RU2788885C2 - Procalcitonin and pro-adm as markers for monitoring of treatment with antibiotics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to a method for control of the treatment with antibiotics in a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, including: taking of the first sample in the specified patient, taking of the second sample in the specified patient in a certain period of time after taking of the first sample and after the beginning of the treatment with antibiotics; determination of levels of procalcitonin (hereinafter – PCT) or its fragment(s) in the first and the second samples, and determination of a level of pro-adrenomedullin (hereinafter – proADM) or its fragment(s) in at least the second sample. Moreover, a lower level of PCT or its fragment(s) in the second sample compared to the first sample and a level of high severity of proADM or its fragment(s) in the second sample, or an increased level of severity of proADM or its fragment(s) in the second sample compared to the first sample, including an increase from a level of low severity to a level of moderate or high severity, or from a level of moderate severity to a level of high severity, indicate the necessity of change in one or more antibiotic agents, while the level of low severity of proADM or its fragment(s) is less than 4 nmol/l, the level of moderate severity of proADM or its fragment(s) is more than 4 nmol/l and less than 6.5 nmol/l, and the level of high severity of proADM or its fragment(s) is more than 6.5 nmol/l. In addition, the present invention also relates to a kit for implementation of the method according to the present invention.

EFFECT: invention provides a method and a kit for control of the treatment with antibiotics in a patient suffering from an infectious disease and receiving the treatment with one or more antibiotic agents.

15 cl, 32 tbl

Description

This invention relates to a method of antibiotic treatment management, stratification and/or control in a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents. In particular, said method includes taking a first sample from said patient, taking a second sample from said patient after taking the first sample and initiating antibiotic treatment, determining the levels of procalcitonin (PCT) or its fragment(s) in the first and second sample, and determining levels of proADM or fragment(s) thereof in at least a second sample, wherein the levels of PCT or fragment(s) thereof in said first and second samples and the level of proADM or fragment(s) in the second sample indicate a need to change treatment one or more antibiotic agents. In a preferred embodiment of the invention, the method includes further determining the level of proADM or its fragment(s) in the first and second samples. In a preferred embodiment of the invention, changes in proADM and PCT levels between the first and second samples indicate a need to change or extend antibiotic treatment. In addition, this invention also relates to a kit for carrying out the method according to this invention.

BACKGROUND OF THE INVENTION

Despite significant improvements in diagnostic and preventive measures, the incidence of sepsis continues to increase rapidly in hospitalized patients (1), with mortality rates ranging from 10% to 54%, depending on the severity of the disease, the definition of organ dysfunction used, and country-specific incidence. (2, 3). Therefore, early and accurate assessment of both infection load and disease severity in terms of the host's overall pathophysiological response is critical in the early stages of sepsis in order to make quick and correct decisions regarding diagnostic testing and treatment strategies, as well as at a later stage. to properly manage patient management, treatment monitoring, and discharge decisions in the presence of clinical recovery.

It is therefore surprising that there is currently no gold standard for diagnosing sepsis (4). The use of procalcitonin (PCT) has partly covered this unmet need for infection load assessment through observational data and antibiotic prescribing interventions (5–7). However, the exact indicator of the severity of the disease has not yet been identified.

Thus, numerous biomarkers and clinical measures have been proposed, including the use of case severity measures such as dynamic organ failure assessment (SOFA), critical illness and chronic disease assessment (APACHE) II, and simplified assessment of critical illness (SAPS) II, however, they are rarely calculated on a daily basis in a routine manner due to the relatively high complexity and time requirements associated with each indicator. The use of new biomarkers may fill this unmet clinical need, but few, if any, are successfully implementing this in general clinical practice (8).

Of these biomarkers, the pro-adrenomedullin medial fragment (SF-proADM) - a peptide produced by multiple tissues to stabilize microcirculation and protect against endothelial permeability and subsequent organ failure (9-16) - has shown significant promise, especially in the fields of sepsis studies (17). lower respiratory infections (18-21), lung transplants (22) and thoracic surgery (23). Indeed, it is widely accepted that the endothelium and microcirculation play a significant role in the pathophysiological host response to sepsis (24,25), with the regulation and distribution of blood flow in each organ being of great importance (25), and therefore may provide an alternative indicator of severity. overall host response versus individual organ dysfunction scores.

Therefore, the present invention uses a range of biomarkers (PCT, lactate, C-reactive protein, SF-proADM) and clinical assessments (SOFA, APACHE II and SAPS II) to (i) provide an accurate assessment of disease severity within a short time frame after staging diagnosis, for example, within 24 hours of diagnosis and within the first ten days of ICU (Intensive Care Unit) therapy, (ii) identify low-risk patients who are eligible for early ICU discharge, and (iii) to identify patients who, despite a reduction in infectious loads, still have a high or increased risk of mortality or other adverse events, and who may require urgent additional diagnostic and therapeutic interventions.

Accordingly, SF-proADM can be used as a tool to identify patients with high disease severity who may require alternative diagnostic and therapeutic interventions, such as a change in antibiotic treatment, and patients with low disease severity who may potentially be eligible for early treatment. transfer from the ICU in combination with no ICU-specific therapy and/or continued treatment with the same antibiotic agents.

In the field of treating patients suffering from an infectious disease, such as patients with sepsis, there is a need for additional tools for monitoring therapy after the start of treatment, as well as tools for antibiotic treatment management, stratification and/or control.

SUMMARY OF THE INVENTION

In light of the difficulties in the prior art, the technical problem underlying the present invention is to provide a means for monitoring therapy in a patient suffering from an infectious disease or a patient with sepsis who is receiving antibiotic treatment, as well as a means for managing antibiotic treatment, stratification and/or control, in particular within a short period of time after the start of treatment.

Therefore, the present invention is directed to providing a method, kit, and additional means for monitoring therapy in patients suffering from an infectious disease, including tools for antibiotic treatment management, stratification, and/or control, as well as tools for predicting, risk assessment, and/or risk stratification of subsequent adverse events. health events in such a patient based on the levels of proadrenomedullin (proADM) determined in the sample obtained from the patient. Thus, one aspect of the invention is the use of a biomarker or combination of biomarkers to distinguish between critically ill patients and/or patients suffering from an infectious disease or sepsis who have received or are undergoing treatment, in particular antibiotic treatment, which have a high risk of adverse events, and severely ill patients and/or patients suffering from an infectious disease or sepsis who have a low risk of an adverse event.

The solution to the technical problem according to the present invention is presented in the independent claims. Preferred embodiments of the invention are presented in dependent claims. The present invention relates to a method for antibiotic treatment management, stratification and/or control in a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, comprising

- taking the first sample from the specified patient,

- taking a second sample from the specified patient at the time point after the first sample was taken and after the start of antibiotic treatment, determining the levels of procalcitonin (PCT) or its fragments in the first and second sample, and

- determining the level of proADM or its fragment(s) in at least the second sample,

wherein the levels of PCT or fragment(s) thereof in said first and second samples and the level of proADM or fragment(s) thereof in the second sample indicate whether a change in treatment with one or more antibiotics is required.

In a preferred embodiment of the method of antibiotic treatment management, stratification and/or control in a patient suffering from an infectious disease and receiving treatment with one or more of the antibiotic agents described herein, the first sample is taken before, at the time of, or after the symptoms of the infectious disease are detected in specified patient.

Patients of the method according to the invention have already been diagnosed with an infectious disease and are already receiving antibiotic treatment. Therefore, the method according to the invention can be used to monitor the success of an initiated antibiotic treatment with an appropriate one or more antibiotic agents. Measured levels of PCT or fragments thereof and proADM or fragments thereof indicate whether a change in ongoing antibiotic treatment is required. In other words, levels of PCT markers or fragments thereof and proADM or fragments thereof can be used as indicators of the likelihood of success of appropriate antibiotic treatment, and thus it can be decided whether treatment should be continued because it is likely to work/improve the patient's health, or need to be changed. In the context of this invention, a change in a patient's antibiotic treatment may include a change in dose, route of administration or schedule of administration, or other parameters of antibiotic treatment, while the changed treatment may still include the same or more antibiotics. agents that were originally used. In addition, a change in antibiotic treatment may also refer to a change in one or more of the antibiotic agents used to treat a patient. Therefore, the change may refer to the addition of an additional antibiotic, or to the replacement of one or more antibiotic agents with one or more other agents.

In some embodiments, antibiotic treatment management, stratification, and/or control preferably includes predicting the success or effectiveness of ongoing antibiotic treatment, including the likelihood of a future adverse event.

Physicians or medical personnel who treat patients suffering from infectious diseases and who are receiving antibiotic treatment may use the method of this invention, preferably in a hospital setting, such as a point of care or intensive care unit (ICU). This method is very useful for monitoring the effect of a therapy that has been started in a patient with an infectious disease and can be used to assess whether the patient being treated is a high-risk patient who should be under intensive medical supervision, while antibiotic treatment can be changed, or that the patient is a low-risk patient with stable or even improving health that may not require a change in antibiotic treatment, possibly because the treatment started successfully improves the patient's condition. Initial antibiotic treatment of patients suffering from infectious diseases can have a direct impact on the likelihood of adverse health events in the patient. Thus, the risk assessment/prediction of a future adverse event provides feedback on the effectiveness of the initiated therapy.

The likelihood of a subsequent adverse event can be assessed by comparing the level of PCT or its fragments and / or the level of proADM or its fragments in the first and / or second sample with a control level (for example, cut-off value or cut-off value and / or group mean), when this control level may correspond to proADM or fragments thereof, and/or PCT or fragments thereof in healthy patients or in patients diagnosed with an infectious disease.

Accordingly, the method of the present invention can assist in predicting the outcome of antibiotic treatment and/or the likelihood of subsequent health problems in a patient. This means that the method according to the invention is able to distinguish between high-risk patients who are more likely to suffer complications, or whose condition will become more severe in the future, despite ongoing antibiotic therapy, and low-risk patients, whose health is stable. or even improved so that they are not expected to suffer from side effects such as death of the patient or worsening of the clinical symptoms or physical parameters of the patient, which may require certain therapeutic measures. A particular advantage of the method of the invention is that a patient with an infectious disease who has been determined to be at low risk by the method of the invention that does not require a change in antibiotic therapy, or at least no change in one or more amount of antibiotics could be discharged from the ICU faster. In addition, for low-risk patients, the intensity and/or frequency of patient monitoring may be reduced. Accordingly, the hospital or other medical institution in charge of the patient can more effectively decide which patients need intensive care and monitoring. Therefore, the respective hospital or facility can, for example, more efficiently allocate intensive care beds for high-risk patients. This will lead to better care for high-risk patients, as medical staff can focus on such patients, while low-risk patients can be discharged from the intensive care unit. This would also lead to significant benefits in avoiding the cost of unnecessary measures that would otherwise be applied to low-risk patients.

In preferred embodiments, the indicated levels of PCT or fragment(s) thereof in said first and second samples and the level of proADM or fragment(s) thereof in the second sample are indicative of a subsequent adverse health event in said patient within 28 days of staging. diagnosis and initiation of treatment. In additional preferred embodiments of the invention, the indicated levels of PCT or its fragment(s) in the specified first and second samples, and the level of proADM or its fragment(s) in the second sample indicate a subsequent adverse health event in the specified patient within 90 days after diagnosis and initiation of treatment. A preferred embodiment of the invention relates to a method for antibiotic treatment management, stratification and/or control in a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, the method comprising

- taking the first sample from the specified patient when determining the symptoms of an infectious disease in the specified patient,

- taking a second sample from said patient after initial identification of said symptoms and initiation of antibiotic treatment,

- determination of the levels of procalcitonin (PCT) or its fragments in the first and second sample, and

- determining the level of proADM or its fragment(s) in at least the second sample,

wherein the levels of PCT or its fragment(s) in said first and second samples and the level of proADM or its fragment(s) in the second sample indicate whether a change in treatment with one or more antibiotic agents is required.

Acquisition in determining the symptom of an infectious disease refers to obtaining shortly after the detection of symptoms of an infectious disease, and may also be referred to as a point in time for determining the symptoms of an infectious disease.

The point in time when patients are diagnosed as being critically ill, or when the symptoms of an infectious disease are identified and the first treatment measures are initiated, is defined as "time 0" or the first sample, which can be a control for the time point of obtaining a subsequent second sample. If a patient's diagnosis or detection of symptoms of an infectious disease and initiation of treatment do not occur at the same time, time 0 is the time when the later of the two events, diagnosis/symptoms and initiation of treatment, occurs. As a rule, the diagnosis of seriously ill patients and/or the identification of symptoms of an infectious disease immediately follow or accompany the initiation of therapy. The first sample is usually taken at or before this time.

It was completely unexpected that the level of proADM or its fragments in a later sample from a patient can provide critical information about the success of ongoing antibiotic treatment and/or the likelihood of a subsequent adverse health event in these patients. There was no suggestion that a single measurement of proADM or its fragments after diagnosis/symptom identification and initiation of treatment in a patient suffering from an infectious disease could provide such important information regarding the likelihood of success of current treatment and prognosis of the patient's health.

It is particularly effective to combine the determination of proADM or fragments thereof (in the second sample) with the determination of PCT or fragments thereof in an earlier sample (first sample) taken from said patient and which can be used to diagnose said patient suffering from an infectious disease at the time of time 0, and determining the level of PCT or its fragments in the second sample taken at a certain point in time after the diagnosis/definition of symptoms and the start of treatment, which is also preferably the same point in time when proADM or its fragments are determined.

This allows you to determine the difference between the level of PCT or its fragment(s) in the second sample compared to the level of PCT or its fragment(s) in the first sample. As can be seen from the data below, determining the difference between the level of PCT or its fragments in the second sample compared to the first sample provides additional information in addition to the information obtained from the levels of proADM or its fragments in the second sample. Based on this pooled information, it is more likely to predict whether antibiotic treatment will be successful in stabilizing or improving the patient's health.

This is a surprising finding because sepsis biomarkers are not usually synergistic or complementary, but merely alternative diagnostic markers.

The combined determination of proADM or its fragments with the determination of PCT or its fragments, whether in the same sample or in samples obtained at different time points, provides a synergistic effect in terms of accuracy and reliability of determining the risk of a subsequent adverse event. These synergistic effects exist for the combined assessment of proADM or fragments thereof with other markers or clinical assessments such as lactate, CRP, SOFA, SAPS II, APACHE II, or other clinical assessments. In a preferred embodiment of the present invention, the second sample is taken from the specified patient within 30 minutes after the symptoms of the infectious disease are identified and antibiotic treatment is started, or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours , 4 days, 7 days, or 10 days after diagnosis and initiation of antibiotic treatment. In other embodiments, the implementation, the second sample is taken from the specified patient 12-36 hours and/or 3-5 days after the definition of symptoms of an infectious disease and the start of antibiotic treatment.

In preferred embodiments of the method according to the invention, said sample is taken from said patient at about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days , 6 days, 7 days, 8 days, 9 days, or 10 days after diagnosis and initiation of antibiotic treatment. In other embodiments, the second sample is taken at time points after said diagnosis is made and antibiotic treatment started, from 30 minutes to 12 hours, 12-36 hours, 3-5 days, 7-14 days, 8-12 days, or 9-11 days .

Ranges between any of the above values can be used to determine when a sample is taken.

According to a preferred embodiment of the present invention, the method includes further determining the level of proADM or its fragment(s) in the first sample.

The fact that the level of proADM or its fragments in a time period as short as about 30 minutes after the detection of symptoms of an infectious disease and the start of treatment can provide such information was completely unexpected.

By determining the level of proADM or its fragments in the first and second sample, it is possible to determine the difference in the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample. This allows you to determine the change in the level of proADM during the course of antibiotic treatment.

It was surprising that determining the change in levels of proADM or its fragments from the time of diagnosis/symptoms of an infectious disease and the initiation of treatment to a later point in time could provide additional information regarding the success of antibiotic treatment and/or the occurrence of a future adverse health event in a patient. . The great advantage of this embodiment of the present invention is that the same sample used to determine the level of PCT or fragments thereof at time 0 can also be used to determine the baseline level of proADM or fragments thereof, which can be compared with the level of proADM or fragments thereof. at a later point in time after diagnosis and initiation of treatment. By determining the change in the level of proADM or its fragments during the treatment of a patient, it is possible to further improve the accuracy of predicting the success of antibiotic treatment and/or the occurrence of an adverse health event in a patient.

In a preferred embodiment of the present invention, an increased level of proADM or fragment(s) thereof in the second sample compared to the first sample indicates a need to change one or more antibiotics.

According to this invention, the term "indicates" in the context of "indicates that a change in one or more antibiotics is required" is intended to measure the likelihood of success of ongoing antibiotic treatment with the corresponding one or more antibiotics. Preferably, "indicating" a change in one or more antibiotics is intended to indicate an increased likelihood that continued antibiotic treatment with one or more antibiotics will not be successful in improving or stabilizing the health of the patient. Therefore, modification of antibiotic agents should be considered to prevent the occurrence of side effects in the patient.

In the context of the present invention, indicating the need to change one or more antibiotic agents used in antibiotic treatment of a patient suffering from an infectious disease is associated with an increased likelihood of future adverse health events in the patient. An indication of the required change in one or more antibiotic agents required is intended to evaluate the effectiveness of antibiotic treatment and should generally not be construed as limiting in terms of unequivocally indicating the absolute success or failure of antibiotic treatment.

In view of the foregoing, proADM and PCT severity levels are nonetheless very reliable in determining whether a change in antibiotic is required when using the thresholds disclosed herein, so a risk assessment based on an assessment of a subsequent adverse event is reliable. determines the appropriate action by the healthcare worker.

Surprisingly, based on the change in the level of proADM or its fragments, one can confidently predict the likelihood of success of ongoing antibiotic treatment and/or the occurrence of an adverse health event in a patient. An increase in the level or severity of proADM or fragments thereof since the detection of symptoms of an infectious disease and the initiation of treatment indicates that one or more antibiotic agents may need to be changed, possibly because an adverse event is likely to occur. Accordingly, based on the change in proADM or fragments thereof during the course of treatment, the clinician can decide whether to change or modify the patient's treatment or to stick with the original treatment.

In the context of the present invention, it is preferred that the patient be diagnosed as suffering from sepsis and/or septic shock. In light of the data presented herein, the predictive power of proADM in samples from these patient populations is particularly accurate in predicting the likelihood of an adverse event in these patients and indicating whether a change in ongoing antibiotic treatment is required.

In addition, the method according to this invention preferably includes determining the level of SP-proADM. The use of the SF-proADM definition is preferred for any proposed embodiment described herein, and may be considered, as appropriate, in the context of each embodiment. In preferred embodiments, the "ADM fragment" can be considered as SF-proADM.

In a preferred embodiment of the method described herein,

- an increased level of PCT or its fragment(s) in the second sample compared to the first sample, and

- the level of moderate or severe proADM or its fragment(s) in the second sample indicates the need to change one or more antibiotic agents,

- while the level of moderate severity of proADM or its fragment(s) is above 4 nmol/l, preferably above 3 nmol/l, more preferably above 2.7 nmol/l, and below 6.5 nmol/l, preferably below 6, 95 nmol/l, more preferably below 10.9 nmol/l, and

- the level of high severity of proADM or its fragment(s) is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l.

In a preferred embodiment of the method described herein,

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample, and

- the level of high severity of proADM or its fragment(s) in the second sample, or an increased level of severity of proADM or its fragment(s) in the second sample compared to the first sample, for example, an increase from a low severity level to a moderate severity level or a high severity level, or from moderate to high severity,

- indicate the need to change one or more antibiotic agents,

- while the level of low severity of proADM or its fragment(s) is below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l,

- the level of moderate proADM or its fragment(s) is above 4 nmol/l, preferably above 3 nmol/l, more preferably above 2.7 nmol/l, and below 6.5 nmol/l, preferably below 6.95 nmol /l, more preferably below 10.9 nmol/l, and

- the level of high severity of proADM or its fragment(s) is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l.

It was quite unexpected that a decrease in PCT levels from the day symptoms were identified and treatment started to a later point in time after treatment initiation may indicate a need to change the antibiotic agents used in the patient's antibiotic treatment. In particular, this increased risk is evident when the level of high severity of proADM or fragments thereof is determined. PCT is believed to be a marker for infectious diseases such as sepsis. Accordingly, it is believed that a decrease in PCT values during treatment indicates an improvement in the patient's health. However, as disclosed herein, it has become apparent that despite a decrease in PCT value, a patient is at risk for future adverse events if the level of proADM or fragments thereof at a later time point is a high severity level, or the severity level of proADM or its fragments increases during antibiotic treatment. Accordingly, the attending physician may adjust the treatment of such a patient, who would not be identified as a high-risk patient, without determining the proADM or its fragments.

It was completely unexpected that the level of proADM or its fragments can correlate with the requirement to change one or more antibiotic agents used in antibiotic treatment. The levels of proADM in samples from patients with infectious diseases according to this invention can preferably be indicated by 3 different levels of severity according to the levels of proADM. High levels of proADM indicate a high severity level, medium levels indicate a moderate severity level, and low levels indicate low severity levels. The appropriate concentrations, which define cut-offs for the respective levels of severity, depend on a variety of parameters such as the time the sample is taken after the patient is diagnosed and treated with the method of this invention, and the method used to determine the level of proADM or its fragments in said sample.

The cut-off values disclosed herein preferably refer to measurements of the level of proADM protein or fragments thereof in a plasma sample taken from a patient by the Thermo Scientific BRAHMS KRYPTOR assay. Accordingly, the values disclosed herein may vary to some extent depending on the detection/measurement method used, and the specific values disclosed herein are also intended to cover corresponding values determined by other methods.

In some embodiments of the invention, the level of low severity of proADM or its fragment(s) is below the threshold in the range of 1.5 nmol/l and 4 nmol/l. Any value within these ranges may be considered as a suitable threshold for low severity levels of proADM or fragments thereof. For example, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2, 7, 2.75, 2.8, 2.85, 2.9, 2.95, 3.0, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9 3.95 , 4.0 nmol/l. In embodiments of the invention, the level of high severity of proADM or fragment(s) exceeds a threshold value in the range from 6.5 nmol/l to 12 nmol/l. Any value within these ranges may be considered as a suitable threshold for high severity levels of proADM or fragments thereof. For example, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7, 7, 7.75, 7.8, 7.85, 7.9, 7.95, 8.0, 8.05, 8.1, 8.15, 8.2, 8.25, 8.3, 8.35, 8.4, 8.45, 8.5, 8.55, 8.6, 8.65, 8.7, 8.75, 8.8, 8.85, 8.9, 8, 95, 9.0, 9.05, 9.1, 9.15, 9.2, 9.25, 9.3, 9.35, 9.4, 9.45, 9.5, 9.55, 9.6, 9.65, 9.7, 9.75, 9.8, 9.85, 9.9, 9.95, 10.0, 10.05, 10.1, 10.15, 10, 2, 10.25, 10.3, 10.35, 10.4, 10.45, 10.5, 10.55, 10.6, 10.65, 10.7, 10.75, 10.8, 10.85, 10.9, 10.95, 11.0, 11.05, 11.1, 11.15, 11.2, 11.25, 11.3, 11.35, 11.4 11.45 , 11.5, 11.55, 11.6, 11.65, 11.7, 11.75, 11.8, 11.85, 11.9, 11.95, 12.0 nmol/l.

All thresholds disclosed herein relating to the level of a marker or biomarker such as proADM or PCT should be understood to be "at or above" a certain threshold, or "at or below" a certain threshold. For example, an embodiment referring to a level of proADM or fragment(s) below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, should be understood as referring to a level of proADM or fragment(s) thereof ) at or below 4 nmol/l, preferably at or below 3 nmol/l, more preferably at or below 2.7 nmol/l. Conversely, an embodiment referring to a level of proADM or fragment(s) greater than 6.5 nmol/l, preferably greater than 6.95 nmol/l, more preferably greater than 10.9 nmol/l, should be understood to refer to a level of proADM or fragment(s) of 6.5 nmol/l or greater, preferably 6.95 nmol/l or greater, more preferably 10.9 nmol/l or greater. In the embodiments described herein, severity levels are preferably determined by thresholds that represent the boundaries between low, moderate, or high severity levels. Any embodiments that provide thresholds may therefore use a single threshold format as the boundary between two severity levels, or a single threshold for each severity level.

In some embodiments, the proADM level threshold between low and moderate severity is:

2.7 nmol/L ± 20% or 2.7 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%,

and between medium and high severity levels:

10.9 nmol/l ± 20% or 10.9 nmol/l ± 15% or ± 12%, ± 10%, ± 8% or ± 5%.

These thresholds are preferably suitable for assessing the level of severity of proADM at baseline, in other words, at the start of diagnosis and/or therapy and/or hospitalization.

In some embodiments, the proADM threshold between low and moderate severity is:

2.80 nmol/L ± 20%, or 2.80 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%,

and between medium and high severity levels:

9.5 nmol/L ± 20%, or 9.5 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%.

These thresholds are preferably suitable for assessing the level of severity of proADM after 1 day, in other words about 24 hours from baseline, in other words about 1 day after diagnosis and/or initiation of therapy and/or hospitalization. For example, in embodiments where proADM is measured one day after the start of therapy, day 1 thresholds may be used. slightly lower (but still relatively high) levels are associated with high risk and are classified as high severity. In some embodiments, the proADM threshold between low and moderate severity levels is:

2.80 nmol/L ± 20%, or 2.80 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%,

and between medium and high severity levels:

7.7 nmol/L ± 20%, or 7.7 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%,

These thresholds are preferably suitable for assessing the level of severity of proADM after 4 days, in other words about 4 days from baseline, in other words about 4 days after diagnosis and/or initiation of therapy and/or hospitalization. For example, in embodiments where proADM is measured 4 days after the start of therapy, day 4 cut-offs may be used. over time, even slightly lower (but still relatively high) levels are associated with high risk and classified as high severity. In some embodiments, the threshold levels to be used in the embodiments described above may be adjusted to an appropriate level depending on the day the measurement is taken. Each of the thresholds is subject to some variability due to the overall variance expected by one skilled in the art. Appropriate threshold levels are determined from extensive data as presented below, but are not meant to be definitive or exact values for all possible embodiments. Using a similar threshold as given, i.e. within ±20%, ±15%, ±12%, ±10%, ±8%, or ±5%, as can be determined by one skilled in the art, similar results can be expected. .

Any embodiment that specifies ±20% of the specified threshold may also be considered to be open to ±15%, ±12%, ±10%, ±8%, or ±5%.

Any embodiment that specifies a specific threshold for a baseline, day 1, or day 4 may also be considered to disclose the corresponding thresholds for other days, e.g., an embodiment describing a baseline threshold may be considered to refer to the same option. implementation that provides a threshold value for day 1 or day 4.

According to a preferred embodiment of the method according to the invention

- more than 50% lower level of PCT or its fragment(s) in the second sample compared to the first sample, and

- the level of moderate severity of proADM or its fragment(s) in the second sample, compared with the level of low severity of proADM or its fragment(s) in the first sample, indicate the need to change one or more antibiotic agents.

It was completely unexpected that the degree of relative change in the PCT level in the first sample to the second sample could provide additional predictive value in relation to the effectiveness and likelihood of success of ongoing antibiotic treatment. As can be seen from the data below, a decrease in PCT levels greater than 50% from time 0 to the second sample, together with an increase in the severity of proADM or its fragments from low to high severity, may be associated with unsuccessful ongoing antibiotic treatment. Accordingly, the attending physician or medical personnel should consider changing one or more of the antibiotic agents currently in use. This is particularly surprising as even a large/drastic decrease in PCT levels greater than 50% may be associated with treatment failure.

In preferred embodiments of the method according to this invention,

- less than 50% lower level of PCT or its fragment(s) in the second sample compared to the first sample, and

- the level of high severity of proADM or its fragment(s) in the second sample, compared with the level of moderate severity of proADM or its fragment(s) in the first sample, indicate the need to change one or more antibiotic agents.

Surprisingly, a decrease in PCT or fragments less than 50% indicates antibiotic failure if it is associated with an increase in the severity of proADM or its fragments from mild or moderate to severe.

In addition, in the context of the method of this intervention, it is preferable that an increased level of PCT or its fragment (s) in the second sample compared to the first sample, and the level of moderate or severe proADM or its fragment (s) in the second sample, indicate the need to change one or more antibiotic agents.

Surprisingly, if PCT levels increase during the course of antibiotic treatment, a change in the antibiotic agents used can only be prescribed if the level of severity of proADM or its fragments in the second sample is moderate or high. In the case of a low severity level of proADM, the person in charge of antibiotic treatment should consider the possibility that one or more antibiotic agents are effective in stabilizing or improving the patient's condition despite an increase in PCT levels. Instead, consideration should be given to changing the dose, route of administration, or other modification of antibiotic treatment without changing the combination of one or more antibiotic agents used. Preferably, the first and second samples of the method according to this invention are selected from the group consisting of a blood sample, a serum sample, a plasma sample and/or a urine sample.

In one embodiment, the invention further includes informing the patient of the results of the diagnostic method described herein.

Additional preferred embodiments of the method according to this invention further include

- determining the level of at least one additional biomarker or its fragment(s) in the first and second sample, and at least one additional biomarker is preferably lactate and/or C-reactive protein,

wherein the levels of at least one additional biomarker in said first and second samples and the level of proADM or fragment(s) thereof in the second sample indicate whether a change in ongoing antibiotic treatment is required.

Preferred embodiments of the method for managing antibiotic treatment, therapy stratification and/or control in a patient suffering from an infectious disease and receiving antibiotic treatment described herein further include

- determining at least one clinical assessment at the time of taking the first sample and at the time of taking the second sample, while at least one clinical assessment is preferably SOFA, APACHE II and/or SAPS II,

wherein at least one clinical assessment at the time of said first and second sample collection and the level of proADM or fragment(s) thereof in the second sample indicate whether a change in ongoing antibiotic treatment is required.

In additional embodiments of the method described herein, the method further includes molecular analysis of a sample from said patient to detect infection. The sample used for molecular analysis to detect infection is preferably a blood sample. In a preferred embodiment, molecular analysis is a method aimed at detecting one or more biomolecules produced by a pathogen. Said one or more biomolecules may be a nucleic acid, a protein, a sugar, a carbohydrate, a lipid and/or a combination thereof, such as a glycosylated protein, preferably a nucleic acid. Said biomolecule is preferably specific for one or more pathogens. In preferred embodiments, such biomolecules are detected by one or more biomolecular assay methods selected from the group consisting of nucleic acid amplification methods such as PCR, qPCR, RT-PCR, qRT-PCR, or isothermal amplification, mass spectrometry, detection of enzymatic activity and detection methods based on immunoassay. Additional methods of molecular analysis are known to the person skilled in the art and include the method according to this invention.

The present invention further relates to a method for administering antibiotic treatment, stratification of therapy and/or control in a patient suffering from an infectious disease and receiving antibiotic treatment, comprising:

- taking the first sample from the specified patient when the symptoms of the infectious disease are detected (time 0) and taking the second sample from the specified patient after the initial identification of the indicated symptoms and the start of treatment,

- determining the level of at least one biomarker or its fragment(s) in the first and second sample and/or determining at least one clinical assessment at the time of taking the first sample and at the time of taking the second sample, wherein at least one biomarker is procalcitonin (PCT), lactate and/or C-reactive protein, and at least one clinical evaluation is SOFA, APACHE II and/or SAPS II,

- determining the level of proADM or its fragment(s) in at least the second sample,

- at the same time, a change in the level of at least one biomarker and / or at least one clinical assessment between the specified first and second sample, and the level of proADM or its fragment (s) in the second sample, indicate whether a change is currently required by the ongoing antibiotic treatment.

This invention also relates to a kit for implementing a method for managing antibiotic treatment, stratifying and/or monitoring therapy in a patient suffering from an infectious disease and receiving antibiotic treatment, and the kit contains

- reagents for determining the level of proADM or its fragment(s) and, optionally, additionally for determining the level of PCT or its fragment(s) in the sample from the subject, and

- control data, such as a control level corresponding to severity levels by proADM levels and possibly PCT levels, said control data being preferably stored on a computer-readable medium and/or used in the form of computer executable code configured to compare certain levels of proADM or its fragment(s) and, optionally, additionally determined levels of PCT or its fragment(s), with the specified control data.

Reagents for determining the level of proADM or its fragment(s), and optionally for determining the level of PCT or its fragment(s), are preferably selected from those necessary for the implementation of the method, for example, antibodies targeting proADM, suitable labels, such as fluorescent labels, preferably two separate fluorescent labels suitable for use in the KRYPTOR assay, sample collection tubes.

In one embodiment of the method described herein, the level of proADM or fragment(s) thereof and optionally PCT or fragment(s) thereof is determined using a method selected from the group consisting of: mass spectrometry (MS), fluorescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (FIA), enzyme immunoassays (ELISA), luminescent bead array assays, magnetic bead array assays, protein microarray assays, rapid form assays such as, for example, immunochromatographic strip assay, rare earth cryptate assay, and automated systems/analyzers.

The method of the invention can furthermore be implemented as a homogeneous method in which the sandwich complexes formed by the antibody/antibodies and the marker, eg proADM or a fragment thereof, to be detected remain suspended in the liquid phase. In this case, it is preferable that when two antibodies are used, both antibodies are labeled with parts of the detection system, which results in signal generation or signal initiation if both antibodies are integrated into one sandwich.

Such methods should be implemented, in particular, as methods for enhancing fluorescence or detecting fluorescence quenching. In particular, a preferred aspect relates to the use of detection reagents that must be used in pairs, such as, for example, those described in US 4882733 A, EP-B 10180492 or EP-B 10539477, and given in the prior art. Thus, measurements are possible in which only reaction products containing both labeling components in one immune complex are detected directly in the reaction mixture.

For example, such technologies are offered under the trademarks TRACE® (Time Resolved Amplified Cryptate Emission) or KRYPTOR®, implementing the principles of the above applications. Therefore, in particular preferred aspects, a diagnostic device is used to implement the method proposed herein. For example, the level of the proADM protein or fragment thereof and/or the level of any other marker of the method proposed herein is determined. In particular, in preferred aspects, the diagnostic device is a KRYPTOR®.

In one embodiment of the method described herein, the method is an immunoassay, and the assay is carried out in a homogeneous phase or in a heterogeneous phase.

In one embodiment of the method described herein, the first antibody and the second antibody are present as dispersed in a liquid reaction mixture, and the first labeling component, which is part of a labeling system based on fluorescent or chemiluminescent quenching or enhancement, is associated with the first antibody, and the second labeling component of said labeling system is coupled to a second antibody such that upon binding of both antibodies to said proADM or detectable fragments thereof, a measurable signal is generated that allows the resulting sandwich complexes to be detected in the analyzed solution.

In one embodiment of the method described herein, the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, particularly of the cyanine type.

In one embodiment of the method described herein, the method further includes comparing the determined level of proADM or fragment(s) thereof with a control level, threshold, and/or group mean corresponding to proADM or fragment(s) in patients who are were diagnosed as seriously ill/suffering from an infectious disease and under therapeutic treatment, while said comparison is performed in the computer processor using computer executable code.

The methods of the present invention may be implemented in part on a computer. For example, the step of comparing a detected level of a marker, eg, proADM or fragments thereof, with a control level may be performed on a computer system. In a computer system, the determined level of marker(s) may be combined with other marker levels and/or subject parameters to compute a score that is indicative of diagnosis, prognosis, risk assessment, and/or risk stratification. For example, certain values may be entered (either manually by a healthcare professional or automatically from the device(s) in which the appropriate marker levels were/were determined) into a computer system. The computer system may be located directly at the point of care (for example, the ICU or ED) or at a remote location connected via a computer network (for example, via the Internet, or through specialized medical cloud systems that can be additionally integrated with other IT systems or platforms such as hospital information systems (HIS)). Typically, a computer system stores values (eg, marker level or parameters such as age, blood pressure, weight, sex, etc., or clinical scoring systems such as SOFA, qSOFA, BMI, etc.) on computer-readable medium and calculates a score based on predetermined and/or pre-stored control levels or control values. The resulting score will be displayed and/or printed to the user (usually by a healthcare professional such as a doctor). Alternatively, or in addition, the appropriate prognosis, diagnosis, assessment, treatment guide, case management guide, or stratification will be displayed and/or printed to the user (typically by a healthcare professional such as a physician).

In one embodiment of the invention, a software system that has a machine learning algorithm can be used, preferably to identify hospitalized patients at risk for sepsis, severe sepsis, and septic shock using data from electronic health records (EMRs). A machine learning model can be trained on a random forest classifier using EHR data (such as laboratory values, biomarker expression, vital signs, and demographics) of patients. An intelligence that gives computers the ability to learn complex patterns of data without being directly programmed, as opposed to simpler rule-based systems. Earlier studies used electronic health record data to trigger warning signals to identify general clinical deterioration. In one embodiment of the invention, the analysis of proADM levels can be included in appropriate software for comparison with existing datasets, for example, proADM levels can also be processed in a program with machine learning algorithms to help diagnose or predict the occurrence of an adverse event.

The combined use of proADM or fragments thereof in combination with another biomarker such as PCT or CRP can be performed either in one multiplex assay or in two separate assays performed on a patient sample. A sample may refer to the same sample or to different samples. The assay used to detect and detect proADM and eg PCT can also be the same or different, eg an immunoassay can be used to detect one of the above markers. More detailed descriptions of suitable assays are provided below.

Thresholds and other reference levels of proADM or fragments thereof in patients who are diagnosed as critically ill and are on treatment can be determined by the previously described methods. For example, those skilled in the art are aware of methods of using the coefficient of variation to assess the variability of assays to establish control values and/or thresholds (George F. Reed et al., Clin Diagn Lab Immunol. 2002; 9(6): 1235-1239).

In addition, the functional sensitivity of the assay can be determined to designate statistically significant values for use as control levels or thresholds in accordance with established methods. Laboratories are able to independently establish the functional sensitivity of the assay using a clinically relevant protocol. "Functional sensitivity" can be thought of as the concentration that results in a coefficient of variation (CV) of 20% (or some other predetermined % CV) and is thus a measure of the accuracy of the assay at low analyte levels. Therefore, CV is a standardization of the standard deviation (SD) that allows estimates of variability to be compared regardless of the magnitude of the analyte concentration over at least most of the working range of the assay.

In addition, methods based on ROC analysis can be used to determine statistically significant differences between two clinical groups of patients. ROC-curves (ROC - Receiver Operating Characteristic) measure the efficiency of sorting the fitted probabilities of the model to sort response levels. Curves-ROC can also help in establishing critical points in diagnostic tests. The higher the curve from the diagonal, the better the match. If a logistic fit has more than two response levels, it creates a generalized ROC curve. On such a graph, there is a curve for each response level, which is the ROC curve for that level versus all other levels. Software is available that allows such analysis to establish suitable reference levels and thresholds, such as JMP 12, JMP 13, Statistical Discovery, from SAS.

Threshold values can be similarly defined for PCT. Literature is available to the skilled artisan to determine the appropriate cut-off value, eg Philipp Schuetz et al. (BMC Medicine. 2011; 9:107) describe that at a cut-off value of 0.1 ng/mL, PCT has a very high sensitivity to rule out infection. Terence Chan et al. (Expert Rev. Mol. Diagn.2011; 11(5), 487-496) described that metrics such as positive and negative odds ratios, which are calculated based on sensitivity and specificity, are also useful for power estimation. diagnostic test. Values are typically displayed for a set of threshold values (CV) as an ROC curve. The area under the curve value is used to determine the best diagnostically meaningful CV. This literature describes variations in CV (cut-off values that depend on analysis and study design) and appropriate methods for determining cut-off values.

Group means for proADM or fragments thereof can also be used as controls, e.g. group means for proADM, where patients diagnosed as "critically ill" can be compared with a control group, with the control group preferably containing more than 10, 20, 30, 40, 50 or more subjects. In one embodiment, the cut-off level for PCT can be a value in the range of 0.01 to 100.00 ng/mL in a serum sample using, for example, the Luminex MAC Pix E-Bioscience assay or the BRAHMS PCT-Kryptor assay. In a preferred embodiment, the PCT threshold may be in the range of 0.01 to 100, 0.05 to 50, 0.1 to 20, or 0.1 to 2 ng/mL, and most preferably >0.05 up to 0.5 ng/ml. Any value within these ranges may be considered as the appropriate threshold value. For example, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 can be used .0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6 , 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng/mL. In some embodiments, PCT levels for healthy subjects are about 0.05 ng/mL. The invention further relates to the medically indicated methods of treatment described herein, wherein the group of patients to be treated is identified, stratified, monitored, predicted, diagnosed, or otherwise assessed using the methods described herein. Suitable treatments for the methods are described herein. Therefore, the present invention is particularly useful for identifying patients at increased risk of developing an adverse event and initiating prophylactic or risk-reducing treatment, or initiating treatment to eliminate the presence of any indicated medical pathology, preferably those designated as severe disease.

Embodiments of the invention relating to the determination of the level of proADM or its fragment(s) in one or more samples of critically ill patients, such as patients with sepsis

Additionally, the invention relates to a method for monitoring therapy, including prediction, risk assessment and/or risk stratification of a subsequent adverse health event in a patient, including

- obtaining a sample of said patient, wherein the patient was diagnosed as seriously ill and therapeutic treatment was initiated, wherein the sample was taken from the patient after the diagnosis and initiation of treatment,

- determination of the level of proADM or its fragment(s) in the specified sample,

wherein said level of proADM or its fragment(s) correlates with the likelihood of a subsequent adverse health event in said patient.

Patients of the method according to this invention have already been diagnosed as seriously ill and have already begun to receive treatment. Thus, the method according to the present invention can be used to monitor the success of an initiated treatment or therapy in terms of determining the likelihood of a subsequent adverse event. Therapeutic monitoring preferably includes prediction of an adverse event and/or risk stratification or risk assessment for the patient in relation to a future adverse event, which risk assessment and determination of said risk should be considered as a means of monitoring the initiated therapy.

In embodiments of the invention, the term "critically ill" may be used interchangeably with "a patient suffering from an infectious disease".

The use of proADM or fragments thereof as a single parameter in embodiments of the present invention is advantageous over the use of other single parameters such as biomarkers or clinical scores because proADM is more accurate in predicting side effects than other markers such as PCT, CRP, lactate, or clinical assessments such as SOFA, SAPS II, or APACHE II. In some embodiments, the invention includes determining additional parameters such as markers, biomarkers, clinical scores, or the like. In another preferred embodiment of the present invention, the patient has been diagnosed using at least one of the biomarkers procalcitonin (PCT), lactate, and C-reactive protein, and/or at least one of the SOFA, APACHE II, and SAPS II clinical assessments. .

In one embodiment, the critically ill patient is a patient diagnosed with an infectious disease, a patient diagnosed with an infectious disease and one or more existing functional organ failures, a patient diagnosed with sepsis, severe sepsis, or septic shock, and/or post-traumatic or postoperative patient. In light of the data presented herein, the predictive power of proADM in samples from these patient populations is particularly accurate in predicting the likelihood of an adverse event in these patients.

In preferred embodiments of the present invention, the adverse health event in said patient is death, preferably death within 28-90 days of diagnosis and treatment, new infection, organ failure, and/or worsening of clinical symptoms requiring debridement procedures, transfusion of components blood, colloid infusion, emergency surgery, invasive mechanical ventilation, and/or kidney or liver replacement.

In preferred embodiments, said level of proADM or fragment(s) thereof correlates with the likelihood of a subsequent adverse health event in said patient within 28 days of diagnosis and initiation of treatment. In further preferred embodiments of the invention, said level of proADM or fragment(s) correlates with the likelihood of a subsequent adverse health event in said patient within 90 days of diagnosis and initiation of treatment.

In some embodiments of the invention, the treatment received by the patient includes one or more of: antibiotic treatment, invasive mechanical ventilation of the lungs, non-invasive mechanical ventilation of the lungs, renal replacement therapy, the use of vasoconstrictor drugs, infusion therapy, apheresis (apheresis) and / or organ protection.

In preferred embodiments of the invention, the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample and/or a urine sample.

Preferably, the method is carried out in some embodiments by determining the level of proADM or fragment(s) thereof, said determination of proADM comprising determining the level of SP-proADM in the sample. The use of the SF-proADM definition is preferred for any proposed embodiment described herein and may be considered, as appropriate, in the context of each embodiment. In preferred embodiments, the "ADM fragment" can be considered as SF-proADM. In a further embodiment of the invention, the level of proADM or fragment(s) thereof is correlated with the likelihood of subsequent health problems in said patient. In a preferred embodiment, the level of proADM or fragment(s) thereof is positively correlated with the likelihood of a subsequent adverse health event occurring in said patient. In other words, the higher the detected level of proADM, the higher the likelihood of a subsequent adverse event.

According to a preferred embodiment of the present invention,

- a low severity level of the proADM or fragment(s) thereof indicates no subsequent adverse event or indicates a low risk of a subsequent adverse event, with the low severity level being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, or

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event or indicates a high risk of a subsequent adverse event, and the level of high severity is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l, or

According to a preferred embodiment of the present invention,

- the level of proADM or its fragment(s) is below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, indicates no subsequent adverse event or indicates a low risk of a subsequent adverse event, or

- the level of proADM or its fragment(s) is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l, indicates a subsequent adverse event or indicates a high risk of a subsequent adverse event.

According to a preferred embodiment of the present invention,

- the low severity level of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the low severity level is below 2.7 nmol/l, or

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 10.9 nmol/l.

This embodiment of the present invention is particularly useful when levels of proADM or fragments thereof are determined in a sample that was taken on the day the patient was diagnosed and treated, in particular about 30 minutes after diagnosis and treatment. This can be seen from the analysis in Example 3.

According to a preferred embodiment of the present invention,

- the low severity level of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the low severity level is below 2.7 nmol/l, or

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 10.9 nmol / l,

- while the levels of proADM or its fragments are determined in the sample, which was taken on the day of diagnosis and start of treatment.

According to a preferred embodiment of the present invention,

- the low severity level of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the low severity level is below 2.8 nmol/l, or

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 9.5 nmol/l.

This embodiment of the present invention is particularly useful when levels of proADM or fragments thereof are determined in a sample taken 1 day after the patient was diagnosed and treated, as seen in Table 12.

According to a preferred embodiment of the present invention,

- the low severity level of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the low severity level is below 2.8 nmol/l, or

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, and the level of high severity is above 9.5 nmol / l,

- while the levels of proADM or its fragments are determined in the sample, which was taken 1 day after the diagnosis and start of treatment.

According to a preferred embodiment of the present invention,

- the level of low severity of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the level of low severity is below 2.25 nmol/l.

This low severity threshold is particularly useful because, in some embodiments, it has been determined to be the optimal threshold between low and moderate levels of proADM or fragments thereof when a patient is sampled on the 4th, 7th, or 10th day after the specified diagnosis and start of treatment. This can be seen from the analysis presented in Table 12.

According to a preferred embodiment of the present invention,

- the level of low severity of proADM or its fragment(s) indicates the absence of a subsequent adverse event, and the level of low severity is below 2.25 nmol / l,

- while the levels of proADM or its fragments are determined in the sample, which was taken 4 or 7 days after the diagnosis and the start of treatment.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 7.7 nmol/l.

This high severity threshold is particularly useful because, in some embodiments, it has been determined to be the optimal threshold between moderate and severe levels of proADM or fragments thereof when a sample is taken from a patient 4 days after said diagnosis and treatment. This can be seen from the analysis presented in Table 12.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, and the level of high severity is above 7.7 nmol / l,

- while the levels of proADM or its fragments are determined in the sample, which was taken 4 days after the diagnosis and the start of treatment.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 6.95 nmol/l.

This high severity threshold is particularly useful because, in some embodiments, it has been determined to be the optimal threshold between moderate and severe levels of proADM or fragments thereof when a sample is taken from a patient 7 days after a specified diagnosis and initiation of treatment. This can be seen from the analysis presented in Table 12.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, and the level of high severity is above 6.95 nmol / l,

- while the levels of proADM or its fragments are determined in a sample that was taken 7 days after the diagnosis and start of treatment.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, with the level of high severity being above 7.45 nmol/l.

This high severity cutoff is particularly useful as it has been determined to be the optimal cutoff between moderate and severe proADM or fragments when a sample is taken from a patient 10 days after a specified diagnosis and start of treatment. This can be seen from the analysis presented in Table 12.

According to a preferred embodiment of the present invention,

- the level of high severity of proADM or its fragment(s) indicates a subsequent adverse event, and the level of high severity is above 7.45 nmol / l,

- while the levels of proADM or its fragments are determined in the sample, which was taken 10 days after the diagnosis and the start of treatment.

Preferably, patients according to the present invention are intensive care unit (ICU) patients, wherein:

- the level of low severity of proADM or its fragment(s) suggests that the specified patient should be discharged from the ICU, or

- the level of high severity of proADM or its fragment(s) suggests a change in the patient's treatment regimen in the ICU.

A particular advantage of this invention is that, based on the classification of certain levels of proADM or its fragments, it is possible to estimate the likelihood of a future adverse health event in an ICU patient. Based on this assessment, it is possible to adjust the following treatment options and decisions.

For example, if the level of proADM or its fragments falls into the category of low severity proADM, the attending physician can more confidently decide to discharge the specified patient from the ICU, since the patient is unlikely to experience an adverse health event, preferably within the next 28 days , more preferably within the next 90 days. Accordingly, it may not be necessary to keep this patient in the ICU. It is also possible to conclude that the current treatment successfully improves the patient's health, as assessed by measuring the risk of an adverse event.

Conversely, if determination of the level of proADM or its fragments in the indicated ICU patient indicates a level of high severity of proADM or its fragments, the attending physician should leave the patient in the ICU. In addition, consideration should be given to adjusting the patient's treatment, as there is a possibility that the current treatment is not improving the patient's health status, so the patient is more likely to suffer from an adverse event in the future.

Modification of treatment in the context of this invention will include, without limitation, adjusting the dose or mode of administration of the current drug. Changing the current treatment to another treatment, adding an additional treatment option to the current treatment, or stopping the current treatment. Various methods of treatment that can be applied to patients in the context of this invention have been disclosed in the detailed description of this patent application.

According to a particularly preferred embodiment of the present invention, the low severity level is below 2.25 nmol/L, said sample is taken from an ICU patient after 1 day or more of said diagnosis and treatment initiation, and the low severity level of the proADM or fragment (s) involves the discharge of the specified patient from the ICU.

Additionally, this invention relates to a method for monitoring therapy, including prediction, risk assessment and/or risk stratification of a subsequent adverse health event in a patient, including

- taking a sample from said patient, where the patient is an ICU patient and therapeutic treatment has been initiated, wherein the sample is taken from the patient after admission to the ICU and initiation of treatment,

- determination of the level of adrenomedulin (ADM) or its fragment(s) in the specified sample,

wherein said level of proADM or its fragment(s) correlates with the likelihood of a subsequent adverse health event in said patient.

In the context of the method of this invention relating to ICU patients (instead of patients who have been diagnosed as seriously ill), reference to the point in time of sampling used to determine proADM or fragments thereof is the point in time when patients enter the ICU and the first measures begin. treatment (time 0). This point in time corresponds to the point in time of diagnosis and initiation of treatment in the method according to this invention, in relation to patients who were diagnosed as "critically ill".

All preferred embodiments of the method of this invention relating to patients who have been diagnosed as seriously ill are also disclosed in the context of the method of this invention relating to ICU patients.

Embodiments of the invention relating to the additional determination of the level of PCT and / or other biomarkers (or clinical scores) in the first and second sample (or at the time of taking the first and second sample) in critically ill patients

A preferred embodiment of the present invention includes further determination of the level of PCT or its fragment(s) in a sample taken from a patient. In a preferred embodiment, a sample for determining the level of PCT or its fragment(s) is taken before, at or after the time of diagnosis and initiation of treatment.

It is particularly advantageous to combine the determination of proADM or fragments thereof with the determination of PCT or fragments thereof in a sample, wherein the sample used to determine proADM may be the same or a different sample used to detect PCT.

The combined determination of proADM or its fragments with the determination of PCT or its fragments, whether in the same sample or in samples obtained at different time points, provides a synergistic effect in terms of accuracy and reliability of determining the risk of a subsequent adverse event. These synergistic effects exist for the combined assessment of proADM or fragments thereof with other markers or clinical assessments such as lactate, CRP, SOFA, SAPS II, APACHE II, or other clinical assessments.

According to a further preferred embodiment of the present invention, the method described herein further includes

- determining the level of PCT or its fragment(s) in the first sample taken from the patient, and said first sample is taken before, during or after the moment of diagnosis and initiation of treatment,

- determination of the level of PCT or its fragment(s) in the second sample taken from the specified patient, and the second sample was taken after the first sample, preferably within 30 minutes after taking the first sample or after 30 minutes, 1 hour, 2 hours, 6 hours , 12 hours, 24 hours, 4 days, 7 or 10 days after the first sample was taken, and

- determination of the difference between the level of PCT or its fragment(s) in the second sample compared to the level of PCT or its fragment(s) in the first sample.

It is particularly advantageous to combine the determination of proADM or fragments thereof in a patient sample with the determination of PCT or fragments thereof in the first sample and the determination of the level of PCT or fragments thereof in a second sample taken after the first sample, wherein the sample used to determine proADM or its fragments may differ from the first sample or the second sample used to determine the PCT or its fragments.

In a preferred embodiment of the method described herein, the method further includes

- determination of the level of PCT or its fragment(s) in the first sample taken from the patient, and the specified first sample is taken at or before the time of diagnosis and treatment (time 0),

- determination of the level of PCT or its fragment(s) in the second sample taken from the specified patient after the diagnosis and initiation of treatment, preferably within 30 minutes after the specified diagnosis and initiation of treatment, or after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after said diagnosis and initiation of treatment, and

- determination of the difference between the level of PCT or its fragment(s) in the second sample compared to the level of PCT or its fragment(s) in the first sample.

It is particularly advantageous to combine the determination of proADM or fragments thereof (in the second sample) with the determination of PCT or fragments thereof in an earlier sample (first sample) taken from said patient and which can be used to diagnose "critically ill" in said patient, at time 0, and determining the level of PCT or fragments thereof in a second sample taken at a certain point in time after diagnosis and initiation of treatment, which is also preferably the same point in time when proADM or its fragments are determined. As can be seen from the data below, determining the difference between the level of PCT or its fragments in the second sample compared to the first sample provides additional information in addition to the information obtained from the levels of proADM or its fragments in the second sample. Based on this combined information, it is more likely to predict whether an adverse health event will occur in a specified patient, compared to predicting the likelihood of an adverse event based solely on information about the level of proADM or its fragments in the second sample. This is a surprising finding because sepsis biomarkers are not usually synergistic or complementary, but merely alternative diagnostic markers.

In a preferred embodiment of the method described herein, the method further includes

- determination of the level of lactate in the first sample taken from the patient, and the specified first sample is taken at or before the time of diagnosis and treatment (time 0),

- determination of the level of lactate in a second sample taken from the specified patient after the specified diagnosis and start of treatment, preferably within 30 minutes or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days , 7 or 10 days after said diagnosis and initiation of treatment, and

- determination of the difference between the level of lactate in the second sample compared to the level of lactate in the first sample.

A preferred embodiment of the present invention includes further determination of the level of CRP or its fragment(s) in a sample taken from a patient. In a preferred embodiment, the sample for determining the level of CRP or its fragment(s) is taken before, at or after the time of diagnosis and initiation of treatment.

It is particularly advantageous to combine the determination of proADM or fragments thereof with the determination of CRP or fragment(s) thereof in a sample, wherein the sample used to determine proADM may be the same or a different sample used to detect CRP or fragment(s) thereof.

According to a further preferred embodiment of the present invention, the method described herein further includes

- determining the level of CRP or its fragment(s) in the first sample taken from the patient, and said first sample is taken before, during or after the moment of diagnosis and initiation of treatment,

- determination of the level of CRP or its fragment (s) in the second sample taken from the specified patient, and the second sample was taken after the first sample, preferably within 30 minutes after taking the first sample or after 30 minutes, 1 hour, 2 hours, 6 hours , 12 hours, 24 hours, 4 days, 7 or 10 days after the first sample was taken, and

- determination of the difference between the level of CRP or its fragment(s) in the second sample compared to the level of lactate in the first sample.

In a preferred embodiment of the method described herein, the method further includes

- determination of the level of CRP or its fragment(s) in the first sample taken from the patient, and the specified first sample is taken at or before the time of diagnosis and start of treatment (time 0),

- determination of the level of CRP or its fragment(s) in the second sample taken from the specified patient after the specified diagnosis and start of treatment, preferably within 30 minutes or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours , 24 hours, 4 days, 7 or 10 days after said diagnosis and treatment, and

- determination of the difference between the level of CRP or its fragment(s) in the second sample compared to the level of CRP or its fragment(s) in the first sample.

A preferred embodiment of the present invention includes the additional definition of SOFA. In a preferred embodiment, SOFA is determined before, at, or after the time of diagnosis and initiation of treatment. It is particularly advantageous to combine the determination of proADM, or fragments thereof, with the determination of SOFA, wherein the sampling time used to determine proADM may be the same or different from the time of SOFA determination.

According to a further preferred embodiment of the present invention, the method described herein further includes

- determination of the first SOFA before, during or after the moment of diagnosis and initiation of treatment,

- determination of the second SOFA within 30 minutes after the determination of the first SOFA or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the determination of the first SOFA, and

- calculation of the difference between two defined SOFAs.

In a preferred embodiment of the method described herein, the method further includes

- determination of SOFA at or before the time of diagnosis and initiation of treatment (time 0),

- determination of SOFA after said diagnosis and initiation of treatment, preferably within 30 minutes or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after said diagnosis and initiation of treatment, and

- calculation of the difference between the SOFA determined after the specified diagnosis and start of treatment, and the SOFA determined at time 0.

The preferred embodiment of the present invention includes an additional definition of SAPS II. In a preferred embodiment, SAPS II is determined before, at, or after the time of diagnosis and initiation of treatment.

It is particularly advantageous to combine the determination of proADM or fragments thereof with the determination of SAPS II, wherein the time of sampling used to determine the proADM may be the same or different from the time of determination of SAPS II.

According to a further preferred embodiment of the present invention, the method described herein further includes

- determination of the first SAPS II before, during or after the moment of diagnosis and initiation of treatment,

- determination of the second SAPS II within 30 minutes after the determination of the first SOFA or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the determination of the first SAPS II, and calculating the difference between two defined SAPS II.

In a preferred embodiment of the method described herein, the method further includes

- determination of SAPS II at or before diagnosis and treatment (time 0),

- determination of SAPS II after said diagnosis and initiation of treatment, preferably within 30 minutes or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after said setting diagnosis and initiation of treatment, and

- calculation of the difference between SAPS II determined after the specified diagnosis and start of treatment, and SAPS II determined at time 0.

A preferred embodiment of the present invention includes the additional definition of APACHE II. In a preferred embodiment, APACHE II is determined before, at, or after the time of diagnosis and initiation of treatment.

It is particularly advantageous to combine the determination of proADM or fragments thereof with the determination of APACHE II, wherein the time of sampling used for the determination of proADM may be the same or different from the time of determination of APACHE II.

According to a further preferred embodiment of the present invention, the method described herein further includes

- determination of the first APACHE II before, during or after the moment of diagnosis and initiation of treatment,

- determination of the second APACHE II within 30 minutes after the determination of the first APACHE II, or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the determination of the first APACHE II, and

- calculation of the difference between two defined APACHE II.

In a preferred embodiment of the method described herein, the method further includes

- determination of APACHE II at or before the time of diagnosis and initiation of treatment (time 0),

- determination of APACHE II after the specified diagnosis and initiation of treatment, preferably within 30 minutes or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the specified statement diagnosis and initiation of treatment, and

- calculation of the difference between APACHE II determined after the specified diagnosis and start of treatment, and APACHE II determined at time 0.

In a preferred embodiment of the present invention,

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample, and the level of low severity of proADM or its fragment(s), with the level of low severity being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, indicates no subsequent adverse event, or

- lower level of PCT or its fragment(s) in the second sample compared to the first sample, and the level of high severity of proADM or its fragment(s), with the level of high severity being above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l, indicates the absence of a subsequent adverse event.

In the context of this invention, the determination of a lower level of the marker in the second sample compared to the first sample may indicate a decrease in the levels of the corresponding marker in the patient during the course of the initiated treatment. Conversely, elevated levels in the second sample compared to the first sample may indicate increasing levels of the marker over the course of treatment.

It is believed that PCT is a marker of severe disease in a patient, in particular a patient with sepsis. Accordingly, it is believed that a decrease in the PCT value during treatment indicates an improvement in the patient's health. However, as disclosed herein, it has become apparent that a decrease in the PCT value may indicate the occurrence of a future adverse event if the level of proADM or its fragments at a later time point is a level of high severity. Accordingly, the attending physician may adjust the treatment of such a patient who would not be identified as a high-risk patient without detecting proADM or fragments thereof in the second sample.

On the other hand, the physician can be sure that an adverse event is unlikely to occur when the PCT level decreases during the course of treatment, while the level of proADM or its fragments in the second sample represents a level of low severity of proADM or its fragments. Accordingly, such patients can be identified as low risk patients. Quite unexpectedly, the combination of determining the change in PCT levels during the treatment of a critically ill patient with the determination of proADM levels at a later point in time leads to improved treatment monitoring, prognosis and assessment of the risk of future adverse health events in the patient.

According to another preferred embodiment of the method described herein,

- increased level of PCT or its fragment(s) in the second sample compared to the first sample, and the level of low severity of proADM or its fragment(s), with the low severity level being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, indicates no subsequent adverse event, or

- increased level of PCT or its fragment(s) in the second sample compared to the first sample, and the level of high severity of proADM or its fragment(s), with the level of high severity being above 6.5 nmol/l, preferably above 6.95 nmol /l, more preferably above 10.9 nmol/l, indicates the occurrence of a subsequent adverse event.

In one embodiment, the invention further includes informing the patient of the results of the diagnostic method described herein.

Embodiments of the present invention relating to the determination of the level of proADM or its fragment(s) in the first and second sample in critically ill patients

A preferred embodiment of the method according to the present invention further comprises

- determining the level of proADM or its fragment(s) in the first sample taken from the patient, and the specified first sample is taken before, during or after the moment of diagnosis and treatment, and

- determination of the level of proADM or its fragment(s) in the second sample taken from the specified patient, and the second sample was taken after the first sample and after the moment of diagnosis and start of treatment, preferably within 30 minutes after taking the first sample or after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the first sample was taken, and

- determining the difference between the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample is obvious.

The first and second samples used to determine the level of proADM or fragment(s) may differ from the first and second samples used to determine the level of PCT or fragment(s) thereof.

A preferred embodiment of the method according to the present invention further comprises

- determining the level of proADM or its fragment(s) in the first sample taken from the patient, and the specified first sample is taken at or before the time of diagnosis and treatment (time 0), and

- determination of the level of proADM or its fragment(s) in the second sample taken after diagnosis and treatment, preferably within 30 minutes, or after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days , 7 or 10 days after said diagnosis and initiation of treatment, and

- determining the difference between the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample is obvious.

A preferred embodiment of the method according to the present invention further comprises

- determining the level of proADM or its fragment(s) in the first sample taken from the patient, and the specified first sample is used to diagnose "critically ill" of the specified patient (time 0), and

- determination of the level of proADM or its fragment(s) in the second sample taken from the specified patient after diagnosis and treatment, preferably within 30 minutes, or after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours , 4 days, 7 or 10 days after the specified diagnosis and initiation of treatment, and

- determining the difference between the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample.

An additional preferred embodiment of the method according to this invention further comprises

- determining the level of proADM or fragment(s) thereof and optionally PCT or fragment(s) thereof in the first sample taken from the patient, said first sample being taken at or before the time of diagnosis and initiation of treatment (time 0), and

- determination of the level of proADM or its fragment(s) and optionally PCT or its fragment(s) in the second sample taken from the specified patient after the specified diagnosis and treatment, preferably within 30 minutes, or after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after said diagnosis and initiation of treatment, and

- determining the difference between the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample.

An additional preferred embodiment of the method according to this invention further comprises

- determining the level of proADM or its fragment(s) and optionally PCT or its fragment(s) in the first sample taken from the patient, and said first sample is used to diagnose "severely ill" to the specified patient (time 0), and determining the level of proADM or fragment(s) thereof and optionally PCT or fragment(s) thereof in a second sample taken from said patient after said diagnosis and treatment, preferably within 30 minutes, or after 30 minutes, 1 hour, 2 hours, 6 hours , 12 hours, 24 hours, 4 days, 7 or 10 days after said diagnosis and treatment, and

- determining the difference between the level of proADM or its fragment(s) in the second sample compared to the level of proADM or its fragment(s) in the first sample. In one embodiment of the method described herein, an increased level of proADM or fragment(s) thereof in the second sample compared to the first sample is indicative of a subsequent adverse event.

In a preferred embodiment of the method according to the invention

- an increased level of proADM or its fragment(s) and an increased level of PCT or its fragment(s) in the second sample compared to the first sample indicates a subsequent adverse event, and/or

- an increased level of proADM or its fragment(s) and a lower level of PCT or its fragment(s) in the second sample compared to the first sample indicates a subsequent adverse event.

In preferred embodiments of the present invention, an increased level of proADM or fragments thereof in the second sample relative to the first sample refers to an increased level of severity in terms of the level of proADM or fragments thereof. Conversely, in preferred embodiments of the present invention, a lower level of proADM or fragments thereof in the second sample compared to the first sample refers to a lower severity level of proADM or fragments thereof in the second sample compared to the first sample.

Additional Embodiments of the Invention in Critically Ill Patients

According to a preferred embodiment of the present invention, the patients are intensive care unit (ICU) patients, and

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample and the level of low severity of proADM or its fragment(s), with the level of low severity being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, suggests that the patient is discharged from the ICU;

- lower level of PCT or its fragment(s) in the second sample compared to the first sample and the level of high severity of proADM or its fragment(s), with the level of high severity being above 6.5 nmol/l, preferably above 6.95 nmol /l, more preferably above 10.9 nmol/l, suggests a change in the treatment regimen of the patient in the ICU;

- increased level of PCT or its fragment(s) in the second sample compared to the first sample, and the level of low severity of proADM or its fragment(s), with the low severity level being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, suggests that the patient is discharged from the ICU; or

- increased level of PCT or its fragment(s) in the second sample compared to the first sample and the level of high severity of proADM or its fragment(s), with the level of high severity being above 6.5 nmol/l, preferably above 6.95 nmol/ l, more preferably above 10.9 nmol/l, suggests a change in the patient's treatment regimen in the ICU.

In an additional embodiment, this invention relates to a kit for carrying out the method according to this invention, and the kit contains

- reagents for determining the level of proADM or fragment(s) thereof, and optionally additionally for determining the level of PCT, lactate and/or C-reactive protein or fragment(s) thereof, in a sample from a subject, and

- control data, such as a control level corresponding to a high and/or low severity level of proADM, where the low severity level is below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, and the level severe is greater than 6.5 nmol/l, preferably greater than 6.95 nmol/l, more preferably greater than 10.9 nmol/l and optionally PCT, lactate and/or C-reactive protein levels, with said data being controls preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare certain levels of proADM or fragment(s) thereof and optionally further determined levels of PCT, lactate and/or C-reactive protein or fragment(s) thereof , with the specified control data.

In an additional embodiment, this invention relates to a kit for carrying out the method according to this invention, and the kit contains

- reagents for determining the level of proADM or its fragment(s) and, optionally, additionally for determining the level of PCT or its fragment(s) in the sample from the subject, and

- control data, such as a control level corresponding to the level of high and/or low severity of proADM, with the level of low severity being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, and the level high severity is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l and optionally PCT levels, said control data being preferably stored on a computer-readable medium and/or used in the form of computer executable code configured to compare the determined levels of proADM, or fragment(s) thereof, and optionally further determined levels of PCT, or fragment(s) thereof, with specified control data.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a method of antibiotic treatment management, stratification and/or control in a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents. As can be seen from the data presented in this document, the probability of occurrence or absence of an adverse health event in a patient is determined by the level of proADM or its fragment(s), which provides information about the possible change in antibiotic treatment. The present invention has the following advantages over conventional methods: The methods and kits of the invention are fast, objective, easy to use and accurate for monitoring therapy in critically ill patients. Methods and kits according to the invention relate to markers and clinical assessments that are easily measured by conventional methods in hospitals, since the levels of proADM, PCT, lactate, C-reactive protein, SOFA, APACHE II, SAPS II can be determined routinely in received blood samples or additional biological fluids or samples obtained from the subject.

As used herein, a "patient" or "subject" may be a vertebrate.

In the context of this invention, the term "subject" includes both humans and animals, in particular mammals, and other organisms. In the context of the present invention, an "adverse health event in a patient" refers to events that indicate a complication or deterioration in the patient's health. Such adverse events include, without limitation, death of the patient, death of the patient within 28-90 days of diagnosis and treatment, occurrence of infection or new infection, organ failure, and worsening of the patient's general clinical signs or symptoms, such as hypotension or hypertension, tachycardia or bradycardia. In addition, examples of adverse events include situations where worsening of clinical symptoms indicates the need for therapeutic measures such as debridement procedure, transfusion of blood products, colloid infusion, invasive mechanical ventilation, non-invasive mechanical ventilation, emergency surgery, replacement therapy organs, such as kidney or liver replacement, and the use of vasoconstrictor drugs.

The patient described in this document who has been diagnosed as "critically ill" may be diagnosed as an intensive care unit (ICU) patient, a patient who requires constant and/or intensive monitoring of his health, a patient diagnosed with sepsis, severe sepsis, or septic shock, a patient diagnosed with an infectious disease and one or more existing organ failures, a patient before or after surgery, a patient with PTSD, a patient with an injury such as a patient from an accident, a patient with a burn, a patient with one or more open lesions. The subject described herein may be in an emergency department or intensive care unit, or in other places of medical care, for example, in an emergency transport vehicle, such as an ambulance, or a general practitioner who is in contact with a patient with these symptoms. Patients with suspected SIRS (systemic inflammatory response syndrome, SIRS) are not necessarily considered critically ill.

The term "OPT patient" refers, without limitation, to a patient who has been placed in an intensive care unit. An intensive care unit may also be referred to as an intensive care unit or intensive care unit (ICU) or intensive care unit (ICU), which is a special department of a hospital or healthcare facility that provides medical care in the form of intensive care. OPT patients usually suffer from severe and life-threatening diseases and injuries that require constant, careful monitoring and support from special equipment and drugs to ensure normal bodily functions. Common pathologies treated in intensive care units include, without limitation, acute or adult respiratory distress syndrome (ARDS), trauma, organ failure, and sepsis.

Used in this document, the term "diagnosis" in the context of this invention refers to the recognition and (early) detection of clinical pathology in a subject associated with an infectious disease. Also, the assessment of the severity of an infectious disease may be covered by the term "diagnosis".

"Prognosis" refers to predicting an outcome or particular risk to a subject based on an infectious disease. This may also include an assessment of the likelihood of recovery or the likelihood of a poor outcome for the specified subject.

The methods of this invention can also be used for surveillance. "Surveillance" refers to tracking an already diagnosed infectious disease, disorder, complication, or risk, for example, to analyze disease progression or the effect of a particular treatment or therapy on disease progression in a critically ill patient or on an infectious disease in a patient.

The term "monitoring therapy" or "control therapy" in the context of this invention refers to monitoring and/or adjusting the therapeutic treatment of the specified subject, for example, by obtaining feedback on the effectiveness of therapy.

In this invention, the terms "risk assessment" and "risk stratification" refer to the distribution of subjects into different risk groups in accordance with their future prognosis. Risk assessment also refers to stratification for the application of preventive and/or therapeutic measures. Examples of risk stratification are the low, intermediate and high risk levels disclosed in this document.

As used herein, the term "treatment management" refers to the application of certain treatment options or medical interventions based on the value of one or more biomarkers and/or clinical parameter and/or clinical assessments.

It is clear that in the context of this invention, "determining the level of proADM or its fragment(s)" or the like. refers to any means of determining a proADM or a fragment thereof. The fragment may be of any length, such as at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as the fragment allows unambiguous determination of the level of proADM or a fragment thereof. In particular, in preferred aspects of the invention, "determining the level of proADM" refers to determining the level of the middle fragment of proadrenomedullin (SF-proADM). SF-proADM is a fragment and/or region of proADM.

The peptide adrenomedullin (ADM) was discovered as a 52 amino acid hypotensive peptide that was isolated from human pheochromocytoma (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a 185 amino acid precursor peptide ("preproadrenomedullin" or "pre-proADM"). An exemplary amino acid sequence of ADM is shown in SEQ ID NO: 1.

SEQ ID NO: 1: pre-pro-ADM amino acid sequence:

1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARIRVKRYRQSMN

101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

ADM contains positions 95-146 of the pre-proADM amino acid sequence and is its splice product. "Proadrenomedullin" ("proADM") refers to pre-proADM without a signal sequence (amino acids 1 to 21), that is, amino acid residues 22 to 185 of pre-proADM. "Proadrenomedullin middle fragment" ("SF-proADM") refers to amino acids 42 to 95 of pre-proADM. An exemplary amino acid sequence of SF-proADM is shown in SEQ ID NO: 2.

SEQ ID NO: 2: SF-pro-ADM amino acid sequence (AS 45-92 pre-pro-ADM):

ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

It is also provided herein that the peptide and its pre-proADM or SF-proADM fragment can be used for the methods described herein. For example, a peptide or fragment thereof may comprise amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre-proADM (mature adrenomedullin, including the biologically active form, also known as bio-ADM). The C-terminal fragment of proADM (amino acids 153 to 185 pre-proADM) is called adrenotensin. ProADM peptide fragments or SF-proADM fragments may contain, for example, at least about 5, 10, 20, 30 or more amino acids. Accordingly, the proADM fragment may, for example, be selected from the group consisting of SF-proADM, PAMP, adrenotensin and mature adrenomedullin, preferably herein the fragment is SF-proADM.

Determining these different forms of ADM or proADM and fragments thereof also includes quantifying and/or detecting specific sub-regions of these molecules, for example by using antibodies or other affinity reagents that target certain portions of the molecules, or by detecting the presence and/or number of molecules. , by measuring the amount of a portion of the protein using mass spectrometry.

Any one or more of the "ADM peptides or fragments" described herein may be used in the present invention. Accordingly, the methods and kits of this invention may also include determining at least one additional biomarker, marker, clinical score, and/or parameter in addition to proADM.

As used herein, a parameter is a characteristic, feature, or measurable factor that can assist in the discovery of a particular system. The parameter is an important element for assessments related to health and physiology, such as the risk of disease/disorder/clinical pathology, preferably organ dysfunction(s). In addition, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. An exemplary parameter may be selected from the group consisting of: Critical Care and Chronic Disease Assessment II (APACHE II), Simplified Critical Care Assessment (SAPSII score), Rapid Dynamic Organ Failure Assessment (qSOFA), Dynamic Organ Failure Assessment (SOFA score), body mass index, weight, age, gender, IGS II, fluid intake, white blood cell count, sodium, potassium, temperature, blood pressure, dopamine, bilirubin, respiration rate, oxygen partial pressure, World Federation of Neurosurgical Societies (WFNS) scale and coma scale Glasgow (GCS).

As used herein, terms such as "marker", "surrogate marker", "predictive marker", "factor" or "biomarker" or "biological marker" are used interchangeably and refer to measurable and quantifiable biological markers ( for example, the concentration of a particular protein or enzyme or fragment thereof, a specific concentration of a hormone or fragment thereof, or the presence of biological substances or fragments thereof) that serve as indicators for health and physiology-related assessments such as risk of disease/disorder/clinical pathology , preferably an unwanted event. A marker or biomarker is defined as a trait that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers can be measured in a sample (in the form of a blood, plasma, urine or tissue test).

At least one additional marker and/or parameter of said subject may be selected from the group consisting of: lactate level in said sample, procalcitonin (PCT) level in said sample, dynamic organ failure score (SOFA score) of said subject, simplified score critical illness assessment (SAPSII) of the specified subject, critical illness and chronic disease assessment II (APACHE II) of the specified subject, and the level of soluble fms-like tyrosine kinase-1 (sFlt-1), histone H2A, histone H2B, histone H3, histone H4, calcitonin , endothelin-1 (ET-1), arginine vasopressin (A VP), atrial natriuretic peptide (ANP), neutrophil gelatinase-associated lipocalin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP) ), lithostatin (PSP), expressed on myeloid cells trigger receptor 1 (TREM1), interleukin-6 (IL-6), interleukin-1, interleukin-24 (IL-24), interleukin-22 (IL-22), inter leukin (IL-20), other ILs, precepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), alpha-1 antitrypsin, matrix metalloproteinase 2 (MMP2), metalloproteinase 2 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP7), placental growth factor (PIGF), chromogranin A, S100A protein, S100B protein and tumor necrosis factor a (TNFa), neopterin, alpha-1 antitrypsin, pro-arginine vasopressin (AVP, proAVP or copeptin) , procalcitonin, atrial natriuretic peptide (ANP, pro-ANP), endothelin-1, 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, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2/MTP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7/Ppbp, CXCL9, IL8/CXCL8, XCL1, XCL2, FAM19A1, FAM19A2 FAM19A3, FAM19A4, FAM19A5, CLCF1, CNTF, IL11, IL31, IL6, 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, IL18-VR, IL1-A, IL1-B, IL1-F10, IL1-F3/IL1-RA, IL1-F5, IL1-F6, IL1-B7, IL1-F8, IL1-RL2, IL1-F9, IL33, or fragments thereof.

As used herein, the term "procalcitonin" or "PCT" refers to a peptide encompassing amino acid residues 1-116, 2-116, 3-116, or fragments thereof, of a procalcitonin peptide. PCT is the peptide precursor of the hormone calcitonin. Thus, procalcitonin fragments are at least 12 amino acids long, preferably greater than 50 amino acids, more preferably greater than 110 amino acids. PCT may contain post-translational modifications such as glycosylation, liposidation, or derivatization. Procalcitonin is the precursor of calcitonin and catacalcin. Thus, under normal conditions, circulating PCT levels are very low (< about 0.05 ng/mL).

The level of PCT in a subject's sample can be determined using an immunoassay as described herein. As used herein, the level of ribonucleic acid or deoxyribonucleic acid encoding "procalcitonin" or "PKT" can also be determined. Methods for determining PCT are known to the skilled person, for example using products obtained from Thermo Fisher Scientific/BRAHMS GmbH.

Lactate, or lactic acid, is an organic compound with the formula CH ₃ CH(OH)COOH that occurs in body fluids, including blood. Blood tests for lactate are performed to determine the state of acid-base homeostasis in the body. Lactic acid is a product of cellular metabolism that can accumulate when cells lack oxygen (hypoxia) and must switch to a less efficient way of producing energy, or when the condition causes excess production or impaired clearance of lactate. Lactic acidosis can be caused by insufficient oxygen to cells and tissues (hypoxia), for example, if someone has a condition that can lead to a decrease in the amount of oxygen delivered to cells and tissues, such as shock, septic shock or congestive heart failure, lactate can be used to detect and assess the severity of hypoxia and lactic acidosis.

C-reactive protein (CRP) is a pentameric protein that can be found in body fluids such as blood plasma. CRP levels may rise in response to inflammation. Measuring CRP values and plotting can be useful in determining disease progression or treatment efficacy. As used herein, the term "dynamic organ failure score" or "SOFA score" is one metric used to track a patient's condition while in an intensive care unit (ICU). The SOFA score is a scoring system that determines the degree of functioning of a human organ or the frequency of failure. The score is based on six different scores, one each for the respiratory, cardiovascular, hepatic, coagulation, renal, and neurological systems. Both average and highest SOFA scores are predictors of outcome. An increase in SOFA score during the first 24-48 hours in the ICU predicts a mortality rate of at least 50% up to 95%. Scores less than 9 give a predicted mortality rate of 33%, and above 14 may be close to or higher than 95%.

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

As used herein, "APACHE II" or "critical and chronic illness assessment II" is a scoring system for classifying disease severity (Knaus et al., 1985). It can be applied up to 24 hours after a patient is admitted to the intensive care unit (ICU) and can be determined based on 12 different physiological parameters: AaDO2 or PaO2 (depending on FiO2), temperature (rectally), mean arterial pressure, arterial pH, 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 Critical Care Score II" refers to a system for classifying the severity of a disease or disorder (see Le Gall JR et al., A new Simplified Acute Physiology Score (SAPS II) based on a European /North American multicenter study JAMA 1993;270(24):2957-63). The SAPS II score consists of 12 physiological parameters and 3 disease-related parameters. The score is calculated based on 12 normal physiological measurements, previous health information, and some information obtained upon admission to the ICU. The SAPS II score can be determined at any time, preferably on Day 2. The "worst" measurement is defined as the value that corresponds to the highest score. The SAPS II score has a range of 0 to 163 points. The classification system includes the following parameters: age, heart rate, systolic blood pressure, temperature, Glasgow Coma Scale, mechanical ventilation or CPAP, PaO2, FiO2, diuresis, blood urea nitrogen, sodium, potassium, bicarbonate, bilirubin, leukocytes, chronic diseases and type of admission. There is a sigmoid relationship between mortality and overall SAPS II score. Subject mortality is 10% with a SAPSII score of 29, mortality is 25% with a SAPSII score of 40, mortality is 50% with a SAPSII score of 52, mortality is 75% with a SAPSII score of 64, mortality is 90% with a SAPSII score of 77 (Le Gall loc. cit.).

As used herein, the term "sample" is a biological sample that is obtained or taken from a patient or subject. As used herein, the term "sample" may, for example, refer to a sample of body fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Preferably herein, the sample is a body fluid sample such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusions, cells, cell extract, tissue sample, tissue biopsy, stool sample, and the like. In particular, the sample is blood, blood plasma, blood serum or urine.

Embodiments of the present invention relate to taking a first sample and taking a second sample. In the context of the method according to this invention, the terms "first sample" and "second sample" refer to the relative definition of the order of sampling used in the method according to this invention. When the terms "first sample" and "second sample" are used in the definition of this method, these samples should not be considered as absolute definitions of the number of samples taken. Therefore, additional samples may be taken from the patient before, during or after the first and/or second sample is taken, or between the first or second samples, and these additional samples may or may not be used in the method of the present invention. Thus, the first sample can be considered as any previously received sample. The second sample may be considered as any additional or subsequent sample.

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

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

As used herein, the term "urine" is a body fluid excreted by the kidneys in a process called urination (or urination) and excreted through the urethra.

In preferred embodiments of the present invention, the patient is diagnosed with sepsis. In particular, the patient may be diagnosed with severe sepsis and/or septic shock.

"Sepsis" in the context of the invention refers to the systemic response to infection. Alternatively, sepsis can be considered as a combination of SIRS with a confirmed infectious process or infection. Sepsis can be characterized as a clinical syndrome defined by the presence of both an infection and a systemic inflammatory response (Levy MM et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med. 2003 Apr; 31(4): 1250-6). As used herein, the term "sepsis" includes, but is not limited to: sepsis, severe sepsis, septic shock.

As used herein, the term "sepsis" includes, but is not limited to: sepsis, severe sepsis, septic shock. Severe sepsis refers to sepsis associated with organ dysfunction, hypoperfusion abnormality, or sepsis-induced hypotension. Hypoperfusion disorders include lactic acidosis, oliguria, and acute change in mental status. Hypotension due to sepsis is defined as systolic blood pressure less than about 90 mmHg or a decrease of about 40 mmHg. or greater than baseline in the absence of other causes of hypotension (eg, cardiogenic shock). Septic shock is defined as severe sepsis with sepsis-induced hypotension persisting despite adequate fluid support, along with the presence of hypoperfusion abnormalities or organ dysfunction (Bone et al., CHEST 101(6): 1644-55, 1992).

The term sepsis can alternatively be defined as life-threatening organ dysfunction caused by an unregulated host response to infection. For clinical practice, organ dysfunction may preferably be represented by an increase in dynamic organ failure score (SOFA) of 2 points or more, which is associated with in-hospital mortality greater than 10%. Septic shock can be defined as a type of sepsis in which particularly profound circulatory, cellular, and metabolic disorders are associated with a higher risk of mortality than sepsis alone. Patients with septic shock can be clinically identified by the need for a vasoconstrictor to maintain a mean arterial pressure of 65 mmHg. or higher and serum lactate greater than 2 mmol/L (>18 mg/dL) in the absence of hypovolemia.

As used herein, the term "sepsis" refers to all possible stages in the development of sepsis.

The term "sepsis" also includes severe sepsis or septic shock, based on the SEPSIS-2 definition (Bone et al., 2009). The term "sepsis" also includes subjects falling under the definition of SEPSIS-3 (Singer et al., 2016). As used herein, the term "sepsis" refers to all possible stages in the development of sepsis.

As used herein, the term "infection" in the context of the invention means a pathological process caused by the entry into normal sterile tissue or fluid of pathogenic or potentially pathogenic agents/pathogens, organisms and/or microorganisms, and preferably refers to infection(s) by bacteria, viruses , fungi and/or parasites. Accordingly, the infection may be a bacterial infection, a viral infection, and/or a fungal infection. The infection may be a local or systemic infection. For the purposes of the invention, a viral infection may be considered as an infection by a microorganism.

In addition, a subject suffering from an infection may be suffering from more than one source(s) of infection at the same time. For example, a subject suffering from an infection may suffer from a bacterial infection and a viral infection; from viral infection and fungal infection; from a bacterial and fungal infection, and from a bacterial infection, a fungal infection and a viral infection, or suffer from a mixed infection that includes one or more of the infections listed herein, including potentially superinfection, such as one or more bacterial infections in addition to one or more viral infections and/or one or more fungal infections.

As used herein, the term "infectious disease" includes all diseases or disorders that are associated with bacterial and/or viral and/or fungal infections.

In one embodiment, the infection to be detected or tested can be selected from Bordetella species such as Bordetella pertussis, Borrelia such as Borrelia burgdorferi, Brucella such as Brucella abortus, Brucella canis, Brucella melitensis or Brucella suis, Campylobacter, such such as Campylobacter jejuni, Chlamydia and Chlamydophila such as Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium such as Corynebacterium diphtheria, Enterococcus, Enterococcus, such as Enterococcus, Enterococcus, such as faecium, Escherichia such as Escherichia coli, Francisella such as Francisella tularensis, Haemophilus such as Haemophilus influenza, Helicobacter such as Helicobacter pylori, Legionella such as Legionella pneumophila, Leptospira such as Leptospira interrogans, Listeria such as Listeria monocytogenes , Mycobacteri um such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma such as Mycoplasma pneumonia, Neisseria such as Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas such as Pseudomonas aeruginosa, Rickettsia such as Rickettsia rickettsia, Salmonella such as Salmonella typhi , Salmonella typhimurium, Shigella such as Shigella sonnei, Staphylococcus such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus such as Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponema such as Treponema pallidum, Vibrio such as Vibrio cholera , Yersinia, such as Yersinia pestis, Yersinia enterocolitica, or Yersinia pseudotuberculosis.

Pathogenic fungi are fungi that cause disease in humans or other organisms. Candida species are important human pathogens that are best known for causing opportunistic infections in immunocompromised hosts (eg, transplant patients, AIDS patients, cancer patients). Infections are difficult to treat and can be very serious: 30-40% of systemic infections are fatal. Aspergillosis is another potential fungal pathogen. Aspergillus can cause disease in three main ways: through the production of mycotoxins; through the induction of allergenic reactions; and through local or systemic infections. For the last two categories, host immune status is key. The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Aspergillus flavus produces aflatoxin, which is both a toxin and a carcinogen and can potentially contaminate food. Aspergillus fumigatus and Aspergillus clavatus can be the cause of the disease. Cryptococcus neoformans can cause disease in humans. Cryptococcus neoformans is a major human and animal pathogen. Cryptococcus laurentii and Cryptococcus albidus are known to occasionally cause moderate to severe disease in immunocompromised patients. Cryptococcus gattii is endemic to tropical parts of the continent of Africa and Australia, and may be the cause of the disease. Histoplasma capsulatum can cause histoplasmosis in humans, dogs, and cats. Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature babies, the elderly, and people with AIDS. Stachybotrys chartarum or "black mold" can cause respiratory damage and severe headaches.

In one embodiment, the infection to be detected or analyzed can be selected from: Acinetobacter baumannii, Klebsiella pneumoniae, Acinetobacter Iwoffii, Listeria monocytogenes, Aeromonas caviae, Morganella morganii, Aeromonas hydrophila, Neisseria gonorrhoeae, Aspergillus flavus, Neisseria meningitidis, Aspergillus nidulans, Pasteurella multocida, Aspergillus niger, Pasteurella pneumotropica, Aspergillus terreus, Propionibacterium acnes, Bacillus anthracis, Proteus mirabillis, Bacillus cereus, Providencia rettgeri, Bacillus subtilis, Pseudomonas aeruginosa, Bacteroides fragilis, Salmonella choleraesuis, Brucella melitensis, Serratia liquefaciens, Burkholderia cepacia, Serratia marcescens , Candida albicans, Staphylococcus aureus, Candida dubliniensis, Staphylococcus epidermidis, Candida glabrata, Staphylococcus haemolyticus, Candida krusei, Staphylococcus hominis, Candida parapsilosis, Staphylococcus sac charolyticus, Candida tropicalis, Staphylococcus warn-eri, Capnoc ytophaga canimorsus, Stenotrophomonas maltophilia, Citrobacter braakii, Streptococcus agalactiae, Citrobacter freundii, Streptococcus anginosus, Clostridium perfringens, Streptococcus bovis, Corynebacterium jeikeium, Streptococcus constellatus, Enterobacter aerogenes, Streptococcus dysgalactiae, Enterobacter cloacae, Streptococcus mutans, Enterobacter sakazakii, Streptococcus pneumoniae, Enterococcus faecalis , Streptococcus pyogenes, Enterococcus faecium, Streptococcus salivarius, Escherichia coli, Streptococcus sanguinis, Shigella sp., Streptococcus suis, Gemella haemolysans, Vibrio vulnificus, Gemella morbillorum, Yersinia enterocolitica, Haemophilus influenzae, Yersinia pestis, Kingella kingae, Yersinia and pseudotuberculosis; Klebsiella oxytoca.

According to the present invention, critically ill patients, such as those with sepsis, may require very strict monitoring of vital signs and/or organ protection monitoring, and may be treated therapeutically.

In the context of this invention, the term "therapeutic treatment" or "treatment" includes various treatments and therapeutic strategies, which include, without limitation, anti-inflammatory strategies, administration of ADM antagonists such as therapeutic antibodies, siRNA or DNA, extracorporeal blood purification or removal of harmful substances by apheresis, dialysis, adsorbers to prevent cytokine storm, removal of inflammatory mediators, plasmapheresis, administration of vitamins such as vitamin C, ventilation such as mechanical ventilation and non-mechanical ventilation to provide the body with sufficient oxygen, for example , procedures for debridement, transfusion of blood products, infusion of colloids, kidney or liver replacement, antibiotic treatment, invasive mechanical ventilation, non-invasive mechanical ventilation, renal replacement therapy, use of vasoconstrictors, fluid resuscitation, apheresis, and measures to I protect organs.

Complementary treatment according to the invention includes administering cells or cell products such as stem cells, blood or plasma and stabilizing the patient's circulation and protecting the endothelial glycocalyx, e.g. by optimal fluid therapy strategies, e.g. to achieve normovolemia, and prevent or treatment of hypervolemia or hypovolemia. In addition, vasoconstrictor drugs or, for example, catecholamine, as well as inhibition of albumin or heparanase with unfractionated heparin or N-desulfated pe-N-acetylated heparin are useful means to maintain circulation and the endothelial layer.

In addition, therapeutic treatment according to this invention includes, without limitation, blood coagulation stabilization, iNOS inhibitors, anti-inflammatory agents such as hydrocortisone, sedatives and analgesics, and insulin.

"Renal replacement therapy" (RRT) refers to therapy that is used to replace the normal blood filtering function of the kidneys. Renal replacement therapy may refer to dialysis (eg, hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration. Such methods are different ways of redirecting the blood into the machine, cleaning it and then returning it to the body. Renal replacement therapy can also refer to kidney transplantation, which is the ultimate form of replacement where an old kidney is replaced with a donor kidney. Hemodialysis, hemofiltration, and hemodiafiltration can be continuous or intermittent, and can use the arteriovenous route (in which blood leaves an artery and returns through a vein) or the venous route (in which blood leaves a vein and returns through a vein). This gives different types of RRT. For example, renal replacement therapy may be selected from, but not limited to, continuous renal replacement therapy (CRRT), continuous hemodialysis (CHD), continuous arteriovenous hemodialysis (CAVHD), continuous venovenous hemodialysis (CVVHD), continuous haemofiltration (CHF) , continuous arteriovenous hemofiltration (CAVH or CAVHF), continuous venovenous hemofiltration (CWH or CVVHF), continuous hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venovenous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis ( IHD), intermittent venovenous hemodialysis (IVVHD), intermittent hemofiltration (IHF), intermittent venovenous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF), and intermittent venovenous hemodiafiltration (IVVHD).

Artificial and mechanical ventilation are effective approaches to improve proper gas exchange and ventilation, and are lifesaving in severe hypoxemia. Artificial ventilation refers to assisting or stimulating the breathing of a subject. Mechanical ventilation may be selected from the group consisting of: mechanical ventilation, manual ventilation, extracorporeal membrane oxygenation (ECMO) and non-invasive ventilation (NIV). Mechanical ventilation refers to the method of mechanically assisting breathing or replacing one's own breathing. This may include an apparatus called a respirator. Mechanical ventilation can be high frequency oscillatory ventilation or partial liquid ventilation. "Infusion therapy" refers to monitoring and controlling a subject's fluid balance and administering fluids to stabilize circulation or organ viability, such as by oral, enteral, or intravenous fluid administration. This includes stabilizing fluid and electrolyte balance, or preventing or correcting hyper- or hypovolemia, and supplying blood products.

Urgent surgery/emergency surgery is required if the patient has an emergency and may require immediate surgery to save life or health. The subject in need of emergency surgery may be selected from the group consisting of subjects suffering from severe trauma, active uncontrolled infection, organ transplant, organ prophylactic or organ stabilizing surgery, or cancer.

Cleaning procedures are hygienic means of preventing infection of subjects, in particular nosocomial infections, including the disinfection of all organic and inorganic surfaces that may come into contact with the patient, such as, for example, skin, objects in the patient's room, medical devices, diagnostic devices or room air. Cleaning procedures include the use of protective clothing and items such as masks, gowns, gloves, or a hygiene airlock, and activities such as restricted visits to the patient. In addition, cleaning procedures include cleaning the patient himself and the patient's clothing.

In the case of severe diseases such as sepsis or severe infections, it is very important to have an early diagnosis, as well as a prognosis and risk assessment for the patient's outcome, in order to find the optimal therapy and management. Therapeutic approaches must be very individual and vary from case to case. Therapeutic monitoring is necessary for advanced treatment and depends on the timing of treatment, the use of combination therapy and the optimization of drug dosing. Incorrect or missed treatment or patient administration tactics will increase the death rate hourly.

The therapeutic treatment according to the present invention may be an antibiotic treatment in which one or more "antibiotics" or "antibiotic agents" may be administered if an infection has been diagnosed or symptoms of an infectious disease have been identified.

The antibiotics or antibiotic agents of this invention also include potentially antifungal or antiviral compounds useful in the treatment of diagnosed infection or sepsis. The antibiotic agents commonly used in the treatment of any given infection are divided into classes of pathogens:

Gram-positive spectrum of action: penicillins (ampicillin, amoxicillin), penicillinase-resistant (dicloxacillin, oxacillin), cephalosporins (1st and 2nd generation), macrolides (erythromycin, clarithromycin, azithromycin), quinolones (gatifloxacin, moxifloxacin, levofloxacin) , vancomycin, sulfanilamide/trimethoprim, clindamycin, tetracyclines, chloramphenicol, linezolid, synercid.

Gram-negative spectrum: broad-spectrum penicillins (ticarcillin, clavulanate, piperacillin, tazobactam), cephalosporins (2nd, 3rd and 4th generation), aminoglycosides, macrolides, azithromycin, quinolones (ciprofloxacin), monobactams (azetreonam), sulfanilamide / trimethoprim, carbapenems (imipenem), chloramphenicol. Pseudomonas spectrum of action: ciprofloxacin, aminoglycosides, some 3rd generation cephalosporins, 4th generation cephalosporins, broad-spectrum penicillins, carbapenems.

Antifungal drugs: alliamins, amphotericin B, fluconazole and other azoles, itraconazole, voriconazole, posaconazole, ravoconazole, echinocandins, flucytosine, sordarins, chitin synthetase inhibitors, topoisomerase inhibitors, lipopeptides, pradimycins, liposomal nystatin, voriconazole, echinocandins, imidazole, triazole, thiazole, polyene.

Antiviral drugs: abacavir, acyclovir, activated caspase oligomerizer, adefovir amantadine, amprenavir (agenerase), ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, dinadosan, double-stranded RNA, doconazole, edoxudin, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliver, famciclovir, fixed dose combination (antiretroviral), fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitor, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type III interferon , interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxidine, metizazon, morpholinos, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, novir, oseltamivir (tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor (pharmacological), raltegravir, reverse transcription inhibitor iptase, ribavirin, ribozyme, rifampicin, rimantadine, ritonavir, RNase H, protease inhibitors, pyramidin, saquinavir, sofosbuvir, stavudine, synergistic enhancer (antiretroviral), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, cyclovala, truvada (Valtrex), Val Ganciclovir, Vikriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zidovudine.

In addition, antibiotic agents containing bacteriophages to treat bacterial infections, synthetic antimicrobial peptides, or iron antagonists/iron chelators can be used. In addition, therapeutic antibodies or an antagonist to pathogenic structures, e.g. anti-VAP antibodies, vaccination against resistant clones, administration of immune cells such as in vitro primed or modulated T effector cells, are antibiotic agents that represent treatment options for critically ill patients. such as patients with sepsis. Other antibiotic agents/treatments or therapeutic strategies against infection or to prevent new infections include the use of antiseptics, disinfectant products, anti-virulence agents such as liposomes, sanitation, wound care, surgery.

It is also possible to combine several of the above antibiotic agents or treatment strategies.

According to the invention, proADM and optionally PCT and/or other markers or clinical scores are used as markers for therapy monitoring, including prognosis, risk assessment, and risk stratification of a subsequent adverse health event in a patient who has been diagnosed as "critically ill". A person skilled in the art is able to obtain or develop means for identifying, measuring, detecting and/or quantifying any of the above proADM molecules or fragments or variants thereof, as well as other markers according to this invention in accordance with standard molecular biological practice.

The level of proADM or its fragments, as well as the levels of other markers, according to this invention, can be determined by any assay that reliably determines the concentration of the marker. In particular, mass spectrometry (MS) and/or immunoassays can be used, as shown in the accompanying examples. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of a macromolecule/polypeptide in solution using an antibody, or antibody binding fragment, or immunoglobulin.

Methods for determining proADM or other markers such as PCT used in the context of this invention are intended for this invention. As an example, a method selected from the group consisting of: mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassays (FIA), enzyme immunoassays (ELISA) can be used. , luminescent bead array assays, magnetic bead array assays, protein microarray assays, rapid form assays such as, for example, immunochromatographic stripe assay, rare earth cryptate assay, and automated systems/analyzers.

Determination of proADM and possibly other markers based on antibody recognition is a preferred embodiment of the invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, that is, molecules that contain an antigen-binding site that specifically binds (immunely reacts with) an antigen. According to the invention, antibodies can be both monoclonal and polyclonal antibodies. In particular, antibodies are used that specifically bind to at least proADM or fragments thereof.

An antibody is considered specific if its affinity for a molecule of interest, such as ADM or a fragment thereof, is at least 50 times higher, preferably 100 times higher, most preferably at least 1000 times higher than for other molecules contained in a sample containing a molecule of interest. It is well known in the art how to design and select antibodies with a given specificity. In the context of the invention, monoclonal antibodies are preferred. An antibody or antibody binding fragment specifically binds to the markers or fragments thereof defined herein. In particular, the antibody or antibody binding fragment binds to the ADM peptides defined herein. Thus, the peptides defined herein may also be epitopes to which antibodies specifically bind. Also, an antibody or antibody binding fragment is used in the methods and kits of the invention that specifically bind to ADM or proADM, in particular SF-proADM.

Also, an antibody or antibody binding fragment is used in the methods and kits of the invention that specifically bind to proADM or fragments thereof, and optionally to other markers of the invention, such as PCT. Illustrative immunoassays can be luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (FIA), enzyme immunoassay (ELISA), luminescent bead array assay, magnetic bead array assay, protein assay. microarrays, analyzes in a fast form, analysis with rare earth cryptates. Also, assays suitable for point-of-care testing and rapid form assays, such as, for example, immunochromatographic strip assays, may be used. Automated immunoassays such as the KRYPTOR assay are also provided.

Alternatively, other capture molecules or molecular scaffolds that specifically and/or selectively recognize proADM may be included in the scope of this invention instead of antibodies. As used herein, the term "capture molecules" or "molecular scaffolds" includes molecules that can be used to bind target molecules or molecules of interest, i.e., analytes (e.g., proADM, proADM, SF-proADM, and PCT) in a sample. Thus, the capture molecules must have an appropriate shape, both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, in order to specifically bind target molecules or molecules of interest. Thus, binding may, for example, be mediated by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic, or hydrogen bond interactions, or a combination of two or more of the above interactions, or covalent interactions between capture molecules, or a molecular scaffold. , and target molecules, or molecules of interest. In the context of this invention, capture molecules or molecular scaffolds, for example, may be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, a peptide, and a glycoprotein. Capture molecules or molecular scaffolds include, for example, aptamers, DARpin (Designed Ankyrin Repeat Proteins). Included are affimers and the like.

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

a) bringing the sample into contact with

i. a first antibody or antigen-binding fragment or derivative thereof specific for the first epitope of said proADM, and

ii. a second antibody or antigen-binding fragment or derivative thereof specific for a second epitope of said proADM; and

b) detection of binding of two antibodies or antigen-binding fragments or their derivatives with the specified proADM.

Preferably, one of the antibodies may be labeled and the other antibody may be associated with the solid phase or may be selectively associated with the solid phase. In a particularly preferred aspect of the assay, one of the antibodies is labeled and the other is either bound to the solid phase or may be selectively bound to the solid phase. The first antibody and the second antibody may be present as dispersed in a liquid reaction mixture, wherein the first labeling component, which is part of a labeling system based on fluorescent or chemiluminescent quenching or enhancement, is associated with the first antibody, and the second labeling component of said labeling system is associated with the second antibody, so that upon binding of both antibodies to said proADM or fragment(s) to be detected, a measurable signal is generated that allows the resulting sandwich complexes to be detected in the analyzed solution. The labeling system may contain a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

In a preferred embodiment, the method is implemented as a heterogeneous sandwich immunoassay, in which one of the antibodies is immobilized on a randomly selected solid phase, for example, on the walls of coated tubes (for example, polystyrene tubes; coated tubes; CT - coated tubes) or plates for microtitrations, for example, consisting of polystyrene, or on particles, such as, for example, magnetic particles, and the other antibody has a group similar to the detectable label, or allowing selective attachment to the label, and which serves to detect the formed sandwich structures. Also possible delayed or subsequent immobilization using suitable solid phases.

The method according to the invention can furthermore be implemented as a homogeneous method in which the sandwich complexes formed by the antibody/antibodies and the marker, proADM or a fragment thereof, to be detected remain suspended in the liquid phase. In this case, it is preferable that when two antibodies are used, both antibodies are labeled with parts of the detection system, which results in signal generation or signal initiation if both antibodies are integrated into one sandwich. Such methods should be implemented, in particular, as methods for enhancing fluorescence or detecting fluorescence quenching. In particular, a preferred aspect relates to the use of detection reagents that must be used in pairs, such as, for example, those described in US 4882733, EP 0180492 or EP 0539477, and given in the prior art. Thus, measurements are possible in which only reaction products containing both labeling components in one immune complex are detected directly in the reaction mixture. For example, such technologies are offered under the trademarks TRACE® (Time Resolved Amplified Cryptate Emission) or KRYPTOR®, implementing the principles of the above applications. Therefore, in particular preferred aspects, a diagnostic device is used to implement the method proposed herein. For example, the level of proADM, or a fragment thereof, and/or the level of any additional marker of the method proposed herein, such as PCT, is determined. In particular, in preferred aspects, the diagnostic device is a KRYPTOR®.

The level of the marker according to this invention, for example, proADM or its fragments, PCT or its fragments, or other markers, can also be determined by methods based on mass spectrometry (MS). Such a method may include determining the presence, amount or concentration of one or more modified or unmodified peptide fragments, e.g., proADM or PCT, in said biological sample or protein cleavage product (e.g., tryptic cleavage) of said sample, and optionally separating the sample from using chromatographic methods, and performing MS analysis with the prepared and optionally separated sample. For example, MS analysis can use selective reaction monitoring mass spectrometry (SRM), multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM), in particular, to determine the amount of proADM or its fragments.

In this document, the term "mass spectrometry" or "MS" refers to an analytical method that allows the identification of compounds by their mass. To improve the mass separation and mass determination capabilities of mass spectrometry, samples can be processed prior to MS analysis. Accordingly, the invention relates to MC detection methods that can be combined with immunoenrichment technologies, sample preparation methods and/or chromatographic methods, preferably liquid chromatography (LC), more preferably high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography. (UHPLC). Sample preparation methods include methods for lysis, fractionation, digestion of the sample to peptides, separation, enrichment, dialysis, desalting, alkylation, and/or reduction of peptides. However, these steps are not mandatory. Selective detection of analyte ions can be performed using tandem mass spectrometry (MS/MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, the term "mass selection" means separating ions having a given m/z or a narrow range of m/z values) followed by fragmentation of the selected ions and mass analysis of the resulting product ions ( fragment).

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

In addition, levels (including control levels) can be determined using mass spectrometry-based methods, such as methods that determine the relative amount or determine the absolute amount of the protein or fragment of interest.

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

1. Determination of increased or decreased amount of target protein by comparing the area of the characteristic SRM peak (selective response control) from a given target peptide fragment found in the sample with the area of the same characteristic SRM peak of the target peptide fragment at least in the second, third , a fourth or more biological samples.

2. Determination of increased or decreased amounts of a target protein by comparing the area of the characteristic SRM peak from a given target peptide found in a sample with the areas of characteristic SRM peaks obtained for peptide fragments from other proteins in other samples obtained from different or different biological sources, where a comparison of the characteristic SRM peak areas between two samples for a peptide fragment is normalized for, for example, the amount of protein analyzed in each sample.

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

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

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

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

2. Peptides can also be quantified using external calibration curves. The bell curve approach uses a constant amount of heavy peptide as an internal standard and a varying amount of light synthetic peptide added to the sample. A representative matrix similar to that of the test samples should be used to generate standard curves to account for the effect of the matrix. In addition, the inverse curve method circumvents the problem of endogenous analyte in the matrix, where a constant amount of light peptide is injected on top of the endogenous analyte to create an internal standard, and different amounts of heavy peptide are injected to create a set of concentration standards. The same amount of standard peptide is added to test samples for comparison with normal distribution curves or inverse curves as the internal standard added to the matrix used to create the calibration curve.

This invention further relates to kits, the use of kits, and methods in which such kits are used. This invention relates to kits for implementing the methods described above and below in this document. The definitions given in this document, for example, proposed in relation to the methods, also apply to the kits according to this invention. In particular, the invention relates to kits for monitoring therapy, including prognosis, risk assessment or risk stratification of a subsequent adverse health event in a patient, while said kit contains

detection reagents for determining the level of proADM or fragment(s) thereof and optionally additionally for determining the level of PCT, lactate and/or C-reactive protein or fragment(s) thereof in a sample from a subject, and detection reagents for determining said level of proADM in the specified sample of the specified subject, and

- control data, such as a control level corresponding to the level of high and/or low severity of proADM, with the level of low severity being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l, and the level severe is greater than 6.5 nmol/l, preferably greater than 6.95 nmol/l, more preferably greater than 10.9 nmol/l and optionally PCT, lactate and/or C-reactive protein levels, with said data being controls preferably stored on a computer-readable medium and/or used in the form of computer executable code configured to compare certain levels of proADM or fragment(s) thereof and optionally further determined levels of PCT, lactate and/or C-reactive protein or fragment(s) thereof , with the specified control data.

As used herein, "control data" includes control level(s) of proADM and optionally PCT, lactate, and/or C-reactive protein. The levels of proADM and optionally PCT, lactate and/or C-reactive protein in a subject's sample can be compared to control levels contained in the control kit data. Control levels are described above in this document and are also illustrated in the accompanying examples. The control data may also include a control sample against which the level of proADM and optionally PCT, lactate and/or C-reactive protein is compared. The control data may also include guidance on the use of kits according to the invention.

The kit may additionally contain items useful for obtaining a sample, such as a blood sample, for example, the kit may contain a container, and the specified container contains a device for attaching the specified container to the cannula or syringe, is syringe-compatible for blood collection, has an internal pressure below atmospheric, one that is suitable for withdrawing a predetermined volume of sample into said container, and/or additionally contains detergents, chaotropic salts, ribonuclease inhibitors, chelating agents such as guanidine isothiocyanate, guanidine hydrochloride, sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate, RNase inhibitory proteins, and mixtures thereof , and / or a filter system containing nitrocellulose, silica matrix, ferromagnetic spheres, infusion return cup, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerin, EDTA, Tris, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pyrol, citrate and mixtures thereof.

As used herein, the term "detection reagent" or the like are reagents that are suitable for detecting the presence of the marker(s) described herein, eg, proADM, PCT, lactate, and/or C-reactive protein. Such exemplary detection reagents are, for example, ligands, such as antibodies or fragments thereof, that specifically bind to the peptide or epitopes of the marker(s) described herein. Such ligands can be used in immunoassays as described above. Additional reagents that are used in immunoassays to determine the level of the marker(s) may also be included in the kit and are referred to as detection reagents in this document. Detection reagents can also refer to reagents that are used to detect markers or fragments thereof by MS-based methods. Thus, such a detection reagent can also be reagents, such as enzymes, chemicals, buffers, etc., that are used to prepare a sample for MS analysis. The mass spectrometer can also be considered as a detection reagent. Detection reagents according to the invention can also be a calibration solution(s), for example, which can be used to determine and compare the level of the marker(s).

The sensitivity and specificity of a diagnostic and/or prognostic test depends not only on the analytical "quality" of the test, but also on the definition of what constitutes an abnormal result. In practice, plots of sensitivity versus false positive rate (ROC curves) are typically calculated by plotting the value of a variable against its relative frequency in “normal” (i.e., apparently healthy people who do not have infection), and “ sick" (eg, subjects having an infection) groups. For any particular marker (eg, ADM), the distribution of marker levels for subjects with and without disease/pathology is likely to overlap. Under such conditions, the test does not definitively distinguish normal from disease with 100% accuracy, and the area of overlap may indicate where the test cannot distinguish normal from disease. A threshold is selected below which the test is considered abnormal, and above which the test is considered normal, or below or above which the test is indicative of a particular pathology, such as an infectious disease. The area under the ROC curve is a measure of the likelihood that the proposed measurement will correctly identify the pathology. ROC curves may be used even if test results do not necessarily give an accurate number. As long as you can rank the results, you can create an ROC curve. For example, test results on samples with "disease" can be ranked according to the degree (for example, 1 = low, 2 = normal and 3 = high). This ranking may be related to the results in the "normal" group and the resulting ROC curve. These methods are well known in the art; see, for example, Hanley et al., 1982. Radiology 143: 29-36. Preferably, the threshold is chosen to provide an ROC area greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to +/-5% of 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 frequency of true positives. Thus, for a particular selected threshold, a value (1-specificity) can be determined and an appropriate sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that a measured marker level will correctly identify a disease or pathology. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

Accordingly, the invention includes administering an antibiotic suitable for treatment based on information obtained by the method described herein.

As used herein, the terms "comprising" and "comprising", or their grammatical variations, should be understood as indicating the claimed features, integers, steps or components, but not excluding the addition of one or more additional features, integers , steps, components, or groups of them. This term encompasses the terms "consisting of" and "consisting essentially of".

Thus, the terms "comprising"/"comprising"/"having" mean that any additional component (or, likewise, features, integers, steps, and the like) may be present. The term "consisting of" means that there is no additional component (or, likewise, features, integers, steps, etc.).

The term "consisting essentially of" or its grammatical variants when used herein should be understood as indicating the claimed features, integers, steps or components, but not excluding the addition of one or more additional features, integers, steps, components or their groups, but only if additional features, integers, steps, components or groups do not significantly change the main and new characteristics of the claimed composition, device or method.

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

The term "method" refers to methods, means, methods and procedures for performing a given task, including, but not limited to: methods, means, techniques and procedures, either already known or easily developed from known methods, tools, methods and procedures. , specialists in the chemical, biological and biophysical field of technology.

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

EXAMPLES

Example ways:

Study design and patients:

This study is a secondary analysis of a placebo-controlled trial of procalcitonin-adjusted sodium selenite antimicrobial therapy in severe sepsis (SISPCT) conducted in 33 multidisciplinary intensive care units (ICUs) across Germany from November 2009 to February 2013 (26 ). Eligibility criteria included adult patients >18 years of age with new severe sepsis or septic shock (<24 hours) as defined by SEPSIS-1 of the ACCP/SCCM Consensus Conference Committee, and were further classified according to the 2016 definitions (sepsis-3 and septic shock). shock-3) (4). Details of the study design, data collection and management have been described previously (26). The Ethics Committee of the University Hospital of Jena and all other centers approved the study, and written informed consent was obtained, if necessary. Biomarker measurements:

Patients were enrolled within 24 hours of diagnosis of severe sepsis or septic shock, and PCT, CRP, and lactate were measured immediately thereafter. PCT was measured on instruments with a measuring range of 0.02-5000 ng/ml and functional assay sensitivity and a lower detection limit of at least 0.06 ng/ml and 0.02 ng/ml, respectively. Collected additional blood samples from all patients and stored them in the central research laboratory in Jena at -80°C. Plasma SF-proADM concentrations were measured retrospectively (Kryptor®, Thermo Fisher Scientific, Germany) with a detection limit of 0.05 nmol/l. At enrollment, a clinical assessment of severity was performed, including dynamic organ failure assessment (SOFA), assessment of critical illness and chronic illness (APACHE) II, and simplified assessment of critical illness (SAPS) II.

Statistical analysis:

Differences in demographic and clinical characteristics in relation to 28-day mortality were assessed using y-test for categorical variables, and Student's t-test or Mann-Whitney U-test for continuous variables, depending on the normality of distribution. Normally and abnormally distributed values were expressed as mean (standard deviation) and median [first quartile-third quartile], respectively. The association between mortality and each biomarker and clinical score at all time points was assessed using the area under the ROC curves (AUROC) and Cox regression analysis, with multivariate analysis adjusted for age and the presence of comorbidities and septic shock. Patients were further divided into three severity subgroups (low, moderate and high) based on the calculation of two AUROC cut-offs across the group for each biomarker and clinical score at each time point, with a target sensitivity and specificity close to 90%. Additionally, a subgroup of clinically stable patients was identified by the absence of any ICU-related procedures or complications (including debridement procedures, emergency surgery, new infections, transfusion of blood products, colloid infusion, invasive mechanical ventilation, kidney/liver replacement, or vasoconstrictor therapy). , and worsening of the patient's general clinical parameters and symptoms), and additionally the group was identified by the corresponding low concentrations of SF-proADM, which did not show any increase since the previous measurement. Mortality and mean length of stay were calculated in both groups and compared with the group of patients who were discharged at each given time point.

As a result, two models were constructed stratifying patients with PCT changes of 20% (from baseline to day 1 based on the average decline in PCT observed over that time period) and 50% (from baseline to day 4 on based on the previously constructed model (26)). Patient subgroups were subsequently defined based on levels of severity based on SF-proADM levels and calculated corresponding mortality rates. Mortality risk in each subgroup was calculated using Cox regression analysis and illustrated with Kaplan-Meier curves. The predicted risk of developing new infections and the need for debridement procedures and emergency surgery within days 4-7 were subsequently explored in the model from baseline to day 4. All data were analyzed using the R software for statistics (version 3.1.2).

Example 1: Patient Characteristics

Characteristics of patients after enrollment in the study are shown in Table 1.

A total of 1089 patients with severe sepsis (13.0%) or septic shock (87.0%) were analyzed, with 445 (41.3%) and 633 (58.7%) patients also meeting the criteria for sepsis - 3 and septic shock - 3, respectively. The admitted patients had a mean age of 65.7 (13.7) years and a mean SOFA score of 10.0 (3.3) points. The 28-day all-cause mortality rate (N=1076) was 26.9% (sepsis - 3: 20.0%; septic shock - 3: 32.1%), with an in-hospital mortality rate of 33.4% (sepsis - 3 : 24.4%; septic shock - 3: 40.4%). Infections arising from a single focus were found in 836 patients (77.7%), with the most common pneumological (N=324; 30.1%), intra-abdominal (N=252; 23.4%), urogenital (N= 57; 5.3%) and in bone/soft tissues (N=50; 4.6%) by the primary source. The corresponding mortality rates were 26.5%, 24.6%, 22.8% and 28.0%, respectively. Infection with multiple foci of origin was found in 240 (22.3%) patients. The most common causes of death included sepsis-induced multiple organ failure (N=132; 45.7%), refractory septic shock (N=54; 18.7%), death from pre-existing disease (N=35; 12.1% ), and acute respiratory failure (N=17; 5.9%). Other causes such as cardiogenic and hemorrhagic shock, pulmonary embolism, cerebral edema, myocardial infarction, and cardiac arrhythmia accounted for a combined mortality rate of 8.6%. Restriction therapy was applied to 3.4% of patients.

Example 2: Association of key biomarkers and clinical scores with mortality

Univariate and multivariate Cox regression analysis showed that SF-proADM had the strongest association with 28-day mortality among the entire group of patients, as well as in subgroups with sepsis-3 and septic shock-3 (Table 2). The corresponding analysis of AUROC revealed significant differences in all comparisons of biomarkers and clinical scores with SF-proADM, with the exception of APACHE II (sepsis-3 patient group).

Similar results were also found for mortality prediction at 7 days, 90 days, ICU, and hospital (Table 3), along with the addition of SF-proADM to all possible combinations of biomarkers and clinical scores (N=63), significantly increasing predictive power (Table 3). four).

Example 3: Identification of High Risk Patients

The entire patient cohort was further stratified based on existing levels of SOFA severity, and the performance of biomarkers and clinical scores in predicting 28-day mortality assessed in each subgroup. SF-proADM showed the highest precision of all parameters in the low (SOFA≤7) and moderate (8≤SOFA≤13) SOFA subgroups (Table 5; Table 6). Subsequently, two corresponding SF-proADM cut-offs were calculated to identify subgroups of low (≤2.7 nmol/L) and high (>10.9 nmol/L) severity at baseline. Compared to SOFA, a more accurate reclassification can be made for both thresholds - low (SF-proADM vs. SOFA: N=265 vs. 232; 9.8% vs. 13.8% mortality), and high (SF-proADM vs. SOFA: N=161 vs. 155; 55.9% vs. 41.3%) severity (Table 7). A subgroup of 94 patients (9.3%) with high SF-proADM concentrations and corresponding low or moderate SOFA had 28 and 90-day mortality rates of 57.4% and 68.9%, respectively, compared with 19.8% and 30. 8% in the remaining group of patients with low and moderate SOFA values. Similar patterns can be found for SAPS II, APACHE II and lactate, respectively (Tables 8-10).

Example 4: Identification of low-risk patients during their stay in a protected area

The study population includes a subgroup of clinically stable patients who have not experienced ICU-related procedures or complications such as debridement procedures, emergency surgery, new infections, transfusion of blood products, colloid infusion, invasive mechanical ventilation, kidney replacement/ liver, worsening of the general clinical indicators and symptoms of the patient. This group of clinically stable patients was classified as low risk.

SF-proADM showed the strongest association with 28-day mortality among all subsequent time points (Table 11) and could provide a stable cut-off value of ≤2.25 nmol/L in identifying the low-risk group of patients, leading to the assignment of more patients to the low-risk group. mortality rate compared with other biomarkers and clinical assessments (Table 12). Accordingly, on day 4, 290 patients could be identified with a low severity level of SF-proADM, of which 79 (27.2%) were clinically stable and had no increase in SF-proADM concentrations compared with the last measurement (Table 13 ). A persistently low concentration of SF-proADM could be detected in 51 (64.6%) patients, while a decrease from moderate to low severity could be observed in 28 (35.4%) patients. The median length of stay in the OPT was 8 [7–10] days, and the mortality rate at days 28 and 90 was 0.0% and 1.4%, respectively. In comparison, only 43 patients were discharged from the OCU on day 4, with 28- and 90-day mortality rates of 2.3% and 10.0%. Analysis of SF-proADM concentrations in this group of patients indicated a range of values, with 20 (52.6%), 16 (42.1%) and 2 (5.3%) patients who had a level of low, moderate and high severity, respectively . Similar results were obtained for patients who remained in the ICU on days 7 and 10. SF-proADM with a stable cut-off value of ≤2.25 nmol/L can identify more low-risk patients with lower mortality rates compared to other biomarkers and clinical assessments. Based on this, more patients can be discharged from the ICU compared to classification systems without ADM. By discharging more patients, the hospital can fill ICU beds more efficiently and benefit from cost avoidance.

Example 5: Additional impact of SF-proADM on procalcitonin-adjusted therapy

Cox's time-dependent regression analysis showed that the earliest significant additional increase in prognostic information relative to baseline SF-proADM values could be observed on day 1, with subsequent single or cumulative measurements leading to significantly stronger associations with 28-day mortality. (Table 14). Thus, two models of algorithms adjusted for PCT were constructed to examine changes in PCT from baseline to either day 1 or day 4, with appropriate subgroup analysis based on severity classification by level of SF-proADM.

In patients with PCT concentration reduction ≥20% from baseline to day 1 (Table 15 and Table 16) or ≥50% from baseline to day 4 (Table 17 and Table 18), 28-day mortality rates were found to be 18 .3% (N=458) and 17.1% (N=557), respectively. This decreased to 5.6% (N=125) and 1.8% (N=111) when patients had consistently low levels of SP-proADM, although it increased to 66.7% (N=27) and 52, 8% (N=39) in patients with persistently high SF-proADM (HR (hazard ratio) [95% CI]: 19.1 [8.0-45.9] and 43.1 [10.1-184 ,0]).

In addition, patients with declining PCT values ≥50% (from baseline to day 4) but persistently high or moderate concentrations of SF-proADM had a significantly higher risk of developing subsequent nosocomial infections (RR [95% CI]: high concentrations : 3.9 [1.5-10.5]; mean concentrations: 2.4 [1.1-5.1] compared with patients with persistently low concentrations; mean concentrations: 2.9 [1.2-6 .8]) compared with a decrease from moderate to low concentrations), or the need for emergency surgery (RR [95% CI]: average concentrations: 2.0 [1.1-3.7] compared with a decrease from moderate to low concentrations). Conversely, patients with increasing from moderate to high concentrations were more likely to require clearance of the site of infection compared with patients with constant average (RR [95% CI]: 3.2 [1.3-7.6]) or decreasing (RR [95 %>] CI]: medium to low: 8.7 [3.1-24.8]); high to medium: 4.6 [1.4 -14.5]) values. When PCT levels were not reduced by ≥50%, a significantly increased risk of requiring emergency surgery was observed if SF-proADM concentrations were either persistently high (RR [95% CI]: 5.7 [1.5–21.9]), or moderate (HR [95% CI]: 4.2 [1.3–13.2]), as opposed to consistently low.

Example 6: Association of key biomarkers and clinical scores with mortality

SF-proADM showed the strongest association in patients with pneumological and intra-abdominal infections, as well as in patients with gram-positive infections, regardless of the origin of the infection (Tables 19-20). When patients were grouped according to ER with surgery, ER with non-operative intervention, and elective surgery leading to ICU admission, SF-proADM provided the strongest and most balanced association with 28-day mortality in all groups (Table 21).

Example 7: Correlation of Biomarkers and Clinical Scores with SOFA at Baseline and Day 1

SF-proADM had the highest correlation of all biomarkers with SOFA score at baseline, which was significantly increased when baseline values were compared to SOFA scores on day 1. The highest correlation can be found between SF-proADM and SOFA at day 10, however, differences were found across the individual sub-scores of SOFA (Tables 22-24).

Example 8: Identification of High Risk Patients

Similar results can be obtained in a subgroup of 124 patients (12.0%) with high SF-proADM concentrations and either low or moderate SAPS II values (high SF-proADM subgroup [54.8% and 65.6% mortality] ; the rest of the SAPS II group [19.7% and 30.0% mortality]), as well as in 109 (10.6%) patients with either low or moderate APACHE II values (high SF-proADM subgroup: [56.9 % and 66.7% mortality]; the rest of the APACHE II group: [19.5% and 30.3% mortality]).

Example 9 Improved Procalcitonin (PCT) Adjusted Therapy with Combination of PCT and ADM

Two models of PCT-adjusted algorithms were built to examine changes in PCT from baseline to either day 1 or day 4, with appropriate subgroup analysis based on severity classification by level of SF-proADM (Tables 25-30).

The previous examples show the addition of ADM in patients with a decrease in PCT <20% or <50%, as well as in patients whose PCT decreased by ≥20% or ≥50%. However, additional analysis shows that ADM can be added regardless of the % reduction or even increase in PCT. Decreased PCT values may account for patients who appear to be working on antibiotic treatment, so the clinician believes they are on the right path to survival (i.e., elimination of the root cause of sepsis—bacteria—should lead to an improvement in the patient's condition).

For example, in some patients PCT levels decrease from baseline (day of admission) to day 1 with a 19% mortality rate on day 28. When ADM is additionally measured, patients with low ADM can be inferred to have a much higher chance of survival or a much lower chance of dying (Table 25; compare the 19% mortality rate for declining PCT alone with the 5% mortality rate for PCT + low ADM). With a reduced risk of death, patients can be discharged from the ICU with more confidence, or fewer diagnostic tests are required (i.e. you know they are on the right path to recovery).

On the other hand, new measures should be considered for those with high ADM values. They are at much higher risk for mortality (compare the 19% mortality rate for decreasing PCT alone with the 58.8% mortality rate for PCT + low ADM). The doctor believes that the patient's condition is improving due to a decrease in the PCT value, but in fact the concentration of ADM remains unchanged. Therefore, it can be concluded that the treatment does not work and needs to be adapted as soon as possible).

Similarly, ADM can help stratify patients with elevated PCT values (Table 25).

Development of new infections

Changes in PCT and SF-proADM were analyzed in two models, either from baseline to day 1 or from baseline to day 4. Patients were grouped by overall PCT changes and severity levels by SF-proADM levels.

The number of new infections during days 1, 2, 3 and 4 (Table 26) and days 4, 5, 6 and 7 (Table 27) was subsequently calculated for each patient who was present on day 1 or day 4, respectively. In some cases, patients were discharged during the observation period. It is assumed that there have been no new infections since discharge. Patients with multiple infections during observation days were counted as having one new infection.

As a clinical conclusion, patients with high concentrations of SF-proADM should potentially be treated with a broad-spectrum antibiotic on admission to the ICU, in combination with others, to stop the development of new infections. Special care should be taken with these patients because of their high susceptibility to new infections.

The need to clean the hearth

Changes in PCT and SF-proADM were analyzed in two models, either from baseline to day 1 or from baseline to day 4. Patients were grouped by overall PCT changes and severity levels by SF-proADM levels.

The number of lesions cleared on days 1, 2, 3, and 4 (Table 28) and days 4, 5, 6, and 7 (Table 29) were subsequently calculated for each patient who was present on day 1 or day 4, respectively. In some cases, patients were discharged during the observation period.

Need for emergency surgery

Changes in PCT and SF-proADM were analyzed in two models, either from baseline to day 1 or from baseline to day 4. Patients were grouped by overall PCT changes and severity levels by SF-proADM levels.

The number of cases/requests for emergency surgery during days 1, 2, 3 and 4 (Table 30) was subsequently calculated for each patient who was present on day 1. In some cases, patients were discharged during the follow-up period.

Example 10: Need to Change or Modify Antibiotic Treatment

Combined with a PCT-adjusted antibiotic treatment algorithm, SF-proADM can stratify those patients who will need to change or modify their antibiotic treatment in the future from those who do not. Changes in PCT and SF-proADM were analyzed in two models, either from baseline to day 1 or from baseline to day 4. Patients were grouped by overall PCT changes and severity levels by SF-proADM levels.

The percentage of day 4 antibiotic changes required for each patient group was subsequently calculated (Tables 31 and 32).

In patients with declining PCT values ≥50%

Patients with increasing levels of SF-proADM, from low to moderate levels, were more likely to require modification of antibiotic treatment on day 4 than those with persistently low levels (odds ratio [95% CI]: 1.5 [0, 6-4.1]).

In patients with declining PCT values <50%

Patients with either increasing levels of SF-proADM, moderate to severe, or persistently high levels, were also more likely to need changes in their antibiotic treatment by day 4 than patients with persistently low levels of SF-proADM (odds ratio [95% CI]: 5.9 [1.9 - 18.1] and 2.9 [0.8 - 10.4], respectively).

Conclusion

Despite an increase in PCT concentrations, either from baseline to day 1 or from baseline to day 4, patients with persistently low SP-proADM concentrations had significantly fewer modifications to their antibiotic treatment than those with persistently moderate or high concentrations.

As a clinical judgment, when faced with increased PCT concentrations, the clinician should check the patient's SP-proADM levels before deciding to change antibiotics. For those with low concentrations of SF-proADM, either increasing the dose or increasing the concentration of the same antibiotic should be considered before considering changing it. For patients with higher concentrations of SF-proADM, earlier antibiotic changes should be considered (ie, on days 1 to 3, compared to day 4).

Discussion of examples

Accurate and rapid assessment of disease severity is critical to initiating the most appropriate treatment as soon as possible. Indeed, delayed or ineffective treatment may lead to an overall worsening of the patient's clinical condition, leading to further treatment becoming less effective and increasing the likelihood of a worse overall outcome (8, 27). As a result, many biomarkers and clinical severity scores have been proposed to address this unmet clinical need, with the Dynamic Organ Failure Assessment (SOFA) currently being highlighted as the most appropriate tool, and as a result playing a central role in the 2016 definition of Sepsis-3. (four). This secondary analysis of the SISPCT study (26) compared stepwise measurements of common biomarkers and clinical assessments such as lactate, procalcitonin (PCT) and SOFA with those of a marker of microcirculatory dysfunction, SF-proADM, for the first time in a large cohort of patients with severe sepsis and septic shock.

Our results show that early use of SF-proADM within the first 24 hours of sepsis diagnosis has, as a consequence, a very strong association with short-, medium- and long-term mortality when compared to all other biomarkers or measures. Previous studies largely support our findings (17, 28, 29), but conflicting results (30) may be partly explained by the smaller sample size analyzed, as well as other factors highlighted in this study, such as microbial species, source of infection, and previous a history of surgery prior to the development of sepsis, all of which may affect the performance of the biomarker, thus increasing the potential variability of results in small study groups. In addition, our study also almost completely confirms the results of a previous study (17), highlighting the superior efficacy of SF-proADM in patients with low to moderate organ dysfunction. Indeed, Andaluz-Ojeda et al. (17) attach great importance to the group of patients with a low level of organ dysfunction, because "this group represents either the earliest manifestation in the clinical course of sepsis, and/or a less severe form of the disease." However, acceptable performance in all severity groups in terms of predicting mortality can be maintained, which also occurred in both groups of patients defined according to the criteria for sepsis-3 and septic shock-3.

The analysis of serial measurements taken after the onset of sepsis made it possible to identify specific groups of patients based on the severity of the disease. The identification of low and high risk patients was of significant interest in our analysis. In many ICUs, the demand for ICU beds may periodically exceed availability, which can lead to inappropriate scheduling, resource allocation, and a subsequent decrease in the likelihood of correct admission to the ICU (32-35). Therefore, an accurate assessment of patients at low risk of hospital death who are eligible for early discharge from the ICU to the intensive care unit can be of significant benefit. In our study, SP-proADM measured at each time point could identify more low-grade patients with the lowest rates of ICU, hospitalization, and 28-day mortality. An additional analysis of a group of patients with low severity and no additional ICU-specific therapies showed that an additional 4 days of ICU stay were observed for each time interval after biomarker measurements were taken. Compared to a group of patients who were actually discharged at each time point, a biomarker approach to accurately identify patients with low severity resulted in a reduction in 28-day and 90-day mortality. Indeed, patients who were discharged had varying concentrations of low, moderate, and high levels of SF-proADM, which was subsequently reflected in a higher mortality rate. However, it is not known whether a few more patients in this group required additional ICU treatment for non-microcirculatory and non-life-threatening complications, or whether those ICU beds were available. However, such a biomarker-based approach for ICU discharge, in addition to physician judgment, may improve proper patient stratification, with associated clinical benefits and potential cost savings.

Conversely, identifying high-risk patients who may require early and targeted treatment to prevent subsequent clinical deterioration may be of even greater clinical relevance. Significant cost savings and reductions in antibiotic use have already been observed with the PCT-adjusted algorithm in SISPCT and other studies (26, 36, 37), but relatively high mortality rates can still be observed even when PCT values appear to , are steadily declining. Our study found that the addition of SF-proADM to a model of PCT decline on subsequent days in the ICU identified low, intermediate, and high-risk patient populations, with increasing and decreasing levels of severity in terms of SF-proADM from baseline to day 1, providing a sensitive and early indication of treatment success. In addition, anticipating the need for future debridement or emergency surgery, as well as susceptibility to developing new infections, can be a significant advantage in initiating additional therapeutic and interventional strategies, thus trying to prevent any future clinical complications at an early stage.

The strength of our study includes a thorough examination of several different low- and high-severity subgroups from a database of randomized trials, adjusting for potential unknowns, and including the largest sample size of patients with sepsis characterized by both definitions of SEPSIS 1 and 3, as well as information on the kinetics of SF-proADM.

In conclusion, SF-proADM is superior to other biomarkers and clinical assessments of disease severity in its ability to determine the risk of death in patients with sepsis, both at initial diagnosis and during ICU treatment. Accordingly, SF-proADM can be used as a tool to identify patients with high disease severity who may require alternative diagnostic and therapeutic interventions, and patients with low disease severity who may potentially be eligible for early ICU discharge in combination with a lack of ICU-specific therapies.

tables

All multivariate p<0.0001 except PCT and CRP for 7-day mortality (0.0015 and 0.0843, respectively).

BIBLIOGRAPHY

1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engi J Med. Apr 17 2003;348(16): 1546-1554.

2. Kaukonen KM, Bailey M, Suzuki S, Pilcher D, Bellomo R. Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand, 2000-2012. JAMA. Apr 02 2014;311(13):1308-1316.

3. Vincent JL, Sakr Y, Sprung CL, et al. Sepsis in European intensive care units: results of the SOAP study. Crit Care Med. Feb 2006;34(2):344-353.

4. Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. Feb 23 2016;315(8):801-810.

5. Kopterides P, Siempos, II, Tsangaris I, Tsantes A, Armaganidis A. Procalcitonin-guided algorithms of antibiotic therapy in the intensive care unit: a systematic review and meta-analysis of randomized controlled trials. Crit Care Med. Nov 2010;38(11):2229-2241.

6. de Jong E, van Oers JA, Beishuizen A, et al. Efficacy and safety of procalcitonin guidance in reducing the duration of antibiotic treatment in critically ill patients: a randomised, controlled, open-label trial. Lancet Infect Dis. Jul 2016;16(7):819-827.

7. Harbarth S, Holeckova K, Froidevaux C., et al. Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis. Am J Respir Crit Care Med. Aug 1 2001;164(3):396-402.

8. Andriolo BN, Andriolo RB, Salomao R, Atallah AN. Effectiveness and safety of procalcitonin evaluation for reducing mortality in adults with sepsis, severe sepsis or septic shock. Cochrane Database SystRev. Jan 18 2017;1:CD010959.

9. Temmesfeld-Wollbruck B, Brell B, David I, et al. Adrenomedullin reduces vascular hyperpermeability and improves survival in rat septic shock. Intensive Care Med. Apr 2007;33(4):703-710.

10. Muller-Redetzky HC, Will D, Hellwig K, et al. Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin. Critcare. 2014; 18(2):R73.

11. Vallet B. Endothelial cell dysfunction and abnormal tissue perfusion. Crit Care Med. May2002;30(5 Suppl):S229-234.

12. Gonzalez-Rey E, Chorny A, Varela N, Robledo G, Delgado M. Urocortin and adrenomedullin prevent lethal endotoxemia by down-regulating the inflammatory response. Am J Pathol. Jun 2006; 168(6): 1921-1930.

13. Carrizo GJ, Wu R, Cui X, Dwivedi AJ, Simms HH, Wang P. Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia-reperfusion. surgery. Feb 2007;141(2):245-253.

14. Brell B, Hippenstiel S, David I, et al. Adrenomedullin treatment abolishes ileal mucosal hypoperfusion induced by Staphylococcus aureus alpha-toxin-an intravital microscopic study on an isolated rat ileum. Crit Care Med. Dec 2005;33(12):2810-2016.

15. Brell B, Temmesfeld-Wollbruck B, Altzschner I, et al. Adrenomedullin reduces Staphylococcus aureus alpha-toxin-induced rat ileum microcirculatory damage. Crit Care Med. Apr2005;33(4):819-826.

16. Vigue B, Leblanc PE, Moati F, et al. Mid-regional pro-adrenomedullin (MR-proADM), a marker of positive fluid balance in critically ill patients: results of the ENVOL study. Critcare. Nov 09 2016;20(1):363.

17. Andaluz-Ojeda D, Cicuendez R, Calvo D, et al. Sustained value of proadrenomedullin as mortality predictor in severe sepsis. J Infect. Jul 2015;71(1): 136-139.

18. Hartmann 0, Schuetz P, Albrich WC, Anker SD, Mueller B, Schmidt T. Time-dependent Cox regression: serial measurement of the cardiovascular biomarker proadrenomedullin improves survival prediction in patients with lower respiratory tract infection. IntJ Cardiol. Nov 29 2012;161(3):166-173.

19. Albrich WC, Dusemund F, Ruegger K, et al. Enhancement of CURB65 score with proadrenomedullin (CURB65-A) for outcome prediction in lower respiratory tract infections: derivation of a clinical algorithm. BMC Infect Dis. 2011;11:112.

20. Albrich WC, Ruegger K, Dusemund F, et al. Optimized patient transfer using an innovative multidisciplinary assessment in Kanton Aargau (OPTIMA I): an observational survey in lower respiratory tract infections. Swiss Med Wkly. 2011;141:wl3237.

21. Albrich WC, Ruegger K, Dusemund F, et al. Biomarker-enhanced triage in respiratory infections: a proof-of-concept feasibility trial. Eur Respir J. Oct 2013;42(4): 1064-1075.

22. Riera J, Senna A, Cubero M, Roman A, Rello J, Study Group Investigators TV. Primary Graft Dysfunction and Mortality Following Lung Transplantation: A Role for Proadrenomedullin Plasma Levels. Am J Transplant. Oct 13 2015.

23 Schoe A, Schippers EF, Struck J, et al. Postoperative pro-adrenomedullin levels predict mortality in thoracic surgery patients: comparison with Acute Physiology and Chronic Health Evaluation IV Score*. Crit Care Med. Feb 2015;43(2):373-381.

24. Tyagi A, Sethi AK, Girotra G, Mohta M. The microcirculation in sepsis. Indian J Anaesth. Jun 2009;53(3):281-293.

25. Hernandez G, Bruhn A, Ince C. Microcirculation in sepsis: new perspectives. Curr Vasc Pharmacol. Mar 01 2013; 11(2): 161-169.

26 Bloos F, Trips E, Nierhaus A, et al. Effect of Sodium Selenite Administration and Procalcitonin-Guided Therapy on Mortality in Patients With Severe Sepsis or Septic Shock: A Randomized Clinical Trial. JAMA Intern Med. Sep 01 2016;176(9):1266-1276.

27. Bloos F, Ruddel H, Thomas-Ruddel D, et al. Effect of a multifaceted educational intervention for anti-infectious measures on sepsis mortality: a cluster randomized trial. Intensive Care Med. May 02 2017.

28. Enguix-Armada A, Escobar-Conesa R, La Torre AG, De La Torre-Prados MV. Usefulness of several biomarkers in the management of septic patients: C-reactive protein, procalcitonin, presepsin and mid-regional pro-adrenomedullin. Clin Chem Lab Med. Jun 17 2015.

29. Christ-Crain M, Morgenthaler NG, Struck J, Harbarth S, Bergmann A, Muller B. Mid-regional pro-adrenomedullin as a prognostic marker in sepsis: an observational study. Critcare. 2005;9(6):R816-824.

30. Suberviola B, Castellanos-Ortega A, Ruiz Ruiz A, Lopez-Hoyos M, Santibanez M. Hospital mortality prognostication in sepsis using the new biomarkers suPAR and proADM in a single determination on ICU admission. Intensive Care Med. Nov 2013;39(11): 1945-1952.

31. Gillmann HJ, Meinders A, Larmann J, et al. Adrenomedullin Is Associated With Surgical Trauma and Impaired Renal Function in Vascular Surgery Patients. J Intensive Care Med. Jan 01 2017:885066616689554.

32. Garrouste-Orgeas M, Montuclard L, Timsit JF, Misset B, Christias M, Carlet J. Triaging patients to the ICU: a pilot study of factors influencing admission decisions and patient outcomes. Intensive Care Med. May 2003;29(5):774-781.

33. Garrouste-Orgeas M, Montuclard L, Timsit JF, et al. Predictors of intensive care unit refusal in French intensive care units: a multiple-center study. Crit Care Med. Apr 2005;33(4):750-755.

34. Mery E, Kahn JM. Does space make waste? The influence of ICU bed capacity on admission decisions. Critcare. May 08 2013;17(3):315.

35. Orsini J, Blaak C, Yeh A, et al. Triage of Patients Consulted for ICU Admission During Times of ICU-Bed Shortage. J Clin Med Res. Dec 2014;6(6):463-468.

36. Kip MM, Kusters R, MJ IJ, Steuten LM. A PCT algorithm for discontinuation of antibiotic therapy is a cost-effective way to reduce antibiotic exposure in adult intensive care patients with sepsis. J Med Econ. 2015;18(11):944-953.

37. Wilke MH, Grube RF, Bodmann KF. The use of a standardized PCT-algorithm reduces costs in intensive care in septic patients - a DRG-based simulation model. Eur J Med Res. Dec 02 2011;16(12):543-548.

Claims (39)

1. A method of administering antibiotic treatment to a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, comprising: - taking the first sample from the specified patient, - taking a second sample from the specified patient at a certain point in time after the first sample was taken and after the start of antibiotic treatment, - determination of the levels of procalcitonin (PCT) or its fragment(s) in the first and second sample, and - determination of the level of proADM (proadrenomedullin) or its fragment(s) in at least the second sample, with a lower level of PCT or its fragment(s) in the second sample compared to the first sample and the level of high severity of proADM or its fragment(s) in the second sample, or an increased level of severity of proADM or its fragment(s) in the second sample compared to the first sample, including an increase from low severity to moderate or high severity, or from moderate to high severity, indicate the need changes in one or more antibiotic agents, while - the low severity level of proADM or its fragment(s) is below 4 nmol/l, - the level of moderate severity of proADM or its fragment(s) is above 4 nmol/l and - below 6.5 nmol/l, and the level of high severity of proADM or its fragment(s) is above 6.5 nmol/l. 2. The method of claim. 1, characterized in that the first sample is taken when the symptoms of an infectious disease in the specified patient, and the second sample is taken after the initial determination of these symptoms and the start of antibiotic treatment. 3. The method of claim. 2, characterized in that the second sample is taken from the specified patient within 30 minutes after the detection of symptoms of an infectious disease and the start of antibiotic treatment, or at least after 30 minutes, 1 hour, 2 hours, 6 hours and / or 12 hours after identifying the symptoms of an infectious disease and starting antibiotic treatment. 4. The method according to claim 2, characterized in that said second sample is taken from said patient 12-36 hours and/or 3-5 days after the symptoms of the infectious disease are identified and antibiotic treatment is initiated. 5. The method according to any one of the preceding claims, characterized in that the patient is diagnosed with sepsis and/or septic shock. 6. The method according to any of the preceding paragraphs, including determining the level of SF-proADM (middle fragment of pro-adrenomedullin). 7. The method according to any of the previous paragraphs, characterized in that - an increased level of PCT or its fragment(s) in the second sample compared to the first sample, and - the level of moderate or severe proADM or its fragment(s) in the second sample indicates the need to change one or more antibiotic agents, - while the level of average severity of proADM or its fragment(s) is above 4 nmol/l, preferably above 3 nmol/l, more preferably above 2.7 nmol/l and below 6.5 nmol/l, preferably below 6.95 nmol/l, more preferably below 10.9 nmol/l, and - the level of high severity of proADM or its fragment(s) is above 6.5 nmol/l, preferably above 6.95 nmol/l, more preferably above 10.9 nmol/l. 8. A method according to any one of the preceding claims, further comprising determining the level of proADM or fragment(s) thereof in the first sample. 9. The method according to any one of the preceding claims, characterized in that an increased level of proADM or its fragment(s) in the second sample compared to the first sample indicates the need to change one or more antibiotic agents. 10. The method according to any of the previous paragraphs, characterized in that - more than 50% lower level of PCT or its fragment(s) in the second sample compared to the first sample, and - the level of moderate severity of proADM or its fragment(s) in the second sample compared to the level of low severity of proADM or its fragment(s) in the first sample indicate the need to change one or more antibiotic agents. 11. The method according to any one of paragraphs. 1-8, characterized in that - less than 50% lower level of PCT or its fragment(s) in the second sample compared to the first sample, and - the level of high severity of proADM or its fragment(s) in the second sample compared to the level of moderate severity of proADM or its fragment(s) in the first sample indicate the need to change one or more antibiotic agents. 12. The method according to any of the previous paragraphs, characterized in that the first and second the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample and/or a urine sample. 13. The method according to any one of the preceding claims, further comprising - determining the level of at least one additional biomarker or its fragment(s) in the first and second sample, and at least one additional biomarker is preferably lactate and/or C-reactive protein, wherein the levels of at least one additional biomarker in said first and second samples and the level of proADM or fragment(s) thereof in the second sample indicate whether a change in ongoing antibiotic treatment is required. 14. A method according to any one of the preceding claims, further comprising - determining at least one clinical assessment at the time of taking the first sample and at the time of taking the second sample, while at least one clinical assessment is preferably SOFA, APACHE II and/or SAPS II, wherein at least one clinical assessment at the time of said first and second sample collection and the level of proADM or fragment(s) thereof in the second sample indicates whether a change in ongoing antibiotic treatment is required. 15. A set for implementing the method according to any one of paragraphs. 1-14 containing: - detection reagents for determining the level of proADM or its fragment(s) and additionally reagents for determining the level of PCT or its fragment(s) in the subject's sample, and - control data including a control level corresponding to severe and/or mild severity of proADM, with low severity being below 4 nmol/l, preferably below 3 nmol/l, more preferably below 2.7 nmol/l and high severity above 6.5 nmol/l preferably above 6.95 nmol/l, more preferably above 10.9 mol/l, and - control data corresponding to PCT levels, wherein said control data is used in the form of computer executable code configured to compare certain levels of proADM or fragment(s) thereof, and additionally determined levels of PCT or fragment(s) thereof, with said control data.

Images