.

Relative Quantity Definition:

1. Determination of increased or decreased presence of a marker by comparing the area of the characteristic SRM peak of a given peptide detected in a biological sample with the area of the same characteristic SRM peak of the same fragmented peptide in at least a second, third, fourth or more biological samples.

2. Determination of increased or decreased presence of the marker by comparing the area of the characteristic SRM peak of a given peptide detected in a biological sample with the areas of the characteristic SRM peak obtained from fragmented peptides of other proteins in other samples obtained from other and separate biological sources, where the area comparison of the characteristic SRM peak of two samples for the fragmented peptide is normalized to the amount of protein analyzed in each sample.

3. Determination of increased or decreased presence of the marker by comparing the characteristic SRM peak area for a given peptide with the characteristic SRM peak areas of other fragmented peptides derived from different proteins in the same biological sample in order to normalize changing levels of the marker to levels of other proteins that do not change their expression levels under different cellular conditions.

4. These assays were applied to both fragmented peptides and modified fragmented peptides, e.g., histone proteins, where the modifications included, but are not limited to, phosphorylation and/or glycosylation, acetylation, methylation (mono-, di-, tri-), citrullination, ubiquitination, and where the relative levels of modified peptides were determined in the same manner as the determination of the relative amounts of unmodified peptides.

Determining the absolute amount of a given peptide:

Comparison of the characteristic SRM/MRM peak area for a given fragmented peptide from a marker in a single biological sample with the characteristic SRM/MRM peak area of a fragmented peptide internal standard added to a protein lysate from a biological sample.

The internal standard was a labeled synthetic version of the fragmented peptide from the test marker protein, or a labeled recombinant protein. This standard was added to the sample in known amounts before or after digestion, and the SRM/MRM characteristic peak area was determined for both the fragmented peptide internal standard and the native fragmented peptide in the biological sample separately, followed by comparison of the areas of both peaks.

This analysis was performed with unmodified fragmented peptides and modified fragmented peptides, where the modifications included, but are not limited to, phosphorylation and/or glycosylation, acetylation, methylation (mono-, di-, tri-), citrullination, ubiquitinylation, and where the absolute levels of the modified peptides were determined in the same manner as the absolute levels of unmodified peptides.

Histone H4 was also measured by immunoassay. The H4 histone immunoassay (H4 AI) consists of a mouse monoclonal antibody (mAb) engineered against a synthetic peptide (amino acids 46-56 of SEQ ID NO: 1) coupled to MagPlex-C microspheres (Luminex, Austin Texas) and a biotinylated polyclonal ovine an antibody (pAb) engineered against the synthetic peptide (amino acids 67-78 of SEQ ID NO 1). A synthetic peptide (amino acids 46-102 of SEQ ID NO: 1) was used as a standard. Samples were measured on a MAgPix using an xPonent 4.2 system (Luminex, Austin, TX). Data were analyzed using 5-parameter logistic regression from JMP-12 (SAS Statistical Software, UK).

Statistical analysis

All analyzes were performed using R 3.0.2 software.

Data are expressed as median and interquartile range [IQR] in brackets.

Because all analyzed biomarkers show significantly right-skewed distributions, values were converted by taking the decimal logarithm prior to inclusion in regression models to reduce the effect of extreme values on model fit.

Values below the limit of quantification (LoQ) have been replaced by a small value below the LoQ. Missing values were not replaced. Each model includes all patients with complete data for all variables in the model.

P-values <0.05 were considered statistically significant.

For survival analysis, follow-up was censored at 3, 7, or 28 days after admission to the ICU (maximum follow-up period (FUP)), depending on the situation. Patients not available for follow-up prior to the FUP being assessed (i.e., due to early discharge or transfer to another unit) were censored on the day of their last ICU visit. Patients alive at maximum FUP were censored on that day. For time-dependent outcome variables, Cox regression models were used. Results shown are χ² likelihood ratio test (L.R. χ² and p-value), C index (Harrel) and standardized risk ratios (HR). Hazard ratios in this application refer to a 2-fold change in biomarker levels (top quartile of biomarkers versus bottom quartile). In the adjusted models, adjustment variables were included in the model to determine the additional impact of biomarkers on model performance.

Results:

The study population was 237 patients. Two patients (one patient without SIRS, one patient with sepsis) had to be excluded from the analysis due to inconsistent mortality data. One hundred and seventy-two patients (73%) had severe sepsis or septic shock, 15 patients (6%) with SIRS and 49 patients (21%) without SIRS. The median age was 67 [59-77 years] years. Most of the patients were male (60%). The most common comorbidities were cardiovascular disease (35%), diabetes mellitus (31%), and malignancy (27%), followed by respiratory disease (16%), liver disease (12%), kidney disease (12%). %) and immunosuppression (7%). The most common sites of infection were the lower respiratory tract (46%) and the urinary tract (45%). SAPS II score (56 [40-69 points] points) and SOFA score (9 [6-12 points] points) were increased at admission. The most frequently observed organ failures were respiratory failure (61%), circulatory shock (56%) and renal failure (41%). Accordingly, many patients required mechanical ventilation (78%), vasopressor agents (68%), and RRT (37%) during their ICU stay. The overall ICU mortality was 32%, and the median length of ICU stay was 5.4 [2.5-10.6] days.

The authors analyzed short-term (e.g., on day 3 and 7, i.e., 3 days and 7 days) and long-term (on day 28, i.e., 28 days) mortality in all 235 critically ill patients using one- and two-dimensional Cox regression models adjusted for age and sex. Twenty-three patients (10%) died by day 3, 49 patients (21%) died by day 7, and 74 patients (32%) died by day 28 after ICU admission. In addition, mortality was analyzed in subgroups of patients with lower respiratory tract infection, urinary tract infection (UTI), and malignancy using univariate Cox regression models adjusted for age and gender. Twenty-seven patients (25%) of 109 patients with lower respiratory tract infection died by day 7, 34 patients (36%) of 94 patients with UTI died by day 28, and 16 patients (25%) of 64 patients with malignancy died by 7 days after admission to the ICU. The ability of each Cox regression model to discriminate between survivors and non-survivors is reflected by a C index ranging from 0 to 1, where the best predictive Cox regression models give a C index close to 1. In addition, HR is calculated, where HR>0 indicates a worse prognosis and HR <0 indicate a protective effect of the variable.

Among all analyzed variables, the SAPS II score at admission to the ICU shows the best forecast of 3-day (index C 0.876, HR at ICR 8.42), 7-day (index C 0.809, HR at ICR 5.15) and 28- daily (C index 0.776, HR at ICR 4.19) death, followed by a SOFA score at ICU admission with a predicted 3-day (C index 0.866, HR at ICR 6.68) and 7-day death (index C 0.778, HR at MCR 3.82) in all 235 critically ill patients (Table 1 - Table 3). Similar results were obtained in the subgroup of patients with sepsis and for predicting 7-day death in critically ill patients with lower respiratory tract infection as assessed by the SAPS II scale (C index 0.773, HR at ICR 3.47) (Table 4).

For comparison, SR-proADM is the best biomarker to discriminate between survivors and non-survivors at day 7 (C index 0.769, HR at ICR 4.53) and at day 28 (C index 0.765, HR at ICR 4.86) after ICU admission. in all critically ill patients. (table 2, table 3). It outperforms the SOFA score in predicting 28-day mortality in all critically ill patients (Table 3). Similar results were obtained in a subgroup of patients with sepsis. In a bivariate analysis, CP-proADM improves the performance of SAPS II or SOFA in predicting 7-day death (C index of 0.832 for the combined SAPS II and CP-proADM model compared to C score of 0.809 for SAPS II alone) and the performance of SAPS II in predicting 28 -day lethal outcome (index C 0.810 for the combined model of SAPS II and SR-proADM compared with the index C 0.776 for SAPS II only) in all patients in critical condition (table 2, table 3). The combined SAPS II or SOFA and SR-proADM model shows no improvement in predictive value in predicting 3-day death in these patients (Table 1).

Compared with other biomarkers and scores, PCT (eg, C index 0.689, HR at ICR 1.90 predicting 28-day death in all critically ill patients) and aldolase B (eg, C index 0.667, HR at ICR 1.47 when predicting 28-day death in all critically ill patients) on admission to the ICU show a modest association with death in all critically ill patients or their subgroups (eg, Table 1). However, in a 2D Cox regression model, PCT improves the predictive value of SAPS II or SOFA in predicting 28-day death in all critically ill patients (e.g., a combined SAPS II and PCT model yields a C index of 0.786 compared to 0.776 for SAPS II in a univariate analysis). ) (for example, table 3). In addition, a 2D Cox regression model including aldolase B improves the association of CP-proADM with 7-day mortality in all critically ill patients (combined model of CP-proADM and aldolase B with a C index of 0.780 compared to a univariate CP-proADM model). with index C 0.769) (Table 2). The prognosis was not further improved with PCT or aldolase B in addition to SAPS II, CP-proADM, or histones in other lethal outcome analyzes (Table 1 - Table 3).

Histone levels H2A, H2B, H3, and H4 at ICU admission were strongly associated with 3-day (eg, C H2B index 0.793, HR at ICR 2.76), 7-day (eg, C index H2B 0.768, HR at ICR 2.40) and 28-day (eg C H2B index 0.752, HR at ICR 2.40) death in all critically ill patients (Table 1 - Table 3). Similar results were obtained in a subgroup of patients with sepsis. Among all histones, H2B is the most effective, followed by H4, H2A and H3 (Table 1 - Table 6). Comparing the efficacy of histones H2A, H2B, H3, and H4 with other biomarkers shows a striking predictive value of histones in assessing short-term (3- and 7-day) mortality, although H2B may be inferior to CP-proADM in predicting 28-day mortality (index C CP-proADM 0.765 compared to C H2B index 0.752). H2B and CP-proADM are comparable to 7-day mortality (H2B C-index 0.793 vs. index C CP-proADM 0.769) in all patients in critical condition (table 1 - table 3).

In 2D Cox regression models, H2A, H2B, H3, and H4 histones improve the performance of SAPS II or SOFA (e.g., a combined H2B and SAPS II model with a C score of 0.811 versus a univariate SAPS II model with a C score of 0.776 in predicting 28-day mortality ) and CP-proADM (combined model of H2B and CP-proADM with a C index of 0.795 compared to a univariate model of CP-proADM with a C index of 0.765 predicting 28-day mortality) (Table 1 - Table 3). In critically ill patients with lower respiratory tract infection, histones H2A, H2B, H3, and H4 are the best predictor of 7-day death among biomarkers at ICU admission (e.g., H2B C index 0.785, HR at ICR 2.56) (Table four). In critically ill patients with UTIs, H2A, H2B, H3, and H4 histones are most strongly associated with 28-day mortality among all ICU admission variables (e.g., H2B C index 0.764, HR at ICR 2.52) (Table 5) . Histones H2A, H2B, H3, and H4 at ICU admission show the strongest association with 7-day mortality (e.g., H2B C index 0.815, HR at ICR 4.79) of all variables in critically ill patients admitted to the ICU with malignant neoplasms (table 6).

Table 1: One- and two-dimensional Cox regression analysis for 3-day mortality in all critically ill patients

Twenty-four of a total (n) 235 critically ill patients died on day 3 after admission to the ICU (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Score; CP-proADM: Regional Mean Proadrenomedullin; PCT: Procalcitonin; Histones represented by histone H2A, H2B, H3, and H4; H4 was also measured by immunoassay (AI )).

Table 1

HR [95% CI] on… Model N Developments LR
χ² df p-value index
C … MKR … 2x
change SAPS II 235 24 55.11 3 <0.001 0.876 8.42 [4.38-16.19] SOFA 235 24 41.78 3 <0.001 0.866 6.68 [3.33-13.39] H2B 235 24 29.31 3 <0.001 0.793 2.76 [1.77-4.29] 1.47 [1.24-1.73] H4 235 24 28.24 3 <0.001 0.786 2.70 [1.72-4.25] 1.45 [1.23-1.72] H2A 235 24 28.14 3 <0.001 0.787 2.45 [1.63-3.66] 1.45 [1.23-1.72] IA H4 228 23 26.61 3 <0.001 0.779 14.35 [3.26-63.19] 1.39 [1.16-1.66] SR-proADM 235 24 24.99 3 <0.001 0.786 4.82 [2.08-11.18] 2.05 [1.40-3.01] H3 235 24 19.68 3 <0.001 0.740 3.45 [1.54-7.70] 1.23 1.08-1.41] Aldolase B 235 24 14.65 3 0.002 0.721 1.65 [1.05-2.59] 1.16 [1.02-1.32] PKT 235 24 12.08 3 0.007 0.705 1.49 [0.85-2.62] 1.09 [0.97-1.23] H2B+SAPS II 235 24 61.95 four <0.001 0.885 H4+SAPS II 235 24 61.83 four <0.001 0.884 H2A+SAPS II 235 24 61.70 four <0.001 0.884 IA H4+SAPS II 228 23 61.51 four <0.001 0.898 H3+SAPS II 235 24 57.02 four <0.001 0.878 SR-proADM+SAPS II 235 24 56.79 four <0.001 0.884 PKT+SAPS II 234 24 55.10 four <0.001 0.875 Aldolase B+SAPS II 234 24 54.97 four <0.001 0.876 SR-proADM+H2V 235 24 36.15 four <0.001 0.828 IA H4+SOFA 228 23 50.05 four <0.001 0.887 H2B+SOFA 235 24 47.98 four <0.001 0.876 H4+SOFA 235 24 47.56 four <0.001 0.876 H2A+SOFA 235 24 47.30 four <0.001 0.874 H3+SOFA 235 24 43.63 four <0.001 0.863 SR-proADM+SOFA 235 24 42.27 four <0.001 0.872 PKT+SOFA 234 24 42.04 four <0.001 0.866 Aldolase B+SOFA 234 24 41.60 four <0.001 0.865 CP-proADM+H4 235 24 35.29 four <0.001 0.826 CP-proADM+H2A 235 24 35.16 four <0.001 0.825 SR-proADM+IA H4 228 23 32.60 four <0.001 0.809 CP-proADM+H3 235 24 29.15 four <0.001 0.806 H2B+Aldolase B 234 24 31.35 four <0.001 0.799 PCT+H2B 234 24 29.29 four <0.001 0.791

Table 2: One- and two-dimensional Cox regression analysis for 7-day mortality in all critically ill patients

Forty-nine out of a total (n) 235 critically ill patients died on day 7 after admission to the ICU (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Score; CP-proADM: Regional Mean Proadrenomedullin; PCT: Procalcitonin; Histones represented by histone H2A, H2B, H3 and H4; H4 was also measured by immunoassay (AI )).

table 2

HR [95% CI] on … Model N Developments LR
χ² df p-value index
C … MKR … 2x
change SAPS II 235 49 68.07 3 <0.001 0.809 5.15 [3.26-8.13] SOFA 235 49 50.64 3 <0.001 0.778 3.82 [2.39-6.09] SR-proADM 235 49 45.88 3 <0.001 0.769 4.53 [2.55-8.04] 1.99 [1.53-2.59] H2B 235 49 45.33 3 <0.001 0.768 2.40 [1.76-3.28] 1.39 [1.24-1.57] H4 235 49 42.81 3 <0.001 0.761 2.32 [1.68-3.18] 1.37 [1.22-1.55] H2A 235 49 40.91 3 <0.001 0.755 2.09 [1.57-2.78] 1.36 [1.21-1.54] H3 235 49 38.22 3 <0.001 0.742 3.62 [2.07-6.34] 1.24 [1.13-1.37] IA H4 228 48 35.98 3 <0.001 0.741 5.57 [2.40-12.96] 1.23 [1.11-1.37] Aldolase B 235 49 26.66 3 <0.001 0.697 1.69 [1.22-2.33] 1.17 [1.06-1.28] PKT 235 49 23.57 3 <0.001 0.698 1.66 [1.12-2.46] 1.12 [1.02-1.22] H2B+SAPS II 235 49 83.39 four <0.001 0.839 H4+SAPS II 235 49 82.29 four <0.001 0.834 H2A+SAPS II 235 49 80.74 four <0.001 0.830 H3+SAPS II 235 49 79.27 four <0.001 0.835 SR-proADM+SAPS II 235 49 76.75 four <0.001 0.832 IA H4+SAPS II 228 48 75.64 four <0.001 0.834 Aldolase B+SAPS II 234 49 70.32 four <0.001 0.809 PKT+SAPS II 234 49 68.47 four <0.001 0.811 H2B+SOFA 235 49 61.45 four <0.001 0.810 H4+SOFA 235 49 60.08 four <0.001 0.804 IA H4+SOFA 228 48 59.58 four <0.001 0.803 SR-proADM+H2B 235 49 59.50 four <0.001 0.804 H2A+SOFA 235 49 58.66 four <0.001 0.800 H3+SOFA 235 49 58.44 four <0.001 0.800 CP-proADM+H4 235 49 57.83 four <0.001 0.801 SR-proADM+SOFA 235 49 57.26 four <0.001 0.800 CP-proADM+H2A 235 49 56.25 four <0.001 0.796 CP-proADM+H3 235 49 55.10 four <0.001 0.795 SR-proADM+IA H4 228 48 52.66 four <0.001 0.780 Aldolase B+SOFA 234 49 51.39 four <0.001 0.781 PKT+SOFA 234 49 50.77 four <0.001 0.778 CP-proADM+aldolase B 234 49 49.84 four <0.001 0.780 PCT+H2B 234 49 46.55 four <0.001 0.769 SR-proADM+PKT 234 49 46.37 four <0.001 0.768 H2B+aldolase B 234 49 46.13 four <0.001 0.765

Table 3: One- and two-dimensional Cox regression analysis for 28-day mortality in all critically ill patients

Seventy-four patients out of a total (n) 235 critically ill patients died on day 3 after admission to the ICU (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Score; CP-proADM: Regional Mean Proadrenomedullin; PCT: Procalcitonin; Histones represented by histone H2A, H2B, H3 and H4; H4 was also measured by immunoassay (AI )).

Table 3

HR [95% CI] on … Model N Developments LR
χ² df p-value index
C … MKR … 2x
change SAPS II 235 74 76.19 3 <0.001 0.776 4.19 [2.89-6.07] SR-proADM 235 74 66.81 3 <0.001 0.765 4.86 [3.05-7.73] 2.06 [1.66-2.54] SOFA 235 74 59.04 3 <0.001 0.753 3.33 [2.27-4.88] H2B 235 74 58.04 3 <0.001 0.752 2.40 [1.84-3.11] 1.39 [1.26-1.54] H4 235 74 53.89 3 <0.001 0.742 2.28 [1.75-2.99] 1.37 [1.23-1.51] H2A 235 74 53.09 3 <0.001 0.742 2.11 [1.66-2.70] 1.37 [1.24-1.52] IA H4 228 70 48.71 3 <0.001 0.734 6.04 [2.97-12.27] 1.25 [1.14-1.36] H3 235 74 45.95 3 <0.001 0.720 3.33 [2.10-5.29] 1.22 [1.13-1.32] PKT 235 74 33.87 3 <0.001 0.689 1.90 [1.37-2.64] 1.15 [1.07-1.24] Aldolase B 235 74 25.91 3 <0.001 0.667 1.47 [1.11-1.95] 1.12 [1.03-1.22] H2B+SAPS II 235 74 100.75 four <0.001 0.811 H4+SAPS II 235 74 98.37 four <0.001 0.806 H2A+SAPS II 235 74 97.64 four <0.001 0.805 SR-proADM+SAPS II 235 74 97.47 four <0.001 0.810 H3+SAPS II 235 74 93.05 four <0.001 0.801 IA H4+SAPS II 228 70 90.73 four <0.001 0.804 SR-proADM+H2B 235 74 83.70 four <0.001 0.795 PKT+SAPS II 234 74 81.63 four <0.001 0.786 CP-proADM+H4 235 74 81.25 four <0.001 0.792 SRproADM+H2A 235 74 80.39 four <0.001 0.789 H2B+SOFA 235 74 77.41 four <0.001 0.785 SR-proADM+SOFA 235 74 77.32 four <0.001 0.783 Aldolase B+SAPS II 234 74 77.24 four <0.001 0.774 CP-proADM+H3 235 74 76.68 four <0.001 0.786 SR-proADM+IA H4 228 70 76.13 four <0.001 0.784 H4+SOFA 235 74 74.89 four <0.001 0.778 H2A+SOFA 235 74 74.08 four <0.001 0.776 IA H4+SOFA 228 70 72.78 four <0.001 0.778 H3+SOFA 235 74 71.09 four <0.001 0.770 CP-proADM+aldolase B 234 74 68.19 four <0.001 0.771 SR-proADM+PKT 234 74 66.57 four <0.001 0.766 PKT+SOFA 234 74 62.80 four <0.001 0.756 PCT+H2B 234 74 62.73 four <0.001 0.757 H2B+aldolase B 234 74 62.54 four <0.001 0.753 Aldolase B+SOFA 234 74 59.13 four <0.001 0.753

Table 4: Univariate Cox regression analysis for 7-day mortality in critically ill patients with lower respiratory tract infection

Twenty-seven of a total (n) 109 critically ill patients with lower respiratory tract infection died on day 7 after ICU admission (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Scale; CP-proADM: Regional Mean Proadrenomedullin; PCT: Procalcitonin; Histones represented by histone H2A, H2B, H3, and H4 were also measured by immunoassay (IA) ).

Table 4

HR [95% CI] on … Model N Developments LR
χ² df p-value index
C … MKR … 2x
change SAPS II 109 27 29.14 3 <0.001 0.773 3.47
[1.86-6.49] H2B 109 27 27.70 3 <0.001 0.785 2.56
[1.59-4.12] 1.39
[1.18-1.64] H4 109 27 26.36 3 <0.001 0.778 2.39
[1.49-3.84] 1.36
[1.15-1.61] H2A 109 27 25.40 3 <0.001 0.774 2.16
[1.40-3.33] 1.35
[1.14-1.60] IA H4 105 26 22.06 3 <0.001 0.752 5.64 [1.66-19.13] 1.23 [1.06-1.41] SOFA 109 27 21.84 3 <0.001 0.742 2.72 [1.38-5.36] 1.05 [1.02-1.09] SR-proADM 109 27 21.54 3 <0.001 0.752 2.86
[1.37-5.94] 1.67
[1.17-2.39] H3 109 27 21.48 3 <0.001 0.760 2.89
[1.36-6.13] 1.19
[1.05-1.35] Aldolase B 109 27 17.16 3 0.001 0.721 1.49
[1.00-2.22] 1.14
[1.00-1.29] PKT 109 27 14.81 3 0.002 0.716 1.47
[0.77-2.80] 1.08
[0.95-1.22]

Table 5: Univariate Cox regression analysis for 28-day mortality in critically ill patients with urinary tract infection

Thirty-four of a total (n) 94 critically ill patients with urinary tract infection died on day 28 after ICU admission (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Score; CP-proADM: Regional Mean Proadrenomedullin; PCT: Procalcitonin; Histones represented by histone H2A, H2B, H3, and H4; H4 was also measured by immunoassay (AI ))

Table 5

HR [95% CI] on … Model N Developments LR
χ² df p-value index
C … MKR … 2x
change H2B 94 34 25.79 3 <0.001 0.764 2.52
[1.75-3.65] 1.39
[1.22-1.59] H4 94 34 25.46 3 <0.001 0.761 2.47
[1.70-3.60] 1.38
[1.21-1.58] H3 94 34 24.98 3 <0.001 0.756 4.52
[2.34-8.75] 1.29
[1.15-1.44] H2A 94 34 24.65 3 <0.001 0.759 2.32
[1.64-3.28] 1.39
[1.21-1.59] SAPS II 94 34 23.69 3 <0.001 0.737 3.00
[1.85-4.89] IA H4 90 31 22.52 3 <0.001 0.751 8.43 [2.79-25.45] 1.29 [1.13-1.47] SOFA 94 34 16.07 3 0.001 0.696 2.28 [1.39-3.76] 1.05 [1.02-1.08] SR-proADM 94 34 14.93 3 0.002 0.689 2.28
[1.36-3.81] 1.65
[1.21-2.25] Aldolase B 94 34 11.96 3 0.008 0.679 1.82
[1.19-2.78] 1.18
[1.05-1.33] PKT 94 34 5.54 3 0.136 0.594 1.30
[0.74-2.29] 1.05
[0.94-1.17]

Table 6: Univariate Cox regression analysis for 7-day mortality in critically ill patients with malignancies

Sixteen patients out of a total (n) 64 critically ill patients with malignancy died on day 7 after admission to the ICU (events). Scale scores or biomarkers measured at ICU admission are included in the models. All 1D or 2D models are adjusted for age and gender. The degrees of freedom (df) reflect the number of variables and adjustments included. Results displayed are χ² likelihood ratio test (L.R. χ² and p-value), C index (Hurrel) and standardized hazard ratios (HR) plus 95% confidence interval (CI), either by interquartile range (ICR) or 2- multiple change. (SAPS II: Simplified Acute Physiological Condition II Scale; SOFA: Dynamic Organ Failure Score; CP-proADM: Mean Regional Proadrenomedullin; PCT: Procalcitonin; Histones are represented by histones H2A, H2B, H3, and H4. H4 was also measured by immunoassay (AI )).

Table 6

HR [95% CI] on … Model N Developments LR
χ² df p-value index
C … MKR … 2x
change H2B 64 16 22.32 3 <0.001 0.815 4.79 [2.44-9.43] 1.60 [1.31-1.96] H2A 64 16 22.28 3 <0.001 0.811 4.35 [2.31-8.20] 1.61 [1.31-1.98] H4 64 16 21.99 3 <0.001 0.812 4.53 [2.33-8.81] 1.60 [1.30-1.97] IA H4 61 16 21.95 3 <0.001 0.803 5.52 [2.24-13.58] 1.64 [1.26-2.13] H3 64 16 17.59 3 0.001 0.767 7.95 [2.70-23.45] 1.40 [1.17-1.67] SAPS II 64 16 15.16 3 0.002 0.755 4.76 [1.99-11.38] 1.02 [1.01-1.03] SR-proADM 64 16 10.20 3 0.017 0.737 3.15 [1.38-7.17] 1.88 [1.20-2.96] SOFA 64 16 9.84 3 0.020 0.722 3.36 [1.42-7.95] 1.06 [1.02-1.11] Aldolase B 64 16 7.30 3 0.063 0.644 2.05 [1.15-3.64] 1.25 [1.05-1.50] PKT 64 16 4.75 3 0.191 0.661 1.95 [0.92-4.14] 1.15 [0.98-1.34]

Example 2: Excellent Accuracy of ProAdrenomedullin in Predicting Death in Sepsis with Different Levels of Disease Severity

Background: The use of new sepsis biomarkers has increased in recent years. However, their predictive value in relation to disease severity has not been studied. In this work, the authors investigated the ability of regional mean proadrenomedullin (SR-proADM) to predict death in patients with sepsis with varying degrees of organ failure compared with procalcitonin, C-reactive protein, and lactate.

Methods: The study was a two-center prospective observational cohort study including patients with severe sepsis or septic shock admitted to the ICU. Plasma biomarkers were measured during the first 12 hours after admission. The association between biomarkers and 28-day mortality was assessed using Cox regression analysis and Kaplan-Meier curves. Patients were divided into three groups according to the assessment of the scale of dynamic assessment of organ failure (SOFA). The accuracy of the biomarkers in relation to lethal outcome was determined by the area under the receiver operating characteristics curve (AUROC).

Results:in The study included 326 patients with severe sepsis (21.7%) or septic shock (79.3%), 28-day mortality was 31.0%. AT in a multivariate analysis, only CP-proADM and lactate were associated with mortality: hazard ratio (HR): 8.5 vs. 3.4 (p < 0.001). CP-proADM showed the best AUROC in predicting death at 28 days in a whole cohort analysis (AUROC [95% CI]: 0.79 [0.74-0.84]) (p <0.001). After stratifying patients according to the degree of organ failure, CP-proADM was the only biomarker that predicted death in all severity groups (SOFA< 6, SOFA=7-12 and SOFA ≥ 13), AUROC [95% CI] 0.75 [0.61-0.88], 0.74 [0.66-0.83], and 0.73 [0 .59-0.86], respectively (p 0.05). All patients with SR-proADM concentrations< 0.88 nmol/l survived up to 28 days. In patients with SOFA< 6 Adding CP-proADM to the SOFA score increased SOFA's ability to detect non-survivors, AUROC [95% CI]: 0.70 [0.58-0.82] and 0.77 [0.66-0.88], respectively (p < 0.05 for both).

Conclusions: The effectiveness of prognostic biomarkers in sepsis strongly depends on the severity of the disease. The accuracy of SR-proADM in predicting death does not depend on the degree of organ failure. As a consequence, it is a good candidate for early detection of patients with moderate sepsis but with an increased risk of death.

Prerequisites

Sepsis remains the leading cause of death in intensive care unit (ICU) patients despite improvements in antibiotic treatment and early hemodynamic correction. In Europe, sepsis in patients with acute illness results in ICU mortality of 27% to 54% depending on severity [1]. In the United States, the Center for Disease Control estimates that 500,000 people develop sepsis and 200,000 die each year [2] [3]. Therefore, rapid diagnosis and evaluation of patients with high-risk sepsis is highly desirable, increasing the likelihood of initiating early and specific treatment. Thus, clinical scales for assessing severity, such as the Dynamic Organ Failure Assessment (SOFA) scale, may play a critical role [4]. However, the stand-alone application of these scoring systems to aid decision making in sepsis has been subject to significant criticism [5]. A standardized assessment tool for the early identification of patients with sepsis at presentation with different levels of severity will be of great value in facilitating clinical decision making and optimizing the use of healthcare resources. Accordingly, a number of predictive biomarkers in the field of sepsis have been proposed over the past few decades - many more than in other diseases. Most of these molecules are hormones, cytokines, or circulating proteins associated with inflammation or the coagulation system, and their measurement can be time-consuming, effortful, and costly [6].

Adrenomedullin (ADM) is a hormone-capable peptide produced by several tissues during physiological and infectious stress with various physiological functions, including vasodilating, anti-inflammatory, and antimicrobial activity, which is further enhanced by its regulation and modulation of complementary activity [7]. Thus, ADM is considered a "hormokine" characterized by a hormone-like behavior in non-inflammatory conditions, when it is produced only by endocrine cells, and a cytokine-like behavior in sepsis, when it is ubiquitously overexpressed. In addition, exogenous ADM has been shown to reduce acute lung injury, vascular permeability, and death in animal models of sepsis; while endogenous overexpression similarly attenuates the lesion resulting from sepsis [8] [9]. The measurement of circulating ADM is complicated by rapid breakdown and clearance from the circulation and is additionally masked by a binding protein (complement factor H), which prevents its detection by standard immunoassay. The mid-regional fragment of proadrenomedullin (SR-proADM), consisting of amino acids 45-92, is more stable and directly reflects the levels of the rapidly degrading active peptide ADM [10]. Elevated concentrations of CP-proADM have been identified in the plasma of patients with community-acquired pneumonia (CAP) and are widely used in assessing the risk and severity of this condition [11] [12] [13]. However, few data are available for patients with severe sepsis and septic shock. In addition, the effect of disease severity on the performance of predictive biomarkers in sepsis is not well understood.

In this study, the authors aimed to evaluate the ability of CP-proADM levels to predict 28-day mortality in patients with sepsis compared with other standard biomarkers (procalcitonin (PCT), C-reactive protein (CRP), and lactate) at three different severity levels. diseases as measured by the SOFA scale.

Methods

Patients, Inclusion and Exclusion Criteria : This study was a prospective observational study of a cohort of patients recruited consecutively from two intensive care units (ICUs) in Spain and France. Adult patients ≥ 18 years of age admitted to the ICU between April 2013 and January 2016 were admitted within 12 hours of meeting the criteria for severe sepsis or septic shock based on the American College of Thoracic Surgeons definition of SEPSIS-2. Medicine/Consensus Conference of the Society for Critical Care Medicine [14]. Patients included in the study also had a SOFA score > 2 and therefore met the criteria for the new definition of sepsis, SEPSIS-3 [15]. Patients with human immunodeficiency virus (HIV) infection and patients who received radiation therapy or received immunosuppressive drugs, including chemotherapy or systemic steroids, in the last 3 months prior to admission to the ICU were considered to be immunosuppressed. Exclusion criteria were age < 18 years, pregnancy, no blood sample available for biomarker profiling within the first 12 hours of ICU admission, or lack of informed consent. Clinical data recorded from medical records included demographics, comorbidities, laboratory tests, microbiological analysis, and biomarker levels. The severity of the disease was assessed at admission by calculating the score on the scale of dynamic assessment of organ failure (SOFA).

Biomarker assessment

Plasma samples for biomarker profiling were collected as close to ICU admission as possible and always within the first 12 hours. Plasma CP-proADM was measured by TRACE technology (time-resolved enhanced cryptate emission) using a novel sandwich immunoassay (Kryptor Compact Plus Analyzer, BRAHMS, Hennigsdorf, Germany); the detection limit was 0.05 nmol/l. PCT was measured using an electrochemiluminescent immunoassay (ECLIA) on a biochemical analyzer (Cobas 6000, Roche Diagnostics, Mélan, France); the limit of detection was 0.02 ng/mL. Serum CRP and lactate were measured by particle-enhanced immunoturbidimetric assay and colorimetric assay, respectively (e501 modular analyzer, Roche Diagnostics, Mélan, France); the limit of detection was 0.15 mg/dl and 0.2 mmol/l, respectively.

Statistical analysis

Differences in demographic and clinical characteristics between survivors and non-survivors were assessed using a χ2 test for categorical variables. To compare continuous variables based on the presence or absence of a normal distribution, Student's t-test or Mann-Whitney U-test were used, respectively. The association between biomarkers and risk of death was assessed using Cox regression analysis, adjusted for confounding factors. Time was censored 28 days after ICU admission. The first 24 hours after admission to the ICU was considered the first day of analysis. Variables givingp < 0.05 in univariate regression analysis were additionally included in multivariate analysis. Biomarkers were log-transformed to achieve a normal distribution. The effect of biomarkers on mean survival time was assessed using Kaplan-Meier curves and the Mantel-Haenszel log-rank test. As with the Cox regression analysis, time was censored 28 days after ICU admission. The accuracy and predictive value of biomarkers in determining mortality was assessed by calculating the area under the receiver operating characteristics curve (AUROC). Patients were divided into three groups based on disease severity as assessed by the SOFA score using two predefined cutoffs, one with a sensitivity close to 90% and one showing a specificity close to 90% to detect non-survivors after 28 days. (Fig. 4). Data was analyzed using IBM SPSS 20.0 software (SPSS, Chicago, IL).

results

Patient characteristics and biomarker concentrations :three hundred twenty-six patients (326) with severe sepsis (21.7%) or septic shock (79.3%) were included in the study, with a 28-day mortality rate of 25.5% and 34.9% in Valladolid and Dijon , respectively, and the overall mortality in both sites of the study was 31.0% (table 7). The median age was 65 years and 54.3% of patients were male. Compared with survivors, non-survivors were older and had higher SOFA scores and an increased incidence of septic shock, mechanical ventilation, renal replacement therapy, neoplasms, cardiovascular disease, chronic renal failure, immune suppression, and respiratory disease (allR<0.05). The most common source of infection was infection of respiratory and urological origin, regardless of outcome. Mortality rates depending on the source of infection were as follows: 37.2% in patients suffering from respiratory infection, 32.4% in patients with urological infection, 28.6% in patients with abdominal infection, 35.5% in patients with primary or secondary bacteremia and 25.6% in patients with infection of another origin. With regard to microbiological identification, both surviving and non-surviving patients showed a proportionate presence of gram (-), gram (+) and viral pathogens. Fungal infections were more common in non-survivors. The most common cause of death was multiple organ failure syndrome (n=58; 57.4%), followed by refractory shock (n=9, 8.9%) and refractory hypoxemia (n=8, 7.9%). Limiting the level of therapeutic intervention was applied to 21 patients. All of CP-proADM, PCT, and lactate were significantly elevated in non-survivors compared to survivors (allp < 0.01), while CRP levels remained similar in both groups. The levels of CP-proADM depending on the source of infection were as follows [median (interquartile range)]: respiratory infection [3.6 nmol/l (5.6)], urological infection [4.6 nmol/l (5.4)] , abdominal infection [4.9 nmol/l (6.5)], bacteremia [3.8 nmol/l (5.1)] and [3.5 nmol/l (5.8)] in infections of other origin. The levels of CP-proADM depending on the infecting microorganism were as follows [median (interquartile range)]: fungal infection [6.1 nmol/l (5.6)], gram (-) bacteria [4.9 nmol/l (5.6) 9)], gram (+) bacteria [4.1 nmol/l (6.2)] or viruses [1.2 nmol/l (3.4)].

Table 7. Clinical characteristics of patients: data are presented as mean (RMS) or median (MQR), as appropriate. Values expressed as a percentage (%) indicate the proportion of survivors and non-survivors at 28 days for specific variables

Survivors
n=225 non-survivors
n=101 Total
n=326 p Patients from Valladolid (n,%) 102 (45.3%) 35 (34.7%) 137 0.071 Patients from Dijon (n,%) 123 (54.7%) 66 (65.3%) 189 Men (n, %) 133 (59.1%) 68 (67.3%) 201 (61.4%) 0.098 Age (years) (mean, SD) 63 (14) 69 (12) 65.4 (14) <0.001 SOFA (average, RMS) 8 (3.4) 11 (3.5) 9 (3.7) <0.001 Septic shock (n,%) 152 (67.5%) 87 (86.1%) 239 (73.3%) 0.020 Artificial ventilation (n,%) 150 (66.7%) 89 (88.1%) 239 (73.3%) <0.001 RRT (n,%) 40 (17.7%) 45 (44.6%) 85 (26.2%) <0.001 ICU stay (days) (mean, RMS) 12.9 (18) 7.7 (6.7) 11.2 (15.6) 0.012 Neoplasm (n, %) 47 (21%) 35 (34.7%) 82 (25.2%) 0.007 Diabetes (n, %) 58 (25.8%) 29 (28.7%) 87 (26.7%) 0.330 COPD (n, %) 33 (14.7%) 16 (15.8%) 49 (15%) 0.450 Cardiovascular disease (n, %) 56 (25%) 41 (40.6%) 97 (29.8%) 0.030 Chronic renal failure (n, %) 16 (7.1%) 16 (15.8%) 32 (9.8%) 0.014 Immune suppression (n, %) 21 (9.3%) 25 (24.8%) 46 (14.1%) <0.001 Respiratory infection (n, %) 98 (43.6%) 58 (57.4%) 156 (48%) 0.014 Urological infection (n, %) 75 (33.3%) 36 (35.6%) 111 (34%) 0.380 Abdominal infection (n, %) 25 (11.1%) 10 (9.9%) 35 (10.7%) 0.450 Other infection (n, %) 32 (14%) 11 (10.9%) 43 (13%) 0.40 Primary or secondary bacteremia (n, %) 69 (30.7%) 38 (37.6%) 107 (32.8%) 0.130 Gram (-) bacteria (n, %) 62 (27.6%) 28 (27.7%) 90 (27.6%) 0.975 Gram (+) bacteria (n, %) 47 (20.9%) 22 (21.8%) 69 (21.2%) 0.855 Mushrooms (n, %) 3 (1.3%) 5 (5%) 8 (2.5%) 0.050 Virus (n, %) 15 (6.7%) 5 (5%) 20 (6.1%) 0.550 CP-proADM (nmol/l) (median, MKR) 2.68 (3.56) 7.44 (6.84) 3.62 (5.6) <0.001 Lactate (mmol/l) (median, mcr) 2.00 (1.54) 3.60 (5.53) 2.12 (2.28) <0.001 CRP (mg/dL) (median, IQR) 147.8 (193.6) 163.0 (181.9) 155.0 (189) 0.200 PCT (ng/ml) (median, IQR) 2.9 (17.5) 5.8 (36.7) 3.54 (27.5) 0.001

Survival analysis

CP-proADM, PCT, and lactate showed a significant association with mortality in univariate Cox regression analysis (Table 8). After adjusting for confounding factors, and compared with PCT, CRP, and lactate, CP-proADM showed the strongest independent association with the risk of death (hazard ratio: 8.5; 95% CI: 4.2-17.4; p < 0.001 ) (Table 8). In addition, Kaplan-Meier analysis showed that none of the patients with a CP-proADM < 0.88 nmol/L died in the first 28 days after admission to the ICU (Fig. 1). This cutoff was chosen because it provided 100% sensitivity in detecting non-survivors in AUROC (FIG. 2).

Table 8 Univariate and multivariate Cox regression analyzes for predicting mortality 28 days after ICU admission.

one dimensional Multidimensional HR (95% CI) p HR (95% CI) p SR-proADM 11.2 (6.3-19.8) < 0.001 8.5 (4.2-17.4) < 0.001 lactate 3.8 (2.6-5.5) < 0.001 3.4 (2.0-5.8) < 0.001 SRP 1.3 (0.8-1.9) 0.266 - - PKT 1.4 (1.2-1.8) 0.001 1.1 (0.9-1.4) 0.326 SOFA 1.2 (1.2-1.3) < 0.001 1.2 (1.1-1.3) < 0.001

Adjustments were made for the following variables: age, septic shock, cardiovascular disease, immune suppression, chronic renal failure, neoplasm, respiratory source of infection, renal replacement therapy, hospital (Valladolid/Dijon), the presence of a fungal infection, limiting the level of therapeutic intervention.

Effect of Disease Severity on Biomarker Efficiency

CP-proADM showed the best AUROC for predicting death at 28 days in the whole cohort analysis, even better than the SOFA score (Fig. 2). After stratifying patients according to the degree of organ failure, CP-proADM was the only biomarker able to discriminate non-survivors from survivors at 28 days in patients with the lowest disease severity (SOFA score ≤ 6) (AUROC [95% confidence interval (95% CI):]: 0.75 [0.61-0.88]), ( p =0.006) (Fig. 3). In moderately severely ill patients (SOFA score 7-12), CP-proADM showed a higher AUROC than that observed for lactate (0.74 [0.66-0.83] versus 0.61 [0.52- 0.71], respectively) (Fig. 3). In the most severely ill patients (SOFA score > 13), CP-proADM and lactate had a similar AUROC (0.73 [0.59–0.86] versus 0.72 [0.59–0.86], respectively). ) (Fig. 3). Neither CRP nor PCT predicted 28-day mortality in either severity group based on SOFA score.

The cut-off values (cutoff) of CP-proADM for detecting non-survivors were those showing the highest specificity among those with a pre-fixed sensitivity of at least 0.80. For patients with SOFA scores < 6, 7-12, and > 13, the cut-off threshold for CP-proADM was 1.79, 3.25, and 5.58 nmol/L, respectively (Table 9).

Table 9 . Cut-off threshold of CP-proADM (nmol/l) with the highest accuracy in predicting 28-day mortality based on the SOFA score. PPV - positive predictive value, NPV - negative predictive value, +LR - likelihood ratio for a positive result, -LR - likelihood ratio for a negative result.

Cutoff threshold Sensitivity Specificity PPV NPV +LR -LR SOFA < 6 1.79 83.0 61.0 23.8 96.2 2.14 0.27 SOFA 7-12 3.25 83.0 52.0 43.4 87.0 1.74 0.33 SOFA > 13 5.58 83.8 60.0 75.6 71.4 2.09 0.27

Length of ICU stay in each severity group was [mean, (SD)]: SOFA ≤ 6: 11.0 days (18.3); SOFA 7-12: 12.4 days (16.0); SOFA ≥ 13: 8.4 days (7.6). Mortality for each severity group was 12.8%, 30.6% and 59.7%, respectively.

SR-proADM improves mortality prediction in less severely ill patients

The authors evaluated the combination of CP-proADM and SOFA score in predicting death, such that patients with CP-proADM concentrations > 1.79 nmol/L were considered to have a 1-point increase in SOFA score. In patients with SOFA < 6, the combination of CP-proADM and modified SOFA score (ADM-SOFA) showed an increased ability to detect non-survivors compared to SOFA alone, AUROC [95% CI]: SOFA 0.70 [0.58-0 .82] and ADM-SOFA 0.77 [0.66-0.88].

Discussion

The severity of sepsis depends on the degree of organ failure, assessed by the SOFA scale, which, in turn, is directly related to the risk of death [15]. However, the emergence of an increasing number of biomarkers may provide a new avenue to improve predictive accuracy simply and quickly. In this regard, the authors' study suggests that CP-proADM may be a promising biomarker. However, previous studies evaluating the prognostic role of CP-proADM in sepsis have yielded conflicting results. Christ-Crain et al found that CP-proADM resulted in an AUROC of 0.81 in determining ICU death in a group of 53 sepsis patients [16]. In contrast, Suberviola et al found limited value for CP-proADM in predicting hospital death in 137 patients with sepsis, where the AUROC was 0.62 [17]. However, Marino et al showed that in 101 patients with sepsis, severe sepsis, or septic shock, plasma adrenomedullin levels were strongly associated with disease severity, need for vasopressor agents, and 28-day mortality. [eighteen]. These conflicting results regarding the predictive role of SR-proADM may be explained by differences in patient characteristics, disease severity, source of infection, surgical or medical treatment, and small sample sizes across studies.

In the present study, the authors demonstrated for the first time that the effectiveness of biomarkers in predicting death in sepsis strongly depends on the degree of organ failure upon admission to the ICU. Patient stratification based on their SOFA score allowed the authors to demonstrate that CP-proADM was the only biomarker capable of detecting non-survivors across all severity groups. This is especially important for less severely ill patients (SOFA score < 6) as this group represents either the earliest clinical presentation of sepsis and/or less severe sepsis in the ICU setting. Thus, CP-proADM may be a good candidate for inclusion in an early-stage sepsis management protocol, as it can provide rapid predictive value and help guide diagnostic interventions and treatment decisions, similar to the role of troponin in myocardial infarction or d dimer in pulmonary embolism. In this regard, the SR-proADM cut-off determined for this group of patients (1.79 nmol/l) can be very useful. This cut-off is capable of detecting death with good sensitivity and a high negative predictive value. Finally, SR-proADM may help stratify patients in clinical trials where novel treatments for sepsis are being investigated.

SR-proADM showed higher predictive value in determining the risk of death than other more commonly used biomarkers, including lactate, in patients with moderate organ failure (SOFA score 7-12). At the same time, both CP-proADM and lactate showed the same efficacy in the most seriously ill patients (SOFA ≥ 13). Thus, the results obtained by the authors confirm the importance of taking into account the degree of organ failure in the development of studies for the detection of prognostic biomarkers in sepsis.

The SEPSIS-3 consensus has recently proposed the use of SOFA organ failure scores to identify high-risk patients with suspected infection [15]. The results obtained by the authors show that a “positive” CP-proADM value can improve the ability of SOFA to predict a lethal outcome in sepsis. Interestingly, the combination of CP-proADM with clinical scores such as PSI or CURB-65 has also shown better results than clinical scores alone in patients with community-acquired pneumonia (CAP) or lower respiratory tract infections (LRTI) [12] [19 ] [20] [21]. Thus, CP-proADM could be used as a reliable risk stratification tool capable of predicting death or adverse events and guiding clinical decisions. Therefore, further clinical studies are needed to evaluate strategies that combine CP-proADM with other classical severity scales and/or biomarkers to improve the diagnosis and prognosis of sepsis [22] [23].

Finally, the authors found that a CP-proADM value of less than 0.88 nmol/L could "rule out" death within 28 days of admission to the ICU. This cut-off threshold can be particularly effective for determining early clinical decisions when the clinical manifestations of overt organ failure are not yet apparent.

Monitoring of SR-proADM over time can further illustrate a trend over time, which may indicate the success of specific treatments and therapies and therefore increase its predictive value in predicting outcomes [25]. Finally, in the cohort studied by the authors, the levels of CP-proADM differed slightly depending on the source of infection. Fungal infections caused the highest levels of CP-proADM, while viral infections caused the lowest. This is likely because fungal infections resulted in higher disease severity (median SOFA score of 12 versus 9 in patients without fungal infection), while viral infections resulted in more moderate severity. disease severity (median SOFA score of 6.5 versus 9 in patients without viral infection).

conclusions

The results obtained by the authors demonstrate that the effectiveness of biomarkers in determining the risk of death in sepsis is affected by the severity of the disease. In patients with moderate severity, CP-proADM outperformed other standard biomarkers. As a consequence, CP-proADM may help in the early identification of patients with sepsis requiring urgent ICU admission, as well as facilitate the subsequent clinical management of these patients.

List of abbreviations

ADM: adrenomedullin

SR-proADM: mid-regional proadrenomedullin

CRP: C-reactive protein

PCT: procalcitonin

SOFA: dynamic organ failure score

CI: confidence interval

AUROC: area of receiver operating curve

PPV: positive predictive value

NPV: negative predictive value

LR: likelihood ratio

HR: risk ratio

PSI: Pneumonia Severity Index

RRT: renal replacement therapy

All sources cited in this application are incorporated herein by reference in their entirety.

Albrich, W. C. and S. Harbarth (2015). "Intensive Care Med 41(10): 1739-1751.

Bone, R. C., R. A. Balk, et al. (1992). Chest 101(6): 1644-1655.

Bouch, D. C. and J. P. Thompson (2008). Continuing Education in Anaesthesia, Critical Care & Pain 8(5): 181-185.

Breslow, M. J. and O. Badawi (2012). Chest 141(1): 245-252.

Ferreira, A. M. and Y. Sakr (2011). Semin Respir Crit Care Med 32(5): 543-551.

Fine, MJ; Auble, T.E.; Yealy, D.M.; Hanusa, BH; Weissfeld, L.A.; Singer, D.E.; Coley, C. M.; Marrie, TJ; Kapoor, WN; et al. (1997). N Engl J Med. 336(4):243-250

Hodkinson, HM (1972). Age and Aging 1(4): 233-8

Halpern, N. A. and S. M. Pastores (2010). Crit Care Med 38(1): 65-71.

Kaneko-Wada Fde, J., G. Dominguez-Cherit, et al. (2015). Gac Med Mex 151(5): 628-634.

Le Gall, J. R., S. Lemeshow, et al. (1993). JAMA 270(24): 2957-2963.

Lim WS, van der Eerden MM, Laing R, et al. (2003). Thorax. 58(5): 377-82

Mayr, V. D., M. W. Dunser, et al. (2006). Crit Care 10(6): R154.

Vincent, J. L. (2008). Langenbecks Arch Surg 393(6): 817-824.

Vincent, J. L., R. Moreno, et al. (1996). Intensive Care Med 22(7): 707-710.

Sources cited in example 2 and in the description:

ADDIN ZOTERO_BIBL {"custom":[]} CSL_BIBLIOGRAPHY 1. Vincent J-L. Clinical sepsis and septic shock--definition, diagnosis and management principles. Langenbecks Arch. Surg. Dtsch. Ges. Fur Chir. 2008;393:817-24.

2. Jean-Baptiste E. Cellular mechanisms in sepsis. J. Intensive Care Med. 2007;22:63-72.

3. Hoyert DL, Xu J. Deaths: preliminary data for 2011. Natl. vital stat. Rep. Cent. Dis. Control Prev. Natl. Cent. health stat. Natl. vital stat. Syst. 2012;61:1-51.

4. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonça A, Bruining H, et al. The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med. 1996;22:707-10.

5. Vincent JL, Opal SM, Marshall JC. Ten reasons why we should NOT use severity scores as entry criteria for clinical trials or in our treatment decisions. Crit. Care Med. 2010;38:283-7.

6. Pierrakos C, Vincent J-L. Sepsis biomarkers: a review. Crit. Care London. English 2010;14:R15.

7. Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y, Amaha K, et al. Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J.Clin. Endocrinol. Metab. 1996;81:1449-53.

8. Müller-Redetzky HC, Will D, Hellwig K, Kummer W, Tschernig T, Pfeil U, et al. Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin. Crit. Care London. English 2014;18:R73.

9. Pugin J. Adrenomedullin: a vasodilator to treat sepsis? Crit. Care London. English 2014;18:152.

10. Struck J, Tao C, Morgenthaler NG, Bergmann A. Identification of an Adrenomedullin precursor fragment in plasma of sepsis patients. Peptides. 2004;25:1369-72.

11. Huang DT, Angus DC, Kellum JA, Pugh NA, Weissfeld LA, Struck J, et al. Midregional proadrenomedullin as a prognostic tool in community-acquired pneumonia. Chest. 2009;136:823-31.

12. Albrich WC, Dusemund F, Rüegger K, Christ-Crain M, Zimmerli W, Bregenzer T, et al. Enhancement of CURB65 score with proadrenomedullin (CURB65-A) for outcome prediction in lower respiratory tract infections: derivation of a clinical algorithm. BMC Infect. Dis. 2011;11:112.

13. Krüger S, Ewig S, Giersdorf S, Hartmann O, Suttorp N, Welte T, et al. Cardiovascular and inflammatory biomarkers to predict short- and long-term survival in community-acquired pneumonia: Results from the German Competence Network, CAPNETZ. Am. J. Respir. Crit. Care Med. 2010;182:1426-34.

14. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. 1992 Chest. 2009;136:e28.

15. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315:801-10.

16. Christ-Crain M, Morgenthaler NG, Struck J, Harbarth S, Bergmann A, Müller B. Mid-regional pro-adrenomedullin as a prognostic marker in sepsis: an observational study. Crit. Care London. English 2005;9:R816-824.

17. Suberviola B, Castellanos-Ortega A, Ruiz Ruiz A, Lopez-Hoyos M, Santibañez M. Hospital mortality prognostication in sepsis using the new biomarkers suPAR and proADM in a single determination on ICU admission. Intensive Care Med. 2013;39:1945-52.

18. Marino R, Struck J, Maisel AS, Magrini L, Bergmann A, Di Somma S. Plasma adrenomedullin is associated with short-term mortality and vasopressor requirement in patients admitted with sepsis. Crit. Care London. English 2014;18:R34.

19. Christ-Crain M, Morgenthaler NG, Stolz D, Müller C, Bingisser R, Harbarth S, et al. Pro-adrenomedullin to predict severity and outcome in community-acquired pneumonia [ISRCTN04176397]. Crit. Care London. English 2006;10:R96.

20. Courtais C, Kuster N, Dupuy A-M, Folschveiller M, Jreige R, Bargnoux A-S, et al. Proadrenomedullin, a useful tool for risk stratification in high Pneumonia Severity Index score community acquired pneumonia. Am. J. Emerg. Med. 2013;31:215-21.

21. Renaud B, Schuetz P, Claessens Y-E, Labarère J, Albrich W, Mueller B. Proadrenomedullin improves Risk of Early Admission to ICU score for predicting early severe community-acquired pneumonia. Chest. 2012;142:1447-54.

22. Guignant C, Voirin N, Venet F, Poitevin F, Malcus C, Bohé J, et al. Assessment of pro-vasopressin and pro-adrenomedullin as predictors of 28-day mortality in septic shock patients. Intensive Care Med. 2009;35:1859-67.

23. Travaglino F, De Berardinis B, Magrini L, Bongiovanni C, Candelli M, Silveri NG, et al. Utility of Procalcitonin (PCT) and Mid regional pro-Adrenomedullin (MR-proADM) in risk stratification of critically ill febrile patients in Emergency Department (ED). A comparison with APACHE II score. BMC Infect. Dis. 2012;12:184.

24. Bello S, Lasierra AB, Mincholé E, Fandos S, Ruiz MA, Vera E, et al. Prognostic power of proadrenomedullin in community-acquired pneumonia is independent of aetiology. Eur. Respir. J. 2012;39:1144-55.

25. Andaluz-Ojeda D, Cicuéndez R, Calvo D, Largo E, Nogales L, Muñoz MF, et al. Sustained value of proadrenomedullin as mortality predictor in severe sepsis. J. Infect. 2015;71:136-9.

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<---

Claims (20)

1. A method for predicting death in a subject suffering from sepsis, comprising determining the level of proadrenomedullin (proADM) in a sample of a given subject, where the proADM level is below 0.88 nmol/l indicates the survival of the subject for approximately 28 days, and the subject has a score on the dynamic organ failure assessment scale (SOFA scale) ≤ 6. 2. The method of claim 1, wherein the proADM is mid-regional proadrenomedullin (SR-proADM) or mature ADM. 3. The method according to claim 1 or 2, further comprising determining at least one marker and/or parameter of a given subject selected from the group consisting of the level of at least one histone, the level of procalcitonin (PCT) in the given sample, assessing by a simplified Acute Physiological Conditions Scale (SAPSII score), Accelerated SOFA (qSOFA) scores, Subject Acute and Chronic Health II (APACHE II) scores, Subject Pneumonia Severity Index (PSI) scores, and CURB scores –65 of this subject. 4. The method of claim 3, wherein the level of the marker or parameter is determined within 12 hours of the subject's admission to the intensive care unit (ICU) of the emergency department. 5. The method according to any one of paragraphs. 1-4, where the proADM level is determined within 12 hours of the subject's admission to the intensive care unit (ICU) of the emergency department. 6. The method according to any one of paragraphs. 1-5, where this sample is a biological fluid, blood, blood plasma, blood serum or urine. 7. The method according to any one of paragraphs. 1-6, where a given level of proADM is determined using a method selected from the group consisting of mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (EIA), solid phase methods enzyme-linked immunosorbent assay (ELISA), bead-based arrays with luminescent labels, magnetic bead-based arrays, protein microarray assays, accelerated assays and rare earth cryptate assays. 8. The method of claim 7, wherein the method is an immunoassay, and wherein the assay is carried out in a homogeneous phase or in a heterogeneous phase. 9. The method of claim 8, wherein the method is an immunoassay comprising the following steps: a) bringing the sample into contact with (i) a first antibody or antigen-binding fragment or derivative thereof specific for the first proADM epitope, and (ii) a second antibody, or an antigen-binding fragment or derivative thereof, specific for a second proADM epitope; and b) detecting the binding of the first and second antibodies or their antigen-binding fragments or derivatives with proADM. 10. The method of claim 9, wherein one of the first or second antibodies is labeled and the other antibody is or can be selectively bound to the solid phase. 11. The method according to claim 9 or 10, wherein the first antibody and the second antibody are present in dispersed form in the liquid reaction mixture and where the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, is associated with the first antibody and the second the labeling component of this labeling system is coupled to a second antibody such that once both antibodies bind to this detectable proADM, a measurable signal is generated allowing the resulting sandwich complexes to be detected in the measurement solution. 12. The method of claim 11 wherein the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, particularly of the cyanine type. 13. The method according to paragraphs. 1-12, where the determined proADM value is used in calculating a score indicative of a given prognosis of a subject's death. 14. The method of claim 13, wherein additionally at least one additional marker and/or subject parameter is used to calculate the score. 15. The method according to claim 14, where at least one additional marker and/or parameter of this subject is selected from the group consisting of the level of at least one histone, the level of procalcitonin (PCT) in this sample, the score on a simplified scale of acute physiological conditions (SAPSII scale), accelerated SOFA (qSOFA) scores, subject's Acute Physiological and Chronic Health Condition II (APACHE II) scores, subject's Pneumonia Severity Index (PSI) scores, and subject's CURB–65 scores .

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