HK40029756B - Workflow for risk assessment and patient management using procalcitonin and midregionalproadrenomedullin - Google Patents

Description

Workflow for risk assessment and patient management using procalcitonin and mid-zone pro-adrenergic lipoprotein

Technical Field

This invention pertains to the field of clinical diagnostics. Specifically, this invention relates to assessing the severity of suspected or infected subjects by determining the levels of procalcitonin (hereinafter: PCT) and/or pro-adrenal medullaris (hereinafter: proADM) (SEQ ID No: 3) or their partial peptides or fragments, particularly mid-region pro-adrenal medullaris (MR-proADM) (SEQ ID No: 2) in patient samples, said subjects may have physiological signs of infection or increased risk factors, particularly from infectious diseases, and this invention relates to its workflow. Furthermore, this invention relates to infection-related assessments such as patient exclusion/determination and stratification, risk assessment, particularly for avoiding readmission and, for example, death in or after hospital discharge.

Depending on the severity assessed, clinical decisions should be made, such as administering medication or moving to the clinical setting and initiating and/or changing or discontinuing treatment (e.g., antibiotics, oxygen, fluids) and/or improving monitoring and/or initiating further diagnostic pathways to improve the assessment of the patient's diagnosis and related treatment.

Background Technology

PCT is known to be a marker of bacterial infection and sepsis. High blood or serum levels of this propeptide hormone indicate a serious infection.

PCT reflects the severity of bacterial infection and is particularly useful for monitoring the progression of infection to sepsis, severe sepsis, or septic shock. PCT can be used to measure the activity of the infection-associated systemic inflammatory response, the success of controlling antimicrobial therapy, and to estimate prognosis (Assicot et al., 1993. Lancet 341:515-8; Clec'h C et al., 2004. Crit Care Med 32:1166-9; Lee et al., 2004. Yonsei Med J 45:29-37; Meisner et al., 2005. Curr Opin CritCare 11:473-480; Wunder et al., 2004. Inflamm Res 53:158-163). Increased PCT levels in patients with sepsis are associated with mortality (Oberhoffer et al., 1999. Clin Chem Lab Med 37:363-368).

During bacterial infections, plasma PCT concentrations are typically above 0.25 ng/ml, and PCT concentrations can rise above the normal range but below this concentration. To date, this concentration is known to be associated with bacterial infections requiring antimicrobial treatment, and these PCT concentrations are associated with adverse event outcomes in these patients (Sinnig et al., 2011. Circ J 75:1184-1191; Kelly et al., 2010. Biomarkers 15:325-331). Furthermore, a PCT level >0.1 ng/ml is used as a cutoff value for the presence of bacterial infection or sepsis.

PCT has been used to guide antibiotic treatment in patients with symptoms of infection (e.g., impaired breathing or respiratory rate (e.g., shortness of breath or cough), abnormal body temperature (below 36°C or above 38°C (fever)), digestive system impairment (e.g., diarrhea, nausea, or vomiting), urinary problems, or impaired circulation (e.g., rapid pulse, pain, signs of inflammation)). In patients presenting to the emergency department (ED) with symptoms of lower respiratory tract infection, PCT is measured, and antibiotic treatment is initiated only in patients with PCT concentrations >0.25 ng/mL or >0.5 ng/mL (Christ-Crain et al., 2004. Lancet 363:600-7). In patients with community-acquired pneumonia (CAP), antibiotic treatment is based on serum PCT concentrations (strongly discouraged when PCT concentrations are <0.1 ng/mL; discouraged when PCT concentrations are <0.25 ng/mL; encouraged when PCT concentrations are >0.25 ng/mL, and strongly encouraged when PCT concentrations are >0.5 ng/mL; a 50-80% decrease in PCT indicates the time point for discontinuing antibiotic use) (Christ-Crain et al., 2006. Am J Resp Crit Care Med 174:84-93; Schuetz et al., 2009. JAMA 302(10)1059-1066). PCT guidance significantly reduces antibiotic use in CAP without worsening patient outcomes. Similarly, PCT-guided therapy using the same decision threshold as described above also significantly reduced antibiotic use for acute respiratory infections in primary care without worsening patient outcomes (Briel et al., 2008. Arch Intern Med 168:2000-7; Burkhardt et al., 2010. Eur Resp J Express; doi:10.1183/09031936.00163309).

Therefore, it is recommended to measure PCT levels in bodily fluid samples from patients with infectious diseases before initiating potentially harmful antibiotic therapy. High blood or serum PCT levels indicate a serious bacterial infection or even sepsis, requiring antibiotic treatment for the corresponding patient. PCT values below 0.1 ng/mL indicate a non-infectious, non-bacterial infection, but also indicate an early stage of bacterial infection. It is recommended to repeat the measurement over the next 6–12 hours, or at least the next 24 hours, to rule out bacterial infection in the decision to admit the patient. This patient group has been previously overlooked.

Furthermore, existing techniques describe how to measure pro-adrenomedullin (proADM) and adrenomedullin (EP0622458B1, Lewis LK, Smith MW, Yandle TG, Richards AM, Nicholls MG. Adrenomedullin (1-52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage. Clin Chem 1998; 44:571-7; Ueda S, Nishio K, Minamino N, Kubo A, Akai Y, Kangawa K et al. Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome. Am J Respir Crit Care Med 1999; 160:132-6; Kobayashi K, Kitamura K, Etoh T, Nagatomo Y, Takenaga M, Ishikawa T et al., Increased plasma adrenomedullin levels in chronic congestive heart failure. Am Heart J 1996; 131:994-8; Kobayashi K, Kitamura K, Hirayama N, Date H, Kashiwagi T, Ikushima I et al., Increased plasma adrenomedullin in acute myocardial infarction. Am Heart J 1996; 131:676-80), particularly for the purpose of diagnosing sepsis (EP1121600B1).

EP1488209B1 discloses MR-proADM for diagnostic purposes (Struck J, Tao C, Morgenthaler NG, Bergmann A. Identification of an Adrenomedullin precursor fragment in plasma of sepsis patients. Peptides 2004; 25:1369-72; Morgenthaler NG, Struck J, Alonso C, Bergmann A. Measurement of mid-regional pro-adrenomedullin in plasma with an immunoluminometric assay. Clin Chem 2005; 51:1823-9; Christ-Crain M, M orgenthaler NG, Stolz D, Muller C, Bingisser R, Harbarth S et al., Pro-adrenomedullin to predict severity and outcome in community-acquiredpneumonia [ISRCTN04176397]. Crit Care 2006; 10: R96; Chr ist-Crain M, Morgenthaler NG, Struck J, Harbarth S, Bergmann A, Muller B. Mid-regional pro-adrenomedullin asa prognostic marker in sepsis: an observational study. Crit Care 2005;9:R816-24).

Combined with additional risk assessment and prognostic support, PCT focuses on the overall patient condition unrelated to infection, which can maximize patient safety and physician confidence in guiding appropriate therapy and treatment (Schuetz P, Mueller B. The role of immune and metabolic biomarkers for improved management of sepsis patients. Expert Rev Clin Immunol. 2014 Sep; 10(9):1255-1262).

In the emergency department (ED) and intensive care unit (ICU), MR-proADM and its precursor proADM provide a more accurate and rapid risk assessment of a patient's sepsis status (Travaglino F, De Berardinis B, Magrini L, et al., Utility of Procalcitonin (PCT) and Mid-regional pro-Adrenomedullin (MR-proADM) in riskstratification of critically ill febrile patients in Emergency Department (ED). A comparison with APACHE II score. BMC Infect Dis. 2012; 12:184), as well as additional assessment of the risk of further clinical complications that may arise from the initial sepsis status, which may include blood clot formation, tissue death, hypoxia, impaired organ blood flow, and ultimately organ dysfunction and failure. In fact, tissue hypoxia can occur even when normal organ blood flow is observed, making it extremely difficult to detect.

However, there is a need to improve, and in particular to rapidly assess, patients present in clinical settings such as primary care or ambulances, preferred EDs or ICUs, in order to improve patient clinical safety and provide support to physicians regarding patient treatment, particularly treatment interventions or management or guidance.

Detailed Implementation

definition:

Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms and additional definitions are set forth throughout the specification.

The terms “a,” “an,” and “the” do not indicate a quantity limitation, but rather that there is “at least one” of the said items.

As used herein, the terms “about” and “approximately” are used as equivalents. Any numbers used in this application, with or without “about”/“approximately”, are intended to cover any normal fluctuations as understood by one of ordinary skill in the art. As used herein, the terms “about” or “about” when applied to one or more target values refer to values similar to the reference value. In some embodiments, unless otherwise stated or obvious from the context, the terms “about” or “about” refer to a range of values falling within 5% of the reference value in either direction (greater or less) (unless the number exceeds 100% of the possible value).

Patient management in emergency departments (EDs) or other clinical settings places significant responsibility on clinicians to make rapid and accurate decisions regarding patient treatment, particularly treatment interventions or management and guidance. Patients often present with clinical signs of infection that can be very similar to other non-infectious conditions or illnesses, such as breathing problems and other issues that may be related to non-infectious conditions like asthma or poisoning. Identification of infection or infection risk, as well as management of further treatment options and medical classifications related to disease severity, are crucial. Patients at increased risk of infection or complications (e.g., those with comorbidities, especially elderly individuals or children with compromised immune responses) are often difficult to manage. Failure to assess a patient can increase the risk of death and serious events such as sepsis or organ dysfunction. It is important to discharge patients, especially those with low-grade severe illness states associated with mortality risk, to avoid complications and/or readmission (post-discharge phase), including readmission.

Therefore, complex decision trees or matrices supported by a combination of biomarkers with one or more critical levels (synonyms: threshold levels) may be needed to provide a workflow for the management of such patients.

The purpose of this invention relates to improved assessment of the severity of patient infections, including risk assessment and related clinical decisions, particularly regarding post-discharge, hospitalization, and readmission of patients.

The objective of this invention is achieved by the claims made herein.

Surprisingly, it has been found that by using PCT and pro-ADM, especially MR-proADM, in a complementary manner to assess severity, and to determine/exclude infection and the severity of disease status, the management of clinical decision-making has been improved by categorizing subjects in order to establish their workflow.

Therefore, the present invention relates to a method for assessing the severity of infection, particularly a method for assessing the severity of infection in subjects suspected of or having an infection, the method comprising the following steps:

(i) Provide bodily fluid samples from the subject;

(ii) Determining the level of procalcitonin (PCT) (SEQ ID No. 1) or a fragment or precursor thereof of at least 12 amino acid residues in the sample, and

The levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2), or a precursor or fragment thereof of at least 12 amino acid residues in length, and

(iii) The severity of infection, particularly the severity of infection in subjects suspected of or having an infection, is determined by comparing the measured PCT and proADM levels, especially MR-proADM levels or fragments thereof, with threshold levels.

Surprisingly, this invention relates to the synergistic effect of PCT and pro-ADM or fragments thereof for managing patients with infection or suspected infection, and the severity of infection. If PCT levels are below 0.1 ng/mL and pro-ADM or fragments thereof, particularly MR-proADM, are below 0.88 nmol/L, such patients should be advantageously discharged from the clinical setting, particularly outpatient settings, due to the assessed severity of illness and correspondingly low mortality. Specifically, if PCT is equal to or greater than 0.1 ng/mL and pro-ADM or fragments thereof, particularly MR-proADM, are below 0.88 nmol/L, the serious risk of infection-independent mortality remains low, and the decision is made to discharge the patient. In this case, the treatment is manageable in an outpatient setting, for example, by prescribing oral or topical medications such as antibiotics, rather than invasive application.

A preferred embodiment of the present invention relates to cases where the severity of an infection is assessed. Therefore, the present invention relates to a method for assessing the severity of an infection, particularly the severity of a suspected infection or infection in a subject, the method comprising the steps of:

(i) Provide bodily fluid samples from the subject;

(ii) Determine the level of procalcitonin (PCT) or a fragment or precursor thereof of at least 12 amino acid residues in the sample, and

The levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly the levels of mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2) or a precursor or fragment thereof of at least 12 amino acid residues in length, and

(iii) The severity of infection, particularly in subjects suspected of or with infection, is determined by comparing the measured PCT and proADM levels, especially MR-proADM levels or fragment levels thereof, with threshold levels.

(iv) Wherein the subject has a non-serious infection when the threshold level of the PCT is below 0.06 ng/ml, more preferably below 0.08 ng/ml, and most preferably below 0.1 ng/ml, and the threshold level of the proADM or its fragment is below 0.6 nmol/L, more preferably below 0.75 nmol/L, and most preferably below 0.88 nmol/ L .

and/or

(v) Wherein the subject has a nearly non-serious infection when the threshold level of the PCT is below 0.06 ng/ml, more preferably below 0.08 ng/ml, most preferably below 0.1 ng/ml and the threshold level of the proADM or its fragment is equal to or higher than 0.6 nmol/L, more preferably equal to or higher than 0.75 nmol/L, most preferably equal to or higher than 0.88 nmol/L and not equal to or higher than 1.1 nmol/L, more preferably not equal to or higher than 1.2 nmol/L, most preferably not equal to or higher than 1.28 nmol/L.

and/or

(vi) wherein the subject has a low level of severe infection when the threshold level of the PCT is below 0.06 ng/ml, more preferably below 0.08 ng/ml, and most preferably below 0.1 ng/ml, and the threshold level of the proADM or its fragment is equal to or higher than 1.1 nmol/L, more preferably equal to or higher than 1.2 nmol/L, and most preferably equal to or higher than 1.28 nmol/L.

and/or

(vii) Wherein the subject has an extremely low incidence of severe infection when the threshold level of the PCT is equal to or greater than 0.06 ng/ml, more preferably equal to or greater than 0.08 ng/ml, and most preferably equal to or greater than 0.1 ng/ml, and the threshold level of the proADM or its fragment is less than 0.6 nmol/L, more preferably less than 0.75 nmol/L, and most preferably less than 0.88 nmol/L.

and/or

(viii) Wherein the threshold level of the PCT is equal to or higher than 0.06 ng/ml, more preferably equal to or higher than 0.08 ng/ml, most preferably equal to or higher than 0.1 ng/ml and the threshold level of the proADM or its fragment is equal to or higher than 0.6 nmol/L, more preferably equal to or higher than 0.75 nmol/L, most preferably equal to or higher than 0.88 nmol/L, and not equal to or higher than 1.1 nmol/L, more preferably not equal to or higher than 1.2 nmol/L, most preferably not equal to or higher than 1.28 nmol/L, the subject has a low degree of severe infection .

and/or

(ix) Wherein the subject has a serious infection when the threshold level of the PCT is equal to or higher than 0.06 ng/ml, more preferably equal to or higher than 0.08 ng/ml, most preferably equal to or higher than 0.1 ng/ml and the threshold level of the proADM or its fragment is equal to or higher than 1.1 nmol/L, more preferably equal to or higher than 1.2 nmol/L, most preferably equal to or higher than 1.28 nmol/L.

and/or

(x) Wherein the subject has a serious infection when the threshold level of the PCT is below 0.06 ng/ml, more preferably below 0.08 ng/ml, and most preferably below 0.1 ng/ml, and the threshold level of the proADM or its fragment is equal to or higher than 1.30 nmol/L, more preferably equal to or higher than 1.40 nmol/L, and most preferably equal to or higher than 1.50 nmol/L.

and/or

(xi) Wherein, when the threshold level of the PCT is below 0.06 ng/ml, more preferably below 0.08 ng/ml, and most preferably below 0.1 ng/ml, and the threshold level of the proADM or its fragment is equal to or higher than 2.25 nmol/L, more preferably equal to or higher than 2.50 nmol/L, and most preferably equal to or higher than 2.75 nmol/L, the subject has a serious infection .

and/or

xii) Wherein, when the threshold level of the PCT is equal to or higher than 0.06 ng/ml, more preferably equal to or higher than 0.08 ng/ml, and most preferably equal to or higher than 0.1 ng/ml, and the threshold level of the proADM or its fragment is equal to or higher than 1.30 nmol/L, more preferably equal to or higher than 1.40 nmol/L, and most preferably equal to or higher than 1.50 nmol/L, the subject has a high degree of severe infection .

and/or

xiii) Wherein the subject has an extremely high level of severe infection when the threshold level of the PCT is equal to or higher than 0.06 ng/ml, more preferably equal to or higher than 0.08 ng/ml, most preferably equal to or higher than 0.1 ng/ml and the threshold level of the proADM or its fragment is equal to or higher than 2.25 nmol/L, more preferably equal to or higher than 2.50 nmol/L, most preferably equal to or higher than 2.75 nmol/L.

The cases refer to the progression of severity from less severe to more severe and most severe, which is related to the patient's classification.

Therefore, based on the severity assessed, the cases (iv-xiii) can be distinguished from each other to exclude or determine patients.

therefore,

(iv) For patients with non-serious infections , serious infections should be ruled out and hospitalization and/or discharge should be avoided.

(v) For patients with almost non-serious infections , they should be assessed as having a possible serious infectious or non-infectious illness and requiring hospitalization without isolation.

(vi) For patients with low-severity infections , it should be determined that they have a possible infectious or non-infectious severe illness and require hospitalization without isolation.

(vii) For patients with very low severity of infection , severe infection should be ruled out and hospitalization and/or discharge should be avoided.

(viii) Patients with low-grade severe infection should be classified as having severe infection and require hospitalization and isolation.

(ix) Patients with severe infections should be classified as having severe infections and require strict hospitalization, isolation, and more frequent monitoring.

(x) Patients with severe infections should be classified as possibly infected or at high risk of infection or as having a serious non-infectious illness with a high risk of complications (25% risk of death) and requiring hospitalization without isolation.

(xi) Patients with severe infections should be classified as having a possible infection or a high risk of infection, or as having a serious non-infectious illness with an extremely high risk of complications (35% risk of death) and requiring hospitalization, especially in special wards such as ICUs requiring isolation.

(xii) Patients with very severe infections should be assessed as having a high risk of severe infection and complications (25% risk of death) and require strict hospitalization and isolation.

(xiii) Patients with extremely severe infections should be assessed as having a very high risk of severe infection and complications (35% risk of death) and require very strict hospitalization and special wards such as ICU and isolation.

A method for treating a patient suspected of having an infection is also provided, the method comprising the steps described above, and furthermore...

(ii) If the patient is designated as (x) a severely infected patient, (xi) a severely infected patient, (xii) a very severely infected patient, or (xiii) a very severely infected patient , antibiotics are administered to the patient in the ICU setting.

A method for treating a patient suspected of having an infection is also provided, the method comprising the steps described above, and furthermore...

(ii) If the patient is designated as (vi) a patient with low severity of infection or (viii) a patient with low severity of infection , antibiotics should be administered to the patient in the hospital but outside the ICU setting.

A method for treating a patient suspected of having an infection is also provided, the method comprising the following steps:

(i) Provide bodily fluid samples from the subject;

(ii) Determine the level of procalcitonin (PCT) or a fragment or precursor thereof of at least 12 amino acid residues in the sample, and

The levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly the levels of mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2) or a precursor or fragment thereof of at least 12 amino acid residues in length, and

(iii) If the PCT level is higher than 0.06 ng/ml and the proADM level is higher than 0.6 nmol/L, then administer antibiotics to the patient.

In a preferred embodiment of the invention mentioned above, in order to control possible infection states (v, vi, x, xi) or to monitor response to treatment with antibiotics, for example (vi, vii, viiii, xii, xiii), the PCT measurement must preferably be repeated at least 6-12 hours or 24 hours after the initial measurement. This method allows for the identification of false-negative patient groups in the early stages of infection, or for monitoring subjects who have an increased risk of infection or complications.

In another preferred embodiment of the invention relating to the aforementioned hospitalized cases (v, vi, vii, viii, ix, x, xi, xii, xiii), periodic remeasurements of PCT and/or proADM or fragments thereof are performed to monitor the severity and treatment management of the same sample.

Furthermore, the cases (iv-ix) can be further distinguished from each other as follows:

If (iv) applies, antibiotics should not be used on the subject.

If (v) applies, antibiotics are not used on the subject, and optionally, for clarification or condition testing, the patient’s sample should be further measured for PCT within at least 6–12 hours or 24 hours.

If (vi) applies, antibiotics are administered to the subject, and optionally, for clarification or examination of the condition, PCT is further measured to identify the early stage of a viral or fungal infection or bacterial infection.

If (vii) applies, it is possible to not use antibiotics in subjects, particularly when PCT values are below the typical threshold for initiating antibiotic action (e.g., 0.25 ng/mL), or when PCT values are above the typical threshold for initiating antibiotic action, or in patients at increased risk of serious infection. Possible antibiotic action may be administered non-invasively (e.g., orally or topically).

If (viii) applies, it may be possible to not use antibiotics on the subject, particularly when the PCT value is below the typical threshold for initiating antibiotic treatment (e.g., 0.25 ng/mL), or when the PCT value is above the typical threshold for initiating antibiotic treatment, or in patients at increased risk of serious infection, and optionally for clarification or condition assessment, the patient's sample should be further measured within at least 6–12 hours or 24 hours.

If any of (ix x, xi, xii, xiii) applies, the antibiotic shall be administered to the subject immediately.

If one of (x, xi, xii, xiii) is applicable, antibiotics should be administered to the subject immediately, and for clarification or condition assessment, further assays of PCT and/or proADM or fragments thereof should be performed on the patient's sample at least 6–12 hours or 24 hours later. Patients are at high risk of complications (25%–35% risk of death), therefore further monitoring is required, especially regarding organ dysfunction, diagnostic interventions (more biomarkers or parameters), and treatments to stabilize the patient, such as oxygen, fluids, intravenous antibiotics, organ protection measures such as kidney replacement, and mechanical ventilation. Remeasurement supports evaluation of treatment efficacy.

The term "may not use" means that it is preferred and recommended not to use, i.e., the administration of antibiotics should be avoided, or, if necessary, low-dose antibiotics or topical treatment are preferred. For further clarification or to examine the condition, a blood culture test may be performed on the patient's sample.

The term "potentially administerable" means that administration is preferred and recommended; that is, antibiotics should be administered, preferably at low doses or for local treatment in cases of low-severity infection, if necessary. Blood cultures may be performed on patient samples for further clarification or to assess the condition.

In cases of low-severity infections (e.g., regarding complications and mortality), topical or oral treatment with medications such as antibiotics or anti-inflammatory drugs is necessary. Clinicians may choose to discharge the patient. For more severe infections, such as moderate or severe infections, systemic administration, such as intravenous or intramuscular administration, is preferred, but at least oral or topical, or a combination thereof, is acceptable.

However, in all cases (iv-xiii), PCT and proADM or fragments thereof can be measured to predict increases or decreases in PCT and/or proADM or fragments thereof levels. These measurements can be performed individually or simultaneously. Preferably, remeasurements of PCT and/or proADM or fragments thereof should be performed routinely to avoid false negatives in infected patients and/or to monitor treatment response.

Therefore, in another preferred embodiment of the invention, the method for assessing the severity of infection is suitable for making clinical decisions, particularly for the use of medicines and other health products or additives, especially antibiotics, oxygen, fluids for advanced treatment and therapy, and for initiating and/or changing or stopping treatment, particularly in intensive care units or emergency rooms or other special care wards, to include decisions to hospitalize patients, including administering medication or moving patients to the clinical setting, including improved monitoring and/or initiating further diagnostic pathways to improve the assessment of patient diagnosis and related treatment, and including isolating or not isolating patients to protect other patients, particularly hospitalized patients.

In particular, in all cases (vi., ix., x., xi., xii., and xiii), where proADM or a fragment thereof is equal to or greater than 1.28 nmol/L, one or more of the following measures i.)–iv.) apply:

i) Blood culture tests are performed from the central venous line and/or from various sources and samples (e.g., blood, serum, plasma, urine, CSF, body fluids, etc.).

ii) Administer antibiotics orally or intravenously according to guidelines, preferably using a combination of antibiotics.

iii) If the test result in i) is negative and PCT is below 0.1 ng/ml, discontinue antibiotic administration.

iv) Monitor blood test results, such as lactate, FBC (complete blood cell count), U&E (urea and electrolytes), LFT (liver function test), CRP, coagulation, etc.

However, PCT and/or proADM or fragments thereof can be measured to predict increases or decreases in PCT and/or proADM or fragments thereof. These measurements can be performed individually or simultaneously. Preferably, remeasurements of PCT and/or proADM or fragments thereof should be performed routinely to avoid false negatives in infected patients and/or to monitor treatment response.

In summary, this invention relates to a workflow that can advantageously manage risk assessment of suspected or infected patients, particularly those at increased risk, presenting in clinical settings such as primary care or ambulances, preferably EDs or ICUs. The workflow is illustrated in Figure 1.

Furthermore, the inventors have discovered that, regardless of the determination of PCT levels, the following threshold levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2) or a precursor or fragment thereof of at least 12 amino acid residues in length, allow for assessment for further clinical decision-making.

Therefore, in another aspect, the present invention provides a method for assessing the severity of infection in a subject, the method comprising the following steps:

(i) Provide bodily fluid samples from the subject;

(ii) Independent of PCT level determination

The levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly the levels of mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2) or a precursor or fragment thereof of at least 12 amino acid residues in length, and

(iii) The severity of infection is determined by comparing the measured proADM levels, particularly MR-proADM levels or fragment levels thereof, with threshold levels.

a.) Wherein, a threshold level equal to or higher than 0.88 nmol/L refers to the clinical decision-making process for assessing the admission of subjects/patients, and/or

b.) Wherein, a threshold level equal to or higher than 1.50 nmol/L refers to identifying high-risk infected or non-infected patients with an approximate mortality rate of at least 25%, and/or

c.) Wherein, a threshold level equal to or higher than 2.75 nmol/L refers to the identification of extremely high-risk infected or non-infected patients with an approximate mortality rate of at least 35%, and/or

d.) Wherein, a threshold level equal to or higher than 2.75 nmol/L refers to the clinical decision to assess the admission of subjects/patients to the ICU.

In another aspect, the present invention provides a method for treating a patient with a severe infection, the method comprising the following steps:

(i) Provide bodily fluid samples from the subject;

(ii) Independent of PCT level determination

The levels of pro-adrenomedullin (proADM) (SEQ ID No:3) or a partial peptide or fragment thereof of at least 12 amino acid residues in length, particularly the levels of mid-region pro-adrenomedullin (MR-proADM) (SEQ ID No:2) or a precursor or fragment thereof of at least 12 amino acid residues in length, and

(iii) The severity of infection is determined by comparing the measured proADM levels, particularly MR-proADM levels or fragment levels thereof, with threshold levels.

(iv) If the MR-proADM level is equal to or higher than 1.50 nmol/L, the patient should be treated with antibiotics.

In some implementations, if the MR-proADM level is equal to or higher than 2.75 nmol/L, the patient is treated with antibiotics in the ICU.

In particular, in all cases where proADM or a fragment thereof is equal to or greater than 1,50 nmol/L, one or more of the following measures (i.)–vi.) apply:

i) Provide oxygen via mask (approximately 15 L/min)

ii) Administer fluid bolus therapy, especially intravenous bolus, to rapidly administer Hartmann's solution (also known as Ringer's lactate solution).

iii) Blood culture tests are performed from the central venous line and from various sources and samples (e.g., urine, CSF, body fluids, etc.).

iv) Administer antibiotics orally or intravenously according to guidelines, preferably using a combination of antibiotics.

v) Monitor blood test results, such as lactate, FBC (complete blood cell count), U&E (urea and electrolytes), LFT (liver function test), CRP, coagulation, etc.

vi) Monitor urine output/fluid balance.

In particular, in all cases where proADM or a fragment thereof is equal to or higher than 2.75 nmol/L, the following measure vii.) also applies:

vii) Monitor organ dysfunction, including checking clinical parameters and other life-sustaining measures.

However, PCT and/or proADM or fragments thereof can be measured to predict increases or decreases in PCT and/or proADM or fragments thereof. These measurements can be performed individually or simultaneously. Preferably, remeasurements of PCT and/or proADM or fragments thereof should be performed routinely to avoid false negatives in infected patients and/or to monitor treatment response.

In summary, this invention relates to a workflow that can advantageously manage risk assessment of suspected or infected patients, particularly those at increased risk, presenting in clinical settings such as primary care or ambulances, preferably EDs or ICUs. The workflow is illustrated in Figure 2.

Within the scope of this invention, "procalcitonin (PCT)" should be understood as a human protein or polypeptide or fragment thereof having an amino acid sequence of 1-116 amino acids, 2-116 amino acids (PCT 2-116), or 3-116 amino acids (PCT 3-116), as described in EP0656121B1 and DE10027954A1. Therefore, the length of the PCT fragment is at least 12 amino acids, preferably greater than 50 amino acids, and more preferably greater than 110 amino acids. PCT may include post-translational modifications, such as glycosylation, lipidation, or derivatization. PCT itself is a precursor to calcitonin and katacalcin. The amino acid sequences of PCT1-116 are provided as SEQ ID NO:1.

Adrenal medullaris (ADM) is encoded as a precursor peptide (“pro-adrenal medullaris” or “pre-proADM”) comprising 185 amino acids, provided herein as SEQ ID NO:4. ADM, and especially the biologically active form of ADM, comprises amino acid positions 95-146 of the pre-proADM sequence and is its splice product.

"Pro-adrenergic medullaris" ("Pro-ADM") refers to pre-proADM without the signal sequence (amino acids 1 to 21), i.e., amino acid residues 22 to 185 of pre-proADM. "Mid-region pro-adrenergic medullaris" ("MR-proADM") refers to amino acids 45 to 92 of pre-proADM. The amino acid sequence of MR-proADM is provided as SEQ ID NO:2. Peptides and fragments of pre-proADM or MR-proADM are also contemplated herein for use in the methods described herein. For example, peptides and fragments of pre-proADM may contain amino acids 22-41 (PAMP peptide) or amino acids 95-146 (mature adrenergic medullaris). The C-terminal fragment of proADM (amino acids 153 to 185 of pre-proADM) is referred to as adrenaline. A proADM peptide or fragment of MR-proADM may contain, for example, five or more amino acids. Therefore, the proADM fragment can be selected, for example, from the group consisting of MR-proADM, PAMP, adrenaline and mature or biologically active adrenomedullin, and preferably, the fragment is MR-proADM.

Therefore, in the context of this invention, the proADM fragment may preferably be selected from the group consisting of MR-proADM, PAMP, adrenaline and mature adrenaline, and most preferably, the fragment is MR-proADM.

MR-proADM (EP 1488209B1) exhibits excellent plasma stability, which is particularly advantageous. Furthermore, the MR-proADM fragment is at least 12 amino acids long, preferably more than 25 amino acids, and more preferably more than 40 amino acids.

Furthermore, this invention covers precursors of MR-proADM, namely proADM (SEQ ID No. 3) and preproADM (SEQ ID No. 4), and fragments thereof. Within the scope of this invention, "pro-adrenal medullarin (proADM)" should be understood as a human protein or polypeptide having a sequence of 1-185 amino acids. Therefore, the length of the proADM fragment is at least 12 amino acids, preferably greater than 80 amino acids, and more preferably greater than 150 amino acids. proADM may include post-translational modifications, such as glycosylation, lipidation, or derivatization.

This precursor peptide contains a 21-amino acid signal sequence at its N-terminus, known as "pre-pro-adrenal medullarin" (Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H, Eto T. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin, Biochem Biophys Res Commun 1993:194:720-725). Pre-pro-ADM contains 185 amino acids and has the sequence according to SEQ ID No:4. Known fragments of pre-pro-ADM include PAMP (AA 22-41), MR-pro-ADM (central adrenal medullaris pro-ADM) (AA 45-92) (SEQ ID No:2), ADM (adrenal medullaris) and its biologically active form (AA 95-146), CT-pro-ADM (adrenaline) (AA 153-185), and pro-adrenal medullaris (proADM) (AA22-185) (SEQ ID No:3).

To date, only a small number of peptide fragments formed in the cleavage of pre-proADM have been characterized more precisely, particularly the physiologically active peptides adrenomedullin (ADM) and “PAMP”, a 20-amino acid peptide (22-41) that follows the 21-amino acid signal peptide in pre-proADM.

EP0622458B1 also describes the N-terminal fragment of (pre)adrenal medullarin propeptide used for diagnosis, such as PAMP (Hashida S, Kitamura K, Nagatomo Y, Shibata Y, Imamura T, Yamada K et al., Development of an ultra-sensitive enzyme immunoassay for human pro-adrenaledullin N-terminal peptide and direct measurement of two molecular forms of PAMP in plasma from healthy subjects and patients with cardiovascular disease. Clin Biochem 2004; 37:14-21).

EP2111552B1 also describes the C-terminal fragment of (pre)adrenal medullarin pro-D, namely CT-pro-ADM (adrenaline), used for diagnosis.

As used herein, the term "subject or synonymous patient" refers to a living human or non-human organism who is receiving or should receive medical care due to disease, particularly infection. This includes individuals without a known disease undergoing examination for pathological signs. Therefore, the methods and assays described herein are applicable to both human and veterinary diseases.

In the context of this invention, the term "patient with increased risk" specifically refers to a subject with a higher predisposition to infection or complications. Such patients may have comorbidities or physical or mental limitations, such as autoimmune diseases, diabetes; or host response defects, such as patients with cancer or loss of function of parts of the immune system (e.g., tonsils) or patients receiving immunosuppressive therapy (e.g., after transplantation or HIV); or certain elderly populations, such as the elderly or young children or infants; or patients with: organ defects such as cardiovascular defects, such as heart failure, myocarditis, pericarditis, valvular heart defects, atherosclerosis, or renal failure; respiratory conditions such as COPD, asthma, or mucoidosis; skin defects, such as burns or more extensive skin inflammation; gastrointestinal defects, such as Morbus Crohn's disease, gastritis, or gallstones; or liver defects, such as hepatitis, cirrhosis, or drug/alcohol dependence or addiction; or cognitive impairment, such as unconscious patients or dementia, or those taking medications that increase the risk of serious adverse events; or obesity.

In the context of this invention, the term "therapeutic intervention or management or guidance" refers to decisions regarding medical activities based on clinical imagery combined with biomarker values or clinical parameters. Medical interventions are taken if measured PCT and/or ADM values indicate a serious condition or infection or an increased risk of infection or complications. These interventions include the administration of medications such as antibiotics and/or pharmaceutical products and/or minimally invasive or invasive procedures such as surgical procedures and/or organ support measures such as the application of fluids such as Hartmann's solution, transfusion of blood or blood fragments, fluid bolus to maintain or restore fluid balance, circulation and metabolism, and/or support for respiration, ventilation (e.g., mechanical ventilation), and/or organ support measures such as hemodilution, blood ablation, renal replacement therapy such as dialysis, immunosuppressive drugs such as steroids, pain management, or isolation of the patient from other patients.

In the context of this invention, the term "infection" refers to the invasion of an organism's body tissues by pathogenic agents such as bacteria, viruses, fungi, or parasites, and the host tissue's response to the infectious agent and its toxins. Infectious diseases, also known as transmissible diseases or infectious diseases, are illnesses caused by infection. Such infectious diseases can preferably be treated by administering antibiotics.

The term “infection” as used in vi-xiii includes the description of the above explanation, and especially for cases with PCT below 0.1 ng/mL, early stages of infection, early stages of bacterial infection, non-bacterial infection, or other non-infectious diseases or conditions, which can be assessed as serious by combined measurements of proADM or fragments thereof, particularly equal to or greater than 0.88 nmol/L, 1.28 nmol/L, 1.5 nmol/L, or 2.75 nmol/L.

In a preferred embodiment, the present invention relates to an infection caused by bacteria, i.e., a bacterial infection. The ailment and/or symptoms vary depending on the type of bacteria and the location of the bacterial infection. These ailments and/or symptoms often overlap with other illnesses or conditions and may be selected from the group comprising: headache, pain in a specific part of the body (e.g., abdomen), temperature dysregulation (>38°C (fever), >36°C), respiratory symptoms selected from the group comprising cough, sputum production, dyspnea, shortness of breath, and pleural pain; a finding during auscultation (e.g., rales, crackles) and a sign of infection (core temperature >38°C, shivering) and a sign of gastrointestinal infection (nausea, vomiting, diarrhea), urinary problems, circulatory disturbances such as rapid pulse, blood pressure disturbances, age, compromised immune system, and other signs or risk factors for infection.

Typical symptoms of infection include localized redness, heat, swelling, and pain. One of the hallmarks of bacterial infection is localized pain, that is, pain in a specific part of the body. For example, if there is a cut and it becomes infected with bacteria, pain will occur at the site of infection. Bacterial sore throat is often characterized by pain that worsens on one side of the throat. If the pain only occurs in one ear, an ear infection is more likely to be a bacterial infection.

As mentioned above, symptoms of bacterial infections vary depending on the type of infection. Symptoms can also vary depending on the area of infection. However, symptoms will always appear even if the area is only mildly infected. When a bacterial infection is found in the respiratory tract, symptoms related to the throat and breathing are observed. Throat infections are very common in people living in highly polluted areas. Pneumonia is common in children and the elderly because their natural immunity is often weakened. Bronchitis, sinusitis, and pharyngitis have also been found in people with bacterial infections. When a bacterial infection is in the respiratory tract, colored nasal discharge and headache are common symptoms.

When an infection is found in the digestive tract (such as gastroenteritis), the symptoms are mostly related to digestive problems. Stomach inflammation and pain are common. Diarrhea and vomiting are other symptoms indicating gastrointestinal infections. Severe bacterial infections can also cause nausea, metabolic imbalances such as excessive stomach acid, and dehydration.

A foul or fishy odor in the vaginal area is a symptom of a vaginal infection. A woman's vagina contains several types of bacteria that are beneficial to the organs. However, if the production of these bacteria is irregular, it can lead to infection. Symptoms of bacterial urinary tract infections include itching and pain in the urinary tract. Vaginal and urinary tract infections should not be ignored because they can lead to further inflammation of internal organs such as the kidneys.

Meningitis is a serious consequence of bacterial infection of the membranes that cover the brain and spinal cord. Although it can also occur in adults, infants are more susceptible to this problem. Common symptoms of bacterial meningitis include stiffness in the body and neck, headache, irritability, fever or below-normal body temperature, and a rash.

Erysipelas is an acute bacterial infection of the dermis, causing inflammation. Patients typically develop symptoms within 48 hours of initial infection, including high fever, chills, headache, and vomiting. Erythematous skin lesions rapidly expand and have well-defined, raised edges. It presents as a red, swollen, hot, hardened, and painful rash, resembling orange peel in firmness. More severe infections may cause vesicles, bullae, and ecchymoses, and may lead to skin necrosis. Lymph nodes may swell and lymphedema may occur. Occasionally, red streaks extending to the lymph nodes may be seen. The infection can occur anywhere on the skin, including the face, arms, fingers, legs, and toes, but it tends to favor the extremities. Adipose tissue is most susceptible to infection, and it is typically present in the facial areas around the eyes, ears, and cheeks.

Peritonitis is an inflammation of the peritoneum, the serous membrane that surrounds the abdominal cavity and visceral parts. Peritonitis can be localized or widespread and can be caused by infection (often by rupture of a hollow organ, as can occur in abdominal trauma or appendicitis). The main manifestations of peritonitis are acute abdominal pain, abdominal tenderness, and abdominal wall tension. The location of these manifestations depends on whether the peritonitis is localized (e.g., pre-perforated appendicitis or diverticulitis) or widespread throughout the abdomen. In either case, the pain typically begins as a widespread abdominal pain (involving localized poor innervation of the visceral peritoneum) and may later become localized (involving the peritoneum innervated by somatic nerves). Perforation of a portion of the gastrointestinal tract and peritoneal rupture are the most common causes of infectious peritonitis.

Cholangitis is an inflammation of the bile ducts. The most common cause is bacterial infection. The typical triad of symptoms in cholangitis is fever, jaundice, and right upper quadrant pain.

Cholecystitis is an inflammation of the gallbladder and usually presents as pain in the right upper quadrant of the abdomen. It is often accompanied by a low-grade fever, vomiting, and nausea.

Osteomyelitis is an infection of the bone or bone marrow. Generally, microorganisms can infect bone through one or more of three basic methods: via the bloodstream, adjacent to a localized area of infection, or through penetrating trauma, including iatrogenic causes such as internal fixation of a joint replacement or fracture, or teeth after root canal filling. Signs and symptoms of osteomyelitis include fever, pain in the infected area, swelling, heat, and redness in the infected area.

Thus, the most dangerous bacterial infection leads to sepsis, a severe condition that causes organ dysfunction and death. Fever and severe chills are symptoms of bacterial infection in sepsis. Patients with sepsis also experience joint pain. This requires immediate treatment to stop the infection from spreading to internal organs and to prevent or overcome a cytokine storm and its fatal consequences. In cases of sepsis, patients will be hospitalized for intensive care. The term sepsis can alternatively be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. For clinical procedures, organ dysfunction is preferably indicated by a Sequential Organ Failure Assessment (SOFA) score increase of 2 points or more, which is associated with an in-hospital mortality rate greater than 10%. Septic shock can be defined as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities result in a higher risk of death compared to sepsis alone. In the absence of hypovolemia, patients with septic shock can be clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mmHg or higher and serum lactate levels greater than 2 mmol/L (>18 mg/dL). Severe sepsis is defined as sepsis associated with organ dysfunction, hypoperfusion abnormalities, or sepsis-induced hypotension. Hypoperfusion abnormalities include lactic acidosis, oliguria, and acute changes in mental status. Sepsis-induced hypotension is defined as a systolic blood pressure below approximately 90 mmHg or a decrease of approximately 40 mmHg from baseline in the absence of other causes of hypotension (e.g., cardiogenic shock). Septic shock is defined as severe sepsis in which septic hypotension persists despite adequate fluid resuscitation, along with hypoperfusion abnormalities or organ dysfunction (Bone et al., CHEST 101(6):1644-55, 1992). The term “sepsis” as used herein encompasses all possible stages of sepsis development.

Therefore, the severity of infection is related to symptoms, disease, and adverse events or outcomes such as patient death, organ dysfunction, or other life-threatening conditions. In addition to PCT and pro-ADM, the methods and kits of the present invention may also include the determination of at least one other biomarker, biomarker, clinical score, and/or parameter.

As used in this article, parameters are characteristics, features, or measurable factors that help define a particular system. Parameters are important elements used in health and physiological-related assessments such as disease/symptom/clinical condition risk and preferred organ dysfunction. Furthermore, parameters are defined as characteristics that are objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Exemplary parameters can be selected from the following groups: Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplified Acute Physiology Scale (SAPS II score), Rapid Sequential Organ Failure Assessment (qSOFA) score, Sequential Organ Failure Assessment (SOFA score), body mass index, weight, age, sex, IGS II, fluid intake, white blood cell count (especially neutrophils, segmented neutrophils, eosinophils, basophils, monocytes, lymphocytes), red blood cell count, platelet count, hemoglobin, hematocrit, sodium, potassium, body temperature, blood pressure, dopamine, bilirubin, respiratory rate, partial pressure of oxygen, World Federation of Neurosurgical Societies (WFNS) classification, and Glasgow Coma Scale (GCS).

As used herein, terms such as “marker,” “surrogate,” “prognostic marker,” “factor,” or “biomarker” or “biological marker” are used interchangeably and refer to measurable and quantifiable biomarkers (e.g., a specific concentration of a protein or enzyme or a fragment thereof, a specific concentration of a hormone or a fragment thereof, or the presence of a biological substance or a fragment thereof) used as indicators of health and physiological assessments such as risk of disease/symptom/clinical condition, preferred adverse events. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. Biomarkers can be measured in samples (e.g., blood, plasma, urine, or tissue tests).

At least one other biomarker and/or parameter of the subject may be selected from the group consisting of: lactate level, CRP, the subject's Sequential Organ Failure Assessment score (SOFA score), the subject's Simplified Acute Physiology Score (SAPSII), the subject's Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and the levels of the following substances: soluble fms-like tyrosine kinase-1 (sFlt-1), histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelin-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP), neutrophil gelatinase-associated lipocalcin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic lithoprotein (PSP), trigger receptor 1 (TREM1) expressed on myeloid cells, interleukin-6 (IL-6), interleukin-1, and interleukin-24 (IL-24). Interleukin-22 (IL-22), interleukin (IL-20), other ILs, Presepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), α-1-antitrypsin, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 2 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP7), placental growth factor (PlGF), chromogranin A, S100A protein, S100B protein and tumor necrosis factor α (TNFα), neopterin, proarginine vasopressin (AVP, proAVP or Copeptin), atrial natriuretic peptide (ANP, pro-ANP), E-selectin, ICAM-1, VCAM-1, IP-10, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, C CL18, CCL19, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CC L3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXC L12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2/MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7/Pp bp, CXCL9, IL8/CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, CLCF1, CNTF, IL1 1. IL31, IL6, ICAM, leptin, LIF, OSM, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5/IFNaG, IFNω/IFNW1, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L/CD178, EDA-A1, TNFSF14, LTA/TNFB, LTB, TNFa, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33 or fragments thereof.

Lactate, or lactic acid, is an organic compound with the chemical formula _CH3CH (OH)COOH, present in body fluids, including blood. Blood lactate testing is performed to determine the body's acid-base homeostasis. Lactate is a product of cellular metabolism and accumulates when cells lack sufficient oxygen (hypoxia) and must convert to less efficient energy production methods, or when conditions lead to excessive lactate production or impaired clearance. Lactic acidosis can be caused by insufficient oxygen in cells and tissues (hypoxia). For example, if someone has conditions that may reduce the amount of oxygen delivered to cells and tissues, such as shock, septic shock, or congestive heart failure, lactate testing can help detect and assess the severity of hypoxia and lactic acidosis.

C-reactive protein (CRP) is a pentamer that can be found in body fluids, such as blood plasma. CRP levels can rise in response to inflammation. Measuring and mapping CRP values can prove useful in determining disease progression or treatment effectiveness.

As used herein, the Sequential Organ Failure Assessment (SOFA) score is a scoring system used to track a patient's condition during their stay in the Intensive Care Unit (ICU). The SOFA score is a scoring system used to determine the extent of an individual's organ function or rate of failure. The score is based on six different scores, each specifically for the respiratory, cardiovascular, hepatic, coagulation, renal, and nervous systems. Both the average and highest SOFA scores are predictors of outcomes. During the initial 24 to 48 hours in the ICU, an increase in the SOFA score predicts mortality of at least 50% to as high as 95%. A score below 9 predicts mortality of 33%, while a score above 14 may be close to or higher than 95%.

As used in this article, the rapid SOFA score (qSOFA) is a scoring system that indicates a patient's risk of organ dysfunction or death. The score is based on three criteria: 1) altered mental status; 2) a decrease in systolic blood pressure of less than 100 mmHg; and 3) a respiratory rate greater than 22 breaths per minute. Patients with two or more of these conditions have a greater risk of organ dysfunction or death.

As used in this article, “APACHE II” or “Acute Physiology and Chronic Health Evaluation II” is a disease severity classification scoring system (Knaus et al., 1985). It can be applied within 24 hours of a patient’s admission to the intensive care unit (ICU) and can be determined based on 12 different physiological parameters: AaDO2 or PaO2 (depending on FiO2), temperature (rectal), mean arterial pressure, pH arterial, heart rate, respiratory rate, sodium (serum), potassium (serum), creatinine, hematocrit, white blood cell count, and Glasgow Coma Scale.

As used herein, “SAPS II” or “Simplified Acute Physiology Score II” refers to a system for classifying the severity of a disease or condition (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 score consists of 12 physiological variables and 3 disease-related variables. Scores are calculated from 12 routine physiological measurements, information about prior health conditions, and some information obtained upon ICU admission. The SAPS II score can be determined at any time, preferably on day 2. The “worst” measurement is defined as the measure associated with the highest score. SAPS II scores range from 0 to 163. The classification system included the following parameters: age, heart rate, systolic blood pressure, body temperature, Glasgow Coma Scale, mechanical ventilation or CPAP, PaO2, FiO2, urine output, blood urea nitrogen, sodium, potassium, bicarbonate, bilirubin, white blood cell count, chronic disease, and admission type. A sigmoid relationship was observed between mortality and the total SAPS II score. At an SAPS II score of 29, the mortality rate was 10%; at 40, 25%; at 52, 50%; at 64, 75%; and at 77, 90% (LeGall loc.cit.).

As used herein, the term "comparison" in relation to the use of PCT and/or proADM or fragments thereof as biomarkers refers to comparing the presence or amount of a biomarker in a patient with the presence or amount of a biomarker in a person known to have a given condition or known to be at risk for a given condition. Biomarker levels in a patient sample can be compared with levels known to be associated with a specific prognosis. Biomarker levels in a sample are considered prognostic; that is, technicians can use biomarker levels to determine whether a patient has a specific risk of experiencing an adverse event and respond accordingly. Alternatively, biomarker levels in a sample can be compared with levels of biomarkers known to be associated with good outcomes (e.g., a lower risk of experiencing an adverse event).

As used herein, the term "sample" refers to a bodily fluid sample obtained for the purpose of assessing, diagnosing, prognosing, or evaluating a target subject, or for guiding treatment, such as a patient. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. Furthermore, those skilled in the art will recognize that some test samples become easier to analyze after fractionation or purification procedures, such as separating whole blood into serum or plasma components.

Therefore, in a preferred embodiment of the invention, the sample is selected from the group consisting of blood samples, serum samples, plasma samples, cerebrospinal fluid samples, saliva samples, and urine samples, or extracts of any of the above samples. Preferably, the sample is a blood sample, and most preferably a serum sample or a plasma sample.

In the context of this invention, the term "plasma" refers to a virtually cell-free supernatant of blood containing an anticoagulant, obtained after centrifugation. Exemplary anticoagulants include calcium ion-binding compounds such as EDTA or citrate, and thrombin inhibitors such as heparin or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrate, EDTA, or heparinized blood) at 2000 to 3000 g for, for example, at least 15 minutes.

In the context of this invention, the term "serum" refers to the liquid portion of whole blood collected after the blood has been clotted. Serum is obtained as a supernatant when the clotted blood (clotted blood) is centrifuged.

The term "urine" refers to the fluid product of the human body that is secreted by the kidneys through a process called urination (or urination) and excreted through the urethra.

In the context of this invention, the term "periodic remeasurement" refers to measurements taken over a repetitive time frame, such as every 6 hours, every 12 hours, very preferably every 24 hours, every 2 days, every 3 days, every 4 days, or preferably during a decision-making process, such as before changing hospital location, discharge, admission, or discontinuation or modification of treatment.

In the context of this invention, the term "severity assessment" refers to the diagnosis and monitoring of the progression of severity and the onset of associated infections, particularly the detection and labeling of infectious diseases at different stages due to their severity. Furthermore, the term "severity assessment" involves grouping subjects into different risk groups based on their further prognosis, particularly as provided in the defined case (ibid.). Thus, risk assessment also involves stratification for the application of preventive and/or therapeutic measures.

Therefore, in the context of this invention, the term "diagnosis" refers to the identification and detection of infection in a subject and/or the determination or exclusion of infection, as well as the associated severity, particularly severe infection.

The term "surveillance" involves tracking a diagnosed infection, related symptoms or complications or risks, such as analyzing disease progression or the effect of specific treatments on infection progression or related symptoms or complications or risks, and assessing treatment response. A positive response can shift biomarker values to the normal range and generally improve a patient's health, such as reducing pain, improving breathing, eliminating fever, etc., and indicate the need for appropriate treatment and/or changes, discontinuation or increase in medical interventions or forms of application (e.g., from intravenous to oral, or vice versa), changes in treatment procedures/medications or medical settings (from a normal workstation to the ICU, or vice versa, or discharge).

This invention also relates to kits, uses of the kits, and methods of using such kits. The invention relates to kits for performing the methods provided above and below. Definitions provided herein, such as those relating to the methods, also apply to the kits of this invention. In particular, the invention relates to kits for assessing, risk-stratifying, monitoring, guiding, and/or controlling infection or suspected infection in said subjects.

The sensitivity and specificity of a diagnostic test depend not only on the "quality" of the analysis but also on the definition of what constitutes an abnormal result. In practice, a receiver operating characteristic (ROC) curve is typically calculated by plotting the relative frequencies of variable values in the "normal" (i.e., seemingly healthy) and "diseased" (i.e., patients with an infection) groups. Depending on the specific diagnostic problem being addressed, the reference group does not necessarily have to be "normal" but can be a group of patients with another disease or symptom, which should be distinguished from the target disease group. For any given biomarker, the distribution of biomarker levels in subjects with and without the disease may overlap. Under these conditions, the test cannot absolutely distinguish between normal and disease with 100% accuracy, and the overlapping area represents where the test cannot distinguish between normal and disease. A threshold is chosen above which the test is considered abnormal (or below which, depending on how the biomarker changes with the disease), and below which it is considered normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow for the correct identification of the symptom. ROC curves can be used even when test results do not necessarily provide precise numbers. ROC curves can be generated whenever the results can be ranked. For example, test results for “disease” samples can be ranked according to severity (e.g., 1 = low, 2 = normal, and 3 = high). This ranking can be correlated with results in the “normal” population and produce ROC curves. These methods are well known in the art (see, for example, Hanley et al., 1982. Radiology 143:29-36). Preferably, a threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7. In this context, the term “about” means +/- 5% for a given measurement.

The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a specific cutoff value, the value of (1-specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured biomarker level will allow for the correct identification of a disease or symptom (e.g., prognosis). Therefore, the area under the ROC curve can be used to determine the validity of the test.

In some embodiments, markers and/or groups of markers are selected to exhibit at least about 70% sensitivity, more preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, even more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity, combined with at least about 70% specificity, more preferably at least about 80% specificity, even more preferably at least about 85% specificity, even more preferably at least about 90% specificity, and most preferably at least about 95% specificity. In particularly preferred embodiments, both sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%. In this context, the term "about" means +/- 5% for a given measurement result.

Threshold levels can be obtained, for example, from a Kaplan-Meier analysis, where the probability of disease occurrence or adverse outcome and/or death is associated with, for example, the quintiles of various biomarkers in the population. According to this analysis, subjects with biomarker levels above the 80th percentile had a significantly increased risk of adverse events according to the invention. Cox regression analysis adjusted for classical risk factors further supported this result. Compared with all other subjects, the highest quartile was significantly associated with an increased risk of disease or the probability of adverse outcome and/or death according to the invention.

Other preferred threshold values are, for example, the 90th, 95th, or 99th percentile of the reference population. Using a higher percentile than the 80th percentile can reduce the number of false positives identified, but may miss subjects who, while at moderate risk, still increase their risk. Therefore, the threshold value can be adjusted based on whether it is considered more suitable for identifying the majority of at-risk subjects at the cost of also identifying "false positives," or more suitable for primarily identifying high-risk subjects at the cost of missing some subjects at moderate risk.

Other mathematical probabilities for calculating individual risk using individual biomarker levels and other prognostic laboratory and clinical parameters include, for example, the NRI (Net Reclassification Index) or IDI (Integrated Discriminant Index). These indices can be calculated according to Pencina (Pencina MJ et al.: Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. Stat Med. 2008; 27:157-172).

Thus, the cutoff values for the mentioned PCT and pro-ADM and their fragments can vary by as much as 5%, 10%, 15%, 16%, and 17%, and are due to diagnostic methods and equipment as well as normal biological variations within the patient population. For example, the cutoff value of 0.88 nmol/L for the mentioned pro-ADM or its fragments has a 10% variation, ranging from 0.792 to 0.968 nmol/L, and shows no overlap with other mentioned cutoff values.

Preferred detection methods include various forms of immunoassays, such as mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence and fluorescence immunoassays, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), luminescent bead arrays, magnetic bead arrays, protein microarray assays, rapid test formats (e.g., immunochromatographic band assays), rare cavitation compound assays, and automated systems/analyzers (e.g., KRYPTOR assays).

The assay can be homogeneous or heterogeneous, competitive or non-competitive. In a particularly preferred embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified binds to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as the surface of beads, pores or other containers, chips or strips, and the second antibody is, for example, an antibody labeled with a dye, a radioactive isotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedure associated with the “sandwich assay” are well-established and known to those skilled in the art (The Immunoassay Handbook, ed. David Wild, Elsevier LTD, Oxford; 3rd edition (May 2005), ISBN-13:978-0080445267; Hultschig C et al., Curr Opin Chem Biol. February 2006; 10(1):4-10. PMID:16376134, incorporated herein by reference).

In a preferred embodiment, the assay comprises two capture molecules, preferably antibodies, both present in dispersion form in a liquid reaction mixture, wherein a first labeling component is attached to a first capture molecule, wherein the first labeling component is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second labeling component of the labeling system is attached to a second capture molecule so as to generate a measurable signal upon binding of the two capture molecules to the analyte, which allows detection of the formation of a sandwich complex in a solution containing the sample.

Even more preferably, the labeling system comprises a combination of rare earth cavitary compounds or rare earth chelates with fluorescent dyes or chemiluminescent dyes, particularly anthocyanin-type dyes.

In the context of this invention, fluorescence-based determinations involve the use of dyes, which may be selected, for example, from the group consisting of: FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700/800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xaton, 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE), N,N,N′,N′-tetramethyl-6-carboxyrhodane Rhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 5-carboxy-Rhodamine-6G (R6G5), 6-carboxy-Rhodamine-6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzoimides such as Hoechst 33258; phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polyacetylenes dyes, etc.

In the context of this invention, chemiluminescence-based determinations involve the use of dyes based on the physical principles described in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th Edition, Executive Editor J.I. Kroschwitz; Editor M. Howe-Grant, John Wiley & Sons, 1993, Vol. 15, pp. 518-562, concerning chemiluminescent materials, which is incorporated herein by reference, including citations to pp. 551-562. A preferred chemiluminescent dye is acrylamide ester.

As mentioned herein, a “assay” or “diagnostic assay” can be of any type used in the diagnostic field. Such an assay can be based on the binding of the analyte to be detected to one or more capture probes with specific affinities. Regarding the interaction between the capture molecule and the target molecule, the affinity constant is preferably greater than ^{10⁸ M⁻¹} .

In the context of this invention, a "capture molecule" is a molecule that can be used to bind a target molecule, i.e., an analyte from a sample (i.e., in the context of this invention, PCT and its fragments and/or pro-ADM or its fragments). Therefore, the capture molecule must be suitably shaped spatially and in terms of surface characteristics (e.g., surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and/or acceptors) to specifically bind the target molecule. Thus, binding can be mediated, for example, by ionic, van der Waals, π-π, δ-π, hydrophobic, or hydrogen bonding interactions, or combinations of two or more of these interactions, between the capture molecule and the target molecule. In the context of this invention, the capture molecule can be, for example, selected from the group consisting of nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides, or glycoproteins. Preferably, the capture molecule is an antibody, comprising a fragment having sufficient affinity for the target molecule, and includes recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibody or fragments derived from a variant chain of at least 12 amino acids in length.

Furthermore, the present invention relates to a computer-based tool for inputting and evaluating data, processing relevant workflow content or the decision tree or matrix, and outputting measurements and management recommendations, for example, to support the management of clinical decisions by using artificial intelligence and/or including machine learning, wherein reference data is stored on a computer-readable medium and/or used in the form of computer-executable code configured to compare the measured levels of PCT or fragments thereof and/or the measured levels of proADM or fragments thereof with the reference data, and wherein the reference data refers to a threshold level as indicated above in the embodiments disclosed in this invention.

Examples and Figures 3-7

The invention is further described with reference to the following embodiments and accompanying drawings. These embodiments and drawings are not intended to limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention provided to better illustrate the invention described herein.

patient:

The patients mentioned had physiological signs or increased risk factors for infection upon admission to the ED. Blood samples were collected upon admission to the ED (time point 0). Plasma (for MR-proADM) and serum (for PCT) concentrations were measured using TRACE (time-resolved amplified cavitation compound emission) technology combined with a novel sandwich immunoassay (Kryptor Compact Plus analyzer, BRAHMS, Hennigsdorf, Germany). The endpoints of the study were, in particular, treatment with antibiotics and 28-day mortality.

Statistical analysis:

Differences in clinical characteristics regarding 28-day mortality were assessed using the ^χ² test for categorical variables and the Student t-test or Mann-Whitney U test for continuous variables, depending on the normality of the distribution. Normally and non-normally distributed variables were expressed as mean (standard deviation) and median [first quartile - third quartile], respectively. Associations between antibiotic guidance, disease status (especially infection) severity, and 28-day mortality prediction with other clinical parameters and scores for each biomarker were assessed using, for example, area under the recipient operating characteristic curve (AUROC), logistic, and Cox regression analyses. Logistic regression models were created individually using the biomarker or score, or adjusted for variables such as mortality or treatment, and expressed as, for example, odds ratio (OR) and 95% confidence interval [95% CI]. A two-sided p < 0.05 was considered statistically significant. All data were analyzed using recognized statistical software. Software capable of performing such analyses to establish appropriate reference levels and critical values, such as JMP 12, JMP 13, and Statistical Discovery from SAS, can be used.

Group:

Statistical calculation is based on

In Group 1, which involves (iv)

PCT < 0.1 and MR-proADM < 0.88

N = 129

Hospitalization/Admission Rate: 33.1%

Antibiotic application rate: 65.3%

Intravenous antibiotic administration rate: 22.6%

Oral antibiotic usage rate: 41.9%

28-day mortality rate: 0.0%

In the second group involving (v)

PCT < 0.1 and MR-proADM ≥ 0.88

N = 68

Hospitalization/Admission Rate: 83.8%

Antibiotic application rate: 67.7%

Intravenous antibiotic administration rate: 46.2%

Oral antibiotic usage rate: 23.1%

28-day mortality rate: 8.9%

In group 3 involving (v)

PCT < 0.1 and MR-proADM ≥ 0.88 and < 1.28

N = 29

Hospitalization/Admission Rate: 82.5%

Antibiotic application rate: 60.0%

Intravenous antibiotic administration rate: 35.9%

Oral antibiotic administration rate: 20.5%

28-day mortality rate: 5.0%

In group 1b involving (vii)

PCT ≥ 0.1 and MR-proADM < 0.88

N = 106

Hospitalization/Admission Rate: 61.3%

Antibiotic application rate: 87.4%

Intravenous antibiotic administration rate: 32.6%

Oral antibiotic usage rate: 38.9%

28-day mortality rate: 0.0%

Group 2b, referring to (viii):

PCT < 0.1 and MR-proADM ≥ 0.88

N = 419

Hospitalization/Admission Rate: 96.2%

Antibiotic application rate: 92.8%

Intravenous antibiotic administration rate: 81.6%

Oral antibiotic usage rate: 10.4%

28-day mortality rate: 16.0%

Group 3b, referring to (viii):

PCT ≥ 0.1 and MR-proADM ≥ 0.88 and < 1.28

N = 99

Hospitalization/Admission Rate: 90.0%

Antibiotic application rate: 84.3%

Intravenous antibiotic administration rate: 55.1%

Oral antibiotic usage rate: 29.2%

28-day mortality rate: 0.0%

Group 4: PCT ≥ 0.1 and MR-proADM ≥ 1.50

N = 262

Hospitalization rate: 97.3%

Antibiotic application rate: 96.0%

Intravenous antibiotic administration rate: 92.2%

Oral antibiotic usage rate: 4.1%

28-day mortality rate: 24.7%

Group 5: PCT ≥ 0.1 and MR-proADM ≥ 2.75

N = 102

Hospitalization rate: 98.6%

Antibiotic application rate: 98.1%

Intravenous antibiotic administration rate: 95.9%

Oral antibiotic administration rate: 2.0%

28-day mortality rate: 34.0%

RRT demand: 15.0%

Oxygen supplementation: 61.6%

Group 4: PCT < 0.1 and MR-proADM ≥ 1.50

N=18

Hospitalization rate: 94.4%

Antibiotic application rate: 88.9%

Intravenous antibiotic administration rate: 72.2%

Oral antibiotic usage rate: 22.2%

28-day mortality rate: 22.2%

Group 5: PCT < 0.1 and MR-proADM ≥ 1.50

N=4

Figure 3 shows the Kaplan-Meier curves using an optimized MR-proADM critical value of 0.88 nmol/L, which demonstrates the complete exclusion of 28-day mortality.

Figure 4 shows the ROC analysis of ADM (black) and PCT (grey) in patients entering the ED to predict 28-day mortality.

Table 1: Area under the curve (AUC and 95% confidence interval (CI)), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), and diagnostic odds ratio (OR) of MR-proADM and PCT in patients at ED for 28-day mortality.

Table 1 shows that the critical value of MR-proADM is 1.5 nmol/L, the AUC is 0.84 (0.78-0.89), the sensitivity is 0.8 [0.61-0.92], the specificity is 0.79 [0.75-0.82], the PPV is 0.15 [0.12-0.18], the NPV is 0.99 [0.98-0.99], the LR+ is 3.74 [2.96-4.72], the LR- is 0.25 [0.12-0.52], and the OR is 14.7 [5.9-36.7]. Table 1 shows that the PCT cutoff value was 2.0 ng/ml, the AUC was 0.68 (0.59-0.77), the sensitivity was 0.8 [0.61-0.92], the specificity was 0.49 [0.45-0.53], the PPV was 0.07 [0.06-0.08], the NPV was 0.98 [0.96-0.99], the LR+ was 1.58 [1.30-1.91], the LR- was 0.41 [0.2-0.83], and the OR was 3.9 [1.6-9.6].

Figure 5 shows the ROC analysis of ADM (black) and PCT (grey) upon entry into ED to identify patients with an increased level of disease severity, as determined by existing organ function (SOFA equal to or greater than 2 points).

Table 2 : Area under the curve (AUC and 95% confidence interval (CI)), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), and odds ratio (OR) of MR-proADM and PCT in patients with increased disease severity upon entering ED.

Table 2 shows that the critical value of MR-proADM is 1.28 nmol/L, the AUC is 0.78 (0.69-0.87), the sensitivity is 0.87 [0.66-0.97], the specificity is 0.6 [0.56-0.64], the PPV is 0.07 [0.06-0.09], the NPV is 0.99 [0.98-1.00], the LR+ is 2.18 [1.82-2.63], the LR- is 0.22 [0.08-0.62], and the OR is 10.1 [2.0-34.3]. Table 2 shows that the PCT cutoff value was 0.47 ng/ml, the AUC was 0.71 (0.61-0.82), the sensitivity was 0.7 [0.47-0.87], the specificity was 0.66 [0.63-0.70], the PPV was 0.07 [0.05-0.08], the NPV was 0.98 [0.97-0.99], the LR+ was 2.07 [1.55-2.77], the LR- was 0.46 [0.25-0.85], and the OR was 4.5 [1.8-11.1].

Figure 6 shows the ROC analysis of ADM (black) and PCT (grey) in patients who require antibiotic administration after entering the ED.

Table 3 : Area under the curve (AUC and 95% confidence interval (CI)), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), and odds ratio (OR) of MR-proADM and PCT in patients who require antibiotic administration upon entry into ED.

Table 3 shows that the critical value of MR-proADM is 0.88 nmol/L, the AUC is 0.77 (0.72-0.81), the sensitivity is 0.78 [0.74-0.82], the specificity is 0.64 [0.56-0.71], the PPV is 0.86 [0.84-0.88], the NPV is 0.50 [0.45-0.56], the LR+ is 2.16 [1.76-2.65], the LR- is 0.34 [0.28-0.42], and the OR is 6.4 [4.4-9.3]. Table 3 shows that the PCT cutoff value was 0.1 ng/ml, the AUC was 0.73 (0.69-0.78), the sensitivity was 0.85 [0.81-0.88], the specificity was 0.50 [0.42-0.57], the PPV was 0.83 [0.81-0.85], the NPV was 0.53 [0.47-0.60], the LR+ was 1.69 [1.45-1.97], the LR- was 0.30 [0.24-0.39], and the OR was 5.6 [3.8-8.2].

Figure 7 shows the ROC analysis of ADM (black) and PCT (grey) among patients who required hospitalization after entering the ED.

Table 4 : Area under the curve (AUC and 95% confidence interval (CI)), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), and odds ratio (OR) of MR-proADM and PCT in patients who decided to be hospitalized upon admission to the ED.

Table 4 shows that the critical value of MR-proADM is 0.88 nmol/L, the AUC is 0.78 (0.74-0.82), the sensitivity is 0.79 [0.76-0.83], the specificity is 0.65 [0.57-0.72], the PPV is 0.86 [0.83-0.88], the NPV is 0.54 [0.49-0.59], the LR+ is 2.26 [1.84-2.77], the LR- is 0.32 [0.26-0.39], and the OR is 7.1 [4.9-10.4]. Table 4 shows that the PCT cutoff value was 0.19 ng/ml, the AUC was 0.73 (0.69-0.77), the sensitivity was 0.63 [0.59-0.67], the specificity was 0.73 [0.66-0.79], the PPV was 0.86 [0.83-0.89], the NPV was 0.42 [0.39-0.46], the LR+ was 2.31 [1.8-2.95], the LR- was 0.51 [0.44-0.59], and the OR was 4.5 [3.1-6.6].

The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention beyond those described herein will become apparent to those skilled in the art, based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all values are approximate and provided for descriptive purposes.

sequence

SEQ ID NO:1 (Amino acid sequence of PCT):

SEQ ID NO:2 (Amino acid sequence of MR-pro-ADM):

SEQ ID NO:3 (amino acid sequence of proADM)

SEQ ID NO:4 (Amino acid sequence of pre-proADM)

sequence list

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<120> Workflow for risk assessment and patient management using procalcitonin and mid-zone pro-adrenergic medullaris

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<141> 2017-12-20

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Claims (17)

1. Use of at least two capture molecules in the preparation of a kit for assessing the severity of infection in a subject, said capture molecules being used to: (a) determine the level of procalcitonin (PCT), and (b) determine the level of mid-zone pro-adrenal medullaris (MR-proADM). The kit is configured to compare the measured levels of (a) PCT and (b) MR-proADM with thresholds for (a) PCT and (b) MR-proADM contained in the kit. The threshold value is selected from the values of features (iv) to (xiii) below. The kit described therein is made as follows: (i) Measure PCT levels in bodily fluid samples from subjects, and (ii) Measure the level of MR-proADM in bodily fluid samples from the subjects, and (iii) The severity of infection in subjects suspected of or having an infection is determined by comparing the measured PCT and MR-proADM levels with threshold levels. (iv) Where a PCT threshold level below 0.1 ng/ml and an MR-proADM threshold level below 0.88 nmol/L indicate that the subject has a non-serious infection. and/or (v) Where a PCT threshold level below 0.1 ng/ml and an MR-proADM threshold level equal to or higher than 0.88 nmol/L and not equal to or higher than 1.28 nmol/L indicate that the subject has a nearly non-serious infection. and/or (vi) Where a PCT threshold level below 0.1 ng/ml and an MR-proADM threshold level equal to or above 1.28 nmol/L indicate that the subject has a low level of severe infection. and/or (vii) Where a PCT level equal to or higher than the threshold level of 0.1 ng/ml and an MR-proADM level lower than the threshold level of 0.88 nmol/L indicate that the subject has an extremely low level of severe infection. and/or (viii) Where a PCT level equal to or higher than a threshold level of 0.1 ng/ml and an MR-proADM level equal to or higher than a threshold level of 0.88 nmol/L and not equal to or higher than a threshold level of 1.28 nmol/L indicate that the subject has a low level of severe infection. and/or (ix) Where a PCT level equal to or higher than a threshold level of 0.1 ng/ml and an MR-proADM level equal to or higher than a threshold level of 1.28 nmol/L indicate that the subject has a serious infection. and/or (x) Where a PCT threshold level below 0.1 ng/ml and an MR-proADM threshold level equal to or above 1.50 nmol/L indicate that the subject has a severe infection. and/or (xi) Where a PCT threshold level below 0.1 ng/ml and an MR-proADM threshold level equal to or above 2.75 nmol/L indicate that the subject has a serious infection. and/or (xii) Where a PCT level equal to or higher than a threshold level of 0.1 ng/ml and an MR-proADM level equal to or higher than a threshold level of 1.50 nmol/L indicate that the subject has a high degree of severe infection. and/or (xiii) Where PCT equal to or higher than the threshold level of 0.1 ng/ml and MR-proADM equal to or higher than the threshold level of 2.75 nmol/L indicate that the subject has an extremely high level of severe infection. 2. The use according to claim 1, wherein the measured PCT level and MR-proADM level are compared with the PCT and MR-proADM threshold levels of at least two of the features (iv) to (xiii). 3. The use according to claim 1, wherein the measured PCT level and MR-proADM level are compared with the PCT and MR-proADM threshold levels of at least one of features (v), (vi), (x), and (xi). 4. The use according to claim 1, wherein a threshold level of MR-proADM equal to or higher than 1.28 nmol/L indicates that one or more of the following measures i)–iv) are applicable: i) Blood culture tests from the central venous line and/or from different sources and samples. ii) Administer antibiotics orally or intravenously. iii) If i) the test result is negative and PCT is below 0.1 ng/ml, discontinue antibiotic administration, or iv) Monitor blood test results. 5. The use according to claim 1, wherein If (iv) applies, antibiotics are not used on the subjects. If (v) applies, antibiotics should not be used on the subject, and for clarification or to examine the condition, the subject's sample should be further tested for PCT within at least 6–12 hours or 24 hours. If (vi) applies, antibiotics shall be administered to the subject. If (vii) applies, it is possible that antibiotics will not be used on the subjects. If (viii) applies, antibiotics may be administered to the subject, and for clarification or to assess the condition, PCT should be further measured on the subject's sample within at least 6–12 hours or 24 hours. If any of (ix), (x), (xi), (xii), or (xiii) applies, the subject shall be given antibiotics immediately. 6. The use according to claim 1, wherein If any of (v), (vi), (viii), (ix), (x), (xi), (xii), or (xiii) applies, then the subject shall be hospitalized. and/or If (iv) or (vii) applies, the subject shall be discharged. 7. Use of the assay reagent for determining the level of mid-zone pro-adrenal medullaris (MR-proADM) in the preparation of a kit for assessing the severity of infection in subjects. The kit is designed to compare the measured level of MR-proADM with a threshold contained in the kit. The threshold value is selected from the value of feature (ii) below. The kit described therein is made as follows: (i) Determining MR-proADM levels independently of PCT levels in body fluid samples from subjects, and (ii) The severity of infection in a subject is determined by comparing the measured MR-proADM level with a threshold level. a) Where the threshold level of MR-proADM equal to or higher than 0.88 nmol/L refers to the clinical decision-making process for assessing the subject's hospital admission, or b) The threshold level for MR-proADM at or above 1.50 nmol/L refers to the identification of high-risk infected subjects with an approximate mortality rate of at least 25%, or c) The threshold level for MR-proADM at or above 2.75 nmol/L refers to the identification of subjects at extremely high risk of infection with an approximate mortality rate of at least 35%, or d) The threshold level for MR-proADM, which is equal to or higher than 2.75 nmol/L, refers to the clinical decision-making process for assessing a subject's admission to the ICU. 8. The use according to claim 1 or 7, wherein the kit comprises a capture molecule for determining the level of MR-proADM. 9. The use according to claim 1 or 7, wherein a threshold level of MR-proADM equal to or higher than 1.50 nmol/L indicates that one or more of the following measures i)–vi) are applicable: i) Provide oxygen through a mask. ii) Perform fluid bolus therapy, iii) Blood culture tests from the central venous line and/or from different sources and samples. iv) Administer antibiotics orally or intravenously. v) Monitor blood test results, vi) Monitor urine output. or Where MR-proADM is equal to or higher than the threshold level of 2.75 nmol/L, the following measures vii) should be applied additionally: vii) Monitor organ dysfunction, including checking clinical parameters and other life-sustaining measures. 10. The use according to claim 1 or 7, wherein the body fluid sample is selected from the group consisting of blood samples, serum samples and plasma samples. 11. The use according to claim 1 or 7, wherein the subject has a bacterial infection, or wherein the subject has a suspected infection, or wherein the subject is at increased risk of infection, or wherein the subject is a discharged patient, or wherein the subject is present in a clinical setting. 12. The use according to claim 11, wherein the subject is present in primary care or an ambulance. 13. The use according to claim 1 or 7, wherein the kit is prepared to measure at least one other biomarker, clinical score and/or parameter in addition to PCT and MR-proADM. 14. The use according to claim 13, wherein the marker is a biomarker. 15. The use according to claim 13, wherein the other parameters are selected from the group consisting of: Acute Physiology and Chronic Health Evaluation II, Simplified Acute Physiology Scale, Rapid Sequential Organ Failure Assessment Scale, Sequential Organ Failure Assessment Scale, Body Mass Index, weight, age, sex, IGSII, fluid intake, white blood cell count, red blood cell count, platelet count, hemoglobin, hematocrit, sodium, potassium, body temperature, blood pressure, dopamine, bilirubin, respiratory rate, partial pressure of oxygen, World Federation of Neurosurgical Societies classification and Glasgow Coma Scale. 16. The use according to claim 13, wherein the other parameters are cell counts of neutrophils, segmented neutrophils, eosinophils, basophils, monocytes, or lymphocytes. 17. The use according to claim 13, wherein the other biomarkers and/or parameters are selected from the group consisting of: lactate levels, the subject's Sequential Organ Failure Assessment score, the subject's Simplified Acute Physiology Score, the subject's Acute Physiology and Chronic Health Evaluation II score, and levels of the following substances: soluble fms-like tyrosine kinase-1, histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelin-1, neutrophil gelatinase-associated lipocalcin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic stone protein (PSP), trigger receptor 1 expressed on myeloid cells, interleukin-6, interleukin-1, interleukin-24, interleukin-22, interleukin-20, presepsin, lipopolysaccharide-binding protein, α-1-antitrypsin, and matrix gold. The following proteins are listed: protease 2, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 7, placental growth factor, chromogranin A, S100A protein, S100B protein, tumor necrosis factor α, neopterin, arginine vasopressin AVP, proAVP or Copeptin, atrial natriuretic peptide ANP, pro-ANP, E-selectin, ICAM-1, VCAM-1, IP-10, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, and CC. L28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2/MIP-2, CXCL 3. CXCL4, CXCL5, CXCL6, CXCL7/Ppbp, CXCL9, IL8/CXCL8, XCL1, XCL2, FAM19A1, FAM19A2 , FAM19A3, FAM19A4, FAM19A5, CLCF1, CNTF, IL11, IL31, ICAM, leptin, LIF, OSM, IFNA1, IFNA1 0. IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5/IFNaG, IFNω/IFNW1, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L/CD178, EDA-A1, TNFSF14, LTA /TNFB, LTB, TNFa, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33.